key: cord- - sdg ll authors: guo, sheng; yang, chengying; diao, bo; huang, xiaoyong; jin, meihua; chen, lili; yan, weiming; ning, qin; zheng, lixin; wu, yuzhang; chen, yongwen title: the nlrp inflammasome and il- β accelerate immunologically mediated pathology in experimental viral fulminant hepatitis date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: sdg ll viral fulminant hepatitis (fh) is a severe disease with high mortality resulting from excessive inflammation in the infected liver. clinical interventions have been inefficient due to the lack of knowledge for inflammatory pathogenesis in the virus-infected liver. we show that wild-type mice infected with murine hepatitis virus strain- (mhv- ), a model for viral fh, manifest with severe disease and high mortality in association with a significant elevation in il- β expression in the serum and liver. whereas, the viral infection in il- β receptor-i deficient (il- r (-/-)) or il- r antagonist (il- ra) treated mice, show reductions in virus replication, disease progress and mortality. il- r deficiency appears to debilitate the virus-induced fibrinogen-like protein- (fgl ) production in macrophages and cd (+)gr- (high) neutrophil infiltration in the liver. the quick release of reactive oxygen species (ros) by the infected macrophages suggests a plausible viral initiation of nlrp inflammasome activation. further experiments show that mice deficient of p (phox), a nicotinamide adenine dinucleotide phosphate (nadph) oxidase subunit that controls acute ros production, present with reductions in nlrp inflammasome activation and subsequent il- β secretion during viral infection, which appears to be responsible for acquiring resilience to viral fh. moreover, viral infected animals in deficiencies of nlrp and caspase- , two essential components of the inflammasome complex, also have reduced il- β induction along with ameliorated hepatitis. our results demonstrate that the ros/nlrp /il- β axis institutes an essential signaling pathway, which is over activated and directly causes the severe liver disease during viral infection, which sheds light on development of efficient treatments for human viral fh and other severe inflammatory diseases. viral fulminant hepatitis (fh) is a clinical syndrome characterized by massive necrosis of hepatocytes along with hepatic encephalopathy during the infections [ ] . despite advances in the development of antiviral drugs, a poor understanding of the immune mechanisms underlying viral fh has largely stalled the identification of effective clinical interventions. fortunately, the recent development of an animal model of fh using murine hepatitis virus strain- (mhv- ) infection has provided insights in understanding the pathogenesis and developing novel therapeutics for the disease [ ] . mhv- is a single-stranded, positive-sense rna virus belonging to the coronavirus family [ ] . the hallmarks of mhv- -induced fh in susceptible balb/cj and c bl/ mice include the appearance of liver sinusoidal thrombosis and hepatocellular necrosis, resulting from over expression of a virus-induced, monocyte/macrophage-specific procoagulant, fibrinogen-like protein- (fgl ). liver accumulation of fgl directly activates the coagulation cascades, a phenomenon known as virus induced procoagulant activity [ ] . mhv- -induced fh exhibits a syndrome that is very similar to the clinical manifestations of patients with viral fh, making it a good animal model for exploring mechanisms underlying the pathogenesis of human viral fh. in addition to fgl , pro-inflammatory mediators such as tnf-α, ifn-γ and complement c a have been proposed to accelerate viral fh pathogenesis [ , ] . nevertheless, the mechanisms on how the inflammatory signaling events that regulate the disease progression are not well understood. recently, it has been shown that dysregulated nlrp (also known as nalp and cryopyrin) inflammasome in macrophages causes the pathogenesis of inflammatory diseases, which highlights the importance of inflammasome in regulating immune-mediated tissue damages [ ] . the generation of biologically active il- β requires cleavage of the inactive precursor proil- β by the nlrp inflammasome, a protein-scaffolding complex consisting of nlrp , caspase- , and the adaptor molecule asc (apoptosis-associated peck-like protein with card domain, pycard) [ , ] . nlrp inflammasome and il- β mediate the host protection against pathogen invasions, whereas, the hyperactivation of nlrp inflammasome contributes to the pathogenesis of certain inflammatory syndromes, including liver injuries such as nonalcoholic/alcoholic steatohepatitis [ , ] , liver fibrosis [ ] , and immune mediated liver injuries [ ] . however, the role of nlrp inflammasome signaling pathway participates in the pathogenesis of viral fh is still unclear. a variety of danger-associated molecular patterns (damps) and pathogen-associated molecular patterns (pamps), including virus rna, nigericin, atp, silica crystals, mitochondrial dna, and aluminum hydroxide, appear to be capable of activating the nlrp inflammasome [ ] . nevertheless, the reactive oxygen species (ros) generated by nicotinamide adenine dinucleotide phosphate (nadph) oxidase are considered to be one of the major factors that activate nlrp inflammasome [ ] . it has been shown that pharmacological inhibition of the nadph oxidase complex (nox) or the down regulation of the nox subunit p phox eliminates nlrp inflammasome activation by preventing ros secretion [ , ] . however, recent studies have also illustrated that mitochondria-originated ros (mitosox) rather than noxderived ros drive nlrp inflammasome activation [ , ] . various stress condition, including increased metabolic rates, hypoxia, or membrane damage, all significantly induce mitosox secretion [ ] . conversely, it remains uncertain for which of the nox-derived ros or mito-sox is responsible for causing nlrp inflammasome-dependent pathology in viral fh development. here, we showed that c bl/ wild type (wt) mice infected with mhv- manifest with high levels of il- β in the serum and liver. conversely, the virus infected il- r -/mice present with much attenuated pathologies, showing with a significant reduction in macrophagederived fgl expression and less liver infiltration of cd + gr- high neutrophils. furthermore, we showed that the in vivo bioactivation of proil- β during mhv- infection is mediated by nlrp inflammasome activation, thereafter, both the nlrp -/mice and the caspase- -/mice display substantial resistance to mhv- -induced il- β production. mechanistically, mhv- infection triggers an acute release of nox-derived ros. blocking ros with diphenyleneiodonium chloride (dpi) inhibits caspase- activation and il- β maturation in vitro. furthermore, nox subunit p phox -deficient mice also exhibited a delayed and moderate viral pathogenesis due to reduction in nlrp inflammasome activation in vivo. these results reveal that the ros/nlrp /il- β axis is a critical signaling pathway leading to the pathogenesis of viral fh. to examine the status of il- β activation in macrophages in response to mhv- infection, primary peritoneal exudative macrophages (pems) and the macrophage line-raw . cells were infected with the virus in vitro. a time course data showed a significant induction of the activated form of il- β (il- β p ) within hours, sustaining to h (fig a) . assessment of the pems isolated from the h of virus infected c bl/ wt mice also revealed a significant increase in proil- β mrna expression ( fig b) . moreover, proil- β mrna expression in the infected livers appeared to be markedly augmented at h (p = . ), sustaining to h (p = . , fig b) . in accordance, western-blotting showed with increases in proil- β and il- β p protein expression at corresponding time points in the infected livers ( fig c) . flow cytometry further validated the patterns of proil- β protein induction in the pems isolated from the virus-infected mice ( fig d) . in agreement, the infected mice also showed significant accumulation of serum il- β during the infection (fig e) . in contrast, serum il- α concentration exhibited little change in mhv- infected mice ( fig f) . these results suggest that il- β significantly elevate in the liver and periphery during viral fh. intervention of il- β signaling reduces mhv- -mediated hepatitis il- β amplifies the pro-inflammatory response via the type-i of il- receptor (il- r ) [ ] . to further investigate whether il- β signaling affects the pathogenesis of viral fh, we infected il- r -/mice with mhv- ( pfu) via intraperitoneal (i.p.) injection. interestingly, il- r -/mice displayed with a significant increase in survival rate with % staying alive for days, as compared to a % death of the wt littermates within days of the viral infection (fig a) . il- r -/mice manifested a significant reduction in hepatocellular damage and a decrease in serum alt/ast levels during the infection (fig b) . the expression of biliary glycoprotein- (bgp ), the receptor for mhv- [ ] , appeared to be significantly lower in the virus infected il- r -/livers comparing to that in the wt controls (fig c) , concurring with the plaque assay data showing with limited virus entrance and amplification in the livers h post-infection ( fig d) . in support, the mhv- infection efficiency in il- r -/-pems dropped more significantly than in the wt counterparts in vivo ( fig e) . obviously, recombinant mouse il- β protein ( ng/ml) is able to significantly induce bgp expression in pems and raw . cells in vitro (fig f) , and in concurrence, il- β treated raw . cells appear to produce more virus than the pbs treated controls post-infection ( fig f) . in validation, we injected the virus-infected wt mice with il- r antagonist (il- ra, mg/kg/day), a naturally occurring cytokine that blocks il- β biologic response [ ] , and observed a significant limitation of il- β secretion (p = . , s a fig mhv- fails to induce fgl production and liver neutrophil infiltration in il- r -/mice fgl plays an essential role in inducing hepatocellular necrosis following mhv- infection [ ] . we firstly examined fgl expression in pems isolated from mhv- infected il- r -/mice and observed substantial lower levels of fgl as compared to the wt controls ( fig a) . the limited fgl expression in macrophages correlates with the low concentrations of fgl observed in the virus infected il- r -/liver and serum (fig b and c ). therefore, in response to mhv- viral infection, il- r -/mice responded with limited fibrinogen formation, leading to a down modulation of liver coagulation and necrosis ( fig d) . similarly, il- ra-treated wt mice displayed with reduction of fgl and fibrinogen deposition in liver tissues, which was followed with decrease in liver damages and enhance the survival time (s b, s d and s e fig) . neutrophils and cd + foxp + regulatory t cells (tregs) have been well recognized as important players in viral fh [ , ] . to determine the role of il- β in regulating these cells during viral fh, we firstly examined liver neutrophil infiltration status. flow cytometry showed that in the liver-tissue samples from and h post mhv- infection, the infiltration of cd + gr- high neutrophils was substantially higher in the wt livers than that in the il- r -/littermates (fig e and f ). the number of cd + foxp + treg in the virus-infected livers appeared to increase significantly after mhv- infection, nevertheless, little difference was observed between il- r -/mice and their wt controls (s fig). similarly, serum concentration of c a, a cytokine that deteriorates the pathogenesis of mhv- -mediated fh [ ] , was not changed dramatically between virus infected il- r -/mice and their wt controls (s a fig). these results suggest that attenuation of viral fh by il- r deficiency could be the consequence of both ineffective fgl production by macrophages and limited cd + gr- high neutrophil infiltration in the affected liver. a reduction of fgl expression was observed in il- r -/mice in response to mhv- infection, together with il- β and fgl were co-expression in pems (fig a) , implying that il- β/ il- r interactions may directly regulate fgl expression in macrophages. to address the issue, we treated raw . cells, a macrophage line capable of expressing fgl , with the recombinant mouse il- β protein ( ng/ml) in vitro. qpcr and western-blotting data showed that il- β alone is incapable of stimulating flg expression, nevertheless, it synergistically enhances tnf-α-induced fgl levels (fig b and c ). the expression of fgl has been proposed to be mediated through the activation of nf-κb and mitogen-activated protein kinase (mapk) signaling pathways under inflammatory conditions [ , ] . to further investigate the molecular mechanisms through which il- β promotes fgl production, we examined these signaling pathways in il- β-treated raw . cells. results showed that either il- β or tnf-α treatment alone, had a minimum stimulation on phosphorylation of the nf-κb chaperone iκbα (p-iκbα) and the nf-κb subunit p (p-p ), appearing only at extended incubation time point ( h). however, synergistic effects of il- β and tnf-α (il- β+tnf-α) seemed to be significant for which substantial increases in phosphorylation of iκbα and p can be detected as early as h post infection ( fig c) . furthermore, the inhibition of nf-κb activation by pyrrolidinedithiocarbamic acid (pdtc) successfully prevented fgl upregulation after il- β+tnf-α treatment ( fig d) . the combination of il- β and tnf-α is capable of potently stimulation the phosphorylation of mapks, including extracellular signal-related kinase (p-erk / ) and p (pp ) (fig c) . nevertheless, the erk inhibitor-pd and the p -mapk inhibitor-sb seemed to be incapable of blocking fgl upregulation. moreover, blocking all of these three pathways did not show additive effect on inhibition of fgl expression ( fig d) . these results suggest that nf-κb rather than the mapk pathways is responsible for il- β+tnf-α-mediated fgl upregulation in viral infected macrophages. it has been established that the caspase- -mediated bio-activation of proil- β is under the control of nlrp inflammasome [ ] . mhv- infected pems and raw . cells exhibited with a significantly enhanced nlrp , asc, pro-caspase- and its activated form (caspase- p ) within h of mhv- infection ( fig a) . in accordance, qpcr analyses illustrated that the mrnas for nlrp and procaspase- were significantly higher in the virus infected livers, this correlates with observation that these virus infected livers also manifest with higher expression of the respective protein ( fig b) . next, we infected nlrp -/mice and caspase- -/mice with mhv- to address the importance of nlrp inflammasome in the causing the virusinduced liver injuries. remarkably, a h viral infection largely failed to induce il- β expression in the livers, which was associated with significant reductions in liver fgl accumulation (fig c) , fibrinogen deposition and local tissue damages, along with significant decreases in serum alt/ast enzymes as compared with the infected wt mice (fig d) . in agreement with these results, we also observed that bgp expression was significantly lower in nlrp -/and caspase- -/livers during infection ( fig c) . meanwhile, nlrp -/mice and caspase- -/mice appeared to produce much less viruses at h of infection as compared to the wt controls ( fig e) . finally, nlrp -/and caspase- -/mice presented with considerably prolonged survival rates toward mhv- infection in comparing to the wt controls ( fig f) . the serum c a in the viral infected nlrp -/and caspase- -/animals was also significantly increased but no different from the wt control mice (s b fig), indicating that c a up-regulation during the viral infection, appears to either additively or synergistically work with other inflammatory factors to cause viral fh. together these observations further validate that the nlrp /caspase- -inflammasome regulates the bio-processing of proil- β for causing the mhv- mediated viral fh. assembly and activation nlrp inflammasome, being critical for bio-processing and activation of il- β, has been suggested to also involve in the bio-activation of il- , another member of the il- superfamily [ ] . the mhv- -infected mice showed a significant up-regulation of proil- mrna in pems and livers (fig a) , as well as enhanced il- protein in serum ( fig b) . however, the recombinant mouse il- protein ( ng/ml) alone, or in the combination with tnf-α and inf-γ, was unable to stimulate fgl mrna transcription in raw . cells or sve- endothelial cells in vitro (fig c) . moreover, mhv- induced liver fgl production remained high in il- -/mice (fig d) , showing with consequentially high levels of fibrinogen deposition, liver damages and hepatocyte necrosis ( fig e) . additionally, liver tissues isolated from il- -/mice appear to up-regulate bgp expression after mhv- infection. in accordance, these mice also manifested with high virus duplication ( fig f) . overall, il- -/mice are still sensitive to mhv- infection (fig g) , suggesting that il- is not essential in mhv- -mediated fulminant hepatitis. many factors contribute to activating the nlrp inflammasome and among which, ros is lately gaining particular attentions [ ] . in order to examine the role of ros in nlrp inflammasome hyperactivation, we first detected the release of nadph oxidase-derived ros by using a permeable dichlorohydrofluorescein (dcfh) upon mhv- infection. flow cytometry showed that the releasing of dcfh from mhv- infected pems and raw . cells significantly increased, especially at h and h post-infection (fig a) . this result correlates with the up-regulation of gp phox , p phox and nox , the subunits that are essential for acute ros secretion in raw . cells (fig b) . however, the dcfh level dropped dramatically at h in addition to nadph oxidase-derived ros, mitochondria may provide an alternate source of ros [ ] . we therefore assessed the functional mitochondrial pool in mhv- infected cells. the viral infection in pems and raw . cells caused an increase in mitochondrial damage, especially at h and h post-infection, as detected by mitotracker green fm, a dye that stains mitochondria with no influence on their membrane potentials (fig a) . similarly, electron microscopy showed with swollen mitochondria in the mhv- infected raw . cells at h and h (fig c) . this sign of mitochondrial damage seemed to strongly correlate with the increase in mitosox release within the same time frame (fig a) . to further elucidate the role of ros in nlrp inflammasome hyperactivation, we treated mhv- infected raw . cells with a ros inhibitor diphenyliodonium chloride (dpi), which is capable of preventing both nox-dependent ros and mitoxos secretion [ ] . noxoriginated dcfh was successfully inhibited by dpi in a dose dependent manner (fig d) . however, mitoxos release was not prevented by the dpi treatment, even at a very high dose ( μm) (fig d) . the efficiency of nox-originated ros inhibition by dpi appeared to correlate with the reduction in il- β activation in the infected raw . cells and pems in dose dependent manners (fig e) . together, these results suggest that the hyperactivation of nlrp inflammasome in macrophage is partially mediated by mhv- induced, nox-derived ros. p phox-/mice are resistance to mhv- induced fh by limiting nlrp inflammasome hyperactivation cells in deficiency of p phox exhibit a reduced capacity in generating ros [ ] . to further investigate the role of nox-originated ros in regulating nlrp inflammasome hyperactivation, we infected p phox-/mice with mhv- and examined the severity of liver pathology. as anticipated, pems isolated from mhv- infected p phox-/mice showed with limited dcfh (fig a) . interestingly, the p phox-/mice also displayed considerable resistance to mhv- infection, presenting with reduced disease severity within the prolonged survival time as compared with the wt controls (p = . , fig b) . the lack of virus-induced ros response, which leads to prohibition of nlrp /caspase- activation and thus reduction in il- β production, seems to be responsible for this effect (fig c and d) . as a result, the virus infection is unable to generate significant fgl accumulation in the liver and serum (fig c and d) . therefore, these mice manifested with less severe fibrinogen deposition, liver injury and hepatocyte necrosis, accompanying with low levels of ast/alt enzymes released by the liver (fig e) . however, the limitation of il- β secretion in these p phox-/mice only slightly affected liver bgp expression (fig c) , and therefore live virus titers were still high at h of infection ( fig f) . conversely, the administration of il- β ( ng/mouse/day) in mhv- infected p phox-/mice was able to reinstate all aspects of disease severity typical in viral fh (figs g and s ) . taken together, these results clearly indicate that the ros/nlrp /il- β axis plays a critical role in the pathogenesis of viral fh. in the present work, we report that mice infected with mhv- , an animal model for viral fh, have significantly elevated levels of il- β in the serum and liver. the accumulation of il- β accelerated liver pathology through synergistically acting with tnf-α, one of the key inflammatory cytokines that has been previously shown to be essential for causing viral fh [ , ] , il- r signaling is responsible for stimulation of fgl expression in macrophages and enhancing infiltration of the inflammatory cd + gr- high neutrophils in the livers. interestingly, mhv- infection in il- r -/mice, or in wt mice treated with il- β signaling inhibitors, such as using il- ra, rescue the otherwise susceptible animals from the viral fh status, presenting with limited virus replication, attenuated disease progression and reduced mortality. we have also shown that the bioprocess of il- β maturation is under the control of a key signaling pathway, involving a mhv- virus inducible, ros-dependent nlrp inflammasome activation. animals lacking of nlrp , caspase- or nadph oxidase subunit p phox that controls acute ros secretion, all exhibited with reduced il- β bio-processing that results in prevention of the mhv- mediated disease severity. to the best of our knowledge, these data provide evidence for the first time showing that the ros/nlrp /il- β axis is an essential contributor for the virus-induced fh. although macrophage-mediated inflammation has been speculated to be critical for gauging the pathological susceptibility of viral fh caused by mhv- infection [ ] , the mechanisms underlying the pathogenesis are not well understood. il- β and il- are two key inflammatory cytokines produced by macrophages which play a pivotal role in antimicrobial immunity [ , ] . previous studies have showed that il- r -/mice appear to have markedly reduced inflammatory pathology in the lung, presumably due to the impaired neutrophil recruitment upon influenza virus infection [ ] . conversely, ramos et al. reported that il- r -/mice exhibited with a higher accumulation of the west nile virus (wnv) in the central nervous system due to a restrained activation of the virus-specific effector cd + t cells [ ] . similarly, il- β -/mice are more susceptible to herpes simplex virus (hsv )-mediated encephalitis due to an increase in viral load [ ] . we here further explored the role of il- β in mhv- mediated fh. interestingly, il- r -/animals display a significant reduction in viral duplication, amelioration of liver damage and a prolonged survival rate against mhv- infection (fig a and b) . these effects are probably due to il- r deficiency lead to limit liver recruitment of cd + gr- high neutrophils and decrease in production of the macrophage-derived fgl , which mediates sinusoidal fibrin deposition and hepatocellular necrosis in response to mhv- infection [ ] . bgp (also called carcinoembryonic cell adhesion antigen a,ceacam a) is the specific receptor for the mouse hepatitis virus (mhv), and down-regulation of bgp by ifn-γ is related to the antiviral state and resistance to mouse hepatitis virus infection [ ] . however, bgp does not appear to be involved in il- and tnf-α secretion from mhv- infected macrophages [ ] . in contrast to ifn-γ treatment, we here showed that the expression of bgp drops significantly in the il- r -/liver during the viral infection, suggesting bgp expression in macrophages is induced by il- β/il- r signaling, and lacking the pathway may compromise virus entrance and amplification. these unpredicted data implies that il- β has double-edge effects on the immune system, in which proper balancing with its signaling extent becomes essential (e) liver fibrinogen deposition was analyzed by immunohistochemistry, the architecture was analyzed by h&estaining and cellular apoptosis was analyzed using tunel staining (left). scale bar μm; arrow indicates positive cells; blue color indicates nuclear staining with dapi, n = per group. serum alt and ast activities were determined with an au automatic biochemistry analyzer (right). *p< . and **p< . , n = per group. (f) liver virus titers at h of infection were analyzed by plaque assay (left), and their levels were compared by statistical analysis (right). *p< . . (g) mhv- infected p phox-/mice were treated with mouse recombinant il- β protein ( ng/day/mouse) and the survival rate was monitored. one of three experiments with similar results is shown. *p< . compared top phox-/-+pbs+mhv- group. for the host in protection against various invading viruses and meanwhile, in prevention of the potential collateral damage. the molecular mechanisms that are responsible for triggering the expression of fgl prothrombinase, which plays a critical role in the development of mhv- mediated fh, are still unclear. mcgilvray et al. found that both erk and p -mapk proteins are activated in mhv- infected pems, and only inhibition of p -mapk can abolish fgl induction and its functional activity [ ] . jia et al. have illustrated that tnf-α upregulates fgl expression via activation of nf-kb and p -mapk in cardiac microvascular endothelial cells [ ] . our recent work also have showed that the inhibition of erk / and p -mapk efficiently block c amediated fgl upregulation [ ] . ning et al., have demonstrated that the hepatocyte nuclear factor- (hnf ) cis-elements and its cognate transcription factor, hnf α, are necessary for mhv- -induced fgl gene transcription [ ] . based on these studies, we further examined the molecular mechanisms underlying il- β-mediated fgl expression. the results show that il- β and tnf-α synergistically induce nf-κb, erk and p -mapk tyrosine-phosphorylation ( fig c) . however, the inhibition nf-κb pathway, but not the erk, or p -mapk signals, markedly prevented fgl expression (fig d) , suggesting that the nf-κb pathways are responsible for il- β+tnf-α-mediated fgl augmentation. the nlrp , rig-i and the aim are three main types of inflammasome complexes that have been shown to control caspase- activity and il- β maturation. it seems that aim is responsible for detecting dna viruses, while both nlrp and rig-i associate with recognition of rna viruses by cells [ , ] . recent evidences suggest that the host protective immunity requires the nlrp inflammasome for fighting against various kinds of viruses, including influenza a virus, modified vaccinia virus ankara, sendai virus, respiratory syncytial virus, encephalomyocarditis viruses, as well as adenoviruses [ ] . our study shows that the mhv- triggered nlrp , asc and caspase- mrna as well as protein expression in pems and raw . cells in vitro (fig a) . nevertheless, loss of either nlrp or caspase- in macrophages reduces il- β secretion upon mhv- challenge (fig c) . additionally, nlrp -/and caspase- -/mice essentially pheno-copied the manifestations of il- r -/mice in response to mhv- infections, these mice evidenced with reduction in mhv- virus-induced il- β production and lessening of disease progression (fig c- f ). these combined data suggest that nlrp -inflammasome acts as a predominant pathway for triggering il- β maturation by mhv- , and probably also by other corona viruses. previous study showed that raw . cells do not release mature il- β because they do not express asc [ ] . conversely, we here show that mhv- promotes il- β secretion from virus infected raw . cells through inducing asc expression. together with the recent work demonstrated that nlrp /asc/caspase- axis participates in the regulation of the generation of il- β in raw . cells, indicating that asc is inducible in the macrophage line raw . cells under circumstances, especially during mhv- infection [ ] . ros plays an essential role in mediating nlrp inflammasome activation [ ] . many different viruses, such as influenza virus, respiratory syncytial virus, and hepatitis c virus, trigger nlrp inflammasome activation through ros-dependent mechanisms [ ] [ ] [ ] . nox is an enzymatic complex consisting mainly of five subunits (p phox , p phox , p phox , p phox and gp phox ) and two gtp-binding proteins (rac /rac ). we here show that mhv- triggers nox-derived ros secretion in macrophages by inducing nox-subunits, including gp phox , p phox and nox- expression in the very early stages of the viral infection (fig a and c) . additionally, preventing nox-derived ros through dpi appeared to successfully down modulate nlrp hyperactivation and il- β maturation in vitro (fig f) . furthermore, virus infected p phox-/macrophages manifested with significant reduction in ros secretion, leading to the control of nlrp hyperactivation, which results in attenuation in severity of the viral fh (fig ) . these results are inconsistent with previous reports that have shown that nadph oxidase-derived ros are not involved in activating nlrp inflammasome [ , ] . one of the discrepancies is the different cell models are used in studies. silica crystals, lps, and uric acid crystals act as the stimulators in these studies, while mhv- virus is the activator in our research. conversely, it is worth mentioning that not all p phox-/mice are completely resistant to mhv- , and these animals eventually still died from the infections (fig b) , together with some virus infected mice still produce high levels of il- β and virus titers, suggesting the presence of other mediators that in response to the virus challenge, are capable of activating nlrp inflammasome in vivo. one of the potential activators is mitosox [ , ] . we have also observed a very high level of the mitosox production in the mhv- infected raw . cells at h and h post-infection in vitro, along with high frequency damage and destruction of mitochondria might simultaneously occur. however, the release of mitosox was unable to be successfully blocked by ros inhibitor-dpi ( um) (fig ) . additionally, dpi is harmful to animals and unsuitable in vivo experiments [ ] . the incapable of completely blocking ros production by using high dose of dpi in vitro suggests the existence of other sources of ros for activating nlrp inflammasome. interestingly, reduced mortality and pathology were seen in mhv- infected p phox-/mice compared to wt littermates despite a lack of significant reduction in virus replication, suggesting that mhv- -mediated pathology is due to inflammation and not direct virus infection. recent studies by warner greene's group demonstrate that hiv can trigger caspase- activation and pyroptosis, a highly inflammatory form of programmed cell death in which dying cells release their cytoplasmic contents, including inflammatory cytokines into the extracellular space where the virus infected cd + t-cells recite [ ] . a similar environment might also explain for the mhv- induced fh status. il- is another member of the il- superfamily that has been indicated to be important in the pathogenesis of mouse models of influenza virus, hbv, rhinovirus and vaccinia virus infection [ ] . for example, il- r -/mice appeared to be protected from influenza viral initiated inflammatory lung damages [ ] . consistent with previous reports, we have detected significantly high levels of matured il- in the serum of mhv- infected wt mice. however, il- deficiency does not prevent bgp expression, virus amplification and fgl accumulation in the liver following mhv- infection, and as the consequence, these mice stay high with fibrinogen deposition, liver damage and hepatocyte necrosis (fig ) . these results suggest that il- is not essential for causing mhv- mediated acute hepatitis. in conclusion, our study elucidates that nlrp inflammasome-dependent il- β production, a primary inflammatory signaling pathway of the host for mounting conventional immunity against pathogen invasions, plays a double-edged role in the host immune system. hepatotropic virus, like mhv- infection in mice, can induce exaggerated inflammation in the liver and cause life-threatening viral fh. these results shed lights on a novel strategy, for which the properly modulation of the il- β signaling pathway, in combination with blocking other inflammatory factors, might benefit the treatment of viral fh and other severe inflammatory diseases in human. the mice approximately weeks of age were used for these experiments. all animals received humane care according to the criteria outlined in the "guide for the care and use of laboratory animals" prepared by the national academy of sciences and published by the national institutes of health (nih publication - revised ). cells raw . cells were provided by the cell institute of the chinese academy of sciences (shanghai, china). peritoneal exudative macrophages (pems) were harvested as described previously [ ] . cells were cultured in -well plates and propagated in dmem supplemented with % fbs, u/ml penicillin, and μg/ml streptomycin. mhv- viruses were expanded in murine cl cells to a concentration of × plaque forming unit (pfu)/ml. the virus-containing supernatants were stored at - °c until use. macrophages were infected with mhv- (multiplicity of infection, moi = ) in vitro and mice were injected with pfu of mhv- via i.p. in some experiments, the virus infected mice were treated with il- r antagonist (il- ra, mg/kg/day) or recombinant mouse il- β protein ( ng/day/mouse) every day. mice were euthanized on the indicated days and the virus titers in liver were determined by plaque assay as described previously [ ] . the sources of antibodies and other reagents are detailed in s text. paraffin-embedded liver tissue blocks were cut into μm slices and mounted onto poly-lysinecharged glass slides, and tissue injury was stained by hematoxylin and eosin (h&e). cellular apoptosis was measured by tunel staining according to the manufacturer's instructions (roche, berlin, germany). the expression of fibrinogen and fgl was detected by immunohistochemistry as described previously [ ] . sections were scored in a blinded fashion for histological diagnosis. total rna was extracted from cultured cells or liver tissues with trizol reagent according to the manufacturer's instructions (invitrogen, ny, usa). first-strand cdna was synthesized with the primescript rt-pcr kit (takara, dalian, china). the expression of mrna encoding for nlrp , caspase- , proil- β and proil- was quantified by real-time quantitative pcr with the sybr premix extaq kit (takara) and was normalized to the expression of β-actin. sequences of the primers are provided in s table. results were calculated and compared by the −ΔΔct method. serum c a, fgl , il- and il- β levels were measured by elisa. the expression of fgl , procaspase- , caspase- -p , nlrp- , p phox , p phox , p phox , nox- , bgp , proil- β and il- β-p in mhv- infected livers or macrophages was detected by western-blotting described previously [ ] . the release of il- β/ros from virus infected macrophages, liver infiltration of cd + gr- high neutrophil and cd + foxp + regulatory t cells (treg), all were detected by flow cytometry (facsaria cytometer, bd, franklin lakes, nj, usa). the death cells were excluded firstly by staining with live/death fixable near-ir ded cell stain kit (life technologies, eugene, oregon, usa). the secretion of nox-derived ros was detected by means of an oxidation-sensitive fluorescent probe-dcfh according to the manufacturer's instructions (beyotime, shanghai, china). moreover, the mitochondria-derived ros was measured in cells stained with mitosox ( μm, invitrogen) for min. to measure mitochondrial damage, cells were stained for min with mitotracker green fm ( nm) and mitotracker deep red fm ( nm), two kinds of dye that stain mitochondria with no influence on their membrane potentials (invitrogen). a total of , live cells were analyzed. all the facs data were analyzed using cell-quest pro software. electron microscopy raw . cells or primary pems isolated from mhv- infected mice were fixed with % (v/ v) glutaraldehyde. sample preparation was conducted as described previously [ ] . mitochondrial morphology and virion was observed with jeol jem hc transmission electron microscopy. all data were analyzed using graphpad prism . software. an unpaired student's t-test (two-tailed) was used to assess comparisons between two groups when the data met the assumptions of the t-test. survival curves were generated by log-rank test. p< . was considered a significant difference. all animal experiments were performed in strict accordance with the guide for the care and use of laboratory animals issued by the ministry of science and technology of the people's republic of china. the protocol was approved by the third military medical university institutional animal care and use committee. supporting information s text. reagents and antibodies. (docx) s table. the primer sequences for qpcr of the indicated genes. acute-on-chronic liver failure: consensus recommendations of the asian pacific association for the study of the liver (apasl) fulminant viral hepatitis: molecular and cellular basis, and clinical implications the fgl /fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis tnf-α and fgl contribute to coagulation and complement activation in virus induced fulminant hepatitis c a/c ar pathway is essential for the pathogenesis of murine viral fulminant hepatitis by way of potentiating fgl /fibroleukin expression intracellular nod-like receptors in host defense and disease interleukin- in the pathogenesis and treatment of inflammatory diseases il- receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice toll-like receptor promotes steatohepatitis by induction of interleukin- beta in mice interleukin- participates in the progression from liver injury to fibrosis type i interferons protect from toll-like receptor -associated liver injury and regulate il- receptor antagonist in mice nlrp inflammasome activation: the convergence of multiple signalling pathways on ros production? signaling by ros drives inflammasome activation innate immune activation through nalp inflammasome sensing of asbestos and silica a role for mitochondria in nlrp inflammasome activation mitochondrial reactive oxygen species promote production of proinflammatory cytokines and are elevated in tnfr -associated periodic syndrome (traps) a mitochondrial love-hate triangle interleukin- beta and the autoinflammatory diseases tissue and cellular distribution of an adhesion molecule in the carcinoembryonic antigen family that serves as a receptor for mouse hepatitis virus tumor necrosis factor α (tnf-α) receptor-i is required for tnf-αmediated fulminant virus hepatitis caused by murine hepatitis virus strain- infection the novel cd + cd + regulatory t cell effector molecule fibrinogen-like protein contributes to the outcome of murine fulminant viral hepatitis tnf-α upregulates fgl expression in rat myocardial ischemia/reperfusion injury il- in inflammatory and autoimmune disease enhanced autoimmunity, arthritis, and encephalomyelitis in mice with a reduced oxidative burst due to a mutation in the ncf gene expression of b and t lymphocyte attenuator (btla) in macrophages contributes to the fulminant hepatitis caused by murine hepatitis virus strain- interleukin- is responsible for acute lung immunopathology but increases survival of respiratory influenza virus infection il- β signaling promotes cns-intrinsic immune control of west nile virus infection tumor necrosis factor-alpha and interleukin- beta play a critical role in the resistance against lethal herpes simplex virus encephalitis down-regulation of bgp (a) viral receptor by interferon-gamma is related to the antiviral state and resistance to mouse hepatitis virus infection macrophage interleukin- and tumour necrosis factor-alpha are induced by coronavirus fixation to toll-like receptor /heparan sulphate receptors but not carcinoembryonic cell adhesion antigen a murine hepatitis virus strain induces the macrophage prothrombinase fgl- through p mitogen-activated protein kinase activation induction of prothrombinase fgl by the nucleocapsid protein of virulent mouse hepatitis virus is dependent on host hepatic nuclear factor- alpha aim recognizes cytosolic dsdna and forms a caspase- -activating inflammasome with asc recognition of rna virus by rig-i results in activation of card and inflammasome signaling for interleukin beta production central roles of nlrs and inflammasomes in viral infection p x receptor differentially couples to distinct release pathways for il- beta in mouse macrophage lipopolysaccharide/adenosine triphosphate induces il- β and il- secretion through the nlrp inflammasome in raw . murine macrophage cells hcv and oxidative stress in the liver inhibition of nox oxidase activity ameliorates influenza a virus-induced lung inflammation suppressing production of reactive oxygen species (ros) for influenza a virus therapy superoxide dismutase regulates caspase- and endotoxic shock reactive oxygen species-independent activation of the il- beta inflammasome in cells from patients with chronic granulomatous disease human nlrp inflammasome activation is nox - independent silica crystals and aluminum salts activate the nalp inflammasome through phagosomal destabilization studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil nadph oxidase cell death by pyroptosis drives cd t-cell depletion in hiv- infection nlrs, inflammasomes, and viral infection interleukin- improves the early defence system against influenza virus infection by augmenting natural killer cell-mediated cytotoxicity gene deletion of gabarap enhances nlrp inflammasome-dependent inflammatory responses we wish to thank dr. dayan cao, huan xu and xi chen for their helpful comments and constructive suggestions. lxz is supported by the intramural research program of the us national institutes of health. key: cord- -s onyix authors: wang, ting; wang, lichun; moreno-vinasco, liliana; lang, gabriel d; siegler, jessica h; mathew, biji; usatyuk, peter v; samet, jonathan m; geyh, alison s; breysse, patrick n; natarajan, viswanathan; garcia, joe g n title: particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation date: - - journal: part fibre toxicol doi: . / - - - sha: doc_id: cord_uid: s onyix background: exposure to particulate matter (pm) is a significant risk factor for increased cardiopulmonary morbidity and mortality. the mechanism of pm-mediated pathophysiology remains unknown. however, pm is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ros generation. objectives: we explored the role of tight junction proteins as targets for pm-induced loss of lung endothelial cell (ec) barrier integrity and enhanced cardiopulmonary dysfunction. methods: changes in human lung ec monolayer permeability were assessed by transendothelial electrical resistance (ter) in response to pm challenge (collected from ft. mchenry tunnel, baltimore, md, particle size > . μm). biochemical assessment of ros generation and ca( +) mobilization were also measured. results: pm exposure induced tight junction protein zona occludens- (zo- ) relocation from the cell periphery, which was accompanied by significant reductions in zo- protein levels but not in adherens junction proteins (ve-cadherin and β-catenin). n-acetyl-cysteine (nac, mm) reduced pm-induced ros generation in ecs, which further prevented ter decreases and atteneuated zo- degradation. pm also mediated intracellular calcium mobilization via the transient receptor potential cation channel m (trpm ), in a ros-dependent manner with subsequent activation of the ca( +)-dependent protease calpain. pm-activated calpain is responsible for zo- degradation and ec barrier disruption. overexpression of zo- attenuated pm-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro. conclusions: these results demonstrate that pm induces marked increases in vascular permeability via ros-mediated calcium leakage via activated trpm , and via zo- degradation by activated calpain. these findings support a novel mechanism for pm-induced lung damage and adverse cardiovascular outcomes. ambient particulate matter (pm) poses a threat to national public health in urban environments and other polluted areas throughout the us and around the world. epidemiological studies have shown associations of exposure to low levels of urban particulate matter with increased cardiopulmonary morbidity and mortality [ , ] . the assessment of pm-induced health effects is challenging. various mechanisms have been proposed to explain the cardiopulmonary health effects of pm including increased pulmonary and systemic oxidative stress and inflammation, enhanced coagulation, and altered cardiac autonomic function [ , ] . airway epithelium represents a well-investigated target for environmental pollutants such as pm. exposure of airway epithelium to airborne pm causes altered cytokine/chemokine gene expression and increased production of il- β, il- , il- and tnf-α [ , ] . now, the lung endothelium is also gaining attention as a viable pm target tissue. exposure of the endothelium to pm or its active components in the systemic circulation induces significant systemic endothelial inflammation and dysfunction, even at low levels of exposure [ , ] . the water soluble fraction of pm (up to - %) can easily diffuse through the epithelium/endothelial barrier to the systemic circulation. bioavailable transition metals present in urban pm catalyze redox reactions in human lung endothelium, which cause oxidative stress, increase the production of inflammatory cytokines, and increase the activation of nf-κb signaling pathways, all of which trigger further endothelial damage [ ] . increased endothelial monolayer permeability is also observed in inflammatory pulmonary conditions such as acute lung injury (ali), acute respiratory distress syndrome (ards), and sepsis; devastating lung disorders with mortality exceeding %, as well as in more subacute and chronic inflammatory disorders such as asthma [ , ] . we recently described a murine asthma model with strong evidence for pm-mediated vascular barrier dysfunction with increased protein leakage into bronchoalveolar lavage (bal), a marker of acute inflammatory lung damage [ ] . by assessment of direct effects on endothelial barrier integrity in vitro, we further demonstrated that the vascular hyperpermeability mediated by intratracheal pm exposure is mainly dependent on acute endothelial barrier disruption by pm [ ] . exposure of human lung ec to pm resulted in significant ros generation, which mediates p beta mapk activation, leading to the phosphorylation of hsp [ ] . phosphorylated hsp facilitates the synthesis of stress fibers and formation of paracellular gaps, which causes protein-containing fluid to leak from the microvessel lumen to the lung alveoli, leading to further pulmonary inflammation [ ] . however, these previous findings did not explain the persistent character of pm-mediated endothelial barrier disruption. to fully examine pm-mediated vascular hyperpermeability, we explored effector targets such as tight junction and adherens junction proteins, which are known to be critical in ec barrier maintenance. we demonstrated that pm exposure induced the relocation of tight junction protein zona occludens- (zo- ) from the cell periphery, which was accompanied by a significant reduction in the level of zo- protein but not in the levels of adherens junction proteins (ve-cadherin and β-catenin). pm also mediated intracellular calcium mobilization via the transient receptor potential cation channel m (trpm ), in a ros-dependent manner with subsequent activation of the ca + -dependent protease calpain. pm-activated calpain is responsible for zo- degradation and ec barrier disruption. these observations not only provide new information as to how pm disrupts endothelial tight junctions, but also represent the first evidence establishing the critical role of calpain signaling in modulating endothelial cell barrier function under oxidative stress. these results increase our understanding of pm-induced adverse cardiopulmonary outcomes. moreover, as the newly characterized signaling cascade of ros/trpm /calpain/ zo- likely has fundamental roles in regulating the cytoskeleton under oxidative stress, these novel observations may have broad applicability to vascular pathophysiology in a variety of cell types. molecular mass standards, polyacrylamide gels, and protein assay reagents were purchased from bio-rad (hercules, ca human lung microvascular ecs obtained from lonza (basel, switzerland) were cultured as previously described [ ] in egmmv- complete medium (lonza). endothelial cells were grown to confluence in polycarbonate plates containing evaporated gold microelectrodes, and ter measurements were continuously obtained using an electrical cell-substrate impedance sensing system (ecis) (applied biophysics, troy, ny) as previously described in detail [ ] . human microvascular lung ecs were transfected with sirna using siport amine (ambion, austin, tx) according to the manufacturer's protocol as we described previously [ ] . ecs plated on glass cover slips were loaded with μm fura- am (invitrogen, carlsbad, ca) in ml of basal medium as previously reported [ ] . the cover slips with ecs were inserted diagonally into cm acrylic cuvettes filled with ml of basal medium at °c. fura- fluorescence was measured with an aminco-bowman series luminescence spectrometer (slm/aminco, urbana, il) at excitation wavelengths of and nm and emission wavelength of nm. the cover slips with ecs were then moved to mm dishes, treated with pm suspension, and incubated for minutes at °c in % o and % co . after every -minute incubation, the coverslips were withdrawn from the dish and inserted back into the acrylic cuvettes for fura- fluorescence measurement, then returned to the dish with the resuspended pm preparation for another incubation period ( - min). male a/j mice ( - weeks of age; jackson laboratories, bar harbor, me) were housed in an environmentally controlled animal facility at the university of illinois at chicago (uic) for the duration of the experiments. all animal procedures follow the guideline of the uic animal care and use committee. pm ( mg/kg, in μl of saline) was delivered via intratracheal aspiration hr after nac or calpeptin treatment, as previously described [ , ] . animals were sacrificed hr after pm treatment, and bronchoalveolar lavage (bal) and lung tissue were collected [ , ] . total bal cells were counted with a hemocytometer. the bal fluid was used for protein and cytokine measurement (bio-rad, hercules, ca) according to the user's manual. data are presented as group means ± sem. we performed statistical comparisons among treatment groups by randomized-design two-way analysis of variance followed by the newman-keuls post hoc test for more than two groups, or by an unpaired student's t-test for two groups. in all cases, we defined statistical significance as p < . . we assessed human lung microvascular ec barrier function as measured by transendothelial electrical resistance (ter), a highly sensitive measurement of permeability. pm challenge ( - μg/ml) induced doseand time-dependent reduction in ter (additional file : figure s ) or increases in fitc-dextran leakage through ec monolayer (additional file : figure s ) [ ] , indicating a loss of ec barrier integrity. at the same time, pm induced a time-dependent ( - hr) reduction of the levels of tight junction proteins zo- and zo- , but did not affect levels of adhesion junction proteins ve-cadherin or β-catenin ( figure a ). no obvious cytotoxicity in the ecs was found after pm challenge ( μg/ml, - hr) with mtt assay [ ] or ldh release assay (additional file : figure s ). pm ( μg/ml) induced substantial time-dependent ros production in microvascular ecs as measured by dcfda oxidation, which peaked around - min (additional file : figure s ). ec pretreatment with n-acetyl-cysteine (nac, mm, hr), an ros scavenger, or peg-catalase (peg-cat, u/ml, hr), which degrades h o , prevented pm-induced dcfda oxidation by ros (additional file : figure s ). pm produced a sustained timedependent decrease in ter, with a maximal effect observed at μg/ml ( % decrease in ter), which is similar to its effects on other endothelial cell types, as we have reported previously [ ] . nac pretreatment ( mm, hr pretreatment) prevented pm-induced zo- degradation ( figure b ), while nac ( mm, - hr) does not change zo- protein levels by itself (additional file : figure s ). after ecs were treated with pm ( μg/ml, hr), zo- was relocated from the cell periphery and degraded, which was followed by gap formation between ecs. ve-cadherin, on the other hand, underwent no relocation or degradation ( figure c ). nac pretreatment ( mm, hr pretreatment) prevented pm-induced zo- relocation and gap formation ( figure c ). these data strongly indicate that pm causes ec barrier disruption selectively via oxidative tight junction protein degradation. nac ( mm) or peg-cat ( u/ml) pretreatment significantly inhibited pm-induced ec barrier disruption as measured by ter ( figure d ). we also examined the effects of another ros scavenger euk- on pmchallenged ecs. as nac, euk ( μm, hr pretreatment) attenuated pm-induced zo- degradation and ter reduction (additional file : figure s ). these results further confirmed that pm induces ros-dependent ec barrier disruption. we previously demonstrated that high levels of ros in endothelial cells activate calpain, a calcium-dependent protease [ ] . we therefore investigated the role of calpain in pm-mediated zo- degradation. pm ( μm, hr) induced a significant increase in calpain activity in ecs, which was inhibited by selective calpain inhibitors alln ( μm, hr pretreatment) and calpeptin ( μm, hr pretreatment) (figure a ). pm-induced calpain activation was also inhibited by bapta-am ( μm, hr pretreatment), a calcium chelator, and by nac ( mm, hr pretreatment) (figure a ). we next determined the role of calpain in pm-induced ec barrier disruption. pm-induced reduction in ter was partially inhibited by calpeptin or alln ( figure b ). in parallel, alln and calpeptin also significantly prevented pm-induced zo- degradation, as did chelation of intracellular calcium via bapta-am ( figure c ). addition of pm ( μg/ml) to human lung microvascular ecs produced slow time-dependent ca + influx ( figure a ; ca + influx is represented by an increase in the / ratio of fura- am.). notably, this finding of increased intracellular ca + is in accordance with our previous finding of increased activated calpain. under oxidative stress, a key member of the transient receptor potential (trp) cation channel, member m (trpm ), is activated and causes slow calcium influx [ ] . we therefore examined the role of activated trpm in pm-stimulated calcium influx. anti-trpm blocking antibody ( μg/ml, hr pretreatment) significantly prevented the pm-induced ca + transients compared to ecs treated with control igg ( figure b ). reductions in trpm protein expression (by sirna) also inhibited the pm-induced ca + influx compared to ecs treated with control sirna ( figure c ). we next investigated whether trpm activation was ros-dependent. depletion of pm-induced ros by nac ( mm, hr pretreatment) significantly prevented pmmediated calcium influx ( figure d ). under oxidative stress, poly adp ribose polymerase (parp) generates adpribose [ , ] , which activates trpm by binding to its carboxyl terminus. the parp inhibitor -aminobenzamide ( -ab, mmol/l, hr pretreatment)significantly reduced pm-induced ca + influx ( figure e ), which further confirmed that trpm is activated by pm via ros and parp. we next investigated the role of trpm activation in ec barrier function and zo- degradation. trpm neutralizing antibody ( μg/ml, hr pretreatment) significantly prevented zo- degradation induced by pm ( figure a ). trpm sirna ( ng/ml), which downregulated trpm protein level ( figure b ), also inhibited pm-induced zo- degradation ( figure c ). in parallel, antagonizing trpm by either trpm antibody ( μg/ml, hr pretreatment) or trpm sirna ( ng/ml) significantly prevented pm-induced ec barrier disruption ( figure d-e) , as indicated by ter measurements. a pm-mediated murine model of pulmonary inflammation has been well established [ ] . we investigated the role of ros and calpain in pm-induced pulmonary inflammation by examining protein leakage, white blood cell infiltration (inflammatory leukocytes), and the release of proinflammatory cytokines into bal fluids ( figure ). pre-administration of nac ( mg/kg) or calpeptin ( mg/kg) hr before pm challenge ( mg/kg, hr) significantly attenuated pm-induced protein leakage into bal fluids (~ % reduction, figure a ). pm challenge ( mg/kg, hr) resulted in an increase in inflammatory leukocytes, e.g. neutrophils and eosinophils [ , ] , in bal fluids ( figure b ). pre-administration of nac ( mg/kg) or calpeptin ( mg/kg) attenuated pminduced inflammatory leukocyte infiltration. furthermore, nac ( mg/kg) or calpeptin ( mg/kg) attenuated the release of pm-induced proinflammatory cytokines il- and thf-α into bal (~ % inhibition, figure c-d) . these results suggest that ros scavenging by nac or figure activated calpain is required for pm-mediated zo- degradation and ec barrier disruption. (a) human lung microvascular ecs grown in -mm dishes to approximately % confluence were treated with alln ( μm), calpeptin (calp, μm), nac ( mm), or bapta-am ( μm) for hr, and then challenged with pm ( μg/ml) for hr. cell lysates were subjected to calpain activity assay by using the calpain activity kit (calbiochem). calpain activity was normalized to protein concentration and expressed as fold changed compared to control. *p < . compared to control. **p < . compared to pm only group. (b) ecs grown on ecis gold electrodes were treated with alln ( μm), calpeptin (calp, μm) for hr, and then challenged with pm ( μg/ml). changes in ter were measured with ecis. *p < . compared to pm only group. (c) ecs grown on -well plates were treated with alln ( μm), calpeptin (calp, μm), or bapta-am ( μm) for hr, and challenged with pm ( μg/ml) for - hr. cell lysates were analyzed by western blotting with antibody to zo- . changes in levels of zo- are expressed as fold changes and normalized to β-actin. shown are representative blots from three independent experiments. *p < . compared to pm- hr group. **p < . compared to pm- hr group. calpain activity inhibition by calpeptin leads to multiple protective effects including enhancement of lung endothelial barrier function, reduction of inflammatory cell infiltration, and prevention of proinflammatory cytokine release in the lungs of pm-challenged mice. we next examined tight junction zo- levels in the pm-exposed lung. pm challenge ( mg/kg, hr) induced a significant reduction of zo- protein levels in the murine lung, while pre-administration of nac ( mg/kg) or calpeptin ( mg/kg) hr before pm challenge attenuated pminduced zo- loss from lung tissues. taken together, these data suggest a crucial role for the ros-calpain-zo- signaling pathway in the regulation of ec barrier disregulation in pm-mediated pulmonary inflammation. to further confirm the critical role of zo- degradation in ec barrier disruption in vitro and pulmonary inflammation in vivo, we next examined the beneficial effects of over-expressing zo- protein in endothelial cells in vitro and in vivo. zo- over-expression ( figure a ) significantly (but not completely) attenuated pm-induced ec barrier disruption ( figure b ). these facts demonstrate that endothelial zo- loss contributes to pm-mediated ec barrier disruption. we next over-expressed zo- in vivo by a liposome delivery system labeled with ace antibody, which successfully over-expressed zo- in human lung microvascular ecs were plated on glass cover slips and loaded with μm fura- am in ml of basic medium. ecs were rinsed twice and treated with pm ( μg/ml). fura- fluorescence was measured at excitation wavelengths of and nm and an emission wavelength of nm ( - min). fura- has excitation wavelengths of nm in its free form and nm when it is bound to calcium; therefore, relative calcium concentrations are represented by the ratio of / . (b) ecs plated on glass cover slips were treated with trpm antibody or control igg ( μg/ml, hr pretreatment). ecs were then rinsed twice and subjected to the same calcium measurement procedures with pm treatment ( μg/ml, - min). *p < . compared to control igg group at the same time point. (c) ecs were transfected with trpm sirna or control sirna for hrs. ecs were then rinsed twice and subjected to the same calcium measurement procedures with pm treatment ( μg/ml, - min). ec cell lysates were analyzed by western blot with antibodies to trpm and β-actin to confirm silencing. *p < . compared to control sirna group at the same time point. (d-e) ecs plated on glass cover slips ( % confluent) were treated with nac ( mm, hr pretreatment) or -ab ( mm, hr pretreatment). ecs were then rinsed twice and subjected to the same calcium measurement procedures with pm treatment ( μg/ml, - min). *p < . compared to pm-only group at the same time point. murine lung tissues ( figure c ). zo- over-expression significantly attenuated bal protein leakage ( figure d) , bal white blood cell infiltration ( figure e) , and the release of proinflammatory cytokine il- into bal ( figure f ), indicating the crucial role of zo- loss in mediating pm-induced pulmonary inflammation and lung vascular hyperpermeability. the most significant finding of the present study is the novel characterization of a ros-dependent pathway that causes calpain-dependent endothelial zo- degradation in response to pm. these data represent the first evidence that calpain signaling, via calcium leakage from activated trpm by ros, plays a critical role in modulating endothelial cell barrier function, resulting in tight junction protein zo- degradation (additional file figure s ). the consequence of zo- degradation is sustained endothelial hyperpermeability and persistent lung inflammation, both of which contribute to variety of acute or chronic cardiovascular disorders [ , ] . these effects were also observed with other types of pm samples ( a from human lung microvascular ecs grown in -well dishes to approximately % confluence were treated with trpm antibody or control igg ( μg/ml) for hr, and then challenged with pm ( μg/ml) for hr. cell lysates were analyzed by western blotting with zo- antibody. changes in levels of zo- are expressed as fold changes and normalized to β-actin. shown are representative blots from three independent experiments. *p < . compared to control. **p < . compared to pm challenge. (b) ecs grown in -well dishes to approximately % confluence were treated with trpm sirna or control sirna ( ng/ml) for hr, and cell lysates were analyzed by western blotting with zo- antibody. (c) ecs grown in -well dishes to approximately % confluence were treated with trpm sirna or control sirna ( ng/ml) for hr, and then challenged with pm ( μg/ml) for hr. cell lysates were analyzed by western blotting with zo- antibody. changes in levels of zo- are expressed as fold changes and normalized to β-actin. shown are representative blots from three independent experiments. *p < . compared to control. **p < . compared to pm challenge. (d) ecs grown on ecis gold electrodes were treated with trpm antibody or control igg ( μg/ml) for hr, and then challenged with pm ( μg/ml). changes in ter were measured with ecis. *p < . compared to pm-challenged group. (e) ecs grown on mm dishes were treated with trpm sirna or control sirna ( ng/ml) for hr, and then plated onto gold electrodes for ecis measurement. hours after replating, the ecs were challenged with pm ( μg/ml) and changes in ter were measured with ecis. *p < . compared to pm-challenged group. national institute of standards and technology, fine pm collected from new york city or baltimore, data not shown), indicating a selective pathogenesis pathway by pm pollution. previous studies report that pm triggers the generation of reactive oxygen species or ros mainly from dysfunctional mitochondria [ , ] , and we also noticed the massive generation of ros by this pm sample is also mainly from mitochondria (unpublished observation). the high iron level of this particular pm might also contribute to the ros generated via fenton reactions. ros released endogenously, have been implicated in the pathophysiology of several lung diseases, including asthma and copd, as the biochemical mechanisms underlying the urban pm-induced airway inflammation and toxicity [ ] . ros are highly reactive and cause deleterious gene, protein, and tissue effects. ros are increased in bal or exhaled breath condensate from patients with inflammatory lung injuries and from people with cardiopulmonary disease who have been exposed to pm [ , ] . this response may reflect the high oxidative potential of fine and ultrafine particulates. residual oil fly ash (rofa) and pm . - . cause pulmonary inflammation mediated by oxidative stress [ , ] . in vivo, exposing rats to pm leads to the formation of free radicals in the lung [ ] . since cardiovascular disease is considered a risk factor of pmrelated mortality and morbidity, it is interesting to note that spontaneously hypertensive rats, when exposed to pm, were more susceptible to pulmonary (inflammatory injury) and cardiovascular complications (acute depression of ecg activity) in an oxidant-dependent manner [ ] . besides ros, pm might trigger adverse outcomes via other potential mechanisms including nonselective phosphatase inhibition (by vanadium) or competitive ion channel inhibition (by nickel) due to the complex and variable chemical components. in this study, we first define a novel pathway that mediates ros-dependent tight junction disruption upon particulate matter challenge. tight junctions, or zonula occludens, are the most apical component of the intercellular junctional complex, which also includes adherens junctions, desmosomes, and gap junctions [ ] . zo- was the first tight junction protein to be identified, and zo- and zo- were later isolated as proteins that coimmunoprecipitated with zo- [ , ] . zo- is a peripheral membrane-associated component of the cytoplasmic plaque of tight junctions and is found ubiquitously within tight junctions of epithelial and endothelial cells [ ] . zo- interacts with many cellular proteins via its multiple protein-binding domains. zo- has been reported to interact with other zo family members or claudins via the pdz domains [ , ] . zo- interacts with the c-terminus of occludin with its guk domain and the acidic domain [ ] . the proline-rich c-terminus of zo- mediates its binding to f-actin in vitro, and thus links it to the cytoskeleton [ ] . clearly, zo- interacts with a wide variety of cell skeleton components and plays a central role in orchestrating tight junction complexes. any dysregulation of zo- in endothelial cells by extracellular stimuli, such as virus shell proteins or alcohol, leads to persistent tight junction disruption and vascular hyperpermeability. calpain is a regulator of endothelial integrity which helps control fundamental cellular processes including cytoskeletal remodeling, membrane fusion, cell proliferation and differentiation, and activation of proteolytical cascades leading to apoptosis [ , ] . under oxidative stress, activated calpain cleaves enos and cytoskeletal proteins and induces apoptosis [ , [ ] [ ] [ ] . particulate matter induces endothelial cell intracellular oxidative stress, which leads to the activation of calpain, one of the major cytoskeletal regulators. here we describe the cleavage of tight junction protein zo- by activated calpain both in vitro and in vivo, indicating that calpain plays a central role in pm-induced endothelial barrier disruption and vascular hyperpermeability. in addition, as activated calpain cleaves other critical cytoskeletal proteins including ezrin and marcks protein, the contribution of the other cytoskeletal proteins to the ec hyperpermeability induced by pm needs to be further investigated. oxidative calcium influx is mediated by plasma membrane cation-permeable ion channels. the transient receptor potential protein (trp) and its homologs are cation channels with a tetramer secondary structure which senses diverse stimuli from the extracellular and intracellular figure over-expression of endothelial zo- attenuates pm-induced ec barrier disruption in vitro and pulmonary inflammation in vivo. (a) human lung microvascular ec were grown to % confluence and treated with zo- expression plasmid with x-fect reagent for hr, and over-expression of zo- protein was confirmed by western blot. (b) the ecs were then challenged with pm ( μg/ml), and changes in ter after hr were measured with ecis. *p < . compared to pm-challenged group. aj mice were treated with zo- expression plasmid with an ace antibody-conjugated liposome delivery system ( mg/kg) for days, then challenged with pm ( mg/kg). after hr of pm exposure, (c) lung zo- levels were analyzed with western blot. shown is one of the three repeated blots. bal was collected and (d) protein content, (e) total white blood cell, and (f) il- levels were measured. n = . *p < . compared to pm-challenged group. **p < . compared to control. environments [ ] . mammalian trps comprise six major subfamilies. trpm , a member of the trp channel m subtype, is a calcium-permeable channel activated by intracellular messengers such as adp-ribose [ ] . massive ros burden induced by pm contributes to dna oxidation and damage, which activates poly-adp ribose polymerase (parp) to initiate dna repair mechanisms. parp binds to single-stranded and double-stranded dna breaks and catalyses the breakdown of nad into nicotinamide and adpribose, the intracellular agonist of trpm [ , , ] . oxidative stress-mediated activation of the parp pathway serves as the major source of free adp-ribose production in endothelial cells [ ] . intracellular adp-ribose activates trpm , allowing calcium ions to enter the cell, which in turn trigger numerous physiological and pathological processes. an important limitation of our study is the high dose of pm that we employed. with - μg/m ambient pm level in the us or europe, it is hardly to achieve a high level of acute pm exposure. while μg/ml (in vitro) or mg/kg (in vivo) are typical doses used in particulate matter toxicology studies [ , , , [ ] [ ] [ ] . with an assumed ambient pm level of μg/m , one man with kg body weight and m /minute respiration rate would receive a dose of mg/kg corresponding to about years of exposure with % deposition rate. as noted, a lot of cities in the developing countries still have high levels of ambient pm. a report by world bank [ ] extensive epidemiologic and experimental evidence has demonstrated that particulate air pollution directly causes cardiopulmonary damage. our observations demonstrate a novel mechanism of pm-mediated disruption of endothelial barrier function which is attributable to zo- degradation by calpain, which is activated by extracellular calcium leakage through oxidant-sensitive trpm channels. therefore, inhibition of ros/trpm /calpain/zo- degradation may provide useful therapeutic strategies for the treatment of endothelial barrier dysfunction and lung inflammation. additional file : figure s . (a-b) pm induces dose-dependent reduction in transendothelial resistance (ter). (c) pm induces dosedependent ( hr) reduction of zo- protein levels. figure s . pm induced fitc-dextran leakage across ec monolayer. figure s . pm ( μg/ml, - hr) does not induce ldh release from human ecs. figure s . nac or peg-cat attenuates pm-induced ros in ecs. figure s . nac ( mm, - hr) does not change zo- protein levels in human ecs. figure s . euk- ( μm, hr pre-treatment) attenuates pm ( μg/ml, hr)-induced zo- degradation and ter reduction. figure s . we hypothesize that pm induces ec barrier disruption in delayed phase (via zo- degradation) and acute phase (via stress fiber formation). fine particulate air pollution and hospital admission for cardiovascular and respiratory diseases coarse particulate matter air pollution and hospital admissions for cardiovascular and respiratory diseases among medicare patients tager i: air pollution and cardiovascular disease: a statement for healthcare professionals from the expert panel on population and prevention science of the american 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rat lung after exposure to an emission source air pollution particle the spontaneously hypertensive rat as a model of human cardiovascular disease: evidence of exacerbated cardiopulmonary injury and oxidative stress from inhaled emission particulate matter regulation of tight junctions and loss of barrier function in pathophysiology zo- , a novel member of the maguk protein family found at the tight junction, interacts with zo- and occludin the tight junction: morphology to molecules zonula occludens- and − are cytosolic scaffolds that regulate the assembly of cellular junctions direct binding of three tight junction-associated maguks, zo- , zo- , and zo- , with the cooh termini of claudins characterization of zo- as a maguk family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin the tight junction protein occludin and the adherens junction protein alpha-catenin share a common interaction mechanism with zo- the tight junction protein zo- establishes a link between the transmembrane protein occludin and the actin cytoskeleton calpains: a physiological regulator of the endothelial barrier? calpain: new perspectives in molecular diversity and physiological-pathological involvement activation of protease calpain by oxidized and glycated ldl increases the degradation of endothelial nitric oxide synthase calpain- induces apoptosis in pulmonary microvascular endothelial cells under septic conditions taurine prevents cardiomyocyte death by inhibiting nadph oxidase-mediated calpain activation trp channels as cellular sensors trpm : a calcium influx pathway regulated by oxidative stress and the novel second messenger adp-ribose calciumdependent modulation of poly(adp-ribose) polymerase- alters cellular metabolism and dna repair poly(adp-ribose) polymerase- (parp- ) controls lung cell proliferation and repair after hyperoxia-induced lung damage parp- inhibition prevents oxidative and nitrosative stress-induced endothelial cell death via transactivation of the vegf receptor ambient particulate matter accelerates coagulation via an il- -dependent pathway airborne particulate matter inhibits alveolar fluid reabsorption in mice via oxidant generation proapoptotic noxa is required for particulate matter-induced cell death and lung inflammation ambient particulate matter concentrations in residential and pollution hotspot areas of world cities: new estimates based on the global model of ambient particulates (gmaps) submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors gratefully acknowledge the contribution of lakshmi natarajan and rebecca sullivan for expert technical assistance. the authors declare that they have no competing interests.authors' contributions tw designed & performed research, analyzed & interpreted data, and wrote the manuscript. lw, lm, gdl, jhs, bm, and pvu performed research and analyzed data. jms, asg, and pnb provided pm sample and reviewed the manuscript. vn interpreted data and reviewed the manuscript. jgng designed research, interpreted data, and wrote the manuscript. all authors read and approved the final manuscript. although the research described in this article has been funded in part by the united states environmental protection agency through grant/ cooperative agreement #rd- , it has not been subjected to the agency's required peer and policy review and therefore does not necessarily reflect the views of the agency and no official endorsement should be inferred. none. key: cord- -ok px z authors: armando, federico; gambini, matteo; corradi, attilio; giudice, chiara; pfankuche, vanessa maria; brogden, graham; attig, friederike; von köckritz-blickwede, maren; baumgärtner, wolfgang; puff, christina title: oxidative stress in canine histiocytic sarcoma cells induced by an infection with canine distemper virus led to a dysregulation of hif- α downstream pathway resulting in a reduced expression of vegf-b in vitro date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: ok px z histiocytic sarcomas represent malignant tumors which require new treatment strategies. canine distemper virus (cdv) is a promising candidate due to its oncolytic features reported in a canine histiocytic sarcoma cell line (dh cells). interestingly, the underlying mechanism might include a dysregulation of angiogenesis. based on these findings, the aim of the present study was to investigate the impact of a persistent cdv-infection on oxidative stress mediated changes in the expression of hypoxia-inducible factor (hif)- α and its angiogenic downstream pathway in dh cells in vitro. microarray data analysis, immunofluorescence for -hydroxyguanosine, superoxide dismutase and catalase, and flow cytometry for oxidative burst displayed an increased oxidative stress in persistently cdv-infected dh cells (dh ond pi) compared to controls. the hif- α expression in dh ond pi increased, as demonstrated by western blot, and showed an unexpected, often sub-membranous distribution, as shown by immunofluorescence and immunoelectron microscopy. furthermore, microarray data analysis and immunofluorescence confirmed a reduced expression of vegf-b in dh ond pi compared to controls. in summary, these results suggest a reduced activation of the hif- α angiogenic downstream pathway in dh ond pi cells in vitro, most likely due to an excessive, unusually localized, and non-functional expression of hif- α triggered by a cdv-induced increased oxidative stress. neoplastic diseases are one of the major causes of death in humans and domestic animals due to disappointing results of many conventional therapies [ , ] . therefore, viral oncolysis represents an interesting potential new option in human as well as veterinary medicine [ ] [ ] [ ] . interestingly, viruses from different families, including members of the paramyxoviridae (canine distemper virus, measles virus and newcastle disease virus), poxviridae (vacciniavirus), reoviridae (reovirus serotype dearing), adenoviridae (adenovirus onyx- and h ), orthomyxoviridae (influenza virus), herpesviridae (herpes simplex virus type ), picornaviridae (coxsackievirus) and rhabdoviridae (vesicular stomatitis virus) possess oncolytic properties [ ] [ ] [ ] . canine distemper virus represents a morbillivirus closely related to human measles virus [ ] , with the latter already described as a promising oncolytic virus in human medicine that has reached the phase of clinical trials [ ] . similarly, the attenuated onderstepoort vaccine strain of canine distemper virus (cdv-ond) represents a potential oncolytic virus for the treatment of canine histiocytic sarcomas [ , ] . canine histiocytic sarcomas are malignant tumors with poor prognosis and limited therapeutic options [ , ] which originate from interstitial dendritic cells or from macrophages [ ] [ ] [ ] [ ] . since its establishment in [ ] , a canine histiocytic sarcoma cell line (dh cells) has been commercially available. dh cells can be infected by cdv-ond [ ] , and have been reported as a promising model for the investigation of viral oncolysis [ , , ] . specifically, acute infection of dh cells with cdv-ond in vitro resulted in a prominent cell death at days post infection [ ] , followed by establishment of persistent infection in tumor cells surviving the acute lytic phase [ ] . in this context, subcutaneous xenotransplantion of persistently cdv-ond infected dh cells resulted in a total regression of neoplasms in a mouse model [ ] . this promising observation was assumed to be related to a decreased vascularization of the transplants [ ] , with the underlying mechanisms not fully understood so far. therefore, additional investigations using persistently cdv-infected dh cells might represent a promising model to study virus-induced alterations of cancer hallmarks [ ] and of the tumor microenvironment [ ] avoiding the confounding effects correlated with ongoing virus-induced cytopathogenic tumor cell death associated with the acute infection [ ] . indeed, as reviewed by lapp et al. [ ] , viral oncolysis mechanisms can be distinguished between primary (i.e., direct virus-induced cytolysis and/or apoptosis) and secondary ones. the latter include a wide range of events leading to tumor cell death, such as modulation of the antiviral and antitumoral immune response, changes in the organization of the tumor-associated extracellular matrix, and alterations of the tumor-associated vasculature and angiogenesis [ , , , , , [ ] [ ] [ ] [ ] . specifically, a reduced vascularization of neoplasms often leads to intratumoral hypoxia [ ] associated with modifications especially of intracellular pathways connected with reactive oxygen species (ros) production and scavenging. ros are highly chemically reactive molecules that can induce damage to cellular macromolecules such as nucleic acid and lipids, when they outnumber scavenging systems [ ] [ ] [ ] . cdv infection can increase ros production and ros-induced damage in vitro and in vivo as shown for spontaneous cdv infection in dogs [ ] [ ] [ ] [ ] [ ] . furthermore, cdv can induce an accumulation of viral glycoproteins in the endoplasmic reticulum (er) of vero cells and primary rat neurons, resulting in increased endoplasmic reticulum stress [ ] , which has been reported as associated with an increased ros production [ ] . nevertheless, ros are physiologically involved in a plethora of different intracellular signaling pathways [ , ] , and play a key role in multiple hallmarks of cancer [ ] . hypoxia-inducible factor -alpha (hif- α) is a transcription factor that after translocation from the cytoplasm to the nucleus, forms a heterodimer with hypoxia-inducible factor -beta (hif- β), which binds to specific dna sequences known as hypoxia response elements (hres) [ , ] . this event induces the expression of numerous genes involved in different cellular responses such as angiogenesis [ , ] , which is driven by several growth factors, including members of the vascular endothelial growth factor (vegf) family. hypoxia, and to a lesser extent ros, represent the most important stimuli for hif- α stabilization and nuclear translocation [ , ] . during normoxia and redox homeostatic state [ ] , hif- α is localized within the cytoplasm and is rapidly degraded by the proteasome after hydroxylation by prolyl hydroxylases (phds) and subsequent ubiquitination by the von hippel-lindau protein (vhl) [ , , ] . in this context, hypoxia and ros directly down-regulate the activity of phds and vhl [ ] , playing therefore a key role in the inhibition of the overall hif- α degradation. in consideration of the above, the hypothesis of the present study was that a persistent cdv-ond infection of dh cells induces oxidative stress followed by a massive inhibition of hif- α degrading pathways. this in turn leads to cytoplasmic, non-functional accumulation of hif- α, which is associated with a reduced expression of hif- α downstream targets, such as vegf-b. based on the aforementioned hypothesis, the aim of the present in vitro study was to demonstrate that histiocytic sarcoma cells (dh cells) persistently infected with cdv-ond show: ( ) an increased oxidative stress status, ( ) an increased hif- α protein expression, ( ) an unusual intracellular distribution of hif- α, and ( ) a reduced expression of hif- α downstream targets, with a special focus on vegf-b. non-infected dh cells obtained from the european collection of authenticated cell cultures (ecacc no. ), and dh cells persistently infected with cdv-ond (dh ond pi) that were established as formerly described [ ] , were cultured according to standard procedures as previously reported [ ] . briefly, cells were cultured in minimal essential medium (mem) with earle's salts (paa, cölbe, germany) supplemented with % fetal calf serum (paa), % penicillin/streptomycin (paa), and % non-essential amino acids (sigma-aldrich, taufkirchen, germany). culture flasks were kept at • c in the presence of % co in a water saturated atmosphere. five formalin-fixed paraffin embedded (ffpe) cell pellets of non-infected dh cells and of dh ond pi cells were produced as previously described [ ] . briefly, cells were scraped and centrifuged at xg for min at • c. afterwards, the supernatant was removed, cells were washed in pbs and centrifuged again. following a second wash and centrifugation step, the pellet was fixed in . ml of % non-buffered formalin overnight at • c, and processed for routine paraffin embedding. in a hypothesis-driven approach, an online available microarray data set of quadruplicates of non-infected dh and dh ond pi cells (arrayexpress; http://www.ebi.ac.uk/arrayexpress; accession number e-mtab- [ , ] ) was investigated for differentially expressed genes related to ros production and scavenging, er-stress and hif- α pathway, with a special focus on the angiogenic downstream targets of the latter. this choice was justified by the results of the functional profiling of the same dataset obtained in a previous study, highlighting a down-regulation of the expression of some of the genes correlated with angiogenesis [ ] . therefore, in the current work, a list of human and murine genes and proteins was manually generated according to the literature [ , , , , [ ] [ ] [ ] [ ] , ] and translated into canine orthologous gene symbols using the web-based hgnc database (hgnc database, hugo gene nomenclature committee (hgnc), european molecular biology laboratory, european bioinformatics institute (embl-ebi), wellcome genome campus, hinxton, cambridge cb sd, united kingdom, www.genenames.org [ ] ). after filtration, the raw expression data of the selected genes were compared between non-infected dh and dh ond pi cells, employing multiple pairwise nonparametric mann-whitney u-tests. statistical analysis was performed with sas enterprise guide (sas version . ; sas institute inc, cary, nc, usa). differential expression was defined as the combination of a fold change (fc) filter (fc ≥ . or ≤ − . ) and of a statistical significance filter (mann-whitney u-test; p ≤ . ) [ ] . to facilitate the interpretation of results, each gene symbol was assigned to at least one of the following functional groups on the basis of the function(s) carried out by its corresponding protein(s): ros production; ros scavenging; er stress; hif- α activation, transcriptional activity and regulation; hif- α angiogenic downstream pathway. immunofluorescence was performed on ffpe pellets of non-infected and persistently cdv infected dh cells as previously described with minor variations [ , ] . briefly, sections were deparaffinized, rehydrated through graded alcohol and pre-treated for antigen retrieval. following blocking of unspecific bindings, sections were incubated with primary antibodies for min at room temperature. after min of incubation with the secondary antibody, nuclei were stained with bisbenzimide (sigma-aldrich chemie gmbh, taufkirchen, germany), and the slides were mounted with dako flourescence mounting medium (dako north america, inc., carpinteria, ca, usa). each reaction was carried out with corresponding positive controls (table ) . for negative controls, the first antibody was replaced with rabbit serum, balb/c ascitic fluid, or goat serum, respectively at corresponding protein concentrations. to verify the persistent infection status of dh ond pi cells (which was set as corresponding to a rate of > % infected cells), an immunolabeling with an anti-cdv nucleoprotein (cdv-np) antibody (clone d ; kindly provided by prof. a. zurbriggen, university of bern, switzerland) was performed. furthermore, pellets were stained with antibodies directed against -hydroxyguanosine/ -hydroxydeoxyguanosine ( ohg/ ohdg, in the following paragraphs simply referred to as ohdg), a marker of ros-damaged rna or dna [ ] ; superoxide dismutase (sod ) and catalase (cat), two ros scavengers; hif- α, a transcription factor; wheat germ agglutinin (wga), a cell membrane marker; cd , directed against tetraspanin- expressed on exosome membranes; and gm- , a marker for golgi apparatus. all details regarding the antibodies used are listed in table . for cdv-np, ohdg, sod , cat, hif- α, and vegf-b, the percentage of immunopositive cells for each group (non-infected dh cells and dh ond pi cells) was assessed manually by counting evenly distributed fields per pellet at a x magnification using an inverted fluorescence microscope (olympus ix- , olympus optical co. gmbh, hamburg, germany) equipped with a olympus dp camera and olympus cellsens standard software version . . additionally, for hif- α the intracellular protein distribution was assessed and calculated as percentage of cells immunopositive within the nucleus, cytoplasm and membrane. for each marker, after calculation of the median percentage of immunopositive cells per pellet, the normality of distribution of the data referring to non-infected and dh ond pi cells was evaluated with the shapiro-wilk test and followed by the mann-whitney u test for pairwise comparison. the difference of the intracellular distribution of hif- α immunopositivity within each group of cells was analyzed with the kruskall-wallis test with post-hoc dunn's test. statistical significance for each analysis was set at p-value ≤ . . all statistical analyses were performed with graphpad prism version . . for windows (graphpad software, la jolla, ca, usa, www.graphpad.com). gar-cy canine fetal brain, liver and kidney bsa, bovine serum albumin; cdv-np, canine distemper virus nucleoprotein; dag-cy , donkey anti goat cyanine -conjugated; dar-cy , donkey anti rabbit cyanine -conjugated; gam-cy , goat anti mouse cyanine -conjugated; gam-cy , goat anti mouse cyanine -conjugated; gar-cy , goat anti rabbit cyanine -conjugated; gar-cy , goat anti rabbit cyanine -conjugated; gm , golgi membrane protein of kda; hif- α, hypoxia-inducible factor α; mdck, madin-darby canine kidney cells; n/a, non applied or non applicable; pbst, phosphate buffered saline tween- ; sod , superoxide dismutase ; vegf-b, vascular endothelial growth factor-b; wga, wheat germ agglutinin; ohdg, -hydroxyguanosine/ -hydroxydeoxyguanosine. non-infected and persistently cdv-ond infected dh cells were treated with ´, ´-dichlorofluorosceindiacetate (dcf, final concentration of µm, sigma aldrich, d ) at • c and % co for min. flow cytometer (attune ® nxt acoustic focusing; laser nm ( mw), filter bl- = / ) analysis was performed measuring mean green fluorescence intensity (x-mean of bl- ) as relative ros production. respective background controls without dcf were included in all assays. threshold was adjusted to unstained cells to remove background. green fluorescence intensity (fitc) of all cells (percentage of positive cells) was recorded by flow cytometry as relative measure of ros production. the following settings were used: acquisition volume of µl/min, stop at , events on all counts; instrument settings: fsc , ssc bl (fitc). were quantified. statistical analyses of measurements were performed with graphpad prism version . . for windows (graphpad software, la jolla california usa, www.graphpad.com) using unpaired t-tests. to evaluate in more detail the intracellular localization of hif- α within dh ond pi cells, immunoelectron microscopy was performed using a % neutral buffered formalin fixed cell pellet as previously described [ ] . ultrathin sections of lr-white embedded samples were immunolabeled with an anti-hif- α antibody ( : dilution; novus biologicals) followed by a goat anti-rabbit igg nm immunogold conjugated secondary antibody (bbi solutions, crumlin, united kingdom). samples were further evaluated using a transmission electron microscope (em a, carl zeiss microscopy gmbh, jena, germany) equipped with a k-ccd-camera (trs) and using image sp professional software. the intracellular hif- α distribution was analyzed by double-labeling immunofluorescence (dl-if). therefore, hif- α was combined with wga as a marker for the cell membrane, cd as an exosomal marker, gm- as a marker for the golgi apparatus, and cdv-np. the evaluation was performed using a leica tcs sp ii fluorescence microscope (leica microsystems, bensheim, germany) with a conventional galvanometer scanner of the leica sp ii tandem scanning system and the leica application suite advanced fluorescent lite . . build (leica, biberach, germany). settings were adjusted using respective control antibodies. images were analyzed using leica las af software (version . . ). cell lysates were prepared by freezing and thawing in ml np- buffer ( mm tris-hcl, mm nacl, % np- , mm edta) with µl protease inhibitor cocktail ( . µm antipain dihydrochloride, . µm aprotinin, . µm leupeptin, . µm pepstatin a in dmso, mm pmsf, µg/ml trypsin inhibitor t ) at ph . (all reagents from sigma-aldrich, st. louis, usa). samples were analyzed by sds-page on % gels and subsequently transferred to a polyvinylidene fluoride (pvdf) membrane as described previously [ ] . immunoblotting was performed using a polyclonal anti-hif- α ( . µg/ml, cayman, ann arbor, usa) and a monoclonal anti-β-actin ( . µl/ml, santa cruz, dallas, usa) antibody, respectively. a polyclonal igg antibody from rabbit serum served as a negative control ( µg/ml, sigma-aldrich, st. louis, usa). secondary anti-rabbit or anti-mouse antibodies conjugated to horseradish peroxidase were used ( . µg/ml, thermoscientific, schwerte, germany). protein bands were visualized using supersignal™ west femto maximum sensitivity western blot chemiluminescence substrate (thermoscientific, schwerte, germany) and a chemidoc mp imaging system (bio-rad, hercules, ca, usa). quantification was performed densitometrically. obtained results for hif- α were displayed as a ratio with the corresponding amount of β-actin. statistical analyses of obtained ratios were performed with graphpad prism version . . for windows (graphpad software, la jolla, ca, usa, www.graphpad.com) using unpaired t-tests. the infection status of dh ond pi cells was assessed via immunofluorescence staining for cdv-np (supplementary figure s ). while immunoreactivity for cdv-np of non-infected dh cell pellets was negative in all cells, dh ond pi cell pellets showed a median percentage of . % (range: . - . %) infected cells (supplementary table s ) . on a molecular level, a manually generated list of canine gene symbols associated with ros production and scavenging, er stress and the hif- α pathway was analyzed using a microarray dataset of dh and dh ond pi cells. this investigation resulted in a list of genes present within the available data set (supplementary table s ). using the combination of a statistical significance filter (mann-whitney u-test; p ≤ . ) and a fold change (fc) filter (fc ≥ . or ≤ − . ), genes were differentially expressed. specifically, canine genes showed a down-regulation, whereas genes were up-regulated (table ) . table . summary of canine gene symbols related to ros production and scavenging, er-stress and hif- α pathway, differentially expressed between non-infected and persistently canine distemper virus infected dh cells, according to the combination of a fold change (fc) filter (fc ≥ . or ≤ − . ) and of a statistical significances filter (p ≤ . ). [ , ] green labeling refers to down-regulated genes; red refers to up-regulated genes. er, endoplasmic reticulum; hif- α, hypoxia-inducible factor α; ros, reactive oxygen species. "hif- α transcription & regulation" is the abbreviation for "hif- α activation, transcriptional activity and regulation" functional group; "hif- α downstream" is the abbreviation for "hif- α angiogenic downstream pathway" functional group. when specifically analyzed according to the functional grouping, genes related to ros production were up-regulated, while nine were down-regulated (table ). among the group of genes related to ros scavenging, five genes were up-and five were down-regulated (table ) . specifically, neutrophil cytosolic factor (ncf ) and thioredoxin interacting protein (txnip), belonging to ros production and ros scavenging functional groups, respectively, were the two most markedly up-regulated genes among the entire set examined. taken together, these findings should be cautiously interpreted as an increased transcription of genes which corresponding proteins are involved in increasing intracellular oxidative stress [ , , , ] . among the group of genes related to er-stress (partially overlapping with both ros production and ros scavenging functional groups), genes were up-regulated while were down-regulated. specifically, among the genes included in the er stress functional group, the xanthine dehydrogenase (xdh) was up-regulated, while among down-regulated genes were included (pdia , pdia and pdia ) out of genes related to protein disulphide isomerases, one (ero l) out of two genes related to endoplasmic reticulum oxidoreductines, and two (canx and ddit ) out of three genes previously related to er-stress induced by acute infection with cdv [ ] . taken together, these results can be cautiously interpreted as indicative of a reduced transcription of genes that are reported to correlate with er-stress [ , [ ] [ ] [ ] [ ] . the hypothesized increased oxidative stress in dh ond pi cells compared to non-infected dh cells was further investigated by means of immunoreactivity for ohdg, sod and cat, as displayed in figure , as well as by determination of oxidative burst by flow cytometry. table s ). immunofluorescence for sod displayed a significantly (p = . ) increased percentage of positive cells in dh ond pi pellets (median = . %, range: . - . %) compared to non-infected dh pellets (median = . %, range: . %- . %) (supplementary table s ). immunofluorescence for cat revealed a significantly (p = . ) increased percentage of positive cells in dh ond pi pellets (median = immunofluorescence for ohdg lacked a significant difference (p = . ) in the percentage of positive cells between non-infected (median = . %, range: . - . %) and dh ond pi pellets (median = . %, range: . - . %) (supplementary table s ). immunofluorescence for sod displayed a significantly (p = . ) increased percentage of positive cells in dh ond pi pellets (median = . %, range: . - . %) compared to non-infected dh pellets (median = . %, range: . %- . %) (supplementary table s ). immunofluorescence for cat revealed a significantly (p = . ) increased percentage of positive cells in dh ond pi pellets (median = . %, range: . %- . %) compared to non-infected dh pellets (median = . %, range: . %- . %) (supplementary table s ). the determination of oxidative burst by flow cytometry demonstrated a significantly (p = . ) increased ros production among dh ond pi cells compared to non-infected dh cells (figure ). table s ). the determination of oxidative burst by flow cytometry demonstrated a significantly (p = . ) increased ros production among dh ond pi cells compared to non-infected dh cells (figure ). despite a lack of difference in ros-induced nucleic acid damage as determined by immunofluorescence of ohdg, these results are collectively indicative of an increased oxidative stress in dh ond pi cells compared to non-infected dh cells, which might lead to an increased level of hif- α and subsequently to an inhibition of its degradation. among the gene symbols referring to the functional group "hif- α activation, transcriptional activity and regulation", three out of genes were down-regulated (table ) . specifically, downregulated gene symbols were those referring to two (engl and engl ) out of three prolyl hydroxylases and to von hippel-lindau (vhl) protein, while hif- α gene symbol (hif a) did not show any significant change (supplementary table s ) . immunoreactivity for hif- α revealed a significant (p = . ) higher percentage of positive dh ond pi cells (median = . %, range . %- . %) (supplementary table s ) compared to non-infected dh cells (median = . %, range: . %- . %), as shown in figure . in non-infected dh cells, hif- α was mainly expressed within nucleus (median = . %, range: . %- . %) and cytoplasm (median = . %, range: . %- . %) and only to a lesser extent in the membrane (median: . %, range: . %- . %), without significant differences (p ranging from . to > . ) between the three localizations. interestingly, dh ond pi cells displayed a significantly higher hif- α expression in the membrane ( figure ) compared to nuclear (p = . ; membrane median = . %, membrane range: . %- . %; nuclear median = . %, nuclear range: . %- . %) but not to cytoplasmic localizations (p = . ; cytoplasm median = . %, cytoplasm range: . %- . %). additionally, the membranous immunopositivity for hif- α in dh ond pi cells was significantly (p = . ) higher when compared to the corresponding localization in non-infected despite a lack of difference in ros-induced nucleic acid damage as determined by immunofluorescence of ohdg, these results are collectively indicative of an increased oxidative stress in dh ond pi cells compared to non-infected dh cells, which might lead to an increased level of hif- α and subsequently to an inhibition of its degradation. among the gene symbols referring to the functional group "hif- α activation, transcriptional activity and regulation", three out of genes were down-regulated (table ) . specifically, down-regulated gene symbols were those referring to two (engl and engl ) out of three prolyl hydroxylases and to von hippel-lindau (vhl) protein, while hif- α gene symbol (hif a) did not show any significant change (supplementary table s ) . immunoreactivity for hif- α revealed a significant (p = . ) higher percentage of positive dh ond pi cells (median = . %, range . %- . %) (supplementary table s ) compared to non-infected dh cells (median = . %, range: . %- . %), as shown in figure . in non-infected dh cells, hif- α was mainly expressed within nucleus (median = . %, range: . %- . %) and cytoplasm (median = . %, range: . %- . %) and only to a lesser extent in the membrane (median: . %, range: . %- . %), without significant differences (p ranging from . to > . ) between the three localizations. interestingly, dh ond pi cells displayed a significantly higher hif- α expression in the membrane ( figure ) compared to nuclear (p = . ; membrane median = . %, membrane range: . %- . %; nuclear median = . %, nuclear range: . %- . %) but not to cytoplasmic localizations (p = . ; cytoplasm median = . %, cytoplasm range: . %- . %). additionally, the membranous immunopositivity for hif- α in dh ond pi cells was significantly (p = . ) higher when compared to the corresponding localization in non-infected dh cells (supplementary table s ). summarized, these results are indicative of an increased level of hif- α in dh ond pi, which is most likely due to a decreased cytoplasmic degradation. to further characterize the intracellular localization of hif- α, immunoelectron microscopy and laser scanning confocal microscopical within non-infected dh cells, hif- α was present within nucleus and cytoplasm without significant differences between the localizations. in contrast, persistently cdv-infected dh cells displayed a significantly higher membranous hif- α expression compared to nuclear (p = . ) but not to cytoplasmic (p = . ) localizations. additionally, the membranous immunopositivity for hif- α in persistently cdv-ond infected dh cells was significantly higher compared to the corresponding localization in non-infected controls. box and whisker plots display median and quartiles with maximum and minimum values. significant differences (p ≤ . , mann-whitney utest (c,d) and kruskall-wallis test with post-hoc dunn's test (d)) are labeled by asterisks (* p ≤ . and ** p ≤ . ). within non-infected dh cells, hif- α was present within nucleus and cytoplasm without significant differences between the localizations. in contrast, persistently cdv-infected dh cells displayed a significantly higher membranous hif- α expression compared to nuclear (p = . ) but not to cytoplasmic (p = . ) localizations. additionally, the membranous immunopositivity for hif- α in persistently cdv-ond infected dh cells was significantly higher compared to the corresponding localization in non-infected controls. box and whisker plots display median and quartiles with maximum and minimum values. significant differences (p ≤ . , mann-whitney u-test (c,d) and kruskall-wallis test with post-hoc dunn's test (d)) are labeled by asterisks (* p ≤ . and ** p ≤ . ). hif- α immunoblotting confirmed the significantly increased protein expression (p = . ) in dh ond pi cells when compared to the non-infected dh cells (figure ). ultrastructural investigation of dh ond pi by immunoelectron microscopy for hif- α revealed that this protein was mostly localized in the sub-membranous compartment as well as within variably sized, round, moderately to highly electrondense vesicles ( figure ). based on the assumptions that many viruses have been shown to induce an increased production of cd + exosomes [ ] , and that viral proteins can be stored within endolysosomal system [ ] , dl-if for hif- α in association with different markers was performed and evaluated by laser scanning confocal microscopy. summarized, these results are indicative of an increased level of hif- α in dh ond pi, which is most likely due to a decreased cytoplasmic degradation. to further characterize the intracellular localization of hif- α, immunoelectron microscopy and laser scanning confocal microscopical analysis of double stainings were performed. ultrastructural investigation of dh ond pi by immunoelectron microscopy for hif- α revealed that this protein was mostly localized in the sub-membranous compartment as well as within variably sized, round, moderately to highly electrondense vesicles ( figure ) . based on the assumptions that many viruses have been shown to induce an increased production of cd + exosomes [ ] , and that viral proteins can be stored within endolysosomal system [ ] , dl-if for hif- α in association with different markers was performed and evaluated by laser scanning confocal microscopy. to verify the specificity of the membranous staining, dl-if for hif- α in association with wga was performed, confirming a membranous to sub-membranous localization of hif- α without overlapping co-staining of the two markers (supplementary figure s ) . to investigate whether hif- α was associated with exosomes, dl-if in association with cd was performed, revealing an occasional co-localization of the two markers ( figure ). to exclude an hif- α storage within the golgi apparatus, dl-if in association with gm- was performed, clearly showing that hif- α was not localized within this cell organelle (supplementary figure s ) . finally, to analyze whether hif- α was associated with cdv-np, dl-if in association with cdv-np was performed, revealing a marked and diffuse co-localization of the two markers ( figure ). in summary, these results confirmed an unexpected localization of hif- α in the sub-membranous compartment of dh ond pi cells, being occasionally associated with cd + exosomes and more frequently with cdv-np. to investigate if this unusual localization of hif- α can affect the expression of its angiogenetic downstream molecules with a special focus on vegf-b, further microarray data and immunofluorescence analyses were performed. ultrastructural investigation of dh ond pi by immunoelectron microscopy for hif- α revealed that this protein was mostly localized in the sub-membranous compartment as well as within variably sized, round, moderately to highly electrondense vesicles ( figure ). based on the assumptions that many viruses have been shown to induce an increased production of cd + exosomes [ ] , and that viral proteins can be stored within endolysosomal system [ ] , dl-if for hif- α in association with different markers was performed and evaluated by laser scanning confocal microscopy. to verify the specificity of the membranous staining, dl-if for hif- α in association with wga was performed, confirming a membranous to sub-membranous localization of hif- α without overlapping co-staining of the two markers (supplementary figure s ) . to investigate whether hif- α was associated with exosomes, dl-if in association with cd was performed, revealing an occasional co-localization of the two markers ( figure ). the intracellular hif- α localization was analyzed by double immunofluorescence with hif- α (cy , green) and cd (cy , red) in persistently canine distemper virus (cdv)-infected dh cells. both proteins were localized within cell membranes and cytoplasm. interestingly, an occasional co-expression (yellow) was noted (arrows; insert) using scanning confocal laser microscopy. (b) a double labeling directed against hif- α (cy , red) and the cdv nucleoprotein (cdv-np; cy , green) revealed a frequent co-localization (yellow) beneath the cell membrane and within the perinuclear area (insert) of persistently cdv-infected dh cells. nuclei were stained with bisbenzimide (blue). bar = µ m. to exclude an hif- α storage within the golgi apparatus, dl-if in association with gm- was performed, clearly showing that hif- α was not localized within this cell organelle (supplementary figure s ) . finally, to analyze whether hif- α was associated with cdv-np, dl-if in association with cdv-np was performed, revealing a marked and diffuse co-localization of the two markers ( figure ). the intracellular hif- α localization was analyzed by double immunofluorescence with hif- α (cy , green) and cd (cy , red) in persistently canine distemper virus (cdv)-infected dh cells. both proteins were localized within cell membranes and cytoplasm. interestingly, an occasional co-expression (yellow) was noted (arrows; insert) using scanning confocal laser microscopy. (b) a double labeling directed against hif- α (cy , red) and the cdv nucleoprotein (cdv-np; cy , green) revealed a frequent co-localization (yellow) beneath the cell membrane and within the perinuclear area (insert) of persistently cdv-infected dh cells. nuclei were stained with bisbenzimide (blue). bar = µm. among the gene symbols referring to the functional group "hif- α angiogenic downstream molecules", six out of genes were up-regulated, whereas genes were down-regulated (table ) . specifically, down-regulated gene symbols included those related to the expression of angiogenetic and anti-angiogenetic macromolecules which transcription is directly induced by the activation of the hif- α downstream pathway (i.e. vascular endothelial growth factor b-vegfb; thrombospondin -thbs ; endothelin -edn /et ; serine peptidase inhibitor e-serpine ; thrombospondin -thbs ; chemokine ligand -cxcl ; cd -nt e; basic fibroblast growth factor -fgf , adrenomedullin-adm; cd ). immunofluorescence for vegf-b revealed a significantly (p = . ) decreased percentage of immunopositive cells in dh ond pi pellets (median = . %, range: . %- . %) (supplementary table s ) compared to non-infected dh pellets (median = . %, range: . %- . %), as shown in figure . table s ) compared to non-infected dh pellets (median = . %, range: taken together, these results are indicative of a reduced activation of the hif- α angiogenic downstream pathway. this is most likely due to an excessive, unusually localized, and nonfunctional protein expression of hif- α, which might be the consequence of a decrease in its cytoplasmic degradation following a virus-induced increased oxidative stress. canine histiocytic sarcoma cells (dh ) persistently infected with cdv-ond display a complete spontaneous tumor regression when xenotransplanted subcutaneously into scid mice [ ] . considered that dh ond pi cells did not show any difference in growth and apoptotic rate compared to non-infected controls in vitro and during the initial phase after transplantation in vivo [ , , ] , it was assumed that tumor regression of dh ond pi xenotransplants was not caused primarily by direct virus-induced cell death alone. indeed, it seems more likely that secondary effects of the viral infection on the tumor microenvironment [ , ] , as similarly reported for reoviruses [ ] , account for the complete regression. specifically, it was estimated that regression of dh ond pi xenotransplants might be related to alterations in cancer-associated angiogenesis [ ] . therefore, the aim of the present in vitro study was to investigate in more detail pathways potentially involved in this regression process, taking advantage of the absence of the confounding effects correlated with ongoing tumor cell death associated with acute cdv-ond infection [ ] . furthermore, to restrict the complex interactions that occur within a living organism, a less complex, highly standardized in vitro model is assumed to facilitate the analysis of specific intracellular pathways. interestingly, the socalled "angiogenic switch" has been reported to be one of the most important hallmarks of cancer [ , ] , thus playing a central role for tumor development and expansion. in this context, the present taken together, these results are indicative of a reduced activation of the hif- α angiogenic downstream pathway. this is most likely due to an excessive, unusually localized, and non-functional protein expression of hif- α, which might be the consequence of a decrease in its cytoplasmic degradation following a virus-induced increased oxidative stress. canine histiocytic sarcoma cells (dh ) persistently infected with cdv-ond display a complete spontaneous tumor regression when xenotransplanted subcutaneously into scid mice [ ] . considered that dh ond pi cells did not show any difference in growth and apoptotic rate compared to non-infected controls in vitro and during the initial phase after transplantation in vivo [ , , ] , it was assumed that tumor regression of dh ond pi xenotransplants was not caused primarily by direct virus-induced cell death alone. indeed, it seems more likely that secondary effects of the viral infection on the tumor microenvironment [ , ] , as similarly reported for reoviruses [ ] , account for the complete regression. specifically, it was estimated that regression of dh ond pi xenotransplants might be related to alterations in cancer-associated angiogenesis [ ] . therefore, the aim of the present in vitro study was to investigate in more detail pathways potentially involved in this regression process, taking advantage of the absence of the confounding effects correlated with ongoing tumor cell death associated with acute cdv-ond infection [ ] . furthermore, to restrict the complex interactions that occur within a living organism, a less complex, highly standardized in vitro model is assumed to facilitate the analysis of specific intracellular pathways. interestingly, the so-called "angiogenic switch" has been reported to be one of the most important hallmarks of cancer [ , ] , thus playing a central role for tumor development and expansion. in this context, the present study focused on pathways correlated with increased levels of intracellular ros. these highly reactive molecules have been reported both as fundamental intermediates in physiological intracellular signaling transduction [ , ] , as well as in the regulation of different cancer hallmarks [ , , ] . specifically, together with hypoxia, ros represent one of the major activators of hif- α [ , , , , ] , a transcription factor involved in the regulation of a wide plethora of cancer features such as invasion, metastasis, and angiogenesis [ , [ ] [ ] [ ] [ ] ] . in the context of the aforementioned considerations, the present study was further directed to investigate the impact of a persistent cdv-ond infection of dh cells on cellular oxidative stress. cdv has been reported as being able to trigger an increase in ros intracellular levels, with the subsequent induction of oxidative stress in different kinds of cells such as microglia, in vitro as well as in vivo [ ] [ ] [ ] [ ] [ ] . similarly, the present study revealed increased ros levels in dh ond pi cells, as demonstrated by an increased oxidative burst, as well as suggested by increased gene transcription of txnip and ncf . specifically, the upregulation of both genes might correlate with an increased intracellular oxidative stress. indeed, ncf encodes for p phox , a protein that is involved in nadph oxidase activation [ , , ] . additionally, thioredoxin-binding protein , encoded by the txnip gene, is an important inhibitor of the thioredoxin ros scavenging system [ , ] . on the other hand, ros-induced nucleic acid damage did not differ in dh ond pi cells compared to non-infected controls. this observation might be interpreted as indicative of an increased oxidative stress associated with the neoplastic nature of dh cells rather than an effect of the viral infection. similarly, increased intracellular ros levels are described in the literature as a common feature of cancer cells [ , , ] . in addition, dh ond pi cells displayed an increased expression of sod and cat compared to non-infected controls. the overexpression of these scavenging enzymes involved in ros detoxification have been correlated with an increased oxidative stress in neoplastic [ , , ] as well as in inflammatory conditions [ ] . the results obtained by microarray analysis of genes correlated with er stress [ , , , , ] are consistent with a reduced transcription of genes correlated with this process. the data in the present study might be interpreted as suggestive of an acquired ability of dh cells to adapt to the persistent infection with cdv-ond. however, a marked protein overexpression of er-stress markers such as calnexin, calreticulin and chop/gadd have been observed in vero cell and primary rat neurons h post-infection with recombinant a / -v cdv [ ] . on the other hand, the aforementioned lack of differences in growth and apoptotic rate between non infected and dh ond pi cells [ , ] is in line with the hypothesis that a persistent infection with cdv-ond might be associated with the activation of adaptive and pro-survival pathways to contrast prolonged oxidative stress, as reported in recombinant hela cells expressing silkworm storage protein [ ] . the hypothesis of the present study is further supported by the finding of an increased expression of ros-scavenging enzymes in dh ond pi cells at both a molecular and protein level, highlighting the plasticity of cancer cells in actively contrasting excessively severe alterations in their redox potential [ , ] . the expression of hif- α was subsequently investigated due to the observation that increased oxidative stress is associated with an increased hif- α stabilization and activation [ , , , , ] . hypoxia has been widely reported as the most powerful inductor of hif- α transcriptional activity [ , , ] ; however, in the present study, cells were cultivated under normoxic conditions. therefore, hypoxia could be excluded as the cause of the increased hif- α protein expression observed in our in vitro model. consequently, it seems more plausible that the increased expression of hif- α in dh ond pi cells was induced by the increased oxidative stress level compared to non-infected controls. the down-regulation of phds as well as of vhl on a molecular level, in association with a lacking regulation of hif- α opposed to an increased expression of the corresponding protein, could imply that the increased protein expression of hif- α in dh ond pi cells does not refer to an increased synthesis, but rather to an inhibition of the degradation pathway. correspondingly, ros have been reported to be directly involved in the inhibition of the aforementioned cytoplasmic enzymes (i.e., phds and vhl) responsible for hif- α hydroxylation and ubiquitination which prelude the rapid degradation of hif- α itself by the proteasome s [ , , ] . in addition to the overall increased expression of hif- α, the present study revealed an unusual localization of the transcription factor in the sub-membranous compartment and, to a lesser extent, within cytosolic vesicles. further investigations aiming to better characterize the aforementioned vesicles, revealed a co-localization of hif- α expression with cd , a marker for the tetraspanin- expressed by exosomal membranes [ ] . interestingly, the presence of hif- α within cd + exosomes has previously been reported in epstein-barr virus-infected np cells [ ] . on the other hand, hif- α only occasionally co-localized with cd + exosomes, while it frequently overlapped with the localization of cdv-np. the measles virus n-protein, which is closely related to cdv-np [ ] , is transported within the cell through the endolysosomal system [ ] , also rendering this a possible mechanism for the canine counterpart. furthermore, this observation displays an interesting basis for future investigations on the exact sub-cellular localization of hif- α within dh ond pi cells. microarray data analysis aiming to investigate the molecular consequences of the unusual localization of hif- α and a prospective loss of function of its transcriptional activity, revealed a significant down-regulation of different genes involved in the hif- α angiogenic downstream pathway, which was further substantiated by a significantly reduced expression of vegf-b on a molecular and protein level. though vegf-b is nowadays recognized as not being directly involved in angiogenesis, this growth factor has been reported as an indirect enhancer of vegf-a (a well-known inducer of angiogenesis), as well as a key promoter of survival of different cell types (including endothelial cells, pericytes and smooth muscle cells) in several pathological conditions [ ] [ ] [ ] . as already reported in the literature [ ] , the markedly reduced expression of vegf-b in dh ond pi cells did not affect cellular growth nor the apoptotic rate [ ] . interestingly, dh ond pi cell xenotransplants displayed a significantly reduced microvessel density compared to non-infected controls [ ] . according to the results of the present study, it can be assumed that hif- α might represent an important mediator of the oncolytic effects described for the in vivo model of dh ond pi xenotransplants as reported previously in another viral oncolysis model [ ] . summarized, the results of the current in vitro study are indicative of a reduced activation of the hif- α angiogenic downstream pathway in dh cells persistently infected with cdv-ond compared to non-infected controls. this is most likely due to an excessive, unusually localized, and non-functional expression of hif- α, which might be the consequence of a decreased cytosolic degradation of this transcriptional factor following a virus-induced increased oxidative stress. future studies are warranted to better characterize the localization of hif- α and the exosomes in which it is contained, as well as to verify the presence of an increased oxidative stress and an aberrant hif- α localization in dh ond pi also in vivo. the latter approach might further substantiate the assumed correlation between reduced angiogenesis, hypoxia and tumor regression in dh ond pi xenotransplants. bar = µm, supplementary table s . summary of statistical analyses depicting median and mean percentage of immunopositive cells for each cell population (i.e. non-infected and dh ond pi cells) or for each specific intracellular localization (i.e., membrane, cytoplasm, or nucleus), with the corresponding minimum-maximum range and standard deviation for each marker investigated. the normality of distribution of each data set as well as the p-value of multiple and/or pairwise comparisons between the groups are also reported kruskall-wallis test, min-max, minimum-maximum range 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cord- -ssrs cwf authors: michaelis, martin; sithisarn, patchima; cinatl jr, jindrich title: effects of flavonoid-induced oxidative stress on anti-h n influenza a virus activity exerted by baicalein and biochanin a date: - - journal: bmc res notes doi: . / - - - sha: doc_id: cord_uid: ssrs cwf background: different flavonoids are known to interfere with influenza a virus replication. recently, we showed that the structurally similar flavonoids baicalein and biochanin a inhibit highly pathogenic avian h n influenza a virus replication by different mechanisms in a lung cells. here, we investigated the effects of both compounds on h n -induced reactive oxygen species (ros) formation and the role of ros formation during h n replication. findings: baicalein and biochanin a enhanced h n -induced ros formation in a cells and primary human monocyte-derived macrophages. suppression of ros formation induced by baicalein and biochanin a using the antioxidant n-acetyl-l-cysteine strongly increased the anti-h n activity of both compounds in a cells but not in macrophages. conclusions: these findings emphasise that flavonoids induce complex pharmacological actions some of which may interfere with h n replication while others may support h n replication. a more detailed understanding of these actions and the underlying structure-activity relationships is needed to design agents with optimised anti-h n activity. highly pathogenic influenza a viruses including h n viruses represent a major pandemic threat. complication rates are much higher in h n patients than in seasonal influenza or pandemic h n / patients [ ] [ ] [ ] [ ] . as of th january , confirmed human h n cases had resulted in deaths (www.who.int). during an initial pandemic phase, matched vaccines will be restricted and antiviral drugs will be critical. the efficacy of the approved anti-influenza drugs (adamantanes, neuraminidase inhibitors) is limited, resistant strains emerge, and h n strains appear to be less sensitive to the established anti-influenza drugs than seasonal influenza strains [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . hence, additional anti-influenza therapies are needed. in , the "who public health research agenda for influenza" expressed a need for additional drugs including those that exert immunomodulatory effects and recommended to investigate natural products for anti-influenza activity (www.who.int). flavonoids are known to exert multiple pharmacological effects including anti-inflammatory and anti-viral activities including inhibition of seasonal influenza a (h n ) viruses [ ] [ ] [ ] [ ] [ ] [ ] . they may interfere with the influenza virus neuraminidase [ ] [ ] [ ] , the virus host cell uptake [ , ] , or cellular signalling events like the activation of nuclear factor kb (nfkb), akt, erk / , p , and/or jnk [ ] [ ] [ ] [ ] [ ] . we showed recently that the flavonoids biochanin a and baicalein interfere with h n replication in lung epithelial cells but that only baicalein inhibited h n replication in primary human monocytederived macrophages [ ] . although biochanin a and baicalein are closely related structures ( figure a) , they differed in their antiviral mechanisms. inhibition of the h n neuraminidase appeared to substantially contribute to the anti-h n effects exerted by baicalein but not by biochanin a. biochanin a interfered in contrast to baicalein with h n -induced activation of constituents of cellular signalling pathways [ ] that are known to be involved in influenza virus replication such as akt, erk / , and nfκb [ , [ ] [ ] [ ] [ ] . notably, the effects of baicalein and biochanin a on h n replication are complex and additional antiviral mechanisms are likely to contribute to their anti-h n activities. flavonoids are known to differ in their effects on the formation of reactive oxygen species (ros). they may display anti-or pro-oxidative effects [ ] . influenza virus replication is influenced by the cellular redox status [ ] . the inhibition of virus-induced ros formation by different strategies including the use of the antioxidant n-acetyl-lcysteine (nac) was shown to inhibit influenza a virus replication including h n strains [ ] [ ] [ ] . here, we investigated the effects of baicalein and biochanin a on h n -induced ros formation and the combined effects of baicalein and biochanin a in combination with the antioxidant nac on h n replication. a cells (human lung carcinoma; atcc, manassass, va, usa: ccl- ) and vero cells (african green monkey kidney; atcc: ccl ) were cultivated as described previously [ ] . human monocytes were isolated from buffy coats of healthy donors (institute of transfusion medicine and immune haematology, german red cross blood donor centre, goethe-university, frankfurt/main, germany) and cd + monocytes were differentiated into mdms as described previously [ ] . cells were infected with h n strain a/thailand/ (kan- )/ (obtained from dr. puthavathana, mahidol university, bangkok, thailand) and virus titres were determined as % tissue culture infectious dose (tcid /ml) as described previously [ ] . flavonoids, nac, or their combinations were present starting from a h pre-incubation period prior to infection. for the identification of statistically significant differences (p < . ), two groups were compared by student's t-test, more groups by anova followed by subsequent stepwise multiple comparison procedure using the student-newman-keuls method. h n infection of a cells at a multiplicity of infection (moi) . resulted in enhanced ros formation compared to control h after infection ( figure b ) as indicated by the use of the image-it live green reactive oxygen species kit (molecular probes, distributed by invitrogen, karlsruhe, germany). baicalein and biochanin a (both obtained from indofine chemical company, hillsborough, nj, usa) did not influence ros levels in non-infected or h n -infected cells in concentrations up to μm. however, at a concentration of μm both compounds increased the ros levels in non-infected as well as h n -infected cells ( figure b) despite the differences in their modes of anti-h n action [ ] . an nac (obtained from alexis, distributed by axxora, germany, dissolved in unsupplemented mem and adjusted to ph . with naoh) concentration of mm was sufficient to reduce the ros levels below the levels observed in non-treated h n -infected a cells ( figure c ). next, we investigated whether the reduction of baicalein-or biochanin a-induced enhanced ros levels in h n -infected a cells by nac influences the antiviral effects of these flavonoids. h n (moi . )-infected a cells were treated with baicalein μm or biochanin a μm in combination with nac in concentrations ranging from . to mm. nac did not affect cell viability alone or in combination with baicalein or biochanin a in the investigated concentrations as indicated by the celltiter-glo® luminescent cell viability assay (promega gmbh, mannheim, germany) (data not shown). while nac mm alone moderately reduced h n titres ( . fold reduction), nac . mm or . mm did not significantly affect virus titres (figure a) . however, nac reduced h n titres in combination with baicalein or biochanin a in a dose-dependent manner in this concentration range in a cells ( figure b) . notably, nac also inhibited baicalein-and biochanin a-induced oxidative stress in h n -infected primary human monocyte- derived macrophages but did not affect h n replication in this cell type (figure ). human monocytes had been isolated from buffy coats of healthy donors, obtained from the institute of transfusion medicine and immune haematology, german red cross blood donor center, johann wolfgang goethe-university, frankfurt am main. in conclusion, we show that two flavonoids that interfere with h n replication by different mechanisms of action exert similar effects at the level of ros induction. baicalein interferes with the h n neuraminidase activity but biochanin does not. biochanin a (but not baicalein) inhibits the activation of signalling molecules involved in h n induced signalling including akt, erk / , and nfκb [ ] . despite these differences in their anti-h n mechanisms, both compounds enhanced h n -induced ros formation in a cells, and the efficacy of both compounds was enhanced by the antioxidant nac. in contrast, inhibition of flavonoid-induced ros formation by nac did not affect virus replication in h n -infected macrophages. these findings emphasise that flavonoids, a class of natural compounds known to exert anti-influenza effects [ ] [ ] [ ] [ ] ] , induce a complex range of pharmacological actions by which they modify influenza a virus replication including highly pathogenic avian h n strains. these actions may be cell type-specific and include pro-and antiviral effects. the overall activity may be the result of the totality of effects exerted by a certain flavonoid in a certain cell type. a more detailed understanding of these actions and the underlying 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proinflammatory cytokine expression and cellular signal transduction in human macrophages infected with different influenza a viruses disruption of virus-host cell interactions and cell signaling pathways as an anti-viral approach against influenza virus infections inhibition of influenza virus-induced nf-kappab and raf/mek/erk activation can reduce both virus titers and cytokine expression simultaneously in vitro and in vivo a prooxidant mechanism for the anticancer and chemopreventive properties of plant polyphenols suppressing production of reactive oxygen species (ros) for influenza a virus therapy (nac) inhibits virus replication and expression of pro-inflammatory molecules in a cells infected with highly pathogenic h n influenza a virus glycyrrhizin exerts antioxidative effects in h n influenza a virus-infected cells and inhibits virus replication and pro-inflammatory gene expression submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution abbreviations nac: n-acetyl-l-cysteine; ros: reactive oxygen species. the authors declare that they have no competing interests.authors' contributions mm and jc designed the study, analysed the data, and wrote the manuscript. ps performed experiments and analysed data. all authors read and approved the final manuscript. key: cord- -mrh pbi authors: dumitrascu, georgiana r.; bucur, octavian title: critical physiological and pathological functions of forkhead box o tumor suppressors date: - - journal: nan doi: . /d. . sha: doc_id: cord_uid: mrh pbi the forkhead box, subclass o (foxo) proteins are critical transcription factors, ubiquitously expressed in the human body. these proteins are characterized by a remarkable functional diversity, being involved in cell cycle arrest, apoptosis, oxidative detoxification, dna damage repair, stem cell maintenance, cell differentiation, cell metabolism, angiogenesis, cardiac development, aging and others. in addition, foxo have critical implications in both normal and cancer stem cell biology. new strategies to modulate foxo expression and activity may now be developed since the discovery of novel foxo regulators and non-coding rnas (such as micrornas) targeting foxo transcription factors. this review focuses on physiological and pathological functions of foxo proteins and on their action as fine regulators of cell fate and context-dependent cell decisions. a better understanding of the structure and critical functions of foxo transcription factors and tumor suppressors may contribute to the development of novel therapies for cancer and other diseases. the forkhead box (fox) proteins represent a wide family of transcription factors that display an extraordinary functional diversity, regulating a variety of critical biological processes , . fox proteins are well known to control the following physiological procceses: apoptosis, cell-cycle, cellular metabolism, immune response, differentiation, development (such as cardiac development) or aging. fox proteins are also involved in various pathologies, such as cancer, diabetes and neurodegenerative diseases , . the first forkhead transcription factor was first identified in , and its function was related to the development of the anterior and posterior gut of the drosophila embryo. the "forkhead" name was given because of the two spiked-head structures found in the embryos of the drosophila melanogaster forkhead mutant presenting modifications of the gut formation . one year later, the sequence comparison between forkhead and mammalian hepatocyte-enriched transcription factor hnf- a showed a conserved -aminoacid dna binding domain, bringing evidence that forkhead proteins are a new class of transcription factors . hence, forkhead transcription factors are defined by their winged-helix dna binding domain, a conserved structure called "forkhead box", the symbol fox being assigned to all vertebrate forkhead transcription factors, according to the revised nomenclature . since its discovery, numerous forkhead genes have been identified in a broad range of organisms, from yeast and worms to humans . interestingly, up to now, fox genes have not been identified in plants . however, an update published in reported that are fox genes identified and classified in the human genome and in the mouse genome . therefore, a standard nomenclature system was required for this extended number of discovered factors. in , daniel e. martinez and his team established fox nomenclature committee, the first step towards an unified nomenclature for the winged-helix / forkhead transcription factors. the committee has changed the initial terms for the forkhead proteins (e.g. freac -forkhead related activator; fkh -drosophila gene fork head) . currently, based on phylogenetic analysis, foxo genes are classified in subclasses that range from foxa to foxs to yield subclasses in total , , , . each subclass has several members noted with an arabic number. while for human forkhead proteins abbreviations have all letters in uppercase (e.g., foxd ), for mouse only the first letter is capitalized (e.g., foxd ) and for all other chordates the first and subclass letter are in uppercase (e.g., foxd ) . one of the largest and the most important subclass of fox family is represented by foxo (forkhead box, subclass o) transcription factors. foxo transcription factors (foxos) are characterized by the same conserved dna binding domain that define the family of forkhead proteins . however, foxos share an additional unique amino acid sequence insertion within the dna binding domain that is not present in other forkhead proteins . only one foxo transcription factor is known in invertebrates (named abnormal dauer formation protein /daf- in the nematode worm caenorhabditis elegans and dfoxo in the fruit fly drosophila melanogaster), while in mammals four foxo proteins encoded by four different genes were identified: foxo , foxo , foxo and foxo . initially, foxo transcription factor (previously known as fkhr-forkhead in rhabdomyosarcoma) was identified through its involvement in chromosomal translocations t( ; ) and t( ; ) in alveolar rhabdomyosarcoma tumors due to pax / -fkhr fusion transcript , . a few years later, foxo (previously known as fkhrl -forkhead in rhabdomyosarcoma like protein ) was characterized and named, based on similarities to fkhr . the gene for foxo was described as being fused to mll transcription factor as a result of the t(x; ) chromosomal translocation in acute lymphoblastic leukemia, therefore, the initial term for foxo was afx (the acute leukemia fusion gene located on chromosome x) . foxo proteins are considered unique cellular targets, regulating a wide variety of critical cellular processes, such as: apoptosis, oxidative stress, dna damage repair, cell cycle, stem cell proliferation and maintenance, metabolism, angiogenesis, vascular tone, cardiovascular development, fertility, immune response and neuronal survival , . the aim of this review is to discuss the most recent advances in elucidating the functions, structure and transcriptional regulation of forkhead box o transcription factors, both in physiological and pathological conditions. the advances in understating the mechanism of foxos regulation of stem cells, cancer stem cells and how non-coding rnas are regulated and regulate the function of foxo genes/protein are presented. in humans, the primary structure of the foxo proteins is characterized by a length of approximately - amino acid residues (aa) for foxo ( aa) and foxo ( aa), and a shorter sequence of approximately amino acids for foxo ( aa) and foxo ( aa) , , , . all members of the foxo family consist of four domains: a forkhead dna-binding domain (dbd), a nuclear localization signal (nls), a nuclear export sequence (nes) and a c-terminal transactivation domain (figure ) . the forkhead dna-binding domain (dbd), also called the forkhead box, is described as a "winged helix" due to the butterfly-like aspect on x-ray crystallography and nuclear magnetic resonance . analysis of the amino acid sequences alignment of foxo proteins revealed a highly conserved dna binding domain among all the members of the broader group of forkhead transcription factors, but also among species, in eukaryotic organisms, from yeast to humans , . moreover, several studies point out that forkhead dbd is not the only region in the foxo molecule that is highly conserved. similarities were also observed in the n-terminal region near the first akt/protein kinase b (pkb) phosphorylation site (thr for foxo and thr for foxo ), the region containing nls, and sequences from c-terminal transactivation domain . as mentioned above, the forkhead domains in foxo subclass of fox family are similar to the forkhead regions of other subclasses. for example, structural similarities have been proven in the core region of foxo compared to foxa , foxk and foxp . the forkhead/winged helix motif is a shared sequence between subclasses of foxo, of around amino acids that folds into three alphahelices (h , h and h ), three beta-strands (s , s and s ) and two wing-like loops (w and w ). the structure has a h -s -h -h -s -w -s -w topology and the strands s , s and s interact with each other forming a beta-sheet. while the n-terminal part of the domain is formed by a cluster of three alpha-helices, the c-terminal half consists of beta strands s and s and two large loops (wings w and w ) . the main dna recognition site is represented by the highly conserved helix h of forkhead dna-binding domain. the stability of forkhead dbd -dna complexes is increased by the more variable regions of the dbd, including h , the region between h and h , wing w and c-terminal segment . although the foxo forkhead box is clearly related to those found in other forkhead genes, all foxo members contain an aditionally segment of five amino acid ( -gdsns- ) between helices h and h , that creates a small extra loop, forming a coil structure in the foxo dbd, and a short helix in the foxo dbd , . this is important in sequence-specific interactions with dna-binding sites . foxo proteins mediate transcriptional activation through binding to the conserved consensus core motif ttgtttac in the dna . they bind dna through a foxo-recognized element with a t/c-g/a-a-a-a-c-a-a consensus sequence in the forkhead dbd (c-terminal) . there are fourteen protein-dna contacts described, which mediate the activation/inhibition of foxo's target genes, such as bim, trail, p , p and catalase. the main recognition site is the α-helix h . forkhead proteins function as transcription factors that bind to dna through their forkhead domain in order to upregulate or downregulate the expression of a tremendous number of target genes . hence, the foxo transcription factors control the expression of a wide spectrum of genes that regulate essential physiological cellular processes, such as cell death/cell survival, cell cycle, cell proliferation, cell differentiation/development, angiogenesis, cell metabolism, stress response and stem cell maintenance , , , , , (figure ) . moreover, foxos are also involved in a wide range of pathological cellular processes, such as cancer, neurodegenerative diseases (parkinson's disease, alzheimer), diabetus mellitus, cardiac failure, atherosclerosis, hypertension and anovulation , , , , , , , . differences between functions and regulatory mechanisms of foxo and foxo proteins are considered to be partially redundant, with several exceptions. for example, akt phosphorylates all foxo family members, inducing their translocation into the cytoplasm and inactivation, while pp a dephosphorylates part of the akt phosphorylation sites in foxo and foxo , reactivating them . several particular differences regarding the regulatory kinases, e ligases or other important enzymes and regulators exist. thus, their activity is in part controlled by different mechanisms . foxo transcription factors regulate the expression of multiple pro-apoptotic and anti-apoptotic proteins, and they have the ability to induce apoptosis by activating either intrinsic or extrinsic pathways of apoptosis , , , , . moreover, the consensus foxo recognition element (fre) -(g/c)(t/a)aa(c/t)aa -which differs from that of other forkhead proteins, seems to have a very important role in both apoptotic pathways, since matching functional fre sites have been identified in the promoters of foxo target genes encoding fas ligand (fasl), insulin like growth factorbinding protein (igfbp ), the apoptotic regulator bcl- interacting mediator of cell death (bim) and others . foxo triggers the mitochondria-dependent intrinsec apoptotic pathway through upregulation of multiple pro-apoptotic bcl- family members, such as puma, bim, noxa, bnip , and downregulation of the anti-apoptotic bcl- family member bcl-xl , , , , . the expression of the anti-apoptotic protein bcl-xl is suppressed by foxo proteins, such as foxo , after increasing the expression of the transcriptional repressor bcl- , in order to trigger apoptosis . both foxo-induced expression of the proapoptotic bcl- family members and foxodownmodulated anti-apoptotic bcl- family members lead to apoptosis due to mitochondrial outer membrane permeabilization (momp), as a response to intracellular stress, growth factor deprivation or other factors . these mitochondrial modifications result in release of cytochrome c, smac/diablo and omi/htra from mitochondria, subsequently triggerring downstream caspases activation , . apaf binds cytochrome c forming a complex named apoptosome, required for caspase activation (initiator caspase) . caspase activation further activates the effector caspases, such , or , triggering caspase cascade . foxo proteins are also able to induce apoptosis by activating the receptor-dependent extrinsic pathway of apoptosis. they induce the upregulation of the death receptor ligands fasl and trail, promoting an autocrine and/or paracrine action of these death ligands on the death receptors . fas signaling pathway is important in apoptosis induction through the extrinsic pathway, since jurkat cells lacking several critical components of fas pathway fail to induce foxodependent cell death . upregulation of tumor necrosis factor related apoptosis inducing ligand (trail) by increased levels of foxo or foxo in cancer cells, such as prostate carcinoma cells, leads to apoptosis . thus, binding of fas ligand (fasl) and trail to their receptors (fas/cd /apo- for fasl and dr , dr for trail) triggers a death-inducing signaling complex (disc) with a subsequent activation of caspases, mainly initiator caspase and effector caspase , leading to apoptosis . moreover, foxo transcription factors directly regulate the expression of tumor necrosis factor receptorassociated death domain (tradd). activation of foxo proteins by pi k-akt pathway inhibitors results in increased expression of tradd protein . tradd is an important adaptor protein interacting with tnfr and fas receptors, mediating apoptosis and nfkb pathway activation . under normal physiological conditions, cyclins and their associate cyclin dependent kinases (cdks) are very important for the progression of the cell cycle . in response to dna damage, foxo transcription factors increase the expression levels of the cdk inhibitors binding to cyclin/cdk complexes, such as p (also known as cdk inhibitor ) and p (kip ) , . cdk inhibitors together with foxo-induced inhibition of cyclins expression act by stopping the cell cycle at different checkpoints in order to repair the dna damage or to remove the damaged cell . for example, a study performed on d murine myeloid cells and on baf murine pre-b-cell lines brings evidence that endogenous foxo proteins are required to enforce cell cycle checkpoints after the cell cycle arrest at g /s transition is induced by foxo by upregulating the negative regulators of the g /s phase, such as the cdk inhibitors: p kip , p kip , p cip /waf (cip/kip family), p ink , p ink (ink family) and the retinoblastoma protein family member p .also, foxo decreases the positive regulators, such as cyclin d , or cyclin d , blocking the g /s transition . additionally, foxo increases the expression of negative regulators such as gadd alpha and cyclin g , resulting in cell cycle arrest at g /m . surprisingly, foxo proteins act as transcription factors for plk expression during cell cycle, plk being critical in promoting g -m phase transition, m phase progression and end of mitosis . a complete knock-down of foxo protein levels induces an arrest in cell cycle, suggesting that a basal, low levels of foxos activity is required for the cell cycle progression . surprisingly, in specific settings, foxo may actually serve as a promoter of proliferation. for example, neutrophils isolated from foxo deficient mice show high levels of fasl expression and apoptosis, revealing that foxo may represses fasl expression in neutrophils, leading to proliferation in this type of cells . foxo was also shown to positively influence proliferation induction in pancreatic β cells in vitro, under low nutritional circumstances. the mechanisms implicated in this process are not completely understood. however, the induction of ccnd gene transcription, which encodes cyclin d , may at least partially be involved. cyclin d represents one of the earliest cell cycle-related events, being critical for the g to s phase progression during the cell cycle . thus, foxo proteins coordinate the expression of multiple important cell cycle regulators, in order to block the g /g , g /s or the g /m transitions during cell cycle when needed, such as after dna damage. however, a basal level of foxo proteins is required for g -m cell cycle transition, when foxo-dependent plk expression seems to be necessary. these results suggest that foxo proteins have to be tightly regulated and are important and sensitive regulators of cell cycle, proliferation and other critical cellular processes. foxo proteins play an important role in regulating differentiation of a wide variety of tissues. it is well known that foxos can control the differentiation of precursor cells into muscle, adipose tissue or blood cells. interestingly, foxos effect on differentiation is context dependent and sometimes foxo isoform dependent. while foxo promotes differentiation of erythroid cells, foxo suppresses the differentiation of precursor cells in adipose and muscle tissues . foxos can also suppress bone formation by inhibiting wnt-β catenin-tcf signaling. wnt signaling is known to stimulate bone formation . moreover, foxo proteins are required for maintenance of somatic/adult stem cells, such as hematopoietic stem cells, and they also regulate cancer stem cells (see details in chapter ). metabolic signaling mediated by forkhead transcription factors is conserved among multiple species, including but no limited to mammals, drosophila melanogaster and caenorhabditis elegans . it was first noticed for the foxo homolog named dauer formation- (daf- ), in the caenorhabditis elegans worm . foxo proteins are involved in critical physiological processes that regulate cellular metabolic activity in many organs, such as liver, pancreas, adipose tissue and hypothalamus , , , . in the liver, foxo forms a complex with another transcription factor, the liver specific pgc alpha, in order to induce gluconeogenesis by upregulating g pase and pepck genes. this is important for maintaining glucose homeostasis . confirming these results, loss of hepatic foxo genes in mice induces a downmodulation of gluconeogenesis and an upregulation of glycolysis . in addition, ectopic expression of foxo in rat primary hepatocytes increases apociii, an inhibitor of lipoprotein lipase, suggesting that foxo is involved in regulating the lipid metabolism . recently, nicotinamide phosphoribosyltransferase (nampt) gene was described to be a new transcriptional target gene of foxos for regulating hepatic triglyceride levels . in pancreas, foxo plays an important role in the maintenance of beta cell function, but also in the development of the pancreas, through repression of the pancreatic transcription factor pdx . aditionally, foxo increases the food intake by upregulating agrp and npy, and by downregulating pomc in hypothalamus, antagonizing the anorexigenic hormone leptin , . recently, g-protein-coupled receptor gpr was described to be a foxo target that activates agrp, suggesting a new mechanism for foxo -agrp mediated food intake that might provide a new treatment for obesity . all these results establish foxo proteins as important regulators of cellular metabolism and potential target in metabolic related diseases. foxo transcription factors function as sensors for reactive oxygen species (ros) and play an important role in cellular resistance to stress, increasing cellular survival. foxo proteins are able to protect the cell from oxidative damage, decreasing the availability of ros. they stimulate the expression of certain genes responsible for the ros inactivation, such as the antioxidant enzymes manganese superoxide dismutase (mnsod), that catalyzes the conversion of superoxide to hydrogen peroxide, and catalase, that converts hydrogen peroxide into water and oxygen . studies on human cardiac fibroblasts revealed that foxo also modulate the antioxidant enzyme peroxiredoxin iii, fighting against cell damage induced by oxidative stress. concomitant peroxiredoxin iii knockdown and foxo knockdown resulted in higher levels of hydrogen peroxide in response to serum starvation, as compared to peroxiredoxin iii knockdown only . other foxo transcriptional targets (figure ), such as sterrole carrier proteins (scps), are now known to play an important role during the defence against oxidative stress. scps are implicated in protecting fatty acids from peroxidation . foxo proteins are also able to increase dna repair, inducing a cell cylce arrest at g /m checkpoint, in order to provide time for repairing the dna damage. one of the transcriptional target of foxo, implicated not only in cell cycle arrest but also in dna repair and cell survival in response to cellular stress, is gadd . similarly, ddb gene protein product is able to repair the dna damage through upregulation by foxo . foxos regulate the longevity of the cell mainly by increasing resistance to stress, modulating the dna repair process and maintaining the stem cells . the lifespan extension is an important function of foxo that is conserved among several species. a well studied example is the role of foxo homologous daf- (caenorhabditis elegans) in aging . loss of foxo in human skin fibroblasts results in aging specific morphological changes, suggesting that foxo is necessary for maintenance or promotion of cellular longevity . moreover, loss of foxo activity leads to decreased mnsod and enhanced cell injury in vascular smooth muscle explanted from aged mice, due to limied ros inactivation . thus, cellular lifespan maintenance requires a certain level of foxos activity and this is mainly achieved by inactivation of akt activity . foxo proteins do not only play an important protective role during senescence/aging, but also during exercise. it was previously suggested that foxos may at least partially be involved in the exerciseinduced beneficial cardiac effects. interestingly, exercise induces an upregulation of foxo and sirt in the heart, with subsequent increased mnsod, catalase and gad alpha, and decreased cyclin d , suggesting the protective role of foxo proteins . interestingly, other studies revealed that, in conditions of oxidative stress, foxo is also able to induce apoptosis by triggering a fas-mediated death pathway in cultured motoneurons, by activating trail, bh -only proteins noxa and bim, and by promoting pro-apoptotic activity of p . additional reports suggest that suppression of foxo proteins expression during oxidative stress can be protective to some extent for cells, since protein inhibition or gene knockdown of foxo and foxo decreases the ischemic infarct size in the brain, protects the metabotropic glutamate receptors during vascular injury, enhances pancreatic β-cell or neuronal survival through nad + precursors and provide trophic factor protection with erythropoietin (epo) and neurotrophins . this suggests that while a certain level of foxo activation is required in cellular stress resistance, sustained activation or activation over a threshold of foxo is detrimental and may induce apoptosis. thus, foxo plays an important role in cellular decision during stress, helping the cells to survive by multiple mechanisms (such as dna repair, ros inactivation etc) and inducing apoptosis when dna or cellular damage can't be repaired. foxo proteins play a central role in maintaining the immune response of the human body. cell type-specific deletions of foxo and/or foxo in mice revealed an important role of foxo proteins in regulating immunological homeostasis and tolerance, by controlling the function and development of b and t lymphocytes. thus, foxo and foxo are essential transcription factors involved in early b cell development and peripheral b cell function, since early deletion of foxo blocked b cell differentiation at the pro-b cell stage. this is due to a defect in interleukin receptor alpha (il- ralpha) expression. deletion of foxo in peripheral b cells was associated with defective expression of both cd l and aid, with subsequent failure in class-switch recombination and reduced igg production upon immunization. moreover, it is known that the pi k-akt-mediated inactivation of foxo is essential for the optimal proliferation of b cells, while ectopic expression of pi k-independent variants of foxo or foxo (active triple mutants at the akt phosphorylation sites) resulted in cell cycle arrest and increased cell death in b cells . another study on mice with t cell-specific deletion of foxo showed a decreased expression of il- receptor on mature t-cells, suggesting that il -r is a transcriptional target of foxo , which is mediating through binding with il- the survival and homeostatic proliferation of peripheral t cell . excesive inflammatory cells may become harmful for the human body through the generation of ros and through the production of cytokines. studies performed on mice deficient of foxo ilustrated lymphoproliferation, inflammation of the salivary glands, lung, and kidney, and increased activity of helper t cells. these observations demonstrate the beneficial role of foxo proteins in human body, by preventing t cell hyperactivity . also, it was demonstrated that mir- has a central role in the late phase of clonal expansion of the helper t lymphocytes, by inducing il- which is able to inactivate foxo . foxo was found to be a suppressor of proliferation in resting helper t lymphocytes and, in order to allow proliferation, t cell activation via tcr/cd and il- r signaling must inhibit foxo . other studies reported that t cells derived from bim and puma deficient mice were resistant to apoptosis after il- deprivation, demonstrating that foxo , through upregulation of bim, puma and p kip is important for the induction of cell cycle arrest and apoptosis of t cells, in the absence of cytokines . foxo proteins may be benefical for autoimmune disorders by inducing a fas mediated apoptosis that target activated t cells, followed by a decrease in cytokine stimulation in patients with autoimmune lymphoproliferative syndromes . foxo proteins play an important role not only during physiological cellular processes, but also in few pathologies, such as cancer. foxos are well known tumor suppressor proteins. although foxo proteins protect the human body by playing a central role in a wide range of mainly physiological functions (as described earlier), under some circumstances, foxo's roles can become harmful for the human cells. this is because foxo is also involved in a number of pathological functions, such as inflammation and muscle atrophy. for example, apoptosis places foxo proteins on the good side when it leads to tumor suppression. however, cellular apoptosis can become itself a significant component for pathology in diseases such as neurodegenerative disease, diabetes mellitus (dm), and cardiovascular injury , , , . cancer: foxo proteins are tumor suppressors foxo proteins are inactivated in a wide variety of malignancies, eiher posttranslational (mainly by pi k-akt mediated phosphorlation) or by fusion mutations (such as pax -foxo ; mll-foxo , mll-foxo ) , . inactivation of foxo by pi k-akt pathway activation is a common feature of many malignancies, such as prostate cancer, breast cancer, leukemia and glioblastoma . conditional deletion of foxo family members in mice leads to the lymphomas and hemangiomas, which suggests that loss of foxos maintains or promotes survival of tumor cells . foxos loss or inactivation is known to play an important role in cancer tumorigenesis or progression, in vivo . for example, a recent study described a significant correlation between low expression of foxo and a poor prognosis for gastric cancer patients, bringing evidence that foxo could be a valuable prognostic biomarker for patients with gastric cancer . in addition, foxo overexpression was shown to reduce motility, invasiveness, and aggressiveness in estrogen receptor α-positive (erα+) breast cancer cells . foxo proteins exert their tumor suppressor functions predominantly by promoting cell cycle arrest, apoptosis, ros inactivation and dna repair, through expression of their target genes , . for instance, foxo -induced expression of bim, a pro-apoptotic member of the bcl- family of proteins induced a caspase-dependent cell death in several types of cancers, such as in chronic leukemias, breast and gastric cancers . similarly, foxo-induced trail and noxa expression induces apoptosis in many malignancies, including leukemias . interestingly, foxo can also inhibit the protooncogene c-myc, indirectly controlling the transcription of a wide set of target genes implicated in cell survival, cell cycle, apoptosis and tumorigenesis. myc is a transcription factor and a well known promoter of survival, proliferation and tumorigenesis, being found upregulated in a large variety of malignancies. noteworthy, activation of foxo proteins not only induces cell cycle arrest or apoptosis, but also a differentiation program. in chronic myeloid leukemia (cml), foxo can induce cml leukemic cells differentiation, by inhibiting the expression of id (inhibitor of dna binding ). cml is characterized by the presence of the bcr-abl fusion protein, which is a constitutive active kinase, controlling many crucial downstream pathways implicated in cell cycle, proliferation, apoptosis and cell adhesion. bcr-abl activates pi k-akt pathway, inactivating foxo proteins and their pro-apopotic and cell cycle arrest signals . the use of the tyrosine kinase inhibitors (tkis) and of the proteasome inhibitor bortezomib results in inhibition of bcr-abl activity and its downstream pathways, with a subsequent activation of foxo proteins . tkis treatment induces a foxo-dependent suppression of id expression, leading to k bcr-abl positive cell line differentiation. thus, bcr-abl can maintain the leukemic state not only by promoting proliferation, cell cycle progression and inhibiting apoptosis, but by also inhibiting foxo-mediated differentiation . inflammation (rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus) it was shown that loss of functional foxo proteins lead to inflammatory cell activation in several disorders, with the subsequent cellular damage, through oxidative stress and excess of cytokines. inactivation of foxo in t lymphocytes, as well as inactivation of foxo and foxo in synovial macrophages in patients with rheumatoid arthritis and osteoarthritis result in inflammatory cell activation. in addition, loss of foxo proteins may be a potential etiology for systemic lupus erythematosus (sle) and rheumatoid arthritis, since foxo gene transcript levels are downregulated in peripheral blood mononuclear cells of these patients . a link between inflammation, insulin resistance and foxo transcription factors was previously suggested when downregulated foxo decreased the levels of c/ebp beta transcription factor in adipocytes, with the subsequent reduction in expression of the pro-inflammatory cytokines ccl (chemokine ligand ) and il- . thus, foxo indirectly might induce an inflammatory status of the adipose tissue, responsible for the insulin resistance in type diabetes. yet, there are studies that describe the ability of foxo proteins to directly induce inflammation through upregulation of the inflammatory cytokine il- b . however, further experiments are required in order to clearly understand the context related mechanisms of foxo-dependent modulation of inflammation. muscle atrophy appears in a variety of diseases, including cancer, diabetes and sepsis, and is characterized by accelerated proteolysis that can be induced through two pathways: the ubiquitinproteasome pathway and through lysosomal pathway, as a consequence of autophagy . studies revealed that foxo proteins can induce muscle atrophy, characterized by decreased muscle function . thus, foxo proteins have been shown to increase the transcription of key regulators of both lysosomal and proteasomal proteolysis: the autophagy followed by the lysosomal proteolysis is stimulated by upregulation of bnip , lc , and gabarapl , while the proteasomal proteolysis is induced by increasing the ubiquitin ligase atrogin . it was also shown that foxo activity is both required and sufficient for induction of autophagy in muscle cells, since studies performed on adult muscle fibers from mice show that the ectopic expression of an active foxo mutant leads to lysosomal proteolysis after formation of autophagosomes, while the knockdown of foxo in these muscles fibers blocked autophagosome formation after starvation , . autophagy has a critical role in maintaining the cellular and metabolic homeostasis. it seems that the metabolic status of the cell strongly influences this process in both normal cells and cancer cells, despite the profound differences in their metabolism . in cancer cells, atp is predominantly produced through the constitutive activation of aerobic glycolysis, process that is modulated by the transcription factor hif α . since p α is required to maintain the levels of hif α target genes, researchers demonstrated that in colorectal cancer cells, the inhibition of p α causes a rapid drop in atp levels, with an acute energy need which activates foxo in an ampkdependent manner, in order to induce autophagy, cell cycle arrest and cell death in these cells , . moreover, the knockdown of foxo was sufficient to induce hypertrophy in cultured neonatal rat cardiomyocytes. in these cells, stimulation with insulin inhibits foxo function, with subsequent downregulation of the antioxidant enzyme catalase and increased levels of ros, that in low levels may act as second messengers for intracellular signaling, making possible the increase in cell size . atrogin , upregulated by foxo and foxo , plays important roles in cardiovascular system, since mice lacking atrogin- are susceptible to cardiac hypertrophy . foxo proteins are inducing atrophy of differentiated cardiac and skeletal muscle cells through protein synthesis inhibition, which leads to a decrease in cell size . in skeletal muscle, this mechanism involves myostatin, a foxo transcriptional target and a secreted molecule that can induce atrophy by protein synthesis inhibition . foxo might be implicated in the development or progression of type diabetes, since increased foxo expression in diabetic mouse liver is associated with increased expression of pepck and g pase, and inhibition of foxo activity downregulated both pepck and g pase expression and normalized the blood glucose levels. thus, foxo -mediated expression of g pase and pepck is critical for gluconeogenesis in the liver during fasting, but its deregulation may be involved in diabetes etiology . physiological functions of foxo take place under certain circumstances, since sometimes the ability to maintain the proper control is overwhelmed . experiments on insulin-producing mouse pancreatic beta cells (betatc- ) show that chronic exposure to high glucose activates foxo transcription factors and leads to upregulation of endogenous inflammatory cytokines interleukin- beta (il- beta) and suppressors of cytokine signalling (socs). these events trigger the activation of caspase- with subsequent apoptosis, suggesting a new mechanism that leads to the destruction of endocrine pancreas in type diabetes . also, exposure to high glucose of the cardiac microvascular endothelial cells (cmecs) isolated from hearts of adult rats show that foxo transcription factors leads to reactive oxygen species (ros) accumulation and apoptosis, suggesting that foxos might be involved in microvascular complications of diabetes . foxo is also involved in insulin resistance and metabolic syndrome, since the activation of foxo in cardiomyocytes leads to increased akt activity and attenuated cellular response to insulin, followed by decreased glucose uptake . interestingly, clinical studies regarding metabolic status profile on age-related diseases, fertility, fecundity and mortality revealed higher hba c levels and increased mortality risk associated with specific haplotypes of foxo . foxo proteins are also activated in an attempt to protect the human body against the oxidative stress resulted due to hyperglycemia that leads to increased production of ros in endothelial cells, liver cells, and pancreatic β-cells. this hyperglycemia-dependent ros increase leads to a subsequent development of insulin resistance and significant neurodegenerative and cardiovascular diseases in the patients with diabetus mellitus . foxos are necessary for endothelial cell development and angiogenesis, since mice that are deficient in foxo lack development of the vascular system and die by embryonic day eleven . in addition, foxo and foxo were shown to be the most abundant foxo isoforms in mature endothelial cells, having also an important role in the regulation of postnatal vessel formation, not only in the embryogenesis . unfortunately, angiogenesis is not only involved in critical physiological processes, such as embryogenesis and postnatal vessel formation, but it is also involved in pathological events, such as chronic inflammation and tumor growth . thus, it is fascinating how the angiogenesis mediated by foxo proteins may become a negative element for the organism, antagonizing the tumor suppresor's main function through new vessel formation, that can lead to tumor cell growth . foxo was associated with both cardiomyocyte survival after oxidative stress and heart muscle loss with subsequent ventricular dysfunction , . foxos are activated after akt inhibition by insulin or other factors, leading to atrogin- induction, which results in a suppression of heart muscle cell size . foxo proteins seem to inhibit the vascular smooth muscle cell proliferation and growth in a rat balloon carotid arterial injury model, suggesting a role of foxos in the regulation of vascular tone and systemic arterial blood pressure, preventing or at least lessening the effects of atherosclerosis and hypertension . also, decreased foxo expression due to high flow states in vessels leads to proliferation of vascular smooth muscle cells, vascular neointimal hyperplasia, and subsequent hypertension . moreover, experiments on lowdensity lipoprotein (ldl) receptor knockout mice resulted in the prevention of atherosclerosis when the triple ablation of foxo , foxo and foxo was induced in endothelial cells . interestingly, the same experiment on myeloid cells lead to more severe atherosclerosis compared to the controls, explained by authors through increased proliferation of granulocyte-monocyte progenitors and high levels of inducible nitric oxide synthase (inos) and oxidative stress, which predispose to atherosclerosis . noteworthy, analysis of mouse oocytes revealed overexpressed foxo transcription factors in primordial and early primary follicles, but downregulated foxo in primary and more developed follicles, suggesting that foxo proteins also have reproductive functions, modulating oocyte and follicular cell maturation. to confirm the hypothesis, constitutively active foxo was induced in transgenic mouse oocytes in primary and more developed follicles, which affected the oocyte growth and follicular development, leading to anovulation and luteinization of unruptured follicles, with subsequent infertility . in addition, foxo and foxo mutations were detected in a small percentage of womens with premature ovarian failure . involvement of foxo family of transcription factors in stem cells self-renewal, survival, proliferation and differentiation is currently under investigation. foxos have been shown to play critical functions in maintaining self-renewal potential and quiescence of hematopoietic stem cells, however, the mechanisms of these processes are not yet well understood , . recent reports show that foxo-mediated regulation of cell cycle, oxidative stress and apoptosis plays an important role in these processes . stem cells are characterized by the capacity of self-renewing and the ability to differentiate . they are necessary in maintenance and propagation of several adult tissues, including but not limited to blood, skin and gastro-intestinal epithelium. adult stem cells or cells with stem cell properties were also found in other critical organs/systems, such as the central nervous system and the lung . previous studies suggested that hematopoietic stem cells are sensitive to reactive oxygen species (ros) levels. foxo family members are known to play a central role in ros detection and in inducing an adaptative response after ros exposure, by inducing the expression of critical enzymes that neutralise ros, such as catalase and manganase superoxide dismutase (mnsod). interestingly, foxo , foxo , and foxo inactivation leads to an upregulation of ros levels in hematopoietic stem cells and their death . deletion of these three foxo family members in mice revealed their importance in controlling ros in stem cells, in vivo . while the number of hematopoietic stem cells in bone marrow of foxo-deficient mice is low, an increase in myeloid progenitor cells in blood is observed. moreover, the repopulation ability is decreased in the absence of foxos and the treatment of foxo-deficient mice with n-acetylcysteine (nac) at least partially rescued these effects . thus, persistent akt activation, which induces inactivation of foxo transcription factors by akt-mediated phosphorylation, results in ros-induced cell death. this is due to the fact that the cells can't synthesize the foxo-dependent ros neutralizing factors catalase and mnsod . between foxo family members, the most important regulator of hematopoietc stem cells survival and self-renewal is foxo , since foxo knockdown induces hematopoietic stem cells depletion . ros neutralizing agent n-acetylcysteine (nac) can rescue hematopoietic stem cells quiescence and at least partially resque their ros-induced loss . all these results suggest that foxo , foxo and foxo -induced resistance to ros is critical in maintaining homeostasis of hematopoietic stem cells in bone marrow . pten tumor suppressor was revealed as an important modulator of hematopoietic stem cells self-renewal and survival. pten is a major inhibitor of akt activation. depletion of pten, induces akt activation and subsequent foxo inactivation. it is likely that foxos mediate at least a part of the pten effects on hematopoietic stem cells . although foxo was established as the most important foxo family member regulator of hematopoietic stem cell's self-renewal, foxo is critical for the human embryonic stem cells (hesc) pluripotency maintenance. foxo induced sox and oct expression is one of the mechanisms responsible for this process. interestingly, in embryonic stem cells akt is not the major regulator of foxo . fascinating, a recent study brings evidence that foxo is a critical regulator of stem cell maintenance in hydra vulgaris, a member of the phylogenetically old animal phyla cnidaria, which has been suggested to be biologically immortal due to the unlimited self-renewal capacity of their stem cells , . foxo transcription factors are not only required for maintenance of somatic stem cells, but they also play an important role in cancer stem cells . recent reports provided evidence that in many types of malignancies, such as leukemia, colon, or gastro-intestinal malignanciens, a small population of cells similar to stem cells exists . these cells, called cancer stem cells, have the potential of forming new tumors. notably, most of the time, the cancer stem cells are resistant to current cancer treatments, and these therapies may result in cancer stem cells enrichment. these cells serve as a starting point in cancer recurrence . similarities between stem cells and cancer stem cells were best described in the hematopoietic system, where similar surface markes and signal transduction patterns were described between the hematopoietic stem cells and leukemia-initiating cells . presented results have not only implications in uncovering the stem cells/cancer stem cells regulation, self-renewal and differentiation, but also for the development of novel therapeutic strategies in cancer, degenerative diseases and many other pathologies . foxo family members are expressed in almost every tissue of the human body, including the nervous system, cardiovascular system, reproductive system of males and females, lung, liver, spleen, pancreas, thymus, and skeletal muscle. however, each member of the foxo family has its own expression pattern, since they are not equally expressed in all tissues . foxo is better represented in adipose tissue . foxo has the highest expression in liver, but it is also being predominantly expressed in heart, brain, kidneys, and ovaries, while foxo is found mainly in the muscle and heart , . the newest member of the transcription family, foxo , is present in the brain. the association of this member with other tissues is still a matter of study . cell lines (immortalized and/or cancer cells) are some of the most used and useful tools for studying the structure, function, regulation and expression of proteins in general, and foxo family members in particular. foxo members expression in different cell lines (including nci group of cell lines) is partially known , . foxo is found to be expressed at high levels in igrov (human ovarian carcinoma cells), astrocyte cells, rl (human follicular lymphoma cells), ht (human colon carcinoma cells) and hek (human embryonic kidney cells). knowing the qualitative and quantitative expression pattern of foxo family members is important in elucidating their functions and regulation. thus, databases summarizing these patterns in various tissues and cell lines are very helpful and necessary. for example, biogps presents experimental results showing the mrna expression levels of a wide number of genes in most of the human tissues and many (mostly human) cell lines , , . regulation of foxos expression is not yet well understood. p was shown to control foxo gene expression by binding to the proximal region of the foxo promoter (cre tandem sites) . non-coding rnas were shown to suppress translation of foxo family members in various tissues and contexts. in particular, the micrornas-dependent inhibition of foxo and foxo expression is better studied and is summariez below, in chapter . however, the main transcription factors implicated in foxos expression are not well understood. interestingly, methylation of fox promotors induces suppression of their expression. as an example, promoter methylation induced by braf results in inactivation of fox genes expression . the mirnas are - nucleotide-long noncoding rna molecules with an important role in post-transcriptional regulation of protein expression that regulate a variety of cellular processes, including cell differentiation, cell cycle progression and apoptosis. these mirnas can function as oncogenes or tumor suppressors, and oncogenic mirnas (oncomirs) are upregulated in cancer cells. in cancer, mirnas were found to be situated both upstream and downstream of the carcinogenesis process and modified expression of some mirnas is the outcome of carcinogenic transformation or progression, revealing that mirnas may be potential diagnostic or prognostic tools in cancer. for instance, micrornas gained a special attention in melanoma studies and the altered pattern of mirna in melanoma seems to be related to apoptosis (mir- b), cell cycle (mir- b) and invasion/metastasis (mir- ) . the microrna (mirna)-mediated regulation of foxo transcription factors was demonstrated by several groups within the last three years. mir- has been shown to specifically target foxo transcription factors irrespective of cell type, since mir- seems to target foxo in melanoma cells, foxo in breast cancer cells and foxo in activated helper t (th) lymphocytes . thus, in melanoma cells, mir- modulate the expression of both foxo and microphthalmia-associated transcription factor (mitf). the inhibition of mir- by anti-mirs (blocking antisense oligonucleotides) hindered melanoma cell migration and triggered their apoptosis . in breast cancer cells, foxo was coordinately targeted by mir- a, mir- , and mir- , while the inhibition of each mirna resulted in induced levels of foxo and reduced breast cancer cell survival . mir- also targets foxo in osteoblasts lineage cells in order to inhibit osteoblast proliferation and differentiation, repressing the osteogenesis . many other micrornas were shown to regulate foxos activity and functions, including apoptosis or cell cycle. mir- has been shown to regulate foxo -induced apoptosis in transitional cell carcinoma . also, increased expression of mir- in breast cancer cells have been shown to downregulate foxo transcription factor with consequent induction of cell proliferation . also, increased mir- , mir- and mir- downregulate the expression of foxo transcription factor in classical hodgkin lymphoma (chl) cell lines, suggesting that decreased foxo expression is involved in lymphomagenesis . moreover, a recent study performed on du and lncap human prostate cancer cells show that upregulated microrna- induces proliferation due to downregulation of the foxo transcription factor . in addition, mir- , which is highly induced in mature activated t and b cells and in treg cells, has recently been shown to target foxo in t cells, but it remains to be shown whether foxo via mir- contributes to the observed phenotypes in b and t lymphocytes . new strategies to modulate foxo expression may now be developed since the discovery of mirnas targeting foxo transcription factors . however, some difficulties may appear in the mirna-based therapeutic manipulation of foxo transcription factors. for example, a single mirna may target hundreds of genes among foxos, and the therapeutic manipulation of a specific mirna could have unanticipated adverse effects by influencing whole gene networks, while having only moderate effects on foxo genes . since delivery of mir- mimics could induce lymphoproliferative disease or other forms of cancer by systemic repression of foxo transcription factors, another challenge in the mirna-foxo therapeutic manipulation is the specific delivery of the mirna into the target cell, in order to avoid adverse effects in other cell types or tissues . several micrornas were identified as being targets of foxo transcription factors. an akt -foxo -mir- d signaling pathway was recently identified. after inhibition of akt, activated foxo leads to upregulation of mir- d. mir- d acts as a tumor suppressor in renal cell carcinoma, further inhibiting the oncoprotein metadherin (mtdh) . another recent study shows that foxo stimulates the expression of a microrna cluster located on a x chromosome, in a direct manner, dependent on rna polymerase ii, but not on the de novo protein synthesis. thus, foxo upregulates mir- , mir- , mir- c, mir- a- , mir- a- . also, the same study shows that inhibition of pi k-akt axis in lncap and mcf human carcinoma cell lines is followed by increased mir- . as suggested by authors, mirnas could be valuable biomarkers of foxo activity . a study performed on primary cultures of neural stem/progenitor cells (nspcs) from adult mice show that the expression levels of the mir- b~ cluster members (mir- b, mir- , and mir- ) is modulated by foxo transcription factors in a complex manner . the precursors of the mir- b~ cluster members are located on mcm gene. foxo directly binds to the first intron of this gene, modulating the expression of the micrornas. thus, foxo transcription factors could be an important tool in preventing the loss of neurogenesis during aging . further studies are needed to completely understand the regulation of micrornas expression by activated foxo proteins. foxo transcription factors and tumor suppressors are ubiquitously expressed in the human body, with some specific differences between its members. as resumed above, foxo proteins are characterized by a remarkable functional diversity, being implicated in regulation of many critical cellular functions, such as cell cycle arrest, apoptosis, oxidative detoxification, dna damage repair, stem cell maintenance, cell differentiation, cell metabolism, angiogenesis, cardiac development, aging and others. foxo proteins play an important role not only during physiological cellular processes, but also in few pathologies, such as cancer. they are well known tumor suppressors proteins. although foxo proteins protect the human body by playing a central role in a wide range of mainly physiological functions, under some circumstances, foxo's roles can become harmful for the human cells. this is because foxo is also involved in a number of pathological functions, such as inflammation, muscle atropy, and a number of physiological functions that become harmful for the organism. for example, apoptosis places foxo proteins on the good side when it leads to tumor suppression, but in some cases cellular apoptosis can become itself a significant component for pathology in diseases such as neurodegenerative disease, diabetes mellitus (dm), and cardiovascular injury. interestingly, while excessive foxo levels induce cell cycle arrest and cell death, complete knock-down of foxos leads to cell cycle progression impairment, suggesting that certain levels of foxo activation are required for cell cycle progression. moreover, foxo proteins play a variety of roles in the cells dependending on the context. in deciphering the role of forkhead transcription factors in cancer therapy foxo tumor suppressors and bcr-abl-induced leukemia: a matter of evasion of apoptosis snapshot: forkhead transcription factors i sly as a foxo": new paths with forkhead signaling in the brain the homeotic gene fork head encodes a nuclear protein and is expressed in the terminal regions of dynamic foxo transcription factors forkhead transcription factors contribute to execution of the mitotic programme in mammals inflammatory arthritis requires foxo a to prevent fas ligandinduced neutrophil apoptosis foxo transcription factors and stem cell homeostasis: insights from the hematopoietic system foxos attenuate bone formation by suppressing wnt signaling foxo is an essential regulator of pluripotency in human embryonic stem cells the fork head transcription factor daf- transduces insulin-like metabolic and longevity signals in c. elegans insulin regulated hepatic gluconeogenesis through foxo -pgc- alpha interaction the forkhead transcription factor foxo links insulin signaling to pdx regulation of pancreatic beta cell growth forkhead transcription factor foxo in adipose tissue tissue regulates energy storage and expenditure forkhead protein foxo mediates agrpdependent effects of leptin on food intake deletion of hepatic foxo / / genes in mice significantly impacts on glucose metabolism through downregulation of gluconeogenesis and upregulation of glycolysis foxo mediates insulin action on apoc-iii and triglyceride metabolism hepatic foxos regulate lipid metabolism via modulation of expression of the nicotinamide phosphoribosyltransferase gene minibrain/dyrk a regulates food intake through the sir -foxo-snpf/npy pathway in drosophila and mammals foxo regulates peroxiredoxin iii expression in human cardiac fibro blasts regulation of sterol carrier protein gene expression by the forkhead transcription factor foxo a dna repair pathway stimulated by the forkhead transcription factor foxo a through the gadd protein daf- /foxo directly regulates an atypical amp-activated protein kinase gamma isoform to mediate the effects of insulin/igf- signaling on aging in caenorhabditis elegans down-regulation of a forkhead transcription factor, foxo a, accelerate s cellular senescence in human dermal fibroblasts down-regulation of manganese-superoxide dismutase through phosphorylation of foxo a by akt in explanted vascular smooth muscle cells from old rats akt negatively regulates the in vitro lifespan of human endothelial cells via a p /p -dependent pathway exercise training promotes sirt activity in aged rats lymphocyte signaling: regulation of foxo transcription factors by micrornas an essential role of the forkhead-box transcription factor foxo in control of t cellhomeostasis and tol erance high glucose induces suppression of insulin signalling and apoptosis via upregulation of endogenous il- beta and suppressor of cytokine signalling- in mouse pancreatic beta cells high glucose induced oxidative stress and apoptosis in cardiac microvascular endothelial cells are regulated by foxo a apo ligand/tumor necrosis factor-related apoptosis-inducing ligand in prostate cancer therapy common mechanism for oncogenic activation of mll by forkhead family proteins applications of post-translational modifications of foxo family proteins in biological functions foxos are lineage-restricted redundant tumor suppressors and regulate endothelial cell homeostasis decreased expression of the foxo a gene is associated with poor prognosis in primary gastric adenocarcinoma patients the dna damage repair protein ku interacts with foxo to coordinate a conserved cellular stress response therapy-resistant acute lymphoblastic leukemia (all) cells inactivate foxo to escape apoptosis induction by trail and noxa induction of mxi -sr alpha by foxo a contributes to repression of myc-dependent gene expression combination of bortezomib and mitotic inhibitors down-modulate bcr-abl and efficiently eliminates tyrosine-kinase inhibitor sensitive and resistant bcr-abl-positive leukemic cells foxo increased pro-inflammatory gene expression by inducing c/ ebpbeta in tnf-alpha-treated adipocytes foxo links insulin resistance to proinflammatory cytokine il beta production in macrophages inhibition of foxo transcriptional activity prevents muscle fiber atrophy during cachexia and induces hypertrophy foxo controls autophagy in skeletal muscle in vivo foxo coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells inhibition of p alpha unveils an ampk-foxo a axis linking autophagy to cancer-specific metabolism foxo a inhibits cardiomyocyte hypertrophy through transactivating catalase regulation of myostatin expression and myoblast differentiation by foxo and smadtranscription factors inhibition of foxo function is associated with improved fasting glycemia in diabet ic mice foxo transcription factors activate akt and attenuate insulin signaling in heart by inhibiting protein phosphatases haplotypes in the human foxo a and foxo a genes; impact on disease and mortality at old age abnormal angiogenesis in foxo (fkhr)-deficient mice multifaceted link between cancer and inflammation constitutive phosphorylation of the foxo transcription factor in gastric cancer cells correlates with microvessel area and the expressions of angiogenesis -related molecules foxo transcription factors promote cardiomyocyte survival upon induction of oxidative stress forkhead transcription factors inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia expanded granulocyte/monocyte compartment in myeloid-specific triple foxo knockout increases oxidative stress and accelerates atherosclerosis in mice mutational screening of foxo a and foxo a in women with premature ovarian failure the pi- kinase pathway in hematopoietic stem cells and leukemia-initiating cells: a mechanistic difference between normal and cancer stem cells mortality pattern suggest lack of senescence in hydra foxos: signalling integrators for homeostasis maintenance modulators of sensitivity and resistance to inhibition of pi k identified in a pharmacogenomic screen of the nci- human tumor cell line collection global proteome analysis of the nci- cell line panel biogps: an extensible and customizable portal for querying and organizing gene annotation resources biogps and mygene.info: organizing online, gene-centric information a gene atlas of the mouse and human protein-encoding transcriptomes control of foxo gene expression by co-activator p braf mutation-specific promoter methylation of fox genes in colorectal cancer tissular and soluble mirnas for diagnostic and therapy improvement in digestive tract cancers immune-related biomarkers for diagnosis/prognosis and therapy monitoring of cutaneous melanoma mir- is a negative regulator of osteoblast proliferation, differentiation, and skeletogenesis through targeting foxo mir- regulates foxo -mediated cell apoptosis in bladder cancer unregulated mir- induces cell proliferation in human breast cancer by downregulating transcriptional factor foxo a foxo is a tumor suppressor in classical hodgkin lymphoma upregulation of mircorna- induces proliferation in human prostate cancer cells by downregulating the transcription factor foxo akt/foxo pathway in renal cell carcinoma foxo regulates expression of a microrna cluster on x chromosome the microrna cluster mir- b~ regulates adult neural stem/progenitor cell proliferation and neuronal differentiation key: cord- -a oy sz authors: yang, shenshu; lian, gaojian title: ros and diseases: role in metabolism and energy supply date: - - journal: mol cell biochem doi: . /s - - - sha: doc_id: cord_uid: a oy sz researches dedicated to reactive oxygen species (ros) had been performed for decades, yet the outcomes remain controversial. with the relentless effort of studies, researchers have explored the role of ros in biosystem and various diseases. ros are beneficial for biosystem presenting as signalling molecules and enhancing immunologic defence. however, they also have harmful effects such as causing tissue and organ damages. the results are controversial in studies focusing on ros and ros-related diseases by regulating ros with inhibitors or promotors. these competing results hindered the process for further investigation of the specific mechanisms lying behind. the opinions presented in this review interpret the researches of ros from a different dimension that might explain the competing results of ros introduced so far from a broader perspective. this review brings a different thinking to researchers, with the neglected features and potentials of ros, to relate their works with ros and to explore the mechanisms between their subject and ros. ros are a set of unstable molecules including hydrogen peroxide (h o ), hydroxyl radical (oh − ), singlet oxygen ( o ) and superoxide (o − ) that are produced by all kinds of cells [ ] . the comprehensive distribution of ros may grant them with a fundamental role in biosystem. although ros play an important role in pathogen resistance and cellular signalling, they are also broadly recognized as harmful reactive particles to cell as they damage intracellular proteins, lipids and nucleic acids. it usually appears in pathological processes when they are not scavenged on time [ ] . the essence that ros are produced in energy demanding conditions where vigorous metabolism is in demand shall not be neglected. the pathogenic role of ros in self-damage and the beneficial role in the immune system may be due to the requirement of energy supply. in these conditions, excessively produced ros bring about oxidative damage to body and pathogens. ros are widely involved in basic mechanisms and pathways. they not only impair cells and tissues with oxidative damage, but also play an important role in many homeostasis processes involving metabolism, immunity, growth and differentiation [ ] . researchers have been regarding ros as byproducts and exploring their effects on organisms, but the fundamental features of ros might illuminate their role in pathologies and biomechanisms. mitochondrial respiratory chain is one of the major sources of cellular ros. atp synthesis produces ros during normal oxygen metabolism. thus, ros are regarded as byproducts during energy perfusion to cell activities in most cases. the primary function of nadph oxidase (nox) enzymes is the generation of ros [ ] . belonging to the nox family, activated nox could promote ros production through ryanodine receptors and thus trigger ca + sparks [ ] . the involvement of nadph and nadh in repiratory chain and cellular metabolisms makes ros produce in all kinds of cells. toll-like receptors (tlr) tlr , tlr and tlr can enhance ros production by recruiting mitochondria to macrophage phagosomes and translocating tumour necrosis factor receptor-associated factor (traf ) to mitochondria to engage in evolutionarily conserved signalling intermediate in toll pathways (ecsit) [ ] . although ros are mostly produced in mitochondria, the detailed mechanisms of the production are still not fully understood. however, the major factors responsible for ros production are respiratory chain complexes (fig. ). nadh-ubiquinone oxidoreductase (complex i) is the major source of mitochondrial ros production in varying diseases. the components responsible for ros production of complex i include ubisemiquinone, flavin mononucleotide, fe-s cluster and nad [ ] . however, ros production by complex i in healthy state is humble presenting little oxidative damage. the major production of ros in this state comes mainly from complex ii through tca cycle. nadh gene mutation which causes deficiency in respiratory complex i could end up in the overproduction of ros and enhance metastasis of tumour cells [ ] . succinate dehydrogenase (sdh) or succinate-coenzyme q reductase (sqr) is composed of sdha, sdhb, sdhc and sdhd. sdha and sdhb are hydrophilic proteins. sdhc and sdhd are hydrophobic proteins that bind to ubiquinone. this oxidoreductase is also known as complex ii that plays an important role in tca cycle and respiratory chain. succinate is the intermediate of tca cycle and also a metabolic signature of ischaemia-reperfusion. it is responsible for ros generation when accumulated from fumarate overproduction and malate/aspartate shuttle during reperfusion. ischaemia injury can be ameliorated by the inhibition of succinate or ros. complex ii turns succinate into fumarate through oxidation in mitochondria with reduced ubiquinone in the membrane [ ] , and succinate could be re-oxidized by sdh, thus increasing ros generation through reverse electron transport in mitochondria. [ ] . ubiquinol-cytochrome c oxidoreductase (complex iii) is encoded by uqcrc (ubiquinol-cytochrome c reductase core protein ) gene and could receive reducing equivalents from complex i and complex ii. the received reducing equivalents are proceeded with ubiquinol and produces semiquinone for further proton transfer. p shc (src homologous-collagen homologue adaptor protein) generates mitochondrial ros as apoptosis signal through oxidation of cytochrome c in mitochondrial electron transfer chain. p mutants could lose the ability to generate ros and induce mitochondrial apoptosis [ ] , but genetic mutation may also contribute to increased generation of ros. isp- and nuo- encode complex iii subunit rieske and complex i subunit ndufb (nadh dehydrogenase [ubiquinone] beta subcomplex subunit ), respectively. mutants in isp- and nuo- are all related with enhanced ros level that leads to lengthened lifespan. ros promotor treatments can lengthen the wild-type lifespan while having no effect on those longevity mutants. and the enhanced ros induces apoptosis pathway fig. generation of ros in mitochondria triggered by ced- that changed the gene expression to protect mitochondrial dysfunction [ ] . tumour necrosis factor alpha (tnfα ) could regulate cell proliferation and death, and the inhibition of nuclear factor kappa-b (nf-κb) makes tnfα bias to cell death. tnfα-induced ros could support c-jun n-terminal kinase (jnk) activation during nf-κb inhibition. the sustained jnk activation enables cytochrome c release and leads to necrotic cell death [ ] . the homeostasis of ros plays an important role in reducing oxidative damage and fulfil energy demand. ros present as signalling molecules in multiple pathways and mechanisms. thus, they are inevitably influenced by proteins and genes involved within. apart from that, other environmental complexes and antioxidants could contribute to ros production according to their redox potential. it is also noted that different mtdna haplotypes may have distinct respiration capacity triggered by varying production of ros [ ] ( table ) . relatively high levels of ros may cause oxidative damage or induce apoptosis during immunological defences or pathological conditions. the mechanisms to survive under such environment are essential for body cells or tumour cells and bacteria. hypoxia inducible factor- alpha (hif- α ) encoded by endothelial pas domain protein (epas ) gene could control ros level in mitochondria through antioxidant enzymes and maintain ros homeostasis [ ] . pparγ coactivator α (pgc- α) is required for antioxidative enzymes including glutathione peroxidase (gpx ) and superoxide dismutase (sod ) [ ] . ros level also could be controlled through degradation of nox on endoplasmic reticulum by protein negative regulator of ros (nrros). this reduces tissue damage and maintains its function upon immunological defence [ , ] . however, ros themselves could activate extracellular signal-regulated kinase (erk) by targeting proteins gαi and gα and protect cardiac cell from oxidative damage [ ] . these proteins present protective effect on body cells and redox balance. it is also noted that the opened potassium channels may reduce ros level [ ] . ros tolerance may be partly involved in the mechanisms behind the tumour cells avoiding immunological defence. cancer cells could produce enough nadph to support vigorous proliferation while maintaining ros homeostasis through gsh (glutathione). enhanced ros in lung cancer cells could inhibit glycolytic enzyme pyruvate kinase m (pkm ). this also allows them to survive under acute oxidative stress and still supports their proliferation [ ] . nuclear factor erythroid- -related factor (nrf ) transcription is increased in tumour cells to suppress ros generation by nrf -keap (kelch-like echassociated protein ) interaction. oncogenic alleles of k-ras, b-raf and myc could increase nrf antioxidant activity and reduce ros level [ ] . researches also indicate that gene ucp (uncoupling protein ) could limit ros production and inflammation in macrophage [ ] . apart from that, antioxidants also include organics like vitamin e, vitamin c and complexes like fhc (ferritin heavy chain). they reduce apoptosis induced by tnfα and jnk activity through suppression of ros accumulation and iron sequestration as a downstream product of nf-κb pathway [ , ] . change cells from proliferation into differentiation ros are important particles involved in immunological defence. overexpressed tnf induces ros in mitochondria through rip -rip -dependent (receptor-interacting protein kinase) pathways. the increased ros leads to both enhanced macrophages killing and necroptosis. this necroptosis relies on mitochondrial cyclophilin d and ceramide [ ] . tlr / / could enhance ros by recruiting mitochondria to macrophage phagosomes and translocating traf to mitochondria to engage ecsit. this further increases the bacterial killing [ ] . ros induces oxidative damage and apoptosis which may contribute to the control of lifespan. p shc generates mitochondrial ros as apoptosis signal through oxidation of cytochrome c in mitochondrial electron transfer chain [ ] . other factors such as matrix metalloproteinase- (mmp- ) could increase cellular ros and stimulate transcription factor snail and epithelial-mesenchymal transition (emt). this process causes dna oxidative damage and genomic instability in breast cancer and turns normal cells into cancer cells [ ] . heart cell stretch could activate nox to produce ros through ryanodine receptors and trigger ca + sparks [ ] . the deficiency of nox inhibitor nrros could lead to elevated oxidative damage. [ ] erythroblastosis virus transcription factor- (ets- ) requires ros to regulate p phox expression. however, this also could contribute to nadph oxidase and ros generation and become an ets-ros positive feedback [ ] . the ros-induced ros-release circle could lead to elevated ros generation as well [ ] . transcription factor upbeat could regulate the balance between cellular proliferation and differentiation through ros. vigorous changes in metabolism may occur during the shift from cell elongation to differentiation to fulfil metabolic demands. upbeat enhances ros level through the repression of peroxidases which could change the pattern of cell from proliferation into differentiation. [ ] . researchers have been trying to elucidate the mechanisms and the role that ros plays in diseases since they were identified. ros influences diseases basically with its function as signalling molecules and oxidants that influence cell survival and oxidative damage. ros could also drive immunity through immunological defence and maintain metabolic balance or heat dissolving. the multiple functions of ros in biosystem may influence each pathema from different aspects ( table ). abnormal cell proliferation and metastasis are common features of cancer. vigorous proliferation demands substantial nadph to produce energy. this process also abundantly increases ros. high levels of ros could induce apoptosis of tumour cells. however, they also protect cells from oxidative damage by suppressing glycolytic enzyme pkm through gsh in cancer [ ] . tumour cells exhibit enhanced nrf transcription. nrf present as antioxidants that control ros level in cancer. the inhibition of nrf could impair tumourigenesis with increased ros level [ ] . oncogenic alleles k-ras, b-raf and myc could contribute to nrf -keap interaction. ros also regulate tumour suppressor protein p and mediate apoptosis in cancer [ ] . stem cells tend to contain lower ros than regularly differentiated cells. cancer stem cells also maintain low levels of ros to avoid apoptosis induced by ros. it also makes them suffer less dna damage from radiation with enhanced ros scavenging systems [ ] . cancer cells resistant to braf and mek inhibitors develop vulnerability to high levels of ros [ ] . thus, the strategy to enhance ros level may seem to present as an important way for cancer chemotherapy. however, researchers also indicated that tumour cells with high metastasis contain nadh gene mutation. the mutation causes deficiency in respiratory complex i and ended up in overproduction of ros, and the metastatic activity could be suppressed with ros scavenger [ ] . increased ros generation could trigger enhanced epidermal growth factor receptor (egfr) signalling and promote tumour progression [ ] . snail and emt stimulated by mmp- could increase ros generation. the elevated ros level could turn normal cells into cancer cells with dna damage and genomic variation. [ ] . ros could also mediate the tumour microenvironment through epithelial-mesenchymal transition that contributes to radioresistance and therapeutic failure [ ] . although suppressing ros signalling to inhibit tumour growth with ros scavenger is not ideal, the process of inhibition impairs ros-mediated oxidative damage and apoptosis [ ] . the uninhibited ros generation and uncontrolled ros level could also promote cancer cell metastasis and the process of canceration. these controversial results bring about hindered exploration of ros-mediated treatments against cancer. rather than focusing on symptoms, the fundamental role of ros and its comprehensive distribution among biosystem may explain these competing results from a broader perspective. ros are highly related with energy production rather than just byproducts. cancer development and tumour metastasis demand larger amounts of energy than normal cells. this energy-acquiring process also produces high levels of ros. the enhancement of ros may also increase energy production to facilitate tumourigenesis. rather than a regulator of the cancer pathology, ros are more likely the representative of energy consumption. it is easy to induce cancer cells death in vitro with oxidative damage. however, the failure to apply oxidative damage to cancer cells clinically seems to be the result of ros homeostasis system in vivo. being part of the mechanisms involved in innate immunity, inflammation eliminates pathogenic factors while causing tissue damage. ros play a similar role in immunity by enhancing immunological defence and causing oxidative damage. nlr family, pyrin domain-containing (nlrp ) inflammasome could enhance inflammation by activating caspase and promoting secretion of il- β and il- . ros are crucial for nlrp activation [ ] . drosophila multipotent haematopoietic progenitors present relatively high levels of ros in in vivo physiological conditions and become low during differentiation. the enhanced ros could promote the differentiation through jnk and the forkhead box o (foxo) pathway, but ros inhibition disabled its differentiation [ ] . although the differentiation prefers low levels of ros, they are still essential for the process. different t-cell subsets also have distinct sensitivity to ros level that may influence their development and function. th cells are involved in autoimmune diseases and inflammatory diseases. experimental autoimmune encephalomyelitis (eae) is a th -mediated autoimmune disease. regulating th cell differentiation by interfering ros level through glutathione metabolism could prevent eae development [ ] . influencing chromatin structure with gls inhibition also enhances ros level and prevents th differentiation [ ] . discovery of brand new t-cell subsets also endows deeper understanding on immune system and provides aspects for the exploration of t-cellregulated autoimmune diseases [ ] . ros could support immune system, but they become cytotoxic while overload [ ] . ros play a role in both activation-induced t-cell death and activated t-cell autonomous death [ ] . oxidative damage leads to cellular damage on dna, protein and lipids. the damage-induced apoptosis plays an important role in inflammatory bowel diseases [ ] . ros also stimulate parasite growth and cause tissue damage to host's organs [ ] . the expression of nadph oxidase is elevated in phagocytic leukocytes upon stimuli. [ ] the ros-mediated autophagy could promote periodontitis and tendinopathy as well [ ] . apart from oxidative damage, ros also serve as signalling molecules and play an important role in homeostasis, metabolism, growth and differentiation [ ] . ucp could limit ros production and inflammation in macrophage and reduce parasitic cysts [ ] . however, ucp relies on ros level required for heat dissipation through thermogenic respiration in brown adipose tissues. the depletion of ros inhibits ucp and heat generation [ ] . cigarette smoking induces oxidative stress in bronchitis and emphysema. inflammation also occurs in these chronic obstructive pulmonary diseases [ ] . the role of ros in bacterial killing appears to be inconsistent among different studies. some research state that increased ros level in bacteria can enhance the killing ability of antibiotics and oxidants [ ] . enhanced ros by excess tnf through rip -rip -dependent pathways in mitochondria lead to both enhanced macrophages killing and necroptosis that relies on mitochondrial cyclophilin d and ceramide [ ] . tnf-α is indicated to contribute to ros production in rheumatoid arthritis [ ] . however, other studies indicate that ros response during bacterial antibiotic killing is dispensable [ ] . ros scavenger and hydroxyl radical inhibitor could suppress antibiotic bacterial killing. antibiotic bacterial killing does not strictly depend on ros [ ] . it is also noted that the level of ros does not influence antibiotics' activity on killing bacteria at all [ ] . antibiotic killing of escherichia coli does not rely on ros [ ] . the enhanced bacterial killing with increased ros level may due to increased metabolism and energy supply that support oxidation and immunity system. however, it applies little effect when they reaches saturation. but moderated metabolism with lower levels of ros surely decreases the ability of bacterial killing. neurons are important cells that control sensory organs and muscle system. the injury of these cells may lead to neuropathy and movement disorder. the relatively low antioxidant activity makes them vulnerable to oxidative damage. the defects in mitochondria may enhance ros generation and thus promote jnk and sterol-regulatory element binding proteins (srebp) activation in neurons that results in neurodegeneration through the accumulation of lipid droplets [ ] . the adipogenesis could also be influenced by ros via signal transducers and activators of transcription (stat ) [ ] . however, antioxidants could rescue the apoptosis [ ] . fhc could suppress ros accumulation and jnk activity through iron sequestration that inhibits tnf-α-dependent apoptosis [ ] . pgc- α could protect neural cells from oxidative damage by reducing ros level via antioxidative enzymes gpx and sod [ ] . methylmercury and manganese could induce neurotoxicity with enhanced ros level [ , ] . and the increased level of ros in the substantia nigra pars compacta leads to neuronal apoptosis of dopaminergic neurons in down syndrome and parkinson's disease. this process may ultimately lead to retardation [ ] . nrros could protect central nervous system from eae by reducing oxidative damage through nox degradation on endoplasmic reticulum [ ] . nevertheless, ros still play an important role in neuronal development and are essential for synaptic plasticity and memory formation with its fundamental role in energy perfusion. the essence that neurons are differentiated cells that lack the potential to proliferate explained these competing results of antioxidative strategies. they maintain a relatively low demand in energy and metabolism. in the heart, angiotensin ii, norepinephrine and tnf-α mediated ros are related with cardiac hypertrophy, myocardial infarction and heart failure. myocardial ischaemia is the most common cause of heart failure. the ischaemia-reperfusion injury leads to apoptosis of cardiomyocytes that is associated with high levels of ros [ ] . the shortage of atp during ischaemia impairs ion pump and causes calcium accumulation. calcium overload and increased ros could rupture plasma membrane and lead to cell death [ ] . cardiac hypertrophy is a compensating process that enables heart to maintain sufficient function. the increased ros during the process is responsive to energy demand caused by insufficient heart function. thioredoxin could reduce cardiac hypertrophy through heat shock protein and class ii histone deacetylases, the latter being a master negative regulator of cardiac hypertrophy [ ] . and it is also noted that ros increased via d-amino acid oxidase in the hearts of rats could directly lead to systolic heart failure without cardiac hypertrophy [ ] . the oxidative damage-mediated apoptosis is the major cause of heart failure as well. the method to fulfil energy demand by using nox to protect heart from failure with improved myocardial energetics via fatty acid oxidation is also proved to be successful [ ] . to reduce oxidative damage, ubia prenyltransferase domain-containing protein presents cardiovascular protective function via antioxidant coenzyme q [ ] . however, the inability to recover from cardiac damage and pathology is also critical for heart failure. postnatal cardiomyocyte cell-cycle arrest is mediated by ros through dna damage response [ ] . heart cell stretch could cause arrhythmogenic ca + sparks based on microtubules [ ] . although the oxidative damage caused by ros is the major reason for heart failure, the role of ros in energy supply is rather important that protect heart from an even sudden failure of insufficient function. ros regulate vascular cell proliferation and apoptosis with their fundamental role in metabolism. oxidative stress could lead to hypertension and promote its pathological process. however, ros are also needed for the relaxation of cerebral arteries [ ] . ets- and angiotensin ii-generated ros play an important role in vascular changes and injury, and no could regulate blood flow homeostasis in blood vessels [ ] . these outcomes seem to be confusing to tell whether ros are beneficial or harmful. the role of ros in biosystem is rather neutral that they mainly respond to energy demand. nox family could influence the neovascularity of tumour and physiological vascular processes [ ] . similar results also presented in ros-mediated wound repair [ ] . ischaemia-reperfusion (ir) causes oxidative damage with increased generation of ros in mitochondria. succinate is the metabolic signature of ischaemia and responsible for ros generation during reperfusion. the reperfusion injury also leads to retinal dysfunction-associated ros production when the blood pressure is low [ ] . succinate accumulates during reperfusion from fumarate overproduction and malate/aspartate shuttle and then re-oxidized by succinate dehydrogenase. this process increases ros generation through reverse electron transport in mitochondria. the inhibition of succinate or ros could ameliorate ir injury [ ] . pre-conditioning protocols could reduce ischaemia-reperfusion injury by regulating ros level [ ] . atherosclerosis could be regulated by ros interacting with transcription factors related with lipid peroxidation and macrophage [ ] . ros induces dna damage and lipid peroxidation in pneumoconiosis and carcinogenesis as well [ ] . increased ros promote thrombus formation in artery and influence other cardiovascular diseases as well [ , ] . the pulmonary vascular lesions and inflammation are broadly recognized pathological changes in acute respiratory distress syndrome (ards) caused by oxidative damage [ ] . taken together, researchers revealed the position of ros in metabolism and energy supply. ros are needed for basic energy demand and vigorous metabolism rather than simply affecting cellular signalling and organism damages. the continuous oxidative damage applied on cell and tissue may lead to severe organic injuries and eventually cause organ failure. the ros level leading to organ failures far exceeds the extent to maintain basic metabolism and thus the balance between energy supply and oxidative damage is tilted. increasing ros grants little beneficial effect in this situation. inhibition of ros could reduce tnf-α-mediated fulminant liver failure. tnfα regulates cell proliferation and death and the inhibition of nf-κb makes tnfα bias to cell death. tnfα-induced ros supports jnk activation during nf-κb inhibition. sustained jnk activation enables cytochrome c release and leads to necrotic cell death [ ] . fhc is a downstream product of nf-κb. they could reduce apoptosis induced by tnfα through suppression of ros accumulation and jnk activity. the suppression of ros is achieved by iron sequestration [ ] . ros are produced by glomerular cells as autacoids [ ] . ros-mediated glomerular basement membrane degradation and altered cell function may contribute to ischaemic renal failure as well [ ] . and the inhibition of ros could decrease caox stone in kidney [ ] . hypoxia-induced requirement of energy supply and metabolism could lead to increased ros response through ca + influx pathway. this mechanism results in physiological, biochemical and molecular changes. the hypoxia-induced ros production is important for respiratory plasticity and sensory plasticity. ros-mediated apoptosis and cellular dysfunction are associated with heart failure [ ] . the arrhythmias caused by elevated ros and altered mitochondrial function may lead to sudden cardiac death [ ] . the comprehensive distribution of ros intrigues researchers to explore the relationship between their subject and ros. the reduced ros level could lower insulin resistance and improve insulin sensitivity in diabetes ii [ ] , and the glucose-stimulated insulin relies on ros signalling [ ] . however, the cellular death owing to ros-mediated oxidative damage also brings about diabetic complications [ ] . mmp activity and transcription factor-β (tgf-β )-induced excessive deposition of extracellular matrix mediated by ros could lead to renal fibrosis [ ] . the process of ageing caused by oxidative damage and muscle dysfunction could lead to sarcopenia. however, ros are also essential for muscle cell development as signalling molecules [ ] . generation of ros in skeletal muscle is enhanced during contractile activity [ ] . ros are increased in the early stage of muscular dystrophy development [ ] . the elevated ros may reduce muscle mass and bring about frailty [ ] . however, ros also plays an important role in muscle remodelling as signalling molecules [ ] . overproduced ros released through mitochondrial permeability transition pore will damage dna and accelerate ageing by reducing cellular nad [ ] . apart from suppressing tumour, p also plays a role in premature ageing by causing reactive damage to dna [ ] . mushroom-contained antioxidants may protect against oxidative damage and ageing [ ] . ros overproduction may contribute to reproductivity issue and infertility through oxidative damage and disturbed hormone balance. the excessive ros may damage spermatogenesis, sperm lipid/protein layer and dna structure. the ros scavenging system to reduce ovarian toxicity is important for follicular development [ ] . the role of ros seems to be similar in different diseases that they are essential for cellular metabolism and become pathogenic while overload. researches of ros have been carried out since last century, the cognition of which varies along elapse of time. ros promote macrophage bacterial killing through oxidative damage and apoptosis. they also engage in multiple cellular pathways as signalling molecules. however, they also have negative effects like inflammation and cytotoxicity. ros can function as intermediates in varying pathways, but they are also widely regarded as etiologic factors for diseases including cancer, inflammation and organ injuries. evidence suggests that the scavenging ros in pathological condition may reduce cell damage and control the pathological process. however, other researches also indicate the positive side of enhancing ros in diseases. thus, the mechanisms of how ros influences diseases remain obscure. europeans have dedicated to the study of ros and made a comprehensive exploration [ ] . yet, it remains controversial in results of ros-targeted strategies applied to clinical research. with the advancement in technique, ros can be measured in living cells of its transient generation with y . eu . vo nanoparticles by illuminating under oxidative conditions [ ] . specific chemical probes and low-temperature electron paramagnetic resonance (epr) technique could monitor ros level of tumour cells in vitro and in vivo [ ] . other environmental factors like ph and ion concentration are also suggested in ros regulation and generation [ ] . also magnetic fields could influence cellular ros level according to its intensity, frequency and exposure time [ ] . the fundamental understandings of ros remain basically the same in the past years. ros are generally regarded as signalling molecules and harmful particles. researchers always focus on ros levels and their results and analyses them from a single perspective. they should be illuminated from different perspectives and with extensive sight. based upon the characteristics of ros discovered by previous researches, i provide a hypothesis here that may explain the competing results so far. ros thrives in conditions that abundant energy is in demand for vigorous metabolism, either in cancer cells' proliferation and inflammatory necrosis, or immunological defence required immune system functioning. thus, ros do not just act as signalling molecules. they may present as basic energy particles like other acknowledged basic nutrient particles including proteins and carbohydrates. and they provide a much more fundamental impact on cellular metabolism. it is more likely that ros respond to elevated metabolism to fulfil energy demand, rather than directly bending itself to oxidative stress, which interprets why different researches demonstrate controversial outcome in regulating ros. the treatment of both inhibition and enhancement of ros in cancer in vitro may due to exhausted energy supply for metabolism and overload of energy supply-induced oxidative damage, rather than just the regulation of a byproduct. of course, it is easier to suppress ros generation with antioxidants or genetic depletion in experimental animal models. but the inability of clinically gene modulation in vivo and the multiple functions of ros in metabolism may lead to limited potrential of direct ros modulation in diseases with ros and metabolic imbalance. the fundamental role of ros grants them with the potential in metabolic regulation. from another aspect, the essence that ros are common particles with comprehensive distribution endows them with more fundamental mechanisms to be explored. the nox family of ros-generating nadph oxidases: 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tnfalpha-induced apoptosis by suppressing reactive oxygen species tnf dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species bissell mj ( ) rac b and reactive oxygen species mediate mmp- -induced emt and genomic instability transcription factor ets- and reactive oxygen species: role in vascular and renal injury mitochondrial rosinduced ros release: an update and review transcriptional regulation of ros controls transition from proliferation to differentiation in the root ros and p : a versatile partnership association of reactive oxygen species levels and radioresistance in cancer stem cells lethally high ros levels thwart resistance the interplay of reactive oxygen species and the epidermal growth factor receptor in tumor progression and drug resistance reactive oxygen species-mediated tumor microenvironment transformation: the mechanism of radioresistant gastric cancer targeting cancer cells by ros-mediated mechanisms: a radical therapeutic approach? nlrp inflammasome activation: the convergence of multiple signalling pathways on ros production? reactive oxygen species prime drosophila haematopoietic progenitors for differentiation glutathione de novo synthesis but not recycling process coordinates with glutamine catabolism to control redox homeostasis and directs murine t cell differentiation distinct regulation of th and th cell differentiation by glutaminase-dependent metabolism a cd (+) t cell population expanded in lupus blood provides b cell help through interleukin- and succinate t cell apoptosis and reactive oxygen species reactive oxygen species and antioxidant defense in human gastrointestinal diseases ros and trypanosoma cruzi: fuel to infection, poison to the heart reactive oxygen species in phagocytic leukocytes sudden death risk in older athletes: increasing the denominator mitochondrial ros regulate thermogenic energy expenditure and sulfenylation of ucp reactive oxygen species in chronic obstructive pulmonary disease potentiating antibacterial activity by predictably enhancing endogenous microbial ros production the role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis fe-s cluster biosynthesis controls uptake of aminoglycosides in a ros-less death pathway antibiotic and ros linkage questioned killing by bactericidal antibiotics does not depend on reactive oygen species cell death from antibiotics without the involvement of reactive oxygen species glial lipid droplets and ros induced by mitochondrial defects promote neurodegeneration mitochondrial stat and reactive oxygen species: a fulcrum of adipogenesis? jak-stat apoptosis and increased generation of reactive oxygen species in down's syndrome neurons in vitro involvement of glutamate and reactive oxygen species in methylmercury neurotoxicity manganese neurotoxicity and the role of reactive oxygen species nitric oxide and reactive oxygen species in parkinson's disease reactive oxygen species, 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oxidative stress and nrf in pancreatic beta-cell function the role of reactive oxygen species in apoptosis of the diabetic kidney reactive oxygen species and matrix remodeling in diabetic kidney reactive oxygen species as mediators of cellular senescence reactive oxygen species and redox-regulation of skeletal muscle adaptations to exercise absence of dystrophin disrupts skeletal muscle signaling: roles of ca + , reactive oxygen species, and nitric oxide in the development of muscular dystrophy control of reactive oxygen species production in contracting skeletal muscle reactive oxygen species are signalling molecules for skeletal muscle adaptation role of reactive oxygen species-mediated signaling in aging ros and senescence in the control of aging reactive oxygen species and antioxidant properties from mushrooms reactive oxygen species and sperm cells single europium-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells teaching the basics of reactive oxygen species and their relevance to cancer biology: mitochondrial reactive oxygen species detection, redox signaling, and targeted therapies extra-cellular but extraordinarily important for cells: apoplastic reactive oxygen species metabolism magnetic fields and reactive oxygen species publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no conflict of interest. key: cord- - b cg authors: aquino-martinez, ruben; khosla, sundeep; farr, joshua n.; monroe, david g. title: periodontal disease and senescent cells: new players for an old oral health problem? date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: b cg the recent identification of senescent cells in periodontal tissues has the potential to provide new insights into the underlying mechanisms of periodontal disease etiology. dna damage-driven senescence is perhaps one of the most underappreciated delayed consequences of persistent gram-negative bacterial infection and inflammation. although the host immune response rapidly protects against bacterial invasion, oxidative stress generated during inflammation can indirectly deteriorate periodontal tissues through the damage to vital cell macromolecules, including dna. what happens to those healthy cells that reside in this harmful environment? emerging evidence indicates that cells that survive irreparable genomic damage undergo cellular senescence, a crucial intermediate mechanism connecting dna damage and the immune response. in this review, we hypothesize that sustained gram-negative bacterial challenge, chronic inflammation itself, and the constant renewal of damaged tissues create a permissive environment for the abnormal accumulation of senescent cells. based on emerging data we propose a model in which the dysfunctional presence of senescent cells may aggravate the initial immune reaction against pathogens. further understanding of the role of senescent cells in periodontal disease pathogenesis may have clinical implications by providing more sophisticated therapeutic strategies to combat tissue destruction. periodontal disease is a bacteria-induced chronic inflammatory condition that gradually deteriorates and destroys the tooth-supporting structures, eventually leading to loss of teeth [ ] . about - bacterial species have been found in samples of human subgingival plaque; however, only - of these species have a causative role in the pathogenesis of periodontal disease [ , ] . a gradual shift in subgingival microbiota, from predominant gram-positive aerobes to gram-negative anaerobic bacteria, has been implicated in the transition from health to disease in periodontal tissues [ ] . in response to subgingival bacterial challenge, a local release of proinflammatory signals is produced leading to the activation of the host immune defense system. although bacteria are essential to initiate inflammation, it is the host immune response against invading pathogens that is the major contributor and ultimate cause of periodontal tissue destruction [ , ] . interestingly, severe gingivitis lesions can remain stable for long periods, even years or decades, until an "unknown factor" disrupts the host-pathogen homeostasis promoting the progression to periodontitis [ ] [ ] [ ] . in order to eliminate invading bacteria, neutrophils are recruited into the infected site, where they use oxidative and non-oxidative mechanisms to destroy pathogens [ ] . although neutrophils and in , hayflick and moorhead identified that the growth of human cells gradually decreased, and eventually arrested, their mitotic capacity when they were cultured for long periods of time in vitro [ ] . this limited proliferative lifespan, also known as hayflick limit, was initially hypothesized as an aging expression at the cellular level [ ] . currently, the original concept of finite proliferation potential is considered a telomere-dependent mechanism, and is associated with the progressive decrease in telomere length as a result of repeated cellular replication ( figure ). in order to eliminate invading bacteria, neutrophils are recruited into the infected site, where they use oxidative and non-oxidative mechanisms to destroy pathogens [ ] . although neutrophils and released reactive oxygen species (ros) rapidly protect against invading bacteria, they can also harm healthy host cells leading to oxidative stress-mediated dna damage over prolonged periods of time [ ] . indeed, it has been recognized that low concentrations of ros, generated during chronic inflammation, can indirectly cause periodontal tissue destruction through the damage to vital cell macromolecules, including dna [ ] . along the same line, recent studies have identified that repeated exposure to lipopolysaccharide (lps), a component of gram-negative bacteria membrane, results in dna damage in different cell types, such as gingival and alveolar bone cells [ , ] . although dna lesions can be repaired, those cells exposed to excessive genomic damage undergo either apoptotic cell death or cellular senescence [ , ] . cells that survive persistent dna damage acquire a senescent phenotype, which can by itself trigger immune cell recruitment through the dysfunctional upregulation of proinflammatory cytokines. most senescent cells overexpress interleukin- (il ), il α, il β, and il , among others, which are collectively known as senescence-associated secretory phenotype (sasp) [ ] . this review focuses on the emerging evidence implicating the potential role of cellular senescence in periodontal tissue deterioration. we emphasize the role of chronic gram-negative bacterial infection in altering the local periodontal environment. in addition, we set forth a hypothesis that long-term bacterial infection and chronic inflammation facilitates a microenvironment for dna damage-driven cellular senescence. finally, we propose a model in which dysfunctional accumulation of senescent cells in periodontal tissues can contribute to exacerbating local immune reaction. in , hayflick and moorhead identified that the growth of human cells gradually decreased, and eventually arrested, their mitotic capacity when they were cultured for long periods of time in vitro [ ] . this limited proliferative lifespan, also known as hayflick limit, was initially hypothesized as an aging expression at the cellular level [ ] . currently, the original concept of finite proliferation potential is considered a telomere-dependent mechanism, and is associated with the progressive decrease in telomere length as a result of repeated cellular replication ( figure ). telomeres gradually shorten as a result of cell division eventually leading to cellular senescence, a phenotype characterized by permanent growth arrest. although mitotic cell division is the main cause of telomere shortening, this process can be affected or accelerated when cells are exposed to oxidative stress or genotoxic agents. indeed, unrelated factors, such as radiation, h o , or certain gram-negative bacteria products, can induce premature senescence. telomeres gradually shorten as a result of cell division eventually leading to cellular senescence, a phenotype characterized by permanent growth arrest. although mitotic cell division is the main cause of telomere shortening, this process can be affected or accelerated when cells are exposed to oxidative stress or genotoxic agents. indeed, unrelated factors, such as radiation, h o , or certain gram-negative bacteria products, can induce premature senescence. given that replicative senescence is a stress-dependent mechanism, cells recognize shortened telomeres as damaged or broken, triggering a dna damage response (ddr). in this mechanism, ataxia telangiectasia mutated (atm)-p axis activation plays a key function [ ] . although telomere shortening itself does not cause growth arrest, it leads to dna double-strand breaks that trigger a p dependent pathway resulting in p activation. in addition to p /p pathway, p ink a is also expressed in senescent cells and has an essential role in establishing and maintaining cell growth arrest [ , ] . although telomere-dependent replicative senescence is a well-recognized cellular process in vitro, it also occurs in vivo in the context of natural chronological aging. gradual telomere shortening, a phenomenon that naturally accompanies the process of aging in humans, mice, and other species, is implicated in decreased tissue renewal and age-related tissue decline as a consequence of stem cell exhaustion [ , ] . although cell division is an essential event that promotes telomere shortening, it is not the only factor implicated in regulating telomere length. oxidative stress can also contribute to accelerated telomere reduction and replicative growth arrest [ ] . although replicative cellular senescence is strongly associated with excessive shortening of telomeres during aging, normal cells exposed to sub-cytotoxic levels of damaging agents can prematurely undergo cellular senescence. cigarette smoke, γ-irradiation, chronic exposure to certain gram-negative bacterial toxins, or moderate concentrations of hydrogen peroxide (h o ), can induce many features of replicative senescence in normal cells [ ] [ ] [ ] [ ] . besides proliferative arrest, these and other harmful physical or biochemical stimuli produce enlarged cytoplasmic morphology, apoptosis resistance, and the striking hypersecretion of proinflammatory and proteolytic factors. this senescentlike phenotype induced by unrelated stressors, which is independent of telomere shortening, is called stress-induced premature senescence [ ] . based on the ability of these agents to cause oxidative stress either directly or indirectly, they represent a useful resource to evaluate the impact of stress on cellular senescence. for this reason, many researchers have used various genotoxic agents to accelerate cell senescence. for example, low doses of irradiation ( . gy) can transiently induce dna damage signaling in human fibroblasts [ ] . of note, such low and transient genotoxic lesions induce a temporal growth arrest, and cells partially recover; however, these cells do not secrete sasp cytokines, such as il [ ] . in contrast to transient dna damage, persistent genomic lesions promote constitutive dna damage signaling and cellular senescence, which is correlated with increased secretion of inflammatory signals [ , ] in agreement with this observation, several studies have reported that premature senescence can also be induced by exposing human cells to subtoxic h o concentrations [ , ] . interestingly, cytolethal distending toxin (cdt), a protein secreted by the periodontal pathogen aggregatibacter actinomycetemcomitans causes a genotoxic effect in different cell types, and also several features observed in cells undergoing replicative or premature senescence [ ] . furthermore, porphyromonas gingivalis lps induces a detrimental effect on dna, and accelerated senescence in microglial cells, adipocyte precursors, dental pulp cells, and alveolar bone cells [ , [ ] [ ] [ ] . it should be noted that stress-induced premature senescence is mostly a response to cellular damage; however, cells can undergo the influence of both telomere-dependent and -independent senescence under specific circumstances. one example of this, in which both mechanisms overlap, can be observed in those tissues under constant tissue renewal and exposed to inflammation-mediated oxidative stress. another example is aging, when oxidative stress induced by low-grade chronic inflammation and age-related replicative senescence can interact and reinforce the senescent phenotype (in part by accelerating telomere shortening) [ , ] . therefore, cells can undergo accelerated senescence independently of telomere shortening as a result of persistent exposure to dna damaging stimuli. although cellular senescence can be induced by repeated mitotic activity and/or several unrelated stressors, a common feature that can occur in most senescent cells is the hypersecretion of multiple signaling molecules, proteolytic enzymes, and other factors. this altered secretome communicates the cellular damage not only to neighboring cells and the surrounding environment, but also alert the innate immune system (see section . ). senescence-associated factors released into the extracellular space can be classified into soluble signals, insoluble proteins, and matrix-degrading factors, which are collectively known as sasp [ ] , or senescence-messaging secretome [ ] . a complex network of interleukins, chemokines, and other soluble families of growth factors constitute a significant proportion of secreted signaling molecules. one of the most significant and consistently secreted cytokines is il [ ] . although il is also strongly secreted, other cytokines such as il α and il β are essential modulators of the senescent-associated cytokine network [ ] . on the other hand, several proteolytic enzymes associated with both irreversible extracellular matrix degradation and cytokine activation are also produced by senescent cells, including matrix metalloproteinase (mmp) , mmp , mmp , and mmp [ , , ] . consistently, the upregulation of the recognized senescent biomarker p ink a is associated with the expression of mmp in cells isolated from the human degenerated intervertebral disc, and chondrocytes from osteoarthritis lesions [ , ] . the secretion of mmps by senescent cells can negatively impact and alter their local environment by promoting the proteolytic cleavage of cell membrane receptors, cytokines, and the permanent degradation of extracellular matrix components, such as collagen [ , ] . in addition, the expression of fibronectin, an insoluble matrix glycoprotein that has an important role in cell adhesion, is increased in senescent cells [ ] . besides soluble, insoluble, and proteolytic factors, the generation of high levels of ros constitutes an integral feature of senescent cells [ ] . in addition to the impact of senescence-associated factors on local inflammation, certain soluble signals can transmit the senescent phenotype to their healthy neighboring cells, a phenomenon known as paracrine senescence or the senescence-induced bystander effect [ , ] . although the impact on other cells is triggered by the combined effect of multiple soluble signals, specific factors and signaling pathways have a relevant role in this process [ , ] . for example, acosta et al. demonstrated that cells targeted by senescent factors displayed an increased small mothers against decapentaplegic (smad)- / and smad / activity. since smad transcription factors are key intracellular targets of transforming growth factor-beta (tgf-β) ligands, this indicates that this pathway plays a relevant role during paracrine senescence [ ] . in agreement with this finding, it has been reported that tgf-β inhibition accelerates liver regeneration by inhibiting paracrine senescence [ ] . furthermore, il and insulin-like growth factor binding protein (igfbp)- / can also produce premature senescence in young cells that contributes to impeded tissue regeneration [ , ] . however, the impact on nearby cells can also be produced independently of cytokines and growth factors. nelson et al. demonstrated that ros generation by senescent cells can be transferred between adjacent cells via gap junctions, producing dna damage in neighboring cells. this genotoxic paracrine effect could explain the formation of scattered clusters of senescent cells observed in hepatocytes [ ] . in addition, increased ros production by senescent cells can also affect nearby cells by activating nuclear factor-κb (nf-κb) [ ] . therefore, senescent cells are a potent source of proinflammatory cytokines, matrix-degrading factors, and ros that disrupt their local environment and affect neighboring cells. however, senescent cells and their sasp can have beneficial roles in certain contexts. from embryonic development to aging, the spatiotemporal presence of senescent cells determines their beneficial or detrimental role. during normal chick embryogenesis, senescent cells have been identified during specific developmental stages, and in particular anatomical structures, such as limbs and pharyngeal arches. intriguingly, embryonic senescent cells disappear before birth [ ] . in this embryological context, senescent cells are eliminated through macrophage-mediated mechanisms followed by tissue remodeling [ ] . of note, human embryos also display these features [ ] . after birth and during adult life, it has been proposed that the essential function of senescent cells is to coordinate a multistep process that begins with their own elimination (clearance of damaged cells) and ends with tissue renewal, a sequence called senescence-clearance-regeneration [ ] . in agreement with this concept, the regenerative effect is largely mediated by sasp factors in part by promoting stemness in surrounding cells [ ] . ritschka et al. also identified that the positive pro-regenerative effect is produced when factors released by senescent cells acted transiently on treated cells. in contrast, the persistent exposure to those factors produced paracrine senescence resulted in impaired regeneration in vivo. consistent with this positive role of senescent cells, demaria and colleagues demonstrated that senescent fibroblasts and endothelial cells contribute to promoting optimal wound healing through the secretion of specific senescence-associated factors [ ] . although plasminogen activator inhibitor- (pai ), chemokine ligand (ccl ), and chemokine ligand (ccl ) had a moderate expression during early wound healing, platelet-derived growth factor a (pdgf-a) and vascular endothelial growth factor (vegf) were highly secreted by senescent cells at this initial stage. interestingly, il and tgf-β were not expressed in cells isolated from wounds. a prominent feature of senescent fibroblasts and endothelial cells was their transient presence during wound healing. therefore, these studies suggest that during adult life the short-term presence of senescent cells can promote an immune-mediated elimination of damaged cells and facilitate tissue regeneration (table ) . in contrast to the beneficial effect of senescent cells observed during embryonic development and wound healing, the number of senescent cells intermittently increases during the lifespan. abnormal accumulation of senescent cells mainly occurs at sites of age-related pathologies and tissues exposed to chronic inflammatory conditions, which is associated with tissue deterioration [ , ] . as a consequence of chronological aging, the burden of senescent cells increases in different tissues in humans, mice, and other species, where they contribute to the development of chronic pathologies including arthritis, osteoporosis, alzheimer's disease, atherosclerosis, cancer, and diabetes [ , ] similar to other agerelated pathologies, the etiology of diabetes may be the result of the impact of different aging mechanism, including stem cell exhaustion, chronic low-grade inflammation, macromolecular damage, and cellular senescence. in this scenario, senescent cells can contribute by affecting pancreatic β-cell function, and sasp-mediated tissue damage [ ] . under hyperglycemic conditions, increased β-cell proliferation is produced, which eventually leads to cellular senescence and contributes to insufficient insulin release [ ] . furthermore, thompson et al. demonstrated that sasp factors have an important role in the immune-mediated senescent β-cell destruction that actively drives type diabetes in mice [ ] . table . the role of senescent cells depends on the context. tumor suppression mechanism [ ] source inflammatory cytokines (il- , tnf-β, il- ) [ , ] embryonic development [ , ] source proteolytic enzymes (mmps) [ , ] tissue remodeling and regeneration [ ] source or ros [ ] wound healing [ , ] stem cell exhaustion and tissue decline [ , ] dysfunctional senescent cells accumulate as a result of defective immune elimination and/or accelerated generation over time [ ] . due to their resistance to apoptosis [ ] , senescent cells persist in tissues and they can promote a negative impact on their environment through various mechanisms. first, as mentioned above, senescent cells become proinflammatory foci scattered throughout tissues promoting immune cell infiltration that results in collateral tissue damage over time [ , ] . besides the exacerbation of local inflammation, senescent cells also constitute an important source of factors that contribute to age-related systemic chronic inflammation [ ] . second, besides contributing to aggravated local chronic inflammation, mitotic arrest of resident stem cells associated with repeated cell division during the lifetime, eventually impairs tissue renewal of damaged cells leading to tissue decline [ , ] . third, the secretion of mmps by senescent cells contribute to producing detrimental changes in the structure and function of the extracellular matrix observed in age-related pathologies [ ] . fourth, the senescence-associated generation of ros can promote dna damage, and subsequently cellular senescence in surrounding cells [ , ] . on the other hand, a reciprocal interaction can be produced between dna damage and chronic inflammatory conditions. that is, dna damage can drive senescence-associated chronic inflammation, and chronic inflammatory conditions by themselves facilitate the onset of oxidative stress-mediated dna damage resulting in a positive feedback loop [ , ] . this concept is supported by the fact that senescent cells are primarily identified in tissues exposed to chronic inflammation, and age-related chronic inflammatory pathologies [ , , ] . therefore, the transient presence of senescent cells can promote the regeneration of damaged tissues. in contrast, dysfunctional senescent cell accumulation contributes to exacerbating local inflammation, disrupted extracellular matrix integrity and deficient tissue regenerative potential. although double-strand breaks are less frequent, they are among the most dangerous and cytotoxic forms of dna damage [ ] . given that genomic instability, a feature of pre-cancerous cells, is produced when dna double-strand breaks are not detected or repaired, cells react to genotoxic insults by activating ddr signaling. a key downstream target of ddr is p that constitutes a barrier against tumor progression [ ] . transiently arresting proliferation, cells can repair dna damage maintaining their viability and limiting the transmission of genomic alterations to daughter cells. in contrast, when excessive or irreparable damage is produced, cells can also undergo either programmed cell death or cellular senescence [ , ] . indeed, cellular senescence and apoptosis are key p -dependent mechanisms mediating the restriction of tumor development, and evidence indicates that both mechanisms could be suppressed in precancerous tissues [ ] . despite some common intracellular signals are shared by senescence and apoptosis, they represent mutually exclusive processes. one the one hand, during apoptosis, excessively damaged cells show structural changes in their morphology and appear rounded and smaller in size [ ] . in addition, apoptotic cells are rapidly eliminated by macrophages via a process that does not produce an inflammatory reaction [ , ] . by contrast, senescent cells are characterized by their resistance to apoptosis, and display enlarged morphology. it seems that the ultimate fate between apoptotic death or survival in a senescent state depends on the severity and duration of dna damage [ ] . excessive damage to dna promotes apoptosis, whereas low and sustained genotoxicity results in cellular senescence [ ] . furthermore, p levels, h o concentrations, and b-cell lymphoma (bcl)- expression are also crucial factors implicated in determining the cell fate decision [ , ] . another important factor in this fate decision is p , a cyclin-dependent kinase inhibitor, which can act as an anti-apoptotic factor (inhibitor of apoptosis) [ ] . therefore, cells that survive irreversible dna acquire a senescent phenotype that protects against malignant transformation. cells that survive severe and/or irreversible dna damage confer a high risk for the survival of the organism, because genomic instability predisposes to oncogenesis [ ] . cellular senescence is a potent defense barrier against cancer progression not only because it arrests the proliferation of damaged cells, but also because it triggers an exacerbated inflammatory reaction that activates the immune system [ , ] . cells that survive irreparable genotoxic stress alert the immune system, attract phagocytic cells and promote their own clearance by triggering a localized proinflammatory cytokine gradient [ , , ] . in this process, sasp mediators act as cell-intrinsic cues that mediate the recruitment of neutrophils, macrophages, and natural killer cells facilitating the detection and elimination of senescent cells. in addition to cytokines, senescent cells continuously produce high mobility group box (hmgb ) in a p -dependent manner [ ] . davalos et al. demonstrated that hmgb is translocated from the nucleus to the extracellular space, where it also attracts innate immune cells stimulating the removal of senescent cells. furthermore, they found that hmgb regulates the expression of sasp, including il , thereby playing an essential role in modulating senescent cells inflammatory activity. this immune-mediated clearance is an important mechanism implicated in restricting the accumulation of senescent cells, which is called senescence surveillance [ , ] . as mentioned above, senescent cells constitute a sustained source of key proinflammatory cytokines, including il α, il β, il , il , tnf-α, and monocyte chemoattractant protein (mcp)- , among others [ , , ] . these studies strongly suggest that cellular senescence is a key intermediate mechanism linking local genotoxic damage and immune cell infiltration [ , ] . therefore, cells that survive irreversible dna damage promote their own elimination by activating the immune defense system, a process that depending on the context can promote tissue regeneration or degeneration. however, with aging immune-mediated elimination of senescent cells is impaired, which contributes to the accumulation of these dysfunctional cells in old tissues. a hallmark of periodontal disease is chronic inflammation, which is triggered in response to pathogenic bacteria in the subgingival area. although the inflammatory reaction is initiated to restrict the propagation of microorganisms and protect host tissues, a paradoxical effect is produced. because the immune-mediated inflammatory response is not specific to kill pathogenic bacteria, this defense mechanism can also damage host periodontal cells. in other words, inflammation does not discriminate between pathogens and healthy resident cells, and consequently, the beneficial effect of repelling infection simultaneously causes undesirable tissue damage. thus, the emphasis on the etiology of periodontal tissue destruction has been focused on the recognition of invading bacteria and/or their products that trigger an immune reaction. however, in the same context of persistent bacterial infection and chronic inflammation, what happens to those cells that survive such detrimental conditions has been overlooked. in addition, how those cells could deteriorate the periodontal microenvironment is currently an emerging topic. clear evidence indicates that sustained exposure to oxidative stress produced during inflammation can produce directly or indirectly dna damage in multiple cell types, including periodontal cells [ , ] . the recent identification of senescent cells in periodontal tissues might provide novel insights into the underlying mechanisms promoting tissue decline [ ] . based on mounting data, there are at least four interconnected factors that could contribute to the accumulation of senescent cells in periodontal tissues. namely, the persistent gram-negative bacterial infection, chronic inflammation itself, continuous renewal of damaged tissues, and bacteria-induced local immunosuppression (figure ). immune cells stimulating the removal of senescent cells. furthermore, they found that hmgb regulates the expression of sasp, including il , thereby playing an essential role in modulating senescent cells inflammatory activity. this immune-mediated clearance is an important mechanism implicated in restricting the accumulation of senescent cells, which is called senescence surveillance [ , ] . as mentioned above, senescent cells constitute a sustained source of key proinflammatory cytokines, including il α, il β, il , il , tnf-α, and monocyte chemoattractant protein (mcp)- , among others [ , , ] . these studies strongly suggest that cellular senescence is a key intermediate mechanism linking local genotoxic damage and immune cell infiltration [ , ] . therefore, cells that survive irreversible dna damage promote their own elimination by activating the immune defense system, a process that depending on the context can promote tissue regeneration or degeneration. however, with aging immune-mediated elimination of senescent cells is impaired, which contributes to the accumulation of these dysfunctional cells in old tissues. a hallmark of periodontal disease is chronic inflammation, which is triggered in response to pathogenic bacteria in the subgingival area. although the inflammatory reaction is initiated to restrict the propagation of microorganisms and protect host tissues, a paradoxical effect is produced. because the immune-mediated inflammatory response is not specific to kill pathogenic bacteria, this defense mechanism can also damage host periodontal cells. in other words, inflammation does not discriminate between pathogens and healthy resident cells, and consequently, the beneficial effect of repelling infection simultaneously causes undesirable tissue damage. thus, the emphasis on the etiology of periodontal tissue destruction has been focused on the recognition of invading bacteria and/or their products that trigger an immune reaction. however, in the same context of persistent bacterial infection and chronic inflammation, what happens to those cells that survive such detrimental conditions has been overlooked. in addition, how those cells could deteriorate the periodontal microenvironment is currently an emerging topic. clear evidence indicates that sustained exposure to oxidative stress produced during inflammation can produce directly or indirectly dna damage in multiple cell types, including periodontal cells [ , ] . the recent identification of senescent cells in periodontal tissues might provide novel insights into the underlying mechanisms promoting tissue decline [ ] . based on mounting data, there are at least four interconnected factors that could contribute to the accumulation of senescent cells in periodontal tissues. namely, the persistent gram-negative bacterial infection, chronic inflammation itself, continuous renewal of damaged tissues, and bacteria-induced local immunosuppression ( figure ). dna damage-driven senescence is probably one of the most underappreciated long-term biological consequences of persistent bacterial infection. the transition from health to disease in periodontal tissues has been associated with a predominantly gram-negative bacterial infection. although some of these bacteria can be identified in healthy individuals, porphyromonas gingivalis, tannerella forsythia, and treponema denticola, are strongly associated with chronic periodontal disease in adults [ ] . at this point, it is important to emphasize that all of these periodontal pathogens, also called "red complex", are gram-negative bacteria identified in subgingival plaque. while it is recognized that pathogens trigger an immunoinflammatory reaction into the infected sites, much less is known about the long-term effects of bacteria and/or their products on dna damage. mounting evidence indicates that persistent exposure to lps, an outer membrane component of gram-negative bacteria, causes a genotoxic effect in macrophages [ ] , and blood mononuclear cells [ ] . consistent with this lps genotoxic effect, a similar response is observed in gingival fibroblasts after the exposure to this gram-negative membrane component for h in vitro [ ] . remarkably, repeated exposure to p. gingivalis lps causes dna damage-driven premature senescence in alveolar bone cells, via a process mediated through the p activation [ ] . likewise, cytolethal distending toxin (cdt), which is secreted by the gram-negative periodontal pathogen a. actinomycetemcomitans, causes irreversible dna damage and mitosis inhibition [ , ] . cdt genotoxicity depends on its internalization from the extracellular space into the nucleus of the host cells, where it causes dna double-strand breaks as a result of its endonuclease activity [ , ] . consequently, human gingival epithelial cells react to cdt intoxication with dna damage in the form of double-strand breaks [ ] . this permanent genotoxic effect induced by cdt results in either apoptotic cell death or cellular senescence [ , ] . intriguingly, fahrer et al. demonstrated that cdt can simulate the effect of relatively low doses of ionizing radiation on dna damage in human fibroblasts [ ] . they also found that cdt not only produced a similar effect as radiation on the expression of several genes, but also that this toxin promoted a higher il , bcl , and mmp expression (in relation to ionizing radiation). based on their results, the authors suggested that cdt treatment could be a useful model to evaluate the cellular reaction to dna damage. therefore, long-term exposure to gram-negative bacteria and/or their toxins might cause a delayed detrimental effect on periodontal tissues by inducing dna damage and leading to cellular senescence. chronic periodontal inflammation creates a permissive environment for dna damage-driven cellular senescence through ros-mediated oxidative stress. during periodontal bacterial infection, recruited neutrophils secrete many proinflammatory cytokines and release ros. the former reinforce further recruitment of phagocytic cells, and the latter is one of the most efficient mechanisms to kill invading bacteria. however, besides this bactericidal effect, ros-mediated oxidative stress generated during chronic inflammation can accelerate cellular senescence through the onset and stabilization of dna damage [ , ] . concentration and duration of ros exposition play a pivotal role in determining cell fate. the biological consequences of oxidative stress can range from cell proliferation to either cellular senescence or apoptotic cell death [ ] . these cellular reactions seem to be conserved in different proliferating mammalian cells [ ] , including mouse and human gingival fibroblasts. kiyoshima et al. demonstrated that murine gingival fibroblasts exposed to lower h o concentrations ( µm) produced increased cell proliferation; however, a gradual decrease in the mitogenic capacity was observed when they were treated with higher concentrations ( µm) [ ] . along with growth arrest, higher concentrations also induced other senescence-like features such as increased senescence-associated β-galactosidase (sa-β-gal) activity, increased p levels, and enlarged cytoplasm. in agreement with these effects, yu et al. found that h o not only inhibits proliferation in human gingival fibroblasts, but also induces mitochondrial stress-mediated cell death [ ] . interestingly, cheng et al. reported that lps increased the generation of ros in gingival fibroblasts [ ] . as a consequence of higher ros production, these experiments showed that lps induced oxidative stress causes dna damage, which is associated with the expression of both anti-apoptotic, e.g., bcl , and other pro-apoptotic proteins. in addition, xia et al. found that rapamycin decreased the expression of il and il in human gingival fibroblasts infected with p. gingivalis by decreasing oxidative stress [ ] . these studies suggest that sustained exposure to low h o concentrations could promote periodontal cell proliferation, whereas intermediate concentrations cause senescence-associated features. in contrast, excessive oxidative stress produced by higher h o concentrations could cause apoptotic cell death in periodontal cells. oxidative stress can indirectly contribute to the pathogenesis of periodontal disease by activating signaling pathways that lead to a "pro-inflammatory state" [ ] . it is recognized that excessive ros generation during inflammation results in periodontal tissue destruction. indeed, patients with severe periodontitis exhibit a lower antioxidant capacity, which is associated with higher oxidative stress [ ] . in addition, increased activity of proinflammatory and oxidative stress markers have been reported in patients with chronic periodontitis compared to healthy controls [ ] . considering the genotoxic effect of ros, another biological consequence of oxidative stress is genomic instability, which is a hallmark of malignant cell transformation. in agreement with this, bacterial infection has been associated with cancer development by promoting chronic inflammation and generating genotoxic products [ ] . these studies raise the possibility that cells that survive long-term exposure to oxidative stress during periodontal inflammation can reach a damage threshold, and eventually activate dna damage response pathways. therefore, oxidative stress generated during chronic inflammation can cause cellular senescence by damaging dna in periodontal cells. senescent cells are not only found in tissues that undergo chronic inflammation, they also reside in tissues with the capacity to be repaired or regenerated [ , ] . since mammalian cells do not proliferate indefinitely, they undergo permanent mitotic arrest when they reach the end of their proliferative lifespan (as discussed above); gingival epithelial cells similarly undergo replicative senescence after around nine passages in culture [ ] . the gingival epithelium acts as a mechanical barrier between bacterial biofilm and gingival tissues, providing a "seal" surrounding the teeth to protect deeper host periodontal tissues against bacterial invasion. when the gingival epithelium is damaged pathogenic microorganisms and/or their toxins gain access into the underlying gingival connective tissue and promote tissue destruction [ ] . besides acting as a physical barrier and secreting antimicrobial agents, the gingival epithelium self-renewal capacity is also a periodontal defense mechanism, as its continuous turnover prevents pathogen invasion [ , ] thus, alterations in gingival epithelium shedding and renewal could contribute to the development of periodontal tissue destruction [ , ] . multiple studies have reported that epithelial damage caused by bacterial infection increases cell turnover not only in gingival tissues, but also in non-gingival tissues [ , ] . indeed, human gingival epithelial cells display accelerated proliferation rates when they are infected with p. gingivalis [ ] . in contrast, damek-poprawa et al. reported that exposure to a. actinomycetemcomitans cdt promoted growth arrest in primary human gingival epithelial cells isolated from healthy donors. they also identified that healthy gingival explants exposed to cdt exhibited disruption of the epithelial layers and dissolution of cell junctions. these morphological changes were similar to those observed in gingival explants from patients with periodontitis [ ] . consistent with this, a. actinomycetemcomitans cdt topical application promotes mitotic inhibition of rat epithelial cells in vivo [ ] . these studies suggest that the self-renewal capacity of gingival epithelium can be impaired directly by certain bacterial toxins, but also indirectly by increasing the proliferating rate eventually leading to accelerated replicative senescence. therefore, we speculate in this context that constant bacterial challenge may lead to senescence-associated epithelial growth arrest, which might compromise the integrity of the epithelial barrier over time. once the gingival epithelial cell barrier has been disrupted, bacteria and/or their virulence factors can reach deeper underlying connective tissues. gingival fibroblasts are the most abundant cells in gingival connective tissues, and similar to epithelial cells display a limited mitotic capacity. they also have a critical role in sustaining periodontal inflammation [ ] . paez et al. recently reported that primary human fibroblasts from young donors displayed senescence features after extensive cell passaging, including increased p ink a and p expression, increased dna damage, and inhibition of proliferation [ ] . intriguingly, in this study, the expression of inflammatory factors, such as il and il , were not identified. these clearly contradictory results are conciliated when we consider that senescence-associated proinflammatory cytokine production is a consequence of persistent dna damage [ ] ; p ink a expression as a result of replicative senescence is not associated with the sasp [ ] . supporting this idea, it has been reported that human gingival fibroblasts stimulated with cdt exhibited growth arrest, and increased il protein levels probably as a consequence of dna damage (as discussed above) [ , ] . in agreement with these studies, xia et al. demonstrated that rapamycin, an mammalian target of rapamycin (mtor) pathway inhibitor, not only delayed the onset of senescence features, but also preserved the mitotic potential of healthy human gingival fibroblasts [ ] . taken together, although gingival fibroblasts have a high self-renewal potential, it could be affected during bacteria-induced inflammation limiting gingival regenerative capacity. in the clinical context, it has been suggested that sustained inflammation during chronic periodontal disease could promote telomere attrition [ ] . indeed, patients with chronic periodontitis displayed shorter leukocyte telomere length, which correlates with disease severity [ ] . in contrast, some studies have reported that periodontitis is not associated to telomere shortening [ ] . for instance, sanders et al. found that patients ( - years), with and without severe chronic periodontitis, had a similar rate of leukocyte telomere length shortening [ ] . these authors suggested that telomere reduction in older individuals could have occurred "earlier in the life course". these studies suggest that telomere dysfunction might play an important role in periodontal disease progression, but additional factors are required to establish tissue destruction. impaired immune activity promotes senescent cell accumulation. besides pathogenic bacteria elimination, the host immune system detects and removes damaged cells, including senescent cells. this process is called senescent cell immune surveillance, in which the release of senescence-associated proinflammatory signals play essential roles in attracting neutrophils, macrophages, lymphocytes and natural killer cells [ , , ] . however, reduced immunosurveillance of senescent cells accelerates their accumulation, resulting in gradual tissue deterioration and contributing to the development of different chronic pathologies [ ] . despite p. gingivalis displays a wide array of invasive mechanisms and different virulence factors, it is important to highlight that its pathogenicity is greatly mediated through the host immunosuppression [ ] [ ] [ ] . surprisingly, a. actinomycetemcomitans cdt was initially identified as an immunosuppressive factor that contributed to the pathogenesis of periodontal disease [ ] . downregulation of the host immunity by a. actinomycetemcomitans could also be mediated through the secretion of leukotoxin (ltxa), a protein that targets and kills polymorphonuclear cells and monocytes [ ] . interestingly, tannerella forsythia attenuates the early host immune response, which results in its impaired recognition and delayed elimination by the immune defense system [ ] . these observations suggest that local host immune suppression caused by bacteria might play an important role in the accumulation of senescent cells by impairing their timely clearance. however, further investigations are required to confirm this hypothesis. nuclear dna damage is intrinsically coupled to the release of proinflammatory signals and innate immune activation. in the context of periodontal inflammation, most studies have been focused on the recruitment of immune cells triggered by bacterial recognition [ , ] . consequently, the emphasis of periodontal tissue destruction has been placed on the immunoinflammatory reaction against bacterial infection. much less explored has been the role of dna damage and cellular senescence on the immune defense activation. however, nuclear dna damage sensors are coupled to a "sterile" inflammatory reaction (non-microbial per se) and the host immune response [ , ] . in fact, dna damage-driven chronic inflammation is not a new concept in aging and cancer research [ ] . recently, our group identified the presence of increased dna damage and abnormal accumulation of senescent cells in young periodontal tissues using a murine model [ ] . these novel insights suggest that cellular senescence could be an important intermediate mechanism between bacterial infection and innate immune response as senescent cells can by themselves activate the host immune reaction. therefore, besides being an additional source of proinflammatory signals, senescent cells can potentially aggravate the initial immune reaction against pathogenic bacteria (figure ). nuclear dna damage is intrinsically coupled to the release of proinflammatory signals and innate immune activation. in the context of periodontal inflammation, most studies have been focused on the recruitment of immune cells triggered by bacterial recognition [ , ] . consequently, the emphasis of periodontal tissue destruction has been placed on the immunoinflammatory reaction against bacterial infection. much less explored has been the role of dna damage and cellular senescence on the immune defense activation. however, nuclear dna damage sensors are coupled to a "sterile" inflammatory reaction (non-microbial per se) and the host immune response [ , ] . in fact, dna damage-driven chronic inflammation is not a new concept in aging and cancer research [ ] . recently, our group identified the presence of increased dna damage and abnormal accumulation of senescent cells in young periodontal tissues using a murine model [ ] . these novel insights suggest that cellular senescence could be an important intermediate mechanism between bacterial infection and innate immune response as senescent cells can by themselves activate the host immune reaction. therefore, besides being an additional source of proinflammatory signals, senescent cells can potentially aggravate the initial immune reaction against pathogenic bacteria (figure ) . cells that survive persistent dna damage acquire a senescent phenotype, which is characterized by the hypersecretion of key proinflammatory cytokines. dna damage-driven senescence is a crucial intermediate mechanism between dna damage and host immune activation. released factors by these dysfunctional cells could aggravate the initial immune reaction against pathogenic bacteria. thus, gradual accumulation of senescent cells in periodontal tissues during lifetime could contribute to the progression of periodontal tissue deterioration. nf-κb is a crucial transcription factor implicated in the coordination of various signaling pathways that modulate different immune and inflammatory processes. consequently, nf-κb regulates the cell response to infection, stress, and damage by modulating the expression of proinflammatory cytokines, chemokines, and adhesion molecules [ , ] . biologically relevant is the fact that different signaling cascades triggered by unrelated stimuli converge at nf-κb. for instance, microbial pathogens and double-strand dna breaks use distinct signaling transduction pathways to activate nf-κb. in fact, lps induced nf-κb activation is mainly mediated by toll-like receptors (tlr), specifically tlr [ ] ; whereas dna damage signaling is initiated in the nucleus figure . potential mechanism by which senescent cells aggravate periodontal tissue deterioration. cells that survive persistent dna damage acquire a senescent phenotype, which is characterized by the hypersecretion of key proinflammatory cytokines. dna damage-driven senescence is a crucial intermediate mechanism between dna damage and host immune activation. released factors by these dysfunctional cells could aggravate the initial immune reaction against pathogenic bacteria. thus, gradual accumulation of senescent cells in periodontal tissues during lifetime could contribute to the progression of periodontal tissue deterioration. nf-κb is a crucial transcription factor implicated in the coordination of various signaling pathways that modulate different immune and inflammatory processes. consequently, nf-κb regulates the cell response to infection, stress, and damage by modulating the expression of proinflammatory cytokines, chemokines, and adhesion molecules [ , ] . biologically relevant is the fact that different signaling cascades triggered by unrelated stimuli converge at nf-κb. for instance, microbial pathogens and double-strand dna breaks use distinct signaling transduction pathways to activate nf-κb. in fact, lps induced nf-κb activation is mainly mediated by toll-like receptors (tlr), specifically tlr [ ] ; whereas dna damage signaling is initiated in the nucleus where atm (ataxia telangiectasia mutated) plays a primary role in the activation of nf-κb [ , ] . these data indicate that unrelated stimuli could reinforce each other, and result in increased inflammatory reaction (figure ). supporting this premise, gölz et al. identified that hypoxia enhanced the nf-κb activation promoted by p. gingivalis lps in periodontal ligament cells [ ] . another example is the glucose boosting effect on lps in mononuclear cells. nareika et al. demonstrated that high glucose produces a strong increment on lps induced nf-κb activation, which results in higher proinflammatory cytokine and mmps expression [ ] . this is consistent with the ability of high glucose to induce oxidative stress by generating ros [ ] . of note, nf-κb activation and accumulation coincide with the acquisition of the senescent phenotype, acting as a major regulator of senescence-associated proinflammatory factors including the expression of il , il , and icam , among others [ , ] . consequently, nf-κb inhibition decreases oxidative stress dna damage and delays the detrimental effects of cellular senescence in mice [ ] . therefore, nf-κb activation might be a unifying intracellular event that potentiates the immunoinflammatory reaction to unrelated stimuli, such as nuclear dna damage itself and bacterial components. these data indicate that unrelated stimuli could reinforce each other, and result in increased inflammatory reaction (figure ) . supporting this premise, gölz et al. identified that hypoxia enhanced the nf-κb activation promoted by p. gingivalis lps in periodontal ligament cells [ ] . another example is the glucose boosting effect on lps in mononuclear cells. nareika et al. demonstrated that high glucose produces a strong increment on lps induced nf-κb activation, which results in higher proinflammatory cytokine and mmps expression [ ] . this is consistent with the ability of high glucose to induce oxidative stress by generating ros [ ] . of note, nf-κb activation and accumulation coincide with the acquisition of the senescent phenotype, acting as a major regulator of senescence-associated proinflammatory factors including the expression of il , il , and icam , among others [ , ] . consequently, nf-κb inhibition decreases oxidative stress dna damage and delays the detrimental effects of cellular senescence in mice [ ] . therefore, nf-κb activation might be a unifying intracellular event that potentiates the immunoinflammatory reaction to unrelated stimuli, such as nuclear dna damage itself and bacterial components. senotherapy is an encouraging therapeutic approach based on the use of synthetic or natural compounds that selectively eliminate senescent cells, or reduce the expression of sasp factors [ ] . since senescent cells and their factors produce a negative impact on surrounding cells and their environment, an additional beneficial effect of senomorphic drugs could be the restoration of the stem cell regenerative potential. thereby, this emerging strategy could ameliorate tissue deterioration and promote tissue regeneration in part by inhibiting key anti-apoptotic pathways, or reinforcing the immune system function [ ] . based on the current evidence, potential strategies targeting senescent cells include at least three different approaches [ ] . first, inhibition of key transcription factors, such as p , or signaling pathways controlling the onset and progression of senotherapy is an encouraging therapeutic approach based on the use of synthetic or natural compounds that selectively eliminate senescent cells, or reduce the expression of sasp factors [ ] . since senescent cells and their factors produce a negative impact on surrounding cells and their environment, an additional beneficial effect of senomorphic drugs could be the restoration of the stem cell regenerative potential. thereby, this emerging strategy could ameliorate tissue deterioration and promote tissue regeneration in part by inhibiting key anti-apoptotic pathways, or reinforcing the immune system function [ ] . based on the current evidence, potential strategies targeting senescent cells include at least three different approaches [ ] . first, inhibition of key transcription factors, such as p , or signaling pathways controlling the onset and progression of cellular senescence. however, given the role of cellular senescence as an anti-neoplastic mechanism, the downregulation of molecular events leading to senescence can potentially promote oncogenic cell proliferation [ ] . second, an alternative strategy to target specific molecular targets is the elimination of tissue-resident senescent cells. senolytics are drugs that selectively kill senescent cells by transiently inactivating senescence-associated anti-apoptotic pathways (scaps), allowing senescent cells to undergo apoptosis [ ] . it has been reported that physical dysfunction and decreased lifespan caused by senescent cells is improved after intermittent senolytic drug administration in old mice [ ] . these beneficial effects of senolytics drugs could also be the result of improving stem cell function [ , ] . third, inhibition of senescence-associated proinflammatory factors by using senomorphic drugs. senomorphics are agents that interfere with the secretion of sasp factors that promote sterile inflammation, which is produced without inducing apoptosis [ , ] . senomorphic compounds include mtor, and nf-κb inhibitors [ , ] . although senomorphic agents can potentially reduce the secretion of senescence-associated factors, a paradoxical effect could also be produced. that is, since immune-mediated senescent cell elimination is modulated by sasp factors, their inhibition could result in higher accumulation of senescent cells [ ] . since the abnormal accumulation of senescent cells negatively impacts their local environment, and recent evidence indicates that pharmacological agents can modulate sasp activity; these findings are of clinical importance as they can represent an additional approach to traditional periodontal therapy. in fact, some experiments have already demonstrated that pharmacological inhibitors can improve oral and periodontal health. an et al. recently demonstrated that short-term treatment with rapamycin, an mtor inhibitor, attenuates gingival and alveolar bone inflammation, and also promotes the regeneration of alveolar bone of old mice [ ] . another recent study has demonstrated that mtor has a central role in regulating lps induced inflammation in microglial cells [ ] . therefore, senotherapeutic drugs are an emerging and promising approach to delay cellular senescence-related tissue dysfunction, but additional studies are required to better understand their mechanisms of action and efficacy, as well as safety profiles. in the context of periodontal regeneration, cell-based tissue engineering constructs have been used as alternative strategy of conventional procedures. this regenerative approach involves the expansion of cells in vitro, which are seeded in three-dimensional scaffolds and subsequently implanted into periodontal lesions. periodontal ligament stem cells (pdlsc) are ideal for periodontal regeneration; however, like other mammalian cells, they undergo senescence. consistent with this concept, zheng et al. demonstrated that the proliferative and differentiation capacity of pdlsc also decreases with age [ ] . interestingly, these authors found that conditioned media from young pdlsc improve the proliferative and differentiation capacity of old pdlsc. in a different study, kuang et al. reported that metformin, a drug used for the treatment of diabetes, inhibits h o damaging effects on human pdlsc resulting in decreased oxidative stress-induced senescence [ ] . as mentioned above in section . , tgf-β ligands have a relevant role in cellular senescence. in agreement with this, it has been reported that tgf-β induces pdlsc senescence by increasing oxidative stress, and that such effect can be reduced by n-acetyl-l-cysteine (nac), a ros inhibitor [ ] of note, honda et al. found that osteoblastic cells undergo stress-induced premature senescence after implantation in calvarial defects. they also reported that systemic administration of the senolytic d+q (dasatinib and quercetin) reduced premature senescence in bone defects [ ] . these studies suggest that delaying the onset and development of cellular senescence may improve the regenerative potential of periodontal tissues. although bacteria are essential to initiate the inflammatory reaction, it is the host immune reaction that ultimately causes tissue destruction. emerging evidence indicates that persistent gram-negative bacterial infection and chronic inflammation itself can facilitate the accumulation of dna damage on those cells that survive under those conditions. although genomic lesions can be repaired, cells that survive irreparable genotoxic insults eventually undergo senescence, which is coupled to the hypersecretion of sasp factors and immune defense activation. since dna damage-driven senescence can by itself induce an immune response, it is presumed that this novel mechanism can potentially contribute to immune-mediated periodontal tissue destruction. senotherapy has been developed to selectively kill senescent cells or decrease the deleterious effects of sasp factors. recently, senescent cells have been identified in the periodontal environment, where they act as an important source of key factors implicated in the etiology of periodontal disease. another recent study has reported that rapamycin, an fda-approved drug used as a senomorphic agent, ameliorates inflammation and "rejuvenates" old periodontal tissues. both studies have established that 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open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors want to thank francesc ventura for his support with the manuscript correction. the authors declare no conflict of interest. key: cord- -c ajfvt authors: sundqvist, martina; holdfeldt, andré; wright, shane c.; møller, thor c.; siaw, esther; jennbacken, karin; franzyk, henrik; bouvier, michel; dahlgren, claes; forsman, huamei title: barbadin selectively modulates fpr -mediated neutrophil functions independent of receptor endocytosis date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: c ajfvt formyl peptide receptor (fpr ), a member of the family of g protein-coupled receptors (gpcrs), mediates neutrophil migration, a response that has been linked to β-arrestin recruitment. β-arrestin regulates gpcr endocytosis and can also elicit non-canonical receptor signaling. to determine the poorly understood role of β-arrestin in fpr endocytosis and in nadph-oxidase activation in neutrophils, barbadin was used as a research tool in this study. barbadin has been shown to bind the clathrin adaptor protein (ap ) and thereby prevent β- arrestin/ap interaction and β-arrestin-mediated gpcr endocytosis. in agreement with this, ap /β-arrestin interaction induced by an fpr -specific agonist was inhibited by barbadin. unexpectedly, however, barbadin did not inhibit fpr endocytosis, indicating that a mechanism independent of β-arrestin/ap interaction may sustain fpr endocytosis. this was confirmed by the fact, that fpr also underwent agonist-promoted endocytosis in β-arrestin deficient cells, albeit at a diminished level as compared to wild type cells. dissection of the barbadin effects on fpr -mediated neutrophil functions including nadph-oxidase activation mediated release of reactive oxygen species (ros) and chemotaxis reveled that barbadin had no effect on chemotactic migration whereas the release of ros was potentiated/primed. the effect of barbadin on ros production was reversible, independent of β-arrestin recruitment, and similar to that induced by latrunculin a. taken together, our data demonstrate that endocytic uptake of fpr occurs independently of β-arrestin, while barbadin selectively augments fpr -mediated neutrophil ros production independently of receptor endocytosis. given that barbadin binds to ap and prevents the ap /β-arrestin interaction, our results indicate a role for ap in fpr -mediated ros release from human neutrophils. neutrophils, the most abundant leukocytes in human peripheral blood, form the frontline of our innate host immune defense, and are rapidly recruited from the circulation to damaged or infected body tissues, where they contribute to bacterial clearance and tissue repair [ ] [ ] [ ] [ ] . the formyl peptide receptor (fpr ), belonging to the family of g protein-coupled receptors (gpcrs), regulates directional neutrophil migration (chemotaxis), granule secretion (degranulation), formation of f-actin filaments (through polymerization of g-actin), and activation of the reactive oxygen species (ros) producing nadph-oxidase [ , , ] . fpr recognizes not only n-formyl peptides of both bacterial and host mitochondrial origin, but also neutrophil surface expression of fpr or cd b was examined by flow cytometry. neutrophils ( × /ml) were equilibrated for min at °c, and then stimulated with either krg (control), an fpr agonist or with barbadin with or without an fpr agonist in the presence of catalase ( units/ml) to avoid potential agonist inactivation via oxidation. cells the plasmid encoding human fpr with an n-terminal snap-tag was obtained from genscript (piscataway, nj, usa); it was constructed by replacing glp r in the previously described pcdna . (+)-flag-snap-glp r plasmid [ ] with human fpr . hek a cells (thermo neutrophil nadph-oxidase activity nadph-oxidase activity was determined by using isoluminol/luminol-enhanced chemiluminescence (cl) [ ] in a six-channel biolumat lb (berthold co., wildbad, germany). polypropylene tubes containing a µl reaction mixture of neutrophils in krg, isoluminol ( × - m) and hrp ( units/ml), were equilibrated at °c for min before addition of µl of stimulus. for latrunculin a or barbadin dilution experiments, neutrophils ( /ml) were pre-incubated at °c with latrunculin a ( ng/ml) or barbadin ( µm) in a separate tube and then µl cell suspension were transferred to polypropylene tubes containing µl °c pre-heated reaction mixture (to obtain cells in the assay system) of krg, hrp and isoluminol in the absence or presence of latrunculin a ( ng/ml) or barbadin ( µm) followed by agonist stimulation ( µl). for phagocytosis-induced intracellular nadph-oxidase activity, yeast particles ( x /ml) were opsonized in % normal human serum ( min, °c) followed by washing and dilution in krg to a concentration of x yeast particles/ml. opsonized yeast solution ( µl) was added to polypropylene tubes containing µl reaction mixture containing x neutrophils in krg, luminol ( × - m), superoxide dismutase ( units/ml) and catalase ( units/ml) in the absence and presence of barbadin ( µm) or latrunculin a ( ng/ml) that had been equilibrated at °c for min; multiplicity of infection (moi): : . the light emission of yeast phagocytosis-induced ros production is expressed as mega counts per min (mcpm). neutrophils ( /ml) were equilibrated for min in the absence and presence of barbadin ( µm) at °c followed by agonist stimulation. after s of treatment, μl cell suspension were transferred to ice-cold fixation/permeabilization solution (bd cytofix/cytoperm solution, . ml) and incubated on ice for min. the cells were then washed twice with bd wash buffer before staining with af -conjugated phalloidin ( min, °c). a minimum of , gated neutrophils (forward scatter; size versus side scatter; density) per sample were collected on an accuri c flow cytometer (bd biosciences, sparks, md, usa). the af -conjugated phalloidin intensity was determined by the geometric mean fluorescence intensity (mfi) as analyzed by flowjo software version . (tree star inc., ashland, or, usa). neutrophils were loaded with fura- -am ( µm) ( min, rt, in darkness) before washing and resuspension in krg. measurements of the transient rise in [ca + ]i were carried out at °c by using a perkinelmer lc fluorescence spectrophotometer with excitation wavelengths of nm and nm, an emission wavelength of nm, and slit widths of nm and nm. the transient rise in intracellular calcium is presented as the fluorescence intensities for both the excitation wavelengths ( and nm) as measured in parallel. for reactivation experiments (fig c) , isoluminol ( × - m) and hrp ( units/ml) were included in the assay system to avoid agonist inactivation by the radicals generated through the myeloperoxidase (mpo)-h o enzyme system [ ] . neutrophils ( × /ml) in krg supplemented with bsa ( . %) were loaded on top of a µm pore size filter and allowed to migrate towards stimuli loaded into the bottom wells of a chemotaxis plate (chemotx, neuro probe, uk) for min at °c, % co . barbadin was present in both upper and lower chambers to avoid any gradient effect. the migrated cells were quantified by measuring myeloperoxidase activity of cell lysates [ ] . all migration (myeloperoxidase activity) values were subtracted from the level of migration without any attractant in the lower compartment (negative control), and the resulting data are presented as the number of cells recovered in the lower compartment, relative to the total number of cells applied to the migration system (neutrophils were added directly to the bottom well of the chemotaxis plate). data analysis was performed using graphpad prism . . a (graphpad software, san diego, ca, usa). curve fitting was performed by non-linear regression using the sigmoidal doseresponse equation (variable-slope). independent bret experiments were normalized to the span of reference compound (wkymvm) as determined by non-linear regression analysis and pooled together. data represent mean values ± sem. statistical analysis was performed using a paired student's t-test (fig d; d, f and h; c and f; b and c; b and d and f) or a repeated measurement one-way anova followed by tukey's multiple comparison test ( fig a, a and h ). statistically significant differences are indicated by *p < . , **p < . , ***p < . . barbadin (structure is shown in fig ) was recently described as a selective inhibitor of the machinery responsible for ap /-arrestin-dependent endocytic internalization of some gpcrs including the vasopressin receptor (v r), the  -adrenergic receptor ( ar) and the angiotensin ii receptor type (at r) [ ] . to study the effect of barbadin on agonist-induced fpr internalization, we used the peptide agonist wkymvm and two lipopeptides, the peptidomimetic cmp and the pepducin f pal that are functionally biased (structures are shown in fig ) . all three agonists (wkymvm, cmp and f pal ) are potent in inducing an fpr -mediated transient rise in [ca + ]i, erk / phosphorylation, and ros production in human neutrophils, but cmp and f pal are biased away from -arrestin recruitment and chemotaxis [ - ]. by using an enhanced bystander bioluminescence resonance energy transfer (ebbret) assay system, we here confirmed that cmp and f pal are poor inducers of arrestin recruitment (fig a) . both wkymvm and f pal triggered fpr internalization but barbadin lacked effect on fpr internalization as determined by ebbret ( fig b) . the inability of barbadin to inhibit agonist-triggered removal of fpr from the hek cell surface was also evident when time resolved-förster resonance energy transfer assay (tr-fret) [ ] was used to study fpr internalization (fig c, d) . these data clearly show that internalization of fpr induced by agonists is unaffected by the presence of barbadin. in contrast to fpr , and in agreement with previously published data [ ] , barbadin reduced v r internalization as measured by ebbret in hek cells that had been transiently transfected with donor-tagged v r (v r-rlucii) and an acceptor anchored to the membrane (rgfp-caax) (fig e) . although barbadin did not appear to block agonist-induced fpr internalization, ebbret experiments investigating fpr trafficking from the plasma membrane (rgfp-caax) to early endosomes (rgfp-fyve) revealed that the internalization was largely (but not completely for wkymvm) dependent on the presence of -arrestin / as illustrated by the reduced responses in hek cells devoid of -arrestin / (arr, fig f, g). in order to confirm that barbadin did indeed block the interaction between -arrestin- and ap , we performed bret experiments to determine the wkymvm and f pal induced interaction between the two proteins. barbadin clearly blocked the wkymvm-induced interaction between -arrestin- and ap , whereas the low level of interaction induced by f pal alone makes it hard to determine the ability/inability of barbadin to inhibit its effect ( fig h) . taken together, these findings demonstrate that barbadin does not affect agonisttriggered internalization of surface-exposed fpr s and that a mechanism independent of arrestin/ap interaction can sustain wkymvm-induced receptor endocytosis. we next determined whether barbadin affects agonist-induced internalization of fpr when it is endogenously expressed in human neutrophils. although ap was clearly present in neutrophils (fig a) , the reduction in the number of surface-exposed fpr s was identical in wkymvm-activated control neutrophils (not treated with barbadin) and in wkymvmactivated cells treated with barbadin ( fig b) . hence, these data strongly imply that barbadin has no effect on agonist-induced fpr internalization in primary human neutrophils in which the receptor is endogenously expressed. neutrophils possess an electron transporting enzyme system, the neutrophil nadph-oxidase, which upon activation generates ros [ ] . despite the insensitivity of the agonist-triggered fpr internalization to barbadin, pre-incubation of neutrophils with barbadin substantially increased the neutrophil release of ros upon wkymvm stimulation (fig c, d) . expectedly, the fpr -selective antagonist rhb-(lys-βnphe) -nh (structure is shown in fig , where it is denoted as fpr ant.) completely blocked the neutrophil response ( fig c) . also, the priming effect (i.e., potentiation of ros production) of barbadin was evident at a concentration of the peptide agonist wkymvm ( nm) too low to trigger ros release in non-primed neutrophils ( fig d) . the priming effect was concentration-dependent, reaching its full effect at m of barbadin with an ec value of  m (fig e) . similarly, barbadin also significantly primed the ros production induced by f pal and cmp ( fig f) . the fact that f pal and cmp cannot induce -arrestin recruitment at these concentrations ([ - ], fig a) , strongly suggests that the priming effect of barbadin on fpr -mediated ros production is independent of the ability of the activating agonist to recruit -arrestin. in addition, the ros release following activation with pma, a compound that bypasses receptors and directly activate protein kinase c (pkc), was unaffected by barbadin ( fig g, h) , which suggests that barbadin lacks a direct effect on the ros-producing nadph-oxidase machinery. it is also important to note that activation of the nadph-oxidase could not be triggered by barbadin alone (fig g, h). collectively, these results suggest that barbadin primes neutrophils in their response to fpr agonists, and the increased nadph-oxidase activation is regulated independently of fpr internalization and fpr -induced -arrestin recruitment. barbadin treatment significantly primed fpr agonist-induced ros production in human neutrophils and further characterization revealed that a very short interaction time between neutrophils and barbadin was needed for barbadin to exert its priming effect. in fact, the increase in neutrophil ros production was the same when barbadin and the fpr agonist wkymvm were added simultaneously, as when the cells were incubated with barbadin prior to addition of the activating peptide wkymvm (fig a) . in addition, we found that the priming effect of barbadin on neutrophils for increased wkymvm response was also rapidly reduced when the barbadin concentration was lowered significantly. neutrophils first incubated for min with an effective priming concentration of barbadin ( µm) and then transferred to two different ros measurement reagents, one containing a new addition of barbadin ( µm) and the other not (the barbadin concentration was thus lowered x to . µm), followed by stimulation with wkymvm. a significantly lower degree of priming effect was observed in cells that were exposed to a reduced concentration (from µm to . µm) than cells incubated constantly to an effective priming concentration ( µm) of barbadin (fig b and c). this suggests that the neutrophil priming induced by barbadin is a process that is reversible, unlike many other priming processes that involve an irreversible process of neutrophil granule secretion [ , , ] . the effect of barbadin on fpr -mediated ros production resembled the effect induced by the f-actin-disrupting agent latrunculin a. barbadin and latrunculin a both prolonged and increased the magnitude of fpr -mediated neutrophil ros production as compared to the corresponding ros production in non-treated control cells ( fig d) . in addition, very similar to barbadin, the priming effect of latrunculin a was rapidly reduced as shown when latrunculin a was removed prior to activation with the fpr agonist wkymvm ( fig e, f ), using the same dilution protocol, to show that the priming effect of barbadin is reversible, as described above. fpr -induced ros production is a process rapidly initiated after addition of an activating agonist, and within a time period of minutes, the ros production is terminated and the cells are homologously desensitized. the desensitized cells are non-responsive to a second stimulation with fpr agonists but fully responsive to an fpr selective agonist or pma [ , ] . the desensitized state could be transferred to an active signaling state to produce ros again (i.e., fpr resensitization or reactivation) when the cytoskeleton was disrupted by latrunculin a (fig a) . the ros production induced by latrunculin a in fpr -desensitized cells was completely abolished by the fpr -selective antagonist rhb-(lys-βnphe) -nh ( fig a) . to determine the ability of barbadin to resensitize desensitized fpr , we reversed the order by which the sensitizer (barbadin) and fpr agonist (wkymvm) were added to the neutrophils. barbadin was added to wkymvm-activated neutrophils at a time point when ros production had returned to a background level; interestingly, these fpr -desensitized cells could be resensitized to produce ros also by barbadin, similar to the effect of latrunculin a ( fig a) . this response was also inhibited by an fpr -selective antagonist (fig a) , clearly demonstrating an fpr -mediated neutrophil resensitization. very similar results were obtained in fpr -desensitized cells when f pal or cmp (at concentrations that could not recruit - fig a) replaced wkymvm as the agonist used to desensitize fpr (fig a) . although the resensitization effects of latrunculin a and barbadin appeared very similar, it should be noted that the lag phase before any ros were generated was shorter when barbadin was used for resensitization, and the time to reach maximal (peak) ros production was also shorter as compared to the response following addition of latrunculin a ( fig b) . however, the amounts of ros produced during resensitization (as measured by the area under the curve) were comparable for barbadin and latrunculin a (fig c) . in summary, we show that barbadin, similarly to latrunculin a, not only potentiates the ros production induced by the different fpr agonists, but also resensitizes fpr signaling when added to fpr desensitized neutrophils. barbadin treatment potentiated and resensitized fpr -mediated signaling leading to ros production. to further investigate the effect of barbadin on fpr signaling and function in neutrophils, we measured the transient rise in cytosolic calcium [ca + ]i mediated through fpr . in contrast to the potentiating effect on ros production, the rise in [ca + ]i induced by wkymvm was not affected by barbadin (fig a, b) . similarly, resensitization by barbadin of agonist-desensitized fpr leading to an activation of the ros producing nadph-oxidase, was not associated with a corresponding rise in [ca + ]i (fig c) . at the functional level, we also observed a biased activity of barbadin in favor of neutrophil ros production over directional cell migration/chemotaxis. neutrophil chemotaxis was measured by using a transwell migration system; neutrophils were placed on top of the filter and allowed to migrate towards different concentrations of fpr agonists that were placed in the bottom well of the chambers. barbadin ( m) was added to both compartments, so that it was present in both the upper chamber together with the cells and the lower chamber containing the agonist. in line with earlier data [ , ], wkymvm, but not f pal , triggered a chemotactic migration of neutrophils. further, neutrophil chemotaxis towards wkymvm was unaffected by barbadin, and the inability of f pal to trigger chemotaxis was retained in the presence of barbadin (fig d) . in summary, these data demonstrate that the effect of barbadin in neutrophils is in favor of fpr -mediated ros over the rise in [ca + ]i and chemotaxis. the functional similarities between barbadin and latrunculin a, both being priming agents affecting the response induced by fpr agonists and agents that resensitize desensitized fpr , promoted us to examine whether barbadin could directly affect the integrity of the actin cytoskeleton and granule secretion. wkymvm induced a rapid polymerization of g-actin monomers into polymerized filamentous actin (f-actin) in human neutrophils (fig a) . however, the presence of barbadin did not affect the formation of f-actin induced by wkymvm (fig a) , indicating that barbadin does not affect the integrity of the actin cytoskeleton. the observation that barbadin does not directly interfere with the integrity of the actin cytoskeleton gained further support from the results obtained with barbadin on two neutrophil responses previously found to be regulated by the actin cytoskeleton, i.e., the atp receptor p y r-mediated ros production and the phagocytosis process [ , ] . it is known that atp upon binding to its neutrophil receptor p y r, triggers ros production provided that the factin structure is disrupted (fig b) . in line with this, latrunculin a treated neutrophils produce ros when activated with atp, but no such effect was obtained with barbadin ( fig b) . activation of the ros-generating nadph-oxidase system during uptake (phagocytosis) of microbes is a process regulated by the cytoskeleton, and accordingly, latrunculin a inhibited the activation process ( fig c) . however, barbadin exerted no inhibitory effect, neither when added before addition of the phagocytosis prey nor when added during the ongoing activation process (fig c, d) . taken together, these data show that barbadin has no direct effects on basic neutrophil functions regulated by the actin cytoskeleton, supporting the conclusion that barbadin lacks a general effect on the assembly of the ros-producing oxidase. our data reveal that barbadin is able to prime neutrophils for enhanced fpr -mediated ros production. neutrophil priming is a well-known process both in vitro and in vivo [ , , , , ] , and an increased exposure of intracellular granule-localized receptors to the plasma membrane as a result of granule secretion has been suggested to be one of the main mechanisms that augments neutrophil nadph-oxidase activity [ , , ] . we next determined whether the receptor mobilizing effect with increased surface fpr expression could account for the barbadin-induced priming effect. however, no increased surface exposure of fpr was induced by incubation of neutrophils with barbadin for up to ten minutes (fig e, f) . in addition to fpr expression, the ability of barbadin to upregulate the surface expression of cd b (complement receptor ; cr ), a marker protein stored in easily mobilized neutrophil granule compartments that can be mobilized to the surface by many secretagogues or priming agents [ ] was investigated. however, similar to the fpr expression, barbadin also lacked effect to upregulate cd b on the plasma membrane, whereas a profound increase of cd b surface expression was induced by the classical secretagogue fmlf (fig g, h) . in summary, our data show that even though barbadin affects fpr signaling in a way that resembles actin cytoskeleton-disrupting agents, barbadin lacks direct effects on the reorganization of the actin cytoskeleton in neutrophils and on receptor mobilization from intracellular granule stores. in the present study, we assessed the role of -arrestin in endocytosis of fpr and in receptor down-stream functional responses in human neutrophils using barbadin, an ap -binding inhibitor that blocks the interaction between -arrestin and ap and prevents agonist triggered endocytosis of many gpcrs [ ] . our data show that the ap protein targeted by barbadin indeed is expressed in neutrophils, yet, barbadin did not block fpr endocytosis. these results imply that fpr can be internalized through a -arrestin/ap -independent process, an assumption in line with the observation that only residual endocytosis of fpr occurs in cells lacking -arrestin. interestingly, barbadin treatment potentiated fpr -mediated ros production and resensitization of fpr -desensitized human neutrophils in a manner similar as an inhibitor of actin polymerization (i.e., latrunculin a). however, barbadin did not interfere with other processes in neutrophils involving the actin cytoskeleton machinery. in addition, the potentiating effect of barbadin on fpr -mediated ros production was found to involve biased functional/signaling as neither fpr -promoted intracellular calcium mobilization nor chemotaxis was affected when ap -binding was inhibited by barbadin. previously, barbadin has been shown to affect several gpcr-mediated functions, including hormone secretion mediated by gonadotropin-releasing hormone (gnrh) receptors [ ] , and uptake/entry of influenza a viruses facilitated by short chain fatty acid receptor (ffar ) signaling [ ] . as described, barbadin prevents ap /β-arrestin-mediated receptor endocytosis, which has been deemed to be the canonical molecular mechanism behind the functional effects of this ap inhibitor. however, the data presented in the current study suggest that alternative endocytosis-and β-arrestin-independent mechanisms can mediate the effects by this ap binding inhibitor with regards to fpr expressing human neutrophils. recent data infer that arrestin appears to be involved in non-canonical and endosomal signaling, besides playing roles in receptor desensitization and endocytosis [ , ] . however, the exact functional role of β-arrestin in fpr signaling needs to be further investigated. it has been suggested that polymerized actin rather than -arrestin constitutes the basis for physical separation of g proteins from activated fprs, resulting in termination of signaling and receptor desensitization [ , , ] . the role of the actin cytoskeleton in the regulation of gpcr signaling in neutrophils was originally defined by measurements of ros generated by the phagocyte nadph-oxidase. involvement of the actin cytoskeleton in the termination/desensitization of fpr signaling became evident from experiments in which actin cytoskeleton disrupting agents prolong fpr signaling and have the ability to resensitize the desensitized receptors [ , , ] . we now show that barbadin lacks effect on agonist-induced endocytosis of fpr as examined in several assay systems. intriguingly, these data strongly indicate that fpr can undergo endocytosis through a -arrestin/ap -independent process. this is an internalization pattern shared with receptors for transferrin and endothelin-a, which are both endocytosed independently of β-arrestin and ap , respectively [ ] . although barbadin did not affect fpr internalization, it convincingly potentiated fpr -mediated ros production and promoted resensitization of desensitized fpr s. a similar augmentation of the ros response was also obtained at concentrations of fpr agonists (wkymvm) that do not recruit -arrestin or by fpr agonists (f pal and cmp ) that are very poor in recruiting -arrestin, suggesting that this novel priming effect of barbadin is achieved without -arrestin recruitment. as mentioned above, the effect of barbadin on fpr -mediated ros production resembled the effect induced by actin cytoskeleton-disrupting agents [ , , ] . despite this deviation from the prototypical mode of action for barbadin, several lines of evidence suggest that barbadin does not directly disrupt the actin cytoskeleton. these findings include that in contrast to latrunculin a, barbadin had no effect on (i) the increase in f-actin polymerization induced by the fpr agonist wkymvm, (ii) the actin cytoskeleton-dependent ros production induced during phagocytic uptake of yeast particles was not affected by barbadin, and (iii) the signals downstream of atp-activated p y rs generated only when the actin cytoskeleton has been disrupted. altogether, these observations indicate that barbadin primes neutrophils and resensitizes/reactivates desensitized receptors through a mechanism resembling that of actin cytoskeleton-disruptive agents. however, as compared to actin cytoskeleton-disruptive agents, the effects exerted by barbadin do not appear to involve a direct effect on the integrity of the actin cytoskeleton. at present, the precise mechanism underlying the influence of barbadin on fpr activity is not known, but as barbadin lacks effect on the fpr -induced transient rise in [ca + ]i, a general modulation of downstream signaling of agonist-occupied fpr is unlikely. regarding assembly and activation of the electron-transporting nadph-oxidase, a large number of stimuli (including many gpcr agonists) can induce ros production in neutrophils, but not all signaling pathways that regulate these activation processes have been identified yet. however, it has been established that there is no direct link between gpcr-mediated activation of the oxidase and the transient rise in [ca + ]i [ , , , , ] , which gains further support from the data presented in this study. hence, even although it is difficult to directly correlate nadph-oxidase activity and calcium signaling during receptor reactivation induced by barbadin or latrunculin a, our data corroborate previous studies demonstrating that a rise in [ca + ]i is not a requirement for activation of the nadph-oxidase. furthermore, our data support the notion that the neutrophil nadph-oxidase can be activated in the absence of arrestin recruitment [ - ]. it follows that barbadin is a biased and functionally selective regulator of fpr signaling as it influences ros production by activating nadph-oxidase without affecting calcium mobilization and neutrophil granule secretion. although -arrestin modulated functions are inhibited by barbadin, the ap inhibitor lacks direct effects on receptor-mediated -arrestin recruitment [ ] . our earlier reports have demonstrated that fpr agonists that are potent stimuli in triggering calcium signaling, erk / phosphorylation and ros production, but differ in their ability to recruit -arrestin, also vary in their ability to induce neutrophils chemotaxis [ ] [ ] [ ] . the present study shows that the functionally selective deviation linked to the ability to recruit -arrestin is retained in the presence of barbadin, further supporting the proposed mode of action of barbadin in that it lacks a direct effect on receptor mediated -arrestin recruitment [ ] . the observation that barbadin potentiates fpr -mediated ros production, no matter whether this was caused by the -arrestin recruiting wkymvm peptide or fpr agonists that are very poor in recruiting -arrestin, suggests that the effects of barbadin on fpr -mediated ros production is not dependent on -arrestin. several in vitro as well as in vivo processes potentiate fpr-mediated ros production, and increased surface receptor expression as a result of granule secretion has been suggested as an important mechanism underlying the potentiation [ , , ] . however, this is not the mechanism involved in the priming effect of barbadin, demonstrated by its inability to induce the mobilization of granules. this conclusion is supported by the fact that while granule mobilization is an irreversible process the effects of barbadin are reversible. thus, the mechanism by which barbadin potentiates fpr -mediated ros remains to be elucidated. given that barbadin binds to ap to prevent -arrestin binding, the role of ap in the observed effects on ros priming needs to be further investigated. with respect to the role of ap , it is interesting to note that a comparison between ec values reveals that the potency of barbadin mediated augmentation of ros production in neutrophils is the same as that found for its inhibition of the -arrestin-ap interaction. this suggests that the barbadin-mediated effect on ros production could be a result of its action on ap (this study and [ ] ). however, other target proteins including other binding partners for ap , such as ap , arh and scr [ ] can, at this point, not be excluded until the modulating effect (if any) of barbadin on these ap binding molecules has been be determined. in summary, this study demonstrates some novel effects of the ap binding compound barbadin. although barbadin did not affect agonist-induced endocytosis of fpr , a process shown to be independent of whether the agonist recruits -arrestin or not, barbadin both increased fpr agonist induced ros production, and resensitized agonist-desensitized fpr to produce ros. notably, these effects of barbadin on fpr also proved independent of whether the agonist recruited -arrestin or not. the effect of barbadin on fpr induced neutrophil ros production is very similar to the actin cytoskeleton-disrupting agent latrunculin a, albeit without altering other neutrophil functions regulated by a dynamic polymerization of the actin cytoskeleton. elucidation of the precise mechanism(s) of barbadin regarding its priming effect on the fpr -mediated ros production in neutrophils would lead to an increased understanding of the underlying molecular mechanisms regulating inflammatory reactions that are dependent on redox reactions. barbadin and structurally related analogs of this ap inhibitor are expected to serve as useful molecular tools for further mechanistic studies of gpcr regulation in neutrophils. neutrophil recruitment and function in health and inflammation formyl peptide receptor orchestrates mucosal protection against citrobacter rodentium infection formyl-peptide receptors in infection, inflammation, and cancer formylpeptide receptor- contributes to colonic epithelial homeostasis, inflammation, and tumorigenesis measurement of respiratory burst products, released or retained, during activation of professional phagocytes basic characteristics of the neutrophil receptors that recognize formylated peptides, a danger-associated molecular pattern generated by bacteria and mitochondria international union of basic and clinical pharmacology. lxxiii. nomenclature for the formyl peptide receptor (fpr) family dual modulation of formyl peptide receptor by aspirin-triggered lipoxin contributes to its anti-inflammatory activity neutrophil chemoattractant receptors and the membrane skeleton direct or c a-induced activation of heterotrimeric gi proteins in human neutrophils is associated with interaction between formyl peptide receptors and the cytoskeleton similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor gpr and the short chain fatty acid receptor ffa r reactivation of formyl peptide receptors triggers the neutrophil nadph-oxidase but not a transient rise in intracellular calcium a new inhibitor of the betaarrestin/ap endocytic complex reveals interplay between gpcr internalization and signalling the g protein-coupled receptor ffar promotes internalization during influenza a virus entry combining elements from two antagonists of formyl peptide receptor generates more potent peptidomimetic antagonists studies on acid stability and solid-phase block synthesis of peptide-peptoid hybrids: ligands for formyl peptide receptors monitoring g protein-coupled receptor and beta-arrestin trafficking in live cells using enhanced bystander bret isolation of mononuclear cells and granulocytes from human blood. isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at g isolation of lymphocytes, granulocytes and macrophages real-time trafficking and signaling of the glucagon-like peptide- receptor translating in vitro ligand bias into in vivo efficacy a methodological approach to studies of desensitization of the formyl peptide receptor: role of the read out system, reactive oxygen species and the specific agonist used to trigger neutrophils lipopolysaccharide-induced granule mobilization and priming of the neutrophil response to helicobacter pylori peptide hp( - ), which activates formyl peptide receptor-like galectin- activates the nadph-oxidase in exudated but not peripheral blood neutrophils the synthetic peptide trp-lys-tyr-met-val-met-nh specifically activates neutrophils through fprl /lipoxin a receptors and is an agonist for the orphan monocyte-expressed chemoattractant receptor fprl p y receptor signaling in neutrophils is regulated from inside by a novel cytoskeleton-dependent mechanism an intact cytoskeleton is required for prolonged respiratory burst activity during neutrophil phagocytosis multiple phenotypic changes define neutrophil priming priming and de-priming of neutrophil responses in vitro and in vivo granulopoiesis and granules of human neutrophils beta-arrestin-dependent signaling in gnrh control of hormone secretion from goldfish gonadotrophs and somatotrophs gpcr-g proteinbeta-arrestin super-complex mediates sustained g protein signaling reactivation of desensitized formyl peptide receptors by platelet activating factor: a novel receptor cross talk mechanism regulating neutrophil superoxide anion production neutrophil signaling that challenges dogmata of g protein-coupled receptor regulated functions after  min stimulation, barbadin ( m) or la ( ng/ml) was added (indicated by an arrow). (e-h) analysis of fpr and cd b surface expression was performed by flow cytometry. (e-f) neutrophils were incubated in the absence (buffer) and presence of barbadin ( m) at °c for different time points as indicated on the x-axis, prior fixation and staining with an anti-fpr antibody. (g-h) neutrophils were incubated in the absence (buffer, min) and presence of barbadin ( m; min) or fmlf ( nm; min) at °c prior staining with an anti-cd b antibody key: cord- -yl emjef authors: moro, loredana title: mitochondria at the crossroads of physiology and pathology date: - - journal: j clin med doi: . /jcm sha: doc_id: cord_uid: yl emjef mitochondria play a crucial role in cell life and death by regulating bioenergetic and biosynthetic pathways. they are able to adapt rapidly to different microenvironmental stressors by accommodating the metabolic and biosynthetic needs of the cell. mounting evidence places mitochondrial dysfunction at the core of several diseases, notably in the context of pathologies of the cardiovascular and central nervous system. in addition, mutations in some mitochondrial proteins are bona fide cancer drivers. better understanding of the functions of these multifaceted organelles and their components may finetune our knowledge on the molecular bases of certain diseases and suggest new therapeutic avenues. mitochondria are semi-autonomous organelles with a double membrane system, namely the inner and the outer mitochondrial membrane that delimit the intermembrane space. the inner mitochondrial membrane demarcates the matrix, a viscous microenvironment that contains several enzymes catalyzing a plethora of anabolic and catabolic reactions. mitochondria contain their own genome, the mitochondrial dna (mtdna), a circular double-stranded dna molecule of , bp in humans, which encodes only mitochondrial proteins belonging to the electron transport chain (etc), transfer rnas and ribosomal rnas needed to carry out the mitochondrial protein synthesis. all the other mitochondrial components are encoded by the nuclear genome. mitochondria are the energy powerhouses of the cell, being responsible for % of energy production in the form of atp by coupling the flux of electrons throughout the mitochondrial respiratory complexes i-iv with oxidative phosphorylation (oxphos). in brief, complete oxidation of nutrients through the tricarboxylic acid cycle (tca) within mitochondria produces reduced coenzymes (nadh, fadh ) that act as electron donors. the flux of electrons through the mitochondrial respiratory chain complexes produces an electrochemical gradient used by the mitochondrial respiratory complex v to generate atp. notably, the function of mitochondria in cell physiology goes beyond their role as energy producers and metabolic regulators. indeed, these multifaceted organelles play a pivotal role in the modulation of cell death pathways and intracellular signaling [ ] . the etc is also the main cellular source of reactive oxygen species (ros), owing to an incomplete reduction of oxygen by complex i and complex iii. mitochondrial ros production can lead to oxidative damage to proteins, membranes and dna, thus impairing the ability of mitochondria to carry out their biosynthetic and catabolic reactions, including the tca cycle, heme synthesis, fatty acid oxidation, the urea cycle and amino acid metabolism [ ] . mitochondrial oxidative damage can also promote permeabilization of the mitochondrial outer membrane (momp), resulting in release of intermembrane space proteins, such as cytochrome c, and activation of the mitochondrial apoptotic pathway. furthermore, mitochondrial ros production promotes the opening of the mitochondrial permeability transition pore (mptp), leading to permeabilization of the inner mitochondrial membrane to small molecules in pathological conditions, such as during ischaemia (loss of blood flow) and subsequent reperfusion [ ] . two mitochondria quality control mechanisms are in place to meet the functional needs of any given cell under different physiological and pathological conditions: (a) mitochondrial biogenesis, fusion and fission [ ] [ ] [ ] ; (b) mitophagy [ , ] . the first mechanism is a balanced process that allows maintenance of the physiological mitochondrial homeostasis when cells face metabolic or microenvironmental stresses [ ] . mitochondrial fission guarantees an adequate distribution of mitochondria in dividing cells. mitochondrial fusion allows complementation between dysfunctional mitochondria within the cell to maximize mitochondrial performance in response to stress. three gtpases, mitofusin (mfn ), mfn , and optic atrophy (opa ), are primarily involved in the regulation of mitochondrial fusion. instead, mitochondrial fission is mainly controlled by the gtpase dynamin-related protein (drp ) [ ] . disruption of the balance between fusion and fission is associated with neurodegenerative diseases, such as parkinson's, and cancer [ , ] . the second mechanism, mitophagy, is a specific form of autophagy that removes damaged mitochondria and reduces the mitochondrial mass upon microenvironmental stresses, such as hypoxia and nutrient starvation, promoting cell survival [ ] . mitophagy dysregulation has been implicated in cancer development and progression [ ] , neurodegeneration [ ] and cardiovascular diseases [ ] . mitochondrial dysfunction can lead to an array of diseases. depending on the nature of the defect leading to mitochondrial dysfunction, primary and secondary mitochondrial diseases can be distinguished. primary mitochondrial diseases develop as a consequence of germline mutations in mtdna and/or nuclear dna genes that encode proteins affecting mitochondrial functionality and energy production, including etc proteins and proteins involved in mtdna replication, such as polg. the first primary mitochondrial disease was described in [ ] and involved a -year-old woman displaying excessive perspiration, polyphagia, polydipsia without polyuria, asthenia and decreased body weight, symptoms that started when she was seven years old. in addition, her basal metabolic rate was + %, and she presented with creatinuria, myopathy and pathological cardiomyogram. she was diagnosed with a disorder of the enzymatic organization of the mitochondria. studies with mitochondria isolated from the skeletal muscle of this hypermetabolic patient revealed oxphos uncoupling [ ] . since then, a range of primary mitochondrial diseases has been described (reviewed in [ ] ). secondary mitochondrial defects can be caused by germline mutations in genes not involved in respiration/oxidative phosphorylation or can be acquired during the lifetime upon environmental insults. notably, environmental stress can induce mtdna alterations leading to mitochondrial dysfunction during aging, inflammatory response, etc. [ , ] . from a pathological point of view, primary and secondary mitochondrial diseases can cause very similar symptoms, sometimes making diagnosis difficult. at the molecular level, mitochondrial dysfunction can affect the levels of key intracellular signaling regulators, such as ros and ca + , that can be transmitted to the nucleus (mitochondria-to-nucleus signaling or retrograde signaling) resulting in changes in gene expression and modulation of a range of cellular functions [ , [ ] [ ] [ ] . in addition, the release of mtdna and peptides from the mitochondrial matrix can activate an immune response that promotes a pro-inflammatory cascade [ ] . mitochondrial metabolites can also act as signaling molecules and epigenetic modulators. in this context, citrate, an intermediate of the tca cycle, represents the major source of acetyl-coa for protein acetylation, a co-and post-translational modification that regulates protein levels and intracellular signaling in physiological and pathological conditions [ ] . emerging data have also provided new evidences of connections between mitochondrial dynamics and physical contacts among mitochondria and the endoplasmic reticulum (er), known as mitochondrial-associated er membranes (mams), which can finetune the mechanisms of regulation of energy production, ca + homeostasis, survival and apoptosis [ ] . here, a synthetic overview of the role of mitochondria in specific physiopathological conditions is provided ( figure ). cardiovascular diseases are a leading cause of death worldwide. this class of diseases comprises several pathologies, including ischemic heart disease, peripheral vascular disease, cardiac arrest, heart failure, cardiomyopathies, hypertension, atherosclerosis, and arrhythmia. mitochondria have been involved at various degrees in the pathological aspects of these diseases. notably, mitochondrial dysfunction of muscle cells represents a key event in the prognosis of peripheral arterial disease. reduced oxphos activity due to etc impairment increases ros levels and ca + release from mitochondria, causing apoptosis [ ] . however, if ros levels remain below a threshold, the cells activate a defense program involving production of antioxidants and increased mitochondrial biogenesis. these mechanisms, known as mitohormesis, can limit the damage caused by repeated cycles of ischemia-reperfusion in peripheral arterial disease [ ] . pharmacological treatments that can improve mitohormesis might be a promising therapeutic approach for peripheral arterial disease and other cardiovascular diseases. disruption of mitophagy also exacerbates the development of cardiovascular diseases [ ] . growing evidence indicates that the pharmacological targeting of the mitochondria with drugs/natural compounds able to modulate mitophagy can ameliorate cardiovascular disorders in patients and be cardioprotective [ , ] . future studies that aim at a better understanding the pathogenesis of some cardiovascular diseases are crucial to develop mitochondria-targeting drugs in the clinic. inflammation is a complex, protective body response to infections and tissue damage. the inflammatory response signals the immune system to repair damaged tissue and defend against pathogens (viruses, bacteria, etc.) or other harmful stimuli through secretion of specific mediators. however, when inflammation persists, it may drive various diseases and tissue damage. mitochondrial-derived ros play a key role in the inflammatory response. notably, mitochondria are considered the main drivers of the nlrp (nod-, lrr-and pyrin domain-containing ) inflammasome [ ] [ ] [ ] [ ] , representing a central hub that controls innate immunity and response to inflammation. among various inflammatory conditions, mitochondria are involved in the hyper-inflammatory response, also reported as cytokine storm, caused by the sars-cov- (covid- ) respiratory cardiovascular diseases are a leading cause of death worldwide. this class of diseases comprises several pathologies, including ischemic heart disease, peripheral vascular disease, cardiac arrest, heart failure, cardiomyopathies, hypertension, atherosclerosis, and arrhythmia. mitochondria have been involved at various degrees in the pathological aspects of these diseases. notably, mitochondrial dysfunction of muscle cells represents a key event in the prognosis of peripheral arterial disease. reduced oxphos activity due to etc impairment increases ros levels and ca + release from mitochondria, causing apoptosis [ ] . however, if ros levels remain below a threshold, the cells activate a defense program involving production of antioxidants and increased mitochondrial biogenesis. these mechanisms, known as mitohormesis, can limit the damage caused by repeated cycles of ischemia-reperfusion in peripheral arterial disease [ ] . pharmacological treatments that can improve mitohormesis might be a promising therapeutic approach for peripheral arterial disease and other cardiovascular diseases. disruption of mitophagy also exacerbates the development of cardiovascular diseases [ ] . growing evidence indicates that the pharmacological targeting of the mitochondria with drugs/natural compounds able to modulate mitophagy can ameliorate cardiovascular disorders in patients and be cardioprotective [ , ] . future studies that aim at a better understanding the pathogenesis of some cardiovascular diseases are crucial to develop mitochondria-targeting drugs in the clinic. inflammation is a complex, protective body response to infections and tissue damage. the inflammatory response signals the immune system to repair damaged tissue and defend against pathogens (viruses, bacteria, etc.) or other harmful stimuli through secretion of specific mediators. however, when inflammation persists, it may drive various diseases and tissue damage. mitochondrial-derived ros play a key role in the inflammatory response. notably, mitochondria are considered the main drivers of the nlrp (nod-, lrr-and pyrin domain-containing ) inflammasome [ ] [ ] [ ] [ ] , representing a central hub that controls innate immunity and response to inflammation. among various inflammatory conditions, mitochondria are involved in the hyper-inflammatory response, also reported as cytokine storm, caused by the sars-cov- (covid- ) respiratory infection ( [ ] and references therein). when macrophages and other immune cells detect viruses, they start secreting cytokines and chemokines to communicate with other immune cells [ ] . strikingly, wuhan's covid- patients with severe clinical symptoms requiring icu admission displayed higher levels of the cytokines/chemokines ccl , tnf-α and cxcl compared to individuals with less severe symptoms [ ] . the release of large quantities of pro-inflammatory cytokines and chemokines by overdriven immune effector cells sustains an aberrant systemic inflammatory response that results in the immune system attacking the body, which in turn causes the acute respiratory distress syndrome [ ] . immune cells under a hyper-inflammatory state metabolically adapt to this stress condition by favoring aerobic glycolysis over oxphos for energy production. this metabolic rewiring allows macrophages to become more phagocytic and favors anabolic reactions for the synthesis and secretion of cytokines and chemokines in a vicious cycle ( [ ] and references therein). side by side, many biosynthetic reactions occurring in mitochondria of hyper-activated macrophages are inhibited as a consequence of oxphos and tca cycle inhibition. melatonin's synthesis is among these reactions: acetyl-coa, a cofactor in the rate-limiting reaction for melatonin synthesis, lacks due to the tca cycle inhibition [ ] . thus, melatonin cannot be synthetized. notably, melatonin is a potent anti-inflammatory and anti-oxidant and its administration to covid- patients has been recently proposed as potential adjuvant treatment strategy to reduce the severity of the covid- pandemic [ ] [ ] [ ] . though clinical evidences are not yet available, several scientific data supports the potential utility of melatonin to attenuate the worst symptoms of covid- infection [ , ] . mitochondrial dysfunction has long been recognized as a driver of the aging process. early studies have linked accumulation of mitochondrial dna mutations and the concomitant decline in etc and oxphos activity to aging [ , ] . furthermore, genetic studies in mice support a causal relation between mtdna depletion and aging [ ] . recent evidences have confirmed that healthy centenarians retain more "intact" mtdna copies than old people and frail centenarians [ ] , suggesting that "healthy" mtdna is a hallmark of healthy aging. besides the mtdna status, activation of mitochondria-to-nucleus signaling pathways, particularly the mitochondrial unfolded protein response (upr mt ), has been implicated in aging. upr mt activation promotes transcription of several nuclear genes, such as those encoding antioxidant proteins and enzymes, which support survival, gain of the mitochondrial functionality and, thus, longevity and lifespan [ ] . it should be noted that if a heteroplasmic mtdna pool is present, upr mt activation could exacerbate mitochondrial dysfunction as it may lead to accumulation of mutant mtdna [ ] . alterations in the removal of damaged mitochondria through mitophagy have also been implicated in aging. mitophagy markedly decreases during aging in mammalian tissues and organs [ , ] and this may be responsible for the known accumulation of damaged mitochondria in aging tissues. notably, genetic manipulations in c. elegans that increase mitophagy also extend the organismal lifespan [ ] , strengthening the connection between altered mitophagy and aging. neurodegenerative diseases are characterized by changes in mitochondrial morphology and biochemical activity. alzheimer's (ad) and parkinson's (pd) disease are the most diffuse neurodegenerative illnesses among older adults. brain cells from ad and pd patients show reduced respiratory activity and mitochondrial biogenesis [ , ] . a prominent pathological feature of ad is the impaired cerebral glucose metabolism, which is reduced by % in the early stages, preceding neurological impairment and atrophy, and further declines in the late stages of the disease [ ] . the decrease in glucose metabolism is associated with reduced expression and activity of mitochondrial enzymes, including pyruvate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, three enzymes of the tca cycle [ ] . in addition, reduced activity of the mitochondrial respiratory complexes i, ii, iii and iv has also been documented [ ] . somatic mutations in the mitochondrial genome have been detected in postmortem brain tissue from ad patients, at levels higher than in healthy brains [ ] . these mutations may not only affect the etc but also trigger other neuropathological consequences, such as increased ros production and oxidative stress in neurons and promotion of amyloidogenic processing of the amyloid precursor protein. mitophagy is also diminished in ad's neurons, and this may contribute to the etiopathogenesis of ad. indeed, mitophagy was able to prevent or reverse the cognitive impairment in several ad models [ ] , confirming the critical involvement of mitochondria in ad. mutations in nuclear genes encoding mitochondrial proteins important for the proper function of mitochondria have been directly linked to pd. notably, mutations in proteins involved in mitochondrial quality control, such as pink , parkin and lrrk , are a frequent cause of monogenic pd [ ] . loss or impaired functionality of these proteins results in mitochondrial fragmentation, dysregulation of calcium homeostasis and changes in mitochondria-endoplasmic reticulum contact sites (mercs). recently, mutations in miro , a protein important for the regulation of the structure and function of mercs, have been causally linked to pd establishing that variants in the gene encoding for miro represent rare genetic risk factors for neurodegenerative diseases like pd ( [ ] and references therein). although there is no doubt about the involvement of mitochondrial dysfunction in ad and pd, still more research is required to identify therapeutic targets that could improve mitochondrial activity and reduce oxidative stress in neurons in the early stages of these neurodegenerative diseases. future studies should be aimed at investigating the chronological sequence of molecular events involved in the pathogenesis of these diseases. further investigations are also needed to assess whether mitochondrial dysfunction represents a primary cause of ad or a consequence of other molecular/genetic events. mitochondrial dysfunction has been involved in different aspects of the pathogenesis of cancer, from the early steps of cancer development to cancer progression to a metastatic phenotype, and resistance to anti-cancer drugs [ , , ] . in this context, mutations in three tca cycle enzymes, namely succinate dehydrogenase, fumarate hydratase and isocitrate dehydrogenase, have been shown to play a causal role in carcinogenesis [ , ] , thus providing compelling evidence for the involvement of mitochondrial metabolic alterations as cancer drivers. indeed, mutations in succinate dehydrogenase predispose to hereditary paragangliomas, pheochromocytomas, neuroblastomas, gastrointestinal tumors, renal cell cancers and thyroid tumors [ ] . sporadic and hereditary mutations of fumarate hydratase trigger accumulation of an oncogenic metabolite, i.e., fumarate, that favors development of hereditary leiomyomatosis and renal cell carcinoma, ewing sarcoma and osteosarcoma, adrenocortical carcinoma, pheochromocytoma, glioma, neuroblastoma, paraganglioma, and ependymoma [ ] . mutations in isocitrate dehydrogenase are only somatic and have been detected in about % of patients with acute myeloid leukemia or angioimmunoblastic t-cell lymphoma, and at lower frequencies in patients with thyroid, prostate, colorectal cancer and b-cell acute lymphoblastic leukemia [ , ] . besides mutations in nuclear-encoded mitochondrial proteins, mutations in mtdna-encoded proteins have also been implicated in the pathogenesis of cancer. the spectrum of somatic mtdna mutations varies among different tissues, and increasing evidence shows that the load of mtdna mutations could have prognostic value. the majority of cancer-related mtdna mutations have been found in prostate cancer, with a total of more than unique somatic mtdna mutations associated with this cancer [ ] . there is increasing evidence that mtdna mutations/depletion may favor cancer progression to a metastatic and drug-resistant phenotype through increased production of ros and/or activation of a mitochondria-to-nucleus signaling that leads to expression of pro-metastatic and pro-survival nuclear genes [ , , [ ] [ ] [ ] . although mtdna damage may not be the first driver of cancer progression, it is likely that it represents a "supporter" event that facilitates and accelerates different steps of the metastatic cascade, probably within a precise time window that remains to be identified. mitochondrial dysfunction is implicated in several pathological conditions, ranging from neurodegenerative and cardiovascular diseases, to aging, cancer and inflammation. each of these conditions shows a peculiar involvement of mitochondria. for example, up to % of pd patients show a defect in miro function, because this protein, located on the mitochondrial surface, fails to detach from depolarized mitochondria resulting in defective mitochondrial locomotion and clearance by mitophagy [ ] . these new results suggest that miro -based therapeutic strategies may provide new avenues to a personalized medicine for pd. the role of mitochondrial dysfunction in other diseases is still somehow controversial. in some cases, it may represent a driver event, like for mutations in the tca cycle enzymes succinate dehydrogenase, fumarate hydratase and isocitrate dehydrogenase that predispose to certain types of tumors. in other cases, a transient mitochondrial dysfunction may support a metabolic rewiring needed by the cells to adapt and survive to microenvironmental stressors. the author declares no conflict of interest. how mitochondria produce reactive oxygen species the 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promotes anoikis resistance and invasion through activation of pi k/akt mitochondrial dna depletion sensitizes cancer cells to parp inhibitors by translational and post-translational repression of brca miro marks parkinson's disease subset and miro reducer rescues neuron loss in parkinson's models this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -ftlb b authors: mroczek, seweryn; kufel, joanna title: apoptotic signals induce specific degradation of ribosomal rna in yeast date: - - journal: nucleic acids res doi: . /nar/gkm sha: doc_id: cord_uid: ftlb b organisms exposed to reactive oxygen species, generated endogenously during respiration or by environmental conditions, undergo oxidative stress. stress response can either repair the damage or activate one of the programmed cell death (pcd) mechanisms, for example apoptosis, and finally end in cell death. one striking characteristic, which accompanies apoptosis in both vertebrates and yeast, is a fragmentation of cellular dna and mammalian apoptosis is often associated with degradation of different rnas. we show that in yeast exposed to stimuli known to induce apoptosis, such as hydrogen peroxide, acetic acid, hyperosmotic stress and ageing, two large subunit ribosomal rnas, s and . s, became extensively degraded with accumulation of specific intermediates that differ slightly depending on cell death conditions. this process is most likely endonucleolytic, is correlated with stress response, and depends on the mitochondrial respiratory status: rrna is less susceptible to degradation in respiring cells with functional defence against oxidative stress. in addition, rna fragmentation is independent of two yeast apoptotic factors, metacaspase yca and apoptosis-inducing factor aif , but it relies on the apoptotic chromatin condensation induced by histone h b modifications. these data describe a novel phenotype for certain stress- and ageing-related pcd pathways in yeast. gene expression in all organisms is regulated at multiple levels, including transcription initiation, mrna stability and turnover, translation and protein degradation. not surprisingly, rapid changes in cell metabolism and most responses to environmental stimuli involve significant flux of many cellular rnas, of which mrna transcriptome profiles have been most extensively studied to date ( , ) . however, also stable rnas, such as ribosomal, transfer, nuclear and nucleolar rnas (rrnas, trnas, snrnas and snornas), are likely to undergo specific transformations in altered conditions. it has been demonstrated that certain pathways of cell death are accompanied by the destruction of nucleic acids. for example, in metazoans the programmed cell death (pcd) called apoptosis, in addition to irreversible dna damage, which is considered an apoptotic hallmark ( ) , also involves specific cleavage of several rna species, including s rrna, u snrna or ro rnp-associated y rnas ( ) . it was proposed that rrna degradation could contribute to cell autodestruction, whereas degradation of anti-apoptotic factors mrnas would accelerate apoptosis. in higher eukaryotes, rna cleavage is probably carried out by rnase l, a -oligoadenylate-dependent endoribonuclease, which functions in rna decay during the interferon-induced response to viral infection, and whose activation in animal cells causes apoptosis ( ) ( ) ( ) . however, rnase l-independent cleavage of s rrna in virus infected cells has also been reported ( ) . the occurrence of apoptosis was assumed to be limited to metazoans, where elimination of single cells does not kill the whole organism. nevertheless, recent studies revealed the existence of cell death pathway in yeast, saccharomyces cerevisiae, and other unicellular eukaryotes, with typical hallmarks of apoptosis: dna fragmentation, externalization of phosphatidyl serine and chromatin condensation. pcd in yeast is triggered by several different stimuli, including ageing, expression of mammalian pro-apoptotic proteins, exposure to low doses of h o , acetic acid, hyperosmotic stress and mating-type a-factor pheromone ( ) ( ) ( ) . unicellular organisms are believed to undergo pcd for a variety of reasons, including elimination of old, infected and damaged cells in growth-limiting conditions for better survival of the remaining population, and adaptation of the more fit subpopulation to the ever changing and challenging environment ( ) . orthologues of core regulators of mammalian apoptosis, such as the caspase-related protease yca , a homologue of mammalian pro-apoptotic mitochondrial serine protease htra (nma ), a yeast endog nuclease nuc , apoptosis inducing factor (aif ) involved in chromatin condensation, aif-homologous mitochondrionassociated inducer of death (amid) ndi and an inhibitor of apoptosis (iap) bir , are conserved in yeast ( ) ( ) ( ) ( ) ( ) ( ) . in addition, in both human and yeast cells, histone modifications (histone h b phosphorylation at serine in human and at serine in yeast and h b deacetylation at lysine in yeast) play an important role in apoptotic chromatin condensation and cell death ( ) ( ) ( ) . however, several apoptotic factors are missing in yeast, including the bcl- /bax family and the apoptosis protease activator factor apaf- . also, there is no good homologue of rnase l to execute possible rna degradation. nevertheless, yeast apoptosis has recently been shown to be activated in mrna decay mutants (dcp and lsm) ( , ) , supporting the notion that rna metabolism and apoptosis are linked. pcd occurs via a number of different mechanisms, e.g. caspase-dependent or independent, however, in all eukaryotes it is thought to be correlated with high levels of reactive oxygen species (ros). ros can either be generated exogenously through respiration or originate from exogenous sources such as exposure to hydrogen peroxide (h o ), superoxide anions or hydroxyl radicals. excessive ros results in damage of cellular components (dna, lipids and proteins), cell cycle arrest, ageing and finally cell death ( ) . cells have developed a complex network of defence mechanisms, both enzymatic and non-enzymatic, against adverse consequences of oxidative stress ( ) . non-enzymatic system comprises a set of small molecules acting as ros scavengers (e.g. glutathione, thioredoxin, glutaredoxin and ascorbic acid), whereas enzymatic system eliminates oxygen radicals by the action of specialized cytosolic or mitochondrial enzymes (e.g. catalases, superoxide dismutases, glutathione peroxidases and thioredoxin peroxidases) ( ) . most genes encoding components of these systems are induced in response to oxidative stress and are under transcriptional control of specific factors, for example yap , msn /msn and skn in budding yeast ( ) . however, it appears that there is no general oxidative stress response. in s. cerevisiae, different response pathways are triggered by specific oxidants and different genes are involved in maintaining efficient cellular resistance to various sources of ros ( , ) . interestingly, recent genomic approaches to identify these genes showed that strains lacking proteins which function in rna metabolism were oversensitive to oxidative stress ( , ). these included genes encoding rrna helicases (dbp , dbp ), rrna processing factors (nop , nsr ), mrna deadenylases (ccr , pop ) and several mitochondrial rna splicing components ( , ) . this indicates that rna processing and degradation may have a role in cellular response to ros. in this study, we examined the effects of elevated ros levels generated by oxidative stress, ageing and other apoptotic-inducing treatments on the status of ribosomal rna in yeast s. cerevisiae and we have shown that mature rrnas become specifically fragmented as a result of the cell response to these conditions. rna degradation coincides with fragmentation of chromosomal dna but occurs considerably earlier and most likely upstream of the activation of major apoptotic regulators, yca and aif . the existence of this mechanism underscores the role of gene expression, namely rrna turnover, in regulating certain pathways of cell death, in this case most likely through destruction of ribosomes and subsequent inhibition of translation in the early stages of apoptosis. yeast strains and plasmids used in this work are listed in supplementary table s . the transformation procedure was as described ( ) . strains were grown at c either in ypd, ypgal or ypgly medium ( % yeast extract, % bacto-peptone, % glucose or % galactose or % glycerol, respectively) or in synthetic complete medium (sc, . % yeast nitrogen base, % glucose or % galactose, supplemented with required amount of amino acids and nucleotide bases). strains w - a-bax, w - a-bcl-x l , w -rdna were grown in sc media without leucine, tryptophan or uracil, respectively. yeast cultures in early logarithmic phase (od $ . ) were stressed with h o ( . - mm), menadione ( . - . mm), cumene hydroperoxide (chp, . - . mm), tert-butyl hydroperoxide (t-bhp, - mm), paraquat ( . - mm), diamide ( . - mm) and linoleic acid hydroperoxide (loaooh, . - . mm) for min (all reagents from sigma). treatment with acetic acid stress was performed as described ( , ) . cells were grown in sc media to early exponential stage, shifted to sc media, ph = , and treated with mm acetic acid (sigma) for min. hyperosmotic shock was achieved by growth of exponential cells in sc complete media containing % (wt/wt) glucose (fluka) or % (wt/wt) sorbitol and % glucose (sigma) for - h ( ). chronological ageing was performed by constant growth of yeast cultures in sc complete medium for - days ( ) . for expression of murine bax or bcl-x l proteins, cells were grown to early exponential phase in sc-leu or sc-trp, respectively. expression of murine bax was induced by shifting the cells grown to early exponential phase in sc-leu medium containing glucose to sc-leu medium containing galactose for h. pre-treatment with ascorbic acid ( mm, fluka) and respiratory chain inhibitors oligomycin a ( . mg/ml, sigma) and sodium azide ( . mm, sigma) was performed for or min prior to treatment with h o . for inhibition of protein synthesis, cells were treated with mg/ml or mg/ml of cycloheximide (sigma) for min. cell fixation was achieved by addition of formaldehyde (final concentration %) or etoh (final concentration %) to exponentially growing yeasts and incubation in room temperature for min or min, respectively. formaldehyde was quenched by addition of glycine to the final concentration of . m for min. following the removal of fixation agents, cells were exposed to h o for min as described earlier. preparation of samples and analysis of chromosomal dna fragmentation by pulsed field gel electrophoresis (pfge) was performed exactly as described ( ) . pfge was conducted in a chef-driii chiller system (bio-rad). one percent agarose gels were run in . % tris borate-edta buffer at c with an angle of with a voltage of v/cm and switch times of - s for h. gels stained in ethidium bromide were analysed after destaining using syngene gene genius bioimaging system. rna extraction, northern hybridization and primer extension were essentially as described ( , ) . lowmolecular weight rnas were separated on % acrylamide gels containing m urea and transferred to a hybond n+ membrane by electrotransfer. high-molecular-weight rnas were analysed on . % agarose gels and transferred by capillary elution. oligonucletides used for rna hybridization and primer extension (w and w ) are listed in supplementary table s . quantification of northern blots was performed using a storm phosohorimager and imagequant software (molecular dynamics). dideoxy-dna sequencing was performed on pcr-templates prepared from genomic yeast dna using the same primers as for primer extension (w and w ) and a fmolseq kit (promega) according to manufacturer's instructions. the race assay was carried out on total rna ( mg) isolated from untreated cells and treated with mm h o . dna 'adaptor' oligonucleotide (w ) carrying aminolinker at the -end was ligated with the -end of total rna using u of t rna ligase (neb). ligation was performed in the presence of % peg (sigma) at c. rna was extracted with phenol: chloroform: isoamyl alcohol (v/v : : ), precipitated and used as a template for cdna synthesis using w primer complementary to the anchor sequence and the enhanced avian hs rt-pcr kit (sigma) according to manufacturer's instructions. cdna was amplified using primers w and w , the resulting pcr product was gel purified, cloned into pgem-t easy vector and sequenced using primer w . rnase h cleavage was performed essentially as described ( ) . samples of mg of total rna were annealed with ng of oligonucleotide complementary to the specific regions within rrna at c for min and digested with . u rnase h at c for h. for detection, samples were separated on polyacrylamide gels and analysed by northern hybridization using probes located upstream of rhase h cleavage. to examine the existence of the rna degradation pathway in yeast under oxidative stress, we have performed treatments with low doses of oxidative agents generating different ros. these included the inorganic h o (concentrations . - mm), superoxide-generating menadione (concentrations . - . mm), paraquat (concentrations . - mm), thiol oxidant diamide (concentrations . - mm), organic chp (concentrations . - . mm), t-bhp (concentrations - mm) and a loaooh (concentrations . - . mm). such concentrations of oxidants result in - % of cell death ( , , ) . total rna from wild-type w or by cells grown to early exponential phase (od = . ) in ypd media and treated with chemical compounds for min was separated on . % denaturing agarose/formaldehyde gels and analysed by northern hybridization using a probe complementary to the -end of mature s rrna (starting at position + ). this rna species was chosen in the first place, since effects on s rrna have been observed in apoptotic mammalian cells ( , , ) . on treatment with two oxidants, h o and menadione, extensive decay of the mature s and accumulation of specific degradation products was observed, whereas little or no degradation occurred for other chemicals tested ( figure a , data shown only for w strain and treatment with h o , menadione, chp and t-bhp). this indicates that rna cleavage accompanies some oxidative stress pathways, as it is known that different oxidants elicit specific cellular responses that, though partly overlapping, induce different groups of genes and require individual sets of specialized defence functions to maintain resistance ( , , ) . in addition to the s rrna, other rrna species were also probed for undergoing specific decay. after treatment with h o , rna damage with accumulation of characteristic breakdown products occurred for . s and, to a much lesser extent, for s, but not for s, (figure b and c; data not shown). in the case of s, hardly any degradation intermediates were detected, there was some decay of the mature rna, however, it was approximately . to -fold weaker than for the mature s. therefore, we conclude that mainly the two components of the large ribosomal subunit, s and . s, undergo specific apoptotic degradation. the oxidants utilized, except for h o , had not been tested for apoptotic effects in yeast. one of the most recognized apoptotic markers is fragmentation of chromosomal dna. internucleosomal dna laddering, typical for mammalian apoptosis, has not been detected during pcd in yeast; nevertheless, a higher order chromatin fragmentation to segments of several hundred kilobases also occurs in yeast ( , ) . this dna breakdown can be monitored either by the terminal deoxynucleotidyl transferase dutp nick-end labelling (tunel) assay or by using pfge of genomic dna. the latter approach was applied to verify which oxidative agents lead to apoptotic phenotypes. chromosomal dna from cells treated with h o ( mm), menadione ( . mm), chp ( . mm), paraquat ( mm), diamid ( mm) and t-bhp ( mm) for min was analysed using pfge ( figure d ). clear dna degradation was observed only for cells exposed to h o and menadione, other treatments did not result in a visible apoptotic fragmentation. this is in a striking agreement with rrna degradation that occurred only in h o -and menadione-treated cells. this strongly indicates that rrna decay phenotype can be related to apoptosis. to confirm this, we have examined other conditions known to provoke apoptosis in yeast, i.e. acetic acid, ageing and hyperosmotic shock ( figure e -h). for treatment with acetic acid, cells were grown in sc complete medium (ph ) to exponential phase and exposed to mm acetic acid for up to min ( , ). hybridizations with probes against s (probe , position + , lanes - ; probe w , position + , lanes - ; probe w , position + , lanes - ; probe w , position + , lanes - ; probe w , position + , lanes - and probe w , position + , lanes - ). asterisks above the arrows indicate the products that were further analysed. arrow marked with a hatch shows a band matching the potential product of the major cleavage, product is marked with one asterisk. (b-c) primer extension analysis for two main cleavage sites in the s rrna in w cells treated with mm h o (a) and in -day old chronologically aged rho w cells (b). primer extensions were performed using primers w for sites around positions + and + and w for sites around position + relative to the end of the mature s. dna sequencing on a pcr product encompassing the end of the s from + to + , using the same primers was run in parallel on % sequencing polyacrylamide gels (lanes - ). the sequences with primer extension stops are shown on the right. secondary structures of the regions in the vicinity of the cleavages, indicated by arrowheads and shown beside corresponding primer extension reactions, were adapted from the website http://rna.icmb.utexas.edu/. (d-e) ends of cleaved-off products for the major cleavage at positions + - were mapped by race. (d) pcr reactions on cdna prepared using total rna from untreated control (lane , c) and cells treated hyperosmotic shock was achieved by growth of exponential cells in sc complete media supplemented with % (wt/wt) glucose or % (wt/wt) sorbitol ( ) . and finally, chronological ageing was performed by constant growth of yeast cultures in sc complete medium for - days ( ) . this analysis revealed that all apoptotic stimuli tested resulted in the s and . rrna fragmentation with the degradation pattern specific for each condition (shown in figure e -g for s in all apoptotic conditions and in figure h for . s during ageing). the accumulating intermediates generated by some factors were comparable (see for example, cleavages mediated by % glucose and % sorbitol, acetic acid and h o , figure e and f); however, the general outcome of each treatment indicated differences in the course of events during each response. the occurrence of rna degradation triggered by h o was monitored during a time course between and min and over a broad range of concentrations ( . - mm for min) ( figure e , lanes - and figure a ). degradation was initiated relatively fast, since it was apparent at min for . mm h o ( figure e , lanes - ), - min for mm acetic acid ( figure e , lanes - ), h for % glucose and % sorbitol ( figure f ) and days for ageing ( figure g ) following the treatment. this onset of rrna degradation distinctly precedes the timing of dna damage characterized in apoptotic yeast exposed to the same stimuli ( ) , indicating that rna decay process is activated early during the response. also, in the case of h o , low doses of the oxidant, starting with . mm and optimal at . - mm, were sufficient to initiate rrna degradation with the appearance of specific bands. when high concentration of h o ( mm), believed to result in cell necrosis, was used, these specific degradation products were absent; however, the level of mature s and s rrnas was also significantly reduced ( figure a ; data not shown). these data show that different ros-generating treatments that lead to yeast apoptosis, namely h o and acetic acid, ageing and hyperosmotic shock, induce rna fragmentation that most likely precedes the dna damage and, as in higher eukaryotes, can be considered a hallmark of the induction of pcd in yeast. cleavages in the s rrna are endonucleolytic and require cellular machinery specific cleavages within the s rrna generated in the presence of hydrogen peroxide were monitored by northern hybridization with probes located along the molecule to narrow down the regions to be further analysed ( figure a ). this analysis showed the accumulation of diverse degradation products, some of which extended from the -end of the molecule (figure a, lanes - ) , whereas others were also truncated at their ends ( figure a, lanes - ) . the striking decrease in the level of the mature s rrna at higher doses of the oxidant ( - mm) indicates that following specific cleavages the majority of rrna becomes degraded, possibly by the exosome complex of ! exonucleases that participates in the decay of rrna precursors and excised transcribed spacers ( ) . the emergence of the characteristic cut-off in the signal at the fragment size corresponding to the position of the probe indicated that major cleavage sites are located around positions + , + and + with respect to the -end of the molecule. two of these cleavages, at positions + - and + - , were mapped for treatment with mm h o by primer extension using primers w and w situated downstream of the expected cleavage sites ( figure b ). similarly, major cleavage sites were analysed in -day old chronologically aged cells using the same primers ( figure c ) and mapped at positions + - and + - . according to the secondary structure of the s rrna taken from ( ), the regions where mapped cleavages occur (shown in figure b and c besides corresponding primer extension reactions) are located at unpaired nucleotides in loops or bulges. this points to the action of single-stranded rna nucleases. to establish the nature of the observed rna fragmentation, -ends of the products generated by the h o mediated cleavage in the s at positions + - and + - were determined by the race. to this end, dna 'anchor' oligonucleotide (w ) was ligated with t rna ligase to total rna from untreated and treated w cells to prepare cdna using a primer specific for the anchor (w ). this served as a template to amplify products containing required fragments using the same primer and a primer that covers the -end of s rna starting at position + (w ). the ensuing pcr fragments ( figure d ) were cloned into pgem-teasy and sequenced. the results of sequenced clones for the cleavage at + - and clones for the cleavage + - are shown in figure e . in the case of the major site (cuts at positions + - ), this analysis confirms that the and ends of this degradation product overlap ( figure e , lower panel), which is consistent with the endonucleolytic mechanism of the cleavage. mapping the and ends at site + - by primer extension and race produced a different pattern: these ends do not match ideally but the products with mm h o (lane ). to generate cdna, total rna that had been ligated to an 'anchor' oligonucleotide (w ) with t rna ligase, was reverse transcribed using a primer specific for the anchor (w ). this was followed by pcr reaction using the same primer and the primer starting at position + in the s rrna (w ). arrows indicate products corresponding to fragments cleaved at + - (lower) and + - (upper). pcr fragments were cloned into pgem-teasy and sequenced. (e) sequences obtained by the race analysis for fragments cleaved at site + - ( independent clones) and site + - ( independent clones). the corresponding regions of the s with cleavage sites mapped by primer extension and indicated with empty arrowheads are shown above in grey. figures in parentheses show the number of identical clones. (f) mapping ends of two major cleavages sites using rnase h cleavage on total rna extracted from wild-type, rrp - and ski d cells treated with mm h o (lanes, - ) and from wild-type untreated control (lane , c). rnase h treatment was performed on rna samples annealed to dna oligonucleotides w and w complementary to positions + and + , respectively. samples were separated on a % acrylamide gel and hybridized with probe w (f-i) and probe w (f-ii) to detect ends of fragments cleaved at + - (f-i) and at + - (f-ii), respectively. arrows show more defined ends of products cleaved at + - for all strains and at + - in the mutants; vertical bar in f-i indicates heterogenous ends of products cleaved at + - in wild-type cells. get progressively shorter pointing at the action of ! exonucleases ( figure e, upper panel) . the most likely candidate is the exosome, a large complex with a ! exonucleolytic activity involved in the processing and degradation of mrna, rrna and other rna substrates ( ) . mutants in the exosome core component rrp , the nuclear subunit rrp and the cytoplasmic cofactor ski , were used to assess the status of the product ends by a specific rnase h cleavage. this cleavage, directed by a dna-rna hybrid between oligonucleotide w and a complementary region in the s starting at residue + , allows higher resolution of analysed rnas. this analysis shows that products generated at site + - in the exosome mutants rrp - and ski d, but not in rrp d, are extended and less heterogenous than corresponding fragments in the wild-type strain (figure f-i; data not shown). in contrast, positions of cleavages at site + - are not affected by mutations in the exosome (fig. f-ii) . this suggests that the cytoplasmic exosome may contribute to rrna decay by digesting ends of at least some cleavage products. to ascertain that rna degradation process is enzymatic and not chemically induced by various reactive compounds, yeast cells were fixed with % formaldehyde for min or with % ethanol for min prior to exposure to increasing concentrations of hydrogen peroxide ( figure a and b) . both fixation procedures preserve cellular structures, however, it is known that most fixatives have harmful consequences, e.g. cause some loss of cellular components, including ribosomes. nevertheless, a similar approach had been used to demonstrate that dna damage in apoptotic yeast cells was an enzymatic process ( ) . also, in the case of rna, the appearance of specific h o -induced degradation products was prevented by fixation, although the overall level of rrna was reduced. some faster migrating rna species were detected in formaldehyde or ethanol fixed cells, however, these were generated also in the absence of the oxidant and did not intensify after treatment ( figure a and b, lane ) . finally, to check whether rrna destruction during apoptotic response is not due to cessation of translation in dying cells, cells were treated for min with the translation elongation inhibitor cycloheximide ( mg/ml) and this did not lead to an apparent rrna degradation (supplementary figure s a) . this is also supported by our earlier observations that exposure of yeast cells to many oxidative agents that cause cell death does not result in rrna decay ( figure a) . together, this strongly suggests that rrna degradation observed in apoptotic and oxidative stress conditions is not simply a result of cell death but is produced in the process that requires enzymatic activity and functional cellular machinery. rrna is most likely cleaved endonucleolytically and in some cases, dictated probably by the rna structure, this is followed by exonucleolytic digestion by the exosome. rrna degradation is strongly correlated with ros levels and is connected with oxidative stress response and apoptosis pathways treatment with oxidative agents and apoptotic stimuli generate elevated levels of ros in the cell. to test whether there is a direct link between rna fragmentation and ros, a potent ros scavenger, l-ascorbic acid (vitamin c), was used ( ) . the presence of mm ascorbic acid prior to treatment with standard doses of h o almost totally abrogated degradation of the s rrna ( figure a ). in addition, the ectopic expression of murine bcl-x l protein of the anti-apoptotic mammalian bcl- family, known to have a protective effect against ros in yeast ( , ) , also strongly safeguarded the s rrna from rapid degradation by h o ( figure b ). in contrast, expression of the mammalian pro-apoptotic bax protein that increases ros level ( , ) , additionally enhanced the degradation phenotype ( figure c ). this confirms the direct correlation between the production of ros and the fate of cellular nucleic acids, leading not only to dna but also rrna damage and destruction of ribosomes. from the data presented so far, it appears that the observed rrna fragmentation may possibly represent a part of the cellular oxidative stress and apoptotic responses. this was assessed by testing the extent of the s rrna degradation in different mutants defective in these pathways. in the first place, yca d strain, lacking the only identified apoptotic metacaspase yca in yeast, and aif d cells not expressing the yeast apoptosis inducing factor aif ( , ), were assayed for h o -induced rna decay, however, no significant differences were observed (supplementary figure s b and c) . similarly, addition of a broad-range caspase inhibitor z-vad-fmk ( mm) that prevents yca -dependent cell death in yeast ( , ) had no effect on rrna fragmentation (data not shown). this indicates that rrna degradation detected in all apoptotic conditions tested is independent of the two major apoptosis mediators, yca and aif , and of other potential yeast caspases. likewise, treatment with translation inhibitor cycloheximide, that has been shown to prevent apoptotic cell death induced by h o and acetic acid ( , ) , had little or no effect on h o -mediated rrna degradation (supplementary figure s a) . however, it appears that events in the course of apoptosis that require protein synthesis are rather late, for example dna fragmentation and chromatin condensation, whereas rrna decay is initiated relatively fast. in contrast, different outcome was observed for mutants inhibiting chromatin condensation during h o -induced apoptosis. phosphorylation of serine and deacetylation of lysine , both in histone h b, were reported to have an essential role for the progress of cell death in yeast ( , ) . in agreement, s a or k q mutations in histone h b that prevent these modifications and abrogate apoptosis resulted in the significant reduction of the s rrna degradation, both the decay of the mature rrna and the amount of degradation products ( figure a ). together, these data show that the destruction of ribosomal rna in cells treated with h o , and possibly with other apoptotic stimuli, is a part of a yeast cell death pathway that involves histone modification and not the caspase-dependent pathway. remarkably, nuc -mediated apoptosis resulting from over-expression of yeast endog homologue nuc , a major mitochondrial nuclease, was also reported to be yca -and aif -independent and related to histone modifications ( ) . alternatively, it can be envisaged that damage of ribosomes triggered by apoptotic stimuli is an early event during the response, does not require protein synthesis, precedes caspase activation and acts as an upstream signal in the apoptotic pathway. this scenario is consistent with most observations so far. as the oxidative stress in yeast proceeds through multiple pathways that involve different response mechanisms ( , ), we tested several known enzymes and factors that regulate these responses. these included two major transcription factors, yap and skn , that control expression of several genes induced by oxidative stress and in this way participate in ros sensing ( , , ( ) ( ) ( ) , as well as components of antioxidant pathways, e.g. superoxide dismutases sod - , glutathione peroxidases gpx - , glutaredoxins grx - , peroxiredoxins tsa - , prx , dot and ahp , thioredoxins trx - and thioredoxin reductases trr - ( , ). these enzymes are required for protection against ros either by catalysing the breakdown of oxidative compounds or by restoring natural intracellular redox equilibrium. in addition, as glutathion protects cells against ros, we also used a gsh d strain lacking a g-glutamylcysteine synthetase, which, when grown on glutathion-free synthetic medium, leads to glutathion depletion and cell death ( , ) . strains lacking these proteins are more sensitive to several oxidants than their isogenic wild-types ( ), and following treatment with h o they showed a marked increase in the s rrna degradation, however, to different degrees depending on the mutant. in figure b -d, yap d, skn d, gpx / / d, sod / d, grx / -d, prxd (tsa / d/prx d/ ahp d/dot d) and gsh d strains are shown, which gave the most evident effects in comparison with their respective isogenic wild-types, particularly when considering the decay rate of the mature s rrna. these data indicate that properly functioning oxidative stress response also protects cellular components such as nucleic acids from the attack by ros and that defects at any step of this defence result in a more severe and faster breakdown. it is noteworthy that multiple anti-oxidant mutants lacking all components of each enzymatic pathway exhibit a stronger effect on the s degradation than single mutants, pointing to the additive protection actions of these systems. striking effects on rrna stability in strains lacking stress response transcription factors yap and skn indicate that the synthesis of new anti-oxidant proteins that are induced by oxidative stress might be required for protection of ribosomes. consistently, blocking protein synthesis by pre-treatment with cycloheximide resulted in somehow stronger rrna degradation (supplementary figure s a, lanes - and - ). taken together, this indicates that targeting rrna degradation during oxidative stress may directly contribute to cell death. to test whether the level of rrna, which reflects the amount of cellular ribosomes, may be somehow linked with cell survival under oxidative stress, we attempted to create a situation where the steady-state level of mature rrnas will be increased or decreased. however, additional copies of rdna present on a multicopy pnoy plasmid under control of the inducible gal promoter ( ) did not affect the amount of any mature rrna species, possibly due to mechanisms that regulate ribosome abundance (data not shown). in contrast, the level of total genomic and plasmid-derived s and s rrnas was, to our surprise, reduced to % in a strain transformed with the multicopy pjv plasmid expressing a tagged rdna gene under the control of the constitutive pgk promoter ( ) , when compared to a strain transformed with vector alone ( figure a ). the basis of this effect is unclear, particularly that it was seen even though the tagged rrna versions were expressed as confirmed by northern blots using probes specific for plasmid borne s rrna ( figure a ). nevertheless, the strain carrying pjv showed a decreased viability already in the absence of oxidant ( . -fold) and even more strikingly reduced following treatment with different concentrations of h o ( . -fold for . mm and . for for mm, respectively) relative to the strain with vector alone ( figure b ). in another approach, we used the noy strain, which carries a temperature sensitive (ts) rna polymerase i ( ) . at c, this strain ceases to grow but it sustains slow growth at c due to reduced levels of mature rrna. expression of additional copies of rdna from pnoy or pjv plasmids improves growth at all temperatures and rescues the ts-lethal phenotype ( , ) . growth of noy expressing additional rdna under the control of gal (pnoy ) or pgk (pjv ) promoters resulted in total rrna levels lower by % in the latter case ( figure c ). this relatively modest difference in rrna abundance led to a % decrease in survival of cells exposed to oxidative stress ( figure d ). this indicates that there may exist a correlation between the quantity of ribosomal subunits and the capacity of the cell to elicit functional defence mechanisms and prevent cell death. it is possible that there is a feedback mechanism that controls this relationship: a healthy cell that contains an adequate number of ribosomes is able to respond more efficiently to stress stimuli to protect cell components from damage, including ribosomes themselves. therefore, provided that the level of ribosomal rna monitors cell fitness, its sudden reduction may act as one of the signals to initiate cell death mechanisms, including apoptosis. rrna degradation depends on the mitochondrial activity in the cell mitochondria are the major source of endogenous ros generated by oxidative phosphorylation. the extent to which mitochondria are involved in mammalian or yeast apoptosis is still questionable, although it appears that mitochondrial ros could be important in some signalling pathways ( , ) and have a central role in some apoptotic pathways and less crucial in others ( ) . for example, apoptotic cell death in yeast induced by acetic acid, pheromone and bax expression was shown to be mediated by mitochondria ( , ) . the correlation between mitochondria and rrna stability was assessed, in the first place, by checking rrna level in respiratory-deficient rho cells lacking mtdna in conditions inducing apoptosis, i.e. exposed to h o ( figure a ), mm acetic acid ( figure b ), hyperosmotic stress ( % glucose, figure c ) and during chronological ageing ( figure d ). all treatments resulted in a remarkably robust degradation of the s and . s rrnas in rho strains when compared to the parental w and by strains (figure , supplementary figure s e ; and data not shown). this points to the importance of the functioning mitochondria in the stressinduced rrna degradation. to test the contribution of the oxidative phosphorylation, two mutants in these pathways were used, op with a point mutation in a major adp/atp carrier aac (arg !his ), and a triple aac / / Á deletion mutant ( ) ( figure e ). both mutants behaved in a similar manner as rho cells and exhibited more pronounced rrna degradation in the presence of h o than the isogenic wild-type; however, the phenotype was stronger for op than upon deletion of the three carrier proteins, possibly due to a dominant negative effect in the point mutant. in addition, treatment with f -f atpase proton-pump inhibitors, oligomycin a ( . mg/ml) and sodium azide (nan , . mm) that block electron transfer and the synthesis of mitochondrial atp ( - ), resulted in a moderate increase in the rrna cleavage ( figure f and supplementary figure s d ). all these experiments indicate that the process of respiration, though generating the endogenous ros, is also vital for counteracting its adverse effects. this was further supported by the degree of rrna protection against oxidative damage caused by h o observed for yeast cells grown on different carbon sources, which are known to affect the level of respiration ( ) . the most extensive rna decay was observed in glucose, where a process called glucose repression discourages respiration. it was less pronounced in galactose and least of all in the nonfermentable source, glycerol, where mitochondrial respiration is forced ( figure g) . these experiments directly correlate functional mitochondria and the process of respiration with the defence against oxidative stress triggered by h o , acetic acid, hyperosmosis and ageing that, among others, prevents destruction of cellular components, including rrna. several cellular responses are regulated at the translational level, particularly by selective translation of specific mrnas or by inhibition of the ribosome and protein synthesis, as these processes consume a large amount of energy. such inhibition can follow various stimuli, including endoplasmic reticulum stress and unfolded protein response (upr), transition into quiescence and different stress-related and cell death-related signals ( ). the most straightforward and fastest way to achieve translation inhibition is to target ribosomal rna. interestingly, it has been proposed that repression of protein synthesis during upr in human cells is due to the cleavage of s rrna by hire b, a second homologue of ire ( ) . also, during apoptosis in some mammalian cells degradation of s rrna by rnasel, but also of other rnas such as y rna or some mrnas, has been suggested to block protein synthesis that contributes to, but could even initiate, cell death. furthermore, damage to the s rrna may act as a ribotoxic stress and induce an early death-committing signal through activation of sap and map kinases ( , ) . these possibilities were not examined in yeast pcd pathways. we have analysed the behaviour of ribosomal rnas during oxidative stress and in apoptotic pathways that are induced by different stimuli. we have observed that cells exposed to all apoptotic conditions tested, such as h o , acetic acid, hyperosmotic stress ( % glucose) and ageing, reveal a significant degradation of the s and some of . s rrnas, with a much lesser effect on the s rrna. the decay of mature rrnas was accompanied by the accumulation of treatment-specific, yet partly overlapping degradation intermediates. although there is no evidence so far that rrna damage during apoptotic conditions in yeast can directly initiate cell death by activating signalling pathways, it is tempting to speculate that this might be the case. such signalling could be conveyed either by a critical decrease of the mature s rrna or, alternatively, by the accumulation of degradation intermediates/products, which can function as signal molecules triggering a specific pcd pathway. in most cases, when ribosomal rnas are depleted (e.g. in pre-rrna processing mutants), rrna degradation is conducted rapidly with no or little rrna fragments detectable; however, lack of lsm proteins has been reported to result in degradation of ribosomal rnas with accumulation of unusual intermediates ( ) . it is noteworthy that one apoptotic pathway that is linked with rna metabolism is triggered by the defect in mrna turnover caused by mutations in enzymes involved in decapping, including components of dcp - and lsm - complexes ( ) . each of the applied apoptosis-inducing conditions resulted in a clear-cut pattern of the s degradation intermediates or products. this points to the endonucleolytic nature of the reactions, though exonucleolytic destruction, with certain rna fragments temporarily protected by compact structures or tight interactions with proteins, cannot be excluded. however, mapping and boundaries of the major h o -induced cleavage product by primer extension and race, respectively, confirmed that both ends strictly overlap and thus result from the endonucleolytic cut. it has been shown that apoptotic stimuli in yeast generate ros that are closely correlated with the onset or progression of pcd ( ) . we saw that, also the degree of rrna decay corresponded to the cellular level of ros, which was modified by using ros scavenger (ascorbic acid) or ectopic expression of pro-or anti-apoptotic proteins (bax and bcl-x l , respectively) known to affect ros generation. also, rrna degradation was more robust in oxidative stress defence mutants, both enzymatic and non-enzymatic, where intracellular ros is not properly neutralized. all these observations argue that there is a direct link between ros production and rrna fragmentation. it can be envisaged that various reactive species themselves are able to produce endonucleolytic nicks in rna molecules that will lead to breakdown, particularly as all mapped cleavages occur in singlestranded regions that constitute loops and bulges and are more accessible to chemical compounds in the solvent. however, this is not the case, given that specific rna fragmentation did not occur in cells fixed with formaldehyde or ethanol prior to treatment with h o . in addition, oxidative agents used in this work generate different forms of ros such as h o , hydroperoxide (loaooh, chp and t-bhp), superoxide anion (menadione and paraquat) and hydroxyl free radical (produced from h o or superoxide anion). although all were applied at toxic doses, only two of them, h o and menadione, mediated rrna degradation, supporting the notion that oxidative compounds as such do not provoke cuts in rna molecules within the cell. taken together, this strongly suggests that active cellular machinery, such as signalling factors and rna degrading enzyme(s), is required for this process. nevertheless, these enzymatic activities are still to be identified. the closest yeast homologue of mammalian rnase l, ire , a sensor of the unfolded protein response, functions in the unconventional splicing of hac pre-mrna and contains protein kinase and endoribonuclease domains similar to those in rnase l ( ) . although our unpublished data show that deletion of ire has no effect on the h o -induced rrna degradation (m.s. and j.k.), it does not exclude its participation in other apoptotic pathways, for example those related to endoplasmic reticulum stress and unfolded protein response. we are currently testing several known yeast endo-an exonucleases for participation in apoptosis-related rrna degradation. preliminary observations suggest that, contrary to expectations, this function may involve not one but a number of unspecific nucleases, including mitochondrial nuc p, that act in a redundant fashion (m.s. and j.k., unpublished data). the reason why treatment with some oxidative agents but not others produce fragmented rna is not entirely clear, however, it is known that different chemicals induce specific responses leading to expression of distinct sets of genes ( , ) , and possibly only a few activate pathways that involve rna destruction. our results suggest that rrna degradation phenotype most likely accompanies apoptosis, and only apoptosis-inducing oxidants result in rrna decay, whereas cell death caused by others probably occurs via a different mechanism. another question is why each apoptotic stimuli resulted in slightly different set of cleavage products. those which were mapped for treatment with h o and in aging cells illustrate that cleavages often occur in loops and bulges in closely located regions within the s rrna, or rather within the accessible rna elements in the compact rnp structures. the difference in cleavage patterns could be due to stress-induced subtle or severe alterations in ribosome particles that change the local accessibility of the rrna components presented for cleavage. alternatively, if rrna decay is indeed carried out by more than one nuclease, these differences may reflect varying enzyme specificities in each death-inducing condition. it is also possible that somehow different set of nucleases is activated or recruited to rrna substrates during apoptosis triggered by oxidative stress, acetic acid treatment and ageing. mitochondrial respiration protects rrna against deleterious consequences of ros certain aspects of apoptosis, such as the change in mitochondrial membrane potential, fragmentation of mitochondria and the requirement of cyt c and aif release to the cytoplasm, are strongly conserved among different organisms and point to the pivotal role of mitochondria. in yeast, pcd pathways triggered by acetic acid, bax expression and pheromone, are strictly correlated with these events and do not proceed in cells devoid of mtdna (rho ) ( , ) . during chronological ageing and hyperosmotic shock, rho strains were reported to have somehow higher survival, which can be attributed to their long doubling time ( . to -fold), but they still die apoptotically ( , ) . however, mitochondrial function is required for resistance to oxidative stress by way of detoxification or repair of the oxidative damage and, consequently, rho cells are more sensitive to several oxidants, have higher level of endogenous ros and undergo apoptosis caused by h o or amino-acid starvation ( , , ( ) ( ) ( ) ( ) . also, mammalian rho cell lines undergo apoptosis in response to some but not all cell death-activating stimuli. it has been postulated that these differences may arise from a distinct mechanism by which rho cells maintain membrane potential by way of atp consumption ( ) . our results show that, in all conditions tested, rrna degradation is tightly connected with mitochondrial function and the active process of respiration. rho cells that are less resistant to oxidative stress suffer severe rrna degradation in the presence of h o , acetic acid and % glucose and during chronological ageing. also, impediment of oxidative phosphorylation by protonpump inhibitors or by mutations in atp/adp carriers gives a similar outcome. in contrast, rrna is markedly more stable when mitochondrial respiration is enhanced (e.g. by growth on glycerol versus glucose). this is consistent with the protective role of active mitochondria against the damaging effects of ros on cellular components, rrna destruction included. to begin with, several anti-oxidant enzymes localize to mitochondria and are more abundant in respiring cells. in addition, it has been proposed that some anti-oxidant activities may require energy ( ) . as some apoptotic pathways depend on the release of mitochondrial factors to the cytoplasm and are not induced in rho cells, this poses an important question regarding the link between rrna degradation and apoptosis. rrna degradation-apoptotic or not? although several apoptotic mediators (yca , aif , nma , bir and ndi ) have been identified in yeast, their networking in regulation of apoptosis is not yet fully understood. they may interact and function in a similar fashion as their mammalian counterparts, however, in contrast to mitochondrial mammalian proteins, nma and bir are located in the nucleus, so in yeast there might be some deviations from the mammalian model ( , , ) . nevertheless, apoptotic cell-death pathways in yeast induced by h o , acetic acid, hyperosmotic shock, ageing and increased mrna stability were reported to require metacaspase yca , nominating them as caspasedependent pathways ( , , ) . still, deletion of yca in the mrna turnover mutant lsm d does not attenuate mrna decay, placing yca action downstream of the signal rising from mrna level ( ) . in contrast, histone phosphorylation at ser , which requires prior deacetylation at lys , has been shown to mediate h o -induced apoptosis independently of yca ( , ) . also, the activity of the major mitochondrial nuclease, nuc , in the celldeath pathway does not require either apoptotic mediators, yca or aif , but is affected by h b modifications ( ) . moreover, two pcd pathways, namely induced by defects in protein n-glycosylation and triggered by ammonia in multicellular yeast colonies, do not rely on yca but on as yet unknown caspase-like activity ( , ) . degradation of the s/ . s rrnas is observed in all conditions inducing apoptosis, however, this process is not dependent on yca and aif . on the other hand, mutations in histone h b that inhibit phosphorylation at ser and block the progress of h o -induced apoptosis also severely affect rrna degradation. more importantly, rrna degradation coincides with apoptotic dna fragmentation; from several compounds that lead to oxidative stress and cell death, only those that provoked dna destruction also triggered rrna decay. furthermore, at least some apoptotic pathways strictly require the involvement of mitochondria and do not occur in yeast lacking mtdna, whereas rrna degradation in all stress conditions tested is more powerful in rho cells. to sum up: rrna decay induced by apoptotic stimuli occurs during cell death pathway that involves ros generation, mitochondrial activity, fragmentation of chromosomes and histone modification but not apoptotic regulators, yca and aif . this could be due to the existence of numerous different but partly overlapping pcd mechanisms, whether caspase-and mitochondria-dependent or independent. ever increasing numbers of such pathways has been described in the literature in recent years. however, we favour a different model, where all these elements function in concert at different steps of the whole scenario. stress stimuli induce signals, possibly via ros, affecting different levels of gene expression, namely chromatin modifications, transcription and translation, which as a result activate defence response. at this level, mitochondrial respiration helps to protect cellular components via adaptive mechanisms with anti-oxidant functions. even so, when the attack is not successfully pacified, generated ros molecules initiate the destruction of cellular machineries, targeting in the first place crucial elements such as protein synthesis (i.e. ribosomes). now, the progression of the response comes to the crossroadsif the conditions are appropriate, the cascade of events leading to apoptosis is triggered (e.g. release of cytc and other mitochondrial factors to the cytoplasm) resulting in cell death with characteristic apoptotic markers. alternatively, when apoptotic prerequisites are not met, cells do not enter this pathway. the more fit cells escape death, whereas others die anyway, maybe less rapidly and through a different pathway. in this scenario, certain events, including generation of ros, histone modifications and possibly also rna fragmentation, occur upstream of subsequent steps, such as activation of caspases and other apoptotic regulators with resulting apoptotic phenotypes. genomic expression programs in the response of yeast cells to environmental changes cells have distinct mechanisms to maintain protection against different reactive oxygen species: oxidative-stress-response genes trashing the genome: the role of nucleases during apoptosis caspase-dependent cleavage of nucleic acids pppa p a p a: an inhibitor of protein synthesis synthesized with an enzyme fraction from interferon-treated cells activation of the ifn-inducible enzyme rnase l causes apoptosis of animal cells the role of - oligoadenylate-activated ribonuclease l in apoptosis rnase l-independent specific s rrna cleavage in murine coronavirus-infected cells programmed death in yeast as adaptation? apoptosis in yeast -mechanisms and benefits to a unicellular organism why yeast cells can undergo apoptosis: death in times of peace, love, and war a caspase-related protease regulates apoptosis in yeast the s. cerevisiae htra-like protein nma p is a nuclear serine protease that mediates yeast apoptosis an aif orthologue regulates apoptosis in yeast yeast amid homologue ndi p displays respiration-restricted apoptotic activity and is involved in chronological aging the inhibitor-of-apoptosis protein bir p protects against apoptosis in s. cerevisiae and is a substrate for the yeast homologue of omi/htra endonuclease g regulates budding yeast life and death apoptotic phosphorylation of histone h b is mediated by mammalian sterile twenty kinase sterile kinase phosphorylates histone h b at serine during hydrogen peroxide-induced apoptosis in histone h b deacetylation at lysine is required for yeast apoptosis induced by phosphorylation of h b at serine a truncated form of kllsm p and the absence of factors involved in mrna decapping trigger apoptosis in yeast yeast caspase links messenger rna stability to apoptosis in yeast complex cellular responses to reactive oxygen species oxidative stress responses of the yeast saccharomyces cerevisiae transcription factors regulating the response to oxidative stress in yeast phenotypic analysis of gene deletant strains for sensitivity to oxidative stress improved method for high efficient transformation of intact yeast cells saccharomyces cerevisiae commits to a programmed cell death process in response to acetic acid characterization of dna damage in yeast apoptosis induced by hydrogen peroxide, acetic acid, and hyperosmotic shock hyperosmotic stress induces metacaspase-and mitochondriadependent apoptosis in saccharomyces cerevisiae chronological aging leads to apoptosis in yeast fungal small nuclear ribonucleoproteins share properties with plant and vertebrate u-snrnps identification and functional analysis of two u binding sites on yeast pre-ribosomal rna nuclear pre-mrna decapping and degradation in yeast require the lsm - p complex oxygen stress: a regulator of apoptosis in yeast genetic analysis of glutathione peroxidase in oxidative stress response of saccharomyces cerevisiae fine mapping of s rrna sites specifically cleaved in cells undergoing apoptosis s ribosome degradation in lymphoid cell apoptosis: evidence for caspase and bcl- -dependent and -independent pathways a yeast mutant showing diagnostic markers of early and late apoptosis degradation of ribosomal rna precursors by the exosome the comparative rna web (crw) site: an online database of comparative sequence and structure information for ribosomal, intron, and other rnas rna-quality control by the exosome cellular functions of ascorbic acid release of cytochrome c and decrease of cytochrome c oxidase in bax-expressing yeast cells, and prevention of these effects by coexpression of bcl-xl modulation of cell death in yeast by the bcl- family of proteins mammalian bax triggers apoptotic changes in yeast yeast cell death during dna damage arrest is independent of caspase or reactive oxygen species yap and skn control two specialized oxidative stress response regulons in yeast discrimination between paralogs using microarray analysis: application to the yap p and yap p transcriptional networks yeast signaling pathways in the oxidative stress response oxidative activation of antioxidant defence low glutathione pools in the original pso mutant of saccharomyces cerevisiae are responsible for its pleiotropic sensitivity phenotype synthesis of large rrnas by rna polymerase ii in mutants defective in rna polymerase i development and application of an in vivo system to study yeast ribosomal rna biogenesis and function gene rrn in saccharomyces cerevisiae encodes the a . subunit of rna polymerase i and is essential only at high temperatures mitochondrial reactive oxygen species in cell death signaling mitochondria, oxidants, and aging. cell cell death: critical control points cytochrome c release and mitochondria involvement in programmed cell death induced by acetic acid in saccharomyces cerevisiae production of reactive oxygen species and loss of viability in yeast mitochondrial mutants: protective effect of bcl-xl application of inhibitors and uncouplers for a study of oxidative phosphorylation effects of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin on nucleotide binding to the three f -atpase catalytic sites measured using specific tryptophan probes the mitochondrial f f -atpase proton pump is required for function of the proapoptotic protein bax in yeast and mammalian cells translational control in stress and apoptosis translational control by the er transmembrane kinase/ribonuclease ire under er stress ribosome inactivating proteins and apoptosis rearrangement of nuclear ribonucleoprotein (rnp)-containing structures during apoptosis and transcriptional arrest lsm proteins are required for normal processing and stability of ribosomal rnas yeast programmed cell death: an intricate puzzle the transmembrane kinase ire p is a site-specific endonuclease that initiates mrna splicing in the unfolded protein response role of mitochondria in the pheromone-and amiodarone-induced programmed death of yeast saccharomyces cerevisiae has distinct adaptive responses to both hydrogen peroxide and menadione mitochondrial function is required for resistance to oxidative stress in the yeast saccharomyces cerevisiae the role of respiration, reactive oxygen species and oxidative stress in mother cell-specific ageing of yeast strains defective in the ras signalling pathway starvation for an essential amino acid induces apoptosis and oxidative stress in yeast cells depleted of mitochondrial dna (rho ) yield insight into physiological mechanisms physiological regulation of yeast cell death in multicellular colonies is triggered by ammonia defects in n-glycosylation induce apoptosis in yeast we thank m. nomura (university of california, irvine) for strain noy and plasmid pnoy ; h. raue´and j.c. vos (university of vrije, amsterdam) for plasmid pjv ; j. kolarov (comenius university, bratislava) for plasmids prs-bcl-x l and yep -bax and strains aac / / -d and op ; y. inoue (kyoto university) for strains yph , grx / -d and gpx / / -d; e.b. gralla (university of california, los angeles) for strains eg and sod / -d; c.d. allis (the rockefeller university, new york) for strains jhy , say and say ; r. wysocki (university of wroclaw) for plasmid yep -yap . this work was funded by the wellcome trust ( /z/ /z). funding to pay the open access publication charges for this article was provided by the wellcome trust. supplementary data are available at nar online.conflict of interest statement. none declared. key: cord- -r nkdeaw authors: bonora, massimo; patergnani, simone; ramaccini, daniela; morciano, giampaolo; pedriali, gaia; kahsay, asrat endrias; bouhamida, esmaa; giorgi, carlotta; wieckowski, mariusz r.; pinton, paolo title: physiopathology of the permeability transition pore: molecular mechanisms in human pathology date: - - journal: biomolecules doi: . /biom sha: doc_id: cord_uid: r nkdeaw mitochondrial permeability transition (mpt) is the sudden loss in the permeability of the inner mitochondrial membrane (imm) to low-molecular-weight solutes. due to osmotic forces, mpt is paralleled by a massive influx of water into the mitochondrial matrix, eventually leading to the structural collapse of the organelle. thus, mpt can initiate outer-mitochondrial-membrane permeabilization (momp), promoting the activation of the apoptotic caspase cascade and caspase-independent cell-death mechanisms. the induction of mpt is mostly dependent on mitochondrial reactive oxygen species (ros) and ca( +), but is also dependent on the metabolic stage of the affected cell and signaling events. therefore, since its discovery in the late s, the role of mpt in human pathology has been heavily investigated. here, we summarize the most significant findings corroborating a role for mpt in the etiology of a spectrum of human diseases, including diseases characterized by acute or chronic loss of adult cells and those characterized by neoplastic initiation. mitochondrial permeability transition (mpt) remains one of the most unusual and poorly characterized aspects of mitochondrial biology. this phenomenon was first reported in the late s and was originally considered an artefact due to the experimental conditions required to investigate isolated mitochondria. however, (as better described below) mpt is triggered by the accumulation of ca + in the mitochondrial matrix that occurs in isolated mitochondria exposed to a [ca + ] significantly higher than that in the cytoplasm. the development of techniques to measure ca + within different compartments later demonstrated that mitochondria can actively uptake ca + even in living cells, prompting the investigation of mpt in cell pathophysiology. mitochondria actively participate in multiple forms of regulated cell death (rcd) through different routes, all involving major alterations in the outer mitochondrial membrane (omm) and/or inner mitochondrial membrane (imm). activation of the mitochondrial pathway of rca causes the redistribution of mitochondrial proteins into the cytoplasm, which activates cell-death effectors, or the dramatic impairment of cell bioenergetics, which ultimately leads to death of the affected cells. these mechanisms are, in general, categorized into two types that function under different conditions: those involving only outer-mitochondrial-membrane permeabilization (momp) and those in which there is also a long-lasting increase in imm permeability, the mpt. momp is due to the formation of a pore composed of the protein b-cell lymphoma (bcl- ) protein family members bax and bak. in response to some apoptotic signals, bax re-localizes from the cytosol into distinct foci on the omm. there, bax oligomerizes into specific structures, such as rings and arc-shaped structures, which can create large-conductance pores in the omm [ ] [ ] [ ] . similarly, bak (which is mostly constitutively located in the omm) can homo-oligomerize ( figure ). biomolecules , , x of mitochondria actively participate in multiple forms of regulated cell death (rcd) through different routes, all involving major alterations in the outer mitochondrial membrane (omm) and/or inner mitochondrial membrane (imm). activation of the mitochondrial pathway of rca causes the redistribution of mitochondrial proteins into the cytoplasm, which activates cell-death effectors, or the dramatic impairment of cell bioenergetics, which ultimately leads to death of the affected cells. these mechanisms are, in general, categorized into two types that function under different conditions: those involving only outer-mitochondrial-membrane permeabilization (momp) and those in which there is also a long-lasting increase in imm permeability, the mpt. momp is due to the formation of a pore composed of the protein b-cell lymphoma (bcl- ) protein family members bax and bak. in response to some apoptotic signals, bax re-localizes from the cytosol into distinct foci on the omm. there, bax oligomerizes into specific structures, such as rings and arc-shaped structures, which can create large-conductance pores in the omm [ ] [ ] [ ] . similarly, bak (which is mostly constitutively located in the omm) can homo-oligomerize ( figure ). major molecular paths in mitochondria-related regulated cell death (rcd). mitochondrial calcium overload and ros levels can trigger either the activation of intrinsic apoptotic pathway (left side) through the recruitment of bcl- family proteins at the mitochondria, or permeability transition pore complex (ptpc) formation which could lead to mitochondrial outer membrane permeabilization (momp), energetic imbalance, and subsequent release of proapoptotic cofactors from the inter membrane space, such as smac/diablo, cytc, and endog (right side). because of the permeabilization of the omm, many proteins normally localized within the intermembrane space (ims) are simultaneously released into the cytosol; these proteins are involved in the effector phase of apoptosis. in particular, (i) cytochrome c (cytc) mediates the organization of the apoptosome and then the activation of the caspase cascade [ ] ; (ii) apoptosis-inducing factor, iii) mitochondria-associated, (aif) induces chromatin condensation; iv) htra serine peptidase (htra ) and diablo iap-binding mitochondrial protein (smac/diablo) bind inhibitor of apoptosis (iap), preventing the inhibition of procaspases; and endonuclease g (endog) mediates dna fragmentation [ ] (figure ) . investigation of the bcl- -mediated control of rcd revealed a role of ca + -mobilization signals. indeed, several signals can induce rcd by the selective transfer of ca + from the endoplasmic reticulum (er, which acts as a store) to mitochondria [ ] [ ] [ ] . when mitochondria are exposed to a pathological overload of ca + , mpt is triggered [ ] . mpt is associated with the opening of the mitochondrial permeability transition pore complex (ptpc), a voltage-dependent, high-conductance mitochondrial calcium overload and ros levels can trigger either the activation of intrinsic apoptotic pathway (left side) through the recruitment of bcl- family proteins at the mitochondria, or permeability transition pore complex (ptpc) formation which could lead to mitochondrial outer membrane permeabilization (momp), energetic imbalance, and subsequent release of proapoptotic cofactors from the inter membrane space, such as smac/diablo, cytc, and endog (right side). because of the permeabilization of the omm, many proteins normally localized within the intermembrane space (ims) are simultaneously released into the cytosol; these proteins are involved in the effector phase of apoptosis. in particular, (i) cytochrome c (cytc) mediates the organization of the apoptosome and then the activation of the caspase cascade [ ] ; (ii) apoptosis-inducing factor, (iii) mitochondria-associated, (aif) induces chromatin condensation; (iv) htra serine peptidase (htra ) and diablo iap-binding mitochondrial protein (smac/diablo) bind inhibitor of apoptosis (iap), preventing the inhibition of procaspases; and endonuclease g (endog) mediates dna fragmentation [ ] (figure ) . investigation of the bcl- -mediated control of rcd revealed a role of ca + -mobilization signals. indeed, several signals can induce rcd by the selective transfer of ca + from the endoplasmic reticulum (er, which acts as a store) to mitochondria [ ] [ ] [ ] . when mitochondria are exposed to a pathological overload of ca + , mpt is triggered [ ] . mpt is associated with the opening of the mitochondrial permeability transition pore complex (ptpc), a voltage-dependent, high-conductance channel assembled at the interface between the imm and the omm [ ] . ptpc opening leads to the redistribution of small solutes (< . kda). the dramatic osmotic influx of water into the mitochondrial matrix during mpt collapses mitochondrial membrane potential (∆ψm) and all related activities (including atp recycling), and is followed by structural collapse (swelling). it results in the release of mitochondrial proteins from ims which triggers the apoptotic pathway. alternatively, the incapability of the affected cell to sustain atp production leads to the irreversible deterioration of ion homeostasis, ultimately resulting in cell death with a necrotic morphology [ ] (figure ). it is believed that ca + is the only trigger for ptpc opening, while other factors manipulate the threshold of [ca + ] required for the occurrence of the event. these include sensitizers (e.g., low ∆ψ m , ros, high matrix ph, long-chain fatty acids, atractyloside, and carboxyatractyloside) [ ] [ ] [ ] [ ] [ ] [ ] and desensitizers (mg + , adp, atp, acidic matrix ph) [ ] . among the many desensitizing factors, the best characterized is cyclosporine a (csa). this small molecule can inhibit the mitochondrial peptidyl-prolyl isomerase cyclophilin d (cypd) and to date has been considered the gold standard for evaluating the involvement of mpt in pathophysiology. the structure of the ptpc has been investigated for decades and is still not well characterized. original studies on isolated proteins and reconstituted liposomes proposed the voltage-dependent anion channel (vdac) on the omm and the adp/atp translocase (ant) on the imm. the inclusion of ant in the model is important as it possibly represents the inhibition site for adp/atp or the activation site for atractyloside. notably, both proteins could be isolated in a complex containing cypd. murine cypd is coded by the gene ppif. the investigation of ppif -ko mice (then ablated for cypd) robustly confirmed the involvement of cypd in ptpc composition [ ] ; in contrast, the genetic deletion of vdac failed to do so. indeed, when cells from vdac −/−; vdac −/− mice were treated with sirna targeting vdac , they displayed comparable sensitivity to mpt [ ] . a similar approach was conducted to investigate ant. the knockout (ko) of the three mouse isoforms of ant indeed showed that ptpc requires a large amount of ca + to open, and that the inhibitory effect of adp was lost [ ] . interestingly, in cells from ant triple-ko animals, mpt was still sensitive to csa, and in cypd-ko cells, mpt was still responsive to adp [ ] . the combined deletion of ants and cypd in a quadruple-ko model conferred resistance of mpt to [ca + ] as high as mm (which is considerably high), potentially indicating that the ptpc did not manifest. these experiments confirmed a role for ant in the ptpc, but also implied that some other partners of cypd participate in the formation of the ptpc. recently, a new candidate was proposed as a pore-forming member of the ptpc-mitochondrial f /fo atp synthase (hereafter referred to as atp synthase). indeed, (i) genetic manipulation of atp synthase subunits markedly affected mpt; (ii) isolated atp synthase, or its c subunit, reconstituted in artificial bilayers generated ptpc-like currents after ca + stimulation; (iii) atp synthase interacts with cypd; and (iv) molecules that target atp synthase impair mpt [ ] [ ] [ ] . furthermore, the mutagenesis of the beta subunit (β-subunit) and the oligomycin-sensitivity-conferring protein (oscp) of atp synthase impaired the effects of ca + and acidic ph, respectively, on the ptpc [ , ] . atp synthase is usually arranged in dimers that further cluster with oligomers on the imm. ptpc-derived currents were obtained from monomers or dimers in different experimental settings, opening a debate on which portions of the complex are required [ , ] . despite this, it was demonstrated that mpt is mediated by the rupture of atp synthase dimers and that it was preventable by mutagenesis of the c subunit, altering the c-ring conformation [ ] . crispr/cas -mediated deletion of the atp synthase c or b subunits was still detectable, suggesting that atp synthase might not be directly involved in the pore formation [ , ] . later studies in cells devoid of the c subunit revealed that conductance of ptpc was significantly reduced and that the remaining current could still be inhibited by csa or compounds targeting ant (adp and bongkrekic acid) [ ] . multiple hypothesis are now under evaluation to explain these apparently conflicting results: (i) atp synthase might only indirectly regulate ptpc, by controlling crista structure and adp levels, (ii) atp synthase and ant might form two independent pores among the imm and, (iii) ant and atp synthase (which can interact in the so-called atp synthasome) might be synergistically required for the proper formation of the ptpc pore. studies on ppif -ko mice demonstrated that cypd-dependent mpt is fundamental for the activation of rcd with necrotic features. indeed, cypd-deficient cells are resistant to necrotic cell death induced by reactive oxygen species (ros) and ca + overload, while stimuli that activate momp are insensitive to a lack of cypd [ , ] . additionally, neuronal cell lines stably overexpressing cypd in mitochondria were prone to necrotic cell death after mpt induction, and were instead more resistant to apoptosis induced by nitric oxide (no) or staurosporine [ ] . this evidence suggests that mpt ultimately results in necrosis and does not induce other forms of cell death ( figure ). as apoptotic cell death is an active, energy-demanding process, this conclusion is logical for all those conditions in which a marked ptpc opening is triggered, reaching the non-return point of energy depletion that engages the mechanism described above. nevertheless, some deviation from this model should be considered. indeed, different reports have shown that in several experimental models, different stimuli that increase intracellular ros elicit markers of intrinsic apoptotic pathways that can be inhibited by csa, including mitochondrial proapoptotic protein release, phosphatidylserine exposure, and dna fragmentation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this suggests that, at least under selected experimental conditions, submaximal mpt might represent an alternative path for apoptosis activation. furthermore, proteins that regulate momp are strictly connected to mpt. several direct protein-protein interactions between bcl- family members and constitutive mitochondrial proteins involved in mpt, such as ant, vdac, and atp synthase, have been confirmed. bax and bak are also required for ptpc-dependent necrotic cell death; in fact, the loss of bax/bak resulted in resistance to mitochondrial calcium overload and swelling [ ] . the confirmation of mpt control by these interactions was demonstrated by the evidence of bax-and bak-induced loss of ∆ψ m , mitochondrial swelling, and cytc release through a ca + -and csa-dependent mechanism [ ] . in addition, it was demonstrated that tbid induced transient openings of the ptpc associated with a conspicuous remodeling of mitochondrial cristae through a bak-independent but csa-inhibitable process [ ] . ant function is under the control of bcl-xl expression: growth-factor-deprived cells avoid apoptosis via an efficient exchange of adp for atp promoted by bcl-xl, permitting mitochondria to adapt to changes in metabolic demand [ ] . furthermore, bcl- positively regulates ant activity, while bax inhibits it by disrupting its interaction with bcl- [ ] . necroptosis is a form of regulated necrosis recently discovered under conditions in which the apoptotic pathway was inhibited, and it presents morphological features of both apoptosis and necrosis. the key upstream kinases involved in the activation of necroptosis are ripk [ ] , ripk , and the substrate mlkl [ ] , which can be inhibited either through genetic or pharmacological methods to block this type of programmed cell death [ ] . ripk , which is essential for tnfa-induced necrosis, can inhibit the adp/atp exchange mediated by ant [ ] , which coincides with the loss of the cypd-ant interaction, reduced atp, and the induction of necrotic cell death, suggesting a role of the ant-cypd interaction in necroptosis [ ] . bax and bak have also been defined as mediators of necroptosis [ ] ; in fact, the elimination of bax/bak or the overexpression of bcl-xl leads to the inhibition of the necroptotic process [ ] . furthermore, necroptosis is associated with mitochondrial cytc release and is partly sensitive to csa inhibition [ ] . the investigation of mpt over the years has revealed its importance in multiple subroutines of rcd, prompting investigation into its involvement in human pathology. in the present manuscript, we review the most recent literature discussing the role of the ptpc in human diseases caused by the dysregulation of cell death. ischemia and consequent reperfusion injury (ri) are pathological manifestations in which the involvement of mpt has been robustly confirmed. ischemia is characterized by the reduced oxygenation of a portion of a tissue (hypoxia), which results in the loss of tissue functions and eventually the activation of multiple forms of rcd. ischemia impacts multiple organs, especially those that are more susceptible to hypoxia, such as the heart, brain, and kidney. interestingly, not all tissues share the same susceptibility to ischemia/ri. in fact, the severity of the injury largely depends on how different types of cells, and therefore tissues, can survive under hypoxic conditions [ , ] . mechanistically, a lack of oxygenation translates into reduced activity of the electron transport chain (etc), and hence results in the blockage of oxidative phosphorylation (oxphos) and a consequent reduction in atp recycling. in this scenario, to meet energy demands, cells upregulate anaerobic glycolysis, producing lactic acid and hydrogen ions, which results in intracellular acidosis. by neutralizing ph via the activation of na + /h + antiporter (nhe), the cell undergoes sodium accumulation that is counterbalanced by the reverse activity of na + /ca + exchanger (ncx). concomitantly, the reduction in available atp depresses the activity of na + /k + atpase, plasma membrane calcium atpase (pmca), and sarco-/endoplasmic reticulum ca + -atpase (serca), which ultimately results in an overload of cytosolic ca + [ ] . additionally, the reduced po causes the accumulation of electrons among different respiratory complexes, leading to the production of ros. the concomitant increase in intracellular ca + and ros and the decrease in ∆ψ m favors the induction of mpt. this mechanism has been demonstrated in cultured neonatal rat cardiomyocytes [ ] , hepatocytes [ ] , and immortalized cells [ ] . among all tissues, the brain exhibits high sensitivity to ischemia due to its glucose-dependent metabolisms [ ] . rapid and csa-dependent mitochondrial depolarization is reported to occur early during experimental stroke in the mouse somatosensory cortex in vivo [ ] . additionally, csa protects the retinal ganglion from cell death during acute intraocular pressure (iop) elevation, a peculiar inducer of ischemia [ ] . furthermore, csa administration attenuates hypoxic-ischemic brain injury in newborn rats induced by unilateral carotid artery ligation [ ] . despite this, mpt is commonly believed to occur to a minimal extent during ischemia. indeed, as described, hypoxia leads to the accumulation of protons and adp, which are strong inhibitors of ptpc. during ischemia, the manifestation of mpt depends on the subtle equilibrium between ptpc inducers (ca + and ros) and inhibitors (protons and adp), which accumulate as a result of impaired mitochondrial respiration and excess glycolysis ( figure ). furthermore, the adaptive response to hypoxia-mediated by hif a affects ptpc opening by regulating hexokinase ii (hkii) levels [ ] , another reported ptpc regulator. indeed, when hif a is stabilized (e.g., following hypoxia or gsk a administration), hkii protein expression significantly increases in the mitochondrial fraction, and this is directly involved in the cytoprotective effect started by hif a stabilization. genetic depletion of hkii completely abolished this path even in the presence of an activated hif a [ ] . nevertheless, hkii is considered an inhibitor of the ptpc only when bound to the omm (especially to vdac), rather than when generally overexpressed in mitochondria. nevertheless, under this condition, ca + overload may induce mpt-independent cell death as a result of the excessive activation of ca + -dependent enzymes such as phospholipases, proteases, and endonucleases [ , ] . noticeably, during myocardial infarction, the activation of calpains, ca +dependent cysteine proteases, results in myofibril disruption, thus promoting hypercontracture in nevertheless, under this condition, ca + overload may induce mpt-independent cell death as a result of the excessive activation of ca + -dependent enzymes such as phospholipases, proteases, and endonucleases [ , ] . noticeably, during myocardial infarction, the activation of calpains, ca + -dependent cysteine proteases, results in myofibril disruption, thus promoting hypercontracture in the heart, which consists of sustained shortening and stiffening of the myocardium [ , ] . the situation dramatically changes during tissue reperfusion. indeed, the restoration of po recovers respiration, atp synthesis, and the activity of plasma membrane pumps, which re-equilibrate intracellular ph. as a result, the inhibition of mpt by adp and protons is removed, lowering the threshold for ptpc opening. in addition, reperfusion stimulates ros production by multiple sources [ ] . at the mitochondrial level, succinate is observed to accumulate in the mitochondrial matrix during ischemia and it was proposed to stimulate ros production from complex i through reverse electron transport (ret), at the time of reperfusion [ ] . other pieces of evidence suggest that etc conditions are not favorable for ret at the beginning of reperfusion, and that a burst of ros production only occurs after a first wave of mpt. besides mitochondria, reperfusion induces ros via other enzymes including (but not limited to) xanthine oxidase, nadph oxidase, and nitric oxide synthase [ ] . these are believed to further stimulate ros generation in the mitochondria, forming a vicious cycle and making mitochondria de facto the largest ros source during reperfusion [ , ] . the elevated superoxide anion (o − ) reacts with nitric oxide (no), producing the highly reactive peroxynitrite. this leads to a reduction in the availability of no (which is a potent vasodilator) and causes the accumulation of neutrophils [ ] . furthermore, oxidative damage causes lipid peroxidation, dna damage, and enzyme denaturation and activates the innate anti-inflammatory response, aggravating reperfusion injury (ri) [ ] . the occurrence of mpt during the reperfusion phase was demonstrated by the experiments of griffith and halestrap in . they showed that mitochondrial accumulation of radioactive deoxy glucose (hot-dog), which can pass through the imm only during mpt, did not occur in isolated hearts undergoing ischemia, but was significantly induced (and inhibitable by csa) during reperfusion [ ] . in the past decade, several in vitro and in vivo studies have confirmed the involvement of mpt in ischemia/ri [ ] . the administration of csa (as well as its analog fk ) has been shown to protect against ischemia/ri in multiple animal models and in multiple tissues, including cardiac and skeletal muscle, brain, kidney, liver, lungs, and testis [ ] [ ] [ ] [ ] [ ] [ ] . ppif -ko mice are significantly protected from ischemia/ri in both cardiac muscle and the brain, in terms of both tissue function and survival rates [ , , ] . in addition to cypd manipulation, genetic interference in mechanisms of ca + homeostasis proved the importance of mpt in ischemia/ri. cardiac-specific ablation of ncx significantly decreased ischemia/ri in isolated hearts [ ] . similarly, mice overexpressing bcl have reduced [ca + ] m accumulation and are therefore protected from myocardial ischemia/ri [ ] . accordingly, tissue-specific overexpression of the mitochondrial na + /ca + exchanger (ter-nclx) in cardiac muscle accentuates the extrusion of ca + from mitochondria to the cytosol and then suppresses [ca + ] m , decreasing the sensitivity of cardiomyocytes to ptpc opening. hearts from ter-nclx mice also display protection from left coronary artery ligation-induced ischemia/ri [ ] . additionally, impairing mitochondrial calcium uniporter (mcu), using the inhibitor ru [ ] or in mcu-ko mice [ ] , lowers the uptake of mitochondrial ca + and is correlated with increased brain and heart function. we recently demonstrated that inhibiting atp synthase via n,n-dicyclohexylcarbodiimide (dccd) partially recovered contractility of isolated heart exposure to ischemia/ri by langhendorff apparatus. most interestingly, we observed that serum levels of the c subunit in patients with st-segment elevation myocardial infarction (stemi) correlated to several surrogate markers of myocardial reperfusion [ ] . renal tissue is also known to be affected by mpt in multiple conditions. acute kidney failure (mostly known as acute kidney injury, aki) is often characterized by extensive necrosis. the protective effect of csa or cypd inactivation on experimental ischemia/ri has been largely reported in kidneys, as previously stated. atherosclerotic renal artery stenosis (aras) is probably the most frequent cause of ischemia/ri-related aki. the outcomes of experimental aras are significantly improved by exposure to the mitochondrial-targeted peptide elamipretide. this peptide displays multiple protective functions, including the buffering of ros and the stabilization of the structural mitochondrial lipid cardiolipin. specifically, in experimental aras, the protective effect of elamipretide is believed to be mediated by desensitization to ptpc opening [ ] . renal tissue can also undergo, at nominal po , conditions resembling manifestation of ri and that appear to be dependent on mpt. for example, an important cause of aki with extended necrosis is exposure to drugs (e.g., fans or chemotherapy) and crystal nephropathies. the etiology of these ischemic-like conditions is not yet fully comprehended, although it is proposed to act through excess ros production, which ultimately triggers ptpc opening. in support of this model, under conditions of oxidative stress, glycogen synthase kinase- β (gsk β), an interactor and regulator of the putative ptpc, translocates from the cytosol to mitochondria in a vdac -dependent manner, promoting ptpc opening [ ] . inhibition of gsk β promotes resistance to mpt in mice undergoing paraquat-or diclofenac-induced nephrotoxicity [ , ] . interestingly, it is now well recognized that cell death during aki is significantly dependent on necroptosis, a finding largely confirmed by investigations on mlkl-ko and ripk -ko animals [ ] . cisplatin-induced aki is protected by the inactivation of both ripk and cypd. ripk /ppif -double-ko models are more protected than the models with either ko [ ] , suggesting that mpt and ripk contribute to rcd by independent but concomitant pathways. a recent study by mulay et al. using cypd-ko and mlkl-ko mice showed that experimental conditions mimicking acute oxalosis (crystal nephropathy characterized by sudden increases in serum oxalate levels) resulted in kidney failure with necroptotic features that was strongly dependent on mpt [ ] . that study, however, did not report significant differences among the ppif -ko, mlkl-ko and ppif /mlkl-double-ko mice in terms of protection from cell death. degenerative disorders are human diseases characterized by the chronic loss of fully differentiated cells, which leads to the progressive impairment of the structure and/or function of the affected tissue/organ. among the most frequent and probably most investigated degenerative disorders are conditions that affect the cns or the skeletal or cardiac muscle. as previously mentioned, these organs all require large amounts of energy; therefore, it is not surprising that the mutations most frequently associated with degenerative disorders are linked to mitochondria. intriguingly, many of these conditions share impaired mitochondrial respiration and atp production, increased ros production, and altered ptpc sensitivity. the involvement of mpt in these disorders appears to be more complex than the involvement of mpt in ischemia/ri in terms of molecular and cellular interactions. we therefore summarize the major observations made in different degenerative conditions. the primary features of neurodegeneration are the abnormal presence and accumulation of mutant and/or damaged proteins. protein aggregation is the main cause of changes in the intracellular environment such as oxidative status, impaired protein quality control system, transcriptional alteration, and mitochondrial dysfunction. all these variations critically contribute to the pathogenesis of nds and culminate in neuronal cell death. among the diverse types of proteins that aggregate, amyloid-beta (aβ), tau, and alpha-synuclein (αsyn) are the most commonly studied and represent the primary cause of sporadic and familial alzheimer's disease (ad) and parkinson's disease (pd), the most prevalent nds. interestingly, aβ, tau protein, and αsyn are found in the mitochondria of patients affected by ad or pd [ ] [ ] [ ] or their related animal models [ ] [ ] [ ] . in particular, these protein aggregates have been observed to colocalize or directly interact with multiple partners of the ptpc, such as cypd, vdac, ant, and atp synthase [ , , , ] . additionally, aβ exposure in cultured cortical neural progenitor cells induces ptpc opening [ ] . in particular, short aβ exposure led to decreased cell proliferation. however, when the exposure and thus ptpc opening were prolonged, csa-inhibitable necrotic rcd was activated. consistent with this result, intravital multiphoton imaging of ad mouse models demonstrated that near senile aβ plaques, mitochondria showed severe structural and functional abnormalities, suggesting that senile plaques are the main source of toxicity in vivo [ ] . aβ plaques and phosphorylated tau interact with vdac, leading to mitochondrial dysfunction [ ] . similarly, a specific tau fragment (nh - - fragment) affects oxphos and mitochondrial dynamics by interacting with ant and impairing ptpc regulation [ ] . furthermore, αsyn oligomers move into mitochondria and colocalize with atp synthase, inducing its oxidation concomitantly with increased ptpc opening, mitochondrial swelling, and necrosis activation [ ] . additionally, a pd mouse model characterized as having a mutant human αsyn (thy -hαsyn-a t tg mice) proved that αsyn associates with neuronal mitochondria and interacts with vdac and cypd in vivo [ ] . this work directly linked motor abnormalities and neuropathology to the ptpc. the study of rare inherited mutations in pd has provided insight into the molecular mechanisms of mitophagy, the regulated delivery of dysfunctional mitochondria to lysosomes via autophagic machinery. multiple pd-related genes have been identified, among which the mitochondrial kinase pink and the cytosolic e ubiquitin ligase parkin are the most characterized. the ptpc may be involved in mitophagy-mediated quality control processes, which play a critical role in conserving neuronal health and function. indeed, it has been suggested that the opening of the ptpc regulates mitochondrial depolarization and subsequent mitochondrial degradation in autophagosomes. furthermore, csa and its analogs block autophagosome formation, and the alteration of ptpc opening has been unveiled in models lacking pink , parkin, and dj , which are components of the best-characterized stress-induced mitophagy pathways [ , ] . consistently, the downregulation of pink in mouse neurons resulted in altered mitochondrial morphology and function, ros production, and finally ptpc opening. all these events were accompanied by the induction of mitochondrial autophagy [ ] . similarly, pink -deficient neurons showed selective increases in mitochondrial ca + , ptpc opening, and defective mitochondrial respiration. in addition, the inhibition of ptpc opening was found to be sufficient to rescue the mitochondrial impairments observed in pink −/− cells [ ] . ros production and ptpc opening were increased in primary mouse embryonic fibroblasts (mefs) and brains from park −/− (the gene coding for dj- ) mice compared with wild-type (wt) samples. in contrast, antioxidant molecules decreased ros levels and ptpc opening. interestingly, in contrast to pink −/− cells, the lack of dj- did not affect mitochondrial respiration and ca + dynamics, suggesting that dj- has a possible antioxidant role. finally, parkinsonian toxins (such as -hydroxydopamine and neurotoxin -methyl- -phenyl- , , , -tetrahydropyridine) are widely employed as in vivo and in vitro chemical models of pd and have been found to be potent activators of both ptpc and mitophagic processes [ , ] . notably, both vdac and ant were demonstrated to be required for proper mitophagy [ , ] . finally, recent pieces of evidence demonstrated that human samples obtained by ad-affected patients displayed inhibited damaged mitochondrial clearance [ ] and that mitophagy activation diminished insoluble aβ and tau hyperphosphorylation to revert cognitive impairments in an ad mouse model [ , ] (figure ). taken together, these findings suggest that the mitochondrial accumulation of disease-specific protein aggregates might favor mpt via direct interactions or mitophagy impairment, which leads to the accumulation of mitochondria prone to ptpc opening and rcd (figure ). als is the most common neuromuscular degenerative disease affecting adults. while several works have suggested a protein-aggregation origin, this progressive and severely disabling fatal neurological disease is generally considered to have multifactorial causes. currently, there are no cures or effective treatments for als, and the molecular pathogenesis of als is poorly understood. recent findings show that mitochondrial perturbations are implicated in the pathogenesis and progression of als. altered fission-fusion dynamics, altered mitochondrial ca + homeostasis, excessive oxidative stress, reduced oxphos activity, and decreased proapoptotic factor release have been found in als models in vitro and in vivo. additionally, the ptpc is emerging as a critical player in als. in a transgenic model of als (g ahigh), a profound alteration in mitochondrial structures with increased ptpc activity was observed [ ] . interestingly, dendritic mitochondria from the same als animal model displayed increased contact sites between the imm and omm. this conformation might favor the formation of the ptpc. furthermore, the deletion of cypd delayed disease onset and extended the survival of transgenic als mice [ ] . consistent with this finding, the exposure of two independent als murine models to the novel ptpc inhibitor gnx- protected against motor neuron degeneration and mitochondrial impairment and promoted their survival nearly -fold [ ] . preclinical studies have shown that olesoxime, a member of the cholesteroloxime family, improves the survival of neural cells and reduces the effects of oxidative stress by modulating the ptpc. in particular, this compound concentrates in mitochondrial compartments, where it binds the ptpc interactors vdac and mitochondrial translocator protein (tspo). following olesoxime binding, the ptpc was desensitized, leading to neural cell protection both in vitro and in vivo [ , ] . accordingly, the potent antioxidant and inhibitor of ptpc edaravone (radicut™) has been approved for the treatment of als [ ] . compounds derived from cinnamic anilides such as gnx- and gnx- also represent an mpt-based treatment of als. in a murine model of als, these compounds delayed the onset of symptoms, increased lifespan, and reduced the inflammatory response [ ] . ms is the most common primary demyelinating disease of the brain. ms is an inflammatory t-cell-mediated autoimmune disease characterized by progressive demyelination, gliosis (scarring), and neuronal loss [ ] . recently, mitochondrial dysfunction has been increasingly linked to the pathogenesis of ms. additionally, impaired mitochondrial enzyme complex activity [ , ] , increased oxidative stress [ ] , altered mitochondrial dna [ ] , impaired quality control systems [ , ] , and abnormal mitochondrial number and morphology have been described in ms patients [ ] and in vivo ms mouse models [ , ] . in this context, it was discovered that the ptpc might also contribute to ms; indeed, cypd-ko mice with experimental autoimmune encephalomyelitis (eae), a commonly used animal model for ms, recovered from their induced disabilities. furthermore, axonal damage was decreased, and the mitochondria of cultured cypd-ko neurons accumulated higher levels of ca + and were more resistant to oxidative stress compared to wt [ ] . subsequent studies confirmed this finding and demonstrated that the selective inhibition of ptpc exerted neuroprotective effects on the eae model by increasing mitochondrial function, reducing oxidative stress, and blocking mitochondrial swelling and ca + -mediated ptpc formation [ ] . the p shc protein may also modulate the mitochondrial dynamics and ptpc opening that occur during neurodegeneration in eae. p shc is a product of the shca gene normally localized in the cytoplasm. however, once phosphorylated by protein kinase c-beta and following interaction with pin , p shc moves to the mitochondria, where it regulates distinct cellular processes such as apoptosis and autophagy [ , ] . furthermore, in the mitochondrial compartment, p shc works as a ros amplifier by generating mitochondrial ros to induce ptp opening. consistent with this function, when eae was induced in p shc-ko (p shc−/−) mice, the clinical symptoms (manifested as limb weakness and paralysis) of these model mice were less severe than those of wt mice [ ] . the fact that the onset and development of eae in p shc/cyc-d-double-ko mice were identical to those observed in p shc−/− mice validates the role of the p shc-ptpc pathway in neurodegeneration and confirms that once activated, p shc interacts with ptpc to promote its opening [ ] . mds refer to a clinically and genetically heterogeneous group of degenerative muscle diseases, manifested primarily as the progressive weakness and degeneration of skeletal muscles that control movement, resulting in severe pain, disability, and ultimately death. some forms of md also affect cardiac muscle [ , ] . current therapies to treat muscle degenerative diseases are still limited by poor targeting, although promising new therapeutic directions remain [ ] . the most common and severe form of human muscular dystrophy is duchenne md (dmd), which is an x-linked recessive genetic disorder associated with respiratory complications and cardiac dysfunction [ ] . the disease is caused by a genetic defect-the absence of the cytoskeletal protein dystrophin, the primary function of which is to link the myofiber cytoskeleton to the extracellular matrix, stabilizing the sarcolemma [ ] [ ] [ ] . although the gene underlying the disorder was identified in [ ] , the pathophysiology leading to disease remains unclear. numerous mitochondrial alterations are correlated with dystrophic conditions, including impaired atp production, substrate handling, ca + buffering capacity, and elevated ros production. the mitochondria in muscular fibers from dystrophic mdx mice (a murine model of dmd) displayed a significantly shorter time to mpt induction in response to ca + than wt mice [ ] . in c. elegans and zebrafish models of dmd, mitochondrial fragmentation is detectable before overt signs of muscular degeneration, and csa feeding delays muscle degeneration [ ] . cyclosporine also inhibits calcineurin, a signaling protein involved in skeletal muscle, and its inhibition was found to worsen muscular dystrophy in an mdx mouse model [ , ] . nonetheless, the deletion of ppif and the administration of debio- (a csa inhibitor with no effect on calcineurin) prevented dystrophic conditions in mdx and d-sarcoglycan-ko (scgd−/−) animals [ , ] . additionally, several lines of evidence have shown that ppif deletion is protective in col a −/− mice [ ] . mutations of collagen vi (colvi) genes encoding the extracellular matrix protein, which is abundant in skeletal muscle, cause three muscle diseases in humans: ullrich congenital muscular dystrophy (ucmd), bethlem myopathy (bm), and the recently identified myosclerosis myopathy (mm) [ , ] . these collagen vi myopathies are inherited muscle diseases that share mitochondrial dysfunction due to altered ptpc opening [ ] . mouse models lacking collagen vi (col a −/−) display an early onset myopathic phenotype correlated with ultrastructure defects in mitochondria and the sarcoplasmic reticulum (sr), altered mitochondria caused by eventual inappropriate ptpc opening, and elevated muscle fiber apoptosis [ , , ] . in addition, the absence of colvi led to a marked decrease in the expression of proapoptotic bcl- , which may synergize with calcium to enhance the opening of the ptpc and eventually promote the release of mitochondrial proapoptotic factors [ ] . in light of this fact, the altered expression of collagen in col a −/− mice and in bm and ucmd patients correlates with enhanced ptpc opening, resulting in the functional and ultrastructural deficiency of mitochondria, followed by impaired autophagy, [ ] . as such, autophagy is altered in mds, and autophagy activation due to low amino acid intake improved the skeletal muscle phenotype of a dmd mouse model (mdx), [ ] . consistently, pharmacological treatment with csa has been reported to dramatically recover myofiber degeneration in a col a −/− mouse model and ucmd patients [ , ] . additionally, it has been demonstrated that mitochondria-mediated cell death can be reduced by csa in ullrich congenital muscular dystrophy models [ ] . mitochondrial diseases are a clinically heterogeneous group of disorders that arise because of mitochondrial respiratory chain dysfunction. these diseases are caused by mutations in nuclear dna (ndna) and in mitochondrial dna (mtdna). although mitochondrial diseases can involve any organ or tissue, they characteristically involve multiple systems, typically affect organs that are highly dependent on aerobic metabolism and are often relentlessly progressive with high morbidity and mortality. like other degenerative diseases, the involvement of mpt in mitochondrial diseases has been proposed. mutations in mtdna are responsible for the etiology of the most frequent mitochondrial diseases, especially leber's hereditary optic neuropathy (lhon); neurogenic muscle weakness, ataxia, and retinitis pigmentosa (narp); mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (melas); and myoclonic epilepsy with ragged red fibers (merrf). these alterations most frequently involve single-base mutations in genes encoding components of respiratory complex i, atp synthase, transfer rna, and dna polymerase, but also may be caused by large deletions of mtdna. hybrids carrying mtdna mutations associated with lhon, narp, melas, and merrf displayed poor resistance to oxidative stress, which could be prevented by the administration of csa or the deprivation of extracellular ca + [ ] . accordingly, many of these mutations lower the threshold for ptpc opening in response to ca + and ros [ , ] . mutations in ndna associated with mitochondrial diseases have also been also related to alterations in mpt activity. mutations in leucine-rich pentatricopeptide repeat containing (lrpprc), a protein involved in the maturation and stability of mitochondrial rna [ ] , cause the french-canadian variant of leigh syndrome. loss of lrpprc results in defects in the assembly of respiratory complexes iv and v, leading to severe metabolic alterations. fibroblasts isolated from patients presenting lrpprc inactivation displayed multiple types of mitochondrial dysfunction, including a reduced threshold for ca + -induced mpt [ ] . interestingly, all the mitochondrial alterations mentioned so far are often associated with impaired or unstable assembly of atp synthase, providing significant clues to its involvement in mpt. direct investigations of atp synthase alterations and mpt in the etiology of mitochondrial disease have not been performed. other significant nuclear genes relating mitochondrial diseases to mpt are optic atrophy (opa ) and spastic paraplegia (spg ). opa is an essential protein involved in the fusion and cristae arrangement of the imm. its mutations manifest clinically as optic atrophy, ataxia, and deafness [ ] . spg (paraplegin) is an atp-dependent zinc metalloprotease located in the mitochondrial matrix, and its mutations are associated with chronic progressive ophthalmoplegia [ ] . interestingly, both proteins positively regulate the mpt threshold in response to ca + induction, and their inactivation significantly inhibits ptpc opening [ , ] . mechanisms by which opa and spg act on ptpc are still to be defined. while the role of opa seems to be dependent on its control of crista morphology [ ] , spg appears to regulate the amount of ca + available for mpt via regulation of mitochondrial calcium uniporter assembly [ ] . taken together, this evidence indicates that ptpc might be a significant target for mitochondrial disease; however, the mechanism by which mpt influences these diseases is still poorly understood and might differ among syndromes, calling for further investigations in more complex experimental models. nonalcoholic fatty liver disease (nafld) is among the most prevalent chronic liver diseases in both children and adults, and is predicted to be the primary cause for liver transplants by [ ] . nafld is characterized by an accumulation of fat (steatosis) in the liver, which can progress to inflammatory nash and into more severe stages: fibrosis, cirrhosis, and hepatocellular carcinoma [ ] . the prevalence of nafld has risen rapidly in western societies, particularly in most of the european countries, due to an increase in mass consumption of highly processed ready-made food, which is rich in fructose and saturated fat. nafld has risen rapidly in parallel with the recent surge in metabolic-related diseases such as obesity and type diabetes mellitus (t dm), which have been indicated as risk factors for the prevalence and progression of nafld. despite the attempts of the liver to recover from fat accumulation, in the long run, mitochondrial adaptation is insufficient to prevent lipotoxicity due to continuous ffa accumulation [ ] . at this later time point, mitochondria present alterations in the oxphos complexes, mitochondrial membrane potential, reduced atp synthesis, and induced ptpc opening [ ] . opening of the ptpc may be the basis of the steatosis-induced apoptosis of hepatocytes observed in vitro, and may be related to the steatosis in nafld of human beings [ ] . it has been postulated that stimulated ptpc opening in a rat model of nafld is the result of increased bax expression and aberrant bcl- /bax ratio. this seems to be an important mechanism of the mitochondrial damage in hepatocytes that occurs in nafld [ ] . wang et al. proposed that the overexpression of mitochondrial hepatic cypd induced mitochondrial stress and could be an early event that leads to the liver steatosis [ ] . they found that overexpression of cypd is manifested in mitochondrial swelling and increased mitochondrial ros production. such mitochondrial perturbations provoke er stress through ca + /p mapk activation, finally resulting in the increase of srebp c-mediated synthesis of triglycerides. interestingly, in mice fed with a high-fat diet, the increased level of cypd was observed earlier than triglyceride accumulation in the liver. moreover, wang et al. speculated that cypd knockout or pharmacological inhibition of cypd could ameliorate triglyceride accumulation in hfd-fed mice [ ] . on the other hand, the observations of lazarin et al. indicated that mitochondria isolated from livers of monosodium l-glutamate obese rats were less susceptible to the opening of ptpc by calcium [ ] . regardless of several pieces of evidence for the involvement of the mpt in the nafld animal models, there is no direct evidence supporting the role of ptpc and especially cypd in nafld in humans. one of the earliest established hallmarks of cancers is the resistance of transformed cells to rcd. considering the discussed role of mpt in rcd, it is straightforward to hypothesize that the alteration of the ptpc machinery is involved in the establishment of neoplasia. according to this hypothesis, mpt is predicted to have a tumor-controlling mechanism, and its suppression is required for tumor development. it is harder to prove that a phenomenon does not occur than it is to prove that it does occur. indeed, strong direct evidence confirming or confuting this hypothesis is lacking. experiments based on the genetic manipulation of cypd for the other disease types discussed should provide significant evidence, but nothing of this kind has yet to be reported. still, reduced mpt in transformed cells could be predicted by a large number of findings, as discussed below. another significant hallmark of cancer is metabolic rewiring, especially the abnormal increase in the glycolytic rate at almost normal po (warburg effect). this large glucose consumption causes the significant conversion of pyruvate into lactate, resulting in intracellular acidification and leading to ptpc desensitization similar to that observed in ischemia [ ] . furthermore, many solid tumors develop a hypoxic area that, analogous to ischemic tissue, also promotes hif a accumulation and hkii-mediated desensitization of ptpc. the warburg effect then allows the hypoxic tumor to limit pi and ca + accumulation, similarly to ischemia, favoring the inhibitory effect of low ph. if, by analogy to ischemia, an increase in [ca + ] could be expected, it is true that transformed cells have lower intracellular ca + (figure ) . indeed, it has been shown that h-ras-driven transformation is concomitant with a progressive reduction in the amount of intracellular ca + [ ] . bcl-xl has been shown to negatively regulate ptpc opening by directly interacting with vdac [ ] . additionally, bcl-xl can interact with the β subunit of atp synthase to promote its synthase activity and inhibit ptpc [ ] . furthermore, many other oncogenes and oncosuppressor genes regulate ca + . among the most characterized is bcl- , which can impair [ca + ] in the er lumen and its transfer to mitochondria via multiple proposed mechanisms [ ] . additionally, many tumors show alterations in the pi k pathway. members of this pathway, akt and pten, can localize at mitochondrion-er contact sites, especially with ip r, to alter its activity [ ] [ ] [ ] [ ] [ ] . this effect is coordinated by the oncosuppressor pml [ ] [ ] [ ] . loss of pml or pten and activation of akt (conditions prototypical of multiple tumor types) lead to the inactivation of ip r, which also limits the ca + available to mitochondria. additionally, akt phosphorylates gsk β (altered in several cancer types), resulting in its inactivation and, as discussed, ptpc desensitization [ ] . finally, the master oncosuppressor p interacts with serca to maintain a high level of er [ca + ]. loss or inactivation of p (one of the most common alterations in cancer) impairs [ca + ] and results in reduced sensitivity to mpt-mediated rcd [ ] [ ] [ ] [ ] [ ] . in addition, p can localize to mitochondria and interact with cypd to favor mpt and necrosis. interestingly, a network of chaperones seems to interact with cypd (which could be considered a chaperone itself) to modulate ptpc. in particular, hsp , hsp , and dnajc , which are often overexpressed in tumors, have been shown to interact with cypd, leading to their inhibition (then mimicking csa) and suppressing rcd initiation [ ] [ ] [ ] . these findings are supportive of the hypothesis, but contradict other strong evidence. first, transformed cells often have increased levels of ros, not of a significant increase in multiple scavenging systems. it is currently accepted that elevated ros can act as a mitogenic signal in tumors to support proliferation [ , ] . in addition, studies in cancer cell lines and tumor models have shown that different ptpc members are overexpressed, especially tspo, vdac, and ant, possibly to favor their specific metabolic condition [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we can, therefore, speculate that tumor cells can survive pressure selection by the highly regulated suppression of mpt, which allows the maintenance of mpt-related features with potential mitogenic effects ( figure ). because of the presented evidence (and much more), mpt has been investigated for the treatment of human disease in multiple clinical trials. csa entered a trial procedure that lasted years and ended with failure at phase iii in cardiac ri. indeed, the circus [ ] and cycle [ ] trials consisting of a single intravenous bolus of csa ( . mg/kg) before revascularization had no effect on st-segment resolution or cardiac enzymes and did not improve clinical outcome. these findings were reconsidered by a study by piot et al., who saw hope in the use of csa to treat ri. similarly, tspo targeting by , -seco- -nor-cholestan- -one oxime- -ol (tro ) was tested in clinical trials, due to promising cardioprotective effects on a rat model of cardiac ischemia. however, the desensitization of ptpc opening seemed to be secondary to its remarkable antioxidant properties [ ] . indeed, studies on tspo-ko mice showed that the protein was dispensable in models of ischemia/ri [ ] . however, the safety and efficacy of this drug were evaluated a few years later in patients undergoing percutaneous coronary intervention (pci). this multicenter, double-blinded, phase ii study (mitocare) showed the inefficacy of the compound in reducing or limiting ri [ ] . additionally, an oral version of csa was tested for the treatment of lhon but failed to reach the primary endpoint of the study, although it delayed the onset of the disease. [ ] . there are multiple reasons that csa-based trials have failed to reproduce the protection reported in preclinical studies, but a discussion of these failures is not the purpose of this review. the investigation of novel compounds able to target the ptpc is ongoing, and many investigations have already yielded promising results in preclinical studies. most of these are designed to be more potent and specific inhibitors of cypd, including the small molecules c- , c- , and c- , which have already been proven to be protective in models of ad, acute pancreatitis, and hepatic injury [ ] [ ] [ ] . library screening has also identified ml- [ ] and n-phenylbenzamide [ ] as cypdindependent, offering the possibility to combine their use with csa to limit its side effects. it is of interest that atp synthase is now the subject of investigation in these terms. oligomycin and dccd, which target the c subunit, displayed powerful mpt inhibition in vitro, but they also depleted mitochondrial atp, causing an additional injury [ ] . we recently generated a library of small molecules that target the c subunit by modifying the functional core of oligomycin and obtained new patented compounds able to notably reduce reperfusion damage in animal models of global ischemia without interfering with atp production [ ] . finally, the natural hormone melatonin is of great interest. this hormone can act as an antioxidant and can modulate the ptpc, although its exact mechanism is currently under investigation [ , ] . currently, melatonin is considered the safest drug that can be used as a ptpc inhibitor, and we will probably see an increasing number of investigations on this molecule in the future. funding: p.p. is grateful to camilla degli scrovegni for continuous support. the signal transduction laboratory is supported by the italian association for cancer research (airc: ig- to p.p. and ig- to c.g.), a-rose, telethon (ggp b to p.p.), progetti di rilevante interesse nazionale (prin e l p to p.p and prin e epy to c.g.), the italian ministry of health (gr- - to c.g.), the european research council (erc, -inflapml to c.g.), local funds from the university of ferrara to p.p. and m.b. s.p. was supported by fondazione umberto veronesi. mrw was supported by the national science centre, poland (umo- / /b/nz / ). moreover, mrw gratefully acknowledge the financial support from the foie gras and mtfoie gras projects. these projects received funding from the european union's horizon research and innovation program under the marie skłodowska-curie grant agreement no. 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transition pore complex by a j-protein, dnajc role of reactive oxygen species in cancer progression: molecular mechanisms and recent advancements mitochondria and reactive oxygen species in aging and age-related diseases targeting mitochondria for cancer therapy characterization of porin isoforms expressed in tumor cells relation of cell proliferation to expression of peripheral benzodiazepine receptors in human breast cancer cell lines specific ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in human colorectal cancer cells expression of oxidative phosphorylation genes in renal tumors and tumoral cell lines glucose catabolism in cancer cells: amplification of the gene encoding type ii hexokinase isoenzyme pattern and subcellular localization of hexokinases in human breast cancer and nonpathological breast tissue cyclosporine before pci in patients with acute myocardial infarction new cardioprotective compound, inhibits mitochondrial permeability transition regulation of the mitochondrial permeability transition pore by the outer membrane does not involve the peripheral benzodiazepine receptor (translocator protein of kda (tspo)) effect of intravenous tro as an adjunct to primary percutaneous coronary intervention for acute st-elevation myocardial infarction: mitocare study results cyclosporine a does not prevent second-eye involvement in leber's hereditary optic neuropathy structure based design, synthesis, pharmacophore modeling, virtual screening, and molecular docking studies for identification of novel cyclophilin d inhibitors small molecule inhibitors of cyclophilin d to protect mitochondrial function as a potential treatment for acute pancreatitis small-molecule inhibitors of cyclophilins block opening of the mitochondrial permeability transition pore and protect mice from hepatic ischemia/reperfusion injury synthesis, and optimization of diarylisoxazole- -carboxamides as potent inhibitors of the mitochondrial permeability transition pore n-phenylbenzamides as potent inhibitors of the mitochondrial permeability transition pore melatonin as a master regulator of cell death and inflammation: molecular mechanisms and clinical implications for newborn care melatonin protects cardiac microvasculature against ischemia/reperfusion injury via suppression of mitochondrial fission-vdac -hk -mptp-mitophagy axis key: cord- -xddikzxs authors: wiseman, a. title: avoidance of oxidative‐stress perturbation in yeast bioprocesses by proteomic and genomic biostrategies? date: - - journal: lett appl microbiol doi: . /j. - x. . .x sha: doc_id: cord_uid: xddikzxs aims: bioprocess oxidative stress caused by many reactive oxygen species (ros) can lead to largely irreversible perturbation of yeast bioprocesses. these include the production of proteins derived from recombinant dna yeast technology (aerobically grown saccharomyces cerevisiae). these proteins include rennin, amyloglucosidases (glucamylases), interferons, interleukins, insulin, monoclonal antibodies, tissue plasminogen activators (t‐pa), sexually transmitted disease antigens, and measles, mumps and rubella antigens, growth hormones, somatotropin, blood clotting factors viii and xiii. in addition, there may be a demand for severe acute respiratory syndrome–coronavirus antigens, hepatitis a, b and c viral‐selected antigens, hiv retroviral antigens, influenza antigens, trypanosomal antigens, and foot and mouth disease antigens. prevention of oxidative stress has been achieved by application of antioxidant redox metalloenzymes such as superoxide dismutases (containing cu/zn cytosolic, mn mitochondrial and fe bacterial) glutathione peroxidases (and other se‐containing proteins and enzymes such as the thioredoxins), catalases (fe‐containing), cytochrome c peroxidases (fe‐containing), ceruloplasmins (cu‐containing), metallothionines (these cysteine thiol‐rich proteins bind ions of cadmium and mercury) and tyrosinases(cu‐containing). methods and results: ros are generated inadvertently by single metal valency couples such as feii/feiii and by feiii/fev present in (including human) isoforms in cytochromes p mixed‐function oxidases (ec · · · ; o( ) : mono‐oxygenase nadph/nadh requiring). in addition, mixed‐metal couples such as valency unmatched forms in cui/feii and feiii/mniv can recycle electrons. moreover, proteins/protein chaperone couples can recycle electrons, often where futile‐recycling systems have been instigated. furthermore, oxidized membrane phospholipids (r) can form rooh (lipid hydroperoxides) and roh (lipid alkoxides) that can generate ros through fenton chemistry (iron‐catalysed) chain reactions. utilization of chain‐breaking antioxidants such as vitamin e (α‐tocopherol) in the lipid phase and vitamin c (ascorbate) in the aqueous phase can terminate these ros‐producing reactions. conclusions: the main significance of the study is that proteomic strategies of relief from bioprocess perturbation by ros of yeast fermentations (used to manufacture proteins required in the food and therapeutic bioindustries) may become possible through addition of selected proteins (including metalloenzymes). the main impact of the study is that the utilization of genetically modified (gm) yeast produced by recombinant dna technology genomic strategies could circumvent the bioprocessing problems that otherwise result from the bioprocess perturbations: this is as a result of oxidative stress caused by ros, which is avoidable by deployment of appropriate antioxidants such as vitamins e, c and d (and antioxidant proteins and enzymes often of microbial origin via recombinant dna technology). where biomolecular injury is caused by reactive-oxidant molecular species, such as reactive oxygen species (ros and toxic metals, see tables and ), redox chemistry plays a dominant role in pathogenesis, through the onset of outcomes resulting from biomolecular injury (see . in relation to this, many metals are able to perform futile one-electron recycling between couples formed between appropriate valency states (wiseman and woods ) . furthermore, hydrogen radical abstractions can occur that can trigger a chain reaction in unsaturated phospholipids of oxidized membranes (both periplasmic membranes and those of cytoplasmic organelles) that releases breakdown products (when heated with feii/ascorbate) such as malondialdehyde that are appropriate for bioassay by the thiobarbituric acid (tbars) procedure (halliwell and gutteridge ) . furthermore, the likelihood of superoxide anion-free radical (aeo ) generation produces an enhanced risk of biomolecular injury as a manifestation of the biohazard of particular metal contamination, both of the environment and in the body. the essential role of oxygen gas, which has built up in the atmosphere as a result of the photosynthetic activity of green plants over a ae -billion-year period is paramount in ros production. the toxic nature of oxygen (abele ) , although essential for aerobic life on earth, has been subject to amelioration of toxicity through evolution of antioxidant enzymes that have developed over the last million years. moreover these antioxidant enzymes in the body, such as superoxide dismutase, glutathione peroxidase and catalase, augment the role of dietary antioxidants such as vitamins e and c (wiseman et al. ; wiseman and woods ) . furthermore, the use of dna recombinant forms may confer unexpected technological advantage in bioprocesses in cases where novel proteins, such as biocatalyst enzymes and cells, are introduced into previously unfavourable environments. where these introductions are associated table toxic and nontoxic metals that may be detectable in yeastbioprocess feedstocks (see table with the biometabolism because of gut microflora of animals, dietary idiosyncrasy considerations are involved in the prediction of harmful outcomes. these predictions therefore need to be evaluated as part of the risk/benefit analysis associated with the necessary toxic hazard/risk recommendation by studies in silico and in real-lab (in petri). there is a major interest in the possibility that bioactive compounds in foods from agriculture, particularly those in plant and microbially fermented foods, can protect against a wide range of diseases . furthermore, bioactive compounds can have gender-differentiated effects that are dependent also on a number of other factors including age, genomics and stage of reproductive development. complacency may be the result of overconfidence in the prediction of 'nontoxicity', to bioprocess and to consumers, but failure to predict the likelihood of a disaster outcome with foodstuffs, has been manifest in the past. examples include turkey x-disease caused by aflatoxin (a hepatotoxin and carcinogen) in groundnuts contaminated in storage by aspergillus flavus (late s africa; see tables and ) ; spanish cooking oil syndrome caused by contamination of cooking oil with aniline ( s); syndromes caused by forms of organic mercury produced by micro-organisms in waterways ( s). natural foodstuffs may also contain toxins (see tables and ) and protectants (see tables and ). molecular toxicology has provided a good mechanistic basis for prediction of future problems, and this approach is supported by the power of computer modelling on a wide range of unusual microbially produced chemicals that reach the body via methods of bioprocessing of foodstuffs that include solid-state fermentation. injury to macromolecules, table prevention of biomolecular damage in bioprocessing through addition of antioxidant biochemicals, enzymes and chaperone proteins (see table ) antioxidant chemicals vitamins such as e and c are free radical production chain breakers phytoestrogens (wiseman et al. free radical scavengers such as other free radicals reducing agents (low redox potentials) some proteins (thiol-containing for example) xenoestrogens such as bisphenol a, alkylphenols (octylphenols and nonylphenols) antioxidant enzymes catalases ( table ) in yeasts and other micro-organisms (antioxidants such as vitamins e and c chain terminate the formation of membrane-degrading ros) relates to more than just structural damage that is sometimes obvious and predictable: functional damage may also follow. moreover, functional damage can be predicted in silico: injury therefore to catalytic proteins (enzymes) and honorary enzymes (many other proteins) may result in functional problems with nucleic acids and other biologically active biomolecules. further investigation is needed on the interactions of macromolecules especially in micro-organisms prior to harnessing these into food bioprocessing. prevention of toxicity by safe choice (or gm control) of micro-organisms is to be recommended. unfortunately, prevention is based upon prediction of toxic responses via risk-prediction analysis usually in silico. cure or at least the 'successful treatment' of manifest bioprocess toxic responses may become the easiest option: these will then become a strategy to ameliorate the undesirable damage caused by biomolecular injury in yeast bioprocesses such as brewing (wiseman ; wiseman and woods a,b) . the recognition of characteristic responses of classes of cellular biomolecules, such as nucleic acids, proteins and phospholipids to injury was developed in the twentieth century. nevertheless, a more holistic approach can advance further our understanding of the behaviour, of these macromolecules under oxidative stress. a web of interactions is evident amongst biomolecules in micro-organisms, disturbance of such biomolecular webs by toxic substances is often the basis of the toxic manifestations seen as loss of homeostasis in pure cultures of micro-organisms. furthermore, stability of microbial species in mixed cultures has been attributed to relatively few species in a web of interaction: perturbation to the web by external forces may be subjected to compensatory restoration of the 'equilibrium position' amongst the microbial participants. moreover, supply of metabolites is attributable to enzyme action within the living micro-organisms. for example, atmospheric nitrogen-fixing (using the nitrogenase system) by soil organisms such as azotobacter vinelandii (aerobic) or clostridium pasteuranum (anaerobic) are controlled by metabolite supply. in the latter case, supply of pyruvate allows a chemostat interpretation of growth kinetics, mediated by the coa pyruvate ferredoxin oxidoreductase-catalysed cleavage of pyruvate to acetyl phosphate (this allows a subsequent production of atp) plus carbon dioxide. such microorganisms occupy an ecological niche that can be subjected to perturbation by the introduction of the end-product of nitrogen fixation (ammonia). linking species webs, are therefore a reflection of the underlying enzyme-catalysed webs of biochemical metabolites that make up both the cellular interior milieu of organisms and the associated biochemical mixtures in the growth and maintenance media, especially in scale-up fermentation bioreactors. immobilized enzyme utilization complicates the biochemical mixes present, and make reductionist approaches in prediction of expected biochemical balance more risky, especially if an unidentified microbial mix is employed as the bioindicator in pilot plant studies. toxicity to one species-web, may however favour stability of a competing species web: the presence of known (or unknown) yeast- phytoestrogen isoflavones include the soya protein-derived glucones daidzin and genistin. these are degraded to the aglycone forms daidzein and genistein by gut microflora: and these initial products can be degraded further by other bacteria in the gut for example to equol derivatives and equol conjugates with glucuronic acid (excreted through the kidney). fermented soya foods, such as miso or tempeh, contain mostly the unconjugated isoflavone aglycones . bioprocess antitoxicants (these are often antioxidants against ros), however, lower the accurate prediction of final outcome in the balance equilibria observed with mixed cultures of micro-organisms including bioprocess yeasts, and their enzymes used in bioprocessing (tucker and woods ) . the dilemma faced by molecular toxicologists for biohazard prediction lies, therefore, in an identification of chemostasis based upon a relatively few named species webs. resilience as opposed to growth perturbation may be predicted wrongly in silico for some mixed cultures of micro-organisms: it is essential, therefore, that real-lab (in petri) studies are also undertaken. species bioindicators and molecular biomarkers should both be subject to continuous assessment by critical biomonitoring to predict such yeast-bioprocess perturbation as opposed to resilience outcomes. harmful free radicals (halliwell and gutteridge ) , such as superoxide anion are produced in micro-organisms during aerobic respiration because of only partial reduction of some oxygen molecules in the electron transport chain. this is because of one-electron reduction of each atom of oxygen, instead of two-electron reduction to form water. similarly, in many micro-organisms the electron transfer chain from nadh to water (with insertion of one oxygen atom into xenobiotic substrates) that uses cytochromes p- may display futile cycling, in the absence of substrate, to produce the superoxide anion (aeo ). reduction of ros, and also of reactive nitrogen species (rns), is a priority for avoidance of production in particular microbial cultures susceptible to oxidative stress. for example, damage to biomolecules can occur by attack on phospholipid cytoplasmic and internal membranes, and also notably on dna: and on the recombinant dna of gm yeasts employed in manufacture of proteins such as insulin and interferon, intended for human therapy. all redox biochemicals, existing in their oxidized and reduced forms, constitute an oxidation/reduction couple, for instance, with polyphenols and some nutraceuticals (ridgway and tucker ) . each of such redox couples displays an experimentally determined redox potential (reduction oxidation potential) of a particular voltage under standard (defined) conditions of temperature and pressure (usually °c, ph ae , atm). the standard redox potential scale (e o ) has good oxidizing agents (pro-oxidants) such as molecular oxygen at the top with the high value of + ae v, and the reducing agents (antioxidants) at the bottom. for example, nadh is at ) ae v (the scale passes through zero) and a voltage difference of ae v therefore is associated with electron flow from nadh to o (in oxygen-utilizing respiratory chains). this potential difference is normally sufficient to allow the production of a total of at least three atp, each one at a particular step in the respiratory chain via known carriers by proton pumping through the inner membrane of yeast mitochondria. for instance, in interconversion of fe(iii) : fe(ii) the latter loses one electron during its oxidation to the feiii ( +) form and thus these two forms of iron are pro-oxidant (fe + ) and antioxidant (fe + ), respectively, but with the extra involvement of perferryl fe + in the cytochrome p mechanism via [feo] + the oxonium cation (lewis ; williams et al. ) . fe + /ascorbate is commonly employed to generate fe + , ostensibly the antioxidant form of the iron iii : iron ii couple to catalyse the breakdown of phospholipid liposomal membrane model systems. fe + reacts through fenton chemistry (halliwell and gutteridge ) with lipid hydroperoxides (rooh) or lipid alkoxides (roh) in the phospholipid mixture after storage in an atmosphere that contains oxygen, to form these peroxyl (roo • ) and alkoxy (ro • ) free radical derivatives of phospholipids. these free radicals initiate a chain reaction of further attack of the phospholipids via degradation reactions that produce large amounts of these free radical species (wiseman et al. ; see figs and ) . avoidance biostrategies against ros and rss in many oxidatively stressed bioprocesses that utilise yeasts (and other micro-organisms) are essential where human therapeutic proteins are expected to be produced in high yield. these proteins intended for therapy include insulin, inter ferons, interleukins, growth hormone and blood clotting factors (viii and xiii). moreover, the renewed demand for antigens of viruses such as hepatitis, severe acute respiratory syndrome (coronaviruses), foot and mouth disease, and hiv retroviruses, has established a greater demand for nonperturbation of yeast bioprocesses by ros (wiseman ) by development of stable phenotype despite variation in gene deletion (stearns ) . moreover, antioxidants, natural and synthetic, are being developed to supply the correct level of yeast-bioprocess resilience (wiseman ) (see tables - ) through a range of genomic and proteomic strategies. furthermore, the coupling in yeast bioprocess of thioredoxin (se) as an electron donor for peroxiredoxins (these use catalytic cysteine-cysteine residues to remove peroxide) can be viewed as an useful extension to other antioxidant enzymes which could overload the yeast bioprocess with unwanted metals such as cu and zn (see table ). nevertheless, se-supplementation could be viewed as health benefiting where soils are deficient in this metalloid antioxidant component of glutathione peroxidases (rayman ) . isoflavone phytoestrogens as functional ingredients in soya products: physiological importance of metabolism and possible indications for their rapid absorption following consumption of textured vegetable protein. functional foods ' th uk-nl nitrogen cycle meeting quantitative analysis of the flavonoid content of commercial tomatoes, onions, lettuce and celery free radicals in biology and medicine guide to cytochromes p structure and function se brought to earth procedure for the partial purification of apple leaf polyphenoloxidase suitable for commercial application safeguards and spurs enzymes in food processing crystal structure of human cytochromes p c with bound warfarin phytochemicals epidemiological factors biosustainability-limit to industrial biocatalysts if no supersedence of bioprocess perturbation by resilience? an enhanced multifunctional food -beer? the brewer international welcome praise for copper deleterious outcome of bioprocesses: unless ecobuffering can be effluent-incorporated by the utilisation of immobilised enzymes safe choice of metals in food bioprocess enzyme mimicry biomolecular free radical toxicity: causes and prevention isoflavone aglycone and glucoconjugate content of high-and low-soy uk foods use in nutritional studies key: cord- -ohjik pf authors: vorobjeva, n. v.; chernyak, b. v. title: netosis: molecular mechanisms, role in physiology and pathology date: - - journal: biochemistry (mosc) doi: . /s sha: doc_id: cord_uid: ohjik pf netosis is a program for formation of neutrophil extracellular traps (nets), which consist of modified chromatin decorated with bactericidal proteins from granules and cytoplasm. various pathogens, antibodies and immune complexes, cytokines, microcrystals, and other physiological stimuli can cause netosis. induction of netosis depends on reactive oxygen species (ros), the main source of which is nadph oxidase. activation of nadph oxidase depends on increase in the concentration of ca( +) in the cytoplasm and in some cases on the generation of ros in mitochondria. netosis includes release of the granule components into the cytosol, modification of histones leading to chromatin decondensation, destruction of the nuclear envelope, as well as formation of pores in the plasma membrane. in this review, basic mechanisms of netosis, as well as its role in the pathogenesis of some diseases including covid- are discussed. neutrophils are the largest population of myeloid leukocytes, which normally comprise % of all white blood cells. neutrophils differentiate in the bone marrow from hematopoietic stem cells and, after full maturation, enter the bloodstream. neutrophils circulate in the bloodstream for no more than h, thereupon, they either return to the bone marrow to be phagocytosed by resident macrophages, or undergo apoptosis in peripher al tissues, where they are also phagocytized. when the host organism is infected, neutrophils immediately migrate from the bloodstream to the site of infection pro viding a "first line" of defense against pathogens. being "professional" phagocytes, neutrophils con tain a huge amount of antimicrobial "weaponry" in their granules that allows them to destroy pathogens in the process of phagocytosis. bactericidal enzymes can be also released from the cells during degranulation. activation of nadph oxidase leads to mass generation of reactive oxygen species (ros), which are involved in the destruc tion of pathogens both inside the phagosomes and outside the cells. finally, there is another antimicrobial mecha nism representing the release of neutrophil extracellular traps (nets), which consist of modified chromatin "dec orated" with bactericidal proteins from granules and cytoplasm. this phenomenon was first described in the work of takei and co workers [ ] and subsequently char acterized in detail in the laboratory of arturo zychlinsky [ ] . since it was initially shown that net formation is accompanied by the cell death, this process was called netosis [ ] . formation of nets can be activated by various pathogens, such as bacteria, fungi, protozoa, viruses, as well as bacterial cell wall components -lipopolysaccha rides (lps). netosis can be induced by antibodies and immune complexes, cytokines and chemokines (il , tnf), microcrystals, and other physiological stimuli [ abbreviations: ards, acute respiratory distress syndrome; cgd, chronic granulomatous disease; copd, chronic obstructive pulmonary disease; gsdmd, gasdermin d; lps, lipopolysaccharide of the bacterial wall; mpo, myeloper oxidase; mptp, nonselective mitochondrial permeability tran sition pore; ne, neutrophil elastase; nets, neutrophil extra cellular traps; pcd, programmed cell death; pkc, protein kinase c; pma, phorbol myristate acetate; ros, reac tive oxygen species. * to whom correspondence should be addressed. in order to induce netosis in in vitro experiments, phorbol esters (in particular, phorbol myristate acetate, pma), which mimic the action of diacylglycerol activating protein kinase c, as well as calcium (iono mycin, a ) and potassium (nigericin) ionophores are often used. currently, two fundamentally different forms of netosis have been described: classical or suicidal netosis, which leads to the cell death, and vital netosis, in which the cell retains not only viability, but also many of its effector functions (see below). classical netosis is a special form of programmed cell death (pcd), which is characterized by the release of granule components into the cytosol, as well as chromatin decondensation associated with histone modification. at the same time, many features characteristic of other forms of pcd (apoptosis, necroptosis, pyroptosis, autophagy, secondary necrosis) are also inherent in netosis. during netosis, like in apoptosis, coordinated changes in the nucleus and in the cytoplasm occur, although the nature of the changes is different, and netosis, unlike apoptosis, does not require activation of caspases. unlike apoptosis, netosis (as the other forms of pcd) is accompanied by disturbance of insulating properties of the plasma membrane, and the mechanisms of permeabilization are similar for netosis, pyroptosis, and necroptosis. the signaling mechanisms of netosis may include activation of phosphoinositide kinase (pi k) [ ] , which also controls the autophagy induction, but assembly of autophagosomes is not required for netosis [ ] . the pathways leading to netosis and necroptosis can be especially closely intertwined [ ] . for a long time, it was believed that mitochondria do not play a significant role in the functioning of neutrophils, since their content in these cells is low, energy supply is supported by glycolysis, and nadph oxidase is the main source of ros. subsequently, however, it turned out that mitochondria are involved in the transmission of signals that determine main responses of the neutrophils to pathogens [ ] . it was found that mitochondrial ros are involved in the activation of nadph oxidase and in the induction of netosis caused by various stimuli [ , ] . nets formation has also been shown in other types of immune cells -eosinophils and mast cells [ ] , basophils [ ] , monocytes [ ] , and macrophages [ ] . interestingly, decondensed chromatin is not only used by animals for protection from pathogens, but also by uni cellular eukaryotes [ ] , as well as plants [ ] . thus, it can be assumed that the use of chromatin to protect the host against pathogens arose quite early in the course of evolution of eukaryotes. in recent years, data has accumulated on the role of netosis in a wide range of pathologies associated with inflammatory processes. this led to a surge in publica tions about netosis. almost , publications on this topic can be found in the pubmed database, of which about a third appears in . such intensive stud ies resulted in significant advances in understanding the nature of netosis, but at the same time have led to the accumulation of data that contradict each other. in this review, we attempted to consider basic concepts of the mechanisms of netosis, which determine its place among other forms of pcd. in addition, we briefly dis cuss the role of netosis in pathogenesis of certain dis eases. apparently, induction of netosis can be associated with suppression of apoptosis, which enhances the overall antimicrobial effect. the main mechanisms of nadph oxidase activa tion during netosis are probably not so much different from those that provide an oxidative burst of neutrophils in response to pathogens and other stimuli. we found in the course of investigation of activation of nadph oxi dase by the peptide chemoattractant n formylmethionyl leucyl phenylalanine (fmlp), which caused oxidative burst and degranulation but not netosis, that mitochon dria played an important role in these processes. mitochondria targeted antioxidant skq in submicro molar concentrations prevented activation of nadph oxidase induced by fmlp but not by pma [ ] . mitochondrial ros (mtros) stimulated nadph oxi dase with participation of pkc, but the primary target of their action remained unknown. this may be the pkc itself, which has two redox sensitive sulfur zinc clusters in the diacylglycerol binding domain. in addition, some members of the src kinase family (in particular, lyn kinase), which stimulate pkc via phosphorylation of its biochemistry (moscow) vol. no. tyrosine residues, are activated by ros [ ] . another redox sensitive mechanism of nadph oxidase activation in neutrophils is based on the ros dependent association of the disulfide isomerase protein with the p phox sub unit, which leads to its translocation to the membrane with subsequent assembly of nadph oxidase [ ] . activation of nadph oxidase by mtros was first demonstrated by dikalov and co workers [ ] in endothelial cells. they showed that mitochondria target ed antioxidant mitotempo and expression of mito chondrial superoxide dismutase sod prevented nadph oxidase activation caused by the hormone angiotensin ii, which stimulated hypertension. subsequent studies showed that mtros in the endotheli um stimulated only one of the four isoforms of nadph oxidase -nox [ ], and this isoform was typical for neutrophils. in neutrophils, the nadph oxidase activa tion by myxotiazole, an inhibitor of complex iii of the respiratory chain, which stimulated production of mtros, was described [ ] . in our study [ ] , an increase in mtros and activation of nadph oxidase was caused by a signal from the g protein coupled fmlp receptor, which initiated the release of ca + from intracellular reticulum, as well as ca + independent activation of pi k. both of these signaling pathways are apparently important for the receptor mediated activation of nadph oxidase in neutrophils [ ] . the ca + dependent generation of mtros could serve as a predominant source of ros in the case of netosis induction by ca + ionophores (a or iono mycin) [ ] and some other stimuli [ , ] . these obser vations promote the concept of two different mechanisms of netosis, one of which is independent of nadph oxi dase (see, the review [ ] ). however, mtros can con tribute to the initiation of netosis due to stimulation of nadph oxidase. for example, as was shown in our recent work [ ] , netosis induced by a was inhib ited both by the mitochondria targeted antioxidant skq and specific nadph oxidase inhibitors. at the same time, we also showed that in neutrophils isolated from the blood of patients with x linked cgd formation of nets in response to a depended on the enhanced gener ation of mtros without participation of nadph oxi dase [ ] . we suggest that in the nadph oxidase defi cient neutrophils, mtros are formed with increased intensity due to excessive accumulation of ca + [ ] and this is enough to trigger netosis. the uncontrolled influx of ca + into the cytoplasm of cgd neutrophils is appar ently due to the lack of electrogenic function of nadph oxidase and membrane depolarization upon activa tion [ ] . our studies demonstrated that generation of mtros caused by both fmlp and a depended on opening of the non selective mitochondrial permeability transi tion pore (mptp) [ ] . this phenomenon went unno ticed for a long time, probably due to the fact that the most well known mptp inhibitor cyclosporin a (csa) was also an inhibitor of cytoplasmic phosphatase cal cineurin involved in the transmission of numerous ca + dependent signals [ ] . in our study, we used the mptp inhibitors sangliferin a and bongkrekic acid, which did not affect calcineurin. interestingly, fmlp did not cause significant decrease in the mitochondrial membrane potential and mitochondrial swelling characteristic of the prolonged opening of mptp. we assume that this short term increase in cytoplasmic ca + caused by fmlp induced temporal reversible pore opening. this mptp opening mode has been described both in isolated mito chondria [ ] and in cell models [ ] . signalling function of the mptp opening and associated production of mtros have been previously described in the endothelial cells [ ] and in neutrophils treated with mixotia zole [ ] . netosis caused by a was also dependent on the opening of mptp [ ] . in this case, electron microscopy revealed a significant swelling of mitochon dria, which was accompanied by chromatin decondensa tion and destruction of the nuclear envelope ( fig. ) . it can be assumed that the significant increase in cytoplasmic ca + induced by a caused a prolonged opening of the pore. ca + dependent opening of the mptp stimulated formation of mtros, while mptp inhibitors prevented their accumulation [ ] . the direct cause of excessive formation of mtros was probably related to the release of the main components of antioxi dant defense, such as nadph [ ] and reduced glu tathione [ ] from mitochondria. along with ca + , oxidative stress is an effective inducer of the mptp open ing [ ] . it is possible that the ros formed by nadph oxidase penetrate the cell, thus stimulating opening of the mptp and creating an amplification loop leading to netosis (fig. ) . opening of the mitochondrial pore has long been considered as one of the steps in the apoptosis program [ ] . initially, it was assumed that the mptp opening resulted in swelling of the matrix, rupture of the outer mitochondrial membrane, and exit of proapoptotic pro teins (in particular, cytochrome c) from the intermem brane space into the cytoplasm. later, this hypothesis was modified taking into account the data on the release of cytochrome c through protein pores in the membrane. it was assumed that opening of the mptp promotes change in the morphology of the inner membrane and release of cytochrome c from the space inside the cristae [ ] . the details of this mechanism remain unclear, but there is no doubt that specific pore inhibitors prevent apoptosis in various models. the mptp opening is also involved in the induction of necrotic death. in particular, mptp opening contributes to the necrosis induced by ischemia/reperfu sion of the heart, and its critical role has been confirmed by the protective effect of the pore inhibitors [ ] . these data allow us to place netosis induced by calcium ionophores into the family of mptp dependent pcd mechanisms. the mtros dependent activation of nadph oxidase during netosis, which is often referred to as "nadph oxidase independent", indicates that it seems more accurate to call it "mitochondria dependent netosis". an important event in netosis is the release of some proteins from granules into the cytosol. azurophil gran ules contain the protein complex "azurosome", which includes eight types of proteins, three of them are highly homologous serine proteases -neutrophilic elastase (ne), cathepsin g and azurocidin, and also myeloperox idase (mpo), an enzyme that produces hypochlorite anion using chlorine and hydrogen peroxide as substrates. it was shown that ros cause dissociation of azurosomes, which led to the release of serine proteases and mpo from the granules into the cytosol [ ] . serine proteases (primarily, ne) break down cytoskeletal elements con tributing to realization of netosis [ ] . subsequently, they migrate to the nucleus, where they break down the lamin and histones contributing to the chromatin decon densation and destruction of the nuclear envelope. mpo plays an important role in dissociation of the azurosome and release of the proteases from the granules [ , ] . interestingly, enzymatic activity of mpo is not required at this stage. it is likely that this heme containing protein serves as an intracellular hydrogen peroxide receptor. however, mpo activity is required for netosis, since hypochlorite anion stimulates activity of neutrophil elas tase [ ] . it is worth noting that the ros dependent release of proteins from the granules during netosis is in many ways similar to the ros dependent permeabiliza tion of lysosomes and release of cathepsins from them, which is typical for many variants of necrosis [ ] . additionally, peptidyl arginine deaminase (pad ) is transferred from the cytoplasm into the nucleus to cat alyze citrullination of histones, which leads to chromatin decondensation. inhibition of pad prevented chro matin decondensation and netosis caused by ca + ionophores or shigella flexneri, and neutrophils isolated from the pad knockout mice did not form nets in response to pma [ ] . peptidyl arginine deaminases are ca + binding proteins and their activity is stimulated by ca + . on the other hand, pad is activated and causes biochemistry (moscow) vol. no. histone citrullination in response to the addition of hydrogen peroxide [ ] . moreover, lps related citrulli nation of histones and netosis depend on the intactness of microtubules [ ] . apparently, combination of all these factors is necessary for activation of pad . along with citrullination, histones can also undergo acetylation dur ing netosis, but its role in this process is still poorly understood [ ] . chromatin decondensation, as well as proteolytic damage of the nuclear lamina leads to the destruction of the nuclear envelope, and release of chro matin into the cytoplasm. recently, it has been shown that netosis can occur due to the activation of cyclin dependent kinases (cdks), which facilitate entry into the cell cycle [ ] . it is possible that parts of the mitosis apparatus, such as lamina phosphorylation and centro somes separation are used to destroy nuclear envelope during netosis. at the final stage of netosis, pores are formed in the plasma membrane and chromatin is released into the environment with nets formation. proteins released from the granules are strongly bound to the decondensed chromatin due to electrostatic interaction. the pores that allow this giant complex to pass through are formed by the gasdermin d protein (gsdmd), which also forms pores in the macrophage membrane during pyroptosis. unlike pyroptosis, where gsdmd is activated through the cleavage by caspase and / (caspase , in mice), during netosis it is cleaved and activated mainly by neu trophil elastase [ , ] . activation of gsdmd probably leads to the formation of pores not only in the plas fig. . scheme illustrating the mechanisms of netosis induced by various stimuli. netosis caused by the calcium ionophore a starts with the mobilization of ca + from the endoplasmic reticulum (er), which leads to the activation of crac channels located in the cyto plasmic membrane, and the entry of extracellular ca + into the cytoplasm. in addition, a catalyzes the transfer of ca + through the plasma membrane into the cytoplasm. subsequent accumulation of ca + in the mitochondrial matrix leads to the activation of non selective mitochondrial pores (mptp) and the formation of mitochondrial reactive oxygen species (mtros). along with ca + overload, oxidative stress is also an inducer of the mptp. mtros released from the mitochondria into the cytosol activate nadph oxidase apparently with the participation of protein kinase c (pkc). pkc can be activated with the phorbol ester (pma), which induces nadph oxidase and netosis independently of mtros and the mptp. nadph oxidase can be also activated by a chemoattractant n formylmethionyl leucyl phenylala nine (fmlp), which by binding to a specific receptor activates phospholipase c (plc) to stimulate the formation of diacylglycerol (dag) and inositol triphosphate (ip ) from the phosphatidylinositol , bisphosphate. the activation of nadph oxidase in this case depends on mtros and on the opening of mptp. for reasons not fully understood, fmlp does not induce netosis [ ] . (color version of the figure is available in online version of the article and can be accessed at: https://www.springer.com/journal/ ) malemma, but also in the nuclear membrane [ ] . caspase dependent activation of gsdmd in neutrophils can also occur as a result of noncanonical activation of the inflammasome [ ] , but the role of this mechanism in netosis requires further investigations. gasdermin e, related to gsdmd, is activated by caspase leading to permeabilization of mitochondria and promotion of apoptosis, as well as secondary necrosis [ , ] . another pore forming protein mlkl (not related to gasdermins) is activated by rip kinases during necroptosis. activation of mlkl in neutrophils can also lead to the release of nets [ ] . physiological modulation of netosis. under physio logical conditions, the ratio of co /hco concentra tions, ph, and o content can modulate netosis. under moderately alkaline conditions and at reduced co /hco ratio, the level of netosis caused by pma, ca + ionophore, uric acid microcrystals, or lps was increased [ ] . an increase of ph in neutrophil cyto plasm caused a raise in the concentration of ca + and enhanced production of ros by both nadph oxidase and mitochondria [ ] . a decrease of ph in the medium causes inhibition of netosis, possibly due to inhibition of glycolysis [ ] . in addition, at low ph, probability of the mptp opening is sharply reduced [ ] , which could lower production of mtros [ ] . it is assumed that ph dependence of netosis leads to the fact that it is maxi mally activated on the periphery of inflammatory lesion protecting the tissue from pathogens, while in the center of the lesion, which is characterized by low ph, netosis is weakened and does not enhance tissue damage [ ] . information on the effect of hypoxia on netosis is con troversial. from the one hand, knockout of hif α (the main transcription factor regulating adaptation to hypox ia) suppressed netosis, while pharmacological stabiliza tion of hif α stimulated it [ ] . on the other hand, netosis induced by pma (but not s. aureus) decreased with hypoxia, and this effect was not dependent on hif α [ ] . in addition to the composition of the medium, netosis depends on its osmolarity, therefore, the hyper tonic medium suppressed ros production and netosis. the ros level in this case was critical, since the addition of hydrogen peroxide restored netosis [ ] . cytokines and inflammatory mediators play an important role in the activation of netosis, while at the same time some anti inflammatory substances can cause the opposite effect. thus, prostaglandin e , which plays an important role in resolving inflammation, inhibits netosis due to increase in the intracellular content of cyclic amp [ , ] . activated c protein (a serine pro teinase with anti thrombotic and anti inflammatory effects) can also inhibit netosis by binding to a specific receptor (epcr) or by interacting with protease activat ed receptor (par ) and cd b/cd integrins (mac ). interestingly, the same activated c protein can cleave histones that are part of nets [ ] . it has been shown that netosis is suppressed by the important anti inflam matory cytokine il [ ] . peptides that effectively sup press nets formation have been found in the cord blood. the most common peptide is called the "neonatal nets inhibitory factor" (nnif). this peptide selectively inhib ited netosis without affecting phagocytosis and other neutrophil functions. its intravenous administration pro tected mice from systemic inflammation caused by lps and from microbial sepsis [ ] . pathogenic microorganisms have a wide range of tools that interfere with the microbicidal action of nets [ ] . many microorganisms produce lytic enzymes (pri marily, endonucleases) that destroy nets. bacteria, such as pseudomonas aeruginosa, mycobacterium tuberculosis, as well as fungi of the aspergillus genus produce protective and masking extracellular envelops. in the case of p. aeruginosa, coating with sialic acids induces produc tion of il , which suppresses netosis. the same mechanism appears to be used by the human immunode ficiency virus (hiv ). hiv virions stimulate produc tion of il by dendritic cells, which protects the virus from the lytic action of nets enzymes [ ] . hepatitis b virus (hbv) inhibits netosis by suppressing production of ros in neutrophils using the hbe envelope protein and hbc core protein [ ] . along with the suicidal netosis described above, there are also mechanisms of dna release, when neu trophils retain their viability and natural effector func tions (for a review, see [ ] ). the term "vital netosis" was used to describe these processes. however, the cell death nomenclature committee in [ ] did not recom mend to use the term netosis for processes not associat ed with cell death. the vital release of chromatin was first described in a system where neutrophils were present together with platelets activated by lps via tlr [ ] . netosis, in this case, occurred much faster than when induced by pma, and the role of nadph oxidase and ros was not studied. unfortunately, this interesting model, which has obvious physiological significance, has not been investigated further. a similar vital release of chromatin was observed in vivo when the skin was infect ed with gram positive bacteria [ ] . induction of netosis in this case required opsonization of bacteria, interaction with tlr , and activation of the complement system. interestingly, after netosis, nuclear free neu trophils were capable for chemotaxis and phagocytosis of bacteria. massive and very fast release of mitochondrial dna (mtdna) without loss of viability was observed in eosinophils and neutrophils primed with pro inflamma tory cytokines il /ifn γ or gm csf, respectively, and stimulated with lps [ , ] . in both types of granulo biochemistry (moscow) vol. no. cytes, this process was dependent on the activity of nadph oxidase. a similar phenomenon was observed in the case of basophils [ ] . recently, the vital mtdna release was discovered in b and t lymphocytes, as well as in natural killer cells (nk cells) in response to oligodeoxynucleotides [ ] . unlike granulocytes, the release of mtdna from lymphocytes was not dependent on nadph oxidase. the question of the functional role of extracellular mtdna remains open. low content of mitochondria in neutrophils and, especially, in eosinophils makes formation of the functional nets extremely unlikely. in the case of lymphocytes, extracel lular mtdna did not contain lytic enzymes and most likely performed a signaling function. in particular, mtdna stimulated expression and secretion of the type i interferons by peripheral blood mononuclear cells [ ] . it should be noted that extracellular mtdna (usually, oxi dized) is found in the blood at a wide range of pathologies including systemic lupus erythematosus, an autoimmune disease in the pathogenesis of which netosis plays an important role [ ] . in the first studies on netosis, physiological protec tive role of this phenomenon was suggested. in particular, structures similar to nets were found in the contents of the appendix [ ] . later, nets were found in numerous organs and tissues [ ] , however, the question of its protec tive effect remains open. netosis is observed in foci of infections and, apparently, slows down the spread of pathogens. thus, it has been shown that nets in the staphylococcal skin infections inhibits penetration of the pathogens into the bloodstream [ ] . mice that are not capable of netosis due to knockout of the pad gene suffered more significantly from necrotic fasciitis caused by the group a streptococcus pyogenes [ ] . undoubtedly, excessive formation of nets associated with both increased netosis and defects in the mechanisms for their elimination can lead to inflammatory and autoim mune pathologies, as well as to vascular or duct occlusion. netosis on the surface of epithelium. microorgan isms constantly attack epithelial barriers inducing forma tion of nets on the surface of eyes, oral mucosa, and skin. therefore, formation of nets and their degradation must be strictly regulated to avoid inflammation in these tissues. a number of diseases have been described in which nets play both antimicrobial and pathogenetic role. on the surface of the eye cornea, netosis is involved in the protection against both bacterial and fungal infec tions. in patients with severe netosis, fungal keratitis progressed much easier [ ] , while keratitis caused by clinical isolates of p. aeruginosa was exacerbated by netosis in the mouse model [ ] . in the case of sterile inflammation of the cornea during dry eye syndrome, nets accumulation was also observed [ ] . one of the reasons for netosis could be the increased osmolarity of the lacrimal fluid washing the cornea in patients with dry eye syndrome [ ] . dry eye syndrome, which often occurs as a manifestation of the "graft versus host" reac tion after bone marrow transplantation [ ] , and sjogren's autoimmune syndrome [ ] are also character ized by accumulation of nets in the lacrimal fluid. interestingly, the mitochondria targeted antioxidant skq (an acting component of the eye drops "visomitin") was proven to be highly effective in the treatment of dry eye syndrome of various etiologies [ ] . it can be assumed that this effect of skq is partly relat ed to the suppression of netosis described in our study [ ] . the role of netosis in thrombosis. netosis plays an important role in the pathogenesis of thrombosis of vari ous origin [ , ] . for example, in the model of stenosis of the inferior vein cava probability of thrombosis was sig nificantly reduced in the pad knockout mice [ ] . the role of nets in thrombosis involves interaction with endothelium and platelets, as well as capture of small blood clots. netosis apparently underlies thrombosis associated with excessive innate immunity reactions ("immunothrombosis") [ ] . immunothrombosis, pre sumably, is a protective reaction of the body against pathogens, facilitating their capture in a fibrin clot. in addition, small blood vessel thrombi can create compart ments, where pathogens can be effectively destroyed. however, consequences of such reaction can be tragic. a recent large scale clinical study revealed correlation between the level of netosis and the severity of ischemic stroke and myocardial infarction [ ] . netosis can be activated by microcrystals of cholesterol and participate in the pathogenesis of atherosclerosis. histones in the nets cause tlr dependent activation of macrophages and release of cytokines that activate t helper cells (th ) [ , ] . the inflammatory process in atheroscle rotic plaques can be one of the causes of thrombosis [ ] . oncological diseases could be another cause of thrombo sis, and netosis is believed to be involved in this case [ ] . on the other hand, therapeutic viral infection of tumors induces neutrophil dependent intratumoral coag ulation and death of cancer cells. however, it remains to be elucidated, whether this process is really caused by netosis [ ] . thrombosis, which is characteristic of var ious viral infections, could be associated with excessive netosis and appears to be involved in the pathogenesis of covid (see below). in the pancreatic ducts, netosis can be caused by microcrystals of calcium carbonate, which leads to block age of the pancreatic duct and pancreatitis [ ] . such crystals cause netosis in the bile ducts and in the gall bladder, which can lead to the formation of gall stones [ ] . biochemistry (moscow) vol. no. the role of netosis in the development of pulmonary diseases. in the respiratory tract, netosis contributes to the protection against infection by increasing viscosity of the mucus and by destruction of pathogens. however, many of pathogens (in particular, streptococcus pneumo niae, haemophilus influenzae) produce endonucleases that break down nets and protect the bacteria (see review [ ] ). at the same time, netosis contributes to the development of complications of the lungs infectious diseases, including acute respiratory distress syndrome (ards), chronic obstructive pulmonary disease (copd), as well as bronchial asthma, etc. the recent clinical trial (conducted prior to the onset of the covid pandemic) showed correlation between the nets con tent and severity of the community acquired pneumonia [ ] . acute lung injury (ali) and ards of various eti ologies are accompanied by excessive netosis (see review [ ] ). nets in these pathologies may be involved in the damage of alveolar epithelium and endothelium. copd, which is usually associated with smoking and air pollution, and severe "neutrophilic" asthma are also characterized by accumulation of nets in the sputum and respiratory tract lavage [ ] . rhinovirus infection in patients with asthma significantly increased nets con tent [ ] . in asthma, formation of the chromatin con taining traps can also occur due to the death of eosinophils, which is similar to netosis [ ] . the severe acute respiratory infection covid (coronavirus disease ), which broke out in december in the chinese city of wuhan and subsequently escalated into a pandemic, has affected up to now more than million people from countries. the disease was caused by a new coronavirus called sars cov (severe acute respiratory syndrome coronavirus ), and was accompanied by viral pneumonia often progressing to ards and multiple organ failure. elevated levels of neu trophils in the blood indicate severity of the disease and poor prognosis [ ] . examination of the patients infected with sars cov revealed an increased level of netosis markers (cell free dna, mpo dna complexes, and citrullinated histone h ) and the marker of cell deathlactate dehydrogenase [ ] . concentration of the cell free dna correlated with the content of neutrophils, c reactive protein (marker of the acute phase of inflamma tion), and the d dimer (marker of thrombosis). serum from the patients with covid induced netosis in the healthy donor blood in the in vitro system. one of the manifestations of covid is kawasaki syndrome, vas culitis that occurs in children, which is accompanied by excessive netosis [ ] . netosis in covid can be caused by epithelial and endothelial cells affected by the virus, by activated platelets, and by inflammatory cytokines. at the same time, excessive netosis is involved in the development of the "cytokine storm" and thrombosis, which are main indicators of the severe course of covid [ ] . netosis and autoimmune diseases. many net com ponents, in particular, double stranded dna, granule proteins, and histones, can stimulate production of anti bodies and development of autoimmune diseases. for the first time, involvement of netosis in pathogenesis of autoimmune diseases was studied in detail for systemic lupus erythematosus (see review [ ] ). a special form of granulocytes called "low density granulocytes" appears in the blood during this disease. these cells produce more inflammatory cytokines (including type i interferons) and are more involved netotsis than the normal neutrophils. their nets contain more autoantigens and oxidized mitochondrial dna [ ] , which makes them stronger immunostimulants. one of the peculiarities of low densi ty granulocytes is the increased production of mitochon drial ros. recently, it was shown that the mitochondria targeted antioxidant mitoq (similar in structure to skq ) suppressed nets accumulation and pathological mani festations in the mouse model of systemic lupus erythe matosus [ ] . in rheumatoid arthritis (ra), the development of pathology correlated with accumulation of the netosis markers, such as dna mpo complexes and antibodies against citrullinated histones [ , ] . in addition, it was found that nets engulfed by fibroblasts stimulated formation of the antibodies against citrullinated histones in the ra model [ ] . similar observations indicate involvement of netosis in the autoimmune diseases, such as vasculitis associated with "anti neutrophil anti bodies" (aav), antiphospholipid syndrome, multiple sclerosis, and psoriasis (see review [ ] ). interestingly, in some diseases, netosis can play an anti inflammatory role. for example, during the gout attacks, netosis induced by uric acid crystals is accompanied by the release of lytic enzymes that break down proinflammato ry cytokines in the foci of inflammation. it is believed that netosis in gout prevents development of the chronic dis ease [ ] . evidences for involvement of netosis in various pathologies has led to intensive study and testing of vari ous therapeutic approaches. one approach includes the use of drugs that prevent netosis. it comprises anticy tokine therapy aimed to prevent accumulation of neu trophils in the foci and their activation, as well as apply ing inhibitors of the components involved in netosis program: ne, pad , and gsdmd. another approach is based on the destroying of nets or attenuating their damaging effects. anticytokine therapy directed against il β is widely used in various inflammatory and autoim mune diseases. one of its targets may be excessive netosis. the recombinant anakinra protein, an il β receptor antagonist, is currently undergoing clinical trials biochemistry (moscow) vol. no. as a potential preparation to treat covid (https://clinicaltrials.gov: nct , nct , nct ). among the netosis inhibitors, the trials of the ne inhibitors have been progressed the farthest. the first of these, sivelestat, was approved for treating ards in japan and south korea, but meta analysis of the clinical data did not confirm its effectiveness [ ] . new generation of ne inhibitors is currently only at the first stage of clinical trials. gsdmd and pad inhibitors are at the preclinical stage of testing. of great interest is the use of existing drugs for inhibition of netosis. so, disulfiram, which is used to treat alcoholism, inhibits gsdmd activation and protects mice in the lethal lps induced sepsis model [ ] . it should be noted that gsdmd is also a critical component of the pyroptosis program and the authors of the study attribute its effect to the prevention of macrophage pyroptosis. the in vitro experiments have shown that netosis can be prevented by microtubule inhibitors [ ] . this group of drugs includes colchicine, and the clinical trials testing efficacy of colchicine against covid are currently underway (https://clinicaltrials.gov: nct , nct , nct , nct ). among the drugs aimed to destroy nets, dnase i and anti histone antibodies are the most studied. recombinant dnase i has been successfully used in almost all models of pathologies associated with netosis. in particular, introduction of dnase i significantly facil itates the course of ards [ ] and copd [ ] in animal models. in patients with cystic fibrosis, dnase inhalation improved lung function, and neutrophil elastase facilitat ed sputum dissolution making it more accessible for dnase [ ] . clinical trials of actin resistant dnase (prx ) go through the second phase and give encour aging results (https://clinicaltrials.gov: nct , nct ). it is possible to hope that dnase will not only liquefy sputum, but will also interrupt the progres sion of ards, as was observed in animal experimental models. our experiments with the mitochondria targeted antioxidant skq have shown its potential effectiveness against netosis [ ] . in the mouse model of systemic inflammatory syndrome, skq prevented lethal effect of the inflammatory cytokine tnf [ ] . the antioxidant mitoq, similar in structure to skq , suppressed netosis in the mouse model of systemic lupus erythematosus [ ] . these results suggest, that drugs based on mito chondria targeted antioxidants will be created soon in order to treat diseases associated with the excessive netosis, and, in particular, to combat covid . the program for formation of neutrophil extracellu lar traps -netosis -is being studied extensively, howev er, many questions regarding its mechanisms and physio logical role remain open. first of all, this refers to the sig naling mechanisms of the initiation of netosis. in par ticular, the processes leading to chromatin modification and decondensation remain unclear. targets for the mtros action and details of the mtros dependent acti vation of nadph oxidase have not yet been determined. excessive netosis has been suggested to play an impor tant role in pathogenesis of many infectious, inflammato ry, and autoimmune diseases, but there is no sufficient evidence to support this. a new impetus to study patho physiological role of netosis may be related to its puta tive role in the pathogenesis of covid . the develop ment of new drugs preventing netosis, including mito chondria targeted antioxidants, appears to be a promising area of pharmacological research. rapid killing of human neutrophils by the potent activator phorbol myristate acetate (pma) accompa nied by changes different from typical apoptosis or necrosis neutrophil extracellular traps kill bacteria unconventional roles of the nadph oxidase: signaling, ion homeostasis, and cell death neutrophil extracellular traps: mechanisms of formation and role in health and disease neutrophil extracellular trap formation: physiology in vivo evidence for extracellular dna trap formation neutrophil extracellular traps and micro crystals neutrophil extracellular traps and their role in the develop ment of chronic inflammation and autoimmunity neutrophil extracellular trap cell death requires both autophagy and superoxide generation neither eosinophils nor neutrophils require atg dependent autophagy for extracellular dna trap formation pma and crystal induced neutrophil extra cellular trap formation involves ripk ripk mlkl signaling necroptosis controls net generation and mediates complement activation, endothelial damage, and autoim mune vasculitis the pseudokinase mlkl activates pad dependent net formation in necroptotic neu trophils the role of mitochondrial ros in antibacterial immunity mitochondrial reactive oxygen species are involved in chemoattractant induced oxidative burst and degranulation of human neutrophils in vitro mitochondrial permeability transition pore is involved in oxidative burst and netosis of human neutrophils phagocytosis independent antimicrobial activity of mast cells by means of extracellu lar trap formation nadph oxidase inde pendent formation of extracellular dna traps by basophils proc. natl. acad. sci. usa neutrophil extra cellular traps enriched in oxidized mitochondrial dna are interferogenic and contribute to lupus like disease diverse stimuli engage different neutrophil extracellular trap pathways nadph oxidase modulates ca + dependent formation of neutrophil extracel lular traps accelerated calcium influx and hyperactivation of neu trophils in chronic granulomatous disease the mitochondrial permeability transition pore: channel formation by f atp synthase, integration in signal trans duction, and role in pathophysiology effect of transient and perma nent permeability transition pore opening on nad(p)h localization in intact cells the mitochondrial death/life regulator in apoptosis and necrosis a distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis mitochondrial non specific pores remain closed during cardiac ischaemia, but open upon reperfusion a myeloperoxidase containing complex regulates neutrophil elastase release and actin dynamics during netosis neutrophil elastase and myeloperoxi dase regulate the formation of neutrophil extracellular traps myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity lysosomal membrane permeabilization in cell death: con cepts and challenges, mitochondrion is essential for antibacterial innate immunity mediated by neutrophil extracellular traps regulation of extracellular chromatin release from neu trophils cell cycle proteins control production of neutrophil extracellular traps gasdermin d exerts anti inflammatory effects by promoting neutrophil death gasdermin d plays a vital role in the generation of neutrophil extracellular traps noncanonical inflamma some signaling elicits gasdermin d dependent neutrophil extracellular traps cleavage of dfna by caspase during apoptosis mediates progres sion to secondary necrotic/pyroptotic cell death gasdermin pores permeabilize mitochondria to augment caspase activation during apoptosis and inflammasome activation ménage à trois: the ratio of bicar bonate to co and the ph regulate the capacity of neu trophils to form nets alkaline ph promotes nadph oxi dase independent neutrophil extracellular trap formation: a matter of mitochondrial reactive oxygen species genera tion and citrullination and cleavage of histone extracellular acidification inhibits the ros dependent formation of neutrophil extracellular traps the impact of hypoxia on neutrophil degranula tion and consequences for the host formation of neutrophil extracellular traps under low oxy gen level hypertonic saline suppresses nadph biochemistry oxidase dependent neutrophil extracellular trap formation and promotes apoptosis inhibition of neutrophil extracellular trap formation after stem cell transplant by prostaglandin e prostaglandin e inhibits neutrophil extracellular trap formation through production of cyclic amp activated protein c inhibits neutrophil extracellular trap formation in vitro and activa tion in vivo neutrophil extracellular traps mediate a host defense response to human immunodefi ciency virus neonatal net inhibitory factor and related peptides inhibit neutrophil extracellular trap formation modulation of neutrophil netosis: inter play between infectious agents and underlying host physiolo gy hepatitis b virus inhibits neu trophil extracellular trap release by modulating reactive oxygen species production and autophagy molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death platelet tlr activates neu trophil extracellular traps to ensnare bacteria in septic blood infection induced netosis is a dynamic process involving neutrophil multitasking in vivo catapult like release of mitochondrial dna by eosinophils contributes to antibacterial defense viable neutrophils release mitochon drial dna to form neutrophil extracellular traps lymphocytes eject interferogenic mitochondrial dna webs in response to cpg and non cpg oligodeoxynucleotides of class c neutrophil extracellular traps involvement in corneal fungal infection distinct susceptibilities of corneal pseudomonas aeruginosa clin ical isolates to neutrophil extracellular trap mediated immuni ty ocular surface extracellular dna and nuclease activity imbalance: a new paradigm for inflammation in dry eye disease hyper osmolar stress induces neutrophil extracellular trap forma tion: implications for dry eye disease neutrophil extracellular traps (nets) contribute to pathological changes of ocular graft vs. host disease (ogvhd) dry eye: implications for novel biomarkers and therapeutic strategies autoantibodies to neutrophil extracellular traps represent a potential serological biomarker in rheumatoid arthritis results of a multicenter, randomized, double masked, placebo con trolled clinical study of the efficacy and safety of visomitin eye drops in patients with dry eye syndrome thrombosis: tangled up in nets the pathway of neutrophil extracellular traps towards atherosclerosis and thrombosis neutrophil histone modification by peptidylarginine deiminase is critical for deep vein thrombosis in mice platelet neu trophil interplay: insights into neutrophil extracellular trap (net) driven coagulation in infection thrombus net content is associated with clinical outcome in stroke and myocardial infarction inflammation. neutrophil extracellular traps license macrophages for cytokine pro duction in atherosclerosis ) histones, dna, and citrullination promote neutrophil extracellular trap inflammation by regulating the localiza tion and activation of tlr neutrophil extracellular traps in breast cancer and beyond: current perspectives on net stimuli, thrombosis and metastasis, and clinical utility for diagnosis and treatment targeting tumor vasculature with an oncolytic virus externalized decondensed neu trophil chromatin occludes pancreatic ducts and drives pancreatitis neutrophil extracellular traps initiate gall stone formation the emerging role of neutrophil extracellular traps in respiratory disease markers of neutrophil extra cellular traps predict adverse outcome in community acquired pneumonia: secondary analysis of a randomised controlled trial the counter intuitive role of the neutrophil in the acute respiratory distress syndrome netopathic inflammation in chronic obstructive pulmonary disease and severe asthma host dna released by netosis promotes rhinovirus induced type allergic asth ma exacerbation biological function of eosinophil extracellular traps in patients with severe eosinophilic asth ma uk ( ) covid : consider cytokine storm syndromes and immunosuppression neutrophil extracellular traps in covid enhanced formation of neutrophil extracellular traps in kawasaki disease targeting potential drivers of covid : neutrophil extracellular traps the role of neu trophils and netosis in autoimmune and renal diseases targeting mitochondrial oxida tive stress with mitoq reduces net formation and kidney disease in lupus prone mrl lpr mice nets are a source of citrullinated autoantigens and stimulate inflam matory responses in rheumatoid arthritis increased levels of neutrophil extracellular trap remnants in the serum of patients with rheumatoid arthritis synovial fibroblast neutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis neutrophil extracellular traps (nets) take the central stage in driving autoimmune responses aggregated neutrophil extra cellular traps limit inflammation by degrading cytokines and chemokines effect of a selective neutrophil elastase inhibitor on mortality and ventilator free days in patients with increased extravascular lung water: a post hoc analysis of the picco pulmonary edema study fda approved disulfiram inhibits pyroptosis by blocking gasdermin d pore formation neutrophil elastase enhances sputum solubilization in cys tic fibrosis patients receiving dnase therapy low con centration of uncouplers of oxidative phosphorylation decreases the tnf induced endothelial permeability and lethality in mice the authors are grateful to the co key: cord- -s eopu y authors: nan title: die pathophysiologie der entzündung date: journal: die akute entz&#x fc;ndung doi: . / - - - _ sha: doc_id: cord_uid: s eopu y nan synthese. histamin entsteht durch decarboxylierung der aminosäure l-histidin durch das enzym l-histidin-decarboxylase. zwei bauelemente setzen histamin zusammen: der imidazolring und die Äthylaminkette, über die jeweils verschiedene wirkungen im entzündungsgeschehen vermittelt werden (abb. ). in mastzellen und basophilen granulozyten fi ndet sich histamin in granula gespeichert, wo es elektrostatisch an die anionischen seitenketten von glykosaminoglykanen gebunden ist. das entsprechende glykosaminoglykan in der mastzelle ist das heparin, das der basophilen granulozyten chondroitin- -sulfat. die bindung bewirkt eine ruhigstellung des histamins. so ist es biolo-gisch unwirksam, aber auch vor abbau geschützt. die freisetzung von histamin aus mastzellen und basophilen granulozyten erfolgt durch exozytose der granula. bei der degranulation werden auch weitere entzündungsmediatoren ad hoc gebildet und abgegeben wie prostaglandine, leukotriene und paf. nach extrusion des granulainhalts nach außen wird histamin über verdrängung durch extrazelluläres na + vom glykosaminoglykan abgekoppelt und damit wirksam, aber auch abbaubar. die lebensdauer von histamin hängt vom gewebe ab, in dem es freigesetzt wird, überschreitet aber nicht den bereich von wenigen minuten. ein teil des freigesetzten histamins gelangt ins blut. nach experimenteller provokation einer mastzellenentspeicherung am menschen ist im strömenden blut die maximale konzentration nach minuten erreicht und nach - minuten der normwert von bis pg/ml wieder hergestellt. dieser sofortige Übertritt ins blut erklärt auch den raschen und schlagartigen eintritt systemischer histaminsymptome beim anaphylaktischen schock (s. ). abbau des histamins. nur wenige prozent des serum-histamins werden unverändert im harn ausgeschieden. der überwiegende teil wird durch oxydative desaminierung unwirksam gemacht und erscheint in dieser form im harn. eine desaminierung kann auf zwei wegen erfolgen: Über die monoaminooxydase (mao) oder über die diaminooxydase (dao), auch als "histaminase" bezeichnet. Über die mao werden etwa bis % des histamins abgebaut. zuvor muss aber histamin durch die histamin-n-methyl-transferase zu methyl-histamin umgewandelt werden. dann erst kann durch die mao die oxydative desaminierung über aldehydbildung zu methyl-imidazol-essigsäure erfolgen, die im harn ausgeschieden wird. histamin-n-methyl-transferase ist reichlich in monozyten/makrophagen enthalten. mao ist ein ubiquitär auftretendes enzym mit besonders hoher konzentration in der leber. --dreißig bis % des histamins werden über die dao, syn. histaminase, abgebaut. die dao desaminiert histamin direkt zu imidazol-essigsäure. etwa ein drittel des über die dao abgebauten histamins erscheint als imidazol-essigsäure im harn, der verbleibende teil wird an ribose gebunden und als imidazol-essigsäure-ribosid ebenfalls im harn ausgeschieden (abb. ). dao fi ndet sich in einer reihe von zelltypen und organen. besonders reichlich ist sie in eosinophilen granulozyten enthalten. die messung des blut-histaminspiegels wird bei verschiedene formen von anaphylaxien, urticaria, bronchospasmus etc. sowie bei mastzellentumoren (mastozytomen) aktuell. die messung der abbauprodukte im harn gibt aufschluss über die gesamtbilanz des kurzlebigen histamins. stimulation der mastzellendegranulation. verschiedenartige reize können eine mastzellendegranulation auslösen wie antigenbindung an ige-antikörper auf mastzellen und basophilen granulozyten, anaphylatoxine, eine reihe von cytokinen, bradikinin, substanz p, und darüber hinaus unspezifi sche physikalisch-chemische reize wie thermische, chemische und mechanische einwirkungen. histaminwirkungen. die wirkung des histamins auf zielzellen erfolgt über spezifi sche rezeptoren, von denen zwei arten bekannt und gut studiert sind. eine dritte rezeptorart, ein h -rezeptor, wurde bei labortieren präsynaptisch an cholinergen nerven festgestellt, wo histamin vermutlich die freisetzung von azetylcholin steuert. eine rolle beim menschen ist noch nicht sicher gestellt. der als h bezeichnete rezeptortyp bindet histamin über die Äthylaminkette. in der medizin werden medikamente verschiedenen chemischen baues verwendet, die h -rezeptoren und die über sie vermittelten wirkungen blockieren. diese h -rezeptorenblocker werden unter der bezeichnung "antihistaminika" häufi g therapeutisch eingesetzt. der andere rezeptortyp, der h -rezeptor, bindet histamin über den imidazol-ring (abb. ). h -blocker enthalten strukturanaloga des imidazols. diese pharmaka besetzen eine wichtige stelle in der therapie des gastrischen und des duodenalen ulkus. h wie h -blocker wirken kompetitiv, verdrängen also den natürlichen liganden histamin vom rezeptor und müssen daher entsprechend hoch dosiert werden. eine länger dauernde blockierung von rezeptoren wird jedoch von der betroffenen zelle mit einer rezeptorvermehrung [up-regulation] beantwortet, was eine dosissteigerung des blockers zur erhaltung der therapeutischen wirkung erfordert. bei plötzlicher aufhebung der rezeptorblockade nach ende einer therapie sind die betroffenen zellen mit einer hohen rezeptorzahl bestückt und reagieren folglich auf histamin verstärkt, was zum massiven wiederauftreten der krankheitssymptomatik führen kann (sog. "rebound-phänomen"). für kompetitiv wirkende medikamente kann als faustregel gelten: therapiebeginn mit doppelter erhaltungsdosis, um die rezeptoren initial mit dem blocker zu sättigen, erhaltungsdosis bei bedarf steigern und bei therapieende die dosis schrittweise zurücknehmen ("ausschleichen"), um einen rebound zu verhindern. je nachdem, über welchen rezeptortyp histamin auf die zielzelle wirkt, kann man zwei wirkungsbereiche des histamins unterscheiden: h und h -wirkungen. die h -wirkungen sind die "klassischen" histaminwirkungen, die bereits nach erkannt und studiert wurden. sie umfassen eine reihe typischer entzündungsphänomene. histamin bewirkt in den meisten geweben eine gefäßerweiterung, die der rezeptorverteilung entsprechend in erster linie die mikrozirkulation (kleine arterien und venolen), weniger die widerstands-und kapazitätsgefäße (mittelgroße arterien und venen) betrifft (abb. ). folgen der vasodilatation: lokal wird eine durchblutungssteigerung erreicht, die an der körperoberfl äche als rubor und calor in erscheinung tritt. gelangt histamin jedoch in massiven mengen in die blutbahn, so führt die systemische gefäßerweiterung zu einem druckabfall, der sich zum "histaminschock" steigern kann. die h -vermittelte wirkung des histamins trifft nicht unmittelbar die zielzelle der gefäßerweiterung, die glatte muskelzelle der gefäße, sondern nimmt einen indirekten weg. histamin stimuliert die h -rezeptoren der endothelzelle, die auf diesen reiz hin eine gefäßerweiternde substanz, stickstoffmonoxyd (no), pro-duziert und freisetzt. das no diffundiert zur muskelzelle und ist für deren erschlaffung verantwortlich (s. ). betrifft die kapillaren und noch stärker die postkapillaren venolen. histamin bewirkt eine kontraktion der fortsätze der endothelzellen und eine lösung der lumenwärts gelegenen festen interzellularverbindungen, die dabei interzelluläre lücken (stomata) mit durchlässigkeit für höhermolekulare substanzen freigeben (s. ). der blutdruck treibt flüssigkeit durch die stomata und die basalmembran in den extravasalraum. da im be-reich der stomata auch der filtrationseffekt des endothels verringert ist, hat entzündliches exsudat etwa denselben eiweißgehalt und somit denselben osmotischen druck wie das blutplasma. die fehlende kolloidosmotische druckdifferenz zwischen intra-und extravasalraum schließt den osmotischen rückstrom extravasaler flüs sigkeit in die gefäßbahn aus, was die flüssigkeitsan sammlung zusätzlich verstärkt und das entzündliche Ödem entstehen lässt. das entzündliche Ödem wird ausschließlich über das lymphsystem entlastet (abb. ). folgen der permeabilitätssteigerung. lokal bildet sich reichlich exsudat, das entzündliche Ödem, das an körpero-berfl ächen als schwellung (tumor) imponiert. eine typische, durch histamin verursachte reaktion der haut ist die urticaria, der "nesselausschlag". da die mastzellen der haut in der oberfl ächennahen papillarschicht (stratum papillare) dicht unter der epidermis konzentriert sind, ist die schwellung scharf begrenzt und die rötung deutlich. Ödeme kön-nen an orten beschwerden verursachen bzw. sogar lebensbedrohlich werden, wo sie lumina einengen, wie in der nasenhöhle, im kehlkopf oder in den tieferen atemwegen. eine permeabilitätssteigerung im gesamten zirkulationssystem hat eine massive plasmaverschiebung aus der gefäßbahn in die gewebe zur folge, die das abb. . strömungsverhältnisse und flüssigkeitsverteilung im bereich der mikrozirkulation eines entzündungsherdes im vergleich zu den verhältnissen im nicht entzündeten gewebe. die flüssigkeitsverteilung im durchlässigen gefäßbereich wird einerseits vom blutdruck (filtrationsdruck -aus der gefäßbahn hinaus) und andererseits vom kolloidosmotischen druck und dem gewebsdruck (bindekraft der plasmaeiweiße, widerstand des umliegenden gewebes -in die gefäßbahn hinein) bestimmt. im nicht entzündeten gewebe (a) wird durch den filtrationsdruck im kapillarbereich eiweißarme blutfl üssigkeit in den extravasalraum gepresst, bis der im kapillarverlauf abnehmende blutdruck den kolloidosmotischen druck der plasmaeiweiße (ca. mm hg) erreicht hat. im folgenden gefäßverlauf wird bei weiterhin abnehmendem blutdruck die ausgetretene flüssigkeit wieder über die osmotische bindekraft der plasmaeiweiße rückresorbiert. im durchschnitt des gesamtorganismus werden auf diese weise rund % der fi ltrierten flüssigkeit direkt in die blutbahn rückgeführt, und nur % nehmen den weg über die lymphbahn. dieser rest geht über die lymphbahn als lymphe ab und gelangt über den ductus thoracicus ins blut zurück (starling-hypothese). in entzündetem gewebe (b) herrscht durch die erweiterung der zuführenden arterien (a) und den dadurch vermehrten bluteinstrom ein erhöhter blutdruck, der zusammen mit der gesteigerten durchlässigkeit des endothels für flüssigkeiten und gelöste höhermolekulare stoffe den verstärkten einstrom einer eiweißreichen flüssigkeit in das entzündete gewebe verursacht (entzündliches Ödem). die zusammensetzung der Ödemfl üssigkeit entspricht etwa der des blutplasmas. im venösen bereich (v) nehmen die strömungsgeschwindigkeit und der blutdruck in einem ausmaß ab, das durch die gefäßweite, den verlust an blutvolumen in das Ödem und von der ausbildung des sludge-phänomens bestimmt wird. durch die fehlende kolloidosmotische druckdifferenz kann die Ödemfl üssigkeit nicht in die blutbahn rückströmen, sondern wird vollständig über die lymphbahnen abtransportiert. in den lymphknoten wird die Ödemfl üssigkeit von schadmaterial gereinigt und das spezifi sche immunsystem aktiviert. zirkulierende plasmavolumen erheblich verringert. die hypovolämie verstärkt den durch die gefäßerweiterung verursachten blutdruckabfall und bildet eine weitere komponente des histaminschocks. histamin benötigt zur vollen entfaltung seiner permeabilitätssteigernden wirkung den synergismus weiterer mediatoren wie bradikinin oder prostaglandine. unter entzündlichem Ödem versteht man die extravasale ansammlung von flüssigkeit im entzündungsbereich. bei entzündung treten in der terminalen gefäßbahn veränderungen auf, die den austritt von blutfl üssigkeit im vergleich zu nicht entzündlichen gefäßstrecken wesentlich erleichtern. durch die dilatation zuführender arteriolen wird über den verstärkten blutfl uss der lokale blutdruck und damit der filtrationsdruck erhöht. die durchlässigkeit der funktionellen gefäßstrecke für plasmabestandteile wird durch lückenbildung im endothelbelag der venolen erhöht (permeabilitätssteigerung für höher molekulare stoffe). das entzündliche exsudat ist dadurch eiweißreich und hat etwa die zusammensetzung des blutplasmas. die galen'schen entzündungssymptome rubor, calor und tumor werden durch die gefäßerweiterung und die permeabilitätssteigerung verursacht. die gefäßerweiterung fördert reichlich sauerstoffreiches, daher hellrotes blut, das bei oberfl ächlichem sitz des entzündungsherdes eine rötung (rubor) verursacht. dieses blut hat die temperatur des körperinneren, die einige grad über der temperatur der körperoberfl äche liegt und den entzündeten bereich gegenüber der umgebung relativ wärmer erscheinen lässt (s. ). zusätzlich wird durch die stoffwechselsteigerung im entzündungsherd --wärme frei (calor). das entzündliche Ödem bewirkt die schwellung (tumor), deren ausmaß auch von der beschaffenheit des betroffenen gewebes abhängt. entzündungen des lockeren bindegewebes verursachen starke schwellungen. das kann in hohlorganen komplikationen verursachen, wenn verkehrswege behindert werden (z.b. glottisödem, schwellung der bronchialschleimhaut). mangelnde ausweichmöglichkeiten führen zu starken drucksteigerungen (gehirn, zahnpulpa, foramina intervertebralia). auch der schmerz (dolor) wird vom erhöhten gewebsdruck mitverursacht. das entzündliche Ödem hat eine reihe von aufgaben: es soll das gewebe aufl ockern, um es für die eigentlichen effektoren der immunabwehr, die entzündungszellen, leichter durchgängig zu machen. durch die vermehrte durchsaftung wird eine verdünnung toxischer produkte (mikrobielle produkte, abbauprodukte körpereigenen materials, Überschüsse von regulatoren und wirkstoffen der entzündung) im entzündungsherd erreicht und damit ihre wirkung herabgesetzt. die entzündliche exsudatfl üssigkeit mit toxischen produkten wird über die lymphbahnen abgeführt. auf diese weise wird der entzündete gewebsbezirk vermehrt durchschwemmt und gereinigt. die makrophagen der lymphknoten befreien die lymphe von toxischen produkten. gleichzeitig wird die spezifi sche abwehr aktiviert: bei erstkontakt wird eine spezifi sche immunreaktion über ag-präsentierende zellen initiiert, bei mehrfachkontakt mit einem ag werden die gedächtniszellen aktiviert. der lymphfl uss schwemmt informationsträger wie mediatoren, cytokine und wachstumsfaktoren aus dem entzündungsherd in das blut aus, die systemische reaktionen in gang setzen. zu den veränderungen der flüssigkeitsverteilung in einem entzündungsherd siehe abbildung . ---Über das entzündliche Ödem werden reichlich inaktive vorstufen von im blut zirkulierenden regulatoren, komponenten der blutgerinnung wie auch effektorstoffe der entzündung (immunglobuline) in das entzündete gewebe eingebracht. die erhöhte durchlässigkeit des gefäßendothels ermöglicht den durchtritt auch hochmolekularer stoffe. dem entzündlichen Ödem mag auch eine gewisse mechanische funktion zukommen. die schwellung bewirkt eine einschränkung der beweglichkeit. gemeinsam mit der wirkung des schmerzes wird damit eine ruhigstellung von entzündeten körperpartien erreicht. anders als die gefäßmuskulatur besitzen die glatten muskelzellen der bronchien und des gastrointestinaltrakts sowie die muskelzellen von drüsenendstücken h -rezeptoren, über die sie zur kontraktion angeregt werden. im gi-trakt bewirkt histamin eine verstärkung der peristaltik mit diarrhoe und erbrechen. histamin kann auch zur bronchokonstriktion im rahmen des asthma bronchiale beitragen, ist aber bei diesem krankheitsbild von untergeordneter bedeutung. so zeigen antihistaminika bei asthma bronchiale nur geringen oder gar keinen therapeutischen effekt (s. ). die wirkung von histamin auf die schmerzfasern ist nur gering. Über h -wirkung wird jedoch an sensiblen nervenfasern ein juckreiz hervor gerufen, der eine typische subjektive begleiterscheinung von histamin-vermittelten erkrankungen wie urticaria, prurigo oder rhinitis und conjunctivitis allergica ist. histamin bewirkt über axonrefl exe von sensiblen nerven eine gefäßerweiterung, die über den unmittelbaren einwirkungsbereich des histamins hinausgeht. injiziert man experimentell einer ver---suchsperson histamin intrakutan, so fällt der bereich des erythems weit größer aus als es der ausbreitung des histamins zukommen sollte. das auslösen neuronaler refl exe im bereich der atemwege kann pathogenetisch bedeutsam sein. experimentelle histamininhalationen beim gesunden führen über h -rezeptoren zu einer bronchokonstriktion, die über refl exbögen des nervus vagus vermittelt wird, wie die blockierbarkeit des effekts mit atropin beweist. solche refl exe können beim chronifi zierten asthma bronchiale eine rolle spielen (s. , abb. ). eine andere verbindung zwischen histamin und nervensystem läuft über den schmerzvermittelnden neurotransmitter substanz p. substanz p stimuliert mastzellen zur degranulierung und damit histaminabgabe. umgekehrt kann histamin die freisetzung von substanz p aus nervenfasern bewirken. histamin regt die produktion und abgabe von schleim in schleimdrüsen an. bei atemwegserkrankungen wird dadurch mehr schleim im nasen-und rachenbereich und in den tieferen atemwegen abgegeben. die ventilationsbehinderung, die durch bronchokonstriktion und schleimhautschwellung gegeben ist, wird dadurch verstärkt (s. ). die kenntnis um diesen teil des wirkungsspektrums von histamin ist wesentlich jünger als die der h -wirkung. klinisch wichtige h -vermittelte effekte betreffen die steuerung von organfunktionen. Über h -rezeptoren werden zellfunktionen von granulozyten, monozyten/ makrophagen, lymphozyten und mastzellen gehemmt. die hemmung erfasst zellleistungen wie migration und chemotaxis, phagozytose und degranulation, sowie die produktion und abgabe von ros und regulatoren der entzündung. die hemmende wirkung läuft über die aktivierungskette besetzung der h -rezeptoren, aktivierung der adenylatzyklase und damit anhebung des intrazellulären camp -spiegels und beruht letzten endes auf einer inaktivierung des cytoskeletts (s. ff, abb. ). mastzellen können so über ihre h -rezeptoren die eigene histaminabgabe einschränken. diese produkthemmung trägt zur regulation der histaminkonzentration in geweben bei. histamin ist somit über die h -wirkung ein hemmer der zellulären entzündungsreaktion. eine ausnahme bilden die eosinophilen granulozyten, auf die histamin in einem gewissen konzentrationsbereich chemotaktisch wirkt. histamin ist, neben anderen faktoren (s. ), für die eosinophilie betroffener gewebe im zuge von allergien und parasitenbefall verantwortlich. histamin erhöht auch die aggressivität der eosinophilen granulozyten gegenüber parasiten. in hoher konzentration lähmt es jedoch die aktivität auch dieser zellen. erhöhte histaminspiegel im blut verursachen tachykardie, die teils als kardiale kompensation des blutdruckabfalls aufzufassen ist, aber auch auf einer direkten kombinierten h -und h -wirkung auf das herz beruht. die infusion zumutbarer histaminmengen in freiwillige testpersonen löst eine tachykardie aus, die durch die kombinierte anwendung von h -und h -blockern, jedoch nicht wesentlich durch die getrennte anwendung dieser blocker verhindert werden kann. am meerschweinchenherzen führt die verabreichung hoher histamindosen anfangs zu einer sinustachykardie (positiv chronotrope wirkung) und zu einer erhöhung des fördervolumens (positiv inotrop), hemmt aber die reizleitung vom av-knoten weiter bis zur ausbildung eines schenkelblocks (negativ dromotrop). die entkoppelung der schlagfolge zwischen vorhof und kammern bewirkt einen drastischen abfall der förderleistung des herzens, blutdruckabfall und schocktod. obwohl beim meerschweinchen diese wirkung vorwiegend h vermittelt ist, können parallelen zum menschen gezogen werden. autoptisch fi nden sich bei im anaphylaktischen schock verstorbenen in der nähe des sinusund av-knotens reichlich degranulierte mastzellen. die stimulation der salzsäure-produktion im magen läuft über einen anstieg der gastrinabgabe aus zellen des apud-systems. gastrin stimuliert die histaminabgabe aus mastzellen, und histamin stimuliert über h -rezeptoren die belegzellen der magendrüsen zur produktion und abgabe von salzsäure. eine der modernen möglichkeiten einer konservativen ulkustherapie besteht in der blockierung dieser rezeptoren durch h -blocker, wodurch eine drosselung der salzsäureproduktion erreicht wird. histamin reguliert auch die aktivität des exokrinen pankreas über h und h -rezeptoren. histamin übt auf den ablauf einer entzündung zwei konträre wirkungen aus: histamin ist als mediator entzündlicher reaktionen maßgeblich am zustandekommen einer reihe von erkrankungen beteiligt, die im klinischen alltag eine bedeutende stellung einnehmen. aus pathogenetischer sicht kann man drei gruppen von histamin-vermittelten erkrankungen unterscheiden. a) anaphylaktische reaktionen b) pseudoallergien c) die histaminvergiftung das sind per defi nitionem ige-vermittelte immunreaktionen, also Überempfi ndlichkeitsreaktionen vom typ nach coombs-gell, auch als Überempfi ndlichkeitsreaktionen vom "soforttyp" oder "reagintyp" bezeichnet. klinisch ist für diese krankheitsbilder die pathogenetisch ungenaue bezeichnung "allergie" gebräuchlich. disponierte personen, die als "atopiker" bezeichnet werden, reagieren auf gewisse antigene (ag) mit der bildung spezifi scher antikörper (ak) vom ige-typ. die atopische veranlagung ist angeboren, tritt oft familiär auf und zeigt eine konvergenz mit gewissen mhc i typen. die von den plasmazellen abgegebenen ige-ak gelangen über den blutweg in den gesamten organismus und binden sich mit ihren fc-teil an spezifi sche rezeptoren (s. , abb. ). solche rezeptoren ( fcε-r) wurden an mastzellen, basophilen granulozyten, eosinophilen granulozyten und an manchen makrophagen festgestellt. in dieser bindung sind ige-ak, deren lebenserwartung frei eine halbwertszeit von zweieinhalb tagen nicht überschreitet, vor dem abbau geschützt und langlebig. vermutlich teilen sie die lebensdauer der jeweiligen trägerzelle. für das verständnis des krankheitsgeschehens ist wichtig, dass trägerzellen im gesamten organismus mit ige aufgeladen werden und nicht nur am eintrittsort des antigens. die nach antigenkontakt erfolgte produktion und abgabe von ige sowie die beladung von fcε-r tragenden zellen mit ige nennt der kliniker sensibilisierung. trifft das ag erneut auf eine solcherart sensibilisierte trägerzelle, so wird es von fab-teil des ige-ak gebunden (abb. ) und übt damit einen degranulationsreiz auf die trägerzelle aus. mastzellen und basophile granulozyten geben darauf hin histamin und lipogene mediatoren ab, eosinophile granulozyten entspeichern ihre zytotoxischen granulainhalte und setzen ros frei, makrophagen sezernieren wirkstoffe, die weitere entzündungszellen anlocken und das entzündungsgeschehen beeinfl ussen. da die hauptträger der anaphylaktischen entzündungsreaktion, die mastzellen, in organen der körperoberfl ächen, nämlich im gi-trakt, im respirationstrakt und in der haut konzentriert sind, werden diese organe auch bevorzugt von der entzündlichen symptomatik getroffen. sensibilisierte atopiker beherbergen überdies einen wesentlich größeren mastzellenpool als gesunde. bedingung für eine degranulierung ist allerdings, dass sich ein bi-oder multivalentes ag an mehrere benachbarte iga-ak gleichzeitig bindet, was als quervernetzung oder brückenbildung, [bridging, patching] bezeichnet wird. erst nach einer dimerisierung von rezeptoren können transmembranöse signale im zellinneren entstehen, die eine granulaabgabe und damit verbundene zellleistungen in gang setzen (s. , abb. ). die notwendigkeit der brückenbildung setzt eine gewisse mindestdichte der rezeptorbesetzung voraus. da die ige-rezeptoren auf der inaktiven mastzelle statistisch gleichmäßig verteilt sind und die bindung der ige-ak an die rezeptoren ebenfalls nach zufallgesetzen erfolgt, ist bei niederer besetzungsdichte die wahrscheinlichkeit, dass zwei benachbarte rezeptoren ige tragen und somit eine quervernetzung ermöglichen, gering. bei einem niederen besetzungsgrad der mastzellen mit ige treten deshalb trotz bereits laufender ige-ak-produktion keine klinischen symptome auf. nach länger dauernder oder wiederholter ag-exposition "beladen" sich die mastzellen jedoch fortlaufend oder schubweise mit ige (sog. "booster-effekt"), bis die für die brückenbildung kritische beset-zungsdichte erreicht ist. daher setzt typischerweise die anaphylaktische symptomatik nach einem neuerlichen boosterschub sehr plötzlich und prägnant "über nacht" ein. herkunft und art der allergene antigene, die Überempfi ndlichkeitsreaktionen (allergien) auslösen, werden als allergene bezeichnet. eine aus praktisch-klinischen gesichtspunkten nutzvolle einteilung der allergene ist die nach dem ort des eindringens in den organismus. es lassen sich so unterscheiden: inhalationsallergene -über die atemwege ingestionsallergene -über den verdauungstrakt kontaktallergene -über die äußere körperoberfl äche injektionsallergene -werden direkt ins blut oder in gewebe inokuliert. herkunft und chemische natur der allergene sind vielfältig. meist sind es höhermolekulare eiweiße, die eine ak-bildung auslösen. kleine moleküle und nicht-eiweiße (z. b. chemikalien oder medikamente) wirken nach bindung an körpereigenes eiweiß als haptene. die häufi gsten ursachen für anaphylaktische erkrankungen im europäischen raum sind pollen von windbestäubern, wie gewisse gräser und bäume. sie wirken im conjunctivalbereich als kontaktallergene, in respirationstrakt als inhalationsallergene. eine reaktion beginnt typischerweise mit dem erscheinungsbild der rhinitis und conjunctivitis allergica. nach längerer oder intensiver allergenexposition kann sich bei entsprechender disposition die klinisch wesentlich problematischere ausprägungsform des asthma bronchiale entwickeln (s. ff). häufi ge ingestionsallergene sind nahrungseiweiße wie fisch, krebstiere, eier, milchkasein, früchte wie erdbeeren, zitrusfrüchte u.a.m. allergische reaktionen gegenüber chemikalien und medikamenten sind entsprechend der ----abb. . histamin-freisetzung. im zuge der sensibilisierung lagern sich ige-antikörper mit dem fc-teil an ige-rezeptoren an. bei bindung mindestens zweier benachbarter antikörper an das antigen (brückenbildung) kann sich der reiz entwickeln, der den mechanismus der mastzellen-degranulation in gang setzt. das in den granula enthaltene histamin wird durch exozytose in die umgebung freigesetzt. die degranulation aktiviert den arachidonsäure-metabolismus und mit dem histamin werden gleichzeitig pg, lt und paf abgegeben. steigenden belastung stark im zunehmen. sie können echte anaphylaxien sein, wobei gewöhnlich das allergen als hapten wirkt, oder auch pseudoallergien hervorrufen (s. ). die applikation solcher chemischer stoffe kann auf verschiedenen wegen erfolgen: oral als ingestionsallergen, oberfl ächlich lokal als kontaktallergen oder parenteral als injektionsallergen. kontaktallergene dringen über die äußere körperoberfl äche ein. sie rufen üblicherweise eine Üer vom typ hervor, jedoch mit einer gewissen histaminbeteiligung, wie der heftige juckreiz nahe legt. reine anaphylaktische reaktionen durch kontaktallergene sind selten. injektionsallergene fi nden sich häufi g unter den antibiotika (penicillin, cephalosporine). in diese gruppe fallen auch insektengifte wie die von bienen und wespen. ingestionsallergene können zu einer recht vielfältigen symptomatik führen. reaktionen treten am eintrittsort der allergene im gi-trakt auf. die h -vermittelte gefäßerweiterung und permeabilitätssteigerung, die aktivierung der drüsen-und muskeltätigkeit äußert sich in abdominalen krämpfen, durchfall und erbrechen. wird reichlich histamin freigesetzt, kann es zu blutdruckabfall bis zum anaphylaktischen schock kommen. in die zirkulation abgegebene entzündungsmediatoren können aber auch unspezifi sche fernwirkungen entfalten wie migräne-artige kopfschmerzen oder rheumatoide beschwerden, deren auslösende ursache oft verkannt wird. häufi g bilden sich heftig juckende quaddeln am stamm, armen oder beinen ( urticaria, "nesselsucht", "verdauungsausschlag"). neben einer symptomatischen lokalen therapie sind auch hier antihistaminika hilfreich. beim allergischen asthma bronchiale wird die histaminwirkung von anderen mediatoren überspielt (s. ). ursache des anaphylaktischen schocks ist stets die massive zufuhr des allergens und seine verteilung auf dem blutweg über den gesamten sensibilisierten organismus, was zu einer schlagartigen histaminfreisetzung und zur histaminüberschwemmung des blutes führt. diese bedingung, den großteil der mastzellen des organismus über die blutbahn zu erreichen, können nur injektionsallergene und ingestionsallergene erfüllen. unter den injektionsallergenen, die für den anaphylaktischen schock verantwortlich sein können, sind insektengifte von bienen und wespen häufi g, unter den medikamenten antibiotika oder auch eine zu rasche dosissteigerung bei hyposensibilisierungskuren. der anaphylaktische schock hat seine ursache in einer auf das ganze kreislauf-system ausgedehnten histaminwirkung. gefäßerweiterung in der mikrozirkulation in verbindung mit einer gesteigerten gefäßpermeabilität führen zum raschen blutdruckabfall. ein Überleitungsblock lässt den kreislauf völlig zusammenbrechen. der zustand ist akut lebensbedrohlich, und therapeutisch steht zunächst die schockbekämpfung im vordergrund: flüssigkeitsersatz und sauerstoffbeatmung. vasopressorische medikamente sind beim anaphylaktischen schock zur kreislaufstabilisierung wirksam. moderne therapieschemata inkludieren kombinierte h und h blockade zur prävention von herzfunktionsstörungen und glucocorticoide zur immunsuppression. bei herzstillstand ist nach möglichkeit eine defi brillation durchzuführen, notfalls kann adrenalin intrakardial injiziert werden. bei der atopischen dermatitis, dem "milchschorf" der kinder, sind anaphylaktische elemente mitbeteiligt, wie die oft sehr hohen ige-spiegel nahe legen. die pathogenese dieser erkrankung ist allerdings unklar. störungen im stoffwechsel lipogener mediatoren scheinen eine rolle zu spielen. in den betroffenen hautarealen laufen übersteigerte entzündungsreaktionen ab. besser als die symptomatische behandlung von anaphylaxien ist eine kausale therapie, nämlich die meidung des verantwortlichen allergens. die suche nach der auslösenden ursache kann eine aufwendige arbeit erfordern, die oft vergeblich ist. hilfsmittel dazu sind der prick-test und die bestimmung der ige-blutspiegel mit dem rist -und rast -test. bei pollenallergien sind im frühstadium hyposensibilisierungskuren aussichtsreich. als "pseudoallergie" wird die situation bezeichnet, wenn wirkstoffe oder ihre metabolite direkt, unter umgehung einer spezifi schen immunreaktion, auf effektorzellen der immunantwort ein-wirken. eine mastzellen-degranulation wird also ohne ige-beteiligung ausgelöst. pseudoallergien sind wie die echten allergien stark im ansteigen. grund dafür ist die starke zunahme chemischer wirkstoffe in allen lebensbereichen. es besteht ein deutlicher zusammenhang solcher Überreaktionen mit der zunehmenden verunreinigung der umwelt, luft, wasser und nahrungsmitteln sowie mit der zunehmenden verwendung von chemikalien im alltag. auch der ansteigende gebrauch von medikamenten fällt ins gewicht. mit einzelheiten zu diesem thema beschäftigt sich eine breite fachliteratur. wie die echten allergien treffen auch die pseudoallergien besonders disponierte personen. eine Überempfi ndlichkeit von mastzellen gegenüber unspezifi schen reizen kann anlagebedingt sein und tritt häufi g familiär auf. so gibt es etwa eine Überempfi ndlichkeit gegenüber thermischen reizen. hitze oder kälte rufen dann am ort der einwirkung auf der körperoberfl äche ein juckendes, urtikarielles Ödem hervor. auch schwitzen (azetylcholinfreisetzung an den schweißdrüsen, abb. ) oder mechanische reize können bei empfi ndlichen personen zu einer mastzellen-degranulation und zur quaddelbildung führen. therapiemöglichkeiten sind antihistaminika und die meidung der auslösenden reize. ist folge einer zufuhr von histamin mit der nahrung. analog zur biosynthese im organismus kann histamin auch durch bakterielle decarboxylierung aus histidin entstehen. mögliche quellen von histamin sind bakteriell verdorbene, histidinreiche nahrungsmittel, wie etwa mangelhaft gekühlter fisch. besonders histidinreich sind makrelenartige wie sardinen, thun oder makrelen (scomber). fischvergiftungen auf histaminbasis werden daher als "scombrotoxismus" bezeichnet. histamin reichert sich auch in lebensmitteln an, bei denen bakterielle gärungspro-zesse bestandteil ihrer herstellung und reifung sind, wie in gewissen käsesorten, würsten, oder in sauerkraut. auch manche traubensorten, besonders wenn sie in wenig sonnigen lagen gezogen werden, sind histidinreich. bei der kelterung entsteht histamin. die symptome einer histaminvergiftung gestalten sich nach der histaminmenge und der individuellen reaktivität verschieden. einer erhöhten empfi ndlichkeit einzelner personen kann eine verringerte ausrüstung mit histaminabbauenden enzymen und eine dadurch verzögerte abbauleistung für histamin zugrunde liegen. die symptomatik entspricht den lokalen und systemischen effekten des histamins: gastrointestinale beschwerden wie durchfall und erbrechen, gesichtsröte, blutdruckabfall, migräneartige kopfschmerzen und urticaria. bedingt durch die kurze halbwertszeit des histamins sind die krankheitserscheinungen nur vorübergehend. die therapie besteht in einer symptomatischen Überbrückung der beschwerden und in antihistaminika. synthese, chemie und abbau serotonin ist ein abkömmling der aminosäure tryptophan, aus der es in zwei syntheseschritten gebildet wird. a) nach der hydroxylierung am c -atom entsteht -hydroxy-tryptophan, aus dem b) nach einer decarboxylierung -hydroxy-tryptamin ( ht, serotonin) wird. der abbau erfolgt durch die monoaminooxydase, die reichlich in monozyten/ makrophagen, in der leber, im lungenepithel und in einer reihe weiterer zelltypen vorkommt. das abbauprodukt -oh-indolessigsäure wird im harn ausgeschieden (abb. ). die verteilung von serotonin im organismus ist sehr speziesabhängig. so enthalten etwa die mastzellen und basophilen granulozyten häufi g verwendeter in den enterochromaffi nen zellen ("basalgranulierte zellen") der darmmucosa ist serotonin in den granula der zellbasis gespeichert. nach seiner abgabe bewirkt es eine kontraktion der lamina muscularis mucosae und dient damit der feinsteuerung der darmmotilität. ins blut gelangtes serotonin wird von den thrombozyten über das oberfl ächliche trabekelnetzwerk aufgenommen und vorwiegend in den dichten granula gespeichert (s. ). bei aktivierung der thrombozyten im zuge der blutgerinnung wird es in die umgebung freigesetzt. im zns ist serotonin ein wichtiger neurotransmitter, der für positive stimmungsqualitäten verantwortlich ist. seine wirkung wird über mindestens drei verschiedene rezeptortypen vermittelt. lysergsäurederivate (lsd) blockieren die wirkung und lösen damit psychotrope effekte aus. auch antihistaminika der älteren generationen können die wirkung von serotonin an den zentralen rezeptoren kompetitiv hemmen und so als unerwünschte nebenwirkung müdigkeit und verstimmung verursachen. eine unspezifi sche bindung wird durch die Ähnlichkeit der molekülstruktur (Äthylamingruppe) ermöglicht. diese nebenwirkungen sind bei modernen antihistaminika weitgehend ausgeschaltet. bei der pathogenese der migräne ist serotonin mitbeteiligt. ht -rezeptor -synergisten werden erfolgreich in der migränetherapie eingesetzt. diese wirkung ist ebenfalls stark spezies betont. beim menschen steht die kontraktion glatter muskulatur im vordergrund. gefäßmuskulatur. hier ist die wirkung unterschiedlich. die arterien von niere, lunge und meningen reagieren mit einer kontraktion, arterien in allen anderen organen jedoch mit einer dilatation, die no vermittelt ist (s. ) . gelangt serotonin massiv ins blut, ergibt sich für die gesamtzirkulation ein blutdruckabfall. eine kontraktion der bronchialmuskulatur führt zu ventilationsstörungen, die kontraktion der darmmuskulatur zu motilitätssteigerung, kolik und diarrhoe. in vitro stimuliert serotonin in menschlichen pmn und monozyten über rezeptoren gi-proteine und senkt damit den intrazellulären camp-spiegel, was eine aktivierung dieser zellen mit verstärkter motilität, phagozytose und granulaabgabe zur folge hat (s. , abb. ). ob dieser effekt in vivo eine nennenswerte rolle spielt, wird bezweifelt. die meisten untersucher messen dem serotonin beim entzündungsgeschehen eine untergeordnete bis keine bedeutung bei. darm-carcinoide können schubweise serotonin in die zirkulation freisetzen und damit systemische reaktionen hervorrufen. eine gefäßerweiterung ist besonders an der gesichtshaut auffällig. die anfallsartige gesichtsrötung wird als "flush-syndrom" bezeichnet. daneben können in verschiedenem ausmaß blutdruckabfall, asthmaartige atemnot und diarrhoe auftreten. der nachweis der carcinoidtätigkeit gelingt mit dem abbauprodukt des serotonins -oh-indolessigsäure im harn. eikosanoide sind abkömmlinge ungesättigter fettsäuren mit einem skelett aus c-atomen, die als "eikosa-säuren" bezeichnet werden (von altgriechisch "eikosa", zwanzig). die wichtigste fettsäure für die eikosanoidsynthese ist die arachidonsäure. andere muttersubstanzen treten ihr gegenüber mengenmäßig an bedeutung zurück. eine metabolisierung der eikosasäuren zu eikosanoiden kann über zwei verschiedene enzymsysteme erfolgen: über den cyclooxygenase-weg, der zur bildung von prostaglandinen, prostacyclin und thromboxan führt, und über den lipoxygenase-weg, der leukotriene und verwandte wirkstoffe liefert (abb. ). die meisten dieser derivate sind hochaktive entzündungsmediatoren. von etlichen unter ihnen wird zunehmend sichergestellt, dass sie auch außerhalb entzündlicher prozesse zell-und organfunktionen regulieren und biologische prozesse steuern. alle eikosanoide wirken über spezifische membranrezeptoren auf zielzellen. mehrfach ungesättigte fettsäuren [poly-unsaturated fatty acids, pufa] sind fast ausnahmslos pfl anzliche produkte. der tierische organismus ist auf ihre zufuhr angewiesen, daher auch die bezeichnung "essentielle fettsäuren". eine umwandlung ungesättigter fettsäuren untereinander ist aber im tierischen organismus möglich. die nomenklatur der eikosanoide ist logisch konstruiert und bezieht sich auf ihren chemischen bau. die ersten beiden großbuchstaben weisen ein eikosanoid als produkt des cyclooxygenaseweges oder des lipoxygenaseweges aus, d.h. als ein prostaglandin (pg) oder thromboxan (tx) bzw. als leukotrien (lt). der dritte großbuchstabe bezeichnet das produkt aufgrund seines chemismus näher (z.b. pge, txa, ltb). die nachfolgende arabische ziffer bezieht sich auf die zahl der enthaltenen doppelbindungen (z.b. pge , txa , ltb ). zur geschichte im jahre stellte von euler fest, dass in versuchstiere injizierte samenfl üssigkeit blutdruckabfall und eine kontraktion glatter muskulatur bewirkt. er hielt die prostata, glandula prostatica, für den bildungsort des verantwortlichen übertragbaren faktors. in ableitung vom vermuteten produktionsort wurden die den aktivitäten zugrundeliegenden substanzen "prostaglandine" benannt. tatsächlich stammen die in der samenfl üssigkeit enthaltenen prostaglandine aus der samenblase. prostaglandine im ejakulat von nagermännchen tragen dazu bei, beim weibchen eine ovulation und tubenperistaltik auszulösen und damit gleichzeitig mit der begattung eine befruchtung sicherzustellen. auch beim menschen bestehen noch enge funktionelle beziehungen der prostaglandine zum weiblichen genitaltrakt. die aa ist allerdings in veresterter form einer metabolisierung nicht zugänglich, sondern muss zuerst durch ezyme der phospholipase a -gruppe (pla ) aus der bindung gespalten werden. isoformen der pla liegen im cytosol in inaktiver form vor. Übersteigt die lokale ca ++ -konzentration einen schwellenwert, bindet sich die pla an zellmembranen und wird dadurch aktiv (abb. ). auslösende reize für eine aktivierung sind: bei der esterspaltung entstehen aa und lysolecithin (abb. ). vom lysolecithin ist bekannt, dass es die oberfl ächenspannung herabsetzt und membranfusionen fördert. es wird angenom-men, dass lysolecithin bei vorgängen mitbeteiligt ist, die vesikelabspaltungen aus oder vesikelintegration in die zellmembran beinhalten, wie phagozytose oder exozytose. darüber hinaus dient es in manchen zelltypen zur synthese des plättchen aktivierenden faktors (paf; s. ). lysolecithin kann auch wieder zu lecithin reacyliert werden (abb. ). das andere bruchstück der esterspaltung, die aa, diffundiert vom ort ihrer freisetzung, der zellmembran, zum endoplasmatischen retikulum (er). dort wird sie vom er-gebundenen enzymkomplex cyclooxygenase (cox) zu den cyclischen endoperoxyden pgg und pgh umgesetzt. unter den begriff cox fallen zwei isoenzyme, die sich durch ihre präsenz unterscheiden. die cox vom typ ist ständig vorhanden und arbeitet "konstitutiv", d.h. sie setzt nach bedarf ständig aa-produkte frei. ihre aufgabe ist in abb. . hydrolytische spaltung des lecithins. phospholipasen vom typ a spalten arachidonsäure (aa) hydrolytisch vom lecithin ab und machen damit beide bruchstücke einer weiteren metabolisierung zugänglich. aa kann zu prostaglandinen und leukotrienen, lysolecithin zum plättchen aktivierenden faktor (paf) umgesetzt werden. phosphorsäure und cholin bilden die polaren anteile des lecithinmoleküls. erster linie die steuerung von drüsenfunktionen, organdurchblutung und der tätigkeit glatter muskulatur im rahmen physiologischer nicht-entzündlicher prozesse. Über diese cox werden prostaglandine, prostacyclin und txa synthetisiert. dagegen ist die cox nicht ständig vorhanden, sondern ihre bildung wird durch cytokine induziert, die bei entzündlichen prozessen freigesetzt werden. diese induzierbare cox kommt in pmn, makrophagen, endothelzellen und fibroblasten vor und erzeugt bevorzugt pg, die entzündliche prozesse steuern. wenngleich dieses schema in seiner drastischen entweder-oder zeichnung nicht ganz richtig ist, kann es als vorstellungsgrundlage dienen. so wird etwa abweichend cox in der niere und im zns auch konstitutiv exprimiert. die weltweit am meisten verwendeten medikamente sind entzündungshemmer auf der basis einer cox-hemmung. die klassischen cox-hemmer drosseln sowohl die cox wie auch die cox und greifen damit störend in wichtige organfunktionen ein. mit den modernen "cox -hemmern" wird versucht, bei weitgehender erhaltung der funktion der cox selektiv die cox und deren entzündungsfördernde wirkung einzuschränken (s. , ) . im herzen und im zns wurde ein weiterer cox-typ, die cox , festgestellt. der cox des zns wird bedeutung bei der fieber-und schmerzerzeugung beigemessen. cyclische endoperoxyde entwickeln bereits biologische wirkungen wie vasokonstriktion und thrombozytenaggregation, werden aber gewöhnlich sofort weiter metabolisiert. thrombozyten (blutplättchen, [platelets] ) geben bei ihrer aktivierung pgg und pgh ab, die auf andere plättchen aggregierend wirken und damit die blutgerinnung in gang halten. von endothelzellen abgegebene cyclische endoperoxyde können aber auch von plättchen aufgenommen und zu weiteren produkten wie tx oder pg umgesetzt werden. cyclische endoperoxyde werden durch enzyme weiter metabolisiert, wobei drei in ihrem chemischen bau verschiedene wirkstoffe bzw. wirkstoffgruppen entstehen können. . prostaglandine im engeren sinn (pgd , pge , pgf α) . prostacyclin (pgi ) . thromboxan (txa ). für die gesamtheit aller enzyme, die zur bildung von pgh aus arachidonsäure abb. . grundzüge des arachidonsäure-metabolismus. zellmembranen sind hochvisköse, fl ächenhaft angeordnete flüssigkeiten aus einer doppellage von phospholipiden, deren hydrophile anteile im wässrigen milieu nach außen gerichtet sind, während die hydrophoben fettsäuren -darunter auch die arachidonsäure (aa) -in das membraninnere gedreht werden. bei der üblichen symbolischen darstellung eines phospholipids vertritt der ring den hydrophilen komplex phosphorsäure + x, wobei für x je nach phospholipid cholin, inosin, serin bzw. Äthanolamin stehen kann. die beiden parallelen geraden symbolisieren die hydrophoben fettsäuren. ein anstieg des cytosolischen kalziums aktiviert die phospholipasen a , die aa vom kohlenstoffatom des glycerinskeletts hydrolytisch abspalten. die aa kann -abhängig vom zelltyp und seinem funktionszustand -über die enzyme cyclooxygenase oder lipoxygenase weiter umgesetzt werden. führen, wird manchmal der begriff "pg-synthase" gebraucht. die schritte dieser synthesen sind in den abb. und dargestellt. bildungsorte mit ausnahme des erythrozyten ist jede zelle in der lage, eikosanoide zu erzeugen. welcher bildungsweg, über cyclooxygenase oder lipoxygenase, beschritten wird und welches spezielle produkt dieser synthesewege letztendlich hergestellt wird, hängt vom typ der zelle, aber auch von ihrem zustand und aktivierungsgrad ab. reich an prostaglandinen sind parenchymatöse organe wie niere, lunge und der gi-trakt. hier werden pg unabhängig von entzündungsvorgängen freigesetzt und wirken als parakrine steuerfaktoren auf die feinregulierung von durchblutung, drüsenfunktionen und muskelmotilität. der anteil, der in die blutbahn gelangt, wird sofort abgebaut und kommt systemisch nicht zur wirkung. die einzelnen schritte werden in den abb. und am beispiel der arachidonsäure gezeigt. die metabolisierung von dgla und epa geschieht in analoger weise. wirkung der prostaglandine pg wirken über spezifi sche membranrezeptoren, wobei die einzelne pg-typen recht unterschiedliche effekte auslösen können. die rezeptoren für ein pg treten zudem in verschiedenen isoformen auf, die oft gegensinnige zellantworten vermitteln. auf diese einzelheiten wird hier nicht eingegangen. alle pg-rezeptoren gehören dem siebenfach transmembranös verankerten, g-protein gekoppelten typ an (abb. , s. ). hervorstechende wirkungen sind die erweiterung von arterien und arteriolen und als folge eine gesteigerte durch-abb. . umsetzung von arachidonsäure zu prostaglandinen (pg) . arachidonsäure (aa) wird durch eine phospholipase a aus phospholipiden hydrolytisch abgespalten. in freier form ist aa um das c -atom gefaltet. bei der synthese von pg werden zwei doppelbindungen gesättigt, wobei sich ein cyclopentanring bildet und die faltung der kohlenstoffkette verändert wird. schlüsselstellen für die funktionen der pg bilden die kohlenstoffatome in position , und . die an c und c lokalisierten hydroxylgruppen bzw. ketogruppen bestimmen den typ des prostaglandins und seine affi nität zu den entsprechenden rezeptoren: pgd , pge und pgf α. von den beiden möglichen stereoisomeren des pgf tritt nur die α-form auf. pgf α kann aus pgh , aber auch durch reduktion der ketogruppe am pge entstehen. die reduktion wird durch das enzym pg- -keto-reduktase katalysiert. die am c -atom lokalisierte oh-gruppe ist für die wirksamkeit des moleküls entscheidend. mit der oxydation der c -oh-gruppe beginnt der abbau der pg. blutung im entzündungsbereich (rubor, calor) . pg von e-typ wirken dabei direkt auf rezeptoren der gefäßmuskulatur, und nicht auf dem umweg über no (s. , abb. ). pg vom e-typ erhöhen die permeabilität der kapillaren und postkapillaren venolen und fördern damit das entzündliche Ödem, allerdings nur im synergismus mit anderen mediatoren. pge bringen die bronchialmuskulatur zum erschlaffen, die muskulatur des gi-trakts dagegen zur kontraktion. exokrine drüsen antworten auf pge-reiz mit einer aktivitätssteigerung und einer kontraktion der drüseneigenen muskulatur, was im darm zu gesteigerter motilität und diarrhöen führt. pge wirken immunsuppressiv. in zellen der spezifi schen wie unspezifi schen abwehr werden zellleistungen wie migration, phagozytose, granulaabgabe, die produktion und freisetzung von ros, mediatoren und cytokinen eingeschränkt. den wirkungsmechanismus zeigt abb. . im temperaturzentrum des zns hebt pge den sollwert und setzt damit eine temperatursteigerung in gang (s. ). pgf α erweitert kaum arteriolen, wohl aber postkapilläre venolen. es fehlt auch ein permeabilitätssteigernder effekt. dagegen ist die wirkung auf die zellen der spezifi schen und unspezifi schen abwehr positiv: migration, phagozytose, granulaabgabe, die produktion von ros, mediatoren und cytokinen werden aktiviert. pgf α stellt in dieser hinsicht einen antagonisten zu den pge dar (abb. ). gehirn und rückenmark sind besonders reich an pgd , das in erster linie von nervenzellen, dagegen nur geringfügig von glia synthetisiert wird. im zns dient pgd als modulator neuronaler funktionen. im temperaturzentrum senkt es den sollwert, bewirkt eine hypothermie und ist somit ein antagonist des pge (s. ). auch konnte im tierversuch seine rolle als schlafmediator gesichert werden. außerhalb des zns wird pgd in wesentlich geringeren konzentrationen angetroffen. im gi-trakt tritt es spärlich auf. in organen wie niere, herz oder samenblase, die ansonsten reich an prostaglandinen anderen typs sind, ist die konzentration äußerst gering. hier und diarrhoe, blutdruckabfall, uterine krämpfe und abortgefahr bei schwangerschaft. eine andere therapiemöglichkeit besteht darin, die körpereigene pg-synthese lokal in der magenschleimhaut anzuregen. dieser weg wird seit alters her, wenn auch ohne die kenntnis der näheren zusammenhänge, in der volksmedizin beschritten. milde reize auf die (gesunde!) magenschleimhaut fördern lokal die pg-synthese und beugen einer gastritis und ulcera vor. solche reize bestehen in der verwendung von gewürzen (paprika, pfeffer), säuren (fruchtsäfte vor dem frühstück) und auch Äthylalkohol in maßvollen mengen ("ein schnaps in der früh auf nüchternen magen" als alte bauernregel). die lokale anregung der pg-synthese in der magenschleimhaut ist ein teil der ulkusprophylaxe und -therapie mit sucrosesulfat. umgekehrt führt eine hemmung der pg-synthese zu einer minderung der cytoprotektion und als folge davon zur ausbildung einer erosiven gastritis und von magenulzera und -blutungen, die eine häufi ge komplikation einer therapie mit cox-hemmern und glucocorticoiden darstellen (abb. ). die unter der deutschen bezeichnung "nichtsteroidale antirheumatika" (nsar) bzw. in der englischsprachigen literatur und international unter "nonsteroidal antiinfl ammatory drugs" ( nsaid) zusammengefasste gruppe von medikamenten hemmt die aktivität der cyclooxygenasen (cox), und zwar sowohl der regulatorischen cox wie auch der infl ammatorischen cox (s. art und ausmaß der nebenwirkungen sind stark von dosis, angriffspunkt und dem wirkungsspektrum des jeweiligen typs von nsaid und auch von der individuellen disposition abhängig. auf diesbezügliche einzelheiten wird hier nicht eingegangen, sondern es sollen nur gefahrenquellen im allgemeinen zusammen gefasst werden. magenschleimhaut: der schutz der magenschleimhaut vor einer schädigung und zerstörung durch salzsäure und pepsin ist stark pg abhängig. unterdrückung der pg-produktion durch nsaid kann zu gastritis und zu magen-und duodenalulcera führen (s. ). längerfristige therapien mit nsaid müssen daher von schleimhaut-schützenden maßnahmen begleitet werden. selektive cox -hemmer reduzieren die schädigung der magenschleimhaut beträchtlich (s. ). niere: während in einer gesunden niere pg keinen unentbehrlichen faktor für die blutzufuhr darstellen, ist die nierendurchblutung bei erkrankungen, bei denen vermehrt vasopressoren ( katecholamine, angiotensin ii) freigesetzt wer-den, "prostaglandin abhängig" (s. ). in diese gruppe fallen erkrankungen mit ungenügender renaler perfusion, bei denen das renin-angiotensin-aldosteronsystem überstimuliert ist ( "sekundärer hyperaldosteronismus"), wie herzinsuffi zienz, lebererkrankungen mit un zureichender albuminproduktion, albuminmangel beim nephrotischen syndrom, hypovolämien und schock, oder bei mangeldurchblutungen auf anatomischer basis wie atherosklerose. bei renalem parenchymverlust sind pg vonnöten, um die verbliebenen nephrone maximal zu perfundieren und auf diese weise die nierenfunktion als ganzes aufrecht zu erhalten. nsaid führen bei diesen risikopatienten über eine mangelhafte nierenperfusion zu natrium-und wasserretention mit Ödemneigung, bluthochdruck und, im extremfall, zum akuten nierenversagen. Über den gleichen mechanismus sind nsaid antagonisten von diuretika und können eine antihypertensive therapie durch die provozierte hypervolämie zunichte machen, sind daher in diesen fällen kontraindiziert. bei einem gewissen personenkreis, dessen risikofaktor nicht näher defi niert ist, löst eine länger dauernde therapie mit nsaid eine chronisch-interstitielle marknephritis mit parenchymverlust hervor, die das krankheitsbild der "analgetika-nephropathie" kennzeichnet. exzessive gewebsuntergänge können zur nekrose ganzer papillen führen. berüchtigt war in dieser hinsicht phenacetin, das wegen dieser gefährlichen nebenwirkung aus dem handel genommen wurde. aber auch ass über längere zeiträume in hohen dosen kann diese schäden verursachen ("phenacetinniere", "salicylatniere"). Über die pathogenese der markschädigung siehe s. f. selektive cox -hemmer stellen keinen schutz für die niere dar, da offensichtlich die cox für die regulation der nierenfunktion unentbehrlich ist (s. ). die hemmung der txa produktion, die ja bei gewissen indikationen angestrebt wird, kann sich andererseits in einer verstärkten blutungsneigung manifestieren. häufi g ist die nsaid-induzierte erosive gastritis und/oder das ulcus ventriculi/ duodenii mit gesteigerter blutungstendenz vergesellschaftet. ass und andere nsaid können anaphylaktische reaktionen und pseudoallergien auslösen. (s. , ). bei länger dauernder einnahme von nsaid, besonders ass, können zerebrale symptome auftreten, besonders dann, wenn gleichzeitig ausscheidungsstörungen (nierenerkrankungen) bestehen. ass kann eine ursache für das "reye-syndrom" sein, eine akute enzephalopathie mit leberdegeneration, die vor allem bei kindern auftritt. die symptomatik besteht in Übererregbarkeit, erbrechen, desorientiertheit und stupor bis koma. nsaid können bei disposition zu knochenmarkhemmung mit anämie, leukound thrombopenie bis zur völligen knochenmarkszerstörung ( aplastisches syndrom, panmyelophthise) führen. erhöhung der transaminasen ist bei länger dauernder und höher dosierter therapie mit nsaid häufi g. gelegentlich können schwere leberschäden entstehen. bei atopikern mit besonderer disposition, für die in manchen fällen eine familiäre komponente gesichert werden konnte, löst die einnahme von nsaid, unter dem begriff omega- -fettsäuren [omega- -fatty acids, ω- -fa] werden fettsäuren zusammengefasst, die eine doppelbindung in der omega- -position tragen (abb. ). im zusammenhang mit entzündungsvorgängen ist vor allem die eikosapentaensäure [eikosapentaenic acid, epa, im englischsprachigen schrifttum auch gelegentlich als timnodon acid bezeichnet] von bedeutung. diese c : ω- -fettsäure wird vor allem vom phytoplankton der meere, aber auch von süßwasseralgen und im geringen maß von landpfl anzen synthetisiert und gelangt über die nahrungskette in tierische organismen, wo sie bevorzugt in triglyceride in der c -position eingebaut und gespeichert wird. die erforschung der ω- -fa begann mit der beobachtung, dass traditionell lebende eskimos trotz massiver zufuhr von nahrungscholesterin kaum je an bluthochdruck oder atherosklerose erkranken, oft jedoch erhöhte blutungsneigung zeigen. eine dänische forschergruppe begann nach und intensiver in den siebziger jahren systematisch diesem phänomen nachzugehen. die untersuchungen ergaben, dass neben gewissen rassisch bedingten schutzmechanismen es vor allem die in der nahrung reichlich enthaltenen epa und dha sind, welche die eskimos, deren bevorzugte diät aus fisch und meeressäugern besteht, vor gefäßkrankheiten schützen. an anderen ethnischen gruppen durchgeführte untersuchungen, wie an polarnahe lebenden indianern oder an japanischen fischern, bei denen fisch eine wesentliche nahrungsgrundlage bildet, konnten die an eskimos erhobenen befunde bestätigen. anhand von migrationsstudien wurde weiterhin gezeigt, dass angehörige fisch essender bevölkerungsgruppen den atherosklerose-und hypertonieschutz verloren und dieselbe häufi gkeit an gefäßerkrankungen wie ihre neue umgebung entwickelten, wenn sie die ernährungsgewohnheiten des gastlandes annahmen, die fisch vernachlässigten. somit scheinen wirkung und wert der ω- -fa ausreichend abgesichert. die wirkung der epa setzt an verschiedenen ebenen an und ist erst teilweise erforscht. eine reihe von effekten konnte gesichert werden: hemmung der synthese von fettsäuren, triglyceriden und cholesterin und eine verminderte vldl-sekretion der leber; stimulation der β-oxydation der fettsäuren und der ketogenese; erhöhung der insulinsekretion; verringerung des peripheren gefäßwiderstandes und damit blutdrucksenkung; verringerung der blutviskosität durch erhöhung der verformbarkeit der erythrozyten als weiterer faktor zur senkung des blutdrucks; herabsetzung der blutgerinnung und stimulation der fibrinolyse; aktivitätsminderung weißer myeloischer zellen wie hemmung der chemotaxis und der bildung von ros. diese effekte summieren sich in richtung auf eine vorbeugung der atherosklerose (s. f). die wirkungsmechanismen, die diesen effekten zugrunde liegen, werden allerdings nur zum geringen teil verstanden. auf abkömmlinge des knochenmarks wie granulozyten, monozyten und thrombozyten sowie auf endothelzellen wirkt epa offenbar dadurch, dass sie anstelle von aa oder dgla in phospholipide der zellmembran eingebaut wird. dadurch wird die membranfl uidität erhöht und möglicherweise die erregbarkeit dieser zellen verändert. gesichert ist, dass epa von der cox zu pg mit drei doppelbindungen, und von der lipoxygenase (lox) zu leukotrienen mit fünf doppelbindungen umgesetzt wird. epa und aa konkurrieren um die umsetzenden enzyme und verdrängen sich kompetitiv. während txa fast völlig unwirksam ist, besitzt pgi die selbewirkung wie pgi . damit wird das wirkungsgleichgewicht in richtung des gefäßerweiternden und gerinnungshemmenden pgi verschoben. die affi nität der aa zur cox ist allerdings wesentlich höher als die der epa, so dass der anteil an pgi und txa an der gesamtmenge produzierten pg auch nach hoher epa zufuhr relativ gering ist. epa scheint auch noch über andere, zur zeit unbekannte angriffspunkte den gefäßtonus herabzusetzen. die wirkung über die lox-aktivität läuft analog ab. epa konkurriert mit aa, und ltb ist weitgehend inert. weiters werden paf und ros vermindert produziert und abgegeben, so dass ein hoher epa-anteil in der nahrung mäßig entzündungshemmend wirkt. in der medizin werden epa-präparate als begleittherapie verwendet. die gerinnungshemmenden, blutdruck und blutlipide senkenden eigenschaften werden zur prophylaxe von coronarinfarkt oder angina pectoris eingesetzt. lebenslanger konsequenter fischkonsum wirkt infarkt vorbeugend, wie eindeutig nachgewiesen ist. die entzündungshemmende komponente der epa-wirkung kann bei chronisch-entzündlichen erkrankungen wie cp, psoriasis und akne genutzt werden. bei psoriasis sind lox-produkte wie ltb und -hete (s. , ) in den befallenen hautarealen stark erhöht. den entsprechenden epa-metaboliten fehlt weitgehend der proinfl ammatorische effekt, was den krankheitsverlauf mildert. eine zusätzliche medikation mit epa kann sich in solchen fällen als unterstützende maßnahme bewähren, ersetzt aber meist nicht eine therapie mit wirkungsvolleren mitteln. bei der bildung der leukotriene geht der metabolisierungsweg der aa, dhgl und epa über die lipoxygenasen (lox) (abb. ). es sind drei typen von lox bekannt, die sich durch den angriffspunkt an verschiedenen c-atomen der fettsäure unterscheiden: die -lipoxygenase ( -lox), die -lipoxygenase ( -lox) und die -lipoxygenase ( -lox), die wiederum in verschiedenen iso formen auftreten. ihre biologisch aktiven produkte sind die leukotriene (lt) und verwandte substanzen. ihnen allen ist gemeinsam, dass sie -wie die cox-produkte -nicht gespeichert vorliegen, sondern unmittelbar bei bedarf synthetisiert und freigesetzt werden. da ihr abbau sofort nach abgabe erfolgt, wirken sie kurzfristig lokal und nicht systemisch. die wirkung der leukotriene und verwandter verbindungen erfolgt über spezifi sche rezeptoren an den zielzellen. steigerung der kapillarpermeabilität. cysteinyl-lt bewirken eine lösung der verbindung zwischen endothelzellen und fördern damit die Ödembildung (s. ). entzündungszellen. cysteinyl-lt aktivieren die migration und damit die einwanderung von eosinophilen granulozyten in den entzündungsherd. schleimsekretion. cysteinyl-lt steigern die sekretion und bewirken eine hypertrophie von schleimdrüsen. der mukoziliare transport wird dagegen beeinträchtigt. gefäßwirkung. die wirkung von cysteinyl-lt auf gefäße hängt von der art und der lokalisation der gefäße ab. so werden pulmonalarterien erweitert, coronararterien, nierenarterien und die venen des lungenkreislaufs dagegen enger gestellt. eine steigerung des venösen gefäßwiderstandes in der lunge kann bedrohlich werden, da sie zusammen mit der erhöhten kapillarpermeabilität zu einer beeinträchtigung der mikrozirkulation im pulmonalkreislauf und zu stase und lungenödem führen kann. eine fortgesetzte und massive vasokonstriktion im kleinen kreislauf belastet über eine pulmonale hypertension das herz und kann über ein cor pulmonale zur dekompensation und herzversagen führen. reiche quellen für cysteinyl-lt in der lunge sind im rahmen anaphylaktischer prozesse mit ige sensibilisierte mastzellen. aber auch unspezifi sche entzündungen wie akute oder chronische bronchitiden können neben histamin und paf eine verstärkte abgabe von ltc -lte aus mastzellen, eosinophilen granulozyten und makrophagen bewirken. solche bronchitiden enthalten in ihrer symptomatik ein starkes bronchokonstriktorisches element ("spastische", "asthmoide" bronchitis, s. ). eine freisetzung von cysteinyl-lt in anderen organen als den lungen, die zu funktionsstörungen führt, ist offenbar weniger bedeutsam, jedenfalls weniger gut studiert. eine im tierversuch provozierbare kontraktion der koronargefäße durch ltc resultiert in einer verringerten leistung des herzens. in den nieren führt eine einschränkung der durchblutung durch ltc zu einer abnahme der glomerulären filtration. wieweit solche experimentellen ergebnisse für die humanmedizin beispielhaft sind, bleibt dahingestellt. beim menschen ist bevorzugt die lunge von lt-wirkungen betroffen. sensibilisierte atopiker weisen gegenüber gesunden einen erhöhten mastzellenpool mit erniedrigten reizschwellen auf, und die lungenmastzellen von atopikern metabolisieren aa bevorzugt über den lox-weg zu ltc . ltb wird durch omega-oxydation inaktiviert, das ist die oxydation am letzten (ω), also am c -atom (abb. manche cox-hemmer unter den nsaid wirken in höherer dosierung auch auf die lox. glucocorticoide drosseln unspezifi sch die lt-produktion, indem sie die freisetzung des muttersubstrats aa einschränken und aktivierungswege blockieren (abb. , ). Über die lipoxygenasen können nicht nur entzündungsfördernde, sondern auch äußerst wirkungsstarke entzündungshemmende metabolite, die lipoxine (lx), gebildet werden. lipoxine werden über mehrere unterschiedliche stoffwechselwege synthetisiert. --lyso-paf besitzt noch keine paf-wirkung. dazu muss in einem zweiten schritt lyso-paf durch eine spezifi sche, in den plasmamembranen lokalisierte acetyl-transferase acetyliert werden. lieferant für die acetylgruppe ist acetyl-coa. neben diesem üblichen synheseweg wurde noch ein anderer beschrieben, der anteilsmäßig aber stark zurücktritt. dabei wird phosphocholin durch eine cholin-phospho-transferase auf -acyl- -acetyl-glycerin übertragen. abbau des paf der abbau ist eine umkehrung der synthese (abb. ). eine im blut und in körperfl üssigkeiten vorhandene acetyl-hydrolase spaltet die acetylgruppe am c -atom ab, so dass der inaktive lyso-paf entsteht. das enzym hat eine hohe aktivität. eine minute nach experimenteller i. v. injektion von paf sind bereits % inaktiviert. die beteiligung von paf an systemischen prozessen ist deshalb wenig wahrscheinlich. lyso-paf kann durch eine membrangebundene acyl-transferase, mit der verschiedene zelltypen ausgerüstet sind, wieder zu lecithin aufgebaut werden. die zur acylierung bevorzugte fettsäure ist wiederum die aa. synthetisierter paf muss nicht in jedem fall sofort nach der bildung freigesetzt werden. manche zelltypen, wie endothelzellen und basophile granulozyten halten gebildeten paf zurück und geben ihn nur verzögert ab. endothelzellen können ihn in die zellmembran einbauen (chemische Ähnlichkeit mit dem membranbaustein lecithin), wo er als rezeptor für die adhäsion und die aktivierung von pmn und thrombozyten dienen kann. bei pmn ist die freisetzung des gebildeten paf von der höhe des extrazellulären ca ++ -spiegels abhängig. höhere konzentrationen fördern die paf-abgabe. solche besonderheiten spielen anscheinend bei der körpereigenen kontrolle der paf-wirkung mit. der "paf" ist keine chemisch einheitliche substanz. im lecithin können fettsäuren verschiedener länge und sättigungsgrades in c -position verestert sein. auch kann die acetylierung an anderen phospholipiden als am lecithin ablaufen, so dass pafs verschiedener chemischer zusammensetzung entstehen können. manche untersucher sprechen daher von "den plättchen-aktivierenden faktoren". diese formulierung entspricht eher den tatsächlichen verhältnissen und ist umso mehr gerechtfertigt, als chemisch verschiedene pafs auch unterschiede in ihrer wirkungsintensität zeigen. am aktivsten scheint der paf mit palmitinsäure an c zu sein. in diesem buch wird der einfachheit halber und dem allgemeinen sprachgebrauch folgend "der paf" verwendet. in versuchstiere intrakutan injizierter paf ruft sofort eine margination von pmn im postkapillaren gefäßbereich hervor. nach etwa minuten haben die ersten zellen die blutbahn verlassen und sind ins bindegewebe übergetreten, wo sie, durch den paf stimuliert, ihre aktivitäten entfalten. der paf ist ein potenter förderer der anfangsphase der akuten entzündung, wobei sein wirkungsmodus sowohl im priming (s. ) wie auch in der stimulation kompetenter zellen zur abgabe von entzündungsmediatoren zu sehen ist, deren wirkung der paf zusätzlich steigert. in vitro ist der paf für eosinophile granulozyten wesentlich stärker chemotaktisch als für pmn. stimulierte eosinophile granulozyten setzen wiederum reichlich paf frei. eosinophile granulozyten von asthmatikern produzieren auf reiz mehr paf als diejenigen von gesunden (s. ). intrakutan injizierter paf ruft beim menschen eine starke arterielle gefäßerweiterung und eine permeabilitätssteigerung vor allem der postkapillaren venen hervor. diese wirkung ist offenbar eine direkte, da h -blocker und cox-hemmer die reaktion nicht abschwächen. in vitro bewirkt der paf eine langanhaltende kontraktion der glatten muskulatur von darm und bronchien. in vivo applizierte aerosolinhalationen von paf rufen eine starke bronchokonstriktion hervor. umgekehrt mildern vor der provokation verabreichte paf-antagonisten die bronchokonstriktion. die schleimproduktion in vitro gezüchteter schleimproduzierender zellen kann durch paf-zusatz gesteigert werden. in vivo wird am versuchstier die produzierte schleimmenge erhöht, der schleimtransport durch hemmwirkung auf das flimmerepithel aber verzögert. die verschlechterung der mukoziliaren clearance kann bis zur mucostase führen. dem paf wird heute eine wesentliche rolle beim pathomechanismus des asthma bronchiale und der copd zugeschrieben, wobei die wirkung auch indirekt, nämlich über eine potenzierung anderer mediatoren wie lt, pg, histamin zu laufen scheint. das zustandekommen der trias, die zur ventilationsstörung führen, hängt mit paf-effekten eng zusammen: bronchokonstriktion, submuköses Ödem und vermehrte produktion eines dyskrinen schleims mit mangelhafter clearance. der paf fördert darüber hinaus die infi ltration der entzündeten atemwege mit pmn, makrophagen und eosinophilen granulozyten, die ihrerseits wieder über freigesetzte wirkstoffe, zu denen auch paf gehört, den entzündungsprozess weiter in gang halten und potenzieren (s. , ). die in vielen untersuchungen nachgewiesene mitbeteiligung des paf beim asthma bronchiale bietet einen ansatz für neue therapiemöglichkeiten dieser krankheit. paf-antagonisten sind in klinischer erprobung. beim lungeninfarkt und bei peripheren gefäßverschlüssen eingesetzt. zur plasminaktivierung stehen zwei ansätze zur verfügung: von den exogenen aktivatoren wird streptokinase, von den endogenen werden tpa und urokinase verwendet (abb. ). von diesen wirkstoffen sind rekombinante und modifi zierte formen im einsatz. der erfolg der beiden wirkungsprinzipien hält sich etwa die waage. ausschlaggebend für eine erfolgreiche thrombolyse sind neben der ausdehnung der betroffenen gebiete der möglichst frühzeitige einsatz der therapie. unter dem cas versteht man die aktivierung von entzündungsmediatoren in verbindung mit dem intrinsischen gerinnungssystem (abb. , abb. ). gemeinsamer ausgangspunkt dieser aktivierung ist der fxii. den sinn einer parallellaufenden aktivierung des entzündungs-und gerinnungssystems kann man darin sehen, dass bei gewebsverlet-zungen die blutgerinnung und blutungsstillung in gang gebracht und gleichzeitig entzündungsvorgänge aktiviert werden, die für die nötige abwehr eindringender keime und für die abräumung und die reparatur des defektes sorgen. das einschließen von mikroorganismen in thromben und das abriegeln von entzündungsherden durch fibrinnetze behindert auch die ausbreitung von krankheitserregern. zwischen dem gerinnungssystem und der unspezifi schen abwehr besteht eine alte phylogenetische verbindung (s. ). plasmaprekallikrein (ppk), high-molecular-weight kininogen (hmw-k) und der faktor xi der blutgerinnung zirkulieren locker aneinander gebunden im blut. im bereich einer entzündung kann dieser großmolekulare komplex (mw etwa kd) zusammen mit dem ebenfalls inaktiven fxii die blutbahn verlassen. beide komponenten, der fxii und der ppk -hmw-k -fxi -komplex binden sich über ihre positive ladungsgruppen an negativ geladene oberfl ächen im bereich der entzündung, weshalb die bezeichnung "kontakt-aktivierungs-system" gewählt wurde. als oberfl ächen mit negativer ladung können in frage kommen: nach einem internationalen Übereinkommen werden die complementfaktoren mit "c" und zu ihrer näheren charakterisierung mit durchlaufenden arabischen zahlen bezeichnet, z.b. "c ". durch spaltung aktivierte bruchstücke (complementfragmente) werden mit nachgestellten lateinischen kleinbuchstaben spezifi ziert. so zerfällt etwa der inaktive faktor c in die aktiven fragmente c a und c b. drei aktivierungswege des cs sind bekannt und gut studiert (abb. ). antigene strukturen eine gewisse mindestdichte an gebundenen igg aufweisen müssen, um die statistische wahrscheinlichkeit einer brückenbildung genügend hoch zu halten. damit wird vermieden, dass geringe ak mengen eine starke abwehrreaktion mit complementbeteiligung in gang setzen. zu den einzelnen igg-subtypen entwickelt c q eine unterschiedliche bindungsaffi -abb. . aktivierung des klassischen weges bis c . startbedingung sind benachbart an antigene gebundene igg moleküle, an denen durch quervernetzung die komponente c q aktiviert wird, die daraufhin c r s durch spaltung aktiviert. c r s spaltet und aktiviert ca ++ -abhängig c und c , wobei das spaltprodukt c b sich kovalent an die antigene struktur bindet und das spaltprodukt c a anlagert. c bc a spaltet und aktiviert als "klassische c -konvertase" (c con) c , dessen c b teil sich kovalent neben der c -konvertase an die antigene struktur bindet. der komplex c bc bc a ist somit fest verankert und kann als "klassische c -konvertase" (c con) c ebenfalls durch spaltung aktivieren und mit c b die bildung des cytolytischen komplexes (mac) einleiten. die aktivierten und komplex gebundenen complementfaktoren sind proteolytische enzyme. durch die spezifi tät der enzymatischen wirkungen wird ein vorgegebener aktivierungsfl uss gewährleistet ("komplement-kaskade") und eine starke amplifi kation der reaktion erreicht. die bruchstücke c a, c a und c a sind als "anaphylatoxine" wirkungsvolle entzündungsmediatoren. der faktor h bindet sich an entstehendes c b und macht es der zerstörung durch den faktor i zugänglich (siehe nebenschlussaktivierung s. ). die c konvertase wird mit unterstützung des decay accelerating factor (daf), des membrane cofactor proteins (mcp) und des c b rezeptors (cr ) durch den faktor i abgebaut. cr ermöglicht auch den abbau der c konvertase durch den faktor i. (genaueres siehe s. f). von der klassischen c konvertase kann die complementaktivierung über den membrane attac complex weiterlaufen. im englischsprachigen schrifttum wird dieser abschnitt des cs als "membrane attac complex" ( mac) bezeichnet. an dieser nomenklatur soll auch hier festgehalten werden. in der deutschsprachigen literatur ist auch der terminus "cytolytischer komplex" gebräuchlich. der mac umfasst die glykoproteine c , c , c , c , und c , die nach ihrer aktivierung einen festen verband bilden, der in die zellmembran von zielzellen eingelagert wird und zur cytolyse der zellen führt. an limitierenden kontrollmechanismen sind in plasma gelöste sowie membranständige inhibitoren bekannt. die anaphylatoxine c a, c a und c a sind entzündungsmediatoren, die bei der complementaktivierung entstehen. die entsprechenden vorstufen c , c und c zerfallen im rahmen ihrer aktivierung in hochmolekulare "b" bruchstücke (mw im höheren bis bereich), die sich an die zielstruktur binden, und in die niedermolekularen "a" anteile, die anaphylatoxine (mw im bis bereich), die in lösung gehen und in der umgebung ihrer entstehung aktiv werden. ihre wirkungsdauer ist allerdings nur kurz, da sie rasch durch carboxypeptidasen des plasmas abgebaut werden. die bezeichnung "anaphylatoxine" wurde gewählt, weil diese mediatoren eine symptomatik ähnlich den anaphylaktischen, d.h. ige vermittelten immunreaktionen, hervorrufen können. diese Ähnlichkeit ist zum teil auf die stimulation von mastzellen durch anaphylatoxine und die freisetzung von histamin und aa-metaboliten zurückzuführen (s. ). anaphylatoxine wirken über spezifi sche membranrezeptoren auf die zielzellen. der c a-rezeptor und der c a-rezeptor (cd ) wurden an weißen blutzellen der myeloischen reihe (pmn, eosinophile und basophile granulozyten, monozyten/makrophagen, reichlich an mastzellen), an b-und t-lymphozyten, sowie an nicht-hämatopoetischen zellen wie glat- cd ist der rezeptor für das anaphylatoxin c a. hier sollen die in den vorigen kapiteln systematisch beschriebenen effekte des cs nach funktionellen gesichtspunkten geordnet und zusammengefasst werden. eine cytolyse von zielzellen ist aber auch ohne aktivierung des mac möglich. phagozyten binden sich über ak an antigene zielzellen und zerstören sie durch abgegebene wirkstoffe wie ros, lysosomale enzyme und basische proteine. dieses spezifi sche erkennen von zielzellen über ak durch phagozyten wird als "antibody dependent cellular cytotoxicity", adcc, bezeichnet. bin-dung und phagozytose werden durch eine complementaktivierung verstärkt, die nur bis c b ablaufen muss, um wirksam zu werden. c b wird dabei leicht zu c bi fragmentiert (abb. ). die bindung von c bi zum cr rezeptor nimmt bei der adcc eine zentrale stellung ein. neben phagozyten werden über adcc auch "killer-zellen" wie die n-killerzellen oder den fc-rezeptor tragende tc-zellen zur cytotoxizität angeregt, bei der sie perforine als cytotoxische waffe einsetzen (s. ). die adcc ist bei der tumorabwehr, transplantatabstoßung, infektabwehr und bei autoimmunerkrankungen von großer bedeutung. ein anderer wirkungsmodus des spezifischen immunsystems, um körperfremde zellen, bakterien und viren unschädlich zu machen, ist ihre immobilisierung durch verklumpung, die aggregation, syn. agglutination, durch ak. auch fremdproteine wie etwa bakterientoxine können durch ak-bindung zu größeren komplexen aggregieren, werden dadurch wasserunlöslich und fallen aus, sie präzipitieren. damit werden sie im organismus lokalisiert festgehalten und können von phagozyten eliminiert werden. das cs kann diese aggregationsprozesse fördern und die phagozytose der aggregate durch die opsonine c b und c bi verstärken (abb. ). das anaphylatoxin c a schwächt die spezifi sche immunantwort ab, indem es die proliferation, migration und die sekretionstätigkeit der t-zellen sowie die ak-produktion der b-zellen hemmt und im gegenzug die proliferation und aktivität der suppressor-t-zellen steigert. darüber hinaus aktiviert c a in phagocyten den aa-metabolismus über die cox und steigert die produktion der immunsuppressiven pge-typen (s. ). c a dagegen verstärkt die spezifi sche immunantwort, indem es die il -produk- ( ) rückt das virus an die oberfl äche der wirtszelle, wo es die bildung virus-spezifi scher glykoproteine induziert ( ), die in die zellmembran eingelagert werden ( ). beim "budding" löst sich das virus aus der wirtszelle und nimmt dabei eine hülle aus zellmembran mit eingelagerten virus-kodierten glykoproteinen mit ( ). das extrazelluläre, freie virus gewinnt kontakt mit einer neuen wirtszelle, an die es sich vermittels der glykoproteine an spezifi sche oberfl ächenstrukturen bindet ( ). nach diesem andockvorgang ist das eindringen in die neue wirtszelle und eine virusvermehrung möglich. abwehrmechanismen: spezifi sche ak ( ) und/oder das cs ( ) erkennen die glykoproteine an der oberfl äche der wirtszelle, binden sich an sie und setzen als opsonine die zerstörung und phagozytose der virus-befallenen wirtszelle in gang. bei aktivierung des mac wird die wirtszelle lysiert und im anschluss phagozytiert ( ). complement kann die bindungsstellen an den glykoproteinen abkappen und so ein andocken an eine wirtszelle verhindern ( ). ak und complement können viren agglutinieren und damit immobilisieren ( ). ak und das auf dem klassischen und/oder alternativen weg aktivierte cs opsonieren freie viruspartikel und virus-aggregate und machen sie so der phagozytose zugänglich ( ). cytokine sind regulatorstoffe der entzündung wie auch normaler zellfunktionen, die von verschiedenen zelltypen produziert und in die umgebung (autokrine oder parakrine wirkung) bzw. ins blut abgegeben werden (endokrine wirkung). im nicht entzündeten organismus sind die plasma-konzentrationen vieler cytokine sehr nieder, oft unter der nachweisgrenze von routinemethoden. die von einem cytokin über membranrezeptoren angesprochenen zellen können in ihrer art äußerst vielfältig sein, wie auch ein cytokin von verschiedenen zelltypen synthetisiert werden kann (multifunktionelle, pleiotrope wirkung). als produk-tionsstätten und zielzellen von cytokinen wurden anfangs monozytäre zellen und lymphozyten erkannt und studiert. daher stammt auch die bezeichnung "monokine" und "lymphokine", die heute verlassen ist, da man eine vielzahl von zelltypen als mögliche produzenten festgestellt hat. die meisten cytokine werden auf einen reiz hin synthetisiert und danach abgegeben. einige können aber in gewissen zellen gespeichert werden. chemisch sind cytokine peptide mit sehr uneinheitlichem bau. es gibt solche mit und ohne kohlenhydrat-anteil. da der kohlenhydratanteil schwanken kann, weisen die aktuell gemessenen molekulargewichte oft beträchtliche unterschiede auf. eine weitere ursache für schwankende angaben kann auch darin liegen, dass manche cytokine nur als dimere oder polymere zirkulieren und aktiv sind. in der folgenden aufl istung wird das molekulargewicht des zuckerfreien, monomeren proteinmoleküls angegeben. die wichtigste, aber nicht die ausschließliche aufgabe der cytokine ist die steuerung von entzündungs-und immunvorgängen. dieses buch konzentriert sich auf ihre rolle bei der unspezifischen entzündung. eine unterteilung der gruppe der cytokine kann nach verschiedenen aspekten getroffen werden. bei der hier vorgenommenen aufgliederung werden das wirkungsspektrum und auch historische aspekte, nicht die chemische struktur berücksichtigt. es wird die aktuelle, international anerkannte nomenklatur verwendet. auf die im laufe der entdeckung der verschiedenen cytokine parallel gebrauchten synonyme wird nicht eingegangen. da ein im versuch in vivo appliziertes cytokin eine kettenreaktion an weiteren freigesetzten cytokinen und infl ammatorischen mittlerstoffen auslöst, wurden die wirkungen in erster linie in vitro an isoliertem zellmaterial studiert. das wiederum verzerrt die natürlichen verhältnisse insofern, als cytokine physiologisch im synergismus mit -oder in opposition zu -anderen lokal abgegebenen regulatoren der entzündung agieren. lebensnahere ergebnisse liefern untersuchungen an isolierten organen und geweben. bei der reihenfolge der cytokin-abgabe nach einem entzündungsreiz in vivo wird eine gewisse zeitliche staffelung eingehalten. zuerst werden interleukin (il- ) und der tumor nekrose faktor alfa (tnfα) freigesetzt, auf deren reiz hin weitere cytokine und regulatoren folgen. manche untersucher sprechen hier von einer "primären" und "sekundären" entzündungsantwort [primary, secondary infl ammatory response]. nach dem schwerpunkt ihrer wirkung lassen sich fünf gruppen von cytokinen unterscheiden: in weiterer folge zeigten tierversuche, dass es besonders die lipopolysaccharid-fraktion von bakterienmembranen ist, die eine hämorrhagische nekrose experimenteller tumore auslöst. in den jahren - wurde der eigentlich verantwortliche körpereigene wirkstoff isoliert und als ein polypeptid -der tnfα -identifi ziert. um gelangen die klonierung des gens und die sequenzierung der proteinstruktur. tnfα kann heute rekombinant hergestellt werden. wegen der auffälligen tumorziden wirkung dieses cytokins wurde der traditionelle name "tumornekrose-faktor" beibehalten. das tnfα-monomer ist ein nicht glykosyliertes protein mit einem mw von . d. die aktive form ist das homotrimer, während monomere nicht aktiv sind. eine therapie von tumoren mit tnfα und ifnγ, die sich daraus logisch anbietet, hat sich wegen der unzumutbaren und auch gefährlichen systemischen nebenwirkungen (stark beeinträchtigtes befi nden, schock, fieber, gewichtsverlust) nicht durchsetzen können. chemokine nehmen innerhalb der cytokinfamilie wegen spezifi scher struktureigenschaften und wegen des allen gemeinsamen chemotaktischen effekts auf zielzellen eine sonderstellung ein. der name "chemokin" wurde aus "chemotaktisch" und "cytokin" zusammengesetzt. die besondere struktureigenschaft besteht in der positionierung von cystein an bestimmten stellen des moleküls. danach unterscheidet man drei untergruppen: α-chemokine mit zwei cysteinmolekülen, die durch eine andere aminosäure getrennt sind (cxc-typ), β-chemokine mit zwei unmittelbar benachbarten cysteinmolekülen (cc-typ), und γ chemokine mit nur einem cystein in kritischer position (c-typ dene chemokine angesprochen werden können. wird bei der interleukin-familie dargestellt (s. ). mw . d. produzenten sind die megakaryozyten des knochenmarks, die pf den thrombozyten mitgeben, in denen es in den α-granula gespeichert vorliegt und bei aktivierung abgegeben wird. funktionen: pf ist ein hemmer des roten knochenmarks, drosselt die produktion von pmn, monozyten und erythrozyten und ist ein antagonist des heparins. in vitro regt es die migration von gefäßendothel an, ist chemotaktisch für fibroblasten und stimuliert die histaminabgabe aus mastzellen und basophilen granulozyten. rezeptoren sind noch nicht näher defi niert. der pf -plasmaspiegel hat als indikator für die intravaskuläre thrombozytenaktivierung diagnostische bedeutung. nap- ist ein fragment, das aus verschiedenen inaktiven vorläuferproteinen durch proteolyse entsteht. die vorläufer werden von aktivierten thrombozyten abgegeben und in erster linie durch die lysosomalen proteasen der pmn und makrophagen zu aktiven nap- produkten gespalten. je nach angriffspunkt der spaltung und ausgangsmolekül entstehen nap- formen mit verschiedenen strukturen und molekulargewichten. die wirkung der nap- -varianten besteht vor allem in der aktivierung von pmn. es ist chemotaktisch und stimuliert pmns zur hinauf-regulation von adhäsinen, phagozytose und abgabe lysosomaler enzyme und ros. bindungsversuche weisen auf zwei verschiedene rezeptoren hin. zielzellen sind eosinophile und basophile granulozyten, auf die eotaxin stark und spezifi sch chemotaktisch wirkt. von entzündungszellen freigesetztes eotaxin trägt wesentlich zur massenansammlung von eosinophilen granulozyten an orten anaphylaktischer reaktionen bei. konzentrationsabhängig werden darüber hinaus adhäsion, granulaabgabe und ros-produktion angeregt. eotaxin spricht zielzellen über einen chemokin-rezeptor (ccr ) an, der auch rantes und andere chemokine bindet und über sie entsprechende effekte vermitteln kann. interferone stehen vorwiegend im dienst der regulierung der spezifi schen immunantwort. alle interferone können rekombinant hergestellt werden und kommen therapeutisch zum einsatz. ifnα wird therapeutisch bei gewissen leukämien, dem myelom und bei hepatitis c eingesetzt. mw . d. ifnβ weist mit ifnα zu % eine homologie der aminosäuresequenz auf und wirkt über dieselben rezeptoren, entfaltet daher auch vergleichbare wirkungen. quellen sind fibroblasten und manche epithelien. therapeutisch fi ndet es bei multipler sklerose und gewissen hepatitisformen verwendung. mw . d. quellen sind cd -und cd -positive lymphozyten und nk-zellen. wirkungen: der rezeptor (cd ) fi ndet sich auf einer reihe hämatopoetischer zellen wie t-und b-lymphozyten, nk-zellen, makrophagen, pmn, thrombozyten, aber auch auf endothelzellen, manchen epithelien und auf verschiedenen tumorzelltypen. ifnγ ist ein stark pleiotropes cytokin, das auf vielen ebenen (lymphozyten, makrophagen) in die spezifi sche immunabwehr eingreift. besonders hervorzuheben ist hier die aktivierung von makrophagen zur phagozytose, ag-aufbereitung und ag-präsentation (abb. , abb. ). im bereich der unspezifi schen abwehr steigert ifnγ die tätigkeit von makrophagen und pmn. es verstärkt den antiviralen und antitumor-effekt von ifnα, ifnβ und tnfα und steuert wachstum, differenzierung und aktivierung von gefäßendothel und fibroblasten. therapeutisch wird es wegen seiner antiproliferativen wirkung bei manchen tumoren und bei leukozytosen eingesetzt. wachstumsfaktoren sind wirkstoffe unterschiedlicher protein-oder polypeptidnatur, deren benennung historisch begründet ist. ihre existenz wurde zuerst dadurch nachgewiesen, dass sie in in-vitro oder in-vivo versuchsansätzen zellen zum wachstum und/oder zu bestimmten differenzierungen anregten. Über dieses gemeinsame charakteristikum hinaus können sie aber noch eine fülle unterschiedlicher effekte auf autokriner, parakriner und endokriner ebene entfalten. unter anderem vermitteln sie auch informationen im entzündungsgeschehen; nur auf diesen angriffspunkt soll im folgenden eingegangen werden. die wachstumsfaktoren werden, abhängig vom subjektivem gesichtspunkt des beschreibenden autors, systematisch verschiedenen wirkstoffgruppen zugeteilt, wie auch ihre erforschung noch in fluss ist. hier wird einer klassischen einteilung gefolgt, in die auch die "hämatopoetischen hormone" (colony stimulating factors, erythropoetin und thrombopoetin) aufgenommen werden. die international üblichen englischsprachigen bezeichnungen werden beibehalten. mw . d. das molekül besteht aus α, β und γ untereinheiten. quellen: ngf wurde aus der glandula submaxillaris und aus speichel, aus der prostata und aus dem zns isoliert. rezeptoren fi nden sich auf sensorischen und sympathischen neuronen und auf neuralleisten-abkömmlingen wie melanozyten und paraganglien, auf schwann'schen zellen, monozyten, b-lymphozyten und mastzellen. funktionen: ngf steuert das wachstum und die differenzierung von nervenzellen. es ist für auswachsende neurone chemotaktisch und steuert deren wachstums- die hauptvertreter der fgf-familie sind der acidic fi broblast growth factor (afgf) und der basic fi broblast growth factor (bfgf). zwischen den beiden besteht zu % eine strukturhomologie. weitere verwandte spielen vor allem während der embryonalen und fetalen entwicklung ihre rolle. die fgf sind regulatoren der zellbewegung, des zellwachstums und der differenzierung mesenchymaler gewebe sowie steuerfaktoren bei der angiogenese. sie zeigen eine hohe affi nität zu heparin und verwandten glykosaminoglykanen, an die sie in der extrazellulären gewebsmatrix gebunden vorliegen. klinik: rhtpo wird bei thrombozytopenien zur erhöhung der thrombozytenzahl eingesetzt. es wurden genetisch angelegte strukturvarianten von cytokinen festgestellt, welche die effektivität des wirkstoffmoleküls in positivem wie in negativem sinn beeinfl ussen können. untersuchungen dazu liegen in breiterem umfang für den tnfα und die interleukine il- , il- , il- , il- , il- und den il- ra vor. solche varianten sind für die klinische medizin deshalb von großem interesse, weil hier eine ursache für die beträchtlichen individuellen unterschiede gegeben sein könnte, mit der sich die entzündliche reaktivität auszeichnet. ein besseres verständnis für die individuelle neigung zu infekten, autoimmunprozessen u.ä. wird von erkenntnissen auf diesem sektor erwartet. die forschung ist in fluss. verschiedene zellmembranständige rezeptoren für cytokine zirkulieren gelöst im blut. diese formen werden mit dem präfi x "s" (soluble) gekennzeichnet, wie z.b. "sil- r" für den gelösten il- in der therapie erlangen lösliche rezeptoren bzw. deren analoga zunehmend bedeutung. so wird ein modifi zierter stnfr bereits erfolgreich bei der chronischen polyarthritis (cp) eingesetzt. die in die therapie des sirs und des septischen schocks gesetzten erwartungen wurden durch den stnfr dagegen nicht erfüllt. die blockade des tnfα führte im gegenteil zu einer verschlimmerung des entzündungsgeschehens, wie auch bei der therapie der cp mit stnfr-präparaten infekte als nebenwirkung auftreten können. offenbar ist der tnfα für eine intakte immunabwehr unverzichtbar. allgemeines akutphase-proteine (app) sind proteine oder protein enthaltende strukturen, die normale bestandteile des blutplasmas sind, aber während entzündlicher prozesse vermehrt gebildet werden und vermehrt im blut auftreten. Übereinkommend wurde zur defi nition festgelegt, dass der blutspiegel um mindestens % des normalwertes ansteigen muss. aufgaben der app: generell gesehen helfen die app bei der um-und einstellung des organismus auf die besonderheiten der situation "entzündung" mit. starke entzündliche noxen erfordern vom organismus entsprechend starke gegenmaßnahmen. der "organismus in entzündung" befi ndet sich in einer art von ausnahmezustand, dem mit einem veränderten energetischen und metabolischen niveau entsprochen werden muss. die app tragen zur stabilisierung einer neuen homöostase und zur regulation erforderlicher begleitreaktionen bei und leiten zu gegebener zeit die reparatur-und heilungsphase ein. die aktivität der app soll ein dämpfendes gegengewicht zum entzündungsprozess bilden und sein örtliches wie zeitliches ausufern verhindern. eine wichtiger funktionsbereich der app ist die eingrenzung des entzündungsprozesses auf das erforderliche maß und die benötigte lokalisation. dem entsprechend sind einige app ausgeprägte entzündungshemmer, welche die chemischen wirkstoffe der entzündung unter kontrolle halten. sie können ros neutralisieren oder ihre bildung beschränken (s. f), oder sie sind als anti-proteasen tätig (s. ) . ziel eines regulativen eingriffs durch den organismus ist es dabei, die entzündlichen kampfstoffe im entzündungsherd selbst aktiv sein zu lassen, ihre wirkung in der umgebung der entzündung und im blut jedoch außer kraft zu setzen. auf diese weise wird ein entzündungsherd funktionell abgegrenzt, "lokalisiert", "demarkiert", "sequestriert". zur morphologischen abgrenzung siehe s. . diese ambivalente aufgabe -hemmung der entzündung in der peripherie, förderung der entzündung im zentrum -wird gelöst, indem die app ihre zielstoffe durch bindung neutralisieren. oft sind es stöchiometrische : bindungsverhältnisse zwischen app und entzündungsförderndem wirkstoff. der entstandene komplex ist unwirksam und wird phagozytiert und abgebaut. im entzündungsherd, wo die wirkstoffkonzentration hoch ist, wird das hemmende app verbraucht und der wirkstoff kann seinen effekt entfalten. in der peripherie liegen die verhältnisse umgekehrt: die konzentration der app ist höher als die des wirkstoffs, dessen wirkung somit ausgeschaltet wird. die gefahr dieses regelmechanismus besteht allerdings darin, dass sich bei massiver systemischer wirkstofffreisetzung die app-vorräte im blut erschöpfen (depletion), und die wirkstoffe ohne ausgleichende antagonisten den organismus schädigen oder zerstören können (s. , ). andere app wiederum fördern die abräumung geschädigten oder zerstörten zell-und gewebsmaterials, indem sie das material für die phagozyten des scavengersystems markieren: sie sind opsonine (s. ). wieder andere wirken wachstumsanregend auf fibroblasten, fördern die kollagenbildung und regen das gefäßwachstum an. so tragen sie zum heilungsprozess bei. chemisch sind app glykoproteine mit schwankendem kohlenhydratanteil. ein großer teil der app wandert elektrophoretisch in der α und α -globulin fraktion. quellen der app sind in erster linie die hepatozyten, aber auch die makrophagen der leber ( kupffer zellen) und anderer standorte, sowie endothelzellen, fibroblasten und adipozyten tragen geringfügig zur bildung bei. die stimulation zur bildung der app erfolgt durch stoffe, die im bereich der entzündung freigesetzt werden und mit dem blut zur leber gelangen. eine sol-che endokrine wirkung ist für il- , il- und den tnfα gesichert, wobei der einfl uss von il- dominiert. glucocorticoide (cortisol) fördern die bildung der app, indem sie die rezeptoren für diese cytokine an den leberzellen hinaufregulieren. ein cytokin stimuliert bevorzugt die produktion gewisser app, so dass die relative zusammensetzung der app je nach cytokin-palette sehr unterschiedlich sein kann. zur vollen entfaltung der app ist das zusammenwirken aller cytokine und von cortisol nötig (abb. ). die synthese und abgabe der app erfolgt nicht gleichzeitig mit der entzünd- fibrinogen-und fibrin-bruchstücke aus dem abbau durch plasmin sind immunsuppressiv, indem sie die proliferation von lymphozyten hemmen. unter der bezeichnung "sludge-phänomen" versteht man die tendenz der erythrozyten, sich bei verlangsamter oder unterbrochener blutströmung mit ihren flächen aneinander zu legen. die so entstandenen gestapelten aggregate werden wegen ihrer Ähnlichkeit treffend auch als "geldrollen", und der vorgang der erythrozytenaggregation als "geldrollenbildung" bezeichnet. die englischsprachige bezeichnung "sludge" (= schlamm, matsch) bezieht sich auf die starke und sprunghafte viskositätserhöhung, die blut erfährt, wenn die strömungsgeschwindigkeit aus dem newton'schen in den nicht-newton'schen bereich absinkt und die einzelpartikel sich durch aggregati- "crusta phlogistica" (= entzündliche obere schicht) entspricht der heutigen bezeichnung "buffy coat", und die beurteilung ihrer dicke ist eine grobe meßmethode einer leukozytose. mittelalter und neuzeit übernahmen das antike wissen eines galen und weiteten es aus. erhöhte blutsenkung und crusta phlogistica wurden richtig als krankheitszeichen unddurch die fibrinogenvermehrung -als erhöhtes risiko für thrombosen und embolien, den sog. "schlagfl uss", gedeutet. die mittel zur herabsetzung der gerinnungsfähigkeit des blutes waren einmal die "blutverdünnung" durch blutentnahme, den "aderlass", wobei das fehlende blutvolumen durch den nachstrom eiweißarmer flüssigkeit aus geweben und aus der nahrung ersetzt wird. eine andere möglichkeit der thromboseprophylaxe war der einsatz von blutegeln. beide behandlungsprinzipien fi nden heute noch anwendung. die therapie mit aderlass wurde im zuge mancher medizinischer modetrends exzessiv und zum schaden der patienten betrieben. fehlschlüsse wurden auch aus der erhöhten blutsenkung während schwangerschaft gezogen. dem vermeintlichen Übel versuchte man durch vermehrten aderlass zu begegnen. die so anämisch gemachten frauen überstanden häufi g den blutverlust durch die geburt nicht. unter dieser fehltherapie hatten besonders die ärztlich gut betreuten frauen der oberen gesellschaftsklassen zu leiden. heparin ist weder ein protein noch tritt es bei entzündungen verstärkt im blut auf. da es aber bei der lokalen regulation der entzündung und am heilungsprozess mitwirkt, erfüllt es aufgaben der app und soll deshalb an dieser stelle erwähnt werden. mw: beim menschen je nach bildungsort zwischen und . d, bei manchen spezies bis zu . d. bau: heparin ist ein glykosaminoglykan aus glukosamin und glukuronsäure, die α-glykosidisch miteinander verbunden sind, und sulfat. herkunft: wie der name andeutet, wurde es zuerst in der leber gefunden und beschrieben. heparin wird von mastzellen und basophilen granulozyten gebildet, in deren granula es mit histamin einen komplex bildet. nach abgabe der granula wird der komplex im gewebe durch kationenaustausch gesprengt und sowohl histamin wie heparin werden frei (s. ). heparin fi ndet sich daher hemmung der atheroskleroseentwicklung und -folgen: heparin hemmt die proliferation der glatten muskelzellen der gefäßwand. es aktiviert die endothelständige lipoproteinlipase des skelettmuskels und bewirkt damit eine senkung des triglyzeridspiegels des blutes. deshalb und wegen der antikoagulatorischen wirkung wird heparin zur prophylaxe und therapie von atherosklerose und infarkten eingesetzt (s. ). der abbau des heparins erfolgt enzymatisch durch α-glykosidasen und durch ros. entzündung und blutgerinnung gingen in der phylogenese gemeinsame wege und sind auch beim säuger noch eng miteinander verbunden. viele der involvierten wirkstoffe üben doppelfunktionen aus, indem sie im entzündungsgeschehen wie bei der blutgerinnung in fördernder wie in hemmender weise effektiv sein können. das hämostatische system unterstützt und ergänzt die protektiven aufgaben des immunsystems. faktoren der blutgerinnung können als opsonine dienen, und das einschließen in thromben immobilisiert mikroorganismen und macht sie für das immunsystem erkennbar und zugänglich. die bildung stabiler fibrinschichten grenzt entzündungsherde gegen gesunde bereiche ab und behindert die ausbreitung belebter wie unbelebter noxen in den gesamtorganismus. funktionsgemäß ist in der akutphase mit systembeteiligung -bei infekten und bei weitreichenden gewebszerstörungen mit erhöhten anforderungen an das scavengersystemauch die gerinnungsfähigkeit des blutes erhöht. um jedoch die gerinnungsbereitschaft auf den entzündungsbereich zu beschränken und nicht im gesamtorganismus wirksam werden zu lassen, ist parallel dazu die aktivität der antagonisten der blutgerinnung gesteigert. etliche der synergisten wie auch antago-nisten der blutgerinnung treten als app auf (tabelle ). ausgedehnte chirurgische eingriffe stellen wegen des hohen anfalls an scavengermaterial einen starken entzündlichen reiz dar, der zu einem anstieg der app im blut führt. fibrinogen ist postoperativ nach drei tagen deutlich erhöht und erreicht bei komplikationsfreiem verlauf zwischen dem siebenten bis . tag ein maximum (abb. ). es kann dabei auf das zwei-bis dreifache des normalwertes ansteigen, was die thrombose-und emboliegefahr erhöht. besonders betroffen sind bei bettlägrigen die tiefen beinvenen, in denen sich durch die verlangsamte blutzirkulation bevorzugt thromben bilden. als prophylaxe kann die zirkulation beschleunigt werden, indem man durch kompressionsstrümpfe den blutstrom aus den oberfl ächlichen beinvenen in die tiefen umleitet und die patienten möglichst früh zum gehen veranlasst. routinemäßig wird postoperativ niedermolekulares heparin verabreicht, das im vergleich zu unfraktioniertem, nativem heparin nicht so stark koagulationshemmend wirkt, was die blutungsgefahr herabsetzt (s. ). der erhöhung liegt das physikalische gesetz zugrunde, dass die viskosität mit der zahl und größe der gelösten oder suspendierten partikel (steigerung der inneren reibung) zunimmt. nach dem hagen-poiseuille'schen gesetz steigt der druck, der nötig ist, um eine flüssigkeitssäule in einer röhre zu bewegen, linear mit der viskosität. eine reihe von einzelfaktoren führt während entzündlicher prozesse zu einer erhöhung der blutviskosität und des strömungswiderstandes: in welche richtung sich undifferenzierte stammzellen weiterentwickeln, hängt von verschiedenen steuerfaktoren ab, unter denen die hämatopoetischen hormone, zu denen auch die colony stimulating factors (csf) gehören, gut studiert sind (s. f). man unterscheidet drei typen von granulozyten: neutrophile, eosinophile und basophile granulozyten. für den neutrophilen granulozyten hat sich international die englischsprachige bezeichnung und ihre abkürzung, "polymorphonuclear leukocyte", pmn, eingebürgert, die auch in diesem buch verwendung fi nden soll, wobei bei gegebenheit aber auch der klassische ausdruck "neutrophiler granulozyt" gebraucht wird. bei suche in elektronischen dateien ist es ratsam, unter beiden stichworten nachzufragen, da keine einheitliche nomenklatur eingehalten wird. die "klassische" unterscheidung der granulozyten untereinander erfolgt anhand der unterschiedlichen färbbarkeit ihrer lysosomalen granula mit sauer-basischen farbstoffgemischen, auf die sich auch die namensgebung bezieht. diese methoden -am gebräuchlichsten ist die färbung nach may-grünwald -reichen auch heute noch für den routinegebrauch aus. differenzierter und neuer ist der nachweis der verschiedenen enzymausstattungen der zellen, die sich mit histochemischen methoden darstellen lassen. moderne ansätze bedienen sich der immunfl uoreszenz und markieren typische oberfl ächenstrukturen spezifi sch mit fl uoreszenzmarkierten monoklonalen antikörpern. damit können nicht nur die einzelnen zelltypen exakt voneinander getrennt, sondern auch unterschiedliche funktionszustände, alters-und reifezustände auseinander gehalten werden. viele dieser antigenen oberfl ächenstrukturen sind einheitlich defi niert und werden weltweit in einem nomenklatursystem, dem cd-system ("cluster designation"), zusammengefasst. granulozyten durchlaufen während ihrer existenz drei markante aufenthaltsorte, die ihre entwicklung, aktivitäten und aufgabenbereiche prägen: das knochenmark, die blutbahn und das gewebe. dementsprechend unterscheidet man im leben eines granulozyten eine medulläre phase im knochenmark, eine blutphase und eine gewebsphase. die entwicklung der granulozyten erfolgt beim gesunden erwachsenen aus- nach einer gängigen auffassung befi ndet sich der stammzellenpool in zwei aktivierungsformen im "ruhenden pool", der sich teilt, aber undifferenziert bleibt, und so den stammzellenpool aufrecht erhält, und im "mitotischen pool", welcher der einwirkung der csf zugänglich ist. aus ihm erfolgt über teilung und reifung die bildung der differenzierten formen myeloischer und lymphatischer zellen. steigerung der granulopoese. von allen csf sind die struktur und die kodierenden gene bekannt, und csf können rekombinant hergestellt werden. gm-csf und vor allem g-csf werden klinisch breit eingesetzt. häufi ge indikationen sind myelodepression durch immunsuppressive therapien, z.b. nach organtransplantation oder tumor-chemotherapie. die applikation von m-csf und il- stimuliert makrophagen zur massiven freisetzung von cytokinen wie il- , il- , tnfα u. a. und löst damit starke systemische reaktionen wie fieber, schock, Übelkeit und erbrechen aus, welche die anwendung einschränken oder verbieten. die gefahr einer therapie mit dem gut verträglichen g-csf besteht in einer Überstimulation und erschöpfung des ruhenden stammzellenpools dadurch, dass die gesamtheit der stammzellen in den mitotischen pool übergeführt und damit verbraucht wird ("ausbrennen" des knochenmarks). eine andere gefahr ist die der induktion eines tumorwachstums, vor allem von myeloischen leukämien. csf sind auch in der lage, "schlafende" tumorklone zum wachstum anzuregen. in gefäßen bewegt sich die blutsäule in schichten mit verschiedener geschwindigkeit, als laminare strömung, die eine folge der reibung des blutes an der gefäßwand ist. aus dem verhältnis von volumen zu oberfl äche ergibt sich, dass der reibungseffekt umso mehr anteile der blutsäule erfasst, je geringer der durchmesser des gefäßes ist. in engen blutgefäßen von etwa mm lumen abwärts verteilt sich die geschwindigkeit der einzelnen strömungsschichten in der form einer parabel mit der geringsten geschwindigkeit an der gefäßwand (randstrom, marginalstrom), und der höchsten in der strommitte ( zentralstrom, abb. heitsbildern sirs, ards und mof (s. ff). im postkapillaren venenbereich erweitert sich das strombett beträchtlich, was eine starke verlangsamung der blutströmung zur folge hat. die scherspannung zwischen den erythrozyten verringert sich dadurch so stark, dass bindungstendenzen zwischen den einzelnen zellen wirksam werden, die sich mit ihren flächen aneinander lagern. diese ag-gregatbildung, die sogenannte "geldrollenbildung" [sludged blood] wird durch positiv geladene bindungsbrücken vermittelt, die sich zwischen die negativ geladenen oberfl ächen der erythrozyten einlegen und zur bildung von aggregaten aus mehreren zellen führen. als bindungsbrücken werden höher molekulare plasmaeiweiße wirksam, in erster linie fibrinogen, haptoglobin und immunglobuline (s. f, abb. eine emigration fi ndet in großem umfang nur im bereich der postkapillaren venenstrecke statt. hier sind die rheologischen, räumlichen und morphologischen voraussetzungen gegeben. der langsame blutfl uss begünstigt die aggregation der erythrozyten, wodurch weiße blutzellen an das endothel gedrängt werden, während der blutstrom zentral ungehindert weiterzieht. die geringe scherspannung durch den langsamen blutfl uss sowie die dichte bestückung der endothelzellen mit adhäsinen ermöglicht es den leukozyten, fest am endothel zu haften, aktiv am endothel kriechend kontaktstellen zwischen endothelzellen aufzusuchen und die blutbahn zu verlassen, zu emigrieren. der bau der postkapillaren venen begünstigt die emigration dadurch, dass dieser gefäßtyp keine geschlossene muskellage aufweist, sondern die einzelnen muskelfasern in lockeren zügen mit weiten zwischenräumen angeordnet sind. die bedingungen der emigration sind für die zellen der myeloischen und lymphatischen reihe grundsätzlich gleich. die lockere bindung wird auf der pmn-seite durch l-selektin (cd l) und durch sialoproteine (z.b. sialyl-lewis x, cd s), auf der endothel-seite durch e-selektin (cd e) und p-selektin (cd p) vermittelt. die bindung über selektine bewirkt das rollen marginierter myeloischer zellen. das rollen von lymphozyten erfolgt über andere mechanismen. mit zunehmender zirkulationsdauer verringert sich am pmn der bestand an l-selektin, indem diese proteine durch in den pmn-membranen lokalisierte proteasen von ihrem transmembranösen teil abgespalten werden, während parallel dazu der besatz an β -integrinen durch nachschub aus den sekretvesikeln zunimmt (abb. ). integrine sind locker aneinander gebundene doppelmoleküle, die aus einem αund einem β anteil bestehen. die integrine, die das "sticking" vermitteln, sind aus dem gleichbleibenden β -anteil zahl und mobilität in der membran eingeschränkt wird (abb. , ) . adhäsine sind einem natürlichen turnover unterworfen, indem extrazelluläre teile enzymatisch von der mutterzelle abgespalten werden; sie können im blut nachgewiesen werden. diese bruchstü-cke werden mit dem präfi x "s" (soluble) bezeichnet, also z.b. sicam . da sich gelöste adhäsine ebenfalls an den liganden binden, kompetieren sie mit den zellgebundenen adhäsinen und wirken so als physiologische blocker des liganden (vgl. s. , abb. ). aber auch eine potenzierende wirkung ist möglich, indem gelöste adhäsine ligandenmoleküle aggregieren und damit aktivieren. der wirkungsmodus wird so wesentlich von der art und konzentration der gelösten adhäsine bestimmt. . b) . wenn weiße blutzellen die gefäßwand durchwandern, entstehen notgedrungen lücken in der basalmembran. diese lücken schließen sich offenbar sofort hinter der zelle nach ihrem durchtritt. es ist noch nie gelungen, ultramikroskopisch einen entsprechenden defekt in der basalmembran nachzuweisen. die zahl und relative zusammensetzung der weißen blutzellen im strömenden blut wird im "weißen blutbild" [white blood cell count] erfasst und ist unter normalen verhältnissen bei einem individuum ziemlich konstant, wobei die interindividuelle streubreite allerdings beträchtlich ist. als norm für die gesamtzahl der leukozyten gilt beim erwachsenen der bereich von . bis . zellen pro mikroliter blut. zahlen darunter werden als leukopenie, darüber als leukozytose bezeichnet. der anteil an pmn beträgt normal zwischen und %, als absoluter normbereich kann eine zahl zwischen . und . /µl angenommen werden. bei werten unterhalb dieser norm spricht man von einer neutropenie oder granulopenie, darüber von einer granulozytose. eine neutropenie unter pmn/µl wird als risiko, unter /µl als hohes risiko für infekte angesehen, wobei natürlich auch die dauer solcher neutropenien berücksichtigt werden muss. im frühen kindesalter gelten andere normwerte. bei neugeborenen wird die norm für die gesamtleukozytenzahl mit . bis . /µl, beim kleinkind bis zum ersten lebensjahr mit . bis . angesetzt. einer der gründe für die hohe zahl ist der noch mangelhafte ausbildungsgrad der adhäsine an myeloischen zellen, der verhindert, dass pmn in größerem umfange marginieren (s. ). im gegensatz zu den erythrozyten besteht bei der zahl zirkulierender leukozyten kein geschlechtsunterschied. das weiße blutbild ist einem zirkadianrhythmus unterworfen. in den späten nachtstunden und am frühen morgen ist die zellzahl am höchsten. ein grund dafür ist in dem in diesem zeitraum erhöhten blutspiegel an glucocorticoiden zu sehen, die sowohl eine mobilisierung der knochenmarkspeicher bewirken wie auch die margi-nation myeloischer zellen herabsetzen (s. ). nach nahrungsaufnahme ist die zahl der weißen blutzellen erhöht ("verdauungsleukozytose", s. ). die totalzahl an zirkulierenden leukozyten beträgt beim gesunden erwachsenen um x zellen, wobei die einzelnen leukozytentypen eine unterschiedliche dynamik aufweisen. t-lymphozyten halten sich nur kurz in der blutbahn auf, treten rasch ins gewebe über und rezirkulieren über die lymphe ins blut. da eine solche passage blut-gewebe-lymphsystem-blut etwa eine halbe stunde dauert, wird der organismus täglich etwa mal von diesen zellen durchmustert. pmn sind dagegen "ein-weg-produkte", die nach ihrer blutphase von durchschnittlich sechs bis sieben stunden ins gewebe oder auf körperoberfl ächen auswandern und dort zugrunde gehen. eine rezirkulation in die blutbahn fi ndet nicht statt. die gesamtzahl an pmn im strömenden blut beträgt beim gesunden erwachsenen um die bis x zellen. bei einer rund viermaligen erneuerung des blutpools innerhalb von stunden ergibt sich ein täglicher verschleiß von bis milliarden, also im schnitt etwa pmn, die physiologisch im zuge ihrer scavenger-und abwehrtätigkeit verbraucht werden oder -offensichtlich ineffektiv -apoptotisch zugrunde gehen (s. f chemotaxis kann positiv oder negativ sein, je nachdem ob sich eine zelle in den gradienten hinein oder von ihm fort bewegt. weiße blutzellen sind ausschließlich zu positiver chemotaxis befähigt. negative chemotaxis ist bei einzellern ausgeprägt und stellt als primitive fluchtreaktion eine Überlebensstrategie dar. bei wirbeltieren hat negative chemotaxis während der embryonalentwicklung bei der verteilung von zellmaterial bedeutung. melanoblasten z.b. geben nach einer vermehrungsperiode, während der die tochterzellen nebeneinander zu liegen kommen, negativ wirkende chemotaxine ab, über die sie sich voneinander abstoßen und auf diese weise gleichmäßig in der haut verteilen. auch zellfortsätze können sich chemotaktisch orientieren. so fi nden etwa die neuriten der vorderhorn-wurzelzellen während ihres auswachsens aus den neuroblasten ihren weg über chemotaxine (nerve growth factor s. ), die von den zugehörigen myotomen abgegeben werden. analog zur chemotaxis kann chemokinetik positiv oder negativ ausgeprägt sein, je nachdem ob die ortsveränderung durch einen wirkstoff beschleunigt oder verlangsamt wird. die geschwindigkeitsveränderungen können die spontanbewegung wie auch die chemotaktische bewegung betreffen. chemotaxis und chemokinetik werden über verschiedene molekularbiologi- in diesem prozess der nukleation entstehen kurze oligomere, meist trimere. oligomere können sich nun an bereits vorhandene aktin-polymere ankoppeln, ein vorgang, der wesentlich schneller als die nukleation abläuft. diese verlängerung geschieht wiederum beschleunigt am plus-ende des fadens, so dass eine bevorzugte wachstumsrichtung des aktinfadens vorgegeben ist. fertig polyme- aktin-molekülen zu polymerem aktin. die anlagerung von ca ++ und mg ++ -ionen bewirkt eine konformationsänderung des aktin-monomers, welche die bindung von atp begünstigt (a). aktin und atp bilden zuerst trimere (nukleation von g-aktin), die sich weiter zu hochpolymerem fi lamentösem f-aktin verlängern können. durch die asymmetrie des aktin-atp-komplexes bekommt ein aktin-filament eine bevorzugte wachstumsrichtung (plus-ende, barbed end) und eine gegenseite (minus-ende, pointed end), von der die depolymerisation ausgeht (b). ein fertiges f-aktin-filament besteht aus zwei spiralig umeinander gedrehten einzelfäden (protofi lamenten) und hat einen durchmesser von - nm und eine steigungshöhe von nm, in der sich die helix einmal um ihre achse dreht (c). die länge des fadens kann unterschiedlich sein. risiertes, fi lamentöses (f-) aktin besteht aus zwei spiralig umeinander gedrehten einzelfäden aus atp-aktin (abb. c). ein doppelfaden hat einen durchmesser von bis nm und kann eine unterschiedliche länge erreichen. im elektronenmikroskop erscheint das plus-ende eines aktin-fadens etwas aufgelockert, das minus-ende dagegen eher spitz. wegen der Ähnlichkeit mit einem pfeil wird das plus-ende auch als "barbed end", die gegenseite als "pointed end" bezeichnet. das wachstum wird durch die anlagerung von capping-proteinen oder durch kontakt mit anker-proteinen abgeschlossen. die lebensdauer eines aktin-polymers ist durch die hydrolyse des atp zu adp limitiert. filamente aus adp-aktin besitzen wenig bindungsstärke und zerfallen spontan. nach lösung vom adp ist ein aktinmolekül einer erneuten polymerisation zugänglich. auf-und abbau von aktin können sehr rasch erfolgen. so ist ein pmn in der lage, innerhalb von sekunden sein aktin-cytoskelett umzubauen. im experiment kann der auf-und abbau der aktin-polymere beeinfl usst werden. cytochalasine verhindern die polymerisation, phalloidin stabilisiert die polymere. capping-proteine beenden die polymerisation von aktinfäden. so stoppt gelsolin den anbau am plus-ende, acumentin dagegen am minus-ende. profi lin verhindert die polymerisation. es liegt auf der hand, dass intrazellulär gebildete filamente untereinander und auch mit anderen intrazellulären strukturen verbindungen eingehen müssen, um ein funktionelles gerüst zu bilden. so verbinden filamin-querbrücken aktinfäden untereinander, während α-actinin aktinfäden direkt miteinander bündelt. vinculin kann aktinfäden an zellstrukturen verankern. myosin schafft bewegliche verbindungen zwischen benachbarten ak-tinfäden und zwischen aktinfäden und anderen zellstrukturen. myosin verbindet sich mit aktinfi lamenten zu einer funktionellen einheit, zum aktinomyosin. myosin tritt in zwei formen auf, als myosin i und myosin ii. myosin i ist ein monomeres molekül mit einem charakteristischen kopf-und schwanzteil. der kopfteil bindet sich an ein aktinfi lament und besitzt atpase-aktivität. die bei der hydrolyse von atp frei werdende energie wird in bewegung entlang dem aktinfaden vom minus-zum plus-ende umgesetzt. der schwanzteil kann sich an membranen und verschiedene makromoleküle binden. myosin i transportiert vesikel und anderes material das aktin-zytoskelett entlang und sorgt so für deren verteilung in der zelle, oder verschiebt aktinfi lamente gegenüber der zellmembran und kann auf diese weise verankerte rezeptoren oder ionenkanäle an der zelloberfl äche positionieren. myosin ii liegt im pmn als dimer vor, dessen monomere mit dem schwanzteil verbunden sind. die beiden köpfe des dimers verbindet sich mit je einem aktinfi lament entgegengesetzter wachstumsrichtung. beim gleiten zum plus-ende werden die aktinfäden gegeneinander verschoben (abb. ). in der muskelzelle liegt myosin dagegen ständig in hochpolymerer form vor. mikrotubuli sind bestandteil aller eukaryoten zellen. die hochpolymeren röhrenförmigen gebilde werden aus dimeren bausteinen aufgebaut. ein dimer wiederum besteht aus zwei strukturell ähnlichen proteinen von jeweils kd molekulargewicht, einem αund einem β-tubulin. es bestehen etliche analogien zu den verhältnissen beim aktin. die bausteine treten ebenso in zwei pools auf, die sich durch ihren polymerisationsgrad unterscheiden, im niedermolekularen, dimeren pool (sol-zustand), und einem -hochmolekularen pool, in dem die dimere zu mikrotubuli polymerisiert sind (gel-zustand). die beiden pools verschieben sich je nach funktionszustand der zelle zueinander. der polymerisationsprozess beginnt nicht an beliebigen orten, sondern verlangt gewisse induktionsstrukturen wie bereits vorhandene mikrotubuli oder eine spezielle induktions-organelle, das -zentrosom, das in einer zelle in der einoder mehrzahl auftreten kann. am zentrosom sind es ringförmige bindungsorte aus γ-tubulin, an denen der polymerisationsprozess in gang kommt. voraussetzung für die polymerbildung ist die bindung von gtp an ein tubulin-dimer. der aufbauprozess fi ndet in einer weise statt, dass sich die gtp enthaltenden dimere so zu einer protofi lament-kette vereinigen, dass das β-tubulin-molekül in die wachstumsrichtung zu liegen kommt (plus-ende), während das α-tubulin am freien β-tubulin der wachstumskante andockt. dreizehn solcher protofi lament-ketten lagern sich seitlich aneinander, so dass sich eine röhre von nm durchmesser bildet. da die αbzw. β-tubulin -moleküle benachbarter ketten dabei in mäßiger versetzung aneinander binden, entsteht der eindruck einer spiralstruktur. ein mikrotubulus ist somit ein polares gebilde mit einer wachstumsrichtung, dem plus-ende, und einer bevorzugten abbaurichtung, dem minus-ende, die beide durch die lagerung der dimere in der struktur vorgegeben sind (abb. ). das gtp im mikrotubulus wird laufend hydrolytisch zu gdp gespalten, womit die bindungskräfte zwischen den dimeren abnehmen. solange am plus-ende tubulin-dimere mit gtp vorhanden sind, ist die röhre stabil. diese situation wird einmal durch wachstum gesichert, bei dem sich immer neue, gtp tragende dimere anlagern. eine andere situation, welche den zerfall eines mikrotubulus verhindert, ist seine verankerung an zellstrukturen wie organellen, membranen oder membranproteinen. bildet sich aber aus irgend einem grund am plus-ende gdp, so zerfällt der mikrotubulus rasch in die richtung des minus-endes. mikrotubuli können so in intervallen von mehreren minuten auf-und abgebaut werden. nach der wiederbeladung mit gtp steht ein dimer zum erneuten einbau zur verfügung. eine reihe von steuersubstanzen ("maps", microtubule associated proteins) regelt den aufbau und die organisation der mikrotubuli. ein weiterer steu-abb. . verschiedene funktionen des aktinomyosins. ein myosin-molekül besteht aus einem globulärem kopfteil mit atpase-eigenschaft und einem schwanzteil, mit dem es sich an verschiedenen zellulären strukturen anheften kann. myosin gleitet unter atp-verbrauch auf einem aktin-filament vom minus-zum plusende. wenn myosin-einzelmoleküle (myosin i) an der zellmembran befestigt sind, können sie an aktin gebundene rezeptoren in der membran bewegen und positionieren (a). der schwanzteil kann auch strukturen wie makromoleküle oder vesikel binden und entlang aktin intrazellulär transportieren (b). verbinden sich zwei myosin-moleküle zu dem doppelmolekül myosin ii, werden aktin-filamente gegeneinander bewegt und bewirken eine kontraktion des aktinomyosin-komplexes (c). erfaktor ist der redox-zustand im zytoplasma, insbesondere des intrazellulären puffers glutathion. ein hoher anteil oxydierten glutathions hemmt den aufbau der mikrotubuli, und vice versa. vergleichbar mit dem myosin des aktinomyosin-bewegungssystems sind dem tubulussystem motorproteine beigesellt, die sich entlang der mikrotubuli bewegen und so frachten transportieren können. je nach der bewegungsrichtung werden zwei gruppen von motorproteinen unterschieden kinesine bewegen sich in richtung des plus-endes dyneine bewegen sich in richtung des minus-endes eines mikrotubulus. die motorproteine sind sich in ihrem bauplan ähnlich. wesentliche merkmale sind --zwei globuläre kopfstücke, die atpase-aktivität besitzen und durch energie verbrauchende Änderung ihrer haftorte am mikrotubulus die fortbewegung bewirken, und ein schwanzstück, an das die zu transportierende struktur gebunden wird. die beladung geschieht allerdings spezifi sch, so dass man eine beträchtliche zahl solcher transportproteine kennt, die sich in der affi nität zu ihrer fracht unterscheiden. als frachtstücke kommen etwa vesikel, sekretgranula oder zellorganellen in frage (abb. ). mikrotubuli stellen die verkehrsadern einer zelle dar. sie entlang werden wirkstoffe an den ort ihrer aktivität transportiert, und durch sie werden zellbestandteile in ihrer funktionell sinnvollen position gehalten. vom zentrosom aus, das gewöhnlich in der zellmitte angeordnet ist, können mikrotubuli spontan in alle richtungen der zellperipherie wachsen. finden sie keinen anschluss an stabilisierende strukturen, zerfallen sie wieder, um sich erneut zu bilden. finden sie entsprechenden halt, ist die lebensdauer verlängert. der aufgabenbereich im einzelnen hängt vom zelltyp ab. im abb. . aufbau monomeren tubulins zu mikrotubuli. mikrotubuli setzen sich aus dimeren aus αund β-tubulin zusammen (a). die dimere bilden zuerst protofi lament-ketten, von denen sich ringförmig zusammen lagern, so dass sich eine röhre von nm durchmesser bildet (b). da die einzelnen ketten in der längsrichtung zueinander versetzt sind, entsteht der eindruck einer spiraligen anordnung (c). die wachstumsrichtung, in welcher der anbau von neuen dimeren erfolgt, liegt auf der seite des freien β-tubulins (plus-ende), während der abbau bevorzugt von der gegenseite (freies α-tubulin, minus-ende) erfolgt. auf diese weise sind mikrotubuli zu einem treadmilling fähig. freies cytosolisches kalzium ist der bestimmende second messenger für die aktivitäten eines pmn und darüber hinaus generell von weißen blutzellen und regelt auch den aufbau und die funktion des cytoskeletts. hohe camp-spiegel fördern die aufnahme von ca ++ in die intrazellulären vorratsspeicher und senken damit die konzentration frei verfügbaren cytosolischen ca ++ . maßnahmen zur anhebung des intrazellulären camp bewirken auf diese weise eine hemmung der zellaktivität und werden therapeutisch zur immunsuppression eingesetzt. die lähmung des aktin-umbaues ist teil dieser hemmwirkung (s. , abb. ). dieser fibrillentyp fi ndet sich in allen eukaryoten zellen, ist aber zellspezifi sch ausgebildet und äußerst variabel. im gegensatz zu den mikrotubuli und mikrofilamenten ist für diese proteinstrukturen charakteristisch, dass sie vorwiegend in polymerer, und kaum in monomerer form auftreten. typische vertreter der intermediärfi lamente sind z.b. das keratingerüst in epithelien, die neurofi lamente und das fasergerüst der glia und der sertoli-zellen. intermediär-filamente dienen der mechanischen festigkeit einer zelle, deren zytoplasma sie in statisch sinnvoller anordnung durchziehen. zumeist sind sie in den desmosomen verankert. eine keratinfi brille etwa ist kabelartig aus mehreren untereinheiten verdrillt, die zusammen einen durchmesser von etwa nm aufweisen. zellkerne besitzen unter ihrer oberfl äche ein fl ächenhaftes stützgerüst, die kernlamina. im pmn treten intermediärfilamente vom vimentin-typ auf, die ein lockeres netzwerk in kernnähe und im schwanzteil migrierender zellen bilden. ihre funktion ist unbekannt, mag aber auch stützaufgaben übernehmen. die gesamtheit aller intrazellulärer fi brillärer strukturen wird als cytoskelett bezeichnet. dieser ausdruck ist insofern irreführend, da diese strukturen, die in-termediärfi lamente ausgenommen, alles andere als ein festes "skelett" bilden, sondern ausgesprochen fl üchtige gebilde darstellen, die sich ständig und je nach bedarfslage reorganisieren können. die niedermolekularen anteile des cytoskeletts befi nden sich im cytosol gelöst und können sich zu den hochmolekularen strukturen des cytoskeletts organisieren, man spricht auch von einem Übergang der sol-phase in die gel-phase. in einem inaktiven pmn sind etwa bis % des aktins in der polymeren f-form vorhanden, das meiste davon im zellkortex, und ebenso sind nur rund % des tubulins zu mikrotubuli aufgebaut. bei einer chemotaktischen aktivierung vergrößert sich der polymere anteil im kopfteil der migrierenden zelle zu lasten des niedermolekularen anteils. der umbau des aktin-cytoskeletts eines pmn benötigt nur wenige sekunden. wesentlich ist auch, dass diese fi brillären strukturen vielfach durch besondere proteine, die quervernetzungs-proteine [cross-linking proteins] untereinander verbunden sind, von denen bereits eine beträchtliche zahl charakterisiert werden konnte. zusätzlich bestehen verbindungen zu zellmembranen, membranen von zellorganellen und sekretgranula, die bei bedarf gebildet und gelöst werden können. neben funktionell bedingten intrazellulären transporten und verschiebungen wird auch die lokalisierung der organellen im zellleib fi xiert und die zellform beeinfl usst. der polymerisationsgrad des cytoskeletts bestimmt die viskosität von zellbezirken und darüber hinaus der gesamten zelle. bei einer granulaabgabe etwa muss das kortikale aktin depolymerisiert werden, damit die granulamembran in kontakt mit der zellmembran treten kann (abb. ). im in-vitro experiment führt die depolymerisation des zellkortex mit cytochalasin b zu einer spontanen degranulierung. das xanthinderivat pentoxyphyllin führt über eine steigerung des intrazellulären camp-spiegels zu einer zunahme der sol-phase des cytoskeletts in zirkulierenden leukozyten (s. , abb. ), was sie geschmeidiger macht und eine verminderung ihres widerstandes gegenüber verformung in der mikrozirkulation bewirkt (s. f). das medikament wird klinisch zur verringerung des peripheren druckwiderstandes eingesetzt. das cytosol ist nicht ideal fl üssig, sondern proteine, allen voran nicht polymerisiertes g-aktin, gehen untereinander schwache bindungen ein und bilden fl üchtige fädige strukturen. manche untersucher sprechen hier von einem mikrotrabekulären netzwerk [microtrabecular lattice]. dieses lockere fadennetz verschafft dem cytosol eine gewisse zähfl üssigkeit, die mithilft, zellorganellen in ihren positionen zu halten. die bindungen lösen sich unter scherstress aber auf, so dass sie bewegungsvorgängen innerhalb der zelle keinen nennenswerten widerstand entgegen setzen ( thixotropie). die aufgaben des cytoskeletts lassen sich zu mehreren funktionsbereichen zusammenfassen: lokomotion, das ist zellbewegung mit ortsveränderung. für die bewegung weißer blutzellen ist der ausdruck migration gebräuchlich. "on-spot motility": die meisten zellen sind zur ortsveränderung nicht fähig, aber fast alle zellen können sich insofern bewegen, als sie die form des zellleibes verändern und sich damit aktiv an ihre umgebung anpassen. zellviskosität, funktionelle verteilung der intrazellulären strukturen und zellform. intrazellulärer transport von material über größere strecken, die durch diffusion allein nicht bewältigt werden können, und funktionsgerechte verteilung dieser materialien. vesikel-und granulatransport. beteiligung am membranfl uss. endocytose im vorderende des lamellopodiums wird hochpolymeres f-aktin aus aktin-oligomeren aufgebaut. die induktion zur polymerisation geht von den besetzten chemotaxin-rezeptoren und haftenden adhäsinen aus. die entstehenden aktinfäden werden durch quervernetzungs-proteine zu einem räumlichen maschenwerk verwoben. während an der kante des lamellopodiums die aktinfäden an ihrem vorderende (barbed end) verlängert werden, werden sie an ihrem hinterende (pointed end) abgebaut, so dass das aktingerüst bei etwa gleichbleibender ausdehnung in der bewegungsrichtung der zelle wandert ( treadmilling, abb. ). der anteil des f-aktins am gesamt-aktinpool kann je nach funktionszustand des pmn zwischen und % betragen. dimeres myosin ii kann eine verschiebung der aktinfi lamente zueinander bewirken und so dem lamellopodium zusätzlich form verleihen. zugleich mit den aktin-filamenten bilden sich züge von mikrotubuli aus, die sich vom inneren der wandernden zelle bis zum kortex des lamellopodiums erstrecken. sie stellen fl exible transportstraßen dar. Über diese straßen werden vesikel und granula in den kopfteil der wandernden zelle verlagert und ein lamellopodium mit membranmaterial und rezeptoren versorgt (abb. ). nach heutiger vorstellung spielen mehrere faktoren beim aufbau des lamellopodiums und beim zustandekommen einer gerichteten bewegung zusammen. durch das treadmilling schiebt sich das aktingerüst in das lamellopodium vor. auch ein verquellen gebildeten f-aktins wird als mitursache für eine volumsvergrößerung diskutiert. gleichzeitig wird im bereich des lamellopodiums durch den membranfl uss das membranmaterial vermehrt, so dass cytoskelett und cytoplasma in diesen sich ständig erweiternden sack "hineinfl ießen". mit den sekretvesikeln und granula werden an der vorderkante des lamellopodiums auch frische rezeptoren und adhäsine sowie ionenkanäle eingebaut, die an diesem stoffwechsel-intensiven pol der zelle die empfangsbereitschaft für neue chemotaktische reize und den kontakt zu strukturen der umgebung aufrechterhalten. darüber hinaus werden im zuge des membranfl usses eine reihe zellulärer signalstoffe freigesetzt. durch die aktivierung des arachidonsäure-metabolismus im lamellopodium wird einerseits lyso-lecithin frei, das membranfusionen erleichtert (s. ), andererseits entstehen metabolite wie ltb und paf, die autokrine und parakrine informationen an nachbarzellen übermitteln. sekretgranula extrudieren ihren inhalt an lytischen enzymen in die unmittelbare umgebung abb. . der aktin-umbau als eine grundlage der amöboiden fortbewegung. polymere f-aktin-filamente verlängern sich an der vorderkante des lamellopodiums durch anbau oligomerer g-aktin-moleküle, während an der gegenseite des aktinfadens oligomere abgekoppelt werden. oligomere diffundieren durch das aktin-gerüst nach vorne zum plus-ende der aktin-filamente und stehen hier erneut zum anbau zur verfügung. auf diese weise wandert das aktin-cytoskelett in das lamellopodium hinein ("treadmilling"). sich verlängernde f-aktin-filamente gehen bindungen mit den rezeptoren und adhäsinen ein, die durch den membranfl uss an der vorderkante des lamellopodiums ständig ergänzt werden. besetzung der rezeptoren und adhäsine mit liganden verstärkt ihre anbindung an das cytoskelett. darüber hinaus vernetzen sich f-aktin-filamente untereinander und mit zytoplasmatischen strukturen, wodurch das lamellopodium die nötige form und viskosität erhält. des sich ausweitenden lamellopodiums, lockern damit gewebsstrukturen auf und erleichtern so der zelle ein vorwärtskommen ( "enzymatisches buschmesser"). die bei der membranfusion aktivierte nadph-oxydase unterstützt diese wegbahnung durch die bildung von ros. für die orientierte chemotaktische bewegung ist die beteiligung der mikrotubuli unentbehrlich, während die spontanbewegung auch bei blockierten mikrotubuli (z.b. durch colchizin) abläuft. ein sich amöboid bewegender pmn ist mit einem kettenfahrzeug vergleichbar, das in der bewegungsrichtung kettenmaterial auslegt und es am hinterende wieder einzieht. ros und enzyme helfen hindernisse zu beseitigen (abb. ). mit der rezeptorkonzentration und der bildung fi brillärer proteinstrukturen im lamellopodium hat die zelle eine eindeutige morphologische und funktionelle orientierung erhalten. in diese richtung konzentrieren sich alle aktivitäten wie migration, phagozytose und abgabe von granula und ros. diese polarisierung bedeutet aber keine fi xe strukturveränderung einer zelle, sondern ist ein reversibler funktionszustand, der bei bedarf verändert und neuen verhältnissen angepasst werden kann. bietet man z.b. experimentell einem pmn, der in-vitro in einem chemotaktischen gradienten wandert, an seiner hinterseite das che-motaxin in höherer konzentration an, so wendet die zelle nicht etwa wie ein fahrzeug, sondern an der seite des nunmehr höheren gradienten beginnt der prozess der polarisation von neuem mit rezeptorkonzentration, fi lamentösen strukturen und bildung eines lamellopodiums, während die strukturen des alten kopfstücks aufgelöst werden. pmn und allgemein weiße blutzellen, die in einem chemotaktischen gradienten wandern, erfahren demnach mehrere einschneidende Änderungen ihres verhaltens und ihrer stoffwechselsituation. polarisierung. die zelle bewegt sich mit ihrem vorderen pol in richtung der höheren konzentration des gradienten -positive chemotaxis steigerung des stoffwechsels. die zelle erhöht ihr bewegungstempo -positive chemokinetik die zelle entwickelt am vorderen pol eine gesteigerte reaktivität gegenüber aktivierungsreizen -erhöhtes priming als folge bewegt sich die zelle orientiert und beschleunigt in richtung des zielobjekts und ist in erhöhter bereitschaft, schadmaterial anzugreifen und zu beseitigen. bei der wanderung eines pmn in einen chemotaktischen gradienten hinein können folgende situationen vorliegen: wenn die konzentration eines chemotaxins eine kritische schwelle nicht überschreitet, erreicht der migrierende pmn das zentrum des chemotaktischen gradienten, in dem das chemotaxin in homogener konzentration vorliegt, und bewegt sich hier ungerichtet. da weiße blutzellen zu keiner negativen chemotaxis fähig sind, bleiben sie im zentrum des chemotaxins gefangen, bis sie auf material stoßen, das sie phagozytieren (abb. a). ---■ ein wandernder pmn kann mit einem bulldozer verglichen werden, der sich durch auslegen seiner ketten an der vorderseite und dem einziehen der ketten an der rückseite weiter bewegt. die fortbewegung wird durch wegräumen von hindernissen erleichtert. wenn die konzentration eines chemotaxins einen maximalen grenzwert überschreitet, wird die zellbewegung gehemmt, wobei pmn einen funktionszustand höheren grades einnehmen: sie gehen über hinaufregulierte adhäsine verbindungen mit benachbarten pmn ein, beginnen zu phagozytieren und granula und ros in die umgebung freizusetzen. für diese homotypische adhäsion (adhäsion unter zellen des gleichen typs) sind verbindungen von β -integrinen (cd a/cd und cd b/cd ) zu icam und andere, noch nicht näher defi nierte liganden verantwortlich (s. , ; tabelle und ). der vorgang wird als aggregation bezeichnet. die verständigung der pmn untereinander, über welche diese aktivierungsvorgänge gesteuert werden, geschieht vorwiegend über die von den pmn abgegebenen mediatoren ltb , paf und il . ist der ausgangspunkt des chemoattraktants etwa ein bakterieller herd, so wird dieser herd in einem abstand, der durch die kritische konzentration des chemotaktischen reizes bestimmt wird, von aggregierenden pmn umgeben. die aggregate entstehen zunächst multizentrisch, vereinigen sich bei fortschreiten zu einem geschlossenen wall von granulozyten, die ihre chemischen wirkstoffe in das umschlossene zentrum abgeben. damit werden wesentliche ziele erreicht: der organismus wird von der entzündlichen noxe abgeschirmt der umschlossene bezirk wird mit der noxe vernichtet in diesem abgegrenzten, "demarkierten" bereich wird das geschädigte gewebe mitsamt der auslösenden noxe aus dem gesunden organismus herausgetrennt, "sequestriert", und durch die wirkung der freigesetzten enzyme und ros zerstört und aufgelöst. der gewebsdetritus füllt zusammen mit abgestorbenen pmn und gegebenen falls getöteten mikroor-■ --ganismen den hohlraum als "eiter". ein entzündliche prozess dieses ablaufs wird als abszess bezeichnet (abb. ). von praktischer bedeutung ist, dass dieser kritische maximalwert, ab dem ein chemotaxin die chemotaxis hemmt, nicht konstant ist, sondern wiederum vom primingzustand der zellen abhängt. wenn pmn bereits in der blutbahn stark geprimt werden, ist die chemotaxis dieses kollektivs gehemmt, die adhäsionsbereitschaft und chemische aktivität dagegen gesteigert. in solchen fällen emigriert ein pmn-kollektiv verzögert, pmn heften sich dagegen vermehrt ans endothel, bilden miteinander aggregate (abb. ) und geben chemische wirkstoffe im gefäßbereich ab, die zu abb. . demarkation eines entzündungsherdes und abszessbildung. pmn wandern in richtung des ansteigenden chemotaktischen gradienten, bis sie einen kritischen konzentrationsbereich des gradienten erreichen, bei dem sie die migration einstellen und untereinander bindungen eingehen. es bilden sich anfänglich multizentrische pmn-aggregate, die sich schließlich zu einem geschlossenen wall vereinigen können. aggregierte pmn phagozytieren, degranulieren und geben reichlich enzyme und ros in das zentrum des entzündungsherdes ab, in dem mikroorganismen getötet und geschädigtes gewebe lytisch zerstört werden (eiter). nekrotisches gewebe wird in gleicher weise demarkiert und abgebaut. während sich zentral gelegene pmn im zuge ihrer tätigkeit aufl ösen, wird der granulozytenwall durch nachströmende zellen von außen ergänzt. schäden an gesunden geweben und organen führen. diese schwerwiegenden komplikation kennzeichnen das sirs, ards und das mof (s. ). die molekularbiologischen vorgänge hinter diesen verschiedenen aktivierungsniveaus von pmn, die über ein und denselben rezeptor ausgelöst werden können, werden erst ansatzweise verstanden. sicherlich spielen auch hier die konzentrationen freien cytosolischen ca ++ eine entscheidende rolle. während offenbar kalzium aus den zelleigenen speichern genügt, um niedrige aktivierungsebenen wie migration und adhäsion zu ermöglichen, ist für die erreichung eines hohen aktivierungsgrades wie degranulation und ros-produktion zusätzlich der einstrom extrazellulären kalziums notwendig. die abb. gibt einen Überblick über die heutige vorstellung der zusammenhänge. ein phagozyt hat zwei möglichkeiten, schadmaterial mittels chemischer wirkstoffe zu bekämpfen: durch phagozytose, das ist die aktive aufnahme fester partikel von einer mindestgröße von . µm durchmesser in den zellleib. diese größenordnung ist im lichtmikroskop gerade noch sichtbar. die aufnahme fester partikel unter der sichtbarkeitsgrenze wird als endozytose bezeichnet, die aufnahme fl üssiger stoffe als pinozytose. nach einverleibung der partikel erfolgt deren abbau mittels chemischer wirkstoffe. durch degranulation, das ist die abgabe chemischer wirkstoffe in die umgebung. wenn ein partikel für eine vollständige aufnahme in den zellleib zu groß ist, werden die wirkstoffe nach außen abgegeben. diese abgabe kann dosiert in form der exozytose einzelner lysosomaler granula erfolgen, oder auch massiv, indem die zelle sich selbst zerstört und ihr gesamtes chemische arsenal mit einem schlag freisetzt. allgemeines phylogenetisch hat sich phagozytose allem anschein nach aus dem fressakt von einzellern entwickelt. in höheren tieren ist die fähigkeit zur phagozytose in spezialisierten zellen des immunsystems ausgeprägt entwickelt (fresszellen, phagozyten). der potenteste phagozyt des immunsystems ist der makrophage, dessen benennung diese besondere fähigkeit hervorhebt. aber auch pmn sind wirkungsvolle phagozyten (gelegentlich als "mikrophagen" bezeichnet). wenn in der fachliteratur von "phagozyten" gesprochen wird, sind gewöhnlich diese beiden zelltypen gemeint. daneben sind noch ■ ■ abb. . aggregatbildung von pmn. bei geeigneter aktivierung gehen pmn untereinander über adhäsine bindungen ein, wobei sie sich breitfl ächig aneinander lagern. diese aggregatbildung ist am ort der entzündung eine grundlage der demarkation von geschädigtem gewebe und ein wichtiger mechanismus, den gesunden organismus gegenüber seinen erkrankten bereichen abzugrenzen. pmn-aggregate können aber auch pathologisch werden, wenn sie sich bei ausbreitung des entzündungsprozesses auf das gesamtsystem in der blutbahn bilden, störungen in der mikrozirkulation hervorrufen und durch wirkstoffabgabe (elastase, ros) toxisch werden. gehen dagegen von einem partikel chemotaktische reize aus, sucht der phagozyt die quelle dieses reizes orientiert und beschleunigt auf ( chemotaxis und chemokinetik, s. ). bei stärkeren phagozytosereizen spielen immer chemotaktische momente mit, da angeregte phagozyten in solchen fällen benachbarte zellen zur unterstützung herbeirufen. dazu geben pmn bevorzugt die chemotaxine ltb , paf und il ab, über die weiter pmn angelockt werden. vor der eigentlichen phagozytose muss sich ein partikel an den phagozyten binden. diese bindung ist der mechanismus, über den die zelle ein partikel erkennt und von material unterscheidet, das nicht phagozytiert werden soll, und der den phagozytosereiz auslöst. eine bindung kann über verschiedene mechanismen erfolgen. wenn ein partikel von sich aus positiv geladen ist, haftet es elektrostatisch an ----der negativen zellmembran des phagozyten. ungeladene partikel können sich durch hydrophobe bindung in die zellmembran einlagern und eine phagozytose auslösen. ein spezifi sches erkennen von phagozytose-objekten erfolgt über oberfl ächenrezeptoren der phagozyten, welche diese objekte entweder direkt, oder indirekt binden. immunkompetente zellen besitzen genetisch festgelegte rezeptortypen, die molekulare oberfl ächenstrukturen von körperfremden zellen, zellprodukten oder auch von scavengermaterial erkennen und binden (s. f, tabelle ). entscheidend für den erfolg einer solchen differenzierung ist, dass die molekularen erkennungsmuster auf gesunden, zu erhaltenden körpereigenen zellen und materialien nicht vorkommen. geeignete zielstrukturen auf mikroorganismen sind spezifi sche, hochkonservierte baubestandteile [ pathogen associated molecular patterns, pamp], für die ein phagozyt die passenden rezeptoren besitzt [ pathogen recognition receptors, prr]. zu solchen pathogen-spezifi schen oberfl ächenmolekülen gehören: mannose als charakteristisches kohlenhydrat in membranen von mikroorganismen, lipopolysaccharide (lps) als bausteine der zellmembran gram-negativer bakterien, teichonsäuren [teichoic acid] und peptidoglykane als membranbausteine grampositiver bakterien und hefen, flagellin und pilin als bestandteil des bewegungsapparates mancher pathogene, gewisse für bakterielle dna typische sequenzen (die nicht methylierte cytosin-guanin sequenz), doppelt helikale rna in viren u. a. zu dieser gruppe von rezeptoren gehören auch die toll-like receptors (tlr), von denen beim menschen bisher zehn gesichert sind. tlr sind phylogenetisch hoch konservierte rezeptoren, die zuerst bei drosophila festgestellt wurden und dort bei der embryogenese und bei der infektabwehr aufgaben übernehmen. sie sind als homo-oder heterodimere aktiv (abb. ) und induzieren in phagozyten und anderen zelltypen über nfkb die synthese von cytokinen und wirkstoffen (abb. ) . die fähigkeit zur unterscheidung mittels spezifi scher rezeptoren hat sich im zuge einer langen evolutionären anpassung und bewährung entwickelt. neben dem reiz zur phagozytose können prr auch oder zusätzlich weitere zellaktivitäten ermöglichen oder die freisetzung von cytokinen und wachstumsfaktoren aus phagozyten stimulieren. neben der fähigkeit von phagozyten, schadmaterial direkt an molekularen oberfl ächenmerkmalen zu erkennen, bestehen einrichtungen, über die der organismus selbst zellen und partikel für die phagozytose markiert. diese körpereigenen markermoleküle nennt man opsonine, der vorgang der markierung wird als opsonisation bezeichnet. phagozyten besitzen für opsonine rezeptoren, über die zellaktivierungen und phagozytose in gang gesetzt werden. ein teil der opsonine oder ihrer vorstufen wird während der akutphase reaktion vermehrt gebildet, systemisch auf dem blutweg verbreitet und über das entzündliche Ödem im entzündungsherd angereichert. opsonine entstammen unterschiedlichen humoralen systemen: complementsystem: die opsonine c b und c bi können dem klassischen wie dem alternativen aktivierungsweg entstammen. bindungspartner an phagozyten sind der c b-rezeptor (cr , cd ), cd b/cd und cd c/cd (s. , tabelle ). spezifi sches immunsystem: der fc-teil von igg und igm wird von mehreren fc-rezeptoren erkannt. (tabelle , s. ). die opsonine des complementsystems und der immunglobuline potenzieren sich in ihrer wirkung (abb. ). aktive pmn beziehen ihre energie in erster linie aus der energetisch ungünstigen anaeroben glykolyse. die glukose dazu stammt fast ausschließlich aus den intrazellulären glykogenreserven (s. ). bei der anaeroben glykolyse anfallende milchsäure wird in die umgebung der zelle freigesetzt. die elektronen, die für die bildung von ros benötigten werden, stammen letztendlich aus dem pentosephosphat-shunt, also ebenfalls aus dem glukose-abbau. die folge ist, dass bei dem hohen bedarf und dem unrationellen umgang mit energie die glykogenreserven bald erschöpft sind, was die lebensdauer aktiver pmn stark begrenzt. der bei phagozytosevorgängen schlagartig einsetzende massive sauerstoffbedarf zur synthese von ros wird als oxidative burst bezeichnet (s. ). der daneben häufi g verwendete ausdruck respiratory burst ist irreführend, da der sauerstoff nicht für die zellatmung (aerobe glykolyse) verwendet wird. der nötige molekulare sauerstoff wird aus der umgebung abgezogen. im gegensatz zum pmn sind makrophagen auch in der lage, glucose aerob zu verwerten (s. ). eine degranulation geringen umfangs fi ndet bereits bei der spontanen und chemotaktischen bewegung eines pmn statt. die lysosomalen membranen dienen dabei der vergrößerung der zelloberfl äche bei der bildung des lamellopodiums (s. f) und begünstigen die verformbarkeit der zelle. freigesetzte enzyme und ros helfen einen weg bahnen (s. f). die freisetzung der gra--nula und vesikel erfolgt kontrolliert unter einbeziehung des cytoskeletts (abb. ). bei kontakt mit partikeln, die für eine phagozytose zu groß sind ( "frustrierte phagozytose" abb. ), oder auch bei einwirkung starker nicht partikulärer reize wird der inhalt lysosomaler granula nach außen abgegeben. der transport der granula an die zelloberfl äche erfolgt kinesin-vermittelt entlang von mikrotubuli (s. ). das kortikale aktingerüst bildet jedoch eine barriere, die eine fusion der granulamembran mit der zellmembran und damit eine exozytose verhindert; es wird daher an der kritischen stelle depolymerisiert (abb. ). diese kontrollierte abgabe von granula kann bei starken reizen in eine unkontrollierte form übergehen, bei der schließlich eine zelle in einem zug ihr gesamtes chemisches arsenal freisetzt. die dabei massiv produzierten ros können nicht mehr ausreichend durch die zelleigenen antioxydantien neutralisiert werden (s. ) und zerstören zelleigene membranen durch peroxydation des lipidanteils. ge-bildete ros und der gesamte enzymbestand werden schlagartig in das umfeld abgegeben und kommen konzentriert zur wirkung. die selbstzerstörung ist charakteristisch für die arbeitsweise von pmn und stellt die intensivste form ihrer aktivität dar. in-vitro kann durch wirksubstanzen, die eine depolymerisation des kortikalen aktin-cytoskeletts herbeiführen wie z.b. cytochalasine, eine massive degranulation ausgelöst werden. bei einem entzündlichen prozess in-vivo laufen die verschiedenen aktivierungsphasen von entzündungszellen parallel nebeneinander ab. der granulozytenwall um einen entzündungsherd wird peripher ständig durch neu ankommende pmn ergänzt, die zunächst aggregieren und fest aneinander schließen und so eine demarkationsschicht bilden, weiter zum zentrum jedoch phagozytieren und schließlich unter selbstzerstörung degranulieren und ros freisetzen (abb. ). die lebensdauer maximal aktivierter pmn ist extrem verkürzt und bewegt sich im bereich von minuten (s. ). im zentrum eines solchen von einem granulozytenwall umschlossenen entzündungsherdes ( abszess) bauen enzyme gewebe ab, wodurch die zahl kleiner, osmotisch aktiver bruchstücke stark zunimmt, die in diesem hohlraum einen beträchtlichen osmotischen druck aufbauen können. die druckentlastung erfolgt in die richtung des geringsten widerstandes. daher entleeren sich abszesse häufi g spontan auf äußere und innere oberfl ächen, was den heilungsverlauf meist begünstigt. als therapie wird die entleerung chirurgisch durch "spaltung" des abszesses unterstützt. bei der chronischen polyarthritis setzen ag-ak -komplexe einen immunprozess in gang, in dessen verlauf überaktivierte phagozyten mittels ihrer wirkstoffe den gelenksknorpel schädigen. von --■ aktivierten makrophagen abgegebene cytokine (il , tnfα) stimulieren chondrozyten und osteoklasten zum knorpelund knochenabbau (s. ). bei der gicht lösen im gelenksbereich abgelagerte uratkristalle heftige entzündungsvorgänge aus, in deren verlauf vorwiegend überaktivierte pmn den gelenksknorpel arrodieren und zerstören (s. ). bei verschiedenen vaskulitiden schädigen in der blutbahn aktivierte pmn und monozyten endothel und gefäßwand (s. ff). ständig im zuge einer chronisch-obstruktiven bronchitis einwirkende entzündliche reize halten die phagozyten der atemwege im zustand einer andauernden aktivierung, was schlussendlich zu einer zerstörung der wände der bronchien und bronchiolen und zu bronchiektasien, stenosen und lungenemphysem führt (s. f). chemotaktische bewegung, phagozytose und granulaabgabe hängen von einem funktionstüchtigem cytoskelett ab. Überstürzte aktivitäten von pmn bei gicht und familiärem mittelmeerfi eber abb. . degranulation. sekretgranula werden entlang mikrotubuli zur zelloberfl äche transportiert ( ). das kortikale aktingerüst wird lokal depolymerisiert, so dass die granulamembran mit der zellmembran fusionieren kann ( ). die granulamembran wird mit den enthaltenen adhäsinen, rezeptoren und ionenkanälen in die zellmembran integriert, der granulainhalt nach außen gestülpt ( ). der granulainhalt löst sich und diffundiert in die umgebung ( ). können durch colchizin eingedämmt werden (s. ). wegen der geringen therapeutischen breite wird colchizin üblicherweise in niederer dosierung mit anderen antiphlogistika kombiniert. glucocorticoide greifen auf verschiedenen ebenen in immunreaktionen ein (s. f). Über eine verstärkte expression von lipocortin hemmen sie die aktivität der phospholipasen a und als folge die bildung von lysolecithin und aa. vermutlich werden auf diesem weg membranfusionen bei phagozytose und exozytose erschwert. die verringerte bildung lipogener mediatoren (ltb , paf) schränkt die parakrine stimulation von phagozyten ein. nfkb-vermittelte synthesen werden blockiert (abb. ). glucocorticoide in hohen dosen senken unspezifi sch die membranfl uidität und hemmen damit die zellaktivität (abb. ). aktivierte pmn gewinnen ihre energie zur bewegung, phagozytose und granulaabgabe in erster linie aus der anaeroben glykolyse, deren endprodukt milchsäure in die umgebung abgegeben wird und zur gewebssäuerung beiträgt. die glukose stammt vorwiegend aus den endogenen glykogenreserven, die der pmn zur gänze während seiner reifung im knochenmark speichert (s. ). der glykogenanteil beträgt bis % des nassgewichtes eines pmn, das entspricht etwa dem gehalt in leberzellen. die verwertung von glykogen ist vorteilhaft, da bei der phosphorolyse des glykogens glukosephosphat direkt durch bindung an anorganisches phosphat entsteht, also atp gespart wird. dagegen ist die anaerobe glykolyse eine äußerst unrationelle form der energiegewinnung. im vergleich zum aeroben weg, der mol atp aus einem mol glukose liefert, werden anaerob nur mol atp gewonnen. daher sind bei gesteigerter aktivität diese reserven bald aufgezehrt und die zelle geht zu grunde (s. ). die unabhängigkeit vom sauerstoff hat hingegen den vorteil, dass pmn in schlecht mit blut versorgten, "sequestrierten" entzündungsherden ihre energieversorgung aufrecht erhalten können. molekularer sauerstoff ist andrerseits zur herstellung von ros unentbehrlich (s. ). der glykogengehalt eines pmn kann situationsbedingt schwanken. der gehalt ist bei infekten erhöht, bei manchen leukämien und auch bei diabetes mellitus dagegen verringert. manche erbliche glykogendefekte betreffen auch das glykogen der pmn und können an ihnen nachgewiesen werden. gegenüber der anaeroben glykolyse treten andere möglichkeiten der energiegewinnung weit zurück. die aerobe glykolyse ist mit einem anteil von rund % bedeutungslos. exogen aufgenommene glukose und fruktose können über eine hexokinase verwertet werden. dieser weg ist nur in pmn niederen aktivierungsgrades von bedeutung. desgleichen werden fettsäuren nur wenig genutzt. die mangelhafte ausstattung mit enzymen der oxydativen energiegewinnung (citratzyklus, atmungskette, β-oxydation langkettiger fettsäuren) manifestiert sich im spärlichen auftreten der trägerorganelle mitochondrium. der hohe sauerstoffbedarf der pmn im rahmen von entzündungen dient fast ausschließlich der bildung von ros (s. ). glykosidasen, wie glucuronidasen, galaktosidase und glukosaminidasen spalten glykosaminoglykane und proteoglykane der grundsubstanz und in bakteriellen schutzhüllen. sie sind bei scavengeraufgaben und in der keimabwehr tätig. die ph-optima der meisten von ihnen liegen im beträchtlich sauren bereich von . bis . lysozym ist eine muramidase, die muraminsäure-glykoside der bakterienwand spaltet. außer in den granula von phagozyten kommt lysozym auch frei im speichel, in der tränenfl üssigkeit und laktoferrin bindet eisen und entzieht so bakterien einen wichtigen wachstumsfaktor. auch kann es selbst mikroorganismen binden und dem immunsystem zugänglich machen (opsonin-wirkung). im komplex mit laktoferrin ist eisen nicht mehr als elektronenüberträger wirksam und katalysiert nicht mehr die bildung von oh • . so ist laktoferrin indirekt als antioxydans aktiv. ein molekül laktoferrin kann bis zu sechs eisenionen binden, die wieder an transferrin und weiter an zellen des res abgegeben werden können, wo sie als ferritin gespeichert werden (s. ) . laktoferrin ist auch in vielen drüsensekreten und besonders reichlich in der milch vorhanden; daher der name. granulamembranen der pmn enthalten auch reserven an oberfl ächenproteinen und adhäsinen, die bei der exozytose der zellmembran einverleibt werden. einige gut studierte und bekannte dieser membranproteine sind in tabelle angeführt. ihre bedeutung im einzelnen wird in den jeweiligen sachkapiteln besprochen. der pmn ist ein "ein-weg-artikel", der produziert und im zuge seiner tätigkeit "verbraucht" wird. seine lebenserwartung im entzündungsherd hängt einmal von seiner aktivierung ab. stoffwechselaktive pmn brauchen je nach aktivierungsgrad mehr oder weniger schnell ihren glykogenvorrat auf. sie sind dann nicht mehr in der lage, das nötige atp für die k-na-pumpe bereitzustellen und zerfallen osmolytisch. bei sehr starker stimulation, wie sie etwa durch cytokine, aktivierte mediatoren oder bakterientoxine ausgelöst wird, zerstören sich pmn mit selbst produzierten ros und entlassen dabei schlagartig ihr gesamtes chemisches potential in die umgebung. dieses "selbstmordkommando" ist die intensivste form einer wirkungsentfaltung von pmn. die lebensdauer im gewebe ist dabei drastisch verkürzt und bewegt sich im bereich von wenigen minuten. am ende der perakuten phase, mit dem rückgang eines entzündungsprozesses erhöht sich die lebensdauer der pmn beträchtlich, da eine auto-peroxydation und phagozytosetätigkeit, welche ihre lebensdauer drastisch verkürzt, immer mehr zurücktritt. in dieser phase der entzündung wird die lebenserwartung eines pmn von der apoptose bestimmt, die eine wichtige rolle bei der regression und lösung des entzündungsprozesses einnimmt. ein mit antiapoptotischen faktoren angereichertes milieu kann jedoch die apoptose hinauszögern, verändert pmn auch phänotypisch und macht sie zu zellen, die regulierend auf den entzündungsablauf einwirken (s. f). mit der abgabe von il- sind solche pmn auch in der lage, die spezifi sche immunität zu beeinfl ussen (s. ). an einem tiermodell konnten folgende daten erhoben werden: pmn erreichten im entzündungsherd am höhepunkt eines akuten entzündlichen prozesses stunden nach setzen einer noxe eine lebensspanne von minuten, nach der sie sich durch peroxydation ihrer eigenen membranen aufl östen. mit dem weiteren verlauf des entzündungsvorganges verlängerte sich die lebensdauer auf reife pmn stellen nicht nur speicher für präformierte, im knochenmark gebildete wirkstoffe und oberfl ächenstruktu-abb. . signalübermittlung durch g-protein -gekoppelte rezeptoren. ein rezeptor diesen typs ist eine proteinkette, die aus der extrazellulären bindungsstelle für den liganden, sieben transmembranösen schleifen und einem intrazellulären teil mit der koppelungsstelle für das g-protein besteht ( ). das hetero-trimere g-protein ist aus den drei untereinheiten α, β und γ zusammengesetzt und enthält guanosin-diphosphat (gdp) gebunden. bei besetzung mit einem liganden entwickelt der intrazelluläre teil des rezeptors durch konfi gurationsänderung affi nität zum g-protein -komplex und bindet ihn ( ). am rezeptor-gebundenen g-protein wird gdp gegen guanosin-triphosphat (gtp) ausgetauscht ( ). damit dissoziiert das g-protein vom rezeptor, zerfällt in einen α und einen β-γ -komplex, die beide entlang der membran zu bindungspartnern (enzyme, ionenkanäle etc.) diffundieren ( ) und diese durch bindung aktivieren oder, abhängig vom typ des g-proteins, auch deaktivieren. solange ein rezeptor an den liganden gebunden ist, kann er erneut g-proteine binden und signale aufrecht erhalten ( ). nach wenigen sekunden wird gtp durch eine gtp-ase zu gdp hydrolysiert. dadurch koppelt sich die α-untereinheit des g-proteins vom bindungspartner ab und vereinigt sich mit den β-γ-dimeren. nach dissoziation des liganden vom rezeptor ist die ausgangslage wieder hergestellt ( ). ( ) g-protein -gekoppelte rezeptoren aktivieren phospholipasen vom typ c (plc), die das phosphatidyl-inositol (pi) der zellmembran über phosphatidyl-inositol-diphosphat (pip ) in inosin-triphosphat (ip ) und diacyl-glycerol (dag) spalten. ip aktiviert die freisetzung von ca ++ aus intrazellulären ca ++ -speichern (calciosomen), wodurch die proteinkinasen c (pkc) aktiviert werden, die eine reihe von zellulären effekten in gang setzen. dag verstärkt die wirkung von cytosolischem ca ++ auf die pkc. hohe intrazelluläre konzentrationen von zyklischem adenosin-monophosphat (camp) fördern den rückstrom von ca ++ in die speicher, senken damit cytosolisches ca ++ und dämpfen so zellaktivitäten. ( ) dag kann auch auf dem weniger gut studierten weg über die monomeren low-molecular weight g-proteins (lmw-gp) entstehen, welche die phospholipasen d (pld) aktivieren, die wiederum dag aus phosphatidyl-cholin (pch) herausspalten. ( ) zur vollen aktivierung von pmn-leistungen (degranulation, ros-produktion) ist jedoch zusätzlich ca ++ durch einstrom aus dem extrazellulärraum nötig. die erforderliche Öffnung der kalzium-kanäle ist zeitlich gegenüber der intrazellulären ca ++ -freisetzung verzögert. der einstrom von extrazellulärem ca ++ erhöht sich bei leeren intrazellulären kalziumspeichern. ( ) es werden noch weitere aktivierungswege postuliert. sinn eines "positiven primings" eingesetzt. sinngemäß gibt es auch ein "negatives priming", das heißt eine reaktivität unter derjenigen einer zelle im normalzustand, wie sie z.b. unter dem einfl uss immunsuppressiver therapien oder endogener entzündungs-inhibitoren auftritt. im wissenschaftlichen schrifttum wird jedoch zumeist der ausdruck "negatives priming" vermieden und man gebraucht wendungen wie "mangelhaftes priming" oder "herabgesetzte reaktivität". manche der molekularbiologischen vorgänge, die zum priming führen, sind recht gut erfasst, andere wieder werden nur wenig oder gar nicht verstanden. systematisch gut brauchbar ist die einteilung in kurzzeit-und langzeit-priming, da diesen beiden reaktionsformen verschiedene molekularbiologische voraussetzungen zugrunde liegen. beim kurzzeit-priming werden bereits vorhandene zelluläre einrichtungen aktiviert, während beim langzeit-priming die nötigen einrichtungen erst synthetisiert werden müssen. bei trotz der reichlichen ausstattung mit gut ausgebildeten mitochondrien wird glukose fast ausschließlich anaerob metabolisiert. zur energiegewinnung werden auch fettsäuren herangezogen. die enzyme des pentosephosphat-zyklus, die nadph-oxydase und eine peroxydase sind reichlich vorhanden und aktiv. der eg ist die zelle mit der intensivsten ros-produktion. der golgi-apparat ist gut entwickelt. im gegensatz zum pmn sind eg in der lage, auch noch nach abgeschlossener reifungsphase im knochenmark weiter granula zu produzieren. immunkomplexe werden rasch phagozytiert; durch deren beseitigung wirken eg entzündungshemmend. im vergleich mit pmn werden bakterien weit weniger begierig aufgenommen, auch ist die bakterizide wirkung wesentlich geringer. das chemische potential, nämlich basische proteine und ros, ist in erster linie gegen das hauptzielobjekt der eg, einund mehrzellige parasiten, gerichtet. das physiologische einsatzgebiet der eg umfasst zwei gegensätzliche bereiche: ein teil der leistungen des eg ist dem körpereigenen entzündungshemmenden system zuzurechnen. durch seine ausstattung mit histaminase, mao, kininase i und arylsulfatase b ist der eg in der lage, die enzündungsmediatoren histamin, serotonin, bradykinin und cysteinyl-lt abzubauen. durch die phagozytose von immunkomplexen schränkt der eg immunreaktionen und die aktivierung der zugehörigen infl ammatorischen hilfssysteme ein. Über die reichlich enthaltene -lox werden die antiinfl ammatorischen lipoxine synthetisiert (s. ). mittels einer reihe von kollagenasen bauen eg provisorisch gebildetes typi-kollagen in jungen narben ab, um so für defi nitives, funktionell angeordnetes typiii-kollagen platz zu machen (s. f). eg sind damit an der kontrolle der entzündungsreaktion und am heilungsvorgang aktiv beteiligt. diesen --aufgaben entsprechend treten eg vermehrt während der phase der fibroblastenvermehrung und kollagenbildung in den entstehenden narben auf. ebenso ist das ende von infektiösen erkrankungen häufi g durch eine mäßige bluteosinophilie gekennzeichnet. dieses symptom eines abklingenden entzündlichen prozesses wird und wurde als prognostisch günstig interpretiert und mit dem blumigen ausdruck "morgenröte der entzündung" bedacht. bei einem anteil der eg an den weißen blutzellen von über % liegt eine eosinophilie vor, über deren herkunft sich der behandelnde arzt gedanken machen muss. im europäischen bereich reihen sich die auslösenden ursachen nach häufi gkeit: bei der spärlichen ausstattung mit wirkstoffen kann vom bg keine bedeutende rolle bei der keimabwehr erwartet werden. die aufgaben scheinen eher auf dem regulativen sektor zu liegen. der besatz an fcε-rezeptoren und der gehalt an histamin rücken den bg in die nähe der anaphylaktischen reaktionen. tatsächlich weisen atopiker eine höhere zahl an zirkulierenden bg auf, und die histamin-entspeicherung bei anaphylaktischen reaktionen ist nachgewiesen. bei dem spärlichen auftreten der bg ist es jedoch fraglich, ob solche aktivitäten neben der enormen Überzahl der funktionell gleichgerichteten mastzellen quantitativ überhaupt ins gewicht fallen. so bleibt es offen, in welchem ausmaß bg zum blut-histaminspiegel beitragen. manche untersucher machen wirkungsbereiche außerhalb entzündlicher vorgänge geltend. freigesetzte glykosaminoglykane sollen die heparin-induzierbare lipoprotein-lipase im endothel des skelettmuskels aktivieren und so den bg in den lipid-stoffwechsel einbinden. ein anstieg der zahl zirkulierender bg wird bei anaphylaktischen reaktionen und gelegentlich bei myxödem festgestellt. im übrigen weiß man über diesen zelltyp recht wenig. das spärliche auftreten macht ein studium äußerst schwierig und schreckt potentielle untersucher ab. das geringe vorhandene wissen wurde an leukämischen bg gewonnen, die in großer zahl im blut auftreten können. wieweit solche ergebnisse auf gesunde verhältnisse übertragen werden können, bleibt dahingestellt. monozyten und makrophagen sind angehörige der myeloischen reihe weißer blutzellen. der monozyt stellt die noch unreife, im blut zirkulierende form dieses zelltyps dar, der sich in geweben weiter differenzieren und zum makrophagen spezialisieren kann. die entwicklung des monozyten trennt sich von der mit den granulozyten gemeinsamen ahnenreihe auf der stufe der weißen myeloischen stammzelle (abb. ). Über die reifungsstadien monoblast, promonozyt entsteht der monozyt, der das knochenmark verlässt und auf dem blutweg in die gewebe des organismus gelangt. in dieser phase misst er als "blut-monozyt" bis µm im durchmesser und stellt bis % der zirkulierenden weißen blutzellen. nach seiner emigration aus der blutbahn siedelt er sich in allen geweben des organismus an und differenziert sich zu den formen des makrophagen mit unterschiedlichem aussehen und funktionen (abb. ). dabei lassen sich zwei grundsätzlich verschiedene aufgabenbereiche trennen: allen makrophagen gemeinsam ist die ausgeprägte fähigkeit zur phagozytose, wobei erstaunlich große partikel aufgenommen werden können, was diesem zelltyp zu seinem namen "groß-fresser" verholfen hat. gewebsmakrophagen in hämatomen etwa phagozytieren makrophagen nehmen im spezifi schen immungeschehen schlüsselpositionen ein. die spezifi sche immunantwort beginnt bei makrophagen mit der antigenpräsentation, und mit der phagozytose der ag-ak-komplexe schließen makrophagen eine immunreaktion ab. Über einzelheiten dazu berichtet eine umfangreiche literatur. bei erstkontakt mit einem potentiellen antigen bereiten makrophagen das antigen auf und initiieren die immunantwort (abb. a). bei wiederholtem kontakt mit dem antigen verstärken makrophagen die von den gedächtniszellen ausgehende immunantwort. makrophagen sind produzenten einer langen reihe von modulatoren der spezifi schen wie auch unspezifi schen immunantwort. neben cytokinen setzen sie auch wachstumsfaktoren, komponenten des complementsystems sowie inhibitorsubstanzen frei, welche die spezifi sche immunreaktion steuern können. eine reihe von erregern häufi ger, für den träger bedrohliche erkrankungen die gesamtheit der endothelzellen, das gefäßendothel, kleidet als bis auf wenige ausnahmen einschichtiges plattenepithel das blutgefäßsystem aus. die vom gefäßendothel bedeckten flächen sind gewaltig. für die innere oberfl äche der arterien werden etwa m , für die der venen m , und für die kapillaren m angegeben. das gesamtgewicht des gefäßendothels beträgt etwa . kg. dazu bedeckt endothel auch die innenfl ächen des lymphgefäßsystems. das bindegewebe mit seinen einrichtungen ist der eigentliche bereich, in dem die entzündlichen abwehrvorgänge ablaufen. vergleicht man in einem häufi g verwendeten bild eine entzündung als kriegerische auseinandersetzung mit dem "feind pathogener keim", so entspricht das blut den transport-und versorgungswegen, während der extravasale gewebsbereich das aufmarschgebiet und schlachtfeld darstellt. beschädigtes kriegsgut und feindmaterial werden über den lymphweg abtransportiert und in den lymphknoten gesichtet. der vergleich ist insofern nicht zutreffend, als die gefäße und das anliegende bindegewebe keine passive, bloß erduldende umgebung darstellen, sondern als aktive teilnehmer in den entzündungsprozess involviert sind. für die gestaltung der bindegewebsmatrix ist in erster linie der fibroblast verantwortlich, obwohl auch glatte muskelzellen, epithelien, gefäßendothel und parenchymzellen in der lage sind, matrixmaterial herzustellen. die form des fibroblasten ist gewöhnlich langgezogen oder sternförmig mit mehr oder weniger langen fortsätzen. da sich die zelle der umgebung anpasst, kann die gestalt gewebsspezifi sch stark variieren. der kern sitzt in der zellmitte und ist länglich-walzenförmig. das raue endoplasmatische retikulum ist reichlich entwickelt und weist auf die rege proteinsynthese der zelle hin. der golgi-apparat ist gut ausgeprägt. mitochondrien sind wenig zahlreich, aber groß. einziehungen an der oberfl äche und pinozytotische vesikel sind ausdruck einer regen stoffaufnahme. der fibroblast in stoffwechselruhe wird als fibrozyt bezeichnet. die weitgehende einschmelzung der zytoplasmatischen organellen und der wasserverlust machen die zelle dünn-spindelförmig und unscheinbar. integrine sind adhäsine, mit denen verschiedene zelltypen ausgestattet sind. die heterodimeren strukturen bestehen aus α und β untereinheiten, die nichtkovalent miteinander verbunden sind. neben ihrer haftfunktion steuern integrine auch wachstum, differenzierung, motilität, zellorientierung und zellpolarität. integrine aus β + α untereinheiten dienen vorwiegend als rezeptoren für die verbindung von zellen mit extrazellulärer matrix, aber auch von zellen untereinander. β + α -integrine fi nden sich nur auf leukozyten und vermitteln zell zu zell-verbindungen (s. ff, tabelle ). integrine mit beteiligung von β untereinheiten sind komplex gebaut. die bisher festgestellten acht verschiedenen βund α-untereinheiten ergeben eine vielzahl von kombinationsmöglichkeiten mit spezifi schen funktionen. der fibroblast trägt integrine des β und β -typs, die wesentlich sein verhalten steuern. neben vermehrung, differenzierung und spezialisierung werden migration, adhäsion an bestimmten strukturen, zellform und matrixauf-und -abbau beeinfl usst. form-und größenänderungen während des wachstums wie auch ab-und umbauvorgänge während entzündungsund heilungsprozessen erfordern eine neuordnung bindegewebiger strukturen. bei diesen vorgängen nehmen matrix-metalloproteasen und serinproteasen eine zentrale stellung ein. der anstoß zur wundheilung geht bereits vom gefäßendothel und von thrombozyten aus, die im bereich der läsion aktiviert werden. in geschädigten blutgefäßen gibt das endothel innerhalb von sekunden nach der noxe txa und paf ab (s. , ) , welche die bildung des plättchenthrombus anregen. txa ist überdies ein starker vasokonstriktor. vasokonstriktion und plättchenthrombus sind die am frühesten einsetzenden maßnahmen der blutungsstillung ( hämostase). die intrinsische blutgerinnung mit fibrinbildung folgt erst in einem zweiten schritt (abb. ). auch nicht mechanische, wie z.b. chemische oder thermische noxen lösen im prinzip dieselbe kettenreaktion aus. txa , paf und nap- (s. ) aus thrombozyten sind überdies starke chemotaxine für pmn, die sich rasch dem plättchenthrombus anlagern und sozusagen einen ersten abwehrriegel gegen eindringende mikroorganismen bilden. die immigration der pmn in den geschädigten bereich wird durch die aktivierung und freisetzung von c a, ltb und il- weiterhin verstärkt. der höhepunkt der pmn-einwanderung ist nach einer zeitlich, qualitativ und quantitativ klar defi nierbaren noxe, wie sie z.b. eine operative wunde darstellt, nach etwa sechs stunden erreicht (s. ), der höhepunkt der ansammlung monozytärer zellen im wundbereich dagegen nach zwei bis drei tagen. makrophagen rekrutieren sich dabei aus blut-monozyten sowie aus sessilen makrophagen, die sich lokal vermehren und differenzieren (s. ). diese makrophagen erledigen die abräumung zerstörten gewebes [scavenger activity], bei der sie nach bedarf von pmn unterstützt werden. von ihnen geht auch die weitere organisation des heilungsverlaufes aus, in deren zug fibroblasten in den defekt einwandern, sich vermehren und die angiogenese eingeleitet wird. die steuerung erfolgt vorwiegend durch cytokine und wachstumsfaktoren, für die makrophagen wichtige produzenten darstellen. ein anderes bedeutsames steuerelement für den heilungsverlauf sind t-lymphozyten, die in der späteren entzündungsphase in den entzündungsbereich einwandern (s. ). fibroblasten sind mobile zellen, deren migration durch eine reihe von chemoattraktants gesteuert wird. der wirkungsgrad dieser chemoattraktants ist allerdings je nach gewebszugehörigkeit und herkunft der fibroblasten verschieden. chemotaktisch und chemokinetisch wirksam sind die cytokine tnfα, il- β, il- , die chemokine rantes, mcp, die wachstumsfaktoren pdgf, tgfβ, bfgf, egf, igfi und ii, c a, ltb , fibronectin sowie kollagen-und elastin-bruchstücke. diese faktoren, wie auch substanz p und urokinase (upa), regen darüber hinaus auch die proliferation der fibroblasten und deren produktion von extrazellulärer matrix an. aktivierte fibroblasten wiederum sind selbst bildungsstätten von wirkstoffen, die in die entzündung und wundheilung eingreifen: il β, il- , ifnγ, pdgf, rantes, mcp und die knochenmarkstimulatoren g-csf, m-csf und gm-csf. fibroblasten haften vor allem mittels β und β integrinen an den von ihnen produzierten matrixstrukturen. dieser kontakt bestimmt nicht nur die form und bewegung der fibroblasten, sondern auch wachstum, differenzierung und stoffwechselaktivitäten. umgekehrt können fibroblasten die form der matrix beeinfl ussen, indem sie faserelemente binden und sich kontrahieren. entsprechende adhäsine stehen mit dem cytoskelett in verbindung, das die zellform breinfl usst. so können etwa durch kontraktion der fibroblasten wundränder einander genähert werden. il- und besonders tnfα und β sind für fibroblasten starke chemoattraktants und mitogene. eine unkontrollierte produktion dieser cytokine durch makrophagen und lymphozyten trägt zur entwicklung von fibrosen und zur chronifizierung von entzündungen bei. angiogenese. die vaskularisierung des entzündungsgebietes geht von bestehenden kapillaren der umgebung aus und beginnt schon mit den ersten schritten der entzündung. angiogene signale werden von aktivierten thrombozyten, endothelzellen, fibroblasten, aktivierten makrophagen und anderen zelltypen abgegeben. diese signale bewirken sowohl die steigerung des stoffwechsels, die orientierte migration wie die proliferation der endothelzellen. hemmer der angiogenese halten den prozess im erforderlichen gleichgewicht. prominente angiogene und ihre antagonisten sind in tabelle aufgelistet. das kapillarwachstum beginnt mit der aufl ösung der basalmembran durch metalloproteasen der endothelzellen. darauf folgen die migration in richtung chemotaktischer reize oder entlang haptotaktisch wirkender gewebsstrukturen, und schließlich die vermehrung der endothelzellen unter bildung einer röhre, bei deren ausformung offenbar cadherine eine steuernde funktion übernehmen. in diesem stadium wird eine basalmembran neu gebildet. nach anschluss an das bestehende gefäßnetz ist der kreislauf wiederhergestellt. drei tage nach entzündungsbeginn sind die charakteristika der angiogenese bereits deutlich ausgeprägt. die rückbildung von kapillaren nach narbenheilung läuft über die apoptose der endothelzellen ab. neben ihrer dominanten rolle bei der blutstillung ( hämostase) erfüllen die thrombozyten wichtige aufgaben im rahmen der entzündung und wundheilung. thrombozyten entstehen aus megakaryozyten, indem sich cytoplasmateile von der oberfl äche dieser zellen abschnüren und selbständig werden. thrombozyten sind kernlose, von einer zellmembran umgebene, mäßig gewölbte, rundliche scheibchen ("tablettenform") von bis . µm durchmesser und . bis . µm dicke. die differenzierung des megakaryozyten aus den knochenmarkstammzellen wird durch il- und spezialisiert durch thrombopoetin (s. ) gesteuert und dauert etwa sechs tage. im lichtmikroskop imponiert der thrombozyt bei routinefärbung als scheibchen mit blassblauer peripherie (hyalomer) und stärker getöntem zentrum (zentromer). im elektronenmikroskop zeigt sich dagegen eine hoch differenzierte struktur. ein thrombozyt ist von systemen netzartig verbundener röhren durchzogen. das oberfl ächliche tubuläre netzwerk mündet an der zelloberfl äche, während das zentrale tubuläre netzwerk eine geschlossene struktur im zellinneren bildet. nach ihrem inhalt lassen sich drei typen von granula unterscheiden: α-granula, elektronendichte granula [dense granules] syn. δ-granula, und enzyme enthaltende granula (tabelle ) . diese granula bekommt der thrombozyt bei seiner bildung aus dem megakaryozyten mit; sie sind nicht ergänzbar. mikrotubuli und aktinfi lamente umgeben reifenartig den Äquator und halten den thrombozyten in seiner form. die zellmembran ist mit glykoproteinen besetzt, unter denen rezeptoren und adhäsine charakteristische funktionen dieser zellen vermitteln. daf und hrf die bei der plättchenaggregation freigesetzten chemotaxine txa , nap- und paf aktivieren entzündungszellen, die sich am plättchenthrombus ansammeln (s. ). am plättchenthrombus haftenden pmn wird eine hemmung der plättchenaktivierung und damit eine eindämmung des thrombuswachstums beigemessen. darüber hinaus stellen phagozyten im läsionsbereich wächter gegen eindringende keime dar und unterstützen den abbau beschädigten materials. plättchenaggregate können auch bakterien-und virenkomplexe einhüllen und in zusammenarbeit mit fibrin diese mikroorganismen immobilisieren und vom restorganismus sequestrieren. die komplexe werden von phagozyten beseitigt. die plättchenaggregation ist ein sonderfall, der regulär durch endothel-und gefäßläsionen ausgelöst wird und als hämostatischer schlüsselfaktor physiologischen wert besitzt. normalerweise sind thrombozyten während ihres aufenthaltes in der zirkulation nur sekretorisch tätig, sie aggregieren nicht oder in nicht merklichem ausmaß und werden nach alterung aus dem verkehr gezogen. anders bei defekten der strombahn: an orten mit starken turbulenzen, im bereich geschädigten endothels oder bloßliegender basalmembranen wird die reizschwelle überschritten und es kann zur spontanen plättchenaggregation und thrombusbildung kommen. solche situ-abb. . thrombozyten-aggregation. thrombozyten kontrahieren sich auf starke reize mittels ihres cytoskelettes und setzen dabei die in den netzwerken und granula enthaltenen wirkstoffe frei. Über adhäsine stellen sie feste verbindungen untereinander und mit nachbarstrukturen her. damit können sie nicht nur gefäßlücken abdichten, sondern auch mikroorganismen einschließen und immobilisieren und entzündungsherde von der umgebung abriegeln. ationen sind in hohem maß bei atherosklerotisch veränderten gefäßen gegeben. durch hemmung des aa-metabolismus der thrombozyten durch nsaid kann die bereitschaft zur aggregation und damit die gefahr der thrombusbildung verringert werden (s. , ). eine thrombozytenzahl zwischen . und . /µl blut stellt die norm dar. ein unterschreiten der mindestgrenze wird als thrombopenie bezeichnet. da thrombozyten auf unbekannte weise für die dichtheit und integrität des gefäßnetzes verantwortlich sind, treten bei ihrem mangel blutungen an der haut und an schleimhäuten auf ( thrombozytopenische purpura). die blutungen können je nach ausmaß des mangels punktförmig (petechien) bis fl ächenhaft (ekchymosen) sein. bei zahlen unter . /µl treten die symptome deutlich zutage, unter . besteht lebensgefahr durch ver-abb. . thrombozyten und blutgerinnung. thrombozyten enthalten verschiedene an der blutgerinnung beteiligte faktoren und sind darüber hinaus katalysatoren der hämostase. die faktoren der blutgerinnung müssen durch bindung an phospholipide in räumliche nähe zueinander gebracht werden, um miteinander reagieren zu können. die reaktionsprodukte wirken als proteolytische enzyme, meist als serin-endopeptidasen, die wiederum weitere gerinnungsfaktoren durch proteolyse aktivieren und so die gesamtheit der gerinnungskaskade in gang setzen. kristallisationspunkte der blutgerinnung, die solche phospholipide bereitstellen, sind verletzte zellmembranen, aber auch der plättchenfaktor (pf ), d.i. das membran-phosphatidylserin, das bei aktivierung der thrombozyten an deren oberfl äche exponiert wird. auf diese weise entsteht der komplex aktivierter faktor fxiia und fxi, der wiederum den komplex fix und fviii und dieser weiter fx und fv, thrombin und fibrin aktiviert. bluten über innere körperoberfl ächen. bei der idiopathischen thrombozytopenischen purpura ( werlhof'schen purpura) werden thrombozyten durch autoimmun-antikörper zerstört. hohe thrombozytenzahlen erhöhen das thromboserisiko. Über die lymphozyten und ihre wirksubstanzen, die antikörper, informiert die reichhaltige literatur über die spezifi sche immunität, auf die verwiesen wird. hier sollen nur einige wesenszüge skizziert werden, soweit sie für das verständnis eines akuten, unspezifi schen entzündungsvorganges nötig sind. die lymphozyten sind die repräsentanten der spezifi schen immunabwehr. nach ihrer entwicklung und ihrem späteren aufgabenbereich unterscheidet man lymphozyten der t-reihe und der b-reihe, sowie die natürliche killerzelle (nk-zelle) [natural killer cell]. im gegensatz zur unspezifi schen, angeborenen immunität ist die spezifische immunabwehr nicht zeitlebens und ständig voll funktionsfähig, sondern die bildung ihrer effektorelemente, die antikörper (ak), wird erst durch kontakt mit körperfremdem potentiellem schadmaterial, den antigenen (ag), angeregt. die bezeichnung "induzierbare" oder "adaptive" immunität trifft diesen sachverhalt gut. gebildete ak werden gelöst oder an lymphozyten gebunden ins blut abgegeben und erreichen alle vaskularisierten organe und gewebe. die induktion einer antikörperbildung benötigt bei erstkontakt (primärkontakt) mit dem antigen fünf bis tage, bei wiederkontakt (sekundärkontakt) etwa zwei bis drei tage, wobei die art und applikation des antigens und die individuelle disposition auf die immunantwort starken einfl uss nehmen. bereits vorhandene ak reagieren mit ag sofort und bieten einen entsprechend wirksamen schutz. die lebensdauer von ak ist wie die bereitschaft zu ihrer automatischen nachproduktion zeitlich beschränkt, so dass die blutspiegel der ak nach einem ag-kontakt wieder abfallen und der sofortschutz abnimmt. die halbwertzeit von igg im blut beträgt um tage. die erhaltung der produktion und damit eines blutspiegels eines ak ist sehr von der art des ag abhängig. die impfprogramme zum schutz vor infektionskrankheiten berücksichtigen diese unterschiedlichen bedingungen. eine hauptgruppe der t-lymphozyten stellen die t-helferzellen (th-zellen) dar, die mittlerfunktionen bei der immunantwort ausüben. die zwei klassen von th-zellen, die th und th -lymphozyten, unterscheiden sich durch ihr angebot an mittlerstoffen: th -zellen geben ifnγ, il- und tnfβ ab und steuern in erster linie die makrophagentätigkeit im zusammenhang mit der antigen-präsentation und die bildung zellulärer antikörper. th -zellen setzen vor allem il- , il- , il- , il- und il- frei und steuern vorwiegend die tätigkeit von makrophagen und b-lymphozyten. neben den wirkstoffen der spezifi schen abwehr produzieren t-lymphozyten noch eine große zahl von entzündungsmediatoren, wachstumsfaktoren und csf und greifen so auch wesentlich in das geschehen der unspezifi schen abwehr ein. die cytotoxische t-zelle (tc-zelle) ist ein effektor der spezifi schen immunität und träger cytotoxischer abwehrreaktionen, die sie mit hilfe von zellschädigenden wirkstoffen ausübt ( perforin, syn. cytolysin, und andere, s. ). ein weiterer wirkungsbereich dieser zellen ist die suppression spezifi scher immunreaktionen. th und tc-lymphozyten werden anhand von oberfl ächenstrukturen ("oberfl ächen-marker") unterschieden, die üblicher weise mit immunfl uoreszenz-methoden bestimmt werden. th-zellen tragen als wichtigstes charakteristikum das cd -antigen (cd positive zellen), tc-zellen das cd -antigen (cd +). zur beantwortung spezieller fragestellungen wer-den weitere unterteilungen in subgruppen [subsets] durchgeführt. th-lymphozyten sind ein steuerfaktor bei der wundheilung, obwohl ihre rolle dabei noch nicht gut defi niert ist. offenbar stimulieren sie fibroblasten zur kollagensynthese. lymphozyten-depletierte versuchstiere zeigen eine gestörte wundheilung, mangelhaften kollagenanbau mit bildung leicht zerreißbarer narben. umgekehrt führt eine lang dauernde lymphozytenpräsenz und stimulation nach experimenteller verletzung zu exzessiver fibrosierung und wuchernden narben. beim menschen werden in heilenden wunden sieben bis tage nach der noxe maximale lymphozytenzahlen beobachtet. abb. . das bauschema von immunglobulinen. (a) immunglobuline sind hetero-tetramere aus je leichten ketten mit einem mw von ca. kd (als l, "light", bezeichnet), und schweren ketten mit einem mw von ca. kd (h, "heavy"), die durch disulfi dbrücken zusammengehalten werden. die einzelnen ketten formen schlaufen ("domänen"), die ebenfalls durch disulfi dbrücken stabilisiert werden. die antigen bindenden domänen (fab, "antigen binding") der leichten und schweren ketten werden mit v ("variable"), die konstanten mit c ("constant") bezeichnet; letztere sind mit durchlaufenden nummern versehen. die aus den leichten und den entsprechenden teilen der schweren ketten gebildeten "arme" des immunglobulins können frei schwingen. natürliche killerzellen (nk-zellen) stellen eine gruppe sehr ursprünglicher, noch wenig spezialisierter lymphatischer zellen dar, die zielobjekte über "natürliche" angeborene erkennungsmechanismen erfassen. dazu tragen sie eine reihe von rezeptoren für oberfl ächenstrukturen von viren und tumorzellen, bei deren vernichtung sie einen wichtigen platz einnehmen. durch ihren besatz mit den fc-rezeptoren cd und cd sind nk-zellen auch zur adcc befähigt (s. ). der effektivste wirkstoff zur cytotoxizität ist das perforin, das sie in azurophilen granula gespeichert enthalten. mit der abgabe von cytokinen können sie in die steuerung der spezifi schen wie unspezifi schen abwehr eingreifen. nk-zellen sind keine einheitliche population, sondern der begriff umfasst zellen verschiedener reaktivität und leistungen. der mensch gehört zu den homöothermen lebewesen, die ihre körpertemperatur in engen grenzen konstant halten. das bedeutet, dass wärmeproduktion und wärmeabgabe den äußeren verhältnissen angepasst werden müssen. da sich der mensch gewöhnlich in einer umgebung aufhält, deren temperatur unter der körpertemperatur liegt, erfolgt ein ständiger fluss von wärmeenergie aus dem körper an die außenwelt. diese wärme muss vom körper in entsprechendem maß bereitgestellt werden. eine wärmeproduktion ( thermogenese) fi ndet auf mehreren wegen statt: wie alle homöothermen lebewesen besitzt der mensch zur aufrecherhaltung einer konstanten körpertemperatur zentren, welche die temperatur messen, und mit diesen in verbindung mechanismen, über welche die körpertemperatur beeinfl usst wird. in geringem umfang kommen autonome, periphere regulationsmechanismen der hautgefäße zur geltung, die auf die lokalen temperaturverhältnisse mit einer enger-und weiterstellung reagieren und damit zum temperaturausgleich beitragen. wesentlich effektiver sind jedoch nervöse einrichtungen, welche die bildung bzw. die abgabe von wärme steuern. diese steuerung geht von zwei, in erster linie im hypothalamus lokalisierten zentren aus: dem hinteren "erwärmungszentrum" und dem vorderen "kühlzentrum". das hintere hypothalamische zentrum ("erwärmungszentrum") liegt in der wand des dritten ventrikels. es empfängt reize von den kälterezeptoren, die überall auf der körperoberfl äche, besonders dicht jedoch im gesicht und am stamm lokalisiert sind. dieses zentrum ist "temperaturblind", d.h. es ist selbst nicht in der lage, temperatur zu messen, sondern gibt die empfangenen kältereize weiter und bewirkt maßnahmen, welche die körpertemperatur steigern: einschränkung der wärmeabgabe und erhöhung der wärmeproduktion, die beide durch nervöse und humorale faktoren gesteuert werden. das vordere hypothalamische zentrum ("kühlzentrum") liegt in der präoptischen region des hypothalamus. zusätzliche solcher zentren wurden noch im hinteren hypothalamus, im vorderhirnseptum und im cerebralen cortex festgestellt. das kühlzentrum ist temperatursensibel und stellt sozusagen den thermostaten dar, der die körpertemperatur auf einem gegebenen sollwert hält. dieses zentrum spricht auf die bluttemperatur an und sendet umso mehr nervöse impulse aus, je höher die bluttemperatur liegt. Über diese impulse werden maßnahmen in gang gesetzt, die zu wärmeverlust führen. das kühlzentrum ist dem erwärmungszentrum insofern übergeordnet, als temperatursteigernde impulse das erwärmungszentrum nur verlassen können, wenn das kühlzentrum seine impulse senkt, was erst nach absinken der bluttemperatur eintritt. damit wird einem Überhitzen des organismus etwa nach starken oberfl ächlichen kältereizen vorgebeugt. fieber im rahmen von entzündungen wurde früher vielfach als eine entgleisung des stoffwechsels angesehen, als eine unerwünschte, gefährliche situation, die der arzt grundsätzlich bekämpfen sollte. man unterstellte damit dem natürlichen bauplan, er leiste sich ein besonderes system zur temperaturerhöhung, um sich selber schaden zuzufügen. heute steht man über neue einsichten und erkenntnisse dem fieber wesentlich positiver gegenüber und anerkennt innerhalb gewisser grenzen seinen wert. fieber und die mit ihm verbundenen stoffwechselleistungen stellen eine notfallmaßnahme dar, die dazu dient den organismus in eine erhöhte abwehrbereitschaft gegenüber mikroorganismen und schadmaterial zu versetzen den organismus über eine katabole stoffwechselsituation auf eigenreserven zu schalten, um ihn von einer energie-und stoffzufuhr von außen unabhängig zu machen. eine temperaturerhöhung bis zum kritischen wert von °c bedeutet grundsätzlich eine erhöhung des stoffwechsels, die auch die zellen der spezifi schen und unspezifi schen abwehr betrifft und sie in einen zustand erhöhter leistungsbereitschaft versetzt. folgerichtig lassen sich fähigkeiten von zellen der spezifischen und unspezifi schen abwehr, wie chemotaxis und phagozytose, auch in vitro durch temperaturerhöhung steigern. neben diesen direkten, durch erhöhte temperatur bedingten effekten kommt in vivo im fieber die besondere stimulation der abwehr durch reichlich freigesetzte mediatoren hinzu, von denen cytokine eine schlüsselstellung einnehmen. die umfassende rolle der interleukine und und des tnfα wird woanders beschrieben (s. ff). es seien --hier nur einige wesentliche wirkungsansätze hervorgehoben. im rahmen der unspezifi schen abwehr mobilisieren il- , il- , tnfα wie auch das bei entzündung vermehrt aktivierte c e weiße blutzellen aus dem knochenmark und bewirken damit eine leukozytose (s. ). durch hinaufregulierung von adhäsinen an den weißen zellen und am endothel wird die adhäsion dieser zellen am gefäßendothel erhöht und damit ihre emigration begünstigt (s. ff). im entzündungsherd stimulieren cytokine die tätigkeiten von phagozyten wie chemotaxis, phagozytose, freisetzung von mediatoren und ros, granulaabgabe und damit die weitere aktivierung serogener mediatoren (s. ) . in der leber wird die vermehrte produktion von akutphase-proteinen angeregt, wofür vorwiegend il- verantwortlich ist (s. ff), dessen produktion durch makrophagen und fibroblasten wiederum besonders durch il- stimuliert wird. il- regt fibroblasten zum wachstum und zur produktion von kollagen an und fördert damit heilungsvorgänge. im rahmen der spezifi schen abwehr werden durch il- th-lymphozyten zur proliferation und abgabe von il- angeregt, welches wiederum effektorzellen (tc und b-lymphozyten) zu aktivitäten stimuliert (s. ). die maximale aktivierung von tc zellen durch il- in vitro wurde bei . °c gemessen. auch die tumorabwehr wird durch temperaturerhöhung gesteigert, das wachstum der tumorzellen selbst aber beeinträchtigt. die medizin macht sich diesen effekt mit hyperthermietherapien zunutze. für manche krankheitserreger wird im fieber das temperaturoptimum für ihr wachstum überschritten. die den stoffwechsel während des fiebers bestimmende katabole reaktionslage, in welcher der organismus von eigenen energie-und materialreserven zehrt, läuft auf drei funktionellen etagen ab, die sich in ihren wirkungen zu einem hypermetabolismus vereinigen: auf der ebene des gesteigerten sym-reichlich atp für die energetisch aufwendige gluconeogenese in der leber benötigt. den Überschuss an acetyl-coa kondensiert die leber zu ketonkörpern, die, ins blut abgegeben, das puffersystem bis zur entwicklung einer ketoazidose belasten können. die lunge versucht, der säuerung durch eine verstärkte abatmung von co entgegenzuwirken. der erhöhte sauerstoffbedarf für die ß-oxydation, den citratcyclus und die atmungskette einerseits und die respiratorische kompen sation der ketonkörperbelastung anderer seits erklären die forcierte ( kussmaul'sche) atmung bei fieber. gegebenen falls kann eine hyperventilation auch zu einer alkalose führen. eine temperaturerhöhung bis zu °c steigert alle stoffwechselvorgänge und erhöht damit die abwehrleistung des organismus. somit unterstützt fieber den metabolismus, die belastung durch die entzündung zu meistern. die abb. fasst die steuerung und die auswirkungen des fiebers zusammen. die beträchtliche steigerung der stoffwechsel-und entzündungsvorgänge während fi eberhafter erkrankungen verlangt nach einem gegenprinzip, das ein ausufern der entzündlichen reaktion verhindert und den katabolismus auf das erforderliche maß einschränkt. in einer solchen gegenregulation sind cortisol und insulin faktoren mit schlüsselcharakter. cortisol nimmt einerseits im katabolen stoffwechsel eine zentrale fördernde stellung ein, wirkt aber auf der anderen seite als entzündungshemmer. es drosselt nicht nur die proliferation und die leistungen von zellen der spezifi schen und unspezifi schen abwehr, sondern hemmt auch die freisetzung der cytokine il- und tnfα, die wiederum wirkungsvolle verstärker der entzündung und kataboler reaktionen wie proteolyse im skelettmuskel und lipolyse im fettgewebe sind. auf der anderen seite stimulieren interleukine und tnfα die freisetzung von cortisol und von entzündungshemmenden akutphase-proteinen (s. ). dieses beispiel demonstriert die ambivalente verzahnung von beteiligten steuervorgängen untereinander. indem entzündungsfördernde cytokine und hemmende elemente der entzündung in einem negativen feedback miteinander verbunden sind, wird einerseits die ausbildung einer entzündungsreaktion begrenzt und ein ausufern verhindert, auf der anderen seite aber der entwicklung einer entzündungsreaktion spielraum gelassen. die anpassung der abwehrleistung an das erforderliche maß läuft allerdings nicht immer störungsfrei ab. fehlleistungen des immunsystems in die eine -zu geringe ausprägung mit infektgefahr -wie die andere richtung -zu starke entzündungsreaktion mit selbstschädigung des organismus -sind ein arbeitsfeld der täglichen klinischen routine. der insulinspiegel des blutes ist bei länger anhaltendem fieber erhöht, was an sich für eine katabole stoffwechselsituation paradox ist. es sind vermutlich cytokine (il- u.a.), welche die hemmung der insulinfreisetzung durch katecholamine, die über α-rezeptoren an den pankreatischen b-zellen vermittelt wird, aufheben und im gegenteil eine verstärkte abgabe veranlassen. hier ist ein deutlicher unterschied zwischen nicht-entzündlichem und entzündlichem stress gesetzt. mit einem mehrangebot des anabolen insulins will der organismus den katabolismus eingrenzen und ein ausufern verhindern. bezeichnenderweise laufen entzündliche erkrankungen bei diabetikern komplikationsreicher ab als bei nicht-diabetikern, da sie leicht in katabole stoffwechselentgleisungen münden können ( coma diabeticum es stellt sich die frage, was der organismus mit der massiven bereitstel-abb. . fieberentstehung im rahmen von entzündungsreaktionen. der kontakt mit exogenen pyrogenen stimuliert phagozyten zur abgabe der endogenen pyrogene il- , il- und tnfα, die auf dem blutweg das präoptische temperaturzentrum im hypothalamus erreichen, wo sie die produktion von pge erhöhen. der durch pge ausgelöste anstieg von camp in den zellen des temperaturzentrums hebt den temperatur-sollwert, und über vermittlung des sympathikus werden maßnahmen eingeleitet, welche die körpertemperatur erhöhen: minderdurchblutung der hautgefäße und gesteigerte wärmeproduktion durch muskelzittern. katecholamine und die hormone der hypothalamus -hypophysenvorderlappen -nebennierenrinden -achse lösen einen stoffwechsel-katabolismus aus und setzen damit energieträger und bausteine für die anforderungen der akutphase-reaktion frei. der gebräuchlichste therapeutische eingriff zur fiebersenkung ist die hemmung der pge -synthese durch cox-hemmer. lung von bausteinen und energieträgern bei entzündungen bezweckt. fettsäuren sind wertvolle energielieferanten. aminosäuren dienen einmal als bausteine zur massenproduktion von zellen der spezifi schen und unspezifi schen abwehr und zur herstellung von antikörpern, akutphase-proteinen, mediatorproteinen u.a. ein anderer teil der aminosäuren wird der gluconeogenese zugeführt, da der körper zur glucose-eigenproduktion gezwungen ist. ein krankes tier stellt die futtersuche ein oder jagt nicht mehr, sondern verkriecht sich und versucht, die entzündliche schädigung in ruhe, von den eigenen reserven lebend, zu überwinden. der mensch begibt sich in einer solchen situation gerne ins bett. die ausgeprägte somnolenz, die ihm diesen entschluss erleichtert, ist il- und tnfα vermittelt (s. ). der appetit ist während fi eberhafter erkrankungen gering oder fehlt gänzlich. die energie für die energetisch aufwendige gluconeogenese und für die stoffwechselsteigerung und synthesen wird in erster linie vom fettgewebe geliefert, baustoffe kommen aus dem skelettmuskel. der massive abbau von körpersubstanz äußert sich in einem rapiden gewichtsverfall. die kritische grenze, ab der die aktivitäten der spezifi schen und unspezifi schen abwehr nicht mehr gefördert, sondern im gegenteil eingeschränkt werden, liegt bei . bis °c. eine temperaturerhöhung über dieses kritische limit hinaus beeinträchtigt zunehmend die körpereigenen abwehrmechanismen und begünstigt die ausbreitung von krankheitserregern. hohe körpertemperaturen stellen darüber hinaus gefahren für organe und stoffwechsel dar. die katabole stoffwechselsituation führt über einen raschen verbrauch von depotfett und muskelprotein zu einer starken gewichtsabnahme und zum substanzverlust des körpers. die verstärkte atemtätigkeit hat zusammen mit der gesteigerten sekretion des hypotonen schweißes einen selbstverständlich verteilt sich das hier kursorisch zusammengefasste szenarium an möglichkeiten sehr unter-schiedlich auf die betroffenen individuen. wie soll nun der therapeut im einzelfall die vor-und nachteile des fiebers beurteilen, wann soll er fi ebersenkend eingreifen? fieber stellt eine schutzmaßnahme dar, die einen organisch gesunden körper bei der bekämpfung entzündlicher noxen unterstützt. unter ärztlicher kontrolle soll dieser schutzmechanismus seine wirkung entfalten können. fieber über den richtwert von °c bietet dagegen keinen nutzen mehr, sondern wird in zunehmendem maß zur gefahr und soll daher therapiert werden. neben der höhe der temperatur ist auch die individuelle situation des patienten zu beachten. kinder erreichen oft in kurzer zeit hohe temperaturen, die ebenso schnell wieder fallen können. alte menschen können wiederum auf vergleichsweise geringfügige temperaturerhöhungen mit unverhältnismäßig starken komplikationen reagieren. länger dauernde fieberperioden sind wegen der starken gewichtsabnahme und des damit verbundenen substanzverlustes und der verlängerten regenerationszeit von nachteil. besondere beachtung und eine angepasste betrachtungsweise erfordert das fieber bei personen mit organischen und metabolischen vorschäden. kreislauf-geschädigte personen sind ebenso wie solche mit zentral-nervösen defekten erhöht gefährdet. bei nierenerkrankungen und bei respiratorischer insuffi zienz, wo die pufferkapazität herabgesetzt ist, führt eine ketonkörperbelastung frühzeitig zur ketoazidose. die säurebelastung kann bei gichtkranken zum anfall führen. bei alkoholikern kann fieber ein delir auslösen. eine besondere stellung nimmt wegen seines häufi gen auftretens der diabetes mellitus ein. diabetiker sind erhöht infektanfällig (s. ), antworten häufi g auf entzündungen mit nur geringem temperaturanstieg, dekompensieren jedoch rasch in richtung eines coma diabeticum (s. f). der gebräuchlichste angriffspunkt einer therapie ist die hemmung der pge -synthese mittels cox-hemmern (s. ). neben der temperatur kann damit auch der gewichtsverlust reduziert werden. auf breiterer basis wirken glucocorticoide, welche die effektvollsten entzündungshemmer darstellen. sie unterbinden neben einer prostaglandinfreisetzung die abgabe von pyrogen wirksamen cytokinen aus makrophagen und anderen kompetenten zellen. neben einer reihe unerwünschter wirkungen (s. ) übt diese medikamentengruppe auch einen ausgeprägten immunsuppressiven effekt aus, der die infektentwicklung weiter begünstigt. glucocorticoide sollen daher nur nach strenger indikationsstellung und, wenn nicht umgehbar, unter intensiver antibiotika-abschirmung eingesetzt werden. eine physikalische maßnahme zur temperatursenkung stellen kältepackungen dar. eine wichtige begleitmaßnahme bei fieber ist die kontrolle des flüssigkeitsund elektrolythaushaltes. besondere sorgfalt verlangen kinder und betagte patienten. der schmerz ist eine komplexe sinneswahrnehmung eines individuums, die als warnsignal zu werten ist, um schäden zu vermeiden oder zu erkennen. schmerz kann akut fluchtrefl exe auslösen oder, als chronischer schmerz, die schonung geschädigter körperpartien bis zur heilung sicherstellen. im prozess der schadensbekämpfung und heilung nimmt die entzündung eine zentrale stellung ein, wobei der schmerz den entzündungsgrad refl ektiert und das individuum in einem subjektiv deutlich fassbaren rückfl uss über den grad des schadens und den heilungszustand informiert. wegen der starken beeinträchtigung des wohlbefi ndens und bewusstseins wird schmerz jedoch als negatives phänomen angesehen und therapeutisch entsprechend bekämpft. phylogenetisch entwickelte sich der schmerz mit dem bewusstsein und folglich zusammen mit der ausbildung des telencephalons. wird das bewusstsein ausgeschaltet, fehlt auch die schmerzempfi ndung (zentrale narkose). niederen tieren wird keine mit uns vergleichbare schmerzempfi ndung zugebilligt, sondern den schmerz ersetzen fluchtoder abwehrrefl exe; eine anschauung, die schwer beweisbar und strittig ist. in Übereinstimmung mit dieser ansicht nehmen jedenfalls die tierversuchsgesetze wirbellose von ihren bestimmungen aus. schmerzreize werden über zwei fasersysteme vom ort ihres einwirkens zentralwärts geleitet: einmal über die markhaltigen, schnellleitenden a-delta fasern (bis zu m pro sekunde). die vermittelte empfi ndungsqualität ist der distinkte, gut lokalisierbare schmerz, der abwehr-und fluchtrefl exe auslöst. die c-fasern dagegen sind marklos und langsam leitend (ca. . bis m pro sekunde). die vermittelte empfi ndung ist dumpf und unscharf lokalisierbar. c-fasern übermitteln in erster linie den chronischen schmerz. als "schmerzrezeptoren" werden die vielfach verästelten endverzweigungen dieser zentripetalen nervensysteme, die nozizeptoren, angesehen. sie fi nden sich in verschiedener dichte an allen körperoberfl ächen (haut, schleimhäute) und in der tiefe in muskeln, sehnen und bändern. auch das viszerum wird mit schmerzfasern vor allem vom c-typ versorgt, die auf dehnung, motilitätssteigerung und auf mediatorreize im rahmen von entzündungen, nicht jedoch auf mechanische traumen ansprechen. so sind etwa operationsschnitte im viszeralen abdomen schmerzlos. die afferenten, schmerzleitenden adelta und c-fasern werden im rückenmark vielfach verschaltet. sitz der schaltzellen ist vor allem die substantia gelatinosa. von hier wird der reiz über den tractus spinothalamicus zum thalamus und weiter zentralwärts geleitet. nach der -im Übrigen sehr umstrittenen -"gate control theory" kann eine erregung der schmerzfasern durch zwei "gates" (eingänge) in das bewusstsein eindringen: entweder als schmerz oder als sinneseindruck anderer qualität. welcher dieser wege im jeweiligen fall beschritten wird, hängt neben der intensität von der verarbeitung des reizes in den schaltzellen des rückenmarks ab, die wieder unter dem steuernden einfl uss des gehirns stehen. im tierversuch lassen sich im bereich des zns der thalamus, hypothalamus, der nucleus amygdalae und insbesondere die formatio reticularis als verarbeitungsstellen von schmerzreizen nachweisen. beim menschlichen gehirn mit seiner komplizierten, vom intellekt überlagerten psyche fehlen diesbezüglich detaillierte erkenntnisse. unbestreitbare erfahrungstatsache ist jedoch, dass qualität und intensität einer subjektiven schmerzempfi ndung stark von psychischen faktoren abhängen. bestimmend ist hier die persönlichkeitsstruktur, die wiederum von sozialen (etwa familienvorbild) und kulturellen einfl üssen (etwa kulturkreise mit schmerzunterdrückung als ideal) geprägt ist. ebenso modifi zieren aufmerksamkeit, vorangegangene schmerzerfahrung oder schmerzerwartung das persönliche schmerzerlebnis. es bestehen auch alters-und geschlechtsunterschiede. Ältere menschen ertragen gegenüber jüngeren, frauen gegenüber männern den schmerz besser. schmerzschwelle und schmerzintensität unterliegen einem zirkadianrhythmus mit einem maximum in den nachtstunden. grund dafür ist das vegetativum. der nächtliche parasympathikotonus senkt die schmerzschwelle und erhöht so die schmerzempfi ndlichkeit, der am tage vorherrschende sympathikus hebt dagegen die schwelle an. adrenerge reaktionslagen wie stress lassen "den schmerz vergessen". so senkt auch zigaretten rauchen durch steigerung des sympathikotonus die schmerzempfi ndung. eine zentrale bedeutung für die weiterlei-tung und verarbeitung von schmerzrei-misch soweit verändert, dass ihr anti-in-abb. . hemmung der entzündungsaktivität durch glucocorticoide auf zellulärer ebene. ( ) synthetische wie natürliche glucocorticoide (cortisol) diffundieren durch die zellemembran zum intrazellulären glucocorticoidrezeptor und binden an ihn, wobei das hitzeschockprotein (hsp ) vom rezeptor abgespalten wird. ein transportmechanismus für glucocorticoide durch die zellmembran wird diskutiert, ist aber nicht bewiesen. der glucocorticoid-rezeptor-komplex diffundiert weiter in den zellkern, wo er abhängig vom zelltyp an den "negative glucocorticoid responsive elements" die expression einer reihe von proinfl ammatorischen faktoren hemmt. im gegensatz dazu wird an den "positive glucocorticoid responsive elements" die expression von lipocortin erhöht. lipocortin ist ein kd protein der annexin-familie, das in die zellmembran eingelagert wird und die aktivität der phospholipase a (pla ) hemmt. durch die verringerte freisetzung von arachidonsäure (aa) wird der aa-metabolismus und damit die entzündliche reaktivität der zelle eingeschränkt. das hsp aktiviert gene für die produktion zellprotektiver proteine. ( ) glucocorticoide werden bei hohen konzentrationen direkt in die zellmembran eingelagert und dämpfen die reaktivität und aktivität der zelle durch erhöhung der membranviskosität. gel stimuliert wiederum die insulin-einer breiten anwendung von biologics in der medizinischen praxis stehen heute noch eine reihe von hindernissen im wege. blockierte rezeptoren und cytokine werden von der zelle rasch ersetzt, so dass meist dauerinfusionen über längere zeiträume nötig sind. zudem sind die zur zeit enormen kosten solcher neu entwickelter medikamente kein unbeträchtliches gegenargument. trotzdem muss in diesem konzept gezielter therapien mittels eingriffen auf physiologischer ebene eine zukunft der medizin gesehen werden. diacylglycerol (dag) spaltet. dag aktiviert die ca ++ -abhängigen proteinkinasen, unter ihnen die inhibitor of kappa b kinasen (iκb-kinasen), die den nfκb/iκb komplex spalten. der nfκb, von dem verschiedene subtypen bekannt sind, ist ein proteinkomplex mit einem mw um kd, dessen aufgabe die induktion von transskriptionen im zellkern ist (transskriptionsfaktor) salicyldienen. chemisch sind nsaid sieben hauptgruppen in zwei hauptklassen zuzuordnen. carbonsäuren mit den vertretern salicylsäurederivate (z.b. acetylsalicylsäure), anthranilsäurederivate (z.b. etofenamat), essigsäurederivate (z.b. indomethacin), und propionsäurederivate (z.b. iboprufen) hemmer (s. ) sind dagegen abkömmlinge von sulfonamiden und verwandten schwefelverbindungen lichen metapher die entzündung als kriegerischen akt dar, mit dem sich ein organismus gegen einen feind zur wehr setzt, so sind pmn mit dem landser vergleichbar (wenig spezialisiert und sehr zahlreich), die lymphozyten entsprechen den spezialtruppen, während der makrophage den offi zier darstellt, der das geschehen vor ort und den gesamtablauf im generalstab lenkt. diesem bild entsprechend steuert der makrophage sowohl den lokalen entzündungsvorgang, wie er auch für systemische entzündungsreaktionen wie fieber, leukozytose und das auslösen der apr hauptverantwortlich ist. als mittel dazu steht ihm eine lange reihe von regulatorstoffen zur verfügung, die er selbst freisetzt oder deren freisetzung oder aktivierung er steuern kann. einzelheiten dazu werden in den einschlägigen kapiteln angeführt.im zuge eines entzündungsablaufes ändern makrophagen ihre funktionellen schwerpunkte. zu entzündungsbeginn steht das aggressive element im vordergrund: makrophagen fördern die apoptose von leukozyten und stromazellen und beseitigen deren reste, sie töten und phagozytieren mikroorganismen, immunkomplexe und opsonierte partikel und lösen mit hilfe ihrer enzyme gewebsbestandteile auf. durch abgabe pro-infl ammatorischer cytokine, wachstumsfaktoren und mediatoren aktivieren sie das entzündungsgeschehen. sie selbst werden durch hinaufregulierung entsprechender rezeptoren für signalstoffe der entzündung sensibilisiert.in der regressionsphase einer akuten entzündung wandelt sich der makrophage in eine gewebs-protektive zelle um, die bei der wiederherstellung des gefüges und bei der heilung mitwirkt. die nun freigesetzten cytokine sind antiinfl ammatorisch. es werden apoptosehemmende signale abgegeben, zellvermehrung und wachstum stimuliert, der matrixaufbau und die angiogenese gefördert, stromazellen werden zu synthesen angeregt.die ursachen für diesen wandel sind im detail nicht geklärt. offenbar hat aber das phagozytierte material einfl uss. zu den entscheidenden schritten der vermehrung, differenzierung, reifung von zellen der myeloischen reihe und letztendlich der freisetzung reifer differenzierungsprodukte aus dem knochenmarksspeicher in die blutbahn trägt der makrophage wesentlich bei. der makrophage ist eine quelle von csf (s. f). ebenso ist er ein produzent der "leukozytosefaktoren" il , il , tnfα sowie von c , aus dem bei aktivierung c e abgespalten werden kann (s. ). auf diesem weg wird das zentrum der erzeugung und speicherung über den bedarf am ort der entzündung informiert und dieser bedarf kontrolliert gedeckt.die gesamtheit des retikulo-endothelialen systems (res), rotes knochenmark, milz und lymphoretikuläres gewebe, macht beim erwachsenen über zwei kilogramm aus und ist damit das größte organ des körpers. im dienst des schutzes des organismus gegen äußere und innere noxen stellt es mit der vielfalt an abgegebenen informationsträgern auch ein bedeutendes endokrines organ dar, in dem der makrophage eine zentrale stellung einnimmt. steigt die absolute zahl an monozyten beim erwachsenen über /µl, beim kind über /µl und beim säugling über /µl an, spricht man von einer monozytose. eine "infektiöse monozytose" kann als begleitsymptom einer reihe von infektionskrankheiten auftreten: bei endocarditis lenta, malaria, typhus und besonders stark bei viruserkrankungen wie hepatitis epidemica, mumps und viruspneumonien. als erreger von monozytosen werden häufi g epstein-barr-und webe funktionelle unterschiede zeigen können. auf diese heterogenität wird hier nicht eingegangen. unter den vielfältigen aufgaben des endothels im rahmen von entzündungsvorgängen ist hervorzuheben: lang anhaltender, chronischer schmerz bewirkt eine sympathikotone reaktionslage und über diese einen stoffwechsel-katabolismus und gewichtsverlust. am beginn dieser äußerst komplexen und noch wenig durchschauten verarbeitungsprozesse auf zentraler ebene steht die reizwahrnehmung durch die schmerzfasern. als reize kommen mechanische, thermische und chemische einfl üsse in betracht. zu letzteren gehören die schmerzauslösenden entzündungsmediatoren histamin, serotonin und das hochwirksame bradykinin, die über spezifi sche rezeptoren an den nozizeptoren wirken. grundsätzlich ist zu diesen schmerzvermittlern zu sagen, dass sie allein kaum oder nur geringgradig schmerz auslösen, aber durch weitere mediatoren in ihrer wirkung beträchtlich potenziert werden können. das gilt vor allem für histamin und serotonin, die allein ohne synergismen kaum schmerz erzeugend sind. prominente synergisten sind die prostaglandine e und e , die selbst nicht schmerz erregen, aber bei der gestaltung des chronischen schmerzes eine wichtige stellung einnehmen, indem sie die reizschwelle an den endverzweigungen der c-fasern für schmerzmediatoren senken. im versuch intradermal injiziertes pge verursacht einen kaum wahrnehmbaren schmerzreiz. zusammen mit bradykinin, histamin oder serotonin verabreicht steigt die schmerzempfi ndung deutlich an (s. ). die mit abstand häufi gste form des schmerzes ist der chronische schmerz im rahmen von entzündungen. seine beseitigung oder zumindest linderung geschieht durch einen eingriff in den synergismus unter den entzündungsmediatoren, indem die pg-synthese durch cox-hemmer gedrosselt wird. die schmerzbekämpfung mittels acetylsalicylsäure ist die gebräuchlichste medikation der welt (s. ).anästhetika und narkotika wirken auf anderer ebene. indurative prozesse der lunge und anderer organe die schmerztherapie zur therapie der entzündung werden drei "klassische" gruppen von medikamenten verwendet: auf diesem weg wirken auch native, körpereigene glucocorticoide anti-infl ammatorisch.bei hoher, pharmakologischer dosierung werden glucocorticoide in die zellmembran eingebaut und verursachen, wie auch die muttersubstanz cholesterin, eine erhöhung der membranrigidität und damit eine verlangsamung aller vitalvorgänge der zelle ("membran-stabilisierung"). diese wirkung tritt physiologisch nicht auf, da so hohe konzentrationen durch eine natürliche glucocorticoid-freisetzung nicht erreicht werden (abb. ). eine länger dauernde therapie mit den stark eiweiß-katabolen glucocorticoiden hemmt die leukopoese im knochenmark, so dass nach erschöpfung der knochenmark-reserven eine senkung der leukozytenzahl bis zur leukopenie auftritt (s. ). von die-----sem katabolismus sind in gleicher weise lymphozyten und antikörper des spezifi schen immunsystems betroffen (s. , abb. ). glucocorticoide fördern die produktion von akutphase proteinen, unter denen manche entzündungshemmend wirken (s. ).glucocorticoide werden systemisch (oral, parenteral) und lokal (lösungen, salben, aerosole) appliziert. glucocorticoide sind medikamente mit hohem nebenwirkungs-potential und können, abhängig von dosierung, therapiedauer und individueller empfi ndlichkeit, komplikationen und schäden nach sich ziehen.die beabsichtigte therapeutische wirkung einer entzündungshemmung schließt die negative kehrseite, nämlich die hemmung des immunsystems und eine erhöhte infektgefährdung, mit ein. hemmung der kollagensynthese: dieser effekt kann therapeutisches ziel sein, wenn etwa überschießende narbenbildung oder indurative prozesse in organen unterbunden werden sollen. zum negativen effekt gehören eine verzögerte wundheilung und eine negative knochenbilanz. da durch die eingeschränkte synthese des kalzium-bindenden proteins (cabp) in der darmschleimhaut auch die ca ++ -aufnahme vermindert ist, wird sowohl die neubildung wie auch die mineralisierung des knochens beeinträchtigt ( osteopathie). diesem defi zit in der knochenbilanz muss bei länger dauernder, intensiver corticoid-therapie unbedingt gegengesteuert werden. kinder entwickeln unter corticoid-therapie einen minderwuchs. glucocorticoide aktivieren die gluconeogenese aus eiweiß. dementsprechend zehrt eine therapie an der muskelmasse. der hohe blut-glukosespie--■ ■ ■ freisetzung aus dem inselapparat und in folge die fettsynthese in der leber und die fettspeicherung im fettgewebe. die angehobenen insulinspiegel lenken das fett in pannuszellen am körperstamm und im abdomen, die mit insulinrezeptoren reichlich ausgestattet sind. es entsteht typischerweise eine "stammfettsucht". blutglucose über der resorptionsschwelle der nieren von bis mg% wird im harn ausgeschieden ( "steroid-diabetes"). hohe blutspiegel von glucose und glucocorticoiden können katarakt und glaukom hervorrufen. auch synthetische glucocorticoide üben in höherer dosierung mineralocorticoid-wirkung aus und führen über eine vermehrte retention von na + und wasser zu Ödemen und bluthochdruck. das ödematös durchtränkte stammfett prägt das erscheinungsbild: massiger körperstamm und nacken, aber durch den muskelabbau ■ ■ dünne arme und beine. gesichtsödeme bewirken das "vollmondgesicht". wegen der Ähnlichkeit mit dem morbus cushing, bei dem die glucocorticoide aus endogener Überproduktion vermehrt sind, wird dieser habitus als "cushingoides erscheinungsbild" bezeichnet. die veränderung tritt bei langzeittherapie über einer gewissen glucocorticoid-belastung, der sog. "cushing-schwelle" auf, die individuell verschieden ist. ulcera des magens und des duodenuums ( "steroid-ulcus") entstehen durch eine hemmung der phospholipase a und den daraus resultierenden mangel an cytoprotektivem pge (abb. , abb. langdauernde hemmung der hypothalamus-hypophysen-nebennierenrinden-achse (hhn-achse) kann eine inaktivitätsatrophie der beteiligten drüsen zur folge haben, aus der sie sich nur langsam oder auch gar nicht mehr erholen. um dieses risiko möglichst klein zu halten, werden glucocorticoide nicht als zirkadiane dauerspiegel therapiert, sondern man folgt dem physiologischen tagesrhythmus: morgens hoch, danach abfallend. ein plötzliches absetzen einer langdauernden therapie kann eine insuffi ziente hhn-achse zurücklassen, bis zu deren regeneration eine gluconeogenese nicht ausreichend betrieben werden kann. in katabolen situationen (hunger, körperliche anstrengung, stress, fieber) besteht dann die gefahr einer hypoglykämie. man soll deshalb im bedarfsfall der hhn-achse durch schrittweise reduktion der glucocorticoid-dosis ("ausschleichen") die möglichkeit zur erholung geben.unerwünschte nebenwirkungen treten naturgemäß nach langdauernder, hochdosierter systemischer (oraler) gabe von glucocorticoiden besonders stark in erscheinung. aber auch bei inhalativer applikation von aerosolen werden glucocorticoide über die schleimhäute ins system aufge-nommen und können nebenwirkungen entwickeln. bei äußerlicher anwendung etwa in lotionen oder salben wird die kollagensynthese mit der folge von hautatrophien beeinträchtigt. lokale injektionen von glucocorticoiden gegen entzündung und schmerzen von gelenken wie z.b. in der sportmedizin schwächen das kollagen der sehnen und bänder und erhöhen das risiko von rissen unter belastung. da diese medikamentengruppe bei der therapie der chronischen polyarthritis (cp) einen standardplatz einnimmt, wird im deutschen auch der ausdruck "nicht steroidale antirheumatika" (nostar, auch nsar) verwendet. die cp deckt aber bei weitem nicht das einsatzgebiet dieser entzündungshemmer ab. nsaid sind die am häufi gsten verschriebenen heilmittel überhaupt. in europa machen sie knapp % des medikamentenumsatzes aus.zur geschichte. die äußerliche anwendung von zerquetschten weidenwurzeln, die salicylsäure enthalten (salix abb. . hemmung der pge-synthese durch antiphlogistische therapien. glucocorticoid-therapie hemmt über eine vermehrte lipocortin-expression die phospholipase a (pla ) und damit die arachidonsäure (aa)-freisetzung und pge -synthese. nsaid-therapie wiederum hemmt die cyclooxygenasen (cox) und damit die umsetzung von aa zu pge . beide therapieformen resultieren in niederen pge -konzentrationen in der magen-und darmschleimhaut und der niere, welche die ulkusentstehung begünstigen und nierenschäden verursachen können.abb. . glucocorticoid-therapie und cortisol-synthese. natürliches cortisol sorgt in einem negativen feedback für eine regulierung der cortisol-produktion in der nebennieren-rinde: die freisetzung des corticotropinreleasing hormon (crh) im hypothalamus und des adreno-corticotropen hormons (acth) in der adenohypophyse werden gehemmt. in gleicher weise greifen synthetische glucocorticoide in die cortisol-produktion über die hypothalamus-hypophysen-nebennierenrinden (hhn)-achse ein. hohe und langdauernde glucocorticoidtherapie kann zur inaktivitätsatrophie der hhn-achse und zu cortisol-mangel nach absetzen der therapie führen. allen gruppen der nsaid gemeinsam ist die hemmung der cyklooxygenase (cox). je nach nsaid-typ ist die wirkung auf die cox unterschiedlich.manche nsaid hemmen die cox kompetitiv mit geringer bindungsaf- leukotrien-antagonisten: zur hemmung der wirkung der cysteinyl-leukotriene c d e ("slow reacting substance of anaphylaxis") wurden zwei therapeutische wege begangen:hemmung der lt-synthese durch behinderung der aktivierung der -lipoxygenase (s. ). blockierung der rezeptoren für cysteinyl-lt. diese letztere gruppe von lt-antagonisten ist in europa zugelassen und wird in der therapie des asthma bronchiale eingesetzt.histamin-antagonisten: die entzündlichen wirkungen des histamins werden durch h -rezeptorblocker ( "antihistaminika") neutralisiert. die histamin-abgabe aus den mastzellen kann durch spezifi sche ca-antagonisten ( dinatrium-chromogylzinsäure) gehemmt werden (abb. ). eine mäßige hemmwirkung auf mastzellen entfalten auch β -mimetika (tabelle , s. ). moderne therapiekonzepte versuchen hoch spezifi sch an strategisch günstigen stellen in den entzündungsablauf einzugreifen. dazu werden gentechnisch hergestellte natürliche wirkstoffe bzw. wirkstoffe in geringer abwandlung (analoga) oder auch antikörper gegen biologische wirkstoffe eingesetzt, die unter dem begriff "biologics" zusammengefasst werden. auf diesem weg sollen cytokine, adhäsine und rezeptoren durch antikörper, lösliche rezeptoren und natürliche antagonisten in ihrer wirkung neutralisiert werden. zielobjekte für solche steuernden eingriffe sind vor allem adhäsine an granulozyten, lymphozyten und endothelzellen sowie cytokine, die bei sirs, sepsis, bei transplantatabstoßung u.a. blockiert werden sollen (s. , ) . die unter hohem kraftaufwand der pharmaindustrie meist rekombinant hergestellten wirkstoffe haben die erwartungen häufi g nicht oder nur zum teil erfüllt. vielversprechende ergebnisse konnten dagegen in der therapie der cp mit tnfα-antagonisten erzielt werden.-- zur entzündungshemmung bzw. zur neutralisation bei entzündungen anfallender toxischer produkte können auch wirkstoffe beitragen, die in gewissen nahrungsmitteln vorhanden sind. mehrfach ungesättigte fettsäuren, wie die eikosapentaensäure und dokosahexaensäure werden in nur schwach wirksame endprodukte des cox und lox-stoffwechsels umgesetzt. speisefi sche reichern solche vom phytoplankton produzierten hoch-ungesättigten fettsäuren in ihren fettdepots an (s. f).eine andere gruppe entzündungshemmender nahrungsbestandteile sind die sauerstoffradikalfänger. als "fänger" kommt eigentlich alles in frage, was sich leicht oxydieren lässt: phenole, aro-matische amine, flavonoide, β-carotine und carotinoide, α-tokopherol ( vitamin e), ascorbinsäure ( vitamin c) in gemüse, obst, früchten, tee, rotwein, in der kleberschicht von getreide ("vollkornkost") und vielen anderen natürlichen nahrungsmitteln. der wert einer fisch, obst und gemüse reichlich enthaltenden kost, wie sie in subtropischen klimazonen tradition ist ("mittelmeerkost"), zur prophylaxe der atherosklerose ist wissenschaftlich gesichert. atherosklerose ist eine spezifi sche, chronisch verlaufende entzündung der arteriellen intima, die durch entzündungshemmende alimentäre faktoren günstig beeinfl usst werden kann (s. ). eine derartige ernährung wird darüber hinaus bei chronischen entzündungen wie cp, psoriasis, akne, atopischer dermatitis und als begleitende unterstützung anti-infl ammatorischer therapien empfohlen. es muss aber immer betont werden, dass eine alimentäre regulierung des pro-und antiinfl ammatorischen gleichgewichts nicht durch eine einmal-therapie, sondern nur durch eine konsequente einhaltung des dietätischen regimes über jahre und jahrzehnte erreicht werden kann. da sie sehr schmackhaft sind, können solche kostformen auch dem noch nicht-kranken zur erhaltung der gesundheit empfohlen werden. hoch-ungesättigte fettsäuren sind auch bei schwerem trauma, sepsis und sirs in infusionslösungen mit der erwartung einer entzündungshemmung im einsatz. key: cord- - uaj hmx authors: desmonts de lamache, d.; moges, r.; siddiq, a.; allain, t.; feener, t. d.; muench, g. p.; mckenna, n.; yates, r. m.; buret, a. g. title: immuno-modulating properties of tulathromycin in porcine monocyte-derived macrophages infected with porcine reproductive and respiratory syndrome virus date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: uaj hmx porcine reproductive and respiratory syndrome virus (prrsv) is a positive-stranded rna virus that grows in macrophages and causes acute pneumonia in pigs. prrsv causes devastating losses to the porcine industry. however, due to its high antigenic variability and poorly understood immunopathogenesis, there is currently no effective vaccine or treatment to control prrsv infection. the common occurrence of prrsv infection with bacterial infections as well as its inflammatory-driven pathobiology raises the question of the value of antibiotics with immunomodulating properties for the treatment of the disease it causes. the macrolide antibiotic tulathromycin (tul) has been found to exhibit potent anti-inflammatory and immunomodulating properties in cattle and pigs. the aim of this study was to characterize the anti-viral and immunomodulating properties of tul in prrsv-infected porcine macrophages. our findings indicate that blood monocyte-derived macrophages are readily infected by prrsv and can be used as an effective cellular model to study prrsv pathogenesis. tul did not change intracellular or extracellular viral titers, not did it alter viral receptors (cd and cd ) expression on porcine macrophages. in contrast, tul exhibited potent immunomodulating properties, which therefore occurred in the absence of any direct antiviral effects against prrsv. tul had an additive effect with prrsv on the induction of macrophage apoptosis, and inhibited virus-induced necrosis. tul significantly attenuated prrsv-induced macrophage pro-inflammatory signaling (cxcl- and mitochondrial ros production) and prevented prrsv inhibition of non-opsonized and opsonized phagocytic function. together, these data demonstrate that tul inhibits prrsv-induced inflammatory responses in porcine macrophages and protects against the phagocytic impairment caused by the virus. research in live pigs is warranted to assess the potential clinical benefits of this antibiotic in the context of virally induced inflammation and tissue injury. a a a a a responsible for estimated losses exceeding us$ million/year in the usa alone, porcine reproductive and respiratory syndrome (prrs) is a devastating disease in the swine industry [ ] . first identified in europe and north america in the late s [ ] , this syndrome is currently prevalent in most swine-producing countries [ ] . its causative agent, the porcine reproductive and respiratory syndrome virus (prrsv), is a small enveloped positive-sense singlestranded rna virus, member of the arterivirus genus [ ] . sequence comparison between viral isolates demonstrated that prrsv exists in at least two distinct genotypes, the european genotype (eu type or type i) commonly referred to as prrsv- , and the north american genotype (na type or type ii) known as prrsv- [ ] . prrsv has a very narrow cell tropism, and may induce persistent asymptomatic infections [ , ] . in its natural host, the virus targets alveolar macrophages (am) [ , ] , and is able to infect most cells of the monocyte-macrophage lineage such as intravascular and lymph node macrophages [ , , ] . these cells play a crucial role in immune surveillance, pathogen killing and adaptive immune response stimulation [ ] . prrsv impairs macrophage phagocytic and bactericidal functions, induces host cell death often resulting in an inflammatory response, and perhaps most importantly predisposes the pig to secondary infections [ ] [ ] [ ] [ ] [ ] . indeed, opportunistic pathogens, whether viral-swine influenza virus, pseudorabies virus-or bacterial-streptococcus suis, bordetella brochiseptica-potentiate prrsv-induced pneumonia [ ] . these synergistic effects promote a self-sustaining inflammatory response increasing the severity and the duration of the disease [ ] [ ] [ ] [ ] [ ] . the common occurrence of prrsv infection with bacterial infections combined with the lack of efficient vaccines begs the question of the value of antibiotics for the treatment of prrs. traditionally, antibiotic efficacy is evaluated solely based on their antimicrobial properties. however, some macrolides have been found to modulate ros and pro-inflammatory cytokines such as cxcl- and il- , and to alter the production of lipid mediators that regulate inflammation [ ] [ ] [ ] [ ] [ ] [ ] . these antibiotics accumulate within leukocytes at concentrations that may reach times the systemic levels, which in turn allows them to be transported directly to the site of infection and confers them superior pharmacodynamics [ ] . there is little evidence supporting a direct anti-viral property for macrolides, but their effects on leukocytes support the hypothesis that such macrolides may be beneficial in the context of viral infections such as prrsv [ , ] . in an attempt to uncover new mechanisms whereby macrolides may protect against the detrimental effects of prrsv, the present study investigated the effects of tulathromycin in porcine monocyte-derived macrophages. tulathromycin is a triamilide in which its lactone-ring is comprised of polar amine groups. it is used for the treatment and prevention of swine respiratory diseases associated with actinobacillus pleuropneumoniae a gram-negative bacteria often found in prrsv-infected pigs [ ] . a. pleuropneumoniae exerts cytotoxic effects in macrophage and neutrophils and increases the production of pro-inflammatory il- , cxcl- -also known as interleukin- -and leukotriene b , which ultimately leads to severe pulmonary tissue damage and death [ ] [ ] [ ] [ ] [ ] . recent studies have demonstrated that in addition to its antimicrobial effects, tulathromycin inhibits cxcl- and ltb production in stimulated neutrophils and macrophages [ , , ] . in addition, tulathromycin promotes the apoptotic death of neutrophils and their phagocytic clearance by macrophages -a phenomenon known as efferocytosis-both crucial processes in the resolution of inflammation [ , , [ ] [ ] [ ] . we hypothesized that tulathromycin may generate immunomodulatory benefits in prrsv-infected monocyte-derived macrophages. the findings indicate that tulathromycin, in the absence of a direct anti-viral effect, is able to restore the phagocytic function and to attenuate the pro-inflammatory phenotype of prrsv-infected monocyte-derived porcine macrophages. the african green monkey kidney cell line marc- (crl- ) which is highly permissive to prrsv, was used for viral passage and plaque titration assay, as validated previously [ , , ] marc- cells were cultivated in dulbecco's modified eagle's medium (dmem; thermo fisher scientific, waltham, ma, usa) supplemented with % fbs (invitrogen, carlsbad, ca, usa) and iu/ml penicillin-streptomycin (thermo fisher scientific, waltham, ma, usa). the cells were maintained at ˚c, % co and passaged twice weekly. prrsv- isolate nvsl - (genbank accession no. ay . ) was used in all experiments as previously described [ ] . viral titration was performed via plaque assay. briefly, marc- cells were seeded in well plates (costar; sigma aldrich, saint-louis, mo, usa) and grown until confluency. once at confluency, cells were infected with prrsv, for hour in serum-free dmem to allow attachment of viral particles. following attachment, marc- were overlaid with a solution of x mem diluted : with . % agarose. infection was carried for h and plaques were revealed with neutral red (sigma-aldrich, saint-louis, mo, usa). dr. r. m. yates from university of calgary generously provided both marc- cell line and prrsv- isolate nvsl - . all animal experimental practices and care were conducted according to the standards of the canadian council of animal care guidelines and approved by the university of calgary life and environmental science animal care committee. blood was collected from healthy large white and landrace cross -to weeks old ( -to kg) female and castrated male piglets. the animals were housed at the veterinary science research station (university of calgary) at ˚c ± ˚c with % humidity, light cycles consisted of hours continuous light exposure followed by hours of darkness. piglets were fed twice with the antibiotic-free feed % hog grower (hi-pro feeds, okotoks, ab, canada), water was provided ad libitum. after weeks, animals were euthanized and tissues made available for secondary teaching and research use. in accordance with the standards of the canadian council on animal care, pigs were euthanized by intracardiac injection with sodium pentobarbital. monocytes were obtained and differentiated into macrophages as described previously [ ] . briefly, blood was pooled and centrifuged for minutes at x g, ˚c in a heraeus megafuge r (thermo fisher scientific, waltham, ma, usa). the plasma was removed, and the buffy coat layer was collected into and diluted : in filter-sterilized . % nacl. sterile polysucrose and sodium diatrizoate gradient solution (histopaque; sigma-aldrich, saint-louis, mo, usa) was added into each tube before centrifugation for minutes at x g, ˚c. pbmcs located at the opaque interphase were then collected, washed with sterile-filtered x hank's balanced salt solution (hbss; thermo fisher scientific, waltham, ma, usa) and centrifuged for minutes at x g, ˚c. contaminating erythrocytes were removed by three hypotonic lysis cycles with sterile ice-cold double-distilled water for seconds followed by the addition of x hbss to restore tonicity. pbmcs were then resuspended in serum-free iscove's modified dubelcco's medium (imdm; thermo ficher scientific, waltham, ma, usa) supplemented with iu/ml penicillin-streptomycin. cells were counted using a hemocytometer and viability was assessed by . % trypan blue exclusion (flow laboratories). pbmcs purity was determined by diff-quick staining on cytospin slides (cytospin cytocentrifuge, thermo fisher scientific, waltham, ma, usa). the cells were then plated in tissue-culture treated , , and well plates (costar; sigma aldrich, saint-louis, mo, usa) or in labtek chamber slides (thermo fisher scientific, waltham, ma, usa) at a concentration of . x cells/ ml for two hours to allow attachment. following adhesion, non-adherent mononuclear cells were washed with warm hbss ( ˚c). subsequent adherent monocytes were incubated for days at ˚c, % co in imdm supplemented with % heat inactivated(hi)-pig serum (ge healthcare, chicago, il, usa), iu/ml penicillin-streptomycin and % l supernatant to allow for differentiation into monocyte-derived macrophages (mdms). l -conditionned medium is commonly used to potentiate monocytes to differentiate into homogenous populations of mature macrophages [ , ] . culture media was changed every days. flow cytometry was used to quantify the number of cells expressing cd (a known cluster of differentiation of monocytes and macrophages). more than % of the isolated cells expressed cd (s fig) . on day , as described previously [ ] , macrophage differentiation was monitored by microscopic morphological changes using diff-quick, and esterase staining, a well known feature allowing to distinguish between monocytes and mature macrophages [ ] . at day , more than % of the cell preparations were differentiated macrophages (data not shown). seven days-old differentiated macrophages were incubated with tulathromycin (draxxin; zoetis, parsippany-troy hills, nj, usa) diluted in imdm + % hi-pig serum at a concentration of . mg/ml or mg/ml or with vehicle control (imdm + % pig serum), as established recently [ , ] . at these concentrations and time points, the drug exhibits immunomodulating properties in bovine macrophages without inducing apoptosis [ ] . antibiotics like tulathromycin accumulate within leukocytes at concentrations that may reach > times the systemic levels, which in turn allows them to be transported directly to the site of infection, and hence confers them with superior pharmacodynamics [ , ] . this phenomen is critical to the mode of action of tulathromycin. the drug concentrations used in these present experiments are consistent with this knowledge, and with previous studies that showed that tulathromycin has immunomodulating effects in bovine and porcine neutrophils and macrophages [ , , ] . these recent studies have reproduced the same immunomodulating effects seen in vitro at these drug concentrations than when using live infected cattle and pigs given tulathromycin at the recommended therapeutic dosage [ , , ] . hence the concentrations used here reflect the physiological conditions in which the drug accumulates at high concentrations within these leukocytes. indeed, comparison of intracellular drug concentrations in treated versus untreated animals have been published previously [ ] . using lc/ ms ms, in animals given the recommended dose of . mg/kg body weight, studies have measured the rapid and prolonged distribution of the drug into lung homogenates, pulmonary epithelial ling fluid (pelf), as well as in pelf cells. macrophages are the major constituents of pelf cells in such preparations. drug levels measured in pelf cells reached concentrations times greater than those in plasma [ ] . it is believed that this great affinity for cellular uptake may be related, in part, to the tri-basic chemical structure of the drug and the trapping of ionized drug within acidic phagolysosomes. macrophages were infected with prrsv minutes after tul or vehicle treatment, or not infected (uninfected controls) and incubated for h at ˚c, % co to allow virus attachment and entry (time ; t = ). prrsv was diluted in serum-free dmem to reach a multiplicity of infection (m.o.i) ranging from . to depending on the experiment. culture media was replaced by pre-warmed imdm supplemented with % pig serum for all experimental groups. prrsv infection was performed for another to hours depending on the experiment. all functional assays contained the following experimental groups: untreated and uninfected control (control); tulathromycin-treated (tul); untreated and prrsv-infected (virus); tulathromycin-treated and prrsv-infected (tul+virus); lps-activated; pro-apoptotic positive control (staurosporine; μm) (sts); or pro-necrotic positive control ( . % triton-x) (trit-x) where appropriate. to avoid l cytokine-induced polarization of macrophages, all macrophages activation experiments were performed on monocytes that were grown in l supernatant free. the effects of tulathromycin and prrsv on macrophage differentiation and activation was determined via microscopic observations and cytokine quantification. mdms were treated with tulathromycin ( . or mg/ml) for hour and infected with prrsv (m.o.i. of . ) for , , or h at ˚c, % co . supernantants were collected and frozen at - ˚c until processed and macrophages were stained with diffquick (electron microscopy sciences, hatfield, pa, usa) and observed with a nikon eclipse t microscope to assess morphological changes. images were taken with a retiga x camera (q imaging, surrey, bc, canada) on a leica dmr fluorescent microscope (leica, wetzlar, germany) and analyzed using imagej software. individual macrophage morphology was assessed and classified as "resting" or "fibroblast-like" morphology. at least macrophages per group in independent experiments were assessed. supernatants were processed to measure interleukin- (cxcl- ) and interleukin- (il- ) concentrations using the porcine cxcl- quantikine enzyme-linked immunosorbent assay (elisa; p , r&d systems, minneapolis, mn, usa) and the il- quantikine elisa (p , r&d systems, minneapolis, mn, usa) respectively. samples were processed as per manufacturer's instructions. ros production by mdms following tulathromycin treatment and/or prrsv infection was monitored with the oxiselect intracellular ros assay kit (cell biolabs, san diego, ca, usa). experiments assessed ros production in resting cells, as well as in a group of cells induced by lipopolysaccharide (lps) to determine the effects of the various stimuli under basal conditions in these cells, as well as when they were activated. mdms were infected for , , , or h (m. o.i of . ) or uninfected (uninfected control). the same treatments were performed on macrophage stimulated with lipopolysaccharide ( μg/ml lps from e. coli o :b (sigma-aldrich, saint-louis, mo, usa). mdms were exposed to ', '-dichlorodihydrofluorescin diacetate (dcfh-da) a cell-permeable fluorogenic probe oxidized to highly fluorescent ', '-dichlorodihydrofluorescein (dcf) by ros. fluorescence intensity, proportional to ros levels within the cytosol was measured using a spectramax m e microplate reader (molecular devices, san jose, ca, usa) reading at nm (excitation) and nm (emission). phagocytic capacity of mdms was assessed using non-opsonized zymosan particles and opsonized latex beads. non-opsonized phagocytosis was monitored using fluorescently labelled saccharomyces cerevisiae zymosan a particles (texas red; sigma-aldrich, saint-louis, mo, usa). mdms seeded on labtek chamber slides or on coverslips at x cells/ml were infected for or hours (m.o.i of . ) or not infected (uninfected control). following infection, experimental groups were incubated with zymosan a particles diluted in control media to a final ratio of : (zymosan:cells) for hour. after exposure, extracellular zymosan a particles were washed away with warm pbs and the cells were fixed in ice-cold % acetone solution. actin was stained with the alexa fluor phalloidin antibody (thermo fisher scientific, waltham, ma, usa) and the nucleus was revealed with dapi (thermo fisher scientific, waltham, ma, usa). enumeration of intracellular zymosan was performed using a leica dmr fluorescent microscope. fc-mediated phagocytic index was measured using carboxylate-modified μm diameter latex or silica beads (kisker biotech, steinfurt, germany) covalently coated with bsa and human igg (sigma-aldrich, saint-louis, mo, usa). the beads were subsequently incubated with macrophages for minutes at a : (beads:cells) ratio. following phagocytosis, extracellular beads were washed away with warm pbs and the cells were stained with diffquick (electron microscopy sciences, hatfield, pa, usa) before microscopic observations. macrophages containing one or more zymosan particles or latex beads were considered as 'positive cells', the phagocytic index was calculated as the ratio of positive macrophages versus total macrophages. a minimum of cells per experimental group were counted from randomly selected fields. all pictures were taken using leica dmr fluorescent microscope with a retiga x (q imaging, surrey, bc, canada) and analyzed using imagej software. in order to prevent any counting bias slides labellings were covered with tape prior to microscopic observations. the pro-apoptotic effects of tulathromycin and prrsv were assessed using a cell death detection elisa kit (roche) according to the manufacturer's instructions as previously described [ , ] . absorbance was measured using a spectramax m e microplate reader (molecular devices, san jose, ca, usa) set at nm. mdms were incubated with tulathromycin ( . or mg/ml) for minutes and infected with prrsv (m.o.i. of . ) for , or h at ˚c, % co . similar experimental treatments were conducted with cells stimulated with lps ( μg/ml from e. coli o :b ; sigma-aldrich, saint-louis, mo, usa) to assess apoptosis in activated macrophages. for all experiments, cells incubated with imdm containing % hi-pig serum or staurosporine ( μm) were used as negative and positive controls respectively. annexin v staining (roche) was performed on the same experimental groups to further assess apoptotic cell death. staining was performed as per manufacturer's instructions and fluorescence was observed using a leica dmr fluorescent microscope equipped with a hcx pl fluotar x objective (aperture = . ). images were taken at , and hours post infection (p. i) with a retiga x (q imaging, surrey, bc, canada). importantly, experiments measuring cytokines and ros followed those in which we measured apoptosis. this allowed to adjust concentrations of tulathromycin and virus to levels at which apoptosis was not detected in the cells, and hence cell death would not be a factor in the cellular cytokine and ros responses. necrosis was assessed through the determination of lactate dehydrogenase (ldh) levels using a cytotoxicity detection kit (roche). mdms were treated with vehicle medium alone (control) or with tulathromycin ( mg/ml) for minutes. cells were then infected with prrsv for , , or h (m.o.i. . ) or supplemented with control medium, and % triton x in media was used as positive control. supernatants were collected and processed following manufacturer's instructions. a spectramax m e microplate reader (molecular devices, san jose, ca, usa) was used to measure ldh concentrations in each sample at nm. necrosis was expressed as the absorbance ratios of the experimental cell lysates versus absorbances from controls arbitrarily set at . ( %). all experimental groups were assessed in duplicates. to assess the potential anti-viral effects of tulathromycin, extracellular and intracellular viral particles counts were monitored with plaque titration assays. for extracellular counts, supernatants were harvested at , , and hours p.i and incubated with confluent marc- cells for h as described above. for intracellular counts, macrophages were washed twice with warm ( ˚c) phosphate buffer saline (pbs; sigma-aldrich, saint-louis, mo, usa) and lysed with double distilled water exposure and thorough mixing. cellular debris were spun down at , x g for minutes and supernatants were harvested and incubated with confluent marc- cells as described previously. prrsv staining was performed to further characterize potential antiviral effects. marc- cells were seeded in labtek chamber slides, grown to % confluence and infected with prrsv (m.o.i. . ) for h at ˚c, % co . prrsv foci numbers and size were revealed using the sr- f antibody (rti, llc, brooking, sd, usa). fluorescence ratio was calculated using imagej. five fields of view per well were counted per sample. expression levels of prrsv receptors in mdms in the presence and absence of tulathromycin were assessed by immunofluorescence. seven days old mdms cultivated with or without l conditioned medium were treated with hbss (control) or tulathromycin ( mg/ml for hours). the murine l fibroblast cell line, known to secrete macrophage-colony stimulating factor (m-csf) is widely used to induce macrophage differentiation from monocytes and prevent differentiation into monocyte-derived dendritic cells [ ] . prior to staining, l -grown cells were washed times in ice-cold pbs to remove all l media and then fixed in % paraformaldehyde in pbs for minutes. fixed cells were then washed times in cold pbs and stained for hour with a r-phycoerythrin (rpe) conjugated anti-cd antibody (bio-rad, hercules, ca, usa) and a fluorescein isothiocyanate (fitc) conjugated anti-cd antibody (bio-rad, hercules, ca, usa) at a dilution of to and to respectively. following staining cells were washed times in cold pbs and observed under leica dmr fluorescent microscopy. fluorescence ratios from randomly selected fields were calculated using the software imagej. images were taken with a retiga x camera. to prevent any bias, slide labelling was covered with tape prior to microscopic observations. all statistical analyses were made using prism software and data were expressed as means + standard error from mean (sem). all data sets were tested for normality. data with parametric distribution were compared using student's t-test, or one-way anova with tukey's multiple comparision anlaysis where appropriate. non-parametric data were compared with a kruskal-wallis test. for every assay, a minimum of separate, independent experiments were conducted with all experimental groups assayed in duplicates or triplicates. statistical significance was established at p < . . in order to determine whether blood monocytes and mdms are susceptible to prrsv we isolated blood monocytes from healthy pigs and cultured them for a period of days in medium supplemented with pig serum to mimic biological conditions. after plating, adherent monocytes exhibited a round shape morphology and were approximatively μm in diameter ( fig a) . by day , the cells displayed a larger, macrophage-like, morphology with characterisitic cytoplasmic vacuoles (fig c) . monocyte differentiation was also measured using non-specific esterase (nse) staining. by day more than % cells were esterase-positive cells. oneday-old monocytes were significantly less susceptible to prrsv compared to days old differentiated mdms (fig ) . prrsv viral particle numbers increased by a . log ( -fold increase) in mdms, and by a . log ( -fold increase) in blood monocytes, between and hours p.i. in both cell types, prrsv infection reached a plateau at h p.i. (fig ) . to optimize the macrophage differentiation protocol, mdms were also cultured in a l -conditioned medium. monocytes cultivated in l -conditioned medium showed the same morphology as those cultivated in medium devoid of l -factors. consistent with previous data, microscopic observation and nse staining showed that by day , more than % cells were macrophages. viral titers in monocytes incubated with l were significantly higher at hours p.i. compared to viral titers in monocytes cultivated without l supernatant (fig ) . numbers of prrsv infectious particles were significantly elevated in mdms cultivated with l -supernatants at all time points of the infection (except from the hours p.i. time point) versus mdms cultivated in medium supplemented with hi-pig serum alone (fig ) . in l -cultivated mdms, prrsv infection peaked at h p.i. and declined afterwards, whereas it continued to increase at hours in l -cultivated monocytes (fig ) . based on these observations, we chose to use l -cultivated mdms for functional experiments, unless stated otherwise. considering that l supernatants may contain cytokines other than m-csf (such as il- and il- ) that could influence macrophage polarization and confound our studies, we decided to grow our cells in medium containing only pig serum when assessing macrophage pro-inflammatory signaling. moreover, to limit the impact of tulathromycin and prrsv-induced apoptosis on macrophage numbers and functions, we treated our cells with tulathromycin at a concentration of . mg/ml and decreased prrsv m.o.i from . to . . at these concentrations, neither the virus nor the drugs significantly induced mdm apoptosis at the experimental time points (data not shown). if the cells were treated at a concentration of mg/ml, functional analysis were performed before hours of incubation. prrsv infection induced a sharp fibroblast-like morphological alteration and pseudopod projections in mdms (fig a) . in uninfected cells (control and tul), less than % of cells exhibited this change in morphology, while nearly % of the cells exhibited this phenotype upon prrsv infection (fig b) . tulathromycin pre-treatment significantly inhibited this morphological change in mdms (fig b) . since macrophage shape and function are correlated [ ] , we hypothesized that prrsv-induced morphological changes were associated with a change in macrophage activation. infection of mdms with prrsv caused a -fold increase of cxcl- secretion after hours (fig ) . prrsv-induced cxcl- secretion was significantly inhibited when mdms were pretreated with tulathromycin (fig ) . the positive control lps, also significantly increased the production of cxcl- (fig ) . we then measured the production of mitochondrial ros, a hallmark of pathogenic oxidative damage in inflamed tissues [ , ] . prrsv infection significantly increased intracellular ros (fig ) . tulathromycin treatment abolished prrsv and lps-induced intracellular ros production, however, it did notrestore ros levels to control values in cells exposed to both lps and prrsv (fig ) . interestingly, intracellular ros levels were significantly lower in mdms incubated with lps and prrsv compared to mdms stimulated only with lps (fig ) . as our results indicated that tul inhibited macrophage pro-inflammatory signaling, another set of experiment assessed the effects of the drug on il- , a cytokine with potent anti-inflammatory properties [ ] . resting mdms produced approximately pg/ml il- throughout the course of the experiments (fig ) . prrsv infected cells secreted significantly less il- compared to control cells at and hours p.i. (fig ) . il- levels did not significantly change versus controls when uninfected cells were treated with tulathromycin alone. however, immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages prrsv-induced il- inhibition was abolished when the cells were pre-treated with tulathromycin at and hours post infection (fig ) . tulathromycin ( mg/ml), as well as prrsv alone (m.o.i = . ), or the positive control staurosporine induced mdms apoptosis h post-infection (fig ) . combined pre-treatment with tulathromycin ( mg/ml; h) and prrsv (m.o.i = . ) for hours showed an additive effect to induce further mdms apoptosis versus single treatments (fig a) . to confirm these data, cells were stained with annexin v, a phospholipid-binding protein with high affinity for the early apotptic marker phosphatidylserine (ps) [ ] . at hours, both tulathromycin alone or prrsv alone induced significant levels of apoptosis compared to controls ( -fold increase vs. control) (fig b and c) . when cells were exposed to the combination of tulathromycin immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages and prrsv, levels of apotosis were almost double those measured in cells exposed to single treatments (fig b and c ). prrsv infection (m.o.i = . ) significantly increased the levels of ldh produced during necrosis and hours p.i. (fig ) . treatment with tulathromycin ( mg/ml) significantly reduced prrsv cell necrosis at hours (fig ) . this effect of tulathromycin could no longer be detected at hours. tulathromycin alone did not alter levels of necrosis (fig ) . triton-x (trit-x), used as a pro-necrotic positive control, induced necrosis in mdms (fig ) . another set of experiments assessed the effects of prrsv, and of tulathromycin, on the nonopsonized and opsonized phagocytic functions of mdms. prrsv infection significantly immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages inhibited both phagocytic functions of the cells (figs and ). prrsv-induced phagocytic inhibition was inhibited by tulathromycin (figs b and b ). tulathromycin treatment alone did not alter mdms phagocytosis versus controls (figs and ). during phagocytosis, macrophages can engulf multiple antigens at the same time [ , ] . additional experiments assessed mdms that engulfed less than particles (ie with basal phagocytic indices) versus cells that ingested more than particles (i.e. with elevated phagocytic indices). prrsv infection significantly reduced the number of cells with high phagocytic indeces, an effect that was abolished by tulathromycin (figs c and c) . tulathromycin inhibited the prrsv-induced reduction of basal and high phagocytic indices (figs and ). tulathromycin alone did not change either of the mdms phagocytic indices versus controls. the same results were obtained when the cells were infected for hours (fig ) . another set of experiments assessed whether the effects of tulathromycin described above were associated with direct antiviral properties of the antibiotic, in porcine mdms (fig a) or marc- cells (fig b) . upon incubation with prrsv, extracellular and intracellular viral particles were enumerated via plaque assay. tulathromycin did not change intracellular or extracellular viral titers in either of the cell models (fig a and b ). to verify these results, marc- cells were stained with fitc-conjugated anti-prrsv nucleocapsid antibody sr f antibody. size and numbers of viral foci were calculated in presence or absence of tulathromycin ( fig c) . again, tulathromycin pre-treatment did not alter viral titers compared to exposure to prrsv alone (fig c and d ). to further examine the effects of tulathromycin on prrsv infectivity, experiments measured viral receptor expression in mdms. to date, two major prrsv receptors have been extensively studied (cd and cd ) and it is not entirely clear which one of these two receptors is essential for prrsv infection [ ] [ ] [ ] . since l -conditioned medium increases viral titers, we hypothesized that it might be due to an increase in cell permissivity resulting from an increase in prrsv receptor expression. to test this hypothesis, we cultivated monocytes in medium containing pig serum alone or in l -conditionned medium for days and then treated them with tulathromycin. mdms differentiated in medium devoid of l -supernatant expressed both receptors. approximatively % of cells expressed cd and % of cells expressed cd . tulathromycin treatment did not significantly change the percentage of cd and cd positive cells (respectively % and % of positive cells) (fig a; upper panels; fig b) . mdms incubation in l -supernatant supplemented medium was sufficient to significantly increase the number of cd positive cells (more than % of mdms were cd positive versus less than % in pig serum supplemented medium alone). in addition, following l -supernantant exposure we were not able to detect any cd positive cells (fig a; lower panels; fig b) . tulathromcyin treatment following l -incubation did not have any significant effect on viral receptor expression in these experiments (fig a; lower panels; fig b) . prrs is one of the most devastating diseases of the porcine industry [ , ] . treatment options to control prrs outbreaks are limited and the efficacy of vaccines is thwarted by the antigenic variability of prrsv [ ] . disease severity is closely related to the ability of the virus to dysregulate macrophages functions and induce inflammation. therefore, we hypothesize that targeting either of these components may represent a critical element of novel therapeutic approaches. anti-inflammatory and immunomodulatory properties of macrolides have been well established [ - - ] . whether these effects may be beneficial in the context of viral diseases such as prrs remains obscure. the present study assessed the anti-viral and immunomodulating properties of tulathromycin (tul) in prrsv-infected porcine macrophages. the findings indicate that tul inhibits prrsv-induced inflammatory responses in porcine monocyte-derived macrophages and protects against the phagocytic impairment caused by the virus, in the absence of any direct anti-viral effects. the two most common cellular models are pams and marc- cells [ , ] . both have significant limitations. the isolation of pams requires bronchoalveolar lavages, and the function of these cells depends on the age and environment of the animal [ ] . moreover, shortly after the initiation of a respiratory infection, alveolar macrophages are replaced by monocytederived macrophages, which therefore represent a key cell population in host-prrsv immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages interactions. monkey marc- epithelial cells do not originate from pigs. therefore, the present experiments developed and used a simple porcine monocyte-derived macrophage model system to characterize the impact of tulathromycin on prrsv infection. previous in vitro studies have shown that the virus could infect blood monocyte-derived macrophages (mdms) [ ] . consistent with previous findings, monocytes were less susceptible to prrsv than differentiated monocyte-derived macrophages [ ] . the addition of l supernatant during macrophage differentiation significantly increased their susceptibility to the virus compared to macrophages cultivated in medium supplemented with pig serum alone. it has been well established that l supernatant is a source of m-csf and is used to induce macrophage differentiation [ ] . immunostaining of prrsv receptors showed that the addition of l immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages strongly upregulated cd ( % positive cells to % positive cells) but abolished cd expression. these results indicate that l supernatant modulate the expression of prrsv receptors, and that cd alone is sufficient for prrsv infection. this is consistent with recent observations showing that cd , but not cd , enabled non-permissive cells to become susceptible, and that increased cd correlates with increased susceptibility to prrsv [ , , ] . l supernatant is known to contain m-csf, but very little is known about other cytokines and chemokines present in this supernatant [ ] . considering that cd and cd expression can be induced by il- and ifn-γ respectively, and that il- treatment increases prrsv infectivity while ifn-γ decreases it [ , , ] , the role of these cytokine in the modulation of macrophage susceptibility to infection requires further investigation. the present findings demonstrate that mdms can readily be infected by prrsv, and hence represent a useful cellular model to study prrsv pathogenesis, as suggested recently [ ] . a hallmark of prrsv pathogenesis resides in its ability to alter macrophages survival and function, hence predisposing the host to secondary infections [ , ] . there is correlation between macrophage morphology and function, hence providing an easy way to monitor immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages changes in macrophage polarization [ ] . in this study we found that prrsv infection dramatically altered monocyte-derived macrophage morphology, inducing an elongated phenotype with numerous cytoplasmic pseudopods. recent findings indicate that these pseudopods promote intercellular junctions allowing prrsv to evade host immunity through direct intercellular spread [ ] . tulathromycin pre-treatment was sufficient to prevent the prrsvinduced pseudopod formation and morphological alterations in macrophages. whether tul may prevent intercellular junctions and thus hinder prrsv immune evasion requires more research. to test the hypothesis that change in macrophage morphology was associated with altered function, we measured the production of pro-and anti-inflammatory cytokines (cxcl- and il- respectively) as well as the production of mitochondrial ros. cxcl- is a potent neutrophil chemoattractant secreted by macrophages and other cell types, and is a critical mediator of neutrophil infiltration in inflamed tissues [ ] . the present findings demonstrate that prrsv is a potent inducer of cxcl- in monocyte-derived macrophages. virally induced cxcl- secretion was inhibited by tul. studies in live animals are warranted to assess whether these observations suggest that tul might attenuate prrsv-induced inflammation through cxcl- inhibition. mitochondrial ros production is a hallmark of cell stress and inflammation, and contributes to prrsv-induced tissue damage [ , ] . other reports showed that mitochondrial ros production was implicated in prrsv-induced apoptotic death of marc- cells [ ] . here we demonstrate that prrsv indeed induces ros production in porcine monocyte-derived macrophages, and that this production is inhibited when the cells are pre-treated with the antibiotic. tulathromycin was also able to restore ros levels to control in lps-stimulated cells but not in lps and prrsv exposed to both lps and prrsv. interestingly, in these conditions mdms showed a decrease in ros production compared to cells exposed only to lps. this suggest that prrsv may inhibit intracellular ros production of mdms during bacterial infections.another set of studies sought to determine whether tul inhibition of the viral-induced pro-inflammatory cxcl- coincided with an increase in antiinflammatory signaling. we found that the virus alone was able to inhibit il- secretion, and that tul blocked this effect. these data are in contrast with others from the scientific literature. indeed, it is generally accepted that prrsv induce il- production to increase its infectivity [ ] . in fact, il- activated cells are more permissive to prrsv than unstimulated m -polarized macrophages [ ] . more research is necessary to explain the mechanisms whereby prrsv regulates the production of il- . tulathromycin alone did not induce il- secretion suggesting that cxcl- and mitochondrial ros inhibition by tul was not dependent on il- production. taken together the present findings strongly support the hypothesis that tulathromycin may attenuate prrsv-induced inflammation by inhibiting production of pro-inflammatory cxcl- , and by preventing the suppression of anti-inflammatory il- . consistent with previous studies, we found that prrsv and tul induced macrophage apoptosis [ , , , ] . the present findings also illustrate that tul and prrsv haver additive pro-apoptotic effects. morevoer, the data indicate that prrsv leads to cell necrosis, an effect that was inhibited by tul. necrosis is known to exacerbate local inflammation, to induce the release of cytotoxic molecules, and to lead to extensive tissue damage, while cell apoptosis contributes to the resolution of inflammation [ , ] . more research in live prrsv-infected animals will help determine whether tul is able to promote the resolution of prrsv-induced pulmonary inflammation at least in part via such a mechanism, as well by shifting local cytokine release from pro-inflammatory to anti-inflammatory mediators. it is well established that prrsv infected pigs are often infected by secondary pathogens [ , ] . at present, the mechanisms resulting in the increase of secondary infections during prrsv infections remain incompletely understood. studies have shown that prrsv is directly able to impair macrophage phagocytosis, which in turn may represent a key element of the development of secondary infection [ , , ] . macrophage phagocytosis is triggered when phagocytic receptors including opsonic receptors (fcr) or pattern recognition receptors such as the mannose receptor, are activated [ , ] . using non-opsonized zymosan particles or igg-coated latex beads, the present findings demonstrate that prrsv significantly inhibits both phagocytic pathways. these results are consistent with previous reports showing decreased phagocytosis of latex beads, or live bacteria (streptococcus suis) upon prrsv infection [ , ] . recent findings suggest that prrsv- inhibits phagocytosis through its interaction with sialoadhesin (also referred to as cd ). however, in our model system, l cultivated mdms were negative for cd suggesting either that mechanisms for inhibition of phagocytosis are strain and/or genotype dependent, or that prrsv may inhibit phagocytosis through multiple pathways [ ] . another report recently demonstrated that the same nsvl- - strain as used here may impair phagosomal maturation and nadph oxidasemediated respiratory burst, both implicated in the antimicrobial properties of macrophages [ ] . tul blocked the prrsv-induced inhibition of non-opsonized and igg-mediated macrophage phagocytosis. these observations pave the way towards studies in vivo to assess whether this antibiotic might help control secondary infections during prrsv infections through this mechanisms in addition to its direct anti-microbial properties. the mechanisms whereby tul protects against prrsv-induced inhibition of phagocytosis require further elucidation. some macrolides such as tilmicosin and tylvalosin have been recently demonstrated to possess direct anti-viral effects against prrsv [ , ] , while others like erythromycin do not [ ] . in the experiments described herein, tul did not exhibit any direct anti-viral properties, nor did it significantly alter the expression of the two receptors used by the virus for entry, cd and cd . together, the data indicate that in porcine mdms, tul is able to block prrsvinduced pseudopod formation, necrosis, pro-inflammatory cxcl- and mitochondrial ros production, and inhibition of macrophage phagocytosis. in addition tul also synergized with prrsv to induce pro-resolution cell apoptosis and the production of anti-inflammaotry il- . the results also show that the protective modulation of macrophage structure, function, and behavior by tul occurs in the absence of a direct anti-viral effect. the present observations pave the way towards further studies with a prrsv- strain to determine whether the effects we observed in this study are conserved with the other prrsv genotype. studies in vivo will help determine whether and how these effects may translate into clinical benefits. assessment of the economic impact 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virus (prrsv) infection: a novel quinolone function which potentiates the antiviral cytokine response in marc- cells and pig macrophages the authors thank troy feener, barbara smith, and the staff at the veterinary sciences research station at the university of calgary for their help with animal handling. we also thank dr. constance finney and dr. edina szabo for their help with flow cytometry. buret. key: cord- -slgywe c authors: nunn, alistair v. w.; guy, geoffrey w.; brysch, wolfgang; botchway, stanley w.; frasch, wayne; calabrese, edward j.; bell, jimmy d. title: sars-cov- and mitochondrial health: implications of lifestyle and ageing date: - - journal: immun ageing doi: . /s - - -x sha: doc_id: cord_uid: slgywe c infection with sars-cov- displays increasing fatality with age and underlying co-morbidity, in particular, with markers of the metabolic syndrome and diabetes, which seems to be associated with a “cytokine storm” and an altered immune response. this suggests that a key contributory factor could be immunosenescence that is both age-related and lifestyle-induced. as the immune system itself is heavily reliant on mitochondrial function, then maintaining a healthy mitochondrial system may play a key role in resisting the virus, both directly, and indirectly by ensuring a good vaccine response. furthermore, as viruses in general, and quite possibly this new virus, have also evolved to modulate immunometabolism and thus mitochondrial function to ensure their replication, this could further stress cellular bioenergetics. unlike most sedentary modern humans, one of the natural hosts for the virus, the bat, has to “exercise” regularly to find food, which continually provides a powerful adaptive stimulus to maintain functional muscle and mitochondria. in effect the bat is exposed to regular hormetic stimuli, which could provide clues on how to resist this virus. in this paper we review the data that might support the idea that mitochondrial health, induced by a healthy lifestyle, could be a key factor in resisting the virus, and for those people who are perhaps not in optimal health, treatments that could support mitochondrial function might be pivotal to their long-term recovery. the risk of severe morbidity associated with infection by sars-cov- rises with age and underlying comorbidities, which indicate that up to . billion people, or % of the global population, could be at severe risk; the increased risk seems to be largely associated with an imbalanced and/or an excessive inflammatory response [ ] . one suggestion is that the severity could be related to a failure of inflammation resolution, leading to pulmonary hyper-inflammation and "cytokine storms" [ ] . with increasing age there is often an exaggerated innate immune response to respiratory infections [ ] and rising inflammatory tone [ , ] . overall, it seems that susceptibility to the virus is related to an age-related loss of adaptive immunity combined with an increased innate immune response [ ] . this "inflammaging" seems to be associated with t-cell immunosenescence and thymic atrophy; critically, exercise seems to be protective [ ] . the protective effect of exercise is informative, as the pathological severity of sars-cov- infection seems to be associated with many obesity-related co-morbidities, such as diabetes [ ] [ ] [ ] , in contrast, physical fitness is emerging as a preventative strategy against the virus [ ] . this suggests that as well as age, lifestyle could be important in determining susceptibility to the virus. we have suggested that a modern sedentary lifestyle has effectively removed exogenous hormetic stimuli, such as physical activity, which is leading to an accelerated ageing phenotype [ ] . in short, a modern lifestyle could be accelerating the process of "inflammaging": obesity is associated with a pro-inflammatory state, increased inflammatory macrophages and altered t-cell homeostasis [ ] . in contrast, exercise is largely anti-inflammatory, which is thought to explain its many benefits [ , ] . a key player in this adaptation is the mitochondrion, as mitochondrial stress enhances mitochondrial function not only in muscle, but in multiple other organs with myokines playing a key role [ , ] . for example irisin, which protects mitochondria, can protect against ischaemia/reperfusion (ir) injury in the lung [ ] . irisin has also been found to favourably alter genes in adipocytes that are affected by the sars-cov- [ ] and to modulate macrophage reactive oxygen species (ros), displaying anti-oxidant and anti-inflammatory properties [ ] . critically, exercise can enhance mitochondrial function and capacity in peripheral blood mononuclear cells (pbmcs) [ ] . as mitochondria are pivotal in the immune response and many viruses in turn modulate mitochondria [ , ] , it is possible that altered mitochondrial function may explain at least some of the variance in responses to sars-cov- . as most cells in the body contain mitochondria, including immune cells, this would be expected and is now embraced by the concept of "immunometabolism". this is perhaps most clearly seen in the clinical phenotype of subjects with inherited mitochondrial defects who often display immunodeficiency and a much higher rate of infectionshighlighting the reliance of the immune system on mitochondria [ , ] . although this is relevant to resistance to the virus, it is also perhaps relevant to the efficacy of vaccines; thacker and colleagues, using gene expression assays of pbmcs, have shown that there is an age-related decrease in response to influenza vaccines, which appears to be linked to decreased mitochondrial function [ ] . in short, compromised mitochondrial function, either due to genetic factors, extreme age, or lifestyle, could have a bearing on both resistance to the virus and the ability to mount an effective response to a vaccine. it therefore seems that maintaining "mitochondrial health" is vital, which probably correlates with an effective mitochondrial reserve induced by factors like physical activity, such that when the system is "stressed" (e.g., by a virus), it can cope. although the virus may only infect certain cells, the immune response is global and dependent on mitochondrial function in multiple tissues and organs. what is clear is that severity is associated with the hyperinflammation syndrome and involves dysregulation of many different cell types [ ] . this is to be expected, as throughout evolution, viruses have evolved to manipulate the immune system to hide from it, and can invoke immunosuppression, which in itself can become pathological, for instance, by modulating t-cells [ , ] . it now seems that the spike protein of sars-cov- can bind to t-cell receptors (tcrs), acting as a superantigen and causing excessive activation of the adaptive immune systempotentially resulting in the hyperinflammatory syndrome [ ] . this is perhaps relevant as persistent antigenic stimulation can lead to t-cell exhaustion, which is associated with decreased oxidative phosphorylation and loss of mitochondrial function despite enhanced glycolysisbut can be reversed using anti-oxidants [ ] . data is now showing that covid- patients do have populations of t-cells displaying mitochondrial dysfunction, as well as altered mitochondrial markers in monocyteshinting that immune-metabolic phenotyping could be used to understand disease pathogenesis and possible treatments; this could include targeting mitochondria [ ] . in short, the immune system itself could well be a target for this virus. apart from the virus targeting the tcr as a super antigen, there is evidence that other than it binding to the angiotensin converting enzyme (ace) as its main receptor, it may also bind receptors on immune cells, such as cd and cd [ ] , or neuropilin- (nrp- ) [ , ] . we have structured this paper to first review the now established data on general mitochondrial function and health in relation to "inflammaging", followed by the evidence suggesting that the sars-cov- virus itself manipulates mitochondrial function and what we might learn from batswhich are thought to be its natural host. from this we propose that a poor lifestyle accelerates "inflammaging" which is associated with mitochondrial ill-health, and in some populations this predisposes them to a worse outcome. in the second part of the paper we discuss the implication of this idea in relation to current and suggested drug-based treatments and vaccine efficacy, the "long-covid" syndrome, as well as how environmental factors may make some people more vulnerable. understanding these concepts may help inform clinical strategy. circulating extracellular vesicles (evs) derived from immune cells seem to have emerged as a means of studying immunosenescence. in particular, they show an agerelated decline in mitochondrial functionwhich could be related to dysfunctional mitophagy [ ] . in fact, mice engineered to have dysfunctional t-cell mitochondria display accelerated senescence and "inflammaging", highlighting the point that t-cells can determine organismal fitness and lifespan [ ] . this does support data indicating the importance of a healthy t-cell response in defending against the virus [ , ] . the underlying aetiology for "inflammaging" has long thought to be associated with mitochondrial dysfunction as suggested by nick lane in in his "double agent" theory [ ] , and is now receiving renewed interest, for instance, in how decreasing mitochondrial function can reduce t-cell function and enhance immune senescence, as mitochondria are pivotal in metabolic reprogramming towards the warburg effect [ ] . indeed, as mitochondrial dysfunction can lead to "inflammaging", the observed increase in older people of mitokines could be an attempt by the system to restore homeostasis as many are anti-inflammatory. unfortunately, for many, this response doesn't fully compensate [ ] . this is why "exogenous" factors, such as physical activity or calorie restriction seem to be required to optimise function; these were normal factors during evolution, but are not in our modern sedentary and obesogenic environment. one aspect of ageing is a failure to remove damaged components, for instance, dysfunctional mitochondria via mitophagy, which could lead to immune dysfunction [ ] . it has been suggested that imbalances in mitochondrial mass could be responsible for ageing-related t-cell subset dysfunction [ ] , which would suggest a failure of mitophagy. indeed, activation of mitophagy/autophagy is thought to be a pivotal mechanism in slowing ageing and inhibiting inflammation during calorie restriction (cr) [ ] : cr/intermittent fasting has been suggested as a defence against the sars-cov- as it is antiinflammatory [ ] . in contrast, a modern sedentary lifestyle is also contributing to "inflammaging", which acts as a common mechanism linking sarcopenia, obesity, cardiomyopathy and dysbiosis, with over-activation of nod-like receptor pyrin family domain containing (nlrp ) inflammasomes and mitochondrial dysfunction playing key roles [ ] . overall, this all seems to support a close link between immunosenescence, inflammaging and failing mitochondrial function. does sars-cov- modulate mitochondrial function, either indirectly or directly, and if so, in what cells? the above suggests that there is a close link between mitochondrial dysfunction and immunosenesence, which could lead to an increased chance of an imbalanced immune response to sars-cov- . this could take the form of both an inability to clear it, but also an exaggerated pro-inflammatory response and a "cytokine storm". however there could also be another factor, and that is that the virus is modulating mitochondrial function to help it replicate. one clue to this possibility is that many viruses do appear to manipulate bioenergetics towards aerobic glycolysis (the "warburg effect"); this is a highly energydependent process to help generate substrates to build new virus particles [ ] . aerobic glycolysis does require healthy mitochondria, and is a normal process in multiple cell types, including immune cells [ ] . perhaps tellingly, data suggest that successful clonal expansion of vaccine-elicited t-cells is heavily depending on mitochondrial function [ ] . what this suggests is that any cell forced to produce new viruses, if its mitochondria are not functioning optimally, could rapidly become energy deficient and be more likely to die, and depending on its type and location, could either enhance inflammation and/or compromise the immune response. what are the sars-cov- receptors and where are they found? the direct impact of the virus will depend on which cells it infects. to date, most evidence points towards ace being the primary receptor for this virus. early data suggested ace is predominantly expressed in pulmonary alveolar type progenitor (at ) and respiratory epithelial cells, but is also expressed in myocardial, illium and oesophagus, as well as some kidney cellswith little expression in immune cells [ ] . elevated ace expression has also been found in the olfactory neuroepithelium, potentially explaining the anosmia that some patients have suffered [ ] . more recent data has suggested that ace may be primarily expressed in bronchial transient secretory cells [ ] . perhaps of relevance to the increased risk associated with obesity is that high ace expression has been found in both visceral and subcutaneous adipose tissue; this is important as adipose tissue in obesity is well known to secrete higher levels of angiotensin , an inflammatory component of the renin-angiotensin aldosterone system (raas), which is key in driving many of the pathological complications associated with this condition [ ] . critically, obesity also seems to be associated with increased expression of ace in the lung, and enhanced inflammatory markers and dysregulated lipogenesis; viruses are well known to hijack lipid metabolism as part of their life cycle [ ] . although ace is not highly expressed in immune cells, it is possible that other proteins expressed on immune cells could be acting as sars-cov- receptors, such as cd (also known as dipeptidyl peptidase , dpp ) or cd (also called basigin). cd can be activated by cyclophilins, which are inhibited by cyclosporine a. critically, the expression of these potential receptors changes with age, as well as with co-morbid conditions, such as obesity and hypertension [ ] . thus both cd and cyclophilin a have been suggested as potential targets for treating the virus. for example, cyclosporine is very effective against corona viruses; however, its immunosuppressive actions would limit its usefulness [ ] . cd and ace expression is often increased in lung disease, resulting in excessive activation of the raas and enhancing damage, which could, in part, explain the origins of the cytokine storm. it has been suggested that melatonin, a potent natural anti-oxidant, could suppress the cd inflammatory pathway and help in treating covid- patients [ ] . in silico binding studies do seem to support the possibility that the virus does indeed use cd as a receptor, and could, potentially, explain why lymphopenia is associated with severity of covid- and a loss of t-cell subsets [ ] . data is indicating that this virus may also bind to neuropilin- (nrp- ); this protein is expressed on many cells, including those in the central nervous and immune systems, and is also a receptor for vascular endothelial growth factor a (vegf-a) [ , , ] . apart from suggesting it can thus potentially infect the central nervous system (cns), it also appears that sars-cov- can induce analgesiawhich could aid in increased disease transmission in asymptomatic individuals [ ] . nrp- is also a focus for immunotherapy treatments in oncology, as it is expressed on subsets of regulatory t-cells [ ] . there is also data indicating it is expressed in the cardiovascular system; if its expression is reduced, it results in cardiac mitochondrial dysfunction as it controls the master mitochondrial regulator, peroxisomal proliferator-activated receptor γ coactivator α (pgc α), as well as peroxisomal proliferating activating receptor γ (pparγ) [ ] . this data does suggest that the virus not only modulates essential components of the raas affecting inflammatory balance via ace , but if it is also modulating the t-cell response directly, for instance, via cd , or the tcr, or even, nrp- . in sars-cov- the open reading frame- b (orf- b) encodes for a protein that locates to the mitochondrion. here it induces fusion by triggering degradation of dynamin-like protein (drp- ), while inhibiting mitochondrial anti-viral signalling proteins (mavs). this is thought to underlie its ability to suppress the anti-viral interferon response. it can also induce autophagy and activate nf-κb [ ] . mavs are small proteins that on detection of double stranded rna (dsrna) oligomerise on mitochondria to form a signalling platform and initiate interferon signalling, as well as cell death [ , ] . it also seems that mavs can act as adaptor proteins for nlrp , forming a complex with mitochondria, although the inflammasome can also be activated in a way that doesn't induce an interferon response, but can induce the interleukin beta (il-β) response [ ] . with regards sars-cov- , protein interaction mapping shows that it shares a great deal of homology with sars-cov- , but significantly, several of its proteins are also predicted to directly interact with mitochondria, such as non-structural proteins (nsps) and , and orf c, as well as components of the interferon and nf-κb pathways [ ] . this, because of the well described role of viruses in manipulating mitochondrial function, has led to other groups suggesting that indeed, mitochondrial "hijacking" by sars-cov- could be a key factor in the pathogenesis of this virus [ ] . many viruses also use viroporin proteins that can oligomerise to help viral entry and release, as well as control intracellular signalling ions, such as calcium or potassium. they can also, via direct protein interaction, manipulate signalling pathways. the host cell detects these as changes in ions levels and ros, and via, for instance, the nlrp inflammasome, activates cellular defence [ ] . sars-cov- has at least three viroporins, two of which are essential for replication and virulence [ ] ; the e protein, in particular, not only seems to trigger p mapk activity, but also seems to modulate calcium flux by acting as a permeable ion channel in endoplasmic reticulum-golgi intermediate compartment (ergic)/golgi membranes, activating the inflammasome [ ] . sars-cov- seems to have a similar e viroporin that induces ionic imbalance [ ] . from the calcium and ros signalling perspective this is particularly important, as mitochondria are not only pivotal in calcium buffering and signalling, but are also controlled by calcium [ ] . data suggest that many viruses form viral "factories", which are constructed from host cell membranes, and are often tightly coupled to mitochondria to provide precursors and energythis includes the coranoviridae [ , ] . emerging data is now suggesting that t-cell mediated immunity may be playing a powerful role in protecting against the virus, as many asymptomatic people, or those who have only had mild symptoms, show low levels of anti-sars-cov- antibodies but a strong t-cell mediated response against the virus. in contrast, more severe disease is associated with more rapid seroconversion and the presence of inflammatory markers, such as creactive peptide (crp) [ , ] . in fact, it now appears that the severity of infection positively correlates with a decreased type interferon (ifn ) response, but an exaggerated inflammatory response, characterised by high levels of interleukin (il- ) and tumour necrosis factor alpha (tnfα)possibly related to excessive activity of nuclear factor kappa b (nf-κb). this latter finding could be related to an auto-inflammatory loop in the lungs [ ] . it does seem that in some people that the transcriptional response to sars-cov- is imbalanced, with a less than optimal interferon-i and -iii response, but an exaggerated chemokine one; this may represent an evolved manipulation of the immune system by the virus that worsens the outcomes for older patients with comorbidities as they cannot clear the virus properly [ ] . data from autopsies of deceased covid- patients show that tissue inflammation and organ dysfunction do not map to the cellular distribution of the virus, hinting at tissue-specific tolerance. in fact, severe inflammatory changes seem to be largely restricted to the lungs and the reticulo-endothelial system. this suggested that covid- related deaths were due to immunemediated, rather than pathogen-mediated organ inflammation and injury [ ] . it may therefore be relevant that ifn can also have some anti-inflammatory actions, modulating for instance, nlrp / inflammasomes and inhibiting interleukin- (il- ) production [ ] . type interferons are key in modulating t-cell responses and resistance to viruses [ , ] . it had been suggested that as the virus uses ace as a receptor on the cell surface it could trigger activation of the renin-angiotensin-aldosterone system (raas), which in turn, leads to hyperactivation of the nlrp inflammasome and pyroptosis, a form of cell death that results in inflammatory amplification [ ] . data does now seem to support this and has been shown in various types of human stem cellswhich could potentially affect tissue regeneration [ ] . ace cleaves angiotensin ii to generate angiotensin ( - ), which is largely anti-inflammatory and protective [ ] . critically, mitochondria have a functional angiotensin system [ ] , and ace seems to be mitochondrially protective [ ] . potentially of interest here is that a product of ace , angiotensin-( - ), seems to inhibit mitochondrial fission in the heart, enhancing mitochondrial fusion and calcium buffering and protecting against cardiac hypertrophy [ ] . it is thus possible, by binding to ace , the virus may suppress a counterbalancing anti-inflammatory pathway that affects mitochondrial function. so why would sars-cov- do this? one possible explanation is that the virus affects the most prevalent immune cells in the lungs, monocytes/macrophages, inducing them to shift metabolically to aerobic glycolysis, which favours viral growth. the infection, in the presence of oxygen, seems to achieve this by triggering mitochondrial reactive oxygen species (ros) production, stabilising the hypoxia-inducible factor- α (hif- α), which in monocytes, consequently inhibits t-cell responses and lung epithelial cell death. it seems that high glucose levels induce viral replication [ ] . furthermore, the inflammasome can also modulate glycolysis; in macrophages, this may be a key process in metabolic reprogramming [ ] . critically, inflammasome activation can be inhibited by nuclear factor, erythroid -like (nfe l /nrf ), which is pivotal in enhancing antioxidant defences and suppressing inflammation [ ] ; it therefore counterbalances nf-κb, which is also redox activated, but central to the immune response [ ] . another key factor is that the sars-cov- genome encodes proteins that can target the nf-κb pathway [ ] . sars-cov- therefore seems to induce a warburg shift (aerobic glycolysis), which is a tactic that many other viruses, and cancer cells, use [ ] . it is thus of relevance that that the metabolic reprogramming induced by sars-cov- can be suppressed by melatonin [ ] , which is a powerful antioxidant that protects mitochondria [ ] . in fact sars-cov- also seems to induce activation of pathways like p mitogen activated protein kinase (mapk), which results in cell cycle arrest, inhibition of apoptosis, and results in a feed-forward inflammatory loop [ ] ; the systems it targets therefore do seem have much in common with those that are altered in cancer [ ] . critically, mapks also modulate mitochondrial function, for instance, interacting with the voltage dependent anion channel (vdac ) [ ] . this seems to add up to the virus manipulating several pathways to invoke aerobic glycolysis, which must involve mitochondrial function. diabetes is also associated with activation of p mapk via ros generated by glucose induced mitochondrial dysfunction that can be offset by targeted mitochondrial antioxidants [ , ] . not only is diabetes a risk factor for a worse outcome when infected with sars-cov- , but the virus itself may induce a worsening of the condition [ ] [ ] [ ] . indeed, it now seems that fasting blood glucose is a predictor of mortality for covid- patients [ ] . overall, prediabetes and/or type diabetes (t d) itself is embraced by the concept of the metabolic syndrome in which insulin resistance, mitochondrial dysfunction and inflammation are all components [ ] . metformin, which modulates mitochondrial function, is a key treatment for t d [ ] and has shown some benefit in covid- patients [ , ] . in contrast, evidence indicates that the inflammatory effect of the western diet may induce activation of the nlrp inflammasome [ ] . in light of the emerging data, this could only worsen the potential for an exaggerated inflammatory response. it is therefore likely that sars-cov- does modulate mitochondrial function. so it could be surmised, for instance, that this virus could ensure close tethering of mitochondria, and via calcium flux, stimulate their function. clearly, if this process was too overwhelming, or the mitochondria were already functionally compromised, this would rapidly lead to mitochondrial stress. with regards this, singh and colleagues have highlighted an interesting link with viruses and the production of mitochondrially-derived vesicles (mdvs), which are normally part of a system to remove damaged components from the mitochondrion [ ] . if, like sars-cov- , this new virus also does this, and also induces mitochondrial fusion, it hints at an interesting ability to prevent apoptosis, as well as mitophagy, but stimulate a mechanism to move virus particles around. if it is also inhibiting mav activity, then the mitochondrion might not initiate interferon signalling, but might still continue, potentially by producing higher than normal levels of ros, to stimulate inflammasome activity and metabolic reprogramming towards glycolysis. in effect, the virus repurposes the normal inflammatory metabolic reprogramming towards aerobic glycolysis, which involves modulation of mitochondrial function, but manages to suppress the normal anti-viral interferon response. in many tissues, the system may manage to stay in balance and not cause an overt over activation of the immune system, but in the lungs, it seems that in some people, this balance is lost. figure summarises this. as indicated, if the virus is modulating mitochondrial function in a variety of cell types, either directly, or indirectly, then the more robust the mitochondrial system, fig. viruses like sar-cov- manipulate cellular metabolism leading to the potential for a feed-forward inflammatory loop. viruses have evolved to usurp their host's cellular machinery to make more viruses. one common mechanism is to suppress apoptosis and manipulate the immune system to inhibit specific anti-viral programmes, which usually means interferons, while stimulating a shift towards aerobic glycolysis to provide precursors to build new viruses. however, this latter ability repurposes pathways that are often involved in generalised immunity that both increase the production of pro-inflammatory mediators, while metabolically reprogramming immune cells. in the case of sars-cov- this may well result in a feed-forward pro-inflammatory loop in the lungs, which seems to be driven by monocytes/macrophages switching to aerobic glycolysis and is driven my mitochondrial ros and stabilisation of hif- α; in turn, this metabolic shift suppresses t-cells and the interferon response [ ] . this process is accentuated as the virus may well stimulate inflammasome activation [ ] , while if it is similar sars-cov- , it could also suppress mavs formation and activate nf-kb [ ] ; protein interaction mapping does suggest this is the case [ ] . as it is likely that inflammasome activation can also invoke glycolysis [ ] , then the evolutionary rationale seems sound. of particular importance here is also the balance between nf-kb and nrf , which more or less seem to counter-balance each other, as nrf is pivotal in suppressing excessive oxidative stress [ ] . for more detailed reviews of the role of mitochondria in the immune response see [ , ] the greater the chance of the system being able to resist the virus. in general, hormetic factors, such as exercise, seem to be necessary to maintain mitochondrial health throughout the body; this phenotype is associated with a more balanced immune response and minimisation of "inflammaging". in this section we review why this is, and look at why one of the natural hosts of the virus, the bat, may be able to resist. a robust mitochondrial system and effective immune system may rely on hormesis a key component of effective immunity is now thought to be a healthy mitochondrial system [ ] , while an underlying unifying element to both the ageing process and conditions associated with a poor lifestyle is a degradation in overall mitochondrial function/reserve and a rise in oxidative stress and inflammation [ , ] . an important factor in the maintenance of mitochondrial function is hormesis where low levels of stress induce an over-compensatory response that induces positive adaptations, enabling an organism to better tolerate the stressor next time they encounter it. for example, an effective hormetic response can be induced by sub-lethal doses of physical activity, calorie restriction and many plant polyphenols [ ] , with mitochondrial stress being a key trigger [ ] . this results in an enhanced respiratory reserve and anti-oxidant capacity, and a greater ability to manage the atp/ros ratio when placed under stress [ ] . certainly, small, long-lived species like bats and sparrows, when compared to comparatively much shorter lived species like mice, do demonstrate lower levels of mitochondrial hydrogen peroxide release [ ] . given that mitochondrial dysfunction is strongly correlated to immune dysfunction and chronic inflammation [ ] , then inflammation resolution is probably going to be best achieved by ensuring healthy mitochondrial function as it ensures that ros release does not get out of control. the concept of hormesis suggests that it is important to constantly stimulate the renewal and maintenance of a large population of healthy mitochondria. it may therefore be possible to learn something from one of the natural hosts of sars-cov- , bats [ ] . bats are the only true flying mammal and are exceptionally long-lived for their size. this could be because the evolution of flight has required a whole host of adaptations, including maintaining a large pool of mitochondria that produce very little ros while maintaining a high atp output. this appears to have gone hand-in-hand with changes in the immune system to prevent excessive inflammatory activation by stressed mitochondria, for instance, by dampening nlrp inflammasome activity. the net result is that many bats can tolerate high levels of viruses, like the coronaviridae family [ ] [ ] [ ] [ ] and do show a reduced antibody and inflammatory response, hinting they are using another part of their immune system to control the virus [ ] . the inflammasome may thus be important, as its activation can lead to pyroptosis, an inflammatory form of apoptosis, and can be triggered by excessive mitochondrial stress [ ] . it may well be an essential component in "inflammaging" [ ] . there is some evidence that at least in some species of bat, mitochondrial health, despite bursts of oxidative stress, is maintained by stringent mitochondrial quality control mechanisms, like mitophagy [ ] . mitophagy is in fact a negative regulator of nlrp inflammasome activity, so although mitochondrial damage can activate the inflammasome, it can also activate counter-balancing mitophagy to prevent excessive inflammation [ ] . in short, it seems that powered flight has required the co-evolution of both mitochondria that tightly control ros, and a co-adapted immune system. critically, there is evidence that sars-cov- inhibits autophagy [ ] , suggesting it might also inhibit mitophagy. if this virus does indeed induce mitochondrial fusion, as sars-cov- may do [ ] , then this would fit, as mitochondrial fusion can inhibit mitophagy, and can inhibit cell death and ensure energy production, although prolonged fusion can also initiate cell death in some circumstances [ ] . this latter point suggests another innate anti-viral mechanism. overall, modulation of the inflammasome could be one element in how the virus could result in an "inflammaging" phenotype. the effects of hormesis, certainly for humans, are perhaps most clearly seen in response to exercise training, in particular, aerobic training, where both mitochondrial capacity and function is increased in young and old [ , ] . this is matched by increased survival and healthier ageing in cohorts who undertake plenty of physical activity [ ] . active muscle is generally inflammatory, but commensurately induces counterbalancing powerful anti-inflammatory and anti-oxidant mechanisms throughout the body. exercise thus appears to show a biphasic dose response and the evidence is building that as long as it is not done excessively, in particular, allowing time for recovery, it is highly beneficial: over time the adaptive over-compensation includes an improved anti-inflammatory and anti-oxidant feedback ( ) ( ) ( ) ( ) [ ] . muscle has now been shown to have other functions, like harbouring and supplying anti-viral stem t-cells, hence, antagonising t-cell exhaustion and protecting proliferative potential during inflammation [ ] . in contrast white adipose tissue plays a key role in adaptive immunity, and in excess, contributes to the altered immune function and chronic inflammation often associated with obesity [ ] . in particular, excessive visceral adipose tissue (vat), seems to play a pivotal role in obesity-related pathogenesis; critically, its volume is decreased by exercise [ ] . furthermore, not only does type interferon unlock dormant adipocyte inflammatory potential [ ] , but exercise reduces adipose expression of nlrp [ ] . it therefore seems that adipose tissue and muscle play a yin-yang role in the immune response, whose set point will thus be determined by an individual's fitness and calorie balance, and overall mitochondrial capacity and health, and thus, reserve. in short, mitochondrial reserve, and thus spare respiratory capacity, is pivotal in enhancing the "healthspan", and is greatly improved by exercise [ ] . the key here is that stress can be signalled from mitochondria in any tissue to the rest of the body by way of "mitokines"; muscle activity is a prime inducer of mitochondrial stress [ ] . it therefore seems that control of inflammation is associated with tight control of mitochondrial ros, which is itself dependent on "mitohormesis" by factors such as exercise, plant compounds in the diet, and calorie restriction [ , ] . the basis for this is that life is based on redox and compartmentalised production of ros as part of a signalling system [ , ] . this has led to redox theories of disease and ageing, focussing on the mitochondrion [ ] and their role in generating an agerelated rise in inflammatory tone [ ] , which supports the pivotal role of mitochondria in the immune system [ ] and in resistance to infections including viruses [ ] . in support of this, there is increasing evidence that mitochondria can also act as net sinks of ros and this is linked to lifespan. for instance mitochondria from the long lived naked mole rat (nmr) produce less ros than comparable shorter lived animals [ , ] . furthermore the mitochondria in nmr, and bats, also appear to be able to maintain a depolarisation of the inner membrane for much longer during their life cycle, which is a key mechanism to reduce ros production during ageing [ ] . a key idea that relates to this is the redox-optimised ros balance (r-orb) hypothesis, which stipulates that mitochondrial emission of ros will reach a nadir when respiratory rate reaches a maximumin effect, mitochondria will maximise atp production and minimise ros as they evolved to work at an intermediate redox state [ , ] . thus having a good mitochondrial reserve might suggest that this nadir can be maintained when the system is put under stress. an essential component of mitochondrial control is uncoupling. this is a process whereby the proton gradient in the mitochondrion is uncoupled from atp production, and it initially seemed to be a key process to reduce ros production, as well as generating heat. it was therefore thought to act as a very good safety valve for mitochondria and play a fundamental role in survival and prevention of oxidative damage. in fact, % or more of the energy captured by electron transport is dissipated. however, uncoupling can also be associated with an increase in ros, hence, it is a key component of redox signallingand has led to updated versions of the "uncoupling to survive" hypothesis. it may therefore play a key role in mitohormesis, resulting not just in cell autonomous adaptations, but also systemic adaptation from signals, for instance, sent out from stressed skeletal muscle via mitokines. uncoupling also controls calcium signalling. it now seems that mild uncoupling can, indeed, lead to increased longevity [ ] . it is thus perhaps relevant that a mitochondrial uncoupling protein, ucp , can negatively control the inflammasome [ ] , and in general, seems to suppress immune activity [ ] . uncoupling thus plays an important role in mitochondrial efficiency, which can either be defined as the respiratory control ratio (rcr -ratio of mitochondrial respiration supporting atp synthesis to that required to offset the proton leak) or the atp/oxygen ratio (the amount of atp generated per unit of oxygen consumed) this can lead to some confusion, as it can lead to opposite conclusions about efficiency. however, whichever metric is used, it does describe the capacity to convert resources into atp, and in effect, the coupling efficiency [ ] . in fact a study has shown that skeletal muscle mitochondria in obese, sedentary and insulin resistance women somewhat paradoxically show reduced mitochondrial coupling, but a higher production of mitochondrial hydrogen peroxide. in effect, despite a degree of uncoupling, their mitochondria were showing signs of oxidative stress; this might have been due to nutrient overload. however, an exercise training programme corrected this, and was correlated with an improvement of mitochondrial function, in particular, an enhanced ability to undertake beta oxidation of fats and restoration of metabolic flexibility, the ability to switch between carbohydrate and fat as energy sources, and better insulin sensitivity [ ] . the apparent increase in uncoupling could be part of a homeostatic response to reduce excessive ros production, as ucps can be activated by oxidative stress [ ] . this would further support evidence that exercise induces an adaptive response that enabled the mitochondrial system to cope better. finally, it is perhaps worth emphasising the link between mitochondrial reserve and ability to control oxidative stress. mitochondria can generate ros and are closely linked to nrf , which is a master transcription factor controlling antioxidant responses [ ] . this suggests that exercise will not only induce greater mitochondrial reserve, but greater anti-oxidant capacityand perhaps, a greater reserve ability to uncouple to manage oxidative stress. as previously indicated, like the original sars virus, this new virus also seems to induce worse outcomes in patients who are older, have hypertension and cardiovascular disease, and induces a phenotype characterised by raised inflammatory and coagulation markers, multiorgan failure, as well as neurological complications and myocardial injury [ ] . in short, most things we identify with the ageing process and the metabolic syndrome, both of which are associated with declining mitochondrial function [ , ] . it thus pertinent that the rate of ageing can be modified by lifestyle and disease, and that epigenetics are making it possible to determine, with some degree of accuracy, the biological age and compare it with the chronological age through dna methylation (dnamage), and predict the likelihood of future mortality [ , ] . although mitochondria obviously play a role in this, and reduced mitochondrial dna copy number (mtdnacn) does appear to be a proxy for mitochondrial buffering capacity, and is negatively correlated with dnamage, the precise relationship with biological age is still unclear. for instance, evidence does indicate a clear role for mitochondria in ageing-related disease and mortality, but not necessarily chronological age [ ] . however, data does suggest that inducing mitochondrial dysfunction alone in t-cells can induce premature senescence, driving "inflammaging" and a tendency towards a cytokine storm [ ] . one well known concept in ageing is the idea of declining organ reserve, which at the molecular level, is related to a loss of excess metabolic capacityin particular, bioenergetics and mtdna, as well excess telomere capacity [ ] . in this respect, it could be argued that the immune system could be viewed as an organ, and is also subject to declining reserve. as the immune system ages there is a subclinical accumulation of pro-inflammatory factors, as well as decreased numbers of circulating respiring mitochondria found in extracellular vesicles (evs), which are derived from immune cells [ ] . coupled to this, there is also evidence that with increasing age the monocyte inflammasome-mediated inflammatory response is altered. for instance, this response to influenza a is retained but the anti-viral interferon response declines [ ] . furthermore, ageing is also associated with a gradual loss of anti-oxidant capability that is associated with a decrease in the t helper (th ) anti-viral response, which might underlie some of the anti-viral activity of glutathione and other anti-oxidants [ , ] . this is certainly commensurate with reduced immune system reserve. however, there is still a lot that is not understood about ageing, which is why it has led some authors to categorise it using several separate hallmarks, with mitochondrial function only being one of several integrated systems as the precise cause is still not fully understood [ ] . however, many authors continue to focus on the mitochondrionmainly because it represents an ancient nexus that arose from the endosymbiotic event between a prokaryote and archaean that gave rise to eukaryotes, and understanding this does provide insight into the immune system and inflammation, and the ageing process [ , ] . some have suggested that ageing is actually related to the loss of mitochondrial respiratory reserve capacity [ ] . this, we suggest, does have a great deal of merit, and in relation to resistance to viruses, could be viewed from a reduction in bioenergetic/redox capacity of the immune system as people age. tellingly, reduced skeletal muscle mtdnacn is associated with symptoms of the metabolic syndrome, whereas exercise increases mtdnacn and is negatively associated with markers of the metabolic syndrome and enhanced aerobic capacity [ ] . thus although certainly not the entire story, ageing is associated with declining mitochondrial function, which is likely to be related to reduced immune "reserve" and flexibility. hence, as a proxy for potential severity when infected, mitochondrial function does have its place in the ageing process. the lessons for humans are thus fairly clear: exercise is part of our evolutionary heritage, and plays key role in maintaining optimal mitochondrial health and immune balance (fig. ) . as is becoming clear, maintaining good health, in particular, optimal levels of aerobic fitness and muscle/fat balance is a good preventative strategy, in effect, the results of living a healthy lifestyle. a retrospective study following the hong kong influenza outbreak in found that physical activity was protective and displayed a "u" shaped dose-response curve [ ] . high aerobic fitness is associated with reduced morbidity and mortality [ , ] and physical activity, if not overdone, is generally anti-inflammatory in the longer term [ , ] and results in an enhanced anti-oxidant status [ ] . equally, calorie restriction, which is associated with improved lifespan, is also anti-inflammatory [ , ] , as is a diet high in polyphenols and probiotics [ ] . however, there are still many people, who, for various reasons are not living a particularly healthy lifestylethe effects of which become worse with age. this therefore raises the question, would enhancing/supporting mitochondrial health help in this population if they become infected and would understanding the role of mitochondria help in deciding treatment regimens? there are a number of possibilities, ranging from suppressing the auto-inflammatory loop (e.g., direct targeting of inflammatory pathways, with, say antibodies, or kinase inhibitors), to mitochondrial protection, enhancing mitochondrial turnover and renewal and preconditioning, to direct management of redox. in fact, it seems that many established drugs probably already do modulate mitochondrial function, which may provide us with further insight. the other main strategy and one perhaps with the greatest potential preventative benefit in the long run, is vaccination. the implications of mitochondrial health here could be extremely important in whether or not a vaccine is successful in particular populations, for instance the elderly and those with co-morbidities. many of the compounds now being studied may influence mitochondrial function. for instance research on immunomodulation during influenza infections has fig. resistance to sars-cov- : mitochondrial and redox reserve, hormesis and the metabolic/inflammatory see-saw. data strongly support that people who are aerobically fit, and follow a healthy lifestyle, tend to have a greater metabolic and anti-oxidant reserve related to training-induced mitochondrial adaptation; this is associated with less disease and a longer "healthspan". the underlying principle that leads to this is described by hormesis and is the product of humans having evolved, along with most other animals, in an environment where this adaptation was constantly induced due to the need to move, and food was less available. as a phenotype, they tend to have greater muscle to fat ratio, and exhibit few, if any symptoms of the metabolic syndrome, such as increased liver fat and visceral adipose tissue (vat) volume, insulin resistance, or markers of chronic inflammation. in this context, data suggest that muscle, as an organ, tends to be anti-inflammatory when used regularly, whereas adipose, in particular, in vat is inflammatory if it becomes overloaded. as the virus seems to induce oxidative stress [ ] and aerobic glycolysis to enhance its own replication, it could be argued that in a person with poor mitochondrial and anti-oxidant reserve, infection will tend to tip towards chronic inflammation and incomplete resolution. in this scenario, it is important to suppress either inflammation itself, or provide extra anti-oxidant power to damp down the potential for a feed-forward inflammatory spiral looked at corticosteroids, peroxisomal proliferating activated receptors (ppar) agonists, cyclooxygenase (cox) inhibitors, adenosine monophosphate kinase (ampk) activators, direct antioxidants, and natural products [ ] . all of these can modulate mitochondrial function [ ] [ ] [ ] [ ] [ ] . non-steroidal antiinflammatory drugs (nsaids) in general, to varying degrees, affect mitochondrial function [ ] . critically, network-based drug repurposing has recently identified several candidates, such as irbesartan, paroxetine, sirolimus, melatonin and quinacrine, amongst others [ ] . it has been suggested that angiotensin receptor blockers (arbs) are mitochondrially protective [ ] , while anti-depressants, such as paroxetine, can inhibit mitochondrial function [ ] . sirolimus, or rapamycin, is actually one of the best studied calorie restriction mimetics as it modulates mammalian target of rapamycin (mtor); it is anti-inflammatory and modulates mitochondrial function, and could play a key role in mitohormesis [ ] . it can, in fact, increase mitochondrial respiration and reduce production of hydrogen peroxide [ ] . critically, data does suggest that this new virus can indeed inhibit autophagy and the mtor pathway [ ] . this might suggest that it can enhance atp production while reducing ros, which would obviously benefit it (by at least, initially, suppressing immune activation). overall, it could be argued that compounds that do inhibit mitochondrial function might have a number of effects, such as inducing mitohormesis, so activating mitochondrial turnover and renewal, but they could also disable mitochondrial support for viral replication, and perhaps, enhance apoptosis. however, this has to be balanced with the possibility that they could cause too much damage, and potentially, worsen the situation. one group of drugs that has been investigated as a possible treatment for sars-cov- are the antimalarial aminoquinolones, which have been investigated for decades as immunomodulators and anti-virals. their basic mode of action involves proton capture and deacidification of the lysosomal/endosomal compartment, which interferes with viral replication, autophagy and inflammatory pathways, but they also affect the plasma membrane, map kinases, calcium signalling, as well as dna [ ] . they can also modulate mitochondrial function [ ] [ ] [ ] and have been shown to have antioxidant activity [ ] . paradoxically, they can also induce oxidative stress, which has raised concerns about their use in covid- treatment due to the hypoxia associated with the acute respiratory distress syndrome (ards); one suggested mechanism is increased ros generated by mitochondriaas well as possible direct effects on mitochondria [ ] . a meta-analysis has shown that hydroxychloroquine used in treatment of covid- resulted in a . times greater mortality compared to control groups, whereas its use was associated with a . times improvement in patients with mild to moderate symptoms compared to a control group [ ] . a pharmacovigilance study also found that the use of hydrochloroquine/chloroquine for treatment of covid was associated with higher rates of cardiovascular side effects [ ] . interestingly, in another study, although hydroxychloroquine and chloroquine were not associated with any significant effect on mortality, hydroxychloroquine, but not chloroquine, was associated with a significant reduction in transfer to the intensive care unit of patients admitted to hospital [ ] . critically, a recent review of the literature show these molecules to have biphasic/hormetic effects in multiple models, for instance, they can both stimulate or inhibit cancer cell and virus growth depending on dose [ ] . this not only highlights the role of dose, but also, potentially, the induction of oxidative stress and the patient's underlying health, and whether or not these compounds enhance risk or benefit. another very old anti-inflammatory drug, colchicine, is also being investigated for efficacy in covid- patients, as it seems to inhibit the nlrp inflammasome, perhaps by suppressing the transport of mitochondria [ ] . interestingly, sars-cov- does seem to modulate many proteins related to the cytoskeleton, and can induce filopodial protusions [ ] . colchicine is often used to study autophagy, as it depolarises microtubules, so inhibiting the process. it has now been shown that it can result in impairments in skeletal mitochondrial function, increasing ros, and in older animals, this can result in insulin resistance [ ] . this all suggests that many of these drugs, especially those that might affect mitochondrial function, either directly, or indirectly, and could have an age-dependent effect -especially those that might affect autophagy. an important class of drugs are the mapk inhibitors, many of which have been developed as anti-cancer agents. as indicated previously, mapks can modulate mitochondrial function. they have been proposed as potential treatments as the virus seems to upregulate p activity and inhibit counter-regulatory pathways. although this may well help in viral replication, it can also result in excessive inflammation in some patients [ , ] . interestingly, vemurafenib, a mapk inhibitor, has been shown to inhibit dynamin-related protein (drp ) phosphorylation, reversing excessive mitochondrial fission in melanoma cells and resulting in hyper-fusion and enhancing oxidative phosphorylation and reversal of aerobic glycolysis [ ] . this again highlights the parallels between cancer and viral infection in the sense that both induce extensive metabolic reprogramming and manipulation of the cell cycle, often towards aerobic glycolysis with modulation of mitochondrial function, as well as attenuating/modifying immune responses. data also suggest that cyclophilin inhibitors, such as cyclosporine a, which apart from being immunesuppressants, could also inhibit the replication of related corona viruses. data has already shown that some transplant patients receiving immunosuppressants seemed to have some protection against the virus, although these were observational studies and other factors, such as good hygiene, could be important. but in vitro data does hint at efficacy, especially on other corona viruses, such as middle east respiratory syndrome coronavirus (mers), as does evidence around the importance of the cyclophilins in aiding viral replication. the mode of action is thought to involve inhibition of the calcineurin and suppression of the nuclear factor of activated t cells (nfat) (reviewed in [ ] ). in mice infected with mers-cov, it seems that cyclosporine induces a robust interferon gamma response, which is associated with inhibition of viral replication and release [ ] . mavs are a key component of resistance to viruses, and can activate both interferon and nf-kb pathways, putting mitochondria centre stage in viral defence [ ] ; data indicate that immunophilins are regulators of mavs [ , ] . given the importance of cyclophilin d, a wellknown target of cyclosporine that modulates mitochondrial permeability transition [ ] , it would be interesting to speculate that apart from the well described immunophilin targets of compounds like tacrolimus and cyclosporine, a role for modulation of mitochondria could not be ruled out. in this light, the effectiveness of the pan-cyclophilin inhibitor, alisporivir, which does not have immunosuppressive effects, is potentially interesting as it has high potency against sars-cov- . it has been suggested that its ability to inhibit cyclophilin d, and thus control mitochondrial permeability, maybe of importance in preventing lung damage [ ] . finally, some very promising preliminary data from the recovery trial suggests that low dose dexamethasone could help prevent death of up to % of ventilated patients [ ] . on the th september , the european medicines agency endorsed the use of dexamethasone in covid- patients on oxygen or mechanical ventilation (ema/ / ). the most well-known effect of glucocorticoids is to suppress inflammation, largely through the glucorticoid receptor (gr), but with chronic use they do have side effects, as they are catabolic [ ] . the key point here is that glucocorticoids are generally induced by stress, and in the short term, are highly protective. it is thus relevant that grs also transfer to the mitochondrion and control mitochondrial gene transcription, and have biphasic actions [ ] . it is thus relevant that dexamethasone has been shown to both induce mitochondrial uncoupling and increase oxidative phosphorylation [ ] , but also cause mitochondrial dysfunction [ ] . this is hardly surprising as mitochondria are central to both steroid biosynthesis and action, and thus, stress management [ ] . although it might be surmised that the predominant effect in the recovery trial is through direct suppression of inflammatory pathways, it is not impossible that effects on mitochondria could not be ruled out. a further approach that has been suggested is suppression of oxidative stress by using compounds that are anti-oxidants. direct anti-oxidants, for example n-acetyl cysteine, which, although it has shown some efficacy, has met with limited success due to dose issues [ ] . however vitamin c, which is now known to concentrate in mitochondria and act as a ros scavenger [ ] , could be useful. a retrospective analysis of data has suggested that vitamin c can both reduce the time in the intensive care unit and the time on ventilators, particularly for very ill patients [ , ] . it is now being suggested that it is used in combination with quercetin, which also seems to have efficacy in viral infections [ ] : quercetin is a natural product that has anti-oxidant properties and concentrates in mitochondria and can induce mitochondrial biogenesis [ , ] . another important principle is that many plant compounds seem to have anti-viral properties, as well as anti-cancer properties, and modulate calcium signalling and mitochondrial functionwith common targets, such as vdac. plants suffer both from viral infection and cancer, so it could be that there is some cross over in function from plants to animals [ ] . as viruses seem to hijack their host's cellular machinery, including mitochondrial function, then partially inhibiting mitochondrial function could be an evolved strategy to defeat the virus, especially if it induces apoptosis and/or upregulates mitophagy and mitochondrial renewal and anti-oxidant systems. a good example of this is perhaps salicylic acid, which is a major plant defence signalling compound [ ] and modulates mitochondrial function, both inhibiting the electron transport chain and acting as an uncoupling agent [ , ] , as well as regulating vdac expression [ ] . some plant viruses produce proteins that can inhibit the oxidative burst and salicyclic-acid dependent autophagy [ ] . there is thus, potentially, useful insight provided by the observation that some medicines that are derived from plant (or other organism) defence compounds, also appear to have some benefit in human viral infections. indeed, gurbel and colleagues have suggested that aspirin could be used against sars-cov- , for instance, by reducing its activation of nf-κb [ ] . there is also interest in the potential for compounds such as cannabidiol (cbd) in helping covid- patients, as the cannabinoids do seem to have some antiviral activity, and are anti-oxidant and anti-inflammatory [ ] . cbd, amongst its many identified targets [ ] , does seem to directly modulate mitochondrial function, for instance, it has been shown to bind to vdac and inhibit the electron transport chain [ , ] . there is also evidence that it can inhibit inflammasome activation [ ] . one key mechanism is that many plant compounds activate nrf and are thus hormetic [ ] . furthermore, as many manufactured drugs were developed from defence compounds found in plants and other organisms, this principle could be extended to include them. another example of this could be the statins, which also inhibit mitochondrial function [ ] [ ] [ ] , and one study has indeed shown they can reduce mortality of covid- patients [ ] . another ubiquitous antioxidant molecule, melatonin, which also protects mitochondria [ ] , is also being investigated as an adjuvant to protect against a cytokine storm in sars-cov- infection [ ] . interestingly, it has been shown to reverse the warburg effect in immune cells, potentially having an anti-inflammatory effect, and providing a justification for its use in covid- patients [ ] . likewise, glutathione is also showing promise, in particular, as it seems to help redress the age related th /th imbalance [ ] . of potential relevance is the observation that a modified vitamin e derivative that concentrates in mitochondria has shown benefits in a model of cardiac inflammation induced by sepsis. it seems to do this by suppressing mitochondrial dna damage and its subsequent release [ ] ; mtdna is a potent activator of the inflammatory system [ ] . also of interest here is vitamin d, which has been suggested as a potential adjuvant treatment for patients with the virus, as it may restore immune function. in particular, it may enhance anti-inflammatory cytokine production and so limit the possibility of a cytokine storm. analyses do seem to show that it can have some benefit in people who have low levels of this vitamin [ ] . critically, it modulates mitochondrial function, having diverse affects depending on the tissue; it can stimulate muscle mitochondrial function [ ] , but may also enhance lipid storage and adipogenesis [ ] . interestingly, it has been suggested that covid- morbidity increases with northerly latitude, suggesting a link with ultraviolet light and vitamin d [ ] . vitamin b has also been shown to have some protective effects in mitochondrial myopathy models [ ] , and has been suggested that it could help prevent lung injury in covid- patients [ ] . finally, artificial mitochondrial anti-oxidant molecules, such as mitoq and skq could also provide benefit [ ] . there are also compounds like luminol derivatives, which only become ros scavengers in areas of high oxidative stress and are showing some promise in modulating redox-driven inflammation [ ] [ ] [ ] . however, unlike mitoq and skq , it is likely that luminollike compounds act outside the mitochondrion [ ] . the age related decline in immune function is well described, although not well understood, and affects virtually all components and has a big impact on the success of vaccinationwhich has led to a constant drive to improve vaccines for the older generation, in particular against influenza [ ] . it is, however, recognised to be modifiable by many factors, ranging from exercise, to stress, and chronic infections [ ] . critical in these responses is metabolic flexibility, for instance, the ability to switch between oxidative phosphorylation and glycolysis, and how this effects different sub-populations of cells and the pro-inflammatory/anti-inflammatory balance. for instance, aged b-cells lose oxidative phosphorylation capacity, and rely more on glycolysis and generate more ros. they also infiltrate adipose tissue, heightening inflammation in a process involving the nlrp inflammasome. in relation to the b-cell response, which is key purpose of vaccination, it seems that obesity, and the metabolic syndrome, accelerates immunosenescence and reduces the ability to produce antibodies [ ] . a primary research area is on the development of vaccines for the elderly against influenza; on average, over the age of years, the efficacy drops off rapidly. to study this, immunosenescence related markers in the blood have been correlated with outcome. interestingly, t-cell responses have been found to be a stronger correlates of protection than antibodies. although the biology is immensely complex, and may require a system level "vaccinomics" approach, dysregulated metabolism is clearly part of the problemand it has been suggested that treatments to correct dysregulated metabolic or other physiological processes may be required before administration of vaccines [ ] . it would therefore seem that in order to improve the efficacy of vaccines, it is either necessary to tailor the vaccine to the particular immunosenescence profile a patient shows, or, perhaps to reduce their epigenetic age by ensuring they live a healthy lifestyle and so enhance their immune system. there are a number of factors to consider from this. if, as seems to be the case, this virus is modulating mitochondrial function, and thus, mitochondrial health is important, there are a number of intriguing possibilities. does mitochondrial function explain why morbidity may be greater among men than women? there are obviously confounding behavioural factors, but statistically, men seem to have higher rates of mortality than women when infected with the coronavirus [ , ] . mitochondria in females may be more robust, which could explain why females tend to live longer than males [ ] . would pollution lower resistance to the virus? nitrogen dioxide is oxidative and can induce pulmonary inflammation and reduce function [ ] , oxidise mitochondrial cytochrome c [ ] , while acute inhalation can cause mitochondrial dysfunction in the brain [ ] . data from the united kingdom is now suggesting that high levels of pollution are linked to increased covid- lethality [ ] . linked to this is the very disturbing evidence that iron-rich nanoparticles, largely derived from motor vehicles, are now being found in cardiac mitochondria in the very young, and are causing oxidative stress [ ] . the renin-angiotensin-aldosterone system (raas) and mitochondrial function the coronavirus binds to ace [ , ] and mitochondria have their own angiotensin system [ ] . ace cleaves angiotensin ii to produce antiinflammatory molecules and protects mitochondria [ , ] . this suggests ace / polymorphisms will be a factor in reaction to the virus [ ] . arbs, acei and statins may enhance ace activity. their role in treatment is thus debated [ , ] . during hypoxia, mitochondrial function is inhibited, but then becomes a source of ros during reperfusion. could damaged mitochondria in the lung and/or heart lead to an exacerbation of symptoms if too much oxygen is given to a patient? this is clearly a difficult clinical conundrum, but does suggest that supplementary oxygen should only be used where absolutely necessary. compounds such as melatonin, cbd and curcumin have shown some protective effects ischaemic-reperfusion models [ ] [ ] [ ] curcumin is an uncoupling agent [ ] . cbd modulates mitochondria [ ] . key in this is emerging data that hypoxic preconditioning requires a drop in the mitochondrial proton motive force [ ] . management of etc uncoupling is thus vital for life to control oxidative stress [ ] . ppars may play a key role in controlling uncoupling [ ] . furthermore there is much evidence that anaesthetics can modulate mitochondrial function and could play a role in both preand post-ischaemia protection, and some can act as uncoupling agents [ ] [ ] [ ] . two recent structure-docking studies have indicated that several phytochemicals could inhibit the sars-cov- protease [ , ] . as many phytochemicals can also modulate mitochondrial function [ ] , and primarily evolved to protect the plant, it could be surmised that they are multi-functional and modulate multiple pathways to achieve this. long term effects -"long covid" it is becoming clear that following recovery from the primary infection with sars-cov- , many people are suffering from long term effects, such as fatigue and mental health problems, as well as more obvious lung problems. this has resulted in the formation of a national uk consortium and the launch of the phosp-covid study to investigate the long terms effects on health of this virus (see https://www.phosp.org/) [ ] . one possible consequence of viral infection could be longer term mitochondrial dysfunction, which could lead to a variety of symptoms. mitochondrial function, and their relationship to immunity, is again becoming a focus for research in the chronic fatigue syndrome, which is still not completely understood [ ] . this has been further supported by evidence of mitochondrial dysfunction in pbmcs of people with chronic fatigue syndrome [ ] . can we test the hypothesis that mitochondrial health = immune health and enhanced resistance to the virus? in terms of testing and/or looking for evidence that mitochondria could help explain some of the pathophysiology of this virus, there are several potential ways to look for this relationship, ranging from laboratory based to population studies. this work could be carried out in vitro with cultured cells and/or isolated mitochondria prepared from control and infected individuals. in particular, using imaging to look for co-localisation in "virus factories". it could be predicted that those populations exhibiting the lowest levels of optimal health and the highest levels of the metabolic syndrome and "diabesity", will show the highest susceptibility. for example, it might be revealing to map the case fatality rate to the latest trends in obesity/ diabetes after the necessary confounders are taken into account [ ] . in support of this, the emerging data from new york in relation to sars-cov- infection is that obesity is strongly correlated with critical illness [ ] . in contrast, would those populations showing the highest fitness levels and functioning be more resistant? for instance, would measured vo max show an inverse relationship with morbidity? individuals with known mitochondrial dysfunction are well known to show abnormal susceptibility to infections [ ] . is there a link between mitochondrial haplotype and resistance? there is certainly evidence for different mtdna haplotypes amongst different populations [ ] . although there is an emerging disparity in morbidity between black people and other minorities in the usa, it is thought it may be more to do with socio-economic imbalances and higher rates of lifestyle induced comorbidities [ ] . blood-derived mitochondrial markers of reduced function may correlate with disease severity before, during and after infection. data now show it is possible to determine someone's epigenetic age and compare it with their chronological age. there is a close correlation with this ratio and coexisting morbidity [ ] . thus, would blood-derived epigenetic markers of metabolic age correlate with disease severity before, during and after infection? the main conclusion from this review is that as immune function is dependent on mitochondrial function, and although this does decline with age, the rate it does so can be modified by lifestyle. this is perhaps best 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note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations none.authors' contributions an wrote and edited the manuscript after discussion with gwg, while all other authors contributed equally to critiquing the original drafts and the concepts therein, and approved the final manuscript. no specific source of funding supported this paper.availability of data and materials not applicable.ethics approval and consent to participate none required. all authors have approved the paper for publication. professor geoffrey guy is the founder and chairman of gw pharmaceuticals; professor alistair nunn is a scientific advisor to gw pharmaceuticals; dr. wolfgang brysch is the chief executive officer of metriopharm ag. the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -ti cc m authors: wang, cuixue; zhou, jiedong; wang, jinquan; li, shujing; fukunaga, atsushi; yodoi, junji; tian, hai title: progress in the mechanism and targeted drug therapy for copd date: - - journal: signal transduct target ther doi: . /s - - -x sha: doc_id: cord_uid: ti cc m chronic obstructive pulmonary disease (copd) is emphysema and/or chronic bronchitis characterised by long-term breathing problems and poor airflow. the prevalence of copd has increased over the last decade and the drugs most commonly used to treat it, such as glucocorticoids and bronchodilators, have significant therapeutic effects; however, they also cause side effects, including infection and immunosuppression. here we reviewed the pathogenesis and progression of copd and elaborated on the effects and mechanisms of newly developed molecular targeted copd therapeutic drugs. among these new drugs, we focussed on thioredoxin (trx). trx effectively prevents the progression of copd by regulating redox status and protease/anti-protease balance, blocking the nf-κb and mapk signalling pathways, suppressing the activation and migration of inflammatory cells and the production of cytokines, inhibiting the synthesis and the activation of adhesion factors and growth factors, and controlling the camp-pka and pi k/akt signalling pathways. the mechanism by which trx affects copd is different from glucocorticoid-based mechanisms which regulate the inflammatory reaction in association with suppressing immune responses. in addition, trx also improves the insensitivity of copd to steroids by inhibiting the production and internalisation of macrophage migration inhibitory factor (mif). taken together, these findings suggest that trx may be the ideal drug for treating copd. chronic obstructive pulmonary disease (copd) is a slow-developing, incurable lung disease characterised by a sustaining airflow limitation that further develops into common diseases such as pulmonary heart disease and respiratory failure. copd is caused by a complex interaction between genes and the environment. cigarette smoking is the leading environmental risk factor for copd. fewer than % heavy smoker develop copd, it indicates that genetics may play a role in regulating the risk of copd in smokers. besides genetics, other risk factors are also involved in the development of copd, such as age and gender, , lung growth and development, , exposure to particles, - socioeconomic status, , asthma and airway hyper-reactivity, , chronic bronchitis , and infections. gender may effect whether a person smoke or experiences certain occupational or environmental exposures; socioeconomic status may be related to lung growth and development, and then influence on susceptibility to developing the disease; and long live will allow greater lifetime exposure to risk factors. asthma may be a risk factor for the development of copd. airway hyper-responsiveness is the second risk factor for copd, but airway hyper-responsiveness, as an independent predictor of copd can exist without asthma, suggesting inflammatory profiles of copd different from asthmatic subjects. the pathogenesis of copd remains unclear and has been generally suggested to be related to inflammation, oxidative stress, protease/anti-protease imbalance and decreased immunity. smoking, biofuel smoke-induced oxidative stress and excessive protease production are major factors in copd pathogenesis that cause alveolar cell death, destruction of the extracellular matrix in the alveolar region and loss of alveolar structure. , the primary manifestations in the respiratory tract include airway wall remodelling and mucus retention, and further development leads to a serious decline in the lung function. currently, the main approach is to deal with symptoms of the airflow limitation caused by the above-mentioned symptoms to improve the resulting dyspnoea through medication, oxygen treatment and rehabilitation therapy. however, there is currently no way to prevent the disease progression. drug treatment includes bronchodilators and glucocorticoids, with the main types of bronchodilators including the β receptor agonists and anticholinergic drugs; however, both have many adverse effects. for example, the main side effects of the β receptor agonists are rapid heartbeat, muscle tremors and metabolic disorders. the side-effects of anticholinergic drugs include dry mouth, blurred vision, urinary retention, postural hypotension, cognitive problems and cardiac rhythm disturbance. long-term use of glucocorticoids induces and exacerbates infections, cause hyperglycaemia, osteoporosis and even mental disorders. [ ] [ ] [ ] therefore, a series of new molecular targeted therapeutic drugs to block copd progression is under development. this article introduces the pathogenesis of copd and pharmacology of related anti-copd drugs. specifically, there is a focus on the effective role and mechanism of the small molecule secretory protein thioredoxin (trx) that is widely expressed in lung tissues such as the type ii alveolar cells, macrophages and bronchial epithelium. the occurrence and development of copd is a complex pathological process involving a variety of inflammatory cells, inflammatory mediators and related cell signalling pathways. copd also regulates the goblet cell proliferation, mucoprotein (muc) synthesis and mucus secretion. in recent years, molecular biology has revealed new insights regarding the pathogenesis of copd (fig. ). copd and oxidant/antioxidant imbalance oxidative stress is an important factor in copd pathogenesis. an increased oxidative burden occurs in the lungs of patients with copd, and oxidative stress may be involved in various the pathogenic processes, such as direct injury to lung cells, mucus hypersecretion, inactivation of antiproteases and enhancing lung inflammation through activation of redox-sensitive transcription factors. copd patients suffer from oxidative stress caused by the inhalation of cigarette smoke (cs) or harmful substances which causes an accumulation of pulmonary inflammatory cells (neutrophils, macrophages), leading to large numbers of reactive oxygen species (ros). excessive ros production leads to an oxidative inactivation of anti-proteases, alveolar epithelium damage, increased neutrophil retention and increased expression of various inflammatory mediators in the pulmonary microcirculation, aggravating the development of copd. [ ] [ ] [ ] in addition, ros-activated inflammatory cells, now acting as activated inflammatory cells, also generate ros to further aggravate oxidative stress in tissues. oxidant-generating systems, such as xanthine/xanthine oxidase, can cause airway epithelial mucus secretion. oxidative stress, generated by tobacco smoke and augmented by ros generated from both inflammatory cells and mitochondrial activity by cells resident in the respiratory tract, regulate mucous cell metaplasia and mucin gene expression such as muc ac and muc b. oxidants are also involved in the signalling pathways for epidermal growth factor, which has an important role in mucus production. a relative deficiency of anti-proteases, such as α -antitrypsin, because of their inactivation by oxidants from cigarette smoke or released from inflammatory leucocytes, causes a protease/ anti-protease imbalance in the lungs. cigarette smokeinduced oxidative stress plays role in enhancing inflammation by regulating redox-sensitive transcription factors, such as nuclear factor kappa-b (nf-κb) and activating protein (ap- ), the extra-cellular signal-regulated kinase (erk), c-jun n-terminal kinase (jnk), and p mitogen-activated protein kinase (p mapk) pathways. ros also decrease the activity of histone fig. the pathogenesis of copd is complex and diversified. oxidative stress may participate in various the pathogenic processes, such as direct injury to lung cells, mucus hypersecretion, inactivation of antiproteases and enhancing lung inflammation through activation of redoxsensitive transcription factors. under the stimulation of cigarette smoke, pathogen infection and other factors, oxidative stress is induced and the pulmonary inflammatory cells (neutrophils, cd t lymphocytes, macrophages) accumulate, resulting in a large number of reactive ros. the inflammatory cells are activated by the nf-κb, p mapk and pi k signalling. inflammatory cells (mainly neutrophils) migrate from the circulation to the inflammatory site under sequential regulation involving cytokines and adhesion molecules such as selectin. proteases are involved in tissue remodelling, inflammation and ecm degradation, thereby participating in the pathological process of copd. inflammatory cytokines and chemokines, such as ltb , il- and tnf-α, and other mediators are secreted into the lungs to aggravate the lung tissue damage and promote inflammatory responses. pde decreases camp levels in inflammatory cells and promotes inflammatory cell activity and the release of inflammatory factors. chronic inflammation stimulates the increase of egfr and tgf-β . activated egfr is involved in the proliferation of the airway epithelial goblet cells and mucus production. tgf-β chemoattracts neutrophils, macrophages and mast cells, and activates pi k/akt and/or p mapk signalling to induce pulmonary fibrosis and emt. endothelin- (et- ) produced by endothelial cells, stimulates the contraction and proliferation of vascular smooth muscle cells and the liver to produce more crp, and it also induces the synthesis of vegf. b-type natriuretic peptide (bnp) antagonises renin angiotensin aldosterone system, dilates blood vessels and reduces peripheral vascular resistance, and c-type natriuretic peptide (cnp) dilates blood vessels and inhibits the proliferation of vascular smooth muscle cells deacetylase (hdac) which recoil dna of the histone core to stop transcription, , and probably increase the activity of histone acetyltransferase, which uncoils dna from the histone core to allow transcription. this leads to the further recruitment of inflammatory cells, specifically the neutrophils and macrophages in the alveolar spaces. in addition, oxidative stress extends beyond the lung, contribute to several of the systemic manifestations. peripheral blood neutrophils of copd release more ros than in normal subjects and this is enhanced still further in exacerbation. products of lipid peroxidation are also increased in plasma in smokers with copd, particularly during exacerbations, resulting in dna damage and airway epithelial cell senescence and apoptosis. in addition, the reactive aldehydes such as acrolein and -hydroxy- -nonenal ( -hne) formed from lipid peroxidation cause damage to the epithelial cells, acrolein is unstable and toxic, whereas -hne in high concentrations is capable of inducing caspase (a major promoter of cell apoptosis). , nuclear factor e -related factor (nrf ) is a transcription factor that regulates many antioxidant genes and plays an important role in the body's antioxidant stress. normally, nrf is fixed in the cytoplasm by kelch-like ech-associated protein (keap- ). under oxidative stress, nrf and keap- dissociate, and nrf is transported to the nucleus, activating the transcription of antioxidant genes. , however, the level of nrf in copd patients is reduced, resulting in a reduction of endogenous antioxidants and weakening the body's protection against oxidative stress. sirtuins (sirt ) and sirt are also related to the redox state. sirt is a redox-sensitive protein with a wide range of biological functions, such as inhibition of autophagy, cell aging, emphysema and fibrosis, and inflammation. , , yao et al. demonstrated that sirt can resist the inflammatory response of cigarette smoke to lung cells caused by oxidative stress. inhibition of inflammation may be through deacetylation of nf-κb. , sirt is also associated with redox status and inhibits cell aging and fibrosis. , copd and protease/anti-protease imbalance the pathogenesis of copd is closely related to the imbalance of protease/anti-protease, which leads to the destruction of the elastin framework. proteases are involved in tissue remodelling, inflammation and degradation of the extracellular matrix (ecm) components and the pathology of copd. , in addition, the products of ecm decomposition, such as collagen and elastin, are themselves chemokines of inflammatory cells and cause persistent respiratory system inflammation in copd patients, even leading to systemic autoimmune diseases. elastase is an enzyme that hydrolyses peptides and other proteins, and there are three main types in lung disease: serine proteases, caspases and matrix metalloproteinases (mmps). among them, mmps are a highly conserved family of endopeptidases, consisting of known members, that depend on zinc and calcium ions. macrophages and their macrophagederived mmps may be the dominant factor in the formation of smoking-related emphysema and copd. among the mmps, mmp- and mmp- are most involved in emphysema. mmp- plays an influential role in severity of copd. mmps also play a role in vascular remodelling by modulating the migration and proliferation of smooth muscle cells and endothelial cells and mediating the release of the smooth muscle cell mitogens and growth factors, increasing the risk of copd pulmonary hypertension. [ ] [ ] [ ] [ ] the endogenous inhibitors of mmps are tissue inhibitors of metalloproteinases (timps). timps are a family of low-molecular-weight proteins with four known members. timp- inhibits active mmps, including mmp- , mmp- and mmp- . it has been reported that changes in mmp- poorly correlated with disease intensity and progression in copd. timp- binds to pro-mmp- to prevent the activation of pro-mmp- . however, neutrophil elastase acts by dissociating the binding of timp- to pro-mmp- , which allows mmp- to activate pro-mmp- to become mmp- . in serine proteases, neutrophil elastase (ne), cathepsin g and proteinase- destroy lung tissue by degrading ecm components, and induces epithelial cells and endothelial cells to release a variety of inflammatory factors to activate and chemoattract neutrophils to produce lung inflammation. , alpha-antitrypsin is a protease inhibitor synthesised by liver cells, which control over the proteolytic activity of ne, proteinase- , cathepsin g and neutrophils serine protease- . evidence from experimental models of emphysema and from individuals with genetic deficiency of alpha- antitrypsin provides strong evidence that an imbalance between the enzymes and inhibitors is important in tissue damage and the pathogenesis of copd. protease-anti-proteases imbalance during airway inflammation and airflow restriction may be an important factor affecting airway remodelling and airflow restriction. copd and inflammatory cells, cytokines and chemokines copd pathology is characterised by airway remodelling and inflammatory cell infiltration by the neutrophils, cd t lymphocytes and activated macrophages. neutrophils have been implicated in copd pathogenesis and the extent of neutrophilic infiltration in lung tissues correlates with copd severity. , inflammatory factors play a major role in the onset and development of copd. under the stimulation of cigarette smoke or other harmful substances, the respiratory tract epithelium secretes inflammatory cytokines and chemokines, such as leukotriene b (ltb ), interleukin- (il- ), il- (cxcl ) and tumour necrosis factor-α (tnf-α), and other mediators in the lungs. these inflammatory mediators aggravate the lung tissue damage and promote inflammatory responses. ltb is a lipid mediator derived from arachidonic acid by the sequential action of -lipoxygenase ( -lox), -lipoxygenase-activating protein (flap) and lta hydrolase (lta h) to stimulate leucocyte functions such as cytokines, chemokinesis, lysosomal enzyme release, superoxide anion production, adhesion to endothelial cells, generation of ros and so on. il- activates neutrophils, causing neutrophil infiltration at the inflammatory sites, inducing neutrophils to release elastase and various oxygen free radicals, destroying alveolar surfactants, increasing pulmonary vascular permeability and inducing pulmonary oedema. activated by il- , which is higher in bronchoalveolar lavage fluid (balf) and sputum of copd patients. il- has a similar effect and induces neutrophil migration to the airway and affects degranulation produced by various cell types. tnf-α stimulates the pulmonary microvascular endothelial cells to promote the accumulation, adhesion and migration of the polymorphonuclear leucocytes by inducing the expression of il- and upregulating endothelial adhesion molecules, causes release of lysosomal enzymes, elastase and large quantities of ros; and damages the endothelial cells and alveolar epithelium. tnf-α together with il- β has been identified as a key cytokine that is able to initiate inflammatory cascades during exacerbations of copd. monocyte chemotactic protein (mcp- , ccl- ), macrophage inflammatory protein- α (mip- α, ccl- ) are cc-chemokines, which act as chemoattractants for inflammatory cells like macrophages, lymphocytes. neutrophil-derived ccl- and ccl- are involved in macrophage recruitment into inflamed tissue. in patients with copd, several cc-chemokines like ccl- , ccl- are upregulated to attract specific inflammatory cells, like macrophages, neutrophils and cd (+) t-lymphocytes into the airway, suggesting the contribution of their respective receptor in the pathogenesis of the disease. copd and adhesion factors the inflammatory process of copd is characterised by a continued migration of inflammatory cells (mainly neutrophils) from the blood vessel to the lungs. neutrophil migration is a carefully regulated series of events involving cytokines and adhesion molecules including the selectins (l-, p-and e-selectin), intercellular adhesion molecule- (icam- ) and vascular cell adhesion molecule- (vcam- ). the migration has been described as a multistep process including slow rolling, adhesion strengthening, intraluminal crawling and finally paracellular or transcellular migration through the endothelium. the initial rolling is mediated by l-selectin (cd l) expressed on neutrophils, and eselectin and p-selectin expressed on the endothelium. the main ligand for these selectins is the p-selectin glycoprotein ligand (psgl)- (cd ) expressed on neutrophils and certain endothelial cells. furthermore, e-selectin binds e-selectin ligand (esl- ) and cd on the neutrophil surface, cause the slow rolling of the neutrophil. next, firm adhesion is mediated through, for example, the macrophage antigen- (mac- /cd b) expressed on neutrophils and its ligand icam- expressed on the endothelium. after the neutrophil has been fully arrested, the adhesion of the neutrophil to the endothelial surface is strengthened. stable binding of ligand to vla- (a b -integrin) is rapidly increasing neutrophil infiltration of the inflamed tissue, implicating that internal signals are required for increased adhesion. the third step, intravascular crawling, involves cd b and other β -integrins. prior to the final step, transendothelial migration, vcam- and icam- form the so-called docking structures on the endothelial cells. for the final transendothelial migration, the interaction between two platelet/ endothelial cell adhesion molecules (pecam)- , expressed at the endothelial cell boundary and on neutrophils, is essential for the ultimate transendothelial migration. , selectin l, e and p have been found in copd. psgl- levels were higher in all stable copd patients than those in in healthy controls. increased eselectin and serum icam- have also been reported in copd patients. , during migration, the neutrophils release substantial amounts of proteinases and ros. this process is known as obligate proteolysis and is an important cause for bystander tissue damage in copd. the epidermal growth factor (egf) is a single-chain polypeptide growth factor that promotes the division of epithelial cells and other cells by binding to a specific epidermal growth factor receptor (egfr) on target cells to stimulate and maintain a series of cell growth, proliferation and transformation processes. chronic inflammation increases levels of egfr and its ligands. the expression and activation of egfr are positively correlated with the airway epithelial goblet cell proliferation and mucus production. in the airways, activated neutrophils and their secretions play an important role in an egfr-dependent mucus production. activated neutrophils secrete tnf-α, which upregulates the egfr expression in airway epithelial cells and directly stimulates muc synthesis. the expression of egfr was higher in copd patients than in smokers with normal lung function, which indicated that copd was related to the overexpression of egfr. egfr and its ligand egf binding are the main causes of squamous cell metaplasia, and the growth of epithelial cells is most significant in smokers and copd. these indicate that egfr levels in the small airways of copd patients were associated with decrease in airway functionality. the transforming growth factor-β (tgf-β) family regulates cell proliferation, differentiation and the extracellular matrix synthesis. tgf-β is a chemoattractant for the neutrophils, macrophages and mast cells. tgf-β expression is significantly increased in the airway epithelial cells of copd patients, and an active tgf-β signalling is involved in copd pathogenesis. in copd patients, tgf-β promotes a fibrotic airway remodelling, which can further contribute to a diminished lung function. loss of the alveolar parenchymal tissue may be caused in part by an up-regulation of mmp expression in response to tgf-β signalling, leading to an ecm degradation. , in copd, epithelial to mesenchymal transition (emt) which is associated with airway remodelling and obliteration is activated by canonical pathways such as tgf-β, which induce expression of nuclear transcription factors psmad / and reduced inhibitory smad / expression. , tgf-β also induces and promotes the increased expression of egf, egfr and its related signalling pathways, and their synergism induces emt-related phenotypic changes. , copd and camp cyclic adenosine monophosphate (camp) is a ubiquitous secondary messenger that regulates a variety of essential processes in diverse cell types via camp-dependent effectors such as protein kinase a (pka) and/or the exchange proteins directly activated by camp (epac). epac and pka inhibit the human airway smooth muscle induced by a cigarette smoke extract (cse) by blocking the activation of the nf-κb and erk, respectively, and by releasing neutrophil chemokine il- , which together exert anti-inflammatory effects. camp also mediates the airway smooth muscle relaxation. , camp plays a key role in the functions of many airway cells including controlling ciliary beat frequency (critical for mucus clearance) in airway epithelial cells. in copd, increases in camp levels, activation of pka and enhanced protein phosphorylation have the potential to reduce inflammation and immunomodulation, relax airway smooth muscle, inhibit chemotaxis and abnormal release of inflammatory and cytotoxic mediators, and reduce proliferation and migration of inflammatory cells. phosphodiesterases (pdes) are the only way to degrade cyclic nucleotides in the body, thereby ending the biochemical effects conducted by these second messengers (camp or cgmp). in the balf from copd patients, camp levels are decreased while pde levels are increased. these indicate that camp is regulated via pdes, and two direct downstream effectors of camp (pka and epac). copd and peptide factor copd also includes a gradual increase in pulmonary arterial pressure, and - % (depending on the definition, severity and measurement of copd) have developed pulmonary hypertension. endothelin- (et- ) is an effective vasoconstrictor produced by endothelial cells, which can stimulate the contraction and proliferation of vascular smooth muscle cells. the synthesis and secretion of endothelin in patients with copd increase, and during the exacerbation of copd, the level of endothelin further rises and participates in the formation of pulmonary hypertension. , et- can also stimulate the liver to produce more c-reactive protein (crp) by up-regulating il- . crp further stimulates the release of a variety of biologically active substances, such as endothelin- , il- , etc. amplify the inflammatory effect. , in addition, et- can also participate in the pathological process of copd by inducing the synthesis of vascular permeability factor (vegf). the family of natriuretic peptides includes a-type natriuretic peptide, b-type natriuretic peptide (bnp) and c-type natriuretic peptide (cnp). bnp and cnp play a major role in the occurrence and development of copd. bnp antagonises the renin-angiotensin-aldosterone system, dilates blood vessels and reduces peripheral vascular resistance. cnp also has a strong vasodilator effect and inhibits the proliferation of vascular smooth muscle cells. cnp can block the synthesis of vegf induced by hypoxia and endothelin at the transcriptional level, thereby inhibiting vegf-mediated hyperplasia of endothelial cells. it may play an important role in the development of pulmonary hypertension. copd and the nf-κb pathway the transcription factor nf-κb is studied in systemic inflammation and has been noticed in copd patients. in an inactivated state, nf-κb is located in the cytosol and is complexed with the inhibitory protein inhibitor kappa b (iκb). there are a number of different iκb proteins such as iκbα, iκbβ, iκbγ, iκbɛ and bcl- . iκbβ is only phosphorylated by certain stimuli including lps (lipopolysaccharide) and il- β, whereas iκbα phosphorylation is triggered by most nf-κb activators. when a variety of extracellular stimuli act on receptors of the respiratory epithelium, iκb kinase (ikk) is activated. ikk, in turn, phosphorylates iκbα, resulting in ubiquitination and dissociation of iκbα from nf-κb. nf-κb (p and p ) is then translocated into the nucleus where it binds to specific sequences of dna to cause an inflammatory response. , nf-κb activates proinflammatory genes encoding cytokines and chemokines, such as il- β, il- and tnf-α. the cytokines produced by nf-κb pathway play essential roles in inflammatory cell migration and strengthen oxidative stress during copd development, further aggravating the condition. , both passive smoking and an intratracheal infusion of lps induce copd in rats via the nf-κb signalling pathway. in respiratory tract biopsies from copd patients, activated nf-κb levels were significantly higher than that of normal people, while the level of iκb in the lung tissue from smokers or copd patients was significantly lower than that of non-smoking healthy individuals. overexpression of ikk-β in mouse airway epithelial cells results in an increase in inflammatory mediators and neutrophilic inflammation that is reminiscent of the copd airway following bacterial challenge. in addition, inhibition of ikk-β in vivo and in vitro reduced tnf-α induced muc ac production. this indicates that the nf-κb pathway plays an important role in the occurrence and development of copd. copd and the p mapk p mapks are members of the mapk family activated by a variety of environmental stresses and inflammatory cytokines. it seems to be the most effective mapk, in stabilising, at post-transcriptional level, the mrnas for cytokines and chemokines relevant to copd pathogenesis. p mapks are divided into four subtypes: p α, p β, p δ and p γ. different subtypes are expressed in different tissues, and p α is most abundant in inflammatory cells. cs, lps, inflammatory factors and oxidative stress activate the p mapk pathway. in the airways and sputum of patients with copd, p mapk was significantly increased, and its activation was related to the severity of copd. in addition, the common pathogenic bacteria of copd, nontypeable haemophilus influenzae, contains cytoplasmic proteins that up-regulate human muc ac mucin transcription via a positive p mapk pathway and a negative phosphoinositide -kinase-akt pathway. the activation of p mapk associated with the degree of lung function impairment and alveolar wall inflammation. bacterial or viral infection is a common trigger for copd exacerbations, and exposure to lps induces p mapk activation in rat peritoneal macrophages and dendritic cells as well as an increase expression of inflammatory mediators. , glucocorticoid resistance in copd patients may also be related to the p mapk pathway through phosphorylated glucocorticoid receptor (gr) reducing gr translocation into the nucleus and dna binding, impairing the gr function. , the anti-inflammatory effects of corticosteroids are mediated by gr, and functional impairment of gr is an important mechanism of glucocorticoid resistance. copd and the pi k/akt pathways the phosphatidylinositol kinase (pi k) pathway is a major pathway regulating cell growth, proliferation, metabolism, survival and angiogenesis. serine/threonine kinase (akt), also known as protein kinase b, is an enzyme consisting of a ph domain, a kinase catalytic domain, and a regulatory domain that covalently attaches atp-phosphate groups to the serine/threonine of protein substrates to alter target protein activity. activated pi k phosphorylates phosphatidylinositol diphosphate to produce the secondary messenger phosphatidylinositol , , triphosphate, which binds the ph domains of akt and phosphoinositide dependent kinase- . akt undergoes a conformational change and is transferred to the plasma membrane, leading to akt activation. activated akt regulates cellular functions by phosphorylating various downstream factors including enzymes, kinases and transcription factors. , dysregulation of akt activity impacts on all these essential cellular processes, such as cell growth, survival and inflammation. the pi k/akt signalling pathway plays an important role in copd by regulating inflammatory cell activation, inflammatory mediator release and airway remodelling. the regulation of pi k/akt pathway in neutrophil restore some key copd neutrophil responses. pi k/ akt pathway regulates macrophage polarisation in emphysematous mice generated by cs exposure combined with intraperitoneal injection of cse. ecm proteins promoted proliferation, migration and adhesion of airway smooth muscle cells form rat models of copd through activation of the pi k/akt signalling pathway. ros activates pi k, initiating the pi k/akt signalling pathway. pi k/akt pathway plays an important role in the activation of nrf , which regulates oxidative stress and inflammation in copd. the persistent airway and lung inflammation of copd is related to a decreased histone deacetylase activity (mainly hdac ) caused by oxidative stress. up-regulation of pi kδ/akt signalling reduces the hdac activity, promoting the transcriptional activation of inflammatory genes. another study found that down-regulation of hdac is associated with glucocorticoid resistance. as the inflammatory signalling pathways closely associated with copd development have been elucidated, most candidate therapeutic drugs developed in recent years are molecular drugs targeting these signal transmitting substances. the following sections outline new copd treatment strategies (fig. ). oxidative stress plays a crucial role in the pathogenesis of copd. in the exhaled breath condensate of copd patients, the levels of oxidative stress markers such as hydrogen peroxide (h o ) and isoprostaglandin ( -ip) are significantly increased. through in vivo experiments, antioxidants have been shown to inhibit copd onset. n-acetylcysteine (nac) and other glutamines have been clinically tested in this regard. despite evidence of the beneficial role of nac in copd, its therapeutic efficacy in clinical management of copd has remained controversial due to its reduced bioavailability in an oral form and its acidic nature prohibiting its use in an inhaled form. in addition, the lack of assurance that there is an effective concentration of glutathione in the lung is another major reason for not achieving the desired therapeutic effect. other antioxidant enzymes, such as superoxide dismutase and glutathione peroxidase, have good antiinflammatory effects on smoking-induced lung inflammation in animal models, and clinical trials are underway. copd is also affected by oxidative stress and nitrosative stress. therefore, nitric oxide synthase inhibitors being developed for acute diseases may also be suitable to treat copd. the antioxidant transcription factor nrf downregulates inflammation-associated production of ros and reactive nitrogen species. nrf -deficient mice exposed to cs had more extensive emphysema and more pronounced airway inflammation than wild-type mice. as a small molecule nrf activator, sulforaphane increased the gene expression of nrf , reduced the level of ros, and prevented the damage of cse-treated alveolar epithelial cells. the natural product sulforaphane activated nrf in alveolar macrophage isolated from copd patients denitrify hdac and restore the sensitivity to the glucocorticoid dexamethasone in a glutathione-dependent manner. unfortunately, sulforaphane applied for weeks to patients with copd did not induce the expression of nrf genes or have an effect on oxidative stress, airway inflammation or lung function. resveratrol is a natural sirt activator. in the rat model of copd treated with resveratrol, serum il- and il- levels were decreased and lung inflammation was inhibited. in addition, resveratrol activation of sirt also downregulates the activity of mmp in fibroblasts in copd. in recent years, it has been found that srt is a compound that can activate sirt , which can improve lung function and reduce lung damage caused by cs (table ) . regulating the imbalance between proteases and their inhibitors has attracted much attention as a treatment for copd. mmps which degrade elastin are a target for drug development. nonspecific mmp inhibitors are mainly developed as anticancer agents, but they have been reported to cause side effects such as arthritis, so long-term use as a copd treatment drug may have hidden dangers. mmp- is a member of the mmp family and its endogenous inhibitor is timp- , which binds the carboxyl terminal of the catalytic centre of mmp- to form an enzyme-inhibitor complex through noncovalent bonding. both are key enzymes regulating ecm degradation and synthesis, and changes in their concentrations are closely related to an airway inflammation damage. through culturing balf cells of copd patients with healthy individuals, it was found that mmp- release by macrophages in copd patients was significantly increased while healthy control macrophages released more timps. selective inhibitors of mmp- are also being developed and have proven to be effective in treating copd in animal models, but have not been effective in clinical trials of copd. ne inhibitors are also a valuable potential therapeutic drug, which can not only protect the lung from ne-mediated tissue damage, but also control the exuberant inflammatory response. japan approved sivelestat (ono- ) for the treatment of ali and ards. however, many countries have not approved siveles for clinical use, due to the uncertainty of the randomised doubleblind trial results. other promising ne inhibitors have also been stopped for various reasons. azd ( weeks) combined with budesonide/formoterol has no effect on lung function, quality of life and lung function in copd patients (table ) . the levels of il- β, il- , tnf-α and il- are significantly increased in the sputum and serum of copd patients. they may also be a therapeutic target for copd. as a human anti-il- β monoclonal antibody, canakinumab ( -week treatment) showed no statistical analysis provided for lung function changes. the receptor inhibitor tocilizumab is an available il- inhibitor with confirmed efficacy in rheumatoid arthritis, fig. new molecular targeted drugs. based on the molecular mechanism of copd, many new molecular targeted drugs have been developing in recent years. antioxidants scavenge ros and inhibit oxidative stress in the lungs and reduce cellular damage and inflammation. protease inhibitors restore the balance between protease and anti-protease by inhibiting. proteases. cytokine and chemokine inhibitors play an important role in reducing the inflammatory response. adhesion molecule inhibitors can block inflammatory cells, which continuously migrate from the blood vessels to the tissue. pde inhibitors inhibit pde production to increase the camp activity in cells. in the occurrence and development of copd, the signalling molecules, such as nf-κb, mapk, pi k and vip help regulate inflammation and airway remodellings, and represent plausible targets for the development of therapeutic candidates. candidate drugs include inhibitors of p mapk, nf-κb and pi k, and vasoactive intestinal peptide (vip). the inhibitor of egfr reduces internalisation of egfr but does not reduce mucin stores. tgf-β inhibitor reduces a fibrotic airway remodelling and downregulates mmp expression, endothelin inhibitors prevent the progression of pulmonary hypertension in copd. adenosine a a receptor inhibits neutrophil superoxide production, phagocytosis, adhesion and cytokine release. macrolides reduces the inflammation of copd by regulating the pi k/akt-nrf pathway and control transcription factors such as nf-κb and ap- to inhibit the production of inflammatory cytokines. ppar agonists exert antioxidant and anti-inflammatory effects by downregulating nf-κb and other pro-inflammatory transcription factors but clinical trials in copd require further study. endogenous tnf-α plays an important role in the development of pulmonary fibrosis and causes a secondary interstitial lung disease. as anti-tnf-α therapies, the tnf-α antibody (infliximab) and soluble tnf-α inhibitor (etanercept) have been used as clinical drugs, mainly to treat inflammatory diseases such as rheumatoid arthritis; and the same clinical dose of the tnf-α antibody used in rheumatoid arthritis has a definite effect on bronchial asthma, but has not been confirmed in copd. in contrast, copd patients had significantly increased incidence of airway tumours and lung infections caused by tnf-α antibodies, presenting a difficult problem to be overcome with future anti-tnf-α treatments. inhibitors of the il- receptor cxc chemokine receptor (cxcr ) effectively block neutrophil infiltration in the lung tissue in animal models and clinical trials. cxcr inhibitors block neutrophil invasion and inhibit mucus production and airway remodelling. however, it should be noted that cxcr inhibition triggers side effects similar to those exhibited by the use of glucocorticoids, such as promoting bacterial/ fungal infection and delaying wound healing. currently, there are clinical trials in progress for the cxcr receptor antagonists in copd, bronchial asthma and cystic fibrosis. ccr has an affinity for multiple chemokines, including (mip- α/ ccl ) and regulated on activation, normal t-cell expressed and secreted (rantes/ccl ), which are both elevated in lungs of copd patients. , ccr antagonists have been developed and are in clinical trials for autoimmune diseases. ccr which is the only receptor for (mcp- /ccl recruits inflammatory cells to lungs in copd, and increases synthesis of muc ac and muc b. the levels of both ccr and mcp- /ccl mrna were increased in bronchial epithelium of copd patients. mcp- /ccl production upon cigarette smoke exposure were increased in a mouse model of copd, ccr inhibitors for copd have been studied, but statistical analysis not released. ltb is elevated to some extent in the balf, sputum, serum and lung tissues of copd patients, and is positively correlated with the copd severity. several inhibitors are under development, one of which has entered clinical trials, but no effective anti-inflammatory effect has been demonstrated. in recent years, a -lox inhibitor upstream of ltb has been under development for bronchial asthma, but current -lox inhibitors have problems such as lack of selectivity, structure-activity relationships, methaemoglobin formation, and poor efficiency and oral availability. it should be noted that inhibiting ltb biosynthesis at the level of -lox or flap removes the lta intermediate. however, the latter molecule is an important intermediate in the biosynthesis of anti-inflammatory lipoxins, potentially reducing the net anti-inflammatory effect. in addition, some lta h inhibitors are also being developed , because these have been suggested to block ltb production while preserving lta , allowing shunting into lipoxin a . this features may make lta h inhibitors superior therapeutic molecules as compared with -lox or flap inhibitors (table ) . in the inflammation process of copd, selectins are essential for the migration of inflammatory cells from the bloodstream into the pulmonary tissue. therefore, targeting these molecules may inhibit the inflammatory process of copd. although several selectin inhibitors have been tested in clinical trials for bronchial asthma protect the lung from ne-mediated tissue damage and control the exuberant inflammatory response azd ( weeks) combined with budesonide/ formoterol has no effect on lung function, quality of life and lung function in copd patients patients, they have not proven to be effective. bimosiamose, a pan-selectin antagonist, blocks the adhesion of neutrophils, eosinophils and lymphocytes in vitro, and has anti-inflammatory effect in animal models of lung inflammation. bimosiamose inhalation has good safety and tolerance for copd patients. it reduces the levels of il- and mmp- in the sputum and reduces the number of neutrophils, reduces airway inflammation and improves the lung function, thus warranting further testing. el is an antiselectin monoclonal antibody that recognises specific positions on the e and l selectins to inhibit cell adhesion. el is currently being developed as a therapeutic drug for acute exacerbation of copd. it is clear that further studies are required to demonstrate the true clinical benefits of bimosiamose and el in copd patients. because e, l, p selectins recognise and bind to epitopes containing the carbohydrate sle x of glycoprotein or glycolipidon on a cell surface. a clinical study of -o, -o desulfated heparin (odsh or pgx- ) which is a carbohydrate-based drug was performed in phase ii for copd. however, the trial has been terminated because the interim analysis showed that odsh had no effect on the safety of patients with acute exacerbation of copd (table ) . egfr and tgf inhibitor egfr regulates mucin stores in airway epithelium, which are significantly increased in copd. bibw is the inhibitor of egfr, and inhalation of bibw ( -week treatment) reduced internalisation of egfr but did not reduce mucin stores. inhibition of tgf-β signalling may also be a useful therapeutic strategy in copd. the bone morphogenic protein and activin membrane-bound inhibitor (bambi) is a transmembrane glycoprotein, which acts as a negative regulator of tgf β signalling. bambi is induced by members of the tgf family-²-catenin, smad and smad , acting as a pseudoreceptor. bambi expression as significantly stronger in copd patients and that increased plasma bambi levels in copd patients displayed excellent correlations with enhanced plasma tgf-β levels. the because the mechanisms regulating bambi expression are poorly understood, the clinical trail of bambi is not developed. in addition, small molecule antagonists which inhibit tgf-β receptor kinase or tgf-β activated pathways were studied, , although the long-term safety of such drugs might be a problem, particularly as tgf-β affects tissue repair and is a potent anti-inflammatory mediator (table ) . pde inhibitors pde inhibitors have a selective inhibitory effect on pde specifically expressed in inflammatory, airway smooth muscle and epithelial cells. its main biological effects are selective inhibition of pde , leading to increased camp levels in inflammatory cells, regulating the activity of inflammatory cells and regulating the release of inflammatory factors to exhibit antiinflammatory effects. they have been developed as new antiinflammatory drugs for copd and asthma since the s. the pde inhibitor roflumilast has been approved by the food and drug administration as a copd treatment. it was shown roflumilast reduced the diffuse emphysema induced by cs in mice as compared with that in the control group. other studies have found that roflumilast inhibits bleomycin-induced pulmonary fibrosis in rats and reduces pulmonary vascular remodelling. , clinical trials have proven that roflumilast relieves the symptoms of dyspnoea in copd patients and reduces the frequency of acute attacks. once-daily administration of roflumilast significantly improves forced expiratory volume in s and decreases exacerbations, particularly in patients with severe disease, , but has side effects such as nausea, vomiting and headache. there are mainly four subtypes of pde , a to d. in recent years, the antiinflammatory effect of pde inhibitors has been shown to be mainly related to pde b while pde d is related to side effects such as digestive tract symptoms. the development of new pde b subtype-specific agents is expected, and inhalants that reduce systemic side effects are also being developed. in addition, oher pde inhibitors is being developed now [ ] [ ] [ ] [ ] (table ) . endothelin signalling plays a major role in pulmonary hypertension secondary to copd. endothelin antagonises bosentan (treatment for months) can alleviate the condition of copd patients and prevent the progression of pulmonary hypertension. this effect is more significant in gold iii and iv patients. but for copd patients without pulmonary hypertension, bosentan will aggravate their hypoxaemia. at present, further research on the role of bosentan in patients with gold iii or iv copd and pulmonary hypertension in the acute exacerbation phase is ongoing, but its status is unclear (table ) . vasoactive intestinal peptide vasoactive intestinal peptide (vip) has been characterised as a vasodilatory peptide. it exerts a wide range of biological actions, such as bronchodilation, anti-inflammatory effects, via binding its receptor vpac or vpac to increase significantly camp, adenylate cylase and phospholipase c, which cause different downregulation on a variety of transcription factors. vpac receptor mrna is abundant in lung and t lymphocytes. vpac receptor is mainly distributed in the smooth muscle layer and the base of mucosal epithelium in lung. vpac was particularly elevated in alveolar macrophages of copd patients. vpac receptor was activated by vip, and inhibited the cseinduced cytotoxicity of rat lung alveolar l cells. increased serum vip levels are associated with acute exacerbation of copd patients. vip has significant therapeutic potential in the treatment of copd. how, it clinical application might be limited because of the short half-life of plasma after intravenous administration and the difficulty of routes. (table ) . adenosine a a receptor agonist adenosine is a natural purine nucleoside that is ubiquitous in human tissues and plays a key role in many biological processes, such as energy production and protein metabolism. at present, four subtypes (a , a a , a b and a ) of adenosine receptors have been cloned. the anti-inflammatory effect of adenosine is mainly attributed to occupancy of camp-elevating gs-protein-coupled , [ ] [ ] [ ] [ ] [ ] bosentan blocks endothelin receptor. bosentan ( months) can alleviate the condition of copd patients and prevent the progression of pulmonary hypertension. this effect is more significant in gold iii and iv patients. but for copd patients without pulmonary hypertension, bosentan will aggravate their hypoxaemia. , solithromycin decrease the production of proinflammatory cytokines and chemokines by epithelial and immune cells a macrolide antibiotic. no data of solithromycin ( days) collected for this outcome due to early termination of the trial (nct ) ppar agonists regulates function of multiple cells of the immune system. pparγ agonists includes troglitazone, rosiglitazone, and pioglitazone. pparα agonists includes clofibrate and fenofibrate. patients who took more than two thiazolidinediones ( . % rosiglitazone) had significantly less copd deterioration than patients receiving other diabetes drugs. results information of clinical trail (february, months) has been submitted to clinicaltrials. gov by the sponsor or investigator, but is not yet publicly available on clinicaltrials.gov (nct ) , a a-receptors. the key molecular mechanism is the suppression of nf-κb pathway activated by cytokines such as tnf-α and il- β. a a receptor plays an anti-inflammatory role in specific cells and various inflammation models. knockout of the a a receptor gene will cause mucus production, airway destruction and lung inflammation. currently, several adenosine a a receptor agonists have been proven effective in copd models, but there are cardiovascular side effects. a phase , randomised, double-blind study (nct ) assessed the safety of the selective adenosine a a receptor agonist, regadenoson, compared with placebo in subjects with asthma or copd, and the result showed randomised did not modify repeated forced expiratory volume in s (fev ) when compared to placebo. uk , , which is beneficial in the lungs of anaesthetised guinea pig without any obvious cardiovascular sideeffects. phosphorylated a a receptor agonists are under development to reduce adverse effects such as hypotension (table ) . macrolide antibiotics are secondary metabolites of a variety of actinomycetes bacteria. the molecule contains a - -membered macrolide structure, which has a wide range of functions. it has not only antibacterial function, but also anti-inflammatory effect, inhibition of mucus secretion and immune regulation. , the anti-inflammatory and immunomodulatory functions are mainly -ring and -ring. clinically, they are used for long-term treatment of chronic inflammatory lung diseases such as copd. the anti-inflammatory mechanism of macrolide antibiotics is complex and has not been fully elucidated. it may reduce the inflammation of copd by regulating the pi k/akt-nrf pathway and control transcription factors such as nf-κb and ap- to inhibit the production of inflammatory cytokines. animal models provided further evidence that clarithromycin has an inhibitory effect on the development of emphysema, and its dose is almost the same as the clinical dose. the potential benefit of a new antibiotic, solithromycin was studied for the long-term treatment of copd, but due to the early termination of the study, there were too few subjects and data collected to perform statistical analysis. other macrolides without anti-bacterial activity are being developed as anti-inflammatory drugs and clinical trials are expected in the future (table ) . ppar agonists peroxisome proliferator activated receptors (ppars) are ligandactivated nuclear hormone receptors belonging to the steroid receptor superfamily, including three recognised subtypes (pparα, pparγ and pparδ). the activation of pparγ and pparα may have anti-inflammatory and immunomodulatory effects. pparγ exert antioxidant and anti-inflammatory effects by down-regulating nf-κb and other pro-inflammatory transcription factors. cigarette smoke will down-regulate the expression of pparγ, and the level of pparγ in the lung tissues of copd patients is significantly lower than that of normal people. pparγ agonists, such as the thiazolidinediones, rosiglitazone and pioglitazone, have been shown to reduce lung inflammation in mouse models of tobacco smoke, and studies have found that treating model mice with thiazolidinedione can reverse emphysema. , in addition, pparγ agonists can also inhibit pulmonary fibrosis, which is expected to be a drug to prevent small airway fibrosis in copd. a retrospective epidemiological study showed that diabetes patients treated with pparγ agonists have a significantly reduced risk of copd exacerbation, but their risk of cardiovascular risk events has also increased. in addition, only large doses of thiazolidinediones can produce anti-inflammatory effects, which leads to speculation about the correlation of pparγ stimulation in copd. non-thiazolidinedione pparγ ligands are currently being studied to reduce potential cardiovascular risks. pparα agonists, such as fenofibrate, may have therapeutic potential in the treatment of systemic symptoms of copd (table ) . a japanese drug discovery company is developing a compound code named imd- , which is an ikk β inhibitor developed for the treatment of copd, but has no follow-up information posted since april . it is unclear whether study was performed. bms- is a highly selective ikk inhibitor with good pharmacokinetic characteristics. in the human airway smooth muscle cells, co-incubation with bms- markedly inhibited the nf-κb nuclear translocation induced by tnf-α and il- . ps- induce a dose-dependent inhibition of phosphorylated ikbα and nf-κb activation, and then reduces the expression of adhesion molecules, cytokines and chemokines on airway smooth muscle cells. a small molecule ikk inhibitor is under development as a therapy for inhibiting the nf-κb activity. the effectiveness of ikk inhibitors has been verified in animal models of copd; and clinical trials of ikk inhibitors in patients with bronchial asthma and joint rheumatism have also been conducted. ikk inhibitors are expected to be used as a new therapeutic drug for copd in the future following in-depth research. further developments include nf-κb "decoy" oligonucleotides and antisense and small interfering rna agents. , in addition, nf-κb-deficient mice have been reported to be more prone to sepsis, hence complications such as immunosuppression and infection susceptibility caused by long-term nf-κb inhibition must be considered (table ) . as a new type of anti-inflammatory drug, p mapk inhibitors have attracted much attention from researchers. currently, various small-molecule p mapk inhibitors have been developed and verified in animal models of smoking-induced pneumonia, proving their beneficial anti-inflammatory effects. inhibiting p mapk activation has been found to reduce the cs-induced airway smooth muscle cell proliferation, suggesting that p mapk inhibitors may reduce the progression of copd airway remodelling. p mapk inhibitors also reduce cytokine production by alveolar macrophages. in addition, there is evidence that corticosteroids cannot inhibit p mapk activation, and that p mapk inhibitors combined with corticosteroids enhance the inhibitory effect of corticosteroids on cytokines produced by macrophages in patients with copd mediated by lps. p mapk inhibitors have a unique advantage in patients with a poor hormone response. a days trial of p mapk inhibitor sb in patients with moderate stable copd reduced sputum neutrophils and plasma fibrinogen with improvement in forced vital capacity. the patients with moderate to severe copd receiving p mapk inhibitor ph for week decreased serum crp levels, and induced a significant increase of fev and a concomitant improvement in dyspnoea score. each subtype of p mapk has unique functions due to differential expression across tissue types and different regulatory kinases and downstream genes, hence their targeting comes with adverse effects. although some clinical trials are in progress, due to severe side effects such as those caused by an undesired pharmacological activity, suppression of the innate immune response to viral and bacterial infections, and damage to the central nervous system and liver, , these drugs remain challenging for a clinical application. there is a need to develop inhaled formulations and selective inhibitors of the α-δ subgroups. the inhaled narrow spectrum kinase (p α + src family) inhibitor (jnj /rv ) in patients with moderate to severe copd decreased serum crp levels as well as improved trough fev and dyspnoea index scores. however, p αmapk inhibitors block the upstream mapk kinase kinases, leading to hyperactivation of the transforming growth factor-activated kinase- and mixed-lineage kinase which then hyperactivate the jnk. therefore, other drugs that target more downstream substrates should be also developed (table ) . pi k inhibitors pi k is divided into three categories, namely classes i, ii and iii, among which class i pi k is most widely studied. class i pi k is a heterodimer composed of a regulatory subunit (p ) and a catalytic subunit (p ). there are four types of catalytic subunits: p α, p β, p δ and p γ, and while δ and γ subunits are limited to white blood cells, α and β subunits are widely distributed in various cells. pi k, especially pi k δ and γ subtypes, are closely related to a copd inflammation. pi k inhibitors reduce nitric oxide production by inhibiting carbon monoxide synthase. studies have shown that interruption of the pi k pathway improves severe copd protease imbalance. aerosolized tg - , a compound that selectively blocks pi kγ and pi kδ, inhibits pulmonary neutrophils induced by intranasal lps and smoke in mice with chronic obstructive pulmonary disease. as is a selective inhibitor of pi kγ, which reduces the migration of polymorphonuclear leucocytes in vitro and the infiltration of polymorphonuclear leucocytes in the lungs of mice with lps induced lung injury. the interventional therapy of tg - was successful even in steroid resistant copd induced by smoking in mice. various pi k inhibitors are currently being used in clinical trials, primarily for malignant tumours. , in recent years, inhaled pi kγ/δ inhibitors have been reported to inhibit pneumonia caused by smoking in animal models and have been especially effective and safe for patients with glucocorticoid contraindications. specific pi kδ inhibitors, gsk and rv are being developed, and studies on the effects of such inhibitors in copd are in progress. efficacy data remain limited. , in contrast, even selective p k subtype inhibitors have the risk of immunosuppression and secondary bacterial infections, and reducing the occurrence of side effects will be an important issue (table ) . trx is a multifunctional protein consisting of amino acids with a molecular weight of kda and a highly conserved cys-gly-pro-cys active site. trx exists in two forms: oxidised (trx-s ) and reduced (trx-(sh) ). trx-s can be reduced to trx-(sh) by the exchange reaction of -ss-and -sh under the action of thioredoxin reductase (trxr) and nicotinamide adenine dinucleotide phosphate (nadph). trx cooperates with trxr and nadph to form the trx system. trx catalyses the reduction of disulphide bonds and quenches ros by coupling with trx-dependent peroxidases or peroxiredoxins. in addition to its antioxidant effects, trx has a crucial role in the redox regulation of cellular signalling and activation. trx is involved in various redox-dependent cellular processes such as gene expression, signal transduction, cell growth, apoptosis and interacts with various target molecules. , under stress conditions, trx is released into the extracellular space where it exerts a cytoprotective effect and cytokine-like activities. trx expression in the sputum of copd patients is positively correlated with the degree of hypoxia. mice that overexpress human trx can effectively inhibit a cs-induced emphysema and pulmonary inflammation. intraperitoneal injection of trx suppress a smoke-induced murine pulmonary inflammation by inhibiting the production and release of cytokines, inflammatory mediators, chemokines and ros. trx inducer increases the trx expression in murine lung tissue and improves lung injury. recent research has also shown that inhaled trx also reduces a smoke-induced chronic lung injury. currently, clinical trials targeting acute lung diseases have entered the preparation stage. at the same time, as a pre-clinical trial of copd, intravenous infusion therapy for acute exacerbations, protein inhalation therapy for stable phase, and oral administration of inducers are also underway. adenosine a a receptor exert anti-inflammatory effect by enhancing camp regadenoson group ( months) occurred with higher incidence of dyspnoea, and unable to modify repeated fev when compared to placebo ((nct ). uk , is beneficial in the lungs of anaesthetised guinea pig without any obvious cardiovascular sideeffects. but uk- ( -week inhaled treatment) in dbpcrt showed no significant improvement in fev and quality of life parameters (nct ). , , progress in the mechanism and targeted drug therapy for copd wang et al. adjusting the redox balance trx plays an important role in maintaining the body's redox balance. trx can be used as an electron donor to reduce h o and tertiary butyl hydroperoxide. in addition, when the body's peroxidase reduces hydrogen peroxide, trx is also needed to provide a certain reduction equivalent. further, there are other redox systems similar to the trx system in the body, such as the glutathione (gsh) system. the trx system and the gsh system jointly control the redox system. the equilibrium state of the environment, and the trx and gsh systems cross each other to provide electrons and serve as a compensation system. , in addition, trx and thioredoxin-interacting protein (txnip), a negative regulator, constitute a redox-like protein compound (redoxisome) that regulates a variety of redox-sensitive signals and is essential for maintaining the intracellular and extracellular redox balance and monitoring inflammatory responses. without an oxidative stress, txnip is in a bound state with trx. when the intracellular ros content increases, trx and txnip are dissociated, and trx bind to ros. dissociated txnip combines with and activates the nlrp inflammasome to induce il- β expression in a nlrp -asc-caspase- -dependent manner, thus causing inflammatory reactions. this signalling complex may be a key regulatory mechanism for the body to regulate cellular redox balance and prevent the progression or exacerbation of stressinduced diseases. regulating the protease balance both endogenous and exogenous trx inhibit and improve a protease-induced emphysema. mmp- and mmp- play important roles in copd. oxidative stress upregulates the mmp- expression while trx inhibits mmp- via its antioxidant properties. , the mechanism may be inhibition of p mapk and jnk. in addition to inhibiting mmp, trx also inhibits its inhibitor, timp. studies have found that trx has a differential inhibitory effect on mmp- , mmp- , timp- and timp- , thereby regulating the mmp/timp balance. trx suppresses mmp and timp by reducing their activity but not degrading timp and mmp. , the activity of timp- , timp- or mmp- was not inhibited by a version of trx lacking a disulphide reductase activity or trx with a trxr deficiency. , this indicates that trx regulates the mmp/timp balance by differentially inhibiting the activities of timp and mmp, and this process depends on the redox active sites of trx and trxr. regulating inflammatory cells, cytokines and chemokines trx inhibits the migration and activation of inflammatory cells such as neutrophils, reduces the release of inflammatory factors, and reduces the inflammatory response. both in vivo and in vitro studies have shown that trx inhibits the chemotaxis of macrophages, lymphocytes, and neutrophils. , during copd development, the neutrophils and alveolar macrophages are activated to produce various inflammatory mediators, including il- β, il- , il- and tnf-α. trx significantly inhibits the production and release of il- β, il- , il- and tnf-α induced by lps in the human monocyte-derived macrophages. this is achieved by trx blocking the nf-κb pathway or inhibiting an inflammatory signal activation by cell surface molecules. , the specific mechanism of action has been thoroughly explained in our previous article. regulating adhesion factors and growth factors l-selectin (cd l) is a shedding molecule on neutrophils that plays a vital role in guiding neutrophils to adhere to the vascular endothelium and penetrate the blood vessels. trx inhibited the lpsinduced downregulation of l-selectin exfoliation on neutrophils and reduced the l-selectin attachment to endothelial cells whereas double-mutant trx c s/c s did not inhibit neutrophil adhesion to the endothelial cells. in a rat model of lps-induced inflammation, systemic trx significantly reduced neutrophil infiltration in the bronchus and lung tissues, but did not directly reduce the increased lps-induced icam- present on the endothelial cells. trx undergoes oxidation in response to egf. the local and specific oxidation of trx occurs during the ros signalling produced by egf stimulation. egfr signal transduction requires a special endoplasmic reticulum trx to control receptor expression on the cell surface. intracellular trx inhibits egfr synthesis and activation. tgf-β activates smad / , pi k/akt, erk / , gsk- β and/ or p mapk signalling to induce pulmonary fibrosis and emt. , trx inhibits the mpk -induced tgf-β function in a phosphorylation-dependent mannerm. trx inhibits bleomycininduced skin fibrosis by down-regulating tgf-β. trx overexpression inhibits airway remodelling by inhibiting tgf-β and egfr. regulating camp camp plays a protective role in copd inflammation through its effector proteins epac and pka. in animal models of brain injury, trx protected the astrocytes by activating camp-pka and inhibiting astrocyte apoptosis. down-regulating the trx gene inhibits the camp-pka pathway to cause apoptosis, exacerbating astrocyte damage caused by an oxygen-glucose deprivation/ reoxygenation in vitro. trx is necessary for the nerve growth factor (ngf) signalling through its camp responsive element to drive expression of c-fos, which has been hypothesised to be critical for the function of ngf. pka activity is inhibited by the hydrogen peroxide formed by insulin but activated by trx, which restores the newly formed -ss-bond of pka to the -sh group. we suggest that trx may also protect lung cells by acting on the camp-pka pathway in copd. regulating the nf-κb and mapk pathways trx suppresses the inflammatory response by regulating the nf-κb and mapk pathways. trx modulates nf-κb activity and plays different roles extracellularly and intracellularly. inhibiting the nuclear trx blocks the nuclear activities of nf-κb and ap- dependent transcription factors and reduces neutrophil invasion and tnf-α production. extracellular trx inhibits the production of p and p and promotes iκb synthesis by acting on cell membrane surface receptors to limit nf-κb activation and translocation into the nucleus, thereby blocking the nf-κb pathway. trx inhibits eotaxin-induced phosphorylation of extracellular signal-regulated kinase / and p by reducing the activation of erk / and p mapks. under normal conditions, trx binds to the n-terminal region of the apoptosis signal-regulating kinase (ask ). ask is a member of the mapk kinase family and elicits a wide variety of cellular responses to various types of stress by activating the jnk and p mapk pathways. under oxidative stress, trx separates from ask , activating ask . this indicates that trx acts as an upstream inhibitor of ask , and trx/ask signalling is an upstream modulator of p mapk. trx suppresses p mapk activation in the lps-stimulated neutrophils. in addition, ros exacerbates airway inflammation by activating inflammatory mediators and transcription factors, such as nf-κb, mapk and ap- . this suggests that trx regulates the p mapk pathway by removing ros. adjusting the pi k/akt signalling pathway the pi k/akt signalling pathway is a classic signalling channel that can be activated by various extracellular signals including growth factors, cytokines, and oxidative stress, and plays an important role in copd. trx inhibits the indomethacin-induced ros production and inhibits the expression of phosphorylated akt in rat gastric epithelial cells. in addition, trx deficiency reduces the expression of akt and pakt by no. the main activation signals of pi k/akt signalling are inhibited by trx, and downstream akt phosphorylation was also inhibited by trx. therefore, we suggest that trx likely plays an important role in the pi k/akt signalling pathway. improving glucocorticoid resistance copd is relatively resistant to modulation by corticosteroids; even high doses of glucocorticoid (gc) do not delay or inhibit copd progression. one mechanism of gc resistance is the impairment of gc sensitivity by the macrophage migration inhibitory factor (mif) via map kinase phosphatase- (mkp- ) inhibition. mif is part of a class of pleiotropic immunomodulatory factors with a unique structure that functions similar to chemokines and promotes inflammatory responses, directed cell migration, and release of other cellular inflammatory factors. mif may play a key role in the pathogenesis of airway inflammation. [ ] [ ] [ ] mif has at least two catalytic activities: tautomerase and oxidoreductase activities. the oxidoreductase activity of mif is closely related to the trx family. , - mkp- is an archetypal member of the dual specificity phosphatase family that inactivates the mapk activity in response to pro-inflammatory stimuli. - mkp- is induced by gc to mediate gc inhibition of erk, jnk and p mapk activities and cytokine production induced by proinflammatory stimuli such as lps or il- . [ ] [ ] [ ] it has recently been demonstrated that mif inhibits the gc-induced leucine zipper (gilz) expression via a unique set of effects on transcription factor expression and phosphorylation, and regulation by mif of mkp- and mapk activation are mediated through gilz (fig. ). fig. trx improves gc through mif. one gc resistance mechanism impaired by the mif is the loss of gc sensitivity via inhibition of mkp- . mkp- is induced by gc to mediate gc inhibition of erk, jnk and p mapk activities and cytokine production. mif inhibits gilz expression via a unique set of effects on transcription factor expression and phosphorylation. mkp- and mapk activation are regulated by mif via gilz. both intracellular and extracellular trx bind to mif and form a heterodimer to prevent the mif entry into cells and mif-induced glucocorticoid resistance fig. trx prevents and relieves copd pathogenesis through multiple molecular mechanisms. trx eliminates mif to improve glucocorticoid resistance and eliminates ros and inhibits neutrophil infiltration by regulating adhesion molecules to suppress the production of cytokines to reduce oxidative stress and inflammation. trx exerts its anti-oxidative and anti-inflammatory effects by regulating the nf-κb, mapk, pi k/akt and camp-pka pathways. trx also inhibits the airway neutrophil recruitment by down-regulating the expression of neutrophil l-selectin on circulating neutrophils. trx is subtle in regulating the balance between protease and antiprotease. trx has inhibitory effect on both, but it is asymmetric in its inhibition. trx has stronger inhibitory effects on over-generated proteases, thus maintaining the balance of the protease-antiprotease system. moreover, trx down-regulates the expression of egfr and tgf in the airway to reduce mucus secretion, airway remodelling and pulmonary fibrosis trx regulates mif expression levels and inhibits inflammation caused by mif. , in a mouse asthma model, transgenic overexpression of trx significantly reduced mif expression in the airway epithelial cells and reduced the number of mif-positive inflammatory cells in the lung parenchyma. trx inhibits mif production in human monocytes. mif has a specific affinity for trx; extracellular mif is internalised into cells exclusively by binding to trx on the cell membrane. exogenous trx and intracellular trx combine with mif to form a complex which affects the mif-induced inflammatory response. in addition, some studies have also demonstrated that trx directly bind to glucocorticoid receptor and enhance the cell's response to glucocorticoids. , although there are not the evidence showing the effect of trx on hdac , we suppose that trx may increase hdac activity by regulating cellular redox signalling, and we would like to prove this attractive hypothesis in our next studies. effects on the immune system trx has no inhibitory effect on the immune system while regulating inflammatory responses in various inflammation models. , , there is no significant difference in the population and differentiation of immune cells such as the mast cells, dendritic cells, and lymphocytes between trx overexpression and wt animals. oxidative stress promotes the th type immune response by inducing th cell apoptosis and th cell differentiation, breaking the th /th balance, and causing th airway inflammation. , after the ova challenge, il- expression in balf of trx-tg mice was significantly lower than that in wt mice, while serum levels of il- were comparable. this shows that trx inhibits local th cells to push the balance towards th and suppress local inflammation while having no effect on the th / th balance of the systemic immune system. bronchial ln (bln) cells isolated from the trx-tg mice produced an equal amount of th cytokines il- , il- and il- as the bln cells of wt mice after leaving the high trx environment. this shows that trx does not directly affect the th /th proliferation and differentiation, but rather inhibits inflammation by regulating the production and release of th /th cytokines. copd pathogenesis is mainly related to the overexpression of inflammatory mediators and cytokines, the activation of inflammatory signalling pathways, the protease/anti-protease imbalance, and the oxidation-antioxidation imbalance. these factors are interrelated and it is difficult to achieve the desired treatment results through a single target. owing to the overlapping function of molecular targeted inflammatory signals, the degree to which pathogenesis of copd can be prevented if only one pathway is inhibited remains unknown. at present, some drugs have been proven to be effective in animal tests; some are challenging to be used in clinical trials due to significant side effects while others continue to be in the hypothetical stage and have not been proven effective in treating copd. therefore, further studies of the functions and mechanisms of various target molecules are necessary, and their effectiveness and safety through must be evaluated through animal experiments and clinical trials. trx plays an important role in the treatment of copd (fig. ) . its mechanism of action is highly unified with the pathogenesis of copd, and it effectively inhibits the occurrence and development of copd through a variety of mechanisms. trx also improves the gc resistance of copd. whether used as a supplement to existing therapies or for patients with poor response to hormones, trx has unique advantages. therefore, we suggest that trx has good prospects in treating copd and may be an important drug for copd treatment in the future. copd: the dangerous underestimate of % alpha -antitrypsin deficiency accelerated ageing of the lung in copd: new concepts continuing to confront copd international patient survey: methods, copd prevalence, and disease burden in - relation of birth weight and childhood respiratory infection to adult lung function and death from chronic obstructive airways disease mild 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effects of the p mapk inhibitor sb- on blood biomarkers of inflammation in copd patients efficacy and safety of the oral p inhibitor ph- in chronic obstructive pulmonary disease: a randomised clinical trial phosphoinositide -kinase delta (pi kδ) in leukocyte signaling and function emerging pharmaceutical therapies for copd present status and tasks for future of point of care testing il- induces translocation of nf-kappab in cultured human bronchial smooth muscle cells we deeply appreciate prof. takashi inamoto for pointed advice and discussion for writing up this paper. competing interests: the authors declare no competing interests. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -ec c q authors: hsieh, yi-ting; lin, mei-hui; ho, hung-yao; chen, lei-chin; chen, chien-cheng; shu, jwu-ching title: glucose- -phosphate dehydrogenase (g pd)-deficient epithelial cells are less tolerant to infection by staphylococcus aureus date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: ec c q glucose- -phosphate dehydrogenase (g pd) is a key enzyme in the pentose phosphate pathway and provides reducing energy to all cells by maintaining redox balance. the most common clinical manifestations in patients with g pd deficiency are neonatal jaundice and acute hemolytic anemia. the effects of microbial infection in patients with g pd deficiency primarily relate to the hemolytic anemia caused by plasmodium or viral infections and the subsequent medication that is required. we are interested in studying the impact of bacterial infection in g pd-deficient cells. g pd knock down a lung carcinoma cells, together with the common pathogen staphylococcus aureus, were employed in our cell infection model. here, we demonstrate that a lower cell viability was observed among g pd-deficient cells when compared to scramble controls upon bacterial infection using the mtt assay. a significant increase in the intracellular ros was detected among s. aureus-infected g pd-deficient cells by observing dichlorofluorescein (dcf) intensity within cells under a fluorescence microscope and quantifying this signal using flow cytometry. the impairment of ros removal is predicted to enhance apoptotic activity in g pd-deficient cells, and this enhanced apoptosis was observed by annexin v/pi staining under a confocal fluorescence microscope and quantified by flow cytometry. a higher expression level of the intrinsic apoptotic initiator caspase- , as well as the downstream effector caspase- , was detected by western blotting analysis of g pd-deficient cells following bacterial infection. in conclusion, we propose that bacterial infection, perhaps the secreted s. aureus α-hemolysin in this case, promotes the accumulation of intracellular ros in g pd-deficient cells. this would trigger a stronger apoptotic activity through the intrinsic pathway thereby reducing cell viability when compared to wild type cells. glucose- -phosphate dehydrogenase (g pd) is the key enzyme that catalyzes the first reaction, the oxidation of glucose- -phosphate to -phosphogluconolactone, in the pentose phosphate pathway, thereby providing reducing energy to all cells by maintaining the level of the reduced coenzyme nicotinamide adenine dinucleotide phosphate (nadph). nadph plays an important role in maintaining the supply of reduced glutathione to counterbalance oxidantinduced oxidative stress [ ] . redox imbalance may induce cell apoptosis and necrosis, thus highlighting the role of g pd in defending against oxidative damage [ , ] . g pd deficiency is the most prevalent enzyme defect in humans and affects an estimated million people worldwide, especially in populations historically exposed to endemic malaria [ ] . the most common clinical manifestations are neonatal jaundice and acute hemolytic anemia, which is caused by the impairment of the erythrocyte's ability to remove harmful oxidative stress triggered by exogenous agents such as drugs, infection, or fava bean ingestion [ , ] . hemolytic anemia caused by infection and subsequent medication is a clinically important concern in patients with g pd deficiency. this issue has been a primary focus for many decades in relation to efforts to understand the impact of plasmodium infection (malaria) and antimalarial drugs [ , ] . antimicrobial drug-induced hemolysis is considered the most common adverse clinical consequence of g pd deficiency [ ] . it has also been demonstrated that infections caused by certain viruses, such as hepatitis viruses (a, b, and e) and cytomegalovirus, were associated with hemolytic anemia in patients with g pd deficiency [ , ] . recently, it has been shown that infection by particular viruses, such as enterovirus , dengue virus, and coronavirus, was enhanced in g pddeficient cells [ ] [ ] [ ] . however, the impact of bacterial infection on patients with g pd deficiency still remains to be clarified. most studies have focused on investigating the antibiotic-induced hemolysis after treatment for bacterial infection [ ] . in addition, a case report showed that infection by clostridium difficile may have triggered hemolysis and led to severe jaundice in a g pddeficient neonate, while another case report described hemolysis caused by acinetobacter baumannii infection [ , ] . wilmanski and colleagues demonstrated that hyperinflammation (increasing cytokine levels) caused by acute endotoxemia (induced by the injection of escherichia coli lipopolysaccharide) resulted in increased mortality in g pddeficient mice [ ] . several studies also indicated that g pd deficiency in leukocytes can result in chronic granulomatous disease (cgd) and possibly alter the host defense mechanisms for bacterial infections [ ] [ ] [ ] . thus far, the impact of bacterial infection on patients with g pd deficiency has been found to primarily affect the blood cells, leading to hemolysis or immune weakness based upon the above studies. bacterial infection or treatment with septic plasma might induce mitochondrial dysfunction by the accumulation of reactive oxygen species (ros) and nitric oxide radical (no ． ) in lymphocytes or epithelial cells leading to cell apoptosis [ ] [ ] [ ] . therefore, we propose that cells with g pd deficiency may be less tolerant to the oxidative stress caused by bacterial infection. in the present study, we investigate the direct impact of bacterial infection on g pd-deficient epithelial cells using staphylococcus aureus as a model pathogen. s. aureus, which has long been recognized as a major cause of healthcareassociated infections, can result in sepsis and septic shock leading to vascular damage and multiple organ failure. vancomycin is one of the primary choices to treat infections resulting from multidrug-resistant s. aureus, including methicillin-resistant s. aureus (mrsa). our previous study demonstrated that the vancomycin-treated vancomycinresistant s. aureus (vrsa) strain did enhance cytotoxicity through the activation of σ b and alternation of virulence expression [ ] . whether such enhancement is even stronger in g pd-deficient cells was also investigated in this study. the vancomycin-resistant s. aureus strain sjc was generated by introducing a vancomycin resistance-carrying plasmid (pg ) into strain atcc as described previously [ ] . briefly, the p r vanrsp h haxp y vanyp z vanz gene cluster (the van operon within tn ) in e. faecalis hip was amplified and then cloned into pghl from which the luxab gene was removed to generate pg . all bacterial strains were routinely cultured at °c with the specific required antibiotics (sigma) in bhi broth or on agar plates. a lung carcinoma cell line (a ) obtained from the american type culture collection (atcc) and derivatives of this cell line were used in the present study. the stable g pdknockdown cell line, a - . , and the control cell line transfected with pci-neo vector only, a - s- , were generated, approved and kindly given by dr. hung-yao ho [ ] . briefly, g pd-rnai plasmids were generated by the ligation of complementary oligonucleotides into the pci-neo mammalian expression vector followed by transfection into a cells to generate a - . . the cells were cultured in dmem (gibco brl) supplemented with % fetal calf serum, penicillin ( u/ml), and streptomycin ( u/ml) at °c in a humidified atmosphere of air and % co . before infection, overnight culture of the s. aureus strain sjc was diluted back to an o.d. a of . and subcultured in ml bhi broth without any antibiotic at °c until the o.d. a = . . bacterial infection of cells was performed by inoculating with sjc (at a multiplicity of infection of ; moi= ) in the absence or presence of vancomycin ( μg/ ml). under specified circumstances, the function of α-hemolysin secreted by s. aureus was inhibited by adding oroxylin a ( μg/ml; sigma) [ ] , and the infected cells were cultured at °c in a humidified atmosphere of air and % co for further use. for samples that were to be observed using a microscope, sterile glass cover slips were inserted into each of the wells in advance. the vrsa strain sjc was grown overnight at °c with shaking in bhi broth. the next day, the bacterial culture was diluted ( / ) in fresh bhi broth with or without vancomycin ( μg/ml) and then incubated at °c with shaking. at , , , , , and h, the bacteria were plated on agar for enumeration of the surviving cfus. time-kill curves were then constructed by plotting the mean colony counts (log cfu/ml) versus time from three experiments. cell viability tests were performed using the mtt assay with the cell proliferation reagent mtt ( -[ , -dimethylthiazol- yl]- , -diphenyl tetrazolium bromide; sigma), as described previously [ ] . the mtt tetrazolium ring is cleaved only by active mitochondria, yielding purple formazan crystals whose amount directly correlates with the viable cell count. at h post-infection, μl of a mg/ml mtt solution was added into each well, and the plates were incubated at °c for . h. the purple formazan crystals were dissolved by adding μl of mtt solubilization solution (sigma), and the absorbance at a was spectrophotometrically measured with a reference wavelength of a . the results were expressed as the percent absorbance of each experimental well versus the well containing untreated cells. three wells per experimental condition were counted in three independent experiments. the formation of intracellular ros was visualized by detecting dichlorofluorescein (dcf) derived from the oxidation of dihydrodichlorofluorescein (h dcf) as previously described [ ] . at h post-infection, μm dichlorofluorescein diacetate (h dcfda; invitrogen) was added to each well at °c for min and examined under a fluorescence microscope. quantification of ros formation was performed by flow cytometry. cells were treated as described above followed by two washes in pbs and trypsin treatment. flow cytometric and data analyses were performed as described elsewhere [ ] . the mean fluorescence intensity (mfi) of the dcf channel was determined using cellquest pro software (becton dickinson). the results were expressed as the fold change of the percentage increase in dcf channel. cell apoptosis/necrosis was visualized by annexin v-fitc/ propidium iodide (pi) staining with a commercial apoptosis detection kit (biovision) by microscopy according to the manufacturer's instructions, as well as analysis by flow cytometry. phosphatidylserine translocation in apoptotic cells was stained with annexin v-fitc showing green fluorescence, whereas the nucleic acids in dead (necrosis) cells was stained with pi emitting red fluorescence. dapi was employed to stain the cell nucleus, producing blue fluorescence. at h postinfection, cells were washed three times with pbs. for microscopy, × binding buffer was added to each well together with dapi, annexin v-fitc, and pi ( μl of each) for min. cover slips were removed and observed by confocal microscopy. for flow cytometry analysis, cells were trypsinized following a wash in pbs and resuspended in × binding buffer. five min after the addition of annexin v-fitc and pi ( μl of each), flow cytometry analysis was performed using a facscalilbur (bd bioscience). the cells were excited with the nm laser and green apoptotic cells were detected at nm, while red necrosis cells were detected at nm. no fluorescence could be detected in live cells, and cells showing both green and red fluorescence were considered to be in late apoptosis. biochemical markers of apoptosis were also detected by western blotting analysis. the cells were lysed in lysis buffer described elsewhere and the protein concentration of the lysate was determined by the bradford method [ ] . after resolving the protein lysates by sds-page, the samples were transferred to nitrocellulose membranes and blocked with % milk in tbs containing . % tween (tbst). the membranes were then probed with : primary antibody (mouse multiclonal; sigma) caspase- , caspase- , caspase- or gapdh at °c overnight. after washing in tbst, membranes were probed with : secondary horse antimouse hrp-linked antibody (sigma) for h and developed using ecl reagents (bioman). the blots were photographed using an x-omat (kodak) film processor. a student's t-test and non-parametric tests were used to analyze the experimental data and to compare means. pvalues of less than . were considered statistically significant. to understand whether g pd-deficient epithelial cells were less tolerant than control cells to bacterial infection, the mtt assay was employed to evaluate cell viability upon s. aureus infection. the results shown in figure indicate that cell viability of both a - s- (the scramble control) and a - . (g pd-deficient) cell lines was decreased at h post-infection. however, a - . cell viability ( %) was significantly less (p < . ) than that of a - s- cells ( %). we previously demonstrated that vancomycin could activate σ b in vancomycin-resistant staphylococcus aureus resulting in the enhancement of cytotoxicity [ ] . the effect of vancomycintreated vrsa infection on a cells and their g pd-deficient counterparts was investigated. no additional effect was observed upon vancomycin treatment in either the scramble control or g pd-deficient cells (figure ). we hypothesized that g pd-deficient cells are less tolerant to oxidative stress upon bacterial infection, leading to the accumulation of more intracellular ros when compared to the control scramble cells. this was confirmed by directly observing dcf intensity within cells by fluorescence microscopy. as shown in figure a , the average level of green fluorescence intensity was higher in vrsa-infected cells than in untreated cells of both the a - s- and a - . cell lines. an even stronger fluorescence intensity was observed among vrsa-infected a - . cells than among a - s- cells. treatment with vancomycin showed no obvious effect on the fluorescence intensity of either cell line upon vrsa infection, but a slight increase in ros production was observed in vancomycin-treated control cells when compared to vancomycin-free cells of both cell lines. the production of ros was then further quantified by determining the mfi of the dcf channel. infection by vrsa resulted in a . -and . -fold increase in mfi among a - s- and a - . cells, respectively ( figure b and c). a more significant accumulation of ros production in g pd-deficient cells than in control scramble cells (p < . ) was observed upon bacterial infection. vancomycin treatment of vrsa-infected cells also significantly reduced ros accumulation in a - s- and a - . cells, and vancomycin treatment alone showed a slight but non-significant increase in ros production in both cell lines ( figure b and c). to rule out reduced ros accumulation that might be due to increased bacterial killing upon vancomycin treatment, a bacterial killing kinetic assay was performed. slower bacterial growth was observed in the presence than in the absence of vancomycin for the first hours, and no significant difference was seen for the duration of the experiment ( h; figure s ). in addition, at h post-infection, aliquots of bacteria-infected cell culture media were removed and plated on agar, but no significant difference between the cfus of the different experimental conditions was evident (data not shown). the van operon-carrying plasmid (pg ) was also found in all of the colonies, indicating that the vancomycin resistance construct was stable in sjc (data not shown). whether the accumulation of more intracellular ros resulted in stronger apoptotic activity in g pd-deficient cells was investigated by fluorescence microscopy. the results shown in figure indicate that most of the a - . cells showed apoptosis, necrosis or both phenotypes upon vrsa infection, whereas apoptotic activity was weaker in a - s- cells under the same condition. vancomycin treatment in vrsainfected cells apparently reduced apoptotic activity in both a - s- and a - . cells, as detected by the proportion of pi-stained cells. vancomycin treatment alone had no significant effect on apoptotic activity in either cell line. the quantification assay for apoptotic activity was evaluated by flow cytometry. the percentage of apoptotic and necrotic cells increased to . % in vrsa-infected a - s- cells at h post-infection. the percentage was significantly higher in a - . cells ( . %; p < . ) than in control scramble cells (figure , bottom row). as observed microscopically, vancomycin treatment of vrsa-infected cells significantly reduced the percentage of apoptotic and necrotic cells to . % and . % in a - s- and a - . cells, respectively. we detect a slight and non-significant increase in apoptotic activity upon vancomycin treatment alone in both a - s- and a - . cells (figure ) . in addition to morphological markers, biochemical markers of apoptosis were detected by western blotting analysis. because caspases- , - and - are located at key junctions in apoptosis pathways, their expression in a - s- cells was compared to that in a - . cells upon different treatments [ , ] . the results shown in figure indicate that a similar level of the cleaved caspase- ( and kda) was observed upon vrsa/ vancomycin-treated vrsa infection in both a - s- and a - . cells, suggesting a similar signal strength to trigger the extrinsic apoptotic pathway. a weak cleaved caspase- expression was found in bacteria-free a - . cells, implying that the g pd-deficient cells were less tolerant to extrinsic death ligands during culture. on the other hand, vrsa infection obviously triggered higher expression of the precursor ( kda) and cleaved forms ( and kda) of caspase- , thereafter leading to higher expression levels of pro-caspase- ( kda) and cleaved caspase- ( and kda), which is the effector caspase, in a - . cells than in scrambled control cells (figure ) . the above results suggest that a stronger intrinsically apoptotic signal was triggered in g pd-deficient cells upon bacterial infection. in addition, vancomycin treatment of vrsa-infected cells reduced the expression levels of cleaved caspase- and - in both cell lines (figure ) . trace or non expression of caspase- and - was observed in bacteriafree cell cultures. we previously showed that a decrease in hla (encoding αhemolysin) expression and hemolytic activity was observed in strain sjc upon vancomycin treatment [ ] . to determine whether the reduced ros accumulation and apoptotic activity, particularly in g pd-deficient cells, was due to deceased αhemolysin expression upon vrsa infection in the presence of vancomycin, the production of intracellular ros and cell apoptosis when the α-hemolysin inhibitor oroxylin a was added to the media was quantified by flow cytometry. hemolysis was inhibited when sjc cells were inoculated on blood agar plates containing either oroxylin a ( μg/ml) or vancomycin ( μg/ml) ( figure s ). treatment with oroxylin a or vancomycin, significantly reduced the intracellular ros and apoptotic activity of both cell lines in the presence of infection. oroxylin a treatment alone showed no significant effect on the accumulation of ros or cell apoptosis ( figure ). in the present study, we demonstrate that g pd-deficient epithelial cells were less tolerant to s. aureus infection through the accumulation of more ros leading to stronger apoptotic activity. studies on patients with g pd deficiency have mostly focused on severe hemolytic anemia, extreme hyperbilirubinemia (jaundice), and bilirubin encephalopathy, especially in neonates [ ] . as described, the effect of infection on patients with g pd deficiency was studied mainly with regard to hemolytic anemia caused by plasmodium infection, antimicrobial drugs, and viral and bacterial infections. in addition to hemolytic anemia, the enhancement of both viral infection and immune weakness (such as neutrophil dysfunction) were also addressed. our study demonstrates a direct impact of bacterial infection on g pd-deficient epithelial cells, a result that is in contrast to that found in blood cells. the lack of ros (such as o -) production in phagocytes (neutrophils and monocytes) may cause chronic granulomatous disease-like clinical symptoms in patients with g pd deficiency. such phagocyte dysfunction may lead to recurrent infections by catalase-positive microorganisms (such as staphylococci) because those bacteria could be phagocytosed but not digested [ ] . on the other hand, it has been demonstrated that bacterial infection may induce oxidative stress in lymphocytes, in mammary epithelial cells, or in yeast [ ] [ ] [ ] . consistent with the above findings, our results indicated that intracellular ros were generated in a lung carcinoma cells upon s. aureus infection (figure ) . we further propose that the capacity for ros removal is impaired in g pd-deficient cells following infection-induced oxidative stress. as expected, the accumulation of ros was significantly higher in g pd-deficient cells than in control scramble cells following exposure to vrsa (figure ) . therefore, the impact of bacterial infection on patients with g pd deficiency is twofold. first is the immune weakness due to the dysfunction of phagocytes. second is the impairment of ros removal upon bacterial infection, as we have demonstrated in epithelial cells. it has been well demonstrated that apoptosis signalregulated kinase (ask ), a member in the mitogen-activated protein kinase (mapk) cascades, is activated upon oxidative stress, leading to cell apoptosis [ ] . we demonstrated that the impairment of ros removal in g pd-deficient cells resulted in a higher apoptotic activity than in control scramble cells ( figure ) . the caspases, a family of cysteine proteases, play essential roles in apoptosis. the classical apoptotic machinery involves the activation of initiator caspases such as caspase- and - upon death signals, thereby cleaving the inactive pro-forms of effector caspases such as caspase- to activate them [ , ] .the commencement of the extrinsic apoptotic pathway depends on the activation of caspase- through the binding of extracellular death ligands to transmembrane death receptors. in contrast, the intrinsic pathway is triggered by the release of cytochrome c from mitochondria, thereby activating caspase- . both pathways can lead to the activation of caspase- and other effector caspases [ ] . our results presented here indicate that expression of active caspase- , as well as the downstream caspase- , was much higher in g pd-deficient cells than in control scramble cells upon vrsa infection, suggesting that mitochondrial dysfunction may be the major cause of the increase in cell apoptosis (figure ) . a recent study demonstrated that the incubation of epithelial cells with enterotoxin isolated from aeromonas veronii increased the accumulation of intracellular ros leading to a loss of mitochondrial membrane potential and thereafter undergoing apoptosis through the mitochondrial pathway [ ] . the s. aureus strain sjc used in this study was constructed under the atcc strain genetic background which is known to lack a variety of exotoxins except for α-hemolysin [ ] . therefore, we may propose that α-hemolysin could cause mitochondria dysfunction similar to that found in a. veronii. we further highlight a much worse outcome in g pd-deficient cells upon bacterial infection. unlike the lack of mitochondria in peripheral red blood cells (rbcs), the impact of bacterial infection on rbcs (causing hemolysis) and other cells is different in patients with g pd deficiency. our previous study demonstrated that antibiotic treatment on a drug-resistant s. aureus strain would appear to be an environmental stress that activates σ b , a stress response transcription factor [ ] . in that study, vancomycin induced σ b activity led to the alternation of downstream virulence genes in vrsa strains, as well as the increase in cytotoxicity to the human bronchial epithelial cells (beas- b). the increased expression of bacterial binding capacity, but not the decreased expression of α-hemolysin, associated with increased σ b activity, might be the key contributor to the cytotoxicity. however, similar results were not observed in this study when the a cell line was used, neither in g pd knock down or in control scramble cells. it has been reported that α-hemolysin was an important mediator of cytotoxicity in a cells whereas it was tolerated by beas- b cells [ , ] . as proposed above, α-hemolysin should be the major cause of mitochondria dysfunction through the accumulation of ros in a cells and derivatives. because the expression of α-hemolysin was decreased in sjc upon vancomycin treatment, the impact of vancomycin treatment on the cytotoxicity of vrsa-infected a cells was not obvious in either g pd knock down or control scramble cells (figure ). in fact, vancomycin treatment alleviated the vrsa infection-enhanced ros accumulation and apoptosis in both cells even though cell survival was not improved (figures - ) . this phenomenon may be a result of the increased binding affinity of vrsa to the cells upon vancomycin treatment, which leads to stronger cytotoxicity and possibly bypasses the mitochondria dysfunction pathway, as we reported previously [ ] . therefore, the net impact of vancomycin treatment on a cell survival in the presence of infection was similar to the vancomycin-free condition. nevertheless, we cannot rule out that the misuse of antibiotics in other drug-resistant strains will trigger the expression of other exotoxins that can cause a stronger oxidative stress in g pd-deficient cells. although the susceptibility of different cell types to different bacterial virulence factors is variable, we suppose patients with g pd deficiency are less tolerant to bacterial infection. in conclusion, we demonstrate a cell damage mechanism in g pd-deficient epithelial cells upon bacterial infection. most of the cellular ros are generated from the mitochondria, where there are a variety of mechanisms to reduce the accumulation of ros or protect against oxidative stress [ ] . the loss of mitochondrial membrane potential due to bacterial infection, possibly via s. aureus α-hemolysin in this case, leads to an increase in ros production. in g pd-deficient cells, the impaired reconversion of glutathione disulphide to reduced glutathione caused by the imbalanced nadph/nadp + redox state renders the cell unable to remove ros effectively. therefore, the accumulation of excess ros results in stronger cell apoptosis through the mitochondrial pathway. one of the most important clinical concerns in treating bacterial infection in patients with g pd deficiency is to avoid the usage of antibiotics which can cause hemolytic anemia [ ] . vancomycin treatment did not further enhance vrsa cytotoxicity toward g pd-deficient and control scramble a cells, as opposed to what we had previously observed in beas- b cells [ ] . figure s . time-kill curve of the vrsa strain sjc . strain sjc was incubated without (open diamond) or with vancomycin ( μg/ml; solid circle), and the results are presented as the means±sd of the log cfu/ml from three separate experiments. (tif) figure s . the effects of oroxylin a or vancomycin treatment on the hemolytic activity of sjc cells. sjc cells ( × cfu) were inoculated onto a (a) blank blood agar plate or (b) plate containing vancomycin ( μg/ml) or (c) oroxylin a ( μg/ml) and incubated at °c overnight. 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- journal: damage-associated molecular patterns in human diseases doi: . / - - - - _ sha: doc_id: cord_uid: ztp w yh in this chapter, the role of cell-intrinsic stress responses is examined which include autophagic processes, the oxidative stress response, the heat shock response, the unfolded proteins response, and the dna damage response. autophagy (macroautophagy, microautophagy, and chaperone-mediated autophagy) is a self-digestive process in response to environmental stress to eukaryotic cells, by which cytoplasmic components are delivered to the lysosome for recycling and degradation. the oxidative stress response is directed against any oxidative stress and is mediated by antioxidative defense systems including antioxidant enzymes such as superoxide dismutase, detoxifying enzymes such as glutathione peroxidase, and energy-dependent efflux pumps. the heat shock response is induced upon exposure of cells to any stress condition and characterized by emission of heat shock proteins which operate as damps to maintain and restore homeostasis. the unfolded protein response is induced by any stress of the endoplasmic reticulum that is perceived by three sensor molecules. under remediable endoplasmic reticulum stress conditions, the sensors trigger signalling pathways to resolve this stress. however, in severe irremediable endoplasmic reticulum stress, the unfolded protein response may lead to pro-inflammatory and pro-apoptotic responses resulting in regulated cell death. finally, the dna damage response is induced by any dna damage that occurs in a variety of exogenous and endogenous conditions. when successful, this stress response leads to dna repair and is associated with the emission of various damps which contribute to restoration of homeostasis. when unsuccessful, the dna damage response, like the unsuccessful unfolded protein response, can result in regulated cell death, either in form of apoptosis or necrosis. together, the ultimate goal of all the stress responses is to maintain cellular homeostasis and ensure cell integrity. when they fail, the incidence of regulated cell death is frequently observed. as comprehensively described in part ii, prms are specifically involved in the recognition of mamps and damps. as will be discussed in part vi, each of these recognition receptors can trigger distinct signalling cascades in innate immune cells that modify their gene expression to create and execute efferent innate immune responses that involve ( ) production of inflammatory mediator substances such as cytokines and chemokines, ( ) phagocytosis, and ( ) cytotoxicity, as well as, as described in part viii, may elicit and shape antigen-specific adaptive immune responses. beyond this well-characterized mamp/damp engagement of prms leading to a variety of downstream efferent cellular and humoral responses, the innate immune defense program also depends on cell-autonomous, that is, cell-intrinsic, responses which counteract any stressful insult [ ] . constitutive cell-autonomous immunity mobilizes pre-existing molecules and processes in order to primarily and quickly defend the cell and the host against infectious and sterile injury. hence it can be considered as the very first line of innate immune defense. here, the role of constitutive cell-autonomous responses will be examined, whose involvement in the innate immune defense to stress and injury has only been appreciated within the last few years. the focus of this brief overview will be mainly directed toward cellular stress responses. the term autophagy comes from the greek words "phagy" meaning eat and "auto" meaning self. autophagy is an evolutionarily highly conserved self-digestive process in response to environmental stress to eukaryotic cells, by which cytoplasmic components such as defective/damaged or redundant organelles or protein aggregates are delivered to the lysosome for recycling and degradation. there is convincing evidence indicating that activation of the autophagic process is promoted by mamps and/or damps [ , ] . in more simple words, autophagy is a classical cellprotective and cell-autonomous process of the innate immune system aimed at maintaining and restoring homeostasis at both the cellular (cell-intrinsic) and organismal (cell-extrinsic) level [ ] . although autophagy was initially identified in mammals, a significant breakthrough in our understanding of how autophagy is controlled came from the analysis in the genetically tractable yeast system. pioneering work from ohsumi's group showed that the morphology of autophagy in yeast was similar to that documented in mammals [ ] . (as known, ohsumi received the nobel prize in physiology or medicine .) in fact, the discovery of the autophagyrelated genes in yeast has significantly advanced the understanding of the molecular mechanisms participating in autophagy and the genes involved in regulating the autophagic pathway. many yeast genes have mammalian homologues, confirming that the basic machinery for autophagy has been evolutionarily conserved along the eukaryotic phylum [ ] [ ] [ ] [ ] . notably, a panel of leading experts in the field of autophagy has recently published a new definition of several autophagy-related terms based on specific biochemical features [ ] . accordingly, in the following, three types of autophagy are briefly sketched including macroautophagy, microautophagy, and, in mammals, chaperone-mediated autophagy. each of them fulfils very specific tasks in intracellular degradation. there is general agreement on two main features that characterize bona fide, functional autophagic responses, irrespective of type: ( ) they involve cytoplasmic material; and ( ) they culminate with (and strictly depend on) lysosomal degradation [ ] . thus, although autophagy substrates can be endogenous such as damaged cellular organelles or exogenous such as viruses or bacteria escaping phagosomes, autophagy acts on entities that are freely accessible to cytosolic proteins. this property is essential in order to distinguish between autophagic responses and branches of vesicular trafficking that originate at the plasma membrane, which also culminates in lysosomal degradation. such endocytic processes include phagocytosis, receptor-mediated endocytosis, and macropinocytosis, that is, processes which will be dealt with in part vi, sect. . . of note, however, some forms of autophagy and the endocytic pathway interact at multiple levels, and the molecular machinery responsible for the fusion of late endosomes (also known as mvbs) or autophagosomes with lysosomes is essentially the same [ ] . as stressed [ ] , the strict dependency of autophagic responses on lysosomal activity is necessary to discriminate them from other catabolic pathways that also involve cytoplasmic material, such as proteasomal degradation [ ] . thus, the s proteasome (box . ) degrades a large number of misfolded cytoplasmic proteins that have been ubiquitinated (for (poly)ubiquitination, see box . ) as well as properly folded proteins that expose specific degradation signals, such as the socalled n-degrons [ ] . on the other hand, the proteasome system shares some substrates with different forms of autophagy whereby these two catabolic pathways differ drastically in their final products. thus, proteasomal degradation results in short peptides that are not necessarily degraded further but may flow into additional processes including but not limited to antigen presentation/cross-presentation at the plasma membrane, thereby generating mhc-ii and mhc-i epitopes (compare part viii, chap. ). by contrast, lysosomal proteases fully catabolize polypeptides to their constituting amino acids which eventually become available for metabolic reactions or repair processes. together, as summarized [ ] , bona fide functional autophagic responses navigate cytoplasmic material of endogenous or exogenous origin to degradation within lysosomes (or late endosomes, in specific cases). the binding of many ubiquitin molecules to the same target protein. in its simplest form, ubiquitin can be attached to the target protein as a single moiety resulting in monoubiquitination. ubiquitin itself can be ubiquitinated, resulting in the formation of ubiquitin chains attached to the target protein: polyubiquitination. polyubiquitination of proteins is the triggering signal that leads to subsequent degradation of the protein in the proteasome. ligases play a central role in polyubiquitination. ligases are enzymes that catalyze the synthesis of polyubiquitin chains. ubiquitin conjugation requires typically box the proteasome is a common complex for all living cells, needed to recycle and eliminate unwanted proteins. in analogy, it resembles a chaff-cutter. this molecular machine provides a pathway that is involved in many cellular levels such as protein degradation, antigen processing, cell cycle, apoptosis, and dna repair. the s proteasome that is present in the cytoplasm and nucleus is usually formed by one s proteasome complex and two s proteasome complexes, which are composed of proteases and structural units. the s proteasome is a giant protease responsible for the regulated degradation of polyubiquitylated proteins (see box . ) . it consists of at least distinct subunits and is arranged into two modules: core particle containing catalytic sites and regulatory particles. the cylinder-shaped proteolytic core is the s core particle, which is capped at one or both ends by s regulatory particles. further reading: wehmer m, sakata e. recent advances in the structural biology of the s proteasome. int j biochem cell biol ; : - . basically, the term macroautophagy is often used when describing autophagy in general. the phenomenon is characterized by its typical morphological features which involve dedicated vesicles that can occupy a considerable part of the cytoplasm. typically, macroautophagy is one type of autophagic processes in which the substrates are sequestered within cytosolic double-membrane vesicles termed autophagosomes. the substrates of macroautophagy include superfluous and damaged organelles, cytosolic proteins, and invasive microbes. mechanism of formation and regulation of macroautophagy are very complex and complicated processes that are outlined here in a considerably simplified way. macroautophagy involves the sequestration of cytoplasm via a double-membrane intermediate structure termed the phagophore which matures into an autophagosome; the latter compartment fuses with a lysosome allowing degradation and recycling of the cargo [ ] . in more detail, the process begins with the formation of a membrane of unknown origin, the initial phagophore or isolation membrane. the phagophore then expands, surrounds proteins or organelles, sequesters cytoplasm, and, on completion, develops into a large double-membrane transport vesicle, the autophagosome. subsequently, the autophagosome fuses with a lysosome containing acid hydrolases and releases its contents into the lytic acid hydrolases-containing compartment as part of single-membrane vesicles, termed autophagic bodies. the fused compartment where the autophagic body and its contents are degraded is called an autophagolysosome or autolysosome ( fig. . ) . notably, the process of phagophore expansion three classes of enzymes. e (ubiquitin-activating enzyme) hydrolyzes atp and forms a thioester-linked complex between itself and ubiquitin. e (ubiquitin-conjugating enzyme) receives ubiquitin from e and forms a similar thioester-linked intermediate with ubiquitin. e (ubiquitin ligase) finally binds both the e and a substrate and catalyzes the transfer of ubiquitin to the substrate. ubiquitin itself is often a substrate for further ubiquitylation, which results in the formation of so-called polyubiquitin chains. ubiquitin has seven lysine residues, and depending on the lysine residue used for ubiquitin-ubiquitin chain formation, the polyubiquitin chain can signal different functions. proteins modified by lysine- (k )-or lysine- (k )-linked chains are usually degraded by the proteasome. in contrast, modification by k -linked chains or by a single ubiquitin moiety (monoubiquitylation) seems to trigger other functions, e.g., protein sorting, gene expression, and dna repair. further reading: callis j. the ubiquitination machinery of the ubiquitin system. arabidopsis book ; :e . provides tremendous flexibility and capacity with regard to cargo, allowing entire organelles to be deleted via autophagy; however, this flexibility also means that autophagy must be tightly controlled in order to prevent inappropriate degradation, which could lead to cell death (for relevant papers, see [ ] [ ] [ ] [ ] [ ] [ ] [ ] . intensive studies have been carried out in the past two decades to understand the mechanism and regulation of autophagy. the biogenesis of autophagosomes needs the ordered intervention of autophagy-regulated (atg) proteins that act on different modules. thus, more than atg genes have been identified in human that orchestrate the complex membrane dynamics involved in autophagic sequestration. these atg proteins act sequentially in three macromolecular complexes involved in the three successive stages of autophagy. initiation of autophagy requires the unc- like kinase (ulk )-atg -fip (also known as rb -inducible coiled-coil ) complex, whereby the kinase activity of ulk is controlled by the kinase mammalian target of rapamycin (mtor) in mtor complex (mtorc ), which is sensitive to rapamycin [ ] . the next process, membrane nucleation, requires the beclin / class iii pi k complex, which also plays a major role in membrane trafficking and restructuring involved in autophagy [ , ] ; the final process refers to the elongation, expansion, and closure of the phagophore membrane/autophagosome which mainly relies on atg /microtubule-associated protein light chain (lc ) lipidation. in fact, atg /lc lipidation is regarded as a hallmark of autophagy and is established by a covalent linkage of cytosolic lc to the lipid phosphatidylethanolamine on the surface of the autophagosome [ , ] . of note, in addition to the cytoplasmic ptm of various atg proteins, recent studies have explored the transcriptional and epigenetic control of autophagy [ ] . notably, in human cells, tfeb (for transcription factor eb) and zkscan (for zinc finger with krab and scan domains ) were shown to be implicated in playing a crucial role in autophagy regulation [ , ] . also, there is growing evidence in support of the notion that histone modification/dna methylation acts as an alternative approach for long-term autophagy control [ ] (for histone modification, see part vi, sect. . . ) . also recently, a new ampk→skp →carm (for: ampactivated protein kinase; s-phase kinase-associated protein (p ); coactivatorassociated arginine methyltransferase ) regulatory axis was reported that incorporated cellular nutrient sensing with transcriptional as well as epigenetic control of autophagy [ ] . as concluded by xu and klionsky [ ] , "…this ampk-skp -carm signalling axis integrates the various levels of autophagy regulation including cell signalling, and transcriptional regulation as well as epigenetic modification. epigenetic and transcriptional regulation provides an energy-saving approach for control and also create an enduring memory in preparation for future adverse events. thus, this study has deepened our understanding of how autophagy can be controlled in a holistic manner by pathways linking a multitude of regulation mechanisms. given the extensive involvement of autophagy in human diseases, this work also presents potential directions for novel therapeutic intervention." indeed, besides its beneficial function in controlling cellular homeostasis, macroautophagic pathways when disrupted can have severe consequences leading to major diseases such as cancer, metabolic and neurodegenerative disorders, and cardiovascular and pulmonary diseases [ ] . of note, macroautophagy can be divided into two subtypes depending on the organelle that is targeted for autophagic degradation; thus, the process of mitophagy corresponds to autophagy of mitochondria, whereas the term er-phagy refers to autophagy of the endoplasmic reticulum (er). both processes deserve a few more words in the following subsection. the term mitophagy corresponds to cargo-specific autophagy of mitochondria, a process which mediates the selective removal of mitochondria [ , ] . the aim of mitophagy is to eliminate mitochondria, either to regulate their number to adjust to metabolic demand or to explicitly remove those that are damaged in terms of a quality control. mechanistically, mitochondria are selectively recruited into isolation membranes, which seal and then fuse with lysosomes to eliminate the trapped mitochondria. as discussed [ ] , mitophagy is preceded by so-called mitochondrial fission that divides elongated mitochondria into pieces of manageable size for encapsulation and also controls segregation of damaged mitochondrial material for selective removal by mitophagy. the term er-phagy (also called micro-er-phagy) refers to a process of distinct selective degradation of er membranes and proteins in the lysosome under stress, and this is independent of the core autophagy machinery [ ] [ ] [ ] (for er stress, see sect. . ) . studies on yeast showed that er-phagy is characterized by the fact that stress-induced er whorls are selectively taken up into the vacuole, the yeast lysosome. import into the vacuole was found not to involve autophagosomes but occurs through invagination of the vacuolar membrane, indicating that er-phagy is topologically equivalent to microautophagy [ ] . recent studies on yeast provide evidence suggesting that the atg proteins atg and atg are specific receptors for this pathway of er-phagy [ ] . at this point, it also appears worthwhile to mention that a more recent study on yeast revealed a novel er quality-control pathway, namely, the so-called macro-er-phagy. first results from this study suggest that this pathway delivers an excess of integral-membrane proteins from the er to the lysosome for degradation and, typically, requires the core autophagy machinery [ ] . the brief overview about macroautophagy provides another typical example of innate immune responses which, when controlled, operate in a beneficial homeostatic way but, when uncontrolled, may lead to severe pathologies. for other forms of phagocytic responses, this phenomenon has not been investigated sufficiently. clearly, mitophagy also plays a key homeostatic role in mitochondrial quality control. upregulation of mitophagy has been shown to mitigate excessive mitochondrial accumulation and toxicity to safeguard mitochondrial fitness. hence, mitophagy is a viable target to promote longevity and prevent age-related pathologies [ ] . concerning the two types of er-phagy (micro-and macro-er-phagy), one has to state that research in this exciting field has just begun. several questions remain to be addressed, for example, what is the purpose of er-phagy and what are the underlying mechanisms. future studies will probably provide a clue to elucidating the molecular mechanisms and physiologic roles of er-phagy in other organisms. of note, besides mitophagy and er-phagy, other specific forms of phagocytic pathways have been described. they include -pexophagy as a macroautophagic response preferentially targeting peroxisomes -nucleophagy as an autophagic response selectively targeting portions of the nucleus -ribophagy as a specific autophagic response targeting ribosomes -aggrephagy as an autophagic response specific for protein aggregates -lipophagy in terms of selective autophagic degradation of neutral lipid droplets -bacterial xenophagy as a macroautophagic removal of cytoplasmic bacteria which have escaped the phagosomal compartment upon phagocytosis -viral xenophagy as a macroautophagic response targeting fully formed cytoplasmic virions or components thereof -proteaphagy in terms of macroautophagic responses specific for inactive proteasomes -lysophagy as a specific macroautophagic disposal of damaged lysosomes in mammalian cells for details of these specific forms of phagocytic responses, the reader is referred to the excellent comprehensive review article of galluzzi et al. [ ] . in addition to macroautophagy, two other types of autophagy have been described called microautophagy and chaperone-mediated autophagy (cma). microautophagy together with macroautophagy plays, for example, a role in nutrient recycling under starvation. on the other hand, cma is known to contribute to the maintenance of cellular homeostasis by facilitating recycling of amino acids of the degraded proteins and by eliminating abnormal or damaged proteins, thereby exerting major regulatory functions in different pathophysiological scenarios such as metabolic regulation. here, a few aspects of these two types of autophagic responses are skimpily touched. by contrast to macroautophagy, the process of microautophagy is much less defined in mammals since most studies have been performed in yeast and plants. according to current models, the term refers to a collection of diverse processes. unlike autophagy, microautophagy does not involve the autophagosome-dependent degradation of cytoplasmic components but rather and characteristically relies on the direct engulfment of small portions of cytoplasm into lysosomes or late endosomes by invagination and inward budding of the lysosomal/endosomal membrane, a process that leads to their degradation [ ] [ ] [ ] . though microautophagy is the least studied form of autophagy, a molecular signature of the process has begun to emerge and has led to the definition of microautophagy as a type of autophagy in which the cargo is directly internalized in small vesicles that form at the surface of the lysosome/vacuole or late endosomes (multivesicular bodies), respectively [ ] . in addition to macroautophagy and microautophagy, there is another type of autophagy experiencing increased attention, the cma. characteristically, in cma, cargo delivery also occurs directly at lysosomes, but it does not require formation of vesicles nor membrane invagination. instead, the substrate proteins for this autophagic pathway cross the lysosomal membrane through a protein-translocation complex, that is, a process that requires protein interaction with the chaperone hspa (also known as hsc ) and association of hspa with a specific splicing isoform of lamp- , that is, the lysosomal protein lamp- a. thus, chaperone-bound autophagy substrates bind lamp- a monomers on the cytosolic side of the lysosome, which stimulate the formation of an oligomeric lamp- a translocation complex [ , [ ] [ ] [ ] . essential functions that cma fulfils in cells include a contribution to amino acid recycling during prolonged starvation as well as quality control, directly linked to the ability of this pathway to selectively remove single proteins from the cytosol. for example, cma is up-regulated during oxidative stress where it contributes to the degradation of oxidized proteins (reviewed in [ ] ) (see next sect. . ). of note, growing evidence demonstrates that malfunction of cma plays a vital role in the pathogenesis of severe human disorders. often, the mechanisms underlying the alterations of cma in these pathologies involve perturbations in the functioning of the cma translocation complex. both diminished and enhanced cma activities have been shown to associate with diseases, an observation that emphasizes the importance of a tight regulation of cma activity (highlighted in [ ] ). as argued above, current knowledge about mechanism and physiological relevance of microautophagy in mammalian cells is hard to judge since most findings derive from studies in yeast. however, future studies aimed at identifying proteins controlling microautophagy-related vacuolar membrane changes in yeast will probably allow to search for homologues in mammals and then investigate their contribution to mammalian microautophagy. by contrast, research on cma has already made substantial progress. for example, the recent identification of a plethora of new cma substrates and deficiencies in cma associated with diverse human pathologies has expanded our understanding of the importance of cma in multiple cellular functions. in fact, the growing number of connections between cma and human diseases has already generated interest in modulating cma activity for therapeutic purposes. there is a close relationship between autophagy and mamps and/or damps in the cellular response to injury. in fact, autophagy cannot be restricted to an innate immune mechanism that controls intracellular homeostasis alone but has to be extended in terms of "immunological autophagy" to a process that is committed to control and regulate efferent innate and potential adaptive immune responses. the "medium" for achieving this goal is the mamps and/or damps which operate as a link between intracellular and extracellular events. this scenario is briefly touched in the following. growing evidence indicates that autophagy regulates release and degradation of damps-here in terms of inducible damps-including hmgb , atp, and dna in several cell types [ ] . for example, autophagic mechanisms reportedly promote and regulate the release and secretion of hmgb in a ros-dependent manner in fibroblasts, macrophages, and cancer as well as net-mediated release of hmgb in neutrophils [ , ] . moreover, autophagy has been shown to be required for the liberation/active secretion of atp by dying cancer cells [ , ] . in addition, autophagy was found to contribute to the regulation of the ddr at multiple levels, that is, a process associated with the emission of damps [ , ] (for ddr, see sect. . ). via emission of damps, eventually, together with mamps, autophagy can amplify or even instigate mamp/damp-prm signalling leading to efferent innate immune responses. on the other hand, autophagy can inhibit pro-inflammatory signalling cascades. for example, the atg -atg complex, a key regulator of the autophagic process, was shown to negatively regulate rlr signalling by direct binding to card domains of rig-i and interferon promoter-stimulating factor- (ips- ) [ ] (compare part vi, sect. . . ). moreover, as reviewed elsewhere [ ] , autophagy has been found to inhibit both nlrp and aim inflammasome activation and subsequent production of pro-inflammatory cytokines il- β and il- (for inflammasomes, see part vi, sects. . . and . . ). as a possible mechanism, the authors propose that inflammasome components and pro-il- β are subjected to ubiquitination and subsequent degradation by autophagy, thereby leading to functional inactivation of inflammasomes. conversely, an increasing number of studies suggest that damps, including hmgb , atp, and dna, are powerful stimuli and regulators to elicit autophagic responses [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, hmgb was demonstrated to be an important regulator of autophagy in various types of cancer cells and keratinocytes. mechanistically, the reduced form of the hmgb protein was proposed to be responsible for the promotion of autophagy in an ager/rage-dependent fashion [ , ] . also, and of high interest, in a clinical study on patients with chronic hepatitis b, hmgb -induced autophagy was found to maintain treg function during chronic viral infection [ ] . moreover, in studies on a mini pig lung iri model, evidence was provided indicating that autophagy, when triggered by damps such as hmgb and hsp during iri, amplifies the inflammatory response through enhancing k -linked ubiquitination of traf and activation of the downstream mapk and nf-κb signalling (for traf , mapk, and nf-κb signalling, see part moreover, there is already first evidence suggesting a role of atp in the regulation of autophagy [ ] . in addition, there are accumulating data indicating that cytosolic dna, dislocated as a result of dna damage, may contribute to the regulation of autophagy whereby the dna damage-regulated autophagy modulator (dram ) appears to play a mechanistically crucial role [ ] [ ] [ ] . the mechanisms involved in mamp/damp-activated autophagic responses have only partially been elucidated. in fact, there is convincing evidence suggesting that many mamp/damp-recognizing prms including tlrs (in particular endosomal tlrs), nlrs, and anti-dna receptors can activate autophagic responses by triggering specific signalling pathways (reviewed or discussed in [ , , , ] ). the crosstalk between autophagic responses and damps represents a powerful instrument of the innate immune system to integrate and unify various tools for the promotion and regulation of injury-induced inflammation and, in the presence of nonself-or altered self-antigens, injury-induced adaptive immunity. thus, on the one hand, autophagy is known to promote and regulate the release of damps (though the exact mechanisms are still elusive); subsequently, damps via prmtriggered pathways participate in the regulation of inflammation. on the other hand, activation of prms by mamps and/or damps promotes autophagy activation through a mechanism that has been partially elucidated. in fact, an increasing number of findings suggest that this activation process is triggered by prms following recognition of mamps and/or damps. nevertheless, the precise molecular mechanisms by which prms modulate autophagy remain largely unknown. there is increasing evidence in support of the notion that mamp/damp-activated autophagic responses promote emission of damps which in turn support cellular homeostasis in the course of adaptive stress responses in healthy cells. notably, this cellular homeostatic effect may spread out and affect the whole organism via emission of autophagy-dependent damps. in other words, via damps, autophagy as a cell-intrinsic stress response can fortify its defending capability by providing a link to promotion and regulation of cell-extrinsic efferent innate immune and eventually subsequent adaptive immune responses. however, despite the fact that autophagy is one of the best-known cell-autonomous responses in innate immunity and has clearly been shown to counteract dangerous infectious and sterile cell stress, much is left unclear. one such issue concerns the definition of autophagy-dependent cell death. as discussed and summarized [ ] , autophagy-dependent cell death can be defined as a form of rcd (see next chapter) that can be retarded by pharmacological or genetic inhibition of macroautophagy. in this context, as stressed by galluzzi et al. [ ] , it is important to note that ( ) specificity issues affect most, if not all, pharmacological agents employed so far for suppressing macroautophagic responses and ( ) multiple components of the macroautophagy machinery have autophagy-independent functions. in view of these facts and findings, these authors recommend to favor genetic approaches and to test the involvement of at least two different proteins of the macroautophagy apparatus in a specific instance of rcd before etiologically attributing it to macroautophagy. other unclear issues refer to the specific modulation of autophagy by mamps and/or damps, the precise interaction of autophagy with innate immune signalling cascades, and the cooperation between autophagy and other physiologic cell-intrinsic and cell-extrinsic processes during scenarios of cell stress and tissue injury. efforts to solve these problems are of utmost importance in view of the fact that autophagy-when induced by excessive, chronic, or acute-repetitive emission of damps-can contribute to the pathogenesis of many human diseases, that is, acute and chronic, infectious, or sterile inflammatory disorders. at the respective places, they will often be mentioned in the following chapters as well as in volume . oxidative cell stress and tissue injury reflect most potent and omnipresent threats an organism is exposed to. though there is a robust defense response continuously operating, this kind of injury is known to contribute to the pathogenesis of many human diseases. how can this be? oxidative stress is caused by an imbalance between the production of oxidants such as ros on one side and the biological antioxidative defense system's ability on the other side to counter the oxidant levels with antioxidants, that is, to readily detoxify the toxic reactive species or easily repair the resulting damage. thus, it is the excessive production of ros-overriding the antioxidative capacities-that is pathophysiological and contributes to dysfunction, damage, and even death of cells. by contrast, generation of ros in physiological low/ moderate concentrations-operating as second messenger molecules and causing so-called oxidative "eustress" [ ] -assists in intracellular signalling pathways and, thus, is essential for optimal cell functions and homeostasis of an organism. in other words, the biological effects of ros-beneficial or deleterious-considerably depend on the amounts of ros present and, in action, a phenomenon that is in agreement with the idea that cellular ros generation has characteristics of hormesis implying a dose-response phenomenon that is characterized by beneficial effects at low doses and deleterious effectivity at high toxic doses [ ] . to guarantee this homeostatic function of ros, to keep these molecules within physiological limits, and to prevent their deleterious effects, that is, to maintain hormesis, a smooth running of the oxidative stress response is of utmost importance. accordingly, a few aspects of this critical stress response are addressed in the following. reactive oxygen species are produced from molecular oxygen as a result of normal cellular metabolism. to understand any discussion on a role of ros in host defense or human diseases, one should define free radicals. according to halliwell and gutteridge [ ] , "a free radical is any species capable of independent existence that contains or more unpaired electrons." an unpaired electron is one that occupies an atomic or molecular orbital by itself. radicals can be formed by the loss of a single electron from a non-radical, or by the gain of a single electron by a non-radical. in this sense, superoxide anions (o ·− ), hydroxyl radicals ( · oh), peroxyl radicals (ro · ), and alkoxyl radicals (ro˙) are oxygen radicals. of note, ros is a collective term often used by scientists to include not only the oxygen radicals but also some non-radical derivatives of oxygen such as h o , hypochlorous acid (hocl), ozone (o ), and singlet oxygen ( o ). nitrogen-containing oxidants, such as no · are called rns. generation of ros is generally a cascade of reactions that starts with the production of superoxide anions. superoxide rapidly dismutases to h o either spontaneously (especially at the low ph) or catalyzed by sod. other elements in the cascade of ros generation include the reaction of superoxide with no to form the very toxic peroxynitrite, the peroxidase-catalyzed formation of hocl from h o , and the iron-catalyzed fenton reaction, leading to the generation of hydroxyl radical. the oxidants are produced endogenously as by-products or metabolites of various metabolic processes. multiple enzyme systems produce superoxide radicals and their derivatives including xanthine oxidoreductase (xor), the reduced form of nadph oxidases (noxes), and mitochondrial electron transport chain (etc)associated molecular complexes ( fig. [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] mitochondrial etc is believed to be the main source of ros. in the following, some aspects of these three systems (other systems not mentioned here) are briefly touched, exemplified by and focused on their role as vascular sources of ros production as investigated on models of iri. xanthine oxidoreductase (xor), as a housekeeping enzyme, is probably expressed in all cells but primarily in surface epithelia such as capillary endothelial tissue of various organs. xanthine oxidoreductase, a complex molybdoflavoprotein, is the ratelimiting step in the catabolism of purines, where it catalyzes the last steps of purine metabolism: the conversion of hypoxanthine to xanthine and of xanthine to uric acid, with superoxide/h o generated as by-products. there is a definite role of xor in reperfusion of tissue and organs [ , ] . for example, experiments performed in isolated rat hearts have demonstrated that radical generation and functional injury are decreased by inhibition of xor with oxypurinol. similarly, in human aortic or venous ecs, xor-mediated ros generation has been shown to be a central mechanism of oxygen radical generation upon postischemic reoxygenation [ , ] . the nadph oxidases were initially considered as enzymes expressed only in phagocytic cells involved in host defense and innate immunity; however, recent evidence indicates that there is an entire family of noxes based on the discovery of gp phox homologues. the family comprises seven members, including nox , nox (formerly termed gp phox), nox , nox , nox , duox , and duox [ ] . three members out of the enzyme family are important sources of ros in the vasculature, namely, nox , nox , and nox [ ] [ ] [ ] . today, noxes are perhaps the best-studied enzymes involved in ros production in the blood vessels. remarkably, different members of the nox/duox family engaged in iri are localized in various cells, that is, in vascular cells and phagocytes. this may lead to the notion that noxes in vascular cells are responsible for the first wave of ros production because vascular cells are first confronted with reintroduced molecular oxygen. as generally believed, there is, in fact, no vascular specific nox isoform but rather a complex expression of various nox isoforms in different cells and regions of the vascular system. nevertheless, in arteries from humans and animals, nox , nox , and a shallow level of nox have been consistently found to be present both as messenger rna and as protein [ ] . altogether, the findings and data briefly described here make clear that vascular cells are equipped with efficient machinery able to efficiently produce ros. regarding the different enzymatic sources, superoxide radicals appear to be predominantly generated compared, for example, to hydroxyl radicals. mitochondria have been implicated as potential oxygen sensors by increasing the generation of ros which regulate a variety of hypoxic responses [ ] [ ] [ ] [ ] . in fact, mitochondria are increasingly recognized as lynchpins in the evolution of tissue injury during postischemic reperfusion. it is generally acknowledged that the majority of intracellular ros production is generated in the mitochondrial etc and its associated metabolic enzymes. however, very little is known about which mitochondrial sites are involved in physiological or pathological ros production under native conditions. of note, using inhibitors to manipulate the redox states of particular sites and prevent superoxide generation from others, at least ten different locations of superoxide/h o production in the etc and associated enzymes (krebs cycle, β-oxidation, etc.) have been identified in mammalian mitochondria. in fact, the relative and absolute contributions of specific sites to the production of ros in isolated mitochondria depend very strongly on the substrates being oxidized, and the same is likely valid in cells and in vivo [ ] . for example, superoxide formation occurs on the outer mitochondrial membrane, in the matrix, and on both sides of the inner mitochondrial membrane (fig. . ) . complex i (nadh-ubiquinone oxidoreductase) accepts electrons from nadh; these electrons are carried to complex ii (the succinate dehydrogenase-coq oxidoreductase), where they are used to oxidize succinate to fumarate. afterward, electrons continue to travel down their electrochemical gradient to complex iii (the cytochrome bc complex (ubiquinol-cytochrome c oxidoreductase)), and subsequently to complex iv (cytochrome c oxidase); finally, the electrons are used to reduce molecular oxygen to water. thus, complex i and complex ii oxidize the energy-rich molecules nadh and flavin adenine dinucleotide h , respectively, and then transfer the resulting electrons to ubiquinol that carries it up to complex iii (for competent articles, see [ , ] notably, complexes i and ii generate superoxide within the mitochondrial matrix, whereas complex iii produces superoxide at the qo site, resulting in the release of superoxide into either the intermembrane space or the matrix. regarding complex i, it was recently demonstrated that inhibition of nd , a subunit of complex i, suppresses the activity of this complex and thus ros production [ ] . furthermore, data from another set of studies on complex i showed that stable down-modulation of its subunits grim- and ndufs decreased complex i activity that was associated with a significant reduction in the overall nadh oxidation rate but with an increased production of ros by the target cells [ ] . similar results have been found in studies on complex ii: there is evidence suggesting that inhibition of complex ii on the level of subunits even leads to an increase in ros production. the phenomena can be explained by assuming that, if electrons provided in the course of the etc cannot efficiently be transferred to the next complex, they would leak out from the inhibited complex and generate ros [ ] . the complex iii subunits rieske iron-sulfur protein (risp) encoded by ubiquinol-cytochrome c reductase, rieske iron-sulfur polypeptide (uqcrfs ), and ubiquinol-cytochrome c reductase binding (uqcrb) protein appear to play a crucial role in hypoxia-triggered mitochondrial ros generation (for rieske, see box . ). thus, it was shown that risp promotes the hypoxic stabilization of the transcription factor hif- α protein [ ] and uqcrb was found to mediate hypoxia-induced tumor angiogenesis via mitochondrial ros-mediated signalling [ , ] . also, and of note, a mouse model to permit conditional deletion of the nuclear-encoded risp gene was recently developed to assess its role in hypoxiainduced ros signalling in the pulmonary circulation [ ] . it was found that depletion of risp abolishes the ros response to hypoxia in isolated pulmonary the rieske protein is an iron-sulfur protein (isp) component of the cytochrome bc complex that was first discovered and isolated by john s. rieske and coworkers in . the rieske iron-sulfur protein is an essential subunit of mitochondrial cytochrome bc complexes and, like the majority of mitochondrial proteins, is encoded by a nuclear gene and synthesized on cytoplasmic ribosomes as a precursor with a -residue amino-terminal extension. the iron-sulfur protein is then post-translationally imported into the mitochondria where it is inserted into the bc complex in the inner mitochondrial membrane. at first, the precursor is translocated via translocation contact sites into the matrix. there, cleavage to an intermediate containing an -residue extension occurs. the intermediate is then redirected across the inner membrane, processed to the mature subunit, and assembled into complex iii. arterial smcs and isolated pulmonary artery segments. further, in this article, it was discussed that mitochondria are not the only source of ros during hypoxia. thus, studies using a genetic knockout of p phox suggested that cytosolic nadph oxidase systems may also contribute to a hypoxic pulmonary vasoconstriction response during acute hypoxia [ , ] . according to the authors' conclusion, the blockade of hypoxia-induced ros responses (in these studies observed with depletion of risp) suggests that the mitochondria may act as the initiators of ros production, which could be amplified by engagement of nadph oxidase systems elsewhere in the cell. such "ros-induced ros release" might permit small ros signals generated by mitochondria to activate ros signalling throughout the cell, thereby avoiding mitochondrial damage that might arise if the entire cellular oxidant signal originated from that organelle [ ] (or even leading to excessive ros production?). indeed, the substantial advances in oxidative stress research of recent times, in particular, the specification of hypoxia-sensing ros-producing enzyme systems, will contribute to new therapeutic strategies to be applied in acute and chronic human diseases known to be influenced by oxidative stress. for example, discrimination of oxidative eustress, a fundamental process in maintaining health, from oxidative damage will improve clarity in developing "redox medicine" [ ] . when the redox equilibrium of a cell is upset by pro-oxidant environmental stimuli, that is, when oxidative stress exists, an adaptive stress response takes place which can result in upregulation of antioxidant proteins and detoxification enzymes. these antioxidative defense molecules comprise the following [ ] : ( ) agents that catalytically remove free oxygen radicals and other reactive species, for example, sod, catalase, peroxidase, and thiol-specific antioxidants; ( ) proteins that minimize the availability of pro-oxidants such as iron ions, copper ions, and heme, for example, transferrins, haptoglobins, hemopexin, and metallothionein; ( ) proteins that protect biomolecules against damage (including oxidative damage) by other mechanisms, for example, hsps; and ( ) low-molecular-mass agents that scavenge ros and rns, for example, glutathione, α-tocopherol, and (possibly) bilirubin and uric acid. in the past, it was fashionable to divide the oxidative stress response into three main tiers: ( ) antioxidant enzymes including sod, catalase, glutathione peroxidase, and glutathione; ( ) detoxifying enzymes such as glutathione peroxidase, glutathione s-transferase, aldo-keto reductase, and aldehyde dehydrogenase; and ( ) energy-dependent efflux pumps. as a fourth defense system, the antioxidant nutrients such as vitamins e and c as well as carotenoids were appreciated [ ] . it was generally accepted that the first line of enzymes is of enormous importance in limiting ros-mediated damage to intracellular macromolecules. for example, among the most important regulators of ros levels were the sod enzymes: cu/ znsod in the cytoplasm and outer mitochondrial space and mnsod exclusively in the inner mitochondrial space. mechanistically, superoxide is converted to h o and oxygen (o ·− + o ·− + h + → h o + o ) by sod. peroxiredoxins and abundant catalase enzyme then scavenge h o , converting it to molecular oxygen and water. another example of a first-line defense molecule is trx. thioredoxin contains two adjacent -sh groups in its reduced form which are converted to a disulfide in oxidized trx. notably, it can undergo redox reactions with multiple proteins using the reaction trx however, it then turned out that these antioxidative principles were clearly not % effective at performing this task, as under normal physiological conditions, lipid and dna oxidation products can be detected in blood and urine. because certain compounds of the chemicals generated after an interaction of ros with macromolecules are highly reactive, there must be an equal necessity to detoxify these secondary oxidation products to prevent them from also damaging dna, proteins, and lipids. without the adequate detoxification of such products, an extended chain reaction will occur resulting in the degradation of cellular components and the ultimate death of the cell. this second line of defense against ros is provided by those detoxifying enzymes. finally, detoxified metabolites produced by these enzymes are eliminated from the cell by energy-dependent efflux pumps such as the glutathione s-conjugate transporter, also called the multidrug resistance-associated protein (mrp) [ ] . today, one must state that these previous notions are incomplete. at first, it became apparent that members of the so-called cnc (for cap 'n' collar)-basic region-leucine zipper (bzip) family of transcription factors are principal mediators of defense responses to redox stress. in mammals, the cnc family members nrf and nrf were shown to be involved in the transcriptional upregulation of cytoprotective genes encoding a large number of diverse detoxification, antioxidant, and anti-inflammatory proteins (e.g., glutamate cysteine ligase, nadph-quinone oxidoreductase, glutathione s-transferases, and aldo-keto reductases) as well as enzymes with essential roles in cell metabolism [ ] . more recent studies then revealed that these transcription factors, notably nrf , are activated by keap as the primary negative regulator of nrf , that is, a molecule that simultaneously operates as a sensor protein able to perceive dyshomeostatic subclass iic- damps, for example, in terms of redox changes reflecting electrophilic stress. it is worth to add here that six critical domains have been defined in nfr (neh -neh ), and it is neh , located at the n terminus of nrf that acts as the regulatory domain for the cellular stress response. actually, neh interacts with the cytoplasmic protein keap [ ] . in the following, this very important damp-induced and gene-based antioxidative and cytoprotective system is addressed a bit more in detail. increasing evidence indicates that several redox-regulated gene products serve to protect cells from ros damage. the antioxidant response element (are), a cis-acting dna regulatory element or enhancer sequence is known to be activated by oxidative stress and to be responsible for the transcriptional regulation of several redox-regulated gene products. both nrf and bind to are and regulate are-mediated gene expression and induction. the molecule nrf is more potent than nrf in activation of are-regulated gene expression and is regarded as the principal transcription factor that binds to the are. this transcription factor is ubiquitously expressed and present in various organs and tissues including the kidney, muscle, lung, heart, liver, and brain (for reviews, see [ ] [ ] [ ] [ ] [ ] ). as touched in part ii, sect. . . and part iv, sect. . . , the nrf -triggered antioxidant response is initiated by activation of keap that functions as a substrate adaptor protein for the degradation of nrf and serves as an intracellular sensor for redox changes reflecting the presence of subclass iic- damps (for reviews see [ ] [ ] [ ] ). earlier studies had already shown that nrf is a bzip transcription factor that translocates to the nucleus after liberation under oxidative stress conditions from its cytosolic inhibitor keap [ ] . in the nucleus, nrf was found to form dimers with the proteins maf, jun, fos, activating transcription factors (atf ), and/or creb binding protein (cbp) and, in addition, regulates transcription by binding to the are upstream of a variety of cytoprotective and detoxification target genes to combat the oxidative stress [ ] . thus, established nrf -regulated genes reportedly included cu/zn sod, catalase, trx, trx reductase, glutathione reductase (gr), glutathione peroxidase (gpx), and ferritin l (ftl) [ ] (ftl and ferritin h (fth) subunits are responsible for intracellular iron storage). all of these genes are involved in the response to oxidative stress. there are several other genes also known to be engaged in the response to oxidative stress that are not described here. recently, the molecular signalling mechanism involved in the keap ↔ nrf pathway has been further elucidated and specified. the core can be seen in an axis consisting of redox change (subclass iic- damps)-initiated → keap -induced → nrf -triggered → are-driven expression of antioxidant and detoxifying genes ( fig. . ) (discussed in [ - , , ] ). the complex and complicated sequelae of the pathway are simplified in the following text. under homeostatic and stress-free conditions, binding of keap to the nrf molecule leads to its polyubiquitination and subsequent degradation by the proteasomal pathway, thereby maintaining a consistent generation of nrf and retaining its very low levels in the cytoplasm. in this scenario, keap homodimer binds to a single nrf protein via a high-affinity so-called "etge" motif and low-affinity so-called "dlg" motif. the twosite recognition of nrf by the keap dimer is essential for polyubiquitination of nrf (also see part ii, sect. . . ) (for polyubiquitination, see box . ) . in this sense, the keap ↔ nrf system can be regarded as a vital part of regulating cells under a homeostatic environment. however, in case of dangerous and threatening oxidative (and xenobiotic) stress, the system instigates a stress response that is characterized by a rapid and dramatic cessation of the keap -dependent polyubiquitination process resulting in a rapid increase of nrf abundance. in fact, exposure to ros-mediated stress (or electrophilic stress) is thought to modify the reactive "iic- damps-sensing" cysteine residues in keap , which is associated with a conformational change of the protein resulting in a loss of keap ubiquitination activity (fig. . ) . notably, as a cysteine-rich protein, the human keap possesses cysteine residues, and they are all reactive to stress to varying degrees. among these sensor cysteines of keap , c is best characterized. evidence from a number of studies has suggested that c is the most reactive and critical to the keap ↔ nrf stress-sensing response. even, there is already evidence from a first atomic-level view suggesting that the unique environment of cys (besides cys , cys , and cys ) appears to be the critical residue of keap responsible for detecting increased levels of oxidative stress [ , , ] . oxidative modification of cysteine sensors of keap leads to a loss of its polyubiquitination and degradation activity thereby stabilizing nrf . consequently, fig. . the oxidative stress-induced keap ↔ nrf pathway. under non-oxidative homeostatic conditions, the sensor keap is bound to the nrf molecule resulting in its polyubiquitination and subsequent degradation via the proteasomal pathway. oxidative stress modifies the ros-sensing cysteine residues of keap leading to loss of its polyubiquitination and degradation activity, dissociation of nrf that becomes stabilized and accumulates. then, nrf translocates to the nucleus and forms a heterodimer with the smaf transcription factor. the nrf /smaf heterodimer binds to are and induces transcription of numerous cytoprotective antioxidant and detoxification genes. are antioxidant response element, keap kelch-like erythroid cell-derived protein with cnc homology (ech)-associated protein , nrf nuclear factor-erythroid p -related factors , smaf small musculoaponeurotic fibrosarcoma, ub-ub-ub poly-ubiquitin chain. sources: refs. [ - , , ] stabilized nrf accumulates in the cytoplasm, translocates into the nucleus, and forms a heterodimer with a smaf transcription factor (smaf, small musculoaponeurotic fibrosarcoma). thus, this accumulation of nrf in response to ros (and electrophiles) cannot be regarded as an induction in a strict sense but instead is a mechanism referred to as derepression, that is, from the rapid degradation-based repression. as highlighted and discussed [ ] , there are two models of how to explain the cytoplasmic accumulation process of nrf . the "hinge-and-latch" model holds that the modification of the sensing cysteine residues of keap reduces its affinity for nrf but does not result in release. instead, newly synthesized nrf is translocated to the nucleus to trigger the transcription of nrf -dependent genes. the other model denoted as the "conformation cycling" model claims that keap uses a cyclic mechanism to target nrf for polyubiquitination and proteasomal degradation. an important feature of this cyclic mechanism is that it ensures regeneration of keap which allows the cycle to proceed. modification of specific reactive cysteine residues of keap may block the cycle of keap -dependent nrf degradation allowing de novo synthesized nrf to accumulate. the subsequent transcriptional process in the nucleus has been specified as well. as partially mentioned above, the nrf -smaf heterodimer binds to are or electrophile-responsive element (epre) and induces transcription of numerous cytoprotective genes. of note, recently, an extensive genome-wide analysis of the nrf -smaf-binding sequence, that is, the are/epre, and the maf homodimerbinding sequence (the so-called maf responsive element or "mare") was conducted, and the differences between these elements were clarified. as a result, it was proposed that are, epre, and the nf-e binding sequence be collectively named cnc-smaf-binding elements (csmbe) [ ] . interestingly, prms appear to be co-players in this scenario. thus, tlrs have been observed to induce nrf activation. remarkably, in a recent study, tlr agonists were shown to activate nrf signalling via reduction of keap [ ] . the authors could demonstrate that tlr signalling-induced keap reduction promotes nrf translocation from the cytoplasm to the nucleus, where it activated transcription of its target genes. further, tlr agonists were found to modulate keap at the protein post-translation level through autophagy. in fact, tlr signalling increased the expression of autophagy protein p and lc -ii and induced their association with keap in the autophagosome-like structures. the keap ↔ nrf system as briefly described here is a robust oxidative stress response. its regulatory mechanisms, for example, stress-sensing mechanism, proteasome-based regulation of nrf activity, and selection of target genes have been elucidated mainly in mammals (for proteasome, see box . ). nevertheless, the pathway is now regarded as an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress across the tree of life. thus, the keap ↔ nrf system has been found to be also present in zebrafish, fruit fly, and caenorhabditis elegans indicating that its roles in cellular defense are conserved throughout evolution among vertebrates and suggesting that analogous defense systems are widely conserved throughout the animal kingdom [ , ] . as briefly demonstrated in this subchapter, aerobic organisms have integrated antioxidant systems, which include damp-promoted generation of enzymatic and nonenzymatic antioxidants that are usually effective in blocking harmful effects of ros. however, when ros is produced in excess causing pathological conditions, the stress response against oxidative damage can be overridden. thus, oxidative stress is known to contribute to many pathological conditions, including cancer, neurological disorders, atherosclerosis, hypertension, ards, and chronic obstructive pulmonary disease, just to mention a few of them. plausibly, these disorders are motivation enough to search for new effective therapeutic options by harnessing the new insights into mechanisms of the keap ↔ nrf system. certainly, intense research is essential for a detailed understanding of the precise consequences of targeting keap for disease prevention and treatment. all the more so as the oxidative stress response is integrated into other forms of innate stress responses that will be further outlined in the following subchapters. the heat shock response-one of the most ancient and evolutionarily conserved cytoprotective mechanisms found in nature-is induced upon exposure of living cells to acute, subacute, or chronic stress conditions. this defense response is characterized by the expression of a group of phylogenetically conserved intracellular hsps, which possess the capacity to recognize structures commonly found in the interior of proteins and to bind such structures. thanks to this property, they form a chaperone network involved in correct protein folding, trafficking, and complex assembly (for reviews, see [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). in order to be released from engagement with proteins after folding and take part in further rounds of activity, hsp and other chaperones utilize an intrinsic atpase domain to hydrolyze atp and assume a free conformation [ ] . once released, hsps mediate protective cellular defense mechanisms including regulation of apoptosis, the aim being to maintain and restore cellular protein homeostasis (proteostasis). exposure of almost any cell to heat shock most often leads to the rapid transcription, translation, and accumulation of a variety of hsps that increase to quite considerable levels when the stress is pronounced. in fact, these molecules are induced by various environmental insults that can cause protein denaturation and unfolding within the cells, leading to the formation of nonnative proteins and protein aggregates, thereby emitting dyshomeostatic damps. of note, besides their protective role in different intracellular compartments, hsps in terms of inducible damps can translocate to the cell surface to get exposed or are actively secreted via non-canonical pathways. in case of necrotic cell death, they are passively released in large amounts into the extracellular space to act as constitutively expressed damps (compare part iv, sect. . . and sects . . . and . . . ). once emitted, hsps are sensed by classical recognition receptors and/or non-classical receptors (e.g., cd ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . interestingly, recent knowledge about similarities between allograft and tumor rejection has visualized that the processes of both iri to allografts [ , , ] and therapy-mediated injury to tumors [ ] [ ] [ ] [ ] are characteristically associated with emission of hsps. in all eukaryotes, the hsr is primarily regulated and controlled by the hsfs, in particular, hsf , a sequence-specific factor that binds upstream to heat shock elements in the promoters of target genes [ ] (fig. . ). for example, hsf is activated by environmental stress including oxidative and tumor-associated stress [ , ] . notably, in all eukaryotes, hsf responds to such stress conditions by undergoing a monomer to trimer transition and becomes massively phosphorylated, leading to its acquiring ability to bind to dna rapidly and activate transcription [ ] . there is at least preliminary evidence suggesting that intracellular perturbations reflecting dyshomeostatic damps may activate hsfs [ ] [ ] [ ] [ ] . such molecular alterations reportedly include changes in cytosolic ca + concentration, for example, caused by increase of fluidity in specific membrane domains [ , ] . interestingly, a recent study in support of these earlier findings provided evidence of the existence of a plasma membrane-dependent mechanism of hsf activation in animal cells, which is initiated by specific membrane-dependent trpv calcium channel-like receptors [ ] . these findings lend support to the notion that heat sensing and signalling in mammalian cells are dependent on trpv channels, suggesting that these receptors may act as a major hsr sensor in different epithelial non-cancerous and cancerous cells, capable of triggering the cellular hsr [ ] . in another line of experiments, trpv was demonstrated to mediate the effects of transient heat shock on endocytosis of human monocyte-derived dcs, suggesting a central role of trpv in mediating the cellular action of heat shock on these important cells of the innate immune system [ ] (for trpv channel receptors, also compare part ii, sect. . . ). induction of an hsr is not only mediated by sterile stress conditions but is also believed to be promoted by cell-invading viruses or bacteria. consequent emission of hsps in their role as damps may reflect a mechanism by which pathogens may contribute to sterile inflammation. for example, exacerbation of hepatitis b virus (hbv)-associated liver injury is reportedly characterized by an abnormal immune response that not only mobilizes specific antiviral effects but also poses a potentially lethal non-specific sterile inflammation to the host [ ] . heat shock proteins may be the most extensively studied damps in the context of hbv infection. a number of hsps such as hsp and hsp have been reported to be supportive factors in the process of hbv replication, and selective inhibition of these hsps was proposed to be host-based anti-hbv strategies [ ] [ ] [ ] . thus, as argued [ ] , both infective and sterile inflammation may synergistically contribute to the exaggeration of chronic hepatitis, if the hbv cannot be cleared entirely. likewise, bacterial infections were also reported to promote induction of the hsr [ , ] . for example, in studies on h. pylori and e. coli infection models, the initiation of an hsp stress response could be demonstrated [ , ] . the hsr is recognized and accepted as a classical stress response in nearly all species across the tree of life. it reflects the desperate efforts of a cell to restore homeostasis and survive upon both infectious and sterile insults. its products, the hsps, operate as damps in commission of the innate immune system to reach this goal. notably, via this mechanism, an hsr, induced by infectious damage to a cell, can promote sterile inflammation. whereas a successful hsr upon stress leads to restoration of cellular homeostasis and cell survival, an unsuccessful hsr may result in rcd such as apoptosis [ ] . this phenomenon will be resumed in the following subchapters. the er is a continuous membrane system that forms a series of flattened sacs within the cytoplasm of eukaryotic cells. as a subcellular organelle in the control of proteostasis, it is responsible for calcium storage and lipid biosynthesis as well as the synthesis, correct folding, processing, and maturation of proteins as well as for the orchestration of their transport along the classical/conventional secretory pathway. the er delivers these components to their destination compartments which include the er itself, the golgi apparatus, the plasma membrane, and the extracellular milieu or the endocytic and autophagic pathways. plausibly, the multifunctional nature of this organelle requires a myriad of proteins, unique physical structures, and coordination with and response to perturbations in the intracellular environment. a series of chaperones, folding enzymes, glucosidases, and carbohydrate transferases support and execute these processes. perturbation of er-associated functions such as accumulation of unfolded/misfolded proteins, excessive ros production, hypoxia, calcium and glucose depletion, or viral and bacterial infections reflect stress of the organelle and result in activation of an er stress-coping response, the evolutionary conserved upr [ ] [ ] [ ] [ ] . for example, the processes of both iri-mediated cell damage/cell death [ ] [ ] [ ] and induction of the icd of cancer cells [ ] [ ] [ ] are characterized by demonstration of er stress that is almost always associated with oxidative stress and vice versa [ ] . to cope with er stress, cells have the unique possibility to activate the upr, a dynamic signalling network that orchestrates the recovery of homeostasis or triggers rcd modalities, depending on the level of damage. perception of any perturbation of the er is provided by three sensor molecules of the upr embedded in the er membrane: the perk, ire- , and atf . the upr-mediated recovery of er homeostasis mainly occurs through the perk-eif α-mediated temporary shutdown of protein translation and the activation of a complex genetic program that aims to improve er quality control and adaptive responses [ ] [ ] [ ] [ ] (fig. . ) . accordingly, perk, devoted to perceiving dyshomeostatic damp-emitting er perturbations (so far, no clear evidence for ire- and atf in this respect), may be regarded as a new family of non-classical prms, at least in a broader sense. in nonstressed conditions, the three sensors of the er homeostasis are kept in an inactive state by the er-luminal binding immunoglobulin protein (bip). the protein bip, also known as glucose regulated protein (grp ), is a member of the hsp family of proteins (specifically hspa ). it functions as a chaperone to selectively bind unfolded proteins in the er lumen by interacting with exposed hydrophobic . simplified scenario model illustrating the er stress-induced three arms of the unfolded protein response. perception of any perturbation of the er is provided by three sensor molecules of the unfolded protein response embedded in the er membrane: the perk, ire- , and atf . perk perceives dyshomeostatic damp (subclass iic- damp)-emitting er perturbations (ire and atf still questionable). perk phosphorylates eif a to up-regulate transcription factor atf that induces the expression of transcription factor chop. ire a signals through its rnase via the splicing of xbp mrna. the active transcription factor xbp s translocates to the nucleus. atf is exported from the er to the golgi complex to enter the nucleus as a potent transcription factor. together, these transcription factors of the unfolded protein response determine the cell fate by the regulation of distinct subsets of target genes toward recovery of er homeostasis and cell survival or the induction of regulated cell death in form of apoptosis and ferroptosis. in addition, the transcription factor atf is differentially translated, up-regulating genes participating in autophagy and other homeostatic pathways. atf activating transcription factor, chop cytidine-cytidine-adenosine-adenosine thymidine-enhancer-binding homologous protein, eif α eukaryotic translational initiation factor α, er endoplasmic reticulum, ire α inositol-requiring transmembrane kinase/endoribonuclease α, perk protein kinase-like eukaryotic initiation factor α kinase, upr unfolded protein response, xbp x-box binding protein . xbp s, x-box binding protein whereby the "s" stands for the spliced form of xbp . sources: refs. [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] residues on nascent peptides [ ] . however, in conditions of er stress, bip is detached from these sensors allowing their activation to trigger pathways, collectively included in the term of upr. this stress response acts as a corrective path, capable of both increasing the er folding capacity and decreasing the incoming polypeptide load. of note, this downstream pathway of each of the three upr sensors appears to have an innate preference for a particular type of er stress. moreover, as reviewed [ ] , upon dissociating from bip, each of the three sensors modifies the er to mitigate stress in its own unique way. for example, atf is often the first sensor to respond to er stress. once atf dissociates from bip, it is translocated to the golgi apparatus for cleavage. the cytosolic domain of atf is then free to move to the nucleus, where it moderates increased expression of several proteins involved in lipid biosynthesis and chaperones. this allows an increase in the volume of the er and provides more chaperone proteins to aid in folding, thus relieving some of the er stress. the other two sensors, ire- and perk, remain as integral er proteins but oligomerize and autophosphorylate following bip disassociation (autophosphorylation, a type of post-translational modification of proteins (see part vi, sect. . ); typical for this biochemical process, a phosphate is added to a protein kinase by itself). under remediable er stress conditions, the three sensors trigger signalling pathways to resolve er stress aiming at maintaining cellular integrity (fig. . ) . they are briefly touched in the following (for reviews and original articles, see refs [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] .). for example, misfolded proteins are dislocated in the cytosol where degradation processes such as the er-associated degradation (erad) and autophagy will clear them, thereby reducing their potential toxicity. characteristically, erad is a protein quality control mechanism conserved in all eukaryotic cells and represents a critical arm of the upr, necessary to alleviate er stress. the erad mechanism results in the selective dislocation of unfolded and misfolded proteins from the er to the cytosol via specific membrane machinery. the erad targets are subsequently degraded by the cytosolic ubiquitin proteasome system (ups). the whole process includes transcriptional activation of a variety of er-associated chaperones and folding enzymes which include but are not limited to bip and the lectins calr, calmodulin (cam), and calnexin (cnx). a pathway that represents the most conserved branch of the upr is mediated by ire- , a multifunctional protein that possesses kinase and endonuclease activities. upon activation, ire- aggregates and autophosphorylates, thereby activating its endonuclease activity to catalyze the unconventional splicing of x-box binding protein (xbp ) via removal of a -nucleotide intron. this processing event changes the open reading frame of the mrna, resulting in the translation of the transcription factor now termed xbp s ("s" stands for the spliced form of xbp ) (for splicing, see box . ) . production of xbp s leads to upregulation of several genes involved in the upr's adaptive phase, for example, expression of er-resident molecular chaperones and protein folding enzymes. activation of perk leads to the phosphorylation of eif α that is required for the initiation of translation. this factor inhibits global protein synthesis by inhibiting the assembly of the s ribosome, thereby reducing er load and promoting cellular survival. at the same time and under these conditions, the transcription factor atf is differentially translated, up-regulating genes participating in protein folding, amino acid metabolism and transport, autophagy, and oxidative stress resistance/redox homeostasis. under conditions of prolonged or severe er stress that the upr cannot resolve (see below), atf also contributes to apoptosis through the induction of the transcription factor chop (for cytidine-cytidine-adenosine-adenosine-thymidine-enhancer-binding homologous protein) and by enhancing oxidative stress and protein synthesis. finally, the third branch of the upr is initiated by atf . the sensor atf is retained at the er under homeostatic conditions but translocates to the golgi apparatus under er stress where it is cleaved by the golgi-resident proteases site- protease (sp ) and sp . this event leads to the release of atf n-terminal fragment, a potent transcription factor that moves to the nucleus, where it binds the er stress response element upstream of a subset of upr genes to activate their transcription. together with xbp , this fragment regulates the expression of several genes with functions in protein folding, protein transport, and lipid biosynthesis, that is, genes involved in re-establishing er homeostasis. strikingly, there is a considerable interconnectedness of the er stress-promoted upr with other innate immune processes. for example, an increasing number of studies support the view that oxidative stress has a strong connection with er stress. during the protein folding process, ros are produced as by-products, leading to impaired redox balance conferring oxidative stress. as the protein in molecular biology, splicing is a modification of an rna after transcription in which introns are removed and exons are joined. thus, an intron is any noncoding nucleotide sequence within a gene that is removed by rna splicing during maturation of the final rna product; an exon is any part of a gene that encodes a part of the final mature rna produced by that gene after introns have been removed by rna splicing. this process is needed for the typical eukaryotic messenger rna before it can be used to produce a correct protein through translation. for many eukaryotic introns, splicing is done in a series of reactions catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snrnps), but there are also self-splicing introns. folding process is dependent on redox homeostasis, the oxidative stress can disrupt the protein folding mechanism and facilitate the production of misfolded proteins, causing further er stress [ ] . moreover, this stress response functions as a productive source of damps. typically, hsps and calr, like other er chaperones, can translocate to the cytosol and eventually to the surface of cells. once exposed, these molecules can operate as subclass ib- damps to facilitate engulfment of antigens as mostly described in the context of induction of icd [ , [ ] [ ] [ ] [ ] [ ] . via these mechanisms and by crosstalk with other molecular machines of the innate immune system including nlrp inflammasome activation via txnip (see part iv, sect. . . . and part vi, sect. . . . ) , the upr may contribute to sterile inflammation and immunity (for articles, see refs [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). also, several signalling cascades triggered by the three sensors are apparently potent inducers of autophagy at a cellwide level that-as mentioned above-normally has an adaptive/protective function and consists of the three major types: chaperone-mediated, macro-and microautophagy. interestingly, recent evidence has indicated that upr-induced autophagic processes are capable of alleviating the upr pointing to a crosstalk between these two innate immune defense mechanisms [ , ] . strikingly, a complex relationship reportedly exists between autophagy and damps in cellular adaption to stress and injury and cell death characterized by a crosstalk between autophagy induction and secretion or release of damps. in fact, growing evidence indicates that autophagic mechanisms are involved in regulating release and degradation of damps including calr, hmgb , atp, and dna in several cell types [ , , ] . this scenario may contribute to the observation that autophagy is also able to shape a supportive cellular immune response [ , ] . this kind of innate immune interrelationships is, for example, involved in mechanisms of both reperfusion-mediated cell injury and icd of cancer cells (see refs [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). overall, depending on the duration and intensity of the stress, the upr engages different cellular pathways to restore and maintain cell survival, on the one hand, or trigger apoptosis, on the other hand. in cases of severe irremediable er stress, however, the balance is tipped in favor of pro-death signalling; that is, the upr, now mediated by different biochemical pathways, may lead to pro-inflammatory and pro-apoptotic responses resulting in catastrophic rcd (fig. . ) . while the precise pathways of apoptosis induced by er stress are not known, the up-regulated perk → eif α → atf → chop pathway plays an essential role by reversing translational arrest, increasing generation of ros, and promoting calcium efflux from the er. together, these signals lead to cytochrome c release from mitochondria and loss of membrane potential, resulting in apoptosis [ ] . in this context, it is interesting that one pathway of rn (ferroptosis; see sect. . . ) apparently shares a partially overlapping machinery with er stress, suggesting a molecular interconnectivity between these two events [ ] . the issue of er-stress-promoted upr is a parade example of an injury-induced innate immune response that can decide besides life and death of a cell. placed at the very beginning of defense processes of multicellular organisms upon injury, this stress response nicely reflects a hierarchy of damp emission (see part iv, chap. ) . striking is also the existence of an inter-organelle communication, for example, between upr, inflammasome activation, and autophagic pathways which emerge as a homeostatic network determining the switch from adaptive life-saving programs to cell death under stress conditions, where specialized sentinels are localized at organelle membranes to induce the core apoptosis pathway. as briefly sketched in the next section, this innate immune defense response is not only directed against sterile stress but also against pathogen-mediated stress. in the previous section, induction of a upr was mainly exemplified by referring to reperfusion-mediated cell damage and icd of cancer cells, that is, instances of sterile stress. however, growing evidence is coming to light clearly indicating that viruses and bacteria also induce er stress, thereby activating a robust upr. in fact, the constitution of cellular stress responses is meanwhile regarded as the first line of defense against both viral and bacterial infection. however, the outcome is not always in favor of the host; the pathogen may also profit from this stress response, at least under certain circumstances. an increasing number of reports have recently been published on this emerging topic (such as refs [ ] [ ] [ ] [ ] [ ] [ ] ), the quintessence briefly being addressed here. there are several mechanisms described of how a virus can induce er stress. the central mechanisms of perturbation of the er during virus infection can be seen in the production of large amounts of viral proteins by the virus concerned. such accumulation of viral proteins in the er implies a challenge to the protein folding machinery which may cause er stress and, in turn, activate the upr resulting in restoration of the er homeostasis or apoptosis. so far, at least viruses have been found to be able to induce er stress and activate the three upr stress signalling pathways [ ] . moreover, er stress can be caused by viruses via other mechanisms, for example, as a result of er membrane exploitation, imbalance of calcium concentration, or sabotage/depletion of the er membrane during virion release. viral infections may activate these pathways resulting in the inhibition or promotion of viral replication. for example, the perk-mediated global translation shutdown is a very efficient antiviral mechanism, and a similar shutdown by pkr has been used in the interferon pathway to defend against viral infection [ ] . also, the virus-related upr was found to trigger host inflammatory signalling cascade through innate immune signalling pathways that activate nf-κb and ap- transcription factors as a result of chronic er stress. in fact, overexpression of viral proteins in the er has long been known to activate these transcription factors which induce expression of pro-inflammatory cytokines such as il- [ , ] . increasing evidence supports the notion that the upr signalling synergistically interacts with virus-induced signalling to produce inflammatory cytokines and type i ifns. in addition, other lines of studies have shed light on a role of nod and nod receptors in transducing virus-related er stress signals to elicit inflammation [ ] . so far, however, a possible contribution of damps to the promotion of these signalling pathways has not been investigated. importantly, however, the effects of virus-induced upr have been observed not only to inhibit but also to potentiate viral infection. in fact, manipulation of the upr response has become an asset for many viruses to promote their translation, thereby leading to chronic er stress. in other words, during infection, viruses are capable of hijacking the host translational machinery and fill the er with viral proteins. for example, this is the case for many positive-strand rna viruses, which house the virus replication machinery in the protective er-membrane. in fact, viruses need host er to produce increased quantities of viral proteins to continue replication. intriguingly, as discussed [ ] , many viruses have evolved strategies aimed at continuing the replication cycle. thus, viruses were shown to manipulate the host upr in various ways to stimulate protein synthesis capacity and to improve cell survival by inhibiting cellular apoptosis. in particular, the link between the upr and autophagy are intensely discussed to be involved in this scenario. these two systems may act dependently, or the induction of one system may interfere with the other [ ] . thus, experimental studies could demonstrate that different viruses modulate these mechanisms to allow them to circumvent and bypass the host immune response or, worse, to exploit the host's defense to their advantage. according to current knowledge, rna viruses including influenza virus, poliovirus, coxsackievirus, enterovirus, japanese encephalitis virus, hcv, and dengue virus were shown to regulate these processes. for example, recent studies on hcv-infected hepatocytes confirmed the evidence that virus-associated er stress and upr are linked to cellular autophagy. thus, induction of the cellular autophagic response is reportedly required to improve survival of infected cells by inhibition of cellular apoptosis. moreover, the autophagic response was demonstrated to inhibit the cellular innate antiviral program that usually inhibits virus replication. nevertheless, as argued by the authors [ ] , though hcv induces er stress and autophagy, their cause-effect relationship is not clear. notably, the upr is not only modulated by viruses! recent evidence indicates that this stress response plays multiple roles during bacterial infections as well. thus, as comprehensively reviewed by celli and tsolis [ ] , the upr has been shown to be induced in murine lungs by m. tuberculosis (associated with apoptotic events) and also be correlated with helicobacter-induced gastric carcinogenesis. similarly, in vitro infectious models have revealed upr induction in macrophages and epithelial cells infected with either brucella melitensis and b. abortus or listeria monocytogenes. on the other hand, there is evidence suggesting that bacteria can subvert the upr for their own advantage. nevertheless, indications that bacteria can modulate this response are still somewhat sparse. one example refers to l. pneumophila that recruits components of the erad on its vacuole to mediate turnover of bacterial effectors on the vacuolar surface and uses the proteasome to generate amino acids necessary for its intracellular growth. interestingly, like with virus-induced upr, a bacteria-initiated upr may turn out to be of advantage for the host or in favor of the bacteria, but it is not clear in each case whether this response benefits the host or the pathogen. thus, as argued by celli and tsolis [ ] , "modulation of er function during infection by intracellular bacteria can promote bacterial infection by providing a replicative niche, but at the same time the resulting disruption of the secretory pathway can provide a pattern of pathogenesis that aids the innate immune system in recognizing intracellular infection and in mounting an appropriate defence. however, considering the more rapid evolution of bacterial pathogens compared to their hosts, it is likely that bacteria have evolved to modulate the upr to their advantage during infection." the function of the er stress-induced upr appears to provide another impressive evidence in support of the notion that immunity is induced by both sterile and infectious stressful injury and not primarily by invading nonself. in fact, this stress response is at the forefront of any stressful injury and is dedicated to initiating involvement of further damp-promoted defense mechanisms. however, like all the other innate immune processes, the upr may become uncontrolled. together, this may be reason enough that this stress response is involved in the pathogenesis of many human systemic and organ-specific disorders. in fact, the list of human diseases that are pathogenetically associated with er stress and activation of the upr is steadily growing [ ] [ ] [ ] . they include neurodegenerative and metabolic diseases, autoimmune disorders, and atherosclerosis. given such an ever-increasing list of diseases, it is not a surprise that chemical compounds and inhibitors targeting the upr signalling pathways with a high degree of specificity have been and will be further developed [ ] [ ] [ ] . maintaining genome integrity and transmission of intact genomes is a condition sine qua non for cellular, organismal, and species survival [ ] . this homeostatic integrity is threatened by dna damage that occurs in a variety of conditions including but not limited to ionizing radiation, chemical reactions, and viral infections, two of the most dominant conditions being oxidative stress and replication stress. these exogenous and endogenous factors induce diverse lesions in the dna such as nucleotide alterations (substitution, deletion, and insertion), bulky adducts, collapsed dna replication forks, ssbs, and dsbs (see refs [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). in response to dna damage, cells initiate and activate a complex network of cellular signalling cascades that cooperate to sense and repair lesions in dna, denoted as the ddr. this stress response plays an important role in fighting against detrimental effects of cell stress and injury. it orchestrates many processes, including not only dna repair but also regulation of cell-cycle checkpoints, transcription of ddr genes, and autophagy ( fig. . ). the ddr is controlled by three pi k-related kinases (pikks): the atm, the dna-pk, and the atr kinases (mostly nuclear proteins). all three pikks are enormous polypeptides with similar domain organizations and various common structural features. equipped with the capability to autophosphorylate (see above however, at the beginning of this dance, sensors have recently been identified which, as prms in a wider sense or even as a new group of recognition receptors, are capable of detecting dna damage-as manifested by the toxic dsbs or generation of ssdna to activate these three pikks. as already briefly touched in part ii, sect. . . . , two highly conserved multiprotein complexes, mrn and ku, are considered the primary sensors of dsbs to subsequently activate atm and dna-pk [ , , ] . in addition, ssdna is sensed by the recognition molecule rpa that then, analogous to ku, acts as a signalling and repair platform for downstream factors such as the prp , an e ubiquitin ligase involved in pre-mrna splicing, and atr kinase [ ] . other lines of studies have provided evidence suggesting that the protein prp itself may act as the primary sensor of rpa-ssdna to subsequently activate atr [ ] (fig. . ) . in brief, upon dna damage, dsbs are recognized by the mrn complex. following recognition, mrn recruits atm to this dna lesion where it binds to the c-terminus of nbs as a component of mrn [ ] . following binding, atm kinase is activated. however, the exact mechanism whereby mrn activates atm is still not fully understood (discussed in [ ] ). recognition of dsbs is also realized by ku / heterodimer that has been shown to bind broken dsdna ends preferentially [ ] . it is the ku / then that recruits the catalytic subunit of dna-pk (dna-pkcs) at the site of dsbs to form the dna-pk holoenzyme. the major role of activated dna-pk is to promote a peculiar dna repair mechanism called non-homologous end joining (nhej), a pathway that repairs double-strand breaks in dna [ ] . in fact, nhej repairs most dsbs in mammalian cells. as its name implies, nhej involves ligation of two broken dna ends without needing a repair template [ ] . in contrast to atm and dna-pkcs which respond primarily to dsbs, atr is activated by a much wider range of genotoxic stresses, for example, reflected by exposure of increasing amounts of ssdna as a consequence of compromised activity of replication proteins or nucleolytic processing of various forms of damaged dna. in fact, first evidence suggests that ssdna is initially sensed by rpa which then recruits and activates prp [ ] . in turn, prp facilitates the accumulation of atr-interacting protein (atrip, the regulatory partner of the atr kinase) at dna damage sites, thereby activating atr [ , ] . plausibly, the recent discovery of dna damage-sensing molecules such as mrn, ku / , and rpa calls for the definition of those molecules they recognize, that is, broken dna ends at the site of dsbs and ssdna. as outlined in part iv, sect. . . , they have been tentatively sorted into a subclass of cell-intrinsically emitted damps (subclass iic- ). future studies will have to assign their exact place in the world of damps. apart from those damps emitted in the nucleus, other lines of studies lend support to the suggestion that dna damage or dna replication stress, in case of unsuccessful dna repair, promotes release of aberrant dna structures into the cytosol in the form of ssdna and dsdna breaks/fragments which operate as cell-intrinsically emitted dislocated damps. they are sensed by the dna receptor cgas and probably other as-yet-not-identified dna receptors to activate sting-dependent pathways to promote defense pathways [ , ] (compare part ii, sect. . . ; part iv, sect. . . ; as well as part vi, sect. . . ). as discussed by the authors [ ] , it is conceivable that cytosolic dna is released by dysfunctional mitochondria upon dna damage or generated during repair of damaged genomic dna. moreover, as shown in studies on tumor models, the ddr-through the activation of sting-mediated pathways-induces the expression of another class of constitutive damps which are exposed at the cell surface, namely, the mics and different ulbps [ ] [ ] [ ] [ ] [ ] (compare part iv, sect. . . ). of note, however, the ddr does not always result in a happy end. in fact, when unsuccessful in repairing dna damage, the ddr can lead to cellular senescence or-like a upr in case of irremediable er stress-ultimately induces an rcd, most often in the form of apoptosis, less frequently in the form of necrosis. such subroutines of rcd are presumably aimed at mitigating the propagation of potentially mutated cells leading to cancer or other age-related pathologies (fig. . ) [ , , ]. the dna damage response must be regarded as an efficient cell-intrinsic defense process, which is sophistically connected with other stress responses such as autophagy that also plays a significant role in maintaining genome stability [ ] and the er stress-induced upr [ ] . also, of particular interest is the observation that three tiers of damps are involved in this pathway, starting with broken dna ends at the site of dsbs and ssdna in the nucleus immediately generated upon dna damage; followed by dna fragments dislocated in the cytosol and operating as class iic- damps to promote intracellular innate immune signalling; and finishing with the exposure of subclass ib- damps (e.g., mics) able to activate nk, nkt, and γδ t cells (compare part vii, sects. . . , . . and . . ). this phenomenon may again reflect a particular hierarchy in the work of damps to maintain homeostasis. in fact, genomic integrity is of utmost importance and key to human health, and this is retained by the ddr. this stress response, however, may fail in maintaining and restoring homeostasis. accordingly, dna damage has been observed to play a causal role in numerous human pathologies associated with genome instability or aberrant pikk function such as cancer, leukemia, premature ageing, and certain chronic inflammatory conditions. furthermore, there is growing evidence suggesting that uncontrolled ddr signalling is associated with various neurodegenerative diseases, as documented by recent work showing that atm inhibition alleviates pathologies in models of huntington's disease [ ] . this is reason enough that ddr pathways have been and are being explored therapeutically to induce freedom from these diseases. in particular, small molecule inhibitors of atm, dna-pk, and atr are regarded as potential therapeutic agents, for example, as innovative drugs in cancer treatment [ , ] . an increasing amount of publications in the international literature provides convincing evidence that cell-intrinsic, 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with a senescent phenotype biphasic ros production, p and bik dictate the mode of cell death in response to dna damage in colon cancer cells autophagy regulates dna repair through sqstm /p endoplasmic reticulum stress, genome damage, and cancer targeting atm ameliorates mutant huntingtin toxicity in cell and animal models of huntington's disease phosphatidylinositol -kinase (pi k) and phosphatidylinositol -kinase-related kinase (pikk) inhibitors: importance of the morpholine ring targeting atr in cancer medicine the mechanism of mitochondrial superoxide production by the cytochrome bc complex key: cord- -oo w faj authors: seo, youngsik; park, keunchun; hong, yonggun; lee, eun sik; kim, sang-soon; jung, yong-tae; park, heonyong; kwon, chian; cho, young-sik; huh, young-duk title: reactive-oxygen-species-mediated mechanism for photoinduced antibacterial and antiviral activities of ag( )po( ) date: - - journal: j anal sci technol doi: . /s - - -y sha: doc_id: cord_uid: oo w faj cubic-shaped ag( )po( ) crystals with a mean size of μm were synthesized by a precipitation method from a mixed solution of agno( ), na( )hpo( ), and triethanolamine. the antibacterial activities against escherichia coli, listeria innocua, and pseudomonas syringae dc in both the absence and presence of ag( )po( ) under dark conditions and in the presence of ag( )po( ) under red-light ( nm) and blue-light ( nm) irradiation were examined. the concentrations of reactive oxygen species (ros) were also measured in the antibacterial action of the ag( )po( ) against escherichia coli. the photoinduced enhancement of the ag( )po( ) antibacterial activity under blue-light irradiation is explained by the formation of ros during the antibacterial action of the ag( )po( ). moreover, the antiviral activity of ag( )po( ) against amphotropic a murine leukemia virus enhanced under blue-light irradiation via ros production. these results provide an insight into extended bio-applications of ag( )po( ). photoenhanced catalytic and antibacterial materials have been extensively investigated in efforts to eliminate organic pollutants and microorganisms from wastewater (chatterjee and dasgupta ; lapworth et al. ; mouele et al. ; schwarzenbach et al. ). in the process where electrons from the conduction band recombine with holes from the valence band of photocatalytic materials, reactive oxygen species (ros) such as superoxide anions (o •− ), hydroxyl radicals (•oh), and singlet oxygen ( o ) are produced (dickinson and chang ; . these ros play an important role in the photoenhanced catalytic activities. the ros can also damage biomolecules and regulate cell death of microorganisms (du and gebicki ; overmyer et al. ) . silver phosphate (ag po ), which has an indirect bandgap of . ev, exhibits excellent photoenhanced catalytic activity, with a quantum efficiency as high as % under irradiation at nm (bi et al. ; chen et al. ) . ag po exhibits higher photocatalytic activity than tio in the degradation of organic dyes such as methylene blue and rhodamine b (dong et al. ; liang et al. ) . the antibacterial activity and photoinduced antibacterial activity of ag po have also been investigated (buckley et al. ; piccirillo et al. ; seo et al. ; suwanprateeb et al. ; wu et al. ). however, to the best of our knowledge, there is no report that examines antibacterial or antiviral activities arising from the ros photoinducibly generated by ag po . in this work, we assessed the role of rosmediated behavior in the photoinduced antibacterial and antiviral activities of ag po crystals against various bacteria and amphotropic a murine leukemia virus (mlv), respectively. agno ( %, aldrich), na hpo ( %, aldrich), and triethanolamine (tea; %, aldrich) were used without further purification. ag po was synthesized via a simple precipitation method at room temperature. six milliliters of . m tea aqueous solution was added to ml of . m agno aqueous solution with stirring at room temperature for min. then, ml of mm na hpo aqueous solution was added, and the resulting mixture was stirred for min at room temperature. the product was collected by centrifugation at rpm for min, washed several times with water and ethanol, and then dried for h at room temperature. a small fraction ( μl) of escherichia coli (e. coli) overnight culture was added evenly to fresh ml luria-bertani (lb) medium containing μg/ml of the ag po product with or without mm n-acetylcysteine (nac, aldrich) and then incubated in a °c shaking incubator. pseudomonas syringae (p. syringae) dc was grown at °c in nygb medium ( . % tryptone, % yeast extract, and % glycerol) containing rifampicin. two-day grown p. syringae dc culture was evenly aliquoted into ml fresh nygb medium containing rifampicin and further grown at °c. antibacterial activity of ag po to listeria innocua (l. innocua), which was used as l. monocytogenes surrogate, was measured using the agar-overlay method. bacterial culture incubated in tryptic soy broth (tsb) was inoculated to tryptic soy agar (tsa) or tsa containing ag po ( μg/ml). oxford agar base (oab; difco, sparks, md) with antimicrobial supplement (bacto oxford antimicrobial supplement; difco) was poured into -mm petri dish (bottom agar), overlaid with the inoculated tsa (top agar), and incubated at °c with or without light treatment. after incubation at °c for h, oab images were obtained and typical black colonies were enumerated. for virus production, t human embryonic kidney cells (atcc crl- ) were transiently transfected with a full-length molecular clone pmomlv- a -egfp using the calphos mammalian transfection kit (takara bio, shiga, japan). pmomlv- a -egfp is a replication-competent retroviral vector containing enhanced green fluorescent protein (egfp). to determine the viral titer, ml of virus-containing supernatants and μg/ml ag po were mixed at °c for different irradiation time under the blue and red light sources. ht human fibrosarcoma cells (atcc ccl- ) were infected with ml of viral supernatants at a multiplicity of infection (moi) of in the presence of μg/ ml polybrene. two days after infection, green fluorescent protein (gfp)-positive cells were analyzed by a facscalibur tm flow cytometer (becton, dickinson and company, franklin lakes, nj, usa). intracellular amounts of ros were analyzed by fluorescence spectroscopy after reaction with ′, ′-dichlorodihydrofluorescein diacetate (dcfh-da). briefly, e. coli cells were treated with or without ag po under light irradiation. the cells were then additionally incubated with phosphate-buffered saline (pbs) containing μm dcfh-da for h at room temperature in the dark. finally, the amounts of ros were measured by fluorescence spectrophotometry (synergy htx multimode reader; λ ex = ± nm, λ em = ± nm). to obtain e. coli images, we placed dcfh-da-stained cells on a slide glass, covered them with a cover slip, and then observed them by fluorescence microscopy (axioplan microscope) using a green filter. data are presented as the mean ± sem. statistics were performed by tukey's post hoc test. a p < . is considered statistically significant. the structure and morphology of the ag po product were examined by powder x-ray diffraction (xrd; panalytical x'pert-pro mpd) with cu k α radiation and by scanning electron microscopy (sem; hitachi s- ), respectively. to examine the antibacterial activities of the ag po product, the growth rates of e. coli or p. syringae dc in the absence or presence of ag po without light and in the presence of ag po under blue and red light were determined by measurement of the optical density at nm with a uv-vis spectrophotometer (x-ma v). a blue led (nc led, λ = nm) and a red led (nc led, λ = nm) with equivalent luminescence were used as the blue and red light sources, respectively. results and discussion figure a shows an sem image of the ag po crystals prepared by the precipitation method at room temperature. most of the ag po crystals exhibit a cubic shape with a size of μm. figure b shows the xrd pattern of the as-synthesized ag po crystals. the rietveldrefined cell parameters of the ag po crystals in this work are consistent with those of body-centered cubic ag po with a = . nm (jcpds - ). figure a shows the growth rate of e. coli in the absence and presence of ag po under dark conditions and in the presence of ag po under red-light ( nm) and blue-light ( nm) irradiation. in the control experiment without ag po crystals and under dark conditions, the growth rate of e. coli increases rapidly during the incubation period of h and reaches a fig. a the growth rate of e. coli in the absence (circles) or presence (squares) of ag po under dark conditions and in the presence of ag po under red-light ( nm, triangles) or blue-light ( nm, inverted triangles) irradiation. line graphs represent mean ± sem (n = ). b colony formation of l. innocua in the absence or presence of ag po under dark conditions and in the presence of ag po under red-light or blue-light irradiation. quantified data are shown in bar graphs (mean ± sem; n = ). scale bars are μm in b. c the growth rate of p. syringae dc in the absence (circles) or presence (squares) of ag po under dark conditions and in the presence of ag po under red-light (triangles) or bluelight irradiation (inverted triangles). line graphs represent mean ± sem (n = ) saturation plateau after - h. the incubation time for growth to % is known as the half-maximal growth time. the half-maximal growth time in the control experiment was . h for culturing the e. coli in the absence of ag po and under dark conditions. when ag po crystals were present under dark conditions, the growth rate of e. coli decreased compared with the growth rate in the control experiment and the halfmaximal growth time increased to . h. these results indicate that the ag po crystal exhibits antibacterial activity against e. coli. in the presence of ag po crystals and under red-light ( nm) irradiation, an e. coli growth curve very similar to that for ag po crystals under dark conditions is observed, where the half-maximal growth time is . h. because the indirect bandgap energy of crystalline ag po is . ev ( nm), the red light ( nm, . ev) lacks sufficient energy to transfer the electron from the valance band to the conduction band of the ag po crystal. this suggests that red light does not induce photoenhancement of the antibacterial activity of ag po crystals. however, under blue-light irradiation ( nm, . ev), the growth rate of e. coli is substantially decreased in the presence of ag po crystals and the half-maximal growth time is increased to . h. because the blue light has sufficient energy to transfer electrons from the valance band to the conduction band of the ag po crystals, the photoinduced enhancement of antibacterial activity of ag po is observed only under blue-light irradiation. similar trends were observed for l. innocua, which was used as a surrogate of representative gram-positive foodborne pathogens, l. monocytogenes. figure b shows colonies of l. innocua grown on the selective agar plates in the absence and presence of ag po under dark conditions and red-light or blue-light irradiation. ag po decreases the number of l. innocua colonies by twofold under dark conditions. the colony number is about / cm under blue-light irradiation in the presence of ag po , when compared to /cm and /cm under dark and red-light conditions in the presence of ag po , respectively. this result indicates that blue-light irradiation remarkably and synergistically enhances the antibacterial activity of ag po . photoinduced antibacterial activity on the agar plate gives an insight into applications of ag po in anti-fouling and eco-friendly adhesive industry. we then examined the photoinduced antibacterial activity of ag po against the plant pathogenic p. syringae dc bacterium. in fig. c , the half-maximal growth rates of untreated control and ag po under dark conditions are h and h, respectively. comparatively, the half-maximal growth rates of ag po under red-light and blue-light irradiations are h and undetectable (caused by almost complete inhibition), respectively. accordingly, ag po under blue-light irradiation almost completely inhibits the growth of p. syringae dc , suggesting that ag po under blue-light irradiation can be useful for crop protection from phytopathogenic bacteria. to understand the mechanisms underlying the antibacterial activity of ag po crystals, we examined whether ag po crystals alter the levels of ros in e. coli. interestingly, ag po crystals appeared to increase the level of ros under blue-light irradiation, whereas ag po crystals alone or under red light exhibited no effect, as shown in fig. . quantified amounts of ros and ros-stained e. coli cells are shown in fig. a and b, respectively. in both panels, the level of ros was highest in e. coli cells exposed to ag po crystals in conjunction with blue-light irradiation. these data indicate that the antibacterial activity of ag po under blue-light irradiation corresponds to the amount of ros in e. coli. more convincingly, n-acetylcysteine (nac) known as an ros scavenger reverses the antibacterial activity of ag po under blue-light irradiation as shown in fig. c . we furthermore examined the antiviral activity of ag po under blue-light irradiation. figure a shows that amphotropic a murine leukemia virus (mlv) was more severely inactivated by ag po under bluelight irradiation, when compared to ag po under dark conditions and ag po under red-light irradiation. we assume that inactivation of the mlv by blue-light irradiated ag po might be attributable to the peroxidation of the envelope membrane phospholipids, which is furthermore detrimental to dna (paiva and bozza ) . given that the envelop membrane phospholids are damaged by blue-light irradiated ag po , other enveloped viruses including hiv- , sars-cov, mers-cov, and sars-cov can be inactivated by blue-light irradiated ag po . to understand the antiviral activity of ag po under blue-light irradiation, the possibility of the generation of ros was examined when blue light irradiates on the ag po solution. figure b shows that ros is substantially increased by photoinduction to the ag po solution. this result supports that the ros is detrimental to viral particles. we synthesized cubic ag po crystals with a mean size of μm to investigate their antibacterial and antiviral activities. the ag po crystals showed good antibacterial and antiviral activities against e. coli, l. innocua, p. syringae dc , and amphotropic a mlv. the photoinduced enhancement of the antibacterial and antiviral activities of ag po under blue-light irradiation was observed. the ros mediation process in the antibacterial and antiviral activities was confirmed through measurements of the concentrations of ros. the formation of ros plays an important role in the antibacterial and antiviral activities of ag po . these findings suggest that the photoinduced enhancement of antibacterial and antiviral activities of ag po can be used for the biomedical application including anti-fouling, additives, and crop cultivations. facile synthesis of rhombic dodecahedral agx/ ag po (x = cl, br, i) heterocrystals with enhanced photocatalytic properties and stabilities hydroxyapatite supported antibacterial ag po nanoparticles visible light induced photocatalytic degradation of organic pollutants methods and mechanism for improvement of photocatalytic activity and stability of ag po : a review chemistry and biology of reactive oxygen species in signaling or stress responses a simple way for ag po tetrahedron and tetrapod microcrystals with high visible-light-responsive activity proteins are major initial cell targets of hydroxyl free radicals emerging organic contaminants in groundwater: a review of sources, fate and occurrence mechanism of photogenerated reactive oxygen species and correlation with the antibacterial properties of engineered metal-oxide nanoparticles hierarchical ag po porous microcubes with enhanced photocatalytic properties synthesized with the assistance of trisodium citrate degradation of organic pollutants and microorganisms from wastewater using different dielectric barrier discharge configurations-a critical review reactive oxygen species and hormonal control of cell death are reactive oxygen species always detrimental to pathogens? light induced antibacterial activity and photocatalytic properties of ag/ ag po -based material of marine origin the challenge of micropollutants in aquatic systems photo-enhanced antibacterial activity of ag po preparation and characterization of nanosized silver phosphate loaded hydroxyapatite by single step co-conversion process morphology-controlled synthesis of ag po nano/microcrystals and their antibacterial properties the authors acknowledge financial support from the national research foundation of korea (nrf- r d a b ). all authors have equal contribution to this research work. the author(s) read and approved the final manuscript. availability of data and materials not applicable the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ( ) : page of key: cord- -nqj ctk authors: ogger, patricia p.; byrne, adam j. title: macrophage metabolic reprogramming during chronic lung disease date: - - journal: mucosal immunol doi: . /s - - - sha: doc_id: cord_uid: nqj ctk airway macrophages (ams) play key roles in the maintenance of lung immune tolerance. tissue tailored, highly specialised and strategically positioned, ams are critical sentinels of lung homoeostasis. in the last decade, there has been a revolution in our understanding of how metabolism underlies key macrophage functions. while these initial observations were made during steady state or using in vitro polarised macrophages, recent studies have indicated that during many chronic lung diseases (clds), ams adapt their metabolic profile to fit their local niche. by generating reactive oxygen species (ros) for pathogen defence, utilising aerobic glycolysis to rapidly generate cytokines, and employing mitochondrial respiration to fuel inflammatory responses, ams utilise metabolic reprogramming for host defence, although these changes may also support chronic pathology. this review focuses on how metabolic alterations underlie am phenotype and function during clds. particular emphasis is given to how our new understanding of am metabolic plasticity may be exploited to develop am-focused therapies. the respiratory mucosa is a unique site, as our airways are continually exposed to particulates, viruses, bacteria, and fungi, which challenge the pulmonary immune system . to maintain pulmonary homoeostasis and ensure efficient gas exchange, a complex regulatory system is in place, of which airway macrophages (ams) are a core component. ams are the most numerous immune cell type present in healthy lungs, are strategically positioned at the interface of airways and environment , and critical sentinels of barrier immunity. ams form the first line of defence against inhaled particles, pathogens and antigens . although inherently suppressive, ams exhibit significant functional and phenotypical specialisation, allowing efficient responses to environmental signals and rapid alterations in phenotype. increasing evidence suggests that metabolic alterations provide an additional layer of functional plasticity to am populations. activation of macrophages in vitro with a range of inflammatory stimuli, induce profound metabolic adaptations, such as the switch from oxidative phosphorylation (oxphos) to glycolysis in oxygen-sufficient conditions, similar to the "warburg effect" seen in some cancers . it is now clear that how macrophages utilise energy dictates immune responses, and that manipulating cellular metabolism can alter the inflammatory response . however in vivo, the unique oxygen rich environment of the airways coupled with specific local nutrient availabilities, shapes am phenotype and function. indeed, many recent studies have indicated that in chronic lung diseases (clds), such as asthma, chronic obstructive pulmonary disease (copd), cystic fibrosis (cf), idiopathic pulmonary fibrosis (ipf), and during infection such as with mycobacterium tuberculosis (mtb), there are significant alterations in am metabolic processes and that targeting these pathways could represent an exciting therapeutic approach , . this review focuses on how metabolic adaptations underlie am phenotype and function during clds. particular emphasis is given to how our new understanding of am metabolic plasticity may be exploited to develop am-focused therapies. airway macrophages: guardians of the lung environment to maintain gas exchange, it is critical that ams sustain a naturally hyporesponsive state whilst also reserving the ability to rapidly mount effective inflammatory responses. this balance is achieved through complex am-airway epithelial cell (aec) interactions via cell surface-expressed receptors and secreted products. ams express transforming growth factor beta receptor (tgf-βr), interleukin (il)- receptor (il- r), cd receptor (cd r) and signal regulatory protein-α, key components mediating am: aec crosstalk and in turn, regulating am activation . for example, am-aec contact decreases am phagocytosis and cytokine production in a tgf-β-dependent manner . conversely, loss of the integrin αvβ such as through loss of contact of ams with aec upon toll-like receptor (tlr) activation leads to initiation of the am pro-inflammatory phenotype and inflammatory response . ams are characterised by a distinct cellular phenotype. human ams highly express the lectin-binding transmembrane glycoprotein cd , the adhesion molecule cd , the integrin cd c and mannose receptor cd (fig. ). in mice, expression of the cd + cd + cd c hi cd b lo cell surface phenotype is conserved at steady state [ ] [ ] [ ] , while murine ams also express the mer tyrosine kinase (mertk), sialic acid dependent adhesion molecule siglecf, hormone receptor f / , glycoprotein cd and the cd receptor (fig. ) . recent work in mice has indicated that many tissue resident macrophages, including those in the airways, are foetally derived and self-maintain locally with minimal contribution from circulating monocytes, during steady state conditions [ ] [ ] [ ] [ ] [ ] . during murine prenatal development, foetal liver or yolk sac macrophages are the major contributing pool to am populations and am colonization of the lung occurs in sequential waves in the first week of life . furthermore, post birth and during maturation, circulating monocytes do not significantly contribute to lung macrophage populations at homoeostasis . during pulmonary inflammatory responses however, monocytes are recruited to the lung , and subsequently develop into am-like cells , . thus, post-injury murine airways contain at least two ontologically distinct am populations, tissue resident ams (tr-am) which are prenatally derived and monocytederived ams (mo-ams). several groups have studied samples from lung transplant patients to investigate the origins of ams in the human lung [ ] [ ] [ ] [ ] [ ] . utilizing bronchoalveolar lavage (bal) from sexmismatched lung transplant patients our group recently demonstrated that the majority of ams in human lung post-transplant are derived from peripheral classical monocytes . thus, the unique airway niche combined with distinct ontological origins, age and environmental exposures results in remarkable am plasticity and adaptability [ ] [ ] [ ] . while the recruitment of monocytes to the lungs to replenish the tr-am pool is relatively well understood in mice, the origins of ams in the human lung during healthy aging or clds requires further investigation; in particular clear markers which distinguish mo-ams and tr-am are not well established. airway macrophage metabolic phenotype at homoeostasis beyond delineation of macrophage populations based on anatomical location or ontological origin, macrophage populations are also classified according to their activation status. analogous to the th /th paradigm, in vitro cultured human monocyte derived macrophages (mdms) or murine bone marrow derived macrophages (bmdms) are categorised as m or m macrophages, respectively. seminal studies have demonstrated that pro-wound healing m (il- stimulated) macrophages in vitro rely on fatty acid oxidation (fao), an intact tricarboxylic acid cycle (tca) cycle, high rates of oxphos and increased expression of arginase (arg ), which catalyses the production of ornithine from arginine as precursor for collagen to facilitate wound healing , [ ] [ ] [ ] . conversely, pro-inflammatory m -macrophages rely on glycolysis and breaks in the tca cycle lead to accumulation of metabolites, many of which have signalling functions such as citrate, succinate, fumarate and α-ketoglutarate , , . however, although useful in defining the range of potential macrophage responses, in vitro derived cells do not recapitulate the core aspects of am phenotypes which are shaped by the local niche . as ams are highly adapted to the unique environment of the airway lumen, it is perhaps unsurprising that the metabolic state of ams is also distinct. glucose concentrations in the alveolar lumen are less than % of blood glucose concentrations and ams exhibit extremely low levels of glycolysis ; in stark contrast to bmdms, ams do not undergo glycolytic reprogramming in response to lps . consequently, ams readily engage oxphos and highly express the peroxisome proliferator-activated receptor gamma (pparγ) , which regulates lipid accumulation and promotes fao to sustain oxphos. ams also play a major role in the catabolism of pulmonary surfactant, a monolayer composed mainly of phosphocholinebased lipids, phospholipids and cholesterol which lines the alveoli, lowers surface tension and prevents alveolar collapse during expiration . mice lacking gm-csf and thus the am compartment, develop pulmonary alveolar proteinosis (pap), an inflammatory lung syndrome caused by the defective clearance of surfactant [ ] [ ] [ ] . in humans, mutations in genes encoding for gm-csf receptors, result in hereditary pap as a result of progressive alveolar surfactant accumulation [ ] [ ] [ ] [ ] . am phenotype and behaviour are influenced by surfactant exposure, which has major implications for am-mediated immune responses in pulmonary tissue. there are four principle surfactant proteins (sp-a-d) and sp-a and sp-d have been shown to directly influence am functions such as cell migration, phagocytosis and activation phenotypes . both sp-a and sp-d bind carbohydrates, lipids, and nucleic acids and initiate phagocytosis of inhaled pathogens and apoptotic cells . furthermore, sp-a blocks the binding of tlr ligands to tlr , tlr and tlr co-receptors and furthermore inhibits complement activation , . whilst the alveoli are covered with a monolayer of surfactant, a thin layer of mucus produced by goblet cells and ciliated epithelium protects the airways. mucus serves as a barrier and facilitates clearance of microbes and pollutants. a major component of mucus are mucin glycoproteins, which may be categorized as polymerizing, nonpolymerizing and cell-surface associated. of the cell-surface associated mucins, muc is expressed in ams and contributes to the resolution of inflammation by decreasing phagocytic potential and pro-inflammatory cytokine production . the polymerizing mucins include muc ac and muc b; in particular, muc b deficiency has been linked to particle accumulation in the lung, mucus obstruction and impaired macrophage phagocytosis . pro-inflammatory macrophages induce muc b expression to aid mucociliary clearance . furthermore, a single nucleotide polymorphism in the muc b promotor has been strongly associated with the risk of developing ipf, highlighting the importance of mucins for the pulmonary environment . in addition to low glucose and a lipid rich environment, the airways also have a unique distribution of amino acids and central carbon metabolites. surowiec et al. showed that whilst several glucogenic and ketogenic amino acids were present in the bronchial wash, only alanine is present in bal (fig. ). in addition, the central airways contained key glycolytic and oxphos metabolites such as fructose, glucose- -phosphate, fumarate and malate as well as oxidised gluthathione (gssg, indicating oxidative stress, fig. ) ; interestingly, these could not be detected murine ams express the lectin-binding transmembrane glycoprotein cd , the mer tyrosine kinase (mertk), the integrin alpha x chain protein cd c, the type i membrane glycoprotein cd receptor, the mannose receptor cd , the egf-like module-containing mucin-like hormone receptor-like (f / ), the sialic acid binding lectin siglec-f and the fc receptor cd . human ams express cd , the adhesion molecule cd , cd c, cd as well as mhc class ii receptor hla-dr. in the periphery, suggesting either minimal secretion, high utilisation or as a result of anatomical location (i.e. close proximity to nutrient rich pulmonary capillaries) . recently the lung microbiome has gained attention as a factor which modifies the pulmonary environment and directs immune responses by producing short chain fatty acids (scfa). whilst the airways and alveoli are colonised mainly by proteobacteria, bacteroidetes and firmicutes , , the nasal mucosa additionally hosts actinobacteria , (fig. ) . proteobacteria, bacteroidetes and firmicutes produce large amounts of scfa, including acetate, propionate and butyrate, which influence barrier function by regulating epithelial tight junctions and anti-inflammatory immune responses . recent advances in understanding the pulmonary microbiome during homoeostasis and clds are described in detail elsewhere , , . thus, at homoeostasis ams are exposed to a unique environment, with minimal glucose availability and a distinct distribution of nutrients and scfa, which depend on anatomical location (fig. ) . however, despite the profound influence that local substrate availabilities may exert on macrophage development, activation and function, this is an understudied area. new knowledge, which further defines especially human am substrate dependencies at homoeostasis is required in order to fully understand how local metabolic perturbations during clds may contribute to pathology. this is particularly relevant as already slight changes in nutrient availability during inflammation, such as succinate or citrate, can alter macrophage phenotypes through stabilization of hif α, post-translational modification of proteins and production of no and ros , thereby contributing to a pathological development. am metabolism during clds chronic lung diseases affect a significant proportion of the world's population, killing more than , people in the uk alone, each year . persistent inflammation, impaired repair processes and pulmonary remodelling are cardinal features of clds [ ] [ ] [ ] . there are multiple overlaps in environmental exposures driving clds, such as smoking, pollution and environmental exposures; viral infection can also exacerbate symptoms in each disease , , . interestingly, am metabolic adaptation may play a central role in dictating pathology during clds and present novel therapeutic opportunities (fig. ) . asthma. asthma is a heterogeneous disease of the airways characterized by airway remodelling, mucus production, airway hyperresponsiveness (ahr), and inflammation . although most patients have good control with standard medication, a proportion experience life-threatening, severe disease . ams are central to mediating type- inflammation against allergens and parasitic worms . in vitro, macrophages respond to type- cytokines such as il- that drive an 'alternative' m activation phenotype, linked to wound repair and type- pathology , . manipulation of am phenotype via genetic deletion of the transcription factor interferon regulatory factor (irf ), a master regulator of macrophage activation , promoted pulmonary remodelling, ahr and mucus secretion in mice, in an il- dependent manner . indeed, a recent study has shown that both cd + am (activated by il- and il- ) and pro-inflammatory irf + am are increased in asthmatic patients , highlighting the plasticity of macrophages and heterogeneity of human asthma. roles of ams during antigen induced airway inflammation include phagocytosis of apoptotic cells and eosinophils as well as triggering anti-inflammatory pathways to regulate airway hyper responsiveness, mucus secretion and matrix deposition . in severe asthma however, this protective function is impaired, resulting in the loss of phagocytic ability and anti-inflammatory programme , which can contribute to airway remodelling . thus, ams are uniquely involved in responses to allergen and type- inflammation, and aberrant amphenotypes can directly influence respiratory pathology. numerous lines of evidence suggest that metabolic stress leading to the production of reactive oxygen species (ros) plays a role in asthma. increased ros have been detected in ams of asthmatic patients , and contributes to lung injury and proinflammatory tumour necrosis factor-alpha (tnf-α) and il- β secretion by macrophages . furthermore, heme-oxygenase- (ho- ), which mediates ros production in response to chemical and physical agents, is increased in ams in asthmatics . in addition to these pro-inflammatory characteristics, ams show key features of a more anti-inflammatory phenotype in studies using ovalbumin to model allergic asthma. using this model, al-khami et al. show that expression of carnitine palmitoyltransferase (cpt) is increased in ams, shuttling fatty acids into the mitochondria, as well as increased gene expression of fao related genes . another functional pathway that is altered in asthmatic ams and links to the underlying metabolic phenotype is the eicosanoid pathway, which is induced by th cytokines il- and il- . eicosanoids, including prostaglandins and leukotrienes, are produced from the poly-unsaturated fatty acid arachidonic acid, which is released during asthma . increased production of the eicosanoid -hete and leukotrienes b (ltb ) and e (lte ) has been detected in ams from asthmatic patients stimulated ex vivo . this contributed to bronchial constriction and pro-inflammatory phenotype and failure to generate the anti-inflammatory eicosanoid -hete and prostaglandin e (pge ), which is associated with reduced am phagocytosis . lte has been shown to cause ahr in subjects with aspirin-induced asthma and can be produced in ams by γ-glutamyl transpeptidase , . il- furthermore induces arg , which may further contribute to the asthmatic phenotype via metabolism of collagen precursors ornithine and proline to collagen and airway remodelling , . several of these observed alterations have been targeted therapeutically, attempting to rewire am phenotype. these include the eicosanoid pathway, ros, glycolysis and fao. administration of the anti-inflammatory eicosanoid -hete inhibited leukotriene synthesis and reduced ahr in asthmatics . ex vivo, the corticosteroid dexamethasone decreased levels of thromboxane b and ltb in macrophages and asthmatic ams , while prednisone decreased ltb production in ams . treatment with the antioxidant ad improved ahr and airway inflammation by decreasing ros in the ova-sensitised mouse model of allergic airway disease (aad) , . together, these studies indicate that there is significant disruption of am metabolism during asthma and aad, notably via dysfunction in eicosanoid, glycolysis and fatty acid pathways. in fig. altered metabolic pathways in ams drive key features of chronic lung disease. several metabolic pathways are rewired during chronic lung disease. while this response exists to clear invading pathogens and launch an inflammatory response, long-term activation of these pathways has negative implications. the glycolysis pathway supports inflammatory responses of am, while iron and metabolites produced in the tca cycle can function as bacterial substrates and contribute to pathogen survival. while fatty acid synthesis and oxidation is useful as a way of storing energy and alternative energy source during times of macrophage activation, fatty acid synthesis can also contribute to mucus production. leukotrienes contribute to the am pro-inflammatory phenotype but also cause bronchial constriction and contribute to airway remodelling in asthmatics by causing smooth muscle thickening. the amino acid arginine is a proliferator for collagen via ornithine and proline and can thereby contribute to extracellular matrix deposition. order to evaluate candidate therapies, it is crucial that studies utilise relevant preclinical models and ex vivo patient samples to understand disease. models which more closely recapitulate the complex immune response to allergens, are more likely to reveal viable targets for intervention; in particular the ovalbumin model of aad, which requires an adjuvant and a sensitization phase , is a poor murine model of asthma. furthermore, our new understanding of asthma heterogeneity has allowed the development of biologics which target "type- high" asthma ; delineation of how metabolic changes underlie distinct asthma phenotypes could lead to new treatments for other phenotypes, such as neutrophilic and paucigranulocytic asthma. copd. copd is the th leading cause of death in high income countries , affecting over million people worldwide . copd is a heterogeneous disease, characterised by destruction of the parenchyma and emphysema, narrowing of the airways, remodelling and chronic inflammation driven by chronic exposure to cigarette smoke and particular matter . am numbers are increased in copd bal and contribute to copd pathology through numerous pathways. during copd, ams are found in areas of lung destruction and produce pro-inflammatory cytokines , chemokines and matrix metalloproteases (mmps) with elastolytic properties , . at the same time, tissue inhibitor of metalloproteases (timp)- is decreased in ams in copd and furthermore, decreased phagocytic capacity and impaired bacterial killing have been described in copd-ams [ ] [ ] [ ] . ams of copd patients experience a high level of oxidant burden induced through cigarette smoke and subsequent increased ros production is a key feature . compared to controls, copd-ams secrete increased levels of mitochondrial ros (mtros) , superoxide and hydrogen peroxide , whilst glutamyl cysteine ligase for gsh synthesis is downregulated . cigarette smoking also alters iron homoeostasis and ams in copd show increased sequestering of iron , which can furthermore contribute to ros production. bewley et al. showed recently that increased generation of mtros in copd ams results in impaired bacterial clearance . this study also reported a decrease in the mitochondrial membrane potential , which has recently been linked to am exposure to particulate matter . this may explain the impaired phagocytic capacity of ams in copd as decreased mitochondrial membrane potential results in energy failure in the cell, proton leakage and increased mtros . another study by o'beirne et al. further investigated the metabolic profile of ams from healthy smokers, non-smokers and copd patients. while all groups had similar baseline glycolysis rates, there was a decrease in coupling efficiency, maximal respiration and spare respiratory capacity in copd-ams, while proton leak was significantly increased . in addition, expression of genes related to glutathione metabolism, mitochondrial transport, pyruvate metabolism, tca cycle and electron transport chain were altered in smokers and copd patients, compared to nonsmoking healthy controls . other metabolic alterations in copd ams include increased expression of inducible nitric oxide synthase (inos) contributing to increased levels of nitric oxide (no) and increased levels of the adenosine receptor a br , suggesting increased adenosine metabolism, which might be linked to the increased levels of hif α in copd ams . while excessive ros production through oxidant burden and iron accumulation has been identified as an important regulator of am phenotype in copd, it has only recently been linked to mitochondrial dysfunction and metabolic reprogramming. it would be interesting to follow up on these transcriptomic and metabolic alterations to understand their underlying disease driving role and to identify ways to rewire am metabolism. as corticosteroids have been found to be particularly ineffective in copd, more specific pathways involved in am function and metabolism have been investigated recently, such as the ros pathway and iron accumulation. a study by harvey et al. showed that treatment with sulforaphane in copd ams ex vivo improved bacterial clearance by activating the antioxidant and antiinflammatory nrf pathway , while cloonan et al. found that treatment with an iron chelator or a low iron diet protected mice from cigarette smoke induced copd . furthermore, procysteine, a precursor of gsh, increased am efferocytosis in a mouse model of copd . overall, copd is marked by distinct iron sequestration, ros, no production and energetic dysfunction in ams; further delineation of how mitochondrial phenotype links to inflammatory processes and pathology in copd will allow the identification of molecular targets for modulating mitochondria during the disease. cystic fibrosis. cystic fibrosis (cf) is caused by mutation of the cf transmembrane conductance regulator (cftr), a chloride channel, which regulates fluid homoeostasis in mucosal surfaces. in the lung, cftr mutation and subsequent loss of function results in a reduced aqueous film covering the epithelium and mucus thickening, leading to impaired mucociliary clearance and frequent bacterial infection . cf is furthermore characterised by hyper-inflammation of the lungs, airway obstruction, structural damage and progressive reduction of lung function . during recurring airway inflammation, large numbers of neutrophils, macrophages and t lymphocytes infiltrate the lungs and secrete pro-inflammatory cytokines, while anti-inflammatory il- is reduced , . although am numbers are increased in cf patients , , pathogen clearance is attenuated, leading to colonisation of the airways and chronic inflammation , . meyer et al. report a more pro-inflammatory phenotype of ams in a murine model of cf, even in the absence of infection and mdms differentiated from cf patients show an increased inflammatory profile , while others have shown that monocytes from cf patients had an impairment in activation upon il- stimulation . cf-am phenotype can be heterogenous, depending on infection status and local environment. while ams from p. aeruginosa infected cf patients showed increased expression of mannose receptor cd and augmented arginase activity , in cf sputum ams a decrease in expression of cd and scavenger receptor marco was detected . furthermore, ams are involved in the structural damage in cf airways by secreting serine-and metalloproteases, which subsequently degrade connective tissue components . the lower volume of airway surface liquid in cf airways activates ams to increase their release of mmp , resulting in the cleavage of elastin and degradation of the airway and parenchyma . in cf airways, gsh is depleted , , while levels of iron, transferrin, haem and haemoglobin are increased , resulting in high oxidative stress and ros production. ros in turn can induce tgf-β , which has recently been shown to be increased in cf-bal and ams and inhibits cftr biogenesis and cellular trafficking to the surface of epithelial cells , while also contributing to airway remodelling by recruitment and differentiation of myofibroblasts . however, during infection with bacteria from the burkholderia family, both mdms and ams from cf patients showed reduced superoxide production as well as decreased phosphorylation of nadph oxidase (nox) components p phox and p phox , suggesting an inherent deficit in cf-ams generating oxidative bursts for pathogen defence . p. aeruginosa is one of the most common pathogens to cause recurrent pulmonary infection in cf patients and exploits the host to maintain infection by inducing production of the tca cycle metabolite itaconate in ams. itaconate exerts antimicrobial properties via inhibition of bacterial isocitrate lyase in the glyoxylate shunt and to evade this mechanism p. aeruginosa has developed a way to use itaconate as an energy source . similarly succinate, which is secreted in high levels during cf and especially during bacterial infection , can be utilised by p. aeruginosa and s. aureus as a substrate to generate oxidative stress. changes in lipid metabolism are a hallmark of cf and increased fao, lipid turnover in cell membranes and eicosanoid production in ams have been reported. furthermore, sterol regulatoryelement binding protein (srebp), a regulator of lipid homoeostasis, has been linked to cftr loss of function . this results in altered plasma and tissue fatty acid profiles, and while levels of the omega- fatty acid docosahexaenoic acid (dha) were unchanged in ams upon loss of cftr , ex vivo treatment with dha decreased tnf-α levels . furthermore, in cf ams the antiinflammatory lipoxin a (lxa ), which is synthesised from the fatty acid arachidonic acid, is reduced and the lxa /ltb ratio in cf bal is decreased , while the fatty acid metabolite resolvin d (rvd ) has been suggested as a biomarker . increased energy demand by ams in cf, either by manipulation through bacterial pathogens or to fight sustained infection, results in increased utilization of all available metabolic pathways. recently, lara-reyna et al. reported increased glycolysis, mitochondrial function, and production of tnf-α in cf macrophages is due to an alteration in the serine/threonine-protein kinase/ endoribonuclease ire α pathway and this supports exacerbated inflammation . while this study used pbmcs and monocytederived m macrophages from cf patients, it would be important to detect such a mechanism in ams and to target this pathway specifically. several of the above described pathways have been identified as potential drug targets in cf, however yet there are no treatments targeting ams. delivery of gsh to the lower respiratory tract improves the antioxidant barrier of cf epithelium , while treatment with cysteamine and restoration of microrna (mir ) and mir expression improves disease by restoring autophagy , . several studies administered omega- fatty acids (dha/epa) to cf patients [ ] [ ] [ ] [ ] , although only one trial reported improved fev after months treatment with dha . treatment with dha in a murine model of cf decreased liver inflammation but did not improve lung morphology . in conclusion, am metabolic phenotype during cf is marked by increased energy expenditure to support exacerbated inflammation and is readily exploited by bacterial pathogens, leaving ams deficient of oxidative burst capability during infection. it will be important to clarify the role of fatty acids in cf and furthermore, to target metabolic changes in ams such as increased glycolysis, oxphos and fao specifically to rewire am phenotype and prevent exploitation through bacterial pathogens. idiopathic pulmonary fibrosis (ipf). ipf is a chronic interstitial lung disease characterised by excessive extracellular matrix deposition in the lung parenchyma and has a particularly poor prognosis . repetitive alveolar injury in genetically susceptible individuals causes activation of mesenchymal cells, recruitment of fibroblasts and differentiation into myofibroblasts to replace damaged alveolar epithelial cells and provide a matrix for wound healing and tissue repair . during ipf, the wound healing process is dysregulated, leading to fibrotic plaque formation and excessive build-up of extracellular matrix, resulting in impaired gas exchange. ams have been identified as key contributors to the dysregulated wound healing process, by secreting large amounts of ros and tgf-β . furthermore, ams can shape the extracellular matrix by secreting factors contributing to the matrix (proline, collagen) and breaking down the matrix (plasmin, mmps) [ ] [ ] [ ] . several changes to the central carbon metabolism pathways have been identified recently in ams of ipf patients, including dysmorphic mitochondria . in murine models of pulmonary fibrosis, increased glucose consumption, glycolysis and enhanced expression of key glycolytic mediators was detected , while in ipf ams, expression of the pulmonary glucose transporter glut was increased , which enabled augmented glucose uptake . the increased glucose uptake via glut can furthermore sustain nadph production in the pentose phosphate pathway and tca cycle and is therefore a key substrate for ros production via nox . activation of macrophages results in the accumulation of endogenous metabolites capable of adopting immunomodulatory roles such as succinate and itaconate [ ] [ ] [ ] . recently, our laboratory identified itaconate as an endogenous anti-fibrotic in the human and murine lung. in patients with ipf, there were reduced levels of airway itaconate, and decreased expression of acod (which controls the synthesis of itaconate) in ams compared to healthy controls. acod deficiency in mice leads to more severe disease pathology and exogenous itaconate limits fibroblast activity . these data indicate that am metabolites may play a key role in the pathogenesis of lung fibrosis and may be exploited for the development of anti-fibrotic therapies. ros production is a key feature of ams in ipf and can occur during oxphos, by the membrane bound nox or by reaction of hydrogen peroxide with intracellular iron . nox, and subsequent superoxide production, is activated by binding gtp-bound rac , which is secreted from ams in ipf and can also activate the mtor signalling hub . superoxide produced by nox can further react with no to form peroxynitrite (oono -), another type of ros. at the expense of nadph, no is produced in the mitochondria by inos, which is upregulated in proinflammatory macrophages and in ipf-ams leading to increased levels of the cytotoxic oonoin ipf ams . in the bleomycin mouse model of pulmonary fibrosis, increased levels of superoxide, no and oonowere measured in ams . mtros is furthermore linked to expression of ppar-γ coactivator -alpha (pgc- α), which induces metabolic reprogramming to fao and is regulated by the mitochondrial calcium uniporter (mcu), which is increased in ipf ams . mcu has furthermore been shown to regulate expression of the fatty acid transporter cpt- , which is increased in ams from ipf patients and bleomycin exposed mice . while human ipf-ams have increased levels of mcu, mitochondrial calcium and expression of pgc- a, bleomycin exposed mice utilise increased fao , which is reduced in mice expressing dominant-negative mcu . furthermore, these mice were protected from bleomycin induced pulmonary fibrosis. these findings highlight calcium transport and fao as pathways to target in ipf ams; however, a better understanding of the linking mechanism will be necessary. ipf ams have also been shown to be iron laden , which further induces oxidative stress and ros production . using rna-sequencing, lee et al. show furthermore, that macrophage activation is increased in iron laden ams in ipf, suggesting that iron accumulation plays a role in macrophage activation . the proportion of ams expressing transferrin receptor (cd ), importing transferrin bound iron into the cell, are decreased in ipf ams, leading to an extracellular accumulation of transferrin. furthermore, numbers of cd -negative macrophages are an independent predictor of survival in ipf . iron metabolism is therefore likely a key pathway in ipf-ams and targeting it would be a viable option to decrease ros, oxidative stress and macrophage activation. recently, therapies targeting metabolic processes in ipf are of considerable interest. while antioxidant therapy in ipf was promising in vivo, the double-blind placebo controlled panther trial, administering either n-acetylcysteine or placebo to ipf patients for weeks did not show a change in lung function parameters . another arm of this study investigated the combined potential of corticosteroid prednisone, immunosuppressant azathioprine and n-acetylcysteine but was stopped prematurely due to increased mortality and adverse effects without evidence of benefit . another randomized, double-blind clinical trial assessed the safety and tolerability of n-acetylcysteine in patients already receiving pirfenidone anti-fibrotic therapy. while this trial showed that n-acetylcysteine in combination with pirfenidone was safe, no change in fvc, -minute walk test or occurrence of adverse effects was detected . another promising therapeutic avenue was the use of metformin, a potent metabolic remodelling drug often prescribed for type ii diabetes. while on a global level metformin lowers the amount of blood sugar in diabetic patients, on a cellular level metformin activates ampactivated protein kinase (ampk) leading to inhibition of tgf-β induced nox activity . sato et al., have shown that metformin inhibited tgf-β induced nox activity via ampk leading to inhibition of myo-fibroblast differentiation in vitro and reduced bleomycin induced collagen deposition in vivo . consistent with this, rangarajan et al. showed that metformin treatment reversed bleomycin induced pulmonary fibrosis via ampk activation, while in ipf patients ampk phosphorylation was decreased . a posthoc analysis study of the effect of metformin in ipf patients however showed no change in clinically outcomes , once again showing the difficulty of translating in vitro and in vivo findings into the clinic. another study investigating the nox-nrf imbalance as a therapeutic target showed that in vivo knockdown of nox and nox / inhibition restored the capacity of fibrosis resolution in aged mice . furthermore, treatment with nitrated fatty acids, reversed pulmonary fibrosis in a mouse model by promoting collagen uptake by ams and dedifferentiating myofibroblasts . while these treatment approaches targeted metabolic changes during pulmonary fibrosis, none was specific to ams. targeting macrophage specific metabolic reprogramming, which sustains ros and tgf-β production and contributes to dysregulated wound healing in ipf would therefore be a promising approach. during respiratory tract infections, activation of pattern recognition receptors expressed by am can elicit a variety of proinflammatory host responses . for example, severe coronavirus disease- (covid- ) associated pneumonia patients may exhibit features of systemic hyper-inflammation also known as macrophage activation syndrome or "cytokine storm" which is associated with sustained elevation of macrophage/monocytederived pro-inflammatory cytokines (e.g., il- , il- , tnf-α, il- β) leading to acute respiratory distress syndrome (ards) [ ] [ ] [ ] . using single cell approaches a recent study demonstrated that highly inflammatory, monocyte recruited ams, rather than quiescent pulmonary resident ams, predominate in the bal in covid- patients with severe pathology, implicating these cells in covid- -associated ards . rather than direct infection of ams, am:aec cross-talk has been identified as a major mechanism for control of many respiratory viral infections and aec have been shown to be a key source of pro-inflammatory cytokines, modulating am phenotype , . for example, rhinovirus (rv), the causative agent of the common cold, primarily infects the upper airways, however prior infection with rv attenuates subsequent am antibacterial responses . although ams are susceptible to influenza a viral infection (iav), replication within ams has been shown to be minimal with the exception of several highly virulent strains [ ] [ ] [ ] . here, we will focus on mycobacterium tuberculosis (mtb) infection, as ams are the primary infected cell type and metabolic changes in response to mtb infection are well studied. tuberculosis. tuberculosis (tb) is a contagious, chronic disease and one-third of the world's population is infected with mtb, the causative agent of tb, resulting in~ million deaths per year ( world health organization report) . during infection, mtb colonises ams intracellularly and disables innate intracellular defence mechanisms such as the phagolysosome and inflammasome and accesses macrophage intracellular nutrients . am host defence mechanisms against mtb include production of ros and reactive nitrogen species (rns) for bacterial killing and fusing mycobacteria-containing phagosomes with lysosomes as well as autophagy and apoptosis . however, virulent or multi-drug resistant strains can evade these host responses e.g. by preventing phagolysosome fusion and surviving ros/rns . during mtb infection, ams shift their metabolic programme from oxphos to aerobic glycolysis, which is regulated by hif α and interferon-gamma (ifn-γ). this metabolic shift and subsequent enhanced glycolytic flux in infected ams is crucial to control infection. mice lacking hif α in the myeloid lineage are more susceptible to infection and show decreased cytokine and antimicrobial effector production . to support this metabolic reprogramming, key glycolysis genes are upregulated in the early stages of granuloma formation in mice, supporting the shift towards aerobic glycolysis . mtb furthermore induce ferroptosis, associated with reduced levels of gsh, superoxide and increased free iron. the ferroptosis inhibitor ferrostatin- (fer- ) as well as iron chelation decreased necrotic cell death of mtb-infected macrophages in vitro, while in vivo treatment with fer- reduced bacterial load . mtb can cope in low iron environments however macrophage metabolic reprogramming during chronic lung disease pp ogger and aj byrne by downregulating their non-essential protein content via specific srna . several changes in fatty acid metabolism of mtb infected ams were identified recently. compared to interstitial macrophages during mtb infection, which are reliant on glycolysis, ams utilise fa, which is induced by ppar-α and have a lower burden of mtb infection . to escape host defence, mtb has developed a mechanism inhibiting pathways related to autophagy, lysosomal function and fao in support of replication by inducing microrna- (mir- ) in the host cell. silencing of mir- however induced am lipid catabolism and autophagy and rescued host defence . furthermore, amino acid metabolism is altered during mtb infection. in mice and macaque lungs, indoleamine , -dioxygenase (ido), which is involved in tryptophan catabolism, was increased during mtb infection, while inhibition of ido in a macaque model of tb decreased bacterial burden and pathology, as tryptophan metabolites suppress host immunity . while mtb relies on host lipids as energy source, existing therapies such as targeting ppar transcription factors or cholesterol synthesis have been successful mainly in animal models [ ] [ ] [ ] [ ] , whereas retrospective human studies, which investigated the effect of statins in diabetic tb patients did not show any results . as mtb can also utilise iron as a substrate, another approach is to prevent iron accumulation. treatment of mtb infected human mdms and primary am with iron chelator desferrioxamine (dfx) ex vivo induced the expression of glycolytic enzymes and enhanced glycolysis, as well as il- βα, thereby supporting host defence and offers a novel therapeutic approach, which will need to be investigated in clinical trials. together, these findings highlight the distinct phenotype of ams during mtb infection, which counteracts intracellular infection through aerobic glycolysis, but is also heavily exploited by mtb bacteria feeding on host lipids and iron. targeting metabolism during chronic lung disease many potential targets have been identified recently that could rewire macrophage metabolic and phenotypic changes driving chronic lung disease. since all cells depend on oxidative phosphorylation or cytoplasmic glycolysis to synthesize atp, there is the potential for unwanted side effects by targeting specific metabolic processes. however, it is becoming increasingly apparent that it is possible to safely target metabolic pathways in patients. for example, dimethyl fumarate, a known regulator of macrophage phenotype, is a first-line-treatment for relapsingremitting multiple sclerosis . indeed, metabolic processes are highly plastic with significant redundancy, modulation of these processes may have the added benefit of selectively targeting cells with high metabolic demands . targeted delivery to ams may add another layer of selectivity, improving efficacy, sustained drug release and evading capture by mucus . systems for inhaled am targeted drug delivery include the use of micro-and nanocarriers, including liposomes, which are phagocytosed by ams. rifampicin-loaded microspheres as a therapeutic approach for mtb have been described , and have been further refined to allow a one-step assembly for rifampicin containing microspheres . recently, aerosolised delivery of sirna, which posttranslationally downregulates gene expression, has been developed to target ams specifically , whilst mannose coated microspheres have been developed which exploit the phagocytotic activity of ams . many of these delivery vehicles have been developed to transport antibiotics targeting intracellular am bacterial infections, which are helpful for treating tb, however other drugs could be incorporated into aerosolised micro-or nano delivery systems. specifically, treatment with iron chelators, antioxidants and nitrated fatty acids has shown to rewire am phenotype and improve diverse chronic lung disease; these may be ideal candidates to develop novel, aerosolised vehicle-assisted drug delivery to ams during chronic lung disease. in the last decade enormous strides have been made regarding our understanding of how adaptations in metabolic pathways underlie macrophage phenotype and function. ams are remarkably plastic cells, orchestrating not only pathogen defence and efferocytosis, but also pulmonary tolerance and resolution. it has become increasingly clear that ams tailor their metabolic profile to fit their local niche generating ros for pathogen defence, utilising aerobic glycolysis to rapidly generate cytokines, employing the tca cycle to fuel inflammatory responses and generating metabolites with secondary signalling functions such as citrate, itaconate, succinate and fumarate. work elucidating the complexities of am metabolic alterations in the context of clds has highlighted many potential therapeutic targets (summarized in table ). indeed, a lack of understanding of shared cellular mechanisms, which underlie clds has been a major obstacle in respiratory biology; identification of common am-metabolic pathways/metabolites which directly influence core features of clds would be a significant advance on the route to devising new am-directed strategies to treat pulmonary diseases which affect millions worldwide. lung homeostasis: influence of age, microbes, and the immune system pulmonary macrophages: key players in the innate defence of the airways monocytes and macrophages: developmental pathways and tissue homeostasis metabolic reprograming in macrophage polarization macrophage immunometabolism: where are we (going)? metabolic disorders in chronic lung diseases alveolar macrophage immunometabolism and lung function impairment in smoking and chronic obstructive pulmonary disease alveolar macrophages: plasticity in a tissue-specific context alveolar macrophage in the driver's seat integrin αvβ : structure, function and role in health and disease pulmonary macrophages: a new therapeutic pathway in fibrosing lung disease? flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung identification of myeloid cell subsets in murine lungs using flow cytometry lung environment determines unique phenotype of alveolar macrophages a critical function for cd in lung immune homeostasis and the severity of influenza infection a lineage of myeloid cells independent of myb and hematopoietic stem cells alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via gm-csf tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes yolk sac macrophages, fetal liver, and adult monocytes can colonize an empty niche and develop into functional tissue-resident macrophages the lung environment controls alveolar macrophage metabolism and responsiveness in type inflammation tissue-resident macrophage ontogeny and homeostasis developmental origin of lung macrophage diversity the fate and lifespan of human monocyte subsets in steady state and systemic inflammation monocyte recruitment during infection and inflammation transcriptome analysis highlights the conserved difference between embryonic and postnatal-derived alveolar macrophages cellular chimerism of the lung after transplantation: an interphase cytogenetic study long-term persistence of human donor alveolar macrophages in lung transplant recipients long-term persistence of donor alveolar macrophages in human lung transplant recipients that influences donor specific immune responses the human alveolar macrophage direct evidence for a bone marrow origin of the alveolar macrophage in man dynamics of human monocytes and airway macrophages during healthy aging and after transplant human monocyte subsets are transcriptionally and functionally altered in aging in response to pattern recognition receptor agonists aging is associated with chronic innate immune activation and dysregulation of monocyte phenotype and function agedependent alterations of monocyte subsets and monocyte-related chemokine pathways in healthy adults succinate is an inflammatory signal that induces il- β through hif- α succinate dehydrogenase supports metabolic repurposing of mitochondria to drive inflammatory macrophages cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization metabolic reprogramming in macrophages and dendritic cells in innate immunity tissue-resident alveolar macrophages do not rely on glycolysis for lps-induced inflammation induction of the nuclear receptor ppar-γ by the cytokine gm-csf is critical for the differentiation of fetal monocytes into alveolar macrophages pulmonary surfactant protein a modulates the cellular response to smooth and rough lipopolysaccharides by interaction with cd gm-csf regulates pulmonary surfactant homeostasis and 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resolvin d serves as a marker of lung disease in cystic fibrosis metabolic reprograming of cystic fibrosis macrophages via the ire α arm of the unfolded protein response results in exacerbated inflammation glutathione aerosol suppresses lung epithelial surface inflammatory cell-derived oxidants in cystic fibrosis elevated mirc /mir - cluster expression negatively regulates autophagy and cftr (cystic fibrosis transmembrane conductance regulator) function in cf macrophages cysteamine re-establishes the clearance of pseudomonas aeruginosa by macrophages bearing the cystic fibrosis-relevant f del-cftr mutation an overview of monitoring and supplementation of omega fatty acids in cystic fibrosis fatty acid alterations and n- fatty acid supplementation in cystic fibrosis oral dha supplementation in Δf homozygous cystic fibrosis patients bioavailability and safety of a high dose of docosahexaenoic acid triacylglycerol of algal origin in cystic fibrosis patients: a randomized, controlled study 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function of highly proliferative t cells, but not for dendritic cells or macrophages role of glutamine metabolism in host defense against mycobacterium tuberculosis infection immunometabolism within the tuberculosis granuloma: amino acids, hypoxia, and cellular respiration analyzing the impact of mycobacterium tuberculosis infection on primary human macrophages by combined exploratory and targeted metabolomics competing interests: the authors declare no competing interests.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -gw xnb x authors: yang, mengling; dong, yinmiao; he, qingnan; zhu, ping; zhuang, quan; shen, jie; zhang, xueyan; zhao, mingyi title: hydrogen: a novel option in human disease treatment date: - - journal: oxid med cell longev doi: . / / sha: doc_id: cord_uid: gw xnb x h( ) has shown anti-inflammatory and antioxidant ability in many clinical trials, and its application is recommended in the latest chinese novel coronavirus pneumonia (ncp) treatment guidelines. clinical experiments have revealed the surprising finding that h( ) gas may protect the lungs and extrapulmonary organs from pathological stimuli in ncp patients. the potential mechanisms underlying the action of h( ) gas are not clear. h( ) gas may regulate the anti-inflammatory and antioxidant activity, mitochondrial energy metabolism, endoplasmic reticulum stress, the immune system, and cell death (apoptosis, autophagy, pyroptosis, ferroptosis, and circadian clock, among others) and has therapeutic potential for many systemic diseases. this paper reviews the basic research and the latest clinical applications of h( ) gas in multiorgan system diseases to establish strategies for the clinical treatment for various diseases. molecular hydrogen (h ) is the lightest chemical element in the earth's atmosphere. h is often mixed in gas cylinders for deep-sea divers to breathe, to prevent decompression and nitrogen sickness [ ] . in mammals, h is spontaneously produced by intestinal bacteria in the process of anaerobic metabolism to produce energy and is enzymatically catabolized by hydrogenases to provide electrons. therapeutic applications of h were first described in . dole et al. reported that hyperbaric hydrogen caused marked regression of tumors in mice with skin squamous carcinoma [ ] . however, hyperbaric h is not a clinically feasible option, and h is a physiologically inert gas that seems not to react with any active substances, including oxygen gas, in mammalian cells. thus, h was perceived as being nonfunctional and was disregarded clinically. in , the potential therapeutic benefits of h were described. ohsawa et al. discovered that h has selective antioxidant properties that protect the brain against ischemia/reperfusion (i/r) injury and stroke by specifically neutralizing hydroxyl radicals ( ⋅ oh) and peroxynitrite (onoo-) but not superoxide anion radical ( ⋅ o -), hydrogen peroxide (h o ), and nitric oxide (no) [ ] . the report generated worldwide attention and thrust h into the spotlight of therapeutic medical gas research. many studies using cellular, animal, and clinical experiments in a variety of biomedical fields have explored the therapeutic and preventive effects of h . the collective data have indicated that h is an important pathophysiological regulatory factor with antioxidative, anti-inflammatory, and antiapoptotic effects on cells and organs [ ] [ ] [ ] . it is so convenient to use that h can be easily administered in various ways, including inhalation, injection of h -rich saline (hrs), drinking h -rich water (hw), bathing in hw, and using hrs eyedrops. as well, the production of intestinal h by bacteria can be increased via oral administration of acarbose and lactulose. liu and his colleagues demonstrated that the hydrogen concentration reached a peak of min after oral and intraperitoneal administration, and in only min following intravenous administration [ ] . beginning on december in wuhan, china, illness and pneumonia named coronavirus disease- (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) has spread to become a pandemic. the seventh edition of chinese clinical guidance for covid- pneumonia diagnosis and treatment ( th edition) issued by china national health commission recommended the inhalation of oxygen mixed with hydrogen gas ( . % o and . % h ), bringing h to the forefront of contemporary therapeutic medical gas research. the preventive and therapeutic effects of h have been intensively investigated for various pathological processes. in this review, we summarize the most recently published literature concerning the use of h in respiratory, cardiovascular, nervous, digestive, reproductive, urinary, motor, and sensory system diseases, as well as for the treatment of metabolic syndrome and cancer. we also briefly discuss some known mechanisms underlying the action of h . we hope that this information will increase the understanding of the therapeutic activities of h and inform future h -based therapies. to fully explain the preventive and therapeutic effects of h , biological effects and possible mechanisms are summarized in figures and . . . anti-inflammatory effect of h . the anti-inflammatory effect of h has already been reported in many studies. in the early stage of inflammation, h can reduce the infiltration of neutrophils and m macrophages, and the release of proinflammatory factors by downregulating the expression of intercellular cell adhesion molecule- (icam- ), granulocyte-macrophage colony-stimulating factor (gm-csf), and granulocyte colony-stimulating factor (g-csf) [ ] . h can also inhibit the expression of proinflammatory cytokines during the progress of inflammation and has been revealed in many animal models to decrease the overexpression of early proinflammatory cytokines, such as interleukin-(il-) β, il- , il- , il- , tumor necrosis factor-alpha (tnfα) [ ] , interferon-gamma (inf-γ), and late proinflammatory cytokines, such as high-mobility group box- protein (hmgb ) [ ] . tanaka and colleagues [ ] conducted a gene array analysis of lung grafts from donor rats pretreated with hydrogen ventilation. the authors described that pretreatment with h obviously elevated the expression of two stress-response proteins: heat shock protein a (hspa ) and dual-specificity phosphatase (dusp ). hsp protein nrf- ho- tnf-nf-b inf-fas fasl il- il- il- il- il- encoded by hspa has anti-inflammatory and antiapoptotic properties. dusp can suppress excessive autophagy by inactivating mitogen-activated protein kinases (mapks) to alleviate the inflammatory response. therefore, we speculate that h can positively regulate the expression of stressresponse proteins and improve the anti-inflammatory ability of the organs. another study [ ] demonstrated ventilation with % h in the air significantly reduced the mrna levels of the early growth responsive gene- (egr- ) and chemokine (c-c motif) ligand (ccl ), suggesting that h can affect the progress of inflammation by regulating the transcription of proinflammatory regulatory factors and chemokines. in many diseases, such as airway inflammation and cerebral ischemia, type i hypersensitivity caused by mast cell activation will aggravate the tissue congestion and edema [ , ] . h inhibits the body's inflammatory response by inhibiting th reaction to reduce mast cell activation as well [ ] . h can also regulate inflammation by regulating the physiological pathway of t cells. for example, hydrogen treatment can inhibit the overactivation of the immune system by restoring the loss of regulatory t cells (tregs) [ ] and alleviates inflammation by preventing activation-regulated chemokine-mediated t-cell chemotaxis. the regulation effects of h on programmed cell death, oxidative stress, and mitochondrial function are closely related to the inflammatory response. however, the antiinflammatory effect of hydrogen cannot be overstated. it would be better to be used as a supplementary therapy. many studies have established the antioxidant activity of h . however, recently, a study [ ] has shown that hydrogen can rapidly and slightly increase -hydroxy- ′ -deoxyguanosine ( -ohdg) in urine, a marker for oxidative stress, to a similar level as the increase caused by exercise. exercise-induced generation of reactive oxygen species (ros) is crucial in cell adaptation, and short-term exposure to low levels of ros can protect neurons from oxidative stress that would otherwise be lethal [ ] . h may act as a mitohormetic effector to mediate the beneficial effects on the body through hormetic mechanisms [ ] . the antioxidant effect of h is mainly reflected in several aspects. h was first found to directly eliminate hydroxyl radicals and peroxynitrite. compared with traditional antioxidants, such as superoxide dismutase (sod), catalase, oxidative medicine and cellular longevity and α-tocopherol, h can easily penetrate biofilms and does not affect the normal metabolic redox reaction due to its small molecular weight and antioxidative activity which selectively affects only the strongest oxidant [ ] . h can enhance the expression of the heme oxygenase- (ho- ) antioxidant by activating nuclear factor erythroid -related factor (nrf- ), an upstream regulating molecule of ho- . h can also downregulate ros directly or as a regulator of a gas-mediated signal. further, by upregulating the expression of sod and glutathione (gsh), and downregulating the expression of nadph oxidase (nox ) [ ] , h can significantly reduce ros. another study [ ] showed that h mainly blocks the phosphorylation of ask and its downstream signal molecules p mapk or c-jun n-terminal kinase (jnk), but not the production of ros by nadph oxidase. the effects on the free radical chain reaction of lipid peroxidation may be another important mechanism of hydrogen antioxidation. since otha et al. reported at the th symposium of medical molecular hydrogen at nagoya, japan, in that exposure to a low concentration of hydrogen causes abnormal oxidation of phospholipids [ ] , many studies have established that h can protect cells from lipid and fatty acid peroxidation [ , ] . additionally, h can also reduce the expression of myeloperoxidase (mpo) [ ] or decrease mitochondrial oxidoreductase activity [ ] and stabilize mitochondrial membrane potential [ ] , so as to improve the tissue damage caused by oxidative stress. stress. the accumulation of unfolded protein in the endoplasmic reticulum (er) caused by pathological stress can trigger er stress. zhao et al. [ ] observed that hydrogen inhalation significantly reduced the er stress-related protein and alleviated tissue damage in myocardial i/r injury and later found that a mixture of h and o can block endoplasmic reticulum stress via the pkr-like er-localized eif α kinase-eukaryotic initiation factor alpha-activating transcription factor (perk-eif α-atf ), inositol-requiring enzyme -x-box binding protein (ire -xbp ), and atf pathways. a study of the relationship between h and er stress in rats with i/r injury found that h induced the decrease of grp and tnf receptorassociated factor (traf ) expression [ ] , suggesting that the protective effects of h on myocardial i/r injury are related to decreased er stress. the accumulation of ros can trigger the release of calcium from the er, which results in the depolarization of mitochondria and the loss of the mitochondrial membrane potential [ ] . the negative regulation of ros and the inhibition of programmed cell death by h help maintain the structure and function of mitochondria. it was reported [ ] that hydrogen treatment can block the opening of mitochondrial permeability transition pores in neurons during neurodegenerative disease. however, whether h can indirectly block these pores by reducing the production of ros or acts directly is unclear. early studies [ ] showed that hrs moderated mitochondrial structural damage and simultaneously reduced microrna-(mir-) in hypoxic-reperfusion nerve tissue. however, recent studies have shown that the increase of mir- in injured tissues may be a compensatory action to maintain cell function and reduce ros production [ , ] . whether h can directly inhibit mir- or indirectly reduce it by alleviating inflammation also remains unclear. mitochondrial damage caused by oxidative stress is an important cause of many neurodegenerative diseases. early clinical experiments on parkinson's disease [ ] showed that h can significantly improve neurodegenerative symptoms with a therapeutic effect comparable to that of nonergot dopamine therapy. this cannot be explained by the antioxidative effect of h , and thus, it was suggested that h may target mitochondria to improve the energy metabolism of cells. the observation that h treatment significantly improved the level of sh-sy y atp and Δψm in neuroblastoma [ ] is an indication that h treatment can elevate energy metabolism in mitochondria by activating oxidative phosphorylation. the observations of the conserved structural features shared between hydrogenases and the energy-converting complex i prompted the suggestion that h might serve as both a reductant and oxidant [ ] . the hypothetical function of h in rectifying mitochondrial electron flow can explain the scavenging effect on ros and the ability to slightly improve oxidative stress. . . the effects of hydrogen on the immune system. the main effect of h on the immune system is to reduce the production of immune active substances. evidence suggests that h relieves inflammation in some autoimmune disorders, including rheumatoid arthritis (ra) [ , ] , dermatomyositis [ ] , and psoriasis [ ] . however, whether h directly influences immune cells or organs remains unclear. recent studies have found that h can relieve the dysregulated th /th balance and can influence the number of tregulatory cells (tregs). h was first reported to restore treg loss in a rat model of chronic pancreatitis [ ] and was later proven to increase cd +cd +foxp +treg cells and significantly reduce nasal mucosa damage in animals with allergic rhinitis, which may be secondary to the restoration of th /th balance [ ] . upregulation of tregs has been reported in cerebral i/r models [ ] . this may be caused by the upregulation of tumor necrosis factor-beta (tnf-β ) and downregulation of mir- or mir- . h can also activate peroxisome proliferator-activated receptor-gamma coactivator- alpha (pgc- α) to influence some kinds of t cells [ ] . the specific mechanisms underlying the effects of h on immune cells remain to be defined. upregulates the antiapoptotic factors b-cell lymphoma- (bcl- ) and b-cell lymphoma-extra-large (bcl-xl) [ ] . additionally, terasaki and colleagues reported that h can downregulate the gene expression of proapoptotic bax and inhibit its translocation to mitochondria by an unknown mechanism [ ] . h can also inhibit apoptosis by activating the phosphatidylinositol- -kinase/protein kinase b (pi k/akt) and the janus kinase /signal transducer and activator of transcription (jak -stat ) signaling pathways in rats with myocardial ischemia-reperfusion injury (miri) [ , ] , as well as downregulating the p mapk signaling pathway in rat models with lipopolysaccharide-(lps-) induced acute lung dysfunction [ ] and cerebral ischemia-reperfusion injury (ciri) [ ] . interestingly, wang et al. recently discovered that h inhibited the growth, migration, and invasion of the a and h lung cancer cell lines and promoted cell apoptosis, suggesting that h might play crucial roles in the treatment of lung cancer [ ] . li et al. also revealed the apoptosis-inducing effect of h on kyse- human esophageal squamous cell carcinoma cells [ ] . thus, h may protect normal cells from damage and suppressing cancer cells. although the activation of autophagy can maintain the energy balance of cells through the degradation of macromolecular substances, excessive autophagy or autophagy-related stress triggered by stress stimuli will aggravate the inflammatory damage in tissues and organs. h plays a dual role in the regulation of autophagy. under the regulation of h , autophagy can be activated when protein aggregation becomes toxic and blocked once excessive autophagy causes damage to tissues. zhuang et al. [ ] showed that h treatment downregulated the expression of phosphomammalian target of rapamycin (p-mtor)/mtor and p in lps-treated neuroglial cells and increased the expression of phospho-amp-activated protein kinase (p-ampk)/ampk, light chain (lc ) ii/lc i, triggering receptor expressed on myeloid cells (trem- ), and beclin- to activate autophagy and attenuate neuroinflammation in sepsis. guan et al. [ ] revealed that h can ameliorate chronic intermittent hypoxia-induced kidney injury by decreasing er stress and inducing autophagy by inactivating oxidative stress-dependent p and jnk mapks. h can also inhibit autophagy by downregulating nf-κb, beclin- , and mapk and upregulating the ho- , mtor, and lc b signaling pathways. zhang et al. [ ] found that saturated hydrogen saline alleviated lps-treated lung injury and significantly reduced the expression of autophagy-related proteins, including lc and beclin- (p < : ), suggesting that hydrogen saline can protect lung tissue against excessive autophagy. saturated hydrogen saline can prevent excessive autophagy by eliminating excessive free radicals, reducing the concentration of free radicals in lung tissue, and promoting the expression of mtor. ho- can function as an endogenous cytoprotective protein to assist in the prevention of oxidative stress and excessive cell autophagy. h can increase the tissue expression of ho- by promoting the expression of nuclear erythroid -related factor (nrf ) [ ] . toll-like receptors (tlrs) could be a potential target for h in autophagy regulation. tlr , a key factor in the recog-nition of viral and bacterial factors, can be activated by lps to induce autophagy of macrophages [ ] . the inhibition of an lps-induced inflammatory response by h supports the speculation that tlr may be a potential pathway of hydrogen-induced autophagy. . . . pyroptosis. pyroptosis is an inflammatory programmed cell death pathway that protects multicellular hosts from invasive pathogens, including microbial infections [ ] . human and mouse caspase- , human caspase- and caspase- , and mouse caspase- act as essential activators of pyroptosis. while pyroptosis is normally beneficial for the host, excessive pyroptosis can result in sepsis and septic shock. although there is no experimental data to explain the relationship between h and the pyroptosis pathway, it is conceivable that the regulation of some inflammatory factors and nuclear factors by h will interfere with the triggering of pyroptosis, or at least reduce pyroptosis-related inflammation. in one study [ ] , inhalation of % h reduced the expression of caspase- , a key factor for pyroptosis activation. physical rupture of cells caused by pyroptosis leads to the release of the proinflammatory cytokines il- β and il- , while hydrogen pretreatment can significantly reduce the level of these cytokines [ ] . h has also been shown to regulate the expression of atg , which inhibits pyroptosis [ ] . it has been proposed that hmgb [ ] and ifn-γ [ ] are necessary for caspase- -dependent pyroptosis activation. the negative effect of h on the expression of these two factors may protect cells from pyroptosis. h is able to block the expression of caspase- [ ] , which serves both as the activator of apoptosis, and also blocks pyroptosis by cleaving gasdermin d [ ] . bidirectional crosstalk exists between the caspase- produced in apoptosis and the caspase in pyroptosis. the mechanism of this crosstalk remains unclear. human immunodeficiency virus (hiv) can accelerate the depletion of cd + t cells via interferon-gamma inducible protein -(ifi -) triggered pyroptosis [ ] . thus, the regulation of pyroptosis by h may be a potential mechanism to treat hiv affection. ferroptosis is a form of regulated cell death proposed by dixon et al. [ ] in . ferroptosis is accompanied by a lethal iron-dependent accumulation of lipid hydroperoxides. although laboratory verification has not been forthcoming, we can still speculate that hydrogen can interfere with ferroptosis to alleviate inflammatory necrosis of tissues and organs in the pathological state, given the great deal of overlap between hydrogen regulation and ferroptosis pathways. it has been well-established that hydrogen has a negative regulatory effect on ros. we speculate that the most critical redox imbalance in the process of ferroptosis will be eliminated by hydrogen; thus, ferroptosis will be blocked. in addition, h is able to block mapk pathways, which are to prevent the depletion of reducing substances caused by iron ions and ros [ ] . a recent study [ ] showed that hmgb , which can be downregulated by hydrogen [ ] , can act as a positive regulator of ferroptosis via the ras-jnk/p pathway. oxidative medicine and cellular longevity ho- activity can be increased by hydrogen. ho- is a potential source of intracellular iron, and a recent study [ ] demonstrated that ho- -deficient renal epithelial cells were more sensitive to ferroptosis, indicating that free iron produced by ho- does not promote ferroptosis itself, and that ho- has an antiferroptotic effect. however, the effects of hydrogen on ferroptosis may not always be inhibitory. for example, mir- , an inflammatory mirna that is downregulated by hydrogen, can reduce the occurrence of ferroptosis [ ] . the mechanism underlying the action of hydrogen on ferroptosis is yet to be fully clarified. some of the anti-inflammatory and antioxidation mechanisms of hydrogen are similar to those of gpx [ ] . both molecules have negative effects on the formation of lipid peroxide and nf-κb. the combination of hydrogen and gpx activator may provide a new solution for the treatment of inflammation and other lipid peroxidationmediated diseases. . . . circadian clock. the circadian clock refers to an endogenous oscillator that controls h physiological, metabolic, and behavioral processes. this clock is particularly crucial in maintaining homeostasis [ ] . intestinal microbiota, which regularly produce hydrogen gas in the process of the energy-producing anaerobic fermentation [ ] , undergo diurnal oscillations in composition and function [ ] . in humans, the amount of hydrogen produced varies depending on the individual and the time of the day. wilking et al. suggested that the circadian regulation of protein expression plays an important role in the cellular response to oxidative stress; they concluded that levels of byproducts of oxidative stress, such as protein damage, or lipid peroxidation, also oscillate with circadian rhythmicity, indicating circadian oscillations of oxidative stress responses. thus, this rhythmicity of antioxidant levels can be exploited for a more precise targeting of ros to offer better protection for the cells [ ] . as the antioxidant activity of h has been widely verified, we suggest that h may exert a negative regulatory effect on ros by regulating circadian rhythm, but there is yet no evidence regarding how h is involved in the regulation of circadian rhythm. applications of h h has preventive and therapeutic effects on different system diseases ( due to its small molecular weight, hydrogen in the inhaled gas mixture can reduce airway resistance, increase oxygen dispersion, and increase oxygen flow. covid- can provoke an inflammatory storm by excessively activating the immune system, causing severe inflammatory damage to the lungs and extrapulmonary tissue, which is also the main cause of death [ ] . a study involving patients with ncp showed that patients in the intensive care unit displayed a significantly higher level of inflammatory factors that included il- , il- , il- , and tnf-α and that most of these factors could be downregulated by hydrogen [ ] . we speculate that the application of hydrogen may reduce the risk of inflammatory storm and thus prevent severe effects. many ncp patients need ventilator-assisted therapy. inflammatory changes in the respiratory system make lung tissue prone to ventilatorinduced lung injury (vili), even with low tidal volume [ ] . ventilation with % h was proved to be able to downregulate the mrnas for proinflammatory mediators such as tnf-α, il- β, egr- , and ccl and induced antiapoptotic genes including bcl- and bcl-xl. it is consistent with the histopathological results which showed that inflammatory cell infiltration and bronchial epithelial apoptosis were improved in vili mice after h treatment. h has a potential to protect human lung tissues from vili as well via its anti-inflammatory, antioxidant, and antiapoptotic effects [ ] . moreover, hydrogen can also increase surfactant proteins to prevent further lung injury [ ] . one study reported that chest computed tomography scans performed at the early stage in patients that ultimately developed in severe infection revealed multiple small flake shadows and interstitial changes, suggesting that pulmonary fibrosis affects the prognosis of the disease. hrs has been reported to reverse epithelial-mesenchymal transition (emt) and prevent pulmonary fibrosis by inhibiting oxidative stress and increasing the expression of e-cadherin [ ] . hydrogen also has the potential to protect lung tissues from pulmonary hypertension [ ] , sepsis [ ] , smoke inhalation injury [ ] , hemorrhagic shock and resuscitation [ ] , and other toxic substances/events. in animal models, hydrogen also affects asthma and chronic obstructive pulmonary disease [ ] . system. myriad forms of irreversible damage that occur in nervous system diseases are often caused by neuroinflammation, excessive oxidative stress, mitochondrial dysfunction, and cell death. the therapeutic effects of hydrogen on the nervous system have been verified in animal models and clinical trials. hydrogen can reportedly reduce the loss of dopaminergic neurons and can improve nigrostriatal degeneration a mouse model of parkinson's disease (pd) following treatment with -hydroxydopamine [ ] and methyl- -phenyl- , , , -tetrahydropyridine [ ] . a recent oxidative medicine and cellular longevity clinical trial showed that hw can improve the total unified parkinson's disease rating scale score of pd [ ] . early clinical trials revealed that hydrogen improves pd through antioxidation. more recent research found that hydrogen may slightly increase oxidative stress or act as the rectifier of the mitochondrial electron flow and improve pd by regulating mitochondrial energy [ ] or via hormetic mechanisms. by hormetic mechanisms, h will simulate strenuous exercise to generate a mild increase of ros which will evoke hormesis and then activate the nrf , nf-κb pathways, and heat shock responses to protect neurons and other tissues [ ] . hydrogen also improved cognitive function in alzheimer's patients in a clinical trial [ ] . the collective findings indicate the safety and effectiveness of hydrogen in the treatment of neurodegenerative diseases. hydrogen has also been shown to protect the nervous system of a fetus or newborn. in two case studies, pregnant women who had become infected with covid- displayed a high delivery rate of fetuses with intrauterine distress leading to a higher incidence of perinatal hypoxic-ischemic brain damage [ , ] . the findings indicate the necessity of measures to prevent neonatal encephalopathy or reduce neonatal neurological deficit for pregnant women infected with covid- . h can inhibit the activation of proinflammatory cytokines, microglia [ ] , and -hydroxy dehydrogenase ( -ohdg) [ ] to reduce oxidative damage and neuroinflam-mation in the fetal brain in animal models. the protective effects have been verified by clinical trials. hippocampal damage caused by i/r injury in the uterus on day after birth was reportedly improved for pregnant women who had been treated with hw and was associated with reductions of hydroxy ketone and -ohdg [ ] . the available data support the possibility that hydrogen therapy could protect fetuses of pregnant women infected with covid- . in addition, hydrogen can also protect against spinal cord injury and a variety of brain injuries caused by ischemia, hypoxia, trauma, and hemorrhage. a clinical study of patients suffering from cerebral infarction reported that hydrogen inhalation improved imaging results, national institutes of health stroke scale scores used to quantify the severity of stroke, and physical therapy evaluations based on the barthel index [ ] . a rat model of subarachnoid hemorrhage revealed the influence of hydrogen in lessening neurological deficits [ ] and endothelial cell injury [ ] . in rats, hydrogen can also relieve neuropathic pain [ ] after spinal cord injury and can ameliorate neurotoxicity [ ] . with the acceleration of urbanization and aging of global societies, the risk of cardiovascular diseases (cvd) has increased. the world health organization ranks cvd as the leading cause [ ] sensory system reduces wound area and levels of proinflammatory cytokines [ ] improves auditory brainstem response [ ] metabolic syndrome decreases ldl and increases high-density lipoprotein [ ] decreases glucose and insulin levels [ ] stimulates energy metabolism [ ] cancer controls cancer progression and improves quality-of-life [ ] reduces proportion of terminal pd- + cd + t cells [ ] oxidative medicine and cellular longevity of death globally, accounting for . million deaths annually. two of every five deaths in china are attributed to cvd, which exceeds the death rate due to cancer or other diseases [ ] . however, most clinical trials to date have failed to effectively prevent and treat cvd. thus, novel therapies are urgently required. during the past decade, many basic and clinical studies have provided evidence supporting the view that h treatment protects against cvd and improves cardiac function. ohsawa et al. discovered that consumption of hw for months reduced the oxidative stress level and the volume of atherosclerotic lesions derived from macrophage accumulation in apolipoprotein e knockout mice (apoe-/-mice) [ ] . iketani et al. recently revealed that continuous administration of hw in low-density lipoprotein (ldl) receptordeficient mice decreased the numbers of endothelial cells in the atheroma expressing the senescence factors p ink a and p , as well as suppressing macrophage infiltration and tnf-α expression in the atheroma, suggesting that vascular aging can be suppressed by hw [ ] . another study demonstrated increased flow-mediated dilation in the high-h group in which eight males and females drank hw containing ppm h , indicating that h may protect the vasculature from shear stress-derived ros that is detrimental [ ] . in addition to treating atherosclerosis, h reduces miri, which refers to a heart lesion that develops upon the resumption of the flow of oxygen-rich blood after a period of ischemia and which usually occurs during acute myocardial infarction or open-heart surgery [ , ] . a recent series of studies by li et al. found that hw inhibited cardiomyocyte apoptosis by activating the pi k/akt and jak -stat signaling pathways and can also reduce the level of oxidative stress in myocardial tissue by upregulating the expression of the nuclear erythroid -related factor /antioxidant response element (nrf /are) signaling pathway, which alleviated i/r injury in isolated rat hearts [ , , ] . other studies demonstrated that intraperitoneal injection of hw before reperfusion significantly decreased the concentration of malondialdehyde (mda) and infarct size, as well as reducing myocardial -ohdg and the levels of tnf-α and il- β in an area at risk zones [ , ] . moreover, qian et al. described the hydrogen-mediated protection of myocardium degeneration due to radiation-induced injury in rats [ ] . treatment with hrs was shown to ameliorate vascular dysfunction, including aortic hypertrophy and endothelial function, in spontaneously hypertensive rats by abating oxidative stress, restoring baroreflex function, preserving mitochondrial function, and enhancing nitric oxide bioavailability [ ] . in another study, the intraperitoneal administration of hrs improved hemodynamics and reversed right ventricular hypertrophy in male sprague-dawley rats with pulmonary hypertension induced by monocrotaline [ ] . h inhalation is also a favorable strategy to mitigate mortality and functional outcome of postcardiac arrest syndrome in a rat model [ ] . these collective findings indicate the potential of h in novel therapeutic approaches for the clinical treatment of cvd. diseases. hepatic ischemia-reperfusion injury (hiri) is common in liver sur-gery and liver transplantation. h treatment ameliorated hiri in a mouse fatty liver model by reducing hepatocyte apoptosis, inhibiting macrophage activation and inflammatory cytokines, and inducing ho- and sirt expression [ ] . inhalation of high concentrations of hydrogen significantly improved liver function in a mouse hiri model by activating the a a receptor-mediated pi k-akt pathway [ ] . a recent series of studies also discovered that portal vein injection of hrs in miniature pigs with laparoscopic hiri promoted liver function recovery and liver regeneration by reducing hepatocyte autophagy and apoptosis and inhibited er stress, with significant hepatoprotective effects observed [ ] [ ] [ ] [ ] . hydrogen flush after cold storage refers to an end-ischemic h flush directly to donor organs ex vivo. this approach can significantly protect liver grafts from iri [ ] , providing a potentially effective strategy for organ preservation. other studies demonstrated the protective effects of h in liver damage induced by parasites [ ] , obstructive jaundice [ ] , shock and resuscitation [ ] , sepsis [ ] , doxorubicin [ ] , and aflatoxin b [ ] . in a mouse model of nonalcoholic fatty liver disease (nafld), hw downregulated nrf -mediated mir- expression by targeting the maternally expressed long noncoding rna gene [ ] , providing a rationale for further clinical trials. in a human study, hw also significantly reduced liver fat accumulation in twelve overweight outpatients with nafld [ ] . another in vivo study revealed that oral hw significantly attenuated oxidative stress in patients with chronic hepatitis b [ ] . in recent years, it has become widely accepted that bile acids are a nutrient signaling hormone [ ] . molecular hydrogen was recently demonstrated to participate in the regulation of bile acid metabolism, particularly in the inhibition of bile acid oxidation, in some gut bacteria [ ] . intestinal i/r injury is a multifactorial pathophysiological process with high morbidity and mortality. i/r injury occurs in a variety of clinical settings that include major cardiovascular surgery, trauma, shock, and small intestinal transplantation [ ] . yao et al. recently observed that intraperitoneal injection of h attenuated i/r-induced mucosal injury and apoptosis of epithelial cells in mice by regulating mirnas, in particular by regulating mir- a- p [ ] . furthermore, hw reportedly inhibited intestinal i/r-induced oxidative stress, apoptosis, and inflammation and promoted epithelial cell proliferation in rats, which protected against intestinal contractile dysfunction and damage induced by intestinal i/r [ ] [ ] [ ] . most gastrointestinal microbial species encode the genetic capacity to metabolize h , meaning that h might affect the gut bacterial composition [ ] , and bacterial translocation is an important cause of multiple organ dysfunction syndromes in critical illness. ikeda et al. described that the luminal administration of hw prevented intestinal dysbiosis, hyperpermeability, and bacterial translocation in a murine model of sepsis [ ] . in another study, the inhalation of % h also attenuated intestinal injuries caused by severe sepsis in male nrf ko mice by regulating ho- and hmgb release [ ] . moreover, an in vivo study revealed that h inhalation improved the prognosis in patients with stage iv colorectal cancer by activating pgc- α and restoring exhausted cd + t cells [ ] . oxidative medicine and cellular longevity wang et al. interestingly discovered that cytotoxinassociated gene a cytotoxin, a virulence factor of helicobacter pylori that augments the risk of gastric cancer, can be delivered into host cells by the h -utilizing respiratory chain of the bacterium, extending the roles of h oxidation to include transport of a carcinogenic toxin [ ] . although this study indicated that h may play a role in increasing gastric cancer risk, abundant studies also demonstrated that h is protective in gastric damage induced by oxidative stress [ ] and aspirin [ ] . franceschelli et al. found that electrolyzed reduced water, which is rich in molecular hydrogen, rapidly improved symptoms in patients with gastroesophageal reflux disease [ ] . h treatment also controlled the severity of chronic pancreatitis [ ] and acute necrotizing pancreatitis [ ] . yang et al. recently demonstrated using a mouse model of human endometrial tumor xenograft that hw has an antitumor effect that was sufficient to inhibit xenograft volume and weight of endometrial tumors via the ros/nlrp /caspase- /gsdmd-mediated pyroptotic pathway, indicating a biphasic effect of h on cancer that involves the promotion of tumor cell death and protection of normal cells [ ] . other authors reported that h inhalation reduced the size of the endometrial explants, inhibited cell proliferation, improved sod, and regulated the expression of matrix metalloproteinase and cyclooxygenase in an endometriosis rat model [ ] . hrs is effective in attenuating ovary injury induced by i/r [ ] and cisplatin [ ] . hw improved serum anti-müllerian hormone levels and reduced ovarian granulosa cell apoptosis in a mouse immune premature ovarian failure model induced by zona pellucida glycoprotein [ ] . in a hemisectioned spinal cord injury rat model, hrs inhibited the injury-induced ultrastructural changes in gonadotrophs, ameliorated the abnormal regulation of the hypothalamicpituitary-testis axis, and thereby promoted the recovery of testicular injury [ ] . in irradiated rats, hrs improved testis weight, testis dimensions, sperm count, sperm motility, and serum testosterone levels [ ] . hw stimulated spermatogenesis as well as increased sperm production and sperm motility in mice of different ages [ ] . based on previous studies, begum et al. hypothesized that h may modulate intracellular mapk camp and ca + signals involved in testosterone hormone production to improve male fertility caused by redox imbalance [ ] . finally, h decreased the percentage of sperm abnormalities and improved sperm morphology following the prolonged exposure of mouse low doses of radiation [ ] . the collective data indicate that hydrogen can protect both female and male fertility. . . effects of hydrogen on urinary system diseases. acute kidney injury (aki) is an important risk factor for the development of chronic kidney disease. wu et al. recently found that saturated hydrogen alleviates ccl -induced aki via jak /stat /p signaling [ ] . inhalation of a hydrogen-rich aerosol appears to be very useful for renal protection and inflammation reduction in septic aki, based on the observations of increased anti-inflammatory cytokine (il- and il- ) mrna levels in renal tissues and increased macrophage polarization to the m type, which generates additional anti-inflammatory cytokines (il- and transforming growth factor-beta, tgf-β) [ ] . additionally, h can alleviate aki induced by i/r [ ] , liver transplantation [ ] , burns [ ] , and sodium taurocholate-induced acute pancreatitis [ ] . recently, lu et al. demonstrated that hw can restore a balanced redox status and alleviate cyclosporine a-induced nephrotoxicity by activating the keap /nrf signaling pathway [ ] . h can ameliorate kidney injury induced by chronic intermittent hypoxia by decreasing er stress and activating autophagy through the inhibition of oxidative stress-dependent p and jnk mapk activation [ ] . hw also reportedly can inhibit the development of renal fibrosis and prevent hk- cells from undergoing emt mediated through sirt , a downstream molecule of tgf-β . hrs markedly reduced interstitial congestion, edema, and hemorrhage in renal tissue, prevented renal injury, and promoted renal function recovery after i/r injury in rats through antiapoptotic and antiinflammatory actions in kidney cells [ ] . other authors described that hw significantly reduced the increased postvoid residual volume in obstructed rats and ameliorated bladder dysfunction secondary to bladder outlet obstruction by attenuating oxidative stress [ ] . metabolic syndrome is associated with excess calorie intake and encompasses a range of medical conditions that include obesity, insulin resistance, and dyslipidemia. many studies have demonstrated the protective effects of h in metabolic syndrome. qiu et al. reported that saturated hydrogen decreased total cholesterol, total glyceride, and ldl, increased highdensity lipoprotein in the peripheral blood, and alleviated the activity of isocitrate lyase, suggesting that h could improve lipid metabolism disorders by inhibiting the glyoxylic acid cycle [ ] . glucose and insulin levels in the serum were also significantly lower in h -treated mice, which markedly improved type diabetes mellitus and diabetic nephropathy-related outcomes [ ] . moreover, gut-derived hydrogen production induced by l-arabinose reportedly had beneficial effects on metabolic syndrome in c bl/ j mice fed a high-fat diet [ ] and reduced oxidative stress and the peripheral blood il- β mrna level in sixteen type diabetic patients [ ] . h treatment has also shown positive effects on energy metabolism. in , kamimura et al. reported that prolonged consumption of hw significantly controlled fat and body weights in db/db obese mice by stimulating energy metabolism [ ] . a recent study revealed that h attenuated allergic inflammation in a mouse model of allergic airway inflammation by reversing an energy metabolic pathway switch from oxidative phosphorylation to aerobic glycolysis [ ] . oxidative medicine and cellular longevity the serum -ohdg levels in h -treated race horses were significantly suppressed, strongly suggesting a protective effect of h in exercise-induced, ros-mediated detrimental tissue damage [ ] . additionally, hydrogen bathing attenuated exercise-induced muscle damage and delayed-onset muscle soreness but had no effects on the peripheral neutrophil count and both dynamics and functions of neutrophils [ ] . these findings highlight that further studies are needed to clarify the mechanisms of h . inhalation of h significantly decreased infarct zone and area with loss of tissue structure, attenuated muscle damage, and enhanced functional recovery in a mouse hindlimb i/r injury model [ ] . finally, hasegawa et al. revealed that h improved muscular dystrophy in the mdx mouse model for duchenne muscular dystrophy [ ] . . . effects of hydrogen on sensory system diseases. hydrogen has a therapeutic role in alleviating the damage to some sensory organs, mainly through antioxidation. hydrogen promotes wound healing in tissue or protective barriers including skin and mucosa. for example, preinhalation of hydrogen-containing gas decreased wound healing time in a rat model of radiation-induced skin injury [ ] . other authors reported that h inhalation reduced the wound area and levels of proinflammatory cytokines in pressure ulcers [ ] . moreover, hydrogen can improve skin lesions in some immune disorders by interfering with the immune system or ros removal [ ] . hw also benefits the wound healing process of the oral palate. hydrogen also may protect hearing and vision. kurioka et al. [ ] demonstrated that hydrogen inhalation significantly reduced outer hair cell loss and improved auditory brainstem response after noise exposure, indicating a protective effect for noise-induced hearing loss. hydrogen has been shown to be effective in treating cornea injury caused by alkali [ ] , fluoride, chloropicrin [ ] , and ultraviolet b radiation [ ] . . . effects of hydrogen on cancer. many animal models have established the efficacy of hydrogen against cancers. the attributes of hydrogen include blocking of the regulator for chromosome condensation [ ] , some crucial molecules in stemness [ ] , proliferation [ ] , and angiogenesis [ ] , and the alleviation of oxidative stress. the combination therapy of hydrogen and other novel antineoplastic drugs, such as ly [ ] , which is an inhibitor of pi k, has demonstrated great potential and efficacy. an increasing number of clinical trials are being carried out. a recent survey [ ] on advanced cancer patients exemplified that hydrogen can control cancer progression and improve the quality-of-life. akagi [ ] treated stage iv colorectal carcinoma patients using hydrogen inhalation and documented enhanced mitochondrial activity due to the activation of pgc- α to reduce the proportion of terminal pd- + cd + t cells. the depletion of these cells is associated with improved cancer prognosis [ , ] . this therapeutic effect has also been confirmed in another trial conducted in one patient with metastatic gallbladder cancer [ ] . in a case report in , hydrogen gas therapy resulted in the disappearance of the metastatic brain tumors in a woman diagnosed with lung cancer [ ] . finally, hydrogen can also reduce the side effects of cisplatin [ ] and radiotherapy [ ] . though growing evidences have shown the effects of h on alleviating both cancer progression and side effects of chemotherapeutics, the h therapy applied in cancer is just in a nascent stage. at present, the published researches on the anticancer effects of h mainly focus on lung cancer [ ] , colorectal cancer [ ] , and glioma [ ] . it remains unclear how many cancers can effectively be alleviated by h and how many can not be. at present, patients with covid- pneumonia are usually treated with high flow pure oxygen (without adding h ), although the effect of o when associated with h may give better results [ ] . the production of mucus in these patients reduces the absorption of o , while with a mixture of o and h , the bronchioles and the alveoli of lungs are further expanded, optimizing the absorption of o [ ] . h is used as a catalyst to accelerate the binding of hemoglobin with o and the release of hemoglobin with carbon dioxide [ ] . hydrogen has great potential in the regulation of oxidative stress, inflammation, energy metabolism of organelles, and programmed cell death. many animal experiments and clinical trials have established the protective effects of hydrogen on many organs and systems. research in this area has increased over the past years. however, the details of the specific molecular mechanisms of the therapeutic effects of hydrogen remain unclear. for example, whether hydrogen can truly be used to regulate ferroptosis, pyroptosis, or the circadian clock is not known. since h is not something like rapamycin or leucine only going to have one direction (opposite) effects on autophagy, is it possible to regulate autophagy or apoptosis in a specific direction? previous studies have clearly explained the antioxidative stress effect of hydrogen. however, some recent clinical trials have shown that h can induce oxidative stress in some cases as well. ventilation with h can induce a mild increase of ros to activate the nrf , nf-κb pathways, and heat shock responses. h -induced ros production can also be observed in cancer cells. the specific mechanism underlying the hydrogen-induced increase of oxidative stress should be explained by more experiments. these and other questions concerning the mechanism of hydrogen should be further explored. there are many factors that limit the clinical use of hydrogen. firstly, hydrogen is considered unsafe at concentrations above % because such a high level of h is explosive and might bring cytotoxic effects. previous studies have indicated that the concentration of hydrogen should be stabilized beyond % to enable protection from acute oxidative stress. however, even % of hydrogen is not absolutely safe. most clinical ventilators are equipped with platinum hot manometers, because h and o can overheat the platinum surface at room temperature. secondly, there is a lack of specialized devices that enable the administration of effective hydrogen concentrations, while ensuring that they are safe. thirdly, oxidative medicine and cellular longevity there have been few large-scale controlled human studies on the effects of hydrogen. fourthly, liu and his colleagues demonstrated that the inhalation of h resulted in a slower elevation of the h concentration than that achieved with intraperitoneal, intravenous, or oral administration. however, the elevated h concentrations were maintained for at least minutes after inhalation. thus, it should be deliberated to choose the administration of h [ ] . as a result, the dose-specific effects or side effects of hydrogen in humans remain unclear. the data regarding the known mechanisms underlying the action of hydrogen indicate that hydrogen can alleviate the damage in multiple organs in ncp patients. the comparisons of the different modalities of hydrogen indicate the value of hw in the effective treatment of such patients. hydrogen is inexpensive and safe and can be administered through many ways. we anticipate that as large-scale clinical trials confirm the therapeutic efficacy and safety of hydrogen, its full clinical potential will be realized. novel coronavirus pneumonia h : hydrogen i/r: ischemia/reperfusion hrs: h -rich saline hw: h -rich water ros: reactive oxygen species er: endoplasmic reticulum emt: epithelial-mesenchymal transition cvd: cardiovascular diseases nafld: nonalcoholic fatty liver disease aki: acute kidney injury mda: malondialdehyde nrf : nuclear erythroid -related factor pgc- α: peroxisome proliferator-activated receptorgamma coactivator- alpha. the authors declare no conflict of interest. psychophysiological reactions in humans during an open sea dive to m with a hydrogenhelium-oxygen mixture hyperbaric hydrogen therapy: a possible treatment for cancer hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals hydrogen protects lung from hypoxia/re-oxygenation injury by reducing hydroxyl radical production and inhibiting inflammatory responses effect of shikonin on spinal cord injury in rats via regulation of hmgb /tlr /nf-kb signaling pathway hydrogen-rich saline attenuates cardiac and hepatic injury in doxorubicin rat model by inhibiting inflammation and apoptosis estimation of the hydrogen concentration in rat tissue using an airtight tube following the administration of hydrogen via various routes combined early fluid resuscitation and hydrogen inhalation attenuates lung and intestine injury hydrogen gas reduces hmgb release in lung tissues of septic mice in an nrf /ho- -dependent pathway profiling molecular changes induced by hydrogen treatment of lung allografts prior to procurement hydrogen inhalation ameliorates ventilator-induced lung injury hydrogen inhalation ameliorated mast cell-mediated brain injury after intracerebral hemorrhage in mice allergic airway inflammation induces migration of mast cell populations into the mouse airway hydrogen water ameliorates the severity of atopic dermatitis-like lesions and decreases interleukin- β, interleukin- , and mast cell infiltration in nc/nga mice hydrogen treatment protects mice against chronic pancreatitis by restoring regulatory t cells loss inhalation of hydrogen gas elevates urinary -hydroxy- ′-deoxyguanine in parkinson's disease consumption of hydrogen water reduces paraquat-induced acute lung injury in rats hydrogen and oxygen mixture to improve cardiac dysfunction and myocardial pathological changes induced by intermittent hypoxia in rats molecular hydrogen as an emerging therapeutic medical gas for neurodegenerative and other diseases beneficial biological effects and the underlying mechanisms of molecular hydrogen -comprehensive review of original articles molecular hydrogen suppresses free-radical-induced cell death by mitigating fatty acid peroxidation and mitochondrial dysfunction hydrogen inhalation protects against acute lung injury induced by hemorrhagic shock and resuscitation hydrogen gas inhalation attenuates seawater instillation-induced acute lung injury via the nrf pathway in rabbits therapeutic efficacy of molecular hydrogen: a new mechanistic insight hydrogen gas attenuates myocardial ischemia reperfusion injury independent of postconditioning in rats by attenuating endoplasmic reticulum stress-induced autophagy propofol inhibits parthanatos via ros-er-calcium-mitochondria signal pathway in vivo and vitro inhalation of hydrogen of different concentrations ameliorates spinal cord injury in mice by protecting spinal cord neurons from apoptosis, oxidative injury and mitochondrial structure damages neuroprotective effect of hydrogen-rich saline in global cerebral ischemia/reperfusion rats: up-regulated tregs and down-regulated mir- , mir- and nf-κb expression loading mir- in endothelial progenitor cells derived exosomes boosts their beneficial effects on hypoxia/reoxygeneation-injured human endothelial cells via protecting mitochondrial function microrna- is upregulated in hypoxic cardiomyocytes through akt-and p -dependent pathways and exerts cytoprotective effects pilot study of h therapy in parkinson's disease: a randomized double-blind placebo-controlled trial molecular hydrogen protects against oxidative stress-induced sh-sy y neuroblastoma cell death through the process of mitohormesis molecular hydrogen decelerates rheumatoid arthritis progression through inhibition of oxidative stress molecular hydrogen: new antioxidant and antiinflammatory therapy for rheumatoid arthritis and related diseases open-label trial and randomized, double-blind, placebocontrolled, crossover trial of hydrogen-enriched water for mitochondrial and inflammatory myopathies positive effects of hydrogen-water bathing in patients of psoriasis and parapsoriasis en plaques hydrogen-rich saline ameliorates allergic rhinitis by reversing the imbalance of th /th and up-regulation of cd +cd +foxp +regulatory t cells, interleukin- , and membrane-bound transforming growth factor-β in guinea pigs immunological effect of hydrogen gas-hydrogen gas improves clinical outcomes of cancer patients hydrogen therapy attenuates irradiation-induced lung damage by reducing oxidative stress effects of hydrogen-rich water on the pi k/akt signaling pathway in rats with myocardial oxidative medicine and cellular longevity ischemia-reperfusion injury attenuation of cardiac ischaemiareperfusion injury by treatment with hydrogen-rich water saturated hydrogen saline attenuates endotoxin-induced lung dysfunction hydrogen saline suppresses neuronal cell apoptosis and inhibits the p mitogen-activated protein kinase-caspase- signaling pathway following cerebral ischemia-reperfusion injury hydrogen gas inhibits lung cancer progression through targeting smc influence of hydrogenoccluding-silica on migration and apoptosis in human esophageal cells in vitro molecular hydrogen attenuates sepsis-induced neuroinflammation through regulation of microglia polarization through an mtor-autophagydependent pathway hydrogen protects against chronic intermittent hypoxia induced renal dysfunction by promoting autophagy and alleviating apoptosis effects of hydrogen rich water on the expression of nrf and the oxidative stress in rats with traumatic brain injury tlr mediates the impairment of ubiquitin-proteasome and autophagy-lysosome pathways induced by ethanol treatment in brain molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases hydrogen gas inhalation attenuates sepsis-induced liver injury in a fundc -dependent manner h protects against lipopolysaccharide-induced cardiac dysfunction via blocking tlr -mediated cytokines expression atg deficiency intensifies inflammasome activation and pyroptosis in pseudomonas sepsis the endotoxin delivery protein hmgb mediates caspase- -dependent lethality in sepsis hydrogen-rich saline attenuates isoflurane-induced caspase- activation and cognitive impairment via inhibition of isoflurane-induced oxidative stress, mitochondrial dysfunction, and reduction in atp levels pyroptosis and apoptosis pathways engage in bidirectional crosstalk in monocytes and macrophages ferroptosis: an iron-dependent form of nonapoptotic cell death disulfiram/copper induces antitumor activity against both nasopharyngeal cancer cells and cancer-associated fibroblasts through ros/mapk and ferroptosis pathways hmgb regulates erastininduced ferroptosis via ras-jnk/p signaling in hl- /nras q l cells heme oxygenase- mitigates ferroptosis in renal proximal tubule cells activation of glutathione peroxidase as a novel anti-inflammatory strategy genomics of circadian rhythms in health and disease microbiota diurnal rhythmicity programs host transcriptome oscillations the clinical application of hydrogen as a medical treatment circadian rhythm connections to oxidative stress: implications for human health an overview of sars-cov- (covid- ) infection and the importance of molecular hydrogen as an adjunctive therapy hydrogen/oxygen mixed gas inhalation improves disease severity and dyspnea in patients with coronavirus disease in a recent multicenter, open-label clinical trial clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study clinical features of patients infected with novel coronavirus in wuhan, china regional physiology of ards protective effects of hydrogen-rich saline against lipopolysaccharide-induced alveolar epithelial-to-mesenchymal transition and pulmonary fibrosis oral intake of hydrogen-rich water ameliorated chlorpyrifos-induced neurotoxicity in rats saturated hydrogen saline ameliorates lipopolysaccharide-induced acute lung injury by reducing excessive autophagy protective effects of hydrogen-rich saline on rats with smoke inhalation injury hydrogen gas inhalation ameliorates lung injury after hemorrhagic shock and resuscitation hydrogen-rich saline inhibits tobacco smoke-induced chronic obstructive pulmonary disease by alleviating airway inflammation and mucus hypersecretion in rats molecular hydrogen is protective against -hydroxydopamine-induced nigrostriatal degeneration in a rat model of parkinson's disease hydrogen in drinking water reduces dopaminergic neuronal loss in the -methyl- -phenyl- , , , -tetrahydropyridine mouse model of parkinson's disease effects of molecular hydrogen assessed by an animal model and a randomized clinical study on mild cognitive impairment clinical characteristics and intrauterine vertical transmission potential of covid- infection in nine pregnant women: a retrospective review of medical records clinical analysis of neonates born to mothers with -ncov pneumonia administration of molecular hydrogen during pregnancy improves behavioral abnormalities of offspring in a maternal immune activation model molecular hydrogen affords neuroprotection in a translational piglet model of hypoxic-ischemic encephalopathy maternal molecular hydrogen administration ameliorates rat fetal hippocampal damage caused by in utero ischemia-reperfusion hydrogen gas inhalation treatment in acute cerebral infarction: a randomized controlled clinical study on safety and neuroprotection hydrogen gas therapy improves survival rate and neurological deficits in subarachnoid hemorrhage rats: a pilot study hydrogen inhalation attenuates oxidative stress related endothelial cells injury after subarachnoid hemorrhage in rats hydrogen-rich saline alleviated the hyperpathia and microglia activation via autophagy mediated inflammasome inactivation in neuropathic pain rats china cardiovascular diseases report : an updated summary consumption of hydrogen water prevents atherosclerosis in apolipoprotein e knockout mice administration of hydrogen-rich water prevents vascular aging of the aorta in ldl receptor-deficient mice consumption of water containing over . mg of dissolved hydrogen could improve vascular endothelial function mitochondrial aldehyde dehydrogenase in myocardial ischemic and ischemiareperfusion injury effect of hydrogen-rich water on the nrf /are signaling pathway in rats with myocardial ischemia-reperfusion injury antiinflammatory effect of hydrogen-rich saline in a rat model of regional myocardial ischemia and reperfusion hydrogen-rich saline protects myocardium against ischemia/reperfusion injury in rats the potential cardioprotective effects of hydrogen in irradiated mice chronic hydrogen-rich saline treatment attenuates vascular dysfunction in spontaneous hypertensive rats protective effects of hydrogen-rich saline on monocrotaline-induced pulmonary hypertension in a rat model h gas improves functional outcome after cardiac arrest to an extent comparable to therapeutic hypothermia in a rat model the clinical pathology of severe acute respiratory syndrome (sars): a report from china prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis covid- and cardiovascular disease cardiovascular considerations for patients, health care workers, and health systems during the covid- pandemic prevalence and impact of cardiovascular metabolic diseases on covid- in china molecular hydrogen protects against ischemia-reperfusion injury in a mouse fatty liver model via regulating ho- and sirt expression inhalation of high concentrations of hydrogen ameliorates liver ischemia/reperfusion injury through a a receptor mediated pi k-akt pathway comparative study on protective effect of hydrogen rich saline and adipose-derived stem cells on hepatic ischemia-reperfusion and hepatectomy injury in swine effect of hydrogen-rich saline on apoptosis induced by hepatic ischemia reperfusion upon laparoscopic hepatectomy in miniature pigs influence of hydrogen-rich saline on hepatocyte autophagy during laparoscopic liver ischaemia-reperfusion combined resection injury in miniature pigs hydrogen-rich saline protects against small-scale liver ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress hydrogen flush after cold storage as a new end-ischemic ex vivo treatment for liver grafts against ischemia/reperfusion injury anti-inflammatory properties of molecular hydrogen: investigation on parasite-induced liver inflammation hydrogen-rich saline protects against liver injury in rats with obstructive jaundice hyperoxygenated hydrogenrich solution suppresses shock-and resuscitation-induced liver injury preadministration of hydrogen-rich water protects against lipopolysaccharide-induced sepsis and attenuates liver injury anti-injury effect of hydrogen-enriched water in a rat model of liver injury induced by aflatoxin b high-content hydrogen waterinduced downregulation of mir- alleviates non-alcoholic fatty liver disease by regulating nrf via targeting meg hydrogen-rich water reduces liver fat accumulation and improves liver enzyme profiles in patients with non-alcoholic fatty liver disease: a randomized controlled pilot trial effect of hydrogen-rich water on oxidative stress, liver function, and viral load in patients with chronic hepatitis b bile acid signaling in metabolic disease and drug therapy bile acid oxidation by eggerthella lenta strains c and dsm t therapeutic roles of carbon monoxide in intestinal ischemia-reperfusion injury microrna files in the prevention of intestinal ischemia/reperfusion injury by hydrogen rich saline the effects of hydrogenrich saline on the contractile and structural changes of intestine induced by ischemia-reperfusion in rats luminal injection of hydrogen-rich solution attenuates intestinal ischemia-reperfusion injury in rats the effects of hydrogen-rich saline solution on intestinal anastomosis performed after intestinal ischemia reperfusion injury effects of molecular hydrogen-dissolved alkaline electrolyzed water on intestinal environment in mice hydrogen-rich saline regulates intestinal barrier dysfunction, dysbiosis, and bacterial translocation in a murine model of sepsis hydrogen gas protects against intestinal injury in wild type but not nrf knockout mice oxidative medicine and cellular longevity with severe sepsis by regulating ho- and hmgb release hydrogen gas restores exhausted cd + t cells in patients with advanced colorectal cancer to improve prognosis hydrogen metabolism inhelicobacter pyloriplays a role in gastric carcinogenesis through facilitating caga translocation the protective of hydrogen on stress-induced gastric ulceration protective role of hydrogen-rich water on aspirin-induced gastric mucosal damage in rats modulation of the oxidative plasmatic state in gastroesophageal reflux disease with the addition of rich water molecular hydrogen: a new biological vision hydrogen-rich saline attenuates acute hepatic injury in acute necrotizing pancreatitis by inhibiting inflammation and apoptosis, involving jnk and p mitogen-activated protein kinase-dependent reactive oxygen species hydrogen inhibits endometrial cancer growth via a ros/nlrp /caspase- /gsdmd-mediated pyroptotic pathway effects of hydrogen gas inhalation on endometriosis in rats protective effect of hydrogen rich saline solution on experimental ovarian ischemia reperfusion model in rats hydrogenrich saline attenuates chemotherapy-induced ovarian injury via regulation of oxidative stress hydrogen-rich water exerting a protective effect on ovarian reserve function in a mouse model of immune premature ovarian failure induced by zona pellucida hydrogen-rich saline attenuates spinal cord hemisection-induced testicular injury in rats protection by hydrogen against gamma ray-induced testicular damage in rats combination of korean red ginseng extract and hydrogenrich water improves spermatogenesis and sperm motility in male mice molecular hydrogen may enhance the production of testosterone hormone in male infertility through hormone signal modulation and redox balance protective effects of hydrogen against low-dose long-term radiation-induced damage to the behavioral performances, hematopoietic system, genital system, and splenic lymphocytes in mice saturated hydrogen alleviates ccl -induced acute kidney injury via jak /stat /p signaling aerosol inhalation of a hydrogen-rich solution restored septic renal function hydrogen-rich saline alleviates kidney fibrosis following aki and retains klotho expression hydrogen-rich saline attenuates acute kidney injury after liver transplantation via activating p -mediated autophagy effects of hydrogen-rich saline on early acute kidney injury in severely burned rats by suppressing oxidative stress induced apoptosis and inflammation hydrogen-rich saline attenuates acute renal injury in sodium taurocholate-induced severe acute pancreatitis by inhibiting ros and nf-κb pathway hydrogen-rich water alleviates cyclosporine a-induced nephrotoxicity via the keap /nrf signaling pathway hydrogen rich water attenuates renal injury and fibrosis by regulation transforming growth factor-β induced sirt hydrogen-rich saline promotes the recovery of renal function after ischemia/reperfusion injury in rats via anti-apoptosis and anti-inflammation preventive effect of hydrogen water on the development of detrusor overactivity in a rat model of bladder outlet obstruction saturated hydrogen improves lipid metabolism disorders and dysbacteriosis induced by a high-fat diet subcutaneous injection of hydrogen gas is a novel effective treatment for type diabetes l-arabinose elicits gut-derived hydrogen production and oxidative medicine and cellular longevity ameliorates metabolic syndrome in c bl/ j mice on highfat-diet hydrogen gas production is associated with reduced interleukin- β mrna in peripheral blood after a single dose of acarbose in japanese type diabetic patients molecular hydrogen improves obesity and diabetes by inducing hepatic fgf and stimulating energy metabolism in db/db mice hydrogen attenuates allergic inflammation by reversing energy metabolic pathway switch pilot study: effects of drinking hydrogen-rich water on muscle fatigue caused by acute exercise in elite athletes intravenous infusion of h -saline suppresses oxidative stress and elevates antioxidant potential in thoroughbred horses after racing exercise involvement of neutrophil dynamics and function in exercise-induced muscle damage and delayed-onset muscle soreness: effect of hydrogen bath protective effect of hydrogen gas inhalation on muscular damage using a mouse hindlimb ischemia-reperfusion injury model molecular hydrogen alleviates motor deficits and muscle degeneration in mdx mice protective effect of inhalation of hydrogen gas on radiation-induced dermatitis and skin injury in rats hydrogen gas inhalation protects against cutaneous ischaemia/reperfusion injury in a mouse model of pressure ulcer improvement of psoriasis-associated arthritis and skin lesions by treatment with molecular hydrogen: a report of three cases inhaled hydrogen gas therapy for prevention of noise-induced hearing loss through reducing reactive oxygen species molecular hydrogen effectively heals alkali-injured cornea via suppression of oxidative stress high throughput sirna screening for chloropicrin and hydrogen fluoride-induced cornea epithelial cell injury therapeutic effect of molecular hydrogen in corneal uvb-induced oxidative stress and corneal photodamage molecular hydrogen suppresses glioblastoma growth via inducing the glioma stemlike cell differentiation therapeutic efficacy of hydrogen-rich saline alone and in combination with pi k inhibitor in non-small cell lung cancer a gallbladder carcinoma patient with pseudo-progressive remission after hydrogen inhalation brain metastases completely disappear in non-small cell lung cancer using hydrogen gas inhalation: a case report molecular hydrogen alleviates nephrotoxicity induced by an anti-cancer drug cisplatin without compromising anti-tumor activity in mice hydrogen protects rats from dermatitis caused by local radiation pathological findings of covid- associated with acute respiratory distress syndrome strategies for the prevention and management of coronavirus disease key: cord- - qi pbz authors: benej, martin; danchenko, maksym; oveckova, ingrid; cervenak, filip; tomaska, lubomir; grossmannova, katarina; polcicova, katarina; golias, tereza; tomaskova, jana title: quantitative proteomics reveal peroxiredoxin perturbation upon persistent lymphocytic choriomeningitis virus infection in human cells date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: qi pbz experimental data indicate that during persistent infection, lymphocytic choriomeningitis virus (lcmv) may both directly or indirectly modulate regulatory cellular processes and alter cellular functions that are not critical for survival, but are essential for cell homeostasis. in order to shed more light on these processes, two-dimensional differential in-gel electrophoresis ( d-dige) and maldi-tof tandem mass spectrometry were used to determine the proteome response of the hela cell line to persistent lcmv infection. quantitative analysis revealed differentially abundant proteins. functional analysis showed that lcmv-responsive proteins were primarily involved in metabolism, stress, and the defense response. among identified proteins, we discovered significant changes for peroxiredoxins, a family of antioxidant enzymes. decreased amount of these antioxidant proteins correlated with elevation of reactive oxygen species (ros) in infected cells. increased levels of ros were accompanied by changes in the pattern of telomere restriction fragments (trfs) in infected cells and mediated activation of hypoxia-inducible transcription factor- (hif- ) and phosphatidylinositol -kinase (pi k)/akt signaling pathways. moreover, treatment with antioxidants resulted in reduced levels of viral nucleoprotein, indicating a connection between ros-dependent signaling and viral replication. persistent viral infections are currently one of the most important global health problems in the world. understanding how viral persistence is initiated and maintained, and the pathological consequences of ongoing virus replication, is therefore of high importance. the prototypic mammarenavirus, lymphocytic choriomeningitis virus (lcmv), provides a suitable model system for investigation of the mechanisms of persistent viral infection, virushost interactions, and pathogenesis. studies using this viral model have resulted in significant progress in virology and immunology that is applicable to other human microbial and viral infections (oldstone, ; zinkernagel, ) . in addition, lcmv is a neglected human pathogen of clinical significance that may lead to severe disease and consequences after prenatal infection (barton and mets, ; mets et al., ; barton et al., ) , and life-threatening conditions in immunosuppressed individuals (fischer et al., ; palacios et al., ; macneil et al., ) . moreover, several mammarenaviruses, such as the lassa, junin, and machupo viruses, are associated with severe hemorrhagic fever disease with significant morbidity and mortality in humans, representing serious public health concerns in their endemic regions (geisbert and jahrling, ; buchmeier et al., ) . currently, supportive care and ribavirin (nucleoside analogue) are the only treatment options available (damonte and coto, ; parker, ) . therefore, considerable interest focuses on the development of therapeutic strategies against mammarenavirus infection and disease. understanding how viruses and the hosts interact and how their interactions change over time would help in the rational design of targeting compounds. mammarenaviruses are enveloped viruses with a bisegmented negative-strand rna genome and a non-lytic life cycle restricted to the cell cytoplasm. the s segment of the genome encodes the nucleoprotein (np) that encapsidates viral rna and the glycoprotein precursor (gpc). after post-translational modification, gpc yields gp and gp that mediate virus entry. the l segment encodes the viral rna-dependent rna polymerase (l) responsible for viral rna synthesis, and the zincbinding protein (z) important for viral budding (buchmeier et al., ) . mammarenaviruses are able to establish persistent infection in their natural hosts and in a number of mammalian cells in vitro. during persistence, mammarenaviruses continue to replicate and express viral proteins. at the same time, they interfere with normal host homeostasis, resulting in possible disease without the destruction of the infected cell. additionally, they actively suppress the host's immune response in order to avoid recognition and to allow the spread of infection (oldstone, ) . although research on lcmv has led to many insights into viral persistence, the answers to several crucial questions related to the principles, by which persistence is initiated and maintained, are still lacking. a highly suitable tool to study virus-host cell interactions is proteomics, since viral infection fundamentally affects host cell proteins. it does so at many functional levels, such as affecting the cell signaling pathways, cytokine and growth factor production, apoptosis, phagocytosis, protein degradation, and cytoskeletal rearrangement (coiras et al., ) . so far, several large-scale analyses of host proteome (bowick et al., (bowick et al., , (bowick et al., , , kinome (bowick et al., ) , and transcriptome (djavani et al., (djavani et al., , muller et al., ) have been performed to study the consequences of an acute mammarenavirus infection. moreover, a number of studies employed the proteomic approach to identify interacting partners of mammarenavirus proteins in human cells (khamina et al., ; king et al., ; iwasaki et al., ; loureiro et al., ; ziegler et al., ) . on the other hand, none of them has focused on the host cellular response during persistent infection. herein, we analyzed proteomic changes in a human cell line hela upon persistent infection with the mx isolate of lcmv using quantitative gel-based proteomics. our data reveal significant changes in the proteins associated with metabolism, antioxidant activity, protein folding, rna binding, and immune response. many of the differentially abundant proteins have not been reported previously as virus-responsive upon lcmv infection. thus, these proteins represent potential targets for further in-depth investigations. understanding their roles in persistent lcmv infection would reveal more about the complexity and dynamics of interactions between virus and host cells, and would benefit antiviral research. this study thus offers useful basis for further research into the function of human proteins in persistent infection. human cervical carcinoma cells (hela), human alveolar adenocarcinoma cells (a ) (atcc ccl- ), and baby hamster kidney fibroblast cells (bhk- ) (atcc ccl- ), were grown in dulbecco's modified eagle medium (dmem) containing % fetal calf serum (fcs) supplemented with µg/ml gentamicin in a humidified atmosphere with % co at • c. lcmv mx isolate was continuously propagated in persistently infected hela cells (designated hela-mx). the mx isolate represents a persistent form of the virus capable of propagation through cell-cell contacts without releasing infectious virions (gibadulinova et al., ; reiserova et al., ; tomaskova et al., ) . a cells were infected with mx isolate via cell-free extract from persistently infected bhk- cells (bhk-mx) as described in laposova et al. ( ) and subsequently passaged several times to allow virus spread. whole-proteome samples for d-dige analysis were prepared according to standard protocol. briefly, the cells were washed with ice-cold pbs, and µl of lysis buffer ( mm nacl; m tris, ph . ; % triton x- ; and . % sds) were added to the culture, followed by min incubation on ice. the lysed cells were scraped off, transferred to tubes, passaged through an insulin syringe and centrifuged at , × g for min at • c. next, nine volumes of precipitation solution (acetone:methanol : ) were added, and the resulting solutions were incubated overnight at − • c. the samples were then centrifuged at × g for min at • c. obtained pellets were air-dried and resuspended in µl of utc solution ( m urea, m thiourea, % chaps). final protein concentrations were determined using the bradford method (bio-rad). for the preparative gel, µg of each sample were mixed with the utc solution to the final volume of µl. an equal volume of loading/reswelling buffer [ mm dithiothreitol (dtt); % (v/v) immobilized ph gradient (ipg) buffer, ph - nl (ge healthcare), . % destreak reagent (ge healthcare); adjusted with utc solution to the final volume of µl] was added to the mixture. eighteen centimeters ipg strip with non-linear - ph range (ge healthcare) was passively reswelled in µl of the sample mixture overnight. isoelectric focusing was performed using the multiphor ii unit (ge healthcare), with the following -step protocol: ( ) v, h; ( ) v, h; ( ) v, h; ( ) v, h; ( ) v, h; and ( ) v, h. the current limit was set to ma, with a ramping voltage gradient between the steps. after separation in the st dimension, the strip was equilibrated in sds equilibration solution ( m urea; % sds; % glycerol; . mm tris-hcl ph . ) containing . % (w/v) dtt for min; followed by min incubation with sds equilibration solution in the presence of . % iodoacetamide (iaa). the equilibrated strip was placed on top of a % polyacrylamide gel cast between low-fluorescence glass plates (the backing plate being pretreated with bind silane solution; ml % ethanol, . ml mq water, . ml acetic acid and µl of bind silane, h incubation at room temperature) and fixed by adding . % agarose gel dissolved in mq water with a trace of bromophenol blue. separation in the nd dimension was carried out using the protean xl (bio-rad) chamber. the resulting glass-backed d gel was incubated × min in fixing solution ( ml ethanol, ml acetic acid, ml mq water), the solution being replaced between individual fixing steps. total proteins were stained by overnight incubation with sypro ruby protein gel stain (molecular probes) in the dark. the gel was washed × min in wash solution ( ml methanol, ml acetic acid, ml mq water) and × min in mq water prior to scanning. resulting d map of the proteome was visualized by the pharosfx molecular imager (bio-rad), using µm resolution and appropriate laser/filter wavelengths. in the first step, sample ph was adjusted to fit the range of . - . using m tris. fluorescent dyes cy , cy , and cy were used for minimal sample labeling (ge healthcare). fifty micrograms of each sample were labeled with pmol of either cy or cy , in biological triplicates, using the dye-swap technique between individual replicate pairs. for the internal standard, µg of each sample replicate was mixed with pmol of cy per sample. after min of on-ice incubation in the dark, the labeling reaction was stopped by adding / volume of mm l-lysine to the samples. after a short spin, the labeled mixtures were incubated on ice for min in the dark. subsequently, the samples for each replicate gel were mixed in the following manner cy :cy :cy : : , adjusted with utc solution to the final volume of µl and mixed with an equal volume of the loading/reswelling buffer. ipg strips with non-linear - ph range were passively rehydrated in µl of the sample mixtures overnight in the dark. the d separation protocol was identical to that of the preparative gel with three exceptions -(i) % polyacrylamide gel was cast between two low-fluorescence glass plates, not pretreated with bind-silan; (ii) the proteins in the analytical gels were not fixed, but directly scanned (iii) particular emphasis was put on working in the dark. obtained d gels were immediately scanned between two low-fluorescence glass plates using the pharosfx molecular imager at µm resolution and at appropriate excitation/emission wavelengths corresponding to cy , cy , and cy individual channels. quantitative gel analysis was carried out using decyder . software package (ge healthcare). for those proteins discovered to be different in mock-infected and lcmv-infected samples, analysis of significance was conducted using the student's t-test. only significantly different protein spots (p < . ), with an at least . -fold abundance difference (ratio of mock versus infected sample mean normalized spot signals) were regarded as upor down-regulated. these protein spots of interest were chosen for mass spectrometry detection. target spots of interest were excised from the preparative gel using exquest spot cutter (bio-rad) and destained. after reduction and alkylation, the gel plugs were digested overnight with sequencing grade modified trypsin (promega). the digested peptides were extracted with µl of % acetonitrile (merck) containing . % trifluoroacetic acid (tfa) (merck). samples with digested peptides were evaporated to µl in concentrator plus (eppendorf) and purified using µ-c ziptips (merck millipore). subsequently, µl of peptide mixtures and µl of . mg/ml chca matrix in % acetonitrile, . % tfa, and mm ammonium phosphate were pipetted onto µm anchorchip maldi target (bruker). data were acquired on ultraflextreme tof mass spectrometer (bruker) operated by flexcontrol . (bruker) in positive reflector mode. for each position on the target laser shots were summed in - m/z range. next, we selected the most intense precursor ions per sample for fragmentation with signalto-noise threshold . tandem mass spectra were recorded by accumulation of shots in lift mode using lid (laserinduced dissociation) mechanism. laser power was boosted by % without collision gas, and the detector voltage was increased by %. monoisotopic peak lists were generated in flexanalysis . (bruker) by snap algorithm. precursor spectra with signal-tonoise less than ten were removed, and the remaining masses were externally recalibrated. fragment spectra were smoothed using savitzky-golay algorithm (three cycles with . m/z width), the baseline was subtracted with tophat algorithm, and filtering was done on signal-to-noise level five. subsequent processing for protein identification was done through proteinscape . (bruker) interface connected to mascot server . (matrix science). fixed carbamidomethyl cysteine, variable oxidized methionine, single trypsin miscleavage, and appropriate mass tolerances ( ppm for precursor ions and . da for fragments) were specified. queries were done against homo sapiens proteins downloaded from uniprot in april , containing , sequences. protein identifications were accepted if the ion scores of at least two different matched peptides were higher than the probability of identity threshold ( at p < . ). the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride partner repository (vizcaino et al., ) with the dataset identifier pxd and doi: . /pxd . gene ontology annotation was acquired from the gene ontology project using the panther database for biological procedures and molecular function . functional enrichment analysis was performed by fisher's exact test with false discovery (fdr) correction, with fdr p < . considered significant (mi et al., ) . the protein-protein interaction network was analyzed using the string . database with default settings, while the interaction score was set to medium confidence ( . ) (jensen et al., ). equal amounts of proteins from untreated mock-infected and lcmv-infected cells or cells treated with mm n-acetyl-l-cysteine (nac) for h were separated in % sds-polyacrylamide gel, transferred onto a polyvinylidene difluoride membrane (immobilon-p; millipore), and probed with antibodies specific for lcmv np [in-house generated mab m (tomaskova et al., ) ], alpha-enolase (abcam #ab , : dilution), gp /hsp -beta (millipore #abf , : dilution), prdx (invitrogen #pa - , : dilution), phospho-akt (ser ) (cell signaling technology # , : dilution), akt (cell signaling technology # , : dilution), β-actin (cell signaling technology # , : dilution), and alpha-tubulin (abcam [ab ], : , dilution). detection was performed with hrp-or irdye-labeled secondary antibodies visualized with enhanced chemiluminescence or odyssey clx imager system, respectively. densitometric analysis of protein abundance was performed using imagej software (schindelin et al., ) or image studio lite software, respectively. the generation of reactive oxygen species (ros) was assessed using the fluoroprobe -(and- )-chloromethyl- , -dichlorodihydrofluorescein diacetate (cm-h dcfda). cells ( × cells/well) cultured in -well plates were washed with phosphate-buffered saline supplemented with calcium/magnesium (pbs) and incubated with µm cm-h dcfda (molecular probes) in pbs for min in the dark at • c. after replacement of the reactive agents with pbs, , -dichlorofluorescein (dcf) fluorescence was measured at an excitation wavelength of nm and an emission wavelength of nm using the synergy/h microplate reader (biotec instruments). values were corrected for background auto-fluorescence of non-stained cells and normalized to the number of cells assessed by dapi staining. briefly, at the end of the experiment, cells were fixed with ice-cold methanol for min at − • c, washed twice with pbs and then incubated with dapi ( µg/ml) for min in the dark. after a subsequent washing step with pbs, dapi fluorescence was measured in each well using nm excitation and nm emission wavelengths. the genomic dna (gdna) was isolated from hela and hela-mx cells using qiaamp dna mini kit (qiagen) according to manufacturer's instructions. three µg of genomic dna were digested using a mixture of restriction enzymes (hhai, hinf , mspi, haeiii, rsai, alui) as described by mender and shay ( ) . the dna fragments were separated in % agarose gel for h at . v/cm and stained with . µg/ml ethidium bromide solution for min (stained gel served as a loading control). the gel was then incubated for min in denaturation solution ( . m nacl, . m naoh), min in neutralization solution ( . m nacl, . m tris, ph . ) and min in × ssc ( m nacl, . m na-citrate, ph . ). the dna was then transferred to immobilon ny + membrane (emd millipore) with a vacugene xl blotter (ge healthcare) in × ssc and fixed by incubating the membrane at • c for h. the membrane was pre-hybridized for min at • c in hybridization buffer ( × ssc, × denhardt solution, . % sds) and hybridized at • c overnight in the same buffer containing radioactively labeled telomere-specific c-rich probe prepared as described by mender and shay ( ) . the membrane was washed once with wash buffer i ( × ssc, . % sds) for min at • c, twice for min in wash buffer ii ( . × ssc, . % sds) at • c and twice for min at room temperature with wash buffer iii ( . × ssc, % sds). the signal was detected by personal molecular imager fx (biorad). hela and hela-mx cells were seeded into a -well plate at × cells per well and transfected with µg of the luciferase vector (hre-luc) containing hypoxia-responsive elements the next day, using the turbofect transfection reagent (thermo fisher scientific) according to the manufacturer's instructions. cells were co-transfected with ng of prl-tk renilla vector (promega) to normalize for the transfection efficiency. luciferase reporter construct containing three hypoxia response elements (hre; -mers) from the pgk- gene was a gift from navdeep chandel (emerling et al., ) (addgene plasmid # ; rrid:addgene_ ). reporter gene expression was assessed h after transfection using the dual luciferase reporter assay system (promega) and the synergy ht reader with gen software (biotec instruments). luciferase activity was normalized against renilla activity. figure | preparative d gel. a total of µg proteins from each sample of lcmv-infected and mock-infected hela cells were resolved by d page. the protein spots were visualized by sypro ruby protein gel stain. this preparative d-gel was used to cut out protein spots that were differentially abundant in analytical gels. arrows indicate the isolated and identified protein spots with at least . -fold up-regulation or down-regulation. spots are numbered according to table . statistical analyses were performed using graphpad prism software for windows, unless stated otherwise. two-tailed unpaired t-test was used to analyze significant differences between two cell groups and one-way anova was used to determine statistically significant differences between three or more independent groups, with p < . considered significant. to investigate global protein changes in hela cells during persistent infection with the mx strain of lcmv, equal amounts of total proteins prepared from mock-infected and lcmv-infected hela cells were subjected to d-dige analysis. we used three independent biological replicates to generate comprehensive and reliable data (supplementary figure s ) . the separation strength was satisfactory; the average yield per gel comprised approximately protein spots. initial betweengel matching was carried out by manual landmarking, followed by automatic matching and extensive manual evaluation of the matched spots between individual gels in the decyder d . software. only protein spots showing significance (p < . ) and at least . -fold difference in abundance were considered as up-or down-regulated. according to our analyses, the abundance of protein spots was significantly altered after lcmv infection. since decyder does not allow identification of proteins present only in one condition and absent in the other, we complemented gel analysis with a manual qualitative study of protein presence between individual samples. in total, protein spots were present only in one condition (mockinfected or lcmv-infected). these protein spots were marked as "proteins of interest (pois)" and matched against the sypro ruby-stained preparative gel. the proteins of interest, which we were able to map on the preparative gel, were excised and analyzed by maldi-tof tandem mass spectrometry. twenty-four protein spots with non-redundant proteins were successfully identified, including up-regulated and down-regulated proteins (figure ) . detailed information on the identified proteins is provided in table . several proteins were present in more than one spot, namely: heterogeneous nuclear ribonucleoprotein k (hnrnp k; spot and , both more abundant upon infection), mitochondrial kda heat shock protein (hsp ; spot and , both less abundant in cells affected by persistent virus), and keratin, type ii cytoskeletal (ck- ; spot and , which showed opposite changes). these proteins are most likely different post-translational or alternatively spliced variants. proteins present only in one condition were: (a) unique upon lcmv infection -mitochondrial hydroxymethylglutaryl-coa synthase, guanine deaminase, annexin a , and rab gdp dissociation inhibitor beta; (b) unique in the mock-infected cell line -cellular retinoic acid binding protein , prostaglandin e synthase , and eukaryotic initiation factor a-i. apart from proteins discussed in more detail below, other accumulated proteins upon infection included galectin and macrophagecapping protein, and less abundant endoplasmin, gtp-binding nuclear protein ran, peptidyl-prolyl cis-trans isomerase fkbp , and adenine phosphoribosyltransferase. to better understand the implications of the cellular response to lcmv infection, the corresponding biological functions of the differentially regulated proteins were categorized according to the gene ontology database, using the panther classification system. most of the proteins with significantly altered abundance were involved in metabolic processes ( . %; proteins), particularly lipid and glucose metabolism; cellular processes, such as cellular communication and cell cycle ( . %; proteins); immune system processes ( . %; proteins); developmental processes ( . %; proteins); localization ( . %; proteins); and cellular component organization or biogenesis ( . %; proteins) (figure ) . functional enrichment analysis revealed that the most overrepresented were proteins with peroxiredoxin activity (go: ; raw p-value = . e- ; fdr = . e- ) and proteins that play a role in rna binding (go: ; raw p-value = . e- ; fdr = . e- ). furthermore, network analysis was conducted to reveal protein-protein interactions. forty-two associations were observed in the protein network analysis generated by string . database. the network had significantly more interactions than expected randomly (six expected interactions; ppi enrichment p-value < . e- ). such an enrichment indicates that the proteins are at least partially biologically connected. the confidence view of the protein-protein associations is shown in figure , where the strength of the association is indicated by the thickness of the connecting line (confidence score > . ). similar to gene ontology analysis, the most connected proteins have important functions in antioxidant activity, protein folding, and rna binding. to validate the changes in protein accumulation patterns in response to lcmv infection, two identified proteins, alphaenolase (eno ; more abundant) and heat shock protein kda beta, member [hsp b (endoplasmin); less abundant] were selected for western blot analysis. the reason to select hsp b was its important functionality, pinpointed by the string network analysis. figure a shows a prominent although not statistically significant increase in protein abundance of eno and a decrease for hsp b in infected cells. these results are consistent with those observed in the d-dige analysis. the densitometric analysis of three biological replicates revealed hela-mx/hela average ratio of . for eno and − . for hsp b , while alpha-tubulin was used as a loading control ( figure b) . the results were very close to the changes observed in the d-dige analysis, which were . for eno and − . for hsp b . in this study, we observed a decreased amount of peroxiredoxin (prdx ), peroxiredoxin (prdx ), and peroxiredoxin (prdx ) upon lcmv infection. peroxiredoxins (prxs) are members of an ubiquitous family of thiol-specific peroxidases that reduce mainly hydrogen peroxide (h o ) to water with the use of reducing equivalents provided through the thioredoxin system (wood et al., ) . to confirm the alteration in the abundance of peroxiredoxins during lcmv infection, we selected the most affected prdx for western blot analysis. figure a shows an almost identical decrease in the expression of prdx (− . ) in infected cells as we observed in the d-dige analysis (− . ). since one of the major functions of prxs is cellular protection against oxidative stress, we hypothesized that decreased levels of these antioxidant proteins may lead to an elevation of the ros content in infected cells. the measurement of ros generation in mock-and lcmv-infected cells supported our prediction. we revealed significantly higher production of ros ( . -fold) in lcmv-infected cells than in mock-infected cells ( figure b) . interestingly, another antioxidant enzyme, thioredoxin reductase (trxr ), was more abundant in infected cells (table ), yet this seems to be insufficient to counterbalance the decrease in the levels of peroxiredoxins. in order to determine whether the increased ros level described above is unique to the mx-infected hela cells or can also be seen in other cell lines, we decided to use a lung epithelial cells, which are commonly used as an in vitro model system for arenavirus infections. similar to hela cells, we detected a . figure s a) . further, we hypothesized that increased levels of ros may have an effect on dna of the infected cells. telomeric sequences composed of arrays of -ttaggg- repeats are particularly vulnerable to oxidative damage due to stretches of g residues that are susceptible to the conversion to -oxoguanine ( -oxog). accumulation of -oxog in telomeric repeats may affect accessibility of chromosomal ends to telomerase, binding efficiency of telomere-binding proteins, and/or formation of protective secondary structures such as telomeric loops (von zglinicki, ; ahmed and lingner, ) . as a result, increased levels of ros yield changes in the length of telomeric tracts. to test this hypothesis, we isolated genomic dna from control and infected cells and measured the length of telomere restriction fragments (trfs). we observed that the infected cells contained a more heterogeneous population of trfs and a higher proportion of shorter fragments compared with the control cells (figure ). this indicates that the infected cells have a compromised ability to maintain the standard length of telomeres. it has become apparent in recent decades that less-reactive ros, especially hydrogen peroxide, can function as intracellular signaling molecules regulating multiple physiological and pathological cell processes. it has been shown that ros mediate activation of several signaling pathways and transcription factors, including the pi k/akt signaling cascade (lee et al., (lee et al., , and hif (patten et al., ) . to assess biological implications of increased ros in infected cells, we investigated whether lcmv infection affected transcriptional activity of hif- . using a luciferase reporter containing hre, we detected a modest, but significant increase in hif- transactivation in infected hela cells in comparison to control cells ( figure a) . further, we evaluated activation of akt by probing cellular protein lysates for phospho-akt (s ), which is a marker for activated akt. figures b,c , phosphorylation of akt was induced in infected cells, while overall akt levels were similar in both infected and uninfected cells. since we assumed that activation of akt was triggered by redox signaling, we further investigated the effect of antioxidants on infected cells. we detected decreased levels of phospho-akt in lcmv-infected hela cells treated with n-acetyl cysteine. moreover, inhibition of ros accumulation by the antioxidant also decreased the levels of viral np (figures b,d) . in addition, we observed similar impact of antioxidant treatment in lcmvinfected a cells, although the reduction in np levels was less pronounced (supplementary figure s b) . . the signal obtained with anti-alpha-tubulin antibody was used as loading control. presence of np confirms lcmv infection. one representative of three biological replicates is shown. (b) densitometric analysis of protein abundance was performed using imagej software. relative quantity of proteins was calculated as the ratio of the intensity of each signal to the intensity of the related tubulin internal standard. values represent the means of three biological replicates. error bars denote the standard deviations. * * p < . (hela-mx vs. hela). these data suggest that lcmv maintains its replication in persistently infected cells by regulating redox signaling through modulation of local levels of h o and subsequent activation of cellular processes that it uses for its own benefit. viruses and host cells have established complex, dynamic interactions that either facilitate the efficiency of viral infection or defend the host against invading pathogens. these interactions are crucial for the regulation of metabolism and other processes in the host cell and their elucidation is critical for a detailed description of mechanisms of viral pathogenesis, eventually leading to effective treatment. in this report, we investigated the host response to persistent lcmv infection using d-dige-based proteomic approach. as the model system of persistent infection we used human hela cells long-term infected with lcmv mx isolate (over passages). the mx isolate likely represents a lcmv variant that has undergone complex adaptations. it is characterized by reduced accumulation of viral glycoproteins on the surface of . the signal obtained with anti-β-actin antibody was used as loading control. one representative of three biological replicates is shown. densitometric analysis of protein abundance (right) was performed using imagej software. relative quantity of proteins was calculated as the ratio of the intensity of each signal to the intensity of the related β-actin internal standard. values represent the means of three biological replicates. error bars denote standard deviations. * * p < . (hela-mx vs. hela). (b) ros generation was assessed in a microplate reader using dcf fluorescence and normalized to the number of cells measured by dapi staining. the results represent the mean from three independent biological experiments, each done in eight replicates. error bars denote standard deviations. data are presented as relative increase compared to control (hela cells), which was set to . * * * * p < . (hela-mx vs. hela). infected cells and by spreading to uninfected cells mainly through cell-cell contact without the release of infectious viral particles, all of which strongly resembles persistent lcmv neuronal infection in its native host (rodriguez et al., ) . the use of hela-mx cells has several advantages, such as the feasibility of use of a large number of cells for proteomic analysis, reproducibility, and viral persistence resembling a life-long chronic infection. however, in terms of natural lcmv infection, hela cells possess some limitations and one needs to be cautious about generalizing the results. therefore, we performed some of the experiments also on the a cell line, a model of type ii alveolar epithelial cells (supplementary figure s ) . this cell type is the first to be in contact with the virus during primary infection. it has also been shown that a cells are capable of antigen presentation (salik et al., ) . although the data demonstrating chronic infection in humans are not available, the persisting and figure | lmcv-infected cells exhibit changes in the pattern of telomeric restriction fragments. (a) total dna was isolated from uninfected (hela) and infected (hela-mx) cells, digested by a cocktail of restriction enzymes and the resulting dna fragments were separated by % agarose gel electrophoresis. (b) dna was transferred to nylon membrane and hybridized with a radiolabeled telomeric probe. trfs (telomeric restriction fragments), dna ladder (generuler kb dna ladder (thermo scientific). a representative of three independent experiments is shown. reactivated virus may be a source of unexplained complications (often fatal) in immunosuppressed transplant recipients, as well as in developing embryos (peters, ; bonthius, ). thus, a better understanding of cellular processes exploited or subverted by viruses during persistent infection can help develop new strategies to treat mammarenavirus infections in humans. our analysis revealed protein spots differentially accumulated upon persistent lcmv infection in hela cells. proteins were identified with high confidence. remarkably, the abundance of several antioxidant enzymes was shown to be significantly altered. namely, the levels of prdx , prdx , and prdx were . - . times lower in lcmv-infected cells (table ). in addition, functional enrichment analysis, as well as network analysis, pointed out that the antioxidant system was in the center of virusinduced host proteome response (figure ) . the key functions of prxs include cellular protection against oxidative stress and modulation of signaling pathways that use hydrogen peroxide as a second messenger (rhee et al., a,b) . a significant amount of published data suggests that prxs are hydrogen peroxide sensors that play a central role in redox signaling (rhee et al., ; latimer and veal, ; netto and antunes, ) . this is implicated in a number of biological processes, including growth factor signaling, cell differentiation, and cytokine production (hampton and o'connor, ) . regulation of prxs modifies the concentration of h o and thereby facilitates its signaling functions. we indeed confirmed a slight but significant elevation of the ros content alongside the downregulation of prxs by lcmv both in hela and a cells (figure b and supplementary figure s a) . ros, in the form of h o , can modify protein function, and/or structure through the mechanism of cysteine oxidation that influences a number of signaling cascades. for example, ros activate pi k either directly or by inactivating the phosphatase pten via oxidizing cysteine residues within its active site, resulting in an increased activation of akt and modulation of its downstream targets (lee et al., ; connor et al., ) . once activated, akt promotes cell survival, growth, metabolism, and proliferation by phosphorylating various effectors (ersahin et al., ) . in line with these findings, we observed enhanced levels of active akt in lcmv-infected cells. in addition, inhibition of ros by n-acetyl cysteine led to suppressed phosphorylation of akt in infected cells, suggesting that akt becomes activated in a ros-dependent manner (figures b,c) . notably, the treatment with antioxidants also resulted in reduced levels of viral np in hela, as well as in a lcmv-infected cells, suggesting a link between ros-dependent signaling and virus replication (figures b,d and supplementary figure s b ). these findings are in accordance with previous studies that have demonstrated that ros generated in response to lcmv play an important role in virus binding and subsequent virus replication (michalek et al., ) . moreover, it has been shown that the pi k/akt pathway plays an important role in different steps of the life cycle of a variety of viruses. for instance, infection with the new world mammarenavirus junin virus has been shown to induce the pi k/akt pathway, and inhibition of this pathway has resulted in a decreased infectious virus yield because of blocking the recycling of the transferrin receptor engaged in junv cell entry (linero and scolaro, ) . furthermore, pi k/akt pathway inhibition reduced budding and, to a lower degree, lcmv rna synthesis, but not cell entry (urata et al., ) . additionally, the sars coronavirus induced weak activation of akt that was crucial for the establishment of persistence (mizutani et al., ) . in contrast, pi k/akt pathway inhibition could not interfere with the establishment of persistent junv infection in vero cells, and this pathway was not crucial for the maintenance of junv persistence (linero and scolaro, ). data are presented as a relative increase compared to control (hela cells), which was set to . * * * * p < . (hela-mx vs. hela). (b) immunoblot analysis of p-akt (s ), total akt, and viral np with specific antibodies using whole-cell extracts prepared from untreated (−) mock-infected hela cells (hela) and lcmv-infected hela cells (hela-mx) or cells treated (+) with mm nac for h. the signal obtained with anti-β-actin antibody was used as loading control. one representative of at least four biological replicates is shown. (c) densitometric analysis of protein abundance was performed using imagej software or image studio lite software. relative quantity of p-akt was calculated as the ratio of the intensity of each signal to the intensity of the related signal for total akt. values represent the means of six biological replicates. error bars denote the standard deviations. data are presented as relative change compared to control (untreated hela cells), which was set to . * p = . (hela vs. hela-mx), * p = . (hela-mx vs. hela-mx nac), p = . (hela vs. hela nac). (d) densitometric analysis of protein abundance was performed using imagej software or image studio lite software. relative quantity of np was calculated as the ratio of the intensity of each signal to the intensity of the related β-actin or tubulin internal standard, respectively. values represent the means of four biological replicates. error bars denote the standard deviations. data are presented as relative change compared to untreated hela-mx cells, which was set to . * * p = . (hela-mx nac vs. hela-mx). reactive oxygen species also regulate the activation of several transcription factors, including hifs. hif- is the main hypoxiainducible transcription factor responsible for cell and tissue adaptation to low oxygen, by controlling cell metabolism, proliferation and survival, erythropoiesis and angiogenesis (semenza, ) . there is increasing evidence demonstrating that non-hypoxic stimuli can also activate hif- . it has been shown that the pi k/akt/mtor signaling cascade directly increases the expression of hif- encoding gene on the transcriptional and translational levels (dery et al., ) . any aberrant stimulation of this pathway leads to activation of hif- α, even in normoxic conditions. an increased ros production has been reported as essential for increased hif translation through the pi k pathway (iommarini et al., ) and for hif stabilization through the inactivation of prolyl hydroxylases in a nonhypoxic system (diebold and chandel, ) . accordingly, we demonstrated enhanced hif- transactivation in lcmv-infected cells (figure a) , as well as increased expression of eno (figure ) , a typical hif- target gene (semenza et al., ) . interestingly, hsp b expression was reduced by persistent lcmv (figure ) , which has also been previously reported in the case of hypoxia in min cells (bensellam et al., ) . pi k/akt signaling, as well as the hif transcription factors, have in common that they play an essential role in regulating cellular metabolism (dery et al., ; yu and cui, ) . in view of that, we observed that a large number of proteins identified in this study, such as the above mentioned alphaenolase or hydroxymethylglutaryl-coa synthase, are involved in metabolism, as indicated by the go analysis (figure ) . moreover, we have shown previously that the exposure of mx-infected hela cells to chronic hypoxia resulted in a hif-dependent increase of viral replication and enhanced formation of infectious virions (tomaskova et al., ) . in addition, there is accumulating evidence that many viruses, through various mechanisms, affect the hif- pathway, causing multiple downstream effects, such as modifying host cell metabolism, stimulating inflammation, and facilitating viral replication (morinet et al., ; cuninghame et al., ) . some viruses, such as the vaccinia virus and hepatitis c virus impair hif- α prolyl hydroxylation, which leads to its stabilization (nasimuzzaman et al., ; mazzon et al., ) . hepatitis b virus stabilizes hif- α by diminishing the interaction between vhl and hif- α (moon et al., ) . influenza virus a h n inhibits the proteasome and decreases fih- expression, thereby activating the hif- pathway by stabilizing hif- α (ren et al., ) . contrary to downregulated prxs, we observed a . fold increased abundance of cytoplasmic trxr (encoded by txnrd ) in lcmv-infected cells ( table ) . the thioredoxin (trx) system consisting of nadph, trxr, and thioredoxin, is a key antioxidant mechanism in protection against oxidative stress. it regulates protein dithiol/disulfide balance through its disulfide reductase activity (arner, ) . as already mentioned, trx provides electrons to peroxiredoxins to remove reactive oxygen and nitrogen species. trx reductase then reduces oxidized trx back using the reducing nadph equivalents. thus, trx reductase plays a crucial role in regeneration of a catalytically active form of prxs (lu and holmgren, ) . we can, therefore, speculate that elevated levels of trxr can maintain proper accumulation of local h o to ensure that it acts as a signaling molecule and does not cause oxidative damage. based on the abovementioned facts, it is possible that lcmv modulates the delicate interplay inside cells between oxidants and antioxidants, and determines the activity profile for a variety of proteins that maintain persistent infection (such as transcription factors, kinases and phosphatases, cytoskeletal proteins, or metabolic enzymes). on the other hand, elevated ros levels in infected cells can be a double-edged sword, as they can negatively affect genomic stability of host cells. telomeres, the nucleo-protein complexes at the chromosomal ends, are essential parts of the genome providing solutions to both end-protection and endreplication problems (shay and wright, ) . the solution to end-protection problem is provided by a specialized protein complex called shelterin (de lange, ) , in combination with the formation of a secondary structure called the telomeric loop (griffith et al., ) . the end-replication problem is solved by various means. in human germ or stem cells, and most cancer cells (including hela), telomeres are preserved by telomerase, a reverse transcriptase using an rna subunit as a template for extension of an array of -ttaggg- repeats (shay and wright, ) . guanine residues in telomeric repeats are particularly susceptible to oxidation resulting in their conversion to -oxog (oikawa and kawanishi, ) . accumulation of this oxidized purine compromises the telomere maintenance system in a quite complex way, including inhibition of binding of the shelterin components trf and trf , the formation of t-loop, and/or the inhibition of telomerase (ahmed and lingner, ) . thus, increased intracellular levels of ros often result in telomere shortening (von zglinicki, ) , as observed in this study in the case of hela cells infected by lcmv (figure ). furthermore, it was shown previously by the lingner group that telomeres physically interact with prdx and prdx (aeby et al., ) . although the levels of prdx were not substantially affected in hela-mx cells (data not shown), prdx exhibited a . -fold decrease ( table ) . as prdx was shown to interact with telomeres in the s phase of the cell cycle (aeby et al., ) , reduction in its amount in hela cells infected by lcmv may contribute to a detrimental effect of ros on the maintenance of their telomeric repeats. nevertheless, our data suggest that lcmv affects redox signaling and metabolic pathways that support an anabolic program required for efficient virus replication and/or maintenance of persistent infection. the precise mechanism of how lcmv regulates antioxidant-scavenging proteins in order to effectively manage amounts of ros still remains unknown and requires further investigation. although research on lcmv has led to many insights into viral persistence, the answers to several crucial questions about host-lcmv interactions during persistent infection are still lacking. this study analyzed proteomic changes in a human cell line upon persistent infection with lcmv using quantitative gelbased proteomics. we identified modulated host proteins, which play important roles in a number of cellular processes. uncovering their roles in persistent lcmv infection can broaden our understanding of the complex and dynamic virus-host cell interactions and be valuable for antiviral research. we provided experimental evidence that lcmv negatively affects the levels of antioxidant enzymes peroxiredoxins leading to elevated intracellular content of ros. these changes were accompanied by activation of hif- and pi k/akt signaling pathways as well as changes in the profile of telomeric restriction fragments. the treatment with antioxidants resulted in reduced level of viral nucleoprotein, indicating that virus replication and rosdependent signaling are interconnected processes. the datasets generated for this study can be found in the proteomexchange consortium via the pride partner repository, dataset identifier pxd . jt and lt conceived and designed the experiments. mb, md, and jt analyzed the data. jt wrote the first draft of the manuscript. mb, md, and lt wrote the sections of the manuscript. all authors performed the experiments, contributed to the manuscript revision, read, and approved the submitted version. this project was supported by grants / / , / / to jt, / / to tg, / / to lt from the scientific grant agency of the ministry of education of the slovak republic and the slovak academy of sciences, and apvv- - to lt from slovak research and development agency. we would like to thank dr. gabriela flores-ramirez from the department of rickettsiology, institute of virology, bmc, for her technical assistance. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fmicb. . /full#supplementary-material figure s | d-dige images of biological triplicates. a µg of each sample were labeled with pmol of either cy or cy dye. preferential binding of the dyes was evaluated using dye-swap technique between individual replicates of the analytical gel. in panels (a,c) the hela protein sample was labeled with cy dye (green channel) while cy labeling (red channel) was used for the hela-mx. panel (b) represents replicate gel no. where the hela sample was labeled with cy and the hela-mx was labeled with cy . intra-and inter-gel variations were normalized according to the internal standard consisting of all analyzed samples in ratio : labeled with cy dye. once normalized, these analytical d gels were used to analyze the differential abundance of proteins between hela and hela-mx samples. figure s | reactive oxygen species production and antioxidant treatment in lcmv-and mock-infected a cells. (a) ros generation was assessed in a microplate reader using dcf fluorescence and normalized to the number of cells measured by dapi staining. the results represent the mean from three independent biological experiments, each done in eight replicates. error bars denote standard deviations. data are presented as relative increase compared to control (a cells), which was set to . * * * * p < . (a -mx vs. a ). (b) immunoblot analysis of viral np with specific antibodies using whole-cell extracts prepared from untreated (−) mock-infected a cells (a ) and lcmv-infected a cells (a -mx) or cells treated (+) with mm nac for h. the signal obtained with anti-β-actin antibody was used as loading control. one of two biological replicates is shown. peroxiredoxin protects telomeres from oxidative damage and preserves telomeric dna for extension by telomerase impact of oxidative stress on telomere biology focus on mammalian thioredoxin reductases-important selenoproteins with versatile functions lymphocytic choriomeningitis virus: pediatric pathogen and fetal teratogen lymphocytic choriomeningitis virus: emerging fetal teratogen hypoxia reduces er-to-golgi protein trafficking and increases cell death by inhibiting the adaptive unfolded protein response in mouse beta cells lymphocytic choriomeningitis virus: a prenatal and postnatal threat differential signaling networks induced by mild and lethal 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shortens telomeres structure, mechanism and regulation of peroxiredoxins proliferation, survival and metabolism: the role of pi k/akt/mtor signalling in pluripotency and cell fate determination a proteomic survey of junin virus interactions with human proteins reveals host factors required for arenavirus replication lymphocytic choriomeningitis virus and immunology the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © benej, danchenko, oveckova, cervenak, tomaska, grossmannova, polcicova, golias and tomaskova. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -f km c z authors: nasi, aikaterini; mcardle, stephanie; gaudernack, gustav; westman, gabriel; melief, cornelis; kouretas, demetrios; arens, ramon; sjölin, jan; mangsbo, sara title: reactive oxygen species as an initiator of toxic innate immune responses in retort to sars-cov- in an ageing population, consider n-acetylcysteine as early therapeutic intervention date: - - journal: toxicol rep doi: . /j.toxrep. . . sha: doc_id: cord_uid: f km c z during the current covid- pandemic, a need for evaluation of already available drugs for treatment of the disease is crucial. hereby, based on literature review from the current pandemic and previous outbreaks with corona viruses we analyze the impact of the virus infection on cell stress responses and redox balance. high levels of mortality are noticed in elderly individuals infected with sars-cov and during the previous sars-cov outbreak. elderly individuals maintain a chronic low level of inflammation which is associated with oxidative stress and inflammatory cytokine production, a condition that increases the severity of viral infections in this population. sars-cov infection can lead to alterations of redox balance in infected cells through modulation of nad + biosynthesis, parp function along with altering proteasome and mitochondrial function in the cell thereby leading to enhanced cell stress responses which further exacerbate inflammation. ros production can increase il- production and lipid peroxidation resulting in cell damage. therefore, early treatment with anti-oxidants such as nac during covid- can be a way to bypass the excessive inflammation and cell damage that lead to severe infection.  sars-cov has been reported to modulate parp function and thereby nad+ biosynthesis  cellular homeostasis and redox imbalances by sars-cov can cause stress responses  antioxidants such as nac could limit ros mediated tissue damage during covid- during the current covid- pandemic, a need for evaluation of already available drugs for treatment of the disease is crucial. hereby, based on literature review from the current pandemic and previous outbreaks with corona viruses we analyze the impact of the virus infection on cell stress responses and redox balance. high levels of mortality are noticed in elderly individuals infected with sars-cov and during the previous sars-cov outbreak. elderly individuals maintain a chronic low level of inflammation which is associated with oxidative stress and inflammatory cytokine production, a condition that increases the severity of viral infections in this population. sars-cov infection can lead to alterations of redox balance in infected cells through modulation of nad+ biosynthesis, parp function along with altering proteasome and mitochondrial function in the cell thereby leading to enhanced cell stress responses which further exacerbate inflammation. ros production can increase il- production and lipid peroxidation resulting in cell damage. therefore, early treatment with anti-oxidants such as nac during covid- can be a way to bypass the excessive inflammation and cell damage that lead to severe infection. keywords: covid- , sars-cov , n-acetylcysteine, antioxidants, oxidative stress coronaviruses are tiny viruses with diameter ranging between and nm, whose name derives from the spikes projected from their surface;in transmission electron microscopy they resemble to solar corona. their genetic material is single-stranded rna and they are divided into subgroups, namely alpha (α), beta (β), gamma (γ) and delta (δ). the first two typically infect humans and the others mostly birds [ ] . coronaviruses mainly cause infections of respiratory tract that may be either mild or lethal. two recent outbreaks in several countries worldwide due to coronaviruses have been reported. the first was observed in because of the severe acute respiratory syndrome coronavirus (i.e., sars-cov or sars-cov- ) and the second ten years later caused by the middle east respiratory syndrome coronavirus (i.e., mers-cov). the third and most recent outbreak for which the severe acute respiratory syndrome coronavirus (i.e., sars-cov- ) is responsible, is ongoing throughout the world.it started in wuhan, the capital city of hubei province in china, with patients reporting symptoms of anatypical pneumonia, in december . this outbreak has now reached a global fatality rate of over reported deaths, likely an underestimated number, and fatalities rise rapidly and continuously.although the virus was initially named as -novel coronavirus ( -ncov) by who, it has been renamed as sars-cov- virus and the disease as the coronavirus disease (covid- ). more and more data support the role of excessive immune activation as the cause of lung destruction by sars-cov- [ - ] . although there is no sex-based skewing to contract the viral infection, there appears to be a skewed mortality towards elderly men with underlying diseases [ - ] in the current pandemic. it is known that pulmonary immunity in elderly persons is diminished/impaired, with inadequate innate and adaptive cellular immune responses and reduced function of the lung itself. however, that does not mean that the innate cellular sensing machinery of infected lung epithelial cells is decreased [ ] . in fact, while overall innate immune responses may decline with age, inflammatory cytokines such as il- and tnf-a, and acute phase reactants such as c-reactive protein have been shown to be elevated in elderly, maintaining a low level of chronic inflammation, known as inflammaging which is also accompanied by immunosenescence [ , ] . inflammaging is associated with increased levels of oxidative stress which drive the sustained levels of inflammation [ , ] and impairment of t cell activation in aged individuals,resulting in increased severity of viral infections in elderly individuals. this phenomenon was particularly apparent in previous outbreak of sars with a mortality of > % in individuals > years of age compared to % survival in individuals younger than years old [ ] . in the perspective of the innate immune response towards coronavirus infections we must understand the virus life cycle. coronaviruses are ssrna viruses and sars-cov- infects alveolar epithelial cells via the receptor ace [ ] , triggering innate response mechanisms that alert the immune system. after introduction of viral proteins and rna into the cytoplasm of the host cells, components known to interact with the host proteins and affect cell metabolism are inserted to the cytoplasm which can lead toinduced stress responses [ ] . as most viruses, sars-cov- have been shown to modulate the cellular anti-viral interferon (ifn) responses.viral proteins can either interact directly with the nlrp inflammasome in macrophages or,in cells lacking nlrp ,the viral sensing machinery can be modulated by viral protein aggregates in the cytosol [ ] [ ] [ ] .. there is also a recent report ofa dysfunctional interferon response in critically ill patients during the current pandemic [ ] .it has been shown that corona-derived viral protein deposits lead to endoplasmic reticulum (er) stress and mitochondrial dysfunction in the affected epithelial cells [ ] . hence, while the viral-derived proteins suppress innate sensing machineries such as toll-like receptor (tlr) induced innate responses, the inflammasome can independently trigger a stress-induced reactive oxygen species (ros) production via the redoxhomeostasis sensing machinery and mitochondrial machinery leading to for example il- production [ ] . in support of a potential role of oxidative stress and inflammaging to the pathology of sars-cov , previous studies have indicated that increased chronic oxidative stress through lipid peroxidation lead to enhancement of pla g d expression, a secretory phospholipase a precursor. pla g d expression was shown to be increased in the lungs of middle aged mice, resulting in decreased survival and impaired t cell responses upon infection with sars-cov [ ] . interestingly, nac administration to aged mice, diminished plag d expression in both lung cells and cd c+ dcs. in addition, increased levels of oxidized phospholipidsare a common feature associated with acute respiratory distress syndrome (ards) caused by viruses including sars and h n . notably,in an experimental mouse system that mimics the initial phases ofardsusing inactivated h n lung challenging, ros levels were increased in alveolar macrophages.this led to the formation of oxidized phospholipids and activation of tlr that exacerbated il- production [ , ] and lung injury. lung injury in response to h n was impaired by usage of mice that lack ncf , a major component of nadph oxidase. to maintain normal homeostasis and counteract ros responses described above, cells rely on a balanced redox status together with an effective dna damage repair machinery. the nicotinamide adenine dinucleotide (nad) + is a crucial electron transporter in mitochondrial respiration and oxidative phosphorylation, and is also the sole substrate for poly (adp-ribose) polymerase (parp), responsible for adp ribosylation, an important step in dna repair. upon viral infection, cells sense the ssrna virus and host-viral interactions occur in the cytoplasm of the infected cell that also affect the dna repair system. nad + is generated from ingested tryptophan by the kynurenine pathway and there is both a hepatic (tryptophan , -dioxygenase (tdo)) and extra-hepatic (indoleamine , -dioxygenase (ido)) nad + generating pathway. the ido driven pathway can be triggered by immune activation via ifnᵧ release, in both immune and non-immune cells at local inflammation sites. also, many of the parp-family membersare regulated by ifnᵧ driven cellular responses [ ] . the half-life of nad + is min- h depending on the tissue, and the liver secretes the precursor nicotinamide (nam), which is taken up by the organs and transformed into nad + in the cytoplasm [ ] . nad + levels are decreasing during increased oxidative stress conditions including aging [ ] .coronaviruses reportedly have the capability to reverse adp ribosylation driven by parp, thereby counteracting the host-virus defense system [ ] . as discussed above, the viral infection will also lead to mitochondrial stress inside the alveolar epithelial cells, which increase ros accumulation and skew the redox balance in the exposed cells. in a recent not yet peer reviewed data, transciptomic analysis, in the lungs of a diseased individual from covid- , showed that both specific members of the parp superfamily and the nad biosynthetic pathways are modulated by the infection [ ] . interestingly, the nsp protein of sars-cov was shown to directly interact with the enzymes of the oxido-reductase system and result in loss of inner mitochondrial potential which regulates release of ros [ , ] .if genetic or environmental factors can impact the anti-oxidant power and if this has an impact on the sars-cov- mortality remains to be shown. in addition to the oxidative stress induced by increasing ros production, ros have also been reported to activate the stat/il- axis [ , ] , spiraling cytokine release and immune cell infiltration in the lung as a result. young healthy individuals most likely have a redox homeostasis in balance [ ] , which is better equipped to respond to acoronavirus infection. some will experience signs of ards, but severe cases are few [ , ] . we hypothesize that with age, the gradual decrease in the capability of maintaining redox homeostasis will increase the risk of excessive immune activation and lung damage in response to a viral infection as has been documented now with sars-cov- / infections [ ] . innate immune responses to sars-cov viruses as positive ssrna viruses differ to negative ssrna based influenza viruses in how they trigger ifn pathways, how the viruses replicate in the cell, accumulation of protein aggregates inside the cell, as well as their mechanism to avoid ifn activation [ , , ] . it will be of great importance to compare innate and adaptive immune responses of sars-cov and the seasonal influenza strains, to develop optimal patient care for young and elderly in future pandemics. it should be noted that the severe clinical symptoms of sars-cov commonly arise first after a week after onset of symptoms, or even later, when virus titers commonly decline. a theoretical explanation for this is that the virus with time build up toxic protein aggregates within the cells [ ] . this overload of aggregates could be the reason for the toxic stress response in the epithelial cells and acute ros release. of interest is that ros production hamper the proteasome function, which leads to impaired protein degradation and further negatively influence mitochondrial function [ ] [ ] [ ] [ ] , this negative spiral triggered by accumulation of viral proteins in the cytoplasm over time can become toxic to the cell. there is an urgent need to further understand the redox homeostasis in a corona viremia. we propose that in-depth investigations for the therapeutic use of anti-oxidant pharmaceutical interventions, such as n-acetylcysteine (nac), or other applicable antioxidant therapies, as early pharmacological interventions. it may be equally important to consider how use of antibiotics can pose a risk of mitochondrial integrity [ ] or proteasome function ,to further aggravate ros induced tissue damage. in countries with an overuse of antibiotics, an increase mortality of sars-cov- may be linked to antibiotic-j o u r n a l p r e -p r o o f induced cellular dysfunction, which may be relieved by introducing n-acetylcysteine in the care of sars-cov- patients [ ] . a number of studies have analyzed of the use of nac as preventive or therapeutic interventionfor respiratory tract infections, and while there is not clear effect on mortality, several reports indicate a shortening of the duration of intensive care unit days along with support of use in a disease preventivemanner [- - ] . future trial designs should consider early use of nac in risk groups,or alternativepatient selectionstrategies, to assess if nac can reduce ards incidences in risk groups or patients with predisposing risks of developing ards. interestingly, dietary nac has been shown to reverse the enteropathogenic effects of the coronavirus porcine epidemic diarrhea virus (pedv) [ ] , a disease that causes large economic losses through the high mortality of the affected pigs. in this study there were also indications of therapy reduced systemic oxidative stress symptoms, as indicated by decrease in plasma and mucosal h o levels [ ] . in another study, nac could inhibit h n infection of lung epithelial cells in vitro and production of pro-inflammatory mediators [ ] . there is clearly an urgent medical need for measures against the devastating pandemic covid- with respect to both treatment of the disease and the further dissemination of sars-cov- . with respect to the latter, major efforts involving many companies and academic institutes are currently ongoing to develop vaccines [ ] , but it may still take a while before clinical studies and production have reached a stage of global vaccine coverage. here, we suggest that early treatment in the form of nac should be evaluated a cost-effective intervention for virus-infected patients with symptoms of lung dysfunction, to provide each patient with the best protection against excessive ros production which negatively impact lung integrity, but also to handle drug-induced ros during severe viremia. pharmacological intervention using nac dosing aims to normalize the redoxhomeostasis, as ros production is also part of the normal innate anti-viral host response, when in balance. the administration route should also be evaluated as both oral, infusion as well as 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an overview key: cord- -u r ip authors: laforge, mireille; elbim, carole; frère, corinne; hémadi, miryana; massaad, charbel; nuss, philippe; benoliel, jean-jacques; becker, chrystel title: tissue damage from neutrophil-induced oxidative stress in covid- date: - - journal: nat rev immunol doi: . /s - - - sha: doc_id: cord_uid: u r ip the high neutrophil to lymphocyte ratio observed in critically ill patients with covid- is associated with excessive levels of reactive oxygen species (ros), which promote a cascade of biological events that drive pathological host responses. ros induce tissue damage, thrombosis and red blood cell dysfunction, which contribute to covid- disease severity. we suggest that free radical scavengers could be beneficial for the most vulnerable patients. covid- is caused by the betacoronavirus sars-cov- and has several unique features compared with other coronavirus infections. in the most vulnerable individuals (for example, older, obese or diabetic individuals), the virus sometimes triggers a cascade of acute biological events that can, unfortunately, lead to patients being ventilated and even dying. a far from negligible number of patients require intensive care, and although most hospital stays are short in duration, this places a huge strain on health systems. it is therefore urgent to gain an in-depth understanding of the critical activators of disease severity in order to reduce mortality and hospitalization rates. the high neutrophil to lymphocyte ratio reported in critically ill patients with covid- has been found to predict in-hospital mortality . lung autopsies of deceased patients have revealed neutrophil infiltration in pulmonary capillaries, their extravasation into the alveolar spaces and neutrophilic mucositis . increased levels of circulating neutrophil extracellular traps (nets), which are indicative of neutrophil activation, have also been described in patients . oxidative stress is the result of an imbalance between oxidant production and antioxidant mechanisms that leads to oxidative damage, including lipid peroxidation and dna oxidation. in addition to the neutrophil infiltration and release of reactive oxygen species (ros), viral infections are associated with a decrease in antioxidant defences. exposure to pro-oxidants usually leads to nuclear translocation of the master redox-sensitive transcription factor nrf , which activates antioxidant defences; however, respiratory viral infections have been associated with inhibition of nrf -mediated pathways and nf-κb signalling activation, which can promote inflammation and oxidative damage during these infections . furthermore, there is evidence of a link between decreased expression of the antioxidant enzyme superoxide dismutase (sod ) in the lungs of elderly patients with covid- and disease severity . interestingly, children -whose neutrophils are less reactive and adherent, with no alteration of redox balance -are less prone to developing severe forms of covid- . the cascade of events triggered by the oxidative stress state in sars-cov- infection undoubtedly contributes to the severity of host disease and needs to be further explored. we postulate that, particularly in vulnerable individuals, neutrophilia generates an excess of ros that exacer bates the host immunopathological response, resulting in more severe disease (fig. ) . the deleterious action of ros on the functions of both pulmonary cells and red blood cells (rbcs) can be seen as a major contributor to the hypoxic respiratory failure observed in the most severe cases of covid- . thus, in addition to its damaging effects on alveolar epithelial and endothelial cells with pro-coagulative endotheliitis , an excess of ros can also affect the rbc membrane and haeme group functionality. neutrophils usually initiate aggressive responses upon encountering danger signals, which leads to their rapid migration to the targeted tissue, release of nets, and their production and release of ros in an oxidative burst. it had been assumed that neutrophil migration from the vascular lumen into extravascular tissues is unidirectional, but recent studies have demonstrated that neutrophils can migrate back into the bloodstream, in a process referred to as neutrophil reverse trans endothelial migration (rtem) . rtem neutrophils are rela tively rigid cells, and this physical characteristic may delay their passage through the tissue's microvasculature and prolong contact with the sinusoids. they may become mechanically entrapped in the microvasculature of major organs, thus contributing to distant organ damage and multiorgan failure. by producing excessive ros, deregulated neutrophils can spread a local inflammatory response so that it becomes systemic, which explains why they have been involved the high neutrophil to lymphocyte ratio observed in critically ill patients with covid- is associated with excessive levels of reactive oxygen species (ros), which promote a cascade of biological events that drive pathological host responses. ros induce tissue damage, thrombosis and red blood cell dysfunction, which contribute to covid- disease severity. we suggest that free radical scavengers could be beneficial for the most vulnerable patients. in whole-body processes such as atherosclerosis and thrombosis . improper activation of neutrophils is also a potential explanation for the diffuse microvascular thrombosis and capillary leak syndrome observed in critically ill patients with covid- (ref. ). in addition, excessive ros production may affect membrane lipids, integrins and cytoplasmic proteins in various circulating cells. these effects are particularly critical for rbcs, which may become dysfunctional. first, excess ros can cause oxidation of polyunsaturated fatty acids in the rbc membrane, bringing about a profound modi fication of the membrane lipids' lateral and transversal distribution and organization at the nanoscale level. this results in biophysical and biomechanical changes in the rbc membrane that affect both the diffusion of oxygen and carbon dioxide and the deformability capability of rbcs in the capillary vessels, thereby favouring thrombocytosis. reactivation of neutrophils in response to modification of the rbc membrane further fuels this vicious circle. in addition, this modification affects the release of atp and nitric oxide, both necessary for adequate oxygen transport and vasodilatation between metabolizing tissues and respiratory surfaces. second, ros excess may upset the fe + /fe + balance and disturb iron homeostasis for which iron must be kept in the fe + state to bind oxygen. the protonation of superoxide ion associated to fe + within the haemoglobin haeme keeps the iron in its higher oxidation state and incapable of binding oxygen, resulting in less efficient oxygen transport despite a high oxygen supply. in conclusion, the presence of oxidative stress markers (for example, lipid peroxidation, rtem and a high neutrophil to lymphocyte ratio) in patients with covid- may help to identify high-risk individuals early in the course of the disease and prevent their sudden deterioration. this approach may also pave the way to new therapeutic approaches. we propose that anti oxidants such as n-acetyl-l-cysteine in combination with elastase inhibitors (for instance, sivelestat) could be used to target rtem neutrophils in patients with severe covid- . the clinical implication of dynamic neutrophil to lymphocyte ratio and d-dimer in covid- : a retrospective study in suzhou china targeting potential drivers of covid- : neutrophil extracellular traps harnessing innate immunity to eliminate sars-cov- and ameliorate covid- disease respiratory viral infections and subversion of cellular antioxidant defenses is low alveolar type ii cell sod in the lungs of elderly linked to the observed severity of covid- ? covid- : the vasculature unleashed neutrophil migration in infection and wound repair: going forward in reverse tissue factor-positive neutrophils bind to injured endothelial wall and initiate thrombus formation confirmation of the high cumulative incidence of thrombotic complications in critically ill icu patients with covid- : an updated analysis combination of sivelestat and n-acetylcysteine alleviates the inflammatory response and exceeds standard treatment for acetaminophen-induced liver injury we thank martin deotto for his valuable help in drafting the graphic summary and franck brouillard and christophe bernard for their careful reading of the manuscript. the authors declare no competing interests. fig. | sars-cov- infection can lead to neutrophilia-induced ros release. a | in not at-risk individuals, an excess of reactive oxygen species (ros) is counterbalanced by an increase in antioxidant defences. b | in subjects with impaired redox balance, ros production is not properly controlled, leading to red blood cell (rbc) membrane peroxidation, which in turn perpetuates neutrophil activation. excessive oxidative stress might be responsible for the alveolar damage, thrombosis and rbc dysregulation seen in covid- . anti-oxidants and elastase inhibitors may have therapeutic potential. key: cord- -bbjxd y authors: xia, tian; zhu, yifang; mu, lina; zhang, zuo-feng; liu, sijin title: pulmonary diseases induced by ambient ultrafine and engineered nanoparticles in twenty-first century date: - - journal: natl sci rev doi: . /nsr/nww sha: doc_id: cord_uid: bbjxd y air pollution is a severe threat to public health globally, affecting everyone in developed and developing countries alike. among different air pollutants, particulate matter (pm), particularly combustion-produced fine pm (pm( . )) has been shown to play a major role in inducing various adverse health effects. strong associations have been demonstrated by epidemiological and toxicological studies between increases in pm( . ) concentrations and premature mortality, cardiopulmonary diseases, asthma and allergic sensitization, and lung cancer. the mechanisms of pm-induced toxicological effects are related to their size, chemical composition, lung clearance and retention, cellular oxidative stress responses and pro-inflammatory effects locally and systemically. particles in the ultrafine range (< nm), although they have the highest number counts, surface area and organic chemical content, are often overlooked due to insufficient monitoring and risk assessment. yet, ample studies have demonstrated that ambient ultrafine particles have higher toxic potential compared with pm( . ). in addition, the rapid development of nanotechnology, bringing ever-increasing production of nanomaterials, has raised concerns about the potential human exposure and health impacts. all these add to the complexity of pm-induced health effects that largely remains to be determined, and mechanistic understanding on the toxicological effects of ambient ultrafine particles and nanomaterials will be the focus of studies in the near future. air pollution is a major global problem associated with human health in both developing and developed countries [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the most common sources of air pollution include particulate matter (pm), ozone, nitrogen dioxide and sulfur dioxide [ , , , ] . pm can be defined by their different size ranges, including coarse (< μm), fine (< . μm) and ultrafine particles (ufps, < nm) [ ] [ ] [ ] ] . because numerous studies have shown that fine pm . particle levels can be linked to premature mortality and adverse health effects, pm . levels are often used as a major surrogate for air pollution [ , [ ] [ ] [ ] [ ] , ] . according to the world health organization (who) ambient air pollution database that comprises urban air-quality data for about cities from countries for the years - , almost % of the urban population endured a pm . concentration exceeding the annual mean values of μg/m in the who air-quality guidelines [ ] . in addition, who reported that, in , around million people died-one in eight of the total global deaths-as a result of air pollution exposure [ ] . the new data revealed a stronger link between both indoor and outdoor air pollution exposure and the development of respiratory diseases, including acute respiratory infections, chronic obstructive pulmonary disease (copd) and lung cancer, as well as cardiovascular diseases such as strokes and ischemic heart disease [ , , ] . this finding further confirmed that air pollution is now the world's largest single environmental health risk factor and reducing air pollution could save millions of lives. globally, we are witnessing two opposing trends in terms of changes in the air pollution levels in different countries. using new high-resolution global review xia et al. satellite maps of air quality indicators, nasa scientists tracked air pollution trends from to in various regions and cities globally [ ] . according to the research findings, the usa, europe and japan have improved air quality owing to strict emission-control regulations. as a result, in the usa, reductions in pm . were associated with lower premature mortality and improvement in life expectancy. long-term improvements in air quality were associated with statistically and clinically significant positive effects on lung-function growth in children [ ] . on the contrary, china, india and the middle east countries, with their rapid population growth, fast-growing economies and vastly expanding industrialization, have shown alarming increases in air pollution, along with increased mortality and disease burden in those countries [ ] . on a positive note, in recent years, china's overall air quality has seen early signs of improvement due to a decrease in coal consumption, increase in renewable energy sources and stricter emission control. it is worth noting that, although air pollution mostly is considered a local or regional problem, studies have shown that air pollution could be transported over long distances, even across continents, in the troposphere [ ] . thus, mitigation of local air pollution could have far-reaching health benefits and a collective effort between every country involved would be important. despite the progress in our understanding of air pollution-induced diseases, much research remains to be done [ , , , , ] . for instance, among the pm fractions, ufps possess the highest particle number and surface area, carrying higher chemical contents than pm . [ , [ ] [ ] [ ] [ ] . studies have shown that ufps have detrimental effects on both the respiratory and cardiovascular systems, and exacerbation of asthma [ ] [ ] [ ] , , ] . however, our understanding of ufps is still incomplete because of a deficiency in extensive ufp-monitoring networks, rapid physicochemical characterization techniques, and limited epidemiological and toxicological studies. in addition, the rapid advances in nanotechnology lead to production of high volumes of nanomaterials and increasing use in commercial products [ , , ] . the use of nanomaterials increases the potential for human exposure to nanomaterials that could generate adverse health effects. for instance, ceria (cerium dioxide) are used in diesel as a catalyst to promote the combustion process, which are released in the diesel exhaust, potentially leading to lung exposure. the addition of ceria to diesel fuel resulted in ceria-concentration-dependent emission reductions of co , co, total particulate mass, formaldehyde, acetaldehyde, acrolein and several polycyclic aromatic hydrocarbons; however, it also led to decreases in the size of emitted particles and a substantial increase in the number of ufps (+ %), together with increases in certain other air pollutants, specifically nox (+ . %) and the particle-phase benzo[a]pyrene toxic equivalence quotient (+ %) [ ] . this shows the complexity of the potential impacts introduced by nanomaterials. given the health concerns related to ufps and increasingly produced and used nanoparticles, further research is needed to evaluate the health risks associated with these tiny particles. evidence that links ambient pm to a decrease in life expectancy and increase in premature mortality came from epidemiological studies that have been extensively reviewed before [ , , , , [ ] [ ] [ ] . in a recent study, chen et al. presented findings that implicated long-term exposure to air pollution particles contributed to enormous loss of life expectancy in china [ ] . these results were based on an experimental design making use of a chinese policy that provided free coal for heating in cities located north of huai river, but not in the south, which produced an arbitrary discontinuity for pm air pollution, where the major difference was coal combustion. as a result, mean life expectancy is about . years ( % conficence interval (ci): . , . ) lower in northern compared with southern china due to an increased incidence of cardiorespiratory mortality [ ] . this finding correlated well with a study by pope et al. that used a temporal difference in pm levels observed since the s, when air quality across cities in the usa improved substantially. they found that there was an association between reductions in pm . and an increase in life expectancy; a reduction of μg/m was associated with an increase of . years in life expectancy [ ] . there was also a strong evidence base for morbidity and mortality associated with both short-term (days to weeks) and long-term (years to decades) pm exposures. early evidence linking ambient pm to mortality came from welldocumented short-term extreme air pollution episodes (that lasted for days) in the s to s [ ] . more recently, numerous daily time-series and case-crossover studies have observed a small but statistically robust relationship between daily mortality and short-term (days to weeks) elevation in pm [ ] . in addition, short-term air pollution exposure could also increase the mortality rate of patients with respiratory diseases. for example, cui et al. evaluated air pollution using the air pollution review figure . the contribution of outdoor air pollution sources to premature mortality on a global scale. the data were derived from a global atmospheric chemistry model that links premature mortality to outdoor air pollution, mostly by pm . . study found that, in , outdoor air pollution, mostly by pm . , leads to . million premature deaths per year worldwide, predominantly in asia. unit of mortality was expressed as deaths per area of km × km (color-coded). in the white areas, annual mean pm . and o are below the concentration-response thresholds where no excess mortality is expected. the figure was originally published by [ ] and has been approved for reuse by nature publishing group. index (api) derived from the concentrations of particulate matter, sulfur dioxide, nitrogen dioxide, carbon monoxide and ground-level ozone and their relationship with the case fatality of severe acute respiratory syndrome (sars) in china [ ] . case fatalities of patients from regions with high apis (api > ) and moderate apis ( - ) were compared with that of patients from regions with low apis (api < ). the study showed that the case-fatality rate increased with the increment of api (case fatality = - . + . × api). the correlation coefficient between api and sars fatality was . (p = . ) [ ] . short-term exposure demonstrated that sars patients from regions with moderate apis had an % increased risk of dying from sars compared with those from regions with low apis (relative risk (rr) = . , % ci: . - . ). similarly, sars patients from regions with high apis were twice as likely to die from sars compared with those from regions with low apis (rr = . , % ci: . - . ). for long-term studies, two large-scale prospective cohort studies in the usa showed that there were statistically robust associations between mortality risk and pm . exposure even after smoking and other risk factors were controlled for [ , ] . long-term air pollution exposure could also increase the mortality rate of patients with respiratory diseases such as sars [ ] . although ecologic fallacy and uncontrolled confounding effects might have biased the results, the possibility of an effect of air pollution on the prognosis of sars patients was indicated [ ] . in a recent study, lelieveld et al. used a global atmospheric chemistry model to investigate the link between premature mortality and ambient pm . concentrations [ ] . the authors found that more than . million deaths per year could be attributed to outdoor pm . exposure. the majority of the mortality happened in asia, which strongly influenced the global mortality rate. the highest number of deaths was in the western pacific, where china was the main contributor ( . million per year). southeast asia had the second highest premature mortality, where india was the main contributor ( . million per year) ( fig. ) [ ] . this was in addition to the estimated . million deaths per year caused by indoor air pollution resulting from biomass or coal combustion for cooking and heating [ ] . the cause of premature mortality includes lung diseases, such as chronic obstructive pulmonary disease (copd), lung infection and cancer (described in detail below), as well as cardiovascular diseases and cerebrovascular diseases, etc. [ ] . in addition, many birth cohort studies have linked pm . exposure to asthma and allergic diseases [ , ] . altogether, a large amount figure . general toxicological pathways linking pm lung exposure to cardiovascular and cerebrovascular diseases that cause morbidity and mortality. the first line of defense against pm is the lung, where pm can induce or exacerbate lung diseases including copd, asthma, lung infection disease and lung cancer. furthermore, ufps could translocate out of the lung into the blood stream and can cause systemic inflammation and oxidative stress that negatively impact blood and blood vessel, heart function and brain. of literature provided evidence that breathing combustion-related pm . , even at exposure levels common to urban populations worldwide, contributed to cardiorespiratory disease mortality and diminished the lifespan [ , , , , ] . there was also encouraging evidence showing that improvement in air quality benefited human health and increased the lifespan [ , ] . for example, long-term improvements in air quality were associated with a significant improvement in lung function in children [ ] . however, there are no conclusive data yet available for the human health impacts of ambient ultrafine particles and engineered nanoparticles, which warrant further studies. the lung is the first target organ for air pollution and pm exposure is associated with reduced lung function, increased lung inflammation, asthma, respiratory infections, lung cancer and exacerbation of copd, which lead to systemic inflammation and oxidative stress affecting blood, vasculature, heart and brain, ultimately contribute to the premature mortality ( fig. ) [ , , , ] . it is notable that the acute adverse health effects of pm are most often found in susceptible populations, including children, elderly people and those with chronic diseases [ , ] , while not obvious for short-term exposure in normal healthy people except at very high concentrations [ ] . however, air pollution exposure could induce adverse health effects to normal healthy people [ ] . a panel study conducted among chinese healthy adults during the beijing olympics found that peak expiratory flow levels increased in % of the participants when compared with the during-and pre-olympics time points, while peak expiratory flow levels decreased in % of participants for the post-and during-olympic periods comparison [ ] . children are more susceptible to air pollution because they have smaller airways, higher breathing rates per body mass, immature detoxification and metabolic systems, and they are more frequently exposed to outdoor air [ , ] . the elderly population and people with chronic diseases are susceptible likely because of less efficient particle clearance in the lung or impairment of immune functions. the adverse effects induced by air pollution are not limited to review the lung, pm could induce systemic inflammation and oxidative stress, and nano-sized particles could even translocate out of the lung to other tissues and organs, which induces pathological changes in blood, vasculature, heart and brain ( fig. ) [ , ] . the main pulmonary diseases that are associated with pms are the following. copd is not only associated with smoking, but also has high incidence among non-smokers [ , , ] . the risk factors for copd among non-smokers include indoor air pollution from biomass combustion and second-hand tobacco smoke, as well as occupational exposures and outdoor air pollution [ ] [ ] [ ] . epidemiological studies from both developing and developed countries have shown the association between outdoor pm exposure and copd. in addition to pm, ozone and no could also exacerbate copd. an increase in ambient pm . could induce acute exacerbations and mortality from copd, while improvement in air quality decreases the incidence of copd [ ] [ ] [ ] . abundant evidence has shown that air pollution could induce and exacerbate asthma [ ] . in a recent meta-analysis of several birth cohort studies, the authors suggested that early childhood exposure ( - years old) to traffic-related air pollution (trap) containing ufps and pm . were associated with increased incidence of asthma and risk of sensitization to common allergens [ ] . furthermore, a -year longitudinal study of , adults from eight european countries showed an association between trap exposure and increased incidence of asthma [ ] . the authors found that adult asthma incidence was positively associated with exposure metrics, including pm , pm . , nitrogen oxides, traffic load and intensity. further research with improved personal-level exposure assessment (vs. residential exposure assessment only) and phenotypic characterization was recommended by the authors [ ] . in october , the international agency for research on cancer (iarc) of who announced that outdoor air pollution and pm were classified as a group i carcinogen, which was determined based on the most recent data from human, animal and mechanistic studies [ ] . in one of the first studies of lung cancer among female non-smokers in china with measured indoor exposure to pm , pm . , pm , pm and total suspended particulate (tsp), mu et al. reported that the pm levels in cases were more than three times higher than those in the controls. every μg/m increase in pm is associated with a % increased risk of lung cancer [ ] . multiple studies in recent years have shown a correlation between air pollutants including pm . and no and lung cancer risk [ , [ ] [ ] [ ] . in alone, , deaths from lung cancer worldwide were associated with air pollution [ ] . recent data showed that air pollution was associated with respiratory infections, due to the impaired immune functions of the lung and the susceptible population including children, elderly people and those with chronic diseases [ , ] . two recent epidemiological studies demonstrated associations between air pollution (trap and ozone) and acute respiratory infections as well as increased emergency department visits in young children [ , ] . the most common sources of air pollution include pm, ozone, nitrogen dioxide and sulfur dioxide [ , , ] . among these components, pm has been shown to play a major role in human morbidity and mortality [ , , ] ; thus, we will focus our discussion on pm. pm comes from natural and anthropogenic activities and processes [ , , , , ] . anthropogenic sources of outdoor pm are hightemperature processes (e.g., welding, smelting), combustion (e.g., power generation, land traffic, residential and commercial heating and cooking), industrial and other processes, including agriculture, dust and biomass burning. indoor pm sources are mostly biomass and coal combustion for cooking and heating purposes [ , , , , ] . although the physiochemical properties (chemical composition, metal content, etc.) of pm are different worldwide, they are all associated with adverse human health effects although at different potency levels [ ] . pm is referred to the mass of particles collected with % efficiency for particles with an aerodynamic diameter equal to or less than μm; it should be noted that all particles down to the ultrafine size range were collected [ ] . at this size range, according to the international standards organization thoracic convention, the mass fraction of inhaled particles could penetrate beyond the larynx to the airways [ ] . pm . refers to the respirable fraction . tem images of ambient ultrafine particles and engineered nanoparticle as well as particle characteristics that promote or contribute to ros generation. (a) photoactivation effects of nps, such as the formation of electron-hole pairs during ultraviolet exposure of tio ; this effect has been associated with the generation of oxidative stress and inflammation by tio ; (b) discontinuous crystal planes and material defects of nps that lead to oxygen radical generation due to the active electronic state of the material surface; (c) redox cycling contributes to ros production. this can occur due to the presence of transition metals or redox-cycling organic chemicals on the pm, ufp and np surfaces. pms and ufps, for example, contain organic compounds such as quinones, which can generate ros through redox cycling. moreover, transition metals can generate hydroxyl radicals through the fenton reaction. the fenton reaction is one of the mechanisms by which metal impurities on the cnt surface can induce ros production. finally, (d) particle dissolution (e.g. zno, cdse, cu) can produce free ions that are capable of inducing ros generation and oxidative stress in cells. metal fume fever is a real-world example of this toxicity, commonly for welders. that also contains the ultrafine component, which penetrates to the unciliated regions of the lung and is now being considered worldwide as the standard [ ] . ultrafine particles are found to a large extent in urban air as both singlet and aggregated particles (fig. ) , and indeed are the predominant particle type by number in urban pm and pm . , although they contribute insignificantly to mass [ , , , ] . pm from combustion processes characteristically has an elemental or organic carbon core carrying trace metals, sulfate, ammonium, and volatile and semi-volatile components [ , ] . the composition of combustion-generated pm usually depends on fuel type, burn conditions and atmospheric conditions. compared with pm and pm . , traffic-derived ufps are challenging to characterize geographically or spatiotemporally, as their concentrations decrease sharply downwind from sources, and ufps shift in size from nucleation mode to accumulation mode with time and distance from their emission point due to agglomeration and condensation [ ] [ ] [ ] . for combustion sources, the fuel, combustion conditions and pollution controls will alter the particle numbers and size distribution of the pm emitted [ , ] . however, studies have shown that ufps carried more organic chemicals due to their significantly larger surface area; many of these compounds were redox-active and had the ability to generate reactive oxygen species (ros) [ , ] . studies have shown that ufps were more toxic than their larger counterparts; however, more research is needed to clarify the role of ufps [ , , ] . nanoparticles (nps) are intentionally created with specific size, shape, surface characteristics and functionality that are required for their applications [ , ] . there are similarities between nps and ufps; however, there are also major differences (table ) . nps and pm including ufps could both generate ros or release toxic ions through dissolution, through similar or different mechanisms (fig. ) [ , , ] . although currently there is no definitive evidence to link np exposure to any human disease, much experimental data indicate that some nps with certain physicochemical properties may be potentially hazardous [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . obviously, more research is needed on this front. the potency of pm in causing an adverse health impact is dependent, in part, on its deposition in the airways and chemical composition [ , ] . the main deposition mechanisms in the respiratory tract for inhaled pm include impaction, sedimentation, interception and diffusion [ ] . pm in different size ranges has drastic differences in distribution and deposition in the lung [ ] . this is clearly demonstrated in fig. , which shows the differential deposition of inhaled particles with different sizes in the nasopharyngeal, tracheobronchial and alveolar regions of the human respiratory tract [ ] . for ufps and nps that are in the size range of - nm, the particles could penetrate deeper into the alveolar region and deposit there at high percentages. for larger particles including pm and pm . , they generally deposit in the nasopharyngeal and laryngeal region and poorly deposit in the alveolar region (fig. ) [ ]. the different deposition profiles among particles with diverse sizes could at least partially explain their different health effects [ , ] . for example, the deposition site determines the clearance rate of particles [ , ] . for large particles including pm and pm . , the majority deposited in the upper airways could be removed with the mucus via the mucociliary escalator (fig. ) . the small frac-tion of particles that could pass through upper airways into the alveolar region (fig. ) , such as pm . fine particles, could be easily endocytosed and removed by alveolar macrophages (fig. ) . however, for ufps and nps, the majority of these particles could pass through the airways into the alveolar region. although they also deposit in the larger airways, this only accounts for a small percentage of review xia et al. figure . scheme for physicological and toxicological events occurring after pm . and ufp/np exposure. pm . mostly sticks to the airway, where they are cleared by mucociliary escalator. the remaining pm . particles that get inside the alveoli will be phagocytosed by resident macrophages. however, for ufps and nps, the majority of the particles will get inside the alveoli, where the cellular uptake of nano-sized particles by macrophages is impaired, leading to delayed clearance of particles and generating oxidative stress and pro-inflammatory effects by macrophages and epithelial cells because of prolonged exposure to particles. the prolonged interaction between particles and the epithelium also lead to extrapulmonary translocation to the lung interstitium and even into the circulation. adapted from [ ] . the total number of particles in the lung. furthermore, the particles are so tiny and in such vast numbers that macrophages could not take them up effectively (fig. ) [ , ] . the interactions among particles, epithelial cells and macrophages could generate oxidative stress and pro-inflammatory effects in the lung, and there are reports showing that nano-sized ufps could be more toxic than their larger counterparts (which will be discussed in later sections) [ , , , ] . in addition, extrapulmonary translocation across the epithelium could occur because of the reduced removal rate and longer retention of ufps and nps that allows transcytosis of these nano-sized particles [ , ] . this could lead to secondary deposition of these particles in other tissues and organs, which may contribute to further adverse health effects (fig. ) [ , ] . emerging evidence has shown that, among different particles, ufps are potentially the most dangerous owing to their small size, deep penetration, large surface area/volume ratio, high content of redox-cycling organic chemicals, alveolar deposition and high rates of retention in the lung [ , , ] . compared with pm and pm . , traffic-derived ufps are capable of carrying more organic chem-icals on their significantly larger surface; many compounds (polycyclic aromatic hydrocarbons (pahs) and quinones) are redox-active and have the ability to generate ros [ , [ ] [ ] [ ] , , ] . while pm and pm . can be easily removed by phagocytosis, the extremely small size of ufps enables them to evade such a process and ufps could have more interactions with other cell types in the lung (fig. ) [ , ] . these specific features of ufps can significantly contribute to the adverse effects through ros over-production by the redox-active organic chemicals and metals on particle surface, resulting in cellular oxidative stress [ , , , , ] . oxidative stress has been identified as a major mechanism for pm -, pm . and ufp-associated health effects, including exacerbation of asthma and copd, and promotion of atherosclerosis [ , , , , , ] . some nps, including carbon nanotubes, silver nanoparticles, zno, cuo, etc., have also been shown to be able to generate ros and oxidative stress [ , , , , , ] . at the cellular level, particleinduced oxidative stress can activate a cascade of signaling pathways that mediate the production of pro-inflammatory cytokines/chemokines and induce apoptosis (figs and ) [ , , ] , resulting in inflammation and tissue injury in the respiratory and cardiovascular systems [ , , ] . pm-induced ros production in biological systems and targeted cells originates from a variety of sources ( fig. ) [ ] [ ] [ ] [ ] , , ] . these include: (i) carbon core of pm and ufps could induce ros generation and oxidative stress; (ii) catalytic conversion of pahs to quinones by cytochrome p in the endoplasmic reticulum; (iii) quinone redox cycling by nadph-dependent p reductase in microsomes; (iv) mitochondrial perturbation leading to electron leakage in the inner membrane; and (v) nadph oxidase activity in the macrophage surface membrane and associated phagosomes. both the carbon cores as well the chemicals coated on their surface play a role in these biological events [ , , ] . this also includes the involvement of transition metals in generating ros by a fenton reaction [ , , ] . the pm backbone is formed by chain-like carbon-based nanoparticles. to see whether the particles themselves could induce ros and adverse effects, ultrafine carbon black was selected as the model particle. studies found that ultrafine review figure . sources of pm-and ufp-induced ros production and their cellular effects. quinones, under the catalytic influence of nadph-cytochrome p reductase, can redox cycle to produce ros in the endoplasmic reticulum. phagocytosis can induce the assembly and activation of nadph oxidase to produce superoxide. ufps can interfere in electron transduction in the mitochondrial inner membrane as well as perturb the pt pore to generate ros. ros induce lipid peroxidation in the cell membrane, cross-linking of protein thiol (sh) groups and dna damage. ros can also deplete glutathione (gsh), resulting in oxidative stress in the cell. depending on the levels of oxidative stress, the response could range from induction of nrf release to the nucleus, activation of mapk and nf-kb signaling cascades, or cytotoxicity. according to the hierarchical oxidative stress hypothesis, nrf interaction with the antioxidant response element (are) leads to heme oxygenase and other phase ii enzyme expression at lower levels of oxidative stress (tier ), while, at a intermediary level of oxidative stress, activation of the mapk and nf-kb signaling cascades can induce pro-inflammatory responses (e.g. cytokine and chemokine production) (tier ). at the highest oxidative stress level (tier ), ros can induce the opening of the mitochondrial pt pore, followed by cytochrome c release, caspase- activation and induction of programmed cell death. carbon black could generate ros in cell-free systems and cause oxidative stress to cultured cells [ , , ] . in addition, ultrafine carbon black caused systemic pro-inflammatory responses in the lung including modest neutrophil influx and protein leak [ , ] . there was evidence of systemic oxidative stress in the plasma and increased production of plasma factor vii, which is an independent risk factor for cardiovascular disease [ , ] . redox-cycling organic chemicals on pm surfaces, such as quinones, are capable of generating ros in cellular targets such as bronchial epithelial cells, macrophages and endothelial cells [ , , ] . quinones are byproducts of fossil fuel combustion, as well as the enzymatic conversion of pah in the lung. redox-cycling quinones undergo one-electron reductions mediated by nadph-cytochrome p reductase to form semiquinones. these semiquinones are metastable and donate electrons to o , leading to the formation of o •− . due to their high content of organic chemicals, ambient ufps contribute proportionally more redox-cycling chemicals than larger particles [ , , , ] . catalytically active metals adsorbed on the pm surface have been shown to contribute to oxidative stress in vitro and in vivo [ , , ] . pm contains a number of transition metals (coarse > fine > ufp) that contribute to ros production. among the transition metals, fe, al, cu, ni, mn, zn, cr, ba and sr are the most abundant [ ] . in the presence of hydrogen peroxide, some of these metals, including fe + , have the ability to generate the hydroxyl radical ( • oh) through catalysis of a fenton reaction: the • oh radical is more reactive than o •− and hydrogen peroxide by several orders of magnitude. besides ros generation, transition metals also have the ability to directly perturb the function of the mitochondrial permeability transition (pt) pore [ , , ] . transition metals may also act synergistically with other pm components in impacting mitochondrial function, ros generation, atp production and cell viability [ , , ] . pm components could perturb mitochondrial function, leading to generation of ros and cell death [ , ] . mitochondria have been shown to be a direct subcellular target for ambient ufps. for example, ambient ufps have been found to lodge in the mitochondria of target cells and cause mitochondrial damage [ ] . although the mechanism of mitochondrial localization is still elusive, we hypothesize that particle size, hydrophobicity and presence of organic chemicals may play a role in the process. in addition to direct effects, studies have found that organic pm chemicals are capable of generating ros by their ability to interfere in these electron transfer events. this includes the effect of polar chemicals such as redox-cycling quinones to disrupt electron transfer in the inner membrane [ ] . this disruption in electron flow could favor the formation of ubisemiquinones, thereby contributing to mitochondrial o •− production. in addition, organic chemicals such as quinones and pahs also have the capability to perturb the mitochondrial pt pore (fig. ) [ , ] . the pt pore is a redox-, ph-, calcium-and m -dependent protein complex, which plays a pivotal role in regulating mitochondrial function and controlling cellular apoptosis. opening of the pt pore could lead to mitochondrial swelling and rupture of the mitochondrial outer membrane, by which various proapoptotic proteins such as cytochrome c, smac, apoptosis-inducing factor (aif), etc., are released . potential mechanisms of pm-, ufp-and np-induced nlrp inflammasome activation and il- β production. activation of nlrp inflammasome complex can be induced by various stimuli including pm and np particles, and the pro-il- β is processed by the inflammasomes to produce mature il- β, while il- β plays a major role in inducing the pro-inflammatory and pro-fibrogenic effects in the lung. nlrp inflammasome activation requires two signals in vitro. for signal , pathogen-associated molecular patterns (pamps) such as lps is recognized by toll-like receptor (tlr ) residing on the cell membrane, which further leads to nf-κb activation and the production of pro-il- β and nlrp proteins. nlrp inflammasome activation mechanisms include ros generation, potassium efflux, lysosomal damage and cathepsin b release. after phagocytosis of pm, ufps and nps, nadph oxidase is activated to generate ros. the over-production of ros may cause the destabilization and permeabilization of lysosomes and cathepsin b release, which will initiate the inflammasome activation cascade. mitochondrion is another important source of ros in cells. over-production of mitochondrial ros could lead to release of mtdna and cardiolipin that could induce nlrp inflammasome activation. in addition, potassium efflux induced by particles could also induce mitochondrial and cellular ros production leading to nlrp inflammasome activation. levels of activated nlrp inflammasomes are tightly regulated by autophagy. into the cytosol where they activate a number of apoptotic pathways, ultimately leading to programmed cell death [ , , , , , ] . pm-, ufp-and np-induced oxygen radical generation can result in cellular and tissue injury responses such as inflammation, apoptosis, necrosis, fibrosis and carcinogenesis [ , , , ] . cellular oxidative stress can generate a wide range of responses that can be experimentally detected, including damaged dna-mainly dna single-strand breaks or generating -oxo- -deoxyguanosine ( oxo-dg) that promote cell turnover and proliferation [ ] . at the lowest level of oxidative stress (tier ), the induction of antioxidant and protective responses is mediated by the transcription factor nrf , which regulates the activation of the antioxidant response element in the promoters of phase ii genes [ , ] . as the levels of oxidative stress increases (tier ), this protective re-sponse may yield to pro-inflammatory responses because ros induces redox-sensitive signaling pathways such as the mitogen-activated protein kinase (mapk) and nuclear factor-kappa b (nf-κb) cascades [ , , , , ] . at the highest level of oxidative stress (tier ), a perturbation of mitochondrial inner membrane electron transfer and the open/close status of the pt pore can trigger cellular apoptosis and cytotoxicity. this outcome is also known as toxic oxidative stress (fig. ) [ , , , , ] . using this three-tier screening platform, we have also shown for ambient airpollution particles that one can link the hierarchical oxidative stress paradigm to the in vivo outcomes in animal disease models [ , , , , ] . oxidative stress is also involved in mutagenesis [ ] . future screening could also include dna damage and mutagenesis tests to assess their carcinogenic potential. nps are intentionally created with specific size, shape, surface characteristics and functionality that are required for their applications [ , , , , , ] . although currently there is no definitive evidence to link np exposure to any human disease, experimental data indicate that many nps may be potentially hazardous [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , , , ] . the physicochemical characteristics of nps that may have health implications include particle size, shape, aspect ratio, composition, surface reactivity, solubility and ability to generate ros [ , , ] . similar to ufps, the nano-scale size can enhance np translocation and deposition by interfering with their clearance [ , ] . np composition or modification may affect particle surface reactivity such as the ability to generate ros, which is an important mediator involved in various human diseases. however, ufps and nps are inherently different in many aspects, such as source, morphology, organic chemical content, homogeneity, ros production, possible exposure route and the potential adverse health effects (table ) . extensive in vitro and in vivo studies have been performed to elucidate the toxic effect of engineered nanomaterials (enms) and physicochemical properties of enms, including morphology, size, dissolution, aspect ratio, surface coating, surface reactivity, bandgap and aggregation, have been suggested to determine their toxicity potentials [ , , , , ] . for example, ceo has excellent antioxidant properties that are enabled by the duality of the cerium ion to easily cycle review between ce + and ce + ; it is widely used as a catalyst or fuel additive in diesel to facilitate the combustion process and is released into the air as exhaust. there are reports over the past few years that utilize the antioxidant property of ceo in treating diseases such as cancer, alzheimer's, cardiac arrest, radiation-induced cell death and aging [ ] . however, a recent study found that ceo nps could lead to a decrease in cell viability and the treated cells exhibited characteristic hallmarks of apoptosis [ ] . in this case, ceo np toxicity is caused by mitochondrial damage leading to aif release, but not caspase activation or ros production. moreover, ceo np exposure leads to autophagy and inhibition of autophagy partially reverses cell death by ceo nps [ ] . obviously, more research should be done to clarify the effects of ceo under in vitro and in vivo conditions. tio is the most widely produced nanomaterial and is found in cosmetics, sunscreen, paint, vitamins, toothpaste, food colorants, nutritional supplements, etc. however, study has shown that tio nanoparticles could cause oxidative stress-mediated acute lung inflammation. oberdorster et al. have shown that, on a mass-dose basis, ultrafine tio is more toxic than fine tio [ ] . however, when the doses of particles were expressed in forms of particle surface area, the responses of ultrafine and fine tio particles fell on the same dose-response curve. this suggests that surface area is an important property for nps when considering their toxic potential. also, there is a study showing that tio in anatase form was more potent in ros generation than in rutile form, revealing the important role of crystal structure [ ] . under low levels of ultraviolet light, tio nanoparticles could generate ros and induce cytotoxicity because of their photoactivation property [ ] . another example is zno nanoparticles, which have received significant attention due to their wide use in sunscreens, electronics, optics and photonics. however, pulmonary exposure of zno nanoparticles could lead to transient increases in acute lung inflammation, similarly to a disease called metal fume fever. it has been shown that the toxicity of zno is dependent on the particle dissolution property and shedding of toxic zn ions, which induce oxidative stress and pro-inflammatory effects in vitro and in vivo [ , , ] . in addition to oxidative stress paradigm, recently studies have found nod-like receptor protein (nlrp ) inflammasome activation plays a major role in pm-and np-induced chronic effects such as lung fibrosis [ ] [ ] [ ] , , , [ ] [ ] [ ] [ ] . nanomaterials including rare earth oxides (reos), high-aspectratio materials including carbon nanotube (singlewalled and multi-walled), tio nanobelts and ceo nanorods, d materials including graphene and graphene oxide could induce nlrp inflammasome activation and a series of events leading to epithelial mesenchymal transition and lung fibrosis. carbon nanotubes (cnts) are a type of long-aspectratio nanomaterial which is drawing wide interest because of their potential applications in electronics, optics, drug delivery and cancer therapy. however, animal studies have shown that they could promote the production of pro-fibrogenic cytokines and growth factors (il- β, tgf-β and pdgf-aa, etc.) that may lead to lung fibrosis [ ] [ ] [ ] [ ] . the mechanism involves cellular uptake of cnts, overproduced ros by nadph oxidase activation, lysosomal damage induced by the surface reactivity of cnts and the release of lysosomal protein, cathepsin b, which activates nlrp inflammasome and produces il- β. il- β plays a major role in inflammation and fibrosis by activating epithelial mesenchymal transition in the lung (fig. ) [ , [ ] [ ] [ ] [ ] . wang et al. showed that the dispersal state, hydrophobicity and purity of multi-walled carbon nanotubes (mwcnts) could affect the pro-fibrogenic cellular responses that also correlate with the extent of pulmonary fibrosis [ ] [ ] [ ] [ ] . furthermore, other long-aspect-ratio enms also showed similar effects. ji et al. demonstrated the toxicological effects of long-aspect-ratio nanomaterials using a library of inhouse-made ceo nanoparticles [ ] . in vitro toxicity study demonstrated that, at lengths ≥ nm and aspect ratios ≥ , ceo nanorods induced progressive pro-inflammatory effects and cytotoxicity [ ] . the relatively low 'critical' length and aspect ratio were associated with small nanorod/nanowire diameters ( - nm), which facilitates the formation of stacking bundles that could pierce through cell membrane, causing the release of cathepsin b and further the activation of nlrp inflammasome. the same ceo nanorods could also induce significantly higher lung fibrosis than spherical particles in vivo [ ] . our recent studies show that rare earth oxide nanoparticles (reos), which are widely used in electronics and upconversion nanoparticles for bioimaging, are unstable in the acidic physiological environment, including the lysosomal compartment of the cells [ ] [ ] [ ] . these reos can dissolve and the dissolved ions could bind to phosphates strongly; as a result, the particles transform from spheres to 'sea-urchin'-shaped or mesh-like structures with composition changes to the repo . the sources of phosphates are not limited to free phosphate ions in the lysosomal compartment; they also include phosphate groups on intracellular proteins and membranes. on the one hand, biotransformation of reos could lead to dephosphorylation of phospholipids on the lysosomal membrane, which destabilizes the membrane, leading to lysosomal damage. released cathepsin b from lysosomes to the cytosol review xia et al. will activate nlrp inflammasomes to produce il- β, which initiate a cascade of events that culminate in pulmonary fibrosis [ , , ] . on the other hand, stripping of phosphate groups from lysosomal proteins will lead to loss of enzymatic activities of the proteins, resulting in lysosome dysfunction, which leads to lysosome and autophagosome fusion inhibition and compromised autophagosome degradation in the autophagy flux. this leads to accumulation of activated nlrp inflammasomes because autophagy is the major homeostatic mechanism to remove activated inflammasomes [ ] . the above examples show that the physicochemical properties of nps determine their toxic potential to human health, and more research is needed to understand these interactions between numerous other types of nps and biological systems occurring at the nanobio interface [ , , , , ] . studies have shown that exposure to ufps and nps has the potential to cause adverse health effects in humans [ , , , , , ] . in order to assess the toxic effects of these nano-sized particles, we advocate a predictive toxicological approach with the goal of linking particle physicochemical properties to their toxic effects [ , , ] . to establish this predictive toxicological paradigm, there are five major requirements. the first is to comprehensively characterize their physicochemical properties that may lead to biological injury. it also requires the establishment of nanomaterial libraries with property variations, which allows building the link between material property and the biological/toxicological activities. the second requirement is to develop in vitro cellular screening assays that reveal particleinduced injury mechanisms and pathways such as oxidative stress and nlrp inflammasome activation. third, where possible, it is important to develop high-throughput screening platforms to assess the large number of material physicochemical properties, dosage and time points that are likely to lead to biological injury. fourth, the in vitro data could be used for in silico modeling to establish hazard ranking and structural-activity relationships that can be used to predict enm toxicity. finally, the in vitro hazard ranking and structural-activity relationships will then need to be validated by limited in vivo animal experiments to establish the 'predictiveness' of this approach. we have successfully demonstrated the usefulness of the predictive toxicological approach for the hazard assessment of over different nano-materials and development of major structure activity relationships for groups of nanomaterials. we are confident that this approach would facilitate research on the toxicological effects induced by pms, ufps and nps [ ] . convincing evidence has established the association between pm and many pulmonary diseases that contribute to early mortality and reduced life expectancy. however, for ambient ufps and nps, although much progress has been made in understanding their toxicological effects and mechanisms of toxicity, there are still many knowledge gaps on their impact on human health. in this review, we have summarized recent major findings on cellular, animal and human research with a focus on the respiratory effects and mechanisms of toxicity induced by pm, ufps and nps. available evidence strongly suggests that ufps and nps may be more potent in causing adverse health effects in humans because of their high deposition rate in the alveolar region, impaired clearance by alveolar macrophages and higher surface reactivity, pro-oxidative and proinflammatory effects than their larger counterparts. thus, it is imperative to focus future research on the health effects of nano-scale pollutants so that preventive strategies and regulatory guidelines can be developed to reduce exposure and improve human health. an association between air pollution and mortality in six u.s. 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sublethal effects key: cord- -m ko yal authors: rouco, lara; gonzález-noya, ana m.; pedrido, rosa; maneiro, marcelino title: pursuing the elixir of life: in vivo antioxidative effects of manganosalen complexes date: - - journal: antioxidants (basel) doi: . /antiox sha: doc_id: cord_uid: m ko yal manganosalen complexes are coordination compounds that possess a chelating salen-type ligand, a class of bis-schiff bases obtained by condensation of salicylaldehyde and a diamine. they may act as catalytic antioxidants mimicking both the structure and the reactivity of the native antioxidant enzymes active site. thus, manganosalen complexes have been shown to exhibit superoxide dismutase, catalase, and glutathione peroxidase activities, and they could potentially facilitate the scavenging of excess reactive oxygen species (ros), thereby restoring the redox balance in damaged cells and organs. initial catalytic studies compared the potency of these compounds as antioxidants in terms of rate constants of the chemical reactivity against ros, giving catalytic values approaching and even exceeding that of the native antioxidative enzymes. although most of these catalytic studies lack of biological relevance, subsequent in vitro studies have confirmed the efficiency of many manganosalen complexes in oxidative stress models. these synthetic catalytic scavengers, cheaper than natural antioxidants, have accordingly attracted intensive attention for the therapy of ros-mediated injuries. the aim of this review is to focus on in vivo studies performed on manganosalen complexes and their activity on the treatment of several pathological disorders associated with oxidative damage. these disorders, ranging from the prevention of fetal malformations to the extension of lifespan, include neurodegenerative, inflammatory, and cardiovascular diseases; tissue injury; and other damages related to the liver, kidney, or lungs. in , a research paper by melov et al. [ ] reported the extension of the lifespan of nematode worms (caenorhabditis elegans) by treatment with different manganosalen complexes which act as synthetic scavenger compounds. this study planned to test the theory that reactive oxidative species cause aging. in announcing this research, melov stated that "the results are the first real indication we have that aging is a condition that can be treated through appropriate drug therapy" [ ] . the research paper and this statement attracted extensive coverage in communication media, where these findings were presented as a sort of search for the elixir of life. the published results indicate that the two tested compounds (named as euk- and euk- ) increased the mean lifespan of the worms by percent over the control group and that treatment of prematurely aging worms resulted in normalization of their lifespan, which means a percent increase. however, the chosen nematode worm is a species that has cells in its body whereas humans have trillion, constituting one of the weaknesses of animal models for translational research [ ] . to investigate the protective activity of the manganosalen catalytic superoxide radical anion is a potential harmful species produced during respiration from the one-electron reduction of molecular oxygen. sod catalyzes the dismutation of two superoxide anions to yield hydrogen peroxide and molecular oxygen [ ] . sods are classified according to their metal ion cofactor as fe-sods, ni-sods, cuzn-sods, and mnsods [ ] . sod , also known as manganesedependent superoxide dismutase, is located within the inner mitochondrial matrix, the main site of free radical production from the electron transport chain [ ] , being pivotal in ros release in humans. the coordination environment around the manganese ion for human sod is depicted in figure . the superoxide disproportionation mechanism by sod involves a cyclic one-electron oxidation and reduction of the manganese ion between mn(ii)/mn(iii) oxidation states ( figure ). the catalysis occurs at a rate approaching the diffusion-controlling region (log ksod ⁓ m − s − ) [ ] . superoxide radical anion is a potential harmful species produced during respiration from the one-electron reduction of molecular oxygen. sod catalyzes the dismutation of two superoxide anions to yield hydrogen peroxide and molecular oxygen [ ] . sods are classified according to their metal ion cofactor as fe-sods, ni-sods, cuzn-sods, and mnsods [ ] . sod , also known as manganese-dependent superoxide dismutase, is located within the inner mitochondrial matrix, the main site of free radical production from the electron transport chain [ ] , being pivotal in ros release in humans. the coordination environment around the manganese ion for human sod is depicted in figure . the superoxide disproportionation mechanism by sod involves a cyclic one-electron oxidation and reduction of the manganese ion between mn(ii)/mn(iii) oxidation states ( figure ). the catalysis occurs at a rate approaching the diffusion-controlling region (log k sod~ m − s − ) [ ] . hydrogen peroxide is generated as a by-product of mitochondrial electron transport of aerobic respiration. as it has been mentioned, it is one of the products of the superoxide radical anion dismutation by sod. although h o is less reactive than superoxide, control of h o levels is also critical [ ] . catalase enzymes catalyze the decomposition of hydrogen peroxide to water and oxygen [ ] . the non-heme dinuclear mn catalase was isolated from bacteria. figure shows the core of the active site of this enzyme in lactobacillus plantarum [ ] . the catalase reaction proceeds via a two- hydrogen peroxide is generated as a by-product of mitochondrial electron transport of aerobic respiration. as it has been mentioned, it is one of the products of the superoxide radical anion dismutation by sod. although h o is less reactive than superoxide, control of h o levels is also critical [ ] . catalase enzymes catalyze the decomposition of hydrogen peroxide to water and oxygen [ ] . the non-heme dinuclear mn catalase was isolated from bacteria. figure shows the core of the active site of this enzyme in lactobacillus plantarum [ ] . the catalase reaction proceeds via a two-electron oxidation-reduction cycle during turnover ( figure ). hydrogen peroxide is generated as a by-product of mitochondrial electron transport of aerobic respiration. as it has been mentioned, it is one of the products of the superoxide radical anion dismutation by sod. although h o is less reactive than superoxide, control of h o levels is also critical [ ] . catalase enzymes catalyze the decomposition of hydrogen peroxide to water and oxygen [ ] . the non-heme dinuclear mn catalase was isolated from bacteria. figure shows the core of the active site of this enzyme in lactobacillus plantarum [ ] . the catalase reaction proceeds via a twoelectron oxidation-reduction cycle during turnover ( figure ) . peroxidases catalyze the oxidation of a broad range of substrates by hydrogen peroxide [ ] . hydrogen peroxide is then reduced by two electrons using glutathione (gsh) as a sacrificial reductant, yielding water and glutathione disulfide (gssg) (figure ). the manganese binding site of the manganese peroxidase is also shown in figure [ ] . peroxidases are not limited to h o as the substrate but also catalyze the conversion of organic peroxide to alcohol. thus, peroxidases can eliminate lipid hydroperoxides, another ros which contributes to the progression of disbalanced redox homeostasis [ ] . lipid hydroperoxides can be generated very easily in neuronal membranes rich in polyunsaturated fatty acids like arachidonic acid, docosahexaenoic acid, or eicosapentaenoic acid. peroxidases catalyze the oxidation of a broad range of substrates by hydrogen peroxide [ ] . hydrogen peroxide is then reduced by two electrons using glutathione (gsh) as a sacrificial reductant, yielding water and glutathione disulfide (gssg) (figure ). the manganese binding site of the manganese peroxidase is also shown in figure [ ] . peroxidases are not limited to h o as the substrate but also catalyze the conversion of organic peroxide to alcohol. thus, peroxidases can eliminate lipid hydroperoxides, another ros which contributes to the progression of disbalanced redox homeostasis [ ] . lipid hydroperoxides can be generated very easily in neuronal membranes rich in polyunsaturated fatty acids like arachidonic acid, docosahexaenoic acid, or eicosapentaenoic acid. the enzymatic antioxidant defense along with other nonenzymatic antioxidants act in an effective way against free radicals and other reactive oxygen species to control their damaging effects to macromolecules and body tissues. however, overproduction of ros leads to oxidative damage of cellular structures due to an imbalance in the oxidant-antioxidant status [ ] . ros excess may be derived from exogenous sources as mentioned above and by faulty regulation of cellular antioxidant defenses, normally associated with aging, that can lead to accumulation of toxic levels of ros [ ] . for this situation, a strategy to recover a balance between ros generation and removal is the use of the enzymatic antioxidant defense along with other nonenzymatic antioxidants act in an effective way against free radicals and other reactive oxygen species to control their damaging effects to macromolecules and body tissues. however, overproduction of ros leads to oxidative damage of cellular structures due to an imbalance in the oxidant-antioxidant status [ ] . ros excess may be derived from exogenous sources as mentioned above and by faulty regulation of cellular antioxidant defenses, normally associated with aging, that can lead to accumulation of toxic levels of ros [ ] . for this situation, a strategy to recover a balance between ros generation and removal is the use of antioxidants as therapy [ ] [ ] [ ] [ ] . administration of exogenous native antioxidant enzymes has not been successful for therapeutic treatment of oxidative stress because of several limitations [ ] : (i) short half-life of the enzymes; (ii) difficulty to enter in the cells due to their high molecular weight; (iii) antigenicity; and (iv) high-manufacturing costs. to overcome these limitations, pharmacological research has pointed at the development of low molecular weight sod/cat mimics [ ] [ ] [ ] [ ] . several supplements as α-tocopherol [ ] , ascorbic acid [ ] , carotenes [ , ] , or other organic molecules [ ] [ ] [ ] [ ] with antioxidant properties have been also used for the therapeutic treatment of oxidative stress-induced diseases. manganosalen complexes are coordination compounds with a chelate bis-schiff base ligand obtained by condensation of salicylaldehyde and a diamine. the acronym salen is due to the reactants used to synthesize the ligand n,n-bis(salicylidene)ethylendiamine, obtained by condensation of salicylaldehyde (sal) and ethylenediamine (en) [ ] . although the latter ligand is strictly speaking the salen ligand, actually the class of compounds known as manganosalen complexes encompasses other bis-schiff base ligands (containing two bisimine groups), obtained from different diamines (propylenediamine, phenylenediamine, butylendiamine, etc.) as well as the group of their derivatives with different substituents both in the phenyl rings and in the diamine spacer [ , ] . figure collects some manganosalen complexes with pharmacological relevance used in in vivo studies, corresponding to the euk series patented by eukarion. this type of ligand has oxygen and nitrogen donor groups, which decrease the mn(iii)/mn(ii) redox potentials upon coordination (e • = . v for [mn(h o) ] + , a rather oxidizing potential), and the resulting complexes constitute suitable systems to catalyze multiple redox reactions [ , [ ] [ ] [ ] [ ] [ ] . redox potentials of manganosalen complexes may be tuned by modifying features other than these substituents. in this sense, the number of ligands or the geometry around the metal ion are factors that influence the manganese redox potential of the final complex. for example, alkoxy substituents in the phenyl rings, particularly -methoxy or -ethoxy groups (see euk- , euk- , euk- , euk- , or euk- in figure ), lower the redox potentials for the manganosalen complexes due to the electron-donor character of these substituents, thus facilitating achievement of higher oxidation states for the manganese ion during enzymatic activity. the chelating bis-schiff base ligand forms a typical almost planar mnn o core where the -or -member chelate rings confer high stability. the introduction of different auxiliary ligands (halides, carboxylates, alcohols, dicyanamides, thiocyanates, etc.) alters the geometry of the compounds [ , ] , giving rise to different behaviors in catalysis. labile auxiliary ligands or solvent molecules in the axial positions favor catalytic activity through an inner-sphere electron transfer mechanism in which a vacant can be generated in this site, where the substrate molecule can be subsequently accommodated [ ] . a short two-carbon chain between the imine groups in the schiff base ligand constricts the chelate ring once the nitrogen atom coordinates to the metal, leading to tetragonally elongated geometries [ , [ ] [ ] [ ] [ ] . on the contrary, if the schiff base ligand has a flexible three-membered alkyl chain between the imine groups, a better stabilization of a high-symmetry octahedral symmetry is achieved and, subsequently, the generation of a coordination site gets difficult. thus, a correlation between the factor of the tetragonal elongation and catalytic activity as enzyme mimics has been reported for manganosalen complexes [ , [ ] [ ] [ ] [ ] . substituents. in this sense, the number of ligands or the geometry around the metal ion are factors that influence the manganese redox potential of the final complex. for example, alkoxy substituents in the phenyl rings, particularly -methoxy or -ethoxy groups (see euk- , euk- , euk- , euk- , or euk- in figure ), lower the redox potentials for the manganosalen complexes due to the electron-donor character of these substituents, thus facilitating achievement of higher oxidation states for the manganese ion during enzymatic activity. supramolecular interactions may play different crucial roles in enhancing the activity as enzyme mimics. on the one hand, hydrogen bonding and other supramolecular contacts may induce self-organization of the complexes to afford dimeric entities [ ] . thus, these supramolecular mechanisms may allow aggregation of the complexes into dimers once the monomers cross the cell membrane. on the other hand, supramolecular interactions also play an essential role in biological processes recognition [ ] . second-sphere effects of the substituents may also modulate the redox potentials of the metal center and the metal-ligand bond strength and may guide their reactivity with superoxide anion radical [ ] . alkoxy substituents on the phenyl rings can also participate in establishing supramolecular interactions through hydrogen bonding, which added to their mentioned effect on redox potentials could explain the high activity as enzyme mimics of alkoxy substituted manganosalen complexes [ ] [ ] [ ] . the relatively planar mnn o core of manganosalen complexes is somewhat similar to that of natural manganese mn-macrocycles or mn-porphirins, and this similarity is shown in their chemical reactivity. however, the manganese ion in manganosalen compounds is coordinated to oxygen and nitrogen atoms, which contrasts with porphyrins where the metal is coordinated to nitrogen atoms only. oxygen and nitrogen are the most common donor atoms in biological systems, so the structure of manganosalen complexes ( figure ) resembles those of various native manganoenzymes. manganosalen compounds are easily obtained from inexpensive precursors, and they are cheaper than mn-porphirins. on the other hand, supramolecular interactions also play an essential role in biological processes recognition [ ] . second-sphere effects of the substituents may also modulate the redox potentials of the metal center and the metal-ligand bond strength and may guide their reactivity with superoxide anion radical [ ] . alkoxy substituents on the phenyl rings can also participate in establishing supramolecular interactions through hydrogen bonding, which added to their mentioned effect on redox potentials could explain the high activity as enzyme mimics of alkoxy substituted manganosalen complexes [ ] [ ] [ ] . the relatively planar mnn o core of manganosalen complexes is somewhat similar to that of natural manganese mn-macrocycles or mn-porphirins, and this similarity is shown in their chemical reactivity. however, the manganese ion in manganosalen compounds is coordinated to oxygen and nitrogen atoms, which contrasts with porphyrins where the metal is coordinated to nitrogen atoms only. oxygen and nitrogen are the most common donor atoms in biological systems, so the structure of manganosalen complexes ( figure ) resembles those of various native manganoenzymes. manganosalen compounds are easily obtained from inexpensive precursors, and they are cheaper than mn-porphirins. one of the most interesting properties of the manganosalen complexes is that they are cellpermeable, with better bioavailability than exogenous antioxidant enzymes [ , ] . manganosalen complexes exhibit high sod, catalase, and peroxidase activities, which has led to their development one of the most interesting properties of the manganosalen complexes is that they are cell-permeable, with better bioavailability than exogenous antioxidant enzymes [ , ] . manganosalen complexes exhibit high sod, catalase, and peroxidase activities, which has led to their development as catalytic antioxidants [ ] [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] ] , a term used for all cases in which one single molecule of catalyst induces the detoxification of numerous ros molecules. malfroy et al. first reported the sod mimetic properties of manganosalen complexes [ ] . based on stopped-flow analysis combined with time-resolved uv/vis spectroscopy and global spectra analysis of superoxide decay, it has been reported that manganosalen complexes possess a sod activity of about × m − s − [ , ] , both in hepes and in phosphate buffers. the mechanism followed by the manganosalen complexes is similar to that of the native sod enzyme, a ping-pong mechanism where a superoxide anion radical reduces the synthetic manganese complex from mn(iii) to mn(ii), which is subsequently oxidizes back to mn(iii) by a second superoxide anion radical ( figure a ) [ , , ] . [ ] . based on stopped-flow analysis combined with time-resolved uv/vis spectroscopy and global spectra analysis of superoxide decay, it has been reported that manganosalen complexes possess a sod activity of about × m − s − [ , ] , both in hepes and in phosphate buffers. the mechanism followed by the manganosalen complexes is similar to that of the native sod enzyme, a ping-pong mechanism where a superoxide anion radical reduces the synthetic manganese complex from mn(iii) to mn(ii), which is subsequently oxidizes back to mn(iii) by a second superoxide anion radical ( figure a ) [ , , ] . while the sod activity of manganosalen complexes hardly varies with derivatization, their catalase activity is highly sensitive to the substituents on the aromatic rings or the length of the alkyl chain in the spacer between the imine groups [ ] , ranging from inactivity to high efficiencies in the hydrogen peroxide disproportionation [ ] [ ] [ ] [ ] ] . the efficiency of these systems seems to be related to the presence of at least one vacancy or a labile coordination position on the manganese core. while the sod activity of manganosalen complexes hardly varies with derivatization, their catalase activity is highly sensitive to the substituents on the aromatic rings or the length of the alkyl chain in the spacer between the imine groups [ ] , ranging from inactivity to high efficiencies in the hydrogen peroxide disproportionation [ ] [ ] [ ] [ ] ] . the efficiency of these systems seems to be related to the presence of at least one vacancy or a labile coordination position on the manganese core. electron donor substituents on the phenyl rings of the salen moiety also increase the catalase activity of manganosalen complexes [ ] . the rates at which manganosalen complexes scavenge hydrogen peroxide are similar to those reported for metalloporphyrins. for instance, compound euk- (see figure ) is reported to have a catalase rate greater than mm o /min [ ] , measured by monitoring the conversion of hydrogen peroxide to oxygen using a clark type oxygen electrode [ ] . the mechanism of these synthetic mimics is proposed to involve mononuclear mn(v)=o species [ , ] (figure b ), although some mimics may follow a mechanism through the formation of dimeric species in solution [ ] . anyhow, the mechanism is different from the native catalase enzyme shown in figure . peroxidase mimics are able to convert h o to h o and to scavenge other peroxides, including lipid hydroperoxides or other organic peroxides [ , ] , so that they could scavenge lipid peroxides in tissues. manganosalen complexes are proposed to behave as peroxidase mimics by a mechanism quite similar to that of their catalase action, through a mn(v)=o intermediate, which is able to oxidize an organic substrate (figure b ), to afford the initial catalyst complex. manganosalen complexes often have multiple antioxidant activities at the same time, showing, for instance, both sod and catalase functions. this reactivity against different ross is attractive since hydrogen peroxide is a product released by sod activity. the sod, catalase, and peroxidase activities shown by manganosalen complexes have attracted attention for their use as catalytic antioxidants. subsequent in vitro studies have confirmed their efficiency in oxidative stress models [ , [ ] [ ] [ ] [ ] [ ] [ ] , , ] and other references cited therein, although the focus of this review is on the evaluation of the in vivo studies with different animal models. these studies are organized below according to the pathology or the oxidative damage produced by ros. the brain is prone to oxidative stress as a result of the high levels of oxygen required, which represents % of oxygen uptake when brain accounts for only % of body weight. neurons consume a high rate of energy ( × atp/minute), meaning large amounts of oxygen to maintain neural intracellular ion homeostasis [ ] . moreover, neural membranes have high concentrations of polyunsaturated fatty acids which generate lipid hydroperoxides. additional factors, as the presence of auto-oxidizable neurotransmitters, increase the sensitivity of this organ to ros-mediated damage. excessive ros levels have been associated with neurodegenerative disorders like alzheimer's disease (ad), parkinson's disease (pd), amyotrophic lateral sclerosis (als), or huntington's disease [ ] . as previously mentioned, the euk- , euk- , and euk- models were injected to sod nullizygous mice to rescue them from oxidative neurodegenerative process [ ] . these compounds had previously shown efficacy in a variety of oxidative stress paradigms [ ] [ ] [ ] . for instance, euk- had been found to protect most of the vulnerable neurons from excitotoxic cell death in sprague-dawley rats [ ] . administration of these three manganosalen compounds extended the lifespan of the mice lacking sod by approximately threefold and eliminated clinical signs of the associated neurobehavioral phenotype previously described. this study also indicated that these catalytic antioxidants could cross the blood-brain barrier, particularly euk- , which is slightly more lipophilic than euk- . in a later study using the same animal model, melov et al. reported that neural cell death in defined regions of the frontal cortex of sod null mice is a consequence of endogenous mitochondrial oxidative stress [ ] . in this study, they could partially rescue neural cell death by treatment with a high dose of euk- . melov et al. also reported the therapeutic effects of euk- in preventing a neurodegenerative phenotype in an ah-transgenic mouse model for alzheimer's disease [ ] . this manganese complex and the cyclic analogue euk- were used in a study with c bl/ n sim middle-age mice [ ] , which usually exhibit a dramatic decrease in learning and memory function between and months of age, associated with oxidative protein damage in the brain [ ] . treatment during a -month period with euk- or euk- resulted in an almost complete reversal of age-related learning and memory deficit, showing a complete reversal in protein oxidation and a % reduction in age-related increase in lipid peroxidation. euk- exhibits longer plasma half-life than euk- and subsequently greater biological stability. two such molecules were also used by baudry et al. in older mice, at a lower dose, and for longer periods of time [ ] , preventing age-dependent cognitive decline in the same way as previously found for middle-aged individuals. their results indicate that the age-associated deficits in learning and memory might be originated by oxidative damage to hippocampus, amygdala, or both. euk- was the first manganosalen complex that showed efficacy for the treatment of a neurodegenerative disease using an animal model (see table ). malfroy et al. reported in [ ] how this synthetic catalytic scavenger reduces the severity of autoimmune encephalomyelitis in guinea pigs. watanabe et al. [ ] used the same compound to treat small bowel ischemia/reperfusion injury in sprague-dawley rats. they compared the protective effects of euk- and a mn(iii)-porphirin sod mimic, manganese-meso-tetrakis(n-methylpyridinium- -yl)porphyrin [ ] . both the manganosalen compound and mn-porphyrin showed similar beneficial properties by the inhibition of o , h o , and no production. xu et al. [ ] reported that euk- and euk- reduced the levels of oxidative stress and prolonged survival in a mouse amyotrophic lateral sclerosis model. in a later study, euk- afforded the best results compared to euk- in reducing brain infarct size after middle artery occlusion in a rat model [ ] . after the neuroprotective effects shown by euk- and euk- , doctrow et al. [ ] compared the in vivo antioxidant activity of different analogues, including the two cited compounds, euk- , euk- , euk- , euk- , euk- , and euk- in a rat stroke model. they concluded that alkoxy substituents on the phenyl rings for this series of compounds confers some advantage toward the biological protective effects of these manganosalen complexes. andersen et al. [ ] demonstrated the efficacy of euk- and euk- in protecting against paraquat-induced dopaminergic cell death in adult mice via inhibition of the activation of jun n-terminal kinases (jnk)-mediated apoptosis. paraquat is an herbicide that induces selective loss of dopaminergic neurons of the substantia nigra. these two manganosalen complexes significantly inhibited caspase- activation, cell death, and dna fragmentation in in vitro paraquat exposed rat cells. euk- was also employed in a mouse model of human prion disease [ ] , giving place to a modest % increase of survival compared to untreated disease controls. this beneficial effect was correlated with reductions in oxidative and, especially, nitrative damage to proteins. the same manganosalen complex, euk- , was used by levine et al. [ ] to treat the neurobehavioral defect in ataxia-telangiectasia mice, showing that this catalytic antioxidant corrected the neurobehavioral abnormality. this effect, that was reproducible over time, involved a reduction in the oxidation of brain fatty acids and a retarded development of thymomas. more recent studies were focused on the manganosalen euk- due to both its ability to suppress oxidative stress and its greater biological stability. thus, raber et al. [ ] reported that this compound mitigated radiation-induced cognitive impairments without affecting cognition of sham-irradiated mice. wang et al. [ , ] used euk- to treat hydrogen peroxide-induced dna damage and senescence phenotype in senescence-accelerated mouse-prone mice, reducing age-related loss of both hearing and hair cell degeneration. in their study, they found that cochleae cells treated with euk- displayed increased levels of foxo a and nrf , two transcription factors that have been previously shown to positively regulate cellular resistance to oxidative stress [ , ] . the neuroprotective effects shown by manganosalen complexes are close related to their ability to attenuate inflammation makers such as cytokines or chemokimes. high amounts of hydrogen peroxide activate a number of transcription factors as nfkb, ap- , and nrf [ ] , for which the levels may be regulated by the administration of manganosalen complexes. the anti-inflammatory effects of these catalytic antioxidants play a beneficial role not only in neurons or in the respiratory system but also in a wide range of pathologies related to inflammation ( table ) . hill et al. [ ] treated sprague-dawley rats with euk- to mitigate dna damage in rat lungs after exposure to - . gy doses of gamma rays. the catalytic antioxidant was effective at reducing micronucleus formation in lung fibroblasts, and this could be attributed to the ability of this compound to suppress the signal that would normally turn on the inflammatory response to repair radiation insult. euk- was used by lawler et al. [ ] as an ros scavenger in myopathy in the diaphragm of the mdx mouse model. this manganosalen complex reduced hydroperoxides, markers of oxidative stress in this muscle, attenuated both by the elevation of inflammatory cell invasion and by the nf-κb activity and p subunit protein levels in the mdx diaphragm. muzykantov et al. [ ] employed polyethylene glycol (peg)-liposomes loaded with euk- to alleviate acute pulmonary inflammation induced by endotoxin in mice. this manganosalen compound was also used by singh et al. [ ] to mitigate zinc-and paraquat-induced toxicity in rat polymorphonuclear leukocytes. other manganosalen compounds were used with efficacy against inflammatory episodes is euk- . hill et al. [ ] reported that this synthetic model mitigated the radiation-induced lung injury in -to -week-old fisher rats by scavenging ros and by reducing activity of the nfkb pathway. in this study, euk- also showed the ability to reduce levels of tgf-β expression, activated macrophages, and fibrosis. a study that deserves a more detailed analysis is the one carried out by kash et al. [ ] with euk- to reduce lung damage and to increase survival during influenza virus infection in mice. this pandemic, caused by the h n influenza a virus, infected about a third of the world's population of and resulted in - million deaths worldwide. this influenza and sars-cov- (severe acute respiratory syndrome coronavirus ) share some similarities in the way that they may lead to respiratory failure. many studies demonstrated that the severe lung pathology provoked by the influenza virus infection was associated with immunopathogenic immune response with excessive inflammatory and cell death responses [ ] . euk- treatment caused a marked reduction in the severity of lung pathology and substantially reduced cell death responses at both the rna and the protein levels. the beneficial effect of this manganosalen complex in an animal model affected by the influenza is related to its ability to reduce the most relevant cytotoxic effects of ros, thereby limiting excess cell death responses and allowing for increased lung repair responses. as the antioxidant effects of euk- regulate the response of the host, it could be a therapeutic alternative to other organism's infections compared to influenza. in this sense, it would be interesting to test this hypothesis with studies in animal models of the response to combined treatments of manganosalen complexes and antiviral against the sars-cov- virus. different previous studies showed the beneficial effects of manganosalen complexes to treat lung injuries. euk- was used by fink et al. to attenuate many of the features of lipopolysaccharide (lps)-induced acute lung injury in a porcine model [ , , ] by detoxifying ros without affecting the release of other important proinflammatory mediators like -keto-prostaglandin f alpha, thromboxane b , or tumor necrosis factor alpha. euk- was the antioxidant catalytic model chosen by kamp et al. [ ] to alleviate asbestos-and h o -induced damage in mice, limiting pulmonary fibrosis. hill et al. [ , ] reported the mitigation of radiation-induced lung injury by euk- in sprague-dawley rats. treatment with this manganese-model decreased hydroxyproline content, -hydroxy- -deoxyguanosine, malondialdehyde levels, and activated macrophages levels. lung levels of the cytokine transforming growth factor-β also decreased. medhora et al. [ ] reported the antioxidant effect of euk- to reduce pneumonitis and pulmonary fibrosis after thoracic irradiation in a rat model. the anti-inflammatory effect of other catalytic antioxidants, inspired by manganosalen complexes but not belonging to the euk series, has also been studied. a bioinspired manganese sod mimic, reported by policar et al. [ ] , demonstrated efficiency as an anti-inflammatory agent for c bl/ mice with dinitrobenzene sulfonic acid (dnbs)-induced colitis. inflammation and tissue damage are closely related to different pathologies, including some cardiovascular diseases. inflammation is common for heart disease and stroke patients. the role of excessive ros production during hemorrhagic shock and reperfusion injury has been well documented [ ] . in this way, some already cited studies of manganosalen complexes treatments for anti-inflammatory models also showed beneficial effects for cardiovascular diseases (table ) . for instance, the commented in vivo study of medhora et al. [ ] with euk- mitigated multiple vascular injuries in irradiated lungs. de windt et al. [ ] used euk- to reduce cardiac oxidative stress in harlequin mutant mice and their wild-type counterparts. the results of this study showed an improvement of the left ventricular end-systolic dimensions and a fractional shortening by using this manganese complex. euk- also attenuated necrotic and apoptotic cell death, prevented myocardial oxidant stress, and attenuated cardiac hypertrophy and fibrosis. euk- increased -day survival in irradiated mice according to a study by whitnall et al. [ ] . this manganosalen complex increased the number of diverse circulating blood elements like total white blood cells, lymphocytes, eosinophils, and platelets. the same catalytic antioxidant was tested by monsalve et al. [ ] to regulate ros homeostasis and to control the vascular endothelial cells function in mice. euk- restored endothelial growth factor-a signaling in peroxisome proliferator-activated receptor γ co-activator α (pgc- α), a process to be relevant in metabolic disorders where microvascular complications are frequent, like diabetic retinopathy. excessive ros appeared as key factor in the alteration of the endothelial growth factor-a signaling and in the capacity of endothelial cells to form stable interactions with other endothelial cells and with the extracellular matrix, but these alterations were partially reversed by administration of euk- . recent studies have shown the efficacy of other manganosalen complexes for the treatment of different cardiovascular models. yamada el al. [ ] reported that euk- prevented the force decrease and the actin modifications in pulmonary hypertension diaphragm bundles in wistar rats. in their study, they found that this manganosalen complex does not alter diaphragm contractile function in normal rats. lindner et al. [ ] evaluated the therapeutic effects of euk- in mice with age-dependent atherosclerosis. long-duration therapy ( weeks) with euk- almost completely suppressed plaque development and macrophage content in thoracic aorta of the treated mice compared with control mice. however, therapy for eight weeks did not affect the area or the macrophage content. in the same way as discussed for endothelial cells, any tissue, including the skin, can suffer oxidative damage both of inflammatory and non-inflammatory origins. several studies have focused on the protective effects of the manganosalen compounds in ros-mediated skin damage (table ) . skin is exposed to solar ultraviolet irradiation, ozone, smoke, and air pollution. all of them are environmental sources of ros that induce damage to lipids, proteins, and dna, playing a role in the skin aging process [ ] . euk- , euk- , and euk- were used by benichou et al. [ ] to delay the rejection of fully allogeneic skin transplants in mice. mice treated with euk- showed the longest skin graft survival and, along with euk- , exhibited the longest delays of graft rejection. the three manganosalen complexes reduced anti-donor cytotoxic responses in skin-grafted mice, and they decreased pro-inflammatory type alloresponse while promoted anti-inflammatory type alloimmunity. declercq et al. [ ] carried out a study of the protective effects of euk- on the human skin of healthy volunteers ( - years of age) over a period of years. euk- had been previously reported to increase cell survival in normal human keratinocytes upon exposure to ultraviolet-b, superoxide, or hydrogen peroxide [ , ] . in the study with the human volunteers, euk- (applied at a concentration of . - . %) reduced the level of skin surface lipid peroxidation in uva-exposed skin. noteworthily, the reduction of squalene hydroperoxide levels at the skin surface was found even when applying the antioxidant after uva exposure. as a consequence of this study, euk- is now commercially available as an antioxidant for the protection of dry or irritated skin. euk- was tested in a study performed by lazar et al. [ ] as a potential mitigating drug on end points relevant to radiation dermatitis, skin wound healing, and chronic oxidative stress in rats. the euk- -treated mice group showed reduced radiation dermatitis severity by days after irradiation and displayed significantly smaller wounds than vehicle-treated rats. this manganosalen complex also reversed and normalized the gene expression pattern in irradiated skin by reducing the oxidation of proteins and nucleic acids. the same compound was used by hill et al. [ ] to mitigate the radiation-induced dna damage and the lipid peroxidation in mice. they found that euk- provided some protection against dna damage only when delivered before irradiation. they also demonstrated significant protecting effects on radiation-induced lipid peroxidation at one or more of the three time points after local skin irradiation. pregnancy is a state of oxidative stress due to high metabolic activity in the fetoplacental compartment. regulation of ros during gestation is a complex process, whereas excessive oxidant levels cause biomolecules damage and leads to fetal malformations as a consequence of the attack by ros formed during the resumption of placental perfusion. on the other hand, the maintenance of a physiological level of oxidant levels is essential for governing life processes through redox signaling [ ] . two studies have been reported about the fetal protection or the reduction of pregnancy complications by manganosalen complexes. zhang et al. [ ] studied the effect of long-term high-altitude hypoxia (a severe lack of oxygen) during gestation in sheep. uterine arteries of pregnant sheep are affected by chronic hypoxia due to an inhibition effect of the large conductance ca + activated k + (bk ca ) channel activity by increasing oxidative stress. treatment of the pregnant sheep with euk- resulted in a mitigation of the hypoxia effects on bk ca channel currents in uterine arteries, alleviating pregnancy complications such as pre-eclampsia and fetal growth restriction. the same manganosalen complex was used by chen et al. [ ] to protect ethanol-induced limb malformations in mice. in vivo treatment with euk- resulted in diminished apical ectodermal ridge cell death as well as parallel reductions in the incidence and severity of limb defects in mouse fetuses (from . % to . %). the forelimb malformations were partially reversed by this manganosalen complex, including postaxial ectrodactyly, metacarpal, and ulnar deficiencies. since the imbalance between free radicals and antioxidants can be suffered by a variety of cells and issues, practically any organ can be affected, leading to a wide variety of pathologies. kidney and liver function can be altered by excessive ros, and again, manganosalen complexes appear as antioxidant therapeutic alternatives. kregel et al. [ ] used euk- to prevent age-related oxidative damage associated with environmental stress. they reported that this catalytic antioxidant blocked the activation of activator protein- (a redox-sensitive early response transcription factor involved in the regulation of cellular stress responses) and enhanced stress tolerance in aged animals by reducing cellular oxidative stress and subsequent accrual of hepatic injury in fischer rats. yazdanparast et al. [ ] reported the amelioration of diet-induced nonalcoholic steatohepatitis in rats by euk- and euk- . these two compounds had hepatoprotective, hypolipidemic, hypocholestorolimic, and hypoglycemic effects on the in vivo model. thus, the authors reported that euk- and euk- reduced the sera aminotransferases, the extent of lipid peroxidation, low density lipoprotein contents, cholesterol, and protein carbonylation. the same research group published another study about the protective effects of euk- , euk- , euk- , euk- , euk- , and euk- against ccl -induced damages in rats [ ] . the manganosalen complexes ameliorated the effects of ccl by decreasing the levels of ros, lipid and protein oxidations, and lipofuscin-like pigments formation on the liver and brain. euk- was used by ghouleh et al. [ ] to attenuate the vascular manifestations of sepsis in lipopolysaccharide-treated pigs. this catalytic antioxidant prevented the fall in renal blow flow, an effect associated with a decrease in nitrosative stress in the kidney supporting a renal protective effect. harman proposed that organisms age because they accumulate oxidative damage that comes from ros [ ] . his free radical theory of aging prompted investigations to look for therapeutic antioxidants to lifespan extension. although this theory was supported by later studies that demonstrate that increased production of ros shortens lifespan [ ] and that oxidative damage increases with age [ ] , other reports clearly contradict the basis of this theory [ ] . the aim of this review is not to assess this controversy but to present the results of the different tests in this regard. as already commented in the introductory section, melov et al. [ ] employed euk- and euk- to increase the mean lifespan of caenorhabditis elegans. on the contrary, hekimi et al. [ ] reported that increased oxidative stress caused by deletion of the mitochondrial superoxide dismutase sod- extended lifespan in the same nematode model. according to this latest study, decreased antioxidant function may extend lifespan. in vivo studies are sensitive to small changes in the environment, as gems et al. [ ] hypothesized to explain their results when they tried to reproduce the lifespan extension of the same nematode using euk- . since they did not find any increase in lifespan upon treatment with this sod mimetic, they conclude that this effect reported by melov et al. should be very sensitive to subtle differences in the manner in which the manganosalen complex is administered. their results raised doubts about the potential utility of euk- in the therapeutic attenuation of aging. a subsequent study by gems et al. [ ] tested euk- and euk- again in caenorhabditis elegans. in this study, they found that the synthetic mimetics elevated in vivo sod activity levels (increases -fold) and exhibited protection when the worms were treated with superoxide generators (paraquat and plumbagin). thus, the manganosalen complexes increased lifespan in nematodes compared to the control study, where superoxide levels were elevated, but they did not retard aging in the absence of superoxide generators. euk- , euk- , and caenorhabditis elegans met again in the study of lithgow et al. [ ] . they reported that these manganosalen complexes extended the lifespan of the worms and conferred resistance to two types of oxidative stress-inducing agents (paraquat and thermal stress). the protective effects of euk- and euk- were independent of insulin/igf-i signaling, and they did not show any detrimental repercussion on development or fertility. definitely, in vivo evaluation of these synthetic mimetics in aging studies involves careful consideration of complex concentration, complex delivery, and biotic environment. partridge et al. [ ] treated drosophila melanogaster, the fruit fly, with euk- and euk- , reporting the antioxidant protective effects to rescue pathologies associated with elevated oxidative stress in sod-deficient flies or normal flies exposed to induced oxidative stress. however, the synthetic antioxidants did not extend lifespan in normal, wild-type animals. in a different study, sohal et al. [ ] did not find lifespan extension administrating the euk- to musca domestica, both under normoxic and hyperoxic conditions. in general, the life expectancy of animal models subjected to oxidative stress or with pathologies associated with this stress is increased with treatment with manganosalen complexes, as reviewed in previous sections (table ). however, the use of these synthetic antioxidants in normal and healthy individuals probably does not have any advantageous effect on lifespan extension. throughout the previous sections, the protective effects of manganosalen complexes to combat oxidative stress and its associated pathologies have been presented and discussed. these compounds showed efficiency to reverse different oxidative damage: neurodegenerative, inflammatory, cardiovascular, adrenal and liver diseases, skin damage, and fetal malformations (figure ). the increase of lifespan has been also reported for organisms exposed to oxidative stress, although far from being any elixir of life. not all compounds have the same activity, as they show different lipophilicities, redox properties, or steric hindrance. reproduce positive results [ ] [ ] [ ] . the synthetic catalytic antioxidants approach may have a plus compared with nonenzymatic antioxidants: they may regulate ros by mimicking the mechanism of the native enzymes. finally, in vivo evaluation of pharmacological responses may be affected by multiple factors. moreover, sometimes, the results are simplified by describing them as beneficial or harmful when, as declared by paracelsus, the dose makes the medicine. ross play physiological roles in cell signaling and in the control of gene expression, processes that could be affected by antioxidant therapies. the catalytic oxidants should be administered just in the right dose to combat excessive ros levels. in vitro studies with different cell cultures have shown higher activities for manganosalen complexes at lower doses than those used in the in vivo tests [ , ] . the antioxidant activity observed of the nearly sixty in vivo trials, only a few of them did not give beneficial effects for the desired quality. thus, wada et al. [ ] reported that euk- was ineffective in promoting the restoration of prolonged low-frequency force depression, a state which may suffer skeletal muscles under vigorous activity. in this case, the antioxidant catalyst showed a positive effect on sarcoplasmic reticulum ca + release in wistar rats but a negative effect on myofibrillar ca + sensitivity. on the other hand, espósito et al. [ ] reported the toxicity of euk- ( - µm dose) toward danio rerio individuals (zebrafish), particularly in terms of brain damage. finally, vanfleteren et al. [ ] found that administration of euk- to starving escherichia coli cells surprisingly enhanced the production of ros, resulting in a massive increase of oxidative damage. much is still unknown about the way these manganosalen complexes work in different pathologies. drugs often have unrecognized effects, so we cannot be confident that the beneficial effects of these compounds were due solely to their antioxidant activities. more research is needed, particularly because of the properties showed by this type of compound that also varies with minor structural modifications. evaluation in animal models continues to be necessary, since it cannot be assumed that the in vivo antioxidant activity of different analogues is similar. in the search to identify the best candidate, an orally available one would be the most suitable. efforts should be directed towards obtaining an effective antioxidant, modulating its lipophilicity and redox potentials, that can be administered orally. despite the protective effect against ros shown by these compounds in oxidative stress models in vitro and in vivo, their practical application in humans remains highly challenge. clinical trials are crucial to assess the efficacy of this approach, especially after the results given by other antioxidants. thus, although coenzyme q , β-carotene, α-tocopherol, or other antioxidant supplements showed highly encouraging results in in vivo animal models, most of their clinical trials in humans failed to reproduce positive results [ ] [ ] [ ] . the synthetic catalytic antioxidants approach may have a plus compared with nonenzymatic antioxidants: they may regulate ros by mimicking the mechanism of the native enzymes. finally, in vivo evaluation of pharmacological responses may be affected by multiple factors. moreover, sometimes, the results are simplified by describing them as beneficial or harmful when, as declared by paracelsus, the dose makes the medicine. ross play physiological roles in cell signaling and in the control of gene expression, processes that could be affected by antioxidant therapies. the catalytic oxidants should be administered just in the right dose to combat excessive ros levels. in vitro studies with different cell cultures have shown higher activities for manganosalen complexes at lower doses than those used in the in vivo tests [ , ] . the antioxidant activity observed in cells can even decrease with increasing concentration [ ] , leading to curves that are not dose dependent. this behavior has been also reported in natural compounds such as curcumin or resveratrol, which present antioxidant effects at low doses but induce oxidative stress and cell death at high concentrations [ , ] . in this way, in vitro studies indicate that manganosalen complexes could also interact with other cellular pathways at high concentrations or with a receptor that could be suffering a threshold effect, that is, it would present higher affinity at low concentrations and would be desensitized at higher doses [ ] . in this regard, one of the challenges for the translation of these antioxidant synthetic catalysts to animal or human studies is the use of drug carriers to effectively reach the target site at the appropriate doses [ ] . interaction with nanocarriers or conjugation of simple manganese complexes to synthetic polymers or proteins represent current and future avenues of research to translate manganosalen complexes to clinical applications. the authors declare no conflict of interest. extension of life-span with superoxide dismutase/catalase mimetics the quest for human longevity: science, business, and public policy why animal studies are often poor predictors of human reactions to exposure lifespan extension and rescue of spongiform encephalopathy in superoxide dismutase nullizygous mice treated with superoxide dismutase-catalase mimetics no increase in lifespan in caenorhabditis elegans upon treatment with the superoxide dismutase mimetic 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ischemia/reperfusion injury in rats using an in vivo and an ex vivo electron paramagnetic resonance technique with a spin probe salen-manganese complexes are superoxide dismutase-mimics ivanovic-burmazovic, i. compartive studies on manganese-based sod mimetics, including the phosphate effect, by using global spectral analysis superoxide dismutase mimics: chemistry, pharmacology, and therapeutic potential catalase and glutathione peroxidase mimics delayed treatment with euk- , a novel synthetic superoxide dismutase (sod) and catalase (cat) mimetic, ameliorates acute lung injury in endotoxemic pigs enhanced catalase-like activity of manganese salen complexes in water: effect of a three-dimensionally fixed auxiliary catalase and epoxidation activity of manganese salen complexes bearing two xanthene scaffolds a new type of manganese-schiff base complex, catalysts for the disproportionation of hydrogen peroxide as peroxidase mimics salen-manganese complexes: combined superoxide dismutase/catalase mimics with broad pharmacological efficacy euk- , a synthetic superoxide dismutase and catalase mimetic, prevents oxidative stress and attenuates kainate-induced neuropathology prevention and suppression of autoimmune encephalomyelitis by euk- , a synthetic catalytic scavenger of oxygen-reactive metabolites endogenous mitochondrial oxidative stress: neurodegeneration, proteomic analysis, specific respiratory chain defects, and efficacious antioxidant therapy in superoxide dismutase null mice mice transgenic for alzheimer disease β-amyloid develop lens cataracts that are rescued by antioxidant treatment. free radic age-related losses of cognitive function and motor skills in mice are associated with oxidative protein damage in the brain reversal of age-related learning deficits and brain oxidative stress in mice with superoxide dismutase/catalase mimetics prevention of cognitive deficits and brain oxidative stress with superoxide dismutase/catalase mimetics in aged mice fridovich, i. the ortho effect makes manganese (iii) meso-tetrakis (n-methylpyridinium- -yl) porphyrin a powerful and potentially useful superoxide dismutase mimic synthetic superoxide dismutase/catalase mimetics reduce oxidative stress and prolong survival in a mouse amyotrophic lateral sclerosis model synthetic combined superoxide dismutase/catalase mimetics are protective as a delayed treatment in a rat stroke model: a key role for reactive oxygen species in ischemic brain injury superoxide dismutase/catalase mimetics are neuroprotective against selective paraquat-mediated dopaminergic neuron death in the substantial nigra a manganese-superoxide dismutase/catalase mimetic extends survival in a mouse model of human prion disease. free radic treatment with a catalytic antioxidant corrects the neurobehavioral defect in ataxia-telangiectasia mice. free radic mitigating effect of euk- on radiation-induced cognitive impairments presbycusis: an update on cochlear mechanisms and therapies decisions on life and death: foxo forkhead transcription factors are in command when pkb/akt is off duty activation of the nrf /antioxidant response pathway increases il- expression study of gene regulation by nf-kappa b and ap- in response to reactive oxygen intermediates partial volume rat lung irradiation: the protective/mitigating effects of eukarion- , a superoxide dismutase-catalase mimetic amplification of proinflammatory phenotype, damage, and weakness by oxidative stress in the diaphragm muscle of mdx mice. free radic endothelial targeting of liposomes encapsulating sod/catalase mimetic euk- alleviates acute pulmonary inflammation the manganese-salen compound euk- and n-acetyl cysteine rescue from zinc-and paraquat-induced toxicity in rat polymorphonuclear leukocytes mitigation of radiation-induced lung injury with euk- and genistein: effects in adolescent rats treatment with the reactive oxygen species scavenger euk- reduces lung damage and increases survival during influenza virus infection in mice. free radic aberrant innate immune response in lethal infection of macaques with the influenza virus euk- , a synthetic superoxide dismutase and catalase mimetic, ameliorates acute lung injury in endotoxemic swine role of oxidant stress in the adult respiratoy distress syndrome: evaluation of a novel antioxidant strategy in a porcine model of endotoxin-induced acute lung injury mitochondrial catalase overexpressed transgenic mice are protected against lung fibrosis in part via preventing alveolar epithelial cell mitochondrial dna damage. free radic mitigation of radiation-induced lung injury by genistein and euk- targeting the renin-angiotensin system combined with an antioxidant is highly effective in mitigating radiation-induced lung damage short-term treatment with a sod/catalase mimetic, euk- , mitigates pneumonitis and fibrosis after single-dose total-body or whole-thoracic irradiation a cell-penetrant manganese superoxide dismutase (mnsod) mimic is able to complement mnsod and exerts an anti-inflammatory effect on cellular and animal models of inflammatory bowel diseases involvement of oxygen radicals in shock related cell injury euk- , a superoxide dismutase and catalase mimetic, reduces cardiac oxidative stress and ameliorates pressure overload-induced heart failure in the harlequin mouse mutant evaluation of euk- , a synthetic superoxide dismutase/catalase mimetic as a radiation countermeasure regulation of endothelial dynamics by pgc- α relies on ros control of vegf-a signaling. free radic superoxide dismutase/catalase mimetic euk- prevents diaphragm muscle weakness in monocrotalin-induced pulmonary hypertension assessment of novel antioxidant therapy in atherosclerosis by contrast ultrasound molecular imaging photoaging and oxidative stress prolongation of alloskin graft survival by catalytic scavengers of reactive oxygen species the sod mimetic euk- reduces oxidative stress-mediated renal dysfunction in the rat in vivo a synthetic superoxide dismutase/catalase mimetic (euk- ) inhibits membrane damage-induced activation of mitogen-activated protein kinase pathways and reduces p accumulation in ultraviolet b-exposes primary human keratinocytes a synthetic superoxide dismutase/catalase mimetic euk- mitigates radiation dermatitis and promotes wound healing in irradiated rat skin investigations of antioxidant-mediated protection and mitigation of radiation-induced dna damage and lipid peroxidation in murine skin the role of oxidative stress in the pathomechanism of congenital malformations direct effect of chronic hypoxia in suppressing large conductance ca + -activated k + channel activity in ovine uterine arteries via increasing oxidative stress protection from ethanol-induced limb malformations by the superoxide dismutase/catalase mimetic, euk- redox modulation of the liver with chronic antioxidant enzyme mimetic treatment prevents age-related oxidative damage associated with environmental stress amelioration of diet-induced nonalcoholic steatohepatitis in rats by mn-salen complexes via reduction of oxidative stress ameliorative action of mn-salen derivatives on ccl -induced destructive effects and lipofuscin-like pigment formation in rats' liver and brain: post-treatment of young rats with euks preservation of renal blood flow by the antioxidant euk- in lps-treated pigs aging: a theory based on free radical and radiation chemistry the free-radical theory of ageing-older, wiser and still alive: modelling positional effects of the primary targets of ros reveals new support protein oxidation and aging the free radical theory of aging is dead. long live the damage theory! antioxid. redox signal deletion of the mitochondrial superoxide dismutase sod- extends lifespan in caenorhabditis elegans superoxide dismutase mimetics elevate superoxide dismutase activity in vivo but do not retard aging in the nematode caenorhabditis elegans. free radic oxidative stress in caenorhabditis elegans: protective effects of superoxide dismutase/catalase mimetics the effects of exogenous antioxidants on lifespan and oxidative stress resistance in drosophila melanogaster effects of superoxide dismutase/catalase mimetics on life span and oxidative stress resistance in the housefly, musca domestica. free radic treatment with euk- improves sarcoplasmic reticulum ca + release but not myofibrillar ca + sensitivity after fatiguing contraction of rat fast-twitch muscle toxicity of manganese metallodrugs toward danio rerio long-term multivitamin supplementation and cognitive function in men: a randomized trial current and experimental treatments of parkinson disease: a guide for neuroscientists is antioxidant supplement beneficial? new avenue to explore hormetic effects of curcumin: what is the evidence? revesratrol alters human endothelial cells redox state and causes mitochondrial-dependent cell death flavonoids, cognition, and dementia: actions, mechanisms, and potential therapeutic utility for alzheimer disease. free radic liposomes: clinical applications and potential for image-guided drug delivery this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -h czy bh authors: koirala, prashamsa; jung, hyun ah; choi, jae sue title: recent advances in pharmacological research on ecklonia species: a review date: - - journal: arch pharm res doi: . /s - - - sha: doc_id: cord_uid: h czy bh the genus ecklonia (lessoniaceae, phaeophyceae), commonly called kelp (brown algae), is abundant on the coasts of japan and korea. during the past few decades, ecklonia species have received tremendous attention for their wide range of therapeutic properties and multiple health benefits, such as great nutritional value and being rich in vitamins, minerals, dietary fiber, proteins, and polysaccharides. several novel functional ingredients with diversified biological activities have been isolated and possess antimicrobial, antiviral, hepatoprotective, cardioprotective, anti-inflammatory, neuroprotective, anticarcinogenic, immunomodulatory, hypolipidemic, anti-diabetic, and antioxidant therapeutic properties. the present review discusses the phytochemical, pharmacological, therapeutic, nutritional, and health benefits of different species of genus ecklonia, as well as their use in the prevention of disease and maintenance of good health. seaweed is used as a vegetable and traditional medicine in east-asian countries such as japan, korea, and china. it is an important resource of the marine ecosystem, containing active metabolites with probable nutraceuticals that are rich sources of novel bioactive secondary metabolites and harbor diverse classes of health-promoting molecules, including essential dietary fiber, fatty acids, essential amino acids, and vitamins a, b, c, and e (kolb et al. ) . taxonomic classification divides seaweed into three major classes based more on their pigments and coloration than their genetics: rhodophyceae (red algae), phaeophyceae (brown algae), and chlorophyceae (green algae, also known as macroalgae) (rajasulochana et al. ). brown algae have been used for thousands of years, but only in modern times have they been recognized to contain bioactive substances such as polysaccharides, lipids, and polyphenols, with various pharmacological properties (kumar et al. ) . ecklonia is a genus of kelp (brown algae) belonging to the family lessoniaceae that has an abundance of eckoltype phlorotannins. there are nine species: ecklonia biruncinata, ecklonia brevipes, ecklonia cava (ec), ecklonia fastigiata, ecklonia kurome (ek), ecklonia maxima (em), ecklonia muratii, ecklonia radiate (er), and ecklonia stolonifera (es) (hornemann ; moon et al. ) . ec, an edible marine brown alga, is used as a food ingredient, animal feed, and fertilizer, as well as a raw material in the production of fucoidan and phlorotannin. ec is also used as an herbal remedy in the form of an extract called seanol, a polyphenolic extract, and ventol, a phlorotannin-rich natural agent with two major constituents, phlorotannins and sterols (kang et al. a) . es (turuarame), ek, and er are edible species traditionally eaten in japan and korea and are rich in phlorotannins and fatty acids. ecklonia species are known to exhibit antioxidant (heo et al. ) , anti-inflammatory , antibacterial , anti-diabetic (jung et al. ) , anticancer (kong et al. ), anti-photoaging (joe et al. ) , anti-hiv (artan et al. ) , anti-hypertensive (jung et al. ) , hepatoprotective (jung et al. a) , and anti-allergic activities (le et al. ). due to these numerous health benefits, they have been a focal point for researchers eager to elucidate their pharmacological potential. plenty of information regarding the pharmacological activities of terrestrial plants is available; however, such information is limited for marine species (shibata et al. ) . a handful of excellent studies are available regarding the pharmacological activities of ecklonia (wijesinghe and jeon ; thomas and kim ; li et al. ; . also, jiao et al. ( ) have reported the chemical structures and bioactivities of marine algae. fourteen years of research and nearly $ million of clinical studies demonstrate the importance of ecklonia species. ecklonia-derived polyphenols are unlike those found in land-based plants and are quite possibly the most powerful antioxidants found in nature, being - times more powerful than other polyphenols. the oxygen radical absorbance capacity (orac) score of such a polyphenol is more than . the water-soluble polyphenols found in land-based plants have a half-life of about min, and their antioxidant powers decrease very quickly (seanol ) . various phlorotannins, polysaccharides, enzymatic extracts, and organic extracts from ecklonia exhibit multifaceted beneficial effects when used in pharmaceuticals, nutraceuticals, cosmeceuticals, and functional foods. thus, this genus has been a target of special attention, and consumer-driven demand has led to the development of marine-derived medicines. our review summarizes the literature on the biological characterization and pharmacological bioactivity of various ecklonia species, focusing on recent developments in the therapeutic application of extracts and isolates. a shift in the balance between oxidants and antioxidants in favor of oxidants is called oxidative stress (table ) . it arises when the balance between the production of reactive oxygen species (ros) and antioxidant defenses changes. human cells have an inherited antioxidative defense system in the form of various enzymatic and non-enzymatic pathways for removing ros. elevated production of ros increases oxidative stress, leading to cellular dysfunction, and it can eventually contribute to many pathological conditions, including neurological disorders (agostinho et al. ) , diabetes (ceriello ) , cancer (perse ) , asthma , and dermal disease (trouba et al. ). an ethanolic extract of ec attenuated h o -induced comet tail formation and phospho-histone ch ax expression. furthermore, it enhanced the level of the phosphorylated form of nuclear factor erythroid -related factor (nrf ) and its nuclear translocation, which was associated with the induction of heme oxygenase- (ho- ) and nad'ph-quinone oxidoreductase- (nqo- ). thus, ec provided cytoprotective effects against oxidative stress in muscle cells via up-regulation of nrf -ho- and nqo- expression through the activation of the mitogen-activated protein kinases (mapk) pathway, which could be due to its phenolic content (choi ) . in another study, es and ek showed high total phenolic content (tpc) and high antioxidant activity, including , -diphenyl- -picryl-hydrazyl (dpph) radical and hydroxyl (•oh) radical scavenging, ferrous reducing power, and superoxide (•o -) radical scavenging activity. those results suggest that macroalgal beach-casts could be used as a new natural source for functional foods, cosmetics, medicines, and fertilizer instead of being processed into landfills or incinerated (kuda and ikemori ) . enzymatic extracts from er demonstrated more potent antioxidant ability in the ferric reducing ability of plasma (frap) and orac assays (tpc of . g) than conventional acidic extracts (tpc of . g), showing their potential for value-added nutritional products (charoensiddhi et al. ) . inflammation is a pathological condition that produces highly reactive species. nitric oxide (no), a small diffusible molecule responsible for vasodilatation, neurotransmission, and inflammation, is produced by organisms at a basal concentration. however, under stimulation by pathogens, no is generated in higher amounts by the inducible nitric oxide synthase (inos) in activated macrophages (moncada et al. ) . nonsteroidal anti-inflammatory drugs are commercially available medications for inflammatory pain, but their side effects have limited their use. anti-inflammatory drugs from natural resources have been sought due to the persistent deleterious side effects of commercial medications. ec dose-dependently suppressed inos and cyclooxygenase- (cox- ) protein expression and subsequently reduced no content in phorbol- myristate -acetate enzymatic extracts nf-jb lee et al. ( c) ( nmol/l) and a ( lmol/l) (pmaci)-stimulated human mast cell line- cells. no production decreased by . and . % with and lg/ml ec treatments, respectively. furthermore, ec dose-dependently inhibited both the mrna and protein expression of tumor necrosis factor (tnf)-a, interleukin (il)- b, and il- in pmacistimulated human mast cell line- cells without any cytotoxic effects. the inhibitory effects of ec on il- b ( . %) and tnf-a ( . %) production were greater than those on il- ( . %) at lg/ml. in addition, ec exerted anti-inflammatory action via the inhibition of the extracellular signal-regulated kinase (erk)/mapk signaling pathway, suggesting a potent and efficacious antiinflammatory agent against mast cell-mediated inflammatory diseases (kim d . the world health organization (who) defines diabetes as a chronic disease that occurs when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces, which leads to an increased concentration of glucose in the blood, i.e., hyperglycemia. type diabetes (previously known as insulin-dependent) is characterized by a lack of insulin production. type diabetes (formerly called non-insulindependent) is caused by the body's ineffective use of insulin, often from excess body weight and physical inactivity. the total number of people with diabetes is projected to rise from million in to million in (rathmann and giani ) . intensive insulin therapy carries a high risk of side effects, especially the occurrence of severe hypoglycemia; therefore, research into antihyperglycemic agents has focused on plants used in traditional medicine because they could provide better treatment than the currently used synthetic drugs (mccall ) . kimchi has been a popular side dish in korea since ancient times. baechu kimchi is a salt fermented cabbage widely consumed in traditional korean foods. a recent study revealed that kimchi with added ec extract showed high inhibition against a-glucosidase and a-amylase, with ic values of . and . mg/ml, respectively. both of those inhibitory activities of kimchi with added ecklonia extracts (ke) were higher than those of kimchi extract alone. the hypoglycemic effect of ke was higher than that of kimchi extract on starch loading. ke suppressed the postprandial blood glucose level in both streptozotocin (stz)-induced diabetic and normal mice, which indicated a delay in the absorption of dietary carbohydrates consumed . in another report, baechu kimchi with added ec extract protected human umbilical vein endothelial cells (huvecs) from damage induced by high glucose by restoring cell viability and reducing lipid peroxidation and intracellular ros in a dose-dependent manner. furthermore, it reduced the overexpression of inos, cox- , and nuclear factor-jb (nf-jb) proteins in huvecs, indicating its potential as a treatment against high glucose-induced oxidative stress . ek inhibited carbohydrate-hydrolyzing enzymes, decreased postprandial blood glucose levels, and improved glucose tolerance, decreasing both fasting blood glucose and insulin levels (xu et al. ) . ek effectively down-regulated blood glucose in both db/db mice and prediabetic c bl/ j mice, indicating the presence of the active compounds in the gametophytes. ek regulated metabolism by manipulating the balance among cytokines, including interferon-gamma (ifn-c) or leptin, resulting in the downregulation of blood glucose (dwiranti et al. ). the liver is involved in the metabolism of internal and external toxic agents. it has an astounding role in the performance, maintenance, and regulation of homeostasis in the body and is engaged in almost all biochemical pathways of growth, fight against diseases, nutrient supply, energy provision, and reproduction. it plays a vital role in the detoxification of xenobiotics and drugs (agrawal et al. ) . hepatic disease is a term that indicates damage to liver cells, tissues, structures, or function. because no drug currently available completely or effectively stimulates hepatic function, offers complete protection to the organ, or aids in regenerating hepatic cells, naturally derived hepatoprotective agents are needed to negate the factors that contribute to liver damage. a polyphenol-rich fraction of ec prepared in gijang prevented diabetes by regulating various metabolic processes, such as lipogenesis, lipolysis, inflammation, and the antioxidant defense system in livers and adipose tissues affected by nonalcoholic fatty liver disease in high fat diet (hfd)-fed mice. magnetic resonance imaging/magnetic resonance spectroscopy (mri/mrs) analysis showed that liver fat and liver volume in hfd-triggered obese mice decreased following gijang extract treatment. furthermore, the treatment reduced the mrna expression levels of inflammatory cytokines and hepatic lipogenesis-related genes and increased the mrna expression level of cholesterol a-hydroxylase , the key enzyme in bile acid synthesis. thus, gijang extract ameliorated hepatic steatosis by suppressing inflammation and improving lipid metabolism . apart from this, ec increased the activity of alcohol dehydrogenase, aldehyde dehydrogenase, and cyclic adenosine monophosphate (camp) concentration, suggesting that it plays a role in the activities of alcohol-metabolizing enzymes and their regulating mechanisms in ethanol-treated hepatocytes. ec inhibited cytochrome p e expression related to the production of ros (yamashita et al. ) . a study investigated the protective effect of es in alcoholic fatty liver and found that es treatment suppressed adipogenesis and increased the expression of fatty acid oxidation-related genes, e.g., peroxisome proliferator-activated receptor (ppar)-a and cpt- , but decreased the expression of sterol regulatory element-binding protein (srebp)- , a triglyceride (tg) synthesis-related gene, suggesting that es extract could be useful in preventing fatty acid oxidation and reducing lipogenesis in ethanol-induced fatty liver (bang et al. ) . neurodegenerative diseases are expected to surpass cancer as the second most common cause of death among the elderly by the s (lilienfeld and perl ) . alzheimer's disease (ad) is characterized by loss of memory and other cognitive functions and accounts for most of the deaths in the elderly. an increase in acetylcholinesterase (ache) level around b-amyloid plaques and neurofibrillary tangles is a common feature of ad neuropathology (lam et al. ). parkinson's disease (pd) is a multidimensional progressive disease with many motor and non-motor features, including cognitive dysfunction. adverse intra-and extracellular effects of toxic a-synuclein are believed to be central to the pathogenesis of pd and other nervous system disorders with lewy body pathology (goldman and postuma ; ingelsson ) . many categories of natural and synthetic neuroprotective agents have been reported. considering the devastating side effects of synthetic neuroprotective agents, there is growing interest in nutraceuticals or other herbal alternatives (pangestuti and kim ) . a study by kang et al. ( d) demonstrated that ec n-buoh extract regulated the expression and activity of csecretase and a-secretase, leading to a reduction in ab production by the stable cells and a reduction in the basal nuclear location of the psen responsible for chromosome mis-segregation in neurodegenerative disease. ec and ek together inhibited ache and bace by . ± . and . ± . %, respectively, reducing neuronal cell death and improving dementia, highlighting their synergistic potential. ek likewise showed the highest result of the , -azino-bis-( -ethylbenzothiazoline- -sulfonic acid) (abts) assay (ic = . ± . mg/ml), which the researchers attributed to its tpc (son et al. ) . kim et al. ( e) further highlighted the importance of ec for its potential analgesic effects in postoperative pain and neuropathic pain. ec extracts ( mg/ kg) significantly increased the mechanical withdrawal therapy value. the number of ultrasonic distress vocalizations in the treated group of rats was reduced at and h after plantar incision operation ( . %). ec also increased the paw withdrawal latency in hot-and coldplate tests in the plantar incision rats. after days of continuous treatment, ec ( mg/kg) alleviated the spared nerve injury-induced hypersensitivity response, which could revolutionize the use of natural resources for therapeutic treatment. matrix metalloproteinases (mmps), known as matrixins, are a large family of similar proteolytic enzymes involved in tissue remodeling associated with various physiological and pathological processes, such as morphogenesis, angiogenesis, tissue repair, arthritis, chronic heart failure, chronic obstructive pulmonary disease, chronic inflammation, and cancer metastasis. as a result, mmps are considered viable drug targets in the therapy of those diseases (dormán et al. ) . according to gelatin zymography results, extracts harvested from ec and ecklonia bicyclis showed higher inhibitory effects on mmp- and - activity than those from the other marine plants at concentrations of , , and lg/ml (bae et al. ) . the global epidemic of bacterial resistance to existing antibiotics such as b-lactams and quinolones necessitates the discovery of potent candidates from natural resources, both terrestrial and marine. the evolution of antibacterial biomolecules alongside bacteria over millions of years allow them to overcome strains such as methicillin-resistant staphylococcus aureus (mrsa) and fluoroquinoloneresistant pseudomonas (ramanan et al. ) . therefore, exploring and developing cheaper and more effective natural antimicrobial agents with better potential and fewer side effects than existing antibiotics, good bioavailability, and minimal toxicity is a public health priority (pérez et al. ) . a recent study showed that ec with ciprofloxacin evinced potent antibacterial activity against enterococcus faecalis, showing synergistic effects. etoac exhibited the strongest antibacterial activity, with a minimum inhibitory concentration (mic) value of lg/ml against e. faecalis strains. furthermore, the combination of ciprofloxacin and the etoac fraction resulted in a p fic min of . and p fic max range of . - , suggesting that the ciprofloxacin-etoac combination resulted in an antibacterial synergy effect against e. faecalis (kim et al. c) . ec led to the strongest growth effects on three lactic acid bacteria and fish pathogenic bacteria in a dose-dependent manner. secondary metabolites produced by ec significantly inhibited the growth of pathogen bacteria. in a further in vivo study, the co-treatment of ec and l. plantarum improved the growth and mortality of edwardsiella tardainfected zebrafish by regulating the expression of inflammatory molecules such as inos and cox- . altogether, ec played an important role as a potential prebiotic and protected against the infection caused by e. tarda injection in zebrafish . ec ( . %) improved the growth and body weight of olive flounder and decreased its mortality from e. tarda without changing its biochemical profile. the supplementation of . % ethanolic extract of ec also enhanced the innate immune response of the fish, as evidenced by a high respiratory burst and increased serum lysozyme and myeloperoxidase activity. thus, ec acted as a prebiotic by improving the innate immune response in fish infected with pathogenic bacteria (lee et al. c ). seanol, a seaweed extract rich in phlorotannins, stimulated mineralization with calcium phosphate, increased antibacterial activity, and increased compressive modulus. seanol and alkaline phosphatase (alp) interacted in a non-covalent manner. seanol exhibited antibacterial activity against mrsa with comparable cytotoxicity toward mg- osteoblast-like cells, suggesting its mineralizability and antibacterial activity (douglas et al. ) . venkatesan et al. ( ) reported the rapid biological synthesis of gold nanoparticles (au nps) using ec by reducing chloroauric acid. fourier transform infrared (ftir) spectroscopic analysis showed that au nps functionalized with biomolecules (a primary amine group, a hydroxyl group, and other stabilizing functional groups) showed good antimicrobial activity and biocompatibility with a human keratinocyte cell line. the results indicate that aunps might have promising applications in drug delivery, tissue engineering, and biosensor development. er extract prepared by using celluclast-assisted extraction induced significantly higher production of butyrate ( . lmol/ml) and promoted the growth of beneficial bacteria such as bifidobacterium and lactobacillus, improving gut health (charoensiddhi et al. ). ec and ek had the strongest inhibitory effects when tested using the agar disk diffusion method, with an mic of . mg/ ml and no cytotoxicity even at lg/ml, indicating their potential as therapeutic agents for acne vulgaris ). obesity is a leading preventable cause of death worldwide, with increasing rates in both developed and developing countries. as defined by who, it is a medical condition in which excess body fat has accumulated to the extent that it can have negative effects on health, and it is often comorbid with diseases such as type diabetes, hypertension, coronary disease, and cancer. in , who announced that obesity had reached epidemic proportions worldwide (kumar and rao ; caballero ) . commercial drugs, such as orlistat; lorcaserin; and a combination of phentermine, topiramate, and bariatric surgery, such as roux-en-y bypass or gastric banding, are available. however, concerns about perioperative mortality, surgical complications, and the frequent need for reoperation mean that those procedures tend to be reserved for morbid obesity (rodgers et al. ). thus, remedies from natural sources are vital, especially those from marine sources. the anti-adipogenic activity of ec was determined by measuring lipid accumulation in adipocytes. the n-buoh fraction particularly reduced lipid accumulation and glucose consumption; the adipogenic transcription factors pparc and srebp- c; and the adipogenic specific genes fatty acid binding protein (fabp)- , fabp- , fatty acid synthase (fas), lipoprotein lipase (lpl), hormone-sensitive lipase (hsl), and acyl-coa synthetase (acs ) . ec polyphenol extract regulated fat metabolism, inflammation, and the antioxidant defense system in hfd-induced obese mice. ec polyphenol extract supplementation reduced body weight gain, adipose tissue mass, plasma lipid profiles, hepatic fat deposition, insulin resistance, and the plasma leptin/adiponectin ratio derived from hfd-induced obesity. furthermore, ec polyphenol extract supplementation selectively ameliorated the hepatic protein levels associated with lipogenesis, inflammation, and the antioxidant defense system, as well as activation of ampk and sirtuin (sirt ) to inhibit obesity (eo et al. ) . there has been a worldwide increase in allergic diseases, including atopic dermatitis, asthma, allergic rhinitis, and food allergies, possibly because environmental factors are interacting with genetic factors to sensitize individuals (tanaka ) . numerous studies have been done in the search for anti-allergens, especially from marine resources. ec extract exhibited excellent inhibitory activity against crude histidine decarboxylase (hdc), reducing overall histamine production by . % and thereby enhancing the safety of mackerel muscle (kim et al. g) . another report presented a % inhibition of hdc at a concentration of mg/ml, reducing histamine poisoning by decreasing histamine production in mackerel (jung et al. b) . ec and ek together inhibited the degranulation of a rat basophilic leukemia cell line (rbl- h ), mitigating allergic symptoms and highlighting their synergistic potential (yoshioka et al. ) . phlorotannin-rich es inhibited enzymatic activity and degranulation in stimulated rbl- h cells in a dose-dependent manner. the meoh: chloroform ( : , v/v) (m/c) extract of es also inhibited enzyme activity and degranulation in stimulated rbl cells in a dose-dependent manner. the active compounds in the m/c extract might be phenolic compounds, such as phlorotannins, because the m/c extract became inactive when the phenolic compounds were removed (sugiura et al. ) . ionizing radiation produces deleterious effects, deterministic or stochastic, on living organisms, though it can have health benefits in the form of radiation therapy for the treatment of cancer or thyrotoxicosis. the benefits of ionizing radiation are compromised by the side effects that result from radiation-induced damage to normal tissue, and the synthetic agents used to combat those side effects, wr (amifostine), ok- , and ethiofos, have their own serious side effects, including decreased cellular function, nausea, hypotension, and death (baliga and rao ; park et al. ) . therefore, investigators have directed their attention toward plants and other natural products. enzymatic extracts of ec exhibited radioprotective properties, including the modulation of apoptosis via inhibition of the nf-jb signaling pathway ). ecklonia species and their constituents exhibit pronounced inhibitory effects against oxidative stress (table ) . ec phlorotannins, including phloroglucinol (pg), eckol, dieckol, eckstolonol, and triphlorethol-a (tpa), scavenged intracellular ros, inhibited lipid peroxidation, and suppressed , -azobis( -amidinopropane) dihydrochloride (aaph)-induced cell death in zebrafish embryos. these phlorotannins maintained the positive changes in morphological phenomena; pericardial edema, yolk sac edema, and growth retardation in zebrafish embryos exposed to aaph were not observed in the groups also exposed to phlorotannins, indicating that the phlorotannins possess prominent antioxidant activity against aaph-mediated toxicity (kang et al. a) . tpa further exhibited a protective effect against oxidative stress-induced dna-base damage, especially -oxoguanine ( -oxog), in v - cells. decreased level of -oxog induced by h o were confirmed by an increase in ogg mrna and ogg protein levels. tpa restored the expression of nuclear nrf , small maf protein, and the nrf -maf complex and also increased nrf binding to are sequences and the resulting ogg promoter activity. sequentially, it maintained the levels of the phosphorylated forms of akt kinase downstream of phosphatidylinositol -kinase (pi k) and erk, which are regulators of ogg , suggesting that ogg induction by tpa involves the pi k/akt and erk pathways . eckol from ec also attenuated the high intracellular ca ? levels stimulated by h o , decreased the augmented levels of mitochondrial ros, recovered h o -diminished atp level and succinate dehydrogenase activity, and induced manganese superoxide dismutase through phosphorylated ampk and forkhead box o a (foxo a), which showed a cytoprotective effect on chang liver cells ). fucoidan extracted from ec exhibited prominent effects on peroxyl radical scavenging activity and , -azobisdihydrochloride-induced oxidative stress in vero cells and reduced ros generation, lipid peroxidation, and cell death in a zebrafish model, proving its antioxidant capacities in vitro and in vivo despite being neither a polyphenol nor a flavonoid. although, it did not contain a benzene ring or conjugated structure, it exhibited antioxidant potential (kim et al. c ). in another study, , bieckol, -phloroeckol, dieckol, and phlorofucofuroeckol (pff-a) isolated from ec significantly inhibited high glucose-induced ros and cell death in zebrafish. dieckol significantly reduced heart rate, ros, no, lipid peroxidation generation, and cell death and also reduced the overexpression of inos and cox- , thereby preventing oxidative stress . es could be used as a natural antioxidant and cytoprotective agent. es inhibited ros even at a concentration of lg/ml, yielding five compounds (pff-a, dieckol, eckstolonol, pg, and eckol) that inhibited total ros, proving its use as a potent scavenger (kang et al. ) . three active compounds were isolated from es, pff-a, dieckol, and dioxinodehydroeckol (dhe), among which pff-a and dieckol significantly suppressed intracellular ros in lipopolysaccharide (lps)induced raw . cells, and dhe scavenged dpph radicals. pff-a also significantly inhibited the lps-induced production of no and prostaglandin e (pge ) through the down-regulation of inos and cox- protein expression ). dieckol and pff-a obtained from boiling water-and organic solvent extracts of ec and es showed almost -and -fold stronger antioxidant activity than the standard butylhydroxytoluene, and -and -fold greater activity than l-ascorbic acid in molar concentration, anti-inflammatory activity ecklonia cava dieckol decreased blood glucose level kang et al. ( b) insulin resistance lee and jeon ( ) akt up-regulation kim et al. ( b) phlorofucofuroeckol-a a-glucosidase and a-amylase fucodiphlorethol g uvb induced oxidative stress kim et al. ( f) respectively (chowdhury et al. ) . three known phlorotannins, eckol, pff-a, and dieckol, along with one new compound, eckstolonol, were obtained from es, and the new compound was found to be a potent radical scavenger through its elimination of dpph radicals (kang et al. b) . eckol suppressed the production of intracellular ros and increased glutathione peroxidase (gsh) level in hepg cells. it inhibited the production of ros in h o treated hepg cells in a dose-dependent manner, and the total relative level of lm eckol was estimated to be . ± . % compared to the non-treated group, making it a much stronger ros scavenger than n-acetylcysteine (nac). the intracellular gsh content in hepg cells was dose-dependently enhanced by eckol treatment, indicating higher antioxidant activity than nac. thus, eckol mediated the expression of ho- in hepg cells, which was regulated by nrf activation via the jun n-terminal kinases (jnk) and pi k/protein kinase b (akt) signaling pathways, suggesting that it is a natural antioxidant and cytoprotective agent (jun et al. (fig ) . ec extract and its major compound (dieckol) significantly increased the survival rate and attenuated liver and kidney damage in mice with a whole-body inflammatory condition by down-regulating pro-inflammatory factors (inos, cox- , tnf-a, il- , and hmgb- ) via a nik/tak / ikk/ijb/nf-jb pathway. additionally, ec increased nrf and ho- expression, reducing inflammation (yang et al. ) . dieckol ( and lm) inhibited the production of a macrophage-derived chemokine, c-c motif chemokine , induced by interferon-c ( ng/ml) in a dose-dependent manner and inhibited the nuclear translocation of signal transducers and activators of transcription (stat ). these results showed that dieckol produced anti-inflammatory effects via the down-regulation of stat activation . in addition, , -bieckol isolated from ec suppressed key inflammatory mediators such as no and pge in raw . macrophages. the inhibition of no occurred by suppressing lps-induced expression of inos at the mrna and protein levels in primary macrophages and raw . cells. likewise, , -bieckol reduced the production and mrna expression of the inflammatory cytokine il- , but not that of tnf-a, in raw . cells. furthermore, , -bieckol significantly reduced mortality in lps-induced septic mice, which indicates that the anti-inflammatory properties of , bieckol are associated with the suppression of no, pge , and il- via negative regulation of the nf-jb pathway and ros production in lps-stimulated raw . cells ). dieckol extracted from ec suppressed lpsinduced inos expression in mouse leukemic macrophage raw . cells, decreasing both lps-induced no production and inos promoter-driven transcriptional activity in a dose-dependent manner. furthermore, it prevented lps-mediated nf-jb activity. it also diminished lpsmediated p nuclear translocation or ijba phosphorylation dose-dependently and reduced lps-induced phosphorylation of mapks, especially p mapk. collectively, these findings suggest that dieckol acts as a negative regulator of lps-mediated inos induction by suppressing nf-jb activity, implying a mechanistic role for dieckol in the regulation of the inflammatory response . the in vivo anti-inflammatory effect of fucoidan from ec was studied using tail-cutting-induced recent advances in pharmacological research on ecklonia species: a review and lps-stimulated zebrafish models; it inhibited tail-cutting-induced and lps-stimulated ros and no generation and also showed a protective effect against the toxicity induced by lps exposure in zebrafish embryos ). pff-a isolated from es showed potential antiinflammatory properties in macrophages stimulated by lps. for this, lm of pff-a significantly inhibited inos and cox- mrna levels induced by lps stimulation. similarly, levels of pro-inflammatory cytokines such as il- b, il- , and tnf-a were significantly reduced. pff-a further inhibited the promoter activities of inflammatory mediators and transcriptional factors. thus, pff-a regulated inos and cox- expression through the nf-jbdependent transcriptional control associated with the inhibition of multiple signaling proteins, suggesting that pg derivatives could be potential treatments for inflammatory diseases . the etoac fraction of es, along with its isolated compounds -phloroeckol, , bieckol, pff-a, pff-b, and -b, inhibited the production of lps-induced no and pge and reduced the expression of inos and cox- in a dose-dependent manner (wei et al. ). most of the investigations on phlorotannins, particularly those derived from brown algae, indicate their promising anti-diabetic effects. for example, , -pg- , -bieckol isolated from ec improved postprandial hyperglycemia through a-glucosidase and a-amylase activity in stz-induced diabetic mice. it showed higher inhibitory activity than the positive control, acarbose (a-glucosidase ic of . lm; a-amylase ic of . lm) (lee et al. a ). in addition, , -bieckol purified from ec at concentrations of or lg/ml significantly inhibited high glucose-induced glucotoxicity and dose-dependently reduced the level of thiobarbituric acid reactive substances (tbars), generation of intracellular ros, and the level of no. furthermore, , -bieckol prevented the apoptosis of rat insulinoma cells under high-glucose conditions, attributed to increased expression of the anti-apoptotic protein bcl- and reduced expression of the pro-apoptotic protein bax, establishing ec as a potential nutraceutical candidate for protection against glucotoxicity (park et al. a ). , -bieckol from ec at concentrations of or lg/ml markedly suppressed high-glucose-induced cytotoxicity and dose-dependently decreased the increased levels of tbars, ros, and no caused by high glucose. in addition, it down-regulated the overexpression of inos, cox- , and nf-jb proteins in huvecs, indicating its therapeutic ability to treat diabetic endothelial dysfunction and related complications ). ec-derived dieckol noticeably decreased blood glucose level, serum insulin level, and body weight. furthermore, it reduced tbars and increased the activities of antioxidant enzymes, including superoxide dismutase (sod), catalase (cat), and gsh-px, in liver tissue. in addition, western blotting analysis revealed that dieckol increased the phosphorylation levels of ampk and akt observed in muscle tissues, suggesting that dieckol could be a therapeutic agent for type diabetes ). dieckol-rich extract from ec led to a significant decrease in postprandial glucose level, insulin, and c-peptide level after weeks without any adverse effects. in other words, ec supplementation significantly contributed to lowering postprandial hyperglycemia and reducing insulin resistance . in addition, dieckol improved blood glucose regulation, hepatic glucose metabolic regulation, and akt up-regulation in alloxan-induced hyperglycemic zebrafish ). pff-a isolated from ec showed prominent inhibitory effects against a-glucosidase and a-amylase activities, with ic values of . and . lm, respectively, which were higher than those of acarbose. moreover, the area under the curve was significantly lower after pff-a administration ( vs. mmol min/l) in diabetic mice, indicating its potent anti-diabetic activity (you et al. ) . the a-glucosidase inhibitory property of em and its isolated compounds pg, dibenzo [ , ] dioxine- , , , -tetraol, and eckol exceeded that of the positive control, suggesting their potency as oral anti-diabetic drugs or functional food ingredients, with a promising role in the formulation of medicines and nutritional supplements (rengasamy et al. ) . es exhibited inhibitory activity on glucose-mediated protein damage, advanced glycation end-products (age), and rat lens aldose reductase (rlar), hinting at potential antidiabetic activity. in spite of negligible activity against age, es phlorotannins, including eckol, dieckol, and -phloroeckol, possessed inhibitory activity on glycation, which indicates that pff-a could be used to prevent diabetic complications (jung et al. ) . likewise, fucosterol from es inhibited rlar, human recombinant aldose reductase, protein tyrosine phosphatase b (ptp b), and a-glucosidase activity (jung et al. a ). pff-a, dieckol, and -phloroeckol isolated from es were potent and non-competitive ptp b inhibitors, with ic values ranging from . to . lm, and a-glucosidase inhibitors, with ic values ranging from . to . lm. interestingly, pff-a and -phloroeckol were non-competitive, whereas dieckol exhibited competitive inhibition in a a-glucosidase assay. thus, isolated phlorotannins from both algae possessed marked ptp b and a-glucosidase inhibitory activity that could contribute to the development of therapeutic agents to control postprandial blood glucose level and prevent diabetic complications (moon et al. ) . published results clearly indicate the anti-diabetic potential of brown seaweed and its derived components, which could be used as nutraceuticals or functional foods to treat diabetes. pyrogallol-phloroglucinol- , '-bieckol fucoidan dieckol from ec exerted cytotoxicity in lx- , hsc-t , and hepg cells, with reduced fibrosis features (large, spread out, and flattened polygonal shapes) in lx- cells compared with the untreated control. in addition, it attenuated the expression of a-sma and tgf-b , increased the sub-g phase population, induced caspase- activation, and cleaved parp in hepatic stellate cells. thus, dieckol suppressed liver fibrosis via caspase activation, micro-rna-mediated jnk activation, and via nf-jb inhibition ). an in vitro study of dieckol showed the strongest protective effect and lowest cytotoxicity against ethanol-induced cell apoptosis in chang liver cells. western blot analysis revealed reduced cell apoptosis through the activation of b cell lymphoma-extra large (bcl-xl) and parp and down-regulation of bax and caspase- , providing evidence for this potential protective agent against ethanol-induced liver diseases. in an in vivo study in a zebrafish model, the dieckol-treated group scavenged intracellular ros and prevented lipid peroxidation and ethanol-induced cell death in embryos (kang et al. c cytochrome c from mitochondria to the cytosol in a dosedependent manner (lee et al. a ). pg isolated from es decreased the formation of lipid peroxide in acetaminophen ( mg/kg, i.p.)-induced rats. though the activities of cytochrome p- , aminopyrine n-demethylase, and aniline hydroxylase were unchanged, pg restored enzyme activity in the livers of pretreated-rats, suggesting that acetaminophen-induced hepatic lipid peroxidation could be reduced by enhancing the activity of glutathione s-transferase (gst) (park ) . all of these data suggest that ecklonia species could be potent hepatoprotective agents. dieckol isolated from ec suppressed the phosphorylation of erk in lps-stimulated bv- microglia ( lg/ml), attenuated akt phosphorylation, and increased the expression of gp phox , a catalytic component of the nicotinamide adenine dinucleotide phosphate (nadph) oxidase complex responsible for microglial ros generation. furthermore, dieckol offered neuroprotection, as confirmed in an enhanced green fluorescent protein-transfected b neuroblastoma cell line (cui et al. ) . dieckol isolated from ec showed potent activity on rotenone-induced oxidative stress in sh-sy y cells, a human dopaminergic neuronal cell line. it reduced rotenone-induced cell death and retarded rotenone-induced a-synuclein aggregation in asynuclein-overexpressing sh-sy y cells, which prevented a-synuclein aggregation, adding to its role in the prevention of pd (cha et al. ) . pg ( , , -trihydroxybenzene) from ec attenuated the increase in ros accumulation induced by oligomeric ab - treatment in the ht- hippocampal cell line and ameliorated the reduction in dendritic spine density induced by ab - treatment in rat primary hippocampal neuron cultures. also, pg attenuated cognitive dysfunction in the hippocampal region, indicating its anti-ad effects (yang et al. b) . another report showed that pff-a from ec had particularly potent inhibitory activity (ic = . lm) for butyrylcholinesterase (bche), more than -fold greater than for ache. other polyphenols (pff-a, eckol, , -bieckol, , -bieckol, and dieckol) inhibited glycogen synthase kinase b, which is related to the formation of hyperphosphorylated tau and generation of ab. additionally, pff-a inhibited amyloid precursor protein biosynthesis and showed very strong bace inhibitory activity, with a submicromolar ic , making it an interesting potential drug candidate for ad ). an oligosaccharide sugar chain derived from ek inhibited the toxicity induced by the ab protein in both primary cortical cells and the sh-sy y cell line, inhibiting apoptosis and fibril formation and indicating its potency for ad (hu et al. ) . em exhibited potent activity against ache, as did its isolated compounds pg, dibenzo [ , ] dioxine- , , , -tetraol, and eckol, highlighting its potential as a functional food ingredient for the management of neurodegenerative disorders (kannan et al. ) . a phlorotannin preparation (pp) containing eckstolonol from es and ec exhibited a hypnotic effect by modulating the benzodiazepine site of the c-amino butyric acid receptor. pp ([ mg/kg) decreased sleep latency and increased non-rapid eye movement sleep (nrems). likewise, eckstolonol significantly decreased sleep latency ([ . mg/ kg) and increased the amount of nrems ( mg/kg). in addition, the hypnotic effects were completely abolished by pretreatment with flumazenil, suggesting that the phlorotannins could potentially be used as an herbal medicine for insomnia and offering a promising structure for the development of novel sedative-hypnotics . fucosterol and fucoxanthin from es showed noncompetitive and mixed-type inhibition against b-site amyloid precursor protein cleaving enzyme (bace ). furthermore, molecular docking simulation results demonstrated the effective binding of isolated compounds by the bace enzyme, suggesting that both compounds could be used beneficially in the treatment of ad and providing potential guidelines for the design of new bace inhibitors . isolated eckol and dieckol attenuated tnf-a induced expression of mmp- and basal expression, though the expression of timp- was not affected. however, they did reduce both nf-jb and ap- reporter gene activity, which strongly indicates their mmp inhibitory potential. one study demonstrated the inhibitory effect of eckol and dieckol isolated from es on mmp- expression in human dermal fibroblasts, suggesting the possibility of developing an agent to prevent and treat skin aging (joe et al. ) . obviously, marine sources outweigh terrestrial sources in abundance in the development of safe mmp nutraceuticals. anticoagulation occurs by inhibiting the key serine proteases thrombin and factor xa, facilitated by accelerating the activity of the major physiological serine protease inhibitor serpin-antithrombin iii. heparin, a highly sulfated polysaccharide present in mammalian tissues, is commercially used as a blood anticoagulant. it has antihemostasis, fibrinolytic potentiation, and anti-lipemic activity in addition to coagulation activity. though it is a primary anticoagulant, difficulty in isolating it and its hemorrhagic side effects limit its use and drive researchers to search for novel anticoagulants from natural resources free from cytotoxicity (shanmugam and mody ) . studies of the anticoagulant bioactivity of brown seaweeds suggest that they have more than one mechanism of action, including the direct and indirect inhibition of thrombin through the activation of thrombin inhibitors (e.g., antithrombin and heparin cofactor). interestingly, the algal fucans were found to have anticoagulant activity through a direct inhibition of thrombin, whereas the invertebrate fucans showed activity through an indirect inhibition of the enzyme, which required antithrombin and heparin cofactor ii, and that has driven the rapid discovery of anticoagulants from marine resources (jiao et al. ) . fucoidan isolated from ek significantly inhibited the generation of thrombin and factor xa in the intrinsic pathway. furthermore, it inhibited the formation of prothrombin-activating complex (i.e., prothrombinase); the ic of thrombin generation was one-tenth to one-seventh that of the activity of the thrombin in plasma, whereas the antithrombin activity of fucoidan was mediated by heparin cofactor ii in plasma. this further highlights the relationship between the molecular weight of sulfated polysaccharides and their anticoagulant activity such that the higher molecular weight fucans show greater anticoagulant activity than sulfates with a lower molecular weight (nishino et al. (nishino et al. , a . sulfated polysaccharides, mainly -linked and , -disubstituted fucopyranosyl residues isolated from ek, exhibited potent anticoagulant activity (nishino et al. b) . some potent and novel antiplasmin inhibitors such as pff-a and eckol isolated from ek inhibited the action of alpha -macroglobulin (ic = . lg/ml) and alpha -plasmin inhibitor (ic = . lg/ml), the main plasmin inhibitors in plasma (fukuyama et al. (fukuyama et al. , . thus, the development of antithrombotic algal polysaccharides would be advantageous because their use would avoid the potential for contamination with prions or viruses present in commercial heparins, which are obtained from pig and bovine intestines. moreover, with more specific activities or targets, algal sulfated polysaccharides could find applications complementary to heparin. dieckol from ec significantly blocked the cleavage of sars-cov cl(pro) in a cell-based assay without showing any toxic effects and exhibited a high association rate in the spr sensorgram, forming strong hydrogen bonds to the catalytic dyad (cys and his ) of the sars-cov cl(pro) ). both phlorotannins from ec and isolated epibiotic bacteria showed potent antibacterial activity in close affiliation with the genus bacillus (kanagasabhapathy et al. ) . another novel bacterial strain, designated ec t, was isolated from ec, and it showed potent antibacterial activity (kim et al. d) . another study suggested that the compounds eckol, dieckol, pff, and -phloroeckol exhibited potent antiviral activity, with ic ranging from . ± . to . ± . lm against porcine epidemic diarrhea. these compounds completely blocked the binding of viral spike protein to sialic acids at concentrations less than . lm by hemagglutination inhibition. pff and dieckol inhibited viral replication with ic values of . ± . and . ± . lm, respectively, in the post-treatment assay and exhibited stronger inhibition of viral rna and viral protein synthesis in later stages ( and h) than in early stages ( and h), suggesting their potential as natural therapeutic drugs against coronavirus infection ). dieckol and , -bieckol (hexamers) isolated from ek were tested against the food-borne pathogenic bacteria campylobacter jejuni with an mic of mg/l and . lmol/ml, respectively, which were effective against mrsa as determined by a broth microdilution method. the bactericidal effects of the phlorotannins were more pronounced than those of the catechins . one study showed that n-buoh fractions with isolated phlorotannins (tpa, eckol, and dieckol) increased glycerol secretion and reduced the regulation of adipogenic transcription factors, pparc, ccaat/enhancer-binding protein (c/ebpa), and tnfa. those phlorotannins also reduced the differentiation-dependent factor /srebp- c and downstream genes such as fabp- , fatty acid transport protein- , fas, leptin, and acs , whereas they increased the mrna expression of hormone-sensitive lipase while suppressing perilipin expression to treat obesity . dieckol from ec down-regulated the expression of pparc, c/ebpa, srebp- , and fabp- , showing anti-adipogenic effects on adipocyte differentiation through the activation and modulation of the ampk signaling pathway to improve obesity . enzyme-treated ec and its isolated compounds (eckol, dieckol and pff-a) exhibited potent adipogenic activity in t -l adipocytes. dieckol was found to be the major compound in the enzymatic extract, with a concentration of mg/g. in addition, lg/ml of ec extract inhibited glucose utilization and tg accumulation, as confirmed by oil red o staining. additionally, it decreased the expression of ccaat/(c/ebp)a, srebp- c, adipocyte-fabp, fas, and adiponectin. thus, ec prevented adipogenesis by affecting the activation of the c/ebpa signaling pathway and the resulting adipogenesis-related gene expression (kim and nam ) . in a similar report, dieckol from ec inhibited lipid accumulation via activation of ampka signaling and cell-cycle arrest in t -l cells and mouse and zebrafish models . seapolynol derived from ec inhibited triglyceride synthetic enzymes such as diacylglycerol acyltransferase , gpat , kruppellike factor (klf ), klf , c/ebpb, c/ebpd, and protein c-ets- , whereas it up-regulated klf , an anti-early adipogenic factor, preventing metabolic disorders . fucosterol was isolated from the strong anti-adipogenic ch cl -soluble fraction from es, with significant inhibition ( . %) of intracellular lipid accumulation at a non-toxic concentration. the anti-adipogenic activities of es, along with the isolated fucosterol, reduced lipid content in a concentration-dependent manner without showing any cytotoxicity. fucosterol treatment also yielded a decrease in the expression of the adipocyte markers pparc and c/ebpa in a concentration-dependent manner. taken together, these results suggest that fucosterol inhibits the expression of pparc and c/ebpa, resulting in a decrease in lipid accumulation in t -l pre-adipocytes, thereby indicating the potential of es and its bioactive component fucosterol as anti-obesity agents (jung et al. b ). similarly, fucosterol isolated from es down-regulated the insulin-triggered pi k/akt and erk pathways, which subsequently decreased the expression of adipogenic transcription factors, including pparc, c/ebpa, and srebp- . in addition, fucosterol enhanced sirt expression and decreased phospho-foxo expression, which resulted in the activation of foxo and revealed that fucosterol inhibited adipogenesis of t -l preadipocytes at concentrations of and lm through the modulation of the foxo signaling pathway (lee et al. b) . compound -b isolated from ek inhibited the differentiation of mouse embryonic fibroblasts and t -l cells into adipose cells by acting as the peptidyl prolyl cis/trans isomerase inhibitor responsible for the uptake of tg and the differentiation of fibroblasts into adipose cells in response to insulin stimulation without inducing cytotoxicity. this finding suggests that -b could be a lead drug candidate for obesity-related disorders (mori et al. ) . altogether, these results suggest that several ecklonia species possess potent activity that might be exploited in adjunct therapy for obesity. dieckol from ec inhibited mast cell activation and mast cell-mediated type i allergic reactions caused by igespecific antigen, mainly through the marked downstream signaling of fceri. a high dose of dieckol suppressed hypersensitive reactions, offering another target molecule for the prevention or treatment of mast cell-dependent allergic diseases (ahn et al. c) . six phlorotannins isolated from ek, pg, an unknown tetramer, eckol, pff-a, dieckol, and , bieckol, inhibited hyaluronidase at concentrations of , , [ , , , and lm, respectively. also, , , the strongest hyaluronidase inhibitor, acted as a competitive inhibitor with an inhibition constant (ki) of . lm . park et al. ( ) described the multi-faceted protection mechanisms of pg against oxidative stress caused by ionizing radiation in mice. pg inhibited apoptosis and strengthened hematopoiesis. it increased the viability of splenocytes without cytotoxicity and significantly enhanced the proliferation of splenocytes by limiting the increment of sub-g( ) dna contents via the inhibition of ros production in gy-irradiated splenocytes. in addition, pg significantly decreased dna damage and the number of apoptotic fragments in lymphocytes during oxidative stress and increased the counts of endogenous spleen colony forming units (cfus) compared with control mice exposed to ionizing radiation. a similar study confirmed that pg protected against small intestine damage caused by ionizing radiation, raising the apoptosis threshold of jejunal crypt cells. pg regenerated the intestinal crypts and down-regulated the expression level of proapoptotic molecules such as p , bax, and bak in the small intestine. pg further augmented antiapoptotic molecules such as bcl- and bcl-x(s/l) (ha et al. ) . cancer is the uncontrolled growth of cells that can invade and spread to distant sites of the body. cancer can have severe health consequences and is a leading cause of death. lung, prostate, colorectal, stomach, and liver cancer are the most common types of cancer in men, whereas breast, colorectal, lung, uterine cervix, and stomach cancer are the most common types among women. more than % of cancer deaths could be prevented by modifying or avoiding key risk factors. cancer results from a mutation in the chromosomal dna of a normal cell, which can be triggered by both external factors (tobacco, alcohol, chemicals, infectious agents, and radiation) and internal factors (hormones, immune conditions, inherited mutations, and mutations occurring in metabolism) (croce ; ferlay et al. ) . in korea, cancer accounts for one in four deaths ( . %), and more than , new cancer cases were diagnosed in . although antineoplastic drugs and chemotherapy are available, the deleterious effects of those medications have driven researchers to derive new drug candidates from natural products. dieckol isolated from ec prevented n-nitrosodiethylamine (ndea)-induced rat hepatocarcinogenesis. dieckol administered orally ( mg/kg body weight) for weeks with . % ndea through the drinking water reversed the activities of hepatic marker enzymes such as aspartate transaminase, alanine transaminase, alp, gamma glutamyl transferase, lactate dehydrogenase, a-fetoprotein, and total bilirubin and increased the elevation of cytochrome p . dieckol also decreased lipid peroxidative markers (tbars, lipid hydroperoxides, protein carbonyl content, and conjugated dienes) and decreased the antioxidant cascade viz enzymatic antioxidants (such as sod, cat, glutathione peroxidase, gst, glutathione reductase) and non-enzymatic antioxidants (such as reduced glutathione, vitamin c, and vitamin e). dieckol was more effective at mg/kg than at and mg/kg body weight and protected the liver from cancer (sadeeshkumar et al. ) . likewise, dieckol showed anti-breast cancer activity by regulating the expression of metastasis-related genes ). ec and its major phlorotannin (dieckol) increased the tumor growth-inhibitory effect of cisplatin and reduced cisplatin-induced nephrotoxicity and weight loss in skov -bearing mice. furthermore, they enhanced cisplatin-induced apoptosis by stimulating caspases in skov and a ovarian cancer cells via down-regulation of akt and nf-jb signaling. thus, ec suppressed cisplatin-induced ros production and cell death in normal hek kidney cells, and its major compound dieckol evidently enhanced the suppression of tumor growth by cisplatin with less weight loss and kidney damage in a mouse model . dieckol from ec exhibited cytotoxic effects on a and skov ovarian cancer cells, induced the apoptosis of skov cells, and suppressed tumor growth without any significant adverse effect in the skov -bearing mouse model. furthermore, it activated caspase- ,- , and - ; caused mitochondrial dysfunction; and suppressed the levels of anti-apoptotic proteins. thus, dieckol enhanced intracellular ros, and the antioxidant n-acetyl-l-cysteine (nac) noticeably reversed the caspase activation, cytochrome c release, bcl- downregulation, and apoptosis caused by dieckol through akt and p ). in another investigation, ahn et al. ( b) showed the anticancer effects of crude polysaccharides isolated from ec enzymatic extracts, using amyloglucosidase (amg), viscozyme, protamex, and alcalase enzymes against a colon cancer cell line. among the tested extracts, crude polysaccharides of protamex showed the highest inhibitory effect against the growth of ct- cells and dose-dependently increased the formation of apoptotic bodies and the percentage of sub-g dna content. furthermore, crude polysaccharides of protamex activated caspase and parp to regulate the expression of bax and bcl- and showed the highest inhibitory effect against the growth of ct- cells, attributed to their high fucose and sulfated group content, demonstrating anticancer effects on colon cancer cells via regulation of the bcl- /bax signal pathway. additionally, dieckol isolated from es reduced the number of viable cells and increased the number of apoptotic cells, increased the expression levels of cleaved caspases-( , , , and ) and cleaved poly(adp-ribose) polymerase, increased the permeability of mitochondrial membranes, and increased the release of cytochrome c from mitochondria into the cytosol with an apoptosis-inducing factor. thus, dieckol induced apoptosis via the activation of both death receptors and mitochondrial-dependent pathways in human hepatocellular carcinoma (hep b) cells (yoon et al. ). the skin is the human body's largest organ and is colonized by diverse microorganisms, most of which are harmless or even beneficial to their host. it acts as an anatomical barrier that is tough when intact and prevents the entry and colonization of many microbes (grice and segre ) . however, continuous exposure to ultraviolet (uv) light leads to various complications correlated with various pathological consequences, such as photo-damage of the skin, which is characterized by distinct alterations in the composition of the dermal extracellular matrix and results in wrinkles, laxity, coarseness, mottled pigmentation, and histological changes that include increased epidermal thickness and connective tissue alteration (rittie and fisher ) . therefore, varieties of anti-photoaging or photoprotective compounds from algae and other marine organisms have been isolated. dhe from ec exerted a preventive effect against uvbinduced apoptosis in human keratinocyte (hacat) cells, indicating its benefit as a repair agent for skin damage caused by uvb (ryu et al. ) . fucodiphlorethol g from ec reduced uvb radiation-induced loss of mitochondrial membrane potential, the generation of apoptotic cells, and active caspase- expression. thus, it prevented oxidative damage-mediated apoptosis induced by uvb irradiation (kim et al. f) . dhe from es inhibited cellular melanin content and the expression of melanogenesis-related proteins, including microphthalmia-associated transcription factor and tyrosinase and tyrosinase-related proteins trp- and trp- , stimulating the phosphorylation of akt in a dose-dependent manner without affecting the phosphorylation of erk (lee et al. b ). angiotensin-i-converting enzyme (ace) plays an important physiological role in the regulation of blood pressure by converting angiotensin i to angiotensin ii, a potent vasoconstrictor. some commercial drugs, such as captopril, ramipril, lisinopril, and enalapril, have unfortunate side effects like cough, taste disturbances, skin rashes or angioneurotic edema, so novel compounds such as phlorotannins have been derived from marine organisms as potential ace inhibitors . jung et al. ( ) reported that, among the seven enzymatically hydroxylated brown algal species, ec was a potent ace inhibitor with an ic value of . lg/ml. eckol, pff-a, and dieckol from es showed potent ace inhibiting activity with ic values of . ± . , . ± . , and . ± . lm, respectively, showing the importance of es. the human immunodeficiency virus (hiv) infects cells of the immune system by destroying or impairing their function, ultimately causing immune deficiency and opening the door to opportunistic infections, thereby causing acquired immune deficiency syndrome (aids). approximately . million people were living with hiv at the end of . as of mid- , . million people were receiving antiretroviral treatment worldwide. seven out of ten pregnant women living with hiv received anti-retroviral treatment (who ). more than approved commercial drugs are available for the treatment of aids. these drugs keep the disease under control; however, they are often associated with the emergence of cross-resistant hiv strains and various side-effects (sánchez and holguín ; haas et al. ). so, the need for potential nutraceuticals, especially from marine organisms, to fight this vicious disease is vital. , -dieckol from ec at noncytotoxic concentrations repressed hiv- activated syncytia formation, lytic effects, and viral p antigen production. furthermore, it selectively inhibited the activity of hiv- reverse transcriptase enzyme with % inhibition at lm. therefore, it showed high potential and could be considered as a drug candidate for the development of a new generation of therapeutic agents (karadeniz et al. ) . other pharmacological activities include immunomodulatory, aphrodisiac, anti-hair loss, hearing repair, urinary tract infection remedy, hair growth performance, cosmetic whitening, osteoarthritis, and bone-related conditions. one study suggested that sulfated polysaccharides from ec induced t and b cell responses via both the jnk and nf-kb pathways . ecklonia bicyclis together with the novel drug tradamixina improved male sexual function in elderly men, particularly libido, mild-moderate erectile dysfunction, ejaculation function, and sexual quality of life (sansalone et al. ). purified polyphenols from ec increased fibroblast survival in human dermal papilla cells, preventing hair loss . dieckol isolated from ec suppressed ea.hy cell proliferation induced by vascular endothelial growth factor, demonstrating its anti-proliferative and anti-migratory effects through a mapk molecular signaling cascade and suggesting its potential as an anti-angiogenic candidate . dhe from ec enhanced osteoblast differentiation, as evidenced by increased cell proliferation, alkaline phosphatase activity, and intracellular cell mineralization, along with enhanced levels of osteoblastogenesis markers at lm in mc t -e pre-osteoblasts. furthermore, dhe up-regulated phosphorylated erk and c-jnk, which were also stimulated by the bmp signaling pathway . a polyphenol-rich extract from ec showed potent radical-scavenging activity and decreased the abr threshold shifts, suggesting its potential as a preventive agent against temporary threshold shift . water-soluble sulfated fucans isolated from ec induced the degradation of ij-b and the phosphorylation of mapk in raw . cells, implying that they might stimulate raw . cells through the activation of the nf-jb and mapk pathways (cao et al. ) . the etoac fraction of ec resulted in elongation of the hair shaft in cultured human hair follicles and activated transition of the hair cycle from the telogen to the anagen phase in the dorsal skin of c bl/ mice. furthermore, it induced an increase in igf- expression in human dermal papilla cells (bak et al. ) . ec and its phlorotannins tpa, eckol, and dieckol attenuated the pathophysiological consequences of osteoarthritis and enhanced osteoblast differentiation, as indicated by increased alkaline phosphatase activity and raised levels of osteoblastogenesis, preventing osteoporosis . undoubtedly, ecklonia species and their active metabolites are potential candidates for drug development, as shown by their plethora of activities against various diseases. our review provides current information on the biological and pharmacological potential of this genus, which could be further used for the development of nutraceuticals. screening and study of the interactions between these algae and their constituents and human systems threatened by disease need to continue. therefore, we recommend rapid screening and isolation to develop novel functional ingredients from ecklonia species. the future of pharmaceuticals based on natural products seems promising. neuroinflammation, oxidative stress and the pathogenesis of alzheimer's disease review: hepatoprotective activity of spirulina species the jnk/nfjb pathway is required to activate murine lymphocytes induced by a sulfated polysaccharide from ecklonia cava dieckol, isolated from the edible brown algae ecklonia cava, induces apoptosis of ovarian cancer cells and inhibits tumor xenograft growth a sulfated polysaccharide of ecklonia cava inhibits the growth of colon cancer cells by inducing apoptosis dieckol, a phlorotannin of ecklonia cava, suppresses ige-mediated mast cell activation and passive cutaneous anaphylactic reaction dioxinodehydroeckol enhances the differentiation of osteoblasts by regulating the expression of phospho-smad / / anti-hiv- activity of phloroglucinol derivative, , -bieckol, from ecklonia cava evaluation of effective mmp inhibitors from eight different brown algae in human fibrosarcoma ht cells ecklonia cava promotes hair growth radioprotective potential of mint: a brief review protective effects of ecklonia stolonifera extract on ethanol-induced fatty liver in rats the global epidemic of obesity: an overview water soluble sulfated-fucans with immune-enhancing properties from ecklonia cava possible role of oxidative stress in the pathogenesis of hypertension dieckol, an edible seaweed polyphenol, retards rotenone-induced neurotoxicity and a-synuclein aggregation in human dopaminergic neuronal cells protective effect of a purified polyphenolic extract from ecklonia cava against noise-induced hearing loss: prevention of temporary threshold shift improved antioxidant activities of brown seaweed ecklonia radiata extracts prepared by microwave-assisted enzymatic extraction impact of extraction processes on prebiotic potential of the brown seaweed ecklonia radiata by in vitro human gut bacteria fermentation marine polyphenol phlorotannins promote non-rapid eye movement sleep in mice via the benzodiazepine site of the gabaa receptor the cytoprotective effects of ethanol extract of ecklonia cava against oxidative stress are associated with upregulation of nrf -mediated ho- and nqo- expression through activation of the mapk pathway in vitro antibacterial and anti-inflammatory properties of seaweed extracts against acne inducing bacteria, propionibacterium acnes suppression of nf-jb by dieckol extracted from ecklonia cava negatively regulates lps induction of inducible nitric oxide synthase gene dieckol, a major phlorotannin in ecklonia cava, suppresses lipid accumulation in the adipocytes of high-fat diet-fed zebrafish and mice: inhibition of early adipogenesis via cell-cycle arrest and ampka activation multifunctional activity of polyphenolic compounds associated with a potential for alzheimer's disease therapy from ecklonia cava comparison of ecklonia cava, ecklonia stolonifera and eisenia bicyclis for phlorotannin extraction oncogenes and cancer dieckol attenuates microglia-mediated neuronal cell death via erk, akt and nadph oxidase-mediated pathways enrichment of enzymatically mineralized gellan gum hydrogels with phlorotannin-rich ecklonia cava extract seanol Ò to endow antibacterial properties and promote mineralization effects of gametophytes of ecklonia kurome on the levels of glucose and triacylglycerol in 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seaweeds and phlorotannins from the brown alga ecklonia stolonifera on glucose-mediated protein damage and rat lens aldose reductase kinetics and molecular docking studies of an antidiabetic complication inhibitor fucosterol from edible brown algae eisenia bicyclis and ecklonia stolonifera inhibitory effects of histamine production in mackerel muscle by medicinal herbs and seaweed extracts protective effect of the edible brown alga ecklonia stolonifera on doxorubicininduced hepatotoxicity in primary rat hepatocytes anti-adipogenic activity of the edible brown alga ecklonia stolonifera and its constituent fucosterol in t -l adipocytes kinetics and molecular docking studies of fucosterol and fucoxanthin, bace inhibitors from brown algae undaria pinnatifida and ecklonia stolonifera antibacterial activities of marine epibiotic bacteria isolated from brown algae of japan antioxidant and anti-inflammatory activities of ventol, a phlorotannin-rich natural agent derived from ecklonia cava, and its effect on proteoglycan degradation in cartilage explant culture a new phlorotannin from the brown alga ecklonia stolonifera inhibitory phlorotannins from the edible brown alga ecklonia stolonifera on total reactive oxygen species (ros) generation protective effect of marine algae phlorotannins against aaph-induced oxidative stress in zebrafish embryo dieckol isolated from brown seaweed ecklonia cava attenuates type ii diabetes in db/db mouse model protective effect of dieckol isolated from ecklonia cava against ethanol caused damage in vitro and in zebrafish model phlorotannin-rich ecklonia cava reduces the production of betaamyloid by modulating alpha-and gamma-secretase expression and activity dieckol, a component of ecklonia cava, suppresses the production of mdc/ccl via down-regulating stat pathway in interferon-c stimulated hacat human keratinocytes acetylcholinesterase inhibitory activity of phlorotannins isolated from the brown alga, ecklonia maxima (osbeck) papenfuss 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-oxoguanine dna glycosylase cytoprotective effect of eckol against oxidative stress-induced mitochondrial dysfunction: involvement of the foxo a/ampk pathway protective effect of fucoidan against aaph-induced oxidative stress in zebrafish model the edible brown seaweed ecklonia cava reduces hypersensitivity in postoperative and neuropathic pain models in rats fucodiphlorethol g purified from ecklonia cava suppresses ultraviolet b radiationinduced oxidative stress and cellular damage inhibitory effects of brown algae extracts on histamine production in mackerel muscle via inhibition of growth and histidine decarboxylase activity of morganella morganii protective effect of marine brown algal polyphenols against oxidative stressed zebrafish with high glucose first evidence that ecklonia cavaderived dieckol attenuates mcf- human breast carcinoma cell migration synergistic antibacterial activity of ecklonia cava extract against anti-biotic resistant enterococcus faecalis winogradskyella 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of ros and proinflammatory enzymes in high-glucose-induced human umbilical vein endothelial cells -bieckol protects insulinoma cells against high glucose-induced glucotoxicity by reducing oxidative stress and apoptosis polyphenol-rich fraction of ecklonia cava improves nonalcoholic fatty liver disease in high fat diet-fed mice antimicrobial action of compounds from marine seaweed oxidative stress in the pathogenesis of colorectal cancer: cause or consequence? antibacterial activity of the extracts of marine red and brown algae algaebacteria interactions: evolution, ecology and emerging applications global prevalence of diabetes: estimates for the year and projections for potential antiradical and alpha-glucosidase inhibitors from ecklonia maxima (osbeck) papenfuss uv-light-induced signal cascades and skin aging anti-obesity drugs: past, present and future dioxinodehydroeckol protects human keratinocyte cells from uvb-induced apoptosis modulated by related genes bax/bcl- and caspase pathway protective effects of dieckol on n-nitrosodiethylamine induced hepatocarcinogenesis in rats drug resistance in the hiv- -infected paediatric population worldwide: a systematic review alga ecklonia bicyclis, tribulus terrestris, and glucosamine oligosaccharide improve erectile function, sexual quality of life, and ejaculation function in patients with moderate mild-moderate erectile dysfunction: a prospective, randomized, placebo-controlled, single-blinded study jp renew distributors lombard street heparinoid-active sulphated polysaccharides from marine algae as potential blood anticoagulant agents inhibitory activity of brown algal phlorotannins against hyaluronidase antioxidant activities of phlorotannins isolated from japanese laminariaceae enhancement of human hair growth using ecklonia cava polyphenols in vitro screening for anti-dementia activities of seaweed extracts inhibitory effect of extracts from the brown alga, ecklonia stolonifera, on enzymes responsible for 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polyphenol has a protective effect against ethanolinduced liver injury in a cyclic amp-dependent manner -bieckol, isolated from edible brown algae, exerts its anti-inflammatory effects through inhibition of nf-jb signaling and ros production in lps-stimulated macrophages brown algae phlorotannins enhance the tumoricidal effect of cisplatin and ameliorate cisplatin nephrotoxicity phloroglucinol attenuates the cognitive deficits of the xfad mouse model of alzheimer's disease protective effect of brown alga phlorotannins against hyper-inflammatory responses in lipopolysaccharide-induced sepsis models dieckol, isolated from ecklonia stolonifera, induces apoptosis in human hepatocellular carcinoma hep b cells inhibitory effect of chlorophyll c from brown algae, sargassum horneri, on degranulation of rbl- h cells isolation and structural determination of two novel phlorotannins from the brown alga ecklonia kurome okamura, and their radical scavenging activities phlorofucofuroeckol a isolated from ecklonia cava alleviates postprandial hyperglycemia in diabetic mice acknowledgements this research was supported by the basic science research program through the national research foundation of korea (nrf), funded by the ministry of education ( r a a ). conflicts of interest the authors declare no conflicts of interest. key: cord- - hiiw authors: keshavarz, mohsen; solaymani-mohammadi, farid; namdari, haideh; arjeini, yaser; mousavi, mohammad javad; rezaei, farhad title: metabolic host response and therapeutic approaches to influenza infection date: - - journal: cell mol biol lett doi: . /s - - - sha: doc_id: cord_uid: hiiw based on available metabolomic studies, influenza infection affects a variety of cellular metabolic pathways to ensure an optimal environment for its replication and production of viral particles. following infection, glucose uptake and aerobic glycolysis increase in infected cells continually, which results in higher glucose consumption. the pentose phosphate shunt, as another glucose-consuming pathway, is enhanced by influenza infection to help produce more nucleotides, especially atp. regarding lipid species, following infection, levels of triglycerides, phospholipids, and several lipid derivatives undergo perturbations, some of which are associated with inflammatory responses. also, mitochondrial fatty acid β-oxidation decreases significantly simultaneously with an increase in biosynthesis of fatty acids and membrane lipids. moreover, essential amino acids are demonstrated to decline in infected tissues due to the production of large amounts of viral and cellular proteins. immune responses against influenza infection, on the other hand, could significantly affect metabolic pathways. mainly, interferon (ifn) production following viral infection affects cell function via alteration in amino acid synthesis, membrane composition, and lipid metabolism. understanding metabolic alterations required for influenza virus replication has revealed novel therapeutic methods based on targeted inhibition of these cellular metabolic pathways. influenza virus infection (ivi) is one of the most common infectious agents, capable of infecting a variety of avian and mammalian species. the virus is responsible for seasonal epidemics, leading to - million severe infections and , - , deaths annually [ , ] . despite the annual vaccination program, the high mortality rate caused by influenza infection and its various complications, including chronic lung disease, cardiac disease, asthma, and metabolic disorders, is yet to be adequately addressed [ ] [ ] [ ] . in , eagle et al. first indicated that the addition of glucose to hela cell medium could promote the generation of poliovirus progeny [ ] . results of a published study showed that the replication of the influenza virus depends on host cellular metabolism such that metabolites including nucleic acids, proteins, glycoproteins, and lipids are crucially required for the life cycle of the influenza virus [ ] . recent research on a mouse model showed that influenza infection could affect more than metabolite markers in serum, lung, and bronchoalveolar lavage fluid [ ] . acquiring the required materials from the host cell to self-replicate, the virus can disrupt biochemical processes such as glycolysis, fatty acid (fa) synthesis, and glutamine pathways [ ] . it is of particular importance for scientists to broaden their horizon on the metabolic changes during influenza infection, which in turn paves the way for preventing life-threatening consequences. influenza infection actively provokes the pro-oxidant condition in the host cell to facilitate viral proliferation and pathogenesis. increased expression of influenza m protein can activate protein kinase c and increase reactive oxygen species (ros) production [ ] . on the other hand, pb -f decreases superoxide anion dismutase (sod ) expression and consequently disrupts the ros scavenging process [ ] . in people with influenza infection, increased levels of dna, lipid, and protein oxidation products are found in plasma and urine [ ] [ ] [ ] . also, increased levels of ros and inducible nitric oxide synthase (inos) have been observed as markers of oxidative stress in the lungs of people who died due to pandemic influenza infection [ ] . ros-producing enzymes induced by influenza infection mainly include nadph oxidase (nox) and xanthine oxidase, upregulation of which causes the impaired defensive function of antioxidants [ ] . an increase in ros production, along with impaired antioxidant function, ultimately leads to a profound change in redox homeostasis of the cell [ ] [ ] [ ] [ ] . nox is a phagocytic enzyme that is involved in the production of ros induced by influenza virus [ ] [ ] [ ] [ ] , and impaired nox expression results in a lack of increased rns and ros production following influenza infection [ ] . xanthine oxidase is also an ros-producing enzyme that is induced by influenza infection [ , ] , and its inhibition can hinder ros increase in the cell. on the other hand, increased expression of sod reduces influenza virus titers within the cell [ ] . it is also reported that influenza infection significantly increases ros production by inducing nox , and the proliferation of this virus in lung epithelial cells is dependent on redox-sensitive pathways activated by nox -derived ros [ ] . glutathione (gsh) is a vital antioxidant in the cell, and its cellular content is inversely related to influenza virus replication in the cell [ , ] . it is indicated that higher levels of gsh, antioxidant enzymes such as glutathione peroxidase, and the anti-apoptotic protein bcl- in the lungs of female mice result in superior resistance of these mice to influenza infection. in contrast, male mice are more susceptible to this infection due to higher expression of nox . this difference is due primarily to the higher ability of female mice to maintain cellular redox homeostasis [ ] . amatore et al. also showed that an increase in gsh content in organs by affecting gsh-dependent antiviral pathways strengthens the immune system, in particular th cell response, and decreases viral replication [ ] . however, gsh depletion results in a deviation of the response towards th cells [ ] . furthermore, oxidative stress following infection can induce the transcription factor nf-kb, which subsequently leads to increased levels of inflammatory cytokines, including interleukin (il)- β, il- , ifn, and tnf [ , ] . ifns are one of these cytokines that trigger and affect t cell metabolism via mediating glucose uptake, glycolysis, and lipid synthesis. ifn can also exert its function on metabolic changes by producing several mediators including indoleamine- , -dioxygenase (ido) and nitric oxide (no), both of which appear to have either an inducible or an inhibitory role in viral replication [ ] . since tryptophan is critical for t cell proliferation, depletion of this amino acid by ido suppresses the immune system through the stimulation of t regulatory cells. no, on the other hand, inhibits viral replication via changes in the structure of viral proteins [ ] . in this review, we first discuss the metabolic abnormalities during influenza infection and then shed light on the role of immunometabolites that regulate cellular metabolism. the following sections summarize recent evidence about the novel therapeutic approaches that target metabolic pathways in influenza infection. viruses take advantage of various cellular mechanisms to replicate efficiently. vital metabolic pathways of host cells are one of the most widely used mechanisms targeted by viruses, resulting in considerable changes. in this context, studies have revealed that various human viruses, such as cytomegalovirus [ ] [ ] [ ] , rubella [ , ] , dengue [ ] , mumps [ ] , poliovirus [ , ] and reovirus [ ] can strongly affect host cell glycolysis, lipid metabolism, and glutaminolysis. furthermore, a review by sanchez and lagunoff delineated the activation of these metabolic processes by several viruses [ ] . as will be discussed in this section, influenza as a highly pathogenic human virus interferes tremendously with the host metabolic cycles and thereby forces them to produce viral particles more efficiently. the metabolism and concentration of glucose in the cell play a cardinal role in the homeostasis of cellular metabolic procedures [ ] . shortly after the onset of ivi, the rate of glucose uptake by infected cells increases continually, and the subsequently enhanced glycolysis results in higher glucose consumption, production of viral particles [ , ] , and extracellular concentration of lactate. the relationship between glycolysis and ivi has shown that influenza infection at a higher multiplicity of infection (moi) raises the glycolytic activity of the cells [ ] . in a study by kohio and adamson, a dose-specific increase in influenza infection was associated with higher glucose levels, whereas the treatment of cells with glycolysis inhibitors remarkably suppressed the viral replication. however, the viral infection could be retriggered by adding atp to the cell environment. this study revealed that enhancing vacuolar-type atpase (a proton pump essential for influenza uncoating) via increasing glucose metabolism and, as a result, higher available atp resources, augments the virus infection [ ] . the observations, as mentioned above, reveal a significant increase in atp and glucose consumption within cells following influenza infection and also highlight the dependence of the influenza virus on the glycolysis pathway for energy production. the viral replication has the highest use of atp during influenza infection, releasing large quantities of energy in the form of heat. this process can increase the temperature of infected cells by - °c. as viral proliferation increases, the cellular atp level drops sharply, resulting in reduced potential and stability of the mitochondrial membrane [ ] . based on available results, patients with metabolic disorders can develop more severe influenza infection compared to healthy hosts. there have been several studies showing that diabetes can increase the risk of influenza infection, the severity of the disease, and the fatal consequences of this infection [ ] [ ] [ ] [ ] . about % of patients with type diabetes are overweight, and obesity is a significant risk factor for severe influenza infection [ ] . this reinforcing effect of diabetes on influenza may be due to the inhibitory effect of hyperglycemia on the immune system [ ] [ ] [ ] . it has been shown that this hyperglycemia-associated immunosuppression and susceptibility to influenza infection can be alleviated by insulin administration and diabetes control [ ] . on the other hand, the pentose phosphate pathway (ppp), as another glucose-consuming pathway reported to be enhanced by ivi [ ] , contributes to a higher yield of nucleotides and atp for viral replication [ ] . significant up-regulation of the ppp key enzymes in influenza-infected cells, including glucose -phosphate dehydrogenase (g pd) and -phosphogluconate dehydrogenase ( pgd), was reported by janke et al. [ ] . g pd enzyme is also responsible for the generation of nadph [ ] , a critical component of fatty acid biosynthesis. the level of g pd activity specifies the ability of the cell to clear the accumulated ros. cells with an average g pd level can retain the appropriate gsh/gssg ratio and keep the ros production at a tolerably low level, indicating that g pd activity has an inverse correlation with cellular ros level [ ] . disruption of redox balance has been shown to contribute to replication and virulence of several viruses [ ] [ ] [ ] , and g pd deficiency can cause this disruption. despite the results reported by janke et al., g pd activity seems to have an inverse relation with some other respiratory viral infections. for example, in an in vitro study, after infection with human coronavirus (hcov) e, the production of viral particles in g pd-deficient or g pd-knockdown cells was higher than in healthy cells, and this was correlated with increased oxidant production [ ] . molecular mechanisms through which the virus can control the metabolic pathways have been thoroughly identified. smallwood et al. have shown that an increase in glucose uptake, glycolysis, and glutaminolysis following influenza infection may be related to the loss of pi k/akt/mtor pathway homeostasis and subsequent increase in c-myc expression in the infected cells [ ] . regarding the available results, the mechanistic target of rapamycin complex (mtorc ) and mtorc signaling can be activated by a variety of influenza virus proteins. the viral hemagglutinin (ha) protein, along with virus replication, can upregulate pdpk -mediated phosphorylation and activate akt, which is required for induction of the mtorc signaling pathway by the influenza virus. on the other hand, influenza m protein is capable of down-regulation of the mtorc inhibitor redd , thereby enhancing the mtorc activation [ ] . mtorc signaling, in turn, promotes c-myc expression at the translational level [ ] . additionally, the ns protein can effectively promote the activity of mtorc , which, in turn, upregulates c-myc through foxo inhibition [ ] . moreover, akt-dependent inactivation of foxos can increase glycolysis [ , ] by removing the suppressive force of c-myc [ ] [ ] [ ] . myc enhances glycolysis by upregulating expression of the glucose transporter glut , glycolytic genes, and lactate dehydrogenase (ldh), as the converter of pyruvate to lactate [ , ] . mtorc also mediates upregulation of hypoxia-inducible factor- α (hif- α), a factor that increases the expression of various genes [ ] , including several glycolytic enzymes, glucose transporters, and ldh [ ] [ ] [ ] . akt is able to promote the expression and membrane localization of glut as well as the function of phosphofructokinase [ , ] . furthermore, akt is demonstrated to activate srebp in an mtorc -dependent manner [ ] and to upregulate srebp by enhancing the stability of its processed form [ ] . srebp is shown to be required for the mtorc induced increase in the expression of g pd, which is the rate-limiting enzyme of the ppp oxidative branch [ ] . in addition to the above-mentioned metabolic pathways, the influenza virus exhibits disruptive effects on some other metabolic processes, which gives rise to metabolic disorders and atp crisis. previous studies have found that the influenza infection increases the cellular synthesis of fatty acids [ ] , with some of their derivatives, including eicosanoids [ ] , and these molecules are natural endogenous ligands and stimulators of peroxisome proliferation-activated receptors (ppars) [ ] [ ] [ ] . ppars are a group of nuclear receptor proteins that act as transcription factors and regulate the expression of different genes [ ] involved in cellular differentiation and metabolism of carbohydrates, lipids, and proteins [ ] . furthermore, all types of ppars discovered so far are able to suppress the activity of the pyruvate dehydrogenase (pdh) enzyme (known as a catalyzer of the oxidative decarboxylation of pyruvate leading to acetyl-coa production) in various organs through the upregulation of pyruvate dehydrogenase kinase (pdk)- [ ] . in this respect, extremely low pdh enzyme activity has been found after influenza infection in vitro. enhanced pdk -mediated inhibition of pdh has been found in the lung tissue of influenza-infected mice. this enzyme inhibition contributes to an erroneous process, which causes significant disruption of glucose oxidation, cellular respiration, and lipid metabolism ( fig. ) [ , ] . following infection, levels of phospholipids and several lipid derivatives undergo perturbations. several lines of evidence have shown that in obese mice due to abnormalities in the fig. metabolic changes caused by influenza infection and related mechanisms. several anabolic and catabolic processes can be affected: higher glucose uptake and metabolism in glycolysis and pentose phosphate pathways, higher nucleotide catabolism, increase in biosynthesis of fatty acids including arachidonic acid, the precursor of proinflammatory lipids, and also enhanced glutaminolysis and protein synthesis. activation of mtorc & signaling and downstream factors by influenza infection may have an essential role in the upregulation of these metabolic processes. in addition, high atp consumption and reduced β-oxidation, as well as glucose oxidation by influenza infection, contribute to the atp crisis and hence influenza-related multi-organ failure [ ] . this influenza-mediated elevation of aa, consistent with inflammation, has also been reported in the same study [ ] . the accumulation of the above-mentioned proinflammatory lipids in the cell leads to the promoted synthesis of eicosanoids and inflammatory mediators, thus exacerbating post-infection necrosis, inflammation, and tissue damage in the lungs [ ] . in a prospective cohort study on influenza-infected subjects, lipid inflammatory mediators in serum samples of patients were mainly aa-derived oxylipins, including txb , -deoxy- , -pgd , -hete, , -dhet, -oxoete, lte , and -hpete. although all of these metabolites were shown to be elevated shortly after the infection, , -dhet and -oxoete levels remained considerably high up to weeks postinfection, indicating a constant pulmonary inflammation [ ] . the pulmonary surfactant system, which is involved in suppressing influenza infection in the respiratory tracts [ ] , can be disrupted due to significant influenza-induced changes in the abundance of different types of pc and pe species as the major components of surfactants [ , ] . tanner et al. proposed a principal correlation between influenza replication and choline lipids metabolism. they found an ivi-mediated reduction in ester-linked pc species as well as an increased level of sphingomyelin (sm) [ ] , probably connected with expending cellular choline stores for sm synthesis. this led to an increase in sm species within the infected cell. sm and short-chain fatty acid-containing ether-linked pc (epc) species were found in higher amounts in both infected cells and virions and therefore appeared to be involved in viral morphogenesis. on the other hand, long-chain fatty acidcontaining epc was increased in infected cells while having low levels in the structure of the virion, highlighting the role of this phospholipid in replication of the virus [ ] . evidently, higher production of these complex lipids in the cell will require increased biosynthesis of fatty acids. in this regard, the results of a study revealed that influenza infection could induce fatty acid biosynthesis and cholesterol metabolism in human lung basal epithelial tumor cells [ ] . since srebps are thoroughly identified as stimulators of expression of many genes involved in lipid and sterol biosynthesis, including fatty acid synthase [ ] [ ] [ ] , their upregulation by the influenza virus (through induction of mtorc signaling, as discussed earlier) may logically explain the increased rate of lipogenesis [ ] . a coincidence between increased fatty acid synthesis and a decline in fatty acid β-oxidation has been found during influenza infection, which is attributed to a variety of mechanisms directly or indirectly related to viral replication. for instance, the sharp increase of proinflammatory cytokines during influenza infection [ ] causes decreased hepatic fatty acid β-oxidation both in vitro and in vivo [ , ] , most likely through excessive nitric oxide and other related free radicals [ ] . in addition, increased temperature of cells during infection (which could be the result of virus replication and fever) causes heat stress which in turn can considerably downregulate carnitine palmitoyltransferase ii (cpt ii) activity and reduce the β-oxidation and atp levels in fibroblasts of influenza-associated encephalopathy patients and healthy volunteers [ ] . a study on influenza-infected mice demonstrated a significant depression in long hepatic chain fatty acid β-oxidation at both the mrna and protein level, as several β-oxidation essential enzymes were reduced by > % [ ] . a significant decrease in mitochondrial fatty acid β-oxidation simultaneously with increased biosynthesis of fatty acids and membrane lipids may reflect the fact that the virus stores structural lipids to produce more infectious particles. in addition to impaired metabolism in mitochondria, influenza infection induces severe peroxisomal lipid metabolism disorders, which can be inferred from abnormal levels of several specific long-chain fatty acids [ ] (fig. ) . the aforementioned metabolic processes are not the only pathways affected by influenza virus infection. this virus has the ability to induce higher consumption rates of glutamine during glutaminolysis, which can be attributed to transient c-myc overexpression [ ] . myc acts to regulate glutamine uptake and its utilization in the cell [ ] . it has been demonstrated that catalytic activity of glutaminase, as the key enzyme in glutaminolysis, greatly increases following the infection [ ] . moreover, essential amino acids, especially tryptophan, are other materials whose quantities have been shown to decline in infected tissues [ ] . mtorc can up-regulate protein synthesis through several downstream factors [ ] . thus, induction of mtorc signaling by the influenza virus leads to higher usage of essential amino acid storages for concurrent production of large amounts of viral and cellular proteins. infection of influenza virus can also alter the cellular level and metabolism of purines and pyrimidines [ , , ] , and is associated with both increased activities of nucleotide catabolism core enzymes including adenosine deaminase (ada) and xanthine oxidase (xo) and elevated levels of inosine, hypoxanthine, xanthine, and uric acid in serum and bronchoalveolar lavage fluid. enhanced catabolic degradation of nucleotides and their metabolites can facilitate the production of superoxide and contribute to the pathogenesis of influenza infection [ ] . interferons are well-known cytokines with a powerful capability of altering the cellular functions following viral infections. these alterations affect protein synthesis, composition of the membrane, cellular proliferation, and nutritional status [ ] . interferon stimulated genes (isgs) are the effector components whose transcription could be induced by type i ifns and ifnγ [ , ] . studies have recently underscored the general effect of ifns on the energy metabolism of cells, mostly by promoting glycolysis. for instance, ifnβ has been shown to induce the glucose uptake of embryonic fibroblasts in a pi /akt-dependent manner, thereby increasing atp production [ ] . it has also been demonstrated that type i ifn can stimulate oxygen consumption in a range of cells, including conventional dendritic cells (dcs), keratinocytes, and memory t cells [ ] . indeed, the high yield of atp and mitochondrial fitness guarantee the host cell's need for energy in plasmacytoid dcs (pdcs) and non-hematopoietic cells following challenges with viral pathogens [ ] . these studies emphasized the mediatory effect of type i ifn on glycolysis induction via ifnar , tyk , and stat . it has also been shown that influenza infection stimulates pdcs to enhance their glycolysis and develop a warburg-like remodeling of energy metabolism. this enhanced glycolysis leads to higher ifn production and, consequently, more potent antiviral activity [ ] . ifnγ induces metabolic reprogramming of m macrophages as a rapid increase in aerobic glycolysis, followed by a reduction in oxidative phosphorylation. this metabolic reprogramming maintains cell viability and the inflammatory response while reducing dependence on mitochondrial oxidative metabolism. excessive production of pro-inflammatory cytokines and chemokines in human monocytes/macrophages can be blocked by inhibition of aerobic glycolysis [ ] . also, activation of macrophages by ifnγ induces expression of the atp-citrate lyase enzyme (acly), and blockage of acly activity reduces the production of ros and nitric oxide [ ] . there is a strong consensus that influenza replication is crucially dependent on fatty acids [ ] , which makes it a fascinating target for therapeutic modalities [ ] . thus, the ability of ifn to channel the fas from biosynthesis to catabolism via fatty acid oxidation (fao) is currently known as a promising antiviral strategy in pdcs [ ] , which requires further research for more elucidation. several lines of current evidence have revealed the antiviral activity of type i ifn to be exerted through hampering glucosederived cholesterol and fatty acid synthesis [ , ] . sterol regulatory binding protein (srebp ), along with srebp , is known as the leading transcription factor which orchestrates the biosynthesis pathway of sterol, whose inhibited transcription and expression can be strongly mediated by ifns via ifnar [ ] (fig. ) . [ ] . this recognition can lead to the induction of an inflammatory response that, in turn, controls the replication and spread of the virus [ ] . h n , h n , and h n were correlated with increased transcription of the cytokine response in mice. severe infection with h n , h n , h n , or virus can lead to upregulation of inflammatory cytokine genes along with downregulation of lipid metabolism and coagulation genes [ ] . this uncontrolled proinflammatory response accompanied by an inadequate anti-inflammatory response is referred to as the cytokine storm [ ] . monocytes/macrophages, neutrophils, and lung epithelial cells have useful roles in the cytokine storm developed by influenza infection [ ] . severe cytokine storm, with greater levels of interferons and tumor necrosis factors, has been recognized in patients hospitalized due to influenza infection [ ] . such influenza-induced cytokine storms, together with viral virulence, can develop severe lung injury in patients [ , ] . it is believed that the level of the cytokine storm is directly associated with the severity of the disease caused by influenza infection [ , ] . some specific polymorphisms in immune system genes have determinative roles in the outcome of influenza infection. our previous studies have shown a relationship between cytokine gene polymorphisms and severity of the influenza disease. several cytokines were evaluated after influenza a/h n virus infection, among which il- rs gg and ag, gg and gt of il- (rs ) and il- (rs ) genotype tt polymorphisms were associated with increased risk of influenza infection. in contrast, il- β (rs ) (gg) and il- (rs ) gg and tg genotypes were associated with reduced risk of infection [ ] . in another study, an association between il- β rs and il- rs genotypes and severe influenza disease was found while il- rs and il- rs polymorphisms were not associated with influenza disease. also, lacking an a allele in il- rs could increase the risk of influenza a (h n ) infection [ ] . such polymorphisms in immune system genes may be associated with some metabolic changes and, in turn, may reinforce the metabolic disorders following influenza infection. however, additional studies are needed in this field to confirm or reject this opinion. ifns are known to be capable of depletion of polyamines to limit virus replication. polyamines are small ornithine-derived polycationic molecules which encompass three molecules: putrescine, spermidine, and spermine. spermidine and spermine depletion is one of the compelling mechanisms through which ifns produce their antiviral effects on the replication of rna viruses. mechanistically speaking, polyamines appear to play a pivotal role in the processes of rna transcription and protein translation of viruses, making them a promising target to combat viral infections [ ] . l-tryptophan is one of the nine essential amino acids with a remarkable role in immunosuppression and tolerance and is also essential in protein, kynurenine, and serotonin synthesis [ ] . ido is an intracellular enzyme that induces production of kynurenine from l-tryptophan, thereby acting to deplete tryptophan and modulate the immune system following viral infections [ ] . having two ifn-stimulated response elements and three ifnγ-activated sites in the promoter of ido, ifnγ acts as the most powerful inducer of ido expression [ ] . ido has also been shown to be expressed during influenza infection [ ] . dendritic cells, macrophages, and epithelial cells can express ido [ , ] , and since the primary target for replication of influenza is primarily found to be respiratory epithelial cells, understanding the role of ido during influenza infection is of particular importance. there exists a coincidence of peak ido and ifn-k expression during influenza infection. also, mouse lung airways considerably express ifn type i and iii following infection with influenza [ ] . these findings emphasize that there is upregulated expression and enhanced function of ido during influenza infection, which is found to be induced by ifn-i. moreover, ifn-i is thought to signal the adjacent cells via ifn-ir and stimulate them to produce ido [ ] . nonetheless, the ifn-mediated ido induction during influenza infection generally has undesirable consequences and establishes immune tolerance [ ] . indeed, an inhibitory effect of tryptophan depletion on t cell responses has been confirmed. also, ido induces kynurenine derivation from tryptophan, leading to stimulation of regulatory t cells [ ] . nowadays, ido is hypothesized to be part of the "metabolic, immune regulation," which plays a protective role in immune responses and inhibits the overreaction of these responses against influenza infection. a pleiotropic role has been attributed to ido during infections, which gives rise to the opposing outcomes (fig. ) [ ] . research on the role of ido in influenza infection has been mainly focused on the murine models of influenza infection, emphasizing the increased ido activity and its maximum expression correlated with increased lymphocyte numbers in the respiratory tract [ ] . nitric oxide (no) is a gaseous free radical with accessible vasodilatory and microbicidal functions [ ] . however, the antiviral effect of no has also been documented, leading to reduced viral load and more efficient clearance of infection [ ] . nitrosylation of viral molecules has been offered as an antiviral mechanism employed by no [ ] . also, no synthase-mediated generation of no leads to the depletion of l-arginine, thereby reducing the level of polyamines. thus, ifn-induced nos represents antiviral activities, and this, in turn, may exhibit another mechanism through which ifns hinder viral infections such as influenza [ ] . despite existing data regarding the antiviral activity of no, many studies have considered no as a double-edged sword with both pathogenic and viricidal effects. the role of no in the pathogenesis of pneumonia caused by influenza virus infection has been described in mice. the ifnγ response induces greatly increased levels of inos in the lungs of infected mice, leading to the production of a significant amount of no and peroxynitrite species, which are among the most important pathogenic factors in influenza virus-induced pneumonia in mice [ ] . uetani et al. also observed overexpression of the inos gene in human airway epithelial cells induced by influenza a virus infection [ ] . in addition, no produced by phagocytic cells has antiviral activity that is simultaneous with nonspecific damage of host cells and viral pathogenesis [ ] . in a survey by nin et al. on pandemic a/h n influenza infection, all cases showed increased levels of inos protein, tyrosine nitration, and oxygen free radicals, indicating the production of peroxynitrite. their results revealed the involvement of oxidative and nitrative stress in the pathogenesis of h n influenza virus-induced acute respiratory distress syndrome (ards) [ ] . influenza-induced cytokines such as ifnγ stimulate no release from human airway epithelial cells [ ] [ ] [ ] . as mentioned previously, the influenza infection induces upregulation of hif- α. interestingly hif- α-knockout macrophages show decreased expression of inos after ifnγ stimulation [ ] , indicating the possible involvement of hif- α in influenza pathogenesis. it has been shown that infection of h n and viruses induces higher levels of no in mice compared to the seasonal h n virus and, as a result, they develop more intense pathogenic outcomes, while mice with inos deficiency showed reduced morbidity, mortality, and cytokine production in the lungs following h n and virus infection. also, systemic administration of nos inhibitor could postpone weight loss and death among virus-infected mice [ ] . in another survey, the delivery of no to influenza-infected mice could not improve the lung infection and survival of mice, indicating that no administration was not a suitable treatment strategy for influenza although this was probably due to the difficulty of determining concentrations of no that are both viricidal and safe in host airways [ ] . also, it is reported that no released from s-nitroso-n-acetylpenicillamine (snap) reduces the replication of influenza virus in a dose-dependent manner. the production of no in airway epithelial cells can lead to antiviral rather than harmful effects following influenza infection provided that its production is precisely controlled [ ] . since the influenza virus affects about % of the world population annually, preventive and therapeutic approaches require much closer attention. therapeutic drugs for influenza infection fall into three groups: ) neuraminidase inhibitors (zanamivir, oseltamivir, laninamivir, peramivir), ) m inhibitors (rimantadine and amantadine), and ) polymerase inhibitors (favipiravir) [ , ] . antiviral drug resistance has recently emerged as a global problem which can bring about a remarkable financial and social burden [ ] . therefore, further research is urgently needed to develop novel and promising antiviral drugs. many of the metabolic pathways in influenza infections are increasingly changing, dampening of which appears to hamper the virus replication. one of the newly developed strategies aiming to hinder influenza infection is targeting metabolic pathways and restoration of hemostasis in cells ( table ). the pi k/mtor signaling pathway has been shown to play a pivotal role in a variety of cellular pathways, including proliferation and nutrient uptake, and its activation increases the glucose uptake through the up-regulation of cell surface glucose transporter [ ] . bez alters glucose metabolism via blockage of the pi k/mtor pathway, and some clinical trials are underway to assess this strategy in cancer therapy (smith et al., ). on the other hand, several lines of evidence have demonstrated that sirna targets the pi k-akt-mtor pathway, thereby warding off influenza infection [ ] . in a new study by smallwood et al., it was found that although bez did not interfere with the early stages of the infection, it could finally reduce the viral progeny and result in prolonged survival in mice challenged by the influenza virus. indeed, bez induced hemostasis in the pi k/mtor pathway via phosphorylation of p and e-bp and through reconstitution of metabolic status, which was already altered by the virus [ ] . it has been found that there is an elevated level of pdk in lung, liver, and heart during influenza infection, while the levels of atp and pdh, a key enzyme in the regulation of glucose, lipid and atp levels in human cells, are shown to be reduced [ ] . furthermore, dichloroacetate (dca) is a pyruvate dehydrogenase kinase inhibitor with anti-tumor activity in a variety of carcinomas. studies have also indicated that diisopropylamine dichloroacetate (dada) could ameliorate the metabolism of hepatocytes in chronic liver disease [ ] . in a study by yamane et al. attempting to evaluate the effect of dada on influenza-infected mice (pr ), oral administration of dada was found to not only restore the activity of pdh and atp in affected organs but also suppress cytokine storm and viral replication [ ] . sterols are intermediate metabolites that play an essential role in a broad spectrum of biological pathways, including inflammation. research has shown that interferon production following the immune response in viral infection regulates sterol production paths. blanc et al. revealed that sterol metabolism pathway regulators such as simvastatin, zometa (zoledronic acid), and fpt inhibitor iii could effectively hinder h n influenza replication and cytokine production, which makes them promising therapeutic candidates in acute patients [ , ] . on the other hand, as mentioned earlier, srebps are transcription factors that have a critical role in the process of lipogenesis. studies have shown that these factors can play a variety of roles, such as energy supply and post-translational protein modification, as well as in the propagation of various groups of viruses such as influenza viruses. a study has shown that the am compound, which is a retinoid derivative, inhibits srebp-linked pathways, and it has antiviral activity against influenza a and coronavirus in vitro and in vivo [ ] . concerning the fact that mitochondria and glycolysis are two sources of energy production, they play vital roles in the regulation of innate immunity responses. during the immune system response, and especially the cytokine storm, following influenza infection, atp synthesis in the mitochondria decreases, leading to weakened innate immune responses (dengbing yao). studies have revealed that traditional herbal medicines have an important role in improving influenza-like symptoms in infected patients. results of a study demonstrated that pre-treatment of infected cells with hochuekkito (a traditional japanese herbal medicine) could activate both mitochondrial and glycolytic energy metabolism and thereby intensify symptoms [ ] . also, the effects of traditional chinese medicine (modified jiu wei qiang huo) on h n infected mice were evaluated in another study. the results showed that this herbal medicine could ameliorate weight loss and inflammatory mediators in infected mice through the regulation of amino acid, fatty acid, and arachidonic acid pathways [ ] . based on recent studies, influenza virus infection can interfere with cellular metabolic pathways either directly or indirectly via stimulation of immune system mediators. through enhancing the activity of the mtorc complex, the influenza virus strengthens several metabolic pathways, including glycolysis, glutaminolysis, pentose phosphate, and fatty acid synthesis, to provide more atp and structural materials for viral replication. on the other hand, β-oxidation suppression following viral infection can help to supply essential fatty acids for the synthesis of structural lipids. however, exhausting cellular atp resources due to virus replication, as well as an increase in pro-inflammatory lipid synthesis, will ultimately lead to irreversible cell damage. innate immune responses following influenza infection play a crucial role in metabolic alterations. ifn is one of these mediators that acts on several metabolites such as ido and no and thereby affects lipid and amino acid pathways. since the drug resistance in influenza infection is a global concern, research on designing novel therapeutic modalities to tackle pandemics is of particular importance. thus, a clear understanding of the metabolic alterations during influenza infection would be tremendously helpful for therapeutic purposes. induction of protective immune response to intranasal administration of influenza virus-like particles in a mouse model association of polymorphisms in inflammatory cytokines encoding genes with severe cases of influenza a/h n and b in an iranian population acute 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pluripotency and cell fate determination inhibition of influenza a virus replication by antagonism of a pi k-akt-mtor pathway member identified by gene-trap insertional mutagenesis superior anti-tumor efficacy of diisopropylamine dichloroacetate compared with dichloroacetate in a subcutaneous transplantation breast tumor model key: cord- - ftjqev authors: xu, yinlan; zheng, jiangang; sun, panpan; guo, jianhua; zheng, xiaozhong; sun, yaogui; fan, kuohai; yin, wei; li, hongquan; sun, na title: cepharanthine and curcumin inhibited mitochondrial apoptosis induced by pcv date: - - journal: bmc vet res doi: . /s - - - sha: doc_id: cord_uid: ftjqev background: porcine circovirus type (pcv ) is an immunosuppressive pathogen with high prevalence rate in pig farms. it has caused serious economic losses to the global pig industry. due to the rapid mutation of pcv strain and co-infection of different genotypes, vaccination could not eradicate the infection of pcv . it is necessary to screen and develop effective new compounds and explore their anti-apoptotic mechanism. the natural compounds were purchased, with a clear plant origin, chemical structure and content and specific biological activities. results: the maximum no-cytotoxic concentration (mntc) and % cytotoxic concentration (cc( )) of tested compounds were obtained by the cytopathologic effect (cpe) assay and ( -( , -dimethyithiazol- -yl)- , -diphenyltetrazolium bromide (mtt) method in pk- cells. the results of qpcr and western blot showed that, compared with the pcv infected group, the expression of cap in paeonol ( . mg/ml and . mg/ml), cepharanthine ( . mg/ml, . mg/ml and . mg/ml) and curcumin ( . mg/ml, . mg/ml and . mg/ml) treated groups were significantly lowered in a dose-dependent manner. the results of annexin v-fitc/pi, jc- , western blot and ros analysis showed that the expression of cleaved caspase- and bax were up-regulated bcl- was down-regulated in cepharanthine or curcumin treated groups, while ros and mmp value were decreased at different degrees and the apoptosis rate was reduced. in this study, ribavirin was used as a positive control. conclusions: paeonol, cepharanthine and curcumin have significant antiviral effect. and the pcv -induced mitochondrial apoptosis was mainly remitted by cepharanthine and curcumin. pcv is a dna virus, which is an important pathogen causing high infection rate and immunosuppression in pigs [ ] . the diseases caused by pcv mainly include postweaning multisystemic wasting syndrome (pmws), porcine dermatitis and nephropathy syndrome (pdns), porcine proliferative and necrotizing pneumonia (pnp), etc., which have caused serious economic losses to the global pig industry for about years [ ] . the capsid protein (cap) encoded by the orf -encoded gene of pcv is a major viral structural protein and an immunogen involving viral replication of pcv [ ] . studies have shown that pcv could induce apoptosis and decrease spleen lymphocytes in immune organs of balb/c mice and pigs [ , ] and apoptosis can be induced through endoplasmic reticulum (er) apoptosis, mitochondrial apoptosis, reactive oxygen species (ros) regulation, etc. [ ] [ ] [ ] [ ] . it has been reported that vaccination could not eradicate the infection of pcv , due to the rapid mutation of pcv strain and co-infection of different genotypes, etc. [ , ] . therefore, it is needed to control infection. chinese herbal medicine has the characteristics of high efficiency, low toxicity and low residue. it can improve the immunity, possess anti-stress and anti-virus ability. a variety of natural compounds or their active ingredients have been used to enhance host immune, prevent and treat viral diseases, such as coronavirus diseases [ ] , hepatitis b (hb) [ ] , infectious bronchitis [ ] , african swine fever (asf) [ ] , influenza [ ] and other viral diseases. however, the research on the anti-pcv action of natural compounds and its mechanism has also become a hotspot. in our previous studies, dozens of natural compounds were screened for their antiviral activity, such as chlorogenic acid [ ] , ligustrazine hydrochloride [ ] , scutellarin [ , ] , dipotassium glycyrrhizinate [ , , ] , sodium tanshinone iia sulfonate [ , ] , tea seed saponins [ ] and matrine [ , , ] . among them, only scutellarin or matrine had anti-pcv effect [ , ] . therefore, in this study, from many natural compounds with clear chemical structure, traditional medicine plant source and specific biological activity, compounds with antiviral potential were selected and tested in the model of pcv infected pk- cell to screen anti-pcv compounds in vitro. furthermore, the potential antiviral mechanism of compounds was explored and explained from the perspectives of apoptosis, mitochondrial membrane potential (mmp) and ros. it provides a theoretical basis for the development of a safely, efficiently new compound with anti-pcv effect. compared with the normal group of normal cells, the results showed that (see additional file ), the degree of cytopathological change were also different after the treatment of different compounds and different concentrations of the same compound. due to the low solubility, the maximum solubility concentration (msc) of . mg/ml formononetin, . mg/ml icariine and . mg/ ml astragaloside were used to treat the cells, respectively and no change in cell morphology was observed. the mntc is the same as msc. the compounds except formononetin, icariine and astragaloside showed different cytopathic changes (see additional file ) and mntc values (table ) . dose-response curves of the tested compounds ( fig. ) and cc value (table ) were generated by graphpad prism . analysis after calculating the cr value. the curves were "s" type, showing a dose-dependent relationship. as the concentration of the compound increased, more cells with change in morphology the was observed. to explore the anti-pcv effect of compounds, the expression levels of cap was detected by qpcr and western blot. the results of qpcr showed that, compared to cell control group, the pcv copy numbers were significantly increased (p < . ), compared to pcv infected group, the pcv copy numbers were significantly decreased in these compound treated with formononetin at . , icariine at . , paeonol at . , . and . mg/ml, cepharanthine at . , . and . mg/ml, curcumin at . , . and . mg/ml, curcumol at . and syringin at . and . mg/ml (p < . ) (fig. a) . based on the results by qpcr, dose-dependent paeonol, cepharanthine and curcumin were selected for western blot. the results showed that the expression level of cap protein treated with the three compounds respectively were significantly lower than pcv infected group (p < . ), except the . mg/ml of paeonol group ( fig. b ) (for original and unedited blots see additional file ). therefore, the cepharanthine and curcumin were selected for the subsequent anti-apoptotic test. to evaluate the anti-apoptosis effect of cepharanthine or curcumin, samples were analyzed after being treated with annexin v/pi by flow cytometer. the results showed that compared to the pcv -infected group, the cell apoptosis rates were significantly decreased in the group treated with cepharanthine, curcumin or ribavirin, demonstrating a dose-dependent response except the group of . mg/ml paeonol (p < . ) ( fig. a and b). our results indicated that cepharanthine, curcumin or ribavirin could significantly inhibit pcv induced apoptosis (p < . ). to evaluate the anti-apoptosis mechanism of cepharanthine or curcumin, samples were analyzed after being treated with jc- kit by flow cytometer. the results showed that compared to the pcv -infected group, the mmp value were significantly decreased in the group treated with cepharanthine, curcumin and ribavirin, demonstrating a dose-dependent response (p < . ) ( fig. a and b) . the results indicated that cepharanthine, curcumin or ribavirin could significantly inhibit pcv induced apoptosis via mitochondrial pathway (p < . ). to evaluate the mechanism of inhibition of pcv induced mitochondrial apoptosis by cepharanthine or curcumin, samples were analyzed by western blot and flow cytometer. the results of western blot showed that compared with the pcv -infected group, the expression of pro-apoptin cleaved caspase- and bax was significantly decreased while the expression of antiapoptotic protein bcl- was significantly increased in the treatment group of cepharanthine, curcumin or ribavirin ( fig. a -h) (the original and unedited blots see additional file ). the results of flow cytometer after being stained with ros kit demonstrated that the value of ros in the curcumin-treated group showed strongly downward trend in a dose-dependent manner, compared with the pcv control group ( fig. i and j) . however, only the group of treated with . mg/ml of cepharanthine could significantly reduce the amount of ros (p < . ). the results demonstrated that cepharanthine or curcumin inhibited the pcv induced increase in mmp through the caspase family and bcl- family mechanisms. curcumin inhibited pcv -induced mitochondrial apoptosis by reducing ros, however, ros reduction is not the main pathway for cepharanthine and ribavrin to inhibit pcv induced mitochondrial apoptosis. pcv is the main pathogen causing porcine circovirus associated diseases (pcvad). in this study, natural compounds with defined chemical structures, useful clinical effect and from medicinal plants were selected. after the primary screening, three compounds with antiviral effect, paeonol, cepharanthine and curcumin were further studied. the anti-apoptotic mechanism of these two compounds was investigated by evaluating the level of key apoptins, mmp and ros in the cellular mitochondrial pathway. ribavirin has a broad antiviral activity and was used as a positive control. cepharanthine is a natural alkaloid extracted from the roots of the stephania japonica, which has antiinflammatory [ ] , anti-hepatitis b virus [ ] , herpes simplex type- virus (hsv- ) [ ] and etc. curcumin is an active ingredient extracted from the rhizome of some plants of the zingiberaceae and araceae. medical research shows that curcumin has low toxicity, little adverse reactions and multiple therapeutic effects, such as anti-inflammatory [ ] , anti-oxidative [ ] , anti-h n influenza virus [ ] , dengue virus [ ] , hepatitis c virus [ ] and so on. found that pcv induced mitochondrial apoptosis of pcv infected pk- cells and t cells by increasing the activities of caspase- and caspase- and accelerating the release of cytochrome c (cyto c) from mitochondria to cytoplasm [ ] ; after pcv infected raw . cells, stress-related molecules such as ros production and no secretion were significantly increased [ ] ; previous studies have shown that pcv infection promotes the accumulation of ros, which in turn inhibits the replication of pcv through the nf-κb pathway [ ] . therefore, we hypothesized that the experimental compounds could inhibit the mitochondrial apoptosis of pk- cells induced by pcv infection via acting on mitochondrial membrane potential. mitochondria have a central role in cell apoptosis. the loss of mmp is the marker of mitochondrial apoptosis, the changes of mmp are caused by bcl- family, caspase family and miochondrial permeability transition pore (mptp) [ ] . cleaved caspase- is the main executive protein of apoptosis. there are many members of the bcl- family, among which bax is the main pro-apoptotic protein and bcl- is the main antiapoptotic protein. the opening and closing of mptp is influenced by ros, ca + , adp, oxidative stress, high ph value and other apoptotic factors [ ] . in this study, at first, the cell apoptosis and change of mmp were detected by flow cytometry after annexin v/pi double staining and jc- assay. the results indicated that cepharanthine, curcumin or ribavirin could significantly inhibit pcv -induced apoptosis via mitochondrial pathway. to further evaluate the mechanism of cepharanthine or curcumin inhibiting mitochondrial apoptosis, the apoptins of caspase family and bcl- family were analyzed by western blot, the values of ros were analyzed by flow cytometry after ros kit staining. the results showed that compared with cell control group, the expression levels of cleaved caspase- and bax were upregulated, and the expression of bcl- was downregulated, the ros values are increased. this result is consistent with the previous reports [ , , ] . compared with pcv -infected group, the expression levels of to summarize, our study revealed that paeonol, cepharanthine and curcumin have significant antiviral effect, and cepharanthine and curcumin could inhibit pcv induced mitochondrial apoptosis by through change the mmp (fig. ) . the study provides a base line for further studies on the anti-pcv effect and anti-apoptosis mechanism of cepharanthine and curcumin in experimental animal models infected with pcv , and may be applied in clinical treatment as anti-virus compounds eventually. pcv-free pig kidney endothelial cell line (pk- ) was preserved in our laboratory. cells were cultured and passed in dulbecco's modified eagle's medium (dmem, hyclone, usa) containing % fetal calf serum (fbs, bi, israel) ( % dmem) and maintenance in dmem containing % fbs ( % dmem). the strain of pcv -sh (genbank: ay . ) was gifted by professor jiang ping of nanjing agricultural university. virus was replicated and harvest in pk- cells infected with pcv . the titer of . tcid / ml was determined by indirect immunofluorescence (ifa). antibodies against cap were purchased from biorbyt llc. (california, britain) cleaved caspase- , bcl- and bax were purchased from abcam (cambridge, usa); gapdh and horseradish peroxidase (hrp)-conjugated secondary antibodies were purchased from wuhan sanying biology technology co., ltd. (wu han, china). ribavirin, the positive control drug, was purchased from beijing solarbio technology co., ltd; syringin was purchased from nanjing puyi biotechnology co., ltd; glycyrrhizin aid was purchased from dosf biotechnology co., ltd; other tested compounds, such as curcumol, paeonol, oxymatrine, caffeic acid, formononetin, cepharanthine, apigenin, psoralen, lcariine, curcumin and astragaloside were purchased from national institutes for food and drug control. compounds information and chemical structure were respectively listed table and fig. . the viability of pk- cell was assessed with mtt. . × cells/ml of cells were seeded into -well plates and incubated until % confluency was reached. the tested compounds were added with two-fold serial diluted and cultured for another h and cell control group was established. four repeated wells were set up for each concentration with μl per well. the cytopathic condition of cells was observed every day and photographed. the compounds and dissolution methods are shown in table . then μl mtt ( mg/ml in pbs) was added to each well and incubated at °c for h. μl of dmso (solarbio, beijing) was added and incubated for min after removing the culture medium. the optical density (od) were measured at nm using a microplate spectrophotometer (spectra max m , molecular devices, usa). cytopathic ratio (cr) was calculated based on cr = [(od control -od test)/ od] × . then mntc and cc on pk- cells were calculated using graphpad prism™ . (inc. california, usa). the anti-pcv effect of compounds in pk- cell was determined by real time quantitative pcr (qpcr). when cell confluency rate of -well plate reached %, . tcid of pcv was incubated for h and discarded the supernatant. mntc of every test compound was in two-fold serial dilution with % dmem into gradients and added to -well plates ( μl/well) ( table ) . meanwhile, the cell control group, pcv -infected group and ribavirin positive control group were applied and incubated for h with % co at °c. dna was extracted according to the instruction of dna extraction kit (tiangen, beijing, china) and the dna concentration was measured using a nucleic acid concentration analyzer (nanodrop technologies, wilmington, de, usa). the copy number of the cap gene was detected by qpcr. the primers ′ tac att tcc agc agt ttg and 'ctc ccg cca tac cat aa were used for the amplification of bp fragment. the anti-pcv effect and anti-apoptotic mechanism of the compound was detected by western blot. when cell confluency of -well plate reached %, . tcid of pcv was incubated for h and discarded the supernatant. paeonol, cepharaanthine and curcumin were added in turn, ml/well. the cell control group, pcv infected group and ribavirin positive control group were applied and incubated for h. total cell protein was extracted and protein concentration was determined using the bca protein assay kit (beyotime biotechnology, jiangsu, china). the protein samples were separated by sds-page, then transferred to the pvdf membrane. the levels of pcv cap protein and apoptin were fig. the pcv -induced mitochondrial apoptosis was mainly remitted by cepharanthine and curcumin. the "red arrow" represents the promoting effect and the red "t-shape" indicates the inhibiting effect. the pcv replication is inhibited by cepharanthine and curcumin, selected from natural compounds, through mitochondrial apoptosis pathway detected using an eecl western blot detection kit (cwbio inc., beijing, china) and chemiluminescence imaging system (bio-rad, california, usa). the anti-apoptotic effect of cepharaanthine and curcumin were detected by annexin v/pi. cells in -well plate were incubated with . tcid of pcv for h and discarded the supernatant, then cepharaanthine and curcumin were added, the cell control group, pcv infected group and ribavirin positive control group were applied and incubated for h. samples were collected, centrifugated and treated using annexin v/pi kit (keygen biotech, nanjing, china), then analyzed by flow cytometry (bd biosciences, usa). mitochondrial membrane potential (mmp) of cells was detected by jc- . cells in the -well plate were incubated with . tcid of pcv for h and discarded the supernatant, then cepharaanthine and curcumin were added. after h incubation, cells were stained with jc- using a commercial kit (beyotime biotechnology, jangsu, china) treated and analyzed by flow cytometry (bd biosciences, usa). the amount of reactive oxygen species (ros) was measured by flow cytometry. cells in the -well plate were incubated with . tcid of pcv for h and discarded the supernatant, cepharaanthine and curcumin were added and incubated for h. cells were treated with ros assay kit and the changes of ros were detected by flow cytometer. cc was calculated using nonlinear regression and the results of "log (inhibitor) vs. response-variable slope" were analyzed using graphpad prism. the gray intensity of protein bolts was analyzed by image j (national institutes of health, usa). data generated were analyzed using "bonferroni: compare all pairs of columns" in the graphpad prism™ . software for one-way anova implemented (graphpad software, inc. california, usa), and were expressed as the mean ± standard error of the mean (sem) of at least repeated experiments. different letters (a, b, c, d, etc.) indicate statistically significant difference to other groups (p< . ). supplementary information accompanies this paper at https://doi.org/ . /s - - - . 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combination of transcription inhibitor k- and other antiretroviral agents in acutely and chronically infected cells pulmonary administration of a water-soluble curcumin complex reduces severity of acute lung injury inhibition of curcumin on influenza a virus infection and influenzal pneumonia, via, oxidative stress, tlr / , p /jnk mapk and nf-κb pathways synergistic effects of thymoquinone and curcumin on immune response and anti-viral activity against avian influenza virus (h n ) in turkeys inhibitory effects of curcumin on dengue virus type -infected cellsin vitro curcumin inhibits hepatitis c virus replication via suppressing the akt-srebp- pathway caspase-dependent apoptosis induction via viral protein orf of porcine circovirus binding to mitochondrial adenine nucleotide translocase effect of total flavonoids of spatholobus suberectus dunn on pcv induced oxidative stress in raw . cells. bmc complement reactive oxygen species regulate the replication of porcine circovirus type via nf-κb pathway parvovirus minute virus of mice induces a dna damage response that facilitates viral replication effect of sophora subprosrate polysaccharide on oxidative stress induced by pcv infection in raw . cells publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank prof. jiang (nanjing agricultural university) for his assistance in the pcv . authors' contributions yx, jz, ns and hl designed all the experiments. yx and jz performed the mtt experiments, qpcr and western blot assays. ys, kf, and wy performed the flow cytometer assays. yx, ps, ns, xz and jg wrote the manuscript. all authors read and approved the final manuscript. this work was supported by national key research and development program (grant no. yfd ). the funding body had a role in the design of the study, collection, analysis, and interpretation of data or in the writing of this manuscript. the datasets used and analysed during the current study are available from the corresponding author on reasonable request. the experiment was performed in according to the experiment operational guideline of shanxi province, china. not applicable. all authors declared no competing conflict of interest.author details key: cord- -re v qt authors: le, thanh ninh; chiu, chiu-hsia; hsieh, pao-chuan title: bioactive compounds and bioactivities of brassica oleracea l. var. italica sprouts and microgreens: an updated overview from a nutraceutical perspective date: - - journal: plants (basel) doi: . /plants sha: doc_id: cord_uid: re v qt sprouts and microgreens, the edible seedlings of vegetables and herbs, have received increasing attention in recent years and are considered as functional foods or superfoods owing to their valuable health-promoting properties. in particular, the seedlings of broccoli (brassica oleracea l. var. italica) have been highly prized for their substantial amount of bioactive constituents, including glucosinolates, phenolic compounds, vitamins, and essential minerals. these secondary metabolites are positively associated with potential health benefits. numerous in vitro and in vivo studies demonstrated that broccoli seedlings possess various biological properties, including antioxidant, anticancer, anticancer, antimicrobial, anti-inflammatory, anti-obesity and antidiabetic activities. the present review summarizes the updated knowledge about bioactive compounds and bioactivities of these broccoli products and discusses the relevant mechanisms of action. this review will serve as a potential reference for food selections of consumers and applications in functional food and nutraceutical industries. over the past twenty years, the heavy dependence of human nutrition on the sustainability of agricultural production has been highlighted owing to rapid population growth and environmental degradation [ ] . increasing food and agricultural productivity has simultaneously imposed external expenses and consequences upon the financial resource, ecological system, and human health by excessive water use, soil occupation, fertilizers, pesticides, herbicides, and food waste treatment [ ] . thus, to meet the united nations' sustainable development goals, the agriculture and food sectors have been confronted with a major challenge: to provide adequate nutrition for global food demand while minimizing the negative impacts on the environment [ ] . furthermore, in recent years, improving public awareness about the healthy lifestyle framework has prompted the search for novel food sources, which are rich in essential nutrients and have positive effects on human health [ ] . as a result, functional foods and nutraceuticals are gaining significant attention since these foods could provide both health benefits to reduce the risk of chronic diseases and basic nutrition [ , ] . besides, many scientific projects and research groups are focusing on the recovery of food wastes and upgrading them into high-value byproducts to serve as functional ingredients in new product development [ ] [ ] [ ] [ ] . particularly, the issues of the food systems, including food safety, food security, and food sustainability, should be significantly addressed in the era of the coronavirus (covid- ) pandemic crisis. the availability of bioactive constituent of food and functional foods may become bioactive compounds are extra-nutritional constituents found in foods, mainly in fruits, vegetables, and grains, which are capable of modulating metabolic processes and providing healthpromoting benefits [ ] . regular consumption of broccoli seedlings could stimulate the natural defense systems and decrease the risk of chronic diseases, owing to their concentration of bioactive compounds several times higher than those of the mature florets [ ] . thus, they are considered a novel plant-derived functional food. previous studies have extensively indicated that broccoli sprouts and microgreens contain a remarkably high amount of glucosinolates, phenolic compounds, and essential nutrients ( figure and tables - ). there are numerous methods employed for the extraction of bioactive compounds from herbs, vegetables, and fruits, including emerging technologies such as pressurized hot water extraction, microwave-assisted extraction, or pulsed electric extraction [ ] [ ] [ ] . the separation, identification, and characterization of these compounds of broccoli seedlings, in particular, and the genus brassica, in general, were investigated by both conventional and non-conventional technologies [ , ] . among these technologies, the broccoli samples were mainly conducted by high-performance liquid chromatography (hplc) coupled to diode array (dad), ultraviolet-visible (uv-vis), electrospray ionization (esi), or mass spectrometry (ms) detectors with c analytical columns. gas chromatography combined with mass spectrometry (gc-ms) was also applied to characterize isothiocyanates, the hydrolysis products of glucosinolates. besides, the spectrophotometric (uv-vis) detection was employed in most of the research to determine the total phenolic and flavonoid contents as the simplest procedure, if it was not required to determine a specific compound (tables - ). it could be summarized that glucosinolates and related compounds are the major group of phytochemicals investigated in broccoli sprouts and microgreens, although phenolic compounds have also been analyzed in many studies ( figure ). bioactive compounds are extra-nutritional constituents found in foods, mainly in fruits, vegetables, and grains, which are capable of modulating metabolic processes and providing health-promoting benefits [ ] . regular consumption of broccoli seedlings could stimulate the natural defense systems and decrease the risk of chronic diseases, owing to their concentration of bioactive compounds several times higher than those of the mature florets [ ] . thus, they are considered a novel plant-derived functional food. previous studies have extensively indicated that broccoli sprouts and microgreens contain a remarkably high amount of glucosinolates, phenolic compounds, and essential nutrients ( figure and tables - ). there are numerous methods employed for the extraction of bioactive compounds from herbs, vegetables, and fruits, including emerging technologies such as pressurized hot water extraction, microwave-assisted extraction, or pulsed electric extraction [ ] [ ] [ ] . the separation, identification, and characterization of these compounds of broccoli seedlings, in particular, and the genus brassica, in general, were investigated by both conventional and non-conventional technologies [ , ] . among these technologies, the broccoli samples were mainly conducted by high-performance liquid chromatography (hplc) coupled to diode array (dad), ultraviolet-visible (uv-vis), electrospray ionization (esi), or mass spectrometry (ms) detectors with c analytical columns. gas chromatography combined with mass spectrometry (gc-ms) was also applied to characterize isothiocyanates, the hydrolysis products of glucosinolates. besides, the spectrophotometric (uv-vis) detection was employed in most of the research to determine the total phenolic and flavonoid contents as the simplest procedure, if it was not required to determine a specific compound (tables - ) . it could be summarized that glucosinolates and related compounds are the major group of phytochemicals investigated in broccoli sprouts and microgreens, although phenolic compounds have also been analyzed in many studies ( figure ). summary of the bioactive compounds analyzed in the last ten years of broccoli sprouts and microgreens. glucosinolates (glss), nitrogen−sulfur compounds (β-d-thioglucoside-n-hydroxysulfates), are important plant secondary metabolites, almost exclusively found in the genus brassica, specific to kale, cabbage, and broccoli [ ] . glss are classified as aliphatic (derived from methionine, isoleucine, leucine or valine), aromatic (derived from phenylalanine or tyrosine), or indole (derived from tryptophan) groups. aliphatic group is the major group of glss in almost all cruciferous seeds and seedlings of b. oleraceae, b. napus, b. rapa, and r. sativus [ ] . in broccoli sprouts and microgreens, compounds of glss were identified (table ) . among these compounds, the most abundant glss are glucoraphanin, glucoiberin, glucoerucin, glucobrassicin, and neoglucobrassicin [ ] . in particular, glucoraphanin accounted for over % of the total glss content, which was quantified in the range of to mg per g of fresh weight (fw) [ ] [ ] [ ] . glucosinolates (glss), nitrogen−sulfur compounds (β-d -thioglucoside-n-hydroxysulfates), are important plant secondary metabolites, almost exclusively found in the genus brassica, specific to kale, cabbage, and broccoli [ ] . glss are classified as aliphatic (derived from methionine, isoleucine, leucine or valine), aromatic (derived from phenylalanine or tyrosine), or indole (derived from tryptophan) groups. aliphatic group is the major group of glss in almost all cruciferous seeds and seedlings of b. oleraceae, b. napus, b. rapa, and r. sativus [ ] . in broccoli sprouts and microgreens, compounds of glss were identified (table ) . among these compounds, the most abundant glss are glucoraphanin, glucoiberin, glucoerucin, glucobrassicin, and neoglucobrassicin [ ] . in particular, glucoraphanin accounted for over % of the total glss content, which was quantified in the range of to mg per g of fresh weight (fw) [ ] [ ] [ ] . notably, glss are not the functional components in cruciferous vegetables, rather their hydrolysis products are the putative bioactive compounds [ ] . when plant tissues are mechanically damaged, glss could be hydrolyzed by the enzyme myrosinase into a variety of degradation products, including isothiocyanates (itcs), thiocyanates, nitriles, epithionitriles, and oxazolidines. in particular, itcs have been proven to present strong anticarcinogenic activities [ ] . as mentioned above, predominant glss in broccoli seedlings are glucoraphanin, glucoerucin, and glucobrassicin, which are enzymatically converted into sulforaphane (sfn), erucin (ern), and iberin, respectively [ ] . sfn is a naturally occurring inducer of phase ii enzymes in human and animal bodies to detoxify cancer-causing chemicals. thus, it can reduce the risk of different cancers, especially those of the bladder, colon, and lung [ ] . to date, sfn was analyzed as a major compound among the total of itcs identified from broccoli sprouts and microgreens ( table ). the total itc content of broccoli seedlings was reported to be around mg per g of fw. additionally, such seedlings were said to contain % sfn [ , ] . uv/vis, ultraviolet-visible spectrophotometry; gc-ms, gas chromatography combined with mass spectrometry; gc−fid, gc with a flame ionization detector. besides glss and itcs, another important group of bioactive constituents present in cruciferous vegetables is the phenolic compounds. they are secondary metabolites produced in plants through the phenylpropanoid and shikimate pathways [ ] . based on their structure, which comprises one or more aromatic rings with attached hydroxyl substituents, phenolic compounds can be categorized into various subgroups, such as phenolic acids, flavonoids, tannins, coumarins, lignans, quinones, stilbenes, and curcuminoids. these compounds have been mainly reported for antioxidant activity. moreover, they are also associated with other health-promoting effects such as anticarcinogenic, antimicrobial, anti-inflammatory, and anti-aging properties [ ] . hydroxycinnamic acids and flavonoid glycosides are among the main phenolic compounds found in broccoli seedlings [ , ] . using quantitative and qualitative analysis, phenolic compounds were characterized in broccoli sprouts and microgreens, including hydroxycinnamic acids and derivatives, and flavonoids and derivatives. their phenolic profile composed mostly of sinapic acid, gallic acid, flavonoids (quercetin and kaempferol), and other hydroxycinnamic acids (chlorogenic, caffeic acid, and ferulic acids) ( table ). in general, total phenolic and flavonoid contents were determined to be in the ranges of - mg per g of fw and - mg per g of fw, respectively [ , , ] . similar to other sprouted seeds, broccoli sprouts and microgreens are known for their high concentration of essential nutrient composition [ ] . essential nutrients are compounds that must be supplied from foods, such as vitamins, minerals, fatty acids, and amino acids. they are required for normal body functions, including dna synthesis, energy production, and biosynthetic pathways [ ] . nonetheless, the number of studies, which determined the nutritional composition of broccoli seedlings and the variations occurring during germination, is significantly smaller than that of glucosinolates and phenolic compounds. these studies indicated that broccoli sprouts are expressly rich in vitamins (a, c, k, and folic acid), minerals (potassium, calcium, magnesium, and selenium), pigments (carotenoids and chlorophylls) and some other important nutrients (amino acids, fatty acids, and dietary fiber) [ ] [ ] [ ] , , , ] . in general, the biochemical composition of broccoli sprouts and microgreens depends mainly on the germination time, and -day-old sprouts displayed the highest nutrient concentration [ ] . among these compounds, carotenoids and chlorophylls were determined in several studies of broccoli sprouts [ , , , , ] . carotenoids are a group of isoprenoid molecules synthesized as secondary metabolites by all photosynthetic plants, including broccoli [ ] . they have lipophilic antioxidant and immunomodulatory activities owing to the conjugated double bonds of the long polyene chain, which are capable of inhibiting reactive oxygen species and reducing oxidative damage [ ] . thus, carotenoids might prevent degenerative diseases, such as cardiovascular diseases, skin damage, diabetes, and several types of cancer [ ] . the major carotenoids found in broccoli are β-carotene, α-xanthophyll (lutein), and β-xanthophylls (zeaxanthin, violaxanthin, and neoxanthin) [ ] . in particular, β-carotene is the most typically studied carotenoids in broccoli sprouts and microgreens, due to its importance in medical science [ ] . it supplies the human diet a substantial amount of vitamin a that is essential for organogenesis, tissue differentiation, immune function, and vision [ ] . the total carotenoid (β-carotene) concentration of broccoli sprouts was reported in several studies with a range of to mg per g of dry weight (dw) [ , ] . chlorophylls are another group of light-absorbing pigments regularly present in broccoli sprout extract. they are primary metabolites that have a porphyrin structure [ ] . similar to carotenoids, they also have antioxidant, anti-mutagenic, and anti-inflammatory potential [ ] . the content of chlorophyll in broccoli sprouts was commonly determined along with total carotenoid content in previous studies, ranging from to mg per g dw [ , ] . additionally, a study showed individual concentrations of carotenoids (lutein and neoxanthin) and chlorophyll (chlorophyll a and b) in broccoli sprouts [ ] . the human consumption of foods or nutraceuticals is not limited to bioactive compounds. in the human body, bioavailability is determined as the fraction of the dose administered that reaches the circulatory system and then distributes into target tissues so that the bioactive compounds are biologically available for exerting health-promoting benefits [ ] . many factors could affect the bioavailability of the dietary phytochemicals, including plant cell wall compositions, chemical structures, processing conditions, environmental stress, as well as an individual's gastrointestinal system [ ] . the effectiveness of a high consumption of broccoli sprouts in reducing the risk of cancer has been attributed mainly to their high content of glss. the daily intake of glss is difficult to estimate owing to the high variability of the constituent in cruciferous vegetables, particularly broccoli. a report for glucosinolate intake was indicated for european people ranging from . - mg/day [ ] . glss are broken down by both the plant enzyme myrosinase in the small intestine and the microbiota in the gastrointestinal tract into itcs, which are reported to have the prominent anticancer effect [ ] . after intake - h, the itcs are absorbed from the small bowel and colon and metabolized by the mercapturic acid pathway. the metabolites are detectable in human urine and blood [ ] . in broccoli sprouts, two of the most abundant glss are glucoraphanin and glucoerucin, and myrosinase hydrolysis of these glss form sfn and ern, respectively. sfn, distinguished itcs in broccoli sprouts, is one of the most potent naturally occurring inducers of phase detoxification enzymes, boost antioxidant status, and protect animals against chemically induced cancer [ , ] . its modes of action are involved with the repressor protein kelch-like ech associated protein (keap ), nuclear factor erythroid p -related factor (nrf ), and genes, which contain an antioxidant responsive element (are) [ ] . thus, it is important for the inclusion of potential cancer chemopreventive agents in dairy foods. moreover, it has shown an inhibitory effect for urease from helicobacter pylori, a human pathogen [ ] . the formation of other breakdown products from glss by intestinal microbiota is not well documented. for example, bifidobacterium strains belonging to the human intestinal microbiota can in vitro metabolize the glss to nitriles. it is also known that several microorganisms are able to convert nitriles into ammonia and organic acids [ ] . phenolic compounds are substantial micronutrients in the human diet and the health benefits of polyphenols depend on the amount consumed and on their bioavailability [ ] . the dietary intake of phenolic compounds has been associated with health-promoting effects, such as antioxidant, anti-aging, antiproliferative, and anti-inflammatory activities [ ] . it has been reported that the average dietary intake of phenolic compounds for humans is about - mg/day, including % of hydroxycinnamic acids, - % of flavonoids, and % of anthocyanins [ ] . bioavailability differs significantly among phenolic compounds so that the most abundant compounds in the human diet are not necessarily those leading to the highest concentrations of active metabolites in target tissues [ ] . there are two directions for the digestion of dietary phenolics, comprising digestion along the gastrointestinal tract and digestion inside the enterocytes. these digestions are based on hydrolase enzymes, which are available in the intestinal lumen, brush border, and enterocyte [ ] . many studies showed that polyphenols are absorbed in both the small intestine and the large intestine by intestinal enzymes after microbial digestions [ ] . in the human small intestine and stomach, gallic acid and caffeic acid are the most well-absorbed compounds ( %), followed by catechins ( %). the least well-absorbed polyphenols are proanthocyanidins and the anthocyanins. they are ph-sensitive, which could be broken down in the stomach and readily absorbable. flavonoid group is one of the group molecules with molecular weights > da so that it has a low bioavailability level ( %) because molecules are improbable to be transported through passive diffusion pathways [ , , ] . the dietary intake of carotenoids, particularly vitamin a, has been linked to the protection of dna, proteins, and lipids from oxidative damage [ ] . digested carotenoids decrease oxidative dna damage so that they could protect colon cells against the stress of reactive oxygen species [ ] . the dietary chlorophylls from fresh fruits and vegetables, which compose of chlorophyll a and b, showed distinct biological potentials, including wound healing, the control of calcium oxalate crystals, the modulation of xenobiotic metabolism, and the induction of apoptosis [ ] . broccoli seedlings and their bioactive components exhibit many potential health-promoting roles. in the last decade, the beneficial effects, including antioxidant, anticarcinogenic, antimicrobial, anti-inflammatory, and several other properties, have been widely investigated in a number of in vitro and in vivo studies and topically applied in clinical trials [ , , ] . particularly, these studies mostly focused on the antioxidant and anticancer activities of broccoli sprouts and microgreens owing to the functions of glucosinolates and phenolic compounds (tables and ). in this section, the findings of different biological activities of broccoli sprouts and microgreens will be discussed with the possible mechanisms of action ( figure ). anti-inflammatory, and several other properties, have been widely investigated in a number of in vitro and in vivo studies and topically applied in clinical trials [ , , ] . particularly, these studies mostly focused on the antioxidant and anticancer activities of broccoli sprouts and microgreens owing to the functions of glucosinolates and phenolic compounds (tables and ). in this section, the findings of different biological activities of broccoli sprouts and microgreens will be discussed with the possible mechanisms of action ( figure ) . the overproduction or incorporation of free radicals, such as reactive oxygen species (ros), cause oxidative damage to biomolecules and consequently lead to many chronic diseases, including neurodegenerative diseases, cardiovascular diseases, and certain age-related cancers [ ] . ros are previously thought to form in mammalian cells almost exclusively as a consequence of mitochondrial metabolism. recently, it has been demonstrated that cellular enzymes known as nicotinamide adenine dinucleotide phosphate (nadph) oxidases produce a considerable amount of ros in the human body. moreover, other cellular sources of ros involve neutrophils, monocytes, endothelial cells, xanthine oxidases, cytochrome p , lipoxygenases, and nitric oxide syntheses [ ] . hence, ros has distinct effects on normal physiological processes, oxidative stress/regulation, metabolic diseases, and chronic inflammation. targeting ros is involved in antioxidant, anti-inflammatory, antidiabetic, and anti-obesity therapeutics [ ] . antioxidants are divided into natural and synthetic compounds, which could remove free radicals, inhibit ros formation, and scavenge ros [ ] . previous studies the overproduction or incorporation of free radicals, such as reactive oxygen species (ros), cause oxidative damage to biomolecules and consequently lead to many chronic diseases, including neurodegenerative diseases, cardiovascular diseases, and certain age-related cancers [ ] . ros are previously thought to form in mammalian cells almost exclusively as a consequence of mitochondrial metabolism. recently, it has been demonstrated that cellular enzymes known as nicotinamide adenine dinucleotide phosphate (nadph) oxidases produce a considerable amount of ros in the human body. moreover, other cellular sources of ros involve neutrophils, monocytes, endothelial cells, xanthine oxidases, cytochrome p , lipoxygenases, and nitric oxide syntheses [ ] . hence, ros has distinct effects on normal physiological processes, oxidative stress/regulation, metabolic diseases, and chronic inflammation. targeting ros is involved in antioxidant, anti-inflammatory, antidiabetic, and anti-obesity therapeutics [ ] . antioxidants are divided into natural and synthetic compounds, which could remove free radicals, inhibit ros formation, and scavenge ros [ ] . previous studies on broccoli sprouts and microgreens have demonstrated that they are recognized for their variety of naturally occurring antioxidants, comprising both nutritional antioxidants like vitamins (especially, ascorbic acid and α-tocopherol), and non-nutritional antioxidants such as carotenoids and phenolic compounds [ ] . it was reported that these compounds are responsible for − % of the total antioxidant capacity in broccoli sprouts [ ] . the antioxidant activity of broccoli sprouts and microgreens has been determined in numerous studies using various methods. the capacity of broccoli sprouts and microgreens for chelating fe + and scavenging free radicals such as dpph ( , -diphenyl- -picrylhydrazyl) and abts ( , -azino-bis- -ethylbenzothiazoline- -sulphonic acid) was demonstrated in numerous studies (table ) . among the possible methods, dpph radical scavenging assay, abts radical cation decolonization assay, and ferric reducing antioxidant power (frap) assay were commonly employed in previous studies ( table ). the dpph method is speedy, simple, and low cost in comparison to other test models. dpph, a dark crystalline molecule, is a stable chromogen radical formed by the delocalization of the spare electron over the molecule. the dpph scavenging assay is based on the electron donation of antioxidants to neutralize dpph radical. the reaction occurs with the loss of the violet color of dpph that is measured at nm, and the discoloration acts as an indicator of the antioxidant effectiveness [ ] . the abts decolonization assay is appropriate for both hydrophilic and lipophilic antioxidants. it uses a spectrophotometer to measure the ability of antioxidants to scavenge the stable radical cation abts + , a blue-green chromophore molecule with absorption at nm. antioxidants can neutralize and decolorize the radical cation abts + by electron or hydrogen atom donations [ ] . the frap assay was originally employed to measure reducing power in plasma, but it has been expanded for other biological fluids and plant extracts. thus, it is applicable for both in vitro and in vivo experiments. the frap mechanism is based on electron transfer rather than hydrogen atom transfer. the assay demonstrates the ability of antioxidants to reduce ferric iron. it measures the reduction of the ferric ion (fe + )-ligand complex to the blue-colored ferrous (fe + ) form at low ph by antioxidants (absorption at nm) [ ] . moreover, another mechanism of antioxidant activity of broccoli sprouts and microgreens have been confirmed in several in vitro and in vivo examinations by inhibiting the activity of prooxidant enzymes such as lipoxygenase (lox), and xanthine oxidase (xo), and activating antioxidant enzymes such as peroxidase (pod), catalase (cat), superoxide dismutase (sod), glutathione peroxidase (gpx), nad(p)h-quinone acceptor oxidoreductase (nqo ), and heme oxygenase- (ho- ) ( table ) . prooxidant enzymes are considered as the main biological source of superoxide radicals, whereas antioxidant enzymes could eliminate the excess of reactive oxygen species and reduce oxidative damage during senescence [ , ] . for example, enzyme activities of cytosolic glutathione peroxidase (gpx ), thioredoxin reductase(tr) in the thyroid, plasma glutathione peroxidase (gpx ), and ferric reducing ability of plasma (frap) significantly increased in response to broccoli sprouts ingestion in -week-old wistar rats [ ] . in summary, previous studies showed that broccoli sprout extracts rich in vitamins, carotenoids, and phenolic compounds showed very high antioxidant activity in both in vitro and in vivo tests (table ) . thus, they could be applied for antioxidant therapeutics to reduce the risk of chronic diseases caused by ros. interestingly, although the antioxidant capacity of broccoli seedlings has been reported comprehensively, it is still attracting considerable attention from investigators and researchers at present [ , , ] . abts, , -azino-bis- -ethylbenzothiazoline- -sulphonic acid decolonization activity; dpph, , -diphenyl - -picrylhydrazy radical scavenging activity; frap, ferric reducing-antioxidant power; teac, trolox equivalent antioxidant capacity; orac, oxygen radical absorbance capacity; pod, peroxidase activity; cat, catalase activity; sod, superoxide dismutase activity; gpx, glutathione peroxidase activity; chel, metal chelating activity; lpo, inhibition of lipid peroxidation; loxi, inhibition of lipoxygenase; xoi, inhibition of xanthine oxidase; nqo , nad(p)h-quinone acceptor oxidoreductase ; ho- , heme oxygenase- ; ma/gc, malonaldehyde/gas chromatography. in the last decade, many studies and projects have supported the protective effects of natural products in cancer prevention. the protective role against cancer has been mostly attributed to the high content of glss, typically found in cruciferous vegetables [ ] . broccoli sprouts and microgreens have been valued as a rich source of glss and their hydrolysis products (icts, particularly sfn), which are a well-known class of cancer chemotherapeutic agents that work by inducing apoptosis and arresting cell cycle progression (tables and ) . the potential mechanisms of action mainly involve the inhibition of proliferation and the induction of apoptosis in cancer (table ) . hence, to demonstrate the anticancer activity of broccoli sprouts and microgreens, many in vitro tests have been carried out to determine the antiproliferative activity. their results indicated that broccoli seedlings exert strong cytotoxicity against different types of cancer cell lines, including hepatocellular carcinoma cells (hepg ), prostate carcinoma cells (pc- , at- , and sum ), lung carcinoma cells (a ), and colorectal adenocarcinoma cells (caco- , and ht- ) ( table ). in these studies, the highly effective ic values were found to be from to µg/ml. on the another hand, the selectivity of broccoli seedlings was reported on normal skin fibroblasts (bj), normal colon fibroblasts (ccd -co), and normal liver cells (fl b) by displaying no toxic effects on their viability after the treatment of broccoli samples (table ) . cancer is essentially a disease of uncontrolled cell division. it is caused by an imbalance between cell proliferation and apoptosis [ ] . its development and progression are generally associated with the disorder of cell cycle regulators' activity. defects in the programmed cell death mechanism also make a key contribution to tumor pathogenesis [ ] . thus, most chemotherapeutic agents exert their cytotoxic activity against cancer cells, since they cause dna damage and activate a complex signaling network resulting in cell cycle arrest and apoptosis induction [ ] . targeting the cell cycle phase and the checkpoint signaling pathway, which leads to the arrests at g /s or g /m phases, would provide a promising opportunity for cancer treatment. besides, triggering cell apoptosis could be an effective strategy for potential chemotherapeutic agents [ , , , ] . to confirm the antiproliferative activity of broccoli sprouts and microgreens, several in vitro studies revealed the mechanism of cell death based on inducing cell cycle arrest and apoptosis (table ) . their results showed cell cycle arrests (obviously at g /g and s phases) and significant increases in cell percentage with subg dna content, which is considered as a marker of apoptosis. besides, mitochondrial changes might activate the intrinsic apoptotic pathway. the loss of mitochondrial membrane potential (mmp) leads to the activation of several proteins linked to apoptosis, such as caspase- and cytochrome c [ ] . hence, a few studies indicated the notable decrease in mmp levels of cancer cells after treatment by broccoli seedlings to prove the mechanism of programmed cell death [ , ] . furthermore, some in vivo carcinogenesis models were employed to demonstrate the anticancer effects and related molecular mechanisms of broccoli sprouts and microgreens, such as c bl/ mice, balb/c mice, skh- hairless mice, and sprague-dawley rats (table ). sfn extracted from broccoli sprouts showed the inhibition of breast cancer stem cells and downregulate the wnt/β-catenin self-renewal pathway in a nonobese diabetic/severe combined immunodeficient xenograft model [ ] . sfn isolated from broccoli seeds and sprouts also exhibited anticancer effects in lung cancer cell lines and nude balb/c mice with lung cancer xenograft by inhibiting the pi k-akt signaling pathway [ ] . the transgenic adenocarcinoma of the mouse prostate model with broccoli sprout intake demonstrated a significant decline in prostate cancer occurrence and hdac protein expression in the epithelial cells of the prostate. hdac is one of the histone deacetylase enzymes that turn off tumor suppressor genes and promote the expression of oncogenes [ ] . the broccoli sprout diet administered to adult her /neu mice showed both preventive and suppressive effects on mammary cancer. these protective effects were associated with tumor-and epigenetic-related gene expression, and changed histone acetylation, dna methylation, and dna hydroxymethylation levels [ ] . the dietary administration of broccoli sprout extract to the rat model inhibited bladder cancer development by inducing glutathione s-transferase and nad(p)h-quinone oxidoreductase in the bladder, which are important enzymes against oxidants and carcinogens [ ] . in vitro ns a , h , h , hcc , h , h cells inhibiting cell proliferation; inducing apoptosis [ ] in vitro ns caco- , ccd -co cells inducing cell cycle arrest and apoptosis; decreasing mmp level; increasing ros generation [ ] in vivo ns female nod/scid mice eliminating breast cscs in vivo; downregulating wnt/β-catenin pathway [ ] in vivo ns female nude balb/c mice inhibiting the pi k-akt signaling pathway [ ] in vivo days female skh- hairless mice stabilizing p ; inducing phase enzyme; inhibiting inos upregulation [ ] in vivo ns male tramp mice in c bl/ background decreasing hdac protein expression [ ] in vivo ns sv and her /neu mice modulating epigenetic pathways; regulating epigenetic-controlled gene expression [ ] in vivo ns female her /neu mice regulating tumor-and epigenetic-related gene expression; increasing tumor suppressor gene expression [ ] in vivo days female sprague-dawley rats inducing gst and nqo [ ] hepg the antimicrobial capacity of broccoli has been reported in several publications; however, most of these studies focused on broccoli florets. there are a few reports that displayed the detrimental effect of broccoli sprouts on pathogenic bacteria. a study investigated broccoli sprouts with high levels of gallic acid, esculetin, ferulic acid, and myricetin have the antibacterial activity against foodborne pathogens, including both gram-positive bacteria (staphylococcus aureus and bacillus subtilis) and gram-negative bacteria (salmonella typhimurium and escherichia coli), with minimum inhibition concentration (mic) values from to µg/ml [ ] . this study revealed that the gram-positive bacteria were more sensitive to broccoli sprout extract than the gram-negative bacteria, similar to the previously published results of other plant extracts [ , ] . the possible reason might be the structural differences in the cell wall between these two classes of bacteria. gram-negative bacteria are surrounded by an additional outer membrane, which is a hydrophilic layer to prevent the access of many substances including natural compounds [ , ] . thus, broccoli sprout extract was demonstrated to be more active on gram-positive bacteria. another in vitro study reported the powerful bactericidal activity of broccoli sprouts against helicobacter pylori by the presence of sulforaphane as the major anti-helicobacter active compound, with highly active inhibition zones (> cm) [ ] . many reports showed that plant-derived bioactive compounds, such as phenolics, flavonoids, and glucosinolates, can inhibit the growth and activity of various microorganisms [ ] . with the diversity in the molecular structure and chemical composition, these compounds can perform distinct antimicrobial effects, such as destabilization of the plasma membrane or inhibition of extracellular enzymes [ ] . the antimicrobial activity against pathogenic bacteria, yeast, and phytopathogenic fungi was also proven by the presence of many antimicrobial peptides in broccoli floret extract [ ] . moreover, the daily intake of sulforaphane-rich broccoli sprouts for months was demonstrated to reduce gastric bacterial colonization and attenuate gastritis in helicobacter pylori-infected mice and patients [ ] . in another clinical trial, the high-sulforaphane broccoli sprouts powder also showed a considerable effect on h. pylori eradication in type diabetic patients with positive h. pylori [ ] . collectively, these findings indicate that broccoli sprout extracts might serve as a potential source of antimicrobial agents in the food and pharmaceutical industry. several studies proved that broccoli sprouts and their bioactive components possess anti-inflammatory activity. hence, the application of broccoli sprouts and microgreens has potential in the prevention and treatment of inflammatory bowel diseases, such as ulcerative colitis and crohn's disease [ ] . the anti-inflammatory mechanisms of broccoli sprouts are probably associated with the nuclear factor-kappa b (nf-κb) and nuclear factor erythroid -related factor (nrf ) signaling pathways [ ] . both in vitro and in vivo experiments, as well as clinical trials, mainly exhibit anti-inflammatory effects of broccoli sprouts, which showed high concentrations of sulforaphane and a group of phenolic compounds, including anthocyanins, isoquercetin, chlorogenic and cinnamic acids, via inhibiting inflammatory mediators such as a nitric oxide (no), decreasing the levels of proinflammatory cytokines such as tumor necrosis factor α (tnf-α), interleukin- (il- ), and il- β, and increasing the levels of anti-inflammatory cytokines such as il- and il- [ , , , , , ] . for example, a study showed that sulforaphane-enriched broccoli sprouts inhibited activation of the nf-κb signaling pathway and the secretions of inflammatory proteins (inducible nitric oxide synthase (inos), cyclooxygenase (cox- ), tnf-α, il- , il- β, and prostaglandin e (pge )) in microglial cells (bv ) and male icr (institute of cancer research) mice. in this study, broccoli samples also upregulated the expression of nrf and heme oxygenase- (ho- ) in normal bv cells and against scopolamine-induced amnesia in mouse brain tissue samples [ ] . to analyze immunological parameters in the wistar rats with iodine deficiency, the significant decrease in il- level, along with no significant differences in il- concentration, were observed after adding broccoli sprouts to the diet [ ] . high-sulforaphane broccoli sprouts also indicated favorable effects on inflammatory markers by reducing concentrations of serum high-sensitive c reactive protein (hs-crp), il- , and tnf-α in type diabetic patients [ ] . broccoli is one of the few vegetables that is considered as a supplementary treatment for type diabetes and for the prevention of its long-term complications [ ] . a few studies investigated the potential benefits of broccoli sprouts and microgreens for patients with diabetes. a study determined the effects of broccoli sprout powder on insulin resistance in type diabetic patients as a new approach by the use of its bioactive constituents. the results showed that broccoli sprout powder containing a high concentration of sulforaphane may significantly decrease in serum insulin concentration and lessen complications of diabetes [ ] . in another report, the potential efficacy of sulforaphane extracted from young broccoli sprouts has been confirmed as an effective option for supplementary treatment in type diabetes. it could induce some peroxisome proliferators-activated receptors, which contribute to glucose homeostasis in hyperglycemia and oxidative conditions [ ] . obesity has become a worldwide health problem and leads to adverse metabolic disorders, such as cardiovascular disease and type diabetes. a couple of studies documented positive actions of broccoli sprouts on obesity by regulating lipid metabolism. in a double-blind clinical trial, broccoli sprout powder as supplementary treatment in type diabetic patients could have beneficial effects on lipid profiles and oxidized low-density lipoprotein ratio (ox-ldl/ldl), as risk factors for obesity and cardiovascular disease [ ] . another mechanism to regulate lipid metabolism of broccoli sprouts may be associated with sulforaphane capacity to induce the nrf pathway [ ] . a recent study revealed that glucoraphanin extracted from broccoli sprouts can decrease the lipid accumulation and increase the nrf activation. it leads to the downregulation of the expression of multiple genes involved in gluconeogenesis and lipogenesis, thereby alleviating obesity and diabetes [ ] . several other health-promoting effects of broccoli sprouts and microgreens have also been explored by in vitro, in vivo, and clinical research. a couple of studies indicated that broccoli sprouts are a good source of bioactive compounds that act as xanthine oxidase (xo) inhibitors [ , , ] . xo is the enzyme that catalyzes the metabolism of hypoxanthine and xanthine into uric acid, so inhibiting xo activity may be potentially useful in the treatment of gout or other xo-induced diseases [ ] . broccoli sprout supplementation during pregnancy and the early newborn period could reduce brain injury following placental insufficiency in the newborn rats. the findings provide a new approach for the prevention of cerebral palsy and disabilities related to placental insufficiency [ ] . the analgesic and antinociceptive effects of broccoli sprout extract were proven by the opioid mechanism using two in vivo experimental models of nociception. this study suggests the potential activity of broccoli sprouts in pain therapy [ ] . in a double-blind study, the short-term ingestion of broccoli sprout homogenates showed the beneficial effects on nasal responses to the influenza virus in smokers by significantly decreasing virus-induced markers of inflammation and reducing the virus quantity. it may be a promising strategy for preventing influenza risk in smokers and other people exposed to airborne pollutants [ ] . in recent years, broccoli sprouts and microgreens are one of the most consumed vegetable products. they have gained recognition as functional foods or nutraceutical foods by the increasing interest of consumers for diets that support health and longevity. consequently, the health-promoting compounds of broccoli seedlings have been extracted and isolated to integrate into food and pharmaceutical product formulations. over the past ten years, the extraction, isolation, and characterization of the functional properties of these broccoli products have been demonstrated by numerous scientific publications. in this review, we summarized for the first time the research findings of the last decade into bioactive constituents, bioactivities, and molecular mechanisms of broccoli sprouts and microgreens. they have been proven to present an abundant source of important natural compounds, including glucosinolates, phenolics, flavonoids, vitamins, minerals, and pigments. in particular, glucosinolates and their hydrolysis products are the main components analyzed, followed by phenolic compounds with a detailed profile. moreover, the previous studies have focused on several biological activities of broccoli seedlings, such as antioxidant, anticancer, antimicrobial, and anti-inflammatory, as well as the potentially beneficial effects for patients with cancers, diabetes, and obesity. in general, they are non-toxic or have low toxicity. hopefully, this updated review, in addition to being a reference for consumers' food selections, might attract more attention to broccoli sprouts and microgreens and their further applications in the food and nutraceutical industries, or even in clinical studies as cancer chemopreventive agents. in the future, more bioactive compounds in broccoli sprouts and microgreens will be isolated, identified, and evaluated. further investigations are required for exploring unrecognized biological functions and their underlying mode-of-action and molecular mechanisms in both in vitro and in vivo models, such as cardiovascular protective, hepatoprotective, neuroprotective, or enzyme inhibitory potentials. besides, well-designed clinical trials should be carried out to confirm these health-promoting benefits of broccoli seedlings on humans. food security and sustainable intensification broccoli microgreens: a mineral-rich crop that can diversify food systems how circular will you eat? the sustainability challenge in food and consumer reaction to either waste-to-value or yet underused novel ingredients in food small-seeded legumes as a novel food source. variation of nutritional, mineral and phytochemical profiles in the chain: raw seeds-sprouted seeds-microgreens relationships between bioactive food components and their health benefits the role of functional foods, nutraceuticals, and food supplements in intestinal health polyphenolic profile and varied bioactivities of processed taiwanese grown broccoli: a comparative study of edible and non-edible parts implementation of phenols recovered from olive mill wastewater as uv booster in cosmetics a facile water-induced complexation of lycopene and pectin from pink guava byproduct: extraction, characterization and kinetic studies phenols recovered from olive mill wastewater as additives in meat products the food systems in the era of the coronavirus (covid- ) pandemic crisis. foods recovery of high added-value components from food wastes: conventional, emerging technologies and commercialized applications separation of functional macromolecules and micromolecules: from ultrafiltration to the border of nanofiltration selecting sprouts of brassicaceae for optimum phytochemical composition microgreens: production, shelf life, and bioactive components micro-scale vegetable production and the rise of microgreens microgreen nutrition, food safety, and shelf life: a review bioactive compounds and bioactivities of germinated edible seeds and sprouts: an updated review broccoli and radish sprouts are safe and rich in bioactive phytochemicals evaluation of the bioaccessibility of antioxidant bioactive compounds and minerals of four genotypes of brassicaceae microgreens alone or combined, redirect the biosynthesis of glucosinolates, phenolics, carotenoids, and chlorophylls in broccoli sprouts biochemical composition of broccoli seeds and sprouts at different stages of seedling development dietary sulforaphane-rich broccoli sprouts reduce colonization and attenuate gastritis in helicobacter pylori-infected mice and humans brassica oleracea l. var. italica) sprouts as the potential food source for bioactive properties: a comprehensive study on in vitro disease models the natural compound sulforaphene, as a novel anticancer reagent, targeting pi k-akt signaling pathway in lung cancer analysis and anti-helicobacter activity of sulforaphane and related compounds present in broccoli (brassica oleracea l.) sprouts sulforaphane-enriched broccoli sprouts pretreated by pulsed electric fields reduces neuroinflammation and ameliorates scopolamine-induced amnesia in mouse brain through its antioxidant ability via nrf -ho- activation effects of broccoli sprout with high sulforaphane concentration on inflammatory markers in type diabetic patients: a randomized double-blind placebo-controlled clinical trial effect of broccoli sprouts on insulin resistance in type diabetic patients: a randomized double-blind clinical trial broccoli sprouts powder could improve serum triglyceride and oxidized ldl/ldl-cholesterol ratio in type diabetic patients: a randomized double-blind placebo-controlled clinical trial extraction, chemical characterization and biological activity 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compounds and antioxidant capacity in broccoli sprouts during growth and storage uvb light doses and harvesting time differentially tailor glucosinolate and phenolic profiles in broccoli sprouts uv-b irradiation changes specifically the secondary metabolite profile in broccoli sprouts: induced signaling overlaps with defense response to biotic stressors photosynthetic pigments, phenolic, glucosinolates content and antioxidant capacity of broccoli sprouts in response to nanoselenium particles supply assessment of the anticancer compounds se-methylselenocysteine and glucosinolates in se-biofortified broccoli (brassica oleracea l. var. italica) sprouts and florets bioavailability of isothiocyanates from broccoli sprouts in protein, lipid, and fiber gels genotypic effects on the phytochemical quality of seeds and sprouts from commercial broccoli cultivars effects of long-term consumption of broccoli sprouts on inflammatory markers in overweight subjects bioavailability and inter-conversion of sulforaphane and erucin in human subjects consuming broccoli sprouts or broccoli supplement in a cross-over study design effect of sucrose and mannitol on the accumulation of health-promoting compounds and the activity of metabolic enzymes in broccoli sprouts sucrose enhances the accumulation of anthocyanins and glucosinolates in broccoli sprouts metabolism and antiproliferative effects of sulforaphane and broccoli sprouts in human intestinal (caco- ) and hepatic (hepg ) cells characterization of products from the reaction of glucosinolate-derived isothiocyanates with cysteine and lysine derivatives formed in either model systems or broccoli sprouts physiological and biochemical metabolism of germinating broccoli seeds and sprouts sulforaphane bioavailability from glucoraphanin-rich broccoli: control by active endogenous myrosinase calcium mitigates the stress caused by znso as a sulphur fertilizer and enhances the sulforaphane formation of broccoli sprouts effect of se treatment on glucosinolate metabolism and health-promoting compounds in the broccoli sprouts of three cultivars increasing antioxidant content of broccoli sprouts using essential oils during cold storage. agriculture (polnohospodárstvo) response surface optimization and identification of isothiocyanates produced from broccoli sprouts free radical scavenging, antiproliferative activities and profiling of variations in the level of phytochemicals in different parts of broccoli (brassica oleracea italica) absorption and chemopreventive targets of sulforaphane in humans following consumption of broccoli sprouts or a myrosinase-treated broccoli sprout extract optimisation of enzymatic production of sulforaphane in broccoli sprouts and their total antioxidant activity at different growth and storage days comparative study of predominant phytochemical compounds and proapoptotic potential of broccoli sprouts and florets selenium enrichment of broccoli sprout extract increases chemosensitivity and apoptosis of lncap prostate cancer cells effect of broccoli sprouts on thyroid function, haematological, biochemical, and immunological parameters in rats with thyroid imbalance antiproliferative effect of bioaccessible fractions of four brassicaceae microgreens on human colon cancer cells linked to their phytochemical composition sorting out the value of cruciferous sprouts as sources of bioactive compounds for nutrition and health phenolic acids: natural versatile molecules with promising therapeutic applications effect of bioaccessibility of phenolic compounds on in vitro anticancer activity of broccoli sprouts influence of sodium and maturity stage on the antioxidant properties of cauliflower and broccoli sprouts phenolic profile and antioxidant activity in selected seeds and sprouts energy regulated nutritive and antioxidant properties during the germination and sprouting of broccoli sprouts (brassica oleracea var. italica) morphometric characteristics, polyphenols and ascorbic acid 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inhibitor administration chemical and biological characterisation of nutraceutical compounds of broccoli potential efficacy of broccoli sprouts as a unique supplement for management of type diabetes and its complications oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options reactive oxygen species: the dual role in physiological and pathological conditions of the human body reactive oxygen species in health and disease analysis and antioxidant activity of extracts from broccoli (brassica oleracea l.) sprouts measurement of antioxidant activity review on in vivo and in vitro methods evaluation of antioxidant activity enhancement of antioxidant abilities and the lipoxygenase and xanthine oxidase inhibitory activity of broccoli sprouts by biotic elicitors induction of the phase response in mouse and human skin by sulforaphane-containing broccoli sprout extracts pharmacological targeting of cell cycle, apoptotic and cell adhesion signaling pathways implicated in chemoresistance of cancer cells sulforaphane, a dietary component of broccoli/broccoli sprouts, inhibits breast cancer stem cells broccoli sprouts delay prostate cancer formation and decrease prostate cancer severity with a concurrent decrease in hdac protein expression in transgenic adenocarcinoma of the mouse prostate (tramp) mice maternal epigenetic regulation contributes to prevention of estrogen receptor-negative mammary cancer with broccoli sprout consumption inhibition of urinary bladder carcinogenesis by broccoli sprouts protection against uv-light-induced skin carcinogenesis in skh- high-risk mice by sulforaphane-containing broccoli sprout extracts temporal efficacy of a sulforaphane-based broccoli sprout diet in prevention of breast cancer through modulation of epigenetic mechanisms antimicrobial activity and phytochemical analysis of organic extracts from cleome spinosa which approach is more effective in the selection of plants with antimicrobial activity? evid plant phenolics and phenolic-enriched extracts as antimicrobial agents against food-contaminating microorganisms antimicrobial activity of broccoli (brassica oleracea var. italica) cultivar avenger against pathogenic bacteria, phytopathogenic filamentous fungi and yeast complementary and alternative medicinal effects of broccoli sprouts powder on helicobacter pylori eradication rate in type diabetic patients: a randomized clinical trial nutraceutical improvement increases the protective activity of broccoli sprout juice in a aqueous extract of glucoraphanin-rich broccoli sprouts inhibits formation of advanced glycation end products and attenuates inflammatory reactions in endothelial cells glucoraphanin: a broccoli sprout extract that ameliorates obesity-induced inflammation and insulin resistance broccoli sprout supplementation during pregnancy prevents brain injury in the newborn rat following placental insufficiency broccoli sprouts in analgesia-preclinical in vivo studies effect of broccoli sprouts on nasal response to live attenuated influenza virus in smokers: a randomized, double-blind study funding: this research received no external funding. the authors declare no conflict of interest. key: cord- -ffi k authors: khan, naseem ahmed; singla, mohit; samal, sweety; lodha, rakesh; medigeshi, guruprasad r. title: respiratory syncytial virus-induced oxidative stress leads to an increase in labile zinc pools in lung epithelial cells date: - - journal: msphere doi: . /msphere. - sha: doc_id: cord_uid: ffi k zinc supplementation in cell culture has been shown to inhibit various viruses, like herpes simplex virus, rotavirus, severe acute respiratory syndrome (sars) coronavirus, rhinovirus, and respiratory syncytial virus (rsv). however, whether zinc plays a direct antiviral role in viral infections and whether viruses have adopted strategies to modulate zinc homeostasis have not been investigated. results from clinical trials of zinc supplementation in infections indicate that zinc supplementation may be beneficial in a pathogen- or disease-specific manner, further underscoring the importance of understanding the interaction between zinc homeostasis and virus infections at the molecular level. we investigated the effect of rsv infection on zinc homeostasis and show that rsv infection in lung epithelial cells leads to modulation of zinc homeostasis. the intracellular labile zinc pool increases upon rsv infection in a multiplicity of infection (moi)-dependent fashion. small interfering rna (sirna)-mediated knockdown of the ubiquitous zinc uptake transporter zip suggests that labile zinc levels are increased due to the increased uptake by rsv-infected cells as an antiviral response. adding zinc to culture medium after rsv infection led to significant inhibition of rsv titers, whereas depletion of zinc by a zinc chelator, n,n,n′,n′-tetrakis( -pyridinylmethyl)- , -ethanediamine (tpen) led to an increase in rsv titers. the inhibitory effect of zinc was specific, as other divalent cations had no effect on rsv titers. both rsv infection and zinc chelation by tpen led to reactive oxygen species (ros) induction, whereas addition of zinc blocked ros induction. these results suggest a molecular link between rsv infection, zinc homeostasis, and oxidative-stress pathways and provide new insights for developing strategies to counter rsv infection. importance zinc deficiency rates in developing countries range from to %, and zinc supplementation trials have been shown to correct clinical manifestations attributed to zinc deficiency, but the outcomes in the case of respiratory infections have been inconsistent. we aimed at understanding the role of zinc homeostasis in respiratory syncytial virus (rsv) infection. infection of lung epithelial cell lines or primary small-airway epithelial cells led to an increase in labile zinc pools, which was due to increased uptake of zinc. zinc supplementation inhibited rsv replication, whereas zinc chelation had an opposing effect, leading to increases in rsv titers. increases in labile zinc in rsv-infected cells coincided with induction of reactive oxygen species (ros). both zinc depletion and addition of exogenous ros led to enhanced rsv infection, whereas addition of the antioxidant inhibited rsv, suggesting that zinc is part of an interplay between rsv-induced oxidative stress and the host response to maintain redox balance. r espiratory syncytial virus (rsv) is a common cause of acute lower respiratory tract infection (alri) in infants, which leads to hospitalization. elderly and immunocompromised individuals are also highly susceptible to severe disease due to rsv. according to estimates for the year , rsv-alri led to . million hospital admissions of children less than months of age, of which , succumbed to infection ( ) . rsv belongs to the paramyxoviridae family and is an enveloped, nonsegmented, negativestrand rna virus. the clinical manifestations of rsv infection vary from mild upper respiratory tract illness (urti) to potentially life-threatening lower respiratory tract involvement (lrti). there is no vaccine or effective antiviral drug available for rsv; the only available treatment is immunoprophylaxis of severe rsv disease in high-risk infants with palivizumab ( , ), which is not an affordable option in many low-and middle-income countries. therefore, there is a need to develop affordable interventions through better understanding of cellular factors that regulate rsv infection. zinc is an essential micronutrient and plays diverse physiological roles in multiple cellular processes, such as the immune response, signal transduction, organelle homeostasis, cell proliferation, and cell death ( , ) . zinc deficiency rates in developing countries range from to %. in india, studies have reported that to % of pregnant women and that between and % of children are zinc deficient ( ) . nearly % of healthy elderly subjects may be zinc deficient in developed countries. as per the world health organization estimates, , people die annually due to zinc deficiency, and more than half of these deaths occur in children under the age of years ( ) . zinc supplementation was shown to reduce the respiratory morbidity of alri in children less than years of age who were zinc deficient ( ) . studies examining the clinical effects of zinc for treating pneumonia in children have shown conflicting results, with some studies showing a beneficial effect on the duration of recovery and severity but with other studies suggesting that zinc has no treatment benefit ( ) ( ) ( ) ( ) ( ) . although the necessary role of zinc as a micronutrient in various physiological functions has been demonstrated, the molecular mechanism underlying the effects of zinc during viral infections has not been elucidated. in this study, we utilized changes in intracellular labile zinc pools as a measure of zinc homeostasis in lung epithelial cell lines and primary small-airway epithelial cells (saecs) and investigated the effect of rsv infection on zinc homeostasis. our results suggest that zinc homeostasis plays a critical role in the host response to rsv infection by regulating oxidative stress and inhibiting virus replication. there are contrasting reports on the role of zinc in viral infections, and we speculated that viral infections, especially those from rna viruses, which replicate in the cytoplasm, may alter the dynamics of cytosolic levels of free zinc. a cells were infected with rsv at a multiplicity of infection (moi) of . , and under these conditions, we observed a time-dependent increase in the percentage of infected cells from % to % from h to h postinfection (p.i.) (see fig. s a in the supplemental material). we utilized two different zinc-binding fluorophores, fluozin- (flz- ) and zinpyr- (zp- ), and measured labile zinc levels by flow cytometry in rsv-infected cells. we observed an increase in labile zinc levels in both flz- and zp- stains in a time-dependent manner. levels of flz- in stains increased from % to % from h p.i. to h p.i. (fig. a) in rsv-infected cells. similarly, zp- staining showed an increase from % to % from h p.i. to h p.i. (fig. b) . to further confirm whether an increase in labile zinc levels is due to active rsv replication, we used uv-inactivated rsv, which failed to show any modulation in free zinc levels ( fig. a and b) . we next confirmed whether this modulation of free zinc levels is specific to rsv by infecting a cells with influenza a virus subtype h n (iav) at an moi of . labile zinc levels were measured using flz- and zp- at h p.i. and h p.i. iav infection led to a significant increase in flz- staining only at h p.i. (fig. a) , whereas zp- staining did not show any significant change (fig. b) . we next tested whether the effect is observed with other nonrespi-ratory rna viruses, such as dengue virus (denv) at an moi of , and measured free zinc levels as described above. we did not observe any change in labile zinc levels in cells infected with denv ( fig. a and b) , suggesting that zinc homeostasis is altered specifically by rsv and iav; rsv infection in particular led to increases in both cytosolic and vesicular labile zinc pools, whereas denv infection had no effect in a cells. cell viability was not compromised under any of these conditions, as verified by staining cells with live-dead stain and examining them by flow cytometry (fig. s b) . we next infected a cells with rsv at mois of . , , and , and labile zinc levels were measured at h p.i. using flz- and zp- . we observed an moi-dependent increase in labile zinc levels. flz- staining increased from % to % at mois from . to , and a % increase in staining was observed at an moi of (fig. c) . similarly, zp- staining increased from % at an moi of . , to % at an moi of , and to % at an moi of (fig. d) . we determined the percentages of cells infected under these conditions by immunofluorescence (if) staining for rsv f protein. as expected, the proportion of rsv-infected cells increased from % at an moi of . to % at an moi of , and % of the cells were infected at an moi of (fig. s c) . these results suggest that rsv infection triggers an increase in cellular free zinc levels that is time and moi dependent; this increase in labile zinc levels is specific to rsv, as dengue infection had no effect. we observed increases in labile zinc levels in a cells infected with rsv. to further verify whether this phenomenon is specific to a cells, we used primary small-airway epithelial cells (saecs) to further confirm these observations. saecs were infected with rsv at an moi of , and cells were stained with zp- at and h p.i. under these conditions, around % of the cells were infected with rsv (fig. s ) . as with a cells, we observed a time-dependent increase in labile zinc levels in saecs infected with rsv without any effect on cell viability ( fig. a and b) . we next isolated nasal epithelial cells from nasopharyngeal washes collected from pediatric patients ( table ) infected with rsv or healthy controls. labile zinc levels were measured by zp- staining, and we observed a -fold increase in the zp- signal in nasal epithelial cells isolated from rsv patients compared to levels in cells from healthy controls (fig. c) , and as observed for a cells and saecs, the viability of nasal epithelial cells that were used for zp- staining was not compromised under the experimental conditions (fig. d) . overall, these data suggest that rsv infection leads to an increase in labile zinc levels in cell lines, primary cells, and nasal epithelial cells isolated from rsv patients. apoptosis has been shown to induce an increase in labile zinc levels ( , ) . although cell viability was not affected under our experimental conditions, we further verified whether increases in labile zinc pools are due to apoptosis induction by rsv. we infected a cells with rsv and denv at mois of . and , respectively, and stained cells with annexin-v to measure the proportions of apoptotic cells under these conditions. as observed earlier, there was no significant difference in the nonviable cell populations between rsv and denv ( . % versus . %). the proportion of annexin-v-positive cells increased from . % to . % in cells infected with rsv and to . % in cells infected with denv, clearly indicating that induction of apoptosis does not contribute to induction of labile zinc levels in rsv infections (fig. e) . as a positive control for this assay, we induced apoptosis in a cells by heat stress as per previous studies ( , ) , which led to annexin-v positivity in % of the cells (fig. s a ). under these conditions, cells stained with flz- showed a -fold increase in staining, whereas cells stained with zp- showed an ϳ -fold increase in signal intensity (fig. s b) . these results suggest that although apoptosis leads to increases in labile zinc levels, it does not contribute to increases in labile zinc levels observed in rsv infection. zip kd upregulates rsv infection. we next evaluated whether the increase in labile zinc pools observed in rsv infection is due to enhanced zinc uptake. to test this, we utilized inductively coupled plasma mass spectrometry (icp-ms) to quantify total zinc content within the cell under rsv infection conditions. a cells were infected with rsv and denv at mois of . and , respectively. total metal content was determined at h p.i. the amount of zn, mg, mn, and cu detected by icp-ms in parts per billion was normalized to the total protein content of the cells to account for any difference in cell numbers. we observed an increase in total zinc content from Ϯ ppb/mg (mean Ϯ standard deviation [sd]) in mock-infected cells to Ϯ ppb/mg in rsv-infected cells, but no significant difference was observed in mg or mn content between these cells. surprisingly, we also observed a significant increase in cu content in rsv infection relative to that in uninfected cells ( . Ϯ . ppb/mg versus . Ϯ ppb/mg) ( fig. a to d). this suggests that an increase in the total zinc content in rsv-infected cells is possibly due to zinc uptake. zinc homeostasis in cells is regulated by influx (slc or zip) and efflux (slc or znt) transporters and also by redistribution between intracellular organelles ( ) ( ) ( ) . because the zip family of transporters plays a key role in zinc uptake, we next performed small interfering rna (sirna)-mediated knockdown (kd) of zip , which is a ubiquitously expressed zinc uptake transporter localized to the plasma membrane, and of zip , whose expression is higher in lungs than in other tissues and is also mostly localized to the plasma membrane. we infected zip and zip kd cells with rsv at an moi of . . rsv titers were measured at h p.i. by plaque assays. sirna transfections led to an approximately to % knockdown efficiency in the respective zip and zip mrnas compared to that of nontargeting control (ntc) sirnas (fig. a ). zip knockdown led to around a -fold increase in rsv titers at h p.i., whereas no difference in rsv titers was observed under the zip kd condition ( fig. b and c). the data suggest that downregulating zip , which may impact zinc uptake from the extracellular medium, leads to better rsv infection, suggesting that zinc may act as an antiviral to perturb rsv infection. the cell viability of npa specimens was determined by staining the cells with the fixable viability dye efluor . there were controls and cases. (e) a cells were infected with rsv and denv at mois of . and , respectively. cells were stained with annexin-v and efluor dye at h p.i. and h p.i. cell populations represent different phases: healthy, early apoptosis, late apoptosis, and necrosis. annexin-v signal was detected in fitc filter and efluor- signal was detected in apc-cy- filter. data are from at least two independent experiments. *, p Ͻ . ; **, p Ͻ . ; ns, differences were not significant. our results indicate that cellular zinc levels increase in rsv-infected cells, suggesting that either increased zinc levels benefit rsv infection or zinc induction is part of a host response to deal with rsv infection. we further investigated the relevance of cellular zinc in rsv infection by testing the effect of zinc supplementation and zinc depletion in a cells. we first determined zinc uptake in a cells by supplementing the cells with m to m zinc in serum-free medium (fig. s a ). next, we determined the efficiency of zinc uptake in serum-free media, % serum-containing media, and % serum-containing media. as expected, zinc uptake by cells, measured as increases in labile zinc levels, decreased with increasing concentrations of serum. zp- signal intensity decreased from ϳ . -fold to . -fold with serum concentrations increasing from % to %. zinc uptake, as measured by changes in the zp- signal, was negligible in medium containing % serum under these conditions (fig. s b ). we then tested whether zinc supplementation has any effect on cell viability by treating a cells with different concentrations of znso from m up to m and assessing cell viability (fig. s c) . similarly, we also determined that m znso had no effect on cell viability in saecs (data not shown). we next investigated the effect of zinc supplementation and zinc chelation on rsv infection. we infected a cells with rsv and denv at mois of . and , respectively. after virus adsorption, cells were treated with m znso . viral titers were measured at h p.i. by plaque assay. we observed close to a % reduction in rsv titers under conditions of excess znso in the medium, whereas denv titers showed no significant rsv replication (fig. c ). we also tested whether this inhibition is specific to zinc or whether other divalent cations are also capable of inducing a similar inhibitory effect with rsv. rsv-infected a cells were cultured in the presence of m znso , magnesium chloride, calcium chloride, or copper chloride and m ferrous sulfate and manganese chloride. these concentrations were determined as noncytotoxic concentrations. no divalent cationic salt besides znso showed any inhibitory effect on rsv infection (fig. d) in rsv titers, whereas addition of znso at h p.i. reduced rsv titers by %. no significant effect of znso addition was observed beyond h p.i. (fig. e ). these data suggest that addition of zinc affects early stages of the rsv life cycle. zinc chelation enhances rsv infection. we have recently reported that zinc depletion negatively affects denv infection in caco- cells ( ) . to address if zinc depletion affects rsv infection, we further standardized zinc depletion conditions in a cells using a cell-permeable zinc chelator, n,n,n=,n=-tetrakis( -pyridinylmethyl)- , -ethanediamine (tpen). we first determined the effect of tpen treatment on cell viability by treating cells with tpen at concentrations starting from . m to m. we found that tpen concentrations above . m affected cell viability. we next measured the extent of reduction in labile zinc levels with tpen treatment under these conditions. zp- levels were down by % with . m tpen for h compared to levels with a vehicle control (dimethyl sulfoxide [dmso]) ( fig. s a and s b) . these data suggest that regulation of zinc homeostasis is closely linked to cell viability and that both excess zinc and low zinc may affect cell fates. we infected a cells with rsv or denv and then treated the cells with . m tpen. tpen treatment led to a -fold increase in rsv titers but a nearly complete inhibition of denv titers at h p.i. (fig. a) . we quantitated rsv and denv genome levels under these conditions by quantitative real-time pcr (qrt-pcr). as with virus titers, we observed an ϳ -fold increase in rsv rna levels and a Ͼ % reduction in denv rna levels at h p.i. under zinc-depleted conditions (fig. b) . these data suggest that the cellular response to rsv infection results in an increase in intracellular zinc, which plays an antiviral role by inhibiting rsv replication. zinc inhibits rsv as an antioxidant. respiratory viruses, such as rsv and influenza virus, induce oxidative stress in infected cells ( ) ( ) ( ) . zinc has been shown to ame- liorate oxidative stress, as some of the players in the antioxidant pathways, such as superoxide dismutase (sod), require zinc for their functions ( ) . we hypothesized that induction of oxidative stress by rsv may be a trigger for increases in intracellular zinc levels and that zinc may exert anti-rsv activity as an antioxidant molecule. we first verified induction of oxidative stress by rsv in a cells. oxidative stress was measured by estimating reactive oxygen species (ros) in the cells by flow cytometry using h dcfda dye. we observed a modest % increase in ros levels at h p.i. however, by h p.i., ros levels increased by -fold in rsv-infected cells relative to levels after a mock infection (fig. a) . we next measured the expression levels of mrnas of some of the antioxidant enzymes and found that rsv infection leads to induction of nadph oxidase- (nox ), which is a ubiquitously expressed enzyme involved in the synthesis of ros. rsv infection also led to inhibition in mrna levels of catalase (cat) and glutathione s-transferase a (gsta ) at later stages of infection which coincide with ros induction. no effect was observed on superoxide dismutase- (sod ) (fig. b) . these data suggest that induction of nox and suppression of antioxidant enzymes during rsv infection leads to upregulation of ros. the kinetics of induction of ros in rsv-infected cells correlated with increases in labile zinc levels observed in these cells. as zinc has been shown to act as an antioxidant ( ), we speculated that the increase in labile zinc levels may be part of a host response to counter the damage by rsv-induced oxidative stress. to test this, we first determined whether modulation of zinc levels by zinc supplementation or chelation could affect basal ros levels. a cells were treated with m znso and . m tpen. intracellular ros levels were measured at h, h, and h posttreatment using h dcfda. although excess zinc in the culture medium had no effect on basal ros levels in the first h of treatment, the continued presence of zinc for h lowered basal ros levels by % (fig. c) . in contrast to this, zinc chelation by tpen led to significant increases in ros levels in a time-dependent manner, and we observed a -fold increase in ros levels by h of tpen treatment (fig. d) . to further confirm the role of oxidative stress in rsv infection, cells infected with rsv were treated with m h o (one of the ros) and virus titers were measured at h p.i. as observed in the case of zinc chelation, addition of h o led to a modest but significant increase in rsv titers at h p.i. (fig. a) . we further determined the importance of oxidative stress by adding mm n-acetyl cysteine (nac), which is a potent antioxidant compound, after virus adsorption and rsv titers in the supernatant were measured at and h p.i. by plaque assay. unlike with the addition of ros, the presence of nac during infection suppressed rsv infection (fig. b) . these results indicate that the induction of ros during rsv infection is beneficial for virus replication and that the cells respond to counter this effect by modulating zinc homeostasis and increase the cellular uptake of zinc, which acts to suppress virus-induced oxidative stress, thereby limiting virus replication. zinc homeostasis is regulated by zinc influx and efflux transporters, which modulate cellular zinc levels in response to various stimuli, such as inflammation, stress responses, and growth factors, and changes in intracellular labile zinc levels constitute an integral part of this modulation ( ) ( ) ( ) . free zinc is proposed to act as a second messenger regulating signal transduction pathways, further underscoring the importance of zinc homeostasis in health and disease ( , ) . we demonstrate that rsv infection leads to increases in labile zinc levels in a cells, primary small airway epithelial cells, and epithelial cells isolated from nasopharyngeal aspirates (npa) of children with rsv infection. in contrast to this, denv infection had no effect on zinc homeostasis in a cells, suggesting that, in lung epithelial cells, modulation of zinc homeostasis is specific to rsv. we demonstrate that the total zinc content in rsv-infected cells is increased, suggesting an increase in zinc uptake by zip family transporters. we show that knockdown of zip but not zip led to increases in rsv titers in the supernatants, suggesting that zinc uptake during rsv infection may act as an antiviral response. there are zip transporters in mammalian cells, and we have not explored the role of other zip family members in our study. we further prove the antiviral effect of zinc ions by zinc supplementation and zinc chelation studies and show that these treatments produce opposing results with rsv titers. supplementation of culture media with excess zinc, but not with any other divalent cation, inhibited rsv infection, while zinc chelation by tpen led to increased virus titers. increases in labile zinc levels in rsv infection coincided with an induction of oxidative stress, suggesting a critical role for zinc homeostasis in the host response to rsv infection. we also show that rsv infection actively induces oxidative stress by upregulation of nox , which is involved in the generation of ros and also in the downregulation of cat and gsta , which are components of antioxidant pathways. furthermore, we show that oxidative stress is a proviral pathway in the case of rsv infection, as induction of ros by zinc chelation or addition of ros (h o ) exogenously led to increases in virus titers, whereas antioxidants such nac inhibited rsv. zinc is a stable divalent cation and does not directly undergo redox reactions but is involved in regulating the oxidant/antioxidant balance ( ) . the cytosolic pool of zinc is regulated by metallothioneins, and the redox status of the cells plays an important role in maintaining this labile zinc pool. therefore, induction of oxidative stress by rsv may also act as a trigger for metallothioneins to release zinc into the cytoplasm to maintain the cellular redox state ( , ) . increases in labile zinc levels appear to be an antiviral response, as zinc supplementation led to inhibition of rsv infection, whereas mimicking zinc deficiency by tpen or zip knockdown proved to be beneficial for rsv infection. increased oxidative stress is one of the clinical manifestations of zinc deficiency, which can be corrected by zinc supplementation ( , ) . oxidative stress may influence viral infections in more than one way depending on the host pathways and compartments that are usurped by the viruses for genome replication, translation, assembly, and egress ( , ) . in saccharomyces cerevisiae, induction of oxidative stress by hydrogen peroxide led to increases in the expression of a number of proteins involved in maintaining the redox homeostasis, such as manganese superoxide dismutase (mnsod), cu/zn-sod, catalase, components of proteasome, and heat shock proteins. it was also observed that under oxidative-stress conditions, carbohydrate metabolism was redirected from glycolysis to generation of nadph to deal with the redox imbalance. therefore, viruses such as denv, which depend on glycolysis as a source of energy during replication ( ) , may avoid induction of ros, while rsv actively downregulates antioxidant pathways ( ) . interestingly, treatment of mice or lung epithelial cells with toll-like receptor (tlr) agonists led to induction of ros, which inhibited influenza virus replication, clearly demonstrating the contrasting roles of ros in respiratory virus infections ( ) . based on our results, we propose that rsv infection induces oxidative stress, which creates a favorable environment for rsv replication and spread. cells counter this response by enhancing cellular zinc levels by increased uptake by zinc transporters (zip and possibly other zips). close to billion people worldwide consume zinc-deficient diets, and to % of the deaths, mostly in infants, occur in zinc-deficient subjects due to compromised immune systems. we speculate that children with zinc deficiency may be more susceptible to infections that induce and thrive under oxidative stress, such as rsv, and therefore, zinc supplementation may have a stronger impact in such infections due to its antioxidant function. in addition, whether transient treatment with antioxidants may also provide the same beneficial effect as zinc in rsv infections remains to be explored. written informed consents were obtained from the parents or guardians of the children. npa samples were screened for rsv infection using a strip-based kit (coris bioconcept) and further confirmed by qrt-pcr. briefly, rna was isolated from npa samples using a qiaamp minelute virus spin kit (qiagen), and rt-pcr was set up using a flu-rsv kit (fast-track diagnostics). cells and viruses. bhk and hep- cells were procured from the american type culture collection (atcc) and cultured as described previously, and virus strains used in the study were described previously ( ) . a cells were procured from the european collection of authenticated cell cultures (ecacc) and cultured in dulbecco's modified eagle medium (dmem) with % fetal bovine serum (fbs), penicillin, streptomycin, glutamine, and nonessential amino acids. respiratory syncytial virus (long strain) was procured from the atcc (atcc vr- ). rsv titers were estimated by plaque assays on hep- cells by following the same procedure as for denv ( ) . for uv inactivation, rsv stock was diluted : in ml of mem and divided in two -cm dishes. one dish was put inside the uv chamber and kept for min ϫ times on ice at , ϫ j/cm with -min intervals between each cycle. the other dish was kept on ice as a control in a biosafety cabinet. after the inactivation process, virus was aliquoted in small volumes and stored at Ϫ °c. uv-irradiated virus showed no plaques in plaque assay. all cell lines were checked routinely for mycoplasma contamination using an rt-pcr-based method ( ) . the madin-darby canine kidney (mdck)-london cells (irr-fr- ) and influenza a virus (iav) strain a/mexico/ / (h n ) pdm (irr-fr- ) used in the study were obtained from the international reagent resource. mdck-london cells were grown in advanced mem (gibco) with % fbs along with penicillin-streptomycin and l-glutamine (gibco). for iav infection, % fbs was replaced with % bovine serum albumin (bsa), and m hepes, ph . ( . %), sodium bicarbonate ( . %), and deae dextran ( g/ml) were added. virus stock generation and hemagglutination units (hau) were estimated as described previously ( , ) . briefly, mdck-london cells were infected at °c for h to allow for viral internalization, followed by the addition of infection medium with . g/ml n-tosyl-l-phenylalanine chloromethyl ketone (tpck)-treated trypsin (sigma, usa). iav titers from supernatants were estimated by plaque assays on mdck cells by the same protocol used for denv and rsv. for labile zinc measurement experiments, a cells were infected with iav at mois of . and for h at °c to allow for viral internalization, followed by the addition of infection medium with . g/ml tpcktreated trypsin. virus titers from supernatants were collected at h p.i. and h p.i., and hau were determined as described previously ( , ) . treatment of cells. cells were treated with m znso or . m n,n,n=,n=-tetrakis( pyridinylmethyl)- , -ethanediamine (tpen) (sigma) in serum-free media for the periods indicated in the figures. labile zinc levels were measured by flow cytometry as described in text s in the supplemental material. to study the effect of zinc and tpen on virus infection, m znso or . m tpen was added in serum-free medium after infection. at time points indicated in the figures, the supernatant was collected for estimating virus titer by plaque assay and cells were collected for quantitative real-time pcr (qrt-pcr) as described previously ( ) . for h o and n-acetyl cysteine (nac) treatment, infected cells were incubated with m h o (merck) in serum-free media or with mm nac for the periods indicated in the figures, and the effect on virus titers was assessed by plaque assay. quantitative real-time pcr. cells were harvested for isolation of total rna as described previously ( ) . briefly, rna was isolated using rnaiso plus (takara) per the manufacturer's instructions and reverse transcribed using random hexamers. the reverse transcription product was further amplified using nox , catalase, gsta , and sod using a powerup sybr kit (applied biosystems). human gapdh (glyceraldehyde- -phosphate dehydrogenase) primer mix was used as the housekeeping control. data were analyzed using the ΔΔc t method, where c t is threshold cycle. the list of primers used in the study is provided in table s . for rsv genome quantitation, cdna was made using the reverse primer with a genomic dna (gdna) eraser kit (takara), followed by qrt-pcr using the premix ex taq master mix (takara). sirna knockdown. sirna transfections were carried out as described previously ( ) . briefly, nm concentrations of zip and zip sirna (dharmacon) were mixed with opti-mem (life technologies) and l of lipofectamine rnaimax to a total volume of l in a -well plate. forty thousand cells were transfected in suspension in triplicates and seeded into -well plates. knockdown efficiency was monitored by rt-pcr at h posttransfection. at forty-eight hours posttransfection, cells were infected with rsv at an moi of . , and at h p.i., viral titers in the supernatants were measured by plaque assays. flow cytometry. (i) labile zinc level measurement. labile zn levels in the cells were estimated as described before ( ) . briefly, cells were washed once with phosphate-buffered saline (pbs) after either treatment or infection for the periods indicated in the figures, following which they were detached using trypsin ( to l) (gibco) and collected after the addition of the defined trypsin inhibitor (gibco). cells were resuspended in staining medium (dmem without phenol red supplemented and with mm l-glutamine; gibco). cells were stained using either m fluozin- -am (molecular probes) or . m zinpyr- (zp- ) (santa cruz) in the staining medium. for zp- staining, medium containing mm edta was added to chelate any extracellular zinc during staining. cells were incubated for min at °c in a co incubator and mixed every min. after min, fixable viability stain efluor (becton, dickinson biosciences) was added to the cells at a : dilution, prepared in the staining medium, and incubated for a further min at °c in the co incubator. cells were washed using fluorescence-activated cell sorter (facs) buffer (pbs containing . % fbs), and samples were acquired in a facs canto ii apparatus (becton, dickinson). the amounts of labile zinc present in the cells are presented as the mean fluorescence intensities of flz- and zp- . (ii) annexin-v staining. apoptosis was induced by treating a cells at oe c for min. cells were treated with accutase for min and collected. cells were then centrifuged at ϫ g for min. cells were then stained with . l of annexin-v-fluorescein isothiocyanate (fitc; ebioscience) in ϫ annexin-v binding buffer for min at room temperature according to the manufacturer's protocol. cells were then stained with efluor- fixable viability dye for min, washed with facs buffer (pbs containing . % fbs), and acquired in the facs canto ii system. (iii) ros measurement. cells were cultured in -well plates and treated with m znso or . m tpen posttreatment for the times indicated in the figures. cells were washed twice with pbs and detached by trypsinization. cells were then incubated with medium containing % fbs and a m concentration of the carboxyl analog of =, =-dichlorodihydrofluorescein diacetate (carboxy-h dcfda; molecular probes) for h at °c. cells were washed with facs buffer, and ros levels were measured using the facs canto ii system. icp-ms. a cells were grown in a -well plate. cells were infected with rsv at an moi of . . at h p.i., cells were washed with pbs. later cells were collected in ml of . % sds solution. the lysates were then filtered with a . -m filter. multielement standards (merck) were used from ppb to . ppb using the -fold dilution method in deionized water. acquisition was carried out using the x series inductively coupled plasma mass spectrometer (icp-ms; thermo fisher scientific). protein estimation was carried out using a bicinchoninic acid (bca) kit (pierce). data were normalized to total protein content and are represented as parts per billion per milligram of protein. data analysis. data were analyzed and graphs were prepared using prism software (graphpad software inc.). all experiments were performed in two or more replicates, and graphs represent results from at least two independent experiments performed with two or more replicates each time; values are presented as means Ϯ sds. statistical significance was estimated by t tests (unpaired, nonparametric), two-way analysis of variance (anova), or the mann-whitney test. supplemental material is available online only. text s , docx file, . mb. global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in : a systematic review and modelling study cotton rat model for testing vaccines and antivirals against respiratory syncytial virus immune 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infants with severe pneumonia flux of intracellular labile zinc during apoptosis (genedirected cell death) revealed by a specific chemical probe correlation of apoptosis with change in intracellular labile zn(ii) using zinquin [( -methyl- -ptoluenesulphonamido- -quinolyloxy)acetic acid], a new specific fluorescent probe for zn(ii) nfkappab regulates hsf and cjun activation in heat stress-induced intestinal epithelial cell apoptosis impact of heat stress on cellular and transcriptional adaptation of mammary epithelial cells in riverine buffalo (bubalus bubalis) understanding the contribution of zinc transporters in the function of the early secretory pathway zinc transporters and the cellular trafficking of zinc physiological roles of zinc transporters: molecular and genetic importance in zinc homeostasis zinc chelation specifically inhibits early stages of dengue virus replication by activation of nf-kappab and induction of antiviral response in epithelial cells respiratory syncytial virus infection induces a reactive oxygen species-msk -phospho-ser- rela pathway required for cytokine expression redox biology of respiratory viral infections respiratory syncytial virus infection up-regulates tlr expression by inducing oxidative stress via the nrf /are pathway in a cells roles of zinc and copper in modulating the oxidative refolding of bovine copper, zinc superoxide dismutase zinc and oxidative stress: current mechanisms. antioxidants (basel) : toll-like receptor-mediated regulation of zinc homeostasis influences dendritic cell function zinc is required for fc epsilon ri-mediated mast cell activation induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc release zinc is a novel intracellular second messenger glutamate triggers preferential zn ϩ flux through ca ϩ permeable ampa channels and consequent ros production critical role of zinc as either an antioxidant or a prooxidant in cellular systems the role of metallothionein in oxidative stress zinc decreases c-reactive protein, lipid peroxidation, and inflammatory cytokines in elderly subjects: a potential implication of zinc as an atheroprotective agent zinc supplementation decreases incidence of infections in the elderly: effect of zinc on generation of cytokines and oxidative stress oxidative stress influences positive strand rna virus genome synthesis and capping oxidative stress during viral infection: a review dengue virus induces and requires glycolysis for optimal replication respiratory syncytial virus and cellular stress responses: impact on replication and physiopathology inducible lung epithelial resistance requires multisource reactive oxygen species generation to protect against viral infections n-desmethylclozapine, fluoxetine, and salmeterol inhibit postentry stages of the dengue virus life cycle multi-primer qpcr assay capable of highly efficient and specific detection of the vast majority of all known mycoplasma distinct susceptibility and applicability of mdck derivatives for influenza virus research propagation and characterization of influenza virus stocks that lack high levels of defective viral genomes and hemagglutinin mutations identification and characterization of the role of c-terminal src kinase in dengue virus replication we thank all the members of the ccv lab for their support and critical input. this work was supported by an intermediate fellowship from the india alliance (ia/s/ / / ) to g.r.m. n.a.k. received a research fellowship from the university grants commission ( ). the funders had no role in study design, data collection, and interpretation or the decision to submit the work for publication. n.a. performed the experiments, analyzed data, and wrote and manuscript. s.s. provided reagents and assisted with h n experiments. m.s. assisted in patient enrollment and tests with patient samples. r.l. was involved at the clinical site, monitored data collection, and analyzed the data. g.r.m. conceived the study, designed and performed experiments, analyzed data, and wrote the manuscript. key: cord- -xv tcugl authors: reina, giacomo; peng, shiyuan; jacquemin, lucas; andrade, andrés felipe; bianco, alberto title: hard nanomaterials in time of viral pandemics date: - - journal: acs nano doi: . /acsnano. c sha: doc_id: cord_uid: xv tcugl [image: see text] the sars-cov- pandemic has spread worldwide during , setting up an uncertain start of this decade. the measures to contain infection taken by many governments have been extremely severe by imposing home lockdown and industrial production shutdown, making this the biggest crisis since the second world war. additionally, the continuous colonization of wild natural lands may touch unknown virus reservoirs, causing the spread of epidemics. apart from sars-cov- , the recent history has seen the spread of several viral pandemics such as h n and h n flu, hiv, and sars, while mers and ebola viruses are considered still in a prepandemic phase. hard nanomaterials (hnms) have been recently used as antimicrobial agents, potentially being next-generation drugs to fight viral infections. hnms can block infection at early (disinfection, entrance inhibition) and middle (inside the host cells) stages and are also able to mitigate the immune response. this review is focused on the application of hnms as antiviral agents. in particular, mechanisms of actions, biological outputs, and limitations for each hnm will be systematically presented and analyzed from a material chemistry point-of-view. the antiviral activity will be discussed in the context of the different pandemic viruses. we acknowledge that hnm antiviral research is still at its early stage, however, we believe that this field will rapidly blossom in the next period. t he current emergence caused by sars-cov- is dramatically changing the everyday life of all of us. due to the high globalization, new viruses can spread all over the world much faster than ever, infecting the communities worldwide. the current technologies and measurements are able to sensibly slow down the infection spread. however, their cost is tremendously high, impacting the healthcare systems and causing the shutdown of industries and the lockdown of the population. the impact of sars-cov- on the worldwide economy is estimated to be a − % decline, making this the biggest crisis since the world wars. a possible vaccine is hopefully expected to come in − years, originating a buffer period pretty much uncertain for many people. vaccination is the only way known to accelerate the flock immunity without causing further death by this pandemic. contemporary history has seen the spread of other viral pandemics such as h n flu ( − ) , h n flu ( ), hiv (peak reached between and ), sars ( ), while mers ( to now) and ebola ( to now) viruses are in a prepandemic phase. the continuous colonization of wild nature lands may touch unknown virus reservoirs causing the spread of contagious epidemics. due to these facts, there is a clear urgency in the development of viral treatments to avoid the risk of new pandemics. in particular, the possibility to have smart antiviral tools able to efficiently disinfect surfaces, block the viral spreading, enhance the survival of infected people, and boost immunization are highly desirable. in particular, more investments in the next years are expected in the antiviral research. hard nanomaterials (hnms) have been extensively studied for many types of applications including drug delivery, bioimaging, and biosensing. − the use of nanomaterials in biomedical research is highly developed, reaching in some cases clinical approval. despite that, nanomaterials have been mainly developed for cancer therapy, while scarce attention has been spent on their application in viral infections. the continuous virology research has more and more increased into viral replication machinery, allowing the preparation and rationalization of more sophisticated vaccine formulations and viral inhibitors. the use of hnms may be one of the keys to provide more effective biomedical agents with a wide spectrum of activity in viral pandemics. an increasing number of reports describe how hnms can be successfully applied to block viral spread. hnms can be used at different stages of viral infection: blocking viral entry, hampering interaction with infected host cells, and modulating immune responses. due to their core composition (e.g., metal oxides, noble metals), hnms can have an important antiviral activity inactivating some specific proteins of the capsid or dysregulating radical homeostasis in the virus particles. additionally, surface functionalization can sensibly increase hnm antiviral activity, enabling the mimicking of host cells or enhancing the targeting efficiency. in this review, we will critically analyze different strategies for the application of hnms as antiviral agents. the rational and the synthetic strategies will be highlighted and correlated to different relevant examples. particular attention will be paid on the mechanisms of antiviral action and on hnm applicability and efficacy. for the sake of clarity, this review is divided into three parts, namely: blocking viral entry, antiviral activity in host cells, and stimulation of immune system. we have focused on the different stages of infection. in the section dedicated to blocking viral entry, the application of hnms for surface disinfection and inactivation of the virus prior to interaction with host cells will be described. subsequently, the interaction of hnms after internalization into host cells will be addressed, stressing their antiviral delivery features and their activity in viral replication blockage, leading to host cell survival. then, activation of immune response induced by hnms, triggering the innate and the adaptive (e.g., nanovaccines) immunity, will be presented. the different adopted strategies will be correlated to the nanomaterial core (e.g., composition, size, shape) and surface (e.g., chemistry, surface charge) properties. finally, limits (e.g., unknown long-term toxicity), advantages (e.g., high and wide spectrum virucidal activity), and perspectives will be discussed with particular attention to the applications in viral pandemics (e.g., hiv, sars, and influenza viruses). this review is addressed to material and biomaterials scientists who are interested in antiviral research. we acknowledge that application of hnms as antiviral agents is still in the early stages; however, we believe that the research on this topic is going to grow soon. thus, with this contribution we genuinely hope to inspire researchers in the preparation of smart and efficient hnm antiviral agents. the last decades have been characterized by increasing investigation on viruses. in particular, their surface charge, protein composition, and host cell entry mechanism have been elucidated, allowing to formulate the first-generation wide spectrum antivirals. blocking the viral entry is one of the most common known antimicrobial procedure to stop infections at the early stage. in this context, antiviral materials have been used for surface disinfection or for epidemic limitation in humans and animals. due to their high surface to volume ratio, composition, and tunable surface chemistry, hnms are now more and more studied as powerful agents in blocking viral entry. as for other biological interactions, the attachment and entry of viruses into host cells are mediated by multivalent interactions between the surface of the virus and cell surface receptors. nanomaterials can display multivalency that makes them able to compete with the host cells on virus attachment, limiting their infectivity. the mechanism of antiviral actions relies on the inactivation of the capsid proteins. as a matter of fact, hnms have been used for: ( ) blocking target proteins for viral entry, ( ) capsid protein oxidation, ( ) mimicking cell surface, and ( ) mechanical rupture of viruses ( figure ). these strategies target proteins and mechanisms of entry common in most of the viruses, thus allowing the preparation of wide spectrum antiviral agents. in this section, the application of different hnms as powerful inhibitors of viral entry will be discussed. noble nanoparticles. noble nanoparticles (nps), made of gold and silver, are attractive as antiviral agents for their surface functionalization versatility and their capacity to cleave disulfide bonds. their use in disinfection has been extensively studied for different types of viruses. the morphology and the size of the nps play a crucial role in their ability to efficiently interact with the capsids and in their toxicity for the organism. these nanomaterials are characterized by a very large specific surface area (inversely proportional to the particle diameter). as the particle size becomes smaller and smaller, the percentage of surface atoms increases, creating many unsaturated bonds due to lack of neighboring atoms. as a consequence, agnps and aunps have unstable atoms with high surface energy. this kind of structure provides a lot of contact adsorption sites and reaction points for further modifications. these chemical features allow to easily combine surface np atoms with other atoms through chemical bonds. besides the composition of the metal core, several studies have pointed out the importance of the control of the surface chemistry. the surface groups can: ( ) stabilize nps in the biological media, ( ) insert targeting agents, and ( ) enhance the circulation time inside the body. antiviral efficiency can be also enhanced by the multivalency effect, where highly branched ligands are used to locally augment the local concentration of the targeting molecules. in this section the main strategies and results for silver (agnps) and gold (aunps) nanoparticles in blocking viral entry will be critically discussed. silver nanoparticles. many studies have shown that naked agnps have a good effect on the control and prevention of a variety of viral diseases (table ) . however, the antiviral mechanism of nanosilver is still unclear. the antiviral action is associated with the following mechanisms: nanosilver can prevent the virus from entering the host cells and inhibit the virus from binding to the cell receptor, thereby stopping the virus from infecting the targeted cells. agnps may be able to bind the viral surface protein and inhibit the interaction between the virus and the cell membrane receptors (figure , left). however, it has been also reported that agnps can inactivate the virus through denaturation of surface proteins containing cysteine and methionine residues present on the viral capsid, in a similar way reported for bacteria. for example, agnps smaller than nm were shown to interact with the sulfur-bearing residues of gp glycoprotein knobs distributed on the lipid membrane of hiv- virus, preventing the virus from binding to cd receptor site on the host cells, thus inhibiting the viral infection. by means of a viral adsorption assay, it was shown that the agnp mechanism of anti-hiv action is based on the inhibition of the initial stages of the hiv- cycle. to demonstrate that the antiviral effect of agnps is due to the particle structure rather than to silver ions present in solution, the antiviral activity of silver sulfadiazine (agsd) and silver nitrate (known antibacterial silver salts) was evaluated. both salts showed a much lower therapeutic index than agnps in vitro, indicating that silver ions themselves are less efficient. these results point out that the antiviral efficacy is not only related to the dose of ag + ions present in solution but is also regulated by different other parameters (e.g., size, charge, and surface functionalization) associated with the nanosize dimension. for instance, in the case of herpesviridae and paramyxoviridae viruses (both enveloped viruses with embedded viral-encoded glycoproteins), agnps can effectively reduce their infectivity, by blocking the interaction between the viral particles and the host cells with an antiviral activity strictly dependent on the size and ζ potential of the agnps. as a general observation, it was reported that smaller nanoparticles have better antiviral effect. this effect was associated with the increase of the surface area, where smaller-sized agnps could bind more efficiently to the viral particles exerting a higher antiviral activity. another study reported the impairment of peste des petits ruminants virus (pprv) replication after incubating infectious viral particles with agnps, which did not exhibit any virucidal effect even up to μg/ml. this result suggested that the anti-pprv activity of the agnps is due to the inhibitory effect alternatively, nanosilver can be combined with viral nucleic acids to change the capsid structure, affect the replication of viral genetic material, and make the virus inactive. for example, tem analyses have shown that nps can cause a change of the structure of the ad virus from a hexahedral shape to an irregular shape, destroying its fibers and capsid proteins, leading to inhibition of the virus from binding to the host cells and destroying the dna structure, preventing adenoviral infection. nanosilver can also bind directly to the doublestranded dna of hepatitis b virus to inhibit its replication. in other studies, it has been demonstrated that silver ions released from nanosilver can directly damage the viruses. based in this property, an interesting application has been proposed. agnps were used as a coating on polyurethane condoms, (hsv) . the hypothesized mechanism is that silver ions are transferred directly from oxidized nps to biological targets, such as viral membrane proteins gp and gp . in addition, a small amount of silver ion is also released from the coated contraceptives to improve the antiviral level. although the studies on naked agnps to reduce viral infectivity have shown their potential as broad-spectrum antiviral agents, the understanding of the specific antiviral action mechanism still needs to be elucidated in depth. many studies have shown that the antiviral performance of naked agnps is related to their size, and smaller nanoparticles have better antiviral activities. in addition to particle size, the antiviral action of agnp morphology has also attracted interest to fight against coronavirus. agnps and two types of silver nanowires were able to significantly cause an inhibitory effect on coronavirus transmissible gastroenteritis (tgev)-induced host cell infection and tgev replication. the mechanism is likely based on a direct interaction of agnps with tgev surface proteins (e.g., tgev glycoproteins) to inhibit the beginning of viral infection. it is possible that agnps and ag nanowires alter the structure of some surface proteins of tgev and then inhibit their recognition and adhesion to the cellular receptor papn. although the potential of agnps as antiviral agents has been commonly recognized, unfortunately, their wide biological applications are limited by the risks of self-aggregation and environmental pollution. silver ions can be released from the surface of agnps and potentially pollute the environment, and their agglomeration into bulkier particles or fibers may change their biological characteristics, diminishing the antiviral effect. in several cases, it has been reported that naked agnps may affect human health. therefore, research and development of agnps whose surface is modified or stabilized by protecting molecular layers is an urgent need to overcome these problems ( table ) . poly(n-vinyl- -pyrrolidone) (pvp) is the most commonly used stabilizer of agnps. the pvp-coated agnps are able to inhibit the activities of hiv- , herpes simplex virus (hsv- ), and respiratory syncytial virus (rsv). , , but compared to foamy carbon, small-sized pvp and bsacoated agnps showed poor antiviral activity to the hiv- virus. for rsv, pvp-coated agnps have a specific binding capacity to the viral surface, evidencing a regular spatial arrangement and a clear interaction with g-protein. in addition, to improve the stability of agnps, their surface modification with antiviral drugs was proved to reduce the drug resistance caused by the drugs administered alone. tannic acid-modified agnps showed good antiviral effects on hsv- infection in vitro and in vivo. the viral infection was inhibited only when these nps directly interacted with hsv- virions. indeed, the pretreatment of host cells with such agnps did inhibit the entry of hsv- . due to the high affinity of tannins to proteins and sugars, tannic acid can bind glycoproteins on the surface of viruses to make them inert, impairing glycoprotein function and preventing viruses from attaching and entering host cells. the surface modification can also exert a synergistic antiviral effect. agnps decorated with polyphosphonium-oligochitosan (pqpoc) exhibited moderate to excellent antiviral activity against hav, nov, and coxb . in addition, agnps could interact with the virion glycoproteins and prevent viral attachment and penetration. pqpoc can also serve as an effective virus inhibitor by blocking the interaction of the targeted virus with the host through the electrostatic interaction between the cationic polymers and the negatively charged binding sites of the virus. surface-modified agnps can also prevent viral infection by competitive adsorption on host cells. the process of infection of cells by herpes simplex virus type (hsv- ) involves the interaction between viral envelope glycoproteins and heparan sulfate (hs) on cell surface. therefore, researchers designed agnps capped with mercaptoethanesulfonate (ag-mes) to compete with the cellular hs through the sulfonate end groups, thereby blocking the virus from entering the cells. a few years ago, it was shown that curcumin could prevent the replication and the budding of rsv, but the disadvantage of poor solubility and low bioavailability limited its clinical application. curcumin was used as a reducing and capping agent to prepare stable curcumin agnps (cagnps) under physiological conditions. cagnps could reduce cytopathic effects induced by rsv and showed efficient antiviral activity against infection by directly inactivating the virus prior to entry into the host cells. its antiviral effect was higher than curcumin alone or unmodified agnps ( figure ). alternatively, zhu et al. prepared agnps surface-modified with oseltamivir, amantadine, and zanamivir (ag@otv, ag@am, and ag@znv ), by chemical methods. the results showed that these nanoparticles can directly interact with the virions, resulting in viral function damages. overall different studies have reported the capacity of agnps to block viral entry. however, there is not a concerted antiviral mechanism, but their activity differs from case to case, based on viral particle adsorption, capsid structure alteration, or surface protein denaturation. for agnps, the antiviral activity can be associated with different parameters including size, shape, surface charge, and functionalization but also to the topical release of silver ions able to disturb the viral cycle replication. as described before, bare agnps can be used as disinfectant agents, however their use in biological media is acs nano www.acsnano.org review limited by their low colloidal stability and potential cytotoxicity. surface functionalization can alleviate cytotoxicity, but it can also mask the nanoparticle surface, reducing their affinity for viral particles, thus reducing agnp antiviral activity. for these reasons, agnps at the moment could find application mainly for surface disinfection and for topical administration. further studies are needed to prepare safer agnp formulations for systemic administration. in particular, the clarification of the antiviral mechanisms and the use of surface functional groups able to stabilize agnps in biological fluids without affecting their prominent antiviral activity are probably the most important challenges to tackle. gold nanoparticles. compared to agnps, aunps exhibit reduced toxicity on healthy cells, making them more attractive for in vivo and clinical applications. indeed, aunps have been successfully tested as inhibitors of viral entry into the host cells. aunps interact with hemagglutinin (ha), where au is able to oxidize the disulfide bond of this glycoprotein causing its inactivation, thus impeding the membrane fusion of the virus with host cells. targeting ha has emerged as an alternative strategy to the actual therapies (e.g., matrix protein and neuramidase), especially to pandemic viruses that show an accelerated mutation speed of their surface proteins, hence a resistance to conventional treatments increasing their infectivity and mortality. this strategy has been applied to influenza (e.g., h n , hcv) and herpes viruses. − the activity of aunps is proportional to the surface area exposed. as a consequence, the size and the morphology of these metal nps play a substantial role in their antiviral activity. recently, kim et al. have reported that porous aunps are able to inhibit influenza a infection more efficiently than nonporous aunps. this effect has been associated with the higher surface area of the porous material that favors their interaction with capsids and thus increases their antiviral activity ( figure ). besides the per se antiviral activity, aunp surface modifications have been developed in order to enhance their overall therapeutic benefits. the engineering of tailored aunps with selected ligands has allowed the preparation of efficient antiviral nanoagents. the target ligands can be introduced directly during the particle synthesis via ligand exchange reactions or ligand modifications. for instance, direct reduction of gold ions in the presence of gallic acid produced homogeneous aunps able to sensibly reduce herpes simplex virus infection in vitro. compared to free ligand nps, functionalized aunps benefit from the multivalency effect and higher circulation times, decreasing the needed therapeutic concentrations. functionalized aunps can present organic groups that mimic host cell surfaces or other specific molecular patterns that selectively target the virus. normally, negative charges are used to mimic cell surfaces and favor the interaction between the particles and the capsid. in particular, sulfonates and organic sulfates have been used for their capacity to attract the virus via capsid protein interaction and block the ha activity. aunps functionalized with sulfonates showed an increasing inhibition of influenza a compared to the nanoparticles capped with succinic acid. this study also demonstrated that there is not a correlation between the negative charge and the antiviral activity, but instead the inhibition depends mainly on the organic groups used. thiolcapped aunps also displayed powerful inactivation of bovine viral diarrhea virus in vitro. multivalency has been exploited in more complex systems using dendrons as capping agents. this strategy allows to generate higher concentrations of the target ligand in close proximity to the aunps and to increase the binding efficiency of the nanoparticles to the capsid. the driving force of the antiviral efficiency relies on the concentration of the targeting agent onto the particles. sulfonated dendrons were grafted to aunps via a sulfide bond and tested for hiv inhibition. the results showed that acs nano www.acsnano.org review the decorated aunps exerted a higher affinity to the virus. additionally, comparing aunps functionalized with different generation dendrons, those with a third generation displayed the highest inhibition performance with an ic below . μmol/ml, thus making them attractive for in vivo translation. it is worth noting that the inhibition efficiency is strictly dependent on the available sulfonate groups present on the surface of the nps, making crucial a thorough characterization of the material. the size of the aunps clearly plays an important role in the concentration of targeting ligands exposed per particle. indeed, too big nps have a limited surface area, while too small would not allow an efficient grafting of the dendrons due to steric hindrance. for instance, it has been shown that dendron-functionalized aunps showed a size-dependent antiviral activity for influenza virus, where nm particles exhibited a higher efficiency than nm aunps. this has been associated with the low functionalization grade of the small nanoparticles and to the inappropriate spatial distribution of the interacting ligand/receptor pairs. the development of viral proteomics has profoundly transformed the antiviral and disinfection strategies. in particular, small molecules and peptides able to target and block the viral biochemical machinery have been developed. however, despite these efforts into the drug design, many of these molecules suffer from poor biological effect, low concentration in the diseased areas, and undesired side effects. in this context, aunps have been coupled to biologically inactive small molecules to create biologically active multivalent aunp therapeutics. a bright example has been reported by bowman et al., where the authors functionalized aunps with sdc- , a small membrane fusion inhibitor of hiv. the results demonstrated that, while pure sdc- has low activity, functionalized aunps are able to inhibit hiv replication at μm concentrations. similar results have been reported using targeting peptides. in particular, it was evidenced that the functionalized aunps can sensibly reduce the ic up to orders of magnitude compared to pure peptides. preliminary results in vivo confirmed the biosafety of the aunps. nanoparticles generating reactive oxygen species. one of the main advantages of using nps compared to oxidized metals relies on the slow release of ions and clusters from these particles, leading to an enhancement of the antiviral activity. additionally, the use of metal nps containing cu or fe in ionic form catalyzes the generation of radicals via fenton and fenton-like reactions oxidizing the capsid proteins and consequently blocking the viral infection at early stage. for instance, copper ions (derived from sulfates or iodide salts) have been widely used as antiviral agents because of their activity on several kinds of enveloped and non-enveloped viruses including influenza virus, − herpes simplex virus − and hepatitis a virus. their mechanism of action relies on the formation of cu + ions (from soluble salts or nanoparticles) that generate hydroxyl radicals. the use of metallic copper nanostructures in the form of particles or sheets has shown only a moderate efficiency due to the low concentration and low release of cu + . for these reasons, cu + salts, where the copper ions are readily present in their active monocationic form, have been favored. in particular, cui nanoparticles (stable at room temperature) have been extensively studied for deactivation of feline calicivirus and h n pandemic influenza virus. however, the use of copper salts at high concentrations can irreversibly alter reactive oxygen species (ros) homeostasis of healthy cells, provoking a general toxicity for the organism, limiting their applications to disinfection. nanostructured cuprous and cupric oxides have been also extensively employed as antiviral agents for in vitro applications. for instance, cuprous oxide nanoparticles (cuonps) were successfully employed against hepatitis c. in particular, it was found that these nps exerted a favorable antiviral activity with no cytotoxic effects. cuonps target the binding and entry step of viral infection to hepatic cells ( figure ). similar results were reported on the use of cuonps against hsv- , however without any profound investigation on the antiviral mechanism. alternatively, zinc salts have been successfully used as antimicrobial agents from research up to clinical trials for viral warts. , more recently, zno nanoparticles (znonps) were developed for the treatment of hsv- . znonps were prepared with a tetrapod morphology. the results showed that they can mimic cell surface interacting with the hs present on the viral capsid. additionally, these particles have been used for photocatalysis showing to efficiently destroy the viral proteins upon uv irradiation. besides all these interesting examples, in vivo applications are still needed to validate this therapeutic modality. due to the generation of high levels of ros, the toxicity of copper nanoparticles has been widely debated. the antiviral activity of copper nanoparticles is generally associated with the release of cu + ions in solution, thus the leakage of cytotoxic cationic species can be modulated by surface functionalization before in vitro and in vivo applications. on the other side, the use of nanomaterials generating ros can find applications in textile and surface coating. the general broad virucidal efficiency of copper oxide nanoparticles shown for h n pandemic influenza should be tested on sars-cov- and might be used for improving mask protection efficiency. carbon nanomaterials. due to their diversity, versatility, and tunable surface chemistry, carbon nanomaterials have been attractive for several types of applications. in particular, the past decade has seen a tremendous raise in the preparation of performant carbon-based nanomaterials in the antiviral field. fullerene and its derivatives are the most studied carbon nanomaterials for their virucidal activity. due to the lack of solubility of pristine fullerene, functionalization strategies have been developed to prepare water-soluble drugs. investigations in the biomedical field evidenced the membranotropic capacity of fullerene derivatives. by modulating shape and functions, fullerene derivatives have been shown to possess antiviral properties through inhibition of viral entry and blockage of viral replication. from these results, the attention has been directed also to other carbon nanomaterials. in particular, functional carbon dots (cds) and graphene oxide (go) have been investigated for their ability to block viral entry into host cells. glycofullerenes. the emerging of mortal viruses, like ebola or zika, and the lack of suitable treatments led the academic and the industrial communities to look for alternative therapeutic routes. most of these pathogens are rna enveloped viruses, and they share common infection mechanisms that can be targeted for the preparation of wide small spacer between core c and surrounding fullerenes. b large spacer between core c and surrounding fullerenes. www.acsnano.org review spectrum antivirals. the external surface of the envelope of these viruses is covered by glycans that tightly interact with lectin receptors on host cells. this strong interaction allows the attachment of the virions to the cells, followed by internalization and infection. blocking lectin receptors is a general strategy used to stop viral infection at an early stage. fullerenes have been widely investigated as antiviral molecules, drug carriers, or tissue scaffolds. fullerene applications have been recently extended to the design of mannosylated derivatives to block the entry of viral particles into host cells. mannose, due to the high affinity with lectin receptors, competes with the virus in the interaction with the host cells. for example, one of the targets is the inhibition of viral particles through the interaction of mannose with the dendritic cell-specific icam-grabbing non-integrin (dc-sign). dc-sign receptors mediate the interactions between dcs and t cells. , to exploit these characteristics, mannose was combined with fullerene in the design of the so-called glycofullerenes to study their capacity to inhibit ebola, dengue, and other pathogens. for this purpose, different glycofullerenes were synthesized by changing the number of mannose units (from to ) and the spacers between the fullerene moieties and by varying steric hindrance in order to obtain a library of molecules. the synthetic route is composed of three steps based on "click chemistry": ( ) assembly of glycodendrons by cu(i)-catalyzed azide−alkyne cycloaddition (cuaac), ( ) synthesis of alkynesubstituted bingel-hirsch hexakis-adducts, and ( ) the coupling between the last two products again by cuaac. to increase the number of mannose moieties up to , the glycodendron core was changed from malonate to trialkynyl pentaerythritol. in order to compare the different derivatives, in vitro studies were performed. jurkat cells (lymphocyte t cd immortalized cells) expressing dc-sign were used to prove the inhibition capacity of the glycofullerenes on viral infection of ebola ( figure , route a). the study revealed an ic in the μm range for the mannose fullerene, a lower efficiency with the mannose fullerene with a short spacer (peg, with ethylene oxide units), while a nanomolar ic was achieved with mannose fullerenes with a longer spacer (peg, with ethylene oxide units) ( table ). this first proofof-concept study was then expanded, aiming to obtain a better antiviral activity by increasing the valence and inserting longer and flexible spacers. based on these studies, another class of multivalent fullerene dendrimers was then designed. a fast and controlled synthetic route was developed to achieve giant globular multivalent fullerenes, containing hundreds of functional groups. the first study was performed with tridecafullerenes containing mannoses. the molecular structure is composed of hexakis c surrounding a c core ( figure , route b). compared to the previous study, an ic orders of magnitude lower was measured on the inhibition of ebola virus (table ) . , in order to present more carbohydrates at the periphery of the dendrimer, a trialkynyl pentaerythritol derivative allowed to afford a tridecafullerene with carbohydrates. in this case, the molecule was synthesized with a c tridecafullerene bearing α( , )mannobioside. the use of this disaccharide was already investigated, showing an increase of affinity with dc-sign receptors by a factor of − . the synthetic strategy exploited also the use of strainpromoted copper-free cycloaddition of azides to alkynes (spaac) for the coupling of the core fullerene to the surrounding fullerenes. spaac allows an easier purification avoiding the removal of cytotoxic copper ions. the inhibition performance of this molecule was studied in vitro with viral pseudoparticles of dengue and zika. the comparison was made between and disaccharides tridecafullerenes and disaccharide monofullerene. the results highlighted a picomolar ic inhibition on both zika and dengue models for the disaccharide glycofullerene ( table ) . the ability to inhibit other types of viruses allows the use of glycofullerenes as broad spectrum antiviral drugs. moreover, the negligible toxicity to other cells proved the biocompatibility of these molecules. following an alternative strategy, a supramolecular assembly of monodisperse glycofullerenes, leading to the formation of micelles, was achieved and tested. these micelles present a uniform and spherical shape ( figure , route c). the aggregation synthetic route is faster compared to a controlled synthesis of giant glycofullerenes, but it might suffer from low reproducibility and batch-to-batch differences between each formulation. this self-assembled c functionalized with or mannoses exposes a large amount of carbohydrate at the surface, leading to an inhibition of ebola virus in the nanomolar range (ic of nm for six mannoses and nm for mannoses, respectively, table ). further in vitro studies evidenced again a good biocompatibility of these glycofullerenes. while there are no in vivo studies yet, these promising results enlarge the panel of molecules in the fight against new emerging viruses. functionalized fullerenes have been used for their ability to compete with viral particles through lectin receptors in host cells. there has been a tremendous advancement in the functionalization of fullerenes leading to the preparation of derivatives with a high amount of mannose, capable to enhance the multivalency effect and thus to increase the therapeutic outcome. first, glycofullerenes do not have an intrinsic virucidal activity. they can reduce the infectivity, but they are not able to completely inactivate the virus. second, the mechanism of action of glycofullerenes relies on their interaction with host cells and not with viral particles. thus, for a therapeutic application, they should be injected at different time points, ensuring that the local concentration is therapeutically relevant to prevent the virus from invading the host cells. in addition, glycofullerenes can be internalized into host cells losing their viral "shield" activity. on the other hand, the well-developed surface chemistry of glycofullerenes can be used for other key receptors involved in viral entry. for instance, in the case of the current sars-cov- pandemic, a similar click chemistry strategy can be used to anchor ligands recognized by human lung ace receptors and so inhibiting viral entry. other carbon nanomaterials. alongside fullerenes, other carbon nanomaterials (nms) have been scrutinized for their ability to block viral entry. cds and go are the most known and studied carbon nms with marked antiviral properties. cds are zero-dimensional carbon nanoparticles. they are generally produced via hydrothermal decomposition of carbon containing "low-cost" precursors. the use of cds in the biomedical field has been encouraged by their easy preparation, low toxicity, fluorescence properties, and easy surface functionalization. pristine cds have shown moderate viral blocking activity for hiv infection in vitro. this has been associated with the surface of the material rich in carboxylic and hydroxyl groups prone to form noncovalent acs nano www.acsnano.org review interaction with viral membranes. moreover, due to the complexity of the biological systems these nonspecific interactions could not be so effective in vivo, likely reducing the antiviral efficacy. therapeutic targeting molecules can be grafted onto a cd surface to enhance their antiviral activity. in this context, the design of multifunctional cd platforms can be obtained through two different strategies. the first consists in a single-step reaction that foresees the insertion of the therapeutic molecule directly into the step of preparation. target molecules are decomposed with the other precursors, generating the desired functional cds. this protocol is fast and efficient, however the drug loading as well as its activity are hard to estimate. indeed, the hydrothermal treatment can alter the chemical structure of the active molecule, thus vanishing its therapeutic effect. for these reasons, the reaction conditions must be carefully controlled. the second method is a twostep reaction and implies the postfunctionalization via amide formation on the surface of the cds rich in carboxylic groups. this strategy offers a better chemical control, but the yield and the drug loading may not be quantitative and high, respectively. different functionalized cds were prepared to hamper host cell viral entry. for instance, benzoxazine (a low water-soluble antiviral agent) was incorporated into the cd structure during their preparation (figure ) . the as-prepared cds showed a broad spectrum viral blocking capacity in vitro for enveloped (e.g., japanese encephalitis virus, dengue virus, and zika virus) and non-enveloped viruses (e.g., porcine parvovirus and adenovirus-associated virus). these positive results were explained by the efficient binding and deactivation induced by the multivalent effect of the cds to the viral particles amino-functionalized cds were also tested for the treatment of human norovirus. in this study, cds were functionalized with , ′-(ethylenedioxy)bis(ethylamine) (eda) and ethoxypropylamine (epa) via amide bond formation. these nms exerted a good viral blockage. in particular, epafunctionalized cds were able to inhibit % of viral infection at concentration of μg/ml, while in the case of cds prepared with the other amines, % of inhibition was reported. these effects have been associated with the higher positive charge of cd-eda compared to cd-epa. another surface group used for viral targeting is boronic acid (ba), which can bind glycosylated surfaces forming boronic esters. this strategy was successfully adopted to treat hiv where the boronic groups, linked to different nanoparticles (e.g., silica nanoparticles and nanodiamonds) can target gp receptors on the viral envelope inhibiting the infection. another recent study proposed the use of cd functionalized with phenylboronic acid for prevention of hiv infection. the functional materials showed good inhibition properties compared to nonfunctionalized cds by preventing the binding to the target cell in vitro. overall, the use of cds for stopping host cell viral entrance has shown good results in vitro. however, there is a lack of proofs in vivo limiting their applications to surface disinfection or masks. in addition, most of the in vitro studies foresee first the contact of the cds with the viral particles and then their incubation with host cells. deeper investigations should be performed adding the nms at other time points (for instance in infected cells) to understand if the antiviral activity is maintained. in addition, cds have been successfully used for photodynamic therapy (generating radicals upon light irradiation) in cancer treatment. the same approach may be used to combat viral infections, where the antiviral activity induced by the surface modification can be sensibly enhanced by ros generation under irradiation. graphene materials, and in particular go and reduced go (rgo), have been used for different biomedical applications including drug delivery, biosensing, and tissue engineering. go platforms have shown also interesting antimicrobial activity. regarding viral infection, go was used to block the virus entrance in host cells. go and rgo can be considered as two-dimensional materials that contain hydrophilic and hydrophobic domains allowing to adsorb many biological molecules including nucleic acids and proteins. go showed low interaction with viruses, however its surface functionalization with target molecules can sensibly enhance its affinity for the viral particles. additionally, go can be used as photothermal agent (generation of heat by nir irradiation) or photodynamic therapy (using visible light irradiation) inactivating the capsids by local thermal shock or by radical formation during irradiation, respectively. the use of phototherapies may significantly augment the antiviral properties of the materials. however, we must keep in mind that these therapeutic modalities can be applied only to disinfection, since the radical/heat production may be harmful for the healthy tissues in vivo. photodynamic therapy has been successfully exploited using bacteriophage ms as a model virus. in this study, go was functionalized with an aptamer recognized by the viral surface. the results showed that this functionalization is able to enhance the binding efficiency of the ms capsids onto the go surface compared to nonfunctionalized go. subsequently, irradiation in the visible light was able to disinfect the solution, while nonfunctionalized go showed much less activity due to the lack of adsorption. despite these interesting results, this pioneer work remains at an early research stage since the use of high light dose ( w for − min) and the lack of material recovery and reuse make its application for surface disinfection difficult. go can be also used as a platform to link antiviral agents. encouraging results were reported using go with hypericin for the treatment of a recently appeared duck reovirus. more recently, deokar et al. reported an original rgo-based multifunctional platform for hsv- treatment. in this work, the authors functionalized the material with organic sulfate groups and iron oxide magnetic nanoparticles (fenps). the rgo functionalized with the sulfate is able to mimic the host cell surface and to bind hsv- . subsequently, the viral particles captured onto the rgo-fenp surface can be concentrated via magnetic precipitation and destroyed via photothermal therapy. this approach is highly efficient for disinfection with low energy ( . w/cm for min) and cost effectiveness. hs is a common entry receptor in various types of viruses (e.g., herpes viruses, human papillomavirus, dengue virus). the use of organic sulfate-functionalized graphene sheets mimicking hs, like go and rgo, has been already explored. however, it is worth noting that these nms are prone to strongly adsorb proteins in culture environments (coronation), likely inhibiting their antiviral efficacy. high loadings of sulfate groups were introduced onto rgo using polyglycerol sulfate. , this approach has been used for inhibition of orthopoxvirus, pseudorabies virus, and african swine fever virus in vitro. , graphene has also been used as antiviral material. polysulfates and fatty amines were grafted onto graphene surface via triazine chemistry for the treatment of herpes simplex virus. this strategy promotes the synergy between the electrostatic and hydrophobic interactions, showing incredibly high inhibition efficacy. overall, graphene materials have shown a good capacity to block host cell viral entry. disinfection with graphene family materials is also promising, offering the possibility to couple high viral binding with phototreatments. regarding the go and rgo activity in cellular environments, different parameters must be considered such as protein coronation, blood circulation time, and activity in vivo. so far, the use of sulfonic groups introduced via diazonium salt decomposition has been largely privileged. we take this opportunity to encourage future studies using other targeting groups (e.g., boronic acids) and grafting methods (e.g., epoxide ring opening or hydroxyl esterification reactions). mechanical disruption of the capsid. the most direct way to suppress viruses and stop the spreading of viral infection is to inactivate them before the attachment to the host cells, by binding to the acceptor proteins. one of the most conserved targets of viral attachment ligands is the heparan sulfate proteoglycan (hspg), previously mentioned. hspgs are expressed on the surface of almost all eukaryotic cell types, and many viruses like hiv- , hsv, human papilloma virus (hpv) exploit hspgs as the target of their viral attachment ligands. bearing in mind this behavior, different studies have used hspg-mimicking materials to target this type of virus−cell interaction and to achieve broad spectrum efficacy. for example, in one key study, aunps were functionalized with mercaptoethanesulfonate (mes) based on its mimicry of hs (au-mes nps). au-mes nps were shown to interfere with viral attachment, viral entry, and cell-to-cell spreading. the importance of the polyvalent interactions with the virus makes these nps a good candidate for antiviral therapy. however, au-mes nps presented virustatic activity, meaning that upon dilution of the nps, the virus recovers its infectivity due to the reversibility of the cell-virion interaction. this problem was solved by stellacci and colleagues who developed nps coated with mercapto- -undecanesulfonate (mus) ligands (au-mus nps). the long aliphatic and flexible linkers provide stronger associations with the viral particles compared to au-mes nps, leading to local distortions and eventually inducing a global deformation and breaking of the capsid that inactivates its contagion irreversibly (figure ). these au-mus nps were tested against different hspgdependent viruses, showing a high viricidal activity over hsv, hpv, and rsv ( figure a) . furthermore, the activity of the au-mus nps was studied in vivo using mice infected with rsv, indicating that the material can prevent pulmonary dissemination of the infection and showing potential use as medically relevant virucidal drugs to fight viral infections. more recently, the concept was further extended to cyclodextrins modified with mercapto- -undecanesulfonate, proposing this system as a broad spectrum virucidal macromolecule. besides au-nps, a similar mechanism of disruption was studied with other materials like graphene or go. in a study, in which the toxicity of graphene was evaluated theoretically, it was also shown that graphene nanosheets can interrupt the hydrophobic protein−protein interaction, which is essential to biological functions. this feature was attributed to the hydrophobic nature of graphene. thus, it seems energetically favorable for graphene to slide between the interface of two proteins in contact, due to hydrophobic interactions. in another study inspired by this behavior, the authors performed molecular dynamics simulations of graphene nanosheets in the proximity of the surface of the ebola viral matrix protein vp showing that the nanosheets can break the hydrophobic interactions in vp , a key protein for the replication and stability of ebola virus (figure ). these findings suggest that graphene nanosheets might have potential antiviral activity against ebola; however, there is a lack of experimental evidence that corroborates this mechanism of disruption. on the other hand, go was tested experimentally against pseudorabies virus and porcine epidemic diarrhea virus (pedv), showing significant decrease in the infectivity. it was found that the negatively charged surface of go is important for the adsorption of the virus, whose surface is positively charged, and that go could directly interact with the viral particles and destroy their structures due to the sharp edges of the material (figures and b) . the mechanical disruption of the capsid is a peculiar antiviral mechanism associated with some nms. in particular, the use of specific sulfonates able to mimic heparan sulfate can be also used to target sars-cov- infection. blocking the viral entry, via liquid/surface disinfection or once in the body, is a powerful strategy to hamper early stage viral contagions. on the other hand, when infections have already spread and reached middle and late stages, alternative pharmacological strategies are required. the study of the viral pathogenesis and machinery inside host cells has allowed the preparation of different drugs. due to the present pandemic, such antiviral drugs are now of top interest in the scientific and medical communities. so far, few antiviral drugs are clinically available, and their mechanisms of action consist on the inhibition of reverse transcriptase (hiv, hepatitis), dna inhibition of polymerase (herpes, hiv), inhibition of protease (hiv), blockage of ion channels (influenza), and inhibition of neuramidase (hiv, influenza, hepatitis). however, these drugs suffer from moderate to severe side effects. additionally, rapid mutations in the viral machinery make them resistant to the treatments, making the control and the stop of the infection challenging. the use of hnms in drug delivery has shown several advantages. first, hnms can increase drug solubility and its circulation time. additionally, they can be functionalized with targeting molecules able to direct the drug to the desired organs and so avoiding side effects and reducing the dosage. more recently, new antiviral mechanisms were discovered. in particular, it was found that different types of hnms are able to change the ros homeostasis in infected host cells, stopping the viral replication and preserving the cell survival. in this section both drug delivery materials and hnms with ros modulation properties will be critically presented ( figure ) . nanomaterials for drug delivery. different hnms have been widely tested for drug delivery applications. the advantages of this strategy are several including enhance drug solubility and the possibility of targeting and multivalent effects. as a potential broad spectral antiviral agent, agnps can prevent the virus from adsorbing to the host cell in the early stage of infection and thus show strong antiviral activity. after the cells are infected by the virus, there are ways to inhibit cell apoptosis (figure , right) . for example, lv et al. studied the anti-tgev activity of agnps in swine testicle cells and explored the possible mechanism of agnp inhibition of tgev infection-induced apoptosis. the results showed that these agnps are able to decrease cell apoptosis through the activation of p /mitochondria-caspase- signaling. although the use of agnps has shown good antiviral action to further improve the therapeutic effects and reduce both side effects and drug resistance, the way of binding drugs or genes and other therapeutic agents to agnps has received particular attention. it has been observed that agnps inhibit the activities of neuraminidase and hemagglutinin, preventing the h n influenza virus from attaching to host cells. at the same time, the potential molecular mechanisms revealed that caspase- -mediated apoptosis was inhibited by ros generation. agnps modified with polyethylenimine (pei) can bind sirna. ag@pei@sirna exhibited superior abilities for enhanced cellular uptake and blocking ev virus infection acs nano www.acsnano.org review and significantly decreased the apoptotic cell population, which prevented the spread of ev virus. in addition to drugs and sirna, neutralizing antibodies in combination with agnps were developed. the results demonstrated that there is an additive effect between the antibody and agnps when combined against cell-associated hiv- infection in vitro. the membranotropic properties of fullerenes were widely exploited. for example, pristine c , after accumulation at the cell membrane, can translocate into the cytoplasm by crossing the membrane through multiple energy-dependent pathways despite its hydrophobic character. fullerenes can be made water dispersible using surfactants, sonication or first dissolving them in appropriate organic solvents (dmso). the use of fullerenes as inhibitors of viruses started in with a study focused on hiv infection. this work revealed the interaction between c derivatives and hiv protease through molecular modeling and experimental verification. hiv protease (hiv-pr) is involved in the mechanism of replication in the maturation of hiv virion, cleaving newly synthesized proteins. the active site of hiv protease has a cavity of Å closed to the diameter of c cage. molecular docking and experiments on hiv-pr catalytic activity revealed a blockage of the active site of the enzyme by van der waals interactions leading to an antiviral effect. the following studies were based on a structure−activity relationship between functionalized fullerenes and hiv-pr with the aim to increase the antiviral activity. the introduction of pyrrolidinium salts onto c was tested against the activity of hiv- strain. in a second study, c bearing two ammonium groups was applied against the activity of hiv- and hiv- strains. the results showed the importance of having two moieties in a precise position on the fullerene cage and the influence of the charge of the different salts sensibly increasing its antiviral activity. further investigations using different functional groups (e.g., amino acid derivatives) were explored. c functionalized with aminobutyric acid and aminocaproic acid was able to inhibit hiv viral replication at subnanomolar concentrations. in another work, anionic and cationic pyrrolidinium salts and amino acid functionalized fullerene derivatives were used for the inhibition of hiv reverse transcriptase (hiv-rt). fullerene compounds were compared to nevirapine, an available drug against hiv-rt, revealing a better inhibition compared to the pure drug. the antiviral property of carboxylated fullerenes was confirmed by another study. results obtained in vitro on cem cell line showed low toxicity and a submicromolar ec against hiv- and hiv- strain viral replication. several mechanisms of hiv protease inhibitors have been hypothesized and simulated. some studies investigated the action of fullerene derivatives on viral replication cycle and the virus maturation ( figure ). , for the latter, it was suggested an impairment due to a strong interaction between fullerene and the immature capsid. other rna viruses share ways of viral replication similar to hiv. c functionalized with an amino acid derivative was investigated against hepatitis c rna polymerase (hcv-rp). this essential enzyme for viral replication was inhibited in a submicromolar range, similarly to benzo- , , thiadiazine, a potent specific inhibitor of hcv-rp. another publication highlighted the effect of a c poly(carboxylic acid) derivative on different strains of influenza virus (e.g., a, h n , h n , and b). the inhibition was comparable to tamiflu, rimantadine, ribavirin, and amantadine, but the mechanism of action remains still unclear. these data emphasize that fullerenes c or c can be potent universal antiviral drugs for rna enveloped viruses. however, clear elucidations of the mechanisms of action and in vivo studies are still a missing point. due to their high surface/weight ratio and capacity to pass through cell membranes, carbon nanotubes (cnts) have been extensively explored for drug and gene delivery applications. cnts have been also successfully used for the delivery of antiviral agents. compared to pristine materials, oxidized cnts (ox-cnts) showed an inhibitory activity for hiv viruses per se. this effect has been associated with the oxygenated groups that increase the hydrophilicity and the colloidal stability of the material. the antiviral efficiency has been correlated to the ox-cnt interaction with host cells. however, it is not clear how and what kind of mechanism blocks the viral machinery in host cells. different anchoring strategies have been explored to link antiviral drugs onto cnt surface. for instance, ox-cnts were covalently linked to amino- -nitro- -( , -dimethylbenzyl)-aniline (chi ) and n-( -aminophenyl- -nitro-)- , -dimethylbenzenesulfonamide (chi ), two active non-nucleoside reverse transcriptase inhibitors for hiv treatment. following the covalent conjugation, only a moderate antiviral effect was observed compared to pure drugs, indicating that most probably the nm cell trafficking played a key role on the virucidal activity. in another study, ox-cnts functionalized with cyclodextrin were used for the delivery of acyclovir (a prodrug inhibitor of the viral dna polymerases) for the treatment of hsv- . preliminary results showed that when acyclovir was delivered via the nanotubes, the viral antireplicative effect was higher than the free drug. more recently, a similar approach was applied to herpes virus using cyclodextrin and pei-functionalized cnts for co-delivery of cidofovir and plasmid dna. however, the antiviral effect of the materials was not explained, and the transfection effect was not satisfactory. functionalized cnts have been also used for delivery of ribavirin in vivo using grass carp as an animal model for the study of grass carp reovirus. ox-cnts were first functionalized via amidation with bsa, and then ribavirin was covalently bound to the protein via esterification (figure ) . in vivo tests demonstrated that, when ribovirin was shuttled by ox-cnts, the antiviral efficiency was significantly increased without any evident toxicity and no significant changes in ros-generating enzymatic activities. the use of cnts in drug delivery is however still controversial. graphene-based materials have been limitedly studied as antiviral drug delivery carriers. only a few examples can be found in the literature. graphene quantum dots (gqds) were used for drug delivery of chi and chi and tested in vitro against hiv similarly to cnts. both the prepared gqd-chi and gqd-chi showed a high antiviral activity once into host cells, with low toxicity. go has been also used for the delivery of dnazyme into hepatic cells allowing to block the hepatitis c infection. this specific dna single strand is able to recognize the viral mrna and to silence its expression. overall, carbon nms proved to be interesting carriers for antiviral drugs. however, several questions need to be answered before their safe application as antiviral materials. different reports have demonstrated that pristine cnts display relevant toxicity for healthy cells, but by oxidation of the tubes, the side effects can be sensibly reduced. in addition, more investigations should be performed on cnt toxicity. these nanocarriers indeed are not "innocent delivery agents", but they play a key role in drug internalization pathway and in host cell machinery that might be averse to the expected therapeutic effects. so far, cnt application in biological systems has been studied for years. however, due to their possible toxicity, their real application in clinics seems to be steeper and difficult to achieve. materials tuning reactive oxygen species. ros homeostasis in infected cells has been studied for both rna and dna viruses. for instance, it was shown that infection of mice with influenza a decreased the concentration of lung glutathione and the antioxidant vitamin c, providing evidence that the viral infection was associated with oxidative stress in vitro as well as in vivo. similarly, in hiv infection, induced oxidative stress in host t cells and high concentrations of antioxidants are able to slow down the cell-to-cell viral spreading. the increase of ros concentration is a common process in most of the viral infections. however, the mechanism of radical generation is different from case to case. several proofs suggest that modulating ros homeostasis in infected cells can slow down or block the infection. hnms have been shown to be powerful allies against viral infections. in particular, metal or metal oxide nms, once internalized, can regulate the radical production into infected host cells. in this scenario, the nms can work following two different mechanisms: ( ) enhancing radical activity or ( ) quenching ros inside cell compartments. in the first case, metal oxide nps are able to convert superoxide ions into more reactive hydroxyl radical species via fenton or fenton-like reactions. the excess of superoxide ions is able to oxidize the viral proteins and the genetic material and therefore efficiently block the infection. this approach has been reported using zno nps. , in these studies, zno nps have been successfully applied for the treatment of h n influenza virus and herpes simplex virus. the preliminary results showed that pegylated zno particles were able to efficiently reduce the viral infection with ic similar to acyclovir. more importantly, toxicity of zno was modulated by functionalization with peg, which allowed a higher colloidal stability and a more controlled release of zn + ions to catalyze ros formation. indeed, ros (e.g., superoxide and hydroxyl radicals) produced by the nps should be highly reactive and should not only damage the exogenous biological molecules but also attack different cell compartments. overproduction of ros may reduce virus spread but also induce cell death. interestingly, this approach is applicable only at the early infection stage (after h incubation of the host cells with a virus), but it loses its activity at later time points. these , this specific mechanism of action restricts zno nps to an application only at the early stage of infections. however, the same approach with other metals able to induce fenton or fenton-like reactions (e.g., fe, cu, and mn) has not been reported yet. the choice of proper capping agents may allow to control the metal ion release and thus tune the ros-mediated antiviral activity. we would like to take an advantage here to suggest the growth of the antiviral research in this direction. another successful approach relies on the reduction of ros concentration in host cells. ros scavenging is able to alleviate the toxicity of the infection enhancing cell viability, giving time to start its endogenous antiviral mechanisms. so, this approach may both block infection and ensure host cell survival. in this context, selenium nps (senps) have been extensively studied for their antiviral activity. the mechanism of action of these nps relies on the quenching of the radicals into host cells due to the infection, stopping the mitochondria depolarization and the consequent apoptotic cascade. additionally, senps can also adsorb onto the viral capsid sensibly reducing their infectivity. senps can be prepared via classical mixing of selenium salt precursors in the presence of a reducing agent. more recently, senps have been instead biosynthesized from actinobacteria showing good stability and capacity to inhibit dengue virus in vitro. moreover, senps were used to carry different antiviral drugs including zanamivir, oseltamivir, amantadine, and ribavirin. their functionalization with the desired drug can be easily achieved adding the molecule during their synthesis through the se ion controlled reduction. these nms have been applied for the treatment of h n virus. notably, senps with ribavirin (administered via intranasal absorption every h for days) showed that infected mice had much less alveolar collapse and perivascular and peribronchiolar edema, compared to the group challenged with the virus (figure ). due to their efficacy and low toxicity, senps can be considered a useful material for the treatment of other viral diseases including sars-cov- . as a matter of fact, oxidative stress as well as chronic inflammation may contribute to the aggravation of the covid- symptoms and to the general spread of the infection. the use of senps could eventually alleviate the toxicity of infected patients, giving time for the immune system to react against the contagion. however, the lack of preclinical studies on senps, together the scarce knowledge of their biosafety and long-term toxicity, still remain the main challenges to tackle for clinical translation. the vast majority of the studies show that human survival to viral attack is based on the stimulation and response of our immune system. when a virus enters and starts infecting tissues, the body reacts and triggers a strong immune response to overcome the pathogen invasion and spread. normally, two different immunological reactions take place. the oxidative stress induced by infection causes the activation of the inflammasome through upregulation of pro-inflammatory cytokines such as il- β, pro-il- , and nlrp . excessive upregulation of this mechanism leads to cell damage and eventually to pyroptosis activated by caspases. it was also shown that generation of radicals is able to depolarize mitochondria, hence affecting host cell respiration and inducing ros-mediated apoptosis at the late stage of infection. besides, activation of pro-inflammatory cytokines can alert the immune system blocking the infection (the innate acs nano www.acsnano.org review immune response). the second immune response is specific (the adaptive immune response). immune cells are trained to attack the virus (cellular response), while specific antibodies are produced by b cells (humoral response). in the last years, hnms were proved to tune the immune responses, demonstrating to be a possible alternative against viral infection. in this section the most relevant strategies for the activation of innate and adaptive immune responses using nms will be described ( figure ) . innate immune response. innate immune response is the first response that takes place in the presence of any type of infection. during this early stage, interferon stimulating genes (isgs) are upregulated in the infected cells. this selfdefense mechanism slows down the viral replication and alerts sentinel immune cells that start producing proinflammatory cytokines and trigger inflammation. however, many viruses are able to escape this complex mechanism, retarding the immune response and spreading the infection. the interaction of hnms with the immune system has been more and more studied. in the case of antiviral hnms, many examples can be found in the literature where the nms not only slow down the infection but also tune the innate immune response. certain hmns can display an intrinsic immune stimulation. we have already mentioned above that in the early stage of infection, agnps mainly prevent the virus from entering the host cell through the interaction with the external capsid, but in vitro cellular experiments lack to understand the complex interaction with primary immune cells. recent studies have shown that agnps can potentially induce the expression of genes involved in innate and adaptive immunity-associated pathways, which are known to play crucial role in immune regulation. for example, toll-like receptor can be upregulated by agnps after h, by recognizing the singlestranded rna of the viruses and regulating the antiviral immune response. another study showed that in rsvinfected mice treated with agnps, the particles reduced the production of pro-inflammatory tnf-α and il- cytokines, but potentiated the anti-rsv activity of neutrophils in an experimental mouse model. however, activation of agnps in the reduction of rsv has been noted only when the nm was intranasally inoculated together with the virus, and no results have been reported on the use of agnps administrated on infected mice. in the case of influenza virus infection of lung epithelial cells, it was found that agnps targeted infected lung epithelial cells and reduced viral replication, by preventing autophagy. however, the blockage of the autophagic flux by agnps does not inhibit viral replication in already infected cells. therefore, agnps are more suitable as viral preventive agents due to their pro-inflammatory response rather than drugs. more recently, agnps were combined with graphene materials and exploited as antiviral material for the treatment of porcine reproductive and respiratory syndrome virus (prrsv). go-agnps were able to clump the virus diminishing its fusion with cell membrane. additionally, once go-agnps were internalized in host cells, they stimulated the isgs that blocked viral budding and its diffusion to other cells in vitro (figure ). similarly, cds used for the treatment of rsv and prrsv were able to activate the innate immune response via an upregulation of isgs in vitro. gold nanorods were applied to boost the innate immune response against rsv in vivo. interestingly, it was shown that these nanorods (when intranasally administered with rsv) were not only able to activate the isgs but also to tune the production of pro-and anti-inflammatory cytokines, resulting in the blocking of the infection with a reduced pulmonary inflammation. as drug carriers, hnms can also affect the internalization pathways, modulate drug efficacy, and the immune cell responses. for instance, it was found that the isoprinosine immunomodulatory antiviral drug displays a much higher antiviral efficacy in vivo when delivered with cnts than as a pure drug (in acs nano www.acsnano.org review zebrafish larvae food administration), probably due to a better cellular uptake and the anti-inflammatory properties of the nanotubes. fullerenes also exhibit immunomodulation properties through the release of cytokines by bovine alveolar macrophages. a study explored the influence of functionalization of c with molecules presenting different surface charges like hydroxyl groups and amino acids. the study concluded that negative charges upregulated tnf-α up-secretion in raw . macrophages, but highly positively or negatively charged surfaces increased cell toxicity. the upregulation of the cytokine tnf-α is a proof that fullerene can promote cellular immune response. despite these preliminary results, the actual mechanisms of interaction between hnms and the immune system are still at early stage of understanding. we must consider that the immune response needs to be proportional to the infection grade. if on one side nanosized immuno-boosters can alert more efficiently the sentinel cells, the use of hnms that trigger an exaggerated immune response can promote excessive inflammation, damaging healthy cells and promoting uncontrolled side effects. in the particular context of sars-cov- , the use of agnps, fullerenes, or other pro-inflammatory hnms at the middle and late infection stage may cause an aggravation of the symptoms due to already diffused inflammation. in particular, most of the studies showed the ability to reduce infection when hnms were first incubated with the pathogenic virus, thus limiting their potential use in the early stage infection. the formulation of hnms able to both alert the immune system (e.g., upregulating cytokine) and control lung inflammation (e.g., ros scavenger) even after the early stage infection may be a possible strategy for treatments against sars viruses. adaptive immune response. adaptive immune response is the specific response that the immune system exerts against pathogens. this mechanism is particularly active toward viral infections where the immune system produces specialized lymphocytes (to fight the virus), called memory b cells (to be effective in case of new infections) and antibodies (corresponding to the humoral response). the stimulation of adaptive responses in case of specific infections can be induced artificially through the introduction of attenuated pathogens, stimulating the production of specific antibodies. this is the principle of vaccination, which is the most common procedure for immunization of large areas of population against many kinds of lethal viruses. besides, the use of viral proteins as antigens in the vaccine formulation leads to neutralizing antibodies, but, due to the low immunogenicity of isolated proteins, does not always stimulate sufficiently the immune system to reach total protection. more recently, nanotechnology has been applied to develop more efficient vaccines (e.g., nanovaccines). the use of nanostructures with a size similar to virus (virus-like nanoparticles) sensibly enhances the response helping to reach immunity. hnms can adsorb viral particles and present them to the immune system. , this method of vaccination has been successfully applied in vivo for the challenge of herpes simplex virus. hsv- starts its spreading in vaginal tissues and then diffuses to the neurons causing death in mice. zno nps (teardrop morphology), after vaginal inoculation with hsv- , are able not only to prevent viral cell adhesion but also to expose viral antigens to t cells and dcs, leading to immunization. the preclinical trials acs nano www.acsnano.org review against hsv- showed a survival to infection higher than %. this approach highlights the possibility to couple cell mimicking nms to other co-adjuvants for the formulation of large spectrum nanovaccines ( figure ). , hnms have been also applied to the delivery of antigens, exposing them to the immune system. fullerenes were found as suitable carriers for the delivery of drugs or nucleic acids. functionalized fullerene can also self-assemble into virus-sized nps. investigated as vaccines in cancer immune therapy, polyhydroxy fullerenes (called fullerenols) display interesting properties for antiviral therapy, based on their capacity to selfassemble into virus-like particles (vlps) and so to enhance the immunogenicity of the antigens. the great advantage of this strategy relies on the easy encapsulation process during the self-assembly making fullerenols versatile for the formulation of different kinds of vaccines. these vlps were investigated against hiv- and hepatitis c viruses. compared to conventional protein-or peptide-based vaccines intended to induce antigen-specific adaptive immune responses, dna vaccines are more stable, cost-effective, easy to manufacture, and safe in handling. however, dna vaccines have the disadvantage of being poorly immunogenic. fullerenol vlps allow to avoid the use of other adjuvants. in the case of a vaccine against hiv- , fullerenol vlps penetrated easily into the cells resulting in an enhancement of dna transfection. this was proved in a study using fullerenol encapsulating dna encoding the hiv- envelope protein gp ( figure ). in vitro assays were performed in human embryonic kidney cells line (hek ) showing good transfection ability. following various immunization routes (e.g., activation of toll-like receptor signaling or effector memory t cell immune response), fullerenol vlps can induce an innate and a cellular immunity. a similar study was performed for hepatitis c using the hcv recombinant protein as antigen, confirming the potential efficacy of using fullerenols as antiviral vaccines. nevertheless, these results require further mechanistic investigations. indeed, in vitro studies also evidenced a suppressive effect of acquired immune response of c pyrrolidine tris-acid and fullerenol c (oh) . the fullerenol had a dose-dependent effect on t cell receptormediated activation and antibody production by b cells under anti-cd /il- stimulation. however, the molecular mechanism is still unknown. other hnms including aunps (two subcutaneous injections in guinea pigs) and nanodiamonds (three subcutaneous injections in balb/c mice) were explored as carriers of viral proteins for immunization of swine transmissible gastroenteritis virus and h n influenza, respectively, with good preliminary results. , in these cases, the vaccine formulation relies on the adsorption of the viral antigen onto the surface of the nanoparticles. however, an effective vaccination depends on several factors. first of all, the size of the nps plays a key role on the immune system. for instance, size-dependent vaccination efficacy has been reported in mice immunization against the foot-and-mouth disease virus (intraperitoneal and subcutaneous injection, every days for weeks) using aunps as antigen carriers. in this study, the most effective activity to stimulate the immune system was exerted by particles with a diameter in the range of nm. both smaller or bigger particles evidenced a drop-off of the immunization effect. this aspect cannot be ascribed to the antigen concentration, but must be associated only to the acs nano www.acsnano.org review nanoparticle size; however, the mechanism of interaction remains unknown. the selected antigen plays also a crucial role in the preparation of wide spectrum vaccines. for instance, in the case of influenza, two major membrane glycoproteins, hemagglutinin and neuraminidase, are generally used as antigens. however, the antibodies produced by this vaccination strategy are selective to the dominant epitope which has a low effectiveness or is totally ineffective against other epitopes or other kinds of influenza viruses. m (a viral protein responsible for the budding and scission of the influenza virus) is commonly expressed in different types of influenza viruses with a high rate of conservation but with low antigenicity. it has been shown that aunps functionalized with m e protein have a high immunization capacity in comparison to the antigen alone. mice immunized with aunps (two intranasal injections), and then challenged, showed a survival rate higher than % to california-h n pdm, victoria-h n , and vietnam-h n infections. this strategy shows that hnms can be used to boost the immune response of low immunogenic molecules, providing a wide spectrum vaccination potential. unfortunately, it has not been determined if the budding process in sars-cov- is mediated by viral proteins or via the host cell's endosomal sorting complex, thus more research on the sars-cov- viral machinery is highly desirable. all these approaches are based on the capacity of the nanoparticles to adsorb the antigens and expose them to the immune system in the appropriate conformation to produce the neutralizing and protective antibodies. however, the adsorption is not an easy process to control. for instance, aunps have been ineffective in the immunization against sars-cov, when s viral proteins were used as antigens. in particular, the immunization with the protein alone was more efficient than when it was adsorbed onto the aunp surface. this failure was associated with a conformational change or denaturation of the antigen, which did not lead to the production of the specific antibody. covalent chemistry strategies offer a higher control on the antigen quantification with higher reproducibility and possibility to bind different groups onto the surface of hnms. for example, calcium phosphate nanoparticles (capnps) were successfully covalently functionalized with hen egg lysozyme as model antigen showing an immunization times higher in vivo compared to antigens alone using (one subdermal injection in mice). a similar approach was used with iron oxide nps using mannose (to target dcs) and hepatitis b antigen showing good immunological activity in vitro (two subdermal injections in mice at days distance). more recently, other types of vaccination strategies have been applied using hnms. a smart example has been reported using multifunctional capnps on herpes virus. in this study, capnps have been covalently functionalized with alum/mpl as the adjuvant and two peptides as antigens selected via reverse vaccination. the nm stimulated the immune system generating highly efficient antibodies able to block cell-to-cell infection of herpes virus in vivo (three intramuscular injections in mice every days), increasing the survival rate of immunized mice to % against the controls ( % survival, figure ). the recent history has shown the spread of different viral pandemics such as h n flu, hiv, and sars. nowadays, the sars-cov- pandemic global lockdown has profoundly changed the daily life of most humans, causing uncertainty in short and middle time perspectives. in this context, the scientific community has responded to protect the population by studying new vaccines and disinfection methods to be applied in the near future. despite this tough work, a sars-cov- vaccination will hopefully be available in − years, making this epidemic transient period gloomy with increased instability. the study of more effective vaccines and the production of a wide range antiviral agents is nowadays an extremely hot topic. hnms comprise a family of materials that share a nanostructured hard core and a tunable surface chemistry. in this contribution, we have methodically reviewed different hnms for antiviral properties. hnms can have antiviral properties per se, blocking the viral replication and diffusion, or their antiviral properties can be tailored, playing with surface chemistry. hnms can be used to block viral entry and arrest infection at the early stage. the mechanisms rely on different actions including breaking of capsid disulfide bonds (e.g., noble metal nanoparticles), capsid oxidation (e.g., cuonps), mimicking of cell surface (e.g., carbon nms), or mechanical disruption (e.g., aunps or graphene). surface functionalization additionally confers a higher specificity and pharmacological activity toward the targeted virus. in particular, the high local concentration of ligands on functionalized hnm surface imparts a high multivalent effect, enhancing the viral trapping efficiency of nms. interestingly, hnms that show an intrinsic antiviral activity can further enhance their antiviral efficacy via surface functionalization. some antiviral hnms are good photosensitizers (e.g., cuonps) or exert photothermal activity (e.g., carbon nms), thus their antiviral activity can be trigged by light stimulation. more importantly, most of the settled strategies target common viral entry mechanisms and can be adopted to fight a wide viral spectrum. hnms have been also applied as antiviral agents by their interaction with host cells. hnms are able to block viral replication machinery in host cells (e.g., cnts and fullerenes), inhibiting endogenous enzyme activity. additionally, hnms can be explored for the delivery of antiviral molecules, showing a better antiviral activity and reducing side effects in vitro and in vivo. some hnms can also regulate the ros homeostasis of host cells, reduce apoptosis, and enhance host cell survival during the infection. finally, we have reviewed the role of hnms on immunity. in particular, hnms can stimulate the innate immune response, mainly inducing overexpression of interferon and cytokines. this effect can alert sentinel cells and generally warn the immune system of the infection. hnms can be also used for the activation of the adaptive immune response, foreseeing vaccination. due to the similar size of a virus, functionalized hnms with antiviral molecules (virus-like particles) can enhance their immunogenicity. certainly a huge effort should be done for translation of the research into clinics. indeed, hnm applications as antivirals are still in the early phase of research. several challenges still need to be tackled before their safe use. at the current stage, some hnms have been approved only for surface disinfection. for instance, cunps have been used in filters for the preparation of highly efficient broad spectrum antiviral masks. some hnms have been already clinically approved. for example, fenps were approved for imaging and as a drug to treat iron deficiency anemia in adult patients with chronic kidney disease, while aunps are in clinical trials for the treatment of prostate cancer (photothermal therapy). , however, at the moment no clinical trials are running for the use of hnms as antiviral agents. in fact, there are still several concerns on the applications of hnms in drug formulations. compared to molecules, where mainly concentration and exposure routes are concerned, solving hnm toxicity issues is much more complicated. composition, size, shape, and surface functionalization must be considered to respond to the requirement for safety regulations. additionally, interaction on hnms with the immune system must be better elucidated. in particular, the activation of the immune system and complement activation-related pseudoallergy must be taken into account. for instance, ferumoxytol (a fenp-based drug) has been reported to generate severe anaphylactic reactions in humans, of which were fatal. this is due to the possible interaction of hnms with mast cells, provoking their degranulation/activation and release of histamine even at the first exposure (pseudoallergic-mediated hypersensitivity). besides, the mechanism of activation of these cells is still unknown, although it was found that it depends on the hnm composition, size, and surface chemistry and on the corona formation. on the other hand, it has been demonstrated that hnms can travel to the draining lymph nodes, targeting resident dendritic cells and macrophages. therefore, they are able to interact with antigen presenting cells to stimulate innate and adaptive immune responses. we believe that a joint venture of different chemists, materials scientists, virologists, toxicologists, and medical doctors can push forward the preparation and safe application of hnms in this field, hoping to prevent and eventually block the rise of new viral pandemics. alberto bianco − cnrs, immunology, immunopathology and therapeutic chemistry, upr , university of strasbourg isis, strasbourg, france; orcid.org/ - - - x; email: a.bianco@ibmc-cnrs.unistra.fr giacomo reina − cnrs, immunology, immunopathology and therapeutic chemistry, upr , university of strasbourg isis, strasbourg, france; orcid.org/ - - - ; email: g.reina@ibmc-cnrs.unistra.fr hard nanomaterial, nonpolymeric organic or inorganic materials with sizes comprised between and nm; antiviral, medical term used for any agent or drug altering virus integrity or process involved in viral infection disease; viral infection, process by which viruses invade the body through multiple pathways and multiply in susceptible host cells; viral pathogenesis, approach in biomedical research to understand the process by which a viral infection leads to disease, including mechanism of infection into the host (e.g., viral entry, viral replication) and factors that affect this mechanism (e.g., virus susceptibility to host defenses); membranotropism, the ability of an organism or an agent to interact with biological barriers; immunogenicity, capacity of 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vaccineshow far from clinical use? dendritic cell delivery of plasmid dna: applications for controlled genetic immunization potential suppressive effects of two c fullerene derivatives on acquired immunity prospects for the use of spherical gold nanoparticles in immunization nanodiamond enhances immune responses in mice against recombinant ha/ h n assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide consensus m e peptide conjugated to gold nanoparticles confers protection against h n , h n and h n influenza a viruses gold nanoparticle-adjuvanted s protein induces a strong antigen-specific igg response against severe acute respiratory syndrome-related coronavirus infection, but fails to induce protective antibodies and limit eosinophilic infiltration in lungs nanoparticle-based b-cell targeting vaccines: tailoring of humoral immune responses by functionalization with different tlr-ligands hbs antigen and mannose loading on the surface of iron oxide nanoparticles in order to immuno-targeting: fabrication, characterization, cellular and humoral immunoassay induction of herpes simplex virus type cell-to-cell spread inhibiting antibodies by a calcium phosphate nanoparticle-based vaccine progress in nanomedicine: approved and investigational nanodrugs gold nanoshell-localized photothermal ablation of prostate tumors in a clinical pilot device study engineered nanomaterials and type i allergic hypersensitivity reactions shiyuan peng − cnrs, immunology, immunopathology and therapeutic chemistry, upr , university of strasbourg isis, strasbourg, france lucas jacquemin − cnrs, immunology, immunopathology and therapeutic chemistry, upr , university of strasbourg isis, strasbourg, france andreś felipe andrade − cnrs, immunology, immunopathology and therapeutic chemistry, upr , university of strasbourg isis, strasbourg, france complete contact information is available at: https://pubs.acs.org/ . /acsnano. c the authors declare no competing financial interest. the authors gratefully acknowledge the financial support from the eu graphene flagship project (no. ). this work was partly supported the agence nationale de la recherche (anr) through the labex project chemistry of complex systems (anr- -labx- _csc). we wish to acknowledge the centre national de la recherche scientifique (cnrs) and the international center for frontier research in chemistry (icfrc). s.p. is indebted to the chinese scholarship council for supporting her ph.d. internship as a visiting ph.d. student. a.f.a. wish to thanks the eur csc graduate school (strasbourg, france) for supporting his master studies. key: cord- -s ua re authors: park, mi hee; jo, miran; kim, yu ri; lee, chong-kil; hong, jin tae title: roles of peroxiredoxins in cancer, neurodegenerative diseases and inflammatory diseases date: - - journal: pharmacol ther doi: . /j.pharmthera. . . sha: doc_id: cord_uid: s ua re peroxiredoxins (prdxs) are antioxidant enzymes, known to catalyze peroxide reduction to balance cellular hydrogen peroxide (h( )o( )) levels, which are essential for cell signaling and metabolism and act as a regulator of redox signaling. redox signaling is a critical component of cell signaling pathways that are involved in the regulation of cell growth, metabolism, hormone signaling, immune regulation and variety of other physiological functions. early studies demonstrated that prdxs regulates cell growth, metabolism and immune regulation and therefore involved in the pathologic regulator or protectant of several cancers, neurodegenerative diseases and inflammatory diseases. oxidative stress and antioxidant systems are important regulators of redox signaling regulated diseases. in addition, thiol-based redox systems through peroxiredoxins have been demonstrated to regulate several redox-dependent process related diseases. in this review article, we will discuss recent findings regarding prdxs in the development of diseases and further discuss therapeutic approaches targeting prdxs. moreover, we will suggest that prdxs could be targets of several diseases and the therapeutic agents for targeting prdxs may have potential beneficial effects for the treatment of cancers, neurodegenerative diseases and inflammatory diseases. future research should open new avenues for the design of novel therapeutic approaches targeting prdxs. peroxiredoxin (prdx)s are thiol-specific antioxidant enzymes that reduce various cellular peroxide substrates using cysteine-containing active sites (fatma, kubo, sharma, beier, & singh, ; wood, poole, & karplus, b; wood, schröder, robin harris, & poole, a) . prdxs refer to a family of small ( - kda) non-seleno peroxidases currently known to possess six isozymes, namely, prdx - in mammalian systems (chae, oubrahim, park, rhee, & chock, ; wood et al., a) . these proteins contain either one ( -cys prdx) or two ( -cys prdx) redox-active cysteine residues (schröder et al., ; wood et al., a) . the -cys group includes prdx to prdx , whereas prdx is the sole member of the -cys group (immenschuh & baumgart-vogt, ; rhee, chae, & kim, ) . despite this difference, peroxidase activities of both -cys and -cys groups commonly contribute to cellular protection against oxidative stress (dayer, fischer, eggen, & lemaire, ; monteiro, horta, pimenta, augusto, & netto, ) . during reaction with oxidant substrates, the cys-residues of prdxs are oxidized (chevallet et al., ; schröder, brennan, & eaton, ) . thioredoxin (trx) provides the electron for reducing the oxidized prdx - , whereas glutathione is likely to be employed to reduce the oxidized prdx (poole, hall, & nelson, ; turner-ivey et al., ) . peroxiredoxins (prdxs) are a ubiquitous family of antioxidant enzymes, known to catalyze peroxide reduction to balance cellular hydrogen peroxide (h o ) levels, which is essential for cell signaling and metabolism and act as a regulator of redox signaling (chen, na, & ran, ; perkins, nelson, parsonage, poole, & karplus, ) . redox signaling is a critical component of cell signaling pathways that are involved in the regulation of cell growth, metabolism, hormone signaling, immune regulation and a variety of other physiological functions (mishra, jiang, wu, chawsheen, & wei, ; shadel & horvath, ; trachootham, lu, ogasawara, valle, & huang, ) . therefore, prdxs are involved in the regulation of cell proliferation, apoptosis, embryonic development, lipid metabolism and immune responses. prdxs are involved in particular pathological condition. prdx isoforms can be considered good therapeutic targets in several cancers such as lung cancer (jo et al., ; kim et al., ; soini & kinnula, ; wei et al., ) , glioblastoma (deighton et al., ; khalil, ) , colorectal cancer (lu et al., a (lu et al., , b song et al., ) , prostate cancer (riddell et al., ; ummanni et al., ) and ovarian cancer (chung et al., ; where they protect tumor cells, prdxs are involved in a variety of signaling pathways such as nf-κb signaling pathway, stat pathway, wnt/β-catenin pathway and mapk kinase pathways (brigelius-flohé & flohé, ; jo et al., ; lu et al., b; riddell, wang, minderman, & gollnick, ; yun, choi, oh, & hong, c; yun, park, kim, & hong, b; yun et al., a) that are involved in cancer development. so, prdxs can be explored as therapeutic targets as well as therapeutic tools depending on their role in particular pathological conditions. several studies have suggested that prdxs are related with the development of neurodegenerative diseases. prdxs are involved in neurodegenerative diseases such as ad and pd that are regulated by oxidative stress and redox signaling (cimini et al., ; yun et al., yun et al., , a . prdxs have distinct functional roles in the brain and provide differential contributions to neuropathologic conditions. the expression patterns of prdxs are in fact highly variable in different regions of the brain during neurodegenerative disease processes (hattori & oikawa, ) . given that oxidative damage is involved in the pathogenesis of neurodegenerative diseases, the alteration in prdx expression would appear to be primarily a consequence of cellular resistance to the oxidative damage. prdxs are also important regulators of inflammatory diseases such as obesity (huh et al., ; murri et al., ) , atherosclerosis (guo et al., ; park et al., ) and rheumatoid arthritis (ra) szabó-taylor et al., ) . inflammation is a complex biological self-defense reaction triggered by tissue damage or infection by pathogens. redox-regulated transcription factors and protein kinases play important roles in the regulation of inflammation. nf-κb is a well-characterized redox-sensitive transcription factor important in the initiation and progression of inflammation through the production of various cytokines and prostaglandins. prdxs induce nf-κb activation that is important for the expression of inflammatory proteins (kim et al., a; kim, lee, rhee, seo, & pak, b; riddell et al., ) . also, prdxs are secreted from cells under mild oxidative stress binds tolllike receptor , suggesting that prdxs are involved in inflammation related disease (kuang et al., ; riddell et al., ; shichita et al., a, b) . although several studies of prdxs have been published, a comprehensive review of the various features and functions of prdxs is lacking. in this review article, we will discuss recent findings regarding prdxs in the development of several diseases and therapeutic approaches. based on its conserved cysteine residues and their catalytic mechanisms, mammalian prdxs can be divided into three subgroup, namely typical -cys, atypical -cys, and -cys prdxs. prdx - belong to typical -cys and prdx belongs to atypical -cys group, within which all members contain two conserved cysteine residues. prdx belongs to -cys prdxs subgroup in which one cysteine residue is conserved. moreover, another subgroup named prdx-q was extensively found in plants, which also contains two cysteine residues. these structures reveal prdxs to be very similar, each containing a thioredoxin fold with a few additional secondary-structure elements present as insertions. the most striking differences involve their oligomeric states. the atypical -cys prdxs are monomeric enzymes, whereas both the typical -cys and the -cys prdxs are domain-swapped homodimers in which the c terminus of one subunit reaches across the dimer interface to interact with the other subunit (hirotsu et al., ; schröder et al., ; wood et al., a) . in the typical -cys prdxs, the resolving cysteine is located in the c-terminal arm. three of the typical -cys prdxs crystallized as toroid-shaped complexes consisting of a pentameric arrangement of dimers ((a ) decamer), consistent with observations that -cys prdx dimers can form discrete higher-order oligomers (kitano, niimura, nishiyama, & miki, ) . ahpc from amphibacillus xylanus, another -cys prdx, also crystallizes as an (a ) decamer, although no crystal structure for this enzyme has been reported. the structure and sequence of the peroxidatic active site is highly conserved among the prdx classes . prdxs contain an active site cysteine that is sensitive to oxidation by h o . mammalian cells express six prdx isoforms that are localized to various cellular compartments. the oxidized active site cysteine of prdx can be reduced by a cellular thiol, thus enabling prdx to function as a locally constrained peroxidase (rhee, woo, kil, & bae, ) . regulation of prdx via phosphorylation in response to extracellular signals allows the local accumulation of h o and thereby enables its messenger function woo et al., ) . the fact that the oxidation state of the active site cysteine of prdx can be transferred to other proteins that are less intrinsically susceptible to h o also allows prdx to function as an h o sensor. prdxs are a ubiquitous family of antioxidant enzymes, known to catalyze peroxide reduction to balance cellular h o levels, which is essential for cell signaling and metabolism. the general function and mechanism of action of the prdx redox system is in fig. . prdx is a multifunctional protein originally identified as an intracellular scavenger of h o (yang et al., ) . it has been also shown to act as a molecular chaperone with the ability to modulate the actions of numerous molecules, a regulator of transcription, or as an immunomodulator (jang et al., ; o'leary et al., ; park et al., ; wang, he, sun, delcuve, & davie, ) . prdx is involved in redox regulation of the cells and reduces peroxides with reducing equivalents provided through the trx system but not from glutaredoxin (lehtonen et al., ; neumann et al., ) . the trx system is comprised of nadph, trx and trxr, and forms the trx peroxidase system with prdxs, and reduces peroxides by nadph (watanabe, nakamura, masutani, & yodoi, ) . prdx is involved in this system. prdx gene is mapped on chromosome p . and is a member of the peroxiredoxin family that contains a consensus site (thr( )-pro-lys-lys) for phosphorylation by cyclin dependent kinases (chang et al., ; gisin, perren, bawohl, & jochum, ) . prdx is phosphorylated on thr- during the m-phase, which leads to a more than % decrease in enzymatic activity through the trx system (jang et al., ; neumann, cao, & manevich, ). prdx is implicated in redox regulation of the cell and plays an important role in eliminating peroxides produced during metabolism that participates in the signaling of growth factors and tumor necrosis factor-alpha (tnf-α) through regulating the intracellular concentrations of h o (jamaluddin et al., ; liou & storz, ) . prdx is overexpressed in several cancer cells including esophageal, pancreatic, lung, thyroid and oral cancer (gong, hou, liu, & zhang, ; jiang, wu, mishra, chawsheen, & wei, b; jiang et al., a; taniuchi et al., ; yanagawa et al., ; zhang, liu, szumlinski, & lew, b; zhang et al., a zhang et al., , . concentrated prdx can be secreted by non-small cell lung cancer cells via unusual pathway (chang et al., ) . prdx has a high reactivity for h o . an active cys on one prdx subunit forms a disulphide bond with the other subunit in the presence of h o (könig et al., ; peskin et al., ) . prdx appears to modulate growth factors and the tumor necrosis factor-alpha-mediated signaling pathway via modulating endogenous h o signaling (zhao & wang, ) . when prdx reacts with peroxide, the peroxidatic cysteine at the active site on one subunit is oxidized to a sulfenic acid . through the reduction process of disulphide by trx, prdx is regenerated and completes the cycle and trx is in turn regenerated by thioredoxin reductase (trxr), with reducing equivalents derived from nadph . prdx reduces hydrogen peroxide and alkyl hydroperoxides involved in cell protection and antiviral activity through the trx system (pillay, hofmeyr, & rohwer, ; sobotta et al., ; watanabe et al., ) . prdx acts as a h o signal receptor and transmitter in transcription factor redox regulation. prdx forms a redox relay with the transcription factor stat in which oxidative equivalents flow from prdx to stat , and the redox relay generates disulphide-linked stat oligomers with attenuated transcriptional activity. cytokine-induced stat signaling is accompanied by prdx and stat oxidation and is modulated by prdx expression levels (sobotta et al., ) . moon et al demonstrated that prdx inhibits general immune cell responsiveness, which may be regulated by scavenging low levels of reactive oxygen species (ros) (moon et al., ) . prdx also enhances the activation of plateletderived growth factor (pdgf) receptor and phospholipase cγ in pdgf signaling via the modulation of h o (choi et al., ) . prdx (mer- , sp , aop- ) is known as c-myc, mir- and mir- b target gene and is required for mitochondrial homoeostasis and neoplastic transformation (pablo et al., ; wonsey, zeller, & dang, ) . prdx contains a mitochondrial localization sequence, which is found exclusively in the mitochondrion, comprises % of the total mitochondrial matrix, and uses mitochondrial trx as the electron donor for its peroxidase activity (gourlay, bhella, kelly, price, & lindsay, ) . prdx has been known to eliminate approximately % of mitochondrial h o and plays a key role in mitochondrial redox regulation . the overexpression of prdx has been shown to protect cells from oxidative stress and apoptosis, and knockout studies show an increase in intracellular ros levels and oxidative stress (mukhopadhyay et al., ; nonn, berggren, & powis, ) . prdx protects radical-sensitive enzymes from oxidative damage by a radical-generating system, and acts synergistically with map k to regulate the activation of nf-kappa-b in the cytosol (lin et al., ) . prdx is involved in the regulation of cellular proliferation, differentiation and antioxidant functions that has been localized in the mitochondria (huh et al., ; song et al., ) . increased expression of prdx was found to be associated with hormonal receptors (estrogen receptor and progesterone receptor) (karihtala, mäntyniemi, kang, kinnula, & soini, ) . prdx (aoe , trank) is a ubiquitously expressed member of the peroxiredoxin family that is mainly localized in the endoplasmic reticulum (er), but is also present in the cytosol, lysosome, nucleus, or secreted (leyens, donnay, & knoops, ) . prdx plays an essential role in the redox balance in the er. the cysteine residue of prdx is first oxidized to sulfenic acid form and then forms intermolecular disulphide bond with another prdx molecule, which can be reversed by the reducing activity of the trx-trx reductase system (mehmeti, lortz, elsner, & lenzen, ; tavender & bulleid, ) . under oxidative stress conditions, however, the cysteine of prdx undergoes further oxidation to sulfinic/sulfonic acid forms which can only be reduced by sulfiredoxin (srx) (jeong, bae, toledano, & rhee, ) . the hyperoxidized form of prdx loses its antioxidant property but may function as molecular chaperone to facilitate protein folding zito et al., ) . prdx diminishes oxidative stress by reducing hydrogen peroxide to water in a thiol-dependent catalytic cycle (schulte, ) and has been linked to the regulation of the key pro-inflammatory transcription factor, nf-κb in the cytosol by a modulation of iκb-α phosphorylation (haridas et al., ; jin, chae, rhee, & jeang, ) . prdx is abundantly expressed in the pancreas, liver and heart, while the lowest expression in blood leukocytes and the brain (schulte, ) . prdx (acr , pmp or aoeb ) is an antioxidant enzyme implicated as a protective role against oxidative stress through reduction of hydrogen peroxide and alkyl hydroperoxides with reducing equivalents provided through the trx system and is involved in intracellular redox signaling (knoops, goemaere, van der eecken, & declercq, ) . prdx is a thiol peroxidase that reduces h o times faster than free cysteine that shows hydrogen bonds in the active sites (stephanie et al., ) . the prdx gene is mapped on chromosome on q . that displays mitochondrial presequence and cysteines involved in antioxidant activity with c-terminal peroxisomal sequence (knoops et al., ; nguyên-nhu et al., ) . prdx interacts with peroxisome receptor which uses translation initiation sites to produce mitochondrial forms. prdx acts as an antioxidant on different tissues under normal conditions and inflammatory processes through trx system. the analysis of the '-flanking region of human prdx revealed the presence of several putative response elements for transcription factors involved in the response of mammalian cells to oxidative stress such as nrf or nf-κb (kropotov, usmanova, serikov, zhivotovsky, & tomilin, ; nguyên-nhu et al., ) . prdx expression is highly increased in vitro in primary macrophages exposed to lipopolysaccharide (lps) and ifn-γ (abbas et al., ; diet et al., ) . downstream of the lps/tlr pathway, p and c-jun n-terminal kinase (jnk) were shown also to be major contributors to prdx up-regulation (diet et al., ) . prdx belongs to the ahpc/tsa family and contains trx domain (poole, hall, & nelson, ) . among the six mammalian members of prdx, prdx has a unique property as a bifunctional enzyme that has glutathione peroxidase and ipla activities (fisher, ) . prdx involved in redox regulation of the cell can reduce h o and short chain organic, fatty acid, and phospholipid hydroperoxides (fisher, ) . prdx plays a role in the regulation of phospholipid turnover as well as in protection against oxidative injury (wang, feinstein, manevich, ho, & fisher, ) . site directed mutation analysis has clearly shown that activities of prdx require two distinct active sites, namely, a cys -dependent peroxidase activity and a ser -dependent ipla activity (chen, dodia, feinstein, jain, & fisher, ) . ipla catalyzes the hydrolysis of the sn- fatty acyl ester bond of glycerophospholipides to produce free fatty acids and lysophospholipids (markova et al., ; shalini, chew, rajkumar, dawe, & ong, ) . prdx prefers phosphatidylcholines as substrates, particularly those with arachidonic acid (aa) or palmitic acid at the sn- position (chen et al., ; manevich & fisher, ) . the ability of prdx to release aa seems important, given the crucial role of aa in phospholipid metabolism and cell signaling (hooks & cummings, ) . prdx also protects cells via reducing peroxidazed membrane phospholipids and return to the cytosolic compartment (wang, feinstein, & fisher, ) . general properties of peroxiredoxin isoforms are briefly summarized in table , and the cellular localization and the roles of prdxs are in fig. . general regulatory pathway of prdx expression is in fig. . prdxs are regulated at multiple levels, including both transcriptional regulation and post-translational modifications (klotz et al., ; riquier et al., ) . prdx peroxidase activity is regulated by pin . pin facilitates the protein phosphatase a-mediated dephosphorylation of prdx , which helps to explain the accumulation of the inactive phosphorylated form of prdx in pin -/-mefs (chu et al., ) . prdx is silenced by a decreased histone h acetylation and dna hypermethylation in aml (agrawal singh et al., ) . a recent study also demonstrated a critical role for foxo a in regulating the gene expression of prdx in human cardiac fibroblasts (chiribau et al., ) . in the promoter of human prdx gene, the binding sites for both nuclear respiratory factor and nuclear respiratory factor have been predicted (kropotov et al., ) . increased transcription of prdx has been reported to be mediated by nrf via an antioxidant response elementdriven mechanism . the mapks can mediate phosphorylation of prdx at thr- with a consequent marked increase in its aipla activity . sp-a and prdx directly interact, which provides a mechanism for regulation of the aipla activity of prdx by sp-a . recent findings suggest that fkbp deficiency diminishes the threshold against os by reducing prdx levels (hirota et al., ) . a recent study also suggested that (phox) binds to phospho-prdx and inhibits its pla activity, an interaction that could function to terminate the pla -mediated nox activation signal (krishnaiah, dodia, feinstein, & fisher, ) . moreover, the expression of prdxs is induced by several factors. prdx is also detected in mouse body fluids, and the secretion of prdx from cultured cells was enhanced following the treatment with cytokines such as tgf-β, oncostatin m and il -β (ishii, warabi, & yanagawa, ). another recent study showed prdx and prdx can be secreted from cells as disulphide homodimers in association with exosomes following stimulation by lps or tnf-α (mullen, hanschmann, lillig, herzenberg, & ghezzi, ) . prdx is up-regulated in cells under oxidative stress conditions. the transcription factor nrf and its inhibitor keap play an essential role in the regulation of the stress-induced prdx gene activation through the antioxidant/electrophile response element (are/ epre) (takeuchi et al., ) . the expression levels of prdx and prdx are also up-regulated under oxidative stress conditions (stresing et al., ) . prdx is an oxidative stress inducible protein regulated by nrf and activation of the gene expression was observed in mouse lungs by hyperoxia and in cultured lung epithelial cells following treatment with h o or paraquat . in addition to oxidative stress, keratinocyte growth factor also regulates prdx gene expression (chowdhury et al., ) . prdx expression is down regulated upon serum deprivation and subsequently induced in a time-dependent manner in response to kgf, dexamethasone and h o (gallagher & phelan, ) . tnf-α regulates the expression of prdx to influence neuroplasticity . diseases involved in abnormal inflammatory and metabolic processes, such as cardiovascular dysfunction, cancer, diabetes mellitus and neurodegeneration, often involve abnormal control of ros (rahman, ) . the hyperproliferative property of cancer cells is known to be associated with increased production of intracellular ros (cerutti, ) . also, cancer cells contain large numbers of abnormal mitochondria and produce large amounts of ros (song et al., ) . in cancer cells, key mitochondrial regulators of cell death and other processes are often altered (gogvadze, orrenius, & zhivotovsky, ) . mitochondria of cancer cells differ structurally and functionally from their normal-cell counterparts (gogvadze et al., ; modica-napolitano & singh, ) . increased mitochondrial ros generation and the disturbance of prdx production in cancer cells may lead to oxidative stress and the induction of apoptosis (song et al., ). the prdx system is a cellular defense system against oxidative stress (powers & jackson, ) . as prdxs are antioxidants, they scavenge the h o in cancer cells and they support survival and tumor maintenance by protecting cells from oxidative stress-induced apoptosis (chatterjee et al., ) . several studies have shown that overexpression of prdxs inhibits the development of cancer or promotes growth of cancers. in this review, we divided the role of prdxs by benefit and non-benefit roles in several cancers. the relationship between cancer development and prdxs is summarized in fig. . cellular localization and roles of prdxs. tyrosine residue of prdx is phosphorylated, which causes the transient inactivation of peroxidase activity. as a result, a local accumulation of h o occurs and sustains phosphorylation signaling from tyrosine kinase for the growth factor receptor via the oxidative inactivation of phosphotyrosine phosphatases. consequently the cell growth proceeds for a prolonged period as the result of the elevated h o during the inactivation of prdx . prdx is localized in the cytosol and scavenges ros in the cytosol and blocks bax mediated cell death by inhibition of cellular apoptosis. prdx is localized in the mitochondria. prdx reduces h o level and protects cells from oxidative damages. prdx possesses a hydrophobic n-terminal signal peptide that leads to its secretion from cells and predominant localization in the er. prdx reduces h o and alkyl hydroperoxides that acts as antioxidant on different tissues under normal conditions and inflammatory processes through trx system in peroxisomes. prdx involved in redox regulation of the cell and can reduce h o and short chain organic, fatty acid, and phospholipid hydroperoxides. prdx plays a role in the regulation of phospholipid turnover as well as in the protection against oxidative injury. first, the anti-tumor effect of prdx is well established in breast cancers. several evidences suggest that prdx may act as a tumor suppressor in breast cancer. prdx interacts with the c-myc oncogene and suppresses its transcriptional activity (egler et al., ) . cao et al. demonstrated that prdx protects the tumor suppressive function of pten phosphatase, likely due to the presence of a ros sensitive cysteine in the catalytic domain, and reduces predisposition of genetically modified mice to develop ras-induced mammary tumors ). prdx -deficient mice suffer from shortened survival due to the development of hemolytic anemia and multiple tumors, including mammary carcinomas (neumann et al., ) . these studies suggest that the breast tumor suppressive role of prdx is mediated via c-myc or pten pathways. next, the tumor promoting effect of prdx is well established in breast cancer, oral squamous cell carcinoma, bladder cancer, lung cancer, prostate cancer, hepatocellular carcinoma, esophageal squamous cell carcinoma and pancreatic cancer. the role of prdx in breast cancer has been controversial. recent studies have shown that overexpression of prdx mrna in human breast carcinoma is associated with higher tumor grade (cha, suh, & kim, ) , and high expression of cytoplasmic prdx protein correlated with an increased risk of local recurrence after radiotherapy (woolston, storr, ellis, morgan, & martin, ) . prdx enhances p -mediated cyclooxygenase (cox)- gene expression in estrogen receptor (er) deficient human breast cancer cells (mda-mb- ), and knockdown of prdx can attenuate cox- expression by reducing the occupancy of nf-κb transactivation potential of nf-κb in er-deficient-breast cancer cells . prdx is also overexpressed in oral squamous cell carcinoma (yanagawa et al., ) . a recent study provides experimental evidence that overexpression of prdx is involved in oral carcinogenesis by upregulation of hemeoxygenase and activation of nf-κb pathway . prdx is also related with bladder cancer. prdx is highly expressed in bladder cancer tissues than in the bladder mucosa of normal controls or in normal mucosa surrounding cancer tissues (quan et al., ) . jiang et al showed that prdx silencing by transfection of prdx shrna significantly suppressed growth, promoted apoptosis and regulated the cell cycle in bladder cancer cells and reduced the phospho-nf-κb p and p protein expression suggesting that prdx is related with bladder cancers by regulation of phosphorylation of nf-κb subunit (jiang et al., a, b) . prdx is also related with lung cancer. abnormal up-regulation of prdx by environmental redox change leads to lung cancer progression and radiotherapy resistance by controlling critical molecules . another recent study suggested that down-regulation of prdx significantly inhibited tumor growth and reduced the incidence of spontaneous pulmonary metastasis (chen et al., ) . prdx provides resistance to docetaxel treatment through the suppression of foxo -induced apoptosis in a xenograft tumors, suggesting prdx may be an attractive target in the development of more effective docetaxel-based therapies in lung cancer treatment (hwang et al., ) . prdx is also related with prostate cancer. prdx interacts with the androgen receptor and enhances its transactivation that plays a critical role in prostate cancer . one previous study reported that hypoxia increases ar activity in prostate cancer cells, and that prdx , which is upregulated by hypoxia, interacts with ar to enhance the expression of androgen-regulated genes (chhipa, lee, onate, wu, & ip, ; park et al., ) . prdx is highly expressed in hepatocellular carcinoma. fig. . general regulatory pathway of prdx expression. nrf induces the expression of prdxs in response to oxidative and electrophilic stresses. under unstressed conditions, nrf is degraded via the ubiquitin (ub)-proteasome pathway in a keap -dependent manner. the keap homodimer binds a single nrf molecule through two-site binding utilizing the dlg and etge motifs. when nrf inducers inactivate keap via the modification of cysteine residues (cys) under oxidative stress and electrophiles modified condition, nrf is stabilized, and de novo synthesized nrf translocates into the nucleus. nrf heterodimerizes with small maf proteins (smaf) and activates the expression of target genes including prdxs through antioxidant response elements (ares), exerting cytoprotective effects against various diseases and toxic insults. phosphorylation of nrf by various kinases has also been implicated in the liberation, stability, and trans-activation of nrf . prdx -positive rate was significantly higher in hepatocellular carcinoma than in adjacent non-tumorous liver tissues (sun et al., ) . actually, prdx immunoreactivity was positively correlated with tumor vascular endothelial growth factor (vegf) expression and microvessel density (sun et al., ) . prdx expression was significantly associated with tumor size, microvascular invasion, edmondson grade, tumor capsula status, serum afp, and tumor-node-metastasis stage (sun et al., ) . prdx is involved in esophageal squamous cell carcinoma tumorigenesis through regulation of the mtor/p s k pathway (gong et al., ) . prdx is also significantly increased in pancreatic cancer by upregulation of angiogenesis (cai et al., ) . these studies indicate that prdx has a tumor promoting effect through nf-κb pathway, foxo mediated pathway and mtor/p s k pathway in breast cancer, oral squamous cell carcinoma, bladder cancer, lung cancer, prostate cancer, hepatocellular carcinoma, esophageal squamous cell carcinoma and pancreatic cancer, which suggest that prdx is a potential therapeutic target in these cancer types. the tumor promoting effect of prdx is well established in colorectal carcinoma, prostate cancer, breast cancer and cervical cancer. prdx is overexpressed in colorectal carcinoma tissues compared with the matched non-cancer colorectal mucosa tissues and that expression is positively associated with tumor metastasis and the tnm stage by regulation of oxidation induced apoptosis (lu et al., a) . other researchers also demonstrated that prdx knockdown using a lentiviral vector-mediated specific shrna inhibited cell growth, stimulated apoptosis, and augmented the production of endogenous ros that led to an altered expression of proteins associated with the wnt signaling pathway (lu et al., b) . prdx is also important in prostate cancer. shiota et al demonstrated that the expression of prdx is most significantly elevated among the prdx family in prostate cancer (shiota et al., ) . they showed that prdx in the nucleus and cytoplasm distinctively regulated ar activity and is involved in the proliferation of arexpressing prostate cancer cells and in the progression to castrationresistant prostate cancer, suggesting prdx to be a key factor in prostate carcinogenesis and in the progression to castration-resistant prostate cancer (shiota et al., ) . prdx is highly inducible by therapeutic radiation and drugs in breast cancer cells (wang, diaz, & yen, b; wang, li, liu, hong, & zhang, a) . several resistant tumor cells have shown upregulated prdx levels in breast cancer. prdx plays a role in protecting the cell membrane from ir-induced oxidative radioresistant breast cancer cells (diaz, tamae, yen, li, & wang, ) . prdx is also significantly upregulated in the early diagnosis of hepatitis b virus (hbv) related fibrosis (lu et al., ) . prdx is markedly upregulated in neutrophils of refractory cytopenia with multilineage dysplasia (rcmd) patients compared to healthy donors. in addition, white blood cell and neutrophil counts in rcmd patients correlated inversely with prdx expression (kazama et al., ) . among prdxs, prdx specifically regulates breast cancer cell colonization in lungs by acting on the oxidative and metabolic stress responses of metastatic cells (stresing et al., ) . prdx has a strong pattern of increased expression with increasing severity of the lesion, thus prdx is up-regulated in response to cervical cancer development . these data indicate that prdx is involved in the proliferation or metastasis of cancer, involved in radio-and drug-resistance, and suggest that designing therapeutics targeting prdx may offer a novel strategy for developing treatment for cancer. the tumor promoting effect of prdx is well established in breast cancer, prostate cancer, cervical cancer, hepatocellular carcinoma and ovarian cancer. in a gene silencing study in breast cancer cells, silencing the prdx gene inhibits cell proliferation and induces cell cycle arrest in breast cancers (chua et al., ) . prdx is also involved in prostate cancer. prdx is upregulated in endocrine-regulated tumors and in particular prostatic intraepithelial neoplasia and prostate cancer (whitaker et al., ) . also, the prdx protein is significantly upregulated in antiandrogen-resistant prostate cancer cell lines, resulting in an increased resistance to oxidative stress and failure to activate proapoptotic pathways (whitaker et al., ) . antiandrogen-resistant prostate cancer cells also possess an upregulation of the tricarboxylic acid (tca) pathway and resistance to h o -induced apoptosis through a failure to activate pro-apoptotic pathways, but knockdown of prdx restored h o sensitivity (whitaker et al., ) . prdx is upregulated in response to the development of cervical cancer. the expression of prdx is increased in cervical carcinoma and the pattern of prdx expression in cervical carcinoma was consistent with that of cell proliferation marker, ki (hu, gao, & li, ) . nonn et al reported that prdx played a drug-resistant role against drug-induced oxidative stress and subsequent apoptosis of thymoma cells (nonn et al., ) . prdx is also involved in hepatocellular carcinoma development. the overexpression of prdx is associated with . % hepatocellular carcinoma, and correlated with poor differentiation (qiao et al., ) . also, prdx is resistant to oxidation-induced apoptosis of hep- b cells (wang et al., a, b) . after a low-dose of h o treatment, the ros level was significantly higher in prdx -knockdown hep- b cells than in controls. in addition, prdx down-regulation resulted in decreased proliferation, increased apoptosis, and increased caspase activity of hep- b cells (wang et al., a, b) . the prdx is also overexpressed in ovarian cancer. downregulation of prdx upregulated proapoptotic proteins bax, caspase and caspase , and the silencing of prdx triggered cisplatin mediated apoptosis in the ovarian cancer cells through suppression of the nf-κb signaling pathway (duan et al., ) . these data indicate that prdx is accordingly up-regulated in cancer cells to remove cellular ros and inhibit apoptosis, which provides a favorable microenvironment for cell proliferation. therefore, prdx can be a therapeutic target for several cancer treatments. the tumor promoting effect of prdx is well established in lung cancer, leukemia, glioblastoma, oral squamous cell carcinoma, cardiovascular disease, hepatic diseases and colorectal carcinoma. prdx is associated with lung cancer. wei et al demonstrated that prdx along with srx plays a very important role in tumor progression and metastasis in lung cancer (wei et al., ) . wei et al. showed that srx preferentially binds to prdx and the srx-prdx axis contributes significantly to the maintenance of lung tumor phenotype in vitro and the formation of metastases in vivo and also they suggested the srx-prdx axis in activation of intracellular phosphokinase signaling including ap- /mmp- axis and mapk signaling (wei et al., ) . the expression of prdx is at least . fold higher in tumor cells compared to control and this finding applies most frequently to adenocarcinoma and modestly to squamous cell carcinoma (lehtonen et al., ) . alteration in expression of prdx results in an alteration to the rate of tumor progression and metastasis which is indicated by anchorage independent colony formation, cell migration and invasion of human lung cancer cells . the ability of prdx to promote tumor progression and metastasis is supposed to be due to its antioxidant properties (dando et al., ) . alteration of prdx expression is proposed to play a role in the development of different types of leukemia (palande et al., ) . palande et al. found that the alteration in genomic sequence and expression level of prdx is rare in acute myeloid leukemia but have found strong reduction in prdx expression in acute promyelocytic leukemia (palande et al., ) . also, it was suggested that due to the alteration in prdx expression, the signal transduction from a myeloid growth factor receptor i.e. the granulocyte colony stimulating factor receptor is affected (palande et al., ) . in acute myeloid leukemia (aml) patients, the prdx gene is fused with the aml gene between exon and of aml and exon of prdx (zhang et al., ) . this fusion of aml gene with the prdx gene is supposed to play a role in the altered expression of prdx in acute myeloid leukemia (zhang et al., ) . ho et al., ; jo et al., ; yun et al., a yun et al., , b yun et al., c yun et al., , b yun et al., , a prdx is supposed to play a role in the most aggressive primary brain malignancy . they found that the knockdown of prdx results in reduced glioblastoma multiform cell growth and radiation resistance along with increased ros level, dna damage and apoptosis in in-vitro model, suggesting the importance of prdx in radiation resistance and tumor maintenance of gbm . prdx is also studied for its role in tumor progression, cell migration and invasiveness in oral cavity squamous cell carcinoma (chang et al., a, b) . along with the prognostic value of prdx , prdx can also be a good therapeutic target in oscc by virtue of its ability to mediate cell migration and/or metastasis (liao et al., ) . also, prdx is secreted into an extracellular environment, therefore, its plasma concentration may be used as a molecular indicator of various cardiovascular disease and other disorders involving oxidative stress (schulte, ) . the increased serum prdx concentration is considered as a good indicator of risk to cardiovascular disease because cardiovascular disease have higher level of oxidative stress and prdx is overexpressed in these conditions (abbasi et al., ) . a study in a rat model of wilson's disease has demonstrated that this disease has a lower level of prdx expression as compared to normal (ito et al., ) . prdx can protect the hepatic tissue against the hydrogen peroxide as well as other ros causing oxidative stress. the same study has proposed that prdx can be used as a potential biomarker of hepatic diseases as the prdx serum concentration in this model was found to be quite low. genomic loss of prdx in mice results in testicular atrophy due to elevated spermatogenic cell death (iuchi et al., ) . prdx is also related with colorectal carcinoma. the expressions of the prdx gene and protein were higher in colorectal carcinoma compared to those in the adjacent normal tissues and the expression intensity of the prdx protein was correlated with depth of invasion and lymph node metastasis in crc, and high prdx expression was correlated with short survival time (yi et al., ) . these studies suggest that prdx is involved in the tumor promoting effect through formation of srx-prdx via ap- /mmp- and mapk signaling, and remove cellular ros which provides a favorable microenvironment for cell proliferation. hence, prdx can be a therapeutic target for several cancer treatments. the anti-tumor effect of prdx is well established in breast cancer. recent data showed that of the various prdx family members, prdx was the only one that was significantly downregulated in tumor samples obtained from sudanese breast cancer patients (elamin, zhu, hassan, xu, & ibrahim, ) . the tumor promoting effect of prdx is established in graves' disease. prdx expression is higher in the thyroid gland of patients with graves' disease compared to multinodular goiters, and the level of expression is directly correlated with the functional status of epithelial cells, being higher in multinodular goiters, and even more pronounced in hyperthyroid tissues, such as graves' disease (gérard, many, daumerie, knoops, & colin, ) . wang et al showed that prdx expression is increased in a degenerative tendon compared with normal tendon (wang et al., ) . they showed that while prdx was localized to fibroblasts in a normal tendon, it was localized to fibroblasts and endothelial cells in a degenerative tendon (wang et al., ) . these data indicate that prdx may inhibit breast cancers, but promotes graves' disease. the tumor promoting effect of prdx is well established in lung cancer, ovarian cancer, liver cancer and gastric cancer. prdx has been found at higher levels in lung squamous cell carcinoma patients compared with healthy controls (zhang et al., ) . prdx is elevated in several lung diseases including lung cancer, mesothelioma and sarcoidosis (yun et al., a, b) . invasion-and metastasis-promoting actions of prdx have been found in lung cancer cells (abbas et al., ) . the evidence indicates that the activity of prdx contributes to the invasion, promotion, and metastatic ability of lung cancer cells (ho et al., ) . we previously found that ipla activity of prdx is critical for lung tumor development in in vivo allograft (yun et al., a (yun et al., , b . our recent study demonstrated that prdx promotes tumor development via the jak /stat pathway in a urethane-induced lung tumor model (yun et al., a (yun et al., , b (yun et al., , c . also, prdx promotes lung tumor progression via its gpx and ipla activities (yun et al., a (yun et al., , b . in a recent study, the overexpression of prdx attenuated cisplatin-induced apoptosis in human ovarian cancer cells, whereas the reduction of prdx expression increased cell death in liver cancer cells (pak et al., ) . prdx is highly upregulated in metastatic gastric cancer cells, which are relatively resistant to trail as compared with primary cancer cells (choi, chang, & jung, ) . overexpression of prdx leads to a more invasive phenotype and metastatic potential in human breast cancer through regulation of the levels of upar, est- , mmp- , rhoc and timp- expression (chang et al., ) . prdx phosphorylation and subsequent phospholipase a activity are required for agonist-mediated activation of nadph oxidase in mouse pulmonary microvascular endothelium and alveolar macrophages (chatterjee et al., ) . these studies, as well as our own, suggest that prdx promotes lung tumor development via jak /stat pathway, its gpx and ipla activities, and is involved in drug resistance of several cancer types, suggesting that prdx can be a therapeutic target for cancer treatment prdx is overexpressed or downregulated in the tissue of various cancer patients. cases exhibiting a low prdx expression level included significantly larger tumor mass cases (p b . ), positive lymph node metastasis (pb . ), advanced stage (pb . ), and poorly differentiated cells (pb . ) in oral squamous cell carcinoma (yanagawa et al., ) . prdx is highly expressed in human bladder cancer tissues than in the bladder mucosa of normal controls or in normal mucosa surrounding cancer tissues which correlates with development, recurrence and progression of bladder cancer (quan et al., ) . sun et al reported that prdx positive rate was significantly higher ( . %) in human hepatocellular carcinoma than in adjacent non-tumorous liver tissues breast cancer tumor promoting effect promotes the metastasis -prdx leads to a more invasive phenotype and metastatic potential in human breast cancer through regulation of the level of upar, est- , mmp- , rhoc and timp- expression. chang et al., ( . %) (sun et al., ) . prdx immunoreactivity was positively correlated with vegf expression and microvessel density and prdx expression was significantly associated with tumor size, microvascular invasion, edmondson grade, tumor capsula status, serum afp, and tumor-node-metastasis stage (sun et al., ) . prdx is upregulated in human colorectal carcinoma tissues compared with the matched non cancer colorectal mucosa tissues (lu et al., a) . prdx is markedly upregulated in neutrophils of refractory cytopenia with rcmd patients compared to healthy donors. in addition, white blood cell and neutrophil counts in rcmd patients correlated inversely with the prdx expression (kazama et al., ) . ihc revealed significant overexpression of prdx in prostate cancer, associated with age, increased prostate specific antigen (psa), tumor stage, or gleason score, and high prdx staining was associated with early age and elevated gleason score at time of radical prostatectomy in african-american patients but not in caucasian patients with prostate cancer (basu et al., ) . prdx is upregulated in human endocrine-regulated tumours and in particular prostatic intraepithelial neoplasia and prostate cancer (whitaker et al., ) . the overexpression of prdx is associated with . % hepatocellular carcinoma, and correlated with poor differentiation (qiao et al., ) . the platinumresistant ovarian cancer patient group had significantly higher prdx protein compared to the platinum-sensitive ovarian cancer patient group, suggesting prdx may be associated with drug resistance in ovarian cancer . the expression of prdx is at least . fold higher in human tumor cells compared to control and this finding applies most frequently to adenocarcinoma and modestly to squamous cell carcinoma (lehtonen et al., ) . prdx expression is elevated in human lung carcinomas compared to nonmalignant tissue, and is mainly associated with adenocarcinoma . prdx is also related with colorectal carcinoma. the expressions of the prdx gene and protein were higher in crc compared to those in the adjacent normal tissues (yi et al., ) . the expression intensity of the prdx protein was correlated with depth of invasion, lymph node metastasis and dukes' classification in crc (yi et al., ) . prdx expression in bladder cancer was significantly higher than in normal tissue, as well as corresponding normal bladder mucosa surrounding the cancer (quan et al., ) . a recent study demonstrated that prdx expression was higher in the thyroid gland of patients with graves' disease compared to multinodular goiters (gérard et al., ) . prdx has also been found at higher levels in lung squamous cell carcinoma patients compared with healthy controls (zhang et al., ) . our recent study also showed that the prdx is overexpressed in lung cancer patients (yun et al., a (yun et al., , b (yun et al., , c . overexpression of prdx leads to a more invasive phenotype and metastatic potential in human breast cancer (chang et al., ) . prdxs are involved in several cancer related signal pathways. several studies suggested that nf-κb is related with redox signaling pathway. during redox signaling, some prdxs have been implicated in the regulation of nf-κb through the initial activation in the cytoplasm by controlling the components affecting iκb phosphorylation and subsequent dissociation. in principle, prdxs could have a different function in the nucleus because nf-κb interactions with dna are governed by a redox-sensitive cysteine (cys ) on the p subunit of the nf-κb dimer. oxidation of cys inhibits nf-κb binding and decreases the effectiveness of nf-κb signaling. also, some prdxs have also been implicated in the regulation of mapk pathway during redox signaling. although inhibiting prdx appears to have different effects on mapk activation depending on the cell or stimuli, in some cases the use of different stimuli, or time-points at which mapk activation is assessed, make comparisons between studies difficult. in any case, the consequence of inhibiting prdx in any given cell is likely to be determined by the levels of intracellular ros and intrinsic features of the cell/compartment, such as the capacity to regenerate reduced prdx, using srx, or trx using trxr and nadph. besides these pathways, prdxs also are involved in several cancer related pathways. fig. illustrates the roles of prdxs in the signaling pathways for the development of cancers. it was shown that overexpression of prdx and prdx suppresses pdgf-and egf-dependent h o production as well as tnf-α-induced nf-κb transcriptional activity (kang et al., ) . similar studies have since followed using other ligands, such as thyrotropin (tsh), tnf-α, and tnf-related apoptosis-inducing ligand (trail), as well as further work on egf dependent h o production, in various cell types (benvenga & koch, ) . collectively, these studies show that -cys prdxs not only eliminate the levels of intracellular h o that were increased upon receptor stimulation, but also suppress the downstream signaling responses, including nf-κb transcriptional activity, jnk activity and apoptosis (yang et al., ) . prdx is involved in differential regulation of different map kinases. under conditions of tnf-α stimulation in which prdx activity was either partially blocked using a dominant negative mutant version or was completely abolished by gene knockout, jnk and p map kinase activation were enhanced whereas the activation of extracellular signal related kinase (erk) was suppressed (hanschmann, godoy, berndt, hudemann, & lillig, ) . it was asserted that the endogenous h o regulated by prdx targets the phosphorylation of the tyrosines at sites / and in the pdgf receptor, but has no effect on any of the other tyrosine sites that are known to be phosphorylated, whereas exogenously added h o was shown to induce the phosphorylation of all possible tyrosine sites (choi et al., ) . it was also shown that the site selective response is controlled solely by prdx and not by other peroxidases, and is achieved through the inactivation of the membrane-associated protein tyrosine phosphatases (ptps) . xi et al reported that the protein levels of prdx are significantly reduced in vhl-deficient clear cell renal cell carcinoma (ccrcc) (xi et al., ) . furthermore, stabilization of hif- α protein, caused either by vhl deficiency under normoxia, or by hypoxia, significantly reduced prdx expression. luciferase-reporter and chromatinimmunoprecipitation assays indicated that hif- α binds to the hypoxia-responsive elements of prdx promoter and represses its transcription. finally, shrna-based assays suggested that prdx downregulation is required for the hif- α-dependent proliferation of ccrcc cells (xi et al., ) . ovarian cancer cells transfected with prdx /small interfering (si) efficiently downregulated the expression of prdx and thus decreased the growth of the ovarian cancer cells in vitro and in vivo. furthermore, the silencing of prdx triggered cisplatin mediated apoptosis in the ovarian cancer cells, which may act through suppression of the nf κb signaling pathway (song et al., ) . in antiandrogen-resistant lncap cell lines, prdx is upregulated and the resistant cells also possess an upregulation of the tca pathway and resistance to h₂o₂-induced apoptosis through a failure to activate pro-apoptotic pathways. also, knockdown of prdx restored h₂o₂ sensitivity, and prdx has been identified as a gene induced by oncogenic c-myc (whitaker et al., ) . its specific localization to mitochondria suggests that prdx , together with its mitochondrion-specific electron suppliers trx and trx reductase (trxr) , might provide a primary line of defense against h o produced by the mitochondrial respiratory chain, as sod does against the superoxide radical. in addition, srx plays a crucial role by reducing hyperoxidized prdx via translocation into mitochondria. noh et al. reported that the overexpression of mitochondrion-targeted srx efficiently promotes the restoration of prdx and results in cellular resistance to apoptosis, with enhanced elimination of mitochondrial h o and decreased rates of ΔΨm collapse (noh, baek, jeong, rhee, & chang, ). thus, a trx-related antioxidant system composed of trx , trxr , and prdx has been closely associated with the regulation of apoptosis and the redox control of mpt pores for the release of cytochrome c (noh et al., ) . prdx is associated with lung cancer. srx preferentially binds to prdx and the srx-prdx axis promotes human lung cancer progression through modulation of receptor tyrosine kinase related signaling (wei et al., ) . the same study has also shown the role of srx-prdx axis in activation of intracellular phosphokinase signaling including ap- /mmp- axis and mapk signaling (wei et al., ) . also, the srx-prdx plays a very important role in tumor progression and metastasis in lung cancer. wei et al demonstrated that an alteration in the expression of prdx results in an alteration in the rate of tumor progression and metastasis which is indicated by anchorage independent colony formation, cell migration and invasion of human lung cancer cells (wei et al., ) . this ability of prdx to promote tumor progression and metastasis is supposedly due to its antioxidant properties. analysis of the promoter region of the prdx gene and reporter gene assays revealed the promoter region critical for prdx gene regulation to which the novel negative transcription regulator, gata binds in human breast cancer cell lines (seo, liu, chang, & park, ) . they showed that knockdown of gata led to an increased expression of prdx and inhibition of apoptosis, suggesting that prdx may protect cells from oxidative stress-mediated apoptosis in a gata -regulated manner. in a recent study, overexpression of prdx attenuates cisplatininduced apoptosis in human ovarian cancer cells (pak et al., ) . pak et al showed that cisplatin-induced cytotoxicity in ovarian cancer cells cytotoxicity was associated with increased accumulation of intracellular (ros) and apoptosis mediated by proteolytically activated caspase and . moreover, overexpression of prdx protein or exposure to n-acetylcysteine (nac) reversed the apoptotic effect of cisplatin by reducing ros levels and suppressing the caspase signaling pathway. in contrast, reduction of prdx expression increased peroxide-induced cell death in liver cancer cells (walsh et al., ) . they showed that the cancerous hepatoma cell line is significantly more resistant to peroxideinduced cytotoxicity than the non-cancerous cell line, and hepatoma cell line expresses approximately -fold prdx than non-cancerous cell line. also, they showed that transient transfection of cancerous hepatoma cell line with prdx sirna led to an increase in peroxideinduced cytotoxicity by apoptosis. the invasion-promoting action of prdx has also been confirmed using lung cancer cells, in which upregulation of prdx results in the activation of akt via phosphoinositide kinase (pi k) and p kinase, which promotes cell invasion by inducing urokinase-type plasminogen activator (upa) (ho et al., ; seung et al., ). our study demonstrated that prdx promoted lung tumor growth in an in vivo allograft model. histopathological and western blotting examination demonstrated that expression of proliferating cell nuclear antigen, vegf, mmp- and - , and cyclin-dependent kinases accompanied by increased ipla and gpx activities were increased in the tumor tissues of prdx -overexpressing nude mice (jo et al., ) . in tumor tissues of prdx -overexpressing mice, the activation of mitogen-activated protein kinases and ap- dna binding were also increased (yun et al., a (yun et al., , b . and the growth of lung cancer cell lines (a and nci-h ) was enhanced by the increase in ipla and gpx activities of prdx . in addition, mutant prdx (c s) fig. . signaling pathway related with cancer development. prdx enhances the transactivation potential of nf-κb in er-deficient breast cancer cells and upregulates phosphorylation of nf-κb subunit in bladder cancers. prdx promotes tumorigenesis of esophageal squamous cell carcinoma through regulating the activity of mtor/p s k pathway. prdx is involved in the regulation of wnt/β-catenin signaling in colorectal cancer cells. specifically, prdx inhibits gsk- β activity, enhances β-catenin translocation into the nucleus, and inhibits the levels of β-catenin phosphorylation, thus resulting in significant up-regulation of transcription of the lef/tcf target genes c-myc and survivin. prdx is associated with nf-κb signaling pathway. prdx enhances i-κb phosphorylation and induces nf-κb signaling pathway. prdx also acts synergistically with map k to regulate the activation of nf-κb in the cytosol. prdx binds with srx and the srx-prdx axis contributes to the maintenance of lung tumor phenotype by ap- and mapk signaling pathway. prdx promotes tumor development via the jak /stat pathway in a urethane-induced lung tumor model. upregulation of prdx results in the activation of akt via pi k and p kinase. attenuated prdx -mediated p , erk / , and ap- activities as well as its enzyme activities in the a and nci-h lines. furthermore, tumor growth and p , erk / , and ap- activities were also inhibited in nude mice bearing mutant prdx (c s) compared to prdx . previously, we showed that urethane ( g/kg)-induced tumor incidence in prdx -tg mice was significantly higher compared to non-tg mice (yun et al., a (yun et al., , b (yun et al., , c . in the tumors of prdx -tg mice, the activation of jak /stat and stat dna binding were also increased, accompanied by increased gpx and ipla activities. prdx was colocalized with jak in tumor tissues and lung cancer cells and also showed physical interaction with jak , and increased levels of prdx increase the activation of the jak /stat pathway. furthermore, prdx -tg mice showed altered cytokine levels in the tumors, especially leading to increased ccl levels. we validated that the activation of jak was also decreased in lung tumors of ccr -/mice, and ccl increased the jak /stat pathway in the lung cancer cells, suggesting that prdx promotes lung tumor development via its mediated and ccl -associated activation of the jak /stat pathway. prdxs are differentially expressed in the brain regions depending on the subfamily. prdx and are expressed in glial cells, whereas prdx , , and are expressed in neurons (hanschmann et al., ) . these data indicate that the two prdxs have distinct functional roles in the brain and provide differential contributions to neuropathologic conditions. the expression patterns of prdxs are in fact highly variable in different regions of the brain during neurodegenerative disease processes (hanschmann et al., ) . given that oxidative damage is involved in the pathogenesis of neurodegenerative diseases, the alteration in prdx expression would appear to be primarily a consequence of cellular resistance to the oxidative damage (perez-pinzon, stetler, & fiskum, ) . several studies suggested that the prdxs are related with the development of neurodegenerative diseases or neuroprotective effects (perez-pinzon et al., ; soriano et al., ) . in this regard, we reviewed the roles of prdxs in the development of neurodegenerative diseases and neuroprotective effects. the relationship between neurodegenerative diseases and prdxs is summarized in table . the neuroprotective role of prdx is well established. prdx has a protective role in counteracting aβ injury by increasing cell viability, preserving neurites, and decreasing cell death (cimini et al., ) . the neurodegenerative role of prdx is also established in neuroblastoma cells . the neuroprotective role of prdx is well established in ad and pd. among the antioxidant prdx enzymes, prdx is the most abundant in mammalian neurons, making it a prime candidate to defend against oxidative stress (fang, nakamura, cho, gu, & lipton, ) . prdx is overexpressed in the brain of alzheimer's disease (ad) indicating that prdx protects the brain from aβ - induced neurotoxicity (yao et al., ) . another study also suggested that prdx exhibited anti apoptotic effects in da neurons via suppression of ask dependent activation of jnk and p pathways which are activated in pa neurons of pd brains (hu et al., ) . the neurodegenerative role of prdx is also established in ad and pd. the role of prdx in pd is controversial. garcia-garcia et al suggested that the oxidative modification of prdx is associated with drug induced apoptosis signaling in the model of parkinson disease (garcia-garcia, zavala-flores, rodriguez-rocha, & franco, ) . they suggested that the oxidative modification of prdx is involved in the regulation of -ohda-induced apoptotic signaling in da induced neuronal cell death. qu et al defined the role of prdx in pd and suggested that phosphorylation of prdx isoform is involved in pd (qu et al., ) . in a histochemical study of brains from ad patients, nitrated prdx was identified. randall et al. investigated the functional consequences of prdx tyrosine nitration, and demonstrated that nitration was on a non-catalytic residue that resulted in increases in peroxidase activity and resistance to over-oxidation (randall et al., ) . fang et al observed increased s-nitrosylation of prdx in human parkinson's disease (pd) brains, and s-nitrosylation of prdx inhibited both its enzymatic activity and protective function from oxidative stress (fang et al., ) . dopaminergic neurons, which are lost in pd, become particularly vulnerable, provided a direct link between nitrosative/oxidative stress and neurodegenerative disorders such as pd (fang et al., ) . these data suggest that not only is prdx involved in neuroprotection via inhibition of aβ - induced apoptosis and suppression of jnk and p pathways, but also involved in neurodegeneration via oxidative modification, phosphorylation and s-nitrosylation of prdx . the neuroprotective role of prdx is well established. prdx is involved in the protection of hippocampal neurons from excitotoxic injury. hattori et al showed that prdx is up-regulated in the mitochondria of damaged nerve cells (hattori, murayama, noshita, & oikawa, ) . because mitochondria is involved in excitotoxic damage of nerve cells, mitochondrial prdx seems to be neuroprotective against oxidative insults (hattori et al., ) . also, prdx is decreased in the frontal cortex of patients with down syndrome (pb . ) and pick's disease (pb . ) (krapfenbauer, engidawork, cairns, fountoulakis, & lubec, ) . the neurodegenerative role of prdx is established in pd. a recent study also suggested that the autosomal dominant mutation of lrrk that is important for pd, increased the phosphorylation of prdx and resulted in the oxidative stress induced neuronal death (angeles et al., ) . these data indicate that prdx is involved in the neuroprotection against oxidative insults in the mitochondria, and involved in neurodegeneration through increased phosphorylation by lrrk mutation. there are not many research reports in prdx and prdx . prdx is significantly increased only in pick's disease frontal cortex (krapfenbauer et al., ) . prdx is involved in pd through mitochondrial redox signaling with ca + influx resulting in the development of pd (davey & bolaños, ) . the neuroprotective role of prdx is established in an experimental autoimmune encephalomyelitis (eae) model. in the recent study, we also showed that the expression of an prdx is markedly increased in spinal cords of mice with eae compared to other prdxs. prdx transgenic mice displayed a significant decrease in clinical severity and attenuated demyelination in eae compared to wild type mice, suggesting that prdx expression may represent a therapeutic way to restrict inflammation in the central nervous system and potentiate oligodendrocyte survival, and suggest a new molecule for neuroprotective therapies in ms (yun et al., a (yun et al., , b (yun et al., , c . the neurodegenerative role of prdx is established in ad and pd. our recent study suggested that overexpression of prdx could accelerate the development of ad through increased amyloidogenesis through independent pla activation and nrf transcription . in a mouse model after amyloid beta infusion, memory impairment in prdx transgenic mice was worse than c bl/ mice. in addition, the astrocytes and microglia cells of amyloid-infused prdx transgenic mice were more activated, lipid peroxidation and protein carbonyl levels were increased and glutathione levels were lower, suggesting that prdx is promoting rather than preventing oxidative stress . our recent study also suggested that prdx exacerbates dopaminergic neurodegeneration in a mptp mouse model of pd (yun et al., a (yun et al., , b (yun et al., , c . these data indicate that prdx induces neurodegeneration through ipla and nrf activation in ad, and induction of mptpinduced dopaminergic neurodegeneration of pd, but inhibits multiple sclerosis by suppressing inflammation and blood brain barrier disruption in eae model. the trx system protects against h o -induced apoptosis, and the trx-prdx system detoxifies peroxides by transferring reducing equivalents from nadph to peroxides with cytoprotective and antioxidative effect (soriano et al., ) . several studies reported that prdx - are implicated in protecting neuronal cells from aβ toxicity (yao et al., ) , peroxide and -methyl- -phenylpyridinium (mpp(+)) toxicity (qu et al., ) , oxygen-glucose deprivation and excitotoxicity (hattori et al., ) . also, other prdxs are also involved in neurodegenerative diseases. fig. illustrates the roles of prdxs in the signaling pathways for the neurodegenerative diseases. overexpression of prdx protects against hydroxydopamine toxicity, prevents p mapk activation and subsequent activation of caspase- in mn d cells, but apoptotic death signals were enhanced by rna interference-targeted reduction of prdx . prdx is involved in neurodegeneration through phosphorylation by cdk , a serine/threonine kinase, and contributes to pd. in a recent study, qu et al. showed that cyclin-dependent kinase (cdk ) inhibits prdx activity, leading to increased levels of ros in pd models (qu et al., ) . they also demonstrated that prdx is a bona fide substrate for the cdk -p complex, which phosphorylated prdx at the threonine residue at position . cyclin-dependent kinase , a serine/threonine kinase, contributes to neurodegeneration in pd. this kinase is shown to phosphorylate prdx , inactivating it, and thereby sensitizing neurons to the deleterious effects of parkinsonian toxins. hu et al. explored the role of prdx and showed it inhibited hydroxydopamine-induced ask activation by modulating the redox state of trx preventing its dissociation from ask (hu et al., ) . they showed that lentivirus-mediated prdx overexpression conferred marked in vitro and in vivo neuroprotection against -ohda toxicity in da neurons, and preserved motor functions involving the dopamine system in mouse. in addition to its role as an antioxidant enzyme, prdx exhibited anti-apoptotic effects in da neurons via suppression of apoptosis signal-regulating kinase (ask )-dependent activation of the c-jun n-terminal kinase/c-jun and p pro-death pathways, which are also activated in da neurons of postmortem pd brains. prdx inhibited -ohda-induced ask activation by modulating the redox status of the endogenous ask inhibitor trx. prdx overexpression maintained trx in a reduced state by inhibiting the cysteine thioldisulphide exchange, thereby preventing its dissociation from ask . in cells expressing the lrrk mutant gene that is responsible for up to - % of pd cases in some ethnic populations, the phosphorylation of prdx is increased but decreased peroxidase activity and increased death in neuronal cells (angeles et al., ) . kim et al suggested that apoptosis was inhibited after amyloid beta treatment in pc cells overexpressing wild-type prdx , but not in cells that overexpressed the c s catalytic mutant. this indicates that the peroxidase activity of prdx protects pc cells from amyloidinduced neurotoxicity (kim et al., a, b) . our recent study also demonstrated that aβ - -induced memory impairment in prdx transgenic mice was worse than c bl/ mice, and the expression of amyloid precursor protein cleavage, c , β-site app-cleaving enzyme protects the brain prdx is overexpressed in the brain of ad patients indicating that prdx protects the brain from aβ - induced neurotoxicity cimini et al., pd neurodegenerative effect increase the risk of pd -prdx is involved in the regulation of -ohda-induced apoptosis signaling in da induced neuronal cell death -prdx phosphorylation is involved in pd -prdx promotes oxidative stress-induced neuronal cell death in pd hu et al., ; qu et al., ; fang et al., prdx increase the risk of pd prdx induces astrocytic activation followed by increased proinflammatory cytokines (tnf-α and il -β), -hne, and prdx hyperoxidation in primary cultured astrocytes. yun et al., c yun et al., , b yun et al., , a , inducible nitric oxide synthase, and cyclooxygenase- was greatly increased . in addition, the astrocytes and microglia cells of aβ-infused prdx transgenic mice were more activated, and aβ also significantly increased lipid peroxidation and protein carbonyl levels, but decreased glutathione levels. furthermore, we found that translocation of nuclear factor erythroid -related factor (nrf ) to the nucleus was increased in aβ-infused prdx transgenic mice. these results suggest that the overexpression of prdx could accelerate the development of ad through increased amyloidogenesis through independent pla activation and nrf transcription. we also recently investigated the effects of prdx on mptp-induced dopaminergic neurodegeneration using prdx tg mice (yun et al., a (yun et al., , b (yun et al., , c . prdx tg mice had a much higher loss of dopaminergic neurons by mptp administration compared to non-tg mice. there was also a much higher behavioral impairment and astrocyte activation in prdx tg mice. mptp-induced gpx activity was not different between prdx tg mice and non-tg mice, which is accompanied by hyperoxidation of prdx . intriguingly, the expression pattern of prdx showed similar distribution and co-localization with astrocytes, but not neurons in the mouse and human brain. furthermore, we demonstrated that ipla activity of prdx induced astrocytic activation followed by increased proinflammatory cytokines (tnf-α and il -β), -hne, and prdx hyperoxidation in primary cultured astrocytes. a recent study suggested the translational potential of blocking tlr /prdx signaling for the treatment of ischemic stroke (kuang et al., ) . they showed that ligustilide (lig) exerted antineuroinflammatory and neuroprotective effects against an ischemic insult, and showed that tlr /prdx pathway is involved in the protective effect of lig against postischemic neuroinflammation and brain injury induced by transient middle cerebral artery occlusion (mcao) in rats. also, lig-induced neuroprotection was accompanied by the improvement of neuropathological alterations, including neuron loss, astrocyte and microglia/macrophage activation, neutrophil and t-lymphocyte invasion, and regulation of inflammatory mediator expression. moreover, lig significantly inhibited the expression and extracellular release of prdx and activation of tlr signaling, reflected by decreased tlr expression, extracellular signal-regulated kinase / phosphorylation, and transcriptional activity of nf-κb and signal transducer and activator of transcription in the ischemic brain. redox-regulated transcription factors and protein kinases play important roles in the regulation of inflammation (adler, yin, tew, & ronai, ) . nf-κb is a well-characterized redox-sensitive transcription factor important in the initiation and progression of inflammation through the production of various cytokines and prostaglandins (kunsch & medford, ) . nrf is another redox-regulated transcription factor important for cellular defense against oxidative and electrophilic stress (trachootham et al., ) . nrf activates the expression of stress-dependent cytoprotective genes such as nad(p)h-quinone oxidoreductase and glutathione s-transferase (gst) through a cisacting element called the are/epre (kobayashi et al., ) . prdxs secreted from cells under mild oxidative stress binds tlr and prdxs induces nrf or nf-κb activation that is important for the expression of inflammatory proteins, suggesting that prdxs are involved in the inflammation related disease (madrigal-matute et al., ; westermann, knoblich, maier, lindschau, & haller, ) . parkinson's disease fig. . possible mechanisms of prdxs related with neurodegenerative disease. ros mediate neurotoxicity of ad through modifying the hallmark protein by oxidation. in ad, ros could also activate c-jun n-terminal kinases (jnk) and p , and deactivate protein phosphatase a (pp a). jnk and p promote the expression of tau, which is inhibited by pp a. the activation of jnk and p further stimulate app cleaving enzyme (bace ), causing aβ - accumulation, which leads to activation of nadph oxidase (nox) to produce additional o , and results in ca + influx to elicit excitatory neurotoxicity. prdx inhibits the generation and accumulation of aβ - , and protects the brain from ad neurotoxicity. prdx accelerates cell dysfunction and cell death proceeded from ros generation. in pd, the α-syn is aggregated and generates ros that also generated from ca + accumulation. prdx induces the ca + accumulation that can cause ros generation. prdx and prdx mediate the neurotoxicity through induction of cell dysfunction and cell death from ros accumulation. pd is also generated from lrrk mutation. this mutation causes prdx phosphorylation, after that ros accumulates, resulting in cellular dysfunction and cellular death. prdxs are related with several inflammatory diseases such as atherosclerosis, ra and obesity. the relationship between inflammatory diseases and prdxs is summarized in table . the beneficial role of prdx in inflammatory diseases is well established. in addition, prdx deficiency enhanced the regulated secretion pathway in endothelial cells by the promotion of excessive release of several proinflammatory components. kisucka et al examined the role of prdx in the apolipoprotein e-deficient (apoe -/-) murine spontaneous model of atherosclerosis (kisucka et al., ) . they showed that prdx -/-/apoe -/mice fed normal chow developed larger, more macrophage-rich aortic sinus lesions than prdx +/+ /apoe -/mice, despite similar amounts and size distributions of cholesterol in their plasma lipoproteins, suggesting that prdx protects against excessive endothelial activation and atherosclerosis. the role of prdx as non-beneficial in inflammatory diseases is also established. prdx is induced by exposing macrophages to oxidized ldl and by laminar shear stress in endothelial cells (conway & kinter, ; mowbray, kang, rhee, kang, & jo, ) . also, transcription factor nrf , one of the erythroid transcription factor nfe subunit factors responsible for the induction of defense proteins including prdx , is activated in oxidized ldl-treated macrophage cells, thus these data suggested that prdx is involved in macrophage activation (kisucka et al., ) . in the ra patient tissue, the prdx is overexpressed by more than fold compared with normal tissues . demasi et al suggested that prdx induction could be considered a crucial part of the cellular adaptive response to the b-cell differentiation process to cope with the additional h o associated with massive disulphide bond formation during immunoglobulin folding in the er of plasma cells (demasi et al., ) . these data indicate that prdx inhibits inflammatory disease by protection of excessive endothelial activation and atherosclerosis, but induces inflammatory disease via activation of nrf . the beneficial role of prdx in inflammatory diseases is well established. prdx , a thiol-specific peroxidase, has been reported to regulate proinflammatory responses, vascular remodeling, and global oxidative stress (park et al., ) . prdx removes transiently produced h o in response to activation of various cell surface receptors. prdx regulates platelet-derived growth factor (pdgf) signaling, including enhanced activation of the pdgf receptor and phospholipase cγ , and vascular remodeling, including pdgf-dependent neointimal thickening of vascular smooth muscle cells (choi et al., ) . yang et al showed that prdx inhibits general immune cell responsiveness by scavenging low levels of ros, modulating lps-induced proinflammatory responses, and protecting against endotoxin-induced lethal shock (yang et al., ) . a recent report showed that prdx is more susceptible than prdx to hyperoxidation in cells subjected to sustained global oxidative stress, which suggests that prdx deficiency may lead to accelerated atherosclerosis due to failure to eliminate ros . it has recently been shown that prdx suppresses the proliferation and migration of smooth muscle cells (smcs) through the siteselective phosphorylation of the pdgf receptor and, furthermore, is involved in the neointimal thickening of smcs in balloon-injured carotidartery (kang et al., ) . the role of prdx in inflammatory diseases as non-beneficial in inflammatory diseases is also established. in a recent study by bayer et al, prdx is increased in the presence of the neutrophils and under acidotic conditions and also oxidation of erythrocyte prdx is increased in a mouse model of endotoxemia (bayer, maghzal, stocker, hampton, & winterbourn, ) . another recent study also showed that the level of intracellular prdx protein content of lymphocytes from ra patients was more than -fold higher compared with healthy lymphocytes suggesting that prdx is involved in the persistence of pro-inflammatory cells in chronic inflammation (szabó-taylor et al., ) . also, prdx is readily detected in the blood of severe acute respiratory syndrome patients, whereas it is undetectable in normal control blood . recombinant prdx induces macrophages to produce tnf-α in the absence of any other stimuli, demonstrating that the trx peroxidase prdx is kisucka et al., ; mowbray et al., rheumatoid arthritis non-benefit effect enhance the risk of ra prdx is overexpressed in ra patient tissue by more than fold compared with normal tissues. li et al., prdx atherosclerosis benefit effect suppresses atherosclerosis prdx suppresses the proliferation and migration of smcs through the site-selective phosphorylation of the pdgf receptor and, furthermore, is involved in the neointimal thickening of smcs in balloon-injured carotidartery kang et al., rheumatoid arthritis non-benefit effect enhance the risk of ra prdx is involved in the persistence of pro-inflammatory cells in chronic inflammation because prdx protein content of lymphocytes from ra patient was more than -fold higher compared with healthy lymphocytes obesity benefit effect protects against atherosclerosis prdx -/macrophages oxidized ldl significantly more than did controls and plasma lipid hydroperoxide levels were higher in atherogenic diet-fed prdx -/mice than in controls. wang et al., prdx rheumatoid arthritis non-benefit effect enhance the risk of ra prdx promotes development of ra through activation of nf-κb/ap- coupled jnk pathway in the caia and aia-induced arthritis development model kim et al., appropriately classified as a danger signal, meaning a molecule that is released by cells undergoing stress and acts as an immune mediator (salzano et al., ) . these data suggest that prdx suppresses inflammatory diseases through inhibition of pdgf signaling, inhibition of immune cell responsiveness and elimination of ros, but induces inflammatory diseases through persistence of pro-inflammatory cells and mediation of tnf-α production. the beneficial role of prdx in inflammatory diseases is well established. prdx deficiency increased abnormal lipid accumulation in adipose tissue, due to increased ros (huh et al., ) . they suggested that endogenous prdx may play an essential role in maintaining normal characteristics of adipocytes and that a defect in prdx alters mitochondrial redox state and function, and adipokine expression in adipocytes leading to metabolic alteration (huh et al., ) . these data indicate that prdx protects the inflammatory diseases through elimination of lipid accumulation. the beneficial role of prdx in inflammatory diseases is well established. it was reported that hfd-induced hepatic steatosis and insulin resistance were prevented in prdx transgenic mice by amelioration of oxidative stress (nabeshima et al., ) . it has been suggested that streptozotocin-treated tg mice, which overexpress prdx in pancreatic islets, can protect pancreatic beta-cells against streptozotocininduced injury (insulitis) by suppressing increased oxidative stress and inflammatory signaling activation (ding et al., ) . the role of prdx in inflammatory diseases as non-beneficial is also established. chang et al demonstrated that the expression of prdx is increased in synovial tissues of ra patients . these data indicate that prdx inhibits inflammatory diseases by suppressing oxidative stress and inflammatory signaling. the beneficial role of prdx in inflammatory diseases is well established. wang et al showed that prdx -/macrophages oxidized ldl significantly more than it did in controls and plasma lipid hydroperoxide levels were higher in atherogenic diet-fed prdx -/mice than in controls (wang et al., ) . a recent study showed that increased prdx expression suppresses liver injury induced by free radicalmediated damage via inflammation (khoontawad et al., ) . likewise, an increased liver injury in prdx -knockout mice occurred via increased mitochondrial generation of h o (eismann et al., ) . the role of prdx in inflammatory diseases as non-beneficial is also established. prdx is also involved in the several inflammation related diseases. our recent study suggested that overexpression of prdx promotes development of ra through activation of coupling nf-κb/ap- with the jnk pathway or nf-κb/ap- coupled with the jnk pathway in the caia and aia-induced arthritis development model . our data and other data suggest that prdx inhibits inflammatory diseases by suppression of free radical-induced damage and mitochondrial generation of h o , and induces inflammatory diseases through activation of nf-κb/ap- coupled with the jnk pathway. prdxs secreted from cells under mild oxidative stress binds tlr and prdxs induces nrf or nf-κb activation that is important for the expression of inflammatory proteins, suggesting that prdxs are involved in inflammation related disease (madrigal-matute et al., ; westermann et al., ) . fig. illustrates the roles of prdxs in the signaling pathways for the development of inflammatory diseases. riddell et al. found that extracellular prdx can induce activation of nf-κb through tlr (riddell et al., ) . they showed that incubation of mouse vascular endothelial cells with recombinant prdx caused an increase of vegf expression that was dependent upon tlr and required hypoxia inducible factor- (hif- ) interaction with the vegf promoter. the induction of vegf was also dependent upon nf-κb; however, nf-κb interaction with the vegf promoter was not required for prdx induction of vegf suggesting that nf-κb was acting indirectly to induce vegf expression. riddell et al. also showed that recombinant prdx supplemented to the culture medium induced secretion of tnfα and il- from mouse thioglycolate-elicited peritoneal macrophages (riddell et al., ) . they showed that secretion of cytokines in response to prdx was dependent upon serum and required cd and md . binding of prdx to tg-macrophages occurred within minutes and resulted in tlr endocytosis. prdx interaction with tlr was independent of its peroxidase activity and appeared to be dependent upon its chaperone activity and ability to form decamers. cytokine expression occurred via the tlr-myd signaling pathway, which resulted in nuclear translocation and activation of nf-κb. these findings suggest that prdx may act as a danger signal similar to other tlr binding chaperone molecules such as hsp . shichita et al. showed that prdx to all bind to tlr and induce il- (shichita et al., a (shichita et al., , b . the authors identified prdxs as key initiators of postischemic inflammation in the brain. they showed that il- production from infiltrated macrophages induces il- -producing t cells, which play a role in the promotion of delayed ischemic brain damages. it was speculated that prdxs released from necrotic cells induce nf-κb activation and the production of il- (salzano et al., ) . the authors identified a domain on prdxs required for the tlr -mediated cytokine production. this domain is amino acid residues between and of prdx and homologous regions in other prdxs that commonly contain β sheet and α helix regions. it is noted, however, that the activity of domain containing gst-prdx fusion proteins induced the il- mrna to a much lesser extent compared to whole prdxs. the interaction of prdxs with tlr should be strictly regulated by a manner different from that of lps. kisucka et al examined the role of prdx in the apoe -/murine spontaneous model of atherosclerosis (kisucka et al., ) . they showed that prdx -/-/apoe -/mice fed normal chow developed larger, more macrophage-rich aortic sinus lesions than prdx +/+ / apoe -/mice, despite similar amounts and size distributions of cholesterol in their plasma lipoproteins, suggesting that prdx protects against excessive endothelial activation and atherosclerosis. prdx deficiency enhanced the regulated secretion pathway in endothelial cells by promotion of excessive release of several proinflammatory components of weibel-palade bodies, such as p-selectin and von willebrand factor (kisucka et al., ) . however, prdx deficiency did not affect transcriptional regulation of receptors such as intercellular adhesion molecule (icam)- and vascular cell adhesion molecule- (vcam- ). one study showed that prdx is highly expressed in endothelial and immune cells in atherosclerotic lesions and blocked the increase of endogenous h o by atherogenic stimulation (park et al., ) . deficiency of prdx in apoe -/mice accelerated plaque formation with enhanced activation of p , c-jun, jnks, and p mitogen-activated protein kinase; and these proatherogenic effects of prdx deficiency were rescued by the administration of the antioxidant ebselen. also, they showed that prdx deficiency resulted in increased expression of vascular adhesion molecule- , intercellular adhesion molecule- , and monocyte chemotactic protein- , which led to increased immune cell adhesion and infiltration into the aortic intima. moreover, compared with deficiency of glutathione peroxidase or catalase, prdx deficiency showed a severe predisposition to develop atherosclerosis. chen et al showed that inflammatory stimuli induce the release of oxidized prdx , a ubiquitous redox-active intracellular enzyme . once released, the extracellular prdx acts as a redoxdependent inflammatory mediator, triggering macrophages to produce and release tnf-α. the oxidative coupling of glutathione (gsh) to prdx cysteine residues occurs before or during prdx release, a process central to the regulation of immunity (salzano et al., ) . salzano et al identified prdx among the glutathionylated proteins released in vitro by lps-stimulated macrophages using mass spectrometry proteomic methods. consistent with being part of an inflammatory cascade, it was found that prdx then induces tnf-α release (salzano et al., ) . importantly, the prdx substrate trx is also released along with prdx , enabling an oxidative cascade that can alter the -sh status of surface proteins and thereby facilitate activation via cytokine and toll-like receptors (salzano et al., ) . prdx deficiency increased abnormal lipid accumulation in adipose tissue, due to increased ros. prdx is a secretory antioxidant protein which can be detected in plasma. by virtue of its antioxidant activity, the extracellular prdx can protect the vascular tissue against ros and hence, it has ability to inhibit the oxidative stress induced inflammation in various tissues and it can also reduce the chances of oxidative stress induced diabetes mellitus in animal models (ding et al., ) . yamada et al demonstrated that streptozotocin-treated tg mice, which overexpress hprdx in pancreatic islets, can protect pancreatic beta-cells against streptozotocin-induced injury (insulitis) by suppressing increased oxidative stress and inflammatory signaling activation (yamada, ding, & sasaguri, ) . these observations indicate that tg mice could become a useful animal model to study the relevance of oxidative stress to inflammation, and that a specific accelerator of prdx might prove to be a potential therapeutic agent for ameliorating various chronic inflammatory diseases. it was also reported that hfd-induced hepatic steatosis and insulin resistance were prevented in prdx transgenic mice by amelioration of oxidative stress (ding et al., ) . they showed that prdx protects against the progression of nonalcoholic steatohepatitis and insulin resistance, particularly in the liver, in a nongenetic mouse model of nonalcoholic fatty liver disease /or type diabetes mellitus. these effects of prdx were achieved by reducing . possible mechanisms of prdxs related with inflammatory disease. prdx can induce activation of nf-κb through tlr . prdx is secreted from cells under mild oxidative stress and binds with tlr which results in induced nf-κb activation that is important for the expression of inflammatory proteins. recombinant prdx supplemented to the culture medium induced secretion of tnf-α and il- from mouse thioglycolate-elicited peritoneal macrophages and bmdm. this effect of prdx is quite similar to that of lps as it required membrane proteins cd and md and was mediated by the tlr -myd signaling leading to the activation of nf-κb. it was speculated that prdxs released from necrotic cells induce nf-κb activation and the production of il- . prdx is a negative regulator of pdgf signalling. prdx suppresses activation of pdgf receptor (pdgfr) and phospholipase cγ , and subsequently decreases cell proliferation and migration in response to pdgf. also, prdx is recruited to pdgfr upon pdgf stimulation, and suppresses protein tyrosine phosphatase inactivation. prdx and prdx are tlr -dependent inducers of infiltrating macrophage activation and the subsequent production of inflammatory mediators from invading t cells in the ischemic brain. overexpression of prdx promotes development of ra through activation of nf-κb/ap- coupled with jnk pathway in the caia and aia-induced arthritis development model. oxidative stress and ameliorating hepatic steatosis, inflammatory reaction, apoptotic activity, and fibrogenesis at systemic as well as local levels. prdx is also involved in the several inflammatory related diseases. wang et al showed that prdx -/macrophages oxidized ldl significantly more than did controls and plasma lipid hydroperoxide levels were higher in atherogenic diet-fed prdx -/mice than in controls (wang et al., ) . recent study showed that increased prdx expression suppresses liver injury induced by free radical-mediated damage via inflammation (khoontawad et al., ) . likewise, an increased liver injury in prdx -knockout mice occurred via increased mitochondrial generation of h o (eismann et al., ) . our recent study suggested that overexpression of prdx promotes development of ra through activation of nf-κb/ap- coupled jnk pathway in the caia and aiainduced arthritis development model . prdxs involved in the several disease related signal pathways. also, prdxs are involved in inhibiting the cancer development or promoting growth of cancers. so, they can be a potential therapeutic target through targeting these signal pathways. prdx has breast tumor suppressive role mediated via c-myc or pten pathways. this study provides that tumour suppressive function of prdx and pten through understanding the mechanistic details of prdx peroxidase activity and pten oxidation. on the other hand, prdx has a tumor promoting effect through nf-kb pathway in breast cancer, oral cancer and bladder cancer. prdx suppressed foxo induced apoptosis in lung cancer, activates mtor/p s k pathway in esophageal squamous cell carcinoma and upregulates pancreatic cancer invasion by modulating p mapk signaling. so, enhanced expression of prdx through cancer related pathways serves as a useful marker for the prognosis of patients with this disease and prdx may be a promising molecular target for the therapeutic intervention of these cancer types. prdx promotes colon cancer through upregulation of wnt/βcatenin pathway, promotes prostate cancer through upregulation of ar activity. so, designing therapeutics targeting prdx may offer a novel strategy for developing treatment for cancer. moreover, several studies demonstrated that overexpression of prdx and prdx has an important role in several drug resistances, and several therapeutic agents targeting prdx and prdx are studied for treatment of cancer. he et al showed that prdx knockdown enhances sensitization to β-lap that is an anticancer agent through modulating ros accumulation and mapk activation (he et al., ) . also, downregulation of prdx by a novel fully human phage display recombinant antibody induces apoptosis and enhances radiation sensitization in lung carcinoma cells (guo et al., ) , indicating that prdx could be targets competing against cancer. the therapeutic agents targeting prdx are studied for treatment of cancer. a recent study demonstrated that theonellasterone, a steroidal metabolite isolated from a theonellasponge targets the prdx and protects its cysteine over-oxidation induced by h o both in vitro and in vivo. this suggested that ths can be used as an anticancer agent for several cancers (margarucci et al., ) . liu et al also demonstrated that adenanthin, a diterpenoid isolated from the leaves of rabdosia adenantha inhibits the tumor growth in the mouse acute promyelocytic leukemia mouse model by directly targeting the conserved resolving cysteines of prdx as well as prdx and inhibits their peroxidase activities . hou et al also investigated the effect of adenanthin on hcc cells, and found that the natural agent can kill malignant liver cells in vitro and xenografts through targeting prdx and prdx , thus increasing ros level . overexpression of prdx has an important role in several drug resistances. the silencing of prdx triggered cisplatin mediated apoptosis in ovarian cancer cells through suppression of the nf κb signaling pathway (duan et al., ) , suggesting that prdx is involved in drug resistance. prdx also promotes breast cancer through upregulation of cell cycle and cell proliferation, enhances the risk of prostate cancer, thymoma and hepatocellular carcinoma through increased resistance to oxidative stress. prolonged drug exposure may result in cumulative toxicity. the clinical efficacy of chemotherapy must be enhanced, its attendant toxicity reduced, and resistance overcome. to overcome multidrug resistance in cancer cells, recent chemotherapeutics could be used in combination with other molecules. so, prdx may be a therapeutic target for the combination treatment of several drug resistant cancers. prdx promotes lung cancer, leukemia, glioblastoma, oral squamous cell carcinoma, cardiovascular disease, hepatic diseases and colorectal carcinoma. prdx is involved in the tumor promoting effect through formation of srx-prdx via ap- /mmp- and mapk signaling, and remove cellular ros which provides a favorable microenvironment for cell proliferation. prx- can be used as an important therapeutic target in this disorder. prdx promotes lung tumor development via jak /stat pathway, its gpx and ipla activities, and is involved in drug resistance of several cancer types, suggesting that prdx can be a therapeutic target for cancer treatment. our recent study suggested that thiacremonone inhibits tumor growth via inhibition of glutathione peroxidase activity of prdx through interaction, suggesting that thiacremonone may have potential beneficial effects in lung cancer (jo et al., ) . these data indicate that prdx could be targets of several cancers and the therapeutic agents for targeting prdx may have potentially beneficial effects in cancer treatment. prdxs could be targets for the treatment of neurodegenerative diseases. prdx has a protective role in counteracting aβ injury by increasing cell viability, preserving neurites, and decreasing cell death (cimini et al., ) . these researches provide the neuroprotective function of prdx and understanding the mechanistic details of prdx peroxidase activity in the neurodegenerative diseases. prdx is involved in neuroprotection via inhibition of aβ - induced apoptosis and suppression of jnk and p pathways, providing the neuroprotective function of prdx and understanding the mechanistic details of prdx peroxidase activity in the neurodegenerative diseases. also, because cdk that inhibits prdx activity leads to increased levels of ros in pd, cdk is used as therapeutic target for treatment of pd. using novel cdk modulators, sun et al further showed the mechanism by which cdk can induce oxidative stress in the disease's early stage and cell death in the late stage (sun et al., ) . cdk inhibition rescues mitochondrial damage upon neurotoxic insults, thereby revealing cdk as an upstream regulator of mitochondrial dysfunction. as oxidative stress and mitochondrial dysfunction play pivotal roles in promoting neurodegeneration, cdk could be a viable therapeutic target for ad. zhang et al found that neurotoxin, mpp(+) induces cdk / p -dependent phosphorylation of prdx , resulting in inhibition of its peroxireductase activity and accumulation of ros (zhang et al., a, b) . on the other hand, prdx is also involved in neurodegeneration through phosphorylation by cdk , a serine/threonine kinase, and contributes to pd, providing that prdx can be used for therapeutic target for treatment of pd through targeting this pathway. prdx is involved in the neuroprotection against oxidative insults in the mitochondria, suggesting that prdx up-regulation might be a useful novel approach for the management of neurodegenerative diseases. on the other hand, prdx is involved in neurodegeneration through increased phosphorylation by lrrk mutation, providing therapeutic target in pd patients carrying lrrk mutations. prdx is involved in pd through mitochondrial redox signaling with ca + influx resulting in the development of pd (davey & bolaños, ) , also can be used for therapeutic target. prdx induces neurodegeneration through aipla and nrf activation in ad, and induces neurodegeneration through induction of mptp-induced dopaminergic neurodegeneration of pd, providing that prdx might be used a useful novel approach for the management of neurodegenerative diseases. on the other hand, prdx inhibits multiple sclerosis by suppressing inflammation and blood brain barrier disruption in eae model, suggesting a new molecule for neuroprotective therapies in multiple sclerosis. prdxs could be targets for the treatment of inflammatory diseases. prdx suppresses atherosclerosis through the site-selective phosphorylation of the pdgf receptor pathways, suggesting upregulation of prdx can be used for treatment of atherosclerosis. du et al examined the expressions of prdx and foxo a that were down-regulated in ischemic brain compared with the normal-control group (du et al., ) . they showed that the combined treatment of probucol or atorvastatin dramatically reduced the brain water content and the infarct volume, along with the decrease of prdx , foxo a and nrf was significantly alleviated in combined treatment group. they suggested that probucol combined with atorvastatin effect in the neuroprotection from the damage caused by mcao through up-regulation of prdx , foxo a and nrf . on the other hand, prdx enhances the risk of ra because prdx is involved in the persistence of pro-inflammatory cells in chronic inflammation, providing prdx can be used as therapeutic target of ra. prdx inhibits the risk of obesity through decreased abnormal lipid accumulation in adipose tissue, due to increased ros, suggesting prdx can be used for the treatment of obesity. prdx protects against atherosclerosis and obesity through inhibiting the oxidative stress induced inflammation in various tissues, suggesting up-regulation might be a useful novel approach for the management of atherosclerosis and obesity. but prdx enhances the risk of ra, providing therapeutic target for treatment of ra. prdx enhances the risk of ra through activation of nf-κb/ap- coupled jnk pathway in the caia and aia-induced arthritis development model, suggesting prdx can be used as therapeutic target for treatment of ra. moreover, several therapeutic agents targeting prdx are studied for treatment of inflammatory diseases. among them, epigallocatechin- -gallate modulates the levels of the antioxidant enzymes prdx , catalase and superoxide dismutase (sod), as well as pcna, in nod (ohno et al., ) . they showed that the early pcna elevation was followed by a decline of prdx protein, in contrast egcgfed mice exhibited normal levels of pcna and prdx , comparable to healthy untreated balb/c mice. in conclusion, we discussed the roles and mechanisms of prdxs on cellular homeostasis, diseases development and drug resistance, and propose potential therapeutic approaches for targeting prdxs. we hope this review provides a foundation for further studies to design novel therapeutic approaches targeting prdxs. the authors declare that there are no conflicts of interest. signaling events leading to peroxiredoxin up-regulation in immunostimulated macrophages peroxiredoxin , a novel circulating biomarker for oxidative stress and the risk of incident cardiovascular disease and all-cause mortality role of redox potential and reactive oxygen species in stress signaling genome-wide analysis of histone h acetylation patterns in aml identifies prdx as an epigenetically silenced tumor suppressor gene mutations in lrrk increase phosphorylation of peroxiredoxin exacerbating oxidative stressinduced neuronal death differential expression of peroxiredoxins in prostate cancer: consistent upregulation of prdx and prdx neutrophil-mediated oxidation of erythrocyte peroxiredoxin as a potential marker of oxidative stress in inflammation molecular pathways associated with aggressiveness of papillary thyroid cancer basic principles and emerging concepts in the redox control of transcription factors expression and clinical value of peroxiredoxin- in patients with pancreatic cancer prdx inhibits tumorigenesis via regulating pten/akt activity oxy-radicals and cancer overexpression of peroxiredoxin i and thioredoxin in human breast carcinoma protein glutathionylation in the regulation of peroxiredoxins: a family of thiol-specific peroxidases that function as antioxidants, molecular chaperones, and signal modulators nuclear envelope dispersion triggered by deregulated cdk precedes neuronal death identification of prdx and p ha as metastasis-associated proteins in oral cavity squamous cell carcinoma by comparative tissue proteomics of microdissected specimens using itraq technology peroxiredoxin iii, a mitochondrion-specific peroxidase, regulates apoptotic signaling by mitochondria identification of proteins with increased expression in rheumatoid arthritis synovial tissues regulation of peroxiredoxin i activity by cdc -mediated phosphorylation peroxiredoxin-i is an autoimmunogenic tumor antigen in non-small cell lung cancer identification of the functional role of peroxiredoxin in the progression of breast cancer peroxiredoxin phosphorylation and subsequent phospholipase a( ) activity are required for agonist-mediated activation of nadph oxidase in mouse pulmonary microvascular endothelium and alveolar macrophages plasma proteome of severe acute respiratory syndrome analyzed by twodimensional gel electrophoresis and mass spectrometry -cys peroxiredoxin, a bifunctional enzyme with glutathione peroxidase and phospholipase a activities inhibition of lung tumor growth and augmentation of radiosensitivity by decreasing peroxiredoxin i expression enhanced defense against mitochondrial hydrogen peroxide attenuates age-associated cognition decline regeneration of peroxiredoxins during recovery after oxidative stress: only some overoxidized peroxiredoxins can be reduced during recovery after oxidative stress prx enhances androgen receptor function in prostate cancer cells by increasing receptor affinity to dihydrotestosterone foxo a regulates peroxiredoxin iii expression in human cardiac fibroblasts peroxiredoxin interferes with trail-induced death-inducing signaling complex formation by binding to death effector domain caspase regulation of pdgf signalling and vascular remodelling by peroxiredoxin ii keratinocyte growth factor and glucocorticoid induction of human peroxiredoxin gene expression occur by independent mechanisms that are synergistic oxidant stress stimulates expression of the human peroxiredoxin (prdx ) gene by a transcriptional mechanism involving an antioxidant response element regulation of prdx peroxidase activity by pin silencing the peroxiredoxin iii gene inhibits cell proliferation in breast cancer proteomic identification of overexpressed prdx and its clinical implications in ovarian carcinoma neuroprotective effects of prxi over-expression in an in vitro human alzheimer's disease model dual role of peroxiredoxin i in macrophage-derived foam cells antioxidant mechanisms and ros-related micrornas in cancer stem cells peroxiredoxin links mitochondrial redox signalling with calcium dynamics: impact on parkinson's disease the peroxiredoxin and glutathione peroxidase families in chlamydomonas reinhardtii interactions among mitochondrial proteins altered in glioblastoma expression of peroxiredoxin i in plasma cells of oral inflammatory diseases enhanced radiation response in radioresistant mcf- cells by targeting peroxiredoxin ii. breast cancer regulation of peroxiredoxins by nitric oxide in immunostimulated macrophages overexpression of peroxiredoxin protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice probucol and atorvastatin in combination protect rat brains in mcao model: upregulating peroxiredoxin , foxo a and nrf expression sirna targeting of prdx enhances cisplatin-induced apoptosis in ovarian cancer cells through the suppression of the nf-κb signaling pathway regulation of reactive oxygen species, dna damage, and c-myc function by peroxiredoxin peroxiredoxin- protects against mitochondrial dysfunction and liver injury during ischemia-reperfusion in mice peroxiredoxin v: a candidate breast tumor marker of population specificity s-nitrosylation of peroxiredoxin promotes oxidative stress-induced neuronal cell death in parkinson's disease impaired homeostasis and phenotypic abnormalities in prdx -//-mice lens epithelial cells by reactive oxygen species: increased expression and activation of tgf peroxiredoxin : a bifunctional enzyme with glutathione peroxidase and phospholipase a( ) activities investigating transcriptional regulation of prdx in mouse liver cells thiol-redox signaling, dopaminergic cell death, and parkinson's disease peroxiredoxin expression in the human thyroid gland rare allelic imbalances, but no mutations of the prdx gene in human hepatocellular carcinomas mitochondria in cancer cells: what is so special about them? peroxiredoxin promotes tumorigenesis through regulating the activity of mtor/p s k pathway in esophageal squamous cell carcinoma structure-function analysis of recombinant substrate protein kda (sp- ): a mitochondrial -cys peroxiredoxin organized as a decameric toroid downregulation of peroxiredoxin i by a novel fully human phage display recombinant antibody induces apoptosis and enhances radiation sensitization in a lung carcinoma cells overexpression of peroxiredoxin attenuates atherosclerosis in apolipoprotein e knockout mice molecular mechanisms and health significance: from cofactors to antioxidants to redox signaling cutting edge: trank, a novel cytokine that activates nf-κb and c-jun n-terminal kinase peroxiredoxins in the central nervous system mitochondrial peroxiredoxin- protects hippocampal neurons from excitotoxic injury in vivo peroxiredoxin knockdown potentiates β-lapachone cytotoxicity through modulation of reactive oxygen species and mitogen-activated protein kinase signals uterine fk -binding protein (fkbp )-peroxiredoxin- (prdx ) signaling protects pregnancy from overt oxidative stress crystal structure of a multifunctional -cys peroxiredoxin heme-binding protein kda/ proliferation-associated gene product phospholipase a activity of peroxiredoxin promotes invasion and metastasis of lung cancer cells role of ca +-independent phospholipase a in cell growth and signaling adenanthin targets peroxiredoxin i/ii to kill hepatocellular carcinoma cells peroxiredoxin is a novel marker for cell proliferation in cervical cancer peroxiredoxin- protects against -hydroxydopamine-induced dopaminergic neurodegeneration via attenuation of the ask signaling cascade peroxiredoxin is a key molecule regulating adipocyte oxidative stress, mitochondrial biogenesis, and adipokine expression elevated prx provides resistance to docetaxel, but is not associated with predictive significance in lung cancer peroxiredoxin as an independent prognostic marker for survival in patients with early-stage lung squamous cell carcinoma peroxiredoxins, oxidative stress, and cell proliferation novel roles of peroxiredoxins in inflammation, cancer and innate immunity measurement of peroxiredoxin- serum levels in rat tissue and its use as a potential marker for hepatic disease peroxiredoxin knockout results in elevated spermatogenic cell death via oxidative stress role of peroxiredoxin and peroxiredoxin in protection of respiratory syncytial virus-induced cysteinyl oxidation of nuclear cytoskeletal proteins phosphorylation and concomitant structural changes in human -cys peroxiredoxin isotype i differentially regulate its peroxidase and molecular chaperone functions two enzymes in one: two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function role of sulfiredoxin as a regulator of peroxiredoxin function and regulation of its expression expression of peroxiredoxin and promotes human lung cancer malignancy proteomic analysis of bladder cancer indicates prx-i as a key molecule in bi-tk/gcv treatment system regulatory role for a novel human thioredoxin peroxidase in nf-κb activation lung tumor growth-promoting function of peroxiredoxin anti-cancer effect of thiacremonone through down regulation of peroxiredoxin mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-α vascular injury involves the overoxidation of peroxiredoxin type peroxiredoxins in breast carcinoma peroxiredoxin expression is increased in neutrophils of patients with refractory cytopenia with multilineage dysplasia biomarker discovery: a proteomic approach for brain cancer profiling proteomic identification of peroxiredoxin for host defence against opisthorchis viverrini infection protective effects of peroxiredoxin overexpression on amyloid β-induced apoptosis in pc cells peroxiredoxin i is a ros/p mapk-dependent inducible antioxidant that regulates nf-κb-mediated inos induction and microglial activation elevated peroxiredoxin , but not nf-e -related factor , is an independent prognostic factor for disease recurrence and reduced survival in stage i non-small cell lung cancer exacerbation of collagen antibody-induced arthritis in transgenic mice overexpressing peroxiredoxin suppression of peroxiredoxin in glioblastoma cells increases apoptosis and reduces tumor growth expression of human peroxiredoxin isoforms in response to cervical carcinogenesis peroxiredoxin prevents excessive endothelial activation and early atherosclerosis stimulation of peroxidase activity by decamerization related to ionic strength: ahpc protein from amphibacillus xylanus redox regulation of foxo transcription factors peroxiredoxin : structure, mechanism, and function of the mammalian atypical -cys peroxiredoxin oxidative and electrophilic stresses activate nrf through inhibition of ubiquitination activity of keap the conformational bases for the two functionalities of -cysteine peroxiredoxins as peroxidase and chaperone aberrant expression of peroxiredoxin subtypes in neurodegenerative disorders expression patterns of antioxidant proteins in brains of patients with sporadic creutzfeldt-jacob disease p (phox) terminates the phospholipase a( )-derived signal for activation of nadph oxidase (nox ) mitochondrial targeting of human peroxiredoxin v protein and regulation of prdx gene expression by nuclear transcription factors controlling biogenesis of mitochondria ligustilide ameliorates neuroinflammation and brain injury in focal cerebral ischemia/reperfusion rats: involvement of inhibition of tlr /peroxiredoxin signaling oxidative stress as a regulator of gene expression in the vasculature peroxiredoxin i regulates the component expression of γ-secretase complex causing the alzheimer's disease oxidative modification of peroxiredoxin is associated with drug-induced apoptotic signaling in experimental models of parkinson disease peroxiredoxins, a novel protein family in lung cancer cloning of bovine peroxiredoxins-gene expression in bovine tissues and amino acid sequence comparison with rat, mouse and primate peroxiredoxins proteomic analysis of synovial fibroblast-like synoviocytes from rheumatoid arthritis search for the tumor-associated proteins of oral squamous cell carcinoma collected in taiwan using proteomics strategy identification of candidate prostate cancer biomarkers in prostate needle biopsy specimens using proteomic analysis adenanthin targets peroxiredoxin i and ii to induce differentiation of leukemic cells peroxiredoxin functions as a noncatalytic scavenger of low-level hydrogen peroxide in the erythrocyte peroxiredoxin is upregulated in colorectal cancer and contributes to colorectal cancer cells' survival by protecting cells from oxidative stress peroxiredoxin knockdown by rna interference inhibits the growth of colorectal cancer cells by downregulating wnt/β-catenin signaling peroxiredoxin : a potential biomarker for early diagnosis of hepatitis b virus related liver fibrosis identified by proteomic analysis of the plasma cell stress proteins in atherothrombosis peroxiredoxin , a -cys peroxiredoxin, functions in antioxidant defense and lung phospholipid metabolism theonellasterone, a steroidal metabolite isolated from a theonella sponge, protects peroxiredoxin- from oxidative stress reactions diversity in secreted pla -iia activity among inbred mouse strains that are resistant or susceptible to apcmin//+ tumorigenesis peroxiredoxin improves insulin biosynthesis and glucose-induced insulin secretion in insulin-secreting ins- e cells the sulfiredoxinperoxiredoxin (srx-prx) axis in cell signal transduction and cancer development mitochondrial dysfunction in cancer reduction of -cys peroxiredoxins by ascorbate changes the thiol-specific antioxidant paradigm, revealing another function of vitamin c t lymphocytes and dendritic cells are activated by the deletion of peroxiredoxin ii (prx ii) gene. i l laminar shear stress up-regulates peroxiredoxins (prx) in endothelial cells: prx as a mechanosensitive antioxidant defective mitochondrial peroxiredoxin- results in sensitivity to oxidative stress in fanconi anemia cysteine oxidation targets peroxiredoxins and for exosomal release through a novel mechanism of redox-dependent secretion proteomic analysis of visceral adipose tissue in pre-obese patients with type diabetes peroxiredoxin protects against nonalcoholic steatohepatitis and type diabetes in a nongenetic mouse model analysis of the peroxiredoxin family: using active site structure and sequence information for global classification and residue analysis peroxiredoxin and its role in cell signaling essential role for the peroxiredoxin prdx in erythrocyte antioxidant defence and tumour suppression human peroxiredoxin gene organization, initial characterization of its promoter and identification of alternative forms of mrna sulfiredoxin translocation into mitochondria plays a crucial role in reducing hyperoxidized peroxiredoxin iii increased expression of mitochondrial peroxiredoxin- (thioredoxin peroxidase- ) protects cancer cells against hypoxia and drug-induced hydrogen peroxide-dependent apoptosis ca and ca peroxiredoxin- protects estrogen receptor α from oxidative stress-induced suppression and is a protein biomarker of favorable prognosis in breast cancer epigallocatechin- -gallate modulates antioxidant and dna repair-related proteins in exocrine glands of a primary sjogren's syndrome mouse model prior to disease onset peroxiredoxin overexpression attenuates cisplatin-induced apoptosis in human ovarian cancer cells the antioxidant protein peroxiredoxin is epigenetically down regulated in acute promyelocytic leukemia hypoxia increases androgen receptor activity in prostate cancer cells proteomic profiling of endothelial cells in human lung cancer peroxiredoxin deficiency exacerbates atherosclerosis in apolipoprotein e-deficient mice peroxiredoxin interacts with androgen receptor and enhances its transactivation novel mitochondrial targets for neuroprotection peroxiredoxins: guardians against oxidative stress and modulators of peroxide signaling the high reactivity of peroxiredoxin with h o is not reflected in its reaction with other oxidants and thiol reagents the logic of kinetic regulation in the thioredoxin system overview of peroxiredoxins in oxidant defense and redox regulation overview of peroxiredoxins in oxidant defense and redox regulation exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production detection and identification of peroxiredoxin as a biomarker in hepatocellular carcinoma by a proteomic approach role of cdk -mediated phosphorylation of prx in mptp toxicity and parkinson's disease enhanced expression of peroxiredoxin i and vi correlates with development, recurrence and progression of human bladder cancer studies on free radicals, antioxidants, and co-factors nitration transforms a sensitive peroxiredoxin into a more active and robust peroxidase multiple functions of peroxiredoxins: peroxidases, sensors and regulators of the intracellular messenger h o , and protein chaperones peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides peroxiredoxin controls prostate cancer growth through toll-like receptor dependent regulation of tumor vasculature peroxiredoxin stimulates endothelial cell expression of vegf via tlr dependent activation of hif- α peroxiredoxin stimulates secretion of pro-inflammatory cytokines by binding to toll-like receptor peroxiredoxin post-translational modifications by redox messengers linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin- , which acts as a danger signal cardiac peroxiredoxins undergo complex modifications during cardiac oxidant stress crystal structure of decameric -cys peroxiredoxin from human erythrocytes at . Å resolution peroxiredoxin : a multifunctional biomarker worthy of further exploration gata-binding protein is a novel transcription regulator of peroxiredoxin in human breast cancer cells mitochondrial ros signaling in organismal homeostasis role of constitutive calcium-independent phospholipase a beta in hippocampo-prefrontal cortical long term potentiation and spatial working memory novel therapeutic strategies targeting innate immune responses and early inflammation after stroke peroxiredoxin family proteins are key initiators of post-ischemic inflammation in the brain peroxiredoxin in the nucleus and cytoplasm distinctly regulates androgen receptor activity in prostate cancer cells peroxiredoxin- and stat form a redox relay for h o signaling high association of peroxiredoxins with lung cancer foxm -induced prx regulates stemness and survival of colon cancer cells via maintenance of mitochondrial function mitochondrial peroxiredoxin iii is a potential target for cancer therapy induction of sulfiredoxin expression and reduction of peroxiredoxin hyperoxidation by the neuroprotective nrf activator h- , -dithiole- -thione peroxiredoxin specifically regulates the oxidative and metabolic stress response of human metastatic breast cancer cells in lungs novel genetic tools reveal cdk 's major role in golgi fragmentation in alzheimer's disease diagnostic and prognostic significance of peroxiredoxin expression in human hepatocellular carcinoma lymphocytes from rheumatoid arthritis patients have elevated levels of intracellular peroxiredoxin , and a greater frequency of cells with exofacial peroxiredoxin , compared with healthy human lymphocytes amp-dependent kinase inhibits oxidative stress-induced caveolin- phosphorylation and endocytosis by suppressing the dissociation between c-abl and prdx proteins in endothelial cells peroxiredoxin promotes pancreatic cancer cell invasion by modulating p mapk activity peroxiredoxin iv protects cells from oxidative stress by removing h( )o( ) produced during disulphide formation redox regulation of cell survival role for prdx as a specific sensor in redox-regulated senescence in breast cancer peroxiredoxins and are overexpressed in prostate cancer tissue and affect the proliferation of prostate cancer cells in vitro overexpression of prdx and resistance to peroxide-induced death in hepa - cells: prdx suppression increases apoptosis the role of peroxiredoxin ii in chemoresistance of breast cancer cells peroxiredoxin as an antioxidant enzyme: protection of lung alveolar epithelial type ii cells from h o -induced oxidative stress peroxiredoxin gene-targeted mice show increased lung injury with paraquat-induced oxidative stress selective association of peroxiredoxin with genomic dna and cox- upstream promoter elements in estrogen receptor negative breast cancer cells peroxiredoxin is resistant to oxidation-induced apoptosis of hep- b cells peroxiredoxin deficiency and atherosclerosis susceptibility in mice: significance of genetic background for assessing atherosclerosis peroxiredoxin iii protein expression is associated with platinum resistance in epithelial ovarian cancer antioxidant enzyme peroxiredoxin is upregulated in degenerative human tendon anti-oxidative, anti-cancer and anti-inflammatory actions by thioredoxin and thioredoxin-binding protein- sulfiredoxin-peroxiredoxin iv axis promotes human lung cancer progression through modulation of specific phosphokinase signaling protein kinase c bound to the golgi apparatus supports the formation of constitutive transport vesicles peroxiredoxin- is overexpressed in prostate cancer and promotes cancer cell survival by protecting cells from oxidative stress the c-myc target gene prdx is required for mitochondrial homeostasis and neoplastic transformation inactivation of peroxiredoxin i by phosphorylation allows localized h o accumulation for cell signaling peroxiredoxin evolution and the regulation of hydrogen peroxide signaling structure, mechanism and regulation of peroxiredoxins expression of thioredoxin system and related peroxiredoxin proteins is associated with clinical outcome in radiotherapy treated early stage breast cancer mitogen-activated protein kinase-mediated phosphorylation of peroxiredoxin regulates its phospholipase a( ) activity interaction of surfactant protein a with peroxiredoxin regulates phospholipase a activity hypoxia inducible factor- α suppresses peroxiredoxin expression to promote proliferation of ccrcc cells peroxiredoxin : critical roles in inflammatory diseases peroxiredoxin i expression in human thyroid tumors peroxiredoxin i expression in oral cancer: a potential new tumor marker peroxiredoxin i expression in tongue squamous cell carcinomas as involved in tumor recurrence inactivation of human peroxiredoxin i during catalysis as the result of the oxidation of the catalytic site cysteine to cysteine-sulfinic acid roles of peroxiredoxin ii in the regulation of proinflammatory responses to lps and protection against endotoxin-induced lethal shock interaction of amyloid binding alcohol dehydrogenase/aβ mediates up-regulation of peroxiredoxin ii in the brains of alzheimer's disease patients and a transgenic alzheimer's disease mouse model high expression of peroxiredoxin affects the survival time of colorectal cancer patients, but is not an independent unfavorable prognostic factor prdx exacerbates dopaminergic neurodegeneration in a mptp mouse model of parkinson's disease acceleration of the development of alzheimer's disease in amyloid beta-infused peroxiredoxin overexpression transgenic mice loss of presenilin is associated with increased ipla activity and lung tumor development prdx controls multiple sclerosis by suppressing inflammation and blood brain barrier disruption prdx promotes lung tumor progression via its gpx and ipla activities prdx promotes tumor development via the jak /stat pathway in a urethane-induced lung tumor model prdx , a member of the peroxiredoxin family, is fused to aml (runx ) in an acute myeloid leukemia patient with a t(x; )(p ;q ) induction of peroxiredoxin by hypoxia regulates heme oxygenase- via nf-κb in oral cancer p , the n-terminal domain of p , protects against cdk /p -induced neurotoxicity lentivirusmediated inhibition of tumour necrosis factor-α improves motor function associated with prdx in spinal cord contusion rats pharmacological induction of heme oxygenase- by a triterpenoid protects neurons against ischemic injury triosephosphate isomerase and peroxiredoxin , two novel serum markers for human lung squamous cell carcinoma the protective effect of peroxiredoxin ii on oxidative stress induced apoptosis in pancreatic β-cells oxidative protein folding by an endoplasmic reticulum localized peroxiredoxin key: cord- - ak hto authors: meftahi, gholam hossein; jangravi, zohreh; sahraei, hedayat; bahari, zahra title: the possible pathophysiology mechanism of cytokine storm in elderly adults with covid- infection: the contribution of “inflame-aging” date: - - journal: inflamm res doi: . /s - - - sha: doc_id: cord_uid: ak hto purpose: novel coronavirus disease (covid- ), is an acute respiratory distress syndrome (ards), which is emerged in wuhan, and recently become worldwide pandemic. strangely, ample evidences have been shown that the severity of covid- infections varies widely from children (asymptomatic), adults (mild infection), as well as elderly adults (deadly critical). it has proven that covid- infection in some elderly critical adults leads to a cytokine storm, which is characterized by severe systemic elevation of several pro-inflammatory cytokines. then, a cytokine storm can induce edematous, ards, pneumonia, as well as multiple organ failure in aged patients. it is far from clear till now why cytokine storm induces in only covid- elderly patients, and not in young patients. however, it seems that aging is associated with mild elevated levels of local and systemic pro-inflammatory cytokines, which is characterized by “inflamm-aging”. it is highly likely that “inflamm-aging” is correlated to increased risk of a cytokine storm in some critical elderly patients with covid- infection. methods: a systematic search in the literature was performed in pubmed, scopus, embase, cochrane library, web of science, as well as google scholar pre-print database using all available mesh terms for covid- , coronavirus, sars-cov- , senescent cell, cytokine storm, inflame-aging, ace receptor, autophagy, and vitamin d. electronic database searches combined and duplicates were removed. results: the aim of the present review was to summarize experimental data and clinical observations that linked the pathophysiology mechanisms of “inflamm-aging”, mild-grade inflammation, and cytokine storm in some elderly adults with severe covid- infection. the covid- , now named sars-cov , spreading in wuhan, china, and now spread globally rapidly [ ] . it is reported that covid- has the same viral genome (above % identity in the genome), and pathophysiology mechanisms with the sars-cov [ ] . the covid- infection affecting all age-groups, but it appears to be more severe in elderly adults [ ] . it seems that very high pro-inflammatory cytokine release, which is described as cytokine storm, is a pivotal pathophysiological mechanism in elderly covid- patients [ ] . aging is related to increased levels of systemic pro-inflammatory cytokines and decreased levels of systemic anti-inflammatory cytokines. hence, a chronic condition of inflammation may be created in aged subjects, known as "inflamm-aging" [ , ] . ample studies have indicated elevated levels of interleukin (il)- , il- , tumor necrosis factor-α (tnf α), as well as c-reactive protein (crp) in aged subjects [ , ] . although, the exact underlying mechanism of cytokine storm in elderly adults with severe covid- infection is far from clear. however, it is likely that dysregulation of the cytokine homeostasis in "inflameaging" phenomenon may play a critical role in the risk of a cytokine storm, and subsequently acute respiratory distress syndrome (ards) in some elderly patients with severe covid- infection. it seems that "cytokine storm" phenomenon in elderly patients with severe covid- infection, is associated with many age-related pathophysiologic processes, including alteration of angiotensin-converting enzyme (ace ) receptor expression [ ] , excess ros production [ ] , alteration of autophagy [ ] , the inflammatory phenotype of senescent cell activity, particularly adipose tissue [ ] , and immune-senescence [ ] , as well as lack of vitamin d content [ ] . here, we are going to review and discuss all above mentioned age-related pathophysiological pathways that appear to contribute to the dysregulation of cytokine networks and possibly a cytokine storm in elderly patients with severe covid- infection. it has been shown that covid- infection has distinctive behavior among elderly adults (severe infection) as compared with children and young adults (none or mild infection). indeed, covid- infection can induce severe infection, including pneumonia and ards in some elderly adults or sick patients, and not in children or young adults [ ] . what is the reason that the deadly cases of covid- mainly seen in elderly patients? here, first we are going to review and compare the possible pathophysiology mechanisms of mild infection and severe infection in young and elderly adults with covid- , respectively. despite increasing evidences on the immune response to pathogens, however, less is known about the exact immunologic mechanism of covid- infections. as shown in fig. , initiation of the immune response against invading coronavirus begins with a direct infection of the bronchi and bronchiole epithelium. first, antigen-independent innate immunity provides the first line of leukocytes defense against microorganisms. innate immune defense involves several cell types, including leukocytes such as neutrophils, eosinophils, basophils, monocytes, macrophages, lung epithelial cells, mast cells, natural killer (nk cells) [ ] . following initial covid- infection, lung-resident dendritic cells (dcs) become activated and change to antigen-presenting cells (apcs). indeed, apcs are the first line of defense in recognizing various pathogens. in the lung, dcs resides in and below the airway epithelium, the alveolar septa, pulmonary capillaries, and airway spaces [ ] . activated apcs ingests, and processes an antigen and migrate to the lymph nodes. then, in the lymph nodes, apc presents the antigen in the form of mhc/peptide complex to naïve circulating t helper cells (th ), inducing the immune response [ ] . following activation of th receptor by mhc/peptide complex, t helper cells become activated, proliferate and differentiated to cd + (t helper lymphocytes) and cd + (cytotoxic t lymphocytes) cells. then, cd + th lymphocytes further differentiated into th and th , with different cytokine profiles [ ] . th cells drive cellular immunity and released pro-inflammatory cytokines such as ifn-γ, il- β, il- , and tnf-α [ ] . it is reported that cytokine ifn-γ can inhibit viral replication and enhance antigen presentation [ ] . th cells activated humoral immunity and antibody production and released anti-inflammatory cytokines such as tgf-β, il- , il- , il- , il- and il- [ ] . in fact, a balanced between th and th lymphocyte activity is observed in healthy adults with covid- infection. furthermore, cd + t lymphocytes cytotoxic t cells, as cytotoxic cells, secrete cytotoxic molecules such as granzyme b that kill infected epithelial cells. indeed, cd + t lymphocytes and natural killer cells (nk) play a critical contribution in viral clearance [ ] . both t and b cell responses against covid- observed in the systemic blood pool week after the initiation of covid- symptoms. the autopsy of a patient with covid- identified an accumulation of mononuclear cells (likely monocytes and t cells) in the lungs, with low levels of hyperactive t cells in the peripheral blood. these findings suggested that likely t cells are attracted away from the systemic blood pool and into the infected site (lung) to control the covid- infection [ ] . generally, activation of different th cells and release of ample cytokines and chemokines recruit more innate, cell-mediated and humoral immunologic responses to control covid- in adults. additionally, it seems that the balance between proinflammatory and anti-inflammatory immune responses in the healthy adult can shut down immune activity at the right moment [ ] . it was observed that ards, pneumonia and multi-organ dysfunction are the main immune-clinical symptoms of covid- infection. it is well accepted that ards and pneumonia in deadly cases are due to severe inflammatory responses to the immune system, this so-called a cytokine storm [ ] . on the other hand, severe multi-organ destruction is due to the cytokine storm rather than a direct damaging effect of the virus itself [ ] . it is noteworthy when covid- pathogen reached to the alveoli in elderly and weak adults, pro-inflammatory immune responses become vigorous and un-controllable active. furthermore, impaired anti-inflammatory responses in elderly adults may correlate to increased activity of pro-inflammatory responses [ ] . hence, some elderly adults with severe covid- infection cannot shut down their pro-inflammatory immune response. several studies reported most patients with severe covid- exhibit markedly increased serum levels of pro-inflammatory cytokines, including; ifn-α, ifn-γ, il- β, il- , il- , il- , il- , il- , tnf-α, g-csf, gm-csf, ip , c-reactive protein (crp), mcp , and mip α [ ] [ ] [ ] . it is necessary to mention that cytokines storm directly may lead to immune cell death, tissue damage, and respiratory shut down [ ] . for example, the autopsy findings of aged subjects revealed spleen atrophy and necrosis, lymph node necrosis, hemorrhage in the kidney, hepatomegaly, and degeneration of the neurons in the central nervous system in covid- patients. the number of immune cells also changed in covid- infection [ , ] . indeed, in patients with severe covid- infection, but not in patients with a mild infection, lymphopenia is a common feature, with significantly reduced numbers of cd + t cells, cd + t cells, b cells and nk cells. furthermore, exhaustion markers, such as nkg a receptors on nk cells and cd + t cells, are up-regulated in patients with covid- [ , ] . histochemical staining showed that cd + t cells and cd + t cells were decreased in spleen and lymph nodes. in addition, in the lung with characteristic diffused alveolar damage, the major infiltrated cells were monocytes and macrophages, but very few lymphocytes [ ] . tian and colleagues in using postmortem biopsies identified alveolar damage, fibrosis of the heart and myocardial hypertrophy, and also lobular infiltration of the liver by small lymphocytes in four died cases of covid- [ ] . in addition to cytokine storm, viral particles of covid- can also directly induce multiple organ dysfunctions. because viral particles of covid- infection were identified in the bronchial and type alveolar epithelial cells, fecal, and urine samples [ , , ] . hence, it is suggested that multiple organ dysfunction in severe covid- patients can also cause by a direct attack of the virus. it is far from clear whether cytokine storm, direct effects of the virus, or the synergistic effects of both, contribute to the multiple organ failures in severe covid- patients [ ] . here, proliferate and differentiated to other cells such as cd + (t helper lymphocytes) and cd + (cytotoxic t lymphocytes) cells. in healthy adults, due to sufficient vitamin d level, vd can decrease the expression of pro-inflammatory genes in immune cells. a balanced between pro-inflammatory and anti-inflammatory activity causes shut down of the immune system at the right moment we are going to review the link between cytokine storm in elderly patients of covid- and "inflame-aging". aging is related to elevated systemic levels of pro-inflammatory cytokines, including il- , il- , tnf-α, il- , ifnγ, as well as acute phase proteins. ample studies reported a chronic mild inflammation in aging, which is described as "inflame-aging". this phenomenon can promote ageassociated disorders, including diabetes mellitus, alzheimer's disease, atherosclerosis, etc. accordingly, it seems that increased generation of pro-inflammatory markers and "inflame-aging" have a critical role in the process of cytokine storm in severe covid- patients and enhanced mortality risk [ , ] . as shown in fig. , several factors, including alteration of ace receptor expression, excess reactive oxygen species (ros) production, senescent adipocytes activity, alteration of autophagy and mitophagy, immune-senescent, as well as vitamin d (vd) deficiency, may associate "inflame-aging" to cytokine storm in elderly patients of covid- . the renin-angiotensin system (ras) is an important regulator of several physiologic events, including cardiovascular and blood volume, natriuresis, diabetes, chronic renal disease, and hepatic fibrosis [ , ] . this system is composed of two different pathways, including angiotensin-converting enzyme (ace)/angiotensin ii (ang ii)/angiotensin receptor type (at ) (ace/ang ii/at ) pathway; and angiotensin-converting enzyme (ace )/ang - /mas receptor (ace /ang - /mas) pathway. these two pathways have opposing effects to accommodate a coordinated response to specific triggers. the activity of ace/ang ii/at pathway related to tissue injury, inflammation and fibrosis [ ] . in contrast, the activity of the ace /ang - /mas pathway exerts anti-inflammatory and anti-fibrosis effects [ , ] . ace degrades ang ii, as a major substrate for ace , and generates ang-( - ) [ ] . recently, it is well accepted that ace on lung epithelial cells are the entry-point receptors for covid- particles [ ] . it is demonstrated that the highest expression of ace is in the lungs (type ii alveolar epithelial cells), kidney, heart, and also vascular beds [ ] . yu and colleagues in revealed that ace /ang-( - )/mas pathway markedly suppressed in pancreatitis by inhibition of the p mapk/nf-κb signaling pathway [ ] . fu and colleagues in transfected ace plasmid in primary cultured human retinal pigment epithelium cells (hrpe) followed by stimulation with amyloid-β (aβ). they observed that overexpression of ace markedly decreased aβ-induced inflammatory response by activating the ace / ang-( - )/mas pathway in hrpe [ ] . in the respiratory syncytial virus, ace protected against severe lung injury both in children and an experimental mouse model. in addition, in the ards model, ace knockout mice displayed more severe symptoms of respiratory shut down compared fig. the link between "inflame-aging" and cytokine storm in in elderly adults with severe covid- . several aging-related factors may associate chronic inflammation to cytokine storm in elderly patients of covid- with wild-type mice [ ] . indeed, treatment strategy using ace analogs or vector containing ace result in beneficial effects in diabetic nephropathy, hypertension, cardiac disease [ ] . specific inhibitors of at receptors, losartan, have been shown to be effective in animal models of septic shock. therefore, the above mentioned studies suggested that ace pathway has anti-inflammatory effects. several studies identified age-related decline of ace expression [ , , ] . for example, in the study of xudong and colleagues in revealed age-related difference of ace expression revealed in rat lung. they observed ace expression is significantly reduced with aging. they are suggesting the more elevated ace in young adults as compared to age groups may contribute to the predominance in sars attacks in this age group [ ] . using gtex gene expression data and analysis, chen and colleagues in found markedly higher expression of ace in asian females compared to males. furthermore, they found an age-dependent decline of ace expression, and also a highly significant decrease in type ii diabetic patients. additionally, they established a negative correlation between ace expression and covid- fatality. interestingly, in severe cases, many vital tissues, including those with little ace expressed are severely damaged by covid- infection [ ] . these evidences may partially suggest that the increase concentration of ace receptors in lung epithelial cells in children and young adults may have a protective effect on severe clinical manifestations due to covid- infection. therefore, it is highly likely that low ace expression and unbalance ang ii/ang - level during aging can lead to cytokine storm and lung shut down [ , ] . however, the genetic basis of ace expression and its function in different individuals is still far from clear [ ] . it is well accepted that ros considered as a signaling molecule (at low concentrations), and also as a mediator of inflammation (at high concentrations) [ ] . the main sources of ros are mitochondrial respiratory chain and nadph oxidase [ ] . garrido and colleagues in identified that the immune cells of pre-maturely aging mice presented lower values of antioxidant defenses and higher values of ros and pro-inflammatory cytokines [ ] . hence, it is suggested that excess ros production during aging can turn on an inflammatory machine and subsequently increased release of pro-inflammatory cytokines, including; tnf-α, il- β, il- , and il- , and adhesion molecules. the excess ros production in aging can initiate the proinflammatory generation through activation of multiple transcription factors, including human polynucleotide phosphorylase (hpnpaseold- ), nuclear factor kappa b (nf-κb), activator protein (ap- ), specificity protein (sp ), peroxisome proliferator-activated receptors (ppars) [ , ] . for example, it is reported that hpnpaseold- , which is up-regulated during senescence, may promote the activation of nf-κb pathway and initiates the production of proinflammatory cytokines, such as il- and il- [ ] . furthermore, the expression of hpnpaseold- itself induces ros production. this suggests that hpnpaseold- could be an upstream signaling molecule that increased ros generation and subsequent pro-inflammatory cytokines during aging. in addition to hpnpaseold- , nf-κb is also an important transcription factor that up-regulated during aging by excess ros production. in resting states, an inhibitory protein, ikb, inactivated nf-κb in the cytoplasm. however, ros production can phosphorylate inhibitory ikb proteins, leading to nuclear translocation of nf-κb and regulation of gene transcription. then, activated nf-κb can initiate the secretion and release of pro-inflammatory cytokines, including tnf-a, il- , il- , il- , ifn-g, inos, cox- [ ] . interestingly, as the excess ros production can increase pro-inflammatory cytokines, the pro-inflammatory cytokines can also increase ros production [ ] . for instance, it has been identified that the pro-inflammatory cytokine il- can increase ros generation by increased expression of nadph oxidase- in lung cancer [ ] . additionally, it has been demonstrated that the pro-inflammatory cytokine interferon-γ and the pro-inflammatory component of the bacterial cell wall, lipopolysaccharide, can synergistically increase ros generation in human pancreatitis by nf-κb-dependent expression of duox , a member of the nadph oxidase family [ ] . hence, excess ros production and inflammation are closely related, which are taking part in the pathogenesis of chronic inflammation and "inflame-aging" in elderly adults. autophagy is a conserved catabolic turnover pathway in eukaryotic cells by which cellular material delivered into the lysosomes for degradation. autophagy process is related to the maintenance of cellular homeostasis, and its dysregulation could lead to the development of several aging-related pathophysiological diseases [ ] . it has been shown that the autophagy process, decrease during aging, leads to the accumulation of damaged macromolecules and organelles. the decline of autophagy during aging can induce dysfunctional mitochondria, and subsequent increased ros production [ ] . mitochondria are the major source of ros. in this context, two major processes are for protection from harmful effects of ros, including mitophagy and antioxidant capacity. in one hand, mitophagy, which is characterized by autophagic degradation of mitochondria, decreased with aging [ ] . on the other hand, decreased mitophagy, together with decreased antioxidant capacity during aging can increased ros levels in the body. the excess production of ros in stress condition leading to memory deficits, anxiety-like behavior and increase pro-inflammatory cytokine secretion during aging [ ] [ ] [ ] [ ] . although, the exact underlying mechanism of how the decline in autophagy and a rise in ros levels during aging can elevate pro-inflammatory cytokine release is far from clear. however, it is well accepted that low activity of autophagy process and high level of ros production during aging, can activate and upregulate nod-like receptors (nlrs) [ ] . the nlrs are a type of intracellular pattern-recognition receptors (prrs) for pathogen recognition. they monitor both inflammation and apoptosis signaling pathways. these receptors expressed in many cell types, including immune cells (lymphocytes, macrophages, dendritic cells) and even epithelial cells [ ] . it is observed that activation of cytosolic nlrs increased during aging and in many age-related diseases such as type diabetes mellitus. for example, from ebersole and colleagues study in identified that expression of nlrs increased with aging in the healthy oral mucosa [ ] . additionally, luan and colleagues in found that nlrp expression increased in concanavalin a-induced hepatitis (as a model of autoimmune hepatitis) [ ] . salminen and colleagues in reported that the decrease of autophagic capacity during aging generates the inflammatory situation by means the activation of pro-inflammatory factors, in particular nlrp [ ] . it is accepted that nlrs activity can increase expression of caspase- , and pro-inflammatory cytokines, including il- β and il- , leading to cell death (fig. ) . for example, nadatani in reported that caspase- can induce pyroptosis, a unique form of programmed cell death, through the conversion of pro-il β and pro-il fig. the role of alteration of mitophagy in "inflame-aging" and subsequent cytokine storm in elderly adults. the decline of mitophagy during aging, increased ros production. on one hand, excess ros production can activate and up-regulate intracellular nod-like receptor type (nlr ). nlrs over-activity increase expression of caspase- , and pro-inflammatory cytokines, including il- β and il- , leading to pyroptosis (cell death) of immune cells. in other hand, the excess ros production can increase the pro-inflammatory molecules release through activation of multiple transcription factors, including human polynucleotide phosphorylase (hpnpaseold- ), nuclear factor kappa b (nf-κb), activator protein (ap- ), specificity protein (sp ), peroxisome proliferator-activated receptors (ppars) in their active forms, which promotes further inflammation. in pyroptosis, the dying cells release their cytoplasmic pro-inflammatory contents into the extracellular fluid [ ] . similarly, wang's and colleagues revealed that treatment of ac-yvad-cmk, an inhibitor of nlrp -caspase- , suppressed isoflurane-induced microglial inflammatory response in aged mice [ ] . this finding is a critical study for supporting that nlrp /caspase- pathway is involved in the pathophysiology of chronic inflammatory disease in elderly adults (fig. ) . furthermore, stranks in found that macrophages from aged mice exhibit markedly reduced autophagic flux as compared to young mice. they also reported that reduced autophagy during aging, increased macrophage populations and their phagocytosis function, decreased surface antigen expression, while the increased the inflammatory cytokine response [ ] . additionally, in animal model studies, increase autophagy by means caloric restriction and also exercise, result in down-regulation of il- β production and improve the aging-related proinflammatory profile. so, it seems that crosstalk between the decline of mitophagy pathways and elevated ros level during aging can imbalance, immune system activity of elderly adults [ ] . senescent cells accumulate with aging in many animal and human tissues, leading to chronic inflammation and organ dysfunction [ ] . senescent cells have lower cell viability, as well as insufficient protection against oxidative stress [ ] . additionally, senescent cells can release pro-inflammatory cytokines, including il- α, il- β, il- , il- , il- , ccl- , tnf-α, granulocyte macrophages colony-stimulating factor (gm-csf), growth regulated oncogene (gro), monocyte chemotactic protein (mcp)- , mcp- , mmp- , mmp- [ , ] . so, the immune effector is not the main source of inflammatory markers. aging studies revealed the importance of adipose tissue inflammation in aged animals by the elevated release of interleukin , il- , il- β, as well as tnf-α [ ] [ ] [ ] . adipose tissue is a dynamic structure that plays an important contribution in modulating of metabolism and inflammation. it is highly likely that adipose tissue dysfunction (for instance obesity during aging) is associated with chronic inflammation in aged subjects [ ] . petrakis and colleagues in reported that age-related obesity leading to increased susceptibility of more serious complications of covid- as compared to younger individuals. in obese covid- patients, the adipose tissue interacts with the immune system and increased the lethality of the infection by fat tissue-associated cytokines (adipokines) release. adipocytes released amyloid-a (an adipokine), which act directly on macrophages and increased generation of pro-inflammatory cytokines. the mortality rate for young adults with covid- (with normal body mass index) was approximately %. however, the mortality rate for obese elderly adults with covid- was approximately % [ ] . in addition to obesity, covarrubias and colleagues found that during aging senescent cells significantly accumulate in visceral white adipose tissue and inflammatory cytokines found in the supernatant from senescent cells, which are induced macrophages to proliferate and to express cd , as a t cell activation marker [ ] . alicka and colleagues in found that adipose-derived stem cells from older groups exhibited increased gene expression of pro-inflammatory gene and mirnas (such as il- , il- β, tnf-α, mir- b- p, and mir- - p), and apoptosis markers (such as p , p , caspase- , caspase- ) [ ] . ghosh and colleagues in reported that decreased autophagy activity during aging associated with increased adipose tissue er stress and inflammation in old adipose tissue-derived stromal vascular fraction cells (svfs) in mice. they also revealed that accumulation of autophagy substrates lc -ii and p increased in old svfs, implicated impaired autophagy activity. furthermore, they reported that old svfs had reduced expression of autophagy markers. they also analyzed that decreased autophagy activity in old svfs is correlated with increased secretion of pro-inflammatory cytokines, including il- and mcp- [ ] . therefore, the elevated release of pro-inflammatory cytokines by senescence adipocytes possibly leads to the elevated risk of the cytokine storm in covid- infection in poor prognosis patients. with progressive age, the function of the innate and adaptive immune system undergoes physiological and morphological alteration throughout a lifetime, which is characterized as an immune-senescence [ , ] . indeed, immune-senescence is described as the progressive loss of all immune effectors in both the innate and cell-mediated immune systems with aging [ ] . a normal and physiologic immunity depends on effective cross-talk between innate and adaptive immune systems, so senescent immune effectors markedly impact on the health of elderly individuals [ ] . here, we are going to briefly report age-related alteration of both innate and adaptive immune cells. macrophages are central effector cells of the innate immune system and have many physiological functions [ ] . macrophage can produce several pro-(tnfα, il- , and il- ) and anti-inflammatory (il- , tgfβ, acute phase proteins, and glucocorticoids) molecules, enzymes, growth factors, nitric oxide, toxic reactive radicals, metalloproteinases and inhibitors of metalloproteinases. as the clearance of pathogens proceeds, the anti-inflammatory effectors of macrophages turn off macrophage inflammatory activities [ ] . therefore, during a pathogen-induced inflammatory episode, the balance of macrophage modulating secretion present in the tissues. during aging, the generation of several macrophage-induced factors is reduced, including fibroblast growth factor, vascular endothelial growth factor, epithelial growth factor, tgfβ, toxic free radicals, and expression of nitric oxide synthase. additionally, phagocytic and chemotactic activity of macrophages also decreased by a decline in production of macrophage-specific chemokines, including macrophage inflammatory protein (mip)- and mip- with advanced age. macrophages can also display antigen-presenting activity by expressing major histocompatibility class (mhc), leading to a cross-talk between innate and cell-mediated immune system. it is reported that with progressive age, the expression of (mhc)-ii, decreased in both mice and humans [ , ] . natural killer (nk) cells are another cytotoxic effector of the innate immune system and involved in the early, and fast, faster than t cells, defense against virus infection and other challengers [ ] . the nk cells, such as macrophages, linking innate and cell-mediated immune system. nk cells regulate immune function by the generation and release of various cytokines [ ] . an increase in numbers of circulating nk cells reported during aging [ ] . one of the important cytokines for cytotoxic activity of nk cells is il- , which increases killing properties and proliferation of nk cells. in a healthy young individual, il- can induce ifn secretion by nk cells, but this effect decreased in elderly [ ] . furthermore, aging can change the nk cells phenotypes, which are an increase in cd dim cells and a decrease in cd bright cells. in addition, aging can increase the expression of the immune-senescence marker, cd , on nk cell populations [ ] . dendritic cells (dcs), are another component of the innate immune system and have a critical role in both immunity and tolerance. the resident dcs are immature, but the capture of pathogens converts them to mature form, known as antigen-presentation cells (apcs), through up-regulation of mhc expression. then, mature apcs monitor th and th function. in contrast, immature dcs induce tolerance to self-antigens [ ] . in physiological condition, dcs take up self-antigen and apoptotic cells and transport them to the lymph nodes [ ] . but, during migration for the presentation of pathogens to th cells, they undergo several phenotype alterations, including up-regulation of mhc class i and ii molecules and down-regulation of adhesion molecules [ ] . furthermore, dcs can generate and release various cytokines, so they can modulate inflammatory responses [ ] . it is reported that increased age-related pro-inflammatory cytokines can induce activation and maturation of dcs. panda and colleagues in identified an increase of pro-inflammatory cytokines released from dcs in elderly adults [ ] . moreover, the activity of dcs in elderly adults was higher than young individuals [ ] . ageing is also accompanied by profound and consistent alterations of t-cell immunity [ ] . although, t-cell numbers do not diminish during aging, however, the t-cell pool exhibit potent age-related alterations, including poor t-cell mitogen responses, an inverted cd +/cd + t-cell ratio, reduced proportion of naive cells, as well as an increased proportion of memory cells, in older animals and human [ ] . additionally, aging is related to the overproduction of pro-inflammatory cytokines by t cells, leading to immune pathology [ ] . the cd +cd − subset of cells in the expanded memory cell population has shortened telomeres, suggesting that they have a longer reflective history [ ] . in humans, almost all t cells express cd at birth, and the proportion of cd + declines by the age [ , ] . the increase in these cells has been observed consistently and is used as a prognostic indicator of immune-senescence in older populations [ ] . it is demonstrated that aging also changes the cytokine profile of th cells (il- and il- ) rather than th type (il- , ifn-g), leading to mild age-related inflammation in elderly adults. aging can also potentially affect other th cells pool. the ratio of th cells/ t regulatory cells increased during aging, leading to a basal inflammatory state in elderly adults [ ] . th cells have the pro-inflammatory phenotype, and they are in balance with tregulatory anti-inflammatory cells. these cells are derived from a common precursor (th ) [ ] . t regulatory cells are a subset characterized by a high expression of cd and foxp , a transcriptional factor for the function and differentiation of t regulatory cells [ ] . in addition to anti-inflammatory effects of t regulatory cells, also recognize self-antigens [ ] . furthermore, ageing is correlated with disruption of lymphocyte telomerase up-regulation [ ] . it is accepted that shortened lymphocyte telomeres are associated with a variety of age-associated pathologies [ ] . furthermore, during aging naïve t-cells show multiple alterations, including the shortening of telomeres, the reduced production of il- and the diminished ability to differentiate themselves into effector-cells. the loss in the number and function of the naïve t-cells, with increasing of t cd +, cd ro+, cd + clones in aged subjects [ ] . cd -cells are responsible for the production of pro-inflammatory cytokines and are resistant to apoptosis. it is proposed that they are undergoing cells to senescence, due to the shortening of telomeres and reduction of the proliferative capacity [ ] . interestingly, pro-inflammatory cytokines have been also implicated in these age-associated alterations of t-cell immune-senescence. indeed, inflammatory conditions in elderly adults lead to alterations in t-cell immunity. for instance, age-related increased cytokine tnfα is a potent stimulator of tcd + cell senescence and t-cell differentiation [ ] . humoral immunity mediated by b lymphocytes has a critical role in the modulation of adaptive immunity responses. b lymphocytes produce different types of antibodies for eliminating of challengers. additionally, b lymphocytes have an important role in the immune system through the presentation of antigens and secretion of cytokines [ ] . b lymphocytes arising from hematopoietic stem cells in the bone marrow as pro-b lymphocytes. then, they differentiate into pre-b and then b lymphocytes [ ] . ample studies identified that aging is accompanied by quantitative and qualitative alteration of b lymphocyte pool [ ] . the number of b lymphocytes and the levels of serum immunoglobulin and antibody analyzed during the aging. the hematopoietic stem cells in the bone marrow from aged mice are less effective at generating both b lymphocytes as compared to young mice. the number and the size of the germinal centers of b lymphocytes also decreased with aging. production of precursor b lymphocytes in the bone marrow and the number of pro-b and pre-b lymphocytes decreased in aged mice and human [ ] . it has been shown that aged pro-b lymphocytes havean impaired ability to respond to il- . also, both human naïve and igm memory b lymphocytes impaired during aging [ ] . the percentage of igm memory b lymphocytes are not markedly decreased, however, the total numbers of b lymphocytes decreased. additionally, the antibody level is decreased in the aged subjects. in addition, the affinity and protective ability of antibodies in aged mice decreased as compared to the young mice [ , ] . it is also reported that the immune response to influenza in old mice has less igg level than in young mice. also, young mice had mostly igg plasma level with high-affinity antibody. in contrast, aged mice mostly had igm plasma cells [ ] . hence, antigen-specific antibody responses decline in old mice. the decreased function of b lymphocytes (such as antigen-specific antibody response and antibody affinity) during aging has been attributed to lack of th function. because the function of b lymphocytes is t-dependent [ , ] . taken together, immune-senescence alterations cannot properly fit cell-mediating and humeral immune response in elderly adults. the immune system appears to maintain a mild inflammatory state in elderly adult. therefore, it is suggested that fragile and mildly overactive immune system in elderly adults cannot turn off the pro-inflammatory machine in covid- infection. clinical findings in severe patients with covid- infection are in consistent with the above mention literature. several manifestations including; lymphopenia, reduced numbers of cd + t cells, cd + t cells, b cells and natural killer (nk) cells, monocytes, eosinophils and basophils reported in severe patients with covid- infection [ ] . schouten and colleagues in identified that increasing pro-inflammatory cytokines during aging also correlated with the severity of ards and may partially explain age-dependent difference [ ] . vd together with vitamin d receptor (vdr) has both classical functions (such as bone and calcium-phosphorus homeostasis), and non-classical function (such as antiinflammatory and immune-regulatory function) [ , ] . vdr is expressed by several types of immune cells, including monocytes, macrophages, b and t lymphocytes, as well as dcs [ ] . additionally, the α- -hydroxilase enzyme, which converts inactive metabolite of vd ( (oh) d ) to the active form ( , (oh) d ), is expressed by the majority of immune cells such as macrophages [ ] . liu and colleagues in reported that the expression of vdr and α- -hydroxylase increased in macrophages following exposure to a pathogen [ ] . this finding suggested that the intracrine immune-regulatory function of vd. as shown in fig. , vd can decrease the expression of pro-inflammatory genes (such as tnf-α, il- , monocyte chemotatic protein (mcp- ), and il- β) in immune cells through suppressing excessive ros production, increasing intracellular glutathione levels, suppressing nf-κb and p map kinase expression. excess ros production can increase nf-kb expression in immune cells, leading to excess secretion of pro-inflammatory cytokines, including tnf-a, il- , il- , il- , ifn-g, inos, cox- [ ] . activation of p map kinase pathway can also increase il- and mcp- generation in immune cells by stimulation of the signal transducer and activator of transcription / (stat / ) [ ] . it seems that pathogen challengers turn macrophages on by stimulation of pprs (such as tlr / or nlrs). activation of tlrs or nlr can increase intracellular vdr and α- -hydroxylase expression. then, a complex of vd and vdr together can decrease the pro-inflammatory cytokines, increase the autophagy activity of macrophages, and antimicrobial product generation, including cathelicidin and β-defensin in macrophages [ ] . in addition, of immune regulatory effects of vd on macrophages, it can also suppress the differentiation, and migration of human dcs, and decrease the expression of mhcii on dcs, which are characterized as the tolerogenic properties [ , ] . tolerogenic dcs can increase the number of il- producing cd + t-cells and t regulatory cells. elevated circulating cd + t regulatory have of anti-inflammatory functions and also attenuated the inflammatory response of t-effector cells. these tolerogenic actions of dcs are mediated by increased expression of foxp transcription factor [ ] . furthermore, vd can also make a complex with vdr on the t lymphocytes, leading to suppression of its proliferation. hoe and colleagues in reported that vd markedly decreased proinflammatory cytokines (tnf-α, ifn-γ, and il- β, il- , ifn-γ) in response to bacterial ligands exposure. vd also increased the level of anti-inflammatory cytokine (il- ) [ ] . hence, vd can modulate both innate and adaptive immune responses. elderly adults are at risk for vd deficiency due to several factors, including decreased pre-vd production, poor skin integrity decreased dietary intake of vd, increasing adiposity, obesity, decreased renal function, as well as less time spent outdoors [ ] . vd deficiency has been linked to various aging-related inflammatory diseases, including rheumatoid arthritis, asthma, inflammatory bowel disease, multiple sclerosis, cardiovascular disease, hypertension, diabetes mellitus, and cancer [ ] . additionally, there is a correlation between vd deficiency and risk of respiratory tract infection such as covid- [ ] . for example, ilie and colleagues in examine the association between the mean levels of vd in european countries and morbidity and mortality caused by covid- . their analysis data identified negative correlations between mean levels of vd (average mmol/l) in each country and the number of covid- cases. in addition, a negative correlation was observed between mean levels of vd and the mortality of covid- cases. they also reported that vd levels are severely low in the aging population especially in spain, italy and switzerland, the most vulnerable countries in relation to covid- [ ] . additionally, ebadi and montano-loza in reported that vd can suppress the expression of pro-inflammatory markers, including il- α, il- β, as well as tnf-α. therefore, vd deficiency during aging related to overexpression of th cytokines. they also reported that % of patients with covid- and about % mortality of covid- observed in african-american population in chicago, who are at a greater risk for vd deficiency [ ] . there are reports that polymorphism of vdr gene, including polymorphisms of foki, apai, and taqi, is associated with vd deficiency and increased risk of inflammatory diseases [ ] . hence, vd and vdr pathway together have an important anti-inflammatory function fig. the role of vitamin d content on pro-inflammatory cytokine release in young and elderly adults with covid- infection. following exposure to covid- particles in young adults, sufficient vitamin d content increasing intracellular glutathione levels, suppressing excessive ros production, suppressing nf-κb (nuclear factor kappa b) and p map kinase expression. in contrast, vitamin d deficiency in elderly adults leads to over-activity of p map kinase/ stat (signal transducers and activator of transcription) and ros/ nf-Κb pathways in the immune cells. so, elderly adults with severe covid- infection cannot turn off their pro-inflammatory immune machine in immune effectors through decreasing pro-inflammatory cytokine generation and increasing anti-inflammatory cytokines in immune cells. furthermore, the lack of vd in aged subjects is associated with the pro-inflammatory phenotype of immune cells, leading to likely increasing the risk of elderly adults with chronic mild inflammation condition [ ] . this chronic inflammatory condition likely leads to cytokine storm in elderly covid- patients. in summary, it seems that young adults have balanced between pro-inflammatory and anti-inflammatory cytokine networks (fig. ) . therefore, their balanced immune system can limit the progression of covid- infection. however, elderly patients do not have the same balanced immune response as young adults. as shown fig. , with advancing age, the immune system appears to maintain a condition of mild inflammation. so, the activation of the body with pathogens, such as covid- infection can exaggeratedly increase the amplitude of the immune response, which is known as a cytokine storm. as mentioned above, alteration of ace receptor expression, oxidative stress, adipose tissue-and immune-senescent cell activity, lack of vd content, as well as decrease of autophagy and mitophagy may contribute to high amplitude of the immune response to external challengers in elderly adults. this high amplitude of the immune response in elderly adults can favor induction of the cytokine storm and death in severe and critical cases of covid- infection. nevertheless, the covid- infection is not deadly in all elderly patients, because the aging process is dependent on several markers, including genes, clinical 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date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: fl l b metabolic abnormalities such as dyslipidemia, hyperinsulinemia, or insulin resistance and obesity play key roles in the induction and progression of type diabetes mellitus (t dm). the field of immunometabolism implies a bidirectional link between the immune system and metabolism, in which inflammation plays an essential role in the promotion of metabolic abnormalities (e.g., obesity and t dm), and metabolic factors, in turn, regulate immune cell functions. obesity as the main inducer of a systemic low-level inflammation is a main susceptibility factor for t dm. obesity-related immune cell infiltration, inflammation, and increased oxidative stress promote metabolic impairments in the insulin-sensitive tissues and finally, insulin resistance, organ failure, and premature aging occur. hyperglycemia and the subsequent inflammation are the main causes of micro- and macroangiopathies in the circulatory system. they also promote the gut microbiota dysbiosis, increased intestinal permeability, and fatty liver disease. the impaired immune system together with metabolic imbalance also increases the susceptibility of patients to several pathogenic agents such as the severe acute respiratory syndrome coronavirus (sars-cov- ). thus, the need for a proper immunization protocol among such patients is granted. the focus of the current review is to explore metabolic and immunological abnormalities affecting several organs of t dm patients and explain the mechanisms, whereby diabetic patients become more susceptible to infectious diseases. the metabolic syndrome is defined by the presence of metabolic abnormalities such as obesity, dyslipidemia, insulin resistance, and subsequent hyperinsulinemia in an individual ( ) . dyslipidemia, the main characteristic of metabolic syndrome, is defined by decreased serum levels of high-density lipoproteins (hdls) but increased levels of cholesterol, free fatty acids (ffas), triglycerides (tg), vldl, small dense ldl (sdldl), and oxidized ldl (ox-ldl) ( table ) ( ) . individuals with the metabolic syndrome are much more likely to develop type diabetes mellitus (t dm), cardiovascular diseases (cvds), and fatty liver disease ( ) ( ) ( ) . t dm, the most common form of diabetes (∼ %), is characterized by a systemic inflammatory disease accompanied by insulin resistance (ir) or decreased metabolic response to insulin in several tissues, including the adipose tissue, liver, and skeletal muscle, as well as by reduced insulin synthesis by pancreatic beta cells ( , ) . studies on immunometabolism have indicated that the metabolic states and immunological processes are inherently interconnected ( ) . in this scenario, metabolites derived from the host or microbiota regulate immunological responses during health and disease ( ) . accordingly, in obese individuals, expanded adipose tissue at different locations, by initiating and perpetuating the inflammation, induces a chronic low-level inflammatory state that promotes ir ( ). every organ system in human body can be affected by diabetes, but the extent of organ involvement depends largely on the severity and duration of the disease (figure and table ). during the progression of diabetes, hyperglycemia promotes mitochondrial dysfunction and induces the formation of reactive oxygen species (ros) that cause oxidative stress in several tissues such as blood vessels and pancreatic beta cells ( ) ( ) ( ) . accumulating damage to the mitochondria, as well as several macromolecules, including proteins, lipids, and nucleic acids by ros promotes the process of aging ( ) . as a result, pancreatic β cells that require functional mitochondria to maintain insulin synthesis fail to generate high enough levels of insulin ( , ) . in the absence of compensatory mechanisms, stress-responsive intracellular signaling molecules are activated and cellular damage occurs. elevated intracellular levels of ros and subsequent oxidative stress play an important role in the pro-atherosclerotic consequences of diabetes and the development vascular complications ( , ) . moreover, the non-enzymatic covalent attachment of glucose and its toxic derivatives [e.g., glyoxal, methylglyoxal (mgo), and deoxyglucosone] to the biological macromolecules such as nucleic acids, lipids, and proteins leads to the formation of advanced glycation end products (ages) ( , ) . accumulated ages block the insulin signaling pathway and promote inflammation ( , ) . in addition, the attachment of ages to their receptors [e.g., cd , galectin- , scavenger receptors types i (sr-a ), and ii (sr-a )] on the surfaces of immune cells in the circulation and tissues activates the expression of pro-inflammatory cytokines and increases free radical generation ( ) . furthermore, due to the chronic exposure of cells to high glucose levels in untreated t dm patients, glucose toxicity might occur in several organs. this will figure | effects of t dm on body organs. t dm is an inflammatory state that affects circulatory system, gastrointestinal tract, pancreatic beta cells, liver, and skeletal muscles and makes them dysfunctional. nfald, non-alcoholic fatty liver disease; nash, non-alcoholic steatohepatitis; er, endoplasmic reticulum. eventually lead to nephropathy, cardiomyopathy, neuropathy, and retinopathy. gut microbiome dysbiosis is another important factor that can facilitate the induction and progression of metabolic diseases such as t dm ( ) . the gut microbiome dysbiosis, by altering the barrier functions of intestine and the host metabolic status, promotes the insulin resistance in diabetic patients ( ) . diabetes also impairs the immune system and increases the susceptibility of patients to serious and prolonged infections ( ) . this is likely to be the case with the severe acute respiratory syndrome coronavirus (sars-cov- ), as well ( , ) . in the current paper we will review recent research to explore the impairment of body organs in t dm patients and explain how diabetic patients become more susceptible to certain infectious diseases. vascular homeostasis is an important function of the endothelium. under homeostatic conditions, the ecs maintain the integrity of blood vessels, modulate blood flow, deliver nutrients to the underlying tissues, regulate fibrinolysis and coagulation, control platelet adherence and patrol the trafficking of leukocytes (figure a ) ( ) . normal ecs also internalize high-density lipoproteins (hdls) and its main protein part apolipoprotein a-i (apoa-i) in a receptor-mediated manner to activate endothelial cell nitric oxide (enos) synthase and promote anti-inflammatory and antiapoptotic mechanisms ( figure b ) ( ) . hdl receptors on the surfaces of ecs include: the atp-binding cassette (abc) transporters a and g , the scavenger receptor (sr)-b and the ecto-f -atpase ( ) . according to the epidemiological studies, diabetes mellitus is considered as one of the main risk factors for cvd (figure ) ( ) . from the beginning of t dm, the functions of ecs are impaired, which is the main cause of disease-related side-effects ( ) . ecs can initiate and perpetuate the inflammatory milieu during the pathogenesis of diabetes. due to the negative impacts of hyperglycemia and subsequent oxidative stress, cvds are more common among diabetic patients ( ) . it has been observed that incubation of human aortal endothelial cells (haecs) with a medium containing high glucose concentrations (hg, mm) increases the intracellular levels of mgo and glycated proteins that in turn activate the unfolded protein response (upr) and trigger inflammatory and prothrombotic pathways ( ) . glycated apoa-i, which is formed during hyperglycemia, modifies its structure, decreases its lipid-binding ability, prevents cholesterol efflux from macrophages and impairs its anti-inflammatory function ( , ) . vaisar et al. have shown that hdls from diabetic patients have a reduced capacity to trigger enos production and suppress tumor necrosis factor-α (tnf-α)mediated inflammatory responses within ecs ( ) . diseases such as t dm that induce high levels of vascular injury are accompanied by an elevated number of circulating endothelial cells (cecs) ( ) . t dm-related risk factors such as dyslipidemia, hyperglycemia, and hyperinsulinemia as well as other conditions (e.g., inadequate physical activity, smoking, and high blood pressure) facilitate the formation of atherosclerotic plaques/lesions ( ) . dyslipidemia, due to the elevated flux of ffa from insulin-resistant tissues and spillover from entry (c) blood vessels in t dm patients. during the progression of the disease, red blood cells become glycated, while activated ecs synthesize elevated levels of adhesion molecules and chemokines that facilitate monocytes recruitment, adhesion, and transmigration across the endothelium toward the subendothelial region. monocytes are then differentiated into macrophages and eventually, by excess lipid uptake, generate foam cells. subsequently, further immune cell infiltration into the atherosclerotic lesion occurs, where their inflammatory cytokines promote platelet activation, ec apoptosis, and increased generation of ros and ox-ldl. (d) interactions between oxldl and its receptor aggravate ros generation, nf-κb activation and inflammation. ec, endothelial cell; rbc, red blood cell; plt, platelet; hdl, high-density lipoprotein; ox-ldl, oxidized low-density lipoprotein; ros, reactive oxygen species; enos, endothelial nitric oxide synthase; no, nitric oxide; lox- , lectin-type oxidized ldl receptor . into adipocytes, is considered as an important risk factor for developing cvd among diabetic patients. this is because dyslipidemia promotes inflammation, endothelial dysfunction, and platelet hyperactivation ( , ) . during the progression of atherosclerosis, lipids, immune cells, and extracellular matrix accumulate in the arterial intima or subendothelial regions ( figure c ) ( ) . advanced plaques can impede blood flow and cause tissue ischemia or might become disrupted and generate a thrombus that stops the blood flow of important organs. vascular complications of diabetes engage either tiny or large blood vessels (micro-and macroangiopathy, respectively). microangiopathies, which can be seen in the kidneys, vasa nervorum and eye tissues, cause nephropathy, neuropathy, and retinopathy. macroangiopathies, by inducing atherosclerosis in the coronary, carotid, and peripheral arteries, increase the risk of myocardial infarction (mi), stroke and peripheral artery disease (pad). macrovascular complications due to ec dysfunction are considered as an important cause of mortality and morbidity among diabetic patients ( ) . oxidative stress has an essential role in the induction of vascular complications during the course of diabetes ( ) . ec dysfunction (e.g., delayed replication, dysregulated cell cycling, and apoptosis), as well as enhanced ox-ldl formation are some consequences of oxidative stress. it has been well-established that sdldl and ox-ldl have an enhanced atherogenic ability and are more useful biomarkers than total ldl for predicting cvd ( , ) . sdldl particles have a smaller size than other ldl particles. thus, sdldl particles are more easily oxidized, and their atherogenic potential is enhanced. during oxidative stress, levels of ox-ldl increase by the excess action of reactive oxygen species (ros) ( ) . subsequently, ox-ldl interaction with scavenger receptors, including cd , sr-a /cd , sr-b , and lectin-like ox-ldl receptor- (lox- ) on the surface of ecs activates the nadph oxidase that in turn increases the expression of ros and activates the transcription factor nf-αb ( ) . afterwards, the expression of lox- , adhesion molecules (e.g., selectins and integrins) and the secretion of pro-inflammatory cytokines and chemokines are increased, while no synthesis is decreased in ecs ( figure d ) ( ) ( ) ( ) . ec-derived chemokines bind to their cognate receptors on the surfaces of monocytes and recruit them toward the inflamed endothelium. following this, selectin-based rolling and integrin-based attachment of monocytes to the ecs cause their migration toward the subendothelial region, where they develop into lipid-laden macrophages or foam cells later on ( ) . the scavenger receptor lox- plays an important role in the uptake of ox-ldl during atherogenesis. it is strongly expressed on the surfaces of ecs, but has an inducible pattern of expression on the surface of macrophages and smooth muscle cells ( ) . the accelerated uptake of ox-ldl by macrophages accounts for their transformation into foam cells, the initial hallmark of atherosclerosis ( , ) . besides, diabetes leads to both quantitative and qualitative defects in circulating angiogenic progenitor cells (capcs) that take part in the repair of injured endothelium ( ) . it has been shown that humans or mice with decreased numbers of cd + cd + cd + cd dim sca- + flk- + capcs have an increased prevalence of t dm, elevated hba c levels and aggravated cvd risk scores ( , ) . in diabetic patients, despite elevated serum levels of proangiogenic molecules, like angiopoietin- / , epo, and vegf-a, angiogenesis is impaired. this is mainly due to the decreased expression levels of vegfr and cxcr on the surfaces of capcs, which makes them unresponsive to the angiogenic factors ( , ) . it has also been shown that circulating proangiogenic granulocytes composed of eosinophils and neutrophils are also impaired in diabetic patients ( ) . besides, elevated levels of ages in t dm cause ec dysfunction and vascular inflammation ( ) . ren et al. have shown that incubation of human coronary artery endothelial cells (hcaecs) with ages causes decreased expression (at both mrna and protein levels) and enzymatic activity of enos, increased levels of ros, diminished mitochondrial membrane potential and declined activity of catalase and superoxide dismutase in treated cells ( ) . another study by lan et al. has shown that ages in the pancreas decrease ec viability and induce their apoptosis in an nfκb signaling-related manner ( ) . however, apigenin ( ′ , , trihydroxyflavone) can protect ecs against oxidative stress and subsequent inflammatory reactions mediated by ages ( ) . apigenin binds to methylglyoxal (mgo) and forms a complex that inhibits age formation. chettab et al. have shown that the expression of icam- as well as the production of il- , are significantly increased in huvecs cultured in hg medium compared to cells cultured in normal glucose (ng, . mm) conditions ( ). bammert et al. found out that incubation of huvecs with hg media promotes the generation of endothelial microparticles (emps) that, when added to normally cultured huvecs, downregulate the expression of anti-apoptotic microrna mir-let a, but enhance the synthesis of active caspase- and cause cell apoptosis ( ) . several micrornas, including mir- , mir- a, mir- , mir- a, mir- , mir- , mir- a, mir- , and mir-let d, regulate vascular homeostasis. it has been shown that the expressions of mir- a and mir- are significantly reduced in circulating mps isolated from diabetic patients compared with normal individuals. this could be involved in making diabetic individuals more susceptible to coronary heart disease ( ) . moreover, hg media upregulate the expression of nadph oxidase that will induce the generation of ros. this leads to subsequent apoptosis of the huvecs through a ros-dependent caspase- pathway ( ). su et al. have demonstrated that argirein medication, by inactivating nadph oxidase, can prevent endothelial cell apoptosis in a rat model of t dm and hence attenuate vascular dysfunction ( ) . hg further increases the permeability of the huvecs in a protein kinase c (pkc)-dependent manner ( , ) . hassanpour et al. showed that incubation of endothelial progenitor cells with the serum of t dm patients inhibits their migration toward bfgf, increases their expression of vegfr- , but reduces their expression of vegfr- and induces their apoptosis ( ) . however, humanin (hn), a mitochondriumderived peptide, is cytoprotective against apoptosis during pathological conditions, such as diabetes mellitus ( ) . it has been demonstrated that simultaneous incubation of h c cells, a line of rat cardiac myoblasts, with h o and hn decreases the intracellular levels of ros, preserve mitochondrial function/structure and decline cellular apoptosis ( ) . wang et al. have indicated that the treatment of huvecs with hn before their incubation with hg medium increases the expression of enos, while decreasing the expression of endothelin (et- ), vcam- , tnf-α, il- β, and e-selectin in a krüppellike factor (klf )-dependent manner. such changes in the expression of integrins prevent the attachment of monocytes to huvecs ( ) . accordingly, hn might be used to prevent the development of hyperglycemia-associated ec dysfunction in t dm. ec activation and expression of adhesion molecules also facilitate activation and adhesion of platelets. this will increase the risk of thrombosis and promote the development of thrombotic angiopathy, typical for diabetic patients. platelets are tiny anucleated cellular fragments generated from megakaryocytes in the bone marrow. they circulate in the blood for ∼ - days and play essential roles in hemostasis and in controlling vascular integrity ( ) . circulating inactive platelets move in the proximity of vessel walls (figure a ) and rapidly get activated in response to vascular injury. at the end of their life, platelets are cleared from circulation with the action of the liver and spleen-resident macrophages. platelets have an essential role in the initiation and progression of inflammation. platelet hyperactivation that occurs during inflammatory states (e.g., t dm) facilitates the pathogenesis of cvds ( figure c ) ( , ) . it has been shown that elevated levels of resistin, an adipokine, in diabetic patients enhances oxidative stress, promotes endothelial dysfunction and facilitates platelet activation ( ) . activated platelets with an increased mean volume [mean platelet volume (mpv)] secrete microparticles (mps) and soluble adhesion molecules (e.g., sp-selectin and scd l) that in turn activate endothelial and immune cells ( ) ( ) ( ) . higher levels of platelet-derived mps, which correlate positively with fasting blood sugar and glycated hemoglobin, have been shown in newly diagnosed t dm patients compared to healthy individuals ( ) . in t dm patients thrombotic microangiopathies can lead to the development of cvds ( ) . platelets in the patients adhere to ecs and aggregate more rapidly than in healthy individuals thereby increasing the risk of thrombosis. in a mouse model of t dm, zhu et al. have shown that ages interact with cd , a member of the type scavenger receptor family, on the surfaces of murine platelets to activate them and induce a prothrombotic state ( ) . elevated levels of the p y receptor on the surface of platelets in t dm expose diabetic patients to a prothrombotic condition. this receptor has an essential role in platelet activation ( ) . zhou et al. have shown that long non-coding rna (lncrna) metallothionein pseudogene (mt p ), which is markedly upregulated in megakaryocytes of t dm patients, enhances the expression of p y receptor in platelets ( ) . they indicated that this is due to the inhibitory action of mt p on mir- . virtually all parts of the human digestive system, including the gastrointestinal tract, pancreas, and the liver are affected by diabetes. the git is populated with a myriad of microorganisms, including principally bacteria but also archaea, viruses, fungi, and protozoans that dynamically influence the health status and homeostasis of the host. the physiological functions of the git resident microbes improve gut integrity, protect against microbial pathogens and regulate immune responses ( ) . mucosal barriers, such as intestinal epithelial cells (iecs) and the mucus layer, spatially isolate the host immune system and gut microbiota to prevent unnecessary immune activation and intestinal inflammation. they also facilitate the uptake of nutrients through receptors and transporters. however, hyperglycemia, in a glut -dependent manner, can influence the mucus and alter the integrity of adherence and tight junctions between intestinal epithelial cells of diabetic mice. this will enhance the permeability of the intestinal barrier leading to so called "leaky gut." subsequently, hyperglycemia may facilitate the dispersal of an enteric infection into a systemic infection (figure ) ( ) . interestingly, the reversal of hyperglycemia, conditional deletion of glut from the iecs and inhibition of glucose metabolism will fix the barrier dysfunction and prevent the spread of bacteria ( ). xu et al. have shown that faecalibacterium prausnitzii, one of the most frequent commensal bacteria in normal individuals with essential roles in gut homeostasis, generates anti-inflammatory molecules that enhance the expression of tight junctions and improve intestinal integrity during diabetes ( ) . however, in some cases, gut microbiota dysbiosis or altered microbial composition of the intestines could induce t dm and lead to its progression ( ) . of interest, the widely used antidiabetic drug metformin can improve barrier integrity and restore the healthy microbiota composition of the gut in diabetic patients ( ) . the intestinal commensal bacterium akkermansia muciniphila can also act as a sentinel to reduce microbial translocation across the gut and prevent the subsequent inflammation in patients with t dm ( ) . hyperglycemia can further decrease the intracellular levels of glutathione (gsh) but increase inos activity and no production in the iecs ( ). zhao et al. have found out that hyperglycemia in a pkcα-dependent manner inhibits the ubiquitination, internalization and degradation of the divalent metal transporter (dmt ) present on the microvillar membranes of iecs. subsequently, intestinal iron uptake is enhanced and accumulated iron ions aggravate diabetes-related complications and increase mortality ( , ) . the pancreas consists of the exocrine and endocrine compartments. the endocrine part is made of different cell types, including α, β, δ, and ε cells that secrete glucagon, insulin, somatostatin, and ghrelin hormones, respectively. these cells are aggregated into specialized structures called islets of langerhans, which play an important role in controlling blood glucose levels through the secretion of insulin and glucagon. in t dm, despite normal levels of β-cell replication and islet formation, β-cell apoptosis is increased so that the number of cells declines by ∼ % (figure ) ( ) . during the progression of t dm, the insulin-resistant state forces β-cells to compensate for the lack of insulin by elevating its synthesis to restore the normal blood glucose level. however, in severe diabetic patients, β-cell exhaustion, and subsequent persistent hyperglycemia occur ( ) . furthermore, chronic elevated serum levels of free fatty acids, seen in obesity and t dm, induce lipotoxicity in beta-cells and suppress their insulin secretion ability ( ) . to alleviate chronic inflammation, overcome insulin resistance (ir) and to prevent β-cell apoptosis, stem cells or stem cell derivatives such as insulin-producing cells (ipcs) and exosomes have been suggested ( ) ( ) ( ) ( ) . their effects are believed to be mainly due to their anti-inflammatory activities. secretagogin (scgn) is predominantly expressed by pancreatic β-cells protecting their normal functions. scgn also acts as an insulin binding protein to make it more stable, avoid its aggregation, improve its functions and enhance its secretion ( , ) . in t dm patients, due to the islet cell dysfunction and endoplasmic reticulum (er) stress, serum levels of scgn are elevated reflecting stress and dysfunctional islet cells ( ) . moreover, in patients with t dm, islet amyloid polypeptide (iapp or amylin), a peptide hormone and one of the main secretory products of pancreatic β-cells, tends to deposit in the islets of langerhans, form insoluble fibrils and impair secretory functions of β-cells ( ) . iapp is costored with insulin in the secretory granules of pancreatic β cells. in steady-state conditions it regulates food intake, insulin secretion, and glucose metabolism ( ). ribeiro et al. have noted that pancreatic extracellular vesicles (evs) from healthy individuals, but not from t dm patients, directly bind to iapps and prevent amyloid formation within the pancreatic islets ( ) . the authors showed that the altered protein-lipid composition of the evs is the main reason for this discrepancy ( ) . however, chatterjee et al. have shown that β-cells from t dm patients have a dysfunctional proteasome complex that fails to degrade pancreatic iapp, whereby amyloid formation is induced ( ) . furthermore, in t dm patients, lipids accelerate the formation of fibrillary iapp, which aggravates islet cell damage ( ) . dhar et al. have demonstrated that chronic use of mgo in sprague-dawley rats increases the expression of nf-αb, mgo-derived ages and their receptors in pancreatic β cells. mgo can also induce apoptosis of islet β cells, increase fasting plasma glucose levels and impair glucose tolerance ( ) . in t dm patients the plasma level of mgo directly correlates with fasting blood sugar and hba c levels ( ) . bo et al. further showed that mgo in a dose-based manner impairs insulin secretion of pancreatic β-cell lines min and ins- through increased generation of ros and by induction of mitochondrial dysfunction ( ). robertson et al. have found out that elevated levels of ros in pancreatic β-cells inhibit the pancreas duodenum homeobox- (pdx- ) transcription factor that is needed for insulin synthesis ( ) . it has been shown that chronic use of mgo in animals could induce t dm, while simultaneous use of alagebrium, which breaks age compounds, attenuates the disease ( ) . it has also been reported that during the course of diabetes dedifferentiation and conversion of β-cells into αand δ-"like" cells occurs ( ) . in conclusion, the pancreatic β cell function is progressively reduced during the progression of t dm. the liver is by far the most important metabolic organ with essential roles in regulating homeostasis and mediating glucose and lipid metabolism. metabolic activities of the tissue are precisely controlled by the actions of metabolic substrates, including free fatty acids (ffas) and hormones ( ) . t dm patients usually suffer from a chronic liver condition called non-alcoholic fatty liver disease (nafld). it is characterized by steatosis that means ectopic fat storage in hepatocytes and subsequent insulin resistance (figure ) ( ) . lipid accumulation in hepatocytes leads to impaired biogenesis of mir- that facilitates insulin signaling and prevents lipogenesis ( ) . several factors such as obesity, increased serum levels of fatty acids, and insulin resistance can increase the risk of fatty liver disease. p y receptor, through the induction of the c-jun n-terminal kinase (jnk) and prevention of insulin signaling, can promote insulin resistance in hepatocytes in t dm ( ) . in some cases, nafld may progress into an aggressive form of inflammatory fatty liver disease called non-alcoholic steatohepatitis (nash), which might cause liver cirrhosis and organ failure ( ). dang et al. have indicated that exosomes released from the adipose tissues of obese mice due to the smaller mir- - p content can promote insulin resistance in the murine hepatocyte cell line aml (alpha mouse liver ) ( ) . the adipokine visfatin that is released from the adipose tissue of obese individuals has also been shown to activate the pro-inflammatory stat signaling pathway and nf-κb in the human liver cell line hepg and promote their insulin resistant state ( ) . nevertheless, the hepatocyte growth factor (hgf) can alleviate the insulin resistance of hepatocytes and control their triglyceride and cholesterol contents ( ) . skeletal muscle (sm) is the main tissue that releases glucose after insulin stimulation. hence, insulin resistance in sm has a pivotal role in the metabolic dysregulation of t dm. insulin resistance in sm is the primary defect of t dm that facilitates the progression of fatty liver disease, deposition of fat in the liver (figure ) ( ) . skeletal muscle from diabetic patients expresses less genes related to insulin signaling and metabolic pathways, but more apoptosis and immune-related genes ( ) . this inflammatory milieu is mainly due to the proinflammatory actions of obesity-related adipose tissue mediators, which are released into the circulation and promote inflammation within the sm ( ). furthermore, obesity causes intermyocellular and perimuscular adipose tissue expansion that acts like adipose tissue depots to enhance sm inflammation ( ) . it has been shown that human skeletal muscle cells (hsmc), isolated from diabetic patients, after a -h culture generate significantly more tnf-α, il- , il- , il- , monocyte chemotactic protein (mcp)- , growth-related oncogene (gro)-α, and follistatin compared to non-diabetic individuals ( ) . this altered secretion of myokines (e.g., cytokines secreted by sms) is an intrinsic feature of sm during the progression of t dm. in sm, glut- , which is quickly translocated to the cell surface, facilitates glucose uptake in response to insulin hormone as well as muscle contraction. pinto-junior et al. have shown that the use of age-albumin in rats increases the expression of the inflammatory molecule nf-κb within the sm. nf-κb binds to the promoter of the glut- gene and suppresses its expression (at both mrna and protein levels) ( ) . accordingly, glut- levels on the surfaces of sm decrease and subsequently, whole-body ir develops. the immune system is generally classified into two main arms, innate and adaptive (or acquired) immunity. adaptive immunity is mediated by b cells, which produce antibodies and t cells, which are classified into cd + helper cells and cytotoxic cd + cells. a considerable literature has discussed the dysfunctional immune responses in diabetic patients ( table ) ( ) ( ) ( ) ( ) ( ) ( ) . abnormal immune cell activation and subsequent inflammatory environment has an essential role in the progression of t dm ( ) . in this regard, chronic inflammation due mainly to the activation of the myeloid cell lineage (e.g., macrophages and neutrophils), is directly related to the induction of ir ( , ). their numbers are elevated, are larger and more granular, express diminished levels of antioxidant genes but elevated levels of pro-apoptotic and pro-inflammatory genes. complement system attachment of c-type lectin proteins to mannose residues is decreased, lectin pathway is impaired, cd activity is reduced, mac deposition in vascular walls is increased. dendritic cells (dcs) their numbers and activity are reduced. their cholesterol efflux is decreased, generate foam cells, have dysfunctional efferocytosis. are activated, constitutively release nets, produce high levels of mpo, ros, and calprotectin (s a /a ), are more susceptible to apoptosis, their migration, phagocytosis and microbial killing are impaired. nk cells their numbers are increased but are usually dysfunctional, express high levels of glut but decreased levels of nkg d and nkp , have reduced degranulation capacity, are more susceptible to apoptosis. nkt cells their numbers are increased, produce high levels of ifn-γ, il- , and il- , express high levels of nkp , nkg d, and nkp but low levels of nkg a and b. innate lymphoid cells (ilcs) ilc s are increased and produce high levels of ifn-γ. humoral immunity (b cells) germinal centers are reduced, ab production and isotype switching is defective, abs become glycated, abs fail to activate complement. functions of osteoclasts, which are bone-resident innate immune cells ( ) . this may affect bone structure and delay bone healing. defects in the innate, as well as adaptive immunity, are supposed to be the main cause of diabetic individuals' susceptibility to infections ( ) . furthermore, some microorganisms, especially bacteria, in hyperglycemic conditions are better nourished and become more virulent, while also having a better milieu to cause infections. complement system the complement system is a first-line defense mechanism against invading microorganisms. it acts via different but interconnected classical, alternative, and lectin pathways ( ) . ilyas et al. have shown that under high glucose conditions, the attachment of ctype lectin proteins to high-mannose containing glycoproteins is substantially decreased in a dose-dependent manner. these carbohydrate-binding proteins include mannose-binding lectin (mbl), surfactant protein d (sp-d), dendritic cell-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign, cd ), and dc-sign-related (dc-signr) protein ( ) . reduced binding of mbl in the presence of high levels of sugar causes a significant reduction in the lectin pathway activity, but does not influence classical or alternative pathway activity ( ) . nevertheless, barkai et al. did not find significant differences in the function of classical or mbl pathways between t dm and healthy individuals ( ) . however, significantly decreased activity of ficolin- -mediated lectin and alternative pathways, as well as decreased levels of c d and soluble complement c b- (sc b- ) were seen in diabetic patients with escherichia coli-mediated urinary tract infections ( ) . this may be linked to a reduced ability of diabetics to protect themselves against bacterial infections. the lipopolysaccharides of certain gram-negative bacteria, like salmonella serotype o , as well as the cell walls of fungi, are rich in mannose. possibly, because of this, in addition to additional provision of nutrients, an increased prevalence of fungal infections is seen in t dm patients ( , ) . patel et al. found a significantly higher prevalence of oral candida carriage in diabetic patients compared to healthy controls ( ) . they found that candida albicans was the most commonly isolated species followed by c. tropicalis, but uncommon species such as c. lusitaniae and c. lipolytica were also isolated ( ) . another study by jhugroo et al. showed that c. albicans is the predominant yeast isolated from oral mucosal lesions of diabetic patients, followed by. c. tropicalis and c. krusei dendritic cells (dcs) are a heterogeneous population of specialized and professional antigen-presenting cells (apcs) that create a crucial link between the innate and adaptive immune responses ( , ) . some studies have shown that the numbers of dcs are reduced in both type and diabetes ( , ). seifarth et al. have found that t dm patients with poor metabolic control have decreased numbers of both myeloid and plasmacytoid dcs compared with healthy controls. this could make them more susceptible to opportunistic infections ( ) . in the case of good blood glucose control, the reduction in dc numbers was less prominent but still significant, especially for myeloid dc (mdc ) cells ( ) . another study by blank et al. demonstrated that women with t dm and poor glycemic control (hba c ≥ %) have fewer numbers of circulating plasmacytoid dcs (pdcs) compared to diabetic women with good glycemic control (hba c < %) or to healthy women ( ). montani et al. have recently shown that hyperglycemic medium and hyperglycemic sera derived from t dm patients prevent the maturation of monocytes into effective dcs and their activation in vitro ( ) . interestingly, quercetin, a flavonoid with antiinflammatory and antioxidant characteristics, prevented such effects ( ) . macrophages are important immune cells that play critical roles through all stages of the pathogenesis of t dmrelated atherosclerosis ( ). swirski et al. have shown a significantly elevated number of pro-inflammatory monocytes in the circulation of apoe −/− mice, an animal model of atherosclerosis, compared to control mice ( ) . modifications of the lipoproteins in the arterial walls of diabetic individuals make them pro-inflammatory and activate the overlying endothelium. in response, monocytes are recruited into the subendothelial region, differentiate into macrophages and internalize the accumulated lipoproteins. finally, cholesterol-laden foam cells are generated. they promote inflammation and progression of the disease through the synthesis and secretion of cytokines, chemokines, ros, and matrix metalloproteinases (mmps) (figure c ) ( ) . foam cells lose their migratory potential, die by apoptosis and generate a necrotic core within the atherosclerotic plaque ( ) . it has been demonstrated that the use of mesenchymal stem cells in apoe −/− mice reduces the numbers of monocytes/macrophages at the site of inflammation, decreases lipid deposition and diminishes plaque size ( ). ma et al. have studied the effects of long-term hyperglycemia in diabetic mice and found out that compared to non-diabetic control mice, the numbers of f / + macrophages isolated from spleen (spms), as well as from peritoneal exudates (pems) of diabetic mice are significantly decreased ( ) . subsequently, sun et al. showed that stimulation of pems from diabetic mice in vitro with ifn-γ and lipopolysaccharide (lps) significantly decreased the expression of intercellular adhesion molecule (icam- or cd ), cd , tnf-α, and il- , while it increased the production of nitric oxide (no) ( ) . they further showed that stimulation of pems isolated from diabetic mice with il- caused an enhanced arginase activity ( ) . kousathana et al. have demonstrated that circulating monocytes isolated from diabetic patients produce higher levels il- , while having an impaired activation of the nlrp inflammasome and subsequently reduced il- β production ( ) . however, they showed that proper glycemic control would restore such modifications. poor inflammatory responses in circulating monocytes, as well as in macrophages, are responsible for elevated susceptibility to infections and their severity in patients with t dm. macrophages play a critical role in tissue repair. early in wound healing, they are pro-inflammatory to clear pathogens and debris but later, they resolve inflammation and promote tissue repair. in pathological conditions, failure to transform from pro-inflammatory to the anti-inflammatory proliferative phase can cause chronic inflammation in the affected tissue ( ). have shown that an impaired wound healing process in animals with t dm is due to high levels of nlrp inflammasome activity, which promotes the generation of il- β and il- in macrophages ( , ) . efficient skin wound healing process is mediated by the up-regulation of the peroxisome proliferatoractivated receptor (ppar)-γ in macrophages that convert their pro-inflammatory phenotype into healing-related. pparγ suppresses cytokine production by macrophages and hence is upregulated in inflamed tissue-resident macrophages. however, in t dm, pparγ expression is down-regulated in skin-resident macrophages that enhance the activity of nlrp- inflammasome and cause chronic inflammation. using myeloid-specific pparγ −/− mice, it has been shown that the absence of ppar-γ in macrophages is sufficient to delay the healing process and extend tissue inflammation ( ) . in t dm patients, chronic hyperglycemia and hyperlipidemia trigger the secretion of a damage-associated s a molecule (calgranulin a) from pancreatic islets that in turn increase macrophage infiltration ( ) . westwell-roper et al. have shown that iapp aggregates in t dm patients polarize islet-resident macrophages toward the m -like f / + cd b + cd c + phenotype that produces pro-inflammatory cytokines, including tnf-α, il- β, and il- . furthermore, m cells promote islet inflammation, cause β-cell malfunction and apoptosis ( ) . in t dm, excess phagocytosis of apoptotic β-cells by macrophages induces their lysosomal permeabilization, generation of ros, inflammasome activation, and pro-inflammatory cytokines secretion ( ) . collectively, these observations reveal that the functions and plasticity of macrophages are compromised during the progression of t dm. neutrophils are the most prevalent circulating leukocytes and one of the main components of innate immunity. they are recruited to the sites of infection through chemotaxis following complement activation, most importantly by c a. activated neutrophils bind via their surface receptors to induced ligands on the surfaces of inflamed endothelial cells to migrate to tissues. there they phagocytose and kill invading microbes with lysosomal enzymes, antimicrobial peptides and by the generation of ros ( ). neutrophils from patients with t dm, but not from healthy individuals, are activated and produce elevated levels of ros. so, it could increase the risk of random organ injury ( ) . in diabetic patients, the plasma levels of homocysteine are elevated, which is mainly due to its impaired clearance rate ( ) . this will induce neutrophils to constitutively release neutrophil extracellular traps (nets) that can cause vascular damage and delays in wound healing ( , ) . it has been shown that the circulating level of hydrogen sulfide (h s) is significantly reduced in fasting blood of patients with t dm compared with healthy individuals as well as in streptozotocin-induced diabetic rats compared with controls ( ) . h s is produced from cysteine by the action of several enzymes. it acts as a regulator of cell signaling and homeostasis ( ) . it is essential to maintain balanced levels of antioxidants and protect tissues from oxidative stress ( ) . the use of h s or the endogenous l-cysteine in vitro blocks the production of il- and monocyte chemoattractant protein- (mcp- ) in the human u monocyte cell line incubated in high-glucose medium ( ) . yang et al. have shown that h s treatment decreases netosis and enhances the healing process of diabetic wounds by preventing ros-dependent erk / and p activation ( ) . it has been shown that the levels of net components, including histones, elastase and proteinase- , are elevated in the sera from patients with diabetic foot ulcers ( ) . wang et al. have recently indicated that hg dramatically enhances nadph oxidasedependent net generation in diabetic rats and humans. it was proposed that this could have a role in the induction of diabetic retinopathy ( ) . indeed, patients with t dm have elevated plasma levels of mgo, which can induce the production of proinflammatory cytokines like tnf-α, il- , and il- by neutrophils and make them more susceptible to apoptosis ( ) . myeloperoxidase (mpo), which is abundantly produced by neutrophils, but only to a small extent by monocytes and macrophages, might be useful as an early biomarker of inflammation in diabetic individuals ( ) . binding of mpo to endothelial cells increases its half-life. thereby, more proinflammatory oxidant hypochloric acid (hclo) is generated that extends the damage to blood vessels ( ) . in t dm patients, neutrophil activities, including migration, phagocytosis and microbial killing are impaired. this makes diabetic individuals more susceptible to infections ( ) . it has been welldocumented that neutrophils isolated in animal models of t dm have an impaired tlr signaling pathway. this is reflected as a diminished cytokine and chemokine production, possibly as a consequence of reduced phosphorylation of nfκb and iκbα ( ) . the half-life of these neutrophils as well as their in vivo migration and myeloperoxidase activity are decreased. during hyperglycemia, neutrophils produce calprotectin (s a /a ), which interacts with the receptor for advanced glycation end products (rage) on the surface of hepatic kupffer cells and promotes the synthesis of il- ( ). subsequently, il- stimulates hepatocytes to increase the generation of thrombopoietin that in turn attaches to its receptor on the surfaces of bone marrow precursor cells and megakaryocytes to enhance their proliferation and expansion. this results in reticulated thrombocytosis, which means elevated megakaryocyte activity and thrombopoiesis. interestingly, diabetes-related thrombocytosis and subsequent atherothrombosis can be reduced by lowering blood glucose, depleting kupffer cells or neutrophils or by preventing the binding of s a /a to rage using paquinimod ( ) . thom et al. have shown that the incubation of human and murine neutrophils with hg medium would cause their cytoskeletal and membrane instability. this will induce the generation of . to µm diameter microparticles and activate the nlrp inflammasome ( ) . microparticles, which are potently pro-inflammatory, are found in the circulation of healthy individuals, but their generation is increased during cell activation in several diseases, including t dm and cardiovascular diseases ( , ) . furthermore, serum levels of soluble fasl (sfasl) are increased in patients with t dm thereby activating neutrophils and aggravating the inflammatory milieu ( , ) . the proinflammatory roles of sfasl are mediated through increased amounts or activity of nfκb, il- β, caspase- , cd b/cd , and ros ( ) . caspase- activation prevents the sfasl-dependent apoptosis of neutrophils and inhibits their expression of fas and caspase- ( ) . accordingly, hyperglycemia disturbs the normal functions of neutrophils and increases the susceptibility to infections by pathogenic microorganisms. the expression level of nkg d is negatively correlated with hba c levels implying that chronic hyperglycemia would cause nk cell dysfunction ( ) . also, hyperglycemia increases the expression of unfolded protein response (upr) genes in nk cells and induces their apoptosis ( ) . nkt cells express simultaneously markers of both t cells (tcr and cd ) and nk cells [cd , cd , cd (nkg d), and cd (nkp )]. nkt cell subsets produce a broad range of cytokines, including gm-csf, ifn-γ, tnf-α, il- , il- , il- , il- , il- , il- , il- , and il- ( ) . they recognize lipids and glycolipids presented by cd d molecules. phoksawat et al. have shown that the frequency of cd + cd + cd null cd + nkg d hi nkt cells, which produce high levels of il- , are increased in diabetic patients and their numbers are directly correlated with hba c levels ( , ) . lv et al. have recently shown that the numbers of cd + cd + nkt cells are higher in diabetic patients compared to healthy individuals ( ) . they further showed that such cells are mostly cd + , produce elevated levels of ifn-γ and il- and express high levels of nkp , nkg d, and nkp but low levels of inhibitory receptors nkg a and b ( ) . the co-culture of these cells with huvecs significantly decreased their proliferation and migration abilities that were mainly il- dependent ( ) . taken together these studies show that diabetic individuals appear to have elevated levels of inflammationpromoting nkt cells. ilcs are critical effectors of innate immunity that produce both regulatory and pro-inflammatory cytokines to promote tissue repair, immunity, and inflammation ( ) . mature ilcs lack the tcrs. based on their cell surface markers, cytokine production as well as expression of transcription factors the ilcs are classified into types , , and ( ) . these correspond to the different types of cd + t helper cells: th , th , and th , respectively. ifn-γ is the cytokine signature of ilc s, while type cytokines (e.g., il- and il- ) are mainly produced by ilc s and the main product of ilc s are il- and il- . regarding transcription factors, t-bet is mainly expressed by ilc s, gata and rorα are mostly expressed by ilc s and rorγt is predominantly expressed by ilc ( ) . in t dm, the numbers of circulating as well as adipose tissue-resident ilc s are increased compared with normal individuals ( , ) . the frequency of circulating ilc s is positively correlated with fasting plasma glucose (fpg), hba c, homeostasis model assessment for insulin resistance (homa-ir), serum-free fatty acids (ffas) and adipose tissue insulin resistance index (adipo-ir) ( , ) . it has also been shown that patients with increased numbers of ilc have an elevated risk of developing t dm ( ) . a study by wang et al. indicated that adipose tissue-resident ilc s, via the production of ifn-γ, promote tissue fibrosis and induce diabetes in obese individuals ( ) . liu et al. have demonstrated that the numbers of ilc s as well as serum cytokine levels of il- , il- , and il- are significantly elevated in diabetic kidney disease patients and have a positive correlation with disease severity ( ) . they further demonstrated that ilc s, through the tgf-β signaling pathway, are involved in renal fibrosis seen in diabetic kidney disease ( ) . however, galle-treger et al. indicated that the engagement of the glucocorticoid-induced tumor necrosis factor receptor (gitr/or tnfrsf ) on the surface of activated ilc s promotes their secretion of il- and il- , ameliorates glucose homeostasis, protects against the onset of and improves established insulin resistance ( ) . the protective role of ilc s during acute metabolic stress has also been well-documented by dalmas et al. ( ) . humoral immunity (b cells) elevated levels of blood glucose generate covalent sugar adducts with several proteins through non-enzymatic glycation. this can impair humoral immunity in many ways, e.g., by modifying the structure and functions of immunoglobulins (igs) ( ) ( ) ( ) ( ) ( ) ( ) . such modifications in the structure of igs can be determined using matrix-assisted laser desorption ionization (maldi) mass spectrometry ( , ) . the molecular mass of igs in diabetic patients is higher than in normal subjects ( ) . this can lead to reduced efficiency of vaccines that stimulate humoral immunity in these patients. it has been shown that immunization with influenza (flu) vaccines in diabetic patients induces normal or even elevated levels of flu-specific antibodies compared with normal individuals ( ) ( ) ( ) ( ) . however, the ability of the dysfunctional glycated antibodies to neutralize viruses is impaired, which will increase the susceptibility to infections. farnsworth et al. have shown that in t dm, class switch defects in the assembly of antibody genes are also present ( ) . in a model system, mice with t dm have decreased amounts of specific anti-staphylococcus aureus antibodies (total as well as igg), which will increase the risk of infection and morbidity of diabetic mice. however, the levels of igm were elevated, but inefficient in protecting against infection, possibly because of their inability to directly promote phagocytosis. in another study, farnsworth et al. have demonstrated that defects in humoral immunity, as shown by decreased levels of total igg and anti-staphylococcus aureus antibody, aggravate foot infections in a murine model of t dm ( ) . this was due to a reduced germinal center induction and decreased numbers of t and blymphocytes within the germinal centers. this causes failures in antibody generation and class-switch recombination ( ) . mathews et al. have shown that the protective levels of antibodies against streptococcus pneumoniae surface protein a are lower in diabetic patients compared to non-diabetic individuals. these antibodies also have a reduced potential to trigger complement activation on the surface of pneumococci, whereby phagocytosis of the bacteria becomes compromised ( ) . they showed that hyperglycemia reduces both the antibody titers as well as the ability to deposit complement on the bacteria. the abovementioned changes in the ability to protect against s. aureus and s. pneumoniae are important, because these bacteria belong to the most common infection-causing pathogens in diabetic patients. another major group is constituted by gram-negative bacteria that commonly cause e.g., urinary tract infections. many studies have shown that t-cell functions are impaired in individuals with t dm ( ) ( ) ( ) ( ) . elevated levels of activated cd + cd + t helper cells, cytotoxic t-cells, and th cells have been observed in obese diabetic patients compared to nonobese ones ( , ) . nevertheless, pbmcs isolated from obese diabetic patients produced smaller amounts of il- , il- , and tnf-α after stimulation with phytohemagglutinin (pha) ( ) . martinez et al. indicated that diabetic patients have reduced pathogen-specific memory th responses as well as decreased numbers of cd + t cells in response to stimulation with streptococcus pneumoniae ( ) . th cells are critical for the recruitment of neutrophils to the infection site and improve the phagocytosis of invading bacteria and yeast ( ) . moura et al. have shown that diabetic patients, particularly those with foot ulcers, have reduced levels of naive t-cells, but an elevated number of effector t cells and a reduction in the tcr-vβ repertoire diversity ( ) . the observed changes are mainly due to an abnormal amount of inflammatory cytokines (e.g., ifn-γ and tnf-α) produced during infection and to subsequent robust stimulation of t-cells. leung et al. have reported that ischemic tissues of t dm patients contain elevated numbers of tnf-α and ifn-γ producing th cells but diminished numbers of regulatory t cells (tregs), which suppress angiogenesis and decrease vascular density ( ) . the high rate of infectious diseases in t dm patients might also be linked to a reduction in the mitochondrial dna function that causes downstream lymphocyte dysfunction and subsequently increased susceptibility to infection ( ) ( ) ( ) ( ) . in support, we have recently shown that the numbers of ifn-γ producing cells against cytomegalovirus (cmv), epstein-barr virus (ebv), and influenza virus are fewer in t dm patients compared to normal controls ( ) . kumar et al. have also investigated the functions of cd + t cells and nk cells in the whole blood of t dm patients infected with mycobacterium tuberculosis (m.tb). compared to controls, the patients exhibited a reduction in cytokine production (ifn-γ, il- , il- a/f, and tnf-α) and decreased expression of cytotoxic molecules (perforin, granzyme b, and cd a) ( , ) . these studies conclude that the functions of both cd + and cd + t-cell are defective in t dm patients. t dm is usually associated with an elevated risk of asymptomatic bacteriuria, urinary tract infections (utis), pyelonephritis and non-sexually transmitted genital infections, such as balanitis and vulvovaginal infections ( ) ( ) ( ) . the incidence of infections with a complicated course is significantly higher in diabetic patients compared to healthy controls ( table ) . it seems that it is principally defects in the innate immune responses of diabetic individuals that are responsible for the increased susceptibility and prevalence of infections ( , ( , ) cd + tcells mycobacterium tuberculosis ( , ) susceptible to the causative pathogen of lyme disease, borrelia burgdorferi ( ) . the disease is mainly due to the ability of the bacteria to escape complement opsonization and attack, which leads to an impaired uptake and killing of bacteria by neutrophils ( ) . neutrophil dysfunction also increases the susceptibility of diabetic animals to staphylococcus aureus ( ) ( ) . during the progression of t dm in human subjects, the basal phenotype of macrophages is altered so their capacity to control mycobacterium tuberculosis is diminished ( ) . martinez et al. have indicated that alveolar macrophages isolated from diabetic mice express decreased levels of macrophage receptor with collagenous structure (marco) and cd that are engaged in the recognition of trehalose , '-dimycolate, a bacterial cell wall component ( ) . diabetes increases the severity of tuberculosis (tb) and enhances the risk of progression to the active form in latent infections ( , ) . diabetic tb patients have elevated frequencies of th and th cells as well as increased serum levels of inflammatory cytokines, including ifn-γ, tnf-α, il- β, il- , il- , il- a, and il- but decreased levels of il- compared to non-diabetic tb patients. this can contribute to dysfunctional immune responses and poor immune control of a tb infection ( ) . a positive correlation between the serum levels of ifn-γ, tnf-α, il- , and il- a with hb-a c levels was also observed. this indicates an association between impaired control of diabetes and the proinflammatory milieu. tripathi et al. have demonstrated that serum levels of il- were significantly decreased in tb-infected t dm mice and humans compared to non-diabetic tb-infected mice and humans ( ) . they revealed that the treatment of tb-infected diabetic mice with recombinant il- or ilc s (cellular source of il- ) increased the survival of mice, prevented the accumulation of neutrophils near alveoli, diminished the generation of neutrophil elastase (ela ) and prevented epithelial cell damage ( ) . tan et al. have shown that b. pseudomallei and m. tuberculosisinfected pbmcs of diabetic patients fail to produce il- . this leads to a decreased ifn-γ production, poor bacterial killing and elevated intracellular bacterial loads ( ) . an impaired il- production is mainly due to decreased intracellular glutathione (gsh) concentrations within the infected cells of diabetic individuals ( ) . such a combination of an inflammatory microenvironment and dysfunctional immune responses enhances the bacterial load and can subsequently amplify lung injury and fibrosis in diabetic tb patients. chellan et al. have further shown that infections caused by enterococcus faecalis, staphylococcus aureus, and pseudomonas aeruginosa are more prevalent in the wounds of diabetic patients ( ) . t dm patients are more susceptible to utis caused by antibioticresistant escherichia coli, proteus spp., klebsiella spp., coagulasenegative staphylococci, enterobacter spp., and enterococci ( , ) . diabetic patients are also more susceptible to helicobacter pylori (h. pylori) infections ( ). cui et al. have recently reported that t dm patients have an increased risk of infection with kaposi's sarcoma-associated herpesvirus (kshv or hhv- ) ( ) . they further showed that the viral load and antibody titers are positively correlated with blood glucose levels ( ) . diabetic patients also have been shown to have an increased risk of infection with the severe acute respiratory syndrome coronavirus (sars-cov) ( ( ) . the influenza virus that usually causes self-limiting infections can induce severe forms of the disease in diabetic patients ( , ) . following the h n influenza pandemic, diabetic individuals suffered from more severe infections compared to non-diabetic people ( , ) . diabetic patients have also a higher prevalence of chronic cytomegalovirus (cmv), herpes simplex virus (especially hsv- ), and varicellazoster virus infections ( ) ( ) ( ) . accordingly, it seems that the immune response against viruses is impaired in diabetics, and these patients need more care during viral infections. coronavirus virions are enveloped positive-strand rna spherical viruses with a diameter of ∼ nm characterized by spike proteins projecting from their surface and with an unusual large rna genome ( ) . the spike (s) protein of the virus binds to its receptor on the surface of cells by which intracellular proteases are induced ( ) ( ) ( ) . subsequently, the s protein priming and cleavage occurs that allow viral fusion to the plasma membrane and entrance of viral genome into the cells ( ) . sars-cov and sars-cov- use angiotensin-converting enzyme (ace ) as their receptor while mers-cov uses dipeptidyl peptidase- (dpp ) to enter the cells ( , ) . ace is strongly expressed in blood vessels, pancreas, intestine, brain, lungs, heart, and testis ( ) . interestingly, nasal epithelial cells, especially goblet, and ciliated cells express the highest levels of ace and the intracellular protease transmembrane serine protease (tmprss ) that facilitates the entrance of the sars-cov- ( ) . furthermore, the expression of ace is significantly up-regulated in diabetic patients and those treated with ace inhibitors ( ) . coronaviruses cause respiratory, enteric and central nervous system (cns) diseases in various animal species except rats and mice ( ) . most coronavirus infections are mild, but major outbreaks of deadly pneumonia have been caused by sars-cov, mers-cov, and sars-cov- in , , and - , respectively ( ) . on march , , the world health organization (who) announced the pandemic of sars-cov- , the etiologic agent of coronavirus disease- (covid- ) ( ) . the novel coronavirus pandemic, which has emanated from wuhan, china, promotes symptoms similar to those caused by the sars-cov outbreak in . the viral pandemic, which has put the world on alert, has caused over . × confirmed human cases and at least × deaths throughout the world (https://www.worldometers.info/coronavirus/) by june , . most of the infected people experience only mild to moderate respiratory disease and recover soon without the need for special treatment. however, aged individuals and those with health problems, including diabetes, obesity, cardiovascular disease (cvd), hypertension, immune deficiency, and chronic respiratory disease are more likely to develop serious illness (https://www.who.int/health-topics/coronavirus#tab= tab_ ). patients death is mainly due to the acute respiratory distress syndrome, disseminated intravascular coagulation, hemorrhage, coagulopathy, acute organ (e.g., kidney, heart, liver) injury, multi-organ failure, and secondary bacterial infections ( ) . elevated levels of adipose-tissue derived adipokines, interferon, and tnf-α in diabetic patients may impair immune-responses against sars-cov- ( , ) . it has been shown that diabetic patients have impaired clearance of sars-cov- from their circulation ( ) . accordingly, diabetic patients due to the diminished viral clearance, impaired t cell function, and accompanied cardiovascular disease are more susceptible to the coronaviruses infection and subsequent cytokine release syndrome (crs) ( , ) . in support, elevated levels of il- β, il- , il- , il- , il- , il- , ifn-γ, interferon gamma-induced protein (ip- ), granulocyte colony-stimulating factor (g-csf), macrophage inflammatory protein α (mip α), serum ferritin, fibrinogen, plasminogen, c-reactive protein (crp), and d-dimer have been observed in patients with covid- ( , , , ) . covid- patients, especially those requiring intensive care unit (icu) have decreased total lymphocytes (lymphopenia), t cells (both cd + and cd +), b cells, and nk cells ( , ) . it should be noted that most of the surviving t cells in such patients have an exhausted phenotype ( ) . consequently, disease severity is mainly because of the host immune response to viral infection. current evidence about the relationship between pathophysiological mechanisms of diabetes and covid- are limited and further research is still needed. patients with t dm have an elevated risk of infection with plasmodium falciparum ( ) , toxoplasma gondii ( ), opisthorchis viverrini ( ), strongyloides stercoralis ( ), cryptosporidium parvum ( ), blastocystis hominis ( ), ascaris lumbricoides ( , , ) , and giardia lamblia ( ) . interestingly, diabetic patients who were treated with metformin had less p. falciparum infections compared to untreated patients ( ) . omaña-molina et al. have shown that in a mouse model of t dm the animals have an increased susceptibility to granulomatous amoebic encephalitis (gae) caused by trophozoites of acanthamoeba culbertsoni ( ) . the possible reasons for the increased risk of diabetics for parasitic infections are metabolic abnormalities and immune dysregulation. chellan et al. have shown a higher prevalence of fungal infections in the wounds of diabetic patients ( ) . the prevalence correlated with the levels of hba c. the most widely observed fungal isolates were c. albicans, candida parapsilosis, c. tropicalis, trichosporon asahii, and aspergillus species. some of them were resistant to antifungal medications ( ) . al mubarak et al. have also demonstrated that diabetic patients with periodontitis are more susceptible to infection with c. albicans, c. dubliniensis, c. tropicalis, and c. glabrata ( ) . the incidence of candidiasis was significantly increased in patients over the age of with hba c > ( ). it has also been shown that diabetic patients are more susceptible to utis caused by c. albicans ( ) . hyperglycemia impairs the normal functions of the circulatory system, gastrointestinal tract, pancreatic beta cells, liver as well as of skeletal muscles to boost systemic insulin resistance. a hyperglycemic environment also leads to immune cells dysfunction. it increases intestinal permeability, which subsequently enhances the risk of infections in t dm patients. accordingly, further research is still needed to find missing links between impaired physiological/immunological mechanisms and increased susceptibility to infections in t dm patients. the information would be important for better therapy and the design of much more effective vaccination strategies in diabetic patients. gd 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the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -wbfnhedy authors: smith, sylvia b.; st jules, robert s.; o'brien, paul j. title: transient hyperglycosylation of rhodopsin with galactose date: - - journal: experimental eye research doi: . / - ( ) -j sha: doc_id: cord_uid: wbfnhedy abstract rhodopsin's oligosaccharide chains contain predominantly two types of sugar residues: mannose and n-acetylglucosamine. in the present work, bovine and rat rhodopsin were analysed biochemically for the presence of a third sugar, galactose. treatment of bovine rod outer segments (ros) with galactose oxidase followed by reduction with tritium-labeled sodium borohydride revealed the presence of existing molecules of galactose on rhodopsin. rats injected intravitreally with [ h]galactose and [ c]leucine and maintained in darkness were killed hr, hr, , , or days following the injection. retinas were collected for subcellular fractionation and rhodopsin from each of the fractions was purified by cona sepharose chromatography and sds-page. during the first hr, galactose selectively labeled rhodopsin in the golgi-enriched fraction resulting in increased [ h] [ c] ratios in both golgi and ros. the data suggested that trimming was occurring at the transition from golgi to ros. furthermore, a decrease in isotope ratio in the ros between hr and day suggested further trimming of rhodopsin after membrane assembly in the ros. additional in vivo experiments demonstrated existing molecules of galactose on rhodopsin's oligosaccharide chain using lectin affinity chromatography. rats injected intravitreally with [ s]methionine were dark-adapted for hr. following subcellular fractionation of retinas, cona purified rhodopsin from ros was applied to one of two additional lectin columns: ricinus communis agglutinin (rca) or griffonia simplicifolia i (gsa). eight to nine per cent of the labeled rhodopsin was bound to and eluted from rca, whereas none bound to gsa, indicating the presence of a β-galactoside. the rca agarose eluted protein co-electrophoresed with a rhodopsin standard and was light sensitive. galactose was shown to be the terminal sugar on this subset of rhodopsin and was not capped by neuraminic acid. binding of rhodopsin's oligosaccharide to rca was abolished by pre-treatment with β-galactosidase. decreased binding of rhodopsin to rca was observed following intravitreal injection of castanospermine but not swainsonine. of those two inhibitors of glycoprotein trimming, only castanospermine would be expected to prevent the addition of galactose to the oligosaccharide. the association of galactose with rat rhodopsin appeared to be a transient one. at hr, – % of rhodopsin contained galactose, at hr only · % had galactose and by hr less than % did. the galactose was trimmed from rhodopsin's oligosaccharide presumably after its role was complete. separation of rhodopsin of the plasma membranes from rhodopsin of discs indicated that % of the galactose-containing rhodopsin was in the plasma membrane and only % was in the discs. these findings suggested a possible role for galactose in new disc formation with subsequent removal after the discs are sealed. rhodopsin contains two oligosaccharide chains linked to asn, and asn,, (hargrave, ) . for the most part, these chains contain only two types of sugar residues : mannose and n-acetylglucosamine (heller, ; shichi et al., ; heller and lawrence, : plantner and kean, ) . the synthesis of asparagine-linked glycoproteins typically involves the rough endoplasmic reticulum for the initiation of oligosaccharide synthesis and both the rough endoplasmic reticulum and golgi complex for modification of the oligosaccharide chain (kornfeld and kornfeld, in more complex glycoproteins prior to the addition of a terminal trisaccharide consisting of n-acetylglucosamine, galactose and neuraminic acid. rhodopsin actually contains the first sugar of the terminal trisaccharide as demonstrated by glucosamine incorporation studies (o'brien and muellenberg, ; bok, hall and o'brien, ) . the remaining sugars, galactose and neuraminic acid have not usually been detected in the rhodopsin oligosaccharide chain. however, o'brien ( ) demonstrated the direct transfer of galactose in vitro to bovine rhodopsin and showed chromatographically that the radiolabeledgalactose was incorporated as galactose since the radioactivity coincided with a galactose standard and was not converted to some other sugar (such as mannose). more recently, galactose was successfully incorporated into rat rhodopsin in vitro following incubation of whole retinas with [ h] galactose (st jules, smith and o'brien, ) . fukuda. papermaster and hargrave ( ) analysed bovine rhodopsin's academic press limited amino terminal glycopeptide using gas chromatography and mass spectrometry and reported that in addition to the major oligosaccharide structure which contained three n-acetylglucosamine residues and three mannose residues, a minor variation of rhodopsin contained . mol galactose per three nacetylglucosamine residues and three mannose residues. these data suggested that up to % of rhodopsin contained galactose. morphologic studies of carbohydrate-containing molecules in photoreceptor cells have invoked a different methodology utilizing lectins for detecting the presence of specific sugars. ricinus communis agglutinin (rca) is a lectin that specifically recognizes terminal galactose moieties associated with either glucosamine or n-acetylglucosamine in a / - linkage (baenziger and fiete, ) . transmission electron microscopic studies of frog, bovine, monkey and rat retinas have shown binding of rca to rod outer segments and in some cases rod inner segments (nir and hall, ; hicks and molday, ; koide et al., i : hicks and barnstable, ) . likewise, similar results were obtained in fluorescent microscopic studies detecting rca binding in frog, monkey, mouse, human, rabbit, and fish retinas (bridges and fong, ; bridges, ; uehara et al., ; blanks and johnson, ) . of great interest was the work by hicks and molday ( ) who published photomicroscopic evidence of post-embedding, direct labeling by rca in the basal area of the ros. in the present study, we used biochemical techniques to characterize the subset of rhodopsin which appears to contain galactose. we present evidence to confirm the incorporation of galactose into rhodopsin in in vivo experiments. additionally. we demonstrate the presence of galactose in bovine rhodopsin by radiolabeling existing moieties. finally, using lectin affinity chromatography, an analytical procedure which is rapid, reliable and highly sensitive (cummings, merkle and stults, ) , we are able to separate the galactose-containing species of rhodopsin and study its transient association with the oligosaccharide chain. for most experiments, sprague-dawley rats, -l g (taconic farms, germantown, ny) were used. one set of experiments utilized bovine retinas dissected from bovine eyes obtained fresh from a slaughterhouse. for studies of in vivo labeling of rhodopsin with galactose, rats were anesthetized with ether and injected intravitreally with /ai per eye of a saline solution containing pci ['"clleucine and . ,uci jq-s-'h]galactose ( . ci mmol '). in other in vivu experiments, eyes were injected with ,li ]-?i)leucine ( ci mmol ') or ,&i [" s]nlethionine ( ci mmol ') or i/ci ["%]methionine ;ind //ci ]:sh]glucosamine ( .x ci mmol ). the rats m'ere maintained in darkness and at various intervals were killed with co,. the retinas were removed by proptosing the eye using a curved forceps, the prongs of which were ensheathed in polyethylene tubing. the cornea was slit with a scalpel blade causing the lens and vitreous humor to be extruded. the retina was lifted free of any other ocular material simply by squeezing and lifting the forceps upwards. the sclera remained attached to the optic nerve. this procedure takes only -l s and the retinas are removed intact and ready for biochemical workup. no elaborate dissection is required. ["h]galactose, ]"h]glucosamine, ["hlleucine and [' 'clleucine were purchased from new england nuclear: [""slmethionine was purchased from american radiolabeled chemicals, inc. isolated retinas were fractionated in dim red light by a method described previously (st jules, smith and o'brien, ) . four to six retinas were suspended in ml % sucrose containing mm nacl, rnlv mgci,, and mm tris acetate ph . . rod outer segments were broken off with -set bursts of a vortex mixer. after a s-min centrifugation at g the floating ros were removed with the supernatant and the retinal debris was treated again as above. the pooled crude ros suspension was diluted with volumes of buffered % sucrose, or in some cases . % nacl, and were sedimented by centrifugation at g for min. both the ros and retinal debris were separately homogenized in ml % sucrose made up in mm nacl, mm mgcl,. and mm tris acetate ph - using seven strokes of a dounce tissue grinder (wheaton ml. b pestle with . - . s-inch clearance). these homogenates were layered over separate linear .- % sucrose gradients made up in the same buffer. after centrifugation for hr in a spinco sw rotor at rpm, bands were removed with a pasteur pipet. diluted with loo/, sucrose and sedimented by centrifugation for min at g. two bands of unsealed and sealed ros were near the top of the ros gradient (godchaux and zimmerman. ) . below these was a zone of fine particles, enriched in golgi. in the retinal debris gradient was a trace of the golgi-enriched fraction and a prominent zone of coarse particles, including the rough endoplasmic reticulum, nuclei and synaptosomes. these fractions are described in more detail in st jules and o'brien ( ) . with the bovine retinas, discontinuous gradients were prepared by overlaying the bovine ros sus-pension with the following sucrose solutions: ml of d . , ml of dl. and ml of dl. . the sucrose solutions were made up in mm tris acetate, ph . , containing . mm mgcl,. after centrifugation for min at rpm in a spinco sw ti rotor, the ros were removed from the interface at dl. lldl. with a pasteur pipette, diluted with % sucrose and sedimented by centrifugation for min at g. rhodopsin-containing membranes from four to six rat retinas were extracted in dim red light for hr at °c with ml % emulphogene bc (general aniline and film) in mm tris acetate buffer, ph . , containing mm mgcl, and mm cacl,. the extract was clarified by centrifugation at g for min and applied to a . ml column of cona sepharose (pharmacia). after washing with ml . % emulphogene in the same buffer, the rhodopsin was eluted with eight applications of ~ aliquots of . m amethylmannoside (sigma) also made up in the same buffer with . % emulphogene. twenty microliters of each -~ fraction were sampled for radioactivity. peak fractions were pooled and aliquots were adjusted to % sodium dodecyl sulfate (sds) and mm edta. samples were incubated at room temperature ( °c) for hr in dim red light. after the addition of one half volume of % glycerol containing . y bromophenol blue (biorad) the samples were applied to % polyacrylamide disc gels with % stacking gels for electrophoresis by the method of laemmli ( ) . gels were stained with coomassie brilliant blue (biorad) and cut into . mm slices with a hoefer gel slicer and the slices containing the stained opsin were noted. slices were digested overnight at °c in ncs protein solubilizer (amersham) with . % water. slices were counted (two per vial) in econofluor- (new england nuclear, boston, ma) using a beckman ls scintillation counter. the mobility of radioactive opsin could be compared directly with that of the coomassie blue stained mature opsin in the same cylindrical gel. those experiments utilizing two isotopes (i.e. ["hlgalactose and [xlleucine) permitted determination of the ratio of these isotopes in the opsin band of the gels. to radiolabel existing galactose moieties on rhodopsin, bovine ros purified by a discontinuous sucrose density gradient procedure, were subjected to enzymatic treatment with galactose oxidase followed by a reduction with tritium labeled sodium borohydride. in these experiments no external source of radioactive galactose was provided. ros from two bovine retinas were washed by centrifugation with pbs for min at g. they were suspended in ml pbs, an additional ml of pbs containing u of galactose oxidase (worthington) was added to one ros sample and ml pbs without the enzyme was added to a second ros sample as a control. five microliters . m pmsf in ethanol were added to each sample. ros were incubated at room temperature with occasional swirling for min in the dark. ros were centrifuged at g for min. then resuspended in pbs and centrifuged for the same length of time and speed. ros were suspended in pbs. to each sample was added . mci of tritium labeled sodium borohydride (new england nuclear) in . n naoh made up in ml pbs. after min at room temperature, additional pbs was added and ros were centrifuged at g for min. subsequently, ros were washed twice by centrifugation in pbs. in a repeat of this procedure, one sample received pretreatment with u neuraminidase from clostridium perfringens (sigma) at ph . for hr at 'c and the subsequent galactose oxidase incubation time was doubled. the ros were solubilized and rhodopsin was analysed by sds-page as described above. to further characterize the subset of rhodopsin which contains galactose (without introducing an external source of galactose) lectin affinity chromatography was used. in this method, cona purified rhodopsin or in some cases the clarified extract of ros from six rat retinas labeled with [ s]methionine was applied to columns of either ricinus communis agglutinin i (rca) or grifsonia simplicijolia i (gsa), specific for p-linked or a-linked galactose residues, respectively. rca agarose was purchased from u.s. biochemical corp., cleveland, oh and gsa agarose was purchased from biocarb chemicals, lund, sweden. columns were washed with . % emulphogene in mm tris acetate buffer, ph . , containing mm mgcl, and itim cacl,. samples were eluted from rca agarose or gsa agarose columns with ,ul aliquots of either . m / -methyl-n-galactopyranoside or amethyl-n-galactopyranoside (u.s. biochemical corp.), respectively. aliquots ( ~ ) were analysed for radioactivity using a beckman ls scintillation counter. aliquots of the peak fractions were applied to sds-polyacrylamide gels and analysed for radioactivity of opsin as described above. to determine if the galactose of rhodopsin was capped with a neuraminic acid, rat ros labeled with [ s]methionine were incubated with u neuraminidase from vibrio cholerae (boehringer-mannheim) at °c for hr. a second ros sample was incubated with no enzyme. the incubated suspension was centrifuged for min at g. the ros were extracted for hr and the clarified extracts were applied to kca agarose columns. aliquots of the peak fractions were applied to sds-polyacrylamide gels and analysed for radioactivity of opsin as described above. cona purified rhodopsin labeled with [%]methionine and ["hlglucosamine was incubated at °c for hr with it of n-glycanase (genzyme, boston, ma) in . % sds, mm /$mercaptoethanol, ,llm pmsf. mm edta, . % np- . ph . . at the end of the incubation the ph of the sample was adjusted to . by the addition of acetic acid. the solution was buffered with mes (sigma). b-galactosidase ( mu) from diplococcus pneumoniae (boehringer-mannheim) were added to half of the sample. both the enzyme treated sample and the control were incubated at °c for hr. the samples were applied to separate rca agarose columns and subsequently eluted with -,ll~ aliquots of p-methyl-ogalactopyranoside as described above. the amount of radioactivity eluted from rca after /%galactosidase treatment was compared to that eluted from rca without the enzyme treatment. to determine the consequences of the interruption of glycoprotein processing on the galactosylation of rhodopsin, two inhibitors of the glycoprotein processing pathway, castanospermine and swainsonine (boehringer-mannheim) were used in in vivo experiments. the concentration of the stock solution of castanospermine was mg ml-'. it was prepared by dissolving mg of the inhibitor in ~ dimethyl sulfoxide (dmso, sigma). the concentration of the stock solution of swainsonine was mg ml-l. it was prepared by dissolving ,ug of the inhibitor in ~ water. rats were injected intravitreally with [ s]methionine as a control or [" s]methionine and either castanospermine ( ,ug per eye) or swainsonine ( . ,ug per eye). since the castanospermine preparation utilized dmso at a final concentration of . ,ul per eye the same amount of dmso was also injected into the eyes of all other rats to control for any effects of dmso on the galactosylation of rhodopsin. after hr of dark adaptation rats were killed, retinas were collected for subcellular fractionation. rhodopsin was purified from the ros fraction and the golgienriched fraction by cona sepharose chromatography. a loo-~ fraction of the cona eluate was saved for sds-page and the remainder was applied to rca agarose columns for elution with /&methyl-ngalactopyranoside as described above. the rca eluates were then applied to sds-page gels. the percentage of cona purified rhodopsin which contained galactose was determined from the radioactivity of the rhodopsin band of the gels of the rca eluate as c.ompared to the cona gels for each treatment group. the data from the three repetitions of this experiment were analysed via a one-way analysis of variance for the ros samples and for the golgi samples. the test of significance used was tukey's multiple comparison procedure. l&jht-sensitivity experiments ros labeled with [ %]methionine and purified on a linear sucrose gradient were extracted with . % emulphogene as described above. prior to purification of rhodopsin over cona sepharose, half of the clarified extract was exposed to light for min at °c. each sample was then applied to a cona sepharose column and chromatographed as described above. the eluted samples were each applied to separate rca agarose columns and eluted according to the previously described procedures. selected eluted fractions were applied to sds polyacrylamide gels along with a ["hlrhodopsin standard for determination of the amount of radioactivity present in opsin under the conditions of light exposure and darkness. in order to determine whether the galactosecontaining rhodopsin was present in photoreceptor discs or plasma membrane a modification of the method of molday and molday ( ) was used. [ "s]methionine-labeled ros from rats were prepared using the linear sucrose gradient procedure described previously. ros were suspended in ml homogenizing buffer consisting of mm tris acetate, ph . . mm mgcl,, % sucrose. neuraminidase ( . u) from anthrobacter ureufaciens (boehringer-mannheim) was added to the ros suspension and incubated for hr at °c. after centrifugation at g for min. the pellet was suspended in ml of homogenizing buffer to which ricin agarose (u.s. biochemical corp.) was added. this mixture was incubated for hr at °c. ricin agarose was used rather than ricin-golddextran because it is sufficiently dense to pellet through the sucrose gradient (boesze-battaglia and albert, ). the ros were pelleted in homogenizing buffer by centrifugation at g for min, hypotonically lysed for min in . m tris buffer, ph . , and washed twice with . m tris buffer ph . (containing mm edta) by centrifugation at g for min. the sample was treated with trypsin (boehringer-mannheim) for min at °c at a final concentration . pg ml -i, soybean trypsin inhibitor (boehringer-mannheim) was then added to a final concentration of pg ml-'. after two additional min washings with . m tris buffer at g, the ros membranes were layered on linear sucrose gradients consisting of ml of a - % (w/v) sucrose underlaid with i ml hoy, (w/v) sucrose. all sucrose solutions were made up in mm nacl, mm mgcl,, and mm tris acetate ph . . after centrifugation for hr in a spinco sw rotor at rpm, the band of discs was drawn off using a pasteur pipet and the plasma membranes at the bottom of the tube were also removed. these two fractions were separately centrifuged at g for min followed by the addition of . % emulphogene to each pellet to extract rhodopsin. extracts were applied to rca agarose columns, washed with . % emulphogene, and eluted according to the previously described procedures. selected eluates were applied to sds-polyacrylamide gels and the amount of radioactivity present in rhodopsin was determined for the discs and plasma membrane. injection, four retinas were collected for subcellular fractionation. rhodopsin from each of the fractions was purified by cona sepharose chromatography and sds-page. table ii provides the ratios of [ h] derived from galactose to [l*c]leucine in rhodopsin from the subcellular fractions enriched in either rough endoplasmic reticulum, golgi or ros. the labeling pattern was complex. galactose was probably converted to glucose and mannose residues of the core . results in the experiments using bovine retinas, purified ros were treated with galactose oxidase to oxidize the sixth carbon hydroxyl group of galactose to a carbonyl group. this treatment was followed by exposure to tritium labeled sodium borohydride which chemically reduced the carbonyl group of galactose back to a hydroxyl with the introduction of a tritium atom (gahmberg and hakomori, ; carubelli and wen, ) . figure shows that without galactose oxidase treatment there was tritium incorporation into rhodopsin. there was a twofold increase in label following the galactose oxidase treatment. the galactose oxidase-dependent label tended to be in a species that migrated slower than rhodopsin as is indicated in the figure by the dashed line representing the difference between the two treatment groups. table i illustrates another experiment in which the galactose oxidase incubation time was doubled and the amount of radioactivity in the galactose oxidase treated ros was . -fold greater (over dpm) than the samples not treated with the enzyme. pretreatment with neuraminidase did not increase the labeling. the non-specific labeling of opsin in the samples not treated with the enzyme was due perhaps to reductions of the retinyl-lysine schiff s base linkage. these experiments demonstrate the presence of galactose in the oligosaccharide chains of native rhodopsin in ros plasma membranes and basal folds. since the enzyme does not have access to the carbohydrate chains within the sealed discs, it is not known whether there are any galactose residues in the disc membrane rhodopsin. of sodium dodecyl sulfate-polyacrylamide (sds-page) gels of bovine ros. the ros were purified by discontinuous sucrose density gradients and were incubated min with ( ) or without (m) galactose oxidase. both samples were then incubated with tritium labeled sodium borohydride. aliquots representing the ros of . retinas were solubilized and applied to gels for electrophoresis. although there was incorporation of tritium into rhodopsin without galactose oxidase treatment, there was a considerable increase in label following the galactose oxidase treatment. the galactose oxidase-dependent label tended to be in a species that migrated slower than rhodopsin as indicated by the dashed line (a) representing the difference between the two treatment groups. the coomassie blue-stained opsin band on a parallel gel coincided with the radioactive profile of the untreated sample. or gsa (@-- ). six rats were injected intravitreally with [?!i]methionine and were killed hr later at which time retinas were collected for subcellular fractionation and rhodopsin purification by cona sepharose chromatography. half of the sample was applied to the rca agarose column and half to the gsa agarose column. the washed rca agarose column was eluted with . m /?-methyl-o-galactopyranoside and the washed gsa agarose column was eluted to . m cc methyl-o-galactopyranoside beginning at fraction in both cases. at hr post-injection. - . x of the labeled rhodopsin bound to and was eluted from the rca column, whereas none bound to gsa. oligosaccharide in the rough endoplasmic reticulum. during the first hr. however, galactose selectively labeled rhodopsin in the golgi-enriched fraction resulting in increased [ h]/[ %] ratios in both golgi and ros. by day the galactose pool was exhausted and carbohydrate labeling that occurred in the rough endoplasmic reticulum-enriched fraction was probably due to glycogen turnover. the decreasing isotope ratios at each time point suggest that trimming was occurring at the transition from golgi to ros. furthermore, the decrease in isotope ratio in the ros between hr and day suggests further trimming of coomassie blue and cut into . mm slices for counting. migration was from left to right. the radioactivity profiles were slightly to the left of the coomassie blue-stained opsin band. as expected (see fig. ) of an opsin having a larger oligosaccharide. rhodopsin after membrane assembly in the ros. some of this lost carbohydrate may have been galactose, particularly in the ros. studies to determine the nature of the linkage of galuctose to rhodopsin rats injected intravitreally with [ s]methionine were maintained in darkness for hr at which time retinas were collected for subcellular fractionation and rhodopsin purification by cona sepharose chromatography. the cona-purified rhodopsin was then applied to one of two lectin columns: rca or gsa. rca specifically recognizes ,/i-linked galactose moieties (baenziger and fiete, ) and gsa is specific for a-linked galactose residues (blake and goldstein, ) . at hr, s- . % of the labeled rhodopsin bound to and was eluted from the rca column, whereas none bound to gsa, thus indicating the presence of a /j'-galactoside (fig. ) . the protein eluted from rca was shown to be rhodopsin by coelectrophoresis with rhodopsin that had been labeled with [ h]leucine in vivo for hr (fig. ) . rats injected intravitreally with [ s]methionine were maintained in darkness for and hr at which time retinas were collected for subcellular fractionation. ros were incubated with and without neuraminidase. the amount of rhodopsin bound to and eluted from the rca agarose columns was not increased by treatment with neuraminidase. the total dpm in the opsin region of sds-polyacrylamide gels for the two conditions and time points are shown in two groups of six rats were injected intravitreally with [%imethionine and killed at the indicated times. ros were prepared and half of each sample incubated with neuraminidase. after extraction, the rhodopsin samples were purified on cona sepharose and subsequently chromatographed on ricin agarose. table iii . these data suggest that although complex asparagine-linked oligosaccharides often have a terminal trisaccharide containing n-acetylglucosamine linked to galactose and capped with neuraminic acid (kornfeld and kornfeld, ) rhodopsin lacks neuraminic acid. demonstration that binding of the rhodopsin oligosaccharide to rca can be decreased with p-galactosidase treatment rats were injected intravitreally with [ s]methionine and [ h]glucosamine to provide markers for the polypeptide and the oligosaccharide, respectively. they were maintained in darkness for hr at which time retinas were collected for subcellular fractionation and rhodopsin purification by cona sepharose chromatography. the cona-purified rhodopsin was treated initially with n-glycanase to hydrolyze the asparagine-linked oligosaccharides from rhodopsin. this treatment was followed by incubation of half of the rhodopsin was incubated at °c for hr with nglycanase to hydrolyze the h-labeled oligosaccharide. p-galactosidase was added to half of the sample. both the / galactosidase treated sample and the control were incubated at °c for hr. each sample was applied to an rca agarose column which was washed and subsequently eluted with p-methyl-u-galactopyranoside beginning at fraction . the amount of radioactivity (dpm h) eluted from rca agarose after ,&galactosidase treatment was significantly less ( . %) than that of the sample eluted from rca agarose without the enzyme treatment ( . %). the sample with p-galactosidase which specifically hydrolyses terminal galactose residues that are pl- linked to n-acetylglucosamine. as shown in fig. , the amount of radioactivity eluted from rca after / galactosidase treatment was significantly less than that of the sample eluted from rca without the enzyme treatment. these same results were obtained in repetitions of this experiment and suggest that removal of galactose from the rhodopsin oligosac- rhodopsin was labeled for hr following simultaneous intravitreal injection of [%]methionine and swainsonine or castanospermine, or neither in three groups of six rats. * data are expressed as the mean percentages of three experiments + s.d. one-way analysis of variance for the ros and golgi samples indicated that there was a significant difference among the three groups (ros: f = . , p = . ; golgi: f = . , p = . ). tukey's paired comparisons test confirmed that the percentages obtained for the castanospermine treated group differed significantly from the control or swainsonine treated group, but these latter two groups did not differ significantly (p < . ). methionine were maintained in darkness for hr at which time retinas were collected for subcellular fractionation. half of a sample of detergent extracted purified ros labeled in vivo with [yg]methionine was exposed to light for min while the remainder of the sample was kept dark. each sample was chromatographed on cona sepharose and eluted with cc-methyl mannoside. the cona sepharose column eluates were applied to rca agarose which was washed with buffer and then eluted with p-methyl-ngalactopyranoside beginning at fraction . as would be expected, the dark sample bound to and was eluted from cona sepharose. . % of that sample was bound to and eluted from rca agarose. in contrast, considerably less radioactivity was eluted when the light-exposed sample was chromatographed on cona. what little was eluted did not bind to rca. ( -e) dark: ( -o) light. charide using this highly specific enzyme dramatically reduces the binding of the oligosaccharide to rca. these data provide further support for the presence of galactose on the oligosaccharide of rhodopsin. rats in groups of six injected intravitreally with [ s]methionine only or [? ]methionine and either catanospermine or swainsonine were dark adapted for hr at which time retinas were collected for subcellular fractionation. rhodopsin was purified from the ros-and golgi-enriched fractions by cona sepharose chromatography and the eluates were applied to kca agarose columns. both the kc'a agarose eluates and a sraction of the cona sepharose eluates were applied to sds-page gels. the data in table iv . representing three replicates of this experiment. show that the mean percentage of cona purified rhodopsin from ros which bound to rc. was greatly reduced in the castanospermine exposed group. a similar effect was seen in the golgi-enriched fraction. the statistical analysis of these data indicated that the percentages obtained in the castanospermine treated ros and golgi samples were significantly different from the control or swainsonine treated group. these data demonstrate that castanospermine. which inhibits glucosidase i, disrupted the binding of rhodopsin to rca, whereas swainsonine, which inhibits golgi mannosidase ii. did not. since rca is specific for p-galactose residues, it appears likely that in the presence of castanospermine. galactose was never linked to the oligosaccharide chain of rhodopsin. rats injected intravitreally with ["%]methionine were maintained in darkness for hr at which time retinas were collected for subcellular fractionation. half of a sample of detergent extracted purified ros labeled in vivo with [ s]methionine was exposed to light while the remainder of the sample was kept dark. each sample was chromatographed on cona sepharose and eluted with z-methyl mannoside. when rhodopsin is bleached it binds to cona. but is not eluted from the lectin by a-methyl mannoside. the cona sepharose column eluates were applied to rca agarose columns and eluted with p-methyl-d-galactopyranoside. the elution patterns from the cona sepharose columns and the rca agarose columns are shown in [fig. (a) and (b)], respectively. as would be expected, the dark sample bound to and was eluted from cona. furthermore. . % of that sample was bound to and eluted from rca. in contrast, much less radioactivity was eluted when the light-exposed sample was chromatographed on cona. what little was eluted did not bind to rca. sds-polyacrylamide gels of the material eluted from rca indicated that the dark sample coincided with a rhodopsin standard labeled with [ h]leucine. whereas there was virtually no radioactivity detected on the gel of the light exposed sample (fig. ) . these data indicate that the cona purified material that binds to rca is light sensitive and is therefore rhodopsin. compartments have galactose-containing rhodopsin n-linked oligosaccharides are typically assembled in the endoplasmic reticulum on dolichylphosphate and are then transported by means of vesicles to the golgi membrane (kornfeld and kornfeld, ) . in the trans golgi. galactose may be added to a terminal n- fig. s(b) ]. the dark sample coincided with a rhodopsin standard labeled in vivo with [ h]leucine, whereas there was virtually no radioactivity detected on the gel of the light-exposed sample. as before (fig. ) the radioactive opsins migrated slightly slower than the coomassie blue-stained mature opsin. . elution profiles from rca agarose columns of bands separated from a linear so- % sucrose gradient over which had been layered a crude ros suspension. the ros suspension was prepared from retinas of six rats which had been intravitreally injected with [%]methionine and dark adapted for hr. this procedure typically yields four bands on the gradient: a faint uppermost band - . cm from the top of the gradient, two bands of unsealed and sealed ros ( and . respectively) and a zone of fine particles, enriched in golgi. pellets of each of these bands were detergent extracted and applied to separate rca agarose columns which were then washed. as shown, the golgi-enriched fraction and both ros fractions (ros and ros ) bound to and were eluted from rca with ,&methyla-galactopyranoside. the uppermost band did not demonstrate rca binding. thus rhodopsin appears to have acquired galactose residues before leaving the golgi. acetylglucosamine of the oligosaccharide chain. in an effort to determine if the golgi apparatus, as well as the ros, of rod photoreceptor cells contained galactose, subcellular fractionation of ros was performed. rats were injected intravitreally with [ s]methionine and were maintained in darkness for hr at which time retinas were collected and crude ros preparations were made using the vortexing procedure described. linear - % sucrose gradients of crude ros suspensions which have been centrifuged for hr typically have four bands. a very faint uppermost band is approximately . - . cm from the top of the gradient. two bands of unsealed and sealed ros are below the . cm band (godchaux and zimmerman, ) . below these is a zone of fine particles, enriched in golgi. pellets of each of these bands were detergent extracted and applied to separate rca columns. as shown in fig. , the golgi-enriched fraction and both ros fractions (ros and ros ) bound to and were eluted from rca. the uppermost band did not demonstrate rca binding. sds-page of these fractions showed that the golgi and ros fractions coelectrophoresed with a rhodopsin standard. although the golgi-enriched fraction is not entirely free of ros, the data suggest that the galactose residue of rhodopsin is present on the oligosaccharide in the golgi as it is in other glycoproteins containing n-linked oligosaccharides. the results of the in vivo experiments in which [ h]galactose was used to label rhodopsin over several days (described above) suggested that galactose might be trimmed from the oligosaccharide of rhodopsin in the transition from golgi to ros as well as in ros over time. to test this, a time course experiment was conducted. rats were injected with [ s]methionine and [ h]galactose and groups of animals were killed , and hr following injection. retinas were collected for subcellular fractionation, ros were extracted and rhodopsin was purified by cona sepharose chromatography. a small fraction of the cona eluate was applied to sds-polyacrylamide gels and the remainder was applied to rca. table v provides the data obtained from gel electrophoresis of the cona-and rca-eluted samples. electrophoresis of the rca agarose eluates revealed that the percentage of the cona-purified rhodopsin that bound to rca decreased over time. at hr, . % of the rhodopsin bound to rca, after hr . % was bound and by hr only . % was bound. electrophoresis of the cona-purified rhodopsin showed that the ratio of the isotopes (that is the ratio of [ h]galactose to [ s]methionine) decreased over tie from . at hr to . by hr. clearly, trimming of galactose from rhodopsin had occurred suggesting that galactose may be an early component of rhodopsin, but as the molecule progresses through the ros the galactose appears to be lost. in contrast, the ratio of the isotopes ([ h]galactose to [ s]methionine) in the rhodopsin that bound to rca was higher than in the total pool eluted from cona and did not change significantly over time. thus, the ratio changes were due to removal of galactose residues. retinas were collected at the indicated times. ros were prepared and rhodopsin was purified on cona sepharose. aliquots were analysed by sds-page and the rest chromatographed on ricin agarose. ricin eluates were also analysed by sds-page. it was of interest to determine whether the galactose-containing rhodopsin was present in photoreceptor discs or plasma membrane. a modification of the method of molday and molday ( ) was used in which nine rats were injected intravitreally with [ s]methionine and were maintained in darkness for hr, at which time ros were prepared using the linear sucrose gradient procedure described previously. after treatment with neuraminidase ros were incubated with ricin-agarose. subsequent to lysis and trypsin treatment, the ros membranes were layered on linear - x sucrose gradients underlaid with % sucrose. following centrifugation, discs and the plasma membranes were removed. detergent extracts of these two components were applied to rca agarose columns. elution patterns from rca revealed significant binding of plasma membrane extracts and a small amount of binding of disc extracts. sdspolyacrylamide gels of the eluates showed that the total amount of radioactivity present in rhodopsin of the plasma membrane ( dpm) was . times greater than the total amount of radioactivity present in disks ( dpm). from these experiments it appears that at hr post-injection, . % of the labeled galactose-containing rhodopsin is in the plasma membrane and . % is in discs. although rhodopsin's oligosaccharide chains have been thought to contain mainly two types of sugar residues [mannose and n-acetylglucosamine (heller, yhx ; shichi et al., : heller and lawrence. : plantner and kean. ) ], there is evidence that a small fraction of rhodopsin also may contain galactose. fukuda et al. ( ) found that % of the oligosaccharides in bovine rhodopsin contained galactose. furthermore, o'brien ( i ) showed that bovine ros preparations support the transfer of galactose from ijdp-galactose to rhodopsin. st jules et al. ( i demonstrated the in vitro incorporation of galactose into rhodopsin and its subsequent removal after rhodopsin had reached the outer segment. these findings suggested that galactose may in fact be associated with rhodopsin of the ros. if only for a short period of time. perhaps the transient nature of its association explains its rather elusive detection by some biochemical techniques. in the current experiments, efforts were focused on demonstrating the presence of galactose by methods which could detect existing molecules, rather than solely by methods to show incorporation of exogenous galactose. (;alactose molecules were shown to exist on bovine rhodopsin using the galactose oxidase-sodium borohydride treatment. the results clearly demonstrated that there were galactose molecules present on rhodopsin which were altered by the highly specific galactose oxidase treatment and were then reduced by the sodium borohydride with the introduction of radiolabel. the bovine model does not permit in vivo studies of oligosaccharide synthesis, however. so it became necessary to investigate the incorporation of galactose in the rat model. the studies in which galactose was injected intravitreally in rats and monitored over several days demonstrated a selective labeling during the first hr in the golgi-enriched and the ros fractions. thus, even though conversion of galactose to other sugars commonly found on rhodopsin's oligosaccharide chain probably occurred, the initial pulse of galactose was apparently incorporated as galactose in the golgi where galactosyl transferase is known to be localized. the results strongly suggested that the galactose had been added to rhodopsin and then trimmed over time. the need to demonstrate the presence of galactose without directly incorporating it into rhodopsin lead to the use of lectin affinity chromatography. this powerful technique allows for the separation of glycoproteins based on the affinity for certain sugar moieties (cummings et al., ) . cona. which has an affinity for mannose residues. has long been used in the purification of rhodopsin. in the present work. a portion of the cona-purified rhodopsin was shown to bind to the lectin rca which specifically recognizes terminal galactose moieties associated with either glucosamine or n-acetylglucosamine in a pl- linkage (baenziger and fiete, ) . whereas no rhodopsin bound to gsa which has an affinity for xlinked galactose (blake and goldstein, ) . methionine was injected intravitreally into rats in order to label the polypeptide chain of rhodopsin. that the radiolabeled protein which bound to rca was indeed rhodopsin was verified by light sensitivity experiments. in addition, the binding of the rhodopsin oligosaccharide to rca could be decreased significantly by cleaving the galactose moiety with the highly specific enzyme / -galactosidase. on the other hand, treatment with neuraminidase, which specifically hydrolyses neuraminic acid from oligosaccharide chains, neither increased nor decreased the binding of rhodopsin to rca. these experiments indicated that the galactose of rhodopsin was not capped with neuraminic acid as is often the case with complex asparagine-linked oligosaccharides whose terminal trisaccharides contain n-acetylglucosamine linked to galactose capped with neuraminic acid (kornfeld and kornfeld, ) . the galactose of terminal trisaccharides of n-linked oligosaccharides are typically added to n-acetylglucosamine in the trans-golgi (kornfeld and kornfeld, ) . in the present work, it was of interest to determine if galactose was associated with rhodopsin purified from the golgi-enriched fraction as well as ros. it was determined by subcellular fractionation and subsequent rca chromatography that the rhodopsin of the golgi-enriched fraction did indeed contain galactose. furthermore, it was shown that the addition of galactose to the n-acetylglucosamine could be inhibited by in vivo exposure to castanospermine which specifically inhibits glucosidase i. glucosidase i is a rough endoplasmic reticulum enzyme that cleaves the terminal glucose from the glc,man,glcnac, (repp et al., ) . with this early step in the processing of the oligosaccharide disrupted, the subsequent steps in the processing pathway could not continue. the addition of galactose to n-acetylglucosamine was prevented because the oligosaccharide chain was never processed to the point where galactose is typically added to n-acetylglucosamine. by way of contrast, the addition of galactose to n-acetylglucosamine was not affected by swainsonine which inhibits the golgi mannosidase ii. this enzyme typically cleaves two mannose residues from one of the branches of the oligosaccharide chain (elbein, ) . the processing of the other branch of the oligosaccharide chain can continue normally. it is to this other chain, which contains a mannose and an nacetylglucosamine, that galactose is typically added. however, it should be noted that a negative result with swainsonine is of limited significance since there is no assurance that it reached and inhibited the target enzyme. to the best of our knowledge, these experiments provide the first evidence in a mammalian system that glycoprotein processing can be modified in rhodopsin following in vivo exposure to the appropriate inhibitory agent. chambers et al. ( ) reported that intraocular injection of frogs with up to ,ug of castanospermine and swainsonine resulted in neither a decrease in rhodopsin content nor a change in the length of photoreceptor outer segments. fliesler. rayborn and hollyfield ( a) demonstrated that in vitro exposure of xenopus retinas to castanospermine resulted in the hyperglycosylation of opsin. the opsin underwent normal intracellular transport and was utilized for the biogenesis of ros membranes having normal disc morphology. the hr to day in vivo galactose labeling studies of rhodopsin purified from rough endoplasmic reticulum-, golgi-enriched fractions and ros, suggested that the amount of galactose-derived label associated with rhodopsin was not constant. it appeared from this work that trimming had occurred in the ros over the course of several hours and also had occurred in transit from one subcellular compartment to another, namely, the golgi-enriched fraction to ros. we chose to investigate the trimming phenomenon which was apparently occurring in the ros using lectin affinity chromatography. two hours after the injection, approximately - % of the labeled rhodopsin bound to rca suggesting that about s- % of newly synthesized rhodopsin contains a / -galactose residue. the percentage of rhodopsin which contained galactose decreased by more than half within hr and by hr of the injection less than % of rhodopsin contained galactose. one might surmise that if the galactose concentration is greatest within hr of injection, perhaps it constitutes a transient component of rhodopsin's oligosaccharide chain. one possibility is that it is associated with the rhodopsin of the plasma membrane and basal folds of the ros and is removed as discs are sealed and move apically. the work of hicks and molday ( ) emphasized the presence at the basal area of the outer segments of ricin-binding compounds. the possible function of a galactose on rhodopsin at this one area is open to speculation. it has been shown that the oligosaccharide of rhodopsin or some other ros protein such as peripherin is essential for the process of disc morphogenesis by fliesler and basinger ( ) and by fliesler, rayborn and hollyfield ( ) who successfully disrupted disc formation with tunicamycin, an inhibitor of oligosaccharide synthesis. mechanisms have been proposed (fliesler, rayborn and hollyfield, b; fliesler, ) showing how the oligosaccharide of rhodopsin could play an important role in disc formation. as new discs are formed, presumably the opposite faces of a newly forming disc must be aligned and brought into close apposition. it might be that the oligosaccharide plays some role in the process in much the same way that a lectin binds a ligand or a hydrolase interacts with an oligosaccharide substrate. if there were an enzymesubstrate interaction, perhaps a carbohydrate residue such as galactose on one surface might be cleaved by a hydrolase such as a galactosidase on the opposing surface. during this process the two membrane surfaces would come into close apposition with the exclusion of extracellular matrix allowing fusion of the new disc. this hypothesis led to the experiment in which ros plasma membranes were separated from discs for the purpose of determining in which compartment the galactose-containing rhodopsin was most prevalent. the experiments showed that the greatest preponderance of the galactose-containing rhodopsin was in the plasma membrane component. this finding lends support to the hypothesis that a transiently present galactose is important in new disc formation in bovine and rat retinas because the galactose appears to be associated with the plasma membrane until the discs are finally fused. this proposed mechanism would not specifically require galactose as the substrate, only that the components of the oligosaccharide, however they may vary among species, be the substrates of the appropriate hydrolases. in the summary, the present work has shown through biochemical techniques that a subset of rhodopsin contains galactose. tn fact, shortly after it is synthesized, approximately - "/( of the rhodopsin in the ros contains this sugar residue. the galactose appears to be added in the golgi complex in the same way that a terminal trisaccharide is assembled on a complex oligosaccharide chain. the galactose is not capped, however, with neuraminic acid. we presented evidence to confirm the incorporation of galactose into rhodopsin in in vivo experiments. additionally, we demonstrated the presence of galactose in bovine rhodopsin by radiolabeling existing moieties. finally, using lectin affinity chromatography, we were able to separate the galactose-containing species of rhodopsin and study its transient association with the oligosaccharide chain. we determined that galactose is trimmed from rhodopsin over the course of about hr. presumably after it has served its function which may be related to new disc formation. structural determinants of ricinus communis agglutinin and toxin specificity for oligosaccharides. resolution of nucleotide sugars and oligosaccharides by lectin affinity chromatography specific binding of peanut lectin to a class of retinal photoreceptor cells: a species comparison fatty acid composition of bovine rod outer segment plasma membrane the biosynthesis of rhodopsin as studied by membrane renewal of rod outer segments lectin receptors of rods and cones : visualization by fluorescent label different distribution of receptors for peanut and ricin agglutinins between inner and outer segments of rod cells in vitro radiolabeling of galactosyl and n-acetylgalactosaminyl moieties ofglycoproteins with carbon-l effects of glycosylation inhibitors on the frog retina separation and analysis of glycoprotein oiigosaccharides y ). inhibitors of glycoprotein synthesis retinal rod outer segment membrane assembly: studies with inhibitors of enzymes involved in n-linked oligosaccharide biosynthesis and processing tunicamycin blocks the incorporation of opsin into retinal rod outer segment membranes membrane morphogenesis in retinal rod outer segments: inhibition by tunicamycin i y a). inhibition of oligosaccharide processing and membrane morphogenesis in retinal rod photoreceptor cells protein-bound carbohydrate involvement in plasma membrane assembly : the retinal rod photoreceptor cell as a model rhodopsin carbohydrate : structure of small oligosaccharides attached at two sites near the nh, terminus external labeling of cell surface galactose and galactosamine in glycolipid and glycoprotein of human erythrocytes soluble proteins of intact bovine rod cell outer segments the amino-terminal tryptic peptide of bovine rhodopsin. a glycopeptide containing two sites of oligosaccharide attachment structure of visual pigments i. purification, molecular weight and composition of bovine visual pigmew structure of the glycopeptide from bovine visual pigment localization of lectin receptors on bovine photoreceptor cells using dextrangold markers lectin and antibody labelling of developing rat photoreceptor cells: an electron microscope immunocytochemical study i:ltrastructural localization of lectin receptors in the monkey retinal photoreceptors and pigment epithelium : application of lectin-gold complexes on thin sections. f;xp assembly of asparagine-linked oligosaccharides cleavage of structural proteins during the assembly of the head of bacteriophage t differences in protein composition of bovine retinal rod outer segments disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method. ultrastructural localization of lectms binding sites on the surface of retinal photoreceptors and pigment epithelium rhodopsin as a glycoprotein : a possible role for the oligosaccharide in phagocytosis the biosynthesis of rhodopsin in vitro acylation of bovine rhodopsin by [ h]palmitic acid carbohydrate composition of bovine rhodopsin the effects of processing inhibitors of n-linked oligosaccharides on the intracellular migration of glycoprotein e of mouse hepatitus virus and the maturation of coronavirus particles biochemistry of visual pigments i. purification and properties of bovine rhodopsin the acylation of rat rhodopsin in vitro and in vivo the localization and timing of post-translational modifications of rat rhodopsin localization of fluorescent-labeled lectin binding sites on photoreceptor cells of the monkey retina key: cord- -elbnzgx authors: mutua, victoria; gershwin, laurel j. title: a review of neutrophil extracellular traps (nets) in disease: potential anti-nets therapeutics date: - - journal: clin rev allergy immunol doi: . /s - - - sha: doc_id: cord_uid: elbnzgx activated neutrophils release neutrophil extracellular traps (nets) in response to a variety of stimuli. netosis is driven by protein-arginine deiminase type , with the release of intracellular granule components that function by capturing and destroying microbes, including viral, fungal, bacterial, and protozoal pathogens. the positive effects of pathogen control are countered by pro-inflammatory effects as demonstrated in a variety of diseases. components of nets are non-specific, and other than controlling microbes, they cause injury to surrounding tissue by themselves or by increasing the pro-inflammatory response. nets can play a role in enhancement of the inflammation seen in autoimmune diseases including psoriasis, rheumatoid arthritis, and systemic lupus erythematosis. in addition, autoinflammatory diseases such as gout have been associated with netosis. inhibition of nets may decrease the severity of many diseases improving survival. herein, we describe netosis in different diseases focusing on the detrimental effect of nets and outline possible therapeutics that can be used to mitigate netosis. there is a need for more studies and clinical trials on these and other compounds that could prevent or destroy nets, thereby decreasing damage to patients. neutrophil extracellular traps (nets) were discovered in [ ] and further detailed by brinkmann et al. who termed the process netosis [ ] [ ] [ ] . neutrophils are short-lived granulocytes that are the initial defense against invading pathogens. they achieve this through phagocytosis, degranulation, production of reactive oxygen species (ros), and production of chemokines and cytokines to recruit other immune cells maximizing the host's immune response [ ] [ ] [ ] . neutrophils enhance their antimicrobial properties by releasing nets, composed of extracellular chromatin decorated with histones and numerous granular proteins [ , ] and were identified as part of innate immune response which can either be beneficial or pathological [ , , ] . net formation starts with the activation of neutrophils through the recognition of stimuli and activation of nadph oxidase (nox) complex through protein kinase c (pkc)-raf/merk/erk [ ] [ ] [ ] which in turn activate myeloperoxidase (mpo), neutrophil elastase (ne), and protein-arginine deiminase type (pad ) [ , ] . pad catalyzes citrullination of histones and promotes chromatin decondensation [ ] [ ] [ ] , while the ros species promote netosis by inducing gradual separation and loss of the nuclear membrane with the release of chromatin outside the cell through membrane pores. cellular lysis with a final release of dna, citrullinated histones (cith ), and other intracellular granules form the extracellular traps [ ] . netosis is induced in response to stimuli promoting pathogen clearance by trapping, and either killing through microbial toxicity or immobilizing microbes facilitating phagocytosis by other neutrophils and phagocytes [ , , , ] . due to the non-specific effects of the released enzymatic proteins, nets may lead to uncontrolled inflammatory response causing tissue pathology. there is direct cell damage, recruitment of other pro-inflammatory cells and proteins, and formation of immune complexes that induce autoantibody production leading to tissue damage [ , ] . nets can capture metastatic tumors aggravating cancerous condition [ ] , and in diabetic cases, they lead to a delay in wound healing [ , ] . neutrophil can also form interactions with platelets mediated by p-selectin [ ] . this leads to induction of platelet-derived high-mobility group protein b (hmgb ) [ ] which stimulates nets [ , ] causing occlusion in the vasculature by promoting thrombosis and obstruction causing organ damage. although nets have been shown to promote inflammation, a study done by christine et al. shows that an accumulation of net aggregates can reduce inflammation in a mouse model of gout through the degeneration of cytokines and chemokines [ ] . this means that there is still much about nets we are not aware of, and thus the need for more studies to understand their specific mechanism and how to harness their benefits while limiting their negative effects. there are certain compounds that have been identified in some studies to either inhibit or disrupt netosis, but there is no available therapeutic that has been researched extensively or approved for human use. we propose that the limited therapeutics have been due to the different effects of nets in different disease conditions; thus, identifying a stand-alone compound might be a challenge. in this review, we briefly mention netosis in different diseases and try to reconcile different aspects of net biology highlighting possible compounds that can be considered therapeutic. new approaches in therapeutic design and efficacy testing will have to be developed to find a truly efficacious treatment. here is a brief discussion on the different researched effects of nets. nets have been shown to have positive effects in controlling bacterial infections. they possess antimicrobial properties with components including histones, cathepsin g, ne, mpo, lactoferrin, antimicrobial peptide-ll , pentraxin , gelatinase, proteinase , and peptidoglycan-binding proteins that are bactericidal [ , , ] . nets limit growth or kill bacterial as reviewed by vidal delgado-rizo et al. which include shigella flexneri, pseudomonas aeruginosa, escherichia coli, shigella sonnei, salmonella enteritidis, salmonella typhimurium, klebsiella pneumoniae, pseudomonas aeruginosa, staphylococcus albus, staphylococcus aureus, and propionibacterium [ , ] . in viral infections including influenza, hiv, and respiratory syncytial virus, there is an excessive neutrophil recruitment [ , ] . these viruses stimulate netosis through tlr , and/or with the release of ros species and the nets trap, contain, and eliminate viruses [ ] [ ] [ ] or inhibit viral replication through the blockade of the pkc pathway. histones are also important for viral aggregation and neutralization leading to a significant decrease in viral replication [ , ] . fungi like aspergillus nidulans, candida albicans, aspergillus fumigatus, and cryptococcus spp. induce netosis through the recognition of β-glucan on hyphae by components of the extracellular matrix or activation of nox [ , , ] . nets have been shown to be important in trapping and clearing large pathogens in vivo, thus being critical for antifungal defense [ , , ] . in parasitic conditions including plasmodium falciparum and toxoplasma gondii, there is activation of platelets, monocytes, and neutrophils. net formation, which is dependent on mek-erk pathway, limits the dissemination of the parasites by trapping and killing them [ , ] . histones reduce the replication of the leishmania spp. [ ] and together with other nets-associated compounds, such as ne, mpo, and collagenase, were shown to kill these pathogens [ ] [ ] [ ] [ ] . most studies on nets have been done in mice and in vitro, but there is still a gap in knowledge on the exact mechanism of nets in vivo. this necessitates the need for more studies to clearly evaluate their effects in in vivo and in humans. the ability to detect nets may be used as a prognostic tool for patients with conditions presenting with a higher rate of net formation, facilitating clinicians to provide personalized treatment. for nets to be used as screening tools, there has to be studies to standardize and define normal from abnormal levels. this could involve measurement of net-associated products in the blood cfdna, cith , ne, and mpo. in colorectal and breast cancer patients, cfdna has been quantified in serum samples via a simple nucleic acid-staining assay [ ] [ ] [ ] [ ] . this can be used to classify the cancer; however, measuring circulating mpo/cfdna conjugates and cith may be more specific for net analysis than evaluation of cfdna alone [ ] . cith is highly specific to netosis making it a possible tool for understanding variances between net levels [ ] . thalin observed that high plasma content of cith was a significant indicator of short-term mortality in some cancer patients [ ] , and some observational studies inform on the significance of nets in progression of colorectal cancer [ ] . further human studies are needed to definitively quantify different levels of nets and associate them with poor cancer/disease outcomes. large amounts of circulating nets demonstrated in septic patients are associated with poor outcome and multiple organ failure [ , , ] . this could be due to increased netosis, apoptosis, and necrosis or decreased clearance of extruded products with studies suggesting that cfdna exacerbate inflammation by inducing tnf-α mrna [ , ] . histones also function as damage-associated molecular patterns and can induce organ damage by promoting pro-inflammatory cytokine release causing endothelial dysfunction by inducing cytotoxicity and increasing ros production [ , , ]. nets have been indicated in pathologic alterations in autoimmune and autoinflammatory diseases [ , ] . here, we discuss in brief a few of these diseases. psoriasis is a chronic immune-mediated disease characterized by demarcated erythematous plaques on the skin. some patients may also suffer from psoriatic arthritis with joint pains and deformities [ ] [ ] [ ] [ ] . studies show that neutrophils are recruited to psoriasis lesions where they cluster to form spongiform pustules and munro's microabscesses and produce pro-inflammatory cytokines including il- , il- , and il- s [ , ] . il- in keratinocytes increases the expression of ll , a cathelicidin-derived antimicrobial peptide, and defensins which mediate net formation in dermatological conditions [ , ] . these inflammatory compounds have been shown to promote netosis and pathology in the absence of infection [ ] in these patients. systemic lupus erythematosus (sle) is an autoimmune disease characterized by immune complexes and high levels of ifn-α with the activation of autoreactive b cells [ , ] . there is a possible production of autoantibodies against nucleic acids released by neutrophils undergoing netosis [ , ] with the generated immune complexes representing a source of self-antigens that enhance the autoimmune and inflammatory process. this in turn results in more injury and inflammation [ , ] . rheumatoid arthritis (ra) is a systemic autoimmune disease characterized by persistent synovial inflammation that leads to cartilage and bone injury in the joints [ ] . the synovial fluid at the synovial cavity of ra patients becomes infiltrated with neutrophils that readily form nets [ , ] . studies have demonstrated that circulating neutrophils of ra patients are more easily stimulated to netosis than those from healthy subjects [ , ] , and as in other autoimmune conditions, nets act as a source of extracellular autoantigens leading to excessive innate and adaptive immune responses in the joints and subsequent tissue injury [ , ] . type diabetes mellitus (t dm) is an autoimmune disease characterized by the destruction of β pancreatic cells leading to hyperglycemia [ ] . this causes production of autoantigens that are recognized by immune cells with production of autoantibodies [ , ] . t dm patients are at a risk of developing neutropenia, and neutrophils can be found within infiltrates in pancreatic islets where elevated tnf-α induces formation of nets [ ] . cytokines produced in this process lead to neutrophil recruitment to sites of inflammation, providing negative feedback and contributing to pathogenesis in autoimmune diabetes [ , ] . small vessel vasculitis (svv) is a systemic disease of unknown etiology where the patients exhibit blood vessel inflammation, with necrotizing inflammation in small blood vessels potentially leading to organ damage [ ] [ ] [ ] . these patients have been shown to have anti-neutrophil cytoplasmic antibodies (ancas) [ , ] . proteins released during netosis are the main cause of anca production by activating the complement system resulting in endothelial damage [ , ] . these studies have shown that α-pr and α-mpo ancas induce netosis during active disease perpetuating a feedback loop [ ] . gout is an autoinflammatory disease characterized by the deposition of monosodium urate (msu) crystals in the joints, stimulating immune responses by attracting leukocytes and inducing nets that promote inflammation [ , , [ ] [ ] [ ] . inflammatory bowel diseases (ibds) are disease affecting the gastrointestinal tract characterized by chronic uncontrolled inflammation. the two major forms of ibd include ulcerative colitis (uc) and crohn's disease (cd), which have different etiologies, pathogenesis, and diagnostic features with the differences not fully understood. cd clinically manifests as gastrointestinal disorders but is a systemic disease involving inflammation of the ileum and colon [ ] [ ] [ ] . net formation in cd has not been well-studied although studies indicate that ros production is enhanced, which could promote netosis [ , [ ] [ ] [ ] . uc is also characterized by inflammation of the gastrointestinal tract mostly restricted to the colon with nets observed in the colon accompanied by exacerbated inflammation [ ] . there are a few studies looking into the mechanism of nets in these conditions, but there are more studies needed to better inform on development of effective treatment options. metabolic diseases have been associated with chronic lowgrade inflammation with activation of the innate immune response and recruitment of mononuclear and polymorphonuclear leukocytes increasing cellular dysfunction [ , ] . this microenvironment favors netosis linking it to immune deregulation and hyperglycemia, oxidative stress, inflammation, and further complications of metabolic diseases. type diabetes is a chronic metabolic condition characterized by glucose level build-up in the bloodstream, hyperglycemia, and cells unresponsive to insulin. studies have shown that hyperglycemia predisposes neutrophils to release nets. net-related bioproducts (ne, mpo, and cfdna) are increased compared with non-diabetic subjects and also positively correlate with increased glycated hemoglobin (hba c) levels [ , ] . this suggests that the chronic proinflammatory conditions present during hyperglycemia promote netosis in both the type and type diabetes [ , ] ; thus, net formation is enhanced in hyperglycemic conditions independent of diabetes type and origin. obesity is a metabolic condition characterized by an excess of adipose tissue deposition, as a result of energy imbalance due to increased energy intake versus expenditure. obesity is frequently associated with other chronic complications including cardiovascular disease and diabetes [ , ] . studies have shown an association between obesity and chronic inflammation with enhanced neutrophil activity, increased superoxide radicals, and net formation [ , ] . moorthy et al. show that the neutrophils of mice fed with high-fat diet are more prone to spontaneous net formation, compared with neutrophils derived from mice fed with low-fat diet [ ] . the same is seen in mice fed with high-fat diet and infected with inlfuenza compared with the mice fed with low-fat diet [ ] . there is an increase in obesity globally, necessitating more studies into this condition and the link to the other metabolic conditions. this will inform on management of conditions caused or exacerbated by obesity and how nets play a role in this. although nets can be beneficial, the detrimental effect of nets can cause excessive tissue damage and pathology. there are studies evaluating the possible effects of certain compounds against nets as illustrated in table , and more studies need to be considered to mitigate the negative effects of netosis. acetylsalicylic acid (aspirin) is a non-steroidal drug with an antithrombotic and an anti-inflammatory effect used in the management of inflammatory symptoms. it functions through the irreversible acetylation of cyclooxygenase enzyme (cox), and suppresses prostaglandin generation [ , ] . it is used as an antiplatelet agent for prevention of arterial thromboses as it inhibits thromboxane a [ , ] . thromboxane a is a vasoconstrictor that activates new platelets increasing platelet aggregation, an important function during tissue injury, inflammation, and healing( [ ] ). platelets are the primary effector cells of hemostasis [ ] , but recent evidence indicates that they play a direct role in innate immunity by interacting with pathogens or recognize pathogen-associated molecular patterns (pamps) [ , ] . they facilitate innate immunity and activate netosis via platelet-neutrophil interaction [ , ] . platelet activation through tlr and tlr leads to the expression of pselectin which binds to neutrophil receptor (psgl- ) inducing netosis as demonstrated in mice [ , , ] . netosis is also mediated by the binding of αmβ (mac- ) on neutrophils to glycoprotein bα (gp bα) on platelets generating nets in liver and lungs during endotoxemia and in septic conditions [ , ] . in addition to lps, platelets can be activated by thrombin and arachidonic acid to form nets [ , ] . upon activation, platelets secrete soluble mediators including high-mobility group box- (hmbg- ) [ ], platelet factor (pf ), and ccl (rantes) that induce netosis via the neutrophil g protein coupled receptors [ ] . the interactions between platelets and neutrophils mediate netosis, and inhibition of this interaction using antiplatelet therapy has the potential to inhibit net formation [ , ] . in a study conducted on endotoxin-triggered acute lung injury, pretreatment of mice with aspirin showed a decreased intravascular net formation and reduced degree of lung injury [ , ] . in another study on effects of nets on transplantation in mice, they discovered that platelet activation was also inhibited by aspirin [ ] . lapponi et al. conducted another study where they treated neutrophils with a steroidal immunomodulatory drug (dexamethasone) or aspirin, and discovered that dexamethasone had no effect, while aspirin prevented net formation [ ] . they demonstrated that aspirin functions by inhibiting nf-κb, an inflammatory transcriptional regulator, that promotes netosis. these results show that aspirin could be a useful therapy in the management of pathologic netosis induced by platelets, but we have to keep in mind the side effects of aspirin. aspirin is a blood thinner and predisposes patients to stomach ulcers, so more studies are needed to find out which conditions could benefit from treatment with aspirin, without the excessive side effects. cyclosporine a is an immunosuppressant drug widely used in post-allogeneic organ transplant to reduce the activity of the patient's immune system, and therefore the risk of organ rejection [ , ] . it causes reversible inhibition of immunocompetent lymphocytes and has been used to manage fungal infections, rheumatoid arthritis, asthma, dermatologic drug, and immunosuppressive agent [ , ] . the mechanism of action of cyclosporine a involves binding to cytophilin, resulting in the downregulation of nfat (nuclear factor of activated t cells) transcription factor and inhibiting the calcineurin pathway subsequently inhibiting net formation [ , ] . gupta [ ] [ ] [ ] [ ] [ ] [ ] chloroquine/hydroxychloroquine mmps, timps antimalarial, inhibits cytokine production, interferes with the stimulatory effect of platelet aggregation, maintaining extracellular matrix homeostasis [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] diphenyleneiodonium chloride (dpi) nadph inhibits nadph oxidase ros production [ ] [ ] [ ] [ ] n-acetylcysteine (nac) inhibits ros production, prevents thrombus formation [ ] [ ] [ ] [ ] [ ] [ ] nucleases recombinant human dnase dna matrixes reduces neutrophil infiltration, cleaves dna matrixes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] staphylokinase plasminogen, alpha-defensins converting nets to deoxyadenosine mediating death of immune cells [ ] [ ] [ ] [ ] notable compounds probiotics pkc pathway dampening ros production [ ] [ ] [ ] [ ] vitamin d reduces endothelial damage, decreases cell apoptosis [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] pge prostagladin e , nfat nuclear factor of activated t cells, pad protein-arginine deiminase , ros reactive oxygen species, pkc protein kinase c, mmps matrix metallopeptidase, timps tissue inhibitors of metallopeptidase, mtorc mechanistic target of rapamycin complex , ampk- ′ amp-activated protein kinase. properties), and rapamycin (a cell anti-proliferative and immunosuppressive agent), on both extra and intracellular calcium pools and their modulation in netosis. their data indicated that a combination of ascomycin and cyclosporine a reduced netosis, but the same effect was not evident following treatment with rapamycin [ ] . this opens up the possibility to therapeutically suppress or modulate netosis using cyclosporine a or a combination therapy with ascomycin. there were no other studies we could find in this area, and this could be due to the fact that cyclosporine a and immunosuppressive drugs may impair normal host immune responses to microbes [ , ] , predisposing patients to frequent infections. therefore, there is a need for more studies into how these drugs could be formulated to manage netosis safely. chlor-amidine (cl-amidine) is a compound designed to irreversibly inhibit protein-arginine deiminase (pad) through covalent modification at the active site of the enzymes [ ] . as described above, pad is an enzyme involved in netosis; thus, inhibition of pad is a possible therapeutic target. avin et al. run a study to evaluate the effect of inhibition of pad in netosis using an antagomir- , a pleiotropic microrna important in the regulation of immune responses, demonstrating a decreased induction of pad mrna and subsequent reduced nets in response to pma challenge [ ] . in a mouse model of lupus, systemic treatment with the pad inhibitor (bb-cl-amidine) showed protection of the mice from developing net-mediated vascular damage, endothelial dysfunction, and kidney injury. the study indicated that pad inhibition markedly downregulates the expression of type i interferon-regulated genes and reduces proteinuria and immune complex deposition in the kidneys, while also protecting against skin disease [ ] . another study found that pad -deficient mice (both diabetic and nondiabetic) possess faster wound healing and re-epithelization processes than their wild-type counterparts. this effect was independent of wound infection suggesting that netosis could hinder wound healing by limiting keratinocyte migration and re-epithelization [ ] . it is therefore possible to target inhibition of pad to inhibit net formation; however, it is important to note that pad may have other important functions in immunity which may be impaired [ , ] . in one study, they reported mixed results of pharmacological pad inhibition using cl-amidine in human neutrophils, where netosis induced by smoking was blocked by inhibiting of pad , but netosis induced by cholesterol crystals was not blocked [ ] . with these results, developing a suitable targeted therapy for pad may be challenging thus the need for more carefully considered human studies on the function of pad before using its inhibition as a strategy for management of netosis. pge -prostaglandins (pgs) are members of the eicosanoid family synthesized from arachidonic acid via cox enzymes and produced by nearly all cells within the body. pge is the most abundant prostaglandin in the human body and has been shown to influence both inflammatory and in some cases anti-inflammatory effects [ , ] . shishikura et al. evaluated the effects of pge , agonists and antagonists of its receptors, and modulators of the camp-pka pathway on the formation of nets in vitro (in isolated neutrophils) and in vivo in a mouse model. they also discovered that pge inhibited pma-induced net formation in vitro through ep and ep . exogenous pge treatment limited netosis of neutrophils collected from normal human volunteers and naive mice in an exchange protein activated by camp-and protein kinase a-dependent manner demonstrating a physiologic inhibition of netosis. incubation with a cell-permeable camp analogue, dibutyryl camp, or various inhibitors of a campdegrading enzyme, rolipram (pde inhibitor), and butaprost, (ep receptor agonist) also suppressed net formation [ ] . interestingly, domingo et al. conducted a study in murine bone marrow transplant mice (bmt) where neutrophils overexpress cox- and overproduce pge , leading to defective intracellular bacterial killing. they wanted to determine whether netosis was defective after transplant and whether this was regulated by pge signaling. treatment of bmt neutrophils with rapamycin resulted in reduced net formation relative to control cells while the ep receptor antagonist (pf- ) or the ep antagonist (ae - ), gαscoupled receptors, restored net formation suggesting that blocking pge -ep or ep signaling pathway restores netosis [ ] . these findings will contribute to the development of novel treatments for netosis-related diseases although more studies need to be done to evaluate the effect of using pge as a therapeutic. although pge is beneficial in management of sle and other ifn-α-dependent, th driven diseases [ ] , it could pose a challenge in conditions like arthritis [ , ] associated with pain. pge is known to contribute to pain as part of the inflammatory response, thus the need for more studies to evaluate its effects in different diseases compared with its benefits. pa-dpeg is a peptide inhibitor of complement c (pic ) which mitigates peroxidase activity of mpo, hemoglobin, and myoglobin through a reversible process [ ] . defective complement action caused by dysregulation and acute and chronic tissue damage or transplants can lead to host cell attack contributing to inflammatory conditions [ , ] . this is more so in the kidney which has been shown to be particularly sensitive to complement-mediated damage [ , ] . it is known that complement effectors including c a and membrane attack complex (sc b- ) interact with and can stimulate human neutrophils to generate nets. subsequently, products of netosis can activate complements causing a destructive loop [ ] . therapeutic complement inhibition is successfully used in paroxysmal nocturnal hemoglobinuria showing a promise in its use in other clinical conditions [ , ] . an article by hair et al. demonstrated that pic showed dose-dependent antioxidant activity, acting via the single electron transport (set) and hydrogen atom transfer (hat) mechanisms interfering with oxidation of cysteine residues. they showed that pa-dpeg achieved complete inhibition with complement effector levels equivalent to background. pa-dpeg was also able to dose-dependently inhibit net formation by human neutrophils, stimulated by pma, mpo, or immune complex activated human sera [ ] . their results suggest that pa-dpeg inhibition of nets occurs by blocking the mpo pathway of net formation. this provides proof that peptides can potentially be developed to inhibit complement-induced netosis and be used to manage conditions worsened by nets, although since complements are important in immune response, this needs to be researched further. antibiotics have been used in the management of bacterial infections and also have immunomodulating effects by influencing the properties of numerous immune cells, including neutrophils [ , ] . bystrzycka et al. conducted a study to investigate the effects of azithromycin and chloramphenicol on degranulation, apoptosis, respiratory burst, and the release of nets by neutrophils. their study indicated that pretreatment of neutrophils with azithromycin and chloramphenicol decreases the release of nets, with azithromycin showing a concentration-dependent effect on the respiratory burst in bystrzycka et al.'s study [ ] . another article by manda-handzlik et al. looked into the effects of cefotaxime and gentamicin on nets and discovered that gentamicin inhibits net release by human neutrophils, while cefotaxime had no impact on this process [ ] . the information that antibiotics can modulate net release can be useful in the management of infectious diseases or patients suffering from net-related diseases. since different antibiotics have different effects, there is need for more studies on their mode of action. this will inform on a possible compound for use in the management of nets without interfering with their antimicrobial function. thrombomodulin is a protein cofactor expressed on endothelial cell surfaces that modifies the substrate specificity of thrombin by an allosteric mechanism [ ] . thrombinthrombomodulin complex activates protein c, initiating an essential anticoagulant pathway [ ] [ ] [ ] . thrombosis is the formation of a blood clot within a blood vessel caused by cytokines and other inflammatory mediators produced during an injury, obesity, and in some cases drugs, e.g., estrogen pills [ ] . large amounts of circulating cfdna, present in nets, can influence thrombus formation by impairing fibrinolysis creating a scaffold for the binding of red blood cells, platelets, fibrin, and coagulation factors [ ] . besides cfdna, other net components also exert procoagulant properties with extracellular histones inducing platelet activation, aggregation, and thrombin generation [ , , ] . immuno-thrombosis-induced coagulopathy may contribute to hypercoagulability which increases occurrence of multiple organ dysfunction and mortality [ ] [ ] [ ] . helms et al. did a study where they looked into the use of a recombinant human thrombomodulin (rhtm) and found out that it can limit procoagulant responses. rhtm was also shown to fully inhibit netosis in neutrophils cultured with platelets and in the presence of lps [ ] . there is not much research in effect of rhtm, but this provides a starting point for further research into its potential use to inhibit nets. activated protein c (apc) is a multifunctional serine protease produced in blood by vitamin k-activating protein c. apc has anticoagulant, cytoprotective, and anti-inflammatory activities [ , ] . protein c has been shown to be an important prognostic indicator in patients with sepsis. during sepsis, there is a reduction in the conversion of protein c to its active form due to the downregulation of thrombomodulin by inflammatory cytokines [ ] . the antithrombotic effects of activated protein c is mediated by its ability to inhibit the formation of clotting factors va and villa and disrupting extracellular histones [ , ] . healy et al. demonstrated that apc cleaves and detoxifies extracellular histones and prevents activated platelets from inducing netosis. the pretreatment of neutrophils with apc before inducing netosis inhibited platelet adhesion to nets. they also used antibodies against the neutrophil receptors endothelial protein c receptor (epcr), protease-activated receptor (par ), and macrophage- antigen (mac- ) which blocked apc inhibition of netosis [ ] . another study demonstrated that the blockade of protein c activation lead to exacerbated sublethal lps challenge to turn lethal, which was reversed by treatment with antibodies to histones [ , ] . these findings suggest that the anti-inflammatory function of apc may include inhibition of netosis. drotrecogin alfa is a recombinant human activated protein c produced by xigris approved for use in septic patients [ , , ] . studies should be done to evaluate if this and other similar compounds would be effective in minimizing netosis in sepsis and other conditions with minimal side effects. heparin is a medication and naturally occurring glycosaminoglycan used as an anticoagulant (blood thinner). specifically, it is used in the treatment of heart attacks and unstable angina, and also antagonizes the effects of histones [ , ] . high levels of circulating histones have been positively correlated with disease severity in many disease conditions as they activate nf-κb pathway inducing the secretion of cytokines that amplify inflammation leading to organ damage [ ] [ ] [ ] . heparin has been shown to significantly suppress histone-induced disease [ , ] . studies have been done to evaluate the effect of unfractionated heparin, low molecular weight heparin, e.g., parnaparin and non-anticoagulant heparin [ , , ] . these studies demonstrate that heparin was able to protect mice from organ damage and death by antagonizing circulating histones attenuating tissue damage. administration of heparin, especially the nonanticoagulant heparin, is a novel and promising approach that may be further developed to treat patients with high levels of circulating histones potentially inhibiting netosis without increasing the risk of bleeding. anti-high-mobility group box (hmgb ) is an abundant protein that regulates chromosome architecture and also functions as a damage-associated molecular pattern molecule [ ] [ ] [ ] . it plays a beneficial role in microbial eradication through its pro-inflammatory actions and modulation of neutrophil chemotaxis [ ] [ ] [ ] [ ] [ ] . platelets are the major source of hmgb within the thrombi and present it to neutrophils promoting netosis [ ] [ ] [ ] ] . vogel et al. determined that platelet-derived hmgb is a critical mediator of thrombosis from their study using generated transgenic mice with platelet-specific deletion of hmgb [ ]. these effects were mediated via tlr -and myd -dependent recruitment of platelet guanylyl cyclase (gc) toward the plasma membrane, followed by myd /gc complex formation and activation of the cgmp-dependent protein kinase i [ , ] . mice lacking hmgb in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/ hemorrhagic shock [ , ] . exposure of neutrophils to hmgb resulted in enhanced formation of nets in vitro through tlr -dependent processes contributing to inflammatory processes and tissue injury [ ] [ ] [ ] [ ] ] . studies show that the use of anti-hmgb antibodies may diminish net formation, as seen in a reduction of histone and free dna in the bal fluid of lps-treated mice that received neutralizing antibodies to hmgb [ , ] . however, decreased levels of cytokines in the lungs after administration of anti-hmgb antibodies to lps-treated mice may not necessarily be a direct result of diminished net formation but could reflect the effects of hmgb on other pro-inflammatory pathways [ , ] . this is a promising area necessitating more research into how anti-hmgb interrupts netosis, and possibly use it as a treatment option. c esterase inhibitor (c inh) is an acute phase protein found in blood and a serine protease inhibitor that targets the complement pathway, coagulation pathway (factor xiia), and the contact system protease kallikrein. it is an endogenous inhibitor of c protein in the complement system [ , , ] . c -inh concentrates are approved for use in the management of hereditary angioedema (hae), an autosomaldominant disease caused by c -inh deficiency due to a mutation in the c -inhibitor gene [ , ] . studies have been done to evaluate whether ci-inh may protect from lung injury in vivo possibly explaining the underlying mechanisms mediating protection. these studies demonstrated that application of c inh alleviates bleomycin-induced lung injury via direct interaction with extracellular histones [ , ] . in vitro, c inh was found to bind all histone types with the interaction being independent of its protease inhibitory activity, but dependent on its glycosylation status [ ] . in vivo, histone-c inh complexes were detected in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome and multiple models of lung injury [ ] . the reactivecenter-cleaved c inh attenuated pulmonary damage evoked by intravenous histones indicating that c inh administration may provide a new therapeutic option for disorders associated with histone release [ ] . wygrecka and his colleagues tested c inh for its ability to bind and neutralize histones and determined that c inh can bind purified histones in vitro reducing epithelial cell death by blocking histone interactions with cell surface proteins [ ] . in another study, there was evidence for active binding of the exogenous c inh to extracellular and citrullinated histones released during netosis suggesting an endogenous mechanism by which histones are potentially neutralized [ ] . this mechanism could be exploited for therapeutic management of excessive netosis in other conditions, but studies need to be done to evaluate the effectiveness of these and other compounds with similar effects. metformin, a widely prescribed blood glucose-normalizing antidiabetic drug, suppresses immune responses. it mainly achieves this through the induction of amp-activated protein kinase. ampk is an enzyme that plays a role in cellular energy homeostasis, by activating glucose and fatty acid uptake and oxidation when cellular energy is low. induction of ampk subsequently inhibits the mammalian target of rapamycin (mtorc ), a pathway that regulates mammalian metabolism and physiology, by inhibiting mitochondrial ros production [ ] . this results in its direct effect on the cellular functions of various pro-inflammatory immune cells. due to the ros inhibitory effect of metformin, studies are underway to evaluate it as a drug for regulating autoimmune diseases, treating chronic autoimmune diseases and gero-protection [ , ] . menegazzo et al. investigated the effect of metformin against netosis and discovered that compared with a placebo, it significantly reduced the concentrations of nets in vitro. they showed a reduction in elastase, proteinase- , histones, and cfdna, whereas glucose control with insulin exerted no significant effect [ ] . metformin was shown to prevent membrane translocation of pkc-βii and activation of nox in neutrophils altering pathologic changes in nuclear dynamics and dna release [ , ] . this resulted in a blunted netosis in response to pma and calcium influx. this provides information for a possible use of metformin on the pkc-nox pathway as an anti-netosis therapy. chloroquine/hydroxychloroquine is an antimalarial drug used to treat malaria and has effects on amoeba (a protozoa) and some viruses [ , ] . studies are currently underway to evaluate its effects on the novel corona- virus (covid ) that is currently causing a pandemic [ , ] . hydroxychloroquine (hdq) is a slightly less potent derivative of chloroquine that is used in the treatment of malaria and as an immunosuppressive drug for management autoimmune conditions including sle and ra [ , ] . it exerts its immunosuppressive effect through inhibition of cytokine production with modulation of co-stimulatory molecules and also inhibits leukocyte phagocytosis [ , ] . hdq interferes with the stimulatory effect of platelet aggregation even in the presence of a thrombin agonist [ ] . mmps are matrix metalloproteinases which are enzymes involved in extracellular matrix remodeling, and timps are counter regulatory tissue inhibitors of mmps [ , ] that have been extensively studied in sle [ , ] . research has shown that hdq modulated mmps-timps interaction assisting in maintaining homeostasis of the extracellular matrix [ , ] and may thus play a role in reducing nets. in another study that looked at tumor-derived extracellular vesicle (ev), transportable vesicles important in the exchange of biological molecules between cells and induce formation of nets [ , ] , hdq was shown to inhibit neutrophil uptake of tumor-derived evs, thus reducing netosis [ , [ ] [ ] [ ] . however, the precise mechanism of inhibiting the uptake is largely unknown. due to the associated complications of nets in autoimmune conditions and cancer metastasis, it is important for future research efforts to focus on further investigation of these drugs and other new specific targets for prevention or control of the detrimental effects of nets formation. diphenyleneiodonium chloride (dpi) is as a hypoglycemic agent able to block gluconeogenesis and respiration by inhibiting many enzymes; nadph oxidase, nitric oxide synthase, xanthine oxidase, nadph cytochrome p oxidoreductase and cholinesterase [ , ] . dpi works by binding the heme group of nadph oxidase, inhibiting of nadph oxidase and thus inhibits ros production [ ] . ostafin et al. evaluated the effect of dpi on ros production in the context of nets and discovered that addition of dpi to the sample led to a reduction of extracellular dna release with the strongest inhibition noticed after adding μm dpi. these findings confirmed that dpi is able to block net creation. however, the addition of dpi together with pma or the addition of inhibitor initially and then washing it out before stimulation resulted in different levels of net formation [ ] . these findings necessitate more studies to look into the mechanism of action of dpi under different conditions and in different diseases as a potential therapeutic for nets. n-acetylcysteine (nac), also known as acetylcysteine, is a medication used to treat acetaminophen overdose [ ] and to loosen thick mucus in individuals with cystic fibrosis or chronic obstructive pulmonary disease [ ] . it also functions as an antioxidant which helps mitigate symptoms for a variety of diseases exacerbated by ros species [ , ] . zawrotniak and his team evaluated the effect of nac, ketoprofen, and ethamsylate on netosis and observed a reduction of ros production in a dose-dependent manner. nac inhibited netosis, but in the presence of hydrogen peroxide, this neutrophil ability was restored indicating that nac influences net formation by modulating ros productivity [ ] . the administration of ethamsylate led to a significant reduction in net formation, but this effect was not restored by hydrogen peroxide suggesting an additional side effect of this drug. ketoprofen seemed to promote ros-independent net release, simultaneously inhibiting ros production [ ] . brianna et al. used an acute pulmonary thrombosis model in vivo where nac reduced thrombus formation to a similar extent as the irreversible platelet inhibitor aspirin [ ] . in vitro analysis of platelet activation revealed that nac reduced thrombin-induced platelet-leukocyte aggregate formation in mice model of mutated janus kinase , a common mutation found in patients with chronic hematologic malignancies (chm), and reduced net formation in primary human neutrophils from patients with chm as well as healthy controls [ ] . these results strongly suggest that the therapeutic strategies applied in many neutrophil-mediated diseases should take into account the net-associated effects and that studies should look at the effect of these compounds in other diseases. recombinant human dnase, marketed as pulmozyme (dornase alfa) by genentech, is a highly purified solution of recombinant human deoxyribonuclease i (rhdnase). this is an enzyme which selectively cleaves dna and has been used to hydrolyze the dna present in sputum/mucus of cystic fibrosis patients and reduces viscosity in the lungs promoting clearance of secretions [ ] . nucleases perform various functions like acquiring nucleotide nutrients, allowing or preventing uptake of foreign dna, controlling biofilm formation/dispersal/architecture, aiding some pathogens in invading host by tissue damage, degrading dna matrixes, and immunomodulating the host immune response [ ] [ ] [ ] . studies have demonstrated the destructive effect of dnase on dna-nucleoprotein, and immune complexes, providing a rational way to interfere with the disease processes in sle and lupus nephritis [ ] . numerous other studies have evaluated the effect of rhdnase on nets, with results showing a reduction of netosis with reduced neutrophil infiltration reducing the inflammatory response [ ] [ ] [ ] . albadawi et al. conducted a study where they observed reduced detection of extracellular traps in post-ischemic muscle but did not alter skeletal muscle fiber injury, levels of pro-inflammatory molecules, or atp level. rhdnase treatment enhanced postischemic hindlimb perfusion, decreased infiltrating inflammatory cells, and reduced the expression of thrombinantithrombin iii [ ] . in addition, dnase i decreases tumor volume in rats when injected intramuscularly or intraperitoneally in conjunction with other proteases (papain, trypsin, and chymotrypsin) [ ] ; however, it is not known whether these effects are due primarily to net inhibition, thus the need for more studies. findings from a different study, showed that early and concurrent treatment with dnase i and antibiotics resulted in improved survival, reduced bacteremia, and organ dysfunction in septic conditions [ ] ) suggesting a possible combination therapy to control netosis. additionally, dnase i injection may have off-target effects that need to be considered in its use for control of nets or they may fail to function as expected in vitro [ ] . staphylokinase is an exoprotein produced by staphylococcus aureus, which activates host plasminogen [ ] . it induces extracellular release of alpha-defensins from polymorphonuclear cells promoting a complex formation between alpha-defensins and staphylokinase. the effect of this interaction is an almost complete inhibition of the bactericidal effect of alpha-defensins [ ] . thammavongsa et al. reported that s. aureus escapes these defenses by converting nets to deoxyadenosine, which triggers the caspase- -mediated death of immune cells [ ] .thus, the pathogenesis of s. aureus infections has evolved to anticipate host defenses and to repurpose them for the destruction of the immune system [ , ] . secretory nucleases also provide means of survival to other bacteria like iron-reducing shewanella and such functions help them adapt and survive proficiently [ ] . other than their pro-pathogen roles in survival, nucleases can be used directly as therapeutics due to their biological functions and medical applications in diagnosis, immunoprophylaxis, and autoimmune therapy. in the future, these enzymes can impact human medicine positively by opening new avenues for therapeutics which have otherwise reached saturation due to multi-drug resistance. probiotics are live microorganisms promoted with claims that they provide health benefits when consumed, generally by improving or restoring the gut flora [ , ] . probiotics are considered generally safe for consumption but may cause unwanted side effects and bacteria-host interactions in rare cases. alterations in the gut microbiota, as well as the presence of local and systemic markers of inflammation, are strongly associated with the manifestation of a spectrum of intestinal disorders [ ] . linda et al. investigated the effects of a nonpathogenic, enteropathogenic, and probiotic bacteria on the dynamics of net formation using murine bone marrow-derived neutrophils and the neutrophil-differentiated human myeloid cell line dhl- . they demonstrate that the probiotic lactobacillus rhamnosus strain gg (lgg) inhibits both pma and s. aureus induced nets by inhibiting pkc pathway and dampening ros production disrupting netosis supporting its antioxidative capacity [ ] . given the presence of nets in inflamed intestine [ ] , it is possible that some of the beneficial effects of lgg are attributable to its action on local neutrophils. probiotics have been shown to protect against bacterial-induced cytotoxicity, but more studies need to be done to highlights the dynamic interaction between beneficial bacteria and neutrophils to inform on the usefulness of probiotics as gut-protective and immunomodulatory compounds. vitamin d is a group of fat-soluble secosteroids important for increasing intestinal absorption minerals including calcium, magnesium, and phosphate. vitamin d has other multiple biological effects including activating the innate immune system while dampening the adaptive immune systems [ ] [ ] [ ] [ ] . in humans, vitamin d (cholecalciferol) and vitamin d (ergocalciferol) are the most important [ ] . there are suggestions indicating the benefits of vitamins d on various conditions, but evidence is lacking on whether supplementation of vitamin d helps to reduce the risk of these diseases including asthma, tuberculosis, irritable bowel disease, depression, and other conditions [ , ] . in the case of netosis, handono et al. evaluated the effect of hypovitamin d on nets in sle patients [ ] . they demonstrated a significant decrease in early apoptosis with a moderate positive correlation between ne externalizations with early apoptosis. they concluded that vitamin d could reduce endothelial damage by decreasing netosis activity [ ] . this result may reveal the possibility of vitamin d as supplementary therapy for sle patients and other patients with hypo-vitamin d to prevent netosis and endothelial damage. investigation of the impact of one or more of the therapeutics discussed above in modification of net formation will vary depending upon the disease and drug of interest. where there are animal models of a target disease (e.g., rheumatoid arthritis or psoriasis) administration of the therapeutic drug can be done in a placebo-controlled study evaluating different doses. tools available include histological identification of nets and comparison of nets formed in placebo versus drugtreated animals manually or using available computer programs. one example from our own work involves using a bovine model of respiratory syncytial virus and examination of the role of ibuprofen, a cox-inhibitor which decreases proinflammatory prostaglandin production and thromboxane [ ] . our theory is that ibuprofen would reduce netosis by decreasing neutrophil-activating cytokines and platelet activation. in our study, lungs are harvested at necropsy, fixed and stained with antibodies against citrullinated histones and neutrophil elastase, to delineate the presence of neutrophil nets. another model is the use of neutrophils incubated in vitro with the drug to be tested and staining to determine if there is an effect on netosis under different drug doses. nets have been implicated in many disease processes, and although they have a positive effect by clearing pathogens, they are also destructive due to the release of enzymes and other proteins that cause tissue injury. control of nets is quickly becoming a target for therapeutics in the management of various disease, but it is clear to see that the different compounds that inhibit or clear nets may have other unwanted effects on the immune system. this makes it challenging to conclude that one compound works better that the other and thus the need for more research. there is a possibility that the management of nets may require using a combination therapy that incorporate conventional treatments such fluid therapy, 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extracellular traps: implications for the establishment of cancer-associated thrombosis vitamin d and immune function vitamin d supplementation, -hydroxyvitamin d concentrations, and safety vitamin d prevents endothelial damage induced by increased neutrophil extracellular traps formation in patients with systemic lupus erythematosus an overview of clinical pharmacology of ibuprofen publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -ogo mvi authors: belinskaia, daria a.; voronina, polina a.; shmurak, vladimir i.; vovk, mikhail a.; batalova, anastasia a.; jenkins, richard o.; goncharov, nikolay v. title: the universal soldier: enzymatic and non-enzymatic antioxidant functions of serum albumin date: - - journal: antioxidants (basel) doi: . /antiox sha: doc_id: cord_uid: ogo mvi as a carrier of many biologically active compounds, blood is exposed to oxidants to a greater extent than the intracellular environment. serum albumin plays a key role in antioxidant defence under both normal and oxidative stress conditions. this review evaluates data published in the literature and from our own research on the mechanisms of the enzymatic and non-enzymatic activities of albumin that determine its participation in redox modulation of plasma and intercellular fluid. for the first time, the results of numerous clinical, biochemical, spectroscopic and computational experiments devoted to the study of allosteric modulation of the functional properties of the protein associated with its participation in antioxidant defence are analysed. it has been concluded that it is fundamentally possible to regulate the antioxidant properties of albumin with various ligands, and the binding and/or enzymatic features of the protein by changing its redox status. the perspectives for using the antioxidant properties of albumin in practice are discussed. the production of reactive oxygen species (ros) and reactive nitrogen species (rns) is an inherent property of all tissues. ros and rns play a significant role in the regulation of the main functions of cells: they participate in the reactions of oxidative phosphorylation, transmission of intracellular signals from various growth factors, modulation of various transcriptional proteins, prostaglandin biosynthesis, mitosis and several other processes [ , ] . the sources of ros in cells are well known. the nad(p)h-oxidase system (nox) is perhaps the foremost since ros production is its main function. in the inflammatory and immune response, nox produces a superoxide anion by electron transfer from nad(p)h to molecular oxygen [ , ] . nox and nox isoforms promote the development of endothelial dysfunction, hypertension and inflammation. nox acts as the main source of skeletal muscle ros during contractions [ ] . nox is the only isoform that generates hydrogen peroxide instead of superoxide radical [ ] . the investigation of nox functions has not lost its relevance in in the context of the covid- pandemic. thus, in the research of violi et al. [ ] , it has been demonstrated that oxidative stress an increase in the concentration of intracellular calcium leads to the activation of calcium-dependent proteases and mitochondrial enzymes (pyruvate dehydrogenase, isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase). seizures require a great amount of atp, but when electron transport and atp synthesis are disrupted, an excess quantity of ros is produced, primarily •О - [ ] . mitochondrial superoxide dismutase (sod ) converts two superoxide radicals to hydrogen peroxide, and both of these ros can leave the mitochondrion. the generation of Н О also occurs during the two-electron reduction in oxygen on the mitochondrial electron transport chain (etc). h o is a natural uncoupling agent of the etc: by decreasing the generation of ros on the etc, hydrogen peroxide acts as a negative feedback regulator [ ] . in addition, calcium overload enhances the work of noxs, mainly nox . nox produces superoxide anion, which is converted into hydrogen peroxide by extracellular superoxide dismutase (sod ) [ ] . •О -and h o can then re-enter the intracellular space through the chlorine channels and aquaporins [ ] . moreover, ca + activates nnos, which normally produces no, but in the case of uncoupling, generates superoxide anion [ ] . in the intracellular space, the superoxide anion binds to no to form peroxynitrite. cytoplasmic superoxide dismutase (sod ) converts two superoxide radicals into an oxygen molecule and hydrogen peroxide: the latter is destroyed by a catalase (cat) or glutathione peroxidase (gpx) cycle [ , ] . on the other hand, hydrogen peroxide can be converted to a hydroxyl radical through the fenton reaction with the participation of fe + cations [ ] . ros accumulation in the intracellular space leads to a number of undesirable consequences, including a redox-dependent modification of ryr, which leads to an even greater release of calcium from sr, an increase in calcium overload and seizures. ops have a similar effect on the heart and an increase in the concentration of intracellular calcium leads to the activation of calcium-dependent proteases and mitochondrial enzymes (pyruvate dehydrogenase, isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase). seizures require a great amount of atp, but when electron transport and atp synthesis are disrupted, an excess quantity of ros is produced, primarily ·o − [ ] . mitochondrial superoxide dismutase (sod ) converts two superoxide radicals to hydrogen peroxide, and both of these ros can leave the mitochondrion. the generation of h o also occurs during the two-electron reduction in oxygen on the mitochondrial electron transport chain (etc). h o is a natural uncoupling agent of the etc: by decreasing the generation of ros on the etc, hydrogen peroxide acts as a negative feedback regulator [ ] . in addition, calcium overload enhances the work of noxs, mainly nox . nox produces superoxide anion, which is converted into hydrogen peroxide by extracellular superoxide dismutase (sod ) [ ] . ·o − and h o can then re-enter the intracellular space through the chlorine channels and aquaporins [ ] . moreover, ca + activates nnos, which normally produces no, but in the case of uncoupling, generates superoxide anion [ ] . in the intracellular space, the superoxide anion binds to no to form peroxynitrite. cytoplasmic superoxide dismutase (sod ) converts two superoxide radicals into an oxygen molecule and hydrogen peroxide: the latter is destroyed by a catalase (cat) or glutathione peroxidase (gpx) cycle [ , ] . on the other hand, hydrogen peroxide can be converted to a hydroxyl radical through the fenton reaction with the participation of fe + cations [ ] . ros accumulation in the intracellular space leads to a number of undesirable consequences, including a redox-dependent modification of ryr, which leads to an even greater release of calcium from sr, an increase in calcium overload and seizures. ops have a similar effect on the heart and respiratory muscles, except that in the heart muscle, dhpr and ryr do not contact each other, antioxidants , , of and ca + ions first enter the cytosol through dhpr, and then ca + -dependent release of calcium occurs from the sarcoplasm through ryr [ ] . by a similar mechanism-via muscarinic receptors, calcium channels and enos-ops lead to endothelial dysfunction, which plays a key role in the pathogenesis of poisoning [ ] . the content of active species in cells is strictly controlled by the antioxidant defence system, which include both enzymatic and non-enzymatic processes. the most important non-enzymatic reaction of radical cleavage is their interaction with low-molecular-weight antioxidants such as β-carotene, vitamin c, vitamin e, uric acid, cysteine, glutathione (gsh), polyphenols, etc. as a result of this interaction, the cascade of free radical formation is broken [ ] . sod and cat, as well as peroxidases, glutathione reductase (gr), glutathione-s-transferase (gst), peroxiredoxin (prxs), thioredoxin system and paraoxonase (pon) are traditionally included into the enzymatic antioxidant system. these enzymes are widely described in the literature [ , ] . briefly, sod (ec . . . ) converts two superoxide radicals into an oxygen molecule and hydrogen peroxide. cat (ec . . . ) catalyses the utilisation of hydrogen peroxide to form molecular oxygen. peroxidases (ec . . .x) are a large group of enzymes that catalyse oxidation reactions according to the general scheme: roor'+ electron donor ( e − ) + h + → roh + r'oh. in particular, glutathione peroxidase (gpx, ec . . . ) ensures the destruction of hydrogen peroxide and lipid hydroperoxides with gsh oxidation. glutathione reductase (gr, ec . . . ) reduces oxidised glutathione (gssg) with the participation of nadph. a significant role in cellular redox-dependent processes belongs to the family of glutathione-s-transferases (gst, ec . . . ), which catalyse the conjugation of gsh with a wide range of xenobiotics, weakening their toxic effect [ ] . peroxiredoxins (prxs, ec . . . ) control the level of cytokine-induced peroxides involved in cellular signaling. thioredoxins (trx) and glutaredoxins (grx) are a family of the proteins that restore disulfide bonds in other oxidised proteins by disulfide exchange, while thioredoxin reductase (tr, ec . . . ) reduces the pool of oxidised trx and grx with the participation of nadph [ ] . paraoxonase (pon, ec . . . ) isoform is associated with high-density and, to a lesser extent, with low-density lipoproteins, protecting them from ros exposure, whereas pon is ubiquitously expressed intracellular protein, localised in mitochondria and the endoplasmic reticulum; pon is localised both intracellullarly and on high density lipoproteins [ ] . pon plays a role in the detoxification of ops by acting as a catalytic scavenger [ ] . the above is not a complete list. antioxidant defence is also represented by the enzymes that metabolise the end products of lipid peroxidation (aldehydes, epoxides, alkenes, alcohol), including epoxyde hydrolases (ec . . . ) and aldose reductase (ec . . . ) [ ] . formaldehyde dehydrogenase (ec . . . ) and lactoylglutathione lyase (ec . . . ) oxidise their substrates to organic acids using gsh as a coenzyme [ ] . quinone reductase (ec . . . ) provides a two-electron reduction in quinones to dihydroquinones, which prevents the formation of harmful one-electron reduction products-semiquinones; epoxide hydrolase hydrates epoxides to form diols [ ] . in addition, aldehyde dehydrogenase (ec . . . ) oxidises malonic dialdehyde [ ] . hepatic acyl-coa thioesterase is worth mentioning, since it has been shown to be involved in promoting oxidative capacity through regulation of fa oxidation [ , ] . oxidative stress is an abnormality of the prooxidant and antioxidant balance, which can be caused by low levels of antioxidants and/or an increase in the concentration of reactive species [ ] . this imbalance causes damage to a wide variety of target structures: lipid membranes, free amino acids, polysaccharides, nucleic acids, receptors and transport proteins. the result of this effect is a change in the functional state of a cell, its transformation or death. currently, oxidative stress is considered as an important pathogenetic link in the development of more than diseases [ ] . blood as a carrier of biologically active compounds is exposed to oxidants to a greater extent than the intracellular environment, but the concentration of antioxidants in plasma is much lower than in cells [ ] , and it is albumin that plays one of the key roles in the antioxidant defence of the body under normal conditions and in oxidative stress [ , ] . we first consider some general information about serum albumin. albumin is synthesised in the liver at a rate of about . mg per hour (i.e., - mg per day); the half-life of human serum albumin (hsa) is about - days [ ] . the molecule of hsa is formed by one polypeptide chain, consisting of amino acid residues. in albumins of other species, the length of the polypeptide chain can vary; in particular, bovine serum albumin (bsa) contains amino acid residues, rat serum albumin (rsa)- residues. the secondary structure of the protein contains about % helical structures next to % of turn and extended chain configurations without any β-sheets [ ] (figure a ). three homologous domains (i, ii, iii), consisting of two subdomains (a, b), form a three-dimensional structure of the protein, which is rather labile ( figure b ). when albumin interacts with different substances, the effects of cooperativity and allosteric modulation occur, which is more prevalent in multimeric macromolecules [ , ] . antioxidants , , x for peer review of we first consider some general information about serum albumin. albumin is synthesised in the liver at a rate of about . mg per hour (i.e., - mg per day); the half-life of human serum albumin (hsa) is about - days [ ] . the molecule of hsa is formed by one polypeptide chain, consisting of amino acid residues. in albumins of other species, the length of the polypeptide chain can vary; in particular, bovine serum albumin (bsa) contains amino acid residues, rat serum albumin (rsa)- residues. the secondary structure of the protein contains about % helical structures next to % of turn and extended chain configurations without any β-sheets [ ] ( figure a ). three homologous domains (i, ii, iii), consisting of two subdomains (a, b), form a three-dimensional structure of the protein, which is rather labile ( figure b ). when albumin interacts with different substances, the effects of cooperativity and allosteric modulation occur, which is more prevalent in multimeric macromolecules [ , ] . (b)-the tertiary structure of albumin: domains i, ii and iii are shown in orange, purple and green, respectively; each domain consists of two subdomains (a and b). to create the figure, a three-dimensional structure of human serum albumin from the pdb database, code jqz [ ] , was used. many extracellular proteins undergo post-translational glycosylation, which is the process of covalent binding of oligosaccharide chains to amino acid side-chains. in contrast to many other plasma proteins, the albumin molecule is not covered with a carbohydrate moiety under normal conditions, and can bind different endogenous and exogenous ligands: water and predominantly divalent metal cations, fatty acids, hormones, bilirubin, transferrin, nitric oxide, aspirin, warfarin, ibuprofen, phenylbutazone, etc. [ ] . ligand binding occurs at two primary sites (sudlow sites i and ii), which were described for the first time by gillian sudlow and co-authors [ ] . additionally, the albumin molecule has the third major binding site (site iii) and several secondary binding centers, the exact number of which is unknown. the albumin molecule contains disulfide bonds and one free thiol group in cys . the latter largely determines the participation of albumin in redox reactions. the number of disulfide bonds and cys are conserved in all types of albumin. the role of cys will be discussed in more detail in sections . and . . the three-dimensional structure of hsa was resolved rather late, only in the s [ ] . previously, it was assumed that the albumin molecule had the shape of an elongated or flattened ellipsoid ("cigar" or "pill"), but x-ray analysis showed that the protein has the shape of a heart. in many extracellular proteins undergo post-translational glycosylation, which is the process of covalent binding of oligosaccharide chains to amino acid side-chains. in contrast to many other plasma proteins, the albumin molecule is not covered with a carbohydrate moiety under normal conditions, and can bind different endogenous and exogenous ligands: water and predominantly divalent metal cations, fatty acids, hormones, bilirubin, transferrin, nitric oxide, aspirin, warfarin, ibuprofen, phenylbutazone, etc. [ ] . ligand binding occurs at two primary sites (sudlow sites i and ii), which were described for the first time by gillian sudlow and co-authors [ ] . additionally, the albumin molecule has the third major binding site (site iii) and several secondary binding centers, the exact number of which is unknown. the albumin molecule contains disulfide bonds and one free thiol group in cys . the latter largely determines the participation of albumin in redox reactions. the number of disulfide bonds and cys are conserved in all types of albumin. the role of cys will be discussed in more detail in sections . and . . the three-dimensional structure of hsa was resolved rather late, only in the s [ ] . previously, it was assumed that the albumin molecule had the shape of an elongated or flattened ellipsoid ("cigar" or "pill"), but x-ray analysis showed that the protein has the shape of a heart. in addition to hsa, three-dimensional structures of bsa [ ] , albumin of horse and rabbit [ ] , sheep and goat [ ] , dogs [ ] and cats [ ] have been obtained so far. however, the three-dimensional structure of albumin of rats-the principal animals used in pharmacological and toxicological experiments-has not been obtained yet. considering the fact that albumin is able to bind almost all known drugs and toxic substances [ , ] , this gap should be filled out. the percentage of identity of the primary structures of hsa and rsa is . %, bsa and rsa- . %. in some studies, it was shown that hsa and rsa share similar characteristics of binding biologically active substances, but binding efficiencies of some xenobiotics are different for hsa and rsa [ ] . therefore, the correct extrapolation of in vivo results obtained in rats to a human organism requires the identification of amino acids involved in protein-ligand interaction, determination of all structural and conformational features of the binding sites and comparison of the obtained characteristics in hsa vs. rsa. it is especially important for developing of antidotal therapy for ops poisoning since the use of other mammals in acute experiments is quite complicated. the three-dimensional structure of rsa is needed for such an analysis. in the absence of crystallographic data, the three-dimensional structure of a protein can be obtained with the help of homologous modeling. the approach allows the construction of a tertiary model of the protein on the basis of its primary sequence and the known three-dimensional structures of homologous proteins [ ] . homologous models of rsa have already been constructed both by our group [ ] and other researchers [ ] . below, when discussing the structure of albumins of different species, we give the numbering of hsa as the reference, and if necessary, the corresponding amino acids of bsa is given in brackets-for example, tyr (tyr ). as can be seen in figures and , sudlow site i is much less conservative than sudlow site ii. thus, lys and lys of hsa are replaced with more branched-chain arginines arg and arg in bsa. in rsa, lys is also replaced with arginine. arg in hsa and in rsa is substituted by lys in bsa. leu and leu in hsa and bsa are replaced with met in rsa. similarly, the isoleucines ile and ile in hsa and bsa correspond to met in rsa. isoleucines are located in the position ( ) in hsa and bsa, while leucine-in rsa. valines at position ( ) in hsa and bsa are replaced with isoleucine in rsa. histidines his (his ) and his (his ) in the primary sequence of hsa and bsa are substituted by asn and gln in the rsa sequence. the latter substitutions are of particular interest since his (his ) and his (his ) are located in very close proximity to the catalytic tyrosine tyr (tyr ). according to our computational experiments [ ] [ ] [ ] [ ] , the imidazole ring of his ( ) can attract the proton of the hydroxyl group of tyr (tyr ) and thus regulate the hydrolytic activity of the tyrosine. it should be expected that interspecies differences in the binding and catalytic properties of albumin will show themselves in the characteristics of sudlow site i. antioxidants , , x for peer review of addition to hsa, three-dimensional structures of bsa [ ] , albumin of horse and rabbit [ ] , sheep and goat [ ] , dogs [ ] and cats [ ] have been obtained so far. however, the three-dimensional structure of albumin of rats-the principal animals used in pharmacological and toxicological experiments-has not been obtained yet. considering the fact that albumin is able to bind almost all known drugs and toxic substances [ , ] , this gap should be filled out. the percentage of identity of the primary structures of hsa and rsa is . %, bsa and rsa- . %. in some studies, it was shown that hsa and rsa share similar characteristics of binding biologically active substances, but binding efficiencies of some xenobiotics are different for hsa and rsa [ ] . therefore, the correct extrapolation of in vivo results obtained in rats to a human organism requires the identification of amino acids involved in protein-ligand interaction, determination of all structural and conformational features of the binding sites and comparison of the obtained characteristics in hsa vs. rsa. it is especially important for developing of antidotal therapy for ops poisoning since the use of other mammals in acute experiments is quite complicated. the three-dimensional structure of rsa is needed for such an analysis. in the absence of crystallographic data, the three-dimensional structure of a protein can be obtained with the help of homologous modeling. the approach allows the construction of a tertiary model of the protein on the basis of its primary sequence and the known three-dimensional structures of homologous proteins [ ] . homologous models of rsa have already been constructed both by our group [ ] and other researchers [ ] . histidines his (his ) and his (his ) in the primary sequence of hsa and bsa are substituted by asn and gln in the rsa sequence. the latter substitutions are of particular interest since his (his ) and his (his ) are located in very close proximity to the catalytic tyrosine tyr (tyr ). according to our computational experiments [ ] [ ] [ ] [ ] , the imidazole ring of his ( ) can attract the proton of the hydroxyl group of tyr (tyr ) and thus regulate the hydrolytic activity of the tyrosine. it should be expected that interspecies differences in the binding and catalytic properties of albumin will show themselves in the characteristics of sudlow site i. sudlow site ii is highly conservative (figure ): there are substitutions only in positions (ile , ile and val in hsa, bsa and rsa, respectively), (gln , gln and thr in hsa, bsa and rsa, respectively), (leu , leu and ile in hsa, bsa and rsa, respectively) and (ala , thr and val in hsa, bsa and rsa, respectively). all the replacements, except for the homologous substitution at position , are located at a sufficient distance from the catalytic tyrosine tyr (tyr ). even more surprising differences can be observed in the structure of the redox site near the amino acid residue cys ( figure ). gln , phe , asp and thr in hsa and bsa are replaced with lys , tyr , glu and asn in rsa, respectively. however, the most remarkable difference is that tyr (tyr ) in hsa and bsa is replaced with his in rsa. previously, we showed that sudlow site i and the redox site of hsa and bsa have a mutual effect on each other: a change in the conformation of one site leads to conformational changes in the other [ , ] . in the redox site, the amino acid residues cys , his , tyr (tyr ) and arg (arg ) and their mutual arrangement (the -sh groups of the cysteine and the -oh groups of the tyrosine relative to the imidazole ring of his , as well as the -oh group of the tyrosine relative to the side chain of arg (arg )) play the main role in this effect. how this system works in rsa, where tyr (tyr ) is replaced with a histidine, and how this replacement affects the behavior and availability of cys are still unknown. it is even possible that in rats this mechanism is more effectual than in humans, since these rodents are incredibly omnivorous and adaptable to the environment. as mentioned above, albumin plays a significant role in the antioxidant defence of the body. the structure of a protein contains a number of amino acids and amino acid sequences that even more surprising differences can be observed in the structure of the redox site near the amino acid residue cys ( figure ). gln , phe , asp and thr in hsa and bsa are replaced with lys , tyr , glu and asn in rsa, respectively. however, the most remarkable difference is that tyr (tyr ) in hsa and bsa is replaced with his in rsa. previously, we showed that sudlow site i and the redox site of hsa and bsa have a mutual effect on each other: a change in the conformation of one site leads to conformational changes in the other [ , ] . in the redox site, the amino acid residues cys , his , tyr (tyr ) and arg (arg ) and their mutual arrangement (the -sh groups of the cysteine and the -oh groups of the tyrosine relative to the imidazole ring of his , as well as the -oh group of the tyrosine relative to the side chain of arg (arg )) play the main role in this effect. how this system works in rsa, where tyr (tyr ) is replaced with a histidine, and how this replacement affects the behavior and availability of cys are still unknown. it is even possible that in rats this mechanism is more effectual than in humans, since these rodents are incredibly omnivorous and adaptable to the environment. as mentioned above, albumin plays a significant role in the antioxidant defence of the body. the structure of a protein contains a number of amino acids and amino acid sequences that even more surprising differences can be observed in the structure of the redox site near the amino acid residue cys ( figure ). gln , phe , asp and thr in hsa and bsa are replaced with lys , tyr , glu and asn in rsa, respectively. however, the most remarkable difference is that tyr (tyr ) in hsa and bsa is replaced with his in rsa. previously, we showed that sudlow site i and the redox site of hsa and bsa have a mutual effect on each other: a change in the conformation of one site leads to conformational changes in the other [ , ] . in the redox site, the amino acid residues cys , his , tyr (tyr ) and arg (arg ) and their mutual arrangement (the -sh groups of the cysteine and the -oh groups of the tyrosine relative to the imidazole ring of his , as well as the -oh group of the tyrosine relative to the side chain of arg (arg )) play the main role in this effect. how this system works in rsa, where tyr (tyr ) is replaced with a histidine, and how this replacement affects the behavior and availability of cys are still unknown. it is even possible that in rats this mechanism is more effectual than in humans, since these rodents are incredibly omnivorous and adaptable to the environment. as mentioned above, albumin plays a significant role in the antioxidant defence of the body. the structure of a protein contains a number of amino acids and amino acid sequences that determine its role in redox processes. in this section, we consider three main activities of albumin associated with redox modulation of blood plasma and interstitial fluid. it is well known that polyvalent metals, primarily copper and iron, are pro-oxidants. copper and iron ions can react with hydrogen peroxide to form toxic hydroxyl radicals (fenton reaction) [ ] . by binding iron and copper cations, albumin heavily reduces their activity: bound ions are still available for reaction, but the radicals formed immediately attack the albumin molecule itself and do not interact with other blood components [ ] . in this case, the albumin molecule is damaged, but due to the high concentration of the protein this damage is biologically insignificant. in recent years, some details of the interaction of polyvalent metals with albumin have been determined. thus, the main binding site for cu(ii) is the n-terminal region of human albumin asp-ala-his-lys (n-terminal site, nts) [ ] ( figure ). binding involves the nitrogen atoms of the backbone and the nitrogen atom of the imidazole ring of the nts histidine [ ] . using spectroscopic and computational methods, sendzik et al. [ ] showed that the imidazole rings of two histidines play a key role in the binding of the cu(i) cation. based on the data obtained, the authors suggested that these histidines can be either his- and his- of the metal-binding site of albumin (mbs) (figure ) or his- and his- (the first is included in the nts, the second is in the nearest environment). normally, albumin is not a physiological carrier of fe, but it can bind fe(ii) and fe(iii) during pathological iron overload [ ] . this binding, however, is apparently non-specific, and takes place somewhere on the surface of the protein and does not involve the nts or mbs. antioxidants , , x for peer review of determine its role in redox processes. in this section, we consider three main activities of albumin associated with redox modulation of blood plasma and interstitial fluid. it is well known that polyvalent metals, primarily copper and iron, are pro-oxidants. copper and iron ions can react with hydrogen peroxide to form toxic hydroxyl radicals (fenton reaction) [ ] . by binding iron and copper cations, albumin heavily reduces their activity: bound ions are still available for reaction, but the radicals formed immediately attack the albumin molecule itself and do not interact with other blood components [ ] . in this case, the albumin molecule is damaged, but due to the high concentration of the protein this damage is biologically insignificant. in recent years, some details of the interaction of polyvalent metals with albumin have been determined. thus, the main binding site for cu(ii) is the n-terminal region of human albumin asp-ala-his-lys (n-terminal site, nts) [ ] (figure ). binding involves the nitrogen atoms of the backbone and the nitrogen atom of the imidazole ring of the nts histidine [ ] . using spectroscopic and computational methods, sendzik et al. [ ] showed that the imidazole rings of two histidines play a key role in the binding of the cu(i) cation. based on the data obtained, the authors suggested that these histidines can be either his- and his- of the metal-binding site of albumin (mbs) ( figure ) or his- and his- (the first is included in the nts, the second is in the nearest environment). normally, albumin is not a physiological carrier of fe, but it can bind fe(ii) and fe(iii) during pathological iron overload [ ] . this binding, however, is apparently non-specific, and takes place somewhere on the surface of the protein and does not involve the nts or mbs. right: cys is represented in yellow; six methionine residues are shown in orange; the cysteines within the disulfide bonds that can be reduced when interacting with low-molecular-weight thiols are shown in green. to create the figure, a three-dimensional structure of hsa from the pdb database, code jqz [ ] , was used. albumin acts like a ros trap due to six methionine residues, but mainly due to the free thiol group of cys residue [ , , ] (figure ). this group of activities probably includes the cyanide detoxification reaction by formation of thiocyanate, catalysed in the iiia subdomain, but without the albumin acts like a ros trap due to six methionine residues, but mainly due to the free thiol group of cys residue [ , , ] (figure ). this group of activities probably includes the cyanide detoxification reaction by formation of thiocyanate, catalysed in the iiia subdomain, but without the participation of tyr [ ] . in physiological conditions, about % of all detected plasma thiols are albumin thiols ( % of gsh is kept in erythrocytes, and about / of extracellular cysteine/cystine is in a bound form) [ , ] . the cys residue is able to neutralise such ros and rns as hydrogen peroxide (h o ), peroxynitrite (onoo − ), superoxide anion and hypochlorous acid (hocl), being oxidised to sulfenic acid (hsa-soh) [ , ] . hsa-soh is a central intermediate in the processes of redox modulation of blood plasma and interstitial fluid [ ] . the final result of the oxidative process depends on what happens next to the sulfenic acid. the hsa-soh form of albumin can be irreversibly oxidised to sulfinic acid (hsa-s(o)o − ) [ ] . in theory, the side radical of cysteine can be irreversibly oxidised to sulfonic acid (cys -s(o)o − o − ); however, according to the literature, the percentage of cys in the form of sulfonic acid in blood plasma is extremely low [ ] . sulfenic acid can also be converted to disulfide (hsa-s-s-r) by interacting with low-molecular-weight blood plasma thiols (gsh, homocysteine, free cysteine), and then reduced to hsa-sh [ ] [ ] [ ] . bocedi et al. [ ] wanted to answer the question of why a healthy person has only percent of oxidised albumin, while for low-molecular-weight thiols this value is from to percent. the hsa-cysteine conjugate (hsa-cys -s-s-cys) was used as a model of oxidised human albumin, since this disulfide is the main form of oxidised albumin in blood plasma. biochemical experiments have shown that cysteine is the strongest reducing agent for albumin of the plasma thiols studied, and cystine (cysteine dimer cys-s-s-cys) is the strongest oxidising agent. however, it turned out that for the reduction reaction the second order rate constant was about m − ·s − , while for the oxidation reaction it was times less: . m − ·s − . the authors have concluded that the ratio of the reduced and oxidised forms of albumin is determined by kinetic equilibrium with the cysteine/cystine ratio. in the case of pathology, when the percentage of cystine increases, albumin acts as a redox buffer, maintaining a safe cys-sh/cys-s-s-cys ratio for the body. moreover, according to the data obtained in this work, the remaining albumin cysteines (forming disulfide bridges) barely undergo cysteinylation even with high concentrations of free cysteine, -fold higher than its normal concentration in the blood plasma [ ] . according to other experimental data, however, the interaction of hsa with low-molecular-weight thiols involves not only cys but also some cysteine residues that form disulfide bonds: cys , cys , cys , cys , cys , cys , cys , cys , cys , cys [ , ] . nakashima et al. [ ] proposed a mechanism of albumin cysteines thiolation. according to this model, the free thiol group cys is thiolated first. as a result of this reaction, the thiolate anion rs − is formed, which attacks one of the disulfide bonds of albumin. as a result, one of the cysteines that forms disulfide bonds is thiolated, and the second cysteine is converted into the thiolate anion hsa-s − and interacts with the next molecule of low-molecular-weight disulfide, etc. according to the authors' assumption, the cascade of reactions is interrupted when there are no more disulfide bonds on the surface of the protein available for the thiolate anion. the partial destruction of disulfide bonds of albumin is a rather dramatic event that can lead to protein aggregation and change its functional characteristics. over the years, it has been shown that albumin has a thioesterase [ ] , glutathione and cysteine peroxidase [ , ] and peroxidase activity towards lipid hydroperoxides [ ] [ ] [ ] [ ] . the authors of the work [ ] , by measuring the outcome of mercaptoethanol (mer), found that human blood serum contains a certain thioesterase that catalyses the hydrolysis of s-lauroylmercaptoethanol (s-lme). the authors concluded that this enzyme is serum albumin. firstly, the unit amount of reaction product per mg of crystalline hsa and per mg of serum albumin was the same. secondly, the rate of mer outcome reduced by about percent with various anionic or non-ionic lauryl derivatives and urea; moreover, the product outcome terminated when hsa was inactivated with various detergents or high temperature. the rate of mer outcome significantly reduced after about moles of mercaptoethanol released per mole of hsa. the authors concluded that there is an irreversible acylation of albumin amino acids. moreover, according to the data obtained, lysines, but not tyrosines, are the most likely amino acid residues responsible for the thioesterase activity of hsa towards s-lme, since during the reaction of albumin with s-lme, no decrease in absorption at nm typical for acylation of tyrosine residues was observed. according to the korean researchers cha and kim [ ] , a kda protein isolated from human blood plasma and identified by the n-terminal amino acid sequence as serum albumin was able to accelerate h o reduction by gsh. the authors did not report on the kinetic characteristics of hsa for peroxide and gsh, but they noted that the rate of glutathione-dependent reduction in h o in the presence of hsa in the reaction mixture was a function of the albumin concentration and had a saturation behavior. the results obtained suggested the presence of glutathione peroxidase (gsh: h o -oxidoreductase) activity in hsa, but the authors, unfortunately, did not report on the stoichiometry of the reaction. if the molar ratio between gsh and h o in their interaction catalysed by hsa would be confirmed as : , then this would allow us to more confidently say that albumin is functionally capable of being a gsh:h o oxidoreductase. later, the same group of researhers demonstrated that activation of thiol-dependent antioxidant activity of hsa at alkaline ph was due to the conformational change favorable for the functional cysteine(s)-mediated catalysis [ ] . later, it was shown that palmitoyl-coa induced the conformational changes of hsa and thus provided thioredoxin-linked lipid hydroperoxide peroxidase activity of the protein [ ] . in r. hurst and co-authors [ ] found that hsa is effective in catalysis of the reduction in -palmitoyl- -( -hydroperoxy-cis- ,trans- -octadecadienoyl)-l- -phosphatidylcholine to the corresponding hydroxy derivative when using thiols such as cysteine, glutathione, cysteinylglycine and homocysteine as oxidisable substrates (listed in decreasing order of their effectiveness in albumin-catalysed hydroperoxide reduction). hsa reduced phospholipid hydroperoxide in the absence of a thiol reducing agent, but at a lower rate than with any of them. the authors evaluated the stoichiometry of the reduction in the phospholipid hydroperoxide to the corresponding hydroxy derivative in the presence of albumin and cysteine. the molar ratio between the resulting -palmitoyl- -( -hydroxycis- ,trans- -octadecadienoyl)-l- -phosphatidylcholine and cystine (cysteine disulfide) was close to : , which confirms the hypothesis that albumin functions as a cysteine peroxidase-i.e., catalyses the reaction according to a scheme similar to that of the glutathione peroxidase reaction: rooh + cys-sh → roh + h o + cys-ss-cys, where cys-sh is cysteine, cys-ss-cys is cystine, roh is a hydroxy derivative and rooh is hydroperoxide. the kinetic characteristics towards cysteine were calculated with a fixed concentration of phospholipid hydroperoxide and vice versa, towards phospholipid hydroperoxide with a fixed concentration of cysteine [ ] . the obtained values of the apparent k m and v max for cysteine were ± µm and . ± . nmol/(min × mg protein), respectively (m ± sd). the same parameters for phospholipid hydroperoxide are . ± . µm and . ± < . nmol/(min × mg protein). treatment of albumin with dithiothreitol (dtt) decreases both apparent k m and increases both apparent v max , while modification with n-ethylmaleimide leads to a decrease in both k m and v max . in general, this means that the presence of free sh-groups in the albumin molecule enhances its catalytic properties. the authors confirm the same conclusion using captopril, which increases the cysteine peroxidase activity of albumin, while the relation between activity and the concentration of captopril has a saturation behavior [ ] . the results using captopril indicate the participation of cys in catalysis, but, apparently, the release of additional thiol groups in the albumin molecule during dtt treatment provides greater catalytic efficiency of albumin. surely, the cysteine peroxidase activity of albumin in relation to phospholipid hydroperoxide is low (and its glutathione, cysteinylglycine and homocysteine peroxidase activities towards the same reducible substrate are, apparently, even lower), but, as the authors fairly note, the low activity should be compensated by its high concentration in plasma [ ] . furthermore, cysteine is a major low-molecular-weight thiol in blood plasma, the physiological concentration of which is - µm [ ] . the total concentration of phosphatidylcholine hydroperoxides in plasma is - nm [ ] . it is probable that albumin makes a certain contribution to the catalysis of thiol-dependent reduction in phospholipid hydroperoxides in blood plasma together with other peroxidases. in any case, the presence of cysteine peroxidase (cysteine: phospholipid-hydroperoxide-oxidoreductase) activity in human serum albumin can be confidently stated. in contrast to the intracellular analogue, the monomeric se-containing protein phospholipid hydroperoxide glutathione peroxidase (also named glutathione peroxidase- ; abbreviations-phgpx, gpx ; ec . . . ), the role of which in the protection of cells, including nervous, from the damaging effect of lipid hydroperoxides can hardly be overestimated [ ] [ ] [ ] , as well as in contrast to the extracellular tetrameric glutathione peroxidase- (gpx ; ec . . . ), the decrease in the activity of which is consistently correlated with the development of oncological diseases [ , ] , monomeric (but multi-domain) serum albumin does not contain selenium. paraoxonase activity of albumin is described in detail in our previous research: albumin is able to operate as a paraoxonase though does not depend on ca + ions [ ] . roche et al. [ ] discuss the ability of albumin to bind polyunsaturated fatty acids (pufas) and bilirubin, and thus indirectly further enhance the antioxidant defence of the body. it is known that albumin-bound bilirubin can inhibit lipid peroxidation. bilirubin binds at site iii of albumin [ ] ( figure ). as for pufas, according to the authors, it is possible that in combination with albumin, they are protected from peroxidation. the amino acids arg , lys and lys are responsible for the interaction of the protein with pufa molecules (figure ). as mentioned above, the structure of albumin is rather labile and tends towards allosteric modulation: binding of a ligand in one site can affect the efficiency of binding in another. thus, the conformational changes occur in the albumin molecule after the binding of a number of endogenous compounds, such as bilirubin [ ] , urea [ ] , estradiol [ ] and glucose [ ] . exogenous compounds might also have an allosteric effect. for example, the binding of lorazepam in sudlow site ii changes the binding efficiency of warfarin in sudlow site i [ ] , the binding of tenoxicam in sudlow site i enhances the binding of diazepam in sudlow site ii and vice versa [ ] . these features suggest that a targeted modulation of albumin with the help of the molecules regulating its structural and functional properties can influence the process of the protein interaction with ros and rns. on the other hand, oxidative stress accompanies many diseases, the level of oxidised albumin increases, which in turn can affect the kinetics of pharmacological and toxic compounds. therefore, it is essential to study the interaction of various activities of albumin and answer the following questions. does oxidation of the thiol group of cys (and other amino acids) affect the binding and catalytic properties of albumin towards its ligands? does this effect depend on the oxidative agents and on the structure of the ligand? do endogenous and exogenous compounds affect the availability and reactivity of the thiol group of cys and, as a consequence, the antioxidant properties of albumin? we now review the effect of cys oxidation on albumin binding and pseudo(esterase) activity. our own computational experiments, performed as a part of investigation of the interaction of albumin with ops, were devoted to the study of the influence of the redox status of hsa on their interaction with paraoxon [ ] . we tested three models of the oxidation state of albumin: cys is reduced (cys -sh), cys is oxidised to sulfenic acid (cys -soh) and cys is oxidised to sulfinic acid similar results were obtained in biochemical in vitro experiments with hsa. bertucci et al. [ ] showed that hsa cys oxidation with ethacrynic acid did not affect the affinity of neither sudlow site i towards phenylbutazone nor sudlow site ii towards diazepam, but improved the binding efficiency of bilirubin in the third major albumin binding site (site iii). in the research of anraku et al. [ ] , human albumin was oxidised in vitro by three different methods: by a metal-catalysed oxidation system (mco), chloramine-t and hydrogen peroxide. it turned out that oxidation (by any means) had practically no effect on the binding of warfarin in sudlow site i. oxidation with hydrogen peroxide did not affect the binding of ketoprofen in sudlow site ii, but oxidation with mco and chloramine-t reduced the affinity of sudlow site ii for ketoprofen. the different effect of different oxidants can be explained by the fact that mco and chloramine-t can oxidise not only cys but also the side chains of lysines and arginines, including arg and arg [ , ] , localised in sudlow site ii. the results of in vivo experiments contradict the data obtained in vitro and in silico. in healthy people, about % of albumin remains in a reduced form, but the level of oxidised albumin can increase in some pathological processes and during the aging process [ , ] . the research of [ ] showed that albumin in patients with liver cirrhosis (a disease in which the content of oxidised albumin is increased) binds ligands of sudlow site ii more weakly than in healthy subjects. nagumo et al. [ ] revealed that the content of cysteinylated albumin (hsa-cys -s-s-cys) increased in patients with chronic kidney and liver disease. the binding activity of albumin towards warfarin (a ligand of sudlow site i) and diazepam (a ligand of sudlow site ii) in these patients was significantly lower than in healthy people. thus, the modification of cys impaired the affinity of sudlow sites i and ii for both warfarin and diazepam, which contradicts in vitro data [ , ] . there are several possible explanations for the conflict between in vitro and in vivo data. one of them is that liver and kidney disease can lead to increased levels of certain molecules in blood plasma, which in turn can inhibit (competitively or non-competitively) the binding of ligands in sudlow sites. for example, it is known that the level of glucose in blood can be increased in liver cirrhosis [ ] . on the other hand, the authors of [ ] showed that oxidation of albumin sh-groups with potassium permanganate led to an increase in the number of the sites on the albumin surface available for glucose binding. one more explanation is that serum albumin is loaded with fatty acids (fas) in blood [ ] , which can affect the binding characteristics of the protein in both reduced and oxidised form. changes in the functional characteristics of albumin caused by a change in its redox status are primarily the result of the structural rearrangements in the protein molecule. a number of spectroscopic studies have been carried out so far to study the structural characteristics of reduced and oxidised albumin. it is interesting to compare the study of maciążek-jurczyk and sułkowska with the paper of sakurama et al. [ ] , who also oxidised hsa with chloramine-t and studied the conformational changes in the protein molecule by the circular dichroism method. this research did not reveal significant structural change in oxidised hsa. in both studies, chloramine-t and hsa were mixed in similar proportions at the same temperature. the possible explanations are that different albumin samples and different ways to interrupt the oxidation reaction were used in these experiments. despite some disagreements regarding the conformational rearrangements of the albumin polypeptide chain after oxidative modification, oxidation of albumin definitely leads to the conformational changes of amino acids in the microenvironment of the modification sites. in the research of pieniazek et al. [ ] , albumin was isolated from the plasma of healthy volunteers and patients with chronic kidney disease (ckd) on hemodialysis. it was demonstrated by the method of electron paramagnetic resonance (epr) that oxidation of albumin of the healthy subjects with hydrogen peroxide and tert-butyl hydroperoxide led to conformational changes in the microenvironment of the binding sites of maleimide and iodoacetamide spin labels, which interact predominantly with the thiol group of cys . the oxidants practically did not affect the structural characteristics of albumin from plasma of the subjects with ckd, since albumin of these patients had been already significantly oxidised. christodoulou et al. [ ] reduced fatty acids free bsa with dtt and oxidised with auranofin, and then studied the structural features of the samples by h nmr. comparing the spectra, the authors suggested that the oxidation of cys led to a change in the conformation of his at the n-terminal site of the protein. in our own studies, we have applied the h nmr method to study how the oxidation of bsa with ethacrynic acid (etac) affects the conformational characteristics of the protein. figure shows the spectra of three samples: phosphate buffered saline (pbs) used to prepare bsa solution; commercial bsa of concentration µm; commercial bsa of concentration µm after incubation with etac in a molar ratio of : (oxbsa). commercial bsa was prepared using the same procedure as in [ ] . oxidised bsa was prepared as described in [ ] with minor modifications. the prepared samples of commercial and oxidised bsa were supplemented with deuterium oxide and scanned at room temperature by the one-dimensional h-nmr water suppression method using excitation sculpting with gradients on a bruker avance iii nmr spectrometer. chemical shifts δ were calibrated to tetramethylsilane; the spectra were accumulated for scans using a . s delay between the first radiofrequency pulses. figure a shows the full spectrum. both samples of bsa contain the impurities associated with the imperfect purity of the supplied pbs tablets (green spectrum). based on the literature, it is highly likely that the peak with a chemical shift of . ppm corresponds to acetic acid (ch cooh) [ , ] and a singlet with a chemical shift of . ppm most likely corresponds to ethylene glycol (ch oh) [ ] [ ] [ ] . additionally, the sample of oxidised bsa contains ethanol (c h oh), which was used to dissolve etac. figure b shows the aliphatic region of the spectrum. the change in the shape of the spectrum in the region . - . ppm (peaks a and a') can probably be associated with a change in conformation of the microenvironment of cys (c β h groups [ ] ) after its oxidation with etac. differences between the two samples can also be observed in the region . - . ppm (peaks b and c) . we suppose that this might be due to a change in the conformation of glutamine gln (signal of the c γ h group [ , ] ) and/or proline pro (signal of the c γ h group [ , ] ) located in the microenvironment of cys ( figure b ). according to the literature, cys oxidised to sulfenic or sulfinic acids can form an intramolecular bond with gln , while these amino acids do not interact in reduced albumin [ ] . figure c shows the aromatic region of the spectrum. the change in the shape of the spectrum in the regions . - . ppm and . - . ppm reflects the change in signals from c ε h and c γ h groups of histidines, respectively [ , , ] . the appearance of a weak signal d and a decrease in the intensity of h' peak in oxbsa probably indicates a change in the conformation of his , which interacts with the sh-group of cys in reduced but not in oxidised albumin [ , ] . stewart et al. also noted the importance of his in the reactivity of the thiol group of cys [ ] . antioxidants , , x for peer review of as we have mentioned above, in [ ] , a change in the signal in this region after bsa oxidation was proposed to be due to a change in the conformation of his in the nts of albumin. so, we think that peak e in oxbsa can correspond to a change in his conformation. as we have mentioned above, in [ ] , a change in the signal in this region after bsa oxidation was proposed to be due to a change in the conformation of his in the nts of albumin. so, we think that peak e in oxbsa can correspond to a change in his conformation. the region at . - . ppm corresponds to the signals of the aromatic rings of tyrosine residues [ , , ] . the change in the shape of the spectrum in this region (peaks g, g' and i) is highly likely associated with a change in the conformation of tyr and its microenvironment. according to abundant evidence, tyr plays a key role in the reactivity of cys [ , ] . additionally, we suppose that peak f in oxidised bsa could be a signal of the benzene ring of etac covalently bound to the sh-group of cys [ ] . the signal of the second aromatic hydrogen of etac in oxbsa might contribute to the intensity of peak g too. thus, according to the data obtained, bsa oxidation leads to a change in the conformation of the microenvironment of cys : gln , pro , his and tyr . it should be mentioned that it is undoubtedly difficult to unambiguously interpret the one-dimensional nmr spectrum of such a composite protein as albumin. our conclusions are rather in the nature of an assumption, but nevertheless the result is in fairly good agreement with the literature data. the tools for studying the structural characteristics of macromolecules are constantly evolving. thus, the solution structure (which is more natural than the crystal one) of some proteins with a molecular weight over kda have been obtained by nmr technique to date [ ] . in future, it probably would be possible to obtain the solution structure of albumin, the molecular weight of which is kda, and to trace how the structure of the protein changes when interacting with various ligands. molecular modeling methods are being developed, too: the mathematical apparatus describing the interaction of atoms is being improved; computer power is growing. currently, the classical molecular dynamics is the main computer method for studying the conformational changes of macromolecules; however, it cannot simulate the changes in the structure of a protein at atomic level (for example, the transfer of a proton from one amino acid to another or the formation of covalent bonds between ligands and proteins). with the development of computing power, it became possible to apply the method of quantum molecular dynamics, which is able to simulate these processes [ ] . additional spectroscopic and computational experiments will help amplify the obtained information about structural rearrangements in the albumin molecule after oxidation or reduction in cys in future. now, we consider the possibility of modulating the antioxidant properties of albumin. first of all, it is obvious that the oxidation of the thiol group of cys or its nitrosylation (cys -s-n=o) reduces the ability of albumin to neutralise ros and rns [ ] . however, in addition to the direct oxidation of cys , albumin can undergo other chemical modifications that affect its structure and conformation, which in turn can lead to modulation of its antioxidant properties. glycation is one of these modifications, which is the covalent binding of glucose or another monosaccharide to the side chains of lysines and arginines [ ] . to date, more than albumin glycation sites have been described, but many researchers agree that lys is the most reactive of them [ ] [ ] [ ] . modifications caused by glycation have an important effect on the functional properties of albumin, mainly associated with the changes in its conformation [ ] [ ] [ ] [ ] . however, fas appear to play the main role in the regulation of the antioxidant properties of albumin. for the first time, this conclusion was made by gryzunov and co-authors [ , ] . according to the data obtained, blocking of cys by n-ethylmaleimide did not affect the fluorescence intensity of probe k- (binding in the sudlow site i) in hsa free of fas [ ] . however, adding fas (oleic and linoleic), firstly, changed the conformations of sudlow sites i and ii, and, secondly, strengthened the reactivity of cys thiol group towards , -dithiobis- -nitrobenzoic acid (dtnb) having increased its steric availability. the authors hypothesised that fas, when bound to albumin, simultaneously regulated both its transport and antioxidant functions, serving as a necessary intermediary between these activities [ ] . a similar result was obtained in the research of torres et al. [ ] . the authors showed that in the presence of fas (palmitic, myristic, lauric, stearic, oleic), the reactivity of hsa towards dtnb increased by about times (with a minor scatter depending on fa structure) compared to fas free albumin. stearic acid doubled the rate of the reaction of cys with hydrogen peroxide and peroxynitrite and strengthened the reactivity of sulfenic acid of hsa towards low-molecular-weight thiols. oxidation of cys thiol group did not change the efficiency of the interaction of hsa with fas. pavićević et al. [ ] showed that the binding of fas (palmitic, docosahexaenoic, stearic, oleic, myristic, eicosapentaenoic and fish oil) with hsa increased the reactivity of cys towards methylglyoxal. subsequent experiments demonstrated that the reaction of dtnb with cys (both reduced and modified with methylglyoxal) was accelerated in the presence of fatty acids too. the same research group demonstrated later that the binding of polyphenols enterolactone and enterodiol with hsa increased the reactivity of the cys sh-group towards dtnb [ ] . it was shown that fatty acids were able to modulate this effect. finally, one of the latest investigations of this group revealed that the binding of copper cations cu(ii) with defatted hsa practically did not affect the reactivity of cys , while the addition of copper to the complex of hsa with fas (oleic, myristic, or fish oil) increased the reactivity of the cysteine cumulatively [ ] . in our recent computational experiments [ ] , we have analysed how the redox status of hsa affects the binding of paraoxon in sudlow sites i and ii. however, an analysis of the effect of paraoxon binding on the conformation of cys and its microenvironment has not been performed. here we fill this gap. figure shows how the conformation of cys with different oxidation level of the thiol group depends on the occupancy of sudlow sites. in the upper row ( figure a-c) , paraoxon is bound in sudlow site i; in the lower one ( figure d-f) , it is bound in sudlow site ii. a similar result was obtained in the research of torres et al. [ ] . the authors showed that in the presence of fas (palmitic, myristic, lauric, stearic, oleic), the reactivity of hsa towards dtnb increased by about times (with a minor scatter depending on fa structure) compared to fas free albumin. stearic acid doubled the rate of the reaction of cys with hydrogen peroxide and peroxynitrite and strengthened the reactivity of sulfenic acid of hsa towards low-molecular-weight thiols. oxidation of cys thiol group did not change the efficiency of the interaction of hsa with fas. pavićević et al. [ ] showed that the binding of fas (palmitic, docosahexaenoic, stearic, oleic, myristic, eicosapentaenoic and fish oil) with hsa increased the reactivity of cys towards methylglyoxal. subsequent experiments demonstrated that the reaction of dtnb with cys (both reduced and modified with methylglyoxal) was accelerated in the presence of fatty acids too. the same research group demonstrated later that the binding of polyphenols enterolactone and enterodiol with hsa increased the reactivity of the cys sh-group towards dtnb [ ] . it was shown that fatty acids were able to modulate this effect. finally, one of the latest investigations of this group revealed that the binding of copper cations cu(ii) with defatted hsa practically did not affect the reactivity of cys , while the addition of copper to the complex of hsa with fas (oleic, myristic, or fish oil) increased the reactivity of the cysteine cumulatively [ ] . in our recent computational experiments [ ] , we have analysed how the redox status of hsa affects the binding of paraoxon in sudlow sites i and ii. however, an analysis of the effect of paraoxon binding on the conformation of cys and its microenvironment has not been performed. here we fill this gap. figure shows how the conformation of cys with different oxidation level of the thiol group depends on the occupancy of sudlow sites. in the upper row ( figure a-c) , paraoxon is bound in sudlow site i; in the lower one ( figure d-f) , it is bound in sudlow site ii. it could be noticed that the occupancy of sudlow sites has the greatest effect on the conformation of cys oxidised to sulfenic acid (cys -soh) ( figure b,e) . when paraoxon is bound in sudlow site i (figure b ), the availability of the -soh group is greater than in the case of paraoxon bound in sudlow site ii. it has been mentioned in section . that hsa-cys -soh is a central intermediate in the processes of redox modulation of blood plasma and intercellular fluid, and the final result of the oxidative process depends on what happens with this sulfenic acid. however, our observation has more theoretical than practical significance, since the concentration of the lethal dose of paraoxon in blood hardly exceeds µm, which can in no way affect the total albumin pool. recently, litus et al. performed multifactorial computational disorder analysis of hsa and bsa [ ] . for all the residues of hsa and bsa, the authors calculated the values of mean predicted disorder scores (mpdss) characterising the flexibility of the amino acids, and then they analysed the mpds values of the phosphorylation, acetylation, ubiquitination, methylation and glycosylation sites. according to the data obtained, serum albumins in their function (including the antioxidant properties) often rely on disordered or flexible residues characterised by mpds ≥ . and . ≤ mpds < . , respectively. for example, the arginines and lysines participating in albumin glycation are characterised by rather high disorder scores ranging from . to . . the amino acids of nts asp-ala-his-lys (binding site for cu(ii)) have mpds values of . , . , . and . , respectively. the researchers concluded that intrinsic disorder and high structural flexibility are important for the functionality of serum albumin. in conclusion of this section, it can be noted that all the papers mentioned above indicate the fundamental possibility of modulating the antioxidant properties of albumin with endogenous and exogenous ligands. another important aspect worth paying attention to is that fas are the key transmitters of information between the sites of binding and antioxidant activity of albumin. this fact must be taken into account in the biochemical studies of the drugs interacting with albumin. albumin is usually one of the first proteins to be influenced oxidative stress; therefore, its redox status is widely used as a biomarker of various pathological conditions. it is known that in chronic liver and kidney diseases, as well as in diabetes mellitus, the percentage of cysteinylated albumin (cys -s-s-cys) is markedly increased [ ] . in recent years, it has been shown that oxidised albumin can be a biomarker of the severity of such diseases as hyperparathyroidism [ ] , acute ischemic stroke [ ] , parkinson's disease [ ] , alzheimer's disease [ ] , duchenne muscular dystrophy [ ] , etc. the possibility of using the covalent binding of the products of ops hydrolysis with cys for developing the biomarkers of intoxication is of particular interest. ops adducts with tyr are widely studied and described in the literature [ ] [ ] [ ] . however, for thioether ops (such as vx), another class of adducts can be identified. kranawetvogl et al. [ ] showed that the thiol formed after hydrolysis of this class of ops can interact with the thiol group of cys , and the resulting adduct can be detected by mass spectroscopic methods. the same group of researchers, in a recent work [ ] , studied a real case of poisoning with demeton-s-methyl (o,o-dimethyl s- -(ethylsulfanyl)ethyl phosphorothioate, odm). odm belongs to the class of dimethylphosphoryl (dmp) pesticides. -(ethylsulfinyl) ethanethiol (esoet) is a product of odm hydrolysis by blood esterases. the patient's blood plasma was treated with pronase (a mixture of proteinases), and then the obtained samples were examined by mass spectroscopy. among the identified adducts of albumin with odm, the adduct dmp-tyr (tyr ) had the weakest peak, and could only be detected within two hours after poisoning. the peak intensity of the esoet-cyspro adduct (cys and pro of albumin) was times higher, and its lifetime was h. fujii et al. [ ] performed a comprehensive study of japanese residents: the ratio of oxidised/reduced albumin, the thickness of the intima-media complex of the carotid arteries and the number of plaques in the carotid arteries (the latter two indicators characterise the risk of atherosclerosis) were measured. an inverse relationship was found between the level of oxidised albumin and the risk of atherosclerosis. violi et al. recently showed that hsa level is independently associated with mortality in covid- [ ] . the researchers suggested that it might be connected with the antioxidant and anticoagulant properties of albumin. attempts are being made to use albumin not only as an informant about the condition of patients but also as a therapeutic agent. an interesting application of the redox properties of albumin was proposed by japanese scientists [ ] . it is known that reactive sulfur species (rss) are able to neutralise ultraviolet radiation products (for example, ros and no) that promote melanin synthesis. however, the instability of rss limits their use as inhibitors of melanin synthesis. the authors proposed a method for using albumin as the rss delivery system. it was shown that thiolated albumin (obtained by the incubation of albumin and sodium polysulfide) significantly inhibited melanin synthesis in b melanoma cells. the researchers also suggested that albumin modified in such a way could be used in cosmetology to whiten the skin. in the research of schneider et al. [ ] , the possibility of using human albumin solution to protect patients of an intensive care unit (icu) from bacterial infections was studied. the polypeptide vasostatin- is known to have antimicrobial properties and play a key role in protecting the body from gram-positive bacteria. however, the oxidised form of vasostatin loses its antibacterial properties. oxidative processes are often developed in icu patients, which means that they are more at risk of infection. the study showed that continuous infusion of % albumin reduced the risk of nosocomial infections. by mixing albumin with oxidised vasostatin- and using a high-performance liquid chromatography (hplc) method, the authors demonstrated that albumin reduced the oxidised form of vasostatin, thereby increasing its antibacterial properties. analysis of the literature data allows us to take a fresh look at the results of our research aimed at the development of adjuvant therapy for op poisoning. in section . , we have reviewed studies demonstrating that many fas and some polyphenols affect the reactivity of hsa cys thiol group. earlier in our experiments, we tested polyphenols of green tea extract (gte) as a component of functional nutrition before and after acute poisoning with paraoxon and demonstrated the weakening effect of gte on the development of delayed symptoms of poisoning [ ] . in biochemical in vitro experiments, we have shown that gte polyphenols have an activating effect on the true esterase activity of the protein in sudlow site i towards paraoxon [ ] and have suggested that gte promotes not only the transport but also the utilisation of ops by albumin in the bloodstream. however, in the light of new data, it is possible that the major polyphenol of gte epigallocatechin gallate (egcg) has an additional effect: by binding to albumin, it affects the reactivity of the cys , enhances its antioxidant properties, weakens the strength of oxidative stress and thereby reduces the intensity of delayed effects of poisoning. this hypothesis requires additional testing. despite some progress in studying the possibility of enhancing the antioxidant properties of albumin, the development of the methods for correcting oxidative stress taking into account this ability of the protein is still in its infancy. recently, such classes of compounds as thiol antioxidants (n-acetylcysteine, carbocysteine and erdosteine), superoxide dismutase mimetics (magnesium-containing porphyrins), nadph oxidase inhibitors (apocinin, diphenyliod), setanaxib traps (disulfenton sodium), activators of the transcription factor nrf (sulforaphane, bardoxelone methyl, dimethylfumarate) have been actively tested or are already being used to reduce oxidative stress [ ] [ ] [ ] [ ] . it might be that in the future it will be possible to create the complex therapy for oxidative stress management taking into account the functional properties of albumin. the literature data analysed and the results of our own research allow us to formulate some concluding remarks. firstly, albumin is a universal molecule in a certain sense, which can bind almost all known endogenous compounds, metal ions and xenobiotics and possesses a number of enzymatic activities: (pseudo)esterase, paraoxonase, phosphotriesterase, thioesterase, glutathione peroxidase, cysteine peroxidase and some others. due to this versatility, albumin is a participant of many biochemical processes in the human organism, including participation in antioxidant defence. of course, albumin takes part in the redox reactions non-specifically due to the fact that its concentration in the extracellular compartment is very high and renewal occurs relatively quickly (about days). at the same time, it is a sacrificial antioxidant, which takes the brunt of the extracellular component of oxidative stress. the second important point to note is that albumin is easily modulated due to its flexible structure. the interaction of albumin with active species and 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mortality in covid- ? antioxidants redox signal a novel s-sulfhydrated human serum albumin preparation suppresses melanin synthesis a pilot study on continuous infusion of % albumin in critically ill patients the rat (rattus norvegicus) as a model object for acute organophosphate poisoning. . a system analysis of the efficacy of green tea extract in preventing delayed effects of poisoning testing of polyphenols and fatty acids as modulators of albumin esterase activity towards organophosphates oxidative stress and antioxidant treatment in patients with peripheral artery disease oxidative stress-based therapeutics in copd novel antioxidant astaxanthin-s-allyl cysteine biconjugate diminished oxidative stress and mitochondrial dysfunction to triumph diabetes in rat model this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - iawbnua authors: ohl, kim; tenbrock, klaus; kipp, markus title: oxidative stress in multiple sclerosis: central and peripheral mode of action date: - - journal: experimental neurology doi: . /j.expneurol. . . sha: doc_id: cord_uid: iawbnua accumulating evidence suggests that oxidative stress plays a major role in the pathogenesis of multiple sclerosis (ms). reactive oxygen species (ros), which if produced in excess lead to oxidative stress, have been implicated as mediators of demyelination and axonal damage in both ms and its animal models. one of the most studied cell populations in the context of ros-mediated tissue damage in ms are macrophages and their cns companion, microglia cells. however, and this aspect is less well appreciated, the extracellular and intracellular redox milieu is integral to many processes underlying t cell activation, proliferation and apoptosis. in this review article we discuss how oxidative stress affects central as well as peripheral aspects of ms and how manipulation of ros pathways can potentially affect the course of the disease. it is our strong belief that the well-directed shaping of ros pathways has the potential to ameliorate disease progression in ms. multiple sclerosis (ms) is the most frequent neurological disease in young adults with a complex and still uncertain pathogenesis. the most widely accepted hypothesis is that auto-reactive t cells and bcells induce myelin damage, neuroinflammation and neurodegeneration (compston and coles, ; fletcher et al., ; trapp and nave, ) . however, primary oligodendrocyte dysfunction has also been considered as a potential disease-promoting or disease-triggering factor (barnett and prineas, ) . whatever the trigger factors for lesion experimental neurology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] abbreviations: apcs, antigen-presenting cells; are, antioxidant response element; dmf, dimethyl fumarate; faes, fumaric acid esters; gsh, glutathione; ho, heme oxygenase; ifn, interferon; il, interleukin; inos, inducible nitric oxide synthase; keap , kelch ech associating protein ; mdsc, myeloid-derived suppressor cells; mmf, monomethyl fumarate; nqo , nad(p)h: quinone oxidoreductase ; nrf , nuclear factor (erythroid-derived )-like ; rns, reactive nitrogen species; ros, reactive oxygen species; s p, sphingosine -phosphate; tgf, transforming growth factor; t h , t helper cell; t reg , regulatory t cell; trx, thioredoxin. formation in ms are, we now know that both, central and peripheral cellular components are critically involved in the disease process. despite being of unknown etiology, the (histo-) pathological hallmarks of ms lesions are well-defined. they include focal as well as diffuse demyelination, oligodendrocyte loss, activation of brain resident immune cells such as microglia and astrocytes, and damage of the neuro-axonal unit. such cellular alterations can be found in various brain regions including diverse white and gray matter areas (bo et al., ; kipp and amor, ) . the fast activation of brain intrinsic cells, in particular microglia followed by the activation of astrocytes, is most frequently linked to the expression and release of oxidative-stress related molecules. in this review article we first give a brief definition of "oxidative stress" and "reactive oxygen species (ros)" and then describe the pathways and factors involved in this ubiquitous cellular state. since ms pathogenesis is characterized by the interplay of central and peripheral cellular elements, we then go on to explain how both compartments are regulated by ros. finally, we will argue that currently approved treatment options, most importantly fumaric acid esters (faes), interfere with oxidative stress pathways and by this mechanism exert their beneficial function. however, modulation of central and peripheral ros pathways might result in side effects. oxygen is pivotal for multicellular life. at the same time, it is one of the most reactive and life-threatening agents known. however, at least for aerobic organisms, oxidation has become the main means of energy generation. to guard against the possible deleterious effects of oxygen, intracellular homeostasis is maintained by a balancing of oxidation and reduction (redox) reactions, the so-called "intracellular redox equilibrium". in extreme cases, when metabolic processes or toxic insults lead to a situation where pro-oxidants outbalance the anti-oxidative counterparts, a state of "oxidative stress" is reached. this breakdown of cellular homeostasis results in oxidation-induced damage to lipids, proteins, carbohydrates and nucleic acids, eventually leading to cell death. the agents inducing oxidative stress are chemical compounds classed as reactive oxygen species (ros) or reactive nitrogen species (rns). ros/rns are both instable, and mostly exist in a radical form, which means that they contain unpaired electrons on the outer orbital. the best-studied ros/rns include radicals of oxygen [superoxide anion (o − ), hydroxyl radicals (oh . ), and peroxyradicals (roo • )] or nitrogen [nitric oxide (no . )] as well as non-radical species, such as hydrogen peroxide (h o ) and singlet oxygen. nitric oxide, itself less reactive and generally non-damaging, can rapidly react with a superoxide anion to form peroxinitrate (onoo − ), one of the most deleterious ros/rns known. ros and rns have long been implicated in the pathogenesis of a plethora of diseases such as stroke, parkinson's disease or alzheimer's disease (lin and beal, ) . on the other hand, and this aspect of ros/rns has been less well studied, low levels of ros/rns can act as second messengers for signal transduction/amplification and fulfill specific intracellular functions (reth, ) . key transcription factors regulated by ros include p , ap- (c-jun, c-fos), nf-ĸb and, as discussed in this review article, the transcription factor nrf . all cells are equipped with an intrinsic mechanism that neutralizes excess ros and protects against oxidative injury. this so-called oxidative stress response is mainly, but not exclusively, controlled by the transcription factor nuclear factor (erythroid-derived )-like (nrf ). nrf plays a vital role in maintaining cellular homeostasis, especially upon exposure of cells to chemical or oxidative stress, through its ability to regulate basal and inducible expression of a multitude of antioxidant proteins, detoxification enzymes and xenobiotic transporters (kensler et al., ) . in anti-oxidative stress responses, nrf upregulates phase ii detoxifying enzymes and antioxidant proteins. this nrf -induced enzymatic machinery includes enzymes mediating glutathione (gsh) synthesis, the thioredoxin (trx) enzyme system and detoxifying enzymes like heme oxygenases (ho), or nad(p)h: quinone oxidoreductase (nqo ). how do these components protect the cell? reduced gsh acts by scavenging oxidative species such as superoxides, hydroxyl radicals, nitrogens, and onoo (forman et al., ) . the crucial cysteine molecule is the key to the protection afforded by gsh. its sulfur atom scavenges destructive molecules (peroxides and free radicals) converting them to harmless compounds, such as water. trx plays an important role in maintaining a reduced environment in the cells through thiol-disulfide exchange reactions and, thus, protects cells and tissues from oxidative stress. nqo and ho are important as catalysts of heme and quinone degradation. free heme is liberated under oxidative conditions and mediates ros production. quinones are highly redox active and also lead to formation of ros. since nqo and ho eliminate heme and quinone, they can exert an anti-oxidative function. besides the induction of anti-oxidative factors, nrf also contributes to different cellular functions such as differentiation, proliferation, inflammation and lipid synthesis, and there is increasing evidence of an association between aberrant expression and/or malfunctioning of nrf and diverse pathologies including cancer, neurodegeneration or cardiovascular disease. having outlined the protective potential of the transcription-factor nrf , we will now describe its mode of action. the nrf cell defense pathway is tightly regulated. under quiescent conditions, nrf is retained and degraded in the cytosol by kelch ech associating protein (keap ) (zhang and hannink, ) (fig. ) . various stressassociated stimuli, such as oxidative stress, induce conformational changes in keap that result in the release of nrf from its keap binding. subsequently, nrf trans-locates into the nucleus where it trans-activates the expression of genes containing an antioxidant response element (are) in their promoter regions (kensler et al., ) . although it is well established that nrf activity is controlled, in part, by the cytosolic protein keap , the nature of this pathway and the fig. . scheme of nrf activation. under basal conditions, nrf interacts with keap , which results in degradation of nrf . in response to cellular stress, nrf is liberated from its cytosolic inhibitor, trans-locates into the nucleus and binds to antioxidant response elements (ares) in the promoters of target genes. nrf -regulated genes mainly include genes coding for antioxidative and detoxifying enzymes. mechanisms by which keap acts to repress nrf activity remain to be fully characterized (nguyen et al., ) . whatever mechanisms are involved in nrf liberation and subsequent are-binding, activation of this promoter region results in increased expression of cytoprotective genes. the first of these to be described under the regulation of are-activity were the two major detoxification enzymes, gsta (glutathione s-transferase a ) and nqo (favreau and pickett, ; friling et al., ) . thus, alteration of the cellular redox status due to elevated levels of ros and electrophilic species and/or a reduced antioxidant capacity (e.g. gsh) appears to be an important signal for triggering the transcriptional response mediated by are. besides its critical role in inducible gene expression, the are site is also pivotal for the low-level constitutive (or basal) expression of several genes under physiological conditions. because ros and other endogenous reactive molecules are continuously generated by normal (i.e. physiological) aerobic metabolism, are appears to be pivotal for the maintenance of cellular redox homeostasis under both stressed and non-stressed conditions. since inflammation is closely linked with oxidative stress, it is hardly surprising that nrf deficient mice have a worse disease outcome in several inflammation-mediated animal models, including experimental asthma (rangasamy et al., ) , acute lung injury (reddy et al., ) , sepsis (thimmulappa et al., ) , t cell-mediated hepatitis (osburn et al., ) , or dextran sulfate sodium-induced colitis (khor et al., ) . interestingly, late adult nrf −/− female mice are prone to develop autoimmune syndromes that closely resemble the human disorder systemic lupus erythematosus . t cells contribute to appearance and progression of inflammation and are associated with the above-mentioned disease models. the possible interaction of ros-nrf and t-cell priming and effector function will be discussed later in this review article. besides being expressed in peripheral organ systems, nrf is recognized as an important regulator of inflammation and cell death in the brain. findings highlighting the relevance of the nrf -keap -are system for neurodegenerative and neuroinflammatory diseases include decreased nuclear nrf -levels in the hippocampus of alzheimer's disease patients, increased nrf nuclear translocation in parkinson's disease (ramsey et al., ) , a prominent decrease in gsh levels in the substantia nigra of parkinson's disease patients (sian et al., ) , and lower nrf paralleled by higher keap levels in amyotrophic lateral sclerosis vs. control samples (sarlette et al., ) . taken together, although only a few studies have been conducted in humans, these do indicate that the nrf system is dysregulated in brains of individuals suffering from neurodegenerative diseases and that this dysregulation may well contribute to chronic neuron degeneration in these disorders. besides its likely impact on pathological progression in classical neurodegenerative disorders, the nrf -keap -are system appears at the same time to be a potent regulator of neuroinflammatory diseases. in nrf deficient mice, the inflammatory response in the brain and magnitude of microglia activation in response to lipopolysaccharide is much more pronounced than in normal animals (innamorato et al., ) , and treatment with sulforaphane, a potent nrf -inducing agent, was found to be effective in reducing neurotoxicity associated with herpes simplex virus-stimulated microglial ros production (schachtele et al., ) . furthermore, nrf deficient mice also show a more severe pathology in experimental autoimmune encephalomyelitis (eae) (johnson et al., ) , the most widely used autoimmune-related ms animal model. although the pathogenesis of ms lesion development is complex and involves the activation of both central and peripheral elements of the immune system, adaptive immune-responses undoubtedly play an important role. dysregulation of various different cell types of the adaptive immune system appears to contribute to lesion formation and progression, as shown for t h and t h (hermans et al., ) , t h -cells (tzartos et al., ) , regulatory t (t reg ) cells (chen et al., ; viglietta et al., ) , b-cells (sekizawa et al., ) , and myeloid-derived suppressor cells (mdscs) (zhang et al., ) . the later constitute a very heterogeneous and plastic cell population that consists of myeloid progenitor cells and immature myeloid cells. the most popular concept of ms lesion formation is that acute demyelinating lesions are generated by phagocytes that internalize and degrade apparently normal myelin sheaths in the presence of infiltrating t cells. immune cell recruitment is an early or even initial event in the formation of ms lesions in this model (frischer et al., ; lucchinetti et al., ) . in sharp contrast to this idea, results of other studies suggest that extensive oligodendrocyte apoptosis with early, focal microglia activation is the major pathological feature in newly forming lesions, and immune cell recruitment is a response to this primary oligodendrocyte pathology (barnett and prineas, ; stys et al., ) . whether or not the initial event of ms lesion formation is recruitment of auto-reactive t cells across the blood brain barrier into the brain parenchyma, without doubt the inflammatory process involves the activation and recruitment of t cells, macrophages and microglia to lesion sites. once active demyelination is established, peripheral immune cells (such as lymphocyte, recruited monocytes, mdsc) and their central counterparts (astrocytes and microglia) contribute to progressive tissue damage in ms. these inflammatory processes critically involve ros-mediated tissue injury. activated microglia and infiltrated macrophages are able to generate vast amounts of proinflammatory mediators and oxidizing radicals, such as superoxide, hydroxyl radicals, hydrogen peroxide and nitric oxide (colton and gilbert, ) . furthermore, the activation of immature myeloid cells (i.e. mdscs) has been linked to the induction of no and ros production (zhang et al., ) . most studies addressing the relevance of oxidative stress for ms lesion formation and progression have focused on brain intrinsic cells and recruited monocytes. for example, it has been demonstrated that in white and gray matter lesions myeloperoxidase, a lysosomal enzyme which produces hypochlorous acid from hydrogen peroxide and chloride anion, is predominantly expressed by macrophages and/or activated microglia (gray et al., a (gray et al., , b , emphasizing the key role of these myeloid cells in the generation of ros. further results from autopsy studies showed that in active lesions of the white matter and cerebral cortex, demyelination and neurodegeneration are closely associated with the presence of oxidized lipids (such as oxidized phospholipids and malondialdehyde) in myelin membranes, in apoptotic oligodendrocytes and in the neuro-axonal compartment (fischer et al., ; haider et al., ) . furthermore, nuclei of dystrophic glia cells and neurons were found to contain oxidized dna , and oxidative injury was associated with inflammation and oxidative burst in activated microglia and macrophages expressing p phox, an essential subunit of nadph oxidases (fischer et al., (fischer et al., , . although this situation appears to be only partly reflected in relevant ms animal models (schuh et al., ) , oxidative injury also takes place there. macrophages and microglial cells, isolated from the cns of lewis rats with clinical signs of eae, exhibited significantly elevated spontaneous and inducible ros levels compared to similar cells isolated from healthy controls, or rats sacrificed before manifestation of clinical signs of eae (ruuls et al., ) . from a functional point of view, treatment of eae-rats with catalase, which scavenges the ros h o , markedly suppressed the severity of the disease. beyond that, the ros protectant heme oxygenase- (ho- ) is expressed by monocytes in eae (schluesener and seid, ) , ros aid phagocytosis of myelin by activated macrophages (van der goes et al., ) , and stabilization of mitochondria (which are a major source of ros) ameliorates axonal damage in eae (forte et al., ; qi et al., ) . although the eae animal model is a heterogeneous group of experimental tolls to study ms pathogenesis (van der star et al., ), these results foster the view that ros are critically involved in autoimmune-mediated tissue damage in ms. ros accumulation is also evident in other in vivo demyelinating models such as in corona-virus-induced inflammatory demyelination (schuh et al., ) . furthermore, our group observed strong induction of ho- after short-term cuprizone exposure (own observation, unpublished). taken together, there is ample evidence that ros actively contribute to tissue damage during ms lesion development and progression, and that the activation of the nrf pathway might play a protective role in the pathogenesis of ms by operating on levels of certain enzymes. such effects might include the induction of several antioxidant enzymes that can directly scavenge ros, increasing levels of antioxidant enzymes that might reduce microglial activation and limit myelin phagocytosis and breakdown, and induction of antioxidant enzymes that might prevent oxidative damage to neurons and oligodendrocytes. however, as we will point out in the next chapter, ros are also potent regulators of the adaptive immune-response. the functionality of ros in ms patients should therefore be reflected in both compartments. cells belonging to the adaptive immune response, and in particular cd + t helper (t h ) cells, play an important role in the pathogenesis of ms lesions. t cell activation relies on the binding between t cell receptors (tcrs) and antigens, which are typically short peptides presented by mhc molecules that are displayed on the surface of antigenpresenting cells (apcs), including macrophages, b cells and dendritic cells (dcs), as the most "professional" apc populations. in addition, t cell activation requires a second signal from co-stimulatory molecules (cd /cd ). stimulation of t cells through the tcr and co-receptor cd (cd / ligand) induces transcriptional programsincluding activation of nf-ĸbwhich initiate the production of cytokines such as il- that in turn are important for t cell proliferation and activation. cytokines are also key regulators of t cell differentiation towards one of several t h cell subtypes, including t h , t h , t h and inducible t reg cells. interleukin (il)- and ifn-γ are two important cytokines for t h differentiation, while il- , il- , il- and thymic stromal lymphopoietin drive t h differentiation. transforming growth factor (tgf) β induces t h differentiation in the presence of il- , il- and il- , while tgf-β in the presence of il- induces t reg cells (rev. in zhu and paul ( ) ). once differentiated, t h cell subtypes are further defined by their pattern of cytokine production and by their distinct functions. t h cells produce interferon (ifn)-γ and are important for protective immune responses to intracellular viral and bacterial infections. in contrast, t h cells are critical for clearance of extracellular parasites, whereas t h cells play an important role in protection from bacterial and fungal infection. beyond their central role in adaptive immune responses against pathogens, t h cells, especially through their autoreactive or exaggerated responses, are also involved in autoimmune reactions. until recently t h cells were thought to be the main effector t cell in ms, but more recent studies have highlighted an additional pathogenic role for t h cells (fletcher et al., ) . autoreactive t cells are mostly deleted in the thymus, but some of them escape this socalled central tolerance. consequently, several mechanisms evolved to control autoreactive t h cells in the periphery (peripheral tolerance). dominant tolerance by t reg cells is one strategy to prevent autoimmune disease and maintain immune homeostasis by suppressing autoreactive and exaggerated t cell responses (campbell and koch, ) . t reg cells are categorized as thymus-derived (tt reg cells) or induced (it reg cells). as the name implies, tt reg cells develop in the thymus, whereas it reg cells differentiate from naive t-cell precursors in the periphery. both t reg -cell types express foxp , the lineage-specific and most important transcription factor for maintenance of the t reg cell phenotype and suppressor function (fontenot et al., ; hori et al., ) . with regard to ms and eae, an imbalance of pro-inflammatory responses such as t h and t h and anti-inflammatory responses mediated by t reg or t h cells appears to be crucial for disease development and progression (fletcher et al., ) (fig. ) . t h and t h can be found in ms lesions (lock et al., ; tzartos et al., ) where they initiate and exacerbate an inflammatory cascade by the release of cytokines and recruitment of further inflammatory immune cells. t reg cells, which one would expect to control these exaggerated t h and t h responses, are characterized by several aberrancies in ms. although there are no numerical deficits in t reg cells in ms, the suppressive function of t reg cells appears to be disturbed (frisullo et al., ; haas et al., ; venken et al., ; viglietta et al., ) . furthermore, t reg cells reveal an impaired capacity to proliferate in relapsing-remitting ms (rrms), and express reduced levels of foxp , which is critical for maintaining function and lineage stability (carbone et al., ; huan et al., ) . recent reports also raise the question of whether defects in t reg function are caused by an enhanced plasticity of t regs towards a proinflammatory, cytokine-producing effector phenotype. patients with untreated rrms have higher frequencies of t h -like, ifn-γ-secreting foxp + t cells, with a reduced suppressive capacity (dominguez-villar et al., ) . a shift of t reg cells towards il- producing cells is associated with psoriasis, autoimmune hepatitis and systemic sclerosis, but so far no direct association has been detected in ms. in vivo studies from rodent eae models demonstrate the central function of t reg cells in autoimmune neuroinflammation. transfer of t reg cells ameliorates eae symptoms (kohm et al., ) and nonspecific t reg cell ablation by anti-cd antibodies exacerbates eae severity (gartner et al., ) . in addition, frequencies of t reg cell population within the cns are elevated during the recovery phase of actively induced eae (mcgeachy et al., ) . although the importance of t reg cells for ms disease development and progression is well appreciated, basic aspects of t reg cell biology remain unresolved. whether t reg cells are suppressive at inflammatory sites or act predominantly to limit the priming of naive t cells in the lymph nodes (lns) remains a controversial issue (fig. ) . with respect to central effects, t reg cells isolated from the cns during eae were capable of suppressing t h but not t h cells (o'connor et al., ) but were not able to suppress effector t cells directly isolated from the acutely inflamed cns (korn et al., ) . with respect to peripheral effects, it has been shown that in the absence of t reg cells, there is an enhanced migration of effector t cells from the periphery (lowther et al., ) . furthermore, t reg cells influence eae course by affecting the priming and polarization of effector t cells (kohm et al., ) and can also set a threshold for activation of autoreactive effector t cells by inhibiting their contacts with antigen-loaded dendritic cells (dcs) in the lymph nodes (tadokoro et al., ) . the interplay of local tissue inflammation and t reg cells is poorly understood but might be an important factor for the resolution of autoimmunity. in addition to t reg cells, innate immune cells are also capable to suppress t cell responses. for example, mdscs have been described to suppress the activation of t cells, which makes them attractive targets for the treatment of autoimmune diseases. high accumulation of mdscs in ms and eae indicates that mdscs play an important role (zhang et al., ) . however, beneficial (moline-velazquez et al., ) as well as pathogenic functions (yi et al., ) of mdscs in eae are reported, therefore their exact role remains to be elucidated. oxygen-derived reactive species have long been known to exert diverse effects on cultured mammalian cells, depending on the cell type considered, the oxidative stimulus, and the intensity and timing of stimulation. these effects are most frequently "positive", or mitogenic, at low levels of oxidative stress and "negative", i.e., toxic or growth-arresting, at greater oxidative burdens (pani et al., ) . in consequence, scavenging of endogenous oxygen species by either antioxidant enzymes or cell-permeant antioxidant and reducing agents would interfere with normal cell responses, attenuating or abolishing their effects. this general concept of ros biology and pathology is also relevant to the immune system. the concept that ros are implicated in body defense is definitely not novel. the killing of bacteria through the generation of superoxide, h o , and hypochlorous acid by the coordinated action of nadph oxidase and myeloperoxidase is a key event in the protective function of neutrophils and "professional" phagocytes (pani et al., ) . ros have also been implicated in mechanisms of target cell killing by natural killer cells and in cytolysis by cd + lymphocytes. however, while well recognized as effectors of innate immunity, oxygen radicals are just starting to be considered as potential regulators of antigen-specific immunity and, by extension, of t-lymphocyte function. today we know that ros and redox states are both central to immunity and t-cell function. first, and maybe most importantly, t-cells can sense redox stress (secchi et al., ) . furthermore, redox stress is important for the functional outcome during experimentally-induced adaptive-immunity responses; e.g. in a graft-versus-host disease mouse model, free radical scavenger therapy with necrox- attenuates disease severity, probably via the induction of t reg cells (im et al., ) and oxidative damage regulates antigen-specific t cell responses in agerelated macular degeneration (cruz-guilloty et al., ) . in this context it is important to note that t-cell receptor activation results in intracellular ros production (devadas et al., ; gutscher et al., ) , and that the simultaneous action of the oxidative signal and ca + influx is indispensable for t-cell activation-induced gene expression. taken together, these findings illustrate that ros and t-cell function are intimately linked. effects of ros on adaptive immune responses probably depend on at least two variables, (i) lymphocyte subset and (ii) intra-or extracellular mode of actions. with respect to the first aspect it has been shown that distinct t-cell subsets have different redox statuses, and differential ros susceptibility. t regs , for example, are more resistant to oxidative stress-induced cell death compared to other t cell populations. this is paralleled by a greater secretion of redox proteins such as trx. furthermore, it has been shown that human t regs express high levels of cell surface thiols, which are important reducing agents, and harbor an increased intracellular antioxidative capacity (mougiakakos et al., ). thus, ros effects studied in one lymphocyte population do not necessarily exert the same effects in other ones (if applied at the same concentration). secondly, the intra-and extracellular modes of ros action need to be studied separately. t cell receptor engagement triggers ros production (devadas et al., ) and these intracellularly produced ros species might impact on t-cell function. obviously, the intracellular redox state needs to be finely regulated, and a dysregulation of this dedicated network might impact on proper t-cell functioning. on a more detailed level, intracellular ros play an important role in the immediate early events of t cell activation (devadas et al., ; los et al., ) . however, under certain conditions the potentiation of oxidative stress by, for example, gsh depletion can result in impaired t cell activation (rajah and chow, ) . with regard to the extracellular mode of ros action, ros can act in mammalian cells as biochemical mediators involved in signal transduction from cell surface to the nucleus; implicated mechanisms are (i) induction of protein phosphorylation, and (ii) activation/inhibition of various redox-sensitive transcription factors, among them, nrf . the extracellular ros milieu is indeed important for proper t-cell effector function, most notably by influencing the equilibrium between oxidized and reduced thiols on exofacial membrane proteins (gelderman et al., ) . impaired functioning of the nadph-oxidase complex, which results in lower ros levels, is associated with an increased number of reduced thiol groups (−sh) on t cell membrane surfaces. this reducing extracellular milieu boosts t cell response and proliferation, thus showing that ros production plays a key role in regulating surface redox levels on t cells and thereby suppresses autoreactivity. among the apcs contributing to this reducing milieu, the action of dcs at the level of the immune synapse is of primary importance (fig. c ) (angelini et al., ; yan et al., ) . in line with these findings, sustained pro-oxidant extracellular conditions have been shown to inhibit t cell activation (matsue et al., ; tse et al., ) and induce apoptosis in t cells (hildeman et al., ; tripathi and hildeman, ) (fig. b) . under auto-immune conditions. in peripheral lymph nodes, dcs activate t cells and induce their differentiation towards inflammatory t h and t h cells by the release of cytokines, like il- and il (t h ) and il- (t h ). in ms, t reg cells eventually fail to control this t cell activation process. activated t cells migrate to and enter the cns where they become reactivated by local apcs, and again are not adequately suppressed by t reg cells. t eff cells then expand and drive cns inflammation. after having successfully entered the cns, t cells are exposed to a completely novel oxidative milieu. there, ros molecules are mainly produced by macrophages, microglia and astrocytes and lead to damage of neurons, axons, myelin and oligodendrocytes (indicated by arrows). mdscs can suppress activated t cells, but can also differentiate into dendritic cells and macrophages in the cns and influence the entire scenario. impaired t-effector cell activation and reactivity in an oxidative environment might also be mediated by the suppressive action of t reg cells. for example, it has been shown that the induction of t reg cells by macrophages is dependent on production of ros (fig. a) , and ros deficiency may lead to decreased t reg induction and may hamper t-cell suppression (kraaij et al., ) . furthermore, it was elegantly demonstrated that t reg cells disturb intracellular redox homeostasis in t effs by interfering with extracellular redox remodeling ( (yan et al., ; yan et al., ) , fig. d ). in addition to that, mdsc-mediated suppression of t cell function involves ros, no and peroxynitrite, which induce post-translational modification of t-cell receptors and may thereby cause antigen-specific t-cell unresponsiveness (gabrilovich and nagaraj, ) . notably, extracellular and intracellular ros-related pathways are not completely separated. the antioxidant gsh serves as an important proliferative signal in t cells (suthanthiran et al., ) . naive t cells require cysteine for gsh synthesis and activation, but are unable to import cystine, which can efficiently be converted to cysteine within the cell. they are thus, dependent on cysteine secretion by apcs. to make the situation even more complicated absolute ros levels also appear to play an important role. prolonged exposure to high ros concentrations can inhibit t-cell proliferation and induce apoptosis, whereas low concentrations of ros in t cells are a prerequisite for cell survival (kesarwani et al., ) . in summary, ros levels can impact on the acquired immune system via a variety of mechanisms and are thus critically involved in the development and progression of auto-immune disorders, including ms. still, we are far away to completely understand this complicated scenario. in the previous chapter we have seen that ros levels and t cell functioning are intimately linked. although somewhat neglected by the research community, it appears that oxidative stress not only regulates disease outcome in ms patients by acting within the cns, but may impact on disease burden by shaping the immune response outside of the cns. such a regulation might, for example, occur at the level of the endothelium. endothelium cells, which are found at the interface between the cns and periphery, are regulated in a ms-relevant manner by ros. high ros levels damage brain endothelium and affect blood-brain barrier (bbb) permeability (imaizumi et al., ; olesen, ) . thus, regulative effects of oxidative stress in ms probably include but are not limited to the cns parenchyma. it may also regulate immune cell recruitment at the level of post-capillary venules. an important question arising here is whether ros can also regulate other peripheral aspects of the disease, namely, t effector cell polarization and proliferation. results from other diseases strongly suggest that this is indeed the case. in many cases, mitochondrial disorders, which are closely linked to a disturbed oxidative environment, have hematological manifestations and result in recurrent infections. complex ideficient patients, in particular, present with a severely impaired immune response. they are prone to concomitant infections that inevitably lead to a worsening of symptoms or can even trigger primary occurrence of the disease. a close link between disturbed immune tolerance and mitochondrial ros generation was found in systemic lupus erythematosus patients where t cells are characterized by a higher mitochondrial mass, persistently hyperpolarized mitochondria and elevated ros levels (gergely et al., ) . in addition, t-cell mitochondria and presumably activation-induced mitochondrial ros release play an important role in the pathology of the acquired-immunodeficiency syndrome (aids) (kaminski et al., ) . thus, ros and auto-immunity are indeed closely linked. with respect to ms patients, a plethora of studies have analyzed changes in oxidative stress parameters. however, these studies were mainly conducted on serum and/or plasma samples (fiorini et al., ) , which somewhat limits their interpretation. in one study, urinary -iso-pgf α levels, which reflect the degree of lipid peroxidation due to ongoing oxidative stress, were found to be significantly higher in ms subjects than in controls (guan et al., ) . similar results have been reported for peripheral blood mononuclear cells . other markers for oxidative stress that are found to be elevated in the serum and/or plasma of ms patients include a reduced serum ferroxidase activity, which can potentially lead to a rise in oxidative stress (cervellati et al., ) , lower antioxidant capacity (karlik et al., ) , and higher advanced oxidation protein products along with lower thiol group levels (pasquali et al., ) . interestingly, ms patients with a favorable disease course show higher antioxidant factors, including coq and anti-oxldl autoantibodies, which may shape the immune system in the periphery (gironi et al., ) . on the cellular level, ros production by macrophages and lymphocytes was reported to be higher in ms vs. control patients, and decreased in (kraaij et al., ) . (b) sustained pro-oxidant extracellular conditions inhibit t cell activation and induce apoptosis in t cells (tripathi and hildeman, ) . (c) interaction of dcs with t cells leads to cysteine accumulation in the extracellular space, which produces an extracellular redox potential promoting t cell proliferation (angelini et al., ) . (d) t reg cells inhibit dendritic cell induced extracellular reduced redox potential (yan et al., ) . peripheral blood mononuclear cells of ifnβ- a-treated patients (koch et al., ; lucas et al., ) . to conclude, these findings suggest a role of ros in the peripheral component of ms lesion pathogenesis. in summary, there is ample evidence for a critical function of redox homeostasis disruption in the pathogenesis of ms, and we suggest that this disrupted redox homeostasis is not limited to the brain but also occurs in the periphery in ms patients. the finding that peripheral immune cells withstand oxidative stress, due to upregulated cellular antioxidant defense mechanisms, suggests that ros formation in ms is not necessarily deleterious but might form part of a finely tuned regulatory network. this delicate network could possibly be targeted by novel therapeutic interventions, as a supplement to established treatments. a number of different therapeutic options are available to ameliorate disease activity in ms patients. more and more evidence suggests that currently approved drugs act both within the brain parenchyma and in the peripheral lymphoid organs. for example, the protective effects of the orally available sphingosine -phosphate (s p) receptor modulator fingolimod (also known as fty or gilenya®), have for a long time been attributed to the hindrance of lymphocyte trafficking to target organs through lymphocyte sequestration in secondary lymphoid structures. this drug internalizes the s p -receptor subtype in t cells with subsequent degradation (i.e. acts as a functional antagonist at s p ), preventing t-cell egress from lymphoid organs into the bloodstream, thus limiting the entry of pathogenic t cells into the cns parenchyma. however, its efficacy in ms and related animal models may in part be due to additional, direct effects within the brain, and other s p receptor subtypes appear to be involved . in particular, it has been shown that due to its lipophilic character, fingolimod is able to access the cns parenchyma through the blood-brain barrier. s p receptors are expressed by neuroectodermal cell types such as oligodendrocytes, neurons or astrocytes, and may thus regulate neuron and/or glia cell morphology, migration, process extension, cell growth, differentiation, apoptosis and/or survival . furthermore, a direct neuroprotective effect of fingolimod has been demonstrated in recent preclinical studies (slowik et al., ) . similarly, ifn-β has been shown to be a potent lymphocyte regulator (i.e. peripheral effect) but at the same time might interact with cells of the cns (esen et al., ; vergara et al., ) . thus, the therapeutic efficacy of established interventions may originate in peripheral lymphoid organ but at the same time be manifested within the brain. dimethyl fumarate (dmf), marketed as tecfidera® from biogen idec, has just recently been granted approval as an indicated treatment for ms by the us food and drug administration (fda). in the european union, the medication received approval by the european medicines agency (ema) in early . dmf is a relatively simple molecule derived from fumaric acid. while fumaric acid itself is poorly absorbed by the gastrointestinal tract, its ester derivatives, monomethyl fumarate (mmf) and dmf, both proved beneficial in treating the skin disease psoriasis when administered either topically or orally (altmeyer et al., ) . in vitro studies have demonstrated that dmf is rapidly metabolized at the level of the gastrointestinal tract to its primary, active metabolite mmf by the abundant esterases present in the tissues. the ultimate mechanism of action responsible for the positive treatment effects of faes is still under investigation. what has become clear so far is that, like other medications such as interferon or fingolimod, faes do not have a single mode of action but instead exert multitude biological effects. immunomodulatory effects include a shift towards a t h response litjens et al., ) , and such a shift could result in decreased t h cell activation and tissue infiltration. this balance plays a key role in ms and a shift from t h towards a t h cytokine profile could have a beneficial effect on the clinical course of disease. however, it is not clear if these effects are indeed t cell intrinsic or mediated by apcs. faes also inhibit dc differentiation and reduce production of those cytokines that drive t h and t h cells (il- and il- , il- ) (ghoreschi et al., ; peng et al., ) . with this regard, it might also be interesting to analyze if and how faes, which have been shown to inhibit dendritic cell maturation (peng et al., ) , regulate mdsc activation and/or differentiation into inflammatory apcs. furthermore, it has been reported that faes might enhance t cell apoptosis (treumer et al., ) , or might interfere with leucocyte migratory properties. in one study faes reduced the migratory activity of lymphocytes in vitro, most probably by changing their activation state, as no effect was seen on the expression of mmps, chemokine receptors, and adhesion molecules . furthermore, faes were shown to decrease the expression of adhesion molecules (cd , hla-dr) in lymphocytes in vitro . thus, fae actions on lymphocytes are diverse. with regard to oxidative stress in ms, another interesting fae property emerging in preclinical trials was its ability to positively impact the natural anti-oxidative stress machinery of cells. as pointed out above, in resting states, nrf is sequestered in the cytoplasm by keap . fae has been shown to bind to keap and enable the nuclear translocation of nrf , resulting in transcription of cytoprotective genes including hemoxygenase- (hmox ) and nqo (chen et al., ) . in moginduced eae, faes have been shown to destabilize the nrf -keap complex, thus promoting anti-oxidative nrf pathway activation . interestingly, in vitro, mmf, the primary metabolite of dmf, protected cultured neurons and astrocytes from h o -induced cell death . beyond, faes also suppressed nitrite production and inducible nitric oxide synthase (inos) levels in human astrocytes, where overproduction contributes to oligodendrocyte and neuronal damage . a decreased synthesis of the proinflammatory mediators inos, tnf-alpha, il- beta and il- was also observed in activated fae treated rat microglia and astrocytes in vitro . these studies demonstrate that faes exert neuroprotective as well as anti-inflammatory effects. while the details of the interaction between faes and its target structures and cell types continue to be unveiled, further studies will be needed to show to what extent faes have a direct neuroprotective action, how immunosuppression is exerted, and whether nrf is a critical link in these events. as an inducer of nrf , faes clearly have the potential to become a classical neuroprotective compound. several earlier studies elaborated on the possible neuroprotective effects of other inducers of nrf , including ceftriaxone (lewerenz et al., ) , sulforaphane (danilov et al., ) , and chitosan (khodagholi et al., ) . furthermore, nrf pathway activation by faes might manipulate the oxidative network in lymphocytes by favoring t h or t reg activation. anti-inflammatory effects of nrf activation and the proinflammatory effects of nrf deletion have been demonstrated in a large number of studies with nrf deficient (nrf −/− ) mice, which also included t cell dependent models (as explained in detail above). furthermore, t cell studies demonstrate that nrf activation inhibits t h cytokine production, while concurrently promoting t h cytokine production (rockwell et al., ) . the role of nrf signaling in t reg cells is less clear. the fact that t reg cells, for example, are more resistant to oxidative stress-induced cell death than conventional t cells (mougiakakos et al., ) , and that this increased resistance is associated with a greater expression of nrf mediated genes (mougiakakos et al., ; pae et al., ) , suggest that nrf signaling critically influences t reg cells. in conjunction with these studies and the present data, we propose a dual mechanism of action for faes in ms: targeting immune cells on the one hand and cns cells on the other (as summarized in fig. ). further exploration of the mechanisms operating during immune effector cell priming will require the use of conditional, tissue-specific knock-out animals. as pointed out above, there is substantial evidence that ros production is important for the regulation of surface redox levels of t cells and thereby suppresses auto-reactivity and auto-immune disease development. thus, ros can exert anti-inflammatory effects! in contrast, boosting of nrf -activity, which antagonizes ros-mediated effectsat least in partcan as well ameliorate inflammation. it thus appears that ros-mediated effects depend on the fine-tuning of a multicellular and multi-cascade network, and we are just beginning to understand these finely tuned events. fumaric acid esters (i.e. dmf/mmf), which are prescribed for the treatment of psoriasis and ms, are considered to have a favorable risk profile. however, treatment-related progressive multifocal leukoencephalopathy (pml) has been reported in association with long-lasting, severe lymphocytopenia (ermis et al., ; nieuwkamp et al., ; rosenkranz et al., ) . since the number of patients who are being treated with dmf has been rapidly increasing since approval of delayed-release dmf (tecfidera) as a first-line treatment for rrms, a better understanding is needed of the relevance of ros to immune-cell function. there is no doubt that rosmediated pathways and cellular effects are involved in immune cell priming in the peripheral lymphoid organs. to exert their deleterious effects, t eff as well as t mem cells travel through the circulation into the brain parenchyma where they are re-activated in ms by local apc such as microglia or macrophages. since the oxidative brain environment is altered in ms patients, t-cell reactivation might be affected depending on the redox status in these tissues. for future studies, we recommend detailed examination of the interaction of recruited immune cells with brain resident cells such as microglia and astrocytes, as this would help to establish the circumstances under which t-cells can promote inflammatory cascades within the brain parenchyma and thus regulate the formation and progression of new ms lesions. antipsoriatic effect of fumaric acid derivatives. results of a multicenter double-blind study in patients antigen-presenting dendritic cells provide the reducing extracellular microenvironment required for t lymphocyte activation relapsing and remitting multiple sclerosis: pathology of the newly forming lesion grey matter pathology in multiple sclerosis phenotypical and functional specialization of foxp + regulatory t cells regulatory t cell proliferative potential is impaired in human autoimmune disease serum 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is neuroprotective after cuprizone-induced cns demyelination will the real multiple sclerosis please stand up? glutathione regulates activation-dependent dna synthesis in highly purified normal human t lymphocytes stimulated via the cd and cd antigens regulatory t cells inhibit stable contacts between cd + t cells and dendritic cells in vivo nrf -dependent protection from lps induced inflammatory response and mortality by cddo-imidazolide multiple sclerosis: an immune or neurodegenerative disorder? dimethylfumarate is a potent inducer of apoptosis in human t cells sensitization of t cells to apoptosis-a role for ros? disruption of innate-mediated proinflammatory cytokine and reactive oxygen species third signal leads to antigen-specific hyporesponsiveness interleukin- production in central nervous system-infiltrating t cells and glial cells is associated with active disease in multiple sclerosis in vitro and in vivo models of multiple sclerosis natural naive cd + cd +cd low regulatory t cell (treg) development and function are disturbed in multiple sclerosis patients: recovery of memory treg homeostasis during disease progression ifn-beta reverses the lipopolysaccharide-induced proteome modifications in treated astrocytes loss of functional suppression by cd +cd + regulatory t cells in patients with multiple sclerosis oxidative stress induced by lipid peroxidation is related with inflammation of demyelination and neurodegeneration in multiple sclerosis dimethylfumarate inhibits microglial and astrocytic inflammation by suppressing the synthesis of nitric oxide, il- beta, tnf-alpha and il- in an in-vitro model of brain inflammation extracellular redox modulation by regulatory t cells regulatory t cells interfere with glutathione metabolism in dendritic cells and t cells mouse cd b+gr- + myeloid cells can promote th cell differentiation and experimental autoimmune encephalomyelitis distinct cysteine residues in keap are required for keap -dependent ubiquitination of nrf and for stabilization of nrf by chemopreventive agents and oxidative stress the role and potential therapeutic application of myeloid-derived suppressor cells in allo-and autoimmunity peripheral cd + t-cell differentiation regulated by networks of cytokines and transcription factors key: cord- - d o xk authors: choi, won hyung; lee, in ah title: the mechanism of action of ursolic acid as a potential anti-toxoplasmosis agent, and its immunomodulatory effects date: - - journal: pathogens doi: . /pathogens sha: doc_id: cord_uid: d o xk this study was performed to investigate the mechanism of action of ursolic acid in terms of anti-toxoplasma gondii effects, including immunomodulatory effects. we evaluated the anti-t. gondii effects of ursolic acid, and analyzed the production of nitric oxide (no), reactive oxygen species (ros), and cytokines through co-cultured immune cells, as well as the expression of intracellular organelles of t. gondii. the subcellular organelles and granules of t. gondii, particularly rhoptry protein , microneme protein , and inner membrane complex sub-compartment protein , were markedly decreased when t. gondii was treated with ursolic acid, and their expressions were effectively inhibited. furthermore, ursolic acid effectively increased the production of no, ros, interleukin (il)- , il- , granulocyte macrophage colony stimulating factor (gm-csf), and interferon-β, while reducing the expression of il- β, il- , tumor necrosis factor alpha (tnf-α), and transforming growth factor beta (tgf-β ) in t. gondii-infected immune cells. these results demonstrate that ursolic acid not only causes anti-t. gondii activity/action by effectively inhibiting the survival of t. gondii and the subcellular organelles of t. gondii, but also induces specific immunomodulatory effects in t. gondii-infected immune cells. therefore, this study indicates that ursolic acid can be effectively utilized as a potential candidate agent for developing novel anti-toxoplasmosis drugs, and has immunomodulatory activity. recently, zoonotic diseases have been consistently occurring in different countries worldwide, causing serious threats in many countries and to humans globally. in this respect, the prevention and education of various zoonotic diseases are becoming a major issue. toxoplasmosis is a parasitic disease that is induced through the infection of toxoplasma gondii in many countries worldwide as a zoonotic parasitosis, causing serious diseases and chronic infection in various sites of the human body in all age groups, including adults and young children as well as pregnant women. in particular, because toxoplasmosis is a zoonotic disease that infects humans through cats, many people that have cats as pets, particularly pregnant women, need to take caution in this regard. in the biological aspect, t. gondii not only has intracellular organelles such as the golgi, endoplasmic reticulum, and mitochondrion, but also unique subcellular organelles such as the conoid, apicoplast, surface antigens (sags), dense granule proteins (gras), rhoptries, and micronemes [ ] . t. gondii has an inner membrane complex (imc) involving the plasma membrane, consisting of a unique double membrane structure which is combined with a cytoskeletal network. the imc acts as a major factor in the proliferation and growth for the survival of t. gondii, as well as in motility and cell division, which has been reported to include the t. gondii-infected cells treated with sf ( figure ). furthermore, it was clearly shown that there were significant differences between the untreated t. gondii-group and the experimental groups. in these aspects, it was demonstrated that ua effectively induced the selective anti-parasitic effect/action compared to the sf-treated group and the untreated group through a direct inhibitory effect on the viability of t. gondii. this shows that ua has the potential to be utilized as an anti-t. gondii candidate agent and/or a synergic compound with the existing drugs for developing novel anti-toxoplasmosis agents. therefore, these results indicate that ua strongly inhibits or blocks the survival of t. gondii by effectively inducing anti-t. gondii activity through the selective inhibitory activity and/or action against the viability of t. gondii. the t. gondii-infected cells treated with sf ( figure ). furthermore, it was clearly shown that there were significant differences between the untreated t. gondii-group and the experimental groups. in these aspects, it was demonstrated that ua effectively induced the selective anti-parasitic effect/action compared to the sf-treated group and the untreated group through a direct inhibitory effect on the viability of t. gondii. this shows that ua has the potential to be utilized as an anti-t. gondii candidate agent and/or a synergic compound with the existing drugs for developing novel anti-toxoplasmosis agents. therefore, these results indicate that ua strongly inhibits or blocks the survival of t. gondii by effectively inducing anti-t. gondii activity through the selective inhibitory activity and/or action against the viability of t. gondii. the normal lung cells were infected with t. gondii (cells: t. gondii = : ), and t. gondii-infected cells were treated with different concentrations ( . - µg/ml) of ursolic acid (ua) and sulfadiazine (sf) at • c for h, respectively; their survival rates were inhibited a dose-dependent manner. the results are presented as a percentage of the untreated normal group. * p < . was considered to be significant. t. gondii causes unique anti-apoptotic features and parasitic survival cycles or stages, such as the inactivation of apoptotic proteins during the parasitic life-cycle in host cells after infection, showing critical roles during the proliferation and survival of t. gondii. in particular, the pvm, that functions as a critical indicator during the proliferation stage of t. gondii, is activated in a time-dependent manner after cell invasion of t. gondii, which causes finally the proliferation and the growth of t. gondii. in this aspect, we investigated the inhibitory action of ua against subcellular organelles such as rhoptries, micronemes, and inner membrane complex that form its intracellular metabolic network as well as the homeostasis of t. gondii. as shown in figure , when t. gondii was treated with different concentrations ( and µg/ml) of ua and sf, respectively, the intracellular organelles (rhoptry protein (rop ), microneme protein (mic ), and inner membrane complex sub-compartment protein (imc sub- )) of t. gondii were markedly decreased in a dose-dependent manner, and their changes were clearly observed under electrophoresis. in particular, it is known that rhoptry plays as a critical factor when t. gondii enters into host cells during invasion stage [ ] [ ] [ ] [ ] [ ] . in these aspects, this result shows clearly that ua not only has the direct inhibitory effect against the survival of t. gondii but also induces the anti-t. gondii effect by strongly blocking or inhibiting the homeostasis and network structure of the intracellular organelles of t. gondii. therefore, these results demonstrate substantial evidence that ua consistently causes anti-t. gondii activity/action by effectively inhibiting the intracellular organelles of t. gondii. °c for h, respectively; their survival rates were inhibited a dose-dependent manner. the results are presented as a percentage of the untreated normal group. * p < . was considered to be significant. organelles of t. gondii t. gondii causes unique anti-apoptotic features and parasitic survival cycles or stages, such as the inactivation of apoptotic proteins during the parasitic life-cycle in host cells after infection, showing critical roles during the proliferation and survival of t. gondii. in particular, the pvm, that functions as a critical indicator during the proliferation stage of t. gondii, is activated in a time-dependent manner after cell invasion of t. gondii, which causes finally the proliferation and the growth of t. gondii. in this aspect, we investigated the inhibitory action of ua against subcellular organelles such as rhoptries, micronemes, and inner membrane complex that form its intracellular metabolic network as well as the homeostasis of t. gondii. as shown in figure , when t. gondii was treated with different concentrations ( and μg/ml) of ua and sf, respectively, the intracellular organelles (rhoptry protein (rop ), microneme protein (mic ), and inner membrane complex sub-compartment protein (imc sub- )) of t. gondii were markedly decreased in a dose-dependent manner, and their changes were clearly observed under electrophoresis. in particular, it is known that rhoptry plays as a critical factor when t. gondii enters into host cells during invasion stage [ ] [ ] [ ] [ ] [ ] . in these aspects, this result shows clearly that ua not only has the direct inhibitory effect against the survival of t. gondii but also induces the anti-t. gondii effect by strongly blocking or inhibiting the homeostasis and network structure of the intracellular organelles of t. gondii. therefore, these results demonstrate substantial evidence that ua consistently causes anti-t. gondii activity/action by effectively inhibiting the intracellular organelles of t. gondii. we evaluated the effect of ros and no production induced by ursolic acid in immune cells infected with t. gondii. the production of no and ros was measured in a dose-dependent manner when t. gondii-infected co-cultured immune cells were incubated with various concentrations ( - µg/ml) of ua and sf for h, respectively. in particular, the production of ros was increased in the t. gondii-infected group treated with ua compared with t. gondii-infected group. however, in µg/ml of ua, ros activity gradually indicated lower concentrations than other ua-treated groups (figure ), and these changes of ros activity show that ua acts as a modulator which consistently inhibits the proliferation and the growth of t. gondii by promoting the production of ros in immune cells after the parasite infection. even though sf-treated groups significantly increased ros production compared to the t. gondii-infected group, there were low ros-productive rates compared to ua-treated groups. furthermore, the rate of no production was strongly increased in t. gondii-infected group treated with ua compared with normal and t. gondii-infected groups, but in the range of µg/ml of ua, the rate of no production were low concentrations compared with other ua-treated groups ( figure ). however, the activities of both ros and no induced by ua were significantly increased more than normal and infection groups. interestingly, the activities of ros and no in t. gondii-infected cells treated with sf were lower than t. gondii-infected groups treated with ua. the activities of ros and no were significantly increased in t. gondii-infected groups treated with ua and sf, respectively, whereas their activities were gradually reduced in high concentrations. these results show that ua not only consistently induces the production of ros and no through the interaction between it and immune cells in t. gondii-infected immune cells, but may also be used as a synergic compound for the immunomodulation after parasitic infection. we evaluated the effect of ros and no production induced by ursolic acid in immune cells infected with t. gondii. the production of no and ros was measured in a dose-dependent manner when t. gondii-infected co-cultured immune cells were incubated with various concentrations ( - μg/ml) of ua and sf for h, respectively. in particular, the production of ros was increased in the t. gondii-infected group treated with ua compared with t. gondii-infected group. however, in μg/ml of ua, ros activity gradually indicated lower concentrations than other ua-treated groups (figure ), and these changes of ros activity show that ua acts as a modulator which consistently inhibits the proliferation and the growth of t. gondii by promoting the production of ros in immune cells after the parasite infection. even though sf-treated groups significantly increased ros production compared to the t. gondii-infected group, there were low ros-productive rates compared to ua-treated groups. furthermore, the rate of no production was strongly increased in t. gondii-infected group treated with ua compared with normal and t. gondii-infected groups, but in the range of μg/ml of ua, the rate of no production were low concentrations compared with other ua-treated groups ( figure ). however, the activities of both ros and no induced by ua were significantly increased more than normal and infection groups. interestingly, the activities of ros and no in t. gondii-infected cells treated with sf were lower than t. gondii-infected groups treated with ua. the activities of ros and no were significantly increased in t. gondii-infected groups treated with ua and sf, respectively, whereas their activities were gradually reduced in high concentrations. these results show that ua not only consistently induces the production of ros and no through the interaction between it and immune cells in t. gondii-infected immune cells, but may also be used as a synergic compound for the immunomodulation after parasitic infection. t. gondii proliferates rapidly in host cells through parasitic interactions and signals for its survival after cell invasion, which finally induces destruction of host cells. we evaluated the synergic effect and the activation of cytokines caused by ursolic acid in co-cultured immune cells (macrophages, t cells, b cells and basophil cells) infected with t. gondii. the production of the cytokines (interferon-β, granulocyte macrophage colony stimulating factor (gm-csf), transforming growth factor beta (tgf-β ), and tumor necrosis factor alpha (tnf-α)) in t. gondii-infected immune cells was notably changed in a concentration-dependent manner when the cells were incubated with different concentrations ( - μg/ml) of ua, and their changes were measured under an elisa reader. in particular, the production of interferon-β and gm-csf was effectively increased in t. gondii-infected cells treated with ua compared with t. gondii-infected cells, and these changes show that ua accelerated the production and release of interferon-β and gm-csf by stimulating the macrophage, b cells, and basophil cells ( figure ). moreover, the production of interferon-β was not changed in sf-treated groups, and the production of gm-csf was gradually decreased. furthermore, the expression of tgf-β and tnf-α was markedly reduced in t. gondii-infected cells treated with ua, whereas t. gondii-infected cells treated with sf did not cause the production of tgf-β and tnfα compared with other groups (figure ) . interestingly, t. gondii-infected cells treated with sf did not significantly produce the expression of interferon-β, tgf-β and tnf-α compared to the infected group. this shows that sf is not involved in immune response for expressing tnf-α, tgf-β , and interferon-β in immune cells. furthermore, the production of tgf-β was decreased in t. gondiiinfected cells treated with ua, but the result shows that ua reduced the expression of tgf-β in immune cells by directly inhibiting t. gondii. these results demonstrate that ua not only promotes defense against parasitic infection by effectively inducing the production of interferon-β and gm-csf from immune cells when the cells are infected with t. gondii, but also inhibits the overexpression of inflammatory cytokine by reducing the expression of tnf-α caused by infection. t. gondii proliferates rapidly in host cells through parasitic interactions and signals for its survival after cell invasion, which finally induces destruction of host cells. we evaluated the synergic effect and the activation of cytokines caused by ursolic acid in co-cultured immune cells (macrophages, t cells, b cells and basophil cells) infected with t. gondii. the production of the cytokines (interferon-β, granulocyte macrophage colony stimulating factor (gm-csf), transforming growth factor beta (tgf-β ), and tumor necrosis factor alpha (tnf-α)) in t. gondii-infected immune cells was notably changed in a concentration-dependent manner when the cells were incubated with different concentrations ( - µg/ml) of ua, and their changes were measured under an elisa reader. in particular, the production of interferon-β and gm-csf was effectively increased in t. gondii-infected cells treated with ua compared with t. gondii-infected cells, and these changes show that ua accelerated the production and release of interferon-β and gm-csf by stimulating the macrophage, b cells, and basophil cells ( figure ). moreover, the production of interferon-β was not changed in sf-treated groups, and the production of gm-csf was gradually decreased. furthermore, the expression of tgf-β and tnf-α was markedly reduced in t. gondii-infected cells treated with ua, whereas t. gondii-infected cells treated with sf did not cause the production of tgf-β and tnf-α compared with other groups (figure ) . interestingly, t. gondii-infected cells treated with sf did not significantly produce the expression of interferon-β, tgf-β and tnf-α compared to the infected group. this shows that sf is not involved in immune response for expressing tnf-α, tgf-β , and interferon-β in immune cells. furthermore, the production of tgf-β was decreased in t. gondii-infected cells treated with ua, but the result shows that ua reduced the expression of tgf-β in immune cells by directly inhibiting t. gondii. these results demonstrate that ua not only promotes defense against parasitic infection by effectively inducing the production of interferon-β and gm-csf from immune cells when the cells are infected with t. gondii, but also inhibits the overexpression of inflammatory cytokine by reducing the expression of tnf-α caused by infection. we evaluated the activation and interaction of various cytokines induced by ursolic acid through co-cultured immune cells (macrophage, t cells, b cells and basophil cells) infected with t. gondii. the changes of the cytokines (interleukin (il)- β, il- , il- , and il- ) in t. gondii-infected immune cells were measured in a dose-dependent manner when the cells were incubated with different concentrations ( - μg/ml) of ua, and their changes were evaluated under an elisa reader. in particular, the production of il- β and il- was reduced in the groups treated with ua compared with the infection group, and the expression of il- and il- was significantly increased in t. the changes of the cytokines (interleukin (il)- β, il- , il- , and il- ) in t. gondii-infected immune cells were measured in a dose-dependent manner when the cells were incubated with different concentrations ( - µg/ml) of ua, and their changes were evaluated under an elisa reader. in particular, the production of il- β and il- was reduced in the groups treated with ua compared with the infection group, and the expression of il- and il- was significantly increased in t. gondii-infected cells treated with ua (figures and ). these changes demonstrate that ua promotes the production and the expression of il- and il- by consistently stimulating the immune cells as well as inhibit the expressions of il- β and il- induced by t. gondii infection. interestingly, the production of il- β in t. gondii-infected cells treated with sf was significantly increased and the expression of il- was gradually decreased compared with the infection group. however, there were no significant changes of the production of il- and il- in t. gondii-infected cells treated with sf. in particular, significant differences in the cytokines were clearly measured between the t. gondii-infected group and ua-treated group, and these changes of cytokines were confirmed in all the groups. furthermore, the results show that this co-culture system through immune cells can be usefully utilized as a co-culture method for measuring various cytokines, and as an effective alternative to in vivo experiments. these results indicate that ua not only effectively induces the production of cytokines between the immune cells by increasing the expression of il- and il- in macrophage and b cells after t. gondii infection, but also suppresses or strongly reduces the expression of the inflammatory cytokines from the immune cells. gondii-infected cells treated with ua (figure and ). these changes demonstrate that ua promotes the production and the expression of il- and il- by consistently stimulating the immune cells as well as inhibit the expressions of il- β and il- induced by t. gondii infection. interestingly, the production of il- β in t. gondii-infected cells treated with sf was significantly increased and the expression of il- was gradually decreased compared with the infection group. however, there were no significant changes of the production of il- and il- in t. gondii-infected cells treated with sf. in particular, significant differences in the cytokines were clearly measured between the t. gondiiinfected group and ua-treated group, and these changes of cytokines were confirmed in all the groups. furthermore, the results show that this co-culture system through immune cells can be usefully utilized as a co-culture method for measuring various cytokines, and as an effective alternative to in vivo experiments. these results indicate that ua not only effectively induces the production of cytokines between the immune cells by increasing the expression of il- and il- in macrophage and b cells after t. gondii infection, but also suppresses or strongly reduces the expression of the inflammatory cytokines from the immune cells. in spite of constant public health efforts worldwide for the past decades, infectious diseases such as malaria, influenza, cholera, yellow fever, mers, sars, and tuberculosis have been consistently causing crises in public health as well as global concerns due to their pandemic potential. these serious infectious pathogens have enhanced adaptability to environmental variation and infectivity to host as well as resistance to existing drugs for their viability, evolving in a time-dependent manner. in these aspects, global nonprofit organizations and/or profit companies such as who and the bill & melinda gates foundation as well as various global leading pharmaceutical companies have continuously supported the study for blocking or treating chronic infectious diseases such as zika, aids, and tuberculosis as well as acute infectious diseases including influenza, malaria, cholera, yellow fever, and ebola, focusing on the development of novel drugs and effective vaccine against pathogens. furthermore, in spite of various efforts for developing anti-parasitic drugs against zoonotic parasitosis, effective novel drugs such as anti-malaria drugs have not yet been developed or launched. many people are living with animals such as cats and dogs. in particular, toxoplasmosis, a zoonotic parasitosis, is transmitted via physical contact with pets and companion animals, including cat and dogs. with respect to these aspects, we carefully focused on the parasitic infectious diseases that are consistently caused through various infection pathways and factors. among various parasites, t. gondii induces serious symptoms such as cerebral calcification and meningoencephalitis in the brain, while causing fetal infection through mother during pregnancy, as it is a zoonotic parasite which affects both humans and animals, resulting in parasitic diseases such as toxoplasmosis [ , ] . in spite of constant public health efforts worldwide for the past decades, infectious diseases such as malaria, influenza, cholera, yellow fever, mers, sars, and tuberculosis have been consistently causing crises in public health as well as global concerns due to their pandemic potential. these serious infectious pathogens have enhanced adaptability to environmental variation and infectivity to host as well as resistance to existing drugs for their viability, evolving in a time-dependent manner. in these aspects, global nonprofit organizations and/or profit companies such as who and the bill & melinda gates foundation as well as various global leading pharmaceutical companies have continuously supported the study for blocking or treating chronic infectious diseases such as zika, aids, and tuberculosis as well as acute infectious diseases including influenza, malaria, cholera, yellow fever, and ebola, focusing on the development of novel drugs and effective vaccine against pathogens. furthermore, in spite of various efforts for developing anti-parasitic drugs against zoonotic parasitosis, effective novel drugs such as anti-malaria drugs have not yet been developed or launched. many people are living with animals such as cats and dogs. in particular, toxoplasmosis, a zoonotic parasitosis, is transmitted via physical contact with pets and companion animals, including cat and dogs. with respect to these aspects, we carefully focused on the parasitic infectious diseases that are consistently caused through various infection pathways and factors. among various parasites, t. gondii induces serious symptoms such as cerebral calcification and meningoencephalitis in the brain, while causing fetal infection through mother during pregnancy, as it is a zoonotic parasite which affects both humans and animals, resulting in parasitic diseases such as toxoplasmosis [ , ] . furthermore, it induces serious complications in immune-deficient patients and hiv patients, which attenuates the interaction of the immune system that specifically responds to pathogens in the host [ , ] . t. gondii inhibits the interaction of cytokines such as il- and ifn-γ that are activated through the immune system, attenuating the production of cytokines from host cells. in addition, it is known that t. gondii blocks parasitic inhibitory system while inhibiting the signal pathways of cell cycle initiators or apoptotic mediators for the apoptotic stage in host cells after invasion [ , ] . t. gondii infection induces an imbalance of immune response by causing changes in cytokines such as tnf-α and tgf-β in the host, resulting in immune deficiency by breaking homeostasis and interaction of the immune system in the host. moreover, t. gondii induces the proliferative and growth cycle of t. gondii by rapidly forming pvm and by regulating cell cycle factors for its survival in host cells, maintaining the survival by accelerating the proliferation of t. gondii in host [ , ] . the subcellular organelles and granules of t. gondii, such as the conoid, gras, sags, the rhoptry, microneme proteins, and the inner membrane complex, were focused upon as major targets for blocking t. gondii infection, which was used as major recombinant proteins or factors for protective immunity including vaccination [ ] [ ] [ ] [ ] . for these reasons, in this study we not only evaluated the anti-parasitic effect of ua which inhibits the survival of parasite after t. gondii infection through a t. gondii-infected co-cultured immune system, but also confirmed its mechanism of action with respect to anti-t. gondii effects and immunomodulatory activity. in the present study, the survival rates of t. gondii were markedly inhibited when the parasites were treated with ua and sf compared with the non-treated t. gondii group, and the expression of subcellular organelles and granules of t. gondii in the infected cells was effectively decreased in t. gondii-infected group treated with ua compared with other groups. furthermore, the production of ros and no was significantly increased in the t. gondii-infected group treated with ua compared with both sf-treated and infection groups. interestingly, although ros production was activated in both t. gondii-infected cells treated with ua and sf, its rate was gradually decreased in the range of high concentrations of the compounds. in these aspects, no and ros production not only activates immune defense systems against various pathogens such as bacteria and viruses but also maintains effectively the changes of the homeostasis in the organism, promoting the production of cytokines induced by the interaction of immune cells. cytokines such as il- β, il- , and tnf-α were increased in the infected cells after t. gondii infection, and their production was significantly suppressed after ua treatment. in particular, although the production of tgf-β was reduced in t. gondii-infected immune cells treated with ua, it was shown that ua suppressed the expression of tgf-β in immune cells by inhibiting the survival of t. gondii. in addition, ua not only effectively increased the production of anti-inflammatory cytokines such as il- and interferon-β in t. gondii-infected cells but also activated the expression of il- and gm-csf, promoting the development of th cells and the production of interferon-γ that is induced by nk cells. taken together, the results of this study showed the anti-parasitic effects of ua against the survival of t. gondii as well as demonstrated its mechanism of action causing anti-t. gondii effect in t. gondii-infected co-cultured cells. furthermore, these results indicated that the expression of subcellular organelles and granules of t. gondii was specifically inhibited or blocked in a concentration-dependent manner after treatment with ua. this study provides evidence that ua not only decreases the inflammatory cytokines (il- β, il- and tnf-α) expressed from the immune cells after t. gondii infection, but also effectively increases the production of anti-inflammatory mediators such as il- , il- , gm-csf, and interferon-β, including the activation of ros and no production. interestingly, it was confirmed in this study that macrophage and other immune cells did not directly inhibit or rapidly decrease the survival of parasite as well as the proliferation of t. gondii through the bactericidal action such as phagocytosis of macrophage after t. gondii infection. this may be the result of the differences between the in vitro system and the in vivo experiment including rats and mice. however, the parasite may not be eliminated by the bactericidal action of macrophages and other immune cells in the infected body because t. gondii is found in various body sites of patients in the clinic. moreover, it may be changed or maintained to the parasite type that indicates characteristics of hibernation like t. gondii me strain (type ii) in the body. in summary, the results of this study demonstrate that ua induces anti-t. gondii effects/action by effectively blocking or inhibiting the survival of t. gondii, and also has anti-parasitic activity that consistently inhibits the proliferation/growth of t. gondii by strongly inhibiting the expression of subcellular organelles and granules of t. gondii, such as rop , mic , and imc sub in t. gondii-infected cells. in addition, these results show clearly that ua has the immunomodulatory action/activity which causes the change of cytokines in immune cells by decreasing the inflammatory cytokines (il- β, il- , and tnf-α) and/or by activating anti-inflammatory cytokines including il- , il- , gm-csf, or interferon-β through the interaction between ua and immune cells. furthermore, the results indicate that ua induces the death of t. gondii by promoting the production of ros and no, such as super oxides and hydroxyl radicals in t. gondii-infected immune cells. therefore, this study indicates that ua can be used effectively as a potent candidate agent or a synergic compound with existing drugs in the development of novel anti-t. gondii drugs of the next generation against toxoplasmosis, with potential as a modulatory substance against the immune response of the parasite in parasite-infected immune cells. moreover, ua requires further study for efficacy and safety against toxoplasmosis in preclinical stages through in vivo animal models in the near future. the anti-toxoplasmosis drug, sulfadiazine (sf), was dissolved in dmso, and ursolic acid (ua) was also dissolved in dmso to a concentration of mg/ml according to the manufacturer's instructions. sulfadiazine was used as a standard drug to evaluate whether or not ursolic acid has an anti-parasitic effect and activity against t. gondii. the compounds were filtered using . -µm membrane syringe filters (roshi kaisha, ltd., tokyo, japan) before use, and were stored at − • c deep-freezer until use. macrophages (raw . ), t cells (el ), b cells (fb ), basophil cells (rbl- h ), and normal lung epithelial cells (l ) were purchased from korean cell line bank at seoul national university and american type culture collection (manassas, va, usa). the cells were cultured in rpmi medium and dmem containing mm l-glutamine, supplemented with % decomplemented fetal bovine serum (fbs), penicillin ( units/ml), and streptomycin ( µg/ml) in a humidified atmosphere containing % co in air at • c. the t. gondii rh was suspended with × pbs, which was injected in the abdominal cavity of each balb-c/mouse. five days after the injection, t. gondii was collected from the peritoneal fluids of mouse kept in the abdominal cavities of the mice before use. in the in vitro study, host cells were infected with tachyzoite of t. gondii at a cell-to-parasite ratio of : . the inhibitory effect of ursolic acid against t. gondii was confirmed by directly evaluating the viability of t. gondii exposed to ua and sf through the mtt assay. briefly, after t. gondii was seeded in a -well plate ( × /well), t. gondii was incubated with different concentrations ( . - µg/ml) of ua and sf for h, respectively. the normal lung cells were infected with t. gondii (cells: t. gondii = : ), and t. gondii-infected cells were treated with different concentrations ( . - µg/ml) of ursolic acid (ua) and sulfadiazine (sf) at • c for h, respectively; then their viabilities were evaluated by mtt assay. it was to be determined whether or not ua has anti-parasitic activity and/or effect against t. gondii. the cells were divided into normal and experimental groups, and the survival rate (sr) of t. gondii was calculated as follows: % of sr = (od drug-tested wells − od blank )/(od control − od blank ) × . the optical density (od) was measured at a wavelength of nm using an elisa leader. the inhibitory effect of ursolic acid against the viability of t. gondii was evaluated by measuring the expression of subcellular organelles of t. gondii in t. gondii-infected cells exposed to ua and sf. after normal lung cells were seeded in a -well plate ( × /well), the cells were infected with t. gondii ( : , cells/parasite ratio), which was incubated with different concentrations ( - µg/ml) of ua and sf for h respectively, and their total rna was isolated from t. gondii strain using rneasy mini kit (qiagen, hilden, germany). total rna was obtained from untreated control and treated groups following the manufacturer's recommended procedure, and the total rna was quantified in an eppendorf biospectrometer (eppendorf, seoul, korea). one pair of primers was designed using the primer (version . ) and ncbi primer-blast according to the sequence of the selected specific gene in ncbi genbank as follows: t. gondii mic (accession number: af , bp), rop (accession number: am , bp), imc sub- (accession number: hq , bp), and β-actin (accession number: nm , bp). the specific genes of t. gondii were synthesized to complementary dna (cdna) through one-step rt-pcr kit (bioneer, daejeon, korea). the cdna was analyzed by electrophoresis in a . % (w/v) agarose gel containing µg/ml ethidium bromide at v for h, which was visualized under ultraviolet illumination. the β-actin was used as an internal standard for the amount of the expression of cdna present in each sample. the groups were divided into the untreated positive group (t. gondii-infected group) and experimental groups (ua and sf-treated groups). the effect of nitric oxide (no) and reactive oxygen species (ros) production of ursolic acid was confirmed by measuring no and ros production in t. gondii-infected co-cultured cells (macrophage and basophil cells) exposed to the compounds. the cells were divided into normal and experimental groups as described above. briefly, after the co-culture cells were seeded in a -well plate ( × /well), the cells were infected with t. gondii ( : , cells/parasite ratio), which was incubated with different concentrations ( - µg/ml) of ua and sf for h, respectively. the no and ros production were measured in the control and treated groups according to the manufacturer's instructions through no and ros production kits (cell biolabs, inc. san diego, ca, usa) respectively. the rates of no and ros production were measured using an elisa microplate leader (biotek instruments, inc., winooski, vt, usa). the effect of ursolic acid regarding immunomodulatory action was evaluated by measuring the expression of cytokines in t. gondii-infected co-culture immune cells (macrophage, t cells, b cells, and basophil cells) exposed to ua and sf, respectively. the cells were divided into normal and experimental groups. first, the macrophage and basophil cells were sequentially seeded in a -well plate ( × /well) in a humidified atmosphere containing % co in air at • c. second, t cells and b cells were additionally seeded in the plate ( × /well) after h. namely, the co-cultured immune cells were seeded in a -well plate ( × /well) and the cells were finally infected with t. gondii ( : , cells/parasite ratio) after h, and incubated with different concentrations ( - µg/ml) of ua and sf for h, respectively. the cytokines (tnf-α, tgf-β , interferon-β, gm-csf, il- β, il- , il- , and il- ) were respectively measured in normal, infected, and treated groups according to the manufacturer's instruction through cytokine elisa kits (thermofisher scientific, san diego, ca, usa) and inflammatory multi-cytokine kits (qiagen, hilden, germany), and the untreated t. gondii-infected cells were used as an positive group. all the results were expressed as mean ± standard deviation (sd) of three independent experiments. statistical analysis of the data was performed using student's t-test and analysis of variance (anova). a value of * p < . was considered to be statistically significant. the authors declare no conflict of interest. the following abbreviations are used in this manuscript: secretory traffic in the eukaryotic parasite toxoplasma gondii: less is more a novel family of toxoplasma imc proteins displays a hierarchical organization and functions in coordinating parasite division the inner membrane complex through development of toxoplasma gondii and plasmodium differential roles for inner membrane complex proteins across toxoplasma gondii and sarcocystis neurona development tgtkl is a unique plant-like nuclear kinase that plays an essential role in acute toxoplasmosis toxoplasma gondii chromosomal passenger complex is essential for the organization of a functional mitotic spindle: a prerequisite for productive endodyogeny signaling during the invasion of host cells by toxoplasma gondii intervacuolar transport and unique topology of gra , a novel dense granule protein in toxoplasma gondii evaluation of anti-toxoplasma gondii effect of ursolic acid as a in vitro anti-toxoplasma gondii activity of root extract/fractions of eurycoma longifolia jack imidazo[ , -b]pyridazines targeting toxoplasma gondii calcium-dependent protein kinase decrease the parasite burden in mice with acute toxoplasmosis licochalcone a: an effective and low-toxicity compound against toxoplasma gondii in vitro and in vivo evaluation of atm kinase inhibitor ku- as potential anti-toxoplasma gondii agent in vitro evaluation of b-carboline alkaloids as potential anti-toxoplasma agents identification of small-molecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen novel pharmacological activity of artesunate and artemisinin: their potential as anti-tubercular agents in vitro and in vivo activities of the nitroimidazole tba- against mycobacterium tuberculosis evaluation of anti-tubercular activity of linolenic acid and conjugated-linoleic acid as effective inhibitors against mycobacterium tuberculosis. asian pac in vitro antileishmanial activity of mexican medicinal plants avian coronavirus infectious bronchitis virus susceptibility to botanical oleoresins and essential oils in vitro and in vivo new hybrid trifluoromethylquinolines as antiplasmo dium agents agave sisalana extract induces cell death in aedes aegypti hemocytes increasing nitric oxide production ursolic acid improves diabetic nephropathy via suppression of oxidative stress and inflammation in streptozotocin-induced rats ursolic acid alleviates inflammation and against diabetes-induced nephropathy through tlr -mediated inflammatory pathway oral supplementation with ursolic acid ameliorates sepsis-induced acute kidney injury in a mouse model by inhibiting oxidative stress and inflammatory responses pharmacokinetics and pharmacodynamics of the triterpenoid ursolic acid in regulating the antioxidant, anti-inflammatory, and epigenetic gene responses in rat leukocytes ursolic acid induces apoptosis in colorectal cancer cells partially via upregulation of microrna- and inhibition of jak /stat phosphorylation ursolic acid promotes the sensitization of rhtrail-resistant triple-negative breast cancer ursolic acid elicits intrinsic apoptotic machinery by downregulating the phosphorylation of akt/bad signaling in human cisplatin-resistant oral cancer car cells ursolic acid activates the apoptosis of prostate cancer via rock/pten mediated mitochondrial translocation of cofilin- the pleiotropic antibacterial mechanisms of ursolic acid against methicillin-resistant staphylococcus aureus (mrsa) ursolic and oleanolic acids as antimicrobial and immunomodulatory compounds for tuberculosis treatment antibacterial and synergistic activity of pentacyclic triterpenoids isolated from alstonia scholaris therapeutic effect of ursolic acid in experimental visceral leishmaniasis anti-trichomonas vaginalis activity of ursolic acid derivative: a promising alternative azithromycin and spiramycin induce anti-inflammatory response in human trophoblastic (bewo) cells infected by toxoplasma gondii but are able to control infection anti-parasitic effect on toxoplasma gondii induced by bnsp- , a lys -phospholipase a homologue from bothrops pauloensis venom sterculic acid and its analogues are potent inhibitors of toxoplasma gondii modulation of host hif- α activity and the tryptophan pathway contributes to the anti-toxoplasma gondii potential of nanoparticles pravastatin and simvastatin inhibit the adhesion, replication and proliferation of toxoplasma gondii (rh strain) in hela cells extracts of tectona grandis and vernonia amygdalina have anti-toxoplasma and pro-inflammatory properties in vitro synthesis and evaluation of novel arctigenin derivatives as potential anti-toxoplasma gondii agents rhoptries are major players in toxoplasma gondii invasion and host cell interaction export of a toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion host-parasite interactions: an intimate epigenetic relationship efficient invasion by toxoplasma depends on the subversion of host protein networks toxoplasma gondii ron binds to heparan sulfate on the host cell surface toxoplasmosis in pregnancy: prevention, screening, and treatment fetomaternal and pediatric toxoplasmosis encephalitis is mediated by rop of toxoplasma gondii, a severe pathogen in aids patients toxoplasma gondii igg seroprevalence in patients with hiv/aids toxoplasma gondii-induced activation of egfr prevents autophagy protein-mediated killing of the parasite toxoplasma gondii infection confers resistance against bims-induced apoptosis by preventing the activation and mitochondrial targeting of pro-apoptotic bax lytic cycle of toxoplasma gondii: years later the toxoplasma centrocone houses cell cycle regulatory factors vaccination with toxoplasma gondii sag- protein isprotective against congenital toxoplasmosis in balb/c mice but not in cba/j mice unique secretory organelles andsource of promising vaccine proteins for immunoprevention of toxoplasmosis research progress on surface antigen (sag ) of toxoplasma gondii targeted delivery of toxoplasma gondii antigens to dendritic cells promote immunogenicity and protective efficiency against this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -uv xdp f authors: marí, montserrat; de gregorio, estefanía; de dios, cristina; roca-agujetas, vicente; cucarull, blanca; tutusaus, anna; morales, albert; colell, anna title: mitochondrial glutathione: recent insights and role in disease date: - - journal: antioxidants (basel) doi: . /antiox sha: doc_id: cord_uid: uv xdp f mitochondria are the main source of reactive oxygen species (ros), most of them deriving from the mitochondrial respiratory chain. among the numerous enzymatic and non-enzymatic antioxidant systems present in mitochondria, mitochondrial glutathione (mgsh) emerges as the main line of defense for maintaining the appropriate mitochondrial redox environment. mgsh’s ability to act directly or as a co-factor in reactions catalyzed by other mitochondrial enzymes makes its presence essential to avoid or to repair oxidative modifications that can lead to mitochondrial dysfunction and subsequently to cell death. since mitochondrial redox disorders play a central part in many diseases, harboring optimal levels of mgsh is vitally important. in this review, we will highlight the participation of mgsh as a contributor to disease progression in pathologies as diverse as alzheimer’s disease, alcoholic and non-alcoholic steatohepatitis, or diabetic nephropathy. furthermore, the involvement of mitochondrial ros in the signaling of new prescribed drugs and in other pathologies (or in other unmet medical needs, such as gender differences or coronavirus disease of (covid- ) treatment) is still being revealed; guaranteeing that research on mgsh will be an interesting topic for years to come. glutathione (γ-l-glutamyl-l-cysteinyl-glycine, gsh), the most abundant thiol found in virtually all cells, is a tripeptide synthesized in the cytosol by two adenosine triphosphate (atp)-consuming enzymatic reactions. the first reaction, the formation of γ-glutamylcysteine from glutamate and cysteine by the enzyme γ-glutamylcysteine synthetase (gcs), is rate-limiting due to the usually low availability of cysteine. of note, the inhibition of this reaction by gsh constitutes a regulatory step for maintaining a proper gsh concentration intracellularly [ , ] . the last step in gsh synthesis, regulated by gsh synthetase (gs), requires γ-glutamylcysteine and glycine as substrates ( figure a ) [ ] . the high concentration of gsh, reaching millimolar levels ( - mm) within cells and micromolar the fact that gsh can directly eliminate free radicals and reduce h o is a first line of defense against reactive oxygen species (ros). on the other hand, a second line of defense is formed by glutathione-dependent enzymes that detoxify by-products generated by ros and therefore help prevent ros propagation. thus, gsh participates as a co-factor in several reactions, including the elimination of peroxides by gsh peroxidases (gpx), the covalent addition to protein cysteines (protein-ssg) predominantly performed by glutaredoxin, and the detoxification of electrophiles by s-glutathionylation (gs-r) formation as catalyzed by the enzymes glutaredoxin and glutathione s-transferase (gst) [ , , ] . in addition, an increase in protein s-glutathionylation through the formation of sulfenic acid or nitrosated cysteine intermediates, or by changes in the gsh redox, is associated to conditions such as hypertension, ischemia-reperfusion, and tachycardia where oxidative and nitrosative stresses are present. the presence of sulfenic acid or nitrosated cysteine the thiol group of the aminoacid cysteine in the backbone of gsh is responsible for its antioxidant capacity. this redox-active thiol residue becomes oxidized when gsh reduces target molecule to form glutathione disulfide (gssg) ( figure b) . the gssg/gsh redox couple, being the most abundant in the cells, can interact with other antioxidant redox couples to properly balance the redox environment in the cells [ ] . the fact that gsh can directly eliminate free radicals and reduce h o is a first line of defense against reactive oxygen species (ros). on the other hand, a second line of defense is formed by glutathione-dependent enzymes that detoxify by-products generated by ros and therefore help prevent ros propagation. thus, gsh participates as a co-factor in several reactions, including the elimination of peroxides by gsh peroxidases (gpx), the covalent addition to protein cysteines (protein-ssg) predominantly performed by glutaredoxin, and the detoxification of electrophiles by s-glutathionylation (gs-r) formation as catalyzed by the enzymes glutaredoxin and glutathione s-transferase (gst) [ , , ] . in addition, an increase in protein s-glutathionylation through the formation of sulfenic acid or nitrosated cysteine intermediates, or by changes in the gsh redox, is associated to conditions such as hypertension, ischemia-reperfusion, and tachycardia where oxidative and nitrosative stresses are present. the presence of sulfenic acid or nitrosated cysteine intermediates promotes reversible s-glutathionylation of strategic proteins involved in cell signaling, ion transport, energy production, and cell death. in fact, recent studies indicate that the s-glutathionylation-deglutathionylation cycle cooperates with other post-translational mechanisms in regulating signal transduction, inflammation, metabolism, and apoptosis; therefore, it is emerging as an important post-translational modification [ ] . gssg can rapidly be recycled back to gsh by nicotinamide adenine dinucleotide phosphate (nadph)-dependent glutathione reductase (gr) in key organelles and the cytosol such that the glutathione pool is largely reduced with little gssg being present [ ] . thus, measuring the ratio gssg to gsh is an indicator of cellular oxidative stress. gsh and gssg are found outside cells, but normally at very low concentration- to times less than intracellular gsh. extracellular gsh is thought to function in detoxification along with providing protection against oxidants. gsh is synthesized exclusively in the cytosol; nonetheless it is found present at different intracellular organelles such as the endoplasmic reticulum (er), nucleus, and mitochondria. this compartmentation results in separate redox pools of gsh, where it performs specific functions [ ] . the independence of these separate gsh pools, for example, is supported by the observation that treatment with l-buthionine-sr-sulfoximine (bso), an inhibitor of gsh synthesis, does not result in a complete reduction in the nuclear gsh, as compared to cytosolic gsh [ ] . nuclear gsh is responsible for the maintenance in the reduced state of protein sulfhydryls crucial for dna expression and repair. in addition, in the active phases of cell proliferation, the nucleus accumulates gsh to much greater concentrations than those present in the cytoplasm [ ] . gsh mostly exists in its reduced form in cytosol, nucleus, and mitochondria, while in the endoplasmic reticulum (er) the ratio gsh/gssg is in the range from : to : [ ] , to properly favor the correct folding of proteins that have essential disulfide bonds. thus, in the lumen of the er, there is a substantially higher concentration of gssg, as compared with the rest of the cell which allows the formation of native protein disulfide bonds and also the isomerization of non-native disulfide bonds. both reactions, which can rarely be formed in the cytosol because of the high concentration of gsh, are mainly catalyzed by protein disulfide isomerase (pdi) [ , ] . within the cells, mitochondria are not only the primary site of oxygen consumption, but also the major source of reactive oxygen species (ros), most of them originating at the electron transport chain (etc). for a recent review on the subject, see [ ] . during mitochondrial respiration some "leakiness", or partial reduction reactions occur, mainly from complexes i and iii even under physiologic conditions ( figure ). this leakiness causes the release of superoxide and hydrogen peroxide mostly to the mitochondrial matrix [ , ] . in fact, it has been estimated that superoxide concentration is five-to ten-fold higher in the matrix than that in the cytosol [ ] . sources of reactive oxygen species (ros) in the electron transport chain. during mitochondrial respiration partial reduction reactions occur, mainly from complexes i and iii that cause the accumulation of superoxide and hydrogen peroxide mainly in the mitochondrial matrix. therefore, mitochondria need constant protection from the toxic action of ros as they constitute an important source. low molecular weight antioxidants, such as gsh, vitamin e, or ubiquinone, as well as enzyme defense systems, are responsible for providing this protection. concerning gsh, it was proposed that depletion of the mitochondrial gsh pool frequently correlated better to toxic cell death than overall loss of intracellular gsh. in addition, the mitochondrial gsh was more resilient to exhaustion, upon inhibition of gsh synthesis, than other intracellular gsh pools [ , ] . mitochondrial gsh (mgsh) regulates mitochondrial atp production by modifying critical protein sulfhydryl redox states that consequently influence nicotinamide adenine dinucleotide (nadh) and flavin adenine dinucleotide (fadh ) generation and electron flow in the electron transport chain (etc). principally, complex i contains central redox active thiols that can be reversibly glutathionylated to regulate electron flux in the event of enhanced oxidative stress [ , , ] . mgsh acts in concert with other antioxidant enzymes such as gpx and gpx , gsts, glutaredoxin- , and atp binding cassette transporters to maintain mitochondrial function [ , ] . the mitochondrial gsh pool is maintained in the reduced state by gr that couples gssg reduction to the matrix nadp+/nadph pool ( figure ) [ , ] . the physiological relevance of keeping mitochondrial oxidative stress and redox status is paramount, as evidenced by the fact that the knock-out mice of the mitochondrial enzymes gpx (also known as phospholipid hydroperoxide glutathione peroxidase) or trxr are embryonic lethal [ ] . gpx , using mgsh as a cofactor, is a lipid repair enzyme critical for the reduction of the lipid hydroperoxides formed by the fenton reaction. this reaction occurs when excess iron, in the ferrous form (fe + ), interacts with h o forming as a consequence the hydroxyl radical, a short-life but highly reactive specie that promotes oxidative dna damage, denaturation of proteins, and lipid peroxidation [ , ] (figure ). failure to control the excess iron and ros can lead to ferroptosis, a programmed form of cell death characterized by massive lipid peroxidation. in fact, the sole inhibition of gpx can trigger ferroptosis [ ] . therefore, mitochondria need constant protection from the toxic action of ros as they constitute an important source. low molecular weight antioxidants, such as gsh, vitamin e, or ubiquinone, as well as enzyme defense systems, are responsible for providing this protection. concerning gsh, it was proposed that depletion of the mitochondrial gsh pool frequently correlated better to toxic cell death than overall loss of intracellular gsh. in addition, the mitochondrial gsh was more resilient to exhaustion, upon inhibition of gsh synthesis, than other intracellular gsh pools [ , ] . mitochondrial gsh (mgsh) regulates mitochondrial atp production by modifying critical protein sulfhydryl redox states that consequently influence nicotinamide adenine dinucleotide (nadh) and flavin adenine dinucleotide (fadh ) generation and electron flow in the electron transport chain (etc). principally, complex i contains central redox active thiols that can be reversibly glutathionylated to regulate electron flux in the event of enhanced oxidative stress [ , , ] . mgsh acts in concert with other antioxidant enzymes such as gpx and gpx , gsts, glutaredoxin- , and atp binding cassette transporters to maintain mitochondrial function [ , ] . the mitochondrial gsh pool is maintained in the reduced state by gr that couples gssg reduction to the matrix nadp+/nadph pool ( figure ) [ , ] . the physiological relevance of keeping mitochondrial oxidative stress and redox status is paramount, as evidenced by the fact that the knock-out mice of the mitochondrial enzymes gpx (also known as phospholipid hydroperoxide glutathione peroxidase) or trxr are embryonic lethal [ ] . gpx , using mgsh as a cofactor, is a lipid repair enzyme critical for the reduction of the lipid hydroperoxides formed by the fenton reaction. this reaction occurs when excess iron, in the ferrous form (fe + ), interacts with h o forming as a consequence the hydroxyl radical, a short-life but highly reactive specie that promotes oxidative dna damage, denaturation of proteins, and lipid peroxidation [ , ] (figure ). failure to control the excess iron and ros can lead to ferroptosis, a programmed form of cell death characterized by massive lipid peroxidation. in fact, the sole inhibition of gpx can trigger ferroptosis [ ] . mgsh together with the thioredoxin system, in particular mitochondrial thioredoxin /thioredoxin reductase (trx /trxr ), maintain thiol redox status within mitochondria [ ] . in addition, peroxiredoxins (prx), a family of thiol-specific peroxidases that reduce lipid hydroperoxides and h o [ ] rely on thioredoxins (trxs) as their hydrogen donor. prx , exclusively located in mitochondria, depends on the trx /trxr system for its reduction ( figure ). of importance, depletion of mgsh causes interference with either the gsh system or the trx system due to oxidation of the dithiol on the active site of trx , thus sensitizing to ros-induced cell death. these data enhance the non-redundant functions in the protection against oxidative stress of the gsh and trx systems [ ] . notably, while protein s-glutathionylation is very common throughout the cell, within the mitochondria the proteins are greatly predisposed to reversible s-glutathionylation. in fact, mitochondria contain a large number of proteins, from those involved in energy metabolism, solute transport, ros production, to inducers of apoptosis, antioxidant defense, and those responsible for mitochondrial dynamics, that are targeted by s-glutathionylation. in addition, defects in the reactions responsible for the conjugation and elimination of gsh in mitochondrial proteins may have direct pathological consequences [ , ] . several aspects indicate that a carrier-mediated process accounts for the transport of gsh into mitochondria. among them, the absence of gsh synthesizing enzymes in mitochondria, the negative charge of gsh at physiological ph and the negative potential of the inner mitochondrial membrane are relevant, despite similar concentrations of gsh found in cytosolic and mitochondrial compartments. in fact, to date, the two reported transporters of mgsh capable of catalyzing the uptake of gsh into the mitochondrial matrix are anion carriers, members of the mitochondrial carrier family (slc ), the mitochondrial dicarboxylate carrier (dic; slc a ), and the -oxoglutarate carrier (ogc; slc a ). these mgsh carriers, which mediate an electroneutral exchange, have been mostly described in liver and kidney cells [ ] [ ] [ ] [ ] [ ] . of pathological interest, the ogc transport of mgsh in liver is dependent on membrane dynamics, since cholesterol mgsh together with the thioredoxin system, in particular mitochondrial thioredoxin /thioredoxin reductase (trx /trxr ), maintain thiol redox status within mitochondria [ ] . in addition, peroxiredoxins (prx), a family of thiol-specific peroxidases that reduce lipid hydroperoxides and h o [ ] rely on thioredoxins (trxs) as their hydrogen donor. prx , exclusively located in mitochondria, depends on the trx /trxr system for its reduction ( figure ). of importance, depletion of mgsh causes interference with either the gsh system or the trx system due to oxidation of the dithiol on the active site of trx , thus sensitizing to ros-induced cell death. these data enhance the non-redundant functions in the protection against oxidative stress of the gsh and trx systems [ ] . notably, while protein s-glutathionylation is very common throughout the cell, within the mitochondria the proteins are greatly predisposed to reversible s-glutathionylation. in fact, mitochondria contain a large number of proteins, from those involved in energy metabolism, solute transport, ros production, to inducers of apoptosis, antioxidant defense, and those responsible for mitochondrial dynamics, that are targeted by s-glutathionylation. in addition, defects in the reactions responsible for the conjugation and elimination of gsh in mitochondrial proteins may have direct pathological consequences [ , ] . several aspects indicate that a carrier-mediated process accounts for the transport of gsh into mitochondria. among them, the absence of gsh synthesizing enzymes in mitochondria, the negative charge of gsh at physiological ph and the negative potential of the inner mitochondrial membrane are relevant, despite similar concentrations of gsh found in cytosolic and mitochondrial compartments. in fact, to date, the two reported transporters of mgsh capable of catalyzing the uptake of gsh into the mitochondrial matrix are anion carriers, members of the mitochondrial carrier family (slc ), the mitochondrial dicarboxylate carrier (dic; slc a ), and the -oxoglutarate carrier (ogc; slc a ). these mgsh carriers, which mediate an electroneutral exchange, have been mostly described in liver and kidney cells [ ] [ ] [ ] [ ] [ ] . of pathological interest, the ogc transport of mgsh in liver is dependent on membrane dynamics, since cholesterol accumulation in the inner mitochondrial membrane (imm) results in decreased mgsh transport, and mitochondrial membrane fluidification restores mgsh uptake [ ] . therefore, in addition to regulating mgsh transport, cholesterol modulates susceptibility to oxidative stress and cell death. in this way, cholesterol is set as an important target in the pathophysiology of various diseases as diverse as steatohepatitis (sh) or alzheimer's disease. [ ] [ ] [ ] [ ] . additional studies have suggested that there are intraorgan differences in the transport of mgsh [ , ] and that dic and ogc are only partially responsible for gsh uptake in rat liver mitochondria [ ] . this implies that other putative mgsh carriers are still unknown ( figure ). antioxidants , , x for peer review of accumulation in the inner mitochondrial membrane (imm) results in decreased mgsh transport, and mitochondrial membrane fluidification restores mgsh uptake [ ] . therefore, in addition to regulating mgsh transport, cholesterol modulates susceptibility to oxidative stress and cell death. in this way, cholesterol is set as an important target in the pathophysiology of various diseases as diverse as steatohepatitis (sh) or alzheimer's disease. [ ] [ ] [ ] [ ] . additional studies have suggested that there are intraorgan differences in the transport of mgsh [ , ] and that dic and ogc are only partially responsible for gsh uptake in rat liver mitochondria [ ] . this implies that other putative mgsh carriers are still unknown (figure ). in contrast, a recent study by booty et al. [ ] using the lactococcus lactis system for overexpression and characterization of members of the mitochondrial carrier family [ ] showed no detectable transport of mgsh by dic and ogc carriers. thus, confirmatory studies in either scenario are needed to better define mgsh carriers in different cell types and organs. interestingly, an additional source of mgsh is the one obtained through s-d-lactoylglutathione (slg), a stable intermediate product of the glyoxalase system which catalyzes the conversion of methylglyoxal into d-lactic acid [ ] . slg can enter the mitochondria, and by the action of mitochondrial glyoxalase ii (glo ), be hydrolyzed to lactate and mgsh without the need for atp [ ] (figure ). however, it has not yet been determined the amount of mgsh obtained by slg and the importance of this pathway as compared to mgsh carriers. related to this, it has also been described in vitro that glo , using slg as a substrate, can induce the s-glutathionylation of metabolic enzymes of different cellular compartmentalization, in particular malate dehydrogenase, cytochrome b, and complex i from the mitochondria [ , , , ] , although the relevance of this observations rests to be determined in vivo. moreover, it has been recently proposed that s-glutathionylation of proteins in response to the oxidation of gsh is a means for the inhibition of catabolic pathways leading to a reduction in ros generation, and consequently as a mechanism for desensitization of h o signals [ ] . hence, protein s-glutathionylation could act as a post-translational modification to associate energy metabolism to redox signaling [ , , ] . in contrast, a recent study by booty et al. [ ] using the lactococcus lactis system for overexpression and characterization of members of the mitochondrial carrier family [ ] showed no detectable transport of mgsh by dic and ogc carriers. thus, confirmatory studies in either scenario are needed to better define mgsh carriers in different cell types and organs. interestingly, an additional source of mgsh is the one obtained through s-d-lactoylglutathione (slg), a stable intermediate product of the glyoxalase system which catalyzes the conversion of methylglyoxal into d-lactic acid [ ] . slg can enter the mitochondria, and by the action of mitochondrial glyoxalase ii (glo ), be hydrolyzed to lactate and mgsh without the need for atp [ ] (figure ). however, it has not yet been determined the amount of mgsh obtained by slg and the importance of this pathway as compared to mgsh carriers. related to this, it has also been described in vitro that glo , using slg as a substrate, can induce the s-glutathionylation of metabolic enzymes of different cellular compartmentalization, in particular malate dehydrogenase, cytochrome b, and complex i from the mitochondria [ , , , ] , although the relevance of this observations rests to be determined in vivo. moreover, it has been recently proposed that s-glutathionylation of proteins in response to the oxidation of gsh is a means for the inhibition of catabolic pathways leading to a reduction in ros generation, and consequently as a mechanism for desensitization of h o signals [ ] . hence, protein s-glutathionylation could act as a post-translational modification to associate energy metabolism to redox signaling [ , , ] . cell death is a regulated process and has been evolutionarily conserved in different species from embryogenesis to the maintenance of homeostasis in adult tissues. the different types of cell death are defined by morphological criteria and occur following different pathways. the two most studied and characteristic modes of mammalian cell death are apoptosis and necrosis. for a more extensive review on the molecular mechanisms of cell death, see [ ] . an integral part of apoptotic and necrotic cell death is mitochondrial ros production. consequently, antioxidants such as mgsh combat oxidative stress and increase cell viability in multiple experimental models [ ] . a rigorous balance between mitochondrial ros generation and inactivation, under physiological conditions, is necessary for the maintenance of cellular functions and viability. loss of this balance can lead to cell death [ ] (figure ). antioxidants , , x for peer review of cell death is a regulated process and has been evolutionarily conserved in different species from embryogenesis to the maintenance of homeostasis in adult tissues. the different types of cell death are defined by morphological criteria and occur following different pathways. the two most studied and characteristic modes of mammalian cell death are apoptosis and necrosis. for a more extensive review on the molecular mechanisms of cell death, see [ ] . an integral part of apoptotic and necrotic cell death is mitochondrial ros production. consequently, antioxidants such as mgsh combat oxidative stress and increase cell viability in multiple experimental models [ ] . a rigorous balance between mitochondrial ros generation and inactivation, under physiological conditions, is necessary for the maintenance of cellular functions and viability. loss of this balance can lead to cell death [ ] (figure ). the balance between mgsh levels and reactive oxygen species (ros) present in the mitochondrial milieu determines cellular susceptibility to death stimuli. under physiological conditions or even in the continuous presence of increased ros production survival is guaranteed due to upregulation of gsh-related antioxidant mechanisms. mitochondrial death only arises when the production of ros is high and/or the levels of mgsh are compromised due to minor synthetic capabilities or problems with mgsh transport. the availability of gsh is limiting for the activity of gsh-dependent antioxidant defense systems. in the context of mgsh, there is ample evidence of its importance for cell survival. in general, mitochondrial thiols have been shown to act as regulators of cell death pathways [ , ] , and, in particular, it has been reported that mgsh depletion is a trigger for cell death. consequently, promotion of cell death correlates more closely to the extent of depletion of mgsh rather than the changes in the gsh cytoplasmic pool in diseases or treatments that deplete cellular gsh [ , ] . selective mgsh depletion is able to sensitize to cell death by promoting oxidative stress and nitrosative stress [ ] . furthermore, mitochondrial gssg resulting from gsh oxidation must be efficiently reduced back to gsh by mitochondrial gr. this, in addition, requires the availability of mitochondrial nadph, which also provides reducing equivalents for trxr and is consequently vital for the functioning of the thioredoxin and peroxyredoxin systems. mitochondrial nadp + -dependent isocitrate dehydrogenase (idpm) and the proton-translocating nicotinamide nucleotide transhydrogenase located in the imm are the enzymes responsible for nadph regeneration [ ] . therefore, as expected, modulation of the activity of both enzymes is inversely related to cellular apoptosis susceptibility [ , ] . of interest, in addition to ros formation, iron overload followed by stimulation of mitochondrial lipid peroxidation may also induce a general suppression of mitochondrial figure . the balance between mgsh levels and reactive oxygen species (ros) present in the mitochondrial milieu determines cellular susceptibility to death stimuli. under physiological conditions or even in the continuous presence of increased ros production survival is guaranteed due to upregulation of gsh-related antioxidant mechanisms. mitochondrial death only arises when the production of ros is high and/or the levels of mgsh are compromised due to minor synthetic capabilities or problems with mgsh transport. the availability of gsh is limiting for the activity of gsh-dependent antioxidant defense systems. in the context of mgsh, there is ample evidence of its importance for cell survival. in general, mitochondrial thiols have been shown to act as regulators of cell death pathways [ , ] , and, in particular, it has been reported that mgsh depletion is a trigger for cell death. consequently, promotion of cell death correlates more closely to the extent of depletion of mgsh rather than the changes in the gsh cytoplasmic pool in diseases or treatments that deplete cellular gsh [ , ] . selective mgsh depletion is able to sensitize to cell death by promoting oxidative stress and nitrosative stress [ ] . furthermore, mitochondrial gssg resulting from gsh oxidation must be efficiently reduced back to gsh by mitochondrial gr. this, in addition, requires the availability of mitochondrial nadph, which also provides reducing equivalents for trxr and is consequently vital for the functioning of the thioredoxin and peroxyredoxin systems. mitochondrial nadp + -dependent isocitrate dehydrogenase (idpm) and the proton-translocating nicotinamide nucleotide transhydrogenase located in the imm are the enzymes responsible for nadph regeneration [ ] . therefore, as expected, modulation of the activity of both enzymes is inversely related to cellular apoptosis susceptibility [ , ] . of interest, in addition to ros formation, iron overload followed by stimulation of mitochondrial lipid peroxidation may also induce a general suppression of mitochondrial metabolism. important mitochondrial functions, such as respiration and oxidative phosphorylation, mitochondrial membrane potential (∆ψ), and mitochondrial ca + buffering capacity can be altered by lipid peroxides [ ] [ ] [ ] [ ] . in addition, mitochondrial lipid peroxidation derivatives can damage membranes by altering their barrier function by either directly interacting with membrane proteins and/or indirectly with lipid moieties [ ] . in recent years, mitochondria have been recognized as regulators of cell death by apoptosis and via necrosis. in aerobic cells, in addition to atp production, mitochondria play an essential role in the regulation of intracellular ca + homeostasis. importantly, a potentially harmful effect of ros production in mitochondria is facilitation of ca + -dependent mitochondrial permeability transition (mpt), a step that contributes to cell death [ ] . thus, oxidative stress significantly sensitizes mitochondria to mpt induction. in fact, it has been reported that mitochondrial-generated ros are directly involved in the induction of mpt [ ] . consequently, both oxidative stress and impaired ca + homeostasis promote mitochondrial-mediated cell damage. mpt leads to mitochondrial failure. if there is substantial atp depletion necrosis will occur, and apoptosis will take place if there is activation of caspases and mpt only ensues in a subpopulation of mitochondria, but the remaining organelles are still able to produce atp and preserve mitochondrial membrane potential. moreover, studies have shown that membrane-bound gst in the inner mitochondrial membrane could interact with mpt regulator proteins, such as adenine nucleotide translocator (ant) and/or cyclophilin d, and could contribute to oxidant-induced mpt pores [ ] . excessive alcohol exposure leads to alcoholic liver disease (ald), one of the most serious consequences of chronic alcohol abuse, and a predominant cause of liver-related morbidity and mortality worldwide. the increased production of ros observed after acute or chronic ethanol treatment reduces cellular antioxidant levels and enhances oxidative stress in many tissues, especially in hepatic tissue. ethanol-induced oxidative stress plays an important role in the mechanisms by which ethanol causes liver damage [ ] . the loss of oxidative phosphorylation and the defective atp generation observed in mitochondria after ethanol treatment indicate that mitochondria are specific targets of the toxic effects of ethanol. studies in animal models of chronic ethanol feeding have shown mitochondrial functional modifications, whereas patients with alcoholic steatohepatitis (ash) had mitochondria with morphological and functional abnormalities [ ] [ ] [ ] . mgsh becomes depleted by alcohol intake [ , ] . of interest, alcohol feeding has been shown to sensitize hepatocytes to tumor necrosis factor (tnf), an important mediator of ash. this sensitization to tnf is due to the limitation of mgsh, as a result of the ethanol-induced mitochondrial cholesterol increase that alters membrane-order parameter and partially inactivates the mgsh carrier [ , ] . in vitro, pharmacologic lessening of mgsh sensitizes hepatocytes to tumor necrosis factor (tnf)-mediated cell death, which parallels the findings observed after alcohol intoxication [ ] . selective decrease in mgsh, but not in cytosolic gsh, after alcohol intake has also been reported by other groups [ ] [ ] [ ] . alcohol feeding causes the accumulation of cholesterol in mitochondrial membranes, and subsequent mgsh depletion, by stimulating the expression of the mitochondrial cholesterol carrier steroidogenic acute regulatory protein (stard ) [ ] . gsh precursors are unproductive in refilling mgsh levels due to the primary defect in cytosolic gsh transport into mitochondria, despite a significant increase in cytosolic gsh. in contrast, s-adenosyl-l-methionine (sam) administered to rats fed alcohol chronically has been shown to be able to replenish mgsh levels due to its effect on the normalization of the physical properties of the imm [ ] . of note, subsequent studies have revealed that the depletion of mitochondrial sam precedes that of mgsh and occurs independently of alcohol-mediated disturbances in membrane dynamics. therefore, refuting that alcohol causes an inherent defect in msam transport, and suggesting that after alcohol feeding early reduction of msam contributes to changes in mitochondrial membrane dynamics and the consequent decrease in mgsh [ ] . interestingly, ethanol metabolism through cyp e (cytochrome p e ) is a fundamental step that contributes to hepatic oxidative stress. cyp e is induced after chronic ethanol ingestion and because is a poorly coupled enzyme formation of ros arises even without substrate. in fact, liver human liver hepatocellular carcinoma cell line (hepg ) cells overexpressing cyp e , where an increase in cellular ros is detected, display a significant rise in cellular gsh levels ( %) that is due to an increased rate of gsh synthesis and an enhanced expression of gcs heavy subunit (gcs-hs) mrna, the rate-limiting enzyme in gsh synthesis [ ] . moreover, these cells also display an enhanced expression of alpha and microsomal gst and of catalase [ ] . these cellular adjustments afford protection against prooxidants and reflect an adaptive response by the cells in front of cyp e -derived oxidative stress. thus, it is conceivable that despite the initial adaptation of hepatic cells to compensate with enhanced gsh synthesis and antioxidant capacity the surge of ros inherent to ethanol metabolism [ , ] , mgsh levels cannot be fully restored because of ethanol-induced rise of mitochondrial cholesterol and the consequent reduced mgsh transport to the mitochondria [ ] . non-alcoholic fatty liver disease (nafld) exists as a continuum of disease ranging from extreme buildup of fat within the hepatic parenchyma (simple steatosis), inflammation (non-alcoholic steatohepatitis, nash) to fibrosis, cirrhosis, end-stage liver disease, and there is also an increased risk of hepatocellular carcinoma (hcc). the main risk factors for nafld are obesity, along with type diabetes, and concurrently, nafld is also a risk factor for the occurrence of type diabetes. obesity synergizes with alcohol consumption in triggering the continuous progression of liver damage. current consensus promotes a change in nomenclature from nafld to 'metabolic associated fatty liver disease' (mafld), to reflect also the associated metabolic abnormalities present in the disease (insulin resistance/type diabetes and metabolic syndrome) [ ] . nafld, affecting a quarter of the population, is the most common cause of liver diseases. current studies suggest that hepatic cholesterol accumulation and changes in its regulation are important for the pathogenesis of nafld. original data suggests that hepatic free cholesterol (fc) is an important lipotoxic molecule critical in the development of nash, although the primary molecular mechanisms responsible for the buildup of fibrosis and inflammation that distinguish progressive nash remain unclear [ ] . moreover, there is reliable evidence for a fundamental role of mitochondrial dysfunction in nash pathophysiology, for review see [ ] . impaired mitochondrial function is involved at various levels in the pathogenesis of nash since it increases oxidative stress and cytokine production, causing cell death, fibrosis, and inflammation. as a result, diminished atp synthesis and increased ros production have been described in the livers of nash patients [ ] [ ] [ ] . these biochemical changes are accompanied by ultrastructural abnormalities with the presence of a lesser number of mitochondria that appear bloated and round, with the presence of paracrystalline inclusions and loss of cristae [ , ] . in fact, and similar to what happens in ald, increased mitochondrial fc reduces the fluidity of the mitochondrial membrane and compromises the function of the ogc carrier [ ] , thus depleting mgsh and favoring the generation of mitochondrial ros [ ] . tnf is found overexpressed in the liver and in the adipose tissue of nash patients. this overexpression is more elevated in patients with more advanced nash, corroborating that the tnf system is involved in the pathogenesis of nash [ ] . thus, the functional consequences of mgsh depletion in nafld are sensitization of hepatocytes to tnf, permeabilization of mitochondrial membrane, cytochrome c release, hepatocyte necrosis, and apoptosis, which promote and perpetuate hepatic inflammation and cause nash progression [ ] . while the importance of mgsh has been clearly assessed in vitro and in experimental models, there are almost no studies evaluating this particular factor in patients. a pilot study recently examined the therapeutic effects of oral administration of gsh in patients with nafld. in this group of patients, following treatment with gsh for months, alt levels significantly decreased, and triglycerides, non-esterified fatty acids, and ferritin levels also decreased demonstrating the potential therapeutic effects of oral administration of gsh in practical dose for patients with nafld [ ] . in parallel, an interesting and innovative study in patients with nafld aimed to elucidate the molecular mechanisms underlying the disease enlisted subjects with variable grades of hepatic steatosis (hs) and acquired experimental data on lipoprotein fluxes. these data were used as personalized restrictions of a metabolic model genome-scale in hepatocytes to examine hepatic metabolic differences, with regards to its relations with other tissues. their analysis predicted that subjects with elevated hs have an altered demand for gsh and nad + . in addition, their metabolomics data exhibited that hs negatively correlated with plasma levels of glycine, serine, and associated metabolites, therefore suggesting that these precursors of gsh metabolism could be limiting [ ] . in fact, an altered de novo gsh synthesis was proposed upon quantification of the hepatic expression levels of the associated enzymes. the addition of precursors for gsh and nad + biosynthesis to an experimental model in mice fed a western diet prevented hs, thus confirming their findings. additionally, in a proof-of-concept human study, they found enhanced liver function and reduced hs after supplementation with serine (a precursor of glycine) and therefore proposed a strategy for the treatment of nafld treatment [ ] . these two studies highlight the relevance of maintaining proper gsh levels in nafld. however, large-scale clinical trials are needed to verify oral gsh efficacy, or of its precursors. it would be also interesting to see how this impacts mgsh levels and mitochondrial functionality. mitochondrial dysfunction and oxidative damage are underlying many neurodegenerative disorders such as alzheimer's disease, amyotrophic lateral sclerosis, friedreich's ataxia, huntington's disease, multiple sclerosis, and parkinson's diseases, which point to mitochondrial oxidative stress as a causative factor of neurodegeneration [ ] . in alzheimer's disease (ad), both amyloid-beta (aβ) and hyperphosphorylated microtubuleassociated protein tau (mapt/tau), the two main pathological hallmarks of ad, accumulate in mitochondria resulting in functional impairment and ros generation [ , ] . furthermore, studies using cell lines and mouse models harboring genetic mutations linked to familial ad, have demonstrated that a compromised mitochondrial antioxidant defense, unable to handle mitochondrial-derived ros, promotes the amyloidogenic pathway and the activity of mapt/tau-related kinases, thus contributing to establish a vicious cycle of oxidative stress and damage [ , , ] . in particular, cholesterol-mediated mgsh depletion associated with higher susceptibility to aβ toxicity has been observed in isolated mitochondria from brains and cortical neurons of transgenic mice overexpressing active srebp- /sterol regulatory element binding transcription factor (srebf ) or niemann-pick type c (npc ) knock-out mice, both animal models displaying enhanced intracellular fc levels [ , ] . similarly, pharmacological reduction of the mitochondrial pool of gsh sensitized human neuronal and glial cell lines to aβ-mediated cell death. of therapeutic interest, neuroinflammation and neuronal damage were enhanced in transgenic srebp- mice intracerebroventricular (icv) infused with aβ and prevented upon mgsh recovery by gsh ethyl ester (gee) coinfusion, which can diffuse through mitochondrial membranes, with a similar protection observed by intraperitoneal administration of gee [ ] . accordingly, an ad mouse model that express mutant amyloid precursor protein (app) and presenilin (ps ) together with srebp- /srebf displayed enhanced aβ accumulation and tau pathology, correlating with early oxidative damage and neuroinflammation [ ] . all these pathological alterations were prevented after in vivo gee treatment [ ] . more recently, enhanced aβ-induced mitochondrial oxidative stress linked to cholesterol-mediated depletion of mgsh has also been shown to disrupt key mechanisms of cellular clearance resulting, contributing to aβ deposition [ , ] . thus, mitochondrial cholesterol accumulation emerges as a novel pathogenic factor in ad and the maintenance of mgsh levels as a potential target of therapeutic intervention. disruption of mitochondrial function is a key factor in parkinson's disease (pd) pathogenesis. the selective degeneration and loss of dopaminergic neurons in the substantia nigra of the ventral mid brain lead to dopamine depletion in the striatum causing oxidative stress and mitochondrial damage. these changes are restricted to the degenerating brain regions in pd and determine regional vulnerability [ ] . of note, mitochondrial impairment occurs early in pd pathogenesis, especially at the level of complex i, and animal models of pd are generated after administration of complex i inhibitors such as -methyl- -phenylpyridinium (mpp+) [ ] . relevantly, familial pd is mainly characterized by mutations in genes involved in mitochondrial dysfunction, such as parkin, α-synuclein, and leucine-rich repeat kinase (lrrk ) [ , [ ] [ ] [ ] . on the other hand, exposure to pesticides that disrupt the mitochondrial function increases the likelihood of developing pd [ ] . loss of glutathione in the substantia nigra ( - %), associated with a high proportion of oxidized glutathione, is a prominent hallmark of pd [ ] [ ] [ ] [ ] [ ] [ ] , and precedes the reduction of respiratory complex i activity and low dopamine levels [ ] . altogether, these findings suggest that therapeutic strategies directed to increase gsh levels may be of clinical significance. in this line, in vitro pretreatment with gee has been shown to exert a protective effect in neurons directly exposed to h o or incubated with respiratory complex i and ii inhibitors mpp+ and malonate, respectively. in addition, in vivo studies in animal models of pd elevation of brain gsh by icv infusion of gee have been reported to provide neuroprotection against oxidative stress caused by chronic mitochondrial impairment due to central delivery of mpp+ [ ] . amyotrophic lateral sclerosis (als) is characterized by a progressive degeneration of motor neurons in the brain and spinal cord. approximately % of als cases are considered familial, while the other % are characterized as sporadic. a tight genetic linkage has been reported between familial als and the gene that encodes the cu/zn-binding superoxide dismutase (sod ), a metalloenzyme that catalyzes the dismutation of the superoxide anion (o •− ) to o and h o . nearly mutant forms of sod have been identified in als patients, which are responsible for approximately % of all the inherited cases [ ] . in the rest, although etiology remains still unknown, oxidative damage associated with mitochondrial dysfunction has been shown to play a contributive role [ , ] . depletion of gsh underlies progression of als. thus, strategies aimed at elevating gsh may yield new therapeutics for als. in this regard, a recent study evaluated the use of a nutritional cystine-rich gsh precursor, whey supplement (immunocal ( ® )), in the mutant hsod (g a) mouse model of ald [ ] . the administration of the gsh precursor significantly delayed the disease onset in the transgenic hsod (g a) mice, but without extending life span, most likely due to the inability to recover the mitochondrial gsh pool in the spinal cord [ ] . of relevance for neurodegenerative disorders, a recent study has uncovered that the gsh redox pathway regulates mitochondria dynamics in axons [ ] . specifically, the study in drosophila identifies a novel glutathione s-transferase (gst), gfzf, homologous to gstt in humans, that inhibits mitochondrial hyperfusion under normal physiological conditions. the authors show that changes in the redox balance due to gst loss have a direct impact on mitochondrial trafficking and neuronal response. remarkably, genome-wide association studies (gwas) have linked polymorphisms in gst genes [ ] to increased risk in developing ad and pd later in life. future studies will be needed to analyze whether changes in the gsh:gssg ratio associated to gst activity can alter mitochondrial dynamics described in neurodegenerative disorders, which will provide new mechanistic insights into how these alterations result in an axonal loss. mitochondrial ros generation is exacerbated during diabetes either by alterations in oxidative phosphorylation, by antioxidant depletion, or both [ ] [ ] [ ] [ ] . in turn, antioxidant depletion, particularly of gsh, may favor peroxidative damage in lipids from mitochondrial membranes [ , ] . these oxidative injuries have a direct effect in the mitochondrial electron transfer, resulting in an enhanced electron leak and ros generation that lead to a vicious circle of mitochondrial dysfunction and oxidative stress. diabetic nephropathy (dn), referring to the deterioration of kidney function associated to both type and type diabetes, can progress to chronic kidney disease and, in fact, is a strong predictor of mortality in diabetic patients. emerging evidence points to the oxidative and nitrosative stress as the underlying mechanism by which chronic hyperglycemia causes renal cellular damage [ ] [ ] [ ] [ ] . low levels of renal gsh have been described associated with dn [ , , ] . in turn, dietary gsh supplementation has been shown to partially protect against many of the pathological changes due to dn [ ] . thus, given these findings, it would be logical to suggest that the mgsh pool may be a good choice as a therapeutic target. of interest, a very recent study using the delivery of gsh to the kidney with liposomal technology (gsh-lip) revealed that the complex with liposomes improves the bioavailability and antioxidant capacity of gsh to scavenge redundant ros induced by oxidative stress. furthermore, in vivo imaging showed that gsh-lip is directed to the kidney and significantly recovers renal function. thus, these studies provide the foundation for study the use of antioxidant-related drugs in dn [ ] . aging is a time-sequential degradation of cellular functions caused by accumulated damage that leads to organ failure, and finally death. a wide number of aging theories have been proposed over the years, but the mitochondrial free radical theory of aging is still the best theoretical framework to explain aging and longevity in mammals [ ] . according to this theory, aging is characterized by the loss of redox homeostasis associated with a reduction of the detoxification capacity of cells, which correlates with an increased risk for age-related diseases [ , ] . mitochondrial dysfunction and decay have been widely related to aging and age-related diseases, such as neurodegenerative disorders [ , ] . increased oxidant leakage, mitochondrial dna damage, and susceptibility to apoptotic pore formation are all features displayed by mitochondria from aged tissues [ ] . particularly important for mitochondrial fitness is the role of mgsh in the regulation of atp production. critical protein sulfhydryl redox states depend on mgsh levels, which in turn influence both nadh and fadh generation and the electron flow through the etc [ , , ] . mgsh decreases up to % with age [ , ] , being more marked in male than female mice in many tissues. this age-related depletion of mgsh content may be attributable to different factors such as an enhanced use due to an increasingly oxidant-rich environment or a defective mitochondrial transport resulting from a progressive loss of the mitochondrial membrane fluidity, and it may also reflect a lower rate of synthesis [ ] . in addition, both plasma glutathione and cysteine, a key precursor amino acid for gsh synthesis become oxidized with aging [ ] . it has also been suggested that the mortality and frailty risk in the elderly associated to low dietary protein intake is mainly due to low cysteine availability [ ] , and that dietary supplementation with cysteine and glycine by promoting gsh synthesis could be notably protective against oxidative stress associated to aging [ ] . a proof of concept for the causal role of gsh in the aging process is the fact that over-expression of the enzyme gcs has been shown to prolong the life span of drosophila by up to % [ ] . in addition, it is known that the number of mitochondria decreases with age in liver cells of mice [ ] , rats [ , ] , and humans [ , ] , concurrent with a decrease in mitochondrial dna copy number and mitochondrial protein levels [ ] . as we have seen, the importance of mgsh has been illustrated by the emergent number of pathologies in which its decrease below a threshold produces cell damage, even leading to cell death. this is why modulation of mgsh levels can influence disease progression, and therapies aimed at recovering mgsh levels may be of medical importance in numerous human diseases. the amount of human pathologies or clinical settings in which mgsh may be playing a critical role is still growing. in fact, any situation where a mitochondrial source of ros is detected, either directly such as drugs interacting with subunits of the respiratory chain, or indirectly as in defective mgsh carriers due to lipid changes in the mitochondrial membrane, mgsh could be responsible for cell demise. recent data point to different topics for future analysis of mgsh involvement, such as: lung diseases-glutathione precursors, particularly n-acetylcysteine, have been prescribed for years to prevent acute pulmonary episodes, bronchitis, or emphysema as an effective method of reducing oxidative stress in clinical settings associated with low gsh levels, such as chronic lung diseases [ ] . more recently, glutathione levels have been associated with covid- disease. since mitochondrial ros act as signal-transducing molecules that upregulate inflammatory cytokines [ ] in conditions with excessive inflammatory response, as happens in severe covid- symptoms, it is expected that mitochondrial antioxidants such as mgsh would play a role during the severe acute respiratory syndrome coronavirus (sars-cov- ) viral infection. chemotherapy-standard chemotherapeutic agents such as doxorubicin and cisplatin are well-known inducers of mitochondrial ros during their anti-tumoral action [ , ] . however, the mitochondrial effects of other more recently approved anti-cancer drugs, such as sorafenib or regorafenib, are being just revealed [ , ] . more importantly, mitochondrial antioxidants may reduce the effectivity of these drugs, while glutathione depressors potentiate their effect in hepatocellular carcinoma cells (cucarull et al., unpublished results) . since chemotherapy-induced side effects are frequently also caused by mitochondrial ros, such as cardiotoxicity or kidney injury after doxorubicin and cisplatin treatments, respectively, these potential redox therapies should be carefully directed to the target cells. therefore, intake of antioxidants or ros modulators should be well controlled in order to avoid undesired effects during cancer treatment. gender perspective-gsh levels are different in males and females as a consequence of hormonal regulation and aging [ , ] . although higher mgsh levels in females are expected, this topic has been poorly pursued, with very few studies in animal models and common pathologies. novel results highlighting the antioxidant differences observed between sexes, frequently reflecting sexual dimorphism in disease incidence will increase the interest in specific mgsh levels and maybe suggest gender-specific biomedical strategies. mgsh plays a center role in the cellular defense from death by being a key regulator of mitochondrial oxidative stress. however, up to now, although our knowledge of mitochondrial redox control systems has increased notably, we still lack the full understanding of how mgsh transport works in the different cells/organs. although several carriers have been identified, they most probably do not account for the totality of mgsh transport. in addition, there are conflicting data regarding the acknowledged role of the known mgsh carriers (dic and ogc) in the transport of gsh, which need to be addressed. thus, more effort is needed in the discovery and characterization of these mgsh carriers. numerous pathologies course with mgsh depletion, being in most cases a causative factor for disease progression. accordingly, novel strategies aimed either at preventing mgsh depletion, such as mitochondrial cholesterol lowering agents for liver pathologies, or drugs capable of increasing mgsh levels need to be pursued from a therapeutic perspective. the authors declare no conflict of interest. abbreviations -oxoglutarate carrier (ogc; slc a ); adenine nucleotide translocator (ant); alcoholic liver disease (ald); alcoholic steatohepatitis (ash); alzheimer's disease (ad); amyloid beta peptides (aβ); amyloid precursor protein/presenilin (app/ps ); amyotrophic lateral sclerosis (als); cu/zn-superoxide dismutase (sod ); diabetic nephropathy (dn); electron transport chain (etc); endoplasmic reticulum (er); free cholesterol (fc); glyoxalase ii (glo ); glutathione (gsh); glutathione disulfide (gssg); glutathione reductase (gr); glutathione s-transferase (gst); gsh ethyl ester (gee); gsh peroxidases (gpx); hepatic steatosis (hs); metabolic associated fatty liver disease (mafld); mitochondrial dicarboxylate carrier (dic; slc a ); mitochondrial glutathione (mgsh); mitochondrial nadp+-dependent isocitrate dehydrogenase (idpm); mitochondrial permeability transition (mpt); non-alcoholic fatty liver disease (nafld); non-alcoholic steatohepatitis (nash); parkinson's disease (pd); peroxiredoxins (prx); protein disulfide isomerase (pdi); reactive oxygen species (ros); s-adenosyl-l-methionine (sam); s-d-lactoylglutathione (slg); sterol regulatory element binding protein (srebp- ); thioredoxin /thioredoxin reductase (trx /trxr ); tumor necrosis factor (tnf). intracellular cysteine and glutathione delivery systems glutathione: subcellular distribution and membrane transport mitochondrial glutathione, a key survival antioxidant redox pathways of the mitochondrion mechanisms of liver injury. iii. role of glutathione redox status in liver injury redox state of glutathione in human plasma. free radic glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress analysis of glutathione: implication in redox and detoxification transport of γ-glutamyl amino acids: role of glutathione and γ-glutamyl transpeptidase redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. free radic selectively addressing mitochondrial glutathione and thioredoxin redox systems protein s-glutathionylation: the linchpin for the transmission of regulatory information on redox buffering capacity in mitochondria protein s-glutathiolation: redox-sensitive regulation of protein function depletion of a discrete nuclear glutathione pool by oxidative stress, but not by buthionine sulfoximine. correlation with enhanced alkylating agent cytotoxicity to human melanoma cells in vitro glutathione is recruited into the nucleus in early phases of cell proliferation oxidized redox state of glutathione in the endoplasmic reticulum the role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress an update on mitochondrial reactive oxygen species production production of superoxide radicals and hydrogen peroxide by nadh-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria the mitochondrial generation of hydrogen peroxide. general properties and effect of hyperbaric oxygen mitochondrial free radical generation, oxidative stress, and aging. free radic visualization of the compartmentalization of glutathione and protein-glutathione mixed disulfides in cultured cells regulation and functions glutathione and mitochondria mitochondrial glutathione: features, regulation and role in disease glutaredoxin systems transgenic mouse models for the vital selenoenzymes cytosolic thioredoxin reductase, mitochondrial thioredoxin reductase and glutathione peroxidase programmed cell-death by ferroptosis: antioxidants as mitigators chemical basis of reactive oxygen species reactivity and involvement in neurodegenerative diseases gpx at the crossroads of lipid homeostasis and ferroptosis mitochondrial thiols in antioxidant protection and redox signaling: distinct roles for glutathionylation and other thiol modifications isoforms of mammalian peroxiredoxin that reduce peroxides in presence of thioredoxin mitochondrial thioredoxin- /peroxiredoxin- system functions in parallel with mitochondrial gsh system in protection against oxidative stress protein s-glutathionylation reactions as a global inhibitor of cell metabolism for the desensitization of hydrogen peroxide signals enrichment and functional reconstitution of glutathione transport activity from rabbit kidney mitochondria. further evidence for the role of the dicarboxylate and -oxoglutarate carriers in mitochondrial glutathione transport role of glutathione transport processes in kidney function hepatic mitochondrial glutathione: transport and role in disease and toxicity plasma membrane and mitochondrial transport of hepatic reduced glutathione mitochondrial glutathione transport: physiological, pathological and toxicological implications sensitivity of the -oxoglutarate carrier to alcohol intake contributes to mitochondrial glutathione depletion mitochondrial free cholesterol loading sensitizes to tnf-and fas-mediated steatohepatitis mitochondrial glutathione: hepatocellular survival-death switch app/ps mice overexpressing srebp- exhibit combined aβ accumulation and tau pathology underlying alzheimer's disease mitochondrial cholesterol loading exacerbates amyloid β peptide-induced inflammation and neurotoxicity hepatic mitochondrial transport of glutathione: studies in isolated rat liver mitochondria and h iie rat hepatoma cells the mitochondrial dicarboxylate and -oxoglutarate carriers do not transport glutathione functional expression of eukaryotic membrane proteins in lactococcus lactis s-d-lactoylglutathione can be an alternative supply of mitochondrial glutathione. free radic synthesis and metabolism of methylglyoxal, s-d-lactoylglutathione and d-lactate in cancer and alzheimer's disease. exploring the crossroad of eternal youth and premature aging protein s-glutathionlyation links energy metabolism to redox signaling in mitochondria molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death role of mitochondria in toxic oxidative stress mitochondrial oxidative stress: implications for cell death redox regulation of apoptosis: impact of thiol oxidation status on mitochondrial function mitochondrial thiols in the regulation of cell death pathways mitochondrial glutathione protects against cell death induced by oxidative and nitrative stress in astrocytes physiological roles of nicotinamide nucleotide transhydrogenase a caenorhabditis elegans mutant lacking functional nicotinamide nucleotide transhydrogenase displays increased sensitivity to oxidative stress. free radic regulation of high glucose-induced apoptosis by mitochondrial nadp +-dependent isocitrate dehydrogenase stimulation of lipid peroxidation increases the intracellular calcium content of isolated hepatocytes hepatic mitochondrial energy production in rats with chronic iron overload iron-induced peroxidative injury to isolated rat hepatic mitochondria perturbation in liver mitochondrial ca + homeostasis in experimental iron overload: a possible factor in cell injury inhibition of adenine nucleotide translocator by lipid peroxidation products. free radic opening of the mitochondrial permeability transition pore by uncoupling or inorganic phosphate in the presence of ca + is dependent on mitochondrial-generated reactive oxygen species mitochondrial glutathione transferases involving a new function for membrane permeability transition pore regulation oxidative stress and alcoholic liver disease. semin. liver dis giant mitochondria in hepatocytes: a diagnostic hint for alcoholic liver disease-pubmed effects of ethanol on proteins of mitochondrial membranes the effect of temperature and chronic ethanol feeding on the proton electrochemical potential and phosphate potential in rat liver mitochondria adenosyl-l-methionine and mitochondrial reduced glutathione depletion in alcoholic liver disease mitochondrial glutathione depletion in alcoholic liver disease mechanism of mitochondrial glutathione-dependent hepatocellular susceptibility to tnf despite nf-κb activation selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor effects of ethanol dose and ethanol withdrawal on rat liver mitochondrial glutathione: implication of potentiated acetaminophen toxicity in alcoholics selective mitochondrial glutathione depletion by ethanol enhances acetaminophen toxicity in rat liver overexpression of manganese superoxide dismutase prevents alcohol-induced liver injury in the rat asmase is required for chronic alcohol induced hepatic endoplasmic reticulum stress and mitochondrial cholesterol loading mitochondrial s-adenosyl-i-methionine transport is insensitive to alcohol-mediated changes in membrane dynamics cyp e overexpression in hepg cells induces glutathione synthesis by transcriptional activation of γ-glutamylcysteine synthetase induction of catalase, alpha, and microsomal glutathione s-transferase in cyp e overexpressing hepg cells and protection against short-term oxidative stress cyp e -dependent toxicity and up-regulation of antioxidant genes mafld: a consensus-driven proposed nomenclature for metabolic associated fatty liver disease the role of cholesterol in the pathogenesis of nash role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis. free radic defective hepatic mitochondrial respiratory chain in patients with nonalcoholic steatohepatitis alterations in liver atp homeostasis in human nonalcoholic steatohepatitis: a pilot study reactive oxygen species, cell signaling, and cell injury. free radic mitochondrial abnormalities in non-alcoholic steatohepatitis in situ detection of lipid peroxidation and oxidative dna damage in non-alcoholic fatty liver diseases gene expression of tumor necrosis factor α and tnf-receptors, p and p , in nonalcoholic steatohepatitis patients efficacy of glutathione for the treatment of nonalcoholic fatty liver disease: an open-label, single-arm, multicenter, pilot study personal model-assisted identification of nad + and glutathione metabolism as intervention target in nafld oxidative stress and altered mitochondrial function in neurodegenerative diseases: lessons from mouse models a nh tau fragment targets neuronal mitochondria at ad synapses: possible implications for neurodegeneration mitochondria are a direct site of aβ accumulation in alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression mitochondrial dihydrolipoyl succinyltransferase deficiency accelerates amyloid pathology and memory deficit in a transgenic mouse model of amyloid deposition. free radic reduction in mitochondrial superoxide dismutase modulates alzheimer's disease-like pathology and accelerates the onset of behavioral changes in human amyloid precursor protein transgenic mice mitochondrial cholesterol in alzheimer's disease and niemann-pick type c disease oxidative inactivation of amyloid beta-degrading proteases by cholesterol-enhanced mitochondrial stress cholesterol impairs autophagy-mediated clearance of amyloid beta while promoting its secretion nexus between mitochondrial function, iron, copper and glutathione in parkinson's disease chronic systemic exposure to low-dose rotenone induced central and peripheral neuropathology and motor deficits in mice: reproducible animal model of parkinson's disease lrrk , a puzzling protein: insights into parkinson's disease pathogenesis partners in crime? neurotherapeutics what can we learn about parkinson's disease pathobiology? j. parkinson's dis paraquat, and parkinson's disease nigral glutathione deficiency is not specific for idiopathic parkinson's disease alterations in the distribution of glutathione in the substantia nigra in parkinson's disease idiopathic parkinson's disease, progressive supranuclear palsy and glutathione metabolism in the substantia nigra of patients alterations in glutathione levels in parkinson's disease and other neurodegenerative disorders affecting basal ganglia glutathione-related enzymes in brain in parkinson's disease reduced and oxidized glutathione in the substantia nigra of patients with parkinson's disease glutathione depletion in a midbrain-derived immortalized dopaminergic cell line results in limited tyrosine nitration of mitochondrial complex i subunits: implications for parkinson's disease characterization of intracellular elevation of glutathione (gsh) with glutathione monoethyl ester and gsh in brain and neuronal cultures: relevance to parkinson's disease mutations in cu/zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis mitochondrial dysfunction in als amyotrophic lateral sclerosis a cystine-rich whey supplement (immunocal ® ) delays disease onset and prevents spinal cord glutathione depletion in the hsod g a mouse model of amyotrophic lateral sclerosis glutathione s-transferase regulates mitochondrial populations in axons through increased glutathione oxidation glutathione s-transferase omega genes in alzheimer and parkinson disease risk, age-at-diagnosis and brain gene expression: an association study with mechanistic implications insulin attenuates diabetes-related mitochondrial alterations: a comparative study rage-induced cytosolic ros promote mitochondrial superoxide generation in diabetes impaired mitochondrial respiratory functions and oxidative stress in streptozotocin-induced diabetic rats ubiquinone (coenzyme q ) prevents renal mitochondrial dysfunction in an experimental model of type diabetes. free radic glutathione correlates with lipid peroxidation in liver mitochondria of triiodothyronine-injected hypophysectomized rats glutathione depletion, lipid peroxidation and mitochondrial dysfunction are induced by chronic stress in rat brain mitochondrial glutathione in diabetic nephropathy effect of antioxidants and ace inhibition on chemical modification of proteins and progression of nephropathy in the streptozotocin diabetic rat susceptibility to diabetic nephropathy is related to dicarbonyl and oxidative stress diabetic nephropathy is associated with oxidative stress and decreased renal nitric oxide production biochemistry and molecular cell biology of diabetic complications diabetes-induced changes in glucose synthesis, intracellular glutathione status and hydroxyl free radical generation in rabbit kidney-cortex tubules dietary glutathione protects rats from diabetic nephropathy and neuropathy effect of glutathione liposomes on diabetic nephropathy based on oxidative stress and polyol pathway mechanism aging: a theory based on free radical and radiation chemistry redox theory of aging redox theory of aging: implications for health and disease mitochondria take center stage in aging and neurodegeneration the role of mitochondria in aging mitochondrial dna oxidative damage and repair in aging and alzheimer's disease regulation of mitochondrial glutathione redox status and protein glutathionylation by respiratory substrates cytoplasmic and mitochondrial nadph-coupled redox systems in the regulation of aging the role of mitochondrial oxidative stress in aging. free radic two subpopulations of mitochondria in the aging rat heart display heterogenous levels of oxidative stress. free radic decline in transcriptional activity of nrf causes age-related loss of glutathione synthesis, which is reversible with lipoic acid an increased need for dietary cysteine in support of glutathione synthesis may underlie the increased risk for mortality associated with low protein intake in the elderly deficient synthesis of glutathione underlies oxidative stress in aging and can be corrected by dietary cysteine and glycine supplementation enhanced catabolism of mitochondrial superoxide/hydrogen peroxide and aging in transgenic drosophila a morphometric study of age-dependent changes in mitochondrial population of mouse liver and heart quantitation of mitochondrial dna and protein in the liver of fischer rats during aging glutathione metabolism in heart and liver of the aging rat liver mitochondrial respiratory functions decline with age age changes in size and number of mitochondria of human hepatic cells quantitation of mitochondrial dna, rna, and protein in starved and starved-refed rat liver the role of glutathione in protecting against the severe inflammatory response triggered by covid- mitochondrial reactive oxygen species drive proinflammatory cytokine production mitochondria-dependent ferroptosis plays a pivotal role in doxorubicin cardiotoxicity mitochondria targeted peptide ss- prevent on cisplatin-induced acute kidney injury via regulating mitochondrial ros-nlrp pathway antiapoptotic bcl- proteins determine sorafenib/regorafenib resistance and bh -mimetic efficacy in hepatocellular carcinoma regorafenib alteration of the bcl-xl/mcl- ratio provides a therapeutic opportunity for bh -mimetics in hepatocellular carcinoma models sexual dimorphism in glutathione metabolism and glutathione-dependent responses gender difference in glutathione metabolism during aging in mice this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - nj vb authors: xie, na; zhang, lu; gao, wei; huang, canhua; huber, peter ernst; zhou, xiaobo; li, changlong; shen, guobo; zou, bingwen title: nad(+) metabolism: pathophysiologic mechanisms and therapeutic potential date: - - journal: signal transduct target ther doi: . /s - - - sha: doc_id: cord_uid: nj vb nicotinamide adenine dinucleotide (nad(+)) and its metabolites function as critical regulators to maintain physiologic processes, enabling the plastic cells to adapt to environmental changes including nutrient perturbation, genotoxic factors, circadian disorder, infection, inflammation and xenobiotics. these effects are mainly achieved by the driving effect of nad(+) on metabolic pathways as enzyme cofactors transferring hydrogen in oxidation-reduction reactions. besides, multiple nad(+)-dependent enzymes are involved in physiology either by post-synthesis chemical modification of dna, rna and proteins, or releasing second messenger cyclic adp-ribose (cadpr) and naadp(+). prolonged disequilibrium of nad(+) metabolism disturbs the physiological functions, resulting in diseases including metabolic diseases, cancer, aging and neurodegeneration disorder. in this review, we summarize recent advances in our understanding of the molecular mechanisms of nad(+)-regulated physiological responses to stresses, the contribution of nad(+) deficiency to various diseases via manipulating cellular communication networks and the potential new avenues for therapeutic intervention. introduction nad + was first described in as a component that could increase the fermentation rate in yeast. years later, nad + was determined to play a vital role for hydrogen transfer in redox reaction. as an essential redox carrier, nad + receives hydride from metabolic processes including glycolysis, the tca cycle, and fatty acid oxidation (fao) to form nadh. nadh, therefore, serves as a central hydride donor to atp synthesis through mitochondrial oxphos, along with the generation of ros. beyond its vital role as a coenzyme in energy metabolism, the important role of nad + has expanded to be a co-substrate for various enzymes including sirtuins, parps, cd , cd , cd and sarm . [ ] [ ] [ ] [ ] recently, it has been found that nad + serves as a nucleotide analog in dna ligation and rna capping. , therefore, the dynamic nad + and its metabolites levels, in response to diverse cellular stress and physiological stimuli, rewire biological processes via post-synthesis modification of fundamental biomolecules, including dna, rna and proteins. [ ] [ ] [ ] [ ] [ ] through these activities, nad + impact energy metabolism, dna repair, epigenetic modification, inflammation, circadian rhythm and stress resistance. nad + deficiency, however, contributes to a spectrum of diseases including metabolic diseases, cancer, aging and neurodegeneration disorders. here, we summarize recent advances in our understanding of the nad + homeostasis in response to growth conditions or environmental stimuli, highlighting the actions of nad + in coordinating metabolic reprogramming and maintaining cellular physiologic biology, which enables the plastic cells to adapt to environmental changes. furthermore, we will discuss the nad + and its metabolites serving as an essential hub in both physiological and pathophysiological processes and explore the potential of nad + modulation in the clinical treatment of diseases. nad + homeostasis nad + , one of the most common metabolites in the human body, is in a homeostatic status of biosynthesis, consumption, recycling and degradation at both cellular and systemic levels. nad + biosynthesis de novo pathway. mammalian cells can generate nad + de novo from dietary tryptophan (trp) by the kynurenine pathway (kp), which is initialized by either tdo or ido. the intermediate acms can cyclize spontaneously to qa. however, acmsd converts acms to picolinic acid, limiting the flux from tryptophan to nad + . another critical step catalyzes the conversion of qa to namn by qprt, which commits the pathway to nad + biosynthesis. , the preiss-handler pathway can convert dietary na to namn by naprt. namn derived from both the tryptophan and na is catalyzed by nmnats to yield naad, which is then amidated to nad + by nad synthase (nadsyn) using glutamine as nitrogen donor (fig. ) . , salvage pathway. rather than generated de novo, most nad + is recycled from nam, na, nr and nmn in the salvage pathway to maintain the cellular nad + levels. among these precursors, nam could be recycled from nad + consumption reactions, including both nad + -dependent deacylation and adp-ribosylation, into nmn by nampt, which catalyzes the rate-limiting reaction in the salvage pathway. the precursor nr is imported by ents and transformed to nmn by nrk / . ultimately, nmn is adenylylated by nmnat to yield nad + , (fig. ) . nad + degradation nad + consumption. as a co-substrate important to various postsynthesis modifications of fundamental macromolecules, nad + can be cleaved by nad + -consuming enzymes including parps, sirtuins, cd and sarm to generate nam and adpribose (adpr) (fig. ) . the sirtuins are nad + -dependent deacetylases that are distributed in the nucleus (e.g., sirt , sirt , and sirt ), the cytoplasm (e.g., sirt ) and mitochondria (e.g., sirt - ), respectively. through mediating the post-translational modification dependent on nad + , sirtuins modulate the adaptation to the altered cellular energetic status, especially the activation of oxidative metabolism and stress resistance in mitochondria in various physiological or pathological conditions. parps catalyze reversible adp-ribosylation of macromolecular targets including proteins, dna and rna, utilizing nad + as a cofactor to provide monomer or polymers of adp-ribose nucleotide. , parp members can be categorized into several groups, the poly-adp-ribosyl transferases (e.g., parp , , and ), the mono-adp-ribosyl transferases (e.g., including parp , , - , and [ ] [ ] [ ] [ ] [ ] [ ] [ ] and rbps (e.g., parp , , and [ ] [ ] [ ] . , parpsmediated adp-ribosylation (adpr) plays an essential role in cellular physiological processes in response to stimuli, particularly oxidative stress-induced dna damage. sustained parp activation triggered by intense insults can cause nad + depletion and subsequent cell death. cd consumes nad + to make the calcium-releasing second messengers including adpr (major product), -deoxy-adpr ( dadpr), naadp and cadpr, contributing to age-related nad + decline. , sarm is an important nad + consumer in neurons. the dimerization of tir domain cleaves nad + into adp-ribose, cadpr, and nicotinamide. [ ] [ ] [ ] nad + -consuming enzymes seem to have a different michaelis constant (k m ) value for nad + . the k m of sirt and sirt ranges fig. overview of the nad + metabolism and its physiological function. mammalian cells can synthesize nad + de novo from tryptophan by the kynurenine pathway or from na by the preiss-handler pathway, while most nad + is recycled via salvage pathways from nicotinamide (nam), a by-production of nad + -consuming reactions. nad + can be reduced into nadh in the metabolic processes, including glycolysis, fatty acid oxidation and the tca cycle. nadh, in turn, drives the generation of atp via oxphos, the production of lactic acid from pyruvate, and the desaturation of pufas. nadph plays an essential role in antioxidant defense and regulates cellular signaling via nadph oxidases (noxs). moreover, nad + is found to decorate various rnas in different organisms as nucleotide analog and serves as an alternative adenylation donor for dna ligation in nhej repair. nad + also acts as a co-substrate for a wide variety of enzymes, including parps, sirtuins, cd /cd and sarm , impacting metabolism, genomic stability, gene expression, inflammation, circadian rhythm and stress resistance. using nad + as a cosubstrate, both parps and sirtuins regulate their target molecules, generating nam as a by-product. the cd /cd and sarm also catalyze nad + to nam, producing adpr and cadpr. abbreviations: idos, indoleamine , -dioxygenase; qa, quinolinic acid; namn, nicotinate mononucleotide; qprt, quinolinate phosphoribosyl-transferase; naprt, nicotinic acid phosphoribosyltransferase; nmnats, nicotinamide mononucleotide adenylyl transferases; nadsyn, nad synthase; nr, nicotinamide riboside; trp, tryptophan; nadks, nad + kinases; parps, poly (adp-ribose) polymerases; nnt, nicotinamide nucleotide transhydrogenase; tdo, tryptophan , -dioxygenase; sarm , sterile alpha and tir motif containing ; nnmt, nicotinamide n-methyltransferase; nmn, nicotinamide mononucleotide; pufas, polyunsaturated fatty acids; nam, nicotinamide from to μm, which renders their activity tightly fluctuating with the dynamic physiological cellular nad + levels. other sirtuins, including sirt , sirt , sirt and sirt , have a k m for nad + below the physiological range, implying that nad + might not necessarily be the rate-limiting of their activity. , - parp- , accounting for approximately % of the nad + used by the parp family, has a lower k m for nad + in the range of - μm. [ ] [ ] [ ] of note, the cd and sarm display k m for nad + in a markedly low micromolar range ( - μm) . based on their different k m values, nad + -consuming enzymes display various potential of reducing nad + . under normal homeostatic conditions, cd is expressed at low levels, whereas rising expression of cd with aging plays a vital role in age-associated nad + reduction. , c, a highly potent and specific cd inhibitor, increases nad + levels, leading to activation of sirtuins and parps. generally, the reported k m of parp and cd for nad + are lower than those of the sirtuins, suggesting that elevated activation of parp or cd may limit endogenous sirt activation by reducing nad + content. this notion is confirmed by the observation that parp and cd inhibition effectively increases total nad + availability, leading to sirt activation. nad + methylation. excess nam that is not recycled is metabolized through two enzymatic systems and eventually excreted from the body. the first system methylates the nam into mnam by nnmt, which utilizes the sam as methyl donor. the mnam together with their oxidized compounds, py and py, are eventually eliminated in the urine. this methylation system is quantitatively by far the predominant nam scavenging pathway under most conditions. while an acute pharmacological dose of nam can be converted by cyp e to nicotinamide n-oxide, which is then excreted to the urine. , , therefore, nnmt and cyp e divert nam from recycling to nad + , restraining nam accumulation and inhibition of nad + -dependent signaling. the km of the human nnmt enzyme for nam (approximately μm) is much higher than the affinity of nampt for nam (< μm), suggesting an unsaturated nnmt under normal conditions. increasing dietary nam intake can lead to a proportional increase in nam methylation. , further, elevated nnmt expression or increased mnam levels in the liver can stabilize sirt , which in turn promotes glucose and cholesterol metabolism (fig. ) . subcellular distribution of nad + nad + /nadh. both the oxidized nad(p) + and the reduced nad (p)h have redox and signaling functions with an uneven subcellular distribution. as listed in box , a portfolio of approaches is developed to map the total quantification or cellular concentrations of the four nad + coenzymes. semisynthetic fluorescent biosensor-based analysis of u os cells exhibits around μm for cytoplasmic nad + ,~ μm for nuclear nad + and~ μm for mitochondrial nad + , respectively. meanwhile, the concentration of free cytosolic nad + detected in cell lines including u os, hek t, nih/ t and hela is ranging from to µm. [ ] [ ] [ ] [ ] the similar depletion rate of free nad + in the cytoplasm and nucleus supports the notion regarding a probable exchange of nad + between these compartments, while a slower rate of free nad + depletion in mitochondrial suggests that the mitochondrial nad + pool is segregated from the cytosolic and nuclear pools. , in agreement with these reports, mounting evidence implies that the distinct fluctuation of nad + in mitochondria may be attributed to the membrane impermeability of nad(h). , controversially, isotope-tracer method analysis shows that mammalian mitochondria are capable of taking up intact nad + as well as its precursors, such as nmn and nr. , [ ] [ ] [ ] although nad + transporter has been identified in bacteria, yeast and plants, no mammalian homolog has been discovered so far to validate the import of nad + into mitochondria. [ ] [ ] [ ] [ ] [ ] the nad + pool in each cellular compartment can also be maintained independently via recycling the nad + from nam, dependent on various forms of nmnats, e.g., the nucleic nmnat , cytosolic nmnat and mitochondrial nmnat . nevertheless, the independent mechanism for maintaining nad + through salvaging nmn in mitochondria is challenged by the controversy around the presence or absence of nampt and nmnat in mitochondria. , the electrons of nadh, rather than nadh itself, generated from glycolysis in the cytoplasm could be transferred across the mitochondrial membrane through the nadh shuttle systems. , the glycerol- -phosphate (g- -p) shuttle and malate-aspartate shuttle transfer the electrons from box nad(p) + / nad(p)h detection assays the biochemical analysis uses enzymatic cycling assays, capillary electrophoresis (ce), or high-performance liquid chromatography (hplc) coupled with mass spectrometry (lc/ms/ms), to detect the nad + and nadh contents and the nad + /nadh ratio. , [ ] [ ] [ ] [ ] [ ] the enzymatic cycling assays is based on a multi-step nad + /nadh enzyme cycling reactions that convert wst- to wst- formazan, which can be easily detected at od nm. this assay is recommended to evaluate the effect of activators and inhibitors on nampt activity using purified protein and to check for contamination and interference for nad + present in the sample, such as immunoprecipitated cell lysates. however, this approach measures the total quantity of cellular nad + or nadh, regardless of the free and protein-bound forms or the differentiated subcellular compartmentation. additionally, the requirement of tissue biopsy and extraction renders the enzymatic cycling assays incompatible with longitudinal studies in intact organs. [ ] [ ] [ ] based on the enzymatic cycling reaction, a ce approach is established to measure nad + and nadh in a single cell in a single run with a capillary electrophoresis laser-induced fluorescence (ce-lif) setup. this method shows good reproducibility and specificity with a detection capability as low as . amol of nad + and amol of nadh. , hplc coupled ms can simultaneously analyze the four coenzymes including nad + , nadh, nadp + , and nadph, and the related metabolites. this approach provides accurate, sensitive, reliable, rapid, and reproducible results, which enables us to map various pathophysiological alterations in nad + metabolism. [ ] [ ] [ ] [ ] [ ] autofluorescence approach the autofluorescence approach is a less invasive optical approach. under ultraviolet excitation, nadh/nadph exhibits identical autofluorescence signals, whereas the related oxidized forms nad + /nadp + are not fluorescent. the autofluorescence intensity has often been microscopic determined as the quantification of nad(p)h. additionally, fluorescence lifetime imaging microscopy (flim) is capable of differentiating the quantitative of free and proteinbound nad(p)h independent on intensity, interpreting as an indirect readout of cellular metabolism. collectively, this method based on intensity and decay times of the autofluorescence allows the analysis of cellular redox state and metabolism in cells and tissues. however, the application of this marker-free approach is limited by its signal ambiguity, limited penetration and trouble in monitoring the autofluorescence signal from deep tissue or organs. , genetically encoded fluorescent redox sensors the highly responsive, genetically encoded fluorescent sensors, including frex, liga-cpvenus, sonar, peredox, rexyfp for nad + /nadh, inap - and apollo-nadp + for nadp + /nadph, can image and monitor nad(p) + /nad(p)h redox state in living cells and in vivo. advantages of the fluorescent redox sensors are able to determine subtle perturbations of the cellular energy metabolism in realtime. meanwhile, it can be adapted to high-throughput chemical screening of potential compounds targeting cellular metabolism in a variety of analytical platforms, including microplate readers, flow cytometry, fluorescence microscopy and high-content imaging systems. , , - p-magnetic resonance spectroscopy ( p-mrs) methods p-mrs based nad + assay is a noninvasive method that could quantitatively measure intracellular nad + contents and redox state in animal and human tissues, such as brains. it provides new approach to investigate intracellular nad + redox state and metabolism in the human tissues with the potential for translation to human application. , [ ] [ ] [ ] [ ] isotope-tracer methods isotope labeling metabolites, including [ , , , - h] nam, [u- c] trp, [u- c] na, and nr (nicotinamide - c, ribose - h), can be intravenous infused into mice or added into the media of cell culture. the labeled metabolites in cells, serum and tissues are analyzed by lc-ms. isotope-tracer methods are applied in quantitative analysis of nad + synthesis-breakdown fluxes, including nad + synthesis and consumption fluxes in cell lines, as well as nad + fluxes in vivo. cytosolic nadh to mitochondrial fadh or nadh, respectively. then, the electrons are finally transferred to the etc [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (fig. ) . nadp + /nadph. approximately % of cellular nad + may be phosphorylated by nad + kinases into nadp + , which can be dephosphorylated to nad + by nadp + phosphatases. , cytosolic nadph, the reduced form of nadp + , is mainly generated in the pentose phosphate pathway (ppp) involving g pd and pgd. the mitochondrial nadph can be produced by me that converts pyruvate to malate and by idh that catalyzes isocitrate to αketoglutarate. , additionally, nadp + can also receive the electrons from nadh to form nadph by nnt that locates in the mitochondrial inner membrane. these distinct synthesis pathways might contribute to the differential subcellular nadph/ nadp + ratio, such as a significantly higher ratio in mitochondria (~ ) than that of the cytosol and the nucleus ( ~ ) in u os cells. nadph is required for both the reductive biosynthetic reactions of cholesterol and palmitate and the oxidative reactions catalyzed by nadph oxidases (noxs), nitric oxide synthases (nos), cytochrome p- , and so on. , most importantly, nadph provides the primary reducing power for the thioredoxin (trx) and glutathione (gsh) systems to eliminate ros (fig. ). in line with that, the free nadph/nadp + ratio that indicates the reduction potential is normally sustained at a high level inside cells and is significantly reduced following pro-oxidant agents or h o exposure. , , given its essential role in metabolism and antioxidant defense, nadph/nadp + ratio in cancer cells exhibiting high metabolic rate and ros contents is much higher than that in the embryonic kidney immortalized cell line hek (~ ). albeit further research is needed, quantification of nadph/nadp + ratios provides an effort to map metabolic and redox state of different cell types and organelles. nad + homeostasis at the systemic level the nad + and its metabolites systemically flux and exchange across tissues, with a tissue-specific distribution of nad + biosynthetic enzymes and a tissue-specific preference for specific nad + precursors. it is reported that the de novo biosynthesis of nad + from tryptophan mainly occur in the liver and, to a lesser extent, kidney, which is attributed to the exclusively expressed the nad + homeostasis is maintained by the biosynthesis, consumption and recycling in differentiate subcellular compartments including the cytosol, the nucleus and the mitochondria. nad + precursors including trp, na, nr, nmn and nam are metabolized into nad + via preiss-handler pathway, de novo pathway and salvage pathway, respectively. nad + can receive hydride to yield the reduced form nadh in the metabolic processes including glycolysis, fao, and the tca cycle. nadh provides an electron pair to drive the mitochondrial oxphos for the generation of atp and the conversion of lactic acid to pyruvate. the cytosolic and mitochondrial nadh is exchanged through the malate-aspartate shuttle and glycerol- -phosphate shuttle, while the cytosolic and mitochondrial nadph is exchanged by the isocitrate-a-kg shuttle. nad + can also be phosphorylated into nadp + by nad + kinases including nicotinamide nucleotide transhydrogenase (nnt) and nad kinases (nadks). cytosolic nadp + is reduced into nadph by g pd and pgd in the pentose phosphate pathway, and by me in the conversion of malate to pyruvate. mitochondrial nadph is produced by idh , glud, nnt and me . the nadph is required for the activation of noxs and the synthesis of palmitate. abbreviation: α-kgdh, alpha-ketoglutarate dehydrogenase; glud, glutamate dehydrogenase; nnt, nicotinamide nucleotide transhydrogenase; g pdh, glyceraldehyde -phosphate dehydrogenase; pgd, phosphogluconate dehydrogenase; g pd, glucose- -phosphate dehydrogenase; gpx, glutathione peroxidases; idh / , isocitrate dehydrogenase and ; mdh, malate dehydrogenase; me / , malic enzyme; nadk, nad + kinase; noxs, nadph oxidases; oxphos, oxidative phosphorylation; ppp, pentose phosphate pathway; prx, peroxiredoxin; sdh, succinate dehydrogenase; sod - , superoxide dismutase type - ; tca cycle, tricarboxylic acid cycle; gsh, glutathione; ldh, lactate dehydrogenase enzymes involved in de novo nad + synthesis in these tissues. therefore, the concentration of tryptophan in the diet affects the liver nad + levels. tryptophan also compensates the nad + biosynthesis when the salvage pathway is blocked. , , nam is the main nad + source in both cell lines and most murine tissues. the circulating nam, % of which is released by the liver, is the main nad + source for the rest of the body. the nam uptake preference differs dramatically, ranging from the highest μm in spleen and small intestine to the lowest - μm in the white adipose and skeletal muscle. besides tryptophan and nam, na is the third nad + precursor with concentrations > . mm in mammalian plasma, which can only be used by spleen, small intestine, pancreas, kidney and liver. accordingly, these tissues have been observed with a remarkable expression of naprt that guides na into nad + biosynthesis. additionally, nmn and nr are capable of efficiently elevating tissue nad + concentrations. given that nmn itself is a non-cell penetrating biosynthetic intermediates, nmn or its metabolites may be actively transported across the membrane. the solute carrier family member (slc a ) has been reported as a specific nmn transporter that is responsible for the uptake of nmn and maintenance of nad + level in the murine small intestine. , however, it has been reported that the dephosphorylation of nmn into nr by extracellular '-nucleotidases is required for the uptake and utilization of nmn in cellular nad + synthesis. similarly, the modification of the phosphate group in nmn allows its transportation to activate sarm in cells. therefore, whether nmn is directly transported by slc a remains unclear, which needs further investigation. nr, which can cross the cell membrane through a dypiridamole-inhibitable nucleoside transporter, is preferentially used by muscle to synthesize nad + . accordingly, the nr-using enzyme, nrk , is usually specifically expressed in the skeletal muscle. beyond the systemic regulation of nad + homeostasis across various tissues, it has recently been described that bacteria contribute to mammalian host nad + biosynthesis, especially following oral intake of the amidated precursors. the oral nam and nr can be deamidated by gut microbiota into na, nar, naad and namn in the small intestine and the colon. these deamidated nad + metabolites are circulated to the liver and kidney, significantly contributing to the bulk of nad + biosynthesis. despite major advances in the acknowledgment of tissuespecific nad + homeostasis, further work will be needed to fully characterize the subcellular and systemic modulation of nad + metabolism, which can improve the preventive and therapeutic strategies based on maintaining healthy nad + homeostasis. serving as crucial co-enzymes for redox reactions and cosubstrates for nad + -dependent enzymes, nad + and its metabolites function as a regulatory hub controlling a broad range of physiological processes, including redox homeostasis, genomic stability, gene expression, rna processing, energy metabolism, immunity and inflammation, and circadian clock. nad + metabolism maintains the redox homeostasis cells continuously generate oxidants and produce antioxidants. an imbalance between the oxidant formation and the antioxidant capacity in favor of the former causes oxidative stress. maintenance of a physiological (low-level) oxidative stress, also denoted as oxidative eustress, is pivotal for governing biological processes and physiological functions including cell cycle and proliferation, circadian clock, innate immunity, self-renewal of stem cells and neurogenesis. [ ] [ ] [ ] [ ] however, a variety of stimuli, including nutrient perturbation, genotoxic stress, infection, pollutants and xenobiotics, trigger ros overproduction, resulting in excessive oxidant challenge (oxidative distress). oxidative distress causes damage to fundamental macromolecules including proteins, lipids, rna and dna at a cellular level, which promotes abnormal cell death and inflammation, often culminating in additional oxidative stress at a systemic level. , through giving rise to fast, barrier-less and non-selective oxidation reactions that are responsible for a severe insult of both cell and systematic tissues, oxidative stress is involved in a myriad of pathologies. of note, nad + deficiency exerts effects on the emergence of oxidative stress in multiple diseases, while boosting nad + has protective effects due to enhancement of antioxidant capacity via increasing the gsh levels and the activity of antioxidant enzymes. to counteract the detrimental effects of oxidants, cells can heighten the production of reducing equivalents such as nadph. moreover, nad + -consuming enzymes, such as sirt , can also manipulate the cellular redox status via regulating the activity of enzymes for ros generation and antioxidant factors for ros eradication. [ ] [ ] [ ] therefore, nad(p) + /nad(p)h represents the switching hub that controls prooxidant-antioxidant balance and determines the redox biology (fig. ) . nadh/nadph as the electron donor in ros generation. major endogenous ros via superoxide radicals is constantly produced by both non-enzymatic reactions such as the mitochondrial respiration that needs nadh and enzyme-catalyzed reactions including noxs that require nadph. in a physiological context, the vast majority of cellular ros is produced in mitochondria using nadh as electron donor. , mitochondrial nadh supplies nadh dehydrogenase (complex i) with electrons, which along with the electrons obtained from fadh via complex ii drive the mitochondrial etc to generate a proton (h + ) gradient across the imm for the production of atp. the complex i and complex iii of etc are able to produce the superoxide anion radical (o -) and release it to both the matrix and the intermembrane space. [ ] [ ] [ ] additionally, nadh or fadh is the electron carrier for the mitochondrial membrane proteins, such as gpdm and fqr, and the metabolic enzymes in matrix, such as ogdh and pdh, all of which contribute to ros production. another significant intracellular source of ros is the noxs, especially in response to physiological stimuli, including growth factors, hormones and cytokines, and pathological impulse, such as bacterial and viral infections. rather than generating ros as a by-product, noxs produce superoxide as a primary product using nadph as the electron donor. the nox proteins, including nox - and duox / , have the conserved nadph-binding site at the c-terminus, which extracts electrons from nadph. the presence of fad-binding region and transmembrane hemes enable noxs to act as an electron-transportation chain that transfers two electrons from cytosolic nadph to extracellular o , resulting in the generation of o -. , beyond mitochondria and the nox family, a variety of enzymes including xanthine oxidase (xo), nos, lipoxygenase and cytochromes p (cyp) can all produce ros using nad(p)h as electron donor. mammalian xanthine oxidoreductase (xor), one enzyme in purine catabolism, can exist in both dehydrogenase (xdh) form, which prefers nad + as the electron acceptor, and xo form, which transfers the electrons directly to o , leading to the formation of o and h o . , receiving electrons from nadph, nos catalyzes the production of no from l-arginine participating in a number of biological processes, including neurotransmission, vasodilation and immune response. , ros are also produced via the metabolism of arachidonic acid by lipoxygenases in the presence of nadh or nadph. [ ] [ ] [ ] mammalian cyps are a family of heme-containing nad(p)h-dependent monooxygenases that metabolize numerous endogenous metabolites, including fatty acids and steroids, and exogenous substrates, including carcinogens, pesticides and drugs, resulting in continuous production of ros. , , nadph as the final reducing power for antioxidant defense. besides functioning as the electron donor for ros production, nadph also supplies the reducing power for antioxidant defense. to fine-tune the redox homeostasis that can either prohibit the damage by oxidative distress or maintain the physiologic ros to sustain normal cellular processes, organisms have evolved a complex antioxidant defense system consisting of both enzymatic and non-enzymatic scavengers. , , intriguingly, both enzymatic and non-enzymatic antioxidant system components exhibit their effects in coordination with each other to contribute to redox homeostasis and cell fate using nadph as the ultimate donor of reductive power. , , two classes of enzymatic components, glutathione reductases (grs) and thioredoxin reductases (trxrs), are homologous flavoenzymes that use electrons from nadph to reduce a disulfide to a dithiol. then, the active site dithiol in grs reduces the oxidized gsh (the disulfide gssg) into reduced gsh, the most important non-enzymatic scavenger. gsh is able to reduce the disulfide bonds and hydroperoxides by glutathione peroxidases (gpxs) or promote the glutathionylation at cysteine residues by glutathione s-transferase (gst) to protect protein from oxidation. , [ ] [ ] [ ] similarly, mammalian trxrs maintain the reduced thioredoxin (trx) concentration that supports peroxiredoxin (prx) to remove h o . , therefore, through supplying electrons for bioreductive synthesis and the regeneration of gsh and reduced thioredoxin, nadph plays critical roles in the maintenance of redox homeostasis and modulating redox signaling. nad + -dependent enzymes control redox homeostasis. sirt acts as an essential modulator of oxidative stress via deacetylation of substrates associated with both ros generation and detoxification. sirt deacetylates and activates the multiple components of etc including ndufa of complex i, sdha of complex ii and core i subunit of complex iii. the alteration of etc, therefore, might contribute to an increased ros generation. [ ] [ ] [ ] sirt also enhances cellular antioxidant capacity through augmenting the reducing power, nadph, and increasing the activity of antioxidant enzymes, such as sod and catalase. sirt mediated deacetylation of idh increases the generation of mitochondrial nadph, which elevates the reduced gsh levels. simultaneously, sirt can not only induce the expression of antioxidant enzymes by activating the foxo a, but also enhance the activation of sod and catalase via nad + -dependent deacetylation. , besides sirt -mediated deacetylation, sirt dependent desuccinylation improves ros detoxification via increasing sod activity. these findings reveal a new redox regulation of nad + by sirt -dependent deacetylation in response to oxidative stress, improving the resistance to the detrimental effects of oxidative damage. nad + sustains genomic stability the constant challenges from endogenous ros/rns or exogenous insults, such as radiation, chemical mutagens and carcinogens, render the dna damage a relatively common cellular event. notably, dna damage and subsequent genome instability are major driving forces for tumorigenesis and aging via driving mutation. to sustain the genome stability, cells have evolved a complicated fine-tuning mechanism, termed as dna-damage response (ddr), to detect and repair dna lesions. [ ] [ ] [ ] [ ] as key regulators of multiple dna repair pathways, parps and sirtuins to maintain the redox homeostasis, both enzymatic and non-enzymatic antioxidant system components exhibit their effects in coordination with each other to contract with the ros. gsh, the most abundant of non-enzymatic antioxidants, is synthesized from glutamate, cysteine and glycine catalyzed by two consecutive cytosolic enzymes, gcl and gs. importantly, nadph serves as the reductive power for ros-detoxifying enzymes including glutathione reductases (gr) and thioredoxin reductases (trxr) to maintain the reduced forms of gsh and trx (sh) in response to ros produced from mitochondria or noxs. abbreviations: pgd, -phosphogluconate dehydrogenase; cyps, cytochromes p ; g pd, glucose- -phosphate dehydrogenase; gcl; gr, glutathione reductases; gs; lox; nad, nicotinamide adenine dinucleotide; noxs, nadph oxidases; nadph, nicotinamide adenine dinucleotide phosphate; oxphos, oxidative phosphorylation; prx, peroxiredoxin; gpx, glutathione peroxidases; sod / , superoxide dismutase and ; trx, thioredoxin; trxr, thioredoxin reductases; xo, xanthine oxidase modulate the post-modification of repair components using nad + as co-substrate (fig. ) . consistently, nad + deficiency leads to an impaired ddr and an increased genomic instability, suggesting an interplay between genomic stability and nad + metabolism. [ ] [ ] [ ] dna ligation. the pathological dsbs are primarily repaired by the nhej, a process involves synapsis, end-processing and ligation. , dna ligases-mediated dna end ligation is initialized by adenylating the ligase with an amp moiety. in prokaryotes, both atp and nad + are adenylation donor for dna ligases, while in eukaryotes, only atp is known to be used by dna ligases for the adenylation. recently, it has been reported that human dna ligase iv, a crucial enzyme in nhej, can acquire amp moiety from nad + for dna end ligation. the brca c-terminal (brct) domain of ligase iv is required to recognize of nad + for subsequently ligation in nhej. although future studies will be required to fully characterize the structure of nad + -associated human dna ligase iv, these findings reveal that like atp, nad + can serve as a provider of adenylation for dna ligation in the nhej dna repair pathway. dna repair. beyond regulating the nhej pathway acting as an adenylation donor, nad + also modulates other dna repair pathways via activating nad + -consuming enzymes, parps and sirtuins. , as sensitive dna damage sensors, paprs are recruited and immediately activated by dna breaks. the dnabound parps, such as parp - , can attach the mono-adp-ribose (mar) or poly-adp-ribose (par) moieties directly to the dna breaks. [ ] [ ] [ ] [ ] meanwhile, parps also catalyze the adpribosylation of various proteins that facilitate the chromatin relaxation and the recruitment of repair factors. [ ] [ ] [ ] [ ] [ ] [ ] the effect of parps to stimulate chromatin decompaction might be exerted via the steric hindrance of par chain, the negative charge of dna and par, or the displacement of core histones. , simultaneously, the accumulated pars at dna break sites are required for the recruitment of dna repairs, including xrcc , ddb , alc , recq , chd , brca , ligase v, mre and nbs , to initiate dna repair. [ ] [ ] [ ] [ ] similarly, dna damage induces the relocalization of nad + -dependent deacetylase sirt to dna breaks, which promotes dna repair via opening the chromatin and recruiting the main dna repair factors including ku , nbs , wrn, kap , xpa and apex . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] additionally, parps and sirtuins also simulate genomic damage-signaling kinases, including atm, p , dna-pk, cirbp and foxos, to accelerate dna repair. [ ] [ ] [ ] [ ] [ ] given that dna damage-activated parps account for up to % of cellular nad + consumption, the dna repair activity is highly dependent on the cellular nad + concentration. , , as expected, decreased nad + levels induce the accumulation of dna damage, whereas replenishing intracellular nad + stimulates the dna repair. [ ] [ ] [ ] in contrast to the positive effect of nad + on dna repair, nadp + suppresses the adp-ribosylation-mediated dna damage repair via functioning as an endogenous inhibitor of parps. the structure of nadp + similar to that of nad + renders its binding to parps. however, nadp + has an additional phosphate group on the ' position of the ribose ring, which abolishes the formation of linear par chain. nad + manipulates the gene expression cellular metabolism, such as nad + metabolism, is directly connected to gene expression through regulating the posttranslational modifications (ptms) of histones, the covalent chemical modifications of dna, the activity of transcription factor and rna processing. , fig. nad + serves as a pivotal regulator of gene expression. nad + and its metabolites are used as substrates and cofactors for reactions that coordinate genomic stability, epigenetic status and rna processing through nad + -dependent enzymes. nad + -dependent histonedeacetylases, especially sirt , possess deacetylase activities on multiple transcription coactivators as well as histones, resulting in epigenome remodeling. the lower activity of sirtuins upon lower level of nad + may contribute to histone hyperacetylation and aberrant gene transcription. using nad + as a (adp)-ribose donor, parps mediate adp-ribosylation on itself or on a variety of nuclear target proteins such as topoisomerases, dna polymerases, histones and dna ligases, playing roles in genome stability and gene regulation, from chromatin to rna biology. recently, it has been found that nad + can also serve as a nucleotide analog in dna ligation and rna capping in response to stresses. abbreviations: ctcf, ccctc-binding factor histone modification. histone modification is one of the most crucial epigenetic modification that affects dna structure and gene expression. the post-translational modifications of histones include acetylation, adp-ribosylation, phosphorylation and methylation. among these modifications, the acetylation and adpribosylation are regulated by nad + -dependent enzymes, sirtuins and parps, respectively. sirtuins, also known as nad + -dependent class iii hdacs, remove the acetyl groups from histone, which restores the electrostatic affinity between dna and histones to stabilize the chromatin architecture. , sirt - maintain the chromatin structure via deacetylation of a crucial histone residue, h k . the reduced intracellular nad + concentration limits the deacetylase activity of sirt , resulting in elevated h k ac and gene expression. , sirt can coordinate nf-κb to deacetylate the h k ac, sequestering the expression of glucocorticoid receptors (grs). sirt is able to selectively deacetylate the h k ac, which represses the expression of a specific set of gene targets that is linked to oncogenic transformation. histones also serve as acceptors of adp-ribose upon dna damage to initiate dna repair. the adp-ribosylation of histones by parp- induces the dissociation of nucleosomes, leading to the decompaction of chromatin. furthermore, parp- -mediated parylation of kdm b prevents the demethylation of h k me , rendering the exclusion of h and the opening of chromatin. the decompensation of chromatin structure, therefore, allows the loading of the transcriptional machinery and facilitates gene transcription. dna modification. dna methylation is another widely studied epigenetic modification that is often involved in the regulation of gene expression. nad + deficiency can promote the dna methylation, resulting in gene silencing. nad + depletion elevates the methylation of bdnf promoter, triggering the dissociation of the dna methylation-sensitive nuclear factor ccctc-binding factor (ctcf) and cohesin from the bdnf locus. the detachment of these factors causes the altered methylation and acetylation of histone at this locus, leading to chromatin compaction and gene silencing. the nad + -consuming enzymes, parps, are associated with the regulation of dna modification. inhibition of the parps-mediated adp-ribosylation causes a chromatin compaction dna hypermethylation. parp- can be activated by the chromatin insulator ctcf even without niched dna and efficiently automodified, dependent on nad + . the pars of parp- compete with dna for the noncovalently binding dnmt , causing suppression of its methyltransferase activity. , therefore, the nad + -dependent enzymatic activity of parp- is a crucial regulator of gene expression via protecting the genome from aberrant hypermethylation. another evidence linking the nad + metabolism with dna methylation is the nnmt, which transfers the methyl group from sam to nam, producing s-adenosylhomocysteine (sah) and a stable metabolites -methylnicotinamide (mna). given that the sam is the universal methyl donor for methylation of substrates including proteins, nucleotide acids and lipids, the nnmt induced methyl sink in the form of mna impairs the genome methylation. , moreover, the methionine metabolism catalyzed by nnmt diverts the nam from the nad + salvage pathway. as a consequence, the reduced cellular nad + content restricts the parp -catalyzed adp-ribosylation and the following dna methylation. collectively, increased nnmt expression inhibits the dna methylation through not only decreasing the cellular nad + , the donor for adp-ribosylation required for methylation, but also reducing the level of sam, the methyl donor for methylation. nad + modulates rna processing decorating of rna as a nucleotide analog. chemical modifications of the rna '-end play a pivotal role in various biological functions including the protection of rna from exonuclease cleavage, the recruitment of proteins for pre-mrna processing and nuclear export and the initiation of protein synthesis. recently, nad + has been found to be incorporated into rnas as an initiating nucleotide during transcription to form nad + cap in different organisms including bacterial, yeast, human and virus. [ ] [ ] [ ] [ ] [ ] unlike the well-characterized m g-capped mrna, which maintains highly stability of mrna for translation, the nad + -capped rnas are vulnerable to decay and are inefficiently translated in cells. , , nad + capping can be catalyzed by eukaryotic nuclear rnap ii using nad + and nadh as non-canonical initiating nucleotides (ncins) in de novo transcription initiation. besides the rnas produced by nuclear rnap ii, rnas synthesized by mitochondrial rnap (mtrnap) can also be nad + capped. the human mtrnap conducts a higher efficiency of nad + capping than the nuclear rnap ii, leading to~ % nad + capping of mitochondrial transcripts. the ′ end nad + cap of rnas in the cytoplasm will be removed by two mammalian hydrolases, dxo and nudt . reported as a denadding enzymes, the nudt contributes to the decapping of rna following exposure to nutrient stress, such as glucose deprivation, while dxo is responsible for the environmental stress, such as heat shock. , [ ] [ ] [ ] in line with that nad + -capped rna levels respond to the cellular total nad + concentrations, the capping efficiencies of nad + capping and nadh capping are also regulated by intracellular nad + /nadh ratio. , these results raise the possibility that rnap ii and mtrnaps might function as both sensors and executors, which sense nad + /nadh ratios and induce the nad + capping to regulate the gene expression, leading to the crosstalk between cellular nad + metabolism and transcriptional activity. adp-ribosylation of rna. beyond decorating rna as a nucleotide analog, nad + also provides the adpr groups for the reversible mono-adp-ribosylation of rna phosphorylated ends. this rna modification is catalyzed by parp with a preference for ′ ends, depending on nad + concentration. in addition to parp , trpt , a parp homolog, also catalyzes the adp-ribosylation of rna. the adp-ribosylation renders rna resistant to phosphatase, which might protect the rna from the nuclease attack. similar to the reversible adp-ribosylation of proteins and dna, the adpribosylation of rna can also be efficiently reversed by several cellular hydrolases including targ , macrod - , parg, nudt and arh viruses. besides human hydrolases, macrodomaincontaining hydrolases from veev and sars can remove the adpribosylation of rna catalyzed by parps, suggesting a potential mechanism of pathogenesis via inhibiting the antiviral activity of ifn-stimulated genes, parps. altogether, the adp-ribose moieties attached to rna end might protect rna against degradation or serve as a platform for recruiting proteins, controlling the functional state of rna. nad + facilitates cellular energy metabolism nad + /nadh as hydride-donating coenzyme for metabolism. acting as a coenzyme, nad + plays pivotal roles in energy metabolism pathways including glycolysis, the tca cycle, oxphos, fao and alcohol (ethanol) metabolism. the glycolysis process begins with one glucose molecule and ends with two molecules of pyruvate, which are subsequently transported into the mitochondria to begin the tca cycle. nad + promotes glycolysis by facilitating the enzymatic reactions catalyzed by gapdh and lactate dehydrogenase (ldh), which use nad + as a coenzyme. , nad + is reduced to nadh coupled with the oxidation of g p to , -bp by gapdh. cytosolic pyruvate can also be converted to lactate by ldh, coupled with the oxidation of nadh to nad + . this process helps maintain the cytosolic level of nad + , thus contributing to the continuity of glycolysis. when transported into the mitochondria, the glycolytic end-product pyruvate is decarboxylated to produce acetyl-coa by pdh complex, which reduces nad + to nadh simultaneously. acetyl-coa then starts the tca cycle, where nad + serves as a coenzyme for three ratelimiting enzymes, α-ketoglutarate dehydrogenase (kgdh), isocitrate dehydrogenase (idh ) and malate dehydrogenase (mdh ), to generate nadh. thus, the tca cycle can convert four molecules of nad + to nadh using one molecule of pyruvate in the mitochondria under aerobic conditions. as an electron donor, nadh produced in the tca cycle plays a crucial role in atp synthesis by oxphos, which generates most of the energy through the h + gradient in animal cells. fao breaks down a long-chain acyl-coa, which is generated from fatty acid and coenzyme a by acyl-coa synthetase in the cytosol, to generate acetyl-coa, nadh and fadh in the mitochondria. this process is performed in repeated cycles, each of which removes a two-carbon acetyl-coa from the acyl-coa via four enzymes, the enoyl-coa hydratase, ketoacyl-coa thiolase, acyl-coa dehydrogenase (acads) and hydroxyacyl-coa dehydrogenase (hadh). the last cycle generates two molecules of acetyl-coa. fadh is generated by acads, while nadh is produced from nad + in the reaction catalyzed by hadh. both nadh and fadh generated in the fao are utilized to synthesize atp by the etc. nad + is also a cofactor in alcohol oxidation metabolism taking place mainly in liver cells. alcohol oxidation is completed in a twostep reaction by two enzymes, alcohol dehydrogenase (adh) and aldehyde dehydrogenase (aldh), which catalyzes the reduction of nad + to nadh. both the sufficient glycolysis and the effective oxidation of alcohol require fast reoxidation of nadh to nad + through the coordinated reduction of pyruvate to lactate by ldh or production of atp by mitochondrial etc. , nad + -dependent modification of metabolic enzymes. beyond serving as a hydride-donating coenzyme for metabolism, nadh/ nad + also acts as co-substrate for the sirtuins-mediated posttranslational modification of metabolic enzymes including acetylation, adp-ribosylation, succinylation and malonylation. a large number of enzymes that participated in cytosolic glycolysis, gluconeogenesis, the urea cycle, nitrogen metabolism, glycogen metabolism, mitochondrial fatty acid oxidation, the tca cycle and amino acid catabolism can be regulated by sirtuins. , the mitochondrial sirtuin-related acetylome covers almost all the mitochondrial metabolism, including the enriched sirt related tca cycle, etc, fao, the sirt -associated anion transporters, the translation and energy metabolism, the sirt -regulated tca cycle and branched chain amino acid catabolism (bcaa) metabolism. , the mitochondria sirt function as a metabolic sensor that links the cellular energy status with the mitochondrial protein acetylation patterns. in healthy mitochondria, sirt interacts with atp o, while the low ph owing to the loss of membrane potential weakens the binding affinity between sirt and atp o, leading to the redistribution of sirt to other mitochondria substrates. the ph-insensitive association between sirt and atp o provides a fundamental role for sirt in resetting mitochondrial acetylation in response to stress. the transition to fasting enhances both the cellular nad + level and the sirt expression, which, in turn, catalyzes the deacetylation of lcad to promote fatty-acid oxidation. sirt orchestrates the metabolism reprogramming via controlling the balance between cytosolic glycolytic metabolism and mitochondrial oxidative metabolism. sirt also plays a regulatory role in proline metabolism via deacetylation of pycr . sirt modulates mitochondria energy homeostasis and longevity based on its lysine deacetylase, lysine deacylase, lipoamidase and ribosylase activity. under nutrient-replete conditions, the deacetylation of malonyl coa decarboxylase (mcd) by sirt plays a pivotal role in lipid homeostasis via suppressing fatty acid oxidation and inducing lipid anabolism. the lysine deacylase activity of sirt is involved in the control of leucine metabolism and insulin secretion through regulating the acylation status of enzymes in these pathways. sirt also acts as a cellular lipoamidase with a preferred catalytic efficiency for lipoyl-and biotinyl-lysine modifications to its deacetylation activity. sirt hydrolyzes the lipoamide cofactors from the e component dihydrolipoyllysine acetyltransferase (dlat), leading to diminished pdh activity. furthermore, sirt uses nad + to adp-ribosylate and reduce gdh activity, thereby inhibits insulin secretion in response to amino acids in β cells. sirt is an nad + -dependent lysine desuccinylase, demalonylase, and deglutarylase. bat specific deletion of sirt exhibits hypersuccinylation of proteins involved in the amino acid metabolism, etc and fao. a bunch of mitochondrial proteins have succinylation modification, such as ucp in thermogenic function, glud in glutamate metabolism, sdh in etc, the tca cycle, gls and cps in glutaminolysis, echa and vlcad in fao, hmgcs in ketogenesis and shmt in serine catabolism. [ ] [ ] [ ] [ ] [ ] sirt -mediated desuccinylation also participates in protection against peroxisome-induced oxidative stress via targeting acox . moreover, sirt functions as a leading regulator of protein malonylation in both cytosolic metabolisms including glycolysis, gluconeogenesis and mitochondria fao. for instance, sirt increases the activity of gapdh by demalonylation, thereby controlling the energetic flux through glycolysis. , , collectively, sirtuins orchestrate an integrated modulation of metabolic pathways via nad + -dependent post-translational regulation in response to diverse nutrient signals. rhythmic nad + oscillates circadian clock organisms have developed internal clocks as a timekeeping mechanism to collaborate biological processes with the exogenous environmental and endogenous factors. nad + functions as a metabolic driver of circadian transcription via epigenetic mechanisms, transducing signals originated by environmental stimuli to the circadian clock. the linkage of nad + metabolism to the internal clocks is firstly evidenced by that the nad(p) + /nad(p)h ratio modulates the dna-binding activity of the core oscillators, such as clock: bmal and npas : bmal heterodimers. the redox state of fad and nadph also displays an oscillation pattern in organotypic slices of suprachiasmatic nucleus (scn). the circadian control of intracellular nad + levels by the clock is attributed to the oscillation expression of nampt, a rate-limiting enzyme in the salvage of nad + with a -hour rhythm. , , [ ] [ ] [ ] the e-boxes in the promoter of nampt gene allow the direct transcriptional control by the clock: bmal chromatin complex. furthermore, the expression of enzymes in the nad + salvage pathway, including nmrk , nampt, and nadk, has circadian oscillation patterns in wt and liver-re mice that exclusively express bmal in the liver, suggesting the circadian clock might reprogram nad + salvage synthesis to maintain the fluctuation of nad + . the oscillation of nad + , in turn, coordinates the transcription and behavior through the circadian clock. the reduction of nad + in old mice dampens the circadian transcription, which can be rescued by nad + repletion to youthful levels with nr. the regulatory effect of nad + on circadian reprogramming is mediated by changing the activity of sirtuins and parps, which determines the transcriptional activity of core oscillators. sirt / can be recruited into the core clock clock: bmal complex, which renders the rhythmic acetylation of bmal and the cyclic h k / ac at circadian promoters on their target genes. , , besides, the oscillation activation of sirt also regulates the circadian dynamics via deacetylation of the core clock repressor per k and mixed-lineage leukemia (mll ), thereby controlling rhythmic chromatin property and the activity of bmal : clock complex. , , , , similar to sirtuins, the activity of parps is also regulated by the circadian clock. the oscillation activation of parp- interacts with and poly(adp-ribosyl)ates clock, leading to suppressed binding of clock: bmal to dna and altered circadian gene expression. moreover, parp interacts with ctcf in a circadian manner, regulating lamina-associated chromatin and circadian oscillations in transcription. , these reports indicate a connection between nad + -dependent epigenetic modification and the core circadian clockwork circuitry. the interplay of nad + /nadp + metabolism with circadian clock is further evidenced by the oscillating redox, in which ros levels display a different liver pattern compared to other tissues due to the unique nad + oscillation in response to the autonomous hepatic clock. circadian disruption in beta-bmal (-/-) mice and arrhythmic clock Δ mice decrease the nrf expression and subsequently impair the antioxidant defense system, contributing to increased ros accumulation, oxidative damage and mitochondrial uncoupling. , prxs, the most critical h o -removing enzymes, exhibit rhythmic cycles of oxidation. the circadian clock system can also regulate the production and consumption of gsh through circadian regulation of the rate-limiting enzymes in gsh biosynthesis and cellular detoxification. the oxidation cycle of both prxs and gsh is directly influenced by the availability of redox cofactor nadph, suggesting that nadph metabolism might play a vital role in controlling redox rhythmic and transcriptional oscillations. in line with this notion, it has been demonstrated that inhibition of nadph production from ppp alters circadian rhythms through changing the activity of clock: bmal . [ ] [ ] [ ] thus, nad(p) + /nad(p)h acts as an important modulator of cellular energetic status, enabling the reset of redox rhythmic and transcriptional oscillations based on metabolic signals. nad + metabolism programs immunity and inflammation nad + , along with citrate and succinate, is a novel class of metabolites with inflammatory signaling capacity, linking the nad + metabolism to the programming of immune responses. restoring the nad + levels via de novo biosynthesis in the liver prevents hepatic lipid accumulation and attenuates inflammation in mice on a high-fat diet (hfd). similarly, increased generation of nad + via the kp in resting, aged or immune-challenged macrophages restores oxphos and homeostatic immune responses, whereas inhibition of de novo nad + synthesis induces an increased inflammation-associated tca-cycle metabolite succinate and elevated mitochondria-generated ros, resulting in rising innate immune dysfunction in aging and age-associated diseases. mitochondrial complex iii produces ros immediately after stimulation, which has an essential role in inflammatory macrophage activation. however, the mitochondrial ros are also responsible for dna damage, which causes the abundant consumption of nad + by parps. the nad + abundance as well as the nad + /nadh ratio, therefore, decline significantly even with the induction of the de novo synthesis from the kp in response to the lipopolysaccharide (lps) challenge. , to maintain the cellular nad + level, nad + salvage enzyme nampt has been activated by lps to boost the salvage pathway. elevated expression of nampt maintains the nad + content to drive the glycolysis, which supports the activation of inflammatory macrophages. while in the mitochondrial respiration-impaired cells, nad + could reduce the exacerbated inflammatory response via improving lysosomal function. the addition of nicotinamide precursor nam in mitochondrial respiration-impaired cells restores the lysosomal function and limits the increased proinflammatory profile. furthermore, endotoxin dose-dependent switch of nad + biosynthesis pathways from nampt-dependent salvage to ido -dependent de novo biosynthesis maintains the nuclear nad + pool, which promotes sirt -directed epigenetic regulation of immune tolerance. , owing to its rate-limiting enzymatic activity in nad + salvage pathway, the elevated expression of nampt in the innate immune cells, including macrophages and dendritic cells, further proposed a link between intracellular nad + levels and inflammation. [ ] [ ] [ ] specific competitive inhibitor of nampt could ameliorate the immunity or inflammatory disorders, including dss-induced colitis, arthritis via reducing intracellular nad + levels in inflammatory cells and circulating inflammatory cytokines, including il- beta, tnfalpha, and il- . - cellular levels of nad + regulated by nampt also impacts nad + -dependent enzymes, such as sirtuins. for example, sirtuins modulated the optimal tnf translation. the elevated nad + levels concomitant with sirt switches the nf-κbdependent transcription into the relb-dependent transcription of the tnf-α in endotoxin tolerant sepsis blood leukocytes. additionally, sirt can modulate tnf production by regulating the tnf mrna translational efficiency. in a pancreatic cell line, sirt induces the production of cytokines including il- and tnf, which promote cell migration. sirtuins control immune responses via directly regulating inflammatory transcription factors, including deacetylation of foxp to repress treg cell responses, deacetylation of rorγt to promotet h cell responses, and suppression of nf-κb to reduce inflammatory responses. besides nad + , nadph also plays essential roles in immunity and inflammation, mainly dependent on the nadph oxidases and redox signaling. in an inflammatory response, activation of epithelial and immune cells triggers noxs to generate ros, which can directly kill microorganisms. [ ] [ ] [ ] noxs-derived ros can also act as a second messenger in signaling transduction. it has been reported that nox directly interacts with tlr , which is pivotal for lps-mediated nf-κb activation. in the nasal airway epithelium, the interaction of tlr and another nox isozyme, duox , induces the ros generation and il- expression in response to flagellin exposure. , the phagocytic nadph oxidase complex can also be activated by rubicon to induce a ros burst, inflammatory cytokine production and potent antimicrobial activities. given the essential regulatory role of nad + in fundamental physiologies, nad + metabolic abnormalities contribute to the pathophysiology of various diseases, such as infection, cancers, metabolic diseases, aging and age-associated neurodegeneration disorders. perturbed nad + metabolism in response to infection microbial infection, including viruses and bacteria, causes an imbalance in the cellular redox environment, thus inducing different responses, e.g., antioxidant defenses, cell signaling, immune response and other processes. nad + or nadph level determines the role of ros in infections, either protecting against invading microorganisms or causing tissue damage during the resulting excessive inflammation (fig. ) . nad + mitigates viral infection-induced oxidative damage. oxidative stress is implicated as a pathogenic factor in viral infection. it can be caused by diverse virus families ranging from dna (i.e., hbv, ebv, hsv- ) to rna viruses (i.e., hcv, rsv, denv, influenza, zika, hiv). [ ] [ ] [ ] [ ] [ ] the increased cellular ros by viral infection cause dna damage, gene mutation, cell death, viral dna integration and tumorigenesis. [ ] [ ] [ ] [ ] [ ] [ ] [ ] for instance, acute phase of hcv infection induces oxidative stress via enhancing noxs expression and activity to generate ros generation and decreasing gsh, which supports the high rates of viral replication and apoptotic cell death. on the other hand, the chronic infection maintains a reduced environment to establish viral persistence. moreover, nox-induced ros play various roles in the mechanisms of oncogenesis by hcv, including genome instability, epigenetic regulation, inflammation and modulation of cell growth and death. in rsv-infected airway epithelial cells, nox-generated ros trigger the activation of the transcription factors irf and stat, thereby inducing the expression of chemokines and cytokines involved in the immune/inflammatory responses of the lung. nqo , an enzyme involved in the elimination of ros, is inhibited by hbx, leading to decreased gsh levels and increased susceptibility of hepatoma cells to oxidative damage, cumulating in hbv-associated pathogenesis of chronic liver diseases. to repair the oxidative stress-induced dna damage, a large amount of nad + were consumed by elevated parps in response to virus infection, i.e., hsv- , zikv and new sindbis virus (sv). , , beyond the important role in dna repair, parp- also acts as a modulator of nf-κb, inducing the downstream ccl -ccr signaling, which is required for the recruitment of nk cell to the infection site and viral control. therefore, parps have antiviral activity against multiple classes of viruses, including retroviruses, alphaviruses, filoviruses, herpesviruses, adenoviruses and coronavirus through enhancing the innate immunity. [ ] [ ] [ ] [ ] sirtuins are another class of nad + -consuming enzyme, which have broad-range antiviral properties on diverse viruses including hcv, hiv- , hcmv and influenza a (h n ) virus. [ ] [ ] [ ] [ ] [ ] besides controlling the virus replication, parps or sirtuins also contributes to the oncogenic virus infection, such as oncogenic gamma herpesviruses kshv and epstein barr virus (ebv) infection, and the tumorigenesis through the epigenetic remodeling. [ ] [ ] [ ] cd is the third nad + -consuming enzyme that is upregulated in response to a number of viral infections. [ ] [ ] [ ] [ ] cd is under the transcriptional control of rsv-induced ifns. the cd generated cadpr, in turn, augments the ifns-induced isgs and nf-κb-mediated inflammation, leading to the antiviral hyperinflammation response. in addition to the excessively increased consumption of nad + , multiple viruses cause a decline in nad + concentration through reducing the protein levels of crucial enzymes in the nad + biosynthesis pathway, including qprt and nampt. , regarding the redox role of nadh/nadph in eliminating ros, the depletion of nad + /nadp + pool exacerbates the oxidative damage during virus infection. , , [ ] [ ] [ ] nad + contributes to the bactericidal activity. bacterial infection induces rapid production of intracellular ros either by noxs or mitochondria that are, in turn, crucially required by macrophages to clear bacteria. , elimination of the ros results in defective bactericidal activity, allowing bacteria to survive and repeatedly colonize various tissue sites. , noxs in immune cells, such as macrophages and neutrophils, are primarily responsible for ros production and termed respiratory bursts during phagocytic bacterial killing. , additionally, nox -generated ros are necessary for lc recruitment to phagosomes, revealing an autophagy-dependent antibacterial activity of nox in phagocytes. mycobacterium tuberculosis (mtb) can trigger the production of ros via depletion of nad + . tnt, a major cytotoxicity factor of mtb, hydrolyzes the cellular nad + to nam and adpr, thereby activating the necroptosis effectors mlkl and ripk . moreover, the nad + depletion or the nad + hydrolysis products induced signaling contributes to the tnt-triggered ros production. moreover, the nox-dependent oxidative burst caused by phagocytosis of bacterial cells activates cd , producing naadp in the maturing phagosome. naadp induces the lysosomal ca + efflux and calcineurin-mediated tfeb activation, which enhances the expression of pro-inflammatory cytokines including il- β, il- α and il- . cd also exerts bactericidal activity in an nad + -dependent manner. , cd controls neutrophil chemotaxis to bacterial chemoattractants via producing cyclic adpribose. in macrophage, high levels of cd induced by lxr agonists reduce nad + levels and interfere with cytoskeletal meanwhile, oxidative stress causes the host dna damage that enhances the consumption of nad + by elevated parps. the intracellular nad + can also be reduced by activation of cd that is required for the inflammation against infection. the nad + deficiency therefore might not be able to support the clearance of microbial by autophagy or phagolysosome, the innate immune and inflammation response. abbreviations: ebv, epstein-barr virus; hcv, hepatitis c virus; hrv, human rhinovirus; hrsv, human orthopneumovirus; inos, inducible nitric oxide synthase; isgs, interferon-stimulated genes; iv, influenza virus; kshv, kaposi's sarcoma-associated herpesvirus; mpo, myeloperoxidase; mtb, mycobacterium tuberculosis; noxs, nadph oxidases rearrangements triggered by invasive bacteria, protecting host macrophages from substantial infection. however, in t-cell, the nad + depletion by elevated cd expression increases the acetylation of ezh by in a sirt -dependent manner, leading to reduced cytotoxicity of cd t cell and enhanced inclination to infections in patients with systemic lupus erythematosus (sle). additionally, the nad + concentration and nad + /nadh ratio are significantly elevated in response to group a streptococcus (gas)infection. the addition of nam remarkably enhances the intracellular nad + content that promotes the autophagosomal acidification and clearance of gas in endothelial cells. therefore, nad(p) + /nad(p)h exerts the bactericidal activity by promoting the ros generation, the pro-inflammatory response and the anti-infection autophagy. nad + deficiency accelerates aging multiply evidence elucidates that nad + and nad + -related metabolites govern biological functions in aging, including metabolism, redox homeostasis, mitochondria function and the circadian clock. the nad + decline during normal aging results in oxidative damage, metabolic disorder, circadian rhythm abnormalities, and mitochondrial dysfunction through regulating signaling pathways, such as p , nf-κb, pgc- α and hif- α, by sirtuins and parps. [ ] [ ] [ ] [ ] accordingly, boosting nad + provides a therapeutic option for improving the health lifespan and treating aging-related diseases. nad + deficiency accelerates aging. nad + levels display steadily reduction in old worms, which causes a further shorter lifespan. similarly, mice and rats exhibit an nad + decline during aging in a variety of tissues, such as muscle, adipose tissue, brain, skin, liver and pancreas. the reduced nad + is also observed in the aged human brain and liver. in line with that, the plasma levels of nad + and its metabolites, nadp + and naad also remarkably decline during aging. the age-dependent decline of nad + might be due to either enhanced consumption or reduced biosynthesis. the nad + levels and nampt expression are severely inhibited in various tissues including liver, skeletal muscle, wat and pancreas in age induced t d models. the decreased nampt might be due to the chronic inflammation and impaired circadian clock during aging. , however, another study describes no alteration of the nampt mrna or protein levels in aged mice and human tissues. thus, these controversial findings of nampt-catalyzed nad + biosynthesis overage might come from the differential cell type and tissue context, which will be elucidated in future studies. another explanation for nad + decline with age is the increased nad + consumption by parp or cd . in contrast to the unchanged levels of parp , both the protein levels and enzymatic activity of cd are enhanced during aging, contributing to the age-related nad + decline in mammals. cd is also responsible for the mitochondrial dysfunction by regulation of sirt activity. nevertheless, cd -deficient old mice preserve the nad + levels, mitochondrial respiration and metabolic functions. cd expression might be induced by chronic inflammation, one characteristic during aging. , the nad + decline is a primary driver for the progressive of biological dysfunction and age-related pathologies. thus, the genetic or pharmacological modulation of nad + provides a therapeutic option for multiple age-related diseases. indeed, genetic and pharmacological replenishment of nad + improves the age-related biologic function and increases lifespan at least in worms and mice. , the increased expression of nampt in aging human smcs prolonged lifespan via delaying senescence and enhancing resistance to oxidative stress. supplementation with nad + precursors, nr and nmn, elevated nad + levels that can maintain the mitochondrial and metabolic functions by activating sirt in mice, leading to an extensive lifespan of mice. , , augmentation of nad + by β-lap, a potent substrate of nqo , effectively prevents arhl and its accompanying harmful effects by preventing oxidative stress and inflammation and improving mitochondrial function in rodents. moreover, mounting evidence has shown that nad + -dependent sirtuins can extend the lifespan of yeast, worms, flies and mice and alleviate many diseases of aging-related pathologies. for instance, both brain-specific or whole-body sirt -overexpressing transgenic mice exhibit a slowed aging and a prolonged lifespan. , aging-related nad + decline causes mitochondrial dysfunction. combining the evidence that mitochondrial dysfunction is a hallmark of aging and nad + plays a crucial role in the maintenance of mitochondrial function. , , we can hypothesize that aging-related nad + decline might be the cause of mitochondria dysfunction. nad + boosters play a preventive role in aging via the early-phase activated upr mt , and the late-phase induced antioxidant defense. regulation of nad + availability by parp inhibitors and nad + precursor modulates mitochondrial function through sir- . in worm to extend lifespan. the parylation is also markedly increased in muscle and the liver of aged mice in parallel with robustly decline of nad + levels. since csb can limit the activity of parp via displacing it from damaged dna, in csb-deficient cells and mice, the paprps-mediated parylation is increased and accounts for the majority of cellular nad + consumption. this aberrant activation of parps represses the sirt activity and mitochondrial dysfunction, which can be rescued by both parp inhibitor and nad + precursors. agingrelated nuclear nad + decline inhibits the mitochondrially encoded genes via the sirt -hif- α-c-myc pathway, while boosting nad + levels rescues the mitochondrial function in old mice in a sirt -dependent manner. nad + also affects the acetylation and activity of oxidative enzymes in mitochondria via altering sirt activity. the circadian activity of sirt induced by nad + oscillation regulates the rhythmic acetylation and activation of oxidative enzymes and respiration in isolated mitochondria. nad + ameliorates the oxidative damage during aging. there is a growing awareness that oxidative damage is an essential driver of age-related deterioration in cell function. , the dna oxidative damage and protein oxidation in the aged human brain are associated with declined antioxidant enzyme activities. , agerelated increase in oxidative stress and cell senescence leads cells/ tissues to be more prone to undergo necroptosis, thereby releasing damps that trigger the chronic inflammation observed with aging. the pro-inflammatory cytokines, in turn, augment both mitochondrial and nox-generated ros, contributing to further accumulation of oxidative damage (fig. ). [ ] [ ] [ ] [ ] nadh/nadph is a powerful reduce source for buffering oxidative stress, thereby protecting cells/tissues from oxidative stress during aging. the remarkable reduction of nad + concentration and nad + /nadh ratio in aged rats occurs in parallel with enhanced oxidative stress and diminished antioxidant capacity. nmn addition in isolated aortas elevates the nad + and mnsod levels, thus enhancing the antioxidant capacity. the overexpression of nmnat efficiently boosts nad + in a variety of murine tissues, which significantly suppresses the ros generation, and protects from aging-related insulin resistance. overexpression of g pd promotes nadph production, preventing tissue from oxidative damage to improve mice health span. nad + also regulates oxidative stress in cellular senescence by regulating sirtuins and parps. nad + -dependent sirt is significantly upregulated in response to oxidative stress, protecting heart from oxidative damage, contributing to retard of aging. nad + deficiency correlates with disturbed circadian clocks during aging. besides mitigating the oxidative damage, nad + can extend lifespan by driving the circadian rhythms. the misalignment of the circadian clocks, the internal timekeeper mechanism that links metabolism with the exogenous and endogenous factors, has been associated with the acceleration of aging. the circadian sirtuins link the nad + metabolism to the circadian clock machinery during aging. sirt induces the circadian transcription of core clock genes, such as cry , per , rorγ, and bmal via either rhythmically deacetylating bmal or per . , sirt also modulates clock-mediated chromatin remodeling at h lys /lys at circadian promoters to control circadian. in the scn of aged mice, sirt level is significantly decreased, resulting in a reduction of bmal and other circadian proteins. the autonomous hepatic clock induces the nad + salvage pathway to partially restore nad + oscillation, driving the sirt circadian function in the liver even without inputs from other clocks. therefore, nad + -dependent sirt regulates the aging-dependent decline in central circadian function. the critical role of nad + pool in tumorigenesis nad + not only acts as a co-enzyme for metabolic redox reactions, but also functions as a co-substrate to modulate the activity of nad + -consuming enzymes that govern the critical steps in tumorigenesis, including genome stability, metabolism, cell growth, cell death, redox homeostasis and immune response. the sirtuins and parps in tumorigenesis exhibit both oncogenic and tumor suppressor activity, which might be determined by their sublocalization and cell type (fig. ) . nad + -related metabolic reprogramming and redox homeostasis in tumorigenesis. cancer cells undergo metabolic reprogramming that provides the substrates and energy for biomass generation, to sustain the stress response and continuous proliferation. the metabolic reprogramming is characterized by shifting glucose metabolism to aerobic glycolysis, including enhanced cytosolic lactate fermentation and ppp and decreased oxphos. this shift not only allows for rapid production of energy but also for maintenance of nadh/nad + redox ratio, which is required for metabolic processes, such as aerobic glycolysis, the tca cycle, oxphos, fao, serine biosynthesis and antioxidant defense. [ ] [ ] [ ] cytosolic nad + is required for glycolysis, in which gapdh converts nad + to nadh. in health cells, cytosolic nadh is shuttled into mitochondria, where it is turned into nad + by oxphos whereas in cancer cells, the conversion of nad + to nadh in mitochondria is not sufficient for the high rate of glycolysis due to reduced oxphos. therefore, cancer cells enhance the cytosolic lactate fermentation to generate nadh by ldha. activation of ldha by oncogenic receptor tyrosine kinase fgfr promotes glycolysis and tumor growth by increasing the nad + /nadh ratio, while the aberration of nad + /nadh due to reduced activity of mitochondrial complex i promotes the aggressiveness of human breast cancer cells. the 'hyper-metabolism' of cancer cells causes the excessive generation of ros. ros contribute to tumorigenesis through multiple processes, including causing oxidative dna damage, genomic instability and inflammatory stress to drive malignant transformation, and acting as a messenger to regulate signaling pathways to support tumor initiation, development, and angiogenesis. , , , [ ] [ ] [ ] [ ] cancer cells build a complicate and powerful antioxidant system, such as the gsh and trx systems, to adapt to the high ros levels. notably, both gsh and trx systems rely on the reducing power of nadph, which is generated fig. nad + deficits in aging-associated dysfunction and cancer. environmental stimuli, including nutrient perturbation, infection, radiation and inflammation, induce oxidative stress, which causes the damage of cellular biomolecules and organelles. nad + and its metabolites function as crucial regulators to maintain cellular redox homeostasis through replenishing the reducing power or modulating the activity of nad + -consuming enzymes including sirtuins and parps. however, disequilibrium of nad + metabolism could disturb physiological processes, including mitochondria function, circadian rhythm, inflammation, dna repair and metabolism, leading to aging-associated dysfunction and cancer. nad + levels could be augmented by dietary nad + precursor, inhibitors of nad + -consuming enzymes, caloric restriction and exercise. nad + boosters restore the bioenergetics, redox balance and signaling pathways, ameliorating the adverse effects of pathophysiological conditions, including infection, aging and cancer. abbreviations: -hg, -hydroxyglutarate; α-kg, α-ketoglutarate; ccgs, clock-controlled genes; foxo , forkhead box o ; gsh, glutathione; idh mt , mutant isocitrate dehydrogenase ; noxs, nadph oxidase; per , period circadian clock ; ppp, pentose phosphate pathway; pgc- α, peroxisome proliferator-activated receptor-gamma coactivator alpha; ros, reactive oxygen species; oxphos, oxidative phosphorylation; tca cycle, tricarboxylic acid cycle by g pd in ppp, me in glutamine metabolism and nnt. in cancer cells, increased ros will oxidize the specific isoform of pyruvate kinase (pkm ), diverting glucose flux towards the ppp and generation of nadph for gsh recycling. , , similarly, nutrition stress or oxidative stress induces the expression of enzymes, including nampt, me and nnt, which augments the nadph to support the cell survival under glucose deprival and anoikis conditions, thereby promoting tumor growth and metastasis. [ ] [ ] [ ] idh mutations in human cancers favor consuming the nadph for -hg synthesis at the expense of other nadph-requiring pathways essential for cell viability even when nadph is limiting. additionally, ampk activation by reduced atp levels maintains nadph through inhibiting nadph consumption in fatty-acid synthesis and enhancing nadph production from fattyacid oxidation instead of ppp to inhibit cell death. however, nad + depletion exacerbates oxidative damage via reducing the antioxidant defense capacity, resulting in impaired cell proliferation and increased cell death. , the nad + /nadh ratio emerges as a fine-tuned signal to regulate redox status through sirtuins. sirtuins manipulate the metabolism reprogramming via directedly altering the activity of metabolic enzymes by nad + -dependent modification or changing their expression by regulating transcription factors. all the enzymes but pgi in the glycolysis and the tca cycle can be acetylated under the control of sirtuins. gapdh and pkm are two major enzymes in glycolysis that regulated by sirtuins. sirt can bind and retain gapdh in the cytosol, but sirt actives gapdh via demalonylation, thereby elevating glycolytic flux. both sirt catalyzed deacetylation and sirt -mediated desuccinylation of pkm reduce the activity of pkm , preventing the carbon entry into the tca cycle. in contrast, sirt deacetylates and activates pdha acetylation, linking glycolysis and oxphos. sirt and sirt stabilize and activate the enzymatic activity of shmt by its deacetylase and desuccinylase activity, respectively, thereby promoting the serine catabolism to drive carcinogenesis. sirt -catalyzed demalonylation and inactivation of sdha block the tca cycle and induce succinate accumulation, promoting tumorigenesis and drug resistance. , additionally, sirtuins also regulate the expression of metabolic enzymes via transcription factors, such as hif- α. sirt enhances the enzymatic activities of sod and idh to limit ros levels, which repress the metabolism reprogramming in cancer cell via destabilizing hif- α, thereby repressing tumor growth. , , , [ ] [ ] [ ] it has also been demonstrated that the enzymes in glycolysis (e.g., glut , hk , gsk b, and gapdh), the enzymes (e.g., pdpr pdha , and pdhx) in carbohydrate metabolism, and most enzymes in etc and atp synthesis are assigned as adp-ribose amino acid acceptor. however, whether the adp-ribosylation of metabolic enzymes contributes to the metabolism reprogramming in cancer cells requires further investigation. nad + -regulated genome stability and gene transcription in carcinogenesis. nad + metabolism is not only essential for metabolic and redox homeostasis, but also required for epigenetic reprogramming in tumorigenesis. genomic instability and altered transcriptional pattern are well-known hallmarks of cancer. sirtuins and parps control the genome stability and gene transcription by regulating the histone modification, dna repair, as well as the recruitment and activation of transcription factors. , sirt is responsible for the histone acetylation patterns, including the h k ac, h k ac, h k ac and h k ac, associated with tight chromatin compaction. the activity of sirt also modulates the formation of h k me , h k me and h k me to regulate chromatin. for instance, sirt alters the acetylation patterns of histones h and h , including h k ac, h k ac and h k ac, to regulate the expression of cancer-related genes in breast cancer. besides the impact on chromatin state, sirt modulates the non-histone proteins to initiate dna repair and gene transcription. sirt induces the recruitment of dna repair factors, including nbs and rad , in keratinocytes to maintain genome stability and gene transcription. oxidative stress-induced sirt can deacetylase hmof to reduce the expression of dna repair proteins, including rad , brca and fanca in human colorectal cancer cells. hmof also plays crucial roles in transcription activation by h k acetylation in hela and glioma cancer cells. , the contradictory role of sirt in cancer is manifested as their overexpression in neoplasms, such as prostate, aml, non-melanoma or melanoma skin cancer, and colon carcinomas, and the reduced expression in other cancers, including breast cancer and hepatic cell carcinomas. , thus, the mechanisms underlying the regulatory role of sirt in dna repair and gene transcription in cancer development need further exploration. as aforementioned, parps govern the genome stability and gene transcription via nad + -dependent adp-ribosylation. parps-induced adp-ribose marks elevate -to -fold in response to the oxidative genome damage by h o in human osteosarcoma cells. , , a variety of cancers have somatic mutations resulting in genomic disability and defective dna repair, including brca / , atm, chk and tp . the loss of double-strand repair pathway due to brca or brca mutations renders cancer cells more dependent on the parpsmediated repair, and more sensitive to parp inhibition, raising a possibility of a wider application of parpi in cancer therapy. [ ] [ ] [ ] nad + metabolism is also linked to epigenetic modification by nnmt that transfers the methyl group of sam to nam. nnmt is increased in a broad range of cancers, such as papillary thyroid cancer, renal clear cell carcinoma, glioblastoma tumors, bladder cancer, colorectal cancers, gastric cancers, and oral squamous cell carcinoma. , [ ] [ ] [ ] [ ] elevated nnmt inhibits the methylation potential of cancer cells through inducing methyl sink in the form of mna. reducing the nnmt expression impairs the cell proliferation and tumor growth of mesenchymal glioblastoma stem cells (gscs), accompanied by reduced methylation ability. besides, nnmt promotes hcc cell invasion and metastasis by changing the h k me patterns and transcriptionally activating cd . nnmt-mediated cd mrna m a methylation produces a cd v splice variant, while mna stabilizes cd protein by inhibiting the ubiquitin-mediated degradation. furthermore, nnmt depletion elevates the nad(h) + levels that result in an enhanced expression of sirtuin target genes and a reduced h k ac. therefore, nnmt acts as a crucial metabolic modulator of epigenetic modification, promoting the migration, invasion, proliferation and survival of cancer cells. the oncometabolite, -hydroxyglutarate ( -hg), also couples the nadp + /nadph to epigenetic modification, including histone and dna demethylases, in tumorigenesis. mutant idhs, accounting for % of lower-grade gliomas and secondary gbm, continuously produce -hg. to support -hg synthesis, cancer cells with idh r h mutation enhance nadph production via the ppp. , interestingly, idh mutants compete for nadph to synthesize -hg with other pathways that are critical for cell viability, resulting in further disruption of cellular metabolism and redox homeostasis in tumorigenesis. nad + -dependent cancer cell proliferation and metastasis. given the massive demand for nad + to support the metabolism reprogramming, the genome integrity and gene transcription in tumorigenesis, cancer cells enhance the capacity of nad + production through various pathways. it has been demonstrated that tumors that arise from cells with highly naprt expression will rely on nad + de novo synthesis for survival. while cancers derived from tissues with normal naprt levels are entirely dependent on the nad + salvage pathway for survival. both the upregulated naprt in ovarian cancer and the high expression of nampt in glioblastoma, colorectal cancer tumors, and breast cancer, increase intracellular nad + levels, contributing to cancer cell metabolism and dna repair process in tumors. moreover, resistant ccrf-cem cells with high qprt activity exploit amino acid catabolism as a substitute pathway for nad + generation. high expression of nampt or naprt is associated with tumor progression, invasion, and drug resistance. [ ] [ ] [ ] [ ] this effect is mediated by nad + -dependent parps and sirt . [ ] [ ] [ ] induces the nampt activity to increase nad + content, thereby preventing oxidative damage. activation of the c-myc-nampt-sirt feedback loop may crucially contribute to the initiation and development of both routes to colorectal cancer. , declining nad + levels reduced the sirt -mediated inhibition of stat , which induces the secretion of il- and tfg-β to sustain the signaling required for emt. cd expression inversely correlates with prostate cancer progression due to its ability to lower intracellular nad + , resulting in cell-cycle arrest, reduced glycolytic and mitochondrial metabolism, and impaired fatty acid and lipid synthesis. these findings demonstrated a pro-tumor activity of nampt, suggesting a promising therapeutic target for cancer treatment. nampt inhibitors, fk , stf- and kpt- , can reduce the viability and growth of different cancer cells and have an additive effect in combination with main current chemotherapeutic drugs. [ ] [ ] [ ] [ ] nad + metabolism and metabolic diseases diabetes. diabetes is a chronic metabolic disease characterized by hyperglycemia. the human pancreas cannot produce enough insulin, or the body cannot effectively use the produced insulin, which causing a pathological increase in blood sugar. pancreatic β-cells maintain systemic glucose homeostasis by controlling the release of insulin, thereby responding to changes in metabolic demand. the high capacity, low-affinity glut and high k m glucokinase (gk) in β-cell ensure the proximal glucose-sensing. the glucose fluxes through the glycolytic pathway and the tca cycle, enable the production of nadh and atp. thus, the elevated blood glucose levels lead to more production of nadh and atp, resulting in closure of atp-sensitive potassium channels, cell depolarization, ca + influx and culminating in insulin secretion. [ ] [ ] [ ] [ ] in addition to nadh produced in the mitochondria by the tca cycle, cytoplasmic nad + generation is essential for insulin secretion. , given that β-cells only have extremely low ldha activity to regenerate nad + for glycolysis, nadh generated by glycolysis must be transferred into the mitochondria to be oxidized by complex i. cytoplasmic nadh from the glycolytic pathway is delivered to mitochondria through two nadh shuttles, g p shuttle and ma shuttle, allowing nad + recycling to sustain glycolytic flux. as evidenced, at maximal glucose stimulation, the rising of nad(p) h levels is estimated to be approximately mm in whole pancreatic islet beta cells, with a mm in the cytoplasmic domain and an approximately second delayed mm in mitochondrial domain. the nadh shuttle thus promotes the increase of ca + after the formation of mitochondrial membrane potential and sufficient atp generation from etc, concomitantly triggering glucose-stimulated insulin secretion (gsis) (fig. ) . [ ] [ ] [ ] sustained high levels of insulin demand will eventually give rise to functionally compromised or physical loss of β-cells, which culminate in hyperglycemia and diabetes. [ ] [ ] [ ] however, β-cells exposed to diabetes and hyperglycemia exhibit striking changes in metabolism. , importantly, the increase of krebs' cycle in the mitochondrial that generally responds to glucose is terminated, which will cause glucose to fail to escalate the nadh and atp content in the pancreatic islets of diabetic patients. rather than producing from mitochondrial tca cycle under controlled conditions, the nadh in diabetic islets is generated by cytoplasmic sorbitol oxidation and mitochondrial pyruvate oxidation in response to diabetes and hyperglycemia stimulate. , in diabetes, when gpdh is inhibited due to the reduced utilization of nad + , about % of glucose is involved in the polyol pathway. when β cells are over-nutrient and hyperglycemic, the excessive nadh produced by the polyol pathway will promote the production of superoxide through the overload complex i of etc, resulting in cell dysfunction and impaired insulin secretion. moreover, the two components of the g p shuttle (including gpd in the cytoplasm and gpd in the mitochondria) are up-regulated in mrna and protein levels in diabetic islets, thus ensuring the transfer of electrons from nadh produced in glycolysis to mitochondria. , in line with that, the overexpression of cytoplasmic malic enzyme (me ) enhanced the gsis and anaplerosis in insulinoma cells. selective reduction of cytosolic me expression and enzyme activity significantly reduces gsis and amino acid-stimulated insulin secretion (aasis). there is growing evidence indicating that cytosolic nadph is one of the effectory metabolic coupling factors, a variety of critical intermediates and cofactors involved in the gsis. although glucose causes a dose-dependent inhibition of pentose phosphate pathway activity in beta cells, the major pathway for nadph production, the cytosolic ratio of nadph/ nadp + increases during glucose-stimulated insulin release. [ ] [ ] [ ] nadph stimulates the exocytotic machinery by the redox proteins glutaredoxin and thioredoxin and has a local redox reaction, thereby accelerating exocytosis and promoting the secretion of insulin in pancreatic β-cells. , the nad + level is associated with insulin resistance. hfd significantly impairs the role of nampt-regulated nad + biosynthesis in metabolic organs. mice that specifically knock out the nampt gene of adipocytes have serious insulin resistance, which is manifested by an increase in plasma free fatty acid content and a decrease in plasma content of the main insulin-sensitive adipokine, adiponectin. these deleterious alterations can be normalized by administering nmn. furthermore, nmn alienates glucose intolerance and lipid profiles by recovering nad + levels in age induced t d mouse model. conversely, overexpression of nmnat in mouse can effectively increase nad + levels in a variety of tissues and prevent aging-related insulin resistance caused by diet. owing to its expression and activity increase with age, cd is essential for age-associated nad + decrease through degradation of nmn in vivo. cd deficiency has improved glucose intolerance with hfd, which could be further ameliorated by supplement of nr. c, as a highly specific and effective cd inhibitor, can reverse age-associated nad + reduction and improve some metabolic and physiological parameters of aging, such as glucose tolerance, cardiac function, muscle function and exercise capacity in both natural aging and accelerated aging mice models. obesity. the pathological expansion of adipose tissue is specifically manifested in the dysregulated production of adipokines and lipid, low-grade inflammation and enrichment of extracellular matrix. insulin resistance is a critical whole-body abnormal metabolism closely related to obesity. , a reduction of nad + levels in cells is observed in many tissues with obesity, like the skeletal muscles, hypothalamus, liver and adipose tissue. , supplementation of nr or nmn can protect against the decrease of nad + levels, and partially inhibit the weight gain of the mice fed with hfd by enhancing energy expenditure. the nad + biosynthesis regulated by nampt in adipocytes plays an important role in the pathogenesis of obesity-related metabolic complications. both the expression of nampt in visceral fat and the level of nampt in serum are positively correlated with the degree of obesity. [ ] [ ] [ ] [ ] [ ] in contrast, obese subjects have lower levels of nampt in subcutaneous fat tissue. [ ] [ ] [ ] the upregulation of nampt by the activation of the hif -α pathway under hypoxic conditions plays a vital role in processing dietary lipids to regulate the plasticity of adipose tissue, whole-body glucose homeostasis and food intake. the deficiency of adipose nampt can partially reduce food intake, thereby preventing obesity caused by diet. in addition, nampt-mediated nad + biosynthesis plays a vital role in adipose by promoting weight gain caused by hfd, which can be proved by the inability of hfd-fed fanko mice that can expand adipose tissue normally. , several studies have shown that in different adipocyte models, such as primary adipocytes, t -l preadipocyte cell and sgbs cell, nampt can be secreted directly into the supernatant through non-classical pathways. these results indicate that the adipose tissue is one of the main sources of secreted extracellular nampt (enampt). treatment with enampt can increase the expression of lipoprotein lipase and pparγ in preadipocytes and promote the expression of fatty acid synthase in differentiated adipocytes, which indicates that enampt may be a positive regulator in adipocytes lipid metabolism. adipose tissuespecific nampt knockin and knockout mice (anki and anko) showed opposite alterations of circulating enampt, which accordingly affected hypothalamic nad + /sirt signaling and physical activity. treatment with nmn can improve physical activity deficits in anko mice. the biosynthesis of nad + in adipocytes is crucial for the extension of hdf-induced white fat depots and may have more specific effects in lipid accumulation and processing. based on these observations, the effect of nampt on obesity depends on its enzymatic activity. increasing nad + levels by supplementing nr in mouse tissues and mammalian cells activates sirt and sirt , which ultimately leads to increased oxidative metabolism and prevents metabolic abnormalities induced by hfd. moreover, adding leucine to hfd can increase the expression of nampt and sirt and elevate the level of nad + in cells, which will reduce the acetylation of foxo and ppar-γ co-activator α (pgc α). fig. pathophysiological role of nad + disarrangement in metabolic diseases. a the liver is a master organ of nad + metabolism and may facilitate the nad + biosynthesis in other tissues. nad + metabolism plays a critical role in the lipid metabolism through modulating the activity of sirtuins. the reduced nampt expression and nad + levels contribute to the development of nafld through manipulating dysmetabolic imbalance, hepatic energy homeostasis, glucose homeostasis, hepatic inflammation and insulin resistance. b decreased nad + / nadh ratio by the mismatch between nadh production and oxidation inhibits the activity of sirtuins in the failing heart. elevated protein acetylation weakens the energy metabolism through negative feedback to oxphos and substrate metabolism, impairing antioxidant defense and sensitizing the mptp to ros or calcium. c the deduced nad + levels in kidney are attributed to the decreased expression of enzymes in nad + de novo synthesis and increased consumption by dna damage activated parps. nad + depletion inhibits the sirt /pgc α mediated mitochondrial quality control, atp production and nad + de novo biosynthesis. the phosphorylation of nad + to nadp + enhances the antioxidant defense against oxidant stress. nad + -dependent defect in fao results in intracellular lipid accumulation. in addition, the defected fao and increased desaturation of pufas to hufas due to nad + deficiency and impaired mitochondrial function result in the accumulation of hufa-containing triglycerides and cellular lipid in renal tubular cells. d the insulin secretion is adjusted by the dynamic glucose concentration in blood. as a master regulator of insulin secretion, glucose is metabolized via the glycolysis and tca cycle to produce nadh and atp. the increased nadh and atp induces the closure of atp-dependent k + channels, the opening of voltage-gated l-type ca + channels, the raising of cytosolic ca + and culminating in insulin secretion in pancreatic β-cells. the activity of mitochondrial shuttles including the glycerophosphate and malate/aspartate shuttles allows the reoxidation of cytosolic nadh into nad + , which is required for maintenance of the glycolysis. purple representants the downregulated proteins or activated biological functions, while brown labels the upregulated proteins and repressed physiological activities. abbreviations: acmsd, alpha-amino-beta-carboxy-muconate-semialdehyde decarboxylase; ar, aldose reductase; etc, electron transport chain; grxs, glutaredoxins; hufas, highly unsaturated fatty acids; kmo, kynurenine -monooxygenase; fao, fatty acid oxidation; pufas, polyunsaturated fatty acids; sdh, sorbitol dehydrogenase; trxs, thioredoxins. deacetylation of pgc α may promote the upregulation of genes related to fatty acid oxidation and biogenesis in mitochondria, thereby restoring mitochondrial function and protecting against hfd-induced obesity. non-alcoholic fatty liver disease (nafld). alterations of hepatic metabolism are critical to the development of liver diseases, in which the nafld is the most common chronic liver disease and is strongly related to metabolic syndrome. nafld might eventually cause more severe liver diseases, such as liver fibrosis, liver cirrhosis, liver failure and hepatocellular carcinoma (hcc). [ ] [ ] [ ] it is reported that reduced nad + concentration caused a dysmetabolic imbalance, leading to the development of nafld. oral administration of nr halts the progression of nafld through rescuing the nad + reduction, reducing the total cholesterol and triglyceride levels, decreasing the ast level, and reversing kupffer cells accumulated and inflammatory in aged group. , troxerutin, as a derivative from natural bioflavonoid rutin, could promote nampt expression to restore the nad + level depleted by oxidative stress in the hfd-induced nafld mouse model (fig. ) . in a transgenic mouse model of dn-nampt, the researchers found that middle-aged mice had a systemic reduction of nad + and showed a moderate nafld phenotype, like triggered inflammation, lipid accumulation, increased oxidative stress and impaired insulin sensitivity of liver. some of these phenotypes are further exacerbated after feeding a high-fat diet. however, oral nr can completely reverse these phenotypes caused by nad + deficiency or high-fat diet. significantly, knockdown of nampt gene increases, while over-expression reduces hepatic triglyceride both in vitro and in vivo models. the expression of nampt in patients with nafld has decreased systemically both in serum and within the hepatic tissue, which is regulated by pparα activation and glucose. meanwhile, the nampt is also a target of foxo transcription factors that control the nad + signaling in the regulation of hepatic triglyceride homeostasis. additionally, the hepatic microrna- a, which is increased in obesity, reduces nad + levels and sirt activity by targeting nampt. the antagonism of microrna- a could moderate inflammation, steatosis and glucose intolerance, and recover nampt/nad + levels in diet-induced obese mouse model. it was found that higher level of nampt in serum in women is correlated with a much lower hepatic de novo lipogenesis (dnl) index, although they do not correlate with the dnl index, but had a correlation with a higher hepatic fat in men, implying a sex-dependent interpretation role of serum nampt level for nafld prognosis. mechanistically, reduced nampt/nad + inhibits sirt 's function, thereby attenuating the deacetylation activity of srebp , leading to the expression of acc and fasn. conversely, stabilization of sirt by increasing nnmt expression or mnam levels improves lipid parameters. , , beyond the metabolic activity, nampt may also participate in nafld's pathogenesis by controlling hepatic inflammation, insulin resistance and glucose homeostasis. , enampt regulates glucose production via the pka/creb signaling in hepg cells. the expression of nampt is closely related to the expression of pro-inflammatory cytokines in the inflammation induced by free fatty acids and is remarkably reduced by the inhibition of nf-κb in hepg cells. to date, the clinical analysis reveals controversy regarding the relationship of circulating nampt with nafld. several studies report no statistically significant difference in nampt levels between nafld and healthy controls, as well as among different histological nafld groups. , another study shows that nafld patients have systemically decreased the expression of nampt in both serum and hepatic tissue. in contrast, the liver and serum nampt of morbidly obese women with nafld are significantly higher than that of morbidly obese women with healthy livers. the serum nampt level and its expression in hepatic tissues are positively correlated with pro-inflammatory factors. moreover, the expression of nampt is notably higher in fibrosis patients and is correlated positively with the stage of fibrosis in nafld patients. elevated serum nampt, together with inflammatory factors, such as il- , il- and tnf-a, is associated with an increased likelihood of exhibiting nafld and nash. given that hepatocytes are only one of the sources of enampt, circulating nampt levels may not represent its actually concentration in local liver or adipose tissues, thus requiring further research to determine its exact role in nafld. nad + metabolism in neurodegenerative disorders neurodegenerative diseases are a heterogeneous group of diseases, including alzheimer's disease (ad), parkinson's disease (pd) and amyotrophic lateral sclerosis (als), which are characterized by progressive degeneration of the structure and function of the peripheral and central nervous system, with the characteristics like the accumulation of misfolded and aggregated proteins that are associated with severe proteotoxic stress (fig. ) . axonal degeneration. axonal degeneration is an early and prominent feature of many neurological disorders, including ad, pd, als, ischemic brain and spinal cord injuries, diabetic neuropathy and traumatic brain injury. , sarm , as the evolutionary conservative executor of degradation cascade, is required for the progression of rapid wallerian degradation. the tir domain of sarm has inherent nadase activity, which can cleave nad + into nicotinamide, cadpr and adpr. the nicotinamide acts as a feedback inhibitor of sarm . axons require the nadase activity of the full length sarm to facilitate axonal degeneration and nad + consumption after injury. , , similarly, the loss of endogenous nmnat is an important cause of axon degeneration after injury. the axon damages caused by sarm or nmnat can be restored by increasing nad + synthesis, like over-expressing the nmnat enzyme. , the naturally occurring mutant mice, wallerian degeneration slow (wld s ) with chimeric gene containing the nterminal aa of ube b and full length nmnat , show a delayed wallerian degeneration phenotype. [ ] [ ] [ ] [ ] [ ] several mechanisms are underlying the protective role of nmnat on severed axons. firstly, nmnat acts as a stressresponse protein that aids the clearance or refolding of misfolded proteins like a chaperone. , secondly, nmnat and wlds proteins facilitate axon preservation by suppressing the accumulation of nmn. the activity of nmnat is essential for axon survival because activity reduced mutants have no axon protection effect. the protection effect can also be abolished by the expression of exogenous nmn and ectopic expression of nmn deamidase. , thirdly, nmnat does not change the nad + biosynthesis, but prevents the sarm -dependent nad + depletion caused by injury, which is important for axon degeneration. furthermore, sir , as a mammalian homolog of sirt , is the downstream effector of increased nmnat activity, which can lead to axon protection in wallerian degeneration slow mice. additionally, sarm protein is required for nmn to promote axon degeneration and ca + influx. sarm and nmn play a role in common signaling, which ultimately leads to the increase and breakage of ca + in axons and the dissociation of mitochondrial dysfunction. although the inhibitor of nmn synthase nampt reduces nad + level, it can still provide strong morphological and functional protection for damaged synapses and axons. alzheimer's disease (ad). ad is a long-term chronic disease in the prodromal and preclinical stages with an average course of to years, which is the most common neurodegenerative disorder. currently, amyloid β peptide (aβ), apoe and microtubule-associated protein tau are three important factors that have sufficient evidence as the etiology of ad. the key pathological features of ad are the accumulation of aβ-enriched neuritic plaques and neurofibrillary tangles (nfts) (consisting of tau protein). aβ and phosphorylated tau (p-tau) neurofibrillary lesions lead to the pathology of neurons that display oxidative damage, impaired ca + processing, reduced dna repair, dysfunction of lysosomal and mitophagy, all of which have a positive correlation with the age-related nad + decline. [ ] [ ] [ ] [ ] [ ] mounting evidence supports the promotion of nad + consumption on the process of ad, whereas the accumulation of nad + suppresses the adrelated pathological progress and the decline of cognitive function in different ad model ranging from c. elegans to mice. , , , the increased activity and expression of cd following aging is responsible for the age-associated decrease in nad + level and defective mitochondrial function, which is at least partially regulated by the nad + /sirt signaling pathway. cd deficiency in apps-weps de mouse reduces soluble aβ concentration and aβ plaques, correlated with improved spatial cognition. the brains of individuals with preclinical ad (pcad) and ad exhibit oxidative damage to a variety of molecules, e.g., accumulation of protein carbonyls (pcs) in regions that are rich in aβ-peptide-containing sps, increased lipid peroxidation in ad and pcad hippocampi and elevated dna damage. recent studies have found that damaged cellular energy expenditure and dna repair are related to the ad's pathogenesis. in the novel ad mice with dna repair defects ( xtgad /polβ +/-), the content of nad + is reduced. increasing nad + by supplementing nr can remarkably normalize dna damage, p-tau, synaptic transmission and neuroinflammation, improve the ability of motor function, memory and learning and increase the activity of sirt in the brain. notably, nad + augmentation improves dna repair through improving the neuronal nhej activity in ad mice. , , , the accumulated dna oxidative damage in ad hyper-activates the dna damage sensor parp- , thereby reducing the concentration of cellular nad + and suppressing the function of nad + -sirt -pgc- α axis, which in turn causes abnormal mitochondria. replenishing cellular nad + can promote dna repair in neurons and restore mitochondrial function through mitophagy. mitophagy diminishes insoluble aβ - , aβ - , and hyper-phosphorylated tau, preventing cognitive or memory impairment in mouse model. , additionally, nad + protects neurons against p-tau pathologies. nad + accumulation may inhibit the phosphorylation of different tau protein sites by inhibiting the activity of cdk -p complex. nicotinamide, as a competitive inhibitor of sirtuins, specifically reduces the phosphorylation of tau (thr ), which is related to microtubule depolymerization in an analogous manner to that of sirt . nmnat, as a binding partner of hsp , can specifically recognize p-tau to inhibit its amyloid aggregation in vitro and reduce its symptoms in the fly tauopathy model, and this effect could be competitively destroyed by its enzymatic substrate. parkinson's disease (pd). pd is a common neurodegenerative disease, mainly including motor and non-motor symptoms. the neurons of pd patients exhibit symptoms, such as mitochondrial dysfunction, oxidative stress and nad + metabolic changes. maintenance of nad + levels is vital for proficient neuronal fig. linkages between nad + depletion and neurodegenerative disorders. most neurodegenerative disorders, including axonal degeneration, alzheimer's disease (ad), parkinson's disease (pd), huntington's disease (pd) and amyotrophic lateral sclerosis (als), are associated with mitochondrial dysfunction, lowered antioxidant capacity and heightened mitophagy, all of which are converged into the age-related nad + depletion induced by either enhanced consumption or impaired biosynthesis. these neural pathologies can be rescued by nad + boosting. purple representants the downregulated proteins or activated biological functions, while brown labels the upregulated proteins and repressed physiological activities in neurodegenerative disorders. abbreviations: mhtt, mutant huntingtin; aβ, amyloid beta; nfts, neurofibrillary tangles; -haa, -hydroxyanthranilic acid; qa, quinolinic acid; wlds, slow wallerian degeneration; -hk, -hydroxykynurenine function. it is reported that lrrk g s dopaminergic neurons exhibit a decreased nad + pool and a reduced sirtuin deacetylase activity, correlating with elevated acetylation of sirtuin substrates p , α-tubulin and sod . in the primary cell (spd cell) derived from patients with sporadic pd, the cytosolic conversion of pyruvate to lactic acid resulted in a significant increase in the nuclear nad + level and cellular nad + /nadh ratio. the alteration of nad + metabolism in spd cells contributes to the activation of sirt and subsequently reduces the acetylation level of α-tubulin. inhibition of the deacetylase function of sirtuin- can enhance the acetylation of α-tubulin and facilitate the clearance and transport of misfolded protein. the redox status of nad + /nadh is remarkably decreased in ipsc neurons from gba mutation associated pd, which may be due to the decrease of nmnat , as evidenced by the significant increase after nmn treatment. furthermore, increasing the level of available nad + by supplementing diet containing nad + precursors or inhibiting the activity of nad + -dependent enzyme (e.g., parp that snatches nad + competing with mitochondria) is a feasible strategy to avoid the neurotoxicity related to mitochondrial dysfunction. [ ] [ ] [ ] besides, through maintaining the rate of nad + production and normalizing the nadh/nad + balance, both pentylenetetrazole (ptz) and niacin (na) exhibit neuroprotective properties. , sarm participates in the occurrence of pd mainly through its enzyme activity of nad + degradation. compared with healthy people, the phosphorylation level of sarm is significantly increased in the neuronal cells from pd patients, with a high sensitivity to oxidative stress. in the case of oxidative stress, jnk increases the phosphorylation of sarm , resulting in enhanced nad + degradation activity, which in turn promotes the suppression of mitochondrial respiration. the binding of sarm and pink can facilitate trak -induced ubiquitination of lysine on pink , which is essential for keeping the stabilized status of pink and bringing it into the outer membrane of mitochondria. downregulation of sarm and traf reduce the level of pink and then recruits parkin to the impaired mitochondria, indicating that sarm plays a crucial role in mitophagy via modulating pink . there is evidence showing that supplementation of high-dose nad + precursors in cells or drosophila can alleviate the pathological phenotype by reducing oxidative stress and mitochondrial damage and improving motor function, which provides a feasible solution for the prevention and improvement of pd. , huntington's disease (hd). hd, also known as huntington's chorea (huntington's chorea), is an autosomal dominant hereditary neurodegenerative disorder that has a tremendous devastating effect on patients and their families, characterized by chorealike movements, cognitive decline and psychotic-like symptoms. it is caused by repeated amplification of the cag trinucleotide in the huntingtin (htt) gene on the short arm of chromosome , which leads to a prolonged polyglutamine stretch at the nterminus of the protein. [ ] [ ] [ ] [ ] mitochondrial defection and increased oxidative stresses are the most prominent features in the cells of hd patients. sirt , as a deacetylase in mitochondria, modulates the transcription of mitochondrial in response to oxidative stress. notably, the expression of the htt mutant reduces the deacetylase effect of sirt . in the hd model, reduced expression and deacetylase activity of sirt , which in turn prevents the deacetylation of lkb and sod , leading to a decrease in the level of nad + , defects in mitochondrial biogenesis and accumulation of ros. however, the overexpression of sirt displays protection against abnormal motor function, cortical and striatal atrophy and loss of striatal neurons in the transgenic hd mice via regulating mitochondrial function. both sirt and sirt exert their function through pgc- α, which acts as a modifier in hd and als patients and other models. sirt / -pgc- α pathway in hd transgenic mice attenuates motor deficits and neurodegeneration by alleviating oxidative stress, eliminating huntingtin aggregates and restoring mitochondrial function. , [ ] [ ] [ ] [ ] [ ] it has been reported that the kynurenine pathway (kp) is closely related to the pathogenesis of hd. the degradation process of tryptophan in kp produces a variety of neuroactive metabolites with amino acid-like structures, such as an n-methyl-d-aspartate (nmda) receptor agonist quinolinic acid (qa), and a neuroprotective nmda receptor agonist kynurenic acid (kyna). , compared with the control group, the ratio of kynurenine (kyn) to kyna in hd increases significantly, which is related to the decrease in the production of kyna in hd patients. genetic and pharmacological inhibition of tdo and kmo can increase kyna, but not the level of neurotoxic product -hk, thereby improving the symptoms of neurodegeneration. amyotrophic lateral sclerosis (als). als is a neurological disorder that causes progressive degeneration of the motor neurons in the brainstem, spinal cord and cerebral cortex. , more than gene mutations have been reported to be closely related to als, of which c orf repetitive amplification mutations and sod mutations are the most common causes. among them, als caused by mutations in the antioxidant enzyme sod accounts for about - % of sporadic als (sals) and % of familial als (fals). [ ] [ ] [ ] [ ] [ ] [ ] astrocytes are specific contributors to spinal motor neuron degeneration in sod -related als. the spinal cord astrocytes isolated from sod g a transgenic rats were cocultured with motor neurons, resulting in the induction of motor neuron death. the reactive astrocytes can promote excitotoxic injury of motor neurons by producing nitric oxide and peroxynitrite, which cause mitochondrial damage and apoptosis in cultured neurons, decreasing glutamate transport, releasing proapoptotic mediators selectively toxic to motor neurons. enhancing nad + availability can abrogate the neurotoxicity of astrocytes from diverse als models. either overexpression of nampt or supplementation of nmn can increase the mitochondria and total cellular nad + levels of als astrocytes, thereby enhancing the ability to resist oxidative stress and restoring the toxicity of astrocytes to motor neurons. the nr repletion increases the levels of uprmt-related proteins and promotes the clearance of mutant hsod neurotoxic protein. moreover, supplementation of nr can reduce the expression of neuroinflammation biomarkers in the spinal cord, alleviate the degeneration of motor neurons and appropriately extend the survival time of hsod g a transgenic mice. , studies have found that als is associated with nad + metabolism through kp pathway damage. the impairment of kp in als is confirmed by the following evidence: higher serum tryptophan, cerebrospinal fluid (csf), kyn and qa and a decrease in serum picolinic acid levels in als patients. moreover, both the inflammation of microglia in the motor cortex and spinal cord of als patients and the expression of ido and qa in neuronal cells and microglia increased significantly. , in parallel with the impaired de novo pathway, the nad + decline in the als might also be due to the inadequate nampt-mediated nad + salvage synthesis pathway. nam, a metabolite in the salvage pathway, is reduced in both csf and serum from als patients compared with healthy people. nr supplement can increase the nad + concentration, dependent on nrks (nr kinases), thereby avoiding the need for nampt in the salvage synthesis pathway. , the protective effect of nad + on als might also be linked to the altered activities of sirts, however, the conclusions of many studies are quite different. it has been found that the expression level and function of sirt is selectively reduced in the spinal cord of sod g a mice at the end of als course, while the level of sirt is increased in the human spinal cord after autopsy. the overexpression of nad + dependent deacetylase sirt and sirt can eliminate the neurotoxicity in the astrocyte cultured in vitro by activating the nrf -mediated antioxidant defenses. interestingly, the expression of sirt in the spinal cord of wt mouse is reduced during normal aging, while the expression of sirt in different regions of the brain (including the spinal cord, hippocampus, thalamus and cerebral cortex) in sod g a mouse is increased. besides, overexpression of sirt in motor neurons slows down the progression of als in severely phenotypic sod g a (with high copy numbers) mice, partly by activating the hsf /hsp i molecular chaperone system. , however, sirt and sirt are generally reduced in als primary motor cortex while they are upregulated in the spinal cord in human post-mortem tissues. in contrast to the neuroprotection role of sirt , sirt upregulation is toxic to neuronal cells. , , [ ] [ ] [ ] [ ] [ ] a preliminary clinical trial has confirmed the importance of sirt activation and nad + metabolism in als. the drug used in this trail is eh (a mixture of pterostilbene and nr), which contains the sirt activator and the precursor of nad + . compared with the placebo control group, eh can significantly alleviate the development of als. , nad + in cardiac and renal diseases nad + and heart failure (hf). hf is a complex clinical syndrome caused by various initial heart damage and subsequent disturbance in compensatory effects and pathogenesis mechanisms. it is manifested through a number of complex molecular and systemic dysfunctions from the subcellular level to the multiorgan system of the whole body. , there are many kinds of evidence indicating that the imbalance of myocardial nad + pool is causally linked to metabolic remodeling and mitochondrial dysfunction in hf. a wide range of tissues display a reduction of nad + in the aging mice model. the down-regulation of nad + levels in the heart is most significant. nad + is reduced by % within to months, and as a compensation, the concentration of nadh has increased by %. , the increased nadh/nad + ratio and protein hyperacetylation are found in the hf patients' hearts and pathologically hypertrophied mice. it is important to note that hyperacetylation of mitochondrial proteins is considered to be an inducer for cardiac dysfunction. [ ] [ ] [ ] increasing nad + levels by activating the nad + salvage pathway can inhibit mitochondrial protein hyperacetylation and cardiac hypertrophy and improve cardiac function under stress. proteomics analysis identified a subgroup of mitochondrial proteins, particularly sensitive to the changes of nadh/nad + ratio. it is reported that the hyperacetylation modification of mitochondrial proteins caused by the imbalance of nad + redox mainly promotes the pathological remodeling of the heart through two different mechanisms. first, the hyperacetylation of the mitochondrial malate-aspartate shuttle protein inhibits the oxidation and transport of nadh in the mitochondria, resulting in an imbalance of redox in the cytoplasm. second, the acetylation modification of the oligomycin-sensitive conferring protein increases its binding to cyclophilin d and enhances the sensitivity of the mitochondrial permeability transition pore. both of these conditions can be restored by regulating the normalization of nad + redox balance at the genetic or pharmacological level. it has been reported that hyperacetylation of cardiac protein in mice with hfd is closely related to the decreased expression of sirt . exogenous supplementation of nad + can maintain the intracellular nad + level and block the symptoms of agonistinduced cardiac hypertrophy in vitro and in vivo by regulating the activation of sirt . as a negative regulator in cardiac hypertrophy, sirt protects the heart by inhibiting intracellular ros. mice lacking sirt become more sensitive to the stimulation of several pharmacological or non-pharmacological stressors, showing symptoms, such as fibrosis, cardiac hypertrophy and high mortality. in the sirt knockout mouse model, the progression of fibrosis and cardiac hypertrophy also accelerated with age. in addition, mitochondrial swelling also appears to increase with age due to the increased opening of mitochondrial permeability transition pore (mptp). similarly, the loss of mitochondrial complex i leads to a decrease in the ratio of nad + /nadh and can inhibit the activity of sirt , thereby enhancing the protein acetylation and mptp sensitivity. supplementing cko mice with nad + precursors can partially normalize the nad + /nadh ratio, acetylation of protein and sensitivity of mptp. it is noteworthy that compared with normal mice, sirt deficient mice can cause increased oxidation of fatty acid in heart, which is due to the high acetylation modification and high activity of β-had and lcad. moreover, in the absence of sirt , the succinylation of protein lysine mainly occurs in the heart. when fasting, sirt knockout mouse has reduced echa (an enzyme regulates the oxidation of fatty acid) activity, increased long-chain acyl-coas content and decreased atp content. compared with control mice in the same litter, after min of ischemia and min of reperfusion, the area of cardiac infarction in sirt knockout mice was larger. the ischemia-reperfusion injury (i/r injury) of sirt knockout mouse heart is restored to normal levels by dimethyl malonate (a succinate dehydrogenase (sdh) inhibitor) pretreatment, which implies that the change of sdh activity is the leading cause of the damage. clinically, i/r injury occurs during myocardial infarction or blood supply stop during cardiovascular surgery. i/r injury is related to the decrease of endogenous nampt and the down-regulated expression of sirt , sirt and sirt . nampt strictly controls the nad + and atp content, thus playing an important part in regulating cell survival by suppressing apoptosis and increasing autophagy flux in cardiomyocyte. [ ] [ ] [ ] sirt reduction can increase the sensitivity of heart-derived cells and the adult heart to i/r injury and may cause more severe i/r injury in the elderly heart. , exogenous expression of nmn can activate sirt -mediated foxo deacetylation, which can protect the heart from i/r injury during ischemia and reperfusion. similarly, calorie restriction promotes nad + to stimulate the nampt-sirt signaling pathway, which can protect the heart from i/r injury by up-regulating antioxidants and down-regulating pro-apoptotic molecules by activating foxo. , , [ ] [ ] [ ] it is reported that the human cardiac fibroblasts with high expression of nox and nox are the main source of cardiac fibrosis related to heart failure and cardiac hypertrophy. nadph produced by g pd increases the level of noxs, thereby producing most of the superoxide during the course of heart failure in patients with ischemic cardiomyopathy. under the acceleration of tgf-β , nox mrna is significantly upregulated and mediates the transformation of fibroblasts into myofibroblasts by activating tgf-β -smad / signaling, which leads to cardiac fibrosis. , nad + and kidney failure. acute kidney injury (aki) is a common clinical syndrome, and its prevalence and mortality increase with age. in the aki mouse model, compared with the kidneys from month-old mice, nad + levels in the kidneys from -month-old mice were significantly reduced. renal ischemia impairs de novo nad + biosynthesis via reducing the renal expression of qprt. knockout of one allele of qprt recapitulates these effects and increases susceptibility to aki compared with control mice, which could be prevented by augmenting nad + metabolism with oral nam supplementation. , the robust finding that the early rise of urinary quinolinate levels and the urinary quinolinate/tryptophan ratio are related to the probability of aki and adverse outcomes in a cohort of > patients indicates that impaired nad + metabolism leads to kidney injury in patients. additionally, boosting nad + levels via inhibiting acmsd (an enzyme restricts the de novo synthesis of nad + from tryptophan) also protects against aki after renal i/r injury. the decrease in enzymes related to nad + de novo synthesis is due to the inhibition of the activity of pparγ coactivator α (pgc- α), which is a crucial determinant of renal recovery from aki. , moreover, due to the decrease of -hydroxykynurenine (a cytotoxic metabolite of kmo), the mice that lack active kynurenine -monooxygenase (kmo) will not develop into aki after i/r injury. the expression of parps in the injured kidney's proximal tubules is upregulated from - h after i/r injury. oxidative stress and dna damage caused by i/r injury lead to excessive activation of parps. a study suggests that the activation of parp may lead to cell death through atp consumption and enhancement of the inflammatory cascade in mice. inhibition of excessive activation of parp can protect renal function from abnormal hemodynamics, renal metabolic disorders and renal cell apoptosis during aki. , meanwhile, knocking out the parp gene can protect mice from ischemic kidney damage. the simultaneous effects of impaired nad + biosynthesis and over depletion of nad + by parps can lead to a decrease in the level of nad + in the kidney during aki. the decline in nad + levels therefore results in impaired metabolism and mitochondrial function via sirts and pgc- α. , , sirt protects against mitochondrial damage in the kidneys by attenuating oxidative stress, inhibiting inflammation and inducing autophagy through regulation of the ampk/mtor pathway. , sirt regulates the gluconeogenesis/glycolysis pathway by executing fasting signals via pgc- α. augmenting nad + induces sirt mediated deacetylation of pgc- α, thereby increasing the production of lipolysis product β-hydroxybutyrate and the production of pge , a prostaglandin to maintain kidney function. , additionally, supplementation with nmn restores the activity of kidney sirt , thereby reducing the stress response by regulating the jnk pathway and protecting mice from cisplatin-induced aki. boosting nad + as a therapeutic strategy in general, intracellular nad + levels are maintained between . and . mm, depending on the cell type or tissue. however, the concentration and distribution of nad + can fluctuate in response to diverse physiological stimuli and cellular stresses. altered nad + homeostasis has been linked to multiple diseases affecting different organs, including the brain and nervous system, liver, heart and kidney. nad + depletion is a hallmark of ageing and numerous agerelated disorders. , , , [ ] [ ] [ ] [ ] therefore, boosting nad + offers a promising option for enhancing resilient to aging or diseases, thereby extending a healthy lifespan. the nad + level can be elevated by dietary supplementation of nad + precursors, such as trp, na, nmn and nr, inhibition of nad + -consuming enzymes, including parp and cd , management of the nad + biosynthesis via controlling nad + -biosynthesis enzymes, or improving nad + bioavailability through exercise and caloric restriction. supplementation of nad + precursors nad + precursors can be used as a nutritional supplement to improve a broad spectrum of physiological functions and pathological processes. [ ] [ ] [ ] [ ] [ ] [ ] as highlighted in table , the therapeutic and preventive efficacy of nad + boosters, especially the soluble and orally bioavailable endogenous molecules nr, nam and niacin, have been assessed in a series of clinical trials in humans. nad + precursor: nmn. nmn administration can effectively and rapidly enhance nad + biosynthesis in various tissues, even in the brain, with a promising safety. aged animals are more responsive to nad + replenishment by nmn treatment than the young one due to the age-related decline in nad + availability. nmn treatment exerts beneficial effects on insulin secretion and insulin sensitivity in age-and diet-induced diabetes by restoring nad + biosynthesis. long-term nmn administration rescues the age-associated decline in physiological function, including mitochondrial function, energy metabolism, gene expression changes, insulin sensitivity and plasma lipid profile, thereby improving physical activities, such as bone density and eye function. nmn treatment improves the neuronal functions, including rescuing the memory and cognition in rodent models for alzheimer's disease, protecting neurons from cell death after intracerebral hemorrhage or ischemia, recovering severe retinal degeneration and restoring the age-associated loss of the neural stem/progenitor pool. besides, the nmn administration also exerts a pleiotropic effect on acute heart failure and renal injury. , , it is worth noting that clinical trials of nam have been initiated in patients with cancers including bladder cancer, non-small-cell lung carcinoma, non-melanoma skin cancer, non-hodgkin's lymphoma and multiple myeloma ( table ) . despite that the nmn bioavailability is evidenced by the rapid absorption and convention of administered nmn to nad + in various organs, including skeletal muscle, kidney and liver, the transportation of nmn into cells remains unclear. nmn may be directly taken up by specific transporters, as the nad + content in peripheral organs, such as the gut, is immediately increased by nmn administration. , however, in vitro studies demonstrate that nrk / depletion abandons the incorporation of nmn into nad + synthesis. moreover, nmn administration can significantly elevate nad + biosynthesis in white adipose tissue, heart and the liver, but not the nad + content in brown adipose tissue and kidney, suggesting a tissue-and cell-type-specific transportation of nmn to cells or tissues for nad + biosynthesis presumably due to the deferent expression pattern of nrk . , therefore, the identification of the putative nmn transporter and its tissue specific expression pattern will help assess the cell type or tissuespecific preference of nmn, paving the way for precious clinical application of nmn in different conditions. nad + precursor: nr. nr is another natural compound that displays a surprisingly robust effect on systemic nad + metabolism. a large phase clinical trial of nr has been registered on a broad range of pathologies, including infection, neoplasms, agingrelated diseases and disorders that occur in the circulatory system, genitourinary system, nervous system and skin (table ) . oral nr supplementation in aged participants elevates the muscle nad + metabolome, ameliorates metabolic dysfunction, depresses levels of circulating inflammatory cytokines and increases the antiinflammatory molecule adiponectin in aged human. , , dietary administration with nr improves cold tolerance, endurance and energy expenditure. nr protects mice from hfd-induced body weight gain, enhances the liver weight regain by promoting hepatocyte replication and increasing hepatic atp content in the regenerating liver. nr exhibits beneficial effects in several muscle disorders through improving mitochondrial function and decreasing the upr mt in heart failure mice. nr boosts the nad + biosynthesis to prevent dna damage and tumorigenesis. nad + repletion with nr may reverse nafld by improving mitochondrial function in both hfhs-fed mice and hfc-fed apoe −/− mice. it has been reported that nr has a variety of compelling benefits in the nervous system, including improving the cognitive function and synaptic plasticity in alzheimer's disease and preventing noise-induced hearing loss. nr restores the age-associated decline in the metabolic cycle and circadian behavioral, including the bmal activity, oscillation mitochondrial respiration, rhythmic transcription and late evening activity to youthful levels. nr can be directly transported by ents into cells and enhance nad + biosynthesis bypassing the nampt-mediated salvage pathway. however, the short stability of nr in circulation and rate-limited utilization by the expression of nrks restrict its clinical application. dihydronicotinamide riboside (nrh), a reduced form of nr with oral bioavailability, is developed to overcome these limitations. nrh provides better efficacy to boost nad + synthesis using an nrk / -independent pathway compared with nr and nmn, preventing cisplatin-induced acute kidney injury. the potent and efficient nrh that serve as an nad + booster, offers a promising option to increase nad + levels. , nad + precursors: nam and na. nam is an uncharged molecule that can diffuse rapidly across the plasma, supporting the nad + biosynthesis for most tissues in vivo. oral administrated nam is converted into na in the small intestine and colon by nicotinamidase pnca of gut microbiota. gut microbiota-mediated deamidation of nam is necessary and responsible for the nad + biosynthesis in various organs, including the kidney, the liver and the colon. na is effective in treating dyslipidemia due to its cholesterol lowering actions. na treatment decreases the serum low-density lipoprotein and triglyceride content and elevates the high-density lipoprotein levels. nevertheless, the clinical application of na is limited because the pharmacological dosing of it induces cutaneous flushing via activation of a g protein-coupled receptor, gpr a. given this undesirable effect, several niacin derivatives with prolonged release time, including enduracin, niaspan and acipimox, have been developed. therefore, niacin has been replaced by its derivatives in the clinical treatment of hyperlipidemia. the acipimox can directly affect mitochondrial function in skeletal muscle of patients with type diabetes. the side effects of nad + boosting. the aforementioned findings suggest that elevating nad + levels by administration of nad + precursors, including nmn, nr, nam, and na, is a rational therapeutic strategy to improve a healthy lifespan. given that nad + -depleting drugs exhibit anti-tumor potential due to their impact on dna repair and inflammation, long-term boosting nad + might increase the risk of driving tumor growth. moreover, the detrimental side effects of nad + and its intermediates may be caused by the nad + -dependent sirtuins that have both oncogenic and tumor suppressive activity in different contexts. consistent with this hypothesis, nmn treatment accelerates pancreatic cancer progression via creating an inflammatory environment. , thus, future clinical studies are necessary to assess the long-term safety of nad + precursors in human therapeutics. inhibition of nad + consumption the excessive activation of parps or cd causes a nad + consumption up to the extent that leads to atp decline, energy loss and cell death. , , , thus, reducing the nad + consumption via suppressing parps or cd is also a strategy to boost nad + . , accumulating evidence demonstrates that aberrant parps activation by dna damage causes nad + depletion, contributing to the progression of tumorigenesis and neurodegenerative disorders that involve dna repair defects. to date, parp inhibitors, including niraparib, rucaparib and olaparib have been approved by us-fda to treat cancers, including prostate cancer, breast cancer and ovarian cancer, through disrupting dna repair and replication pathways. [ ] [ ] [ ] parps-mediated adp-ribosylation accounts for up to % of the cellular intracellular nad + consumption, leading to reduced nad + availability for sirtuins. therefore, genetic ablation or pharmacological inhibition of parp- enhances the sirt activity through restoring the nad + content, providing a protection benefit for various tissues, including the liver, muscle and brown adipose tissue. , nadp + has been demonstrated as an endogenous inhibitor of parps, which extend the therapeutic effect of parp inhibitors on cancers with higher levels of nadp + . a variety of flavonoids, including apigenin, quercetin, luteolin, kuromanin, and luteolinidin, exhibit inhibitory effect on cd activity. c is a highly specific cd inhibitor, which has greater potency than the flavonoids in reversing nad + decline during aging, thereby improving several age-associated physiological functions, including cardiac function, muscle function and glucose tolerance. interestingly, c elevates nad + to a higher level in mouse liver than that in muscle, arguing a tissue specific cd activity. thus, further studies uncovering the tissue specific cd activities will facilitate the development of clinical application of cd inhibitors. controlling nad + -biosynthesis pathway enhancing nad + -biosynthesis is one alternative approach to elevate nad + concentration through either increasing the activity of enzymes in nad + -biosynthetic pathway or inhibiting the activity of enzymes in the side branch pathway. nad + is synthesized from both de novo pathway and the salvage pathway. the distinct expression level of naprt in various healthy tissues determines the choice of nad + biosynthetic pathway for survival. cancers arising from tissue with a highly naprt are expression completely and irreversibly dependent on the naprt-regulated de novo pathway, while cancers deriving from tissues with the low level of naprt mainly rely on the nampt-mediated nad + salvage pathway. this deferent dependence renders cancer cells resistant to inhibition of nampt by other nad + synthesis. in line with this hypothesis, the loss of naprt in both rcc cell lines and emt-subtype gastric cell lines renders the cells hypersensitive to nampt inhibitors, such as fk , and kpt- , suggesting that nampt inhibitors may be a promising strategy for naprt deficient tumors. , moreover, bacteria-mediated deamidated nad + biosynthesis also rescues nampti-induced toxicity in cancer cells and xenograft tumors. the enzymatic ability of nampt can be enhanced by pharmacological agents, p c or sbi- . p c is a nampt enhancer with good bioavailability and nontoxicity. it has been demonstrated that p c and its analogs have neuroprotective efficacy in a broad range of preclinical rodent and nonhuman primate models relying on the activation of nampt. [ ] [ ] [ ] [ ] therefore, the neuroprotective activity of p c offers a new pharmacotherapy for age-associated als, alzheimer's disease and parkinson's disease. , , sbi- activates nampt via stabilizing the nampt phosphorylation at his , enhancing the efficiency of nmn generation, providing another option to raise nad + . together, the nampt enhancers, p c and sbi- , warrant further study for the clinical treatment of neuron related diseases. the nad + -biosynthetic pathway can also be increased by blocking the side branch to prevent the escape of intermediates. overexpressing acmsd reduces the nad + level by dissipating acms from the de novo nad + synthesis into the side branch pathway for acetyl-coa production, while inhibiting acmsd elevates nad + concentration. the high expression of acmsd in the kidney and liver offers the therapeutic potential of acmsd inhibitors, such as tes- and tes- , for renal and hepatic dysfunction. , acmsd may also be a novel target for parkinson's disease, as it inhibits the generation of neurotoxin quinolinic acid in the kynurenine pathway. similarly, nnmt shifts the nam into producing -methylnicotinamide, leading to impaired salvage pathway. wat-and liver-specific knockdown of nnmt prevent diet-induced obesity through enhancing the energy expenditure. the effect of nnmt is achieved by its effect on histone methylation. pharmacological inhibition of nnmt significantly shows benefits diet-induced obese mice, including reducing the body weight gain and adipocyte size, and decreasing serum cholesterol levels. , these results suggest that nnmt is an appealing target for obesity and type diabetes treatment. together, acmsd and nnmt provide novel targets for modulating nad + homeostasis, which will be of great importance to determine whether acmsd and nnmt inhibitors can increase nad + levels and achieve therapeutic effects. increasing nad + bioavailability intracellular nad + levels can also be increased by energy stress, including fasting, glucose restriction, caloric restriction (cr) and exercise. cr-mediated nad + boosting depends on the nad + salvage biosynthesis rather than de novo pathway via elevating the nampt expression. , , - cr restores the age-associated circadian decline via sharpening circadian control of nad + metabolism and nad + /sirt -modulated epigenetic modification. [ ] [ ] [ ] [ ] it has been shown that both long-term and short-term cr rescue the large elastic artery stiffening and endothelial dysfunction. , similarly, crboosted nad + level protects the brain against aging and diseases through attenuating plasma membrane lipid peroxidation, protein carbonyls, nitrotyrosine and oxidative stress. beyond cr, the exercise has attracted growing attention due to its benefits on health. in this context, exercise could also increase the nad + level and sirt activity due to an increase in nampt. it has been reported that the nampt protein levels in athletes' skeletal muscle is much higher than in type diabetic, sedentary obese and nonobese subjects. furthermore, exercise training induces a robust increase in the skeletal muscle of nampt protein in sedentary nonobese subjects. the exercise-mediated nad + boosting by inducing nampt is a response strategy for energy stress, which is abolished by depletion of the energy sensor ampk. intriguingly, the gut microenvironment, especially the host-bacteria interactions also contributes to nad + metabolism. the gut microbiota-derived deamidated pathway facilitates the utilization of nam or nr for hepatic nad + synthesis, suggesting that manipulation of microbiota might offer a new option to manipulate nad + metabolism. taken together, both caloric restriction and exercise provide the potential therapeutic strategies in therapy against pathologies related to nad + decline. the levels and compartmentalization of nad + dictate energy state that impinges on normal physiological and biological responses, as indicated by the regulatory role of nad + in proper redox homeostasis, genomic stability, gene expression, circadian clock, inflammation, metabolism, cellular bioenergetics, mitochondrial homeostasis and adaptive stress responses. a healthy lifestyle and exercise are nonpharmacologic strategies to improve the body's resilience and extend healthy lifespan through enhancing nad + levels. nad + boosters can be applied for a broad spectrum of nad + deficiency related pathologies, such as infection, cancer, metabolic diseases, acute injury, aging and aging-related neurodegenerative disorders. conceivably, this could be achieved by boosting nad + via enhancing the nad + generation and diminishing nad + consumption. despite exciting and emerging strides in nad + biology, there are a variety of outstanding questions that warrant future systematic exploitation to accelerate the translation of remarkable bench work to effective clinical application in humans. the first interesting question is that the precise mechanisms executing the beneficial effects of nad + and its metabolites on pathologies and lifespan remain elusive. further investigation understanding the landscape of nad + in response to diseases and identifying the specific effector molecules for each nad + precursors at different time points provide critical insights into development of effective interventions for various physiologies. secondly, the systemic nad + metabolome is largely unexplored. are there any tissue specificities for nad + boosting, such tissue preferences of distinct nad + precursors? what is the crosstalk with the nad + systems of each organ? what is the distinct nad + metabolome in each tissue? in spite of growing interest in the use of nad + precursors as a strategy for healthy aging, the in vivo pharmacokinetics remain poorly understood. the efficacy of nad + boosters, the therapeutic dosages and favorable administration routes should be optimized for different diseases in humans. it is also essential to fully assess the unforeseen side effects of long-term nad + boosting. this task requires the development of new technologies to enable the simplifying, accurate and reproducible monitoring of dynamic nad + and its metabolites in patients and healthy individuals. the alcoholic ferment of yeast-juice part ii.-the coferment of yeast-juice pyridin, the hydrogen-transferring component of the fermentation enzymes (pyridine nucleotide) nicotinamide mononucleotide activation of new dna-dependent polyadenylic acid synthesizing nuclear enzyme characterization of five human cdnas with homology to the yeast sir gene: sir -like proteins (sirtuins) metabolize nad and may have protein adp-ribosyltransferase activity transcriptional silencing and longevity protein sir is an nad-dependent histone deacetylase the silencing protein sir and its homologs are nad-dependent protein deacetylases human dna ligase iv is able to use nad+ as an alternative adenylation donor for dna ends ligation the mechanism of rna ' capping with nad+, nadh and desphospho-coa lc/ms analysis of cellular rna reveals nad-linked rna nad captureseq indicates nad as a bacterial cap for a subset of regulatory rnas identification of nad+ capped mrnas in saccharomyces cerevisiae end nicotinamide adenine dinucleotide cap in human cells promotes rna decay through dxo-mediated denadding quantifying the rna cap epitranscriptome reveals novel caps in cellular and viral rna nicotinamide and pnc govern lifespan extension by calorie restriction in saccharomyces cerevisiae de novo nad(+) synthesis enhances mitochondrial function and improves health kynurenine pathway of tryptophan metabolism: regulatory and functional aspects structural insights into the quaternary catalytic mechanism of hexameric human quinolinate phosphoribosyltransferase, a key enzyme in de novo nad biosynthesis crystal structure of human nicotinic acid phosphoribosyltransferase it's an nad(+) synthase… it's a chaperone… it's a neuroprotector a novel deamido-nad+-binding site revealed by the trapped nadadenylate intermediate in the nad+ synthetase structure role of nicotinamide adenine dinucleotide and related precursors as therapeutic targets for age-related degenerative diseases: rationale, biochemistry, pharmacokinetics, and outcomes structure of nampt/pbef/visfatin, a mammalian nad+ biosynthetic enzyme therapeutic potential of nad-boosting molecules: the in vivo evidence structure of human nicotinamide/nicotinic acid mononucleotide adenylyltransferase. basis for the dual substrate specificity and activation of the oncolytic agent tiazofurin crystal structure of human nicotinamide mononucleotide adenylyltransferase in complex with nmn the multifaceted functions of sirtuins in cancer novel insights into parps in gene expression: regulation of rna metabolism parps and adp-ribosylation: recent advances linking molecular functions to biological outcomes reversible adp-ribosylation of rna systems-wide analysis of serine adp-ribosylation reveals widespread occurrence and sitespecific overlap with phosphorylation the membrane-bound enzyme cd exists in two opposing orientations adenine nucleotides as paracrine mediators and intracellular second messengers in immunity and inflammation sarm -specific motifs in the tir domain enable nad+ loss and regulate injury-induced sarm activation the sarm toll/interleukin- receptor domain possesses intrinsic nad(+) cleavage activity that promotes pathological axonal degeneration nad+ homeostasis in health and disease sirt regulates circadian clock gene expression through per deacetylation the nad(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity the nad+-dependent deacetylase sirt modulates clockmediated chromatin remodeling and circadian control a continuous microplate assay for sirtuins and nicotinamide-producing enzymes srt , srt , srt , and resveratrol are not direct activators of sirt the camp/pka pathway rapidly activates sirt to promote fatty acid oxidation independently of changes in nad(+) biochemical characterization, localization, and tissue distribution of the longer form of mouse sirt sirt deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome investigating the sensitivity of nad+-dependent sirtuin deacylation activities to nadh a novel continuous assay for the deacylase sirtuin and other deacetylases identification of and molecular basis for sirt loss-of-function point mutations in cancer adp-ribose) polymerase is a catalytic dimer and the automodification reaction is intermolecular parp- , a novel mammalian dna damage-dependent poly(adpribose) polymerase the role of parp- and parp- enzymes in metabolic regulation and disease kinetic competence of the cadp-ribose-cd complex as an intermediate in the cd / nad+ glycohydrolase-catalysed reactions: implication for cd signalling nad + -metabolizing ectoenzymes in remodeling tumorhost interactions: the human myeloma model cd dictates age-related nad decline and mitochondrial dysfunction through an sirt -dependent mechanism a potent and specific cd inhibitor ameliorates age-related metabolic dysfunction by reversing tissue nad(+) decline nicotinamide adenine dinucleotide metabolism and neurodegeneration nicotinamide n-oxidation by cyp e in human liver microsomes human liver nicotinamide nmethyltransferase. cdna cloning, expression, and biochemical characterization n -methylnicotinamide oxidation in a number of mammals fate of nicotinamide in the mouse. urinary metabolites niacin catabolism in rodents identification of evolutionary and kinetic drivers of naddependent signaling weak coupling of atp hydrolysis to the chemical equilibrium of human nicotinamide phosphoribosyltransferase metabolic effects of nicotinamide administration in rats nicotinamide n-methyltransferase regulates hepatic nutrient metabolism through sirt protein stabilization semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides biosensor reveals multiple sources for mitochondrial nad + nad(+) metabolism and the control of energy homeostasis: a balancing act between mitochondria and the nucleus sirt deacetylates carbamoyl phosphate synthetase and regulates the urea cycle the secret life of nad+: an old metabolite controlling new metabolic signaling pathways mitochondrial function in liver cells is resistant to perturbations in nad(+) salvage capacity the membrane of peroxisomes in saccharomyces cerevisiae is impermeable to nad(h) and acetyl-coa under in vivo conditions pathways and subcellular compartmentation of nad biosynthesis in human cells: from entry of extracellular precursors to mitochondrial nad generation sirt reverses aging-associated degeneration nicotinamide adenine dinucleotide is transported into mammalian mitochondria the mitochondrial nad(+) transporter (ndt ) plays important roles in cellular nad(+) homeostasis in arabidopsis thaliana identification of the mitochondrial nad+ transporter in saccharomyces cerevisiae the peroxisomal nad carrier from arabidopsis imports nad in exchange with amp molecular identification and functional characterization of arabidopsis thaliana mitochondrial and chloroplastic nad+ carrier proteins a candidate nad+ transporter in an intracellular bacterial symbiont related to chlamydiae subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms nutrient-sensitive mitochondrial nad+ levels dictate cell survival inhibition of nicotinamide phosphoribosyltransferase: cellular bioenergetics reveals a mitochondrial insensitive nad pool role of nadh shuttle system in glucose-induced activation of mitochondrial metabolism and insulin secretion the function and the role of the mitochondrial glycerol- -phosphate dehydrogenase in mammalian tissues redox shuttles in the brain calcium signaling in brain mitochondria: interplay of malate aspartate nadh shuttle and calcium uniporter/mitochondrial dehydrogenase pathways aminooxyacetic acid inhibits the malate-aspartate shuttle in isolated nerve terminals and prevents the mitochondria from utilizing glycolytic substrates developmental changes in the ca +-regulated mitochondrial aspartate-glutamate carrier aralar in brain and prominent expression in the spinal cord the malateaspartate nadh shuttle member aralar determines glucose metabolic fate, mitochondrial activity, and insulin secretion in beta cells lactate oxidation at the mitochondria: a lactate-malate-aspartate shuttle at work cytosolic ca + regulates the energization of isolated brain mitochondria by formation of pyruvate through the malate-aspartate shuttle quantitative analysis of nad synthesis-breakdown fluxes structure and function of nad kinase and nadp phosphatase: key enzymes that regulate the intracellular balance of nad(h) and nadp(h) a genetically encoded tool for manipulation of nadp(+)/nadph in living cells disulfide reductase systems in liver oxidative stress consumption of nadph for -hg synthesis increases pentose phosphate pathway flux and sensitizes cells to oxidative stress sonar, a highly responsive nad+/nadh sensor, allows highthroughput metabolic screening of anti-tumor agents nrk controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells slc a is a nicotinamide mononucleotide transporter a cell-permeant mimetic of nmn activates sarm to produce cyclic adp-ribose and induce non-apoptotic cell death bacteria boost mammalian host nad metabolism by engaging the deamidated biosynthesis pathway oxidative stress: introductory remarks nutrient sensing and the oxidative stress response ros function in redox signaling and oxidative stress oxeiptosis: a discreet way to respond to radicals proliferative neural stem cells have high endogenous ros levels that regulate self-renewal and neurogenesis in a pi k/akt-dependant manner antioxidant and oxidative stress: a mutual interplay in age-related diseases nad(+) administration decreases doxorubicin-induced liver damage of mice by enhancing antioxidation capacity and decreasing dna damage separating nadh and nadph fluorescence in live cells and tissues using flim garlic activates sirt- to prevent cardiac oxidative stress and mitochondrial dysfunction in diabetes a small molecule activator of sirt promotes deacetylation and activation of manganese superoxide dismutase. free radic sirt regulation of mitochondrial oxidative stress modulation of oxidative stress as an anticancer strategy reactive oxygen species and insulin resistance: the good, the bad and the ugly cellular mechanisms and physiological consequences of redox-dependent signalling adaptive redox switches through oxidative/nitrosative protein modifications the nox family of ros-generating nadph oxidases: physiology and pathophysiology the c-terminal peptide plays a role in the formation of an intermediate form during the transition between xanthine dehydrogenase and xanthine oxidase xanthine oxidoreductase in atherosclerosis pathogenesis: not only oxidative stress tetrahydrobiopterin in nitric oxide synthase nitric oxide synthase domain interfaces regulate electron transfer and calmodulin activation superoxide generation by lipoxygenase in the presence of nadh and nadph bioactive lipoxygenase metabolites stimulation of nadph oxidases and reactive oxygen species lipoxygenase-mediated generation of lipid peroxides enhances ferroptosis induced by erastin and rsl cytochrome p family in a cockroach: molecular cloning and regulation by regulation by hypertrehalosemic hormone mechanisms that regulate production of reactive oxygen species by cytochrome p nox redox signaling maintains essential cell populations in the brain methionine residues as endogenous antioxidants in proteins nadph: new oxygen for the ros theory of aging why antioxidant therapies have failed in clinical trials measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue redox signaling mediated by thioredoxin and glutathione systems in the central nervous system oxidative stress and autophagy: the clash between damage and metabolic needs essential role of selenium in the catalytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine mutations a role for the mitochondrial deacetylase sirt in regulating energy homeostasis sirtuin is required for osteogenic differentiation through maintenance of pgc- ɑ-sod -mediated regulation of mitochondrial function succinate dehydrogenase is a direct target of sirtuin deacetylase activity sirt mediates reduction of oxidative damage and prevention of age-related hearing loss under caloric restriction tumour suppressor sirt deacetylates and activates manganese superoxide dismutase to scavenge ros sirt blocks the cardiac hypertrophic response by augmenting foxo a-dependent antioxidant defense mechanisms in mice emerging roles for sirt in metabolism and cancer sirt is a mitochondria-localized tumor suppressor required for maintenance of mitochondrial integrity and metabolism during stress nuclear genomic instability and aging molecular mechanisms of mammalian dna repair and the dna damage checkpoints current role of mammalian sirtuins in dna repair role of nicotinamide in genomic stability and skin cancer chemoprevention the taming of parp and its impact on nad(+) metabolism non-homologous dna end joining and alternative pathways to double-strand break repair multicomponent assemblies in dna-double-strand break repair by nhej effects of deletion and site-directed mutations on ligation steps of nad+-dependent dna ligase: a biochemical analysis of brca c-terminal domain nad(+)-dependent repair of damaged dna by human cell extracts dual function for poly(adp-ribose) synthesis in response to dna strand breakage reversible mono-adp-ribosylation of dna breaks adp-ribose) polymerases covalently modify strand break termini in dna fragments in vitro insight into dna substrate specificity of parp -catalysed dna poly structural basis of detection and signaling of dna singlestrand breaks by human parp- hpf completes the parp active site for dna damageinduced adp-ribosylation adp-ribose)n participates in dna excision repair effects of nicotinamide on nad and poly (adp-ribose) metabolism in dna-damaged human lymphocytes adp-ribose) synthase is the major endogenous nonhistone acceptor for poly(adp-ribose) in alkylated rat hepatoma cells the multifaceted roles of parp in dna repair and chromatin remodelling adp-ribose) polymerase- antagonizes dna resection at double-strand breaks adp-ribosyl)ation-dependent transient chromatin decondensation and histone displacement following laser microirradiation parp -dependent kinetics of recruitment of mre and nbs proteins to multiple dna damage sites overlapping roles for parp and parp in the recruitment of endogenous xrcc and pnkp into oxidized chromatin the xrcc phosphate-binding pocket binds poly (adp-ribose) and is required for xrcc function the comings and goings of parp- in response to dna damage sirt redistribution on chromatin promotes genomic stability but alters gene expression during aging the role of sirt on dna damage response and epigenetic alterations in cancer xrcc coordinates the initial and late stages of dna abasic site repair through protein-protein interactions stress-dependent regulation of foxo transcription factors by the sirt deacetylase sirt regulates the function of the nijmegen breakage syndrome protein sirtuin-mediated deacetylation pathway stabilizes werner syndrome protein the deacetylase sirtuin regulates human papillomavirus replication by modulating histone acetylation and recruitment of dna damage factors nbs and rad to viral genomes sirt and lsd competitively regulate ku functions in dna repair and mutation acquisition in cancer cells ku inhibition impairs both non-homologous end joining and homologous recombination dna damage repair through shp- induced dephosphorylation of sirt in t-cell acute lymphoblastic leukemia (t-all) sirtuin -mediated deacetylation of xpa dna repair protein enhances its interaction with atr protein and promotes camp-induced dna repair of uv damage ataxia telangiectasia mutated (atm) signaling network is modulated by a novel poly(adp-ribose)-dependent pathway in the early response to dna-damaging agents stimulation of the dna-dependent protein kinase by poly(adpribose) polymerase parp- -dependent recruitment of cold-inducible rna-binding protein promotes double-strand break repair and genome stability suppression of the poly(adp-ribose) polymerase activity by dna-dependent protein kinase in vitro the role of sirtuins in antioxidant and redox signaling serine is a new target residue for endogenous adpribosylation on histones nadp(+) is an endogenous parp inhibitor in dna damage response and tumor suppression extracellular nad(+) enhances parp-dependent dna repair capacity independently of cd activity nad(+) replenishment improves lifespan and healthspan in ataxia telangiectasia models via mitophagy and dna repair nad(+) supplementation normalizes key alzheimer's features and dna damage responses in a new ad mouse model with introduced dna repair deficiency metabolic regulation of inflammation epigenetics: poly(adp-ribosyl)ation of parp- regulates genomic methylation patterns regulation of chromatin by histone modifications histone acetylation: molecular mnemonics on the chromatin nad+-dependent deacetylation of h lysine by class iii hdacs the nad(+)-dependent sirt deacetylase translates a metabolic switch into regulatory epigenetics in skeletal muscle stem cells epigenetic modulation of chronic anxiety and pain by histone deacetylation sirt links h k deacetylation to maintenance of oncogenic transformation nicotinamide adenine dinucleotide (nad)-regulated dna methylation alters ccctc-binding factor (ctcf)/cohesin binding and transcription at the bdnf locus parp inhibitor affects long-term heat-stress response via changes in dna methylation ccctc-binding factor activates parp- affecting dna methylation machinery modulation of dnmt activity by adp-ribose polymers nnmt promotes epigenetic remodeling in cancer by creating a metabolic methylation sink proteomics reveals nnmt as a master metabolic regulator of cancer-associated fibroblasts nad( + )-capped rnas are widespread in the arabidopsis transcriptome and can probably be translated mrna capping: biological functions and applications highly efficient ' capping of mitochondrial rna with nad(+) and nadh by yeast and human mitochondrial rna polymerase new insights into decapping enzymes and selective mrna decay identification, biosynthesis, and decapping of nad-capped rnas in b. subtilis structural and mechanistic basis of mammalian nudt rna denadding targeting metabolic pathways for head and neck cancers therapeutics inhibition of nicotinamide phosphoribosyltransferase (nampt), an enzyme essential for nad+ biosynthesis, leads to altered carbohydrate metabolism in cancer cells aerobic glycolysis: meeting the metabolic requirements of cell proliferation nad(h) and nadp(h) redox couples and cellular energy metabolism nad(+) metabolism: bioenergetics, signaling and manipulation for therapy oxidative phosphorylation at the fin de siècle mitochondrial β-oxidation of saturated fatty acids in humans overview: how is alcohol metabolized by the body? fueling thought: management of glycolysis and oxidative phosphorylation in neuronal metabolism regulation of intermediary metabolism by protein acetylation lysine deacetylases and regulated glycolysis in macrophages mitochondrial sirtuin network reveals dynamic sirt -dependent deacetylation in response to membrane depolarization the mitochondrial acylome emerges: proteomics, regulation by sirtuins, and metabolic and disease implications sirt regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation sirt opposes reprogramming of cancer cell metabolism through hif α destabilization sirt regulates cancer cell proliferation through deacetylation of pycr in proline metabolism sirt represses peroxisome proliferator-activated receptor α activity to suppress hepatic fat oxidation sirt is a lysine deacylase that controls leucine metabolism and insulin secretion sirtuin is a lipoamidase regulating pyruvate dehydrogenase complex activity sirt inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells sirt is a nad-dependent protein lysine demalonylase and desuccinylase metabolomics-assisted proteomics identifies succinylation and sirt as important regulators of cardiac function shmt desuccinylation by sirt drives cancer cell proliferation regulation of ucp and mitochondrial metabolism in brown adipose tissue by reversible succinylation sirt regulates the mitochondrial lysine succinylome and metabolic networks sirt stabilizes mitochondrial glutaminase and supports breast cancer tumorigenesis sirt regulates both cytosolic and mitochondrial protein malonylation with glycolysis as a major target sirt -mediated lysine desuccinylation impacts diverse metabolic pathways regulation of circadian clocks by redox homeostasis circadian clock feedback cycle through nampt-mediated nad+ biosynthesis circadian control of the nad+ salvage pathway by clock-sirt predicted role of nad utilization in the control of circadian rhythms during dna damage response circadian clock: linking epigenetics to aging defining the independence of the liver circadian clock nad(+) controls circadian reprogramming through per nuclear translocation to counter aging partitioning circadian transcription by sirt leads to segregated control of cellular metabolism nad(+)-sirt control of h k trimethylation through circadian deacetylation of mll adp-ribose) polymerase participates in the phase entrainment of circadian clocks to feeding crosstalk between metabolism and circadian clocks parp -and ctcf-mediated interactions between active and repressed chromatin at the lamina promote oscillating transcription bmal and beta-cell clock are required for adaptation to circadian disruption, and their loss of function leads to oxidative stress-induced beta-cell failure in mice the circadian clock regulates rhythmic activation of the nrf /glutathione-mediated antioxidant defense pathway to modulate pulmonary fibrosis mitochondrial h o signaling is controlled by the concerted action of peroxiredoxin iii and sulfiredoxin: linking mitochondrial function to circadian rhythm the pentose phosphate pathway regulates the circadian clock circadian clocks in human red blood cells chromatin landscape and circadian dynamics: spatial and temporal organization of clock transcription early aging and age-related pathologies in mice deficient in bmal , the core componentof the circadian clock succinate: a metabolic signal in inflammation macrophage de novo nad(+) synthesis specifies immune function in aging and inflammation succinate dehydrogenase supports metabolic repurposing of mitochondria to drive inflammatory macrophages inflammatory macrophage dependence on nad(+) salvage is a consequence of reactive oxygen species-mediated dna damage mitochondrial respiration controls lysosomal function during inflammatory t cell responses nad(+) metabolism governs the proinflammatory senescenceassociated secretome switch of nad salvage to de novo biosynthesis sustains sirt -relb-dependent inflammatory tolerance the nicotinamide phosphoribosyltransferase: a molecular link between metabolism, inflammation, and cancer increased expression of nampt in pbmc from patients with acute coronary syndrome and in inflammatory m macrophages pre-b cell colony-enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis nad metabolism fuels human and mouse intestinal inflammation pharmacological inhibition of nicotinamide phosphoribosyltransferase/visfatin enzymatic activity identifies a new inflammatory pathway linked to nad pre-b cell colony-enhancing factor (pbef)/ visfatin: a novel mediator of innate immunity intracellular nad levels regulate tumor necrosis factor protein synthesis in a sirtuin-dependent manner nad + -dependent sirt deacetylase participates in epigenetic reprogramming during endotoxin tolerance tryptophan metabolism in inflammaging: from biomarker to therapeutic target chronic inflammation and cytokines in the tumor microenvironment oxidative stress and cancer nadph oxidases: an overview from structure to innate immunity-associated pathologies cutting edge: direct interaction of tlr with nad(p)h oxidase isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of nf-kappa b dual oxidase is essential for the toll-like receptor -mediated inflammatory response in airway mucosa role of nicotinamide adenine dinucleotide phosphate oxidase in oxidative burst response to toll-like receptor signaling in large intestinal epithelial cells autophagy protein rubicon mediates phagocytic nadph oxidase activation in response to microbial infection or tlr stimulation the role of oxidative stress in viral infections hepatitis b virus x protein (hbx)-induced abnormalities of nucleic acid metabolism revealed by ( )h-nmr-based metabonomics metabolomic profile of hepatitis c virus-infected hepatocytes divergent effects of human cytomegalovirus and herpes simplex virus- on cellular metabolism parp- mediated cell death is directly activated by zikv infection hiv infection decreases intracellular nicotinamide adenine dinucleotide enhancement by tumor necrosis factor alpha of dengue virus-induced endothelial cell production of reactive nitrogen and oxygen species is key to hemorrhage development potential dengue virus-triggered apoptotic pathway in human neuroblastoma cells: arachidonic acid, superoxide anion, and nf-kappab are sequentially involved dengue virus replication in human hepatoma cells activates nf-kappab which in turn induces apoptotic cell death hepatitis c virus infection activates the immunologic (type ii) isoform of nitric oxide synthase and thereby enhances dna damage and mutations of cellular genes hepatitis c virus induces toll-like receptor expression, leading to enhanced production of beta interferon and interleukin- replication-incompetent virions of japanese encephalitis virus trigger neuronal cell death by oxidative stress in a culture system increase in de novo hbv dna integrations in response to oxidative dna damage or inhibition of poly (adp-ribosyl) ation counteraction of hcv-induced oxidative stress concurs to establish chronic infection in liver cell cultures oxidative stress and hepatic nox proteins in chronic hepatitis c and hepatocellular carcinoma reactive oxygen species mediate virus-induced stat activation: role of tyrosine phosphatases epigenetic silencing of nad(p)h:quinone oxidoreductase by hepatitis b virus x protein increases mitochondrial injury and cellular susceptibility to oxidative stress in hepatoma cells temporal changes in chromatin, intracellular calcium, and poly (adp-ribose) polymerase during sindbis virus-induced apoptosis of neuroblastoma cells parp- controls nk cell recruitment to the site of viral infection systematic identification of type i and type ii interferon-induced antiviral factors interferon-stimulated poly(adp-ribose) polymerases are potent inhibitors of cellular translation and virus replication parp suppresses zika virus infection through parp-dependent degradation of ns and ns viral proteins the coronavirus macrodomain is required to prevent parp-mediated inhibition of virus replication and enhancement of ifn expression sirtuins are evolutionarily conserved viral restriction factors inhibition of silent information regulator induces glucose metabolism disorders of hepatocytes and enhances hepatitis c virus replication resveratrol inhibited tatinduced hiv- ltr transactivation via nad(+)-dependent sirt activity quinolinate phosphoribosyltransferase is an antiviral host factor against hepatitis c virus infection sirt modulates the sensitivity of prostate cancer cells to vesicular stomatitis virus oncolysis parp stabilizes ctcf binding and chromatin structure to maintain epstein-barr virus latency type activation of kaposi's sarcomaassociated herpesvirus (kshv) by inhibitors of class iii histone deacetylases: identification of sirtuin as a regulator of the kshv life cycle a novel role of sirt in gammaherpesvirus latency and replication human cd interferes with hiv- fusion through a sequence homologous to the v loop of the viral envelope glycoprotein gp clonally diverse cd (+)hla-dr(+)cd (+) t cells persist during fatal h n disease cd modulates respiratory syncytial virus-driven proinflammatory processes in human monocyte-derived dendritic cells hbv and hiv viral load but not microbial translocation or immune activation are associated with liver fibrosis among patients in south africa nicotinamide phosphoribosyltransferase/sirtuin pathway is involved in human immunodeficiency virus type tat-mediated long terminal repeat transactivation why do viruses cause cancer? highlights of the first century of human tumour virology hbx-elevated sirt promotes hbv replication and hepatocarcinogenesis sirtuin isoform enhances hepatitis b virus rna transcription and dna synthesis through the akt/gsk- beta/beta-catenin signaling pathway tlr signalling augments macrophage bactericidal activity through mitochondrial ros neutrophils to the roscue: mechanisms of nadph oxidase activation and bacterial resistance cutting edge: myd controls phagocyte nadph oxidase function and killing of gram-negative bacteria influenza infection suppresses nadph oxidasedependent phagocytic bacterial clearance and enhances susceptibility to secondary methicillin-resistant staphylococcus aureus infection glycolytic reprograming in salmonella counters nox -mediated dissipation of Δph activation of antibacterial autophagy by nadph oxidases nad hydrolysis by the tuberculosis necrotizing toxin induces lethal oxidative stress in macrophages a novel phox/cd / mcoln /tfeb axis important for macrophage activation during bacterial phagocytosis cd controls the innate immune response against listeria monocytogenes cyclic adp-ribose production by cd regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo the nuclear receptor lxr limits bacterial infection of host macrophages through a mechanism that impacts cellular nad metabolism the cd /nad/sirtuin /ezh axis mitigates cytotoxic cd t cell function and identifies patients with sle prone to infections nad+ and sirtuins in aging and disease resveratrol attenuates hypoxic injury in a primary hepatocyte model of hemorrhagic shock and resuscitation astrocytes protect neurons from aβ - peptideinduced neurotoxicity increasing tfam and pgc- and decreasing ppar-γ and sirt- sirt- is required for the inhibition of apoptosis and inflammatory responses in human tenocytes nad(+) in aging: molecular mechanisms and translational implications age related changes in nad+ metabolism oxidative stress and sirt activity in wistar rats hepatic nad(+) deficiency as a therapeutic target for nonalcoholic fatty liver disease in ageing the plasma nad(+) metabolome is dysregulated in "normal" aging nicotinamide mononucleotide, a key nad(+) intermediate, treats the pathophysiology of diet-and age-induced diabetes in mice specific ablation of nampt in adult neural stem cells recapitulates their functional defects during aging nad (+) biosynthesis, aging, and disease tnf-alpha induced cd expression in human airway smooth muscle cells: role of map kinases and transcription factors nf-kappab and ap- the nad(+)/sirtuin pathway modulates longevity through activation of mitochondrial upr and foxo signaling nad + repletion improves mitochondrial and stem cell function and enhances life span in mice extension of human cell lifespan by nicotinamide phosphoribosyltransferase nicotinamide mononucleotide supplementation reverses vascular dysfunction and oxidative stress with aging in mice declining nad(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging augmentation of cellular nad(+) by nqo enzymatic action improves age-related hearing impairment sirt extends life span and delays aging in mice through the regulation of nk homeobox in the dmh and lh the sirtuin sirt regulates lifespan in male mice a high-fat diet and nad(+) activate sirt to rescue premature aging in cockayne syndrome circadian clock nad+ cycle drives mitochondrial oxidative metabolism in mice oxidative stress in aging-matters of the heart and mind neurodegenerative diseases and oxidative stress impact of long-term rf-emf on oxidative stress and neuroinflammation in aging brains of c bl/ mice the need to incorporate aged animals into the preclinical modeling of neurological conditions the potential role of necroptosis in inflammaging and aging pro-inflammatory cytokines increase reactive oxygen species through mitochondria and nadph oxidase in cultured rpe cells nox contributes to age-related oxidative damage to neurons and the cerebral vasculature the role of mitochondrial ros in the aging brain aging induces cardiac diastolic dysfunction, oxidative stress, accumulation of advanced glycation endproducts and protein modification overexpression of nmnat efficiently increases nad and ngd levels and ameliorates age-associated insulin resistance g pd protects from oxidative damage and improves healthspan in mice sirt regulates aging and resistance to oxidative stress in the heart sirt mediates central circadian control in the scn by a mechanism that decays with aging hallmarks of cancer: the next generation nad(+) salvage pathway in cancer metabolism and therapy the nad(+) salvage pathway modulates cancer cell viability via p interplay between sirt proteins and tumour suppressor transcription factors in chemotherapeutic resistance of cancer tyrosine phosphorylation of lactate dehydrogenase a is important for nadh/nad(+) redox homeostasis in cancer cells mitochondrial complex i activity and nad+/nadh balance regulate breast cancer progression regulation of cancer cell metabolism metastasis and oxidative stress: are antioxidants a metabolic driver of progression? cross talk between eif alpha and eef phosphorylation pathways optimizes translational arrest in response to oxidative nadph oxidase -dependent ros is crucial for tlr signaling to promote tumor metastasis of non-small cell lung cancer inhibition of pyruvate kinase m by reactive oxygen species contributes to cellular antioxidant responses oxidative stress, inflammation and carcinogenesis are controlled through the pentose phosphate pathway by transaldolase nicotinamide nucleotide transhydrogenase-mediated redox homeostasis promotes tumor growth and metastasis in gastric cancer nampt suppresses glucose deprivation-induced oxidative stress by increasing nadph levels in breast cancer me regulates nadph homeostasis to promote gastric cancer growth and metastasis ampk regulates nadph homeostasis to promote tumour cell survival during energy stress sirt interacts with and protects glyceraldehyde- -phosphate dehydrogenase (gapdh) from nuclear translocation: implications for cell survival after irradiation sirt deacetylates and increases pyruvate dehydrogenase activity in cancer cells subcellular compartmentalization of nad(+) and its role in cancer: a serenade of metabolic melodies sirt -mediated sdha desuccinylation promotes clear cell renal cell carcinoma tumorigenesis mcu-dependent mitochondrial ca( +) inhibits nad(+)/sirt / sod pathway to promote ros production and metastasis of hcc cells sirt suppresses hypoxia inducible factor α and tumor growth by inhibiting mitochondrial ros production sirt -mediated dimerization of idh directs cancer cell metabolism and tumor growth regulation of glucose metabolism by nad(+) and adp-ribosylation sirt -dependent regulation of chromatin and transcription: linking nad(+) metabolism and signaling to the control of cellular functions sirt -dependent epigenetic regulation of h and h histone acetylation in human breast cancer modulations of hmof autoacetylation by sirt regulate hmof recruitment and activities on the chromatin glioma-induced sirt -dependent activation of hmof histone h lysine acetyltransferase in microglia promotes a tumor supporting phenotype histone deacetylases in acute myeloid leukaemia show a distinctive pattern of expression that changes selectively in response to deacetylase inhibitors impaired dna damage response, genome instability, and tumorigenesis in sirt mutant mice brca and brca : and beyond parp inhibition in cancer: an update on clinical development delivering widespread brca testing and parp inhibition to patients with ovarian cancer synthetic lethal therapies for cancer: what's next after parp inhibitors? nicotinamide n-methyltransferase: more than a vitamin b clearance enzyme upregulation of tissue and urinary nicotinamide nmethyltransferase in bladder cancer: potential for the development of a urine-based diagnostic test expression profile and prognostic value of nnmt in patients with pancreatic cancer serum levels of nicotinamide n-methyltransferase in patients with lung cancer nicotinamide metabolism regulates glioblastoma stem cell maintenance elevated n-methyltransferase expression induced by hepatic stellate cells contributes to the metastasis of hepatocellular carcinoma via regulation of the cd v isoform proteomics reveals nnmt as a master metabolic regulator of cancer-associated fibroblasts reductive carboxylation supports redox homeostasis during anchorage-independent growth the roles of dna, rna and histone methylation in ageing and cancer nad metabolic dependency in cancer is shaped by gene amplification and enhancer remodelling nicotinic acid phosphoribosyltransferase regulates cancer cell metabolism, susceptibility to nampt inhibitors, and dna repair cancer cell metabolic plasticity allows resistance to nampt inhibition but invariably induces dependence on ldha nampt and naprt, key enzymes in nad salvage synthesis pathway, are of negative prognostic value in colorectal cancer epigenetic regulation of nampt by nampt-as drives metastatic progression in triple-negative breast cancer the c-myc/nampt/sirt feedback loop is activated in early classical and serrated route colorectal cancer and represents a therapeutic target nampt regulates pkm nuclear location through - - zeta: conferring resistance to tamoxifen in breast cancer nampt overexpression induces cancer stemness and defines a novel tumor signature for glioma prognosis nampt is a potent oncogene in colon cancer progression that modulates cancer stem cell properties and resistance to therapy through sirt and parp sirt deacetylase activity regulates nampt activity and nad(p) (h) pools in cancer cells decreased nad activates stat and integrin pathways to drive epithelial-mesenchymal transition cd inhibits prostate cancer metabolism and proliferation by reducing cellular nad(+) pools preclinical efficacy of the novel competitive nampt inhibitor stf- in pancreatic cancer dual and specific inhibition of nampt and pak by kpt- decreases kidney cancer growth inhibition of nampt markedly enhances plasma-activated medium-induced cell death in human breast cancer mda-mb- cells selective cytotoxicity of the nampt inhibitor fk toward gastric cancer cells with markers of the epithelial-mesenchymal transition, due to loss of naprt roles of pyruvate, nadh, and mitochondrial complex i in redox balance and imbalance in β cell function and dysfunction metabolic signaling in fuelinduced insulin secretion mitochondrial signals in glucose-stimulated insulin secretion in the beta cell glucose-sensing mechanisms in pancreatic beta-cells separation of the glucose-stimulated cytoplasmic and mitochondrial nad(p)h responses in pancreatic islet beta cells single-cell transcriptomes identify human islet cell signatures and reveal cell-type-specific expression changes in type diabetes single islet beta-cell stimulation by nutrients: relationship between pyridine nucleotides, cytosolic ca + and secretion nad(p)h and membrane potential changes in pancreatic beta-cells by methyl succinate: comparison with glucose the dynamic plasticity of insulin production in β-cells diabetes causes marked inhibition of mitochondrial metabolism in pancreatic β-cells how, when, and where do human β-cells regenerate? identification of the signals for glucose-induced insulin secretion in ins ( / ) β-cells using metformin-induced metabolic deceleration as a model early neural and vascular dysfunctions in diabetic rats are largely sequelae of increased sorbitol oxidation role for malic enzyme, pyruvate carboxylation, and mitochondrial malate import in glucose-stimulated insulin secretion a role for atp-citrate lyase, malic enzyme, and pyruvate/citrate cycling in glucose-induced insulin secretion metabolic fate of glucose in purified islet cells glucose-regulated anaplerosis in beta cells stimulation of insulin release by glyceraldehyde may not be similar to glucose effects of glucose on the cytosolic ration of reduced/oxidized nicotinamide-adenine dinucleotide phosphate in rat islets of langerhans redox control of exocytosis: regulatory role of nadph, thioredoxin, and glutaredoxin glutaredoxin- mediates nadph-dependent stimulation of calcium-dependent insulin secretion nampt-mediated nad(+) biosynthesis in adipocytes regulates adipose tissue function and multi-organ insulin sensitivity in mice epidemiologic and economic consequences of the global epidemics of obesity and diabetes adipose tissue nad(+) biology in obesity and insulin resistance: from mechanism to therapy elevated microrna- a in obesity reduces nad+ levels and sirt activity by directly targeting nampt physiological and pathophysiological roles of nampt and nad metabolism plasma visfatin concentrations and fat depot-specific mrna expression in humans increased levels and adipose tissue expression of visfatin in morbidly obese women: the relationship with pro-inflammatory cytokines human visfatin expression: relationship to insulin sensitivity, intramyocellular lipids, and inflammation visfatin in overweight/ obesity, type diabetes mellitus, insulin resistance, metabolic syndrome and cardiovascular diseases: a meta-analysis and systemic review visfatin in adipocytes is upregulated by hypoxia through hif alpha-dependent mechanism expression of neuropeptide y, omentin and visfatin in visceral and subcutaneous adipose tissues in humans: relation to endocrine and clinical parameters obesity is associated with low nad(+)/sirt pathway expression in adipose tissue of bmi-discordant monozygotic twins weight loss is associated with increased nad(+)/sirt expression but reduced parp activity in white adipose tissue nampt-mediated nad(+) biosynthesis is indispensable for adipose tissue plasticity and development of obesity visfatin is released from t -l adipocytes via a non-classical pathway visfatin regulates genes related to lipid metabolism in porcine adipocytes sirt -mediated enampt secretion from adipose tissue regulates hypothalamic nad+ and function in mice leucine supplementation increases sirt expression and prevents mitochondrial dysfunction and metabolic disorders in high-fat diet-induced obese mice non-alcoholic fatty liver disease inhibition of nampt aggravates high fat diet-induced hepatic steatosis in mice through regulating sirt /ampkalpha/srebp signaling pathway non-alcoholic fatty liver disease as a cause and a consequence of metabolic syndrome nicotinamide and nafld: is there nothing new under the sun? metabolites nicotinamide riboside exerts protective effect against aginginduced nafld-like hepatic dysfunction in mice metabolomic tissue signature in human non-alcoholic fatty liver disease identifies protective candidate metabolites troxerutin improves hepatic lipid homeostasis by restoring nad(+)-depletion-mediated dysfunction of lipin signaling in high-fat diettreated mice intracellular nicotinamide phosphoribosyltransferase protects against hepatocyte apoptosis and is down-regulated in nonalcoholic fatty liver disease hepatic foxos regulate lipid metabolism via modulation of expression of the nicotinamide phosphoribosyltransferase gene association between nicotinamide phosphoribosyltransferase and de novo lipogenesis in nonalcoholic fatty liver disease nnmt: a bad actor in fat makes good in liver er stress-induced upregulation of nnmt contributes to alcoholrelated fatty liver development association of adipokines with development and progression of nonalcoholic fatty liver disease adipocytokines and hepatic fibrosis extracellular visfatin activates gluconeogenesis in hepg cells through the classical pka/creb-dependent pathway involvement of visfatin in palmitate-induced upregulation of inflammatory cytokines in hepatocytes association of plasma visfatin with hepatic and systemic inflammation in nonalcoholic fatty liver disease adipocytokines and cytokeratin- in patients with nonalcoholic fatty liver disease: introduction of cha index plasma visfatin levels and gene expression in morbidly obese women with associated fatty liver disease liver visfatin expression in morbidly obese patients with nonalcoholic fatty liver disease undergoing bariatric surgery prediction of nonalcoholic fatty liver disease via a novel panel of serum adipokines axonal degeneration as a therapeutic target in the cns axonal pathology in traumatic brain injury dsarm/sarm is required for activation of an injury-induced axon death pathway wallerian degeneration: an emerging axon death pathway linking injury and disease sarm activation triggers axon degeneration locally via nad + destruction wallerian degeneration, wld(s), and nmnat a rise in nad precursor nicotinamide mononucleotide (nmn) after injury promotes axon degeneration nmnat inhibits axon degeneration via blockade of sarm -mediated nad(+) depletion. elife increased nuclear nad biosynthesis and sirt activation prevent axonal degeneration wallerian degeneration of injured axons and synapses is delayed by a ube b/nmnat chimeric gene nmnats, evolutionarily conserved neuronal maintenance factors nad synthase nmnat acts as a chaperone to protect against neurodegeneration nmnat :hsp complex mediates proteostasis in proteinopathies nicotinamide mononucleotide adenylyl transferase-mediated axonal protection requires enzymatic activity but not increased levels of neuronal nicotinamide adenine dinucleotide wallerian degeneration is executed by an nmn-sarm -dependent late ca( +) influx but only modestly influenced by mitochondria alzheimer's disease drug repositioning for alzheimer's disease mitophagy inhibits amyloid-β and tau pathology and reverses cognitive deficits in models of alzheimer's disease nad(+) in brain aging and neurodegenerative disorders the road to restoring neural circuits for the treatment of alzheimer's disease evidence that gamma-secretase mediates oxidative stressinduced beta-secretase expression in alzheimer's disease increased oxidative damage in nuclear and mitochondrial dna in alzheimer's disease global metabolic shifts in age and alzheimer's disease mouse brains pivot at nad+/nadh redox sites nicotinamide forestalls pathology and cognitive decline in alzheimer mice: evidence for improved neuronal bioenergetics and autophagy procession alzheimer's disease pathology is attenuated in a cd -deficient mouse model oxidative stress, dysfunctional glucose metabolism and alzheimer disease genome instability in alzheimer disease nuclear dna damage signalling to mitochondria in ageing defective mitophagy in xpa via parp- hyperactivation and nad(+)/sirt reduction mitophagy and nad(+) inhibit alzheimer disease nicotinamide restores cognition in alzheimer's disease transgenic mice via a mechanism involving sirtuin inhibition and selective reduction of thr -phosphotau nicotinamide mononucleotide adenylyltransferase uses its nad(+) substrate-binding site to chaperone phosphorylated tau parkinson's disease psychosis: presentation, diagnosis and management decreased sirtuin deacetylase activity in lrrk g s ipscderived dopaminergic neurons mitochondrial metabolism regulates microtubule acetylome and autophagy trough sirtuin- : impact for parkinson's disease the nad+ precursor nicotinamide riboside rescues mitochondrial defects and neuronal loss in ipsc and fly models of parkinson's disease enhancing nad(+) salvage metabolism is neuroprotective in a pink model of parkinson's disease parp mutations protect against mitochondrial dysfunction and neurodegeneration in a parkin model of parkinson's disease phenothiazine normalizes the nadh/ nad(+) ratio, maintains mitochondrial integrity and protects the nigrostriatal dopamine system in a chronic rotenone model of parkinson's disease a novel treatment target for parkinson's disease c-jun n-terminal kinase (jnk)-mediated phosphorylation of sarm regulates nad(+) cleavage activity to inhibit mitochondrial respiration sarm and traf bind to and stabilize pink on depolarized mitochondria high doses of nicotinamide prevent oxidative mitochondrial dysfunction in a cellular model and improve motor deficit in a drosophila model of parkinson's disease implications of nad metabolism in pathophysiology and therapeutics for neurodegenerative diseases clinical features of huntington's disease huntington's disease-update on treatments huntington's disease: a clinical review the neuropsychology of huntington's disease pgc- α, sirtuins and parps in huntington's disease and other neurodegenerative conditions: nad+ to rule them all trans-(-)-ε-viniferin increases mitochondrial sirtuin (sirt ), activates amp-activated protein kinase (ampk), and protects cells in models of huntington disease thermoregulatory and metabolic defects in huntington's disease transgenic mice implicate pgc- alpha in huntington's disease neurodegeneration impaired pgc- alpha function in muscle in huntington's disease pgc- α rescues huntington's disease proteotoxicity by preventing oxidative stress and promoting tfeb function nicotinamide improves motor deficits and upregulates pgc- α and bdnf gene expression in a mouse model of huntington's disease inhibition of mtor induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of huntington disease the kynurenine pathway modulates neurodegeneration in a drosophila model of huntington's disease kynurenine pathway measurements in huntington's disease striatum: evidence for reduced formation of kynurenic acid antioxidant alternatives in the treatment of amyotrophic lateral sclerosis: a comprehensive review evaluation of the nad(+) biosynthetic pathway in als patients and effect of modulating nad(+) levels in hsod -linked als mouse models als genes in the genomic era and their implications for ftd is sod loss of function involved in amyotrophic lateral sclerosis oxidative stress in neurodegenerative diseases mutations in cu/zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis differential effects of mutant sod on protein structure of skeletal muscle and spinal cord of familial amyotrophic lateral sclerosis: role of chaperone network the genetics and neuropathology of amyotrophic lateral sclerosis astrocytes expressing als-linked mutated sod release factors selectively toxic to motor neurons increased glutathione biosynthesis by nrf activation in astrocytes prevents p ntr-dependent motor neuron apoptosis a role for astrocytes in motor neuron loss in amyotrophic lateral sclerosis enhancing nad+ salvage pathway reverts the toxicity of primary astrocytes expressing amyotrophic lateral sclerosis-linked mutant superoxide dismutase (sod ) nicotinamide riboside enhances mitochondrial proteostasis and adult neurogenesis through activation of mitochondrial unfolded 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-s protects a rat model of alzheimer's disease from neuropsychiatric deficits and neurodegeneration without altering amyloid deposition or reactive glia p c neuroprotective chemicals function by activating the ratelimiting enzyme in nad salvage boosting nad(+) with a small molecule that activates nampt acmsd: a novel target for modulating nad(+) homeostasis is the enzyme acmsd a novel therapeutic target in parkinson's disease? j. parkinson's dis selective and membrane-permeable small molecule inhibitors of nicotinamide n-methyltransferase reverse high fat diet-induced obesity in mice nicotinamide n-methyltransferase knockdown protects against diet-induced obesity requirement of nad and sir for lifespan extension by calorie restriction in saccharomyces cerevisiae calorie restriction extends yeast life span by lowering the level of nadh calorie restriction extends saccharomyces cerevisiae lifespan by increasing respiration lung adenocarcinoma distally rewires hepatic circadian homeostasis restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus entrainment of the circadian clock in the liver by feeding time of feeding and the intrinsic circadian clock drive rhythms in hepatic gene expression short-term calorie restriction reverses vascular endothelial dysfunction in old mice by increasing nitric oxide and reducing oxidative stress life-long caloric restriction reduces oxidative stress and preserves nitric oxide bioavailability and function in arteries of old mice calorie restriction up-regulates the plasma membrane redox system in brain cells and suppresses oxidative stress during aging intracellular redox state revealed by in vivo ( ) p mrs measurement of nad(+) and nadh contents in brains spectrophotometric determination of oxidized and reduced pyridine nucleotides in erythrocytes using a single extraction procedure nicotinamide phosphoribosyl transferase/pre-b cell colonyenhancing 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assay methods for nicotinamide mononucleotide adenylyltransferase of wide applicability intracellular oxidation-reduction states in vivo in vivo monitoring of cellular energy metabolism using sonar, a highly responsive sensor for nad(+)/nadh redox state analysis of redox landscapes and dynamics in living cells and in vivo using genetically encoded fluorescent sensors genetically encoded fluorescent sensors for intracellular nadh detection separating nadh and nadph fluorescence in live cells and tissues using flim characterization of frex as an nadh sensor for in vivo applications in the presence of nad(+) and at various ph values genetically encoded fluorescent indicator for imaging nad (+)/nadh ratio changes in different cellular compartments apollo-nadp(+): a spectrally tunable family of genetically encoded sensors for nadp(+) genetically encoded fluorescent sensors reveal dynamic regulation of nadph metabolism brain bioenergetics and redox state measured by ( )p magnetic resonance spectroscopy in unaffected siblings of patients with psychotic disorders in-vivo( )p-mrs of skeletal muscle and liver: a way for non-invasive assessment of their metabolism in vivo ( ) p mrs assessment of intracellular nad metabolites and nad(+) /nadh redox state in human brain at t p mr spectroscopic imaging of the human brain at t with a ( ) p receive array: an assessment of ( ) h decoupling, t( ) relaxation times, ( ) h-( ) p nuclear overhauser effects and nad competing interests: the authors declare no competing interests.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/ . /. key: cord- -k uo fxm authors: bradshaw, patrick c.; seeds, william a.; miller, alexandra c.; mahajan, vikrant r.; curtis, william m. title: covid- : proposing a ketone-based metabolic therapy as a treatment to blunt the cytokine storm date: - - journal: oxid med cell longev doi: . / / sha: doc_id: cord_uid: k uo fxm human sars-cov- infection is characterized by a high mortality rate due to some patients developing a large innate immune response associated with a cytokine storm and acute respiratory distress syndrome (ards). this is characterized at the molecular level by decreased energy metabolism, altered redox state, oxidative damage, and cell death. therapies that increase levels of (r)-beta-hydroxybutyrate (r-bhb), such as the ketogenic diet or consuming exogenous ketones, should restore altered energy metabolism and redox state. r-bhb activates anti-inflammatory gpr a signaling and inhibits the nlrp inflammasome and histone deacetylases, while a ketogenic diet has been shown to protect mice from influenza virus infection through a protective γδ t cell response and by increasing electron transport chain gene expression to restore energy metabolism. during a virus-induced cytokine storm, metabolic flexibility is compromised due to increased levels of reactive oxygen species (ros) and reactive nitrogen species (rns) that damage, downregulate, or inactivate many enzymes of central metabolism including the pyruvate dehydrogenase complex (pdc). this leads to an energy and redox crisis that decreases b and t cell proliferation and results in increased cytokine production and cell death. it is hypothesized that a moderately high-fat diet together with exogenous ketone supplementation at the first signs of respiratory distress will increase mitochondrial metabolism by bypassing the block at pdc. r-bhb-mediated restoration of nucleotide coenzyme ratios and redox state should decrease ros and rns to blunt the innate immune response and the associated cytokine storm, allowing the proliferation of cells responsible for adaptive immunity. limitations of the proposed therapy include the following: it is unknown if human immune and lung cell functions are enhanced by ketosis, the risk of ketoacidosis must be assessed prior to initiating treatment, and permissive dietary fat and carbohydrate levels for exogenous ketones to boost immune function are not yet established. the third limitation could be addressed by studies with influenza-infected mice. a clinical study is warranted where covid- patients consume a permissive diet combined with ketone ester to raise blood ketone levels to to mm with measured outcomes of symptom severity, length of infection, and case fatality rate. there are tremendous demands on governments and the private sector to solve the covid- crisis with an effective and timely vaccine or therapy. as time passes, the demand for information grows pertaining to how healthy lifestyle and nutrition may play a role in protection against the detrimental outcomes of the sars-cov- virus. in this review, the intricate and detailed interplay among nutrition, metabolism, and the tightly controlled immune system is highlighted. the data suggest that exogenous ketones can increase cell efficiency and metabolic flexibility to provide significant immune modulation. however, challenges remain in identifying the exact dietary macronutrient combinations that will best influence the immune system. it is important for researchers and clinicians to consider metabolic strategies when attempting to identify novel preventative measures for viral infection, as these therapies can support the patient's immune system while showing minimal toxicities. the mechanisms through which exogenous ketones improve energy and redox metabolism and blunt inflammation likely apply not only to covid- but to any viral or bacterial infection where excessive cytokine production can lead to multiple organ failure and mortality. there are many types of metabolic therapies. however, therapies that increase r-bhb levels, including the consumption of a ketogenic diet or different forms of exogenous ketones, will be the focus of this review. others have also suggested that increasing systemic ketone levels may aid host defenses against respiratory viral infection, in part, by decreasing inflammation [ , ] , including a recent comprehensive review [ ] , while a clinical trial of the effects of a ketogenic diet on intubated sars-cov- patients has recently been registered (nct ). induces the innate and acquired immune responses. sars-cov- infects many cell types including type ii alveolar epithelial cells (aec ii) in the lungs [ ] , where this leads to respiratory infection. aec ii either divide to maintain their levels or differentiate into aec type i, which provide the surface area for the vast majority of gas exchange in the lungs [ ] . other important functions of aec ii include the secretion of surfactants, superoxide dismutase (sod ) [ ] , and type i (α/β) and type iii (λ) interferons [ ] to protect airway function. due to these functions, aec ii have high energy requirements and rely heavily on fatty acid oxidation for energy production [ ] . the partial loss of these functions during infection facilitates viral spread and disrupts the immune response and tissue repair. nearly all nucleated cells, including aec ii, can recognize the presence of viruses and initiate an innate immune response to recruit phagocytic cells to the infection. rna viruses such as sars-cov- are primarily recognized by cytosolic retinoic acid-inducible gene i-like receptors (rlrs), rig- , and melanoma differentiation-associated gene (mda ). the endosomal toll-like receptors (tlrs), tlr / and tlr , also play a role [ ] . excessive signaling through these endosomal tlrs can cause inflammatory pathology [ ] . in a cytokine storm, the number of phagocytic cells, including macrophages and neutrophils, increases along with the levels of proinflammatory cytokines, while the numbers of b and t lymphocytes, mediators of the adaptive immune response, decline [ ] . this results in a failure to clear the virus and facilitates a runaway positive feedback loop that increases the numbers of cytokine-secreting innate immune cells. this cytokine storm is emerging as a major contributor to acute respiratory distress syndrome (ards), multiple organ dysfunction, and patient death in covid- [ , ] . figure summarizes the molecular pathologies that occur during sars-cov- infection that lead to a cytokine storm and ards, while figure summarizes how metabolic therapy with ketone ester and a moderately high-fat diet may intervene in the disease process to protect against pathology. . . metabolic therapy. central metabolism is controlled by four major nucleotide coenzyme couples: adp/atp, nad + /nadh, nadp + /nadph, and acetyl-coa/coa [ ] . the prominent role these couples play in central metabolism is highlighted in figure . metabolic therapy aimed at restoring these ratios is often used as an adjunct to more targeted therapies [ ] . the ketogenic diet as a treatment for childhood epilepsy has drawn focus to (r)-beta-hydroxybutyrate (r-bhb) as a metabolic therapy. recently, exogenous ketones, which are various formulations of bhb, acetoacetate, or their precursors, have made it possible to raise blood r-bhb levels and alter the ratios of the controlling coenzyme couples without implementing a ketogenic diet [ ] . (r)- -hydroxybutyl (r)- -hydroxybutyrate, a type of ketone ester, is one of the several forms of exogenous ketones that increase systemic r-bhb levels. r-bhb-derived metabolites restore flux through the citric acid (krebs) cycle and oxidative phosphorylation when viral-induced changes in enzyme activity prevent glucose [ ] or fatty acids [ , ] from fueling these pathways. increasing r-bhb levels has been shown to normalize adp/atp, nad + /nadh, nadp + /nadph, and acetyl-coa/coa ratios in diseased tissue [ ] . r-bhb has multiple anti-inflammatory signaling roles and functions as an epigenetic modifier to stimulate a program of gene expression that alters metabolism to restore cellular redox function. the focus of metabolic therapy is on the restoration of the coenzyme ratios that largely control metabolic flux through central metabolic pathways. function in humans. in a study of blood cytokine levels in well-trained cyclists who compete in multiday races, the levels of tnf-α, il- , il- , and ifn-γ were raised following intense exercise, indicating increased inflammation, whereas the level of il- β was unchanged [ ] . on the last day of an eighteen-day trial, cyclists given daily ketone ester (r)- hydroxybutyl (r)- -hydroxybutyrate showed a % higher mean power output and % increase in the cd + /cd + (t helper cells/cytotoxic t cells) ratio than controls [ ] . an increased cd + /cd + ratio is associated with increased immune function [ ] , and this ratio declines with aging as immune system function declines [ ] . induced by ionizing radiation in model systems. the same ketone ester used in the cycling studies has also been used in radiation mitigation studies. cytokines are central to the pathophysiology of covid- ; while some are beneficial, others are detrimental (il- β, il- , and tnf-α), at least in the context of the cytokine storm [ ] [ ] [ ] [ ] . exposure to acute doses of radiation results in tissue damage and an activation of cytokine cascades [ ] . several pharmaceutical approaches are being studied to prevent or decrease radiation-induced tissue damage and the cascade of harmful cytokines [ ] . there is interest in using this radiation countermeasure strategy as a model for a viral-induced cytokine storm. ir has been shown to increase the expression of the following cytokines and growth factors including il- , il- , il- [ ] , tgf-β, il- , il- [ ] , type i interferons, il- α, il- β, il- , gm-csf, and tnf-α [ , ] . maximal cytokine production occurs between and hours following exposure to short-term radiation [ , ] . the balance of proinflammatory and anti-inflammatory cytokines synthesized is critical in the determination of outcomes [ ] with several factors figure : mechanisms that lead to acute respiratory distress syndrome (ards) and mortality following sars-cov- infection are shown. the cells of the innate immune response secrete increasing amounts of cytokines. the cells that normally protect against a cytokine storm lose this ability leading to a runaway positive feedback loop of cytokine production. abbreviations: β-hsd and β-hsd : β-hydroxysteroid dehydrogenase types and ; ace : angiotensin-converting enzyme ; aec i and aec ii: alveolar epithelial cell types i and ii; dcs: dendritic cells; foxo : forkhead box o transcription factor; foxo : forkhead box o transcription factor; hif- α: hypoxia-inducible factor alpha; ifn: interferon; il- : interleukin- ; irf : ifn-regulatory factor ; nad(h): nicotinamide adenine dinucleotide; nadp(h): nicotinamide adenine dinucleotide phosphate; onoo -: peroxynitrite; pgc -α: pparg coactivator -alpha; pdk and pdk : pyruvate dehydrogenase kinases and ; rlr: retinoic acid-inducible gene i-like receptors; rns: reactive nitrogen species; ros: reactive oxygen species; sod: superoxide dismutase; tgf-β: transforming growth factor-β; tnf-α: tumor necrosis factor-alpha. oxidative medicine and cellular longevity altering the profiles of the cytokines produced including the specific animal species and tissue studied, the magnitude of the radiation received, and whether whole animals, portions of animals, or only cells were exposed [ ] [ ] [ ] [ ] . chronic exposure to very low-dose nonacute radiation can induce hormesis and alter the levels of several cytokines to improve tissue responses [ , ] . further studies would be needed to determine the mechanism of these acute versus subacute radiation cytokine responses. human polymorphisms in cytokine genes have been shown to be responsible for the differences in the extent of pathology that occurs following radiation damage [ ] . limited ir studies with acute radiation have demonstrated that ketone ester was able to decrease chromosomal damage in mice and increase survival in cells [ ] . ongoing studies are directly measuring the effects of ketone ester on animal survival following radiation and the effects on the radiation-induced cytokine storm. there are several other therapies being tested against a radiationinduced cytokine storm that could be considered for the treatment of covid- as well [ ] . targets to treat a cytokine storm. several planned or recently initiated studies are targeting cytokines or their receptors in an attempt to blunt the cytokine storm of covid- . for example, the il- receptor monoclonal antibody (mab) antagonist tocilizumab has been identified as a strong candidate for treatment [ ] . other potential treatments to limit cytokine signaling include sarilumab (il- r mab antagonist), anakinra (il- r recombinant protein antagonist) [ ] , and emapalumab (ifn-γ mab). some cytokines, such as type i interferons, may be beneficial to reduce the cytokine storm. sars-cov- was shown to be quite susceptible to treatment with type i interferons in vitro [ ] figure : the major pathways of central metabolism and reactions that alter the ratios of coenzyme couples are shown. the mitochondrial matrix and the cytoplasm have independent coenzyme couple ratios. atp synthesized in the mitochondrial matrix is exported to the cytoplasm in exchange for adp by the adenine nucleotide translocase present in the inner mitochondrial membrane. acetyl-coa synthesized in the mitochondrial matrix must also be exported to the cytoplasm to provide two carbon units for fatty acid synthesis and protein acetylation. however, acetyl-coa cannot cross the mitochondrial inner membrane. therefore, the transfer of acetyl units across the inner mitochondrial membrane is accomplished using the citrate-pyruvate shuttle or the citrate-malate shuttle. these shuttles alter coenzyme levels and use inner membrane carrier proteins for the transport of citrate, pyruvate, and malate. the net result of the citratepyruvate shuttle on coenzyme levels is the use of energy from atp hydrolysis and nadh oxidation to reduce nadp + to nadph. there is also a citrate-alpha-ketoglutarate shuttle system that has the net effect of using atp hydrolysis to increase nadph in the cytoplasm instead of increasing nadh or nadph in the mitochondrial matrix. synthesizing fatty acids in the cytoplasm and reducing antioxidants require nadph. the malate-aspartate shuttle transfers reducing equivalents from nadh between the cytoplasm and the mitochondrial matrix. the glycerol -phosphate shuttle transfers reducing equivalents from cytoplasmic nadh to the mitochondrial etc. glucose is catabolized by three pathways including the hexosamine biosynthesis pathway that synthesizes uridine diphosphate (udp) nacetylglucosamine, glycolysis that reduces nad + to nadh and synthesizes atp from adp and p i , and the pentose phosphate pathway (ppp) that reduces nadp + to nadph and synthesizes ribose sugars for nucleotide synthesis. when the pyruvate dehydrogenase complex (pdc) is inhibited, lactate is synthesized from pyruvate to recycle nad + from nadh so glycolysis can continue. however, the lactate is exported from the cell and may contribute to lactic acidosis and multiorgan failure [ ] . r-bhb decreases the reliance of cells on glycolysis leading to reduced cellular lactate export. abbreviations: glut and glut : glucose transporters and ; mct: monocarboxylate transporter; nnt: nicotinamide nucleotide transhydrogenase; pdc: pyruvate dehydrogenase complex. oxidative medicine and cellular longevity , which protects lymphocyte function, has been proposed as a therapy to treat lymphopenia that contributes to the cytokine storm [ ] . therefore, clinical studies to determine the effects of metabolic therapy with exogenous ketones in combination with one of these more targeted therapies should be considered for patients with severe sars-cov- infection. mechanism to blunt the cytokine storm . . increased ros levels stimulate inflammasome activity and cytokine production. reactive oxygen species (ros) and reactive nitrogen species (rns) levels increase in the lungs by at least two different mechanisms during the viralinduced cytokine storm. first, viral rna binding to tlrs leads to decreased expression of mitochondrial electron transport chain (etc) genes, which increases mitochondrial superoxide production [ ] [ ] [ ] . second, phagocytic cells are recruited to the lungs and, together with resident lung phagocytes, are activated through tlr and rig- to increase nadph oxidase (nox ) activity [ , ] , increasing the production of both intracellular and extracellular ros and rns to kill pathogens. however, host cells can be damaged as a byproduct of this response, especially in a cytokine storm. a review on the redox biology of respiratory viral infections has recently been published [ ] . the increased ros production from virus-induced tlr signaling [ ] , rig- signaling [ ] , and altered mitochondrial function [ ] leads to the activation of several transcriptional regulators such as nf-κb, ifn-regulatory factor (irf ), and stat to increase the production of cytokines, including tnf-α, il- , and il- , from aec and macrophages [ ] . the enhanced nf-κb activity also leads to the activation of the nlrp inflammasome that increases il- β and il- production [ ] . the transcription of nf-κb is a necessary step in the two-stage model of nlrp activation [ ] . storm. in the lungs, catalase and extracellular superoxide dismutase (sod ) [ ] are synthesized at high levels by aec ii [ , ] . in addition to its normal peroxisomal localization, catalase is secreted to the extracellular space by alveolar macrophages [ , ] through a mechanism that is distinct from the classical secretory pathway [ , ] . older covid- patients, who have a higher risk of mortality from the disease [ ] , were shown to express much less sod from their aec ii than younger patients [ ] , suggesting an important role of sod in protecting against the cytokine storm. these antioxidant enzymes reduce the concentration of toxic superoxide and hydrogen peroxide in extracellular fluids preventing oxidative damage to extracellular structures. in this regard, nox has been shown to synthesize superoxide and release it into the luminal extracellular space mostly from aec i [ , , ] . consistent with its important antioxidant function, polymorphisms in sod are associated with reduced lung function and chronic obstructive pulmonary disease (copd) [ ] . exogenous administration of catalase has been shown to mitigate respiratory viral infections. intranasal catalase protected against respiratory syncytial virus (rsv) infection [ ] , a virus that can induce a cytokine storm [ ] . catalase treatment led to a significant reduction in the levels of the cytokines il- α, tnf-α, and il- and the chemokines cscl , ccl , and ccl [ ] . during the early stages of other types of respiratory infections, increased ros activates the nuclear factor erythroid -related factor (nfe l commonly called nrf ) transcriptional regulator to induce antioxidant genes such as sod and catalase to protect against the ros-induced proinflammatory gene expression and subsequent cytokine storm. however, rsv infection leads to the proteolytic degradation of nrf , preventing the protective antioxidant response [ ] to facilitate the cytokine storm. in addition, catalase can be inactivated by high levels of ros and rns in the lungs [ ] . influenza a virus (iav) is another virus that induces a cytokine storm [ ] . in mice infected with iav, intranasal administration of the mitochondrialtargeted antioxidant, mitotempo, quenched etc-derived ros in the lungs to decrease proinflammatory gene expression, cytokine storm, and consequent mortality [ ] . a similar study found that intranasal administration of a nox inhibitor had similar protective effects against iav infection in mice [ , ] . therefore, both mitochondria-and nadph oxidase-mediated increases in ros contribute to iav mortality. the role of nadph oxidase in respiratory virus infections has been reviewed [ ] . phagocytes. phagocytes release proteolytic enzymes, ros, and rns into their phagosomes to mediate the killing of endocytosed pathogens. the phagosomal oxidative burst requires cytoplasmic nadph as a coenzyme for membrane-bound nadph oxidase to produce phagosomal superoxide ( figure ). the iron-dependent metalloprotein myeloperoxidase (mpo) is a component of primary granules that fuse with the phagosome [ ] . mpo catalyzes the synthesis of toxic hypochlorous acid from hydrogen peroxide and chloride ion. two gases, nitric oxide and co , are critical for the synthesis of toxic carbonate radicals. for this to occur, first the superoxide radical must bind to the nitric oxide radical to form peroxynitrite, and then, peroxynitrite reacts with carbon dioxide to form nitrosoperoxycarbonate that degrades to the carbonate radical. peroxynitrous acid, having a pka of . , forms physiologically when peroxynitrite binds to a proton and is a major source of hydroxyl radicals [ ] . as co levels increase, the half-life of peroxynitrite decreases from roughly a second down to several milliseconds [ ] . the ros and rns that are produced during viral infection take on specific roles as sentinels, messengers, and oxidizing agents that determine the activity of many classes of proteins including transcription factors [ ] . hydroxyl radicals are short lived and function primarily as oxidizing agents. superoxide is negatively charged and does not diffuse directly across lipid bilayers, but it has been shown to be transported by proteinaceous channels from the mitochondria to the cytoplasm [ , ] . hydrogen peroxide is transported by aquaporins across cellular membranes. the concentration oxidative medicine and cellular longevity of hydrogen peroxide in the cytoplasm is generally indicative of the health of mitochondria, but transient increases can be the result of signaling events. nitric oxide is a free radical that passes through membranes and can potentially signal to nearby cells in its relatively short half-life. peroxynitrous acid can also cross cellular membranes. the half-lives and diffusion limits of different types of ros are shown in figure . the importance of this diffusion of ros and rns between cells is that cells that lack the receptor for the virus can attempt to mount an appropriate response to the infection. virus-induced ros production and cytokine storm induce energy dysfunction and redox imbalance in host cells in the lungs by altering the adp/atp, nad + /nadh, and nadp + /nadph ratios that control central metabolism. ( ) ros produced by the mitochondrial etc damages proximal etc proteins resulting in decreased electron flux, which increases the cellular adp/atp (less atp) ( ) the decreased etc flux also decreases the cellular nad + /nadh (more nadh) as the rate of nadh hydrolysis by etc complex i slows ( ) the increased superoxide produced from the etc is converted by superoxide dismutase (sod ) and sod to hydrogen peroxide. h o is detoxified by glutathione peroxidase through the conversion of reduced glutathione (gsh) to glutathione disulfide (gssg). increased activity of nadph-dependent glutathione reductase is needed to recycle gssg to gsh leading to an increased cellular nadp + /-nadph ratio (less nadph). in a parallel pathway yielding the same result, hydrogen peroxide is detoxified by peroxiredoxins. the oxidized peroxiredoxins are then reduced by thioredoxins, and lastly, the oxidized thioredoxins are reduced by thioredoxin reductase using the reducing power of nadph leading to an increased cellular nadp + /nadph ratio the adp/atp, nad + /nadh, and nadp + /nadph couples control hundreds of cellular reactions. when these levels are altered in cells during a cytokine storm, the cells can no longer effectively perform their primary functions leading to cell dysfunction and death and to pathologies such as ards. blunting the cytokine storm in immune cells, transitioning from an inactive state to an inflammatory and then to a postinflammatory state is accompanied by metabolic reprogramming. this assures that cells have adequate energy and redox potential to perform their new roles, including entering the cell cycle for propagation, performing an oxidative burst, or undergoing regulated apoptosis rather than necrosis. the mitochondrial pdc is well positioned to reprogram metabolism as it is the gatekeeper of carbohydrate flux into mitochondria as well as a major regulator of cellular nad + /nadh. when it is active, it reduces mitochondrial nad + , and when it is inhibited, it redirects pyruvate metabolism to the cytoplasm where lactate dehydrogenase reduces pyruvate by oxidizing nadh. pdc activity, by modulating the nad + /nadh ratio, also affects the flux through glycolysis and the mitochondrial citric acid cycle, fatty acid oxidation, and oxidative phosphorylation. oxidative medicine and cellular longevity likely through type i interferon signaling [ ] , which increases proinflammatory cytokine production. in this regard, treatment with tnf-α was shown to downregulate the expression of peroxisome proliferator-activated receptor-γ coactivator- α (pgc- α), a master regulator of mitochondrial gene expression [ ] , decreasing mitochondrial etc function and oxygen consumption in mouse lung aec [ ] . downregulation of pgc- α would likely increase ros production as pgc- α also induces antioxidant enzymes such as sod and catalase [ ] . unexpectedly, peripheral blood mononuclear cells (pbmcs) from sars patients were shown to have increased expression of mitochondrial-encoded subunits of the etc [ ] , which could lead to increased ros production due to incompletely formed etc complexes when the nuclear-encoded subunits are downregulated. sars-cov- rna has been shown to localize to mitochondria [ , ] , likely to attempt to hide from cellular immune surveillance systems. this could partly explain how sars-cov-type coronaviruses have been shown to be quite effective in blocking the type i interferon β response during the initial stages of the infection [ , ] . targeting the viral-induced decrease in pdc activity. in mice infected with iav, atp levels greatly decreased and the level of a negative regulator of pdc, pyruvate dehydrogenase kinase (pdk ), increased substantially. administration of diisopropylamine dichloroacetate (dada), an inhibitor of pdk , significantly delayed mortality from the infection [ ] . however, the infection led to severe anorexia, which also increases pdk levels in some tissues such as those in the muscle [ ] and liver [ ] due to increased foxo , ppar-α, and glucocorticoid receptor (gr) [ ] transcriptional activity. this inactivation of pdc during starvation likely evolved to save glucose and lactate (which is converted back into glucose in the liver as part of the cori cycle) for neurons, which do not efficiently oxidize fatty acids. the full extent to which the virus directly upregulated pdk levels is unknown. the levels of the proinflammatory cytokines tnfα, il- , and il- β were shown to increase following iav infection [ ] . as stated above, a tlr -and type i interferon-dependent response to viral rna has been shown to reduce the expression of four subunits of mitochondrial etc complex i [ ] . this likely contributes to decreased atp production. dada administration significantly increased pyruvate dehydrogenase (pdh) activity and atp levels in the skeletal muscles, heart, lungs, and liver and tended to normalize plasma levels of glucose, lactate, free fatty acids, and r-bhb [ ] . dada administration also suppressed the iav-induced increase in il- , il- , ifn-α, tnfα, and ifn-γ levels, but not that in ifn-β or il- β [ ] . pdk inhibitors have also been shown to have protective antiinflammatory effects. this may partly result from their effects on t lymphocytes, as proinflammatory th cells have high levels of pdk and show primarily glycolytic metabolism, while anti-inflammatory th and treg cells have low pdk levels and show primarily oxidative metabolism. knockdown of pdk suppressed th cells and increased treg cell numbers to restore immune function in mice with experimental autoimmune encephalomyelitis [ ] . oxidative medicine and cellular longevity in another study of iav infection in mice, glucose administration during the period of anorexia following iav infection was found to decrease the mortality rate [ ] . glucose administration likely stimulated the insulin receptor-akt signaling pathway to decrease foxo activation to blunt the increase in pdk levels resulting from anorexia to maintain energy generation [ ] . other activators of mitochondrial energy metabolism, such as the peroxisome proliferation-activated receptor-gamma (ppar-γ) agonist pioglitazone or rosiglitazone and the amp kinase activator, aicar, have also been shown to protect mouse mortality from iav infection [ ] . these compounds are all known to decrease pdk levels in the muscle and liver [ ] . therefore, foxo hyperactivation may be a pathological event in mouse iav infection as it is induced by both increased ros levels [ ] and the anorexia that occurs following iav infection. a mechanism through which antioxidant administration protects against viral infection may be through preventing foxo induction of pdk . the use of ppar-γ agonists to treat the cytokine storm in covid- has been reviewed [ ] . figure shows the central role of pdc at the gateway between glycolytic and citric acid cycle metabolism. figure shows the multiple transcription factors that control the expression of the kinases and phosphatases that regulate pdc activity as well as controlling the expression of the three enzymes that comprise the pdc. figure also shows that r-bhb is catabolized into two acetyl-coa molecules that enter the citric acid cycle and bypass pdc inhibition. an nadh, which fuels complex i of the etc, is also generated during r-bhb oxidation to acetoacetate. r-bhb has also been shown to increase pgc- α levels [ ] and mitochondrial fusion [ , ] , which are known to increase mitochondrial energy generation. therefore, ketone body catabolism is a substantial source of atp when the cytokine storm leads to the block of the mitochondrial oxidation of carbohydrate catabolites. , and hpo -, which regulate plasma membrane potential and osmotic balance [ ] . atp drives the ion pumps that provide the chemiosmotic potential to (r)- -hydroxybutyrate can restore atp levels, decrease the production of lactate, and decrease dependence on glycolysis. oxidative medicine and cellular longevity maintain the distribution of these ions (figure ) , preventing edema. the major sars-cov- spike (s) protein binds to extracellular ca + to facilitate viral fusion with host cells such as aec ii [ ] and signals for the opening of plasma membrane ca + channels through a protein kinase c-α signaling pathway, which may be triggered by er stress [ ] . the sars-cov- envelope (e) protein is a lipidated viroporin, which forms a cation-selective channel in the endoplasmic reticulum that releases ca + [ ] . blocking the viralinduced increase in cellular ca + levels with a chelator decreased infectivity -fold [ ] . the increased cytoplasmic ca + leads to the activation of the plasma membrane and er ca + pumps, depleting atp levels. increased cytoplasmic ca + stimulates the uptake of ca + into the mitochondrial matrix in a mitochondrial membrane potentialdependent manner. in the presence of high levels of ros caused by viral infection, the rapid uptake of ca + into the mitochondrial matrix may stimulate permeability transition pores to open in the inner membrane [ ] , which uncouple mitochondria leading to further energy depletion and cell death. however, mammals have evolved mechanisms to use the viral-induced increase in cytoplasmic ca + as a signal to upregulate host defenses. an increase in the cytosolic ca + concentration by these mechanisms contributes to the activation of the nlpr inflammasome and elevation of il- β and il- [ ] . increased cellular ca + levels in aec ii lead to increased mitochondrial etc-derived ros and increased ros from nadph oxidases duox and duox , the most abundant isoforms in aec, through ca + binding to their ef-hand motifs [ ] . duox is also upregulated at the gene expression level by the increased interferon-β and tnf-α produced in response to the respiratory viral infection [ , ] . hydrogen peroxide can diffuse from aec into adjacent cells leading to oxidative damage, energy depletion, and osmotic imbalance. increased cytoplasmic ca + levels in airway myocytes induce constriction of the airways. several protein kinase c (pkc) isoforms are activated by ca + , and their activation is an important contributor to bronchoconstriction [ , ] . pkc directly targets and inhibits kv k + channels, which are important for the relaxation of airway smooth muscle [ ] . inhibition of kv channels induces bronchial constriction [ ] , which can contribute to ards. figure : maintaining atp levels during respiratory viral infection is the key to maintain proper ion distribution to avoid edema. the active and passive cotransporters and channels maintain the ion gradients between aec and extracellular fluids. the apical ligand-gated channels function to maintain appropriate osmotic pressure to provide sufficient fluid in the airway without causing edema. aec i express superoxideactivated enac na + channels and nox that regulates them. abbreviations: na + / k + -atpase: sodium potassium atpase; ae: anion exchange chloride bicarbonate exchanger; bcftr: basolateral cystic fibrosis transmembrane conductance regulator-like channel; bhbdh: βhydroxybutyrate dehydrogenase; birc: basolateral inward rectifying channel; bk ca : large-conductance ca + -and voltage-gated big k + channel; borc: basolateral outward rectifying channel; cacc/tmem: calcium-activated chloride channels/transmembrane protein; cftr: cystic fibrosis transmembrane conductance regulator; cng: cyclic nucleotide-gated ion channel; enac: epithelial sodium channel; kca . : calcium-activated potassium channel; kv . : voltage-dependent potassium channel; ncx: sodium calcium exchanger; nhe: sodium-hydrogen exchanger; nkcc: sodium potassium chloride cotransporter; nox : nadph oxidase ; pit / : sodium-dependent phosphate transporters and ; ryr: ryanodine receptor calcium-induced ca + channel; serca: sarcoplasmic/endoplasmic reticulum ca + -atpase; sk : sk calcium-activated potassium channel. oxidative medicine and cellular longevity the use of r-bhb as an energy source may blunt the cellular energy deficit, the increase in cytoplasmic ca + levels, and the osmotic imbalance to improve lung function. high-fat, low-carbohydrate enteral feeding of patients with type ii respiratory failure (the inability to expel co at a normal rate) reduced the mean length of time on a ventilator by %, from hours down to hours, compared to patients on high-carbohydrate, low-fat enteral feeding [ ] . arterial blood co levels, an indicator of patient respiratory distress, decreased to % for patients in the high-fat group at the time of weaning off the ventilator. the high-carbohydrate group had an even higher partial pressure of co at weaning than at the onset of ventilation. a likely contributor to the difference observed between the groups was the amount of co synthesized from metabolizing the different diets. the amount of co synthesized for every molecule of oxygen consumed is defined as the respiratory exchange ratio (rer). the rers for catabolism of fat, glucose, and r-bhb are . , . , and . , respectively. a high-fat diet was also protective in a mouse model of ventilator-induced lung injury [ ] , a model of ards. a high-fat, lowcarbohydrate diet supplemented with fish oil, gammalinolenic acid, and antioxidants was also shown to decrease the time on a ventilator for patients with ards due to sepsis/pneumonia [ ] , trauma, or aspiration injury; the findings may be relevant for ards mediated by viral infection as well. due to the lack of published data on the effects of increased r-bhb levels on the human immune system during viral infection, results obtained from several studies of metabolic therapy on iav-infected mice are described below. a shortterm ketogenic diet was shown to protect mice from iav infection, while racemic r-and s- , -butanediol (bd), an exogenous ketone precursor, supplemented to a normal chow diet did not [ ] . a long-term ketogenic diet that was obesogenic was shown to adversely affect glucose tolerance and immune system function [ ] . as described above, glucose gavage during iav-induced anorexia decreased mouse mortality [ ] , while a more recent study showed that glucose metabolism through the hexosamine biosynthetic pathway stimulated a cytokine storm [ ] . in the sections below, the results from these studies will be described in more detail and analyzed in an attempt to reconcile these findings. the rationale will also be discussed for the consumption of a moderately high-fat, moderate-carbohydrate, ketone estercontaining diet at the onset of viral infection,and transitioning to a moderately high-fat, low-carbohydrate, ketone ester-containing diet if the infection becomes severe to blunt the cytokine storm. activating a γδ t cell response. a recent study showed that mice placed on a ketogenic diet for seven days before infection had decreased mortality from iav [ ] . the ketogenic diet increased the number of protective il- -secreting γδ t cells in the lungs. administration of the ketone precursor bd to mice on a chow diet did not protect the survival or lead to the recruitment of γδ t cells to the lungs in the mice infected with iav, even though the r-bhb blood level was equivalent to that of the ketogenic diet. one point raised regarding the design of this study is that while dietary protein amount (% w/w) was uniform between control and kd mice [ ] , the micronutrient and fiber profiles were not [ ] . while this did not likely impact the conclusions of the study, it should be a point of emphasis for future experiments. bd administration may not have shown protection due to a lack of improvement of the cellular redox environment in the lungs that likely occurred during the ketogenic diet. during times of high r-bhb oxidation in the brain, the cytoplasmic nadp + /nadph becomes more reduced and the cytoplasmic nad + ]/[nadh becomes more oxidized [ ] . the ketogenic diet, which is very high in fat content, may have improved the cytoplasmic redox environment, in part, through inhibition of fatty acid synthesis, an nadphconsuming pathway, by increasing levels of palmitoyl-coa, an inhibitor of fatty acid synthase activity. so, the combination of increased r-bhb metabolism and decreased fatty acid synthesis may lead to a decrease in the cytoplasmic nadp + /-nadph ratio that may lead to the recruitment and enhanced function of γδ t cells in the lungs, which does not occur when r-bhb is catabolized when mice are fed a normal chow diet. a high-fat but nonketogenic diet was shown to be ineffective in decreasing iav-induced weight loss and mortality, even though it increased the recruitment of il- -secreting γδ t cells to the lungs. il- binds to receptors on lung epithelial cells and possibly other lung cell types to increase the expression of il- . il- secretion leads to the recruitment of type innate lymphoid cells (ilc s) to the lungs, where they play a role in regulating inflammation and barrier function by secreting the cytokines il- , il- , il- , and amphiregulin. in lung tissue, the ketogenic diet upregulated mitochondrial etc gene expression and the expression of the oxct gene encoding scot (succinyl-coa: -ketoacid coa transferase), the rate-limiting enzyme for ketolysis, suggesting that r-bhb catabolism in the lungs plays an important role in the protective effects of the ketogenic diet against viral infection [ ] . it is hypothesized that supplementation of ketone ester to mice on a moderately high-fat diet will lead both to the recruitment of γδ t cells to the lungs and to decreased mortality of iav-infected mice. while one week of ketogenic diet prior to iav infection was shown to be anti-inflammatory and decrease mouse mortality, three months of the ketogenic diet in the absence of iav infection increased white adipose tissue (wat) inflammation, decreased γδ t cell recruitment to the wat, and led to obesity and glucose intolerance [ ] . oxidative medicine and cellular longevity therefore, current evidence from mouse studies where the animals were fed obesogenic ketogenic diets suggests that only short-term ketogenic diets will activate γδ t cells to boost immune function [ ] . however, another research group identified a ketogenic diet of a different composition that was shown to induce weight loss in mice [ ] . the dietary components responsible for the different effects on weight are currently unknown, although the leptogenic diet contained only half as much protein and used lard, butter, and vegetable oil as fat sources, while the obesogenic diet used hydrogenated soybean oil as the fat source [ , ] . future studies are needed to determine if a ketogenic diet that induces weight loss can provide long-term preservation of the protective γδ t cell response to provide long-term antiviral immunity. it is also hypothesized that increased r-bhb levels improve cellular energy metabolism and redox status to enhance fatty acid beta-oxidation to overcome the metabolic inflexibility mediated by pdc inhibition. r-bhb is known to inhibit adipose tissue lipolysis [ ] , so a high-fat diet may be needed, along with exogenous ketones, to provide sufficient fatty acid beta-oxidation for increased metabolic flexibility to overcome a cytokine storm. the initiation of this diet in humans for covid- poses challenges because it may take many days to adapt to a ketogenic diet to fully upregulate the expression of genes for ketogenesis, ketolysis, and fatty acid oxidation. proinflammatory cytokines also inhibit ketogenesis [ ] . in addition, starting a ketogenic diet, also called ketoinduction, may be accompanied by flu-like symptoms [ ] [ ] [ ] that may limit its application to covid- patients. however, studies could be performed to determine the ability of covid- patients to tolerate a ketogenic diet supplemented with exogenous ketones, to attempt to decrease the early adverse effects of the diet that likely result from decreased energy production when glucose levels initially decline [ ] . if well tolerated, further studies determining the ability of this diet to activate a protective immune response could follow. further experiments are needed to delineate the molecular mechanisms involved if r-bhb precursors such as ketone esters are to be used for the treatment of iav and sars-cov- infections. to test the effectiveness of exogenous ketone administration on iav infection, mice could be supplemented with or without a ketone ester and fed a moderately high-fat, moderate-carbohydrate diet or a moderately high-fat, low-carbohydrate diet or a control chow diet, and weight loss and mortality could be monitored following iav infection. as explained in more detail below, glucose (from carbohydrate metabolism) stimulates important proinflammatory antiviral functions early in infection [ ] , but these proinflammatory actions also increase the cytokine storm late in infection [ ] . so, it is unknown which of these effects will predominate to affect mortality in the presence of high r-bhb levels. it is hypothesized that the moderately high-fat, moderate-carbohydrate, ketone ester-containing diet will provide the most metabolic flexibility to stimulate host cell defense mechanisms. this flexibility should be able to preserve energy metabolism and redox status to boost immune function to decrease iav titer in the lungs. how-ever, the moderately high-fat, low-carbohydrate diet with ketone ester will likely show a stronger ability to blunt the cytokine storm, as increased glucose and insulin levels have recently been shown to block an important antiinflammatory action of r-bhb [ ] . mortality that is greatly blunted by gavage of glucose, but not fat or protein. as alluded to earlier, mice became anorexic following iav infection and the anorexia contributed to their mortality, as glucose gavage was able to decrease the mortality. in that study, gavage of olive oil (fat) or casein (protein) did not decrease mortality [ ] . the proinflammatory cytokines il- , il- , il- , il- , tnf-α, and ifn-γ, several of which increase following iav infection, have been shown to suppress appetite [ ] . so, it is likely that administering a ketogenic diet simply decreased cytokine levels, allowing for an increase in the amount of food consumed to decrease the mortality of the mice. consistent with this interpretation, mice on the ketogenic diet lost less weight following infection than the chow-fed mice [ ] . these results may be applicable to sars-cov- infection, as % of covid- patients reported lack of appetite as a symptom [ ] . a major research question that arises from these studies is whether a twice-daily isocaloric gavage of ketone ester starting on the day of infection, to mimic the protective effect of the twice-daily gavage of glucose [ ] , can protect mice fed a chow diet from iav infection. this hypothesis is reasonable given that the protective ketogenic diet was composed of roughly % fat, % protein, and only . % carbohydrate [ ] . so, the carbohydrate content of the diet was likely too low to provide adequate glucose for protection, and gavage of fats or protein was unable to provide protection [ ] . the ineffectiveness of the ketone precursor bd against iav infection [ ] suggests that ketone ester alone may be ineffective and that gavage of fat and ketone ester together as a cotherapy, to better mimic a ketogenic diet, may be needed for protection. experiments probing possible additive or synergistic effects among glucose, ketone ester, and fats on increased survival during iav infection in mice would provide valuable insights relevant to the protection against a cytokine storm in humans. so, how may glucose gavage of mice during viral-induced anorexia decrease mortality from iav infection? glucose has been shown to stimulate a proinflammatory antiviral response through increased flux through the hexosamine biosynthesis pathway. the increased flux increases levels of the pathway end product udp n-acetylglucosamine (udp glcnac), which increases the o-glcnacylation of the antiviral protein mavs to increase its function (figure (c) ) [ ] . the sars virus synthesizes the nps protein, which partially inhibits mavs function to block host antiviral signaling [ ] . in addition, viral nucleic acids and type i interferon signaling in macrophages lead to the increased oxidative medicine and cellular longevity expression of the glycolytic activator -phosphofructose- kinase and fructose- , -bisphosphatase (pfkfb ), which are required for the increased engulfment of viral-infected cells [ ] . surprisingly, mortality from iav infection in mice has been linked with viral induction of an er stressinduced apoptotic pathway in the brain [ ] . both glucose and r-bhb are important protective fuels for neurons, potentially reconciling findings of how either glucose or a ketogenic diet is protective. in addition, high glucose levels may compensate for the energy and redox crisis occurring as a result of viral-induced pdc inhibition by increasing flux through glycolysis for the synthesis of atp and by increasing flux through the pentose phosphate pathway (ppp) for the synthesis of nadph. glucose flux through the hexosamine biosynthesis pathway has been shown to stimulate the cytokine storm during iav infection in mice by increasing the o-glcnacylation of the transcriptional regulator irf , which increases its activity to stimulate proinflammatory cytokine production (figure (c)) [ ] . this may explain in part why people with diabetes who are infected with sars-cov- have a higher mortality rate [ ] . therefore, inhibitors of irf or inhibitors of o-glcnacylation, such as osmi- , are potential treatments for the sars-cov- cytokine storm. ketone ester treatment has been shown to decrease blood glucose levels [ , ] , which would likely decrease flux through the hexosamine biosynthesis pathway in immune cells to decrease cytokine production. monocarboxylate transporters are expressed in aec ii [ ] , allowing the entry of r-bhb into the cytoplasm. however, these cells have low expression of ketolytic enzymes, so they may be unable to substantially catabolize the r-bhb produced from the consumption of exogenous ketones [ ] . however, a ketogenic diet was shown to increase the expression of ketolytic genes in the lungs [ ] , so it is possible that these enzymes can be induced in aec ii and are responsible, in part, for the protective effects of the ketogenic diet. even if r-bhb is not catabolized by aec ii, the presence of r-bhb in aec ii may still greatly protect these cells through signaling pathway activation, through enzyme inhibition, and through gene expression pattern alteration as described in detail below. depend upon the metabolic state of the cell. r-bhb inhibits the nlrp inflammasome [ ] . s-bhb, the enantiomer of r-bhb, was also effective, but not butyrate. the molecular target through which r-bhb and s-bhb inhibit the inflammasome is still unknown, but treatment decreased cellular k + efflux and reduced inflammasome activator asc (apoptosis-associated speck-like protein containing a card) oligomerization (figure (b) ). r-bhb-mediated inhibition of the inflammasome did not require ketone body catabolism, since sirna of the ketolytic enzyme scot did not block inhibition. inflammasome inhibition was also shown to be independent of the effects of r-bhb on gpr a gprotein-coupled receptor (gpcr) signaling and histone acetylation. in addition to immune cells, several types of epithelial cells express the genes for a functional nlrp inflammasome; r-bhb likely protects aec ii from a cytokine storm in part through this mechanism [ ] . recently, high insulin or high glucose levels were shown to decrease r-bhb-mediated inhibition of the nlrp inflammasome in macrophages in vitro, and -deoxyglucose, a glycolysis inhibitor, was shown to potentiate nlrp inflammasome inhibition by r-bhb [ ] . therefore, the metabolic state of the cell appears to influence the effect of r-bhb on the nlrp inflammasome. somewhat surprisingly, a single dose of exogenous ketones was shown to increase inflammasome activation in lps-stimulated blood cells and increase plasma il- β and il- levels of healthy young people after a -hour overnight fast [ ] . the mechanisms remain unknown, but increased levels of these proinflammatory markers may have been due to an r-bhb-mediated increase in nadph levels stimulating nadph oxidase activity to increase ros levels and possibly also due to increased mitochondrial ros production that occurs when r-bhb and glucose are oxidized simultaneously, as increased ros stimulates nlrp inflammasome activity [ ] . however, a follow-up study by the same group administering exogenous ketones to obese subjects found no difference in inflammasome activity and the levels of many proinflammatory markers but a slight decrease in il- β and tnf-α levels in the exogenous ketone-treated group [ ] . in a study of well-trained cyclists, acute bd administration was shown to slightly increase interferon-gamma expression in pbmcs, while anti-inflammatory cytokine expression was unaltered [ ] . overall, the lack of nlrp inflammasome inhibition and the lack of strong antiinflammatory effects of exogenous ketones in the above human studies are likely due to the metabolic state of the subjects when the exogenous ketones were administered. it is possible that the glucose levels and insulin levels were too high to allow r-bhb to inhibit the inflammasome [ ] . sars-cov- infection can cause ketosis and ketoacidosis, and these patients with high blood r-bhb levels had longer hospitalization and an increased mortality rate [ ] . also, covid- patients with type i or ii diabetes mellitus (dm) have an increased risk of developing diabetic ketoacidosis (dka), which contributes to mortality [ ] . the molecular basis for these findings is not entirely clear as ketone bodies do not directly cause dka. recent findings, however, indicate that the increased glucose levels in mouse models of diabetes can decrease the expression of the ketolytic genes r-bhb dehydrogenase (bdh ) and oxct in the heart [ ] . if this also occurs in other tissues such as skeletal muscle, it would likely lead to increased blood ketone levels. expressing an exogenous transgene to increase o-glcnacylation in the mice further decreased bdh levels demonstrating a role for the hexosamine biosynthetic pathway in this downregulation of ketolytic gene expression. the scot enzyme (oxct gene product) was shown to be directly modified by o-glcnacylation. therefore, administration of an o-glcnacylation inhibitor together with exogenous ketones to diabetic patients with covid- may be beneficial to prevent decreased ketolysis and dka. oxidative medicine and cellular longevity multiple factors may be contributing to acidosis in covid- patients. respiratory acidosis occurs due to a buildup of carbon dioxide in the body, while lactic acidosis occurs due to mitochondrial etc dysfunction or pdc inhibition. r-bhb metabolism, unlike glucose metabolism, does not raise the levels of lactic acid and may even decrease acidosis by lowering the rate of glycolysis and lactic acid synthesis. differential diagnosis and treatment of acidosis have been reviewed [ ] . consumption of a ketone ester has been shown to lower glycemic response in both healthy and obese people [ ] . fatty acid lipolysis in white adipose tissue is inhibited by ketones [ ] , so in most cases, exogenous ketones will inhibit the synthesis of endogenous ketones. before insulin was available, a ketogenic diet that limited carbohydrates to ≤ g/day was a commonly used effective therapy for type i diabetes [ ] . during dka, there are imbalances in the levels of glucagon and insulin and elevation of the stress hormones epinephrine, cortisol, and growth hormone. these changes can be triggered by a stressful event such as covid- . therefore, care would need to be taken in administering exogenous ketones in a clinical trial for covid- . coadministration of sodium bicarbonate may also be beneficial for diabetic covid- patients to buffer changes in blood ph. in this regard, a recent study showed that cyclists administered ketone ester had a % decrease in blood bicarbonate levels and a slight decrease in blood ph, while blood r-bhb levels rose to - mm. administering bicarbonate together with ketone ester prevented these alterations in the blood and increased blood r-bhb levels another . - . mm, and this resulted in a % increase in power output [ ] . if diabetics are to be included in a covid- trial testing the effects of exogenous ketones, the ph of arterial blood gas (abg) and the blood levels of ketones would need to be monitored by experts in managing dka to identify early-stage ketoacidosis so that interventions according to best practices [ ] could be implemented. to avoid risks, patients with naturally high ketone levels should avoid exogenous ketones, so dka may be an exclusion factor in trials. but ultimately, a clinical trial will likely be necessary to determine the effects of exogenous ketone consumption or a ketogenic diet on covid- in both diabetic and nondiabetic patients. the lack of protective effects of high r-bhb levels under certain metabolic conditions is likely due to the same underlying molecular mechanism that prevented supplementation with the ketone precursor bd from preventing mortality in iav-infected mice [ ] . a protective anti-inflammatory γδ t cell response was likely not initiated in these studies. in the studies with exogenous ketones, this was likely due to the acute nature of the exogenous ketone treatment and to the lack of the high-fat, low-carbohydrate diet that may be necessary to initiate this protective anti-inflammatory response. however, there are several other potential mechanisms that may have also prevented the acute ketone ester treatment from influencing the activation state of the inflammasome. for example, a -hour fast in human subjects has been shown to lead to nlrp inflammasome inactivation, due to increased mitochondrial nad + /nadh activating the nad + -dependent sirt protein deacetylase to decrease ros production [ ] . therefore, the -hour overnight fast may have led to a partial inhibition of nlrp inflammasome activity so that ketone ester treatment was unable to decrease the activity any further. it is also possible that at least five days of a moderately high-fat, low-carbohydrate diet with exogenous ketone treatment may be needed to show large anti-inflammatory effects, as it was shown to take five days to fully upregulate the activity of the fatty acid betaoxidation system after initiating a ketogenic diet [ , ] . decrease inflammation. r-bhb was shown to be a class i and iia histone deacetylase (hdac) inhibitor (k i = − mm) that induced expression of several antioxidant genes and the transcriptional regulator foxo a (figures (a) and (b) ) [ ] . administration of the other hdac inhibitors, butyrate or trichostatin a, showed antiinflammatory effects on lung ilc s, while adding both compounds together showed no additive benefit [ ] . this suggested that hdac inhibition is a protective mechanism through which r-bhb and the ketogenic diet prevent lung inflammation. there is a nuclear pool of pdc that contributes to acetyl-coa synthesis for histone acetylation [ ] . pdk also shows a partial nuclear localization [ ] , so the upregulation of pdk expression during viral infection could disrupt nuclear histone acetylation, which could be restored by hdac inhibitors such as r-bhb. in studies with macrophages, butyrate was shown to function as an hdac inhibitor to decrease il- , il- , and nitric oxide levels, but not tnf-α or mcp- levels [ ] . in a co-culture model of raw . macrophages and t -l preadipocytes, addition of butyrate decreased the production of tnf-α, mcp- , and il- and decreased nf-κb expression in the macrophages [ ] . another study found that hdac inhibition decreases nf-κb transcription, which may be responsible for the anti-inflammatory effects [ ] . therefore, increasing r-bhb levels will likely lead to similar antiinflammatory effects on lung macrophages to dampen a cytokine storm, although butyrate has been reported to be a superior hdac inhibitor compared to r-bhb in some cell types such as myotubes and endothelial cells [ ] . this decreased efficacy of r-bhb as an hdac inhibitor in some cell types may result from different rates of transport into the cell or into the mitochondrial matrix, different rates of r-bhb oxidation, or different endogenous nuclear histone acetyltransferase or hdac activities. dietary therapies that increase both butyrate and r-bhb levels may have additive anti-inflammatory effects [ ] . the antibacterial effect of butyrate on intestinal macrophages was shown to be due to hdac inhibition, not gpr a signaling. hdac inhibition led to a decreased rate of glycolysis and increased flux through the ppp increasing amp levels and amp kinase activity and decreasing mtor activity to stimulate autophagy [ ] . in the lung, butyrate inhibition of the class iia hdac, hdac , decreased bacterial-induced inflammation [ ] . during infections, mitochondrial damage leads to the oxidation and release of the inner membrane phospholipid cardiolipin, leading to ppar-gamma sumoylation and recruitment of hdac to the promoter of il- , an anti- peroxynitrite s-nitrosylates hif- α to inhibit its von hippel-landau factor-induced ubiquitination and proteasomal degradation. this stabilizes hif- α leading to increased pdk expression. this stabilization is likely inhibited by r-bhb, which increases nadph to decrease ros/rns levels. (e) nrf is activated by oxidized dj- , and this is regulated by the redox potential of nadp + /nadph that controls the cellular antioxidant potential. the dj- chaperone protein, which is activated by moderate levels of hydrogen peroxide, stimulates the release of nrf from keap allowing nrf to enter the nucleus and induce antioxidant response element gene expression. (f) pgc- α is the master regulator of mitochondrial biogenesis. it is a coactivator of err-α, foxo , foxo a, ppars, and nuclear respiratory factor (nrf ). abbreviations: ac: acetate; are: antioxidant response element; ca : carbonic anhydrase ; ccr : c-c chemokine receptor type ; err-α: estrogen-related receptor alpha; etc: electron transport chain; k: lysine; hdac: histone deacetylase; fih- : factor inhibiting hif- ; foxo : forkhead box o ; foxo : forkhead box o ; foxp : forkhead box p ; g pc: glucose- phosphatase; g pdh: glucose -phosphate dehydrogenase; γgclm: glutamate cysteine ligase modifier subunit; glut : glucose transporter ; gsrx: glutathione reductase; hif- α and hif- β: hypoxia-inducible factor alpha and beta; hre: hypoxia response element; ifng: interferon gamma gene; il r: interleukin- receptor; inos: inducible nitric oxide synthase; irf : interferon regulatory factor ; keap : kelch-like ech-associated protein ; maf: musculoaponeurotic fibrosarcoma; nrf : nuclear respiratory factor ; nrf : nuclear factor erythroid -related factor (nfe l ); rag and rag : recombination activating genes and ; onoo -: peroxynitrite; onooh: peroxynitrous acid; p /cbp: binding protein /creb-binding protein; pdk and pdk : pyruvate dehydrogenase kinases and ; pgc -α: pparg coactivator alpha; phd : prolyl-hydroxylase domain protein ; pvhl: von hippel-landau tumor suppressor protein; scot/oxct : succinyl-coa- -oxaloacid coa transferase also known as -oxoacid coa-transferase ; sell: selectin l; sod : superoxide dismutase ; ub: ubiquitin; vegf: vascular endothelial growth factor. oxidative medicine and cellular longevity inflammatory cytokine, to decrease gene expression. gene expression of tnf-α was unaffected, so increased inflammation was observed. butyrate administration increased il- gene expression to normalize the level of inflammation [ ] . coronaviruses have been shown to increase the oxidation of phospholipids, which stimulate toll-like receptor (tlr ) signaling on macrophages, leading to cytokine production and acute lung injury [ ] , so hdac inhibition with r-bhb appears to be a viable treatment to decrease cytokine levels and inflammation. . . r-bhb binds to the gpr a gpcr to stimulate anti-inflammatory signaling. the gpr a (hydroxycarboxylic acid receptor (hca ), expressed from the hcar gene) gpcr is bound and activated by r-bhb (ec of . mm [ ] ), s-bhb, or butyrate and is expressed in the lung and many types of epithelial cells, macrophages, neutrophils, and dendritic cells, but not in b or naïve t lymphocytes [ ] . however, gpr a was shown to play a role in the expansion of cd + and cd + t cells [ ] . the expression pattern of gpr a suggests that it could play a role in the protective effects of the ketogenic diet against iav infection [ ] . gpr a has been shown to be activated by zika virus infection and protect cells by inhibiting viral replication [ ] . a major mechanism through which gpr a signaling exerts its anti-inflammatory effects is through suppressing the activation of the transcriptional regulator nuclear factor-kappa b (nf-κb) [ ] , required for the transcription and secretion of several proinflammatory cytokines [ ] . studies with gpr a-knockout mice have identified gpr a signaling as essential for the increase in thermogenesis induced by its ligands [ ] . consistent with this, gpr a-knockout mice were obese, showing hepatic steatosis due to upregulation of enzymes of fatty acid synthesis (acc and fatty acid synthase (fas)) and downregulation of enzymes of fatty acid oxidation (cpt- α). ppar-α, the master regulator of ketogenesis, was decreased in the liver, while ppar-γ, the master regulator of adipogenesis, was increased in wat. so, it is likely that stimulation of gpr a plays an important role in the induction of fatty acid beta-oxidation and weight loss induced by the ketogenic diet. macrophages and dendritic cells from gpr a-deficient mice were defective in inducing naïve t cells to differentiate into treg cells and il- -producing t cells [ ] . lack of gpr a also decreased the expression of il- [ ] . gpr a signaling has been shown to be protective by activating the nrf transcriptional regulator through an amp kinase signaling pathway to decrease oxidative stress [ ] . gpr a was also shown to play an important role in maintaining epithelial barrier function during bacterial sepsis [ ] , so it may play a similar role during viral infection. antiviral peptide and protect it from inactivation. cathelicidins are a class of antimicrobial host defense peptides. ll- is one of two human cathelicidins and released by bronchial epithelial cells, macrophages, and neutrophils as part of the innate immune response against respiratory viral infections [ ] . ll- has many functions, including binding to nucleic acids, strengthening the viral rna-induced tlr signaling response to increase type i interferon production [ ] , stimulating inflammasome activation [ ] , and reducing viral load and virion release [ , ] . the peptide has both proinflammatory and anti-inflammatory properties [ ] , but the anti-inflammatory properties may predominate in the lungs as ll- administration decreased the expression of the proinflammatory cytokines il- and il- and the chemokine ccl in response to respiratory viral infection [ ] . respiratory viral infection increases the expression of peptidyl arginine deiminase (pad ) in the lungs. this class of enzymes catalyzes the removal of a positively charged amino group from protein arginine to form citrulline, through a process called citrullination. ll- has five arginine residues essential for its antiviral function, which are targets of pad function following the viralinduced increase in pad expression in the lungs [ ] . increased levels of nadph decrease the catalytic activity of peptidyl arginine deiminases to limit ll- citrullination [ , ] . hdac inhibitors such as butyrate have been shown to increase the expression of ll- [ ] to decrease pathogen infection [ ] . therefore, r-bhb may mitigate respiratory virus infection both by increasing ll- levels and by increasing nadph levels [ ] that protect ll- from inactivation. diet and its level in tissues is regulated by redox-sensitive coenzyme ratios. cortisol, an adrenal gland-secreted hormone, has anti-inflammatory properties through activation of the glucocorticoid receptors. subjects on either a ketogenic diet [ , ] or a severe calorie restriction diet [ ] show transient increases in cortisol levels. in mice, seven days of ketogenic diet led to the transcriptional activation of targets of the glucocorticoid receptor [ ] . if this transient increase in cortisol levels that occurs in humans also occurs in mice on the ketogenic diet, the increased cortisol levels may contribute to the blunting of the cytokine storm in animals that were fed the ketogenic diet for a week before iav infection [ ] . mice that were fed the ketogenic diet for three months showed increased inflammation, which could have been in part due to the return of cortisol to baseline levels [ ] . as may be expected due to the function of cortisol as a stress hormone, when healthy subjects were administered ketone ester in nonketogenic states, no change in cortisol levels was observed [ , ] . in peripheral tissues, such as the lungs, the level of cortisol is regulated by the βhydroxysteroid dehydrogenase ( β-hsd) system consisting of the two enzymes, β-hsd and β-hsd . the conversion of cortisone to the active steroid hormone cortisol is catalyzed by nadph-dependent β-hsd [ ] , while the reverse reaction that regenerates the precursor cortisone is catalyzed by the nad + -dependent β-hsd enzyme (figures and ) . therefore, the level of active cortisol in tissues is under tight control by the nad + /nadh and nadp + /nadph redox ratios. ketone ester treatment has been shown to normalize these coenzyme ratios in diseased mouse tissue [ ] . corticosteroid hormone administration has been used in an attempt to blunt the cytokine storm in several human respiratory viral infections [ ] . to be successful, this therapy must be administered at the appropriate time late in the infection cycle to allow the immune system to first mount a proper antiviral response. since identifying the appropriate timeframe for treatment for different patients is challenging, glucocorticoid therapy has been largely unsuccessful and may have even contributed to detrimental patient effects when used to treat influenza infection [ ] . however, emerging data suggest that short-term dexamethasone treatment may be beneficial for sars-cov- infection [ ] , and dexamethasone treatment was shown to decrease the mortality of patients with severe sars-cov- infection who were placed on a ventilator [ ] . increasing r-bhb levels, together with a moderately high-fat diet, may be able to stabilize nucleotide coenzyme ratios to allow virally infected tissue to increase endogenous cortisol levels at the appropriate time in the infection cycle to decrease inflammation and blunt the cytokine storm. signaling through hdac inhibition. sars-cov- virions bind to angiotensin-converting enzyme (ace ) receptors [ , ] on the surface of host cells, such as aec ii, as a first step in viral entry [ ] . the level of ace receptors decreases during aging and may also decrease due to endocytosis during sars-cov- infection [ , ] . in this reninangiotensin signaling system, renin catalyzes the conversion of angiotensinogen into angiotensin (ang i). angiotensin-converting enzyme (ace ) then catalyzes the conversion of ang i into angiotensin ii (ang ii), which binds to the at r receptor leading to vasoconstriction and proinflammatory, prooxidative, and profibrotic effects leading to tissue injury. ace is normally able to blunt these effects by cleaving ang i and ang ii into peptides that bind to the at r and masr receptors that signal for vasodilation and anti-inflammatory, antioxidative, and antifibrotic effects leading to tissue protection (figure (a) ) [ ] [ ] [ ] [ ] [ ] . increased levels of ang ii stimulate the synthesis of the proinflammatory cytokines il- , ifn-γ, tnf-α, and il- β [ ] , but also the anti-inflammatory cytokines tgf-β and il- , which may induce m macrophage polarization [ ] and prevent the γδ t lymphocyte activation needed to initiate the antiviral immune response [ ] . butyrate and other hdac inhibitors have been shown to decrease the expression of angiotensinogen, renin, and at r to block this proinflammatory signaling [ , ] . therefore, the use of exogenous ketones could lead to a balancing of signaling through the different arms of the renin-angiotensin system when proinflammatory signaling through ang ii predominates such as in aged individuals and subjects infected with sars-cov- . cytokine production in different cell types. the effects of r-bhb on proinflammatory cytokine production in different cell types can vary greatly. for example, r-bhb, when given to isolated macrophages challenged with streptococcus uberis, was shown to increase the expression of il- β and il- and the chemokines cxcl and ccl but had no effect on the expression of tnf-α and tgf-β [ ] . in another report using isolated m peritoneal macrophages, r-bhb was shown to decrease the expression of il- , but not il- β, tnf-α, or il- [ ] . in calf hepatocytes, r-bhb was shown to increase nf-κb activity and the expression of il- β, tnf-α, and il- [ ] , while ketosis had similar effects in the liver of cows [ ] . the absence of ketolytic enzymes and presence of ketogenic enzymes in the liver may contribute in part to the proinflammatory response. in lps-stimulated bv- microglial cells, r-bhb was shown to decrease nf-κb activation and the expression of tnf-α, il- β, and il- [ ] . when infused into the rat brain prefrontal cortex for days in a model of depression (chronic unpredictable stress paradigm), r-bhb was shown to prevent the increase in tnf-α and the decrease in corticosterone brought about by the depression-inducing stress [ ] . peripheral injection of r-bhb was also able to decrease il- β and tnf-α levels in the hippocampus of rats in this depression model [ ] . in bovine aorta endothelial cells stimulated with lps, r-bhb was shown to decrease the expression of tnf-α and interferon [ ] . the different results in different cell types and conditions clearly indicate that more research needs to be done to understand the complex regulation of cytokine production by r-bhb. sirtuin deacetylases. a major mechanism through which r-bhb restores metabolism and redox balance is through epigenetic regulation of gene expression by increasing histone beta-hydroxybutyrylation (figure (c)) and inhibiting class i and iia histone deacetylases (hdacs) to increase histone acetylation. transcription factors and coactivators such as foxo [ , ] , foxo a [ ] , and pgc- α [ ] are induced. complexly, hdac inhibitors can also lead to increased acetylation of foxo , which reduces its activity at the promoters of genes for gluconeogenic enzymes in the liver to decrease blood glucose levels [ ] . this is likely beneficial for inhibiting the cytokine storm as discussed above. these transcriptional regulators increase mitochondrial etc gene expression to help restore the nad + /nadh. they initiate an antioxidant gene expression program together with the induction of ppp enzymes [ , ] to restore the nadp + /nadph. in tissues such as liver, increased r-bhb levels lead to hdac inhibition at the foxo promoter and increased gene expression decreasing proinflammatory cytokine expression [ ] . amp kinase [ ] and nad + -dependent sirt deacetylase [ ] enzymes also act on foxo to increase its activity. insulin signaling through the akt pathway inhibits foxo activity [ ] . the anti-inflammatory action of foxo may be due in part to its activity in lung macrophages where it binds to irf and stimulates the m state [ ] . however, in tumor-localized macrophages, foxo has been shown to stimulate the proinflammatory oxidative medicine and cellular longevity m state and il- β production [ ] . so, the effects of foxo on macrophage function appear to be dependent upon the environmental conditions. increasing nad + levels in the cytoplasm activates sirt to deacetylate glucose- phosphate dehydrogenase (g pd) to increase ppp flux and nadph production [ ] , linking the ratios of the pyridine nucleotide coenzyme couples. sirt also deacetylates the inflammasome, inhibiting its function [ ] , while sirt function also inhibits inflammasome activity by decreasing mitochondrial ros levels [ ] . increased nucleocytoplasmic nad + level also increases the activity of sirt , which deacetylates pgc- α to stimulate mitochondrial etc function [ ] leading to increased mitochondrial nad + /nadh. the increased mitochondrial nad + /nadh activates mitochondrial sirt to deacetylate and activate mitochondrial sod [ ] , isocitrate dehydrogenase (idh ) [ ] , and the kd subunit of etc complex i [ ] to decrease ros levels and decrease the mitochondrial nadp + /nadph. the decreased mitochondrial nadp + /nadph maintains reduced mitochondrial gssg/gsh to prevent the glutathionylation of etc complex i and the oxidation of cardiolipin that decrease complex i activity and decrease the matrix space nad + /nadh [ ] . other important genes induced by the foxo transcriptional regulator to restore metabolism and decrease inflammation and ros production include gpr a, lactate dehydrogenase b (ldhb), thioredoxin (txn ), pepck , and the nad + synthesis genes nampt and nmnat [ ] . expression of pgc- α and err-α. pgc- α and err-α (estrogen-related receptor-α), a binding partner of pgc- α involved in the induction of mitochondrial etc gene expression [ ] , are the transcriptional regulators that are known to induce oxct gene expression in myotubes to increase the levels of its gene product scot (figure (f)) [ ] . err-α, which is widely expressed, is also required for adipose tissue thermogenesis [ ] and is induced by fasting, calorie restriction, cold exposure, and exercise [ , ] . the ketogenic diet has been shown to increase pgc- α levels in muscle [ ] , neurons [ ] , and brown adipose tissue [ ] . these data suggest that err-α and pgc- α are likely the transcriptional regulators that induce oxct and etc gene expression in the lungs during the ketogenic diet to increase ketolysis and mitochondrial biogenesis [ ] . scot activity has been shown to be decreased by tyrosine nitration [ ] and increased by tryptophan nitration [ ] . scot activity was also inhibited by acetylation and activated by sirt -mediated deacetylation [ ] . high-fat diets can decrease pgc- α levels in the liver, decreasing its suppression of nf-κb and leading to increased cytokine production [ ] . contrary to this result, a relatively highfat diet in the presence of ketone ester was shown to increase pgc- α levels and mitochondrial function in brown adipose tissue to stimulate thermogenesis [ , ] . therefore, the consumption of ketone ester may reverse the effects of a high-fat diet on pgc- α expression in some tissues to stimulate fatty acid oxidation and prevent the accumulation of fat in tissues that is associated with negative health outcomes. uncoupling can restore the nad + /nadh and nadp + /nadph ratios. once the viral-and cytokine storm-induced changes in the mitochondrial nad + /nadh have been partially restored through r-bhb-mediated signaling, enzyme inhibition, and upregulation of gene expression, the nad + -dependent bdh enzyme can more effectively catalyze the conversion of r-bhb to acetoacetate. acetoacetate is then metabolized to acetoacetyl-coa, which is metabolized into two molecules of acetyl-coa. as mentioned above, this pathway of acetyl-coa synthesis becomes especially important under conditions of viral infection because pdk expression is upregulated leading to pdc inhibition. . . . nicotinamide nucleotide transhydrogenase. nicotinamide nucleotide transhydrogenase (nnt) is an enzyme that uses energy from the mitochondrial inner membrane proton gradient to synthesize nadph and nad + from nadp + and nadh. nnt gene expression is likely induced by foxo a, since there are binding sites for foxo a in the nnt promoter [ ] and since the c. elegans nnt homolog nnt- is induced by the c. elegans foxo homolog daf- [ , ] . nnt activity is likely high during times of mitochondrial etc dysfunction, such as during a cytokine storm, as decreased mitochondrial nad + /nadh and increased mitochondrial nadp + /nadph stimulate nnt function in the normal nadph-synthesizing and nadhhydrolyzing direction. status. recent evidence suggests that when glucose levels and ppp activity are low, serine and glycine are catabolized in mitochondria, which stimulates one-carbon metabolism, to generate nadph [ ] . there are mechanisms in place to use the mitochondrial matrix space-synthesized nadph to prevent product inhibition of enzyme function, the most important being the fueling of glutathione reductase and thioredoxin reductase to combat ros. alternatively, the nadph equivalents can be shuttled to the cytoplasm using the citrate-pyruvate shuttle. this involves the catabolism of glutamine and glutamate to alpha-ketoglutarate, which can lead to idh functioning in the opposite direction of its normal citric acid cycle activity to oxidize nadph and form isocitrate in the process called reductive carboxylation [ , ] . isocitrate can then be further metabolized to citrate. this citrate, together with other citrate molecules, such as those derived from r-bhb metabolism, gets shuttled into the cytoplasm though the mitochondrial citrate carrier protein (cic) as part of the citrate-pyruvate shuttle (see figure ). in the cytoplasm, the citrate can be converted to acetyl-coa and oxaloacetate by atp-citrate lyase (acly). the acetyl-coa can function in histone acetylation or fatty acid synthesis, while the oxaloacetate can be converted to malate by malate dehydrogenase (mdh ) to restore the cytoplasmic nad + /nadh. malate can then be converted to pyruvate by nadp + -dependent malic enzyme (me ), which concurrently synthesizes nadph [ ] . in the final step, the pyruvate is shuttled back into the mitochondrial matrix space, where it is metabolized by pyruvate carboxylase to form oxaloacetate. the net result is atp + nadh + nadp + − > adp + nad + + nadph, which contributes to the restoration of the redox state. the result on the redox state is very similar to that which occurs due to the nnt reaction, except the nad + and nadph are formed in the cytoplasm instead of the mitochondrial matrix. increased levels of citrate and acetyl-coa in the cytoplasm, which would likely occur as a result of increased shuttle function, inhibit glycolysis at phosphofructokinase [ ] and pyruvate kinase [ ] , respectively, which would further aid in the restoration of nad + /nadh. in m -polarized macrophages, citrate-pyruvate shuttle function can provide nadph that fuels nadph oxidasemediated ros production and contributes to inflammation. these m macrophages upregulate the expression of cis-aconitate decarboxylase (irg /acod ) to convert the citric acid cycle metabolite cis-aconitate to itaconate, which exerts anti-inflammatory actions to restrain the m response, by inhibiting etc complex ii activity to decrease ros production and by activating the nrf (nfe l ) transcriptional regulator. therefore, inhibitors of cic and acly have been shown to be anti-inflammatory compounds [ ] , but these inhibitors may have deleterious effects on the cytoplasmic and mitochondrial redox states in other cell types. in a related redox shuttle, the citrate-malate shuttle, the cytoplasmic malate is imported into the mitochondrial matrix and therefore no cytoplasmic nadph is synthesized. however, this shuttle is slightly more energy efficient, using the hydrolysis of only one molecule of atp. a third shuttle system that exports citrate from the mitochondrial matrix is the citratealpha-ketoglutarate shuttle. in this shuttle, cytoplasmic citrate is converted into isocitrate and then further into alphaketoglutarate by cytoplasmic aconitase (aco ) and isocitrate dehydrogenase (idh ), respectively, with the latter reaction synthesizing nadph to decrease the cytoplasmic nadp + /-nadph [ ] . the alpha-ketoglutarate can then be transported back into the mitochondrial matrix. idh expression is downregulated in m -polarized macrophages when measured hours after stimulation with lps [ ] to turn off citrate-alpha-ketoglutarate shuttle flux and stimulate citratepyruvate shuttle function. however, two to four hours after lps stimulation of macrophages, the alpha-ketoglutaratedependent histone demethylase genes kdm b [ ] and phf [ ] are induced to increase inflammation [ ] . therefore, the citrate-alpha-ketoglutarate shuttle proteins cic, aco , and idh may play a role early in the m polarization process to provide nucleocytoplasmic alphaketoglutarate for the function of these histone demethylase enzymes before the shuttle is turned off. another important mechanism through which a ketogenic diet restores the mitochondrial nad + /nadh during mitochondrial etc dysfunction is through inducing the expression of mitochondrial uncoupling proteins. the rate of nadh oxidation at complex i is normally limited by matrix space adp levels. the presence of uncoupling proteins removes this limitation by allowing protons to flow back into the matrix space to produce heat. this increases the rate of complex i nadh oxidation to increase the mitochondrial nad + /nadh. the cytoplasmic nad + /nadh can also be altered by mitochondrial redox changes through malate-aspartate shuttle function. another benefit of partial mitochondrial uncoupling is the slight decrease in the mitochondrial membrane potential that greatly decreases the generation of superoxide at etc complexes i and iii [ , ] . one drawback of the increased expression of uncoupling proteins is the decrease in atp generation. the ketogenic diet increases ucp expression in brown adipose tissue [ ] and ucp [ ] , ucp , and ucp [ ] in the brain. increases in uncoupling protein levels have been shown to parallel increases in pgc- α levels. administration of ketone ester to mice has been shown to increase the cytoplasmic nad + /nadh and decrease the cytoplasmic nadp + /nadph [ ] and likely utilizes mitochondrial uncoupling and the other mechanisms listed above to restore redox balance. conditions to restore the redox state. the mildly elevated ros production from r-bhb metabolism from a ketogenic diet leads to the activation of the nrf transcriptional regulator [ ] , which stimulates antioxidant response element (are) gene expression. paraquat, a redox cycling agent, which greatly increases superoxide production from etc complex i, was shown to decrease nrf levels, by a mechanism described in detail below, which was restored by the administration of r-bhb [ ] . activation of nrf blunts the cytokine storm through inducing the expression of antioxidant system enzymes including heme oxygenase- , sod [ ] , nadp(h) quinone oxidoreductase (nqo ), gamma-glutamylcysteine synthetase (gclc), thioredoxin (txn), thioredoxin reductase (txnrd ) [ ] , and multiple enzymes for nadph synthesis including idh , malic enzyme (me ), and four enzymes of the ppp including g pd, -phosphogluconate dehydrogenase (pgd), transaldolase, and transketolase [ ] . nrf can be activated by hydrogen peroxide when the ratio of nadp + /nadph is not too high or too low as shown in figure (e). for nrf activation to occur, superoxide produced by the etc in the mitochondrial matrix is converted into hydrogen peroxide by sod . hydrogen peroxide is then transported out of the mitochondrial matrix through aquaporins present in the inner mitochondrial membrane. in the cytoplasm, the redox-sensitive chaperone dj- is activated when cysteine sulfhydryl is oxidized to sulfenic acid by hydrogen peroxide. this activated form of dj- is able to release keap from nrf allowing nrf to enter the nucleus and induce gene expression. when the nadp + /-nadph is too low, it prevents dj- from being oxidized and activated. when the nadp + /nadph is too high, cysteine in dj- becomes overoxidized to sulfonic acid and dj- is destabilized, ubiquitinated, and degraded by the proteasome, so nrf is not activated [ ] . therefore, r-bhb metabolism likely preserves the function of nrf by providing the proper nadp + /nadph ratio, which maintains low to moderate levels of cytoplasmic hydrogen peroxide. oxidative medicine and cellular longevity . . hif -α stabilization by rns leads to proinflammatory cytokine production. hypoxia-inducible factor- α (hif- α) is the master transcriptional regulator of hypoxic gene expression. during normoxia, hif- α is hydroxylated on prolines, which stimulates its binding to von hippel-lindau (vhl) protein that targets it for proteasomal degradation. hif- α is also hydroxylated on asparagine residues by fih- (factor inhibiting hif- - ) to prevent hif- α from binding to its coactivator cbp/p . during hypoxia, the oxygendependent prolyl and asparaginyl hydroxylases are inactive stabilizing the transcriptionally active form of hif- α. hif- α can also be activated by ros or rns. hif- α induces the expression of glucose transporters, glycolytic enzymes, and pdk to inhibit pdc and shunt glycolysis-derived carbon flux away from mitochondria when oxidative phosphorylation is compromised. figure (d) shows the snitrosylation and activation of hif- α by peroxynitrite. s-nitrosylation of hif- α prevents the interaction with vhl for stabilization and inhibits asparagine hydroxylation for activation [ ] . hif- α can also induce expression of the proinflammatory cytokines tnf-α and il- by upregulating nf-κb [ ] . hif- α has been shown to be stabilized by increased pyruvate levels [ ] , such as those that occur following pdc inhibition, or by increased succinate levels [ ] . the circadian transcriptional regulator bmal , which can be induced by the hormone melatonin [ ] , decreases hif- α stability and levels to increase mitochondrial oxidative metabolism [ ] . in lung aecs, stabilization of hif -α was shown to cause er stress and chop-mediated apoptosis [ ] . iav infection was shown to induce the nuclear translocation of hif- α by activating the c-jun n-terminal kinase (jnk) signaling pathway to increase proinflammatory cytokine expression [ , ] . however, hif- α may also play a role in suppressing iav infection as hif- α deficiency stimulated iav replication in aec ii cells by increasing autophagy [ ] . rsv infection was shown to stabilize hif- α by increasing nitric oxide and peroxynitrite levels [ ] . this resulted in increased nf-κb, proinflammatory cytokine levels, and viral replication [ , ] . therefore, increasing levels of r-bhb to decrease rns levels will likely be able to decrease the activation of hif- α and its downstream inflammatory mediators to mitigate sars-cov- and other respiratory viruses. . the effects of r-bhb on cells of the immune system . . increased nadph can increase or decrease ros levels depending upon the cell type. nadph has roles both in the synthesis of superoxide through its role as a cofactor for nox and in peroxide detoxification through its roles as cofactors for glutathione reductase and thioredoxin reductase. in most cell types, the lower k m of nadph for the reductase antioxidant enzymes allows the antioxidant effect to predominate [ ] [ ] [ ] . however, in some cell types such as macrophages and neutrophils, the high expression level of nox enzymes allows ros production to predominate. lung aecs also have relatively high levels of nox , nox , duox , and duox [ ] . future studies should address how altered nadp + /nadph regulates ros production in these cells and if increased ketone body levels increase nadph levels in macrophages, neutrophils, and lung epithelial cells to increase nadph oxidase activity to stimulate host defenses against pathogens. in lung epithelial cells, knockdown of g pd to decrease nadph synthesis reduced nox activity to decrease the antiviral response [ ] . in mice, it has been shown that decreasing nadph synthesis by inhibiting g pd and ppp flux with -aminonicotinamide decreased lps-induced inflammation in a model of acute lung injury [ ] . this data is consistent with the protective effects of the nox inhibitor for iav infection [ ] . the expression of sod and catalase is induced by r-bhb-mediated hdac inhibition [ ] , so when r-bhb increases nadph levels to stimulate nadph oxidase activity, it also increases ros detoxification enzymes to prevent excessive oxidative stress that may lead to a cytokine storm. increased nadph levels also have been shown to stimulate antiviral immunity by decreasing the level of the nadph sensor protein hscarg, which is a negative regulator of nf-κb transcription. through this mechanism, increased nadph levels were shown to increase expression of the mx and tnf-α genes to decrease human coronavirus infection [ ] . one other potential proinflammatory action of the high nadph levels from r-bhb metabolism is the reduction of dihydrobiopterin (bh ) to tetrahydrobiopterin (bh ). bh is an essential cofactor for nitric oxide synthases and aromatic amino acid hydroxylases [ ] . so, increased nadph levels may increase nitric oxide synthase activity leading to rns production and inflammation. however, when bh levels are low, nitric oxide synthases synthesize superoxide instead of nitric oxide [ ] . in this way, increased nadph decreases ros production as it increases rns production. this function could potentially decrease toxic peroxynitrite levels when superoxide is limiting for its synthesis. function. lung-resident macrophage polarization to either a proinflammatory m state or an anti-inflammatory m state is largely controlled by the cytokines secreted by the other cells present in the environment [ ] . r-bhbmediated hdac inhibition, gpr a signaling, and inflammasome inhibition in these cells likely increase the amounts of anti-inflammatory cytokines, such as il- , produced to stimulate more macrophages to the m state. these cytokines also influence the catabolic pathways that are activated to fuel cellular energy needs. m macrophages have increased glycolytic activity and lactate production due to the presence of a dysfunctional citric acid cycle [ ] . increased nitric oxide levels may inactivate citric acid cycle enzyme aconitase (aco ) and the pdc e subunit dihydrolipoyl dehydrogenase (dld). nitric oxide also may decrease the activities of etc complexes i, ii, and iv in m macrophages [ ] . the dysfunctional citric acid cycle resulted in the accumulation of citrate that increased fatty acid synthesis [ ] and succinate that increased mitochondrial ros production [ ] . this metabolic programming in m macrophages likely oxidative medicine and cellular longevity evolved to increase proinflammatory cytokine production. m macrophages are programmed to oxidize pyruvate and fatty acids into acetyl-coa for normal citric acid cycle function and oxidative phosphorylation [ ] , which has been shown to facilitate anti-inflammatory il- secretion [ ] . since macrophages lack the enzyme bdh , they cannot oxidize r-bhb [ ] , but a ketogenic diet also increases the systemic levels of the ketone body acetoacetate that can be oxidized by macrophages. the r-bhb/acetoacetate ratio produced by the liver is proportional to the mitochondrial nad + /nadh ratio [ ] , which is normally around five [ ] , but varies from three to seven. in this regard, acetoacetate but not r-bhb was shown to be metabolized by macrophages to ameliorate liver fibrosis in mice [ ] . neutrophils, another type of phagocyte contributing to the cytokine storm, have few mitochondria and very low expression of ketolytic genes and therefore likely cannot catabolize r-bhb or acetoacetate to a significant extent. reducing glucose uptake into ilc s. a recent study examined the effects of reducing glucose levels, using a ketogenic diet, on allergen-induced lung inflammation in mice. results showed that ilc s in the lungs must increase their uptake of both fatty acids and glucose from the environment to elicit allergen-dependent inflammation. a ketogenic diet reduced systemic glucose levels to decrease lung ilc glucose uptake to prevent airway inflammation in response to the allergen [ ] . the γδ t cell-ilc response activated by the ketogenic diet in mice [ ] was shown to be active in human infants and children on a normal diet, where it protected them from influenza infection [ ] . however, this response appears to be suppressed starting in adolescence and replaced by the ilc system, until it is likely reawakened by the ketogenic diet. we speculate that the higher activity of the γδ t cell-ilc response in children may be one factor responsible for the less severe symptoms when the sars-cov- virus infects individuals in this age group. the increased mortality rate of infected older adults may also be in part due to the increase in inflammation and decline in mitochondrial function and cellular nad + and nadph levels with aging that decreases metabolic flexibility and the ability to "weather" the cytokine storm. . . hdac inhibition and gpr a signaling have anti-inflammatory effects on dendritic cells. dendritic cells play an important function in presenting antigens to t lymphocytes. dendritic cells have a high level of oxidative metabolism until they are activated through their tlrs, at which point they switch to a primarily glycolytic metabolism [ ] , which is essential for their activation by providing cytoplasmic atp for phospholipid synthesis and signals for the remodeling and expansion of the secretory system for its enhanced function in the activated state [ ] . the increased rate of glycolysis in dendritic cells was also required for their secretion of interferon-α to mitigate iav infection. vaccination against iav was able to increase glycolytic function in the dendritic cells [ ] . human monocyte-derived dendritic cells cultured with butyrate showed decreased pro-inflammatory cytokine and chemokine production [ ] . this is likely due to the ability of hdac inhibition and gpr a signaling in dendritic cells to promote treg cells [ ] . the mechanism may be at least partially metabolic in nature. dendritic cells were activated by exposure to lps in the presence or absence of butyrate. butyrate decreased the oxygen consumption rate and blocked the increase in extracellular acidification rate (due to lactate export) used as an indicator of the glycolytic rate [ ] . butyrate functioning as an hdac inhibitor also inhibited the formation of dendritic cells from bone marrow stem cells [ ] . administration of ethyl pyruvate, a cell-permeable pyruvate precursor with known anti-inflammatory properties [ ] , was shown to inhibit the activation of dendritic cells by single-strand rna that binds to tlr . ethyl pyruvate decreased glycolytic and oxidative metabolism and blocked dendritic cell activation through decreasing erk and akt signaling and decreasing the production of nitric oxide [ ] . therefore, ethyl pyruvate administration may be a potential therapy to block the cytokine storm during the later stages of sars-cov- infection. restore redox and energy levels. increasing r-bhb levels may also stimulate the immune system by enhancing b or t lymphocyte function. recent evidence links the ketogenic diet in mice with increasing levels of δγ t cells in adipose tissue [ ] , where the δγ t cells stimulate a thermogenic program [ ] , in part responsible for the weight loss effects frequently provided by the ketogenic diet. the ketogenic diet did not alter the level of ketone body metabolism genes in lung γδ t cells, but it did upregulate the expression of mitochondrial etc genes in these cells [ ] . cells of the immune system express varied amounts of scot, used specifically for ketolysis, and bdh , used for both ketogenesis and ketolysis. b and t lymphocytes possess the greatest amounts of bdh and scot of the blood cell types [ ] , so the ketogenic diet or exogenous ketone treatment may enhance the energy levels and redox status in these cells more effectively. . . . a ketogenic diet normalizes the increased th /treg ratio that occurs due to disease. there are two important subtypes of cd + t lymphocytes, th and treg cells. a ketogenic diet was shown to normalize the proinflammatory th /treg ratio in the blood from epileptic patients [ ] . a high-fat diet may favor the expansion of treg cells over th cells as tregs can take up and utilize fatty acids from the environment, while th do not have this ability [ ] , so th cells must synthesize fatty acids from glucose, which is present at slightly lower levels when consuming the ketogenic diet [ ] . intermittent fasting that increases r-bhb levels was also able to restore this ratio in mice with experimental autoimmune encephalomyelitis (eae), a model of multiple sclerosis [ ] . hdac inhibition in naïve t cells leads to the expression of the foxp transcriptional regulator and treg conversion [ ] . the longevity-promoting mtor inhibitor and antiaging calorie restriction mimetic rapamycin also decreased the th /treg balance. this occurred through inhibition of glycolysis in th cells and stimulation oxidative medicine and cellular longevity of fatty acid oxidation in tregs. fatty acid oxidation was stimulated by amp kinase activation [ ] . a moderate dose of butyrate only induced differentiation of t cells to treg cells when administered together with tgf-β . as tgf-β level is increased by viral infection, this should not pose a problem for the treatment of sars-cov- with ketone ester. in addition, hdac inhibition induces the expression of tgf-β in epithelial cells. however, administering a high dose of butyrate, even when added together with tgf-β , was ineffective at inducing differentiation into il- -producing treg cells. high doses of butyrate resulted in either normal t cells or ifn-γ-producing tregs [ ] . therefore, butyrate or r-bhb may need to be present in the cell nucleus within a specific narrow concentration range for the partial inhibition of hdac function for the optimal treatment of sars-cov- . . . . r-bhb stimulates the formation of cd + memory t cells. cd + memory t cells readily oxidize fatty acids and, like hepatocytes, have the rare ability to simultaneously express the genes for gluconeogenesis and ketogenesis [ ] . the high level of expression of pepck , the rate-limiting step in gluconeogenesis, resulted in the depletion of cellular oxaloacetate levels. therefore, the acetyl-coa formed from fatty acid beta-oxidation was not able to enter the krebs cycle and was therefore used for ketogenesis. the increased r-bhb levels led to increased beta-hydroxybutyrylation of histone proteins in the foxo and pgc- α promoters leading to increased gene expression. increased gluconeogenesis led to increased glucose levels that stimulated the ppp synthesis of nadph required for the long-term protection of the cd + memory t cells against ros [ ] . metabolic therapy with ketone ester should enhance this endogenous epigenetic program to promote the survival of cd + memory t cells to facilitate immune function when a patient is reexposed to sars-cov- . the data presented here suggest two types of clinical studies with covid- patients that would provide data on the efficacy of a ketone-based metabolic therapy. these include the following: ( ) a determination of forced vital capacity by spirometry of covid- patients consuming ketone ester and a moderately high-fat diet (forced vital capacity is a measure of lung function based on taking a full breath and exhaling as much volume of air as possible) ( ) a randomized trial of covid- patients consuming ketone ester with a moderately high-fat diet with evaluations of length and severity of infection and patient mortality a ketone-based metabolic intervention for covid- patients will likely be initiated at one of the three general stages of disease progression. during all three stages, the basic treatment of raising blood ketone levels to to mm with exogenous ketones, increasing consumption of dietary fats, and taking enteric-coated sodium bicarbonate to buffer blood ph will likely be beneficial. the first stage is the onset of disease symptoms. during this stage, a moderate carbohydrate diet will allow normal blood glucose levels to boost immune cell function. the second stage is when the severity of symptoms requires hospitalization and/or ventilation. at this middle stage of infection, a limited carbohydrate diet would be beneficial to lower glucose metabolism and its associated proinflammatory signaling. the third stage is after ventilator use ceases and difficulty in breathing ensues. once again, a low-carbohydrate diet at this late stage is hypothesized to facilitate beneficial anti-inflammatory processes. the expected outcomes no matter when treatment is initiated are a decrease in the incidence of progression to ards, protection of organs from oxidative and inflammatory damage, and increased clearance of the virus to shorten the duration of the infection. patient data analyzed independently for the different stages of therapy initiation may yield important insights into the relative time when ketone-based metabolic intervention is most effective. the studies suggested above, together with long-term patient monitoring, may also yield important insights into the significant post-covid- patient complications. there is evidence from sars-cov- infections that psychiatric and chronic fatigue issues continued for four years after infection [ ] . unfortunately, there are anecdotes from recovered covid- patients describing similar lingering morbidities. we further hypothesize that the severity of these morbidities including chronic fatigue, depression, posttraumatic stress disorders, panic disorders, and somatoform pain disorders may be blunted by a ketone-based metabolic therapy due to the mitigation of cell death and tissue damage. the sars-cov- virus may become a sustained threat to global health. this review has described many of the molecular mechanisms through which an exogenous ketone-based metabolic therapy together with a moderately high-fat diet may stimulate host cell metabolism and defenses as a possible treatment to blunt the cytokine storm associated with severe sars-cov- infection. a clinical trial testing this therapy on patients with sars-cov- is warranted. in addition, further mouse iav infection studies will aid in the determination of permissive dietary conditions under which exogenous ketone supplementation enhances immune function to facilitate viral clearance and decrease mortality. the authors 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cell development by bacterial fermentation products butyrate and propionate through a transporter (slc a )-dependent inhibition of histone deacetylases pyruvate is an endogenous anti-inflammatory and anti-oxidant molecule γδ t cells producing interleukin- a regulate adipose regulatory t cell homeostasis and thermogenesis the effects of ketogenic diet on the th /treg cells imbalance in patients with intractable childhood epilepsy de novo fatty acid synthesis controls the fate between regulatory t and t helper cells the biochemistry of ketogenesis and its role in weight management, neurological disease and oxidative stress intermittent fasting confers protection in cns autoimmunity by altering the gut microbiota metabolites produced by commensal bacteria promote peripheral regulatory t-cell generation rapamycin modulate treg/th balance via regulating metabolic pathways: a study in mice the microbial metabolite butyrate induces expression of th -associated factors in cd (+) t cells ketogenesis-generated βhydroxybutyrate is an epigenetic regulator of cd + t-cell memory development a pck -directed glycogen metabolic program regulates formation and maintenance of memory cd + t cells mental morbidities and chronic fatigue in severe acute respiratory syndrome survivors: long-term follow-up we would like to thank dr. brianna stubbs for reading the paper and offering several helpful suggestions, david lamps for drawing the cells and organs for the graphics, todd king oxidative medicine and cellular longevity for proofreading the paper, and frank llosa for providing the idea to look into the potential effects of ketones on covid- and offering helpful discussion. key: cord- -x qbqy authors: liu, gengqi; lovell, jonathan f.; zhang, lei; zhang, yumiao title: stimulus-responsive nanomedicines for disease diagnosis and treatment date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: x qbqy stimulus-responsive drug delivery systems generally aim to release the active pharmaceutical ingredient (api) in response to specific conditions and have recently been explored for disease treatments. these approaches can also be extended to molecular imaging to report on disease diagnosis and management. the stimuli used for activation are based on differences between the environment of the diseased or targeted sites, and normal tissues. endogenous stimuli include ph, redox reactions, enzymatic activity, temperature and others. exogenous site-specific stimuli include the use of magnetic fields, light, ultrasound and others. these endogenous or exogenous stimuli lead to structural changes or cleavage of the cargo carrier, leading to release of the api. a wide variety of stimulus-responsive systems have been developed—responsive to both a single stimulus or multiple stimuli—and represent a theranostic tool for disease treatment. in this review, stimuli commonly used in the development of theranostic nanoplatforms are enumerated. an emphasis on chemical structure and property relationships is provided, aiming to focus on insights for the design of stimulus-responsive delivery systems. several examples of theranostic applications of these stimulus-responsive nanomedicines are discussed. one of the challenges of traditional drug delivery systems is the lack of specific targeting capability, which leads to dose-limiting side effects [ , ] . some drugs can be rapidly metabolized, and low bioavailability necessitates large injection doses but unsatisfactory therapeutic effects. for example, it is generally necessary to increase the dosage of anticancer drugs for the desired therapeutic effect, to an extent that also causes undesirable side effects [ , ] . in addition, orally administered medicines need to evade the risk of degradation in the acidic/enzymatic environment in the gastrointestinal tract [ , ] . all of these problems can potentially be addressed with stimulus-responsive nanotechnology. utilizing specific microenvironmental conditions at diseased sites or alternatively by using external stimuli, nanotherapeutics can release active therapeutic ingredients specifically at desired spots in a controlled and targeted manner. as early as , tanaka described the phase transition of polyacrylamide polymers [ ] , and in the same year blumenthal reported thermosensitive liposomes [ ] . findings such as these laid the groundwork for stimulus-responsive drug delivery systems, which have been developed for more than years. stimulus-responsive nanomaterials have been designed for a differences between the microenvironment of diseased tissues (e.g., arthritic tissues, tumor cells, and other diseases) and normal tissues can be used as endogenous stimuli to trigger drug release. endogenous stimuli include lower ph values in tumor cells, high concentrations of glutathione (gsh) and reactive oxygen species (ros) in inflammatory tissues and cancer tissues, and specific enzymatic activity at the target sites [ , ] . in addition to these endogenous stimuli above, exogenous stimuli such as magnet, light, and ultrasound can realize real-time tracking of drug distribution, disease diagnosis and disease management. stimulus-responsive systems responsive to one single stimulus or multiple stimuli have been used to treat or diagnose a variety of diseases, such as cancers (colon cancer [ , ] , breast cancer [ , ] , lymphoma [ , ] ), inflammatory diseases (inflammatory bowel disease [ , ] , neurodegenerative diseases [ ] ), diabetes [ , ] and others. some nanomedicines that could be considered stimulus-responsive have successfully gone into clinical trials. thermodox (thermosensitive doxorubicin liposomes) used heat to release the cargo from liposome [ ] ; other successful examples include opaxio [ ] (involving on enzyme-degradable polymer), nanotherm [ ] (based on magnetic iron oxide nanoparticles), and auroshell [ ] (based on thermosensitive gold nanoshells). stimulus-responsive systems also have potential for bioimaging. they can enhance the contrast in specific diseased area in response to internal or external stimuli for real-time monitoring. imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . [ ] hydrazone int. j. mol. sci. , , x for peer review of imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . [ , ] ester int. j. mol. sci. , , x for peer review of imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . [ ] acetal imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . [ , ] cis-aconityl imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . [ ] orthoester imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . [ ] int. j. mol. sci. , , x for peer review of imine [ ] hydrazone [ , ] ester [ ] acetal [ , ] cis-aconityl [ ] orthoester [ , ] silyl ether [ ] (c) twenty minutes after mice were injected with deionized water, mg micromotors and inert polystyrene (ps) particles, superimposed fluorescent images of the whole stomach of mice were collected (both mg micromotors and ps microparticles were loaded with did dye and encapsulated in a ph-sensitive polymer shell). reproduced with permission from the publisher of corresponding reference [ ] . besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table ). disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amide-bonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on not-carcinogenic tissues were also reduced because of such rational design [ ] . besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table ) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ ] diselenium int. j. mol. sci. , , x for peer review of besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table ) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ , ] thioether selenium tellurium besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table ) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table ) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ , ] vinyldithioether int. j. mol. sci. , , x for peer review of besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems (table ) . disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems ( table ). disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ ] pba/pbe besides the development of drug delivery systems, ph-responsive systems can also be used for tumor detection and image-guided surgery [ ] . by taking advantage of the difference of ph values in different target tissues in vivo, ph-responsive systems represent an intriguing strategy in drug delivery system. however, the difference of ph values between target and healthy tissues may not always differ significantly, so responsive systems that rely only on ph may be limited by low specificity and sensitivity. therefore, ph responsive system can be combined with other stimulus conditions such as light, redox, enzymes and others with the aim of improved selectivity for drug release in diseased tissues [ , ] . redox conditions can be used for another type of stimulus-responsive system. these include gsh-sensitive systems, which have attracted much recent attention. gsh-sensitive systems can have stability in normal tissues but undergo release of cargo in diseased tissues in response to higher concentrations of gsh. for example, the concentration of gsh, as a tumor marker in cancer cells is - fold higher than in normal cells [ , ] . the intracellular concentration of gsh ( - mm) is about three orders of magnitude higher than that in the blood ( - µm) [ , ] . disulfide bonds and diselenium bonds have been widely used in the design of redox stimulus responsive systems ( table ). disulfide bonds have also been used in chemical sensors. shi et al. designed and synthesized a nanodrug carrier based on disulfide bond-doped organosilicon-micelle hybrid nanoparticles, the surface of which was modified with disulfide-bonded peg and amidebonded polyethylenimine (pei). this nano-drug carrier exhibited excellent blood circulation ability and accumulation performance in tumor tissues, and the side effects of anticancer drugs on notcarcinogenic tissues were also reduced because of such rational design [ ] . disulfide [ ] diselenium [ , ] thioether selenium tellurium [ ] [ ] [ ] [ ] [ ] [ ] oxalate ester [ , ] vinyldithioether [ ] thioketal [ ] pba/pbe [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) [ ] compared with sulfur, the electronegativity of selenium is relatively low, and the atomic radius of selenium is larger, so selenium has some unique properties such as high reactivity and sensitivity ( table ). the bond energy of s-s is kj·mol − , whereas the bond energy of se-se bond is only kj·mol − , suggesting that se-se bond may have higher sensitivity than disulfide bond; that is, se-se bond has the potential to release drug faster [ ] . wei et al. demonstrated that nanodrug carriers with diselenides could release more drugs using poly (ester carbamate) triblock copolymers (paur-se-se) with se-se bonds compared with poly (ester carbamate) triblock copolymers (paur-s-s) containing s-s bonds. at similar gsh concentrations ( mm), % of encapsulated dox was released by paur-se-se micelles within h, while % of the drug was released by paur-s-s micelles. in addition, the antitumor effect of dox-loaded paur-se-se was six fold higher than that of s-s analogues due to faster cleavage of the diselenium bond and enhanced drug release [ ] . in a recent study, a new type of redox-responsive drug delivery system was developed based on the nano-metal-organic framework. its core structure was formed by zirconium ions (zr + , metal nodes), , -disulfanylterephthalic acid (bdc-(sh) , organic ligand) and benzoic acid (ba, modulator), the sulfhydryl group on the anticancer drug, -mercaptopurine, formed a disulfide bond with bdc-(sh) . this disulfide-based nanomaterial exhibited excellent intracellular drug release ability, and its cytotoxicity to cancer cells was shown to be three times higher than normal cells [ ] . ros-responsive systems are also an effective strategy to control drug release by utilizing the ros accumulated in inflammation sites or other diseased tissues [ ] . it has been shown that the concentration of ros in inflammatory tissue is - times higher than that in normal tissue [ ] . there are two chemical mechanisms commonly used in ros-responsive systems. one is ros-induced non-breaking hydrophobic-hydrophilic transition and the other is ros-induced structural dissociation ( table ). ros can oxidize chalcogens (e.g., s, se, te) [ , ] , causing oxygen atoms to form covalent bonds with chalcogens, and polarizing groups to form hydrogen bonds with environmental water molecules. thus, hydrophobic-hydrophilic transition of the carrier backbone was induced without damaging the chemical structure of the drug carrier. among the chalcogen elements, tellurium-containing compounds have more potential than other chalcogen elements due to their lower electronegativity and lower toxicity [ ] . additionally, it has been shown that tellurium-containing compounds have higher oxidation responsiveness than selenium-containing compounds and sulfur-containing compounds [ , ] . xu et al. reported a synergistic therapeutic nanoplatform using near-infrared light-responsive cisplatin for cancer therapy. cisplatin (cddp) and indocyanine green (icg) were simultaneously loaded in nanocarriers made of amphiphilic tellurium-containing block copolymer peg-pute-peg. tellurium atoms in nanocarriers can be easily oxidized by ros (stimulated by near-infrared laser) produced by indocyanine green. tellurium oxidation weakens the coordination with cisplatin, thereby releasing cisplatin and achieving better anti-tumor effect [ ] . in addition, ros can react with chemical structures such as thioketal (tk), phenylboronic acid/ester (pba/pbe), oxalate ester [ , ] , vinyldithioether [ ] and proline oligomers, leading to the cleavage of these structures. in general, tk groups are easily oxidized to generate acetone and two other thiol-containing fragments in the presence of ros. a novel amphiphilic peptide-drug conjugate for cancer targeted therapy has recently been reported. hydrophilic cyclic peptides and hydrophobic cytotoxin epothilone b (epo b) were linked by ros responsive tk groups. when nanotherapeutics entered tumor cells, tk groups could be cleaved due to high levels of intracellular ros, thereby releasing epo b, which effectively inhibited cell proliferation [ ] . h o can specifically oxidize pbe/pba and subsequently induce the formation of borate ester and hydroxybenzyl alcohol [ ] . in addition, amino acids such as proline, histidine and arginine are also found to be susceptible to ros-mediated and metal-catalyzed oxidation [ , ] . for example, murthy et al. developed thioketal-based nanoparticles that could degrade selectively in the presence of ros in inflamed intestinal tissue. in responsive to abnormally high levels of ros in the sites of intestinal inflammation, thioketal nanoparticles could effectively deliver and release sirna to diseased sites, protecting mice from dextran sodium sulphate (dss)-induced colitis [ ] . in addition to treating inflammation, ros-responsive systems can also be used for cancer treatment. ge et al. designed ros-responsive nanoparticles made up by a thioketal linker (tl), poly (ε-caprolactone) and poly (n,n-dimethylacrylamide) (pcl-tl-pdma). amphiphilic di-block copolymers with pcl-b-poly ( -guanidinoethyl methacrylate) (pcl-pgema) were used to encapsulate the anticancer drug paclitaxel and a photosensitizer, achieving efficient cell uptake and enhanced anticancer efficacy [ ] (figure ). complexity and diversity of the microenvironment in vivo, delivery systems based on specific redox molecular mechanism that can release cargo in a controllable manner are preferred but not well established yet. further investigations are expected to address these potential issues. enzymes have attracted attention in various nanobiotechnology applications owing to their specific biological targeting and catalytic properties. in the microenvironment of the diseased sites, the levels of some enzymes can be abnormal; therefore, enzyme-responsive systems represent an appealing strategy for the development of responsive drug carriers. enzymes can specifically target the affected sites, thereby regulating drug release. hydrolases, including proteases, lipases and glycosidases, have been widely explored in enzyme-responsive systems. oxidoreductases, such as nad(p)h, quinone oxidoreductase isoenzyme i (nqo ) and glucose oxidase (gox) have also been studied [ ] . proteases play important role in biological processes, such as differentiation, angiogenesis, hormone synthesis, digestion, wound repair, hemostasis, inflammation, coagulation, immune response, necrosis and apoptosis [ ] . some proteases are usually overexpressed in diseased tissues, and their activation can be used to release drugs for the development of protease-targeted therapies. cell uptake under nm light irradiation was improved. a synergistic effect of photodynamic therapy (pdt) and chemotherapy was achieved through efficient cell uptake and drug release. reproduced with permission from the publisher of corresponding reference [ ] . redox stimulus-responsive systems need high sensitivity and accuracy for fast triggered responses in diseased tissues, but stability under normal conditions. however, considering the complexity and diversity of the microenvironment in vivo, delivery systems based on specific redox molecular mechanism that can release cargo in a controllable manner are preferred but not well established yet. further investigations are expected to address these potential issues. enzymes have attracted attention in various nanobiotechnology applications owing to their specific biological targeting and catalytic properties. in the microenvironment of the diseased sites, the levels of some enzymes can be abnormal; therefore, enzyme-responsive systems represent an appealing strategy for the development of responsive drug carriers. enzymes can specifically target the affected sites, thereby regulating drug release. hydrolases, including proteases, lipases and glycosidases, have been widely explored in enzyme-responsive systems. oxidoreductases, such as nad(p)h, quinone oxidoreductase isoenzyme i (nqo ) and glucose oxidase (gox) have also been studied [ ] . proteases play important role in biological processes, such as differentiation, angiogenesis, hormone synthesis, digestion, wound repair, hemostasis, inflammation, coagulation, immune response, necrosis and apoptosis [ ] . some proteases are usually overexpressed in diseased tissues, and their activation can be used to release drugs for the development of protease-targeted therapies. for example, a new type of polymer poly (ethylene glycol) diacrylate (pegda) have been designed that could form spherical polymeric nanoparticles when mixed with peptide. the nanoparticles can be hydrolyzed by the overexpressed matrix metalloproteinase in lung lesions and then release the drugs [ ] . in addition, it has been shown that the use of protease triggered drug release can enhance the therapeutic effect of drugs, and also reduce the toxicity and other side effects of drugs [ ] . cathepsin-b is normally expressed in the lysosomes of human cells, but is also overexpressed in many invasive and metastatic cancers. therefore, cathepsin-b has been used as one of the targets of the enzyme-responsive systems. the amino acid sequence of gly-phe-leu-gly (gflg) can be degraded in the presence of cathepsin-b, so it can be applied for the development of nanodrug carriers. for example, peg, anticancer drug cisplatin and gflg peptide were used to be assembled for cisplatin prodrug units. icg was used to co-assemble with cathepsin-responsive cisplatin multidrug nanoplatforms. after cell uptake, the gflg peptide structure was degraded by cathepsin b in lysosomes, which in turn released icg and cisplatin prodrugs for cancer treatment [ ] . among many kinds of matrix metalloproteinases, mmp has been one of the most widely explored for stimulus-responsive systems because it is overexpressed in cancer tissues. mmp -acting substrates have been used for drug delivery and molecular imaging. for example, torchilin et al. synthesized an octapeptide (gly-pro-leu-gly-ile-ala-gly-gln) as a mmp -sensitive linker for the conjugation of long-chain peg to liposomes and encapsulation of the cell-penetrating peptide tatp. when octapeptide (gplgiagq) was cleaved by mmp , tatp was exposed, giving rise to the increased uptake of liposome particles by tumor cells [ ] . in , similar structures were also used in drug-loaded liposomes for pancreatic cancer treatment [ ] . to improve permeability, antitumor efficacy and biodegradability, a size-shrinkable drug delivery system has been designed based on polysaccharide-modified dendrimers, which contained poly (amidoamine), hyaluronic acid and mmp -sensitive peptide linker (plglag). the system retained good stability in blood circulation, and underwent degradation upon the action of mmp , leading to enhanced uptake in tumor cells [ ] . in addition, polypeptide gplgvrg [ , ] , polypeptide gplgvrgdg [ ] and triglyceride monostearate (tgms) [ ] have also been synthesized for mmp sensitive drug delivery systems. phospholipase is another therapeutic target that can be overexpressed in infectious diseases, inflammatory diseases and tumors. in several inflammatory diseases or cancer cell types, the expression of phospholipase a (spla ) has been shown to be higher [ ] [ ] [ ] [ ] . pla is often used as a trigger condition in liposomal drug delivery systems, which specifically hydrolyzes sn- ester bonds in phospholipids, resulting in direct release of active drugs or hydrolysis of liposomes [ ] . for example, colchicine-containing phosphatidylcholinase-responsive liposomes have been developed that are stable in human blood circulation. at the same time, liposomes could release colchicine-containing fatty acids upon the action of high levels of phospholipase a . the latter further hydrolyzed and released colchicine analogues by non-specific enzymes [ ] . besides, in a recent report, an enzyme reactive liposome containing , -dipalmitoyl-sn-glycerin- -phosphocholine (dppc) and -o-stearyl- -retinoic acid receptor (rar)-c -sn-glycerin- -phosphoglycerol with c -rar as a prodrug was designed. in the absence of spla , the ic of c -rar prodrug in mt- breast cancer cell line was µm, while the ic of c -rar prodrug decreased to µm after adding spla , indicating that the prodrug was hydrolyzed into c -rar and lyso-o-spg by spla , in response to phospholipase [ ] . oxidoreductase has also been used as a potential target of stimulus-responsive systems due to its important role in intracellular oxidative environment. gox is a kind of oxidoreductase tested for stimulus-response systems. it catalyzes the oxidation of β-d-glucose to d-glucose-δ-lactone and hydrogen peroxide with oxygen as electron acceptor [ , ] . glucose responsive systems have been assessed for the treatment of diabetes [ , ] . for example, willner et al. recently used zeolite imidazolium frameworks (zif- nmofs) as a glucose-responsive carrier to release insulin and vascular endothelial growth factor (vegf) aptamers, which inhibited angiogenesis by binding to vegf proteins. zif- was stable under neutral physiological conditions but degraded under acidic conditions. insulin or vegf aptamers and gox were integrated into zif- nmofs as biocatalysts. gox catalyzed the aerobic oxidation of glucose to produce gluconic acid. local acidification of nmofs degraded zif- , thereby releasing insulin and vegf aptamers [ ] . besides, nqo has been widely used in stimulus-responsive therapy systems due to its overexpression in tumors and other diseases [ , ] . in a recent report, a nqo -responsive multifunctional polymeric vesicle covalently bound to a photosensitizer (coumarin and nile blue) was prepared. without being triggered by nqo , both fluorescence emission and photodynamic therapy (pdt) capability were turned off due to the "double quenching" effect. upon cellular uptake, highly expressed nqo triggered the self-immolative cleavage of the quinone trimethyl lock, which then led to the release of photosensitizers, near infrared (nir) emission and pdt activation, thereby realizing real-time monitoring and treatment of cancers [ ] . glucosidase is an enzyme that can hydrolyze glycosidic bonds. its concentration in diseased tissues can be higher than that in normal tissues. therefore, glucosidase can be used to design enzyme-responsive system. it was reported that the concentration of α-amylase could increase by -fold in the tumor environment [ ] , so it is an effective anticancer therapeutic approach to design glycosylated drug carriers to release anticancer drugs upon catalysis of glycosidase. for example, scanlan et al. used glycosylated , -naphthalimide as a fluorescent probe for tumor treatment and diagnosis. the glycosylated -amino- , -naphthalimide derivatives with chemical structures including the glycosidic bond can be selectively hydrolyzed by glycosidase to release naphthalimide. it was shown that naphthalimide was uptaken by cells only after the glycan unit was hydrolyzed, indicating high targeting capability of the delivery system [ ] (figure ) . nmofs degraded zif- , thereby releasing insulin and vegf aptamers [ ] . besides, nqo has been widely used in stimulus-responsive therapy systems due to its overexpression in tumors and other diseases [ , ] . in a recent report, a nqo -responsive multifunctional polymeric vesicle covalently bound to a photosensitizer (coumarin and nile blue) was prepared. without being triggered by nqo , both fluorescence emission and photodynamic therapy (pdt) capability were turned off due to the "double quenching" effect. upon cellular uptake, highly expressed nqo triggered the selfimmolative cleavage of the quinone trimethyl lock, which then led to the release of photosensitizers, near infrared (nir) emission and pdt activation, thereby realizing real-time monitoring and treatment of cancers [ ] . glucosidase is an enzyme that can hydrolyze glycosidic bonds. its concentration in diseased tissues can be higher than that in normal tissues. therefore, glucosidase can be used to design enzyme-responsive system. it was reported that the concentration of α-amylase could increase by fold in the tumor environment [ ] , so it is an effective anticancer therapeutic approach to design glycosylated drug carriers to release anticancer drugs upon catalysis of glycosidase. for example, scanlan et al. used glycosylated , -naphthalimide as a fluorescent probe for tumor treatment and diagnosis. the glycosylated -amino- , -naphthalimide derivatives with chemical structures including the glycosidic bond can be selectively hydrolyzed by glycosidase to release naphthalimide. it was shown that naphthalimide was uptaken by cells only after the glycan unit was hydrolyzed, indicating high targeting capability of the delivery system [ ] (figure ). in spite of extensive development of enzyme-responsive systems, there are many drawbacks for them. the level of enzyme expression might be different in different patients, so whether they are sufficiently expressed in a target population is questionable. in addition, the specificity might be another issue. for example, various types of mmps have cross-reactivity. another consideration is whether the cleavage of the enzyme-responsive substrates and polymers is feasible in complex biological environment. the therapeutic effect of one single enzyme stimulus-responsive system might not be specific enough. in spite of extensive development of enzyme-responsive systems, there are many drawbacks for them. the level of enzyme expression might be different in different patients, so whether they are sufficiently expressed in a target population is questionable. in addition, the specificity might be another issue. for example, various types of mmps have cross-reactivity. another consideration is whether the cleavage of the enzyme-responsive substrates and polymers is feasible in complex biological environment. the therapeutic effect of one single enzyme stimulus-responsive system might not be specific enough. solid tumors have a variety of physiological barriers (such as high interstitial pressure and dense extracellular matrix) which affect the uptake of nanoparticles [ ] . photothermal therapy is a minimally-invasive treatment for cancer, which relies on the thermal stress caused by light irradiation at a specific wavelength [ , ] . pdt is another commonly used light-triggered strategy for disease treatments. some examples of photosensitizers include photofrin, visudyne, chlorin e and oligo (p-phenylene vinylene) derivative (opv). they can be activated by light to produce ros, which can not only directly kill cancer cells, but also induce vascular damage, cause membrane oxidation and affect the permeability of cell membrane, which facilitates the penetration of anticancer drugs [ ] . in a recent report, researchers designed a nano drug carrier that combines photosensitizer protoporphyrin, chemical drug doxorubicin (dox) and apatite (apa). with light stimulation, dox and apa were released and ros was generated owing to porphyrins. apa competed with p-glycoprotein (p-gp) transporter to reduce its enzyme catalytic activity and dox was used to treat tumor tissues; thus, the synergistic treatment of chemotherapy and phototherapy was achieved [ ] (figure ). solid tumors have a variety of physiological barriers (such as high interstitial pressure and dense extracellular matrix) which affect the uptake of nanoparticles [ ] . photothermal therapy is a minimally-invasive treatment for cancer, which relies on the thermal stress caused by light irradiation at a specific wavelength [ , ] . pdt is another commonly used light-triggered strategy for disease treatments. some examples of photosensitizers include photofrin, visudyne, chlorin e and oligo (pphenylene vinylene) derivative (opv). they can be activated by light to produce ros, which can not only directly kill cancer cells, but also induce vascular damage, cause membrane oxidation and affect the permeability of cell membrane, which facilitates the penetration of anticancer drugs [ ] . in a recent report, researchers designed a nano drug carrier that combines photosensitizer protoporphyrin, chemical drug doxorubicin (dox) and apatite (apa). with light stimulation, dox and apa were released and ros was generated owing to porphyrins. apa competed with pglycoprotein (p-gp) transporter to reduce its enzyme catalytic activity and dox was used to treat tumor tissues; thus, the synergistic treatment of chemotherapy and phototherapy was achieved [ ] ( figure ). the mechanism of smart acp-dox + apa micelles against multidrug resistance (mdr), for promoting the synergistic antitumor efficacy. reproduced with permission from the publisher of corresponding reference [ ] . the mechanism of smart acp-dox + apa micelles against multidrug resistance (mdr), for promoting the synergistic antitumor efficacy. reproduced with permission from the publisher of corresponding reference [ ] . in addition to photothermal therapy and pdt, light-responsive strategies have also been applied in the design of prodrug systems and drug delivery carriers. light-responsive structure is generally a robust strategy because it is easy to control with good accuracy and minimal invasiveness. light-responsive systems for drug release are often based on the cleavage of prodrugs with light-sensitive structures or the changes of photosensitive molecules upon light stimulation, so as to release the conjugated or encapsulated drugs [ , ] . lovell et al. developed near-infrared light-responsive liposomes doped with hexyloxyethy-pyropheophorbide (hpph) entrapped into the bilayers. the anticancer drug doxorubicin (dox) was encapsulated inside the light-sensitive liposomes. when nir light was irradiated at the target site, hpph-liposomes opened the bilayer structure and then released dox, but when nir light was off, the stable bilayer structure of hpph-liposomes was reformed [ ] . common photolytic functional groups include o-nitrobenzyl derivatives (onb), anthracene [ ] , coumarin esters [ ] , arylmethyl [ ] and pyrenylmethyl ester [ ] (table ) . among these, o-nitrobenzyl derivatives have been widely used because of their fast photolysis rate and simple synthesis process. for example, willner et al. designed a light-responsive and ph-responsive dna microcapsule. light-responsive capsules are assembled by o-nitrobenzyl phosphate groups with dna layers used to stabilize the microcapsule shell (table ) . when stimulated by light, o-nitrobenzyl groups were cleaved, causing the degradation of microcapsules and the release of the cargo [ ] . another report showed that the o-nitrobenzyl linker containing carbamate bond had a better photolysis rate and a slower hydrolysis rate than other o-nitrobenzyl derivatives (containing an ester bond or amide bond), so that it could maintain higher stability in vivo and can degrade faster after light stimulation [ ] . besides, baker et al. chose onb group as the core of photosensitive protective group and conjugated with poly (amidoamine) (pamam) dendrimer to develop a drug delivery platform for methotrexate (mtx) loading. it was demonstrated that this photochemical approach can be used for in vivo delivery of anticancer drugs [ ] . ultraviolet-visible (uv-vis) light could be used for the trigger of o-nitrobenzyl derivatives and aryl methyl photolysis functional groups; however, it is difficult to achieve tissue penetration for better therapeutic performance. compared with uv and visible light, near-infrared light with wavelengths in the - nm range can penetrate into deeper tissues. nir could be used to cleave, isomerize or rearrange molecules through the two-photon absorption (tpa) process and nir-uv upconversion (uc) process. the theoretical analysis on the principle of tpa process was first proposed in s [ ] . however, the generation of tpa requires high excitation energy, and there are still some problems in practical applications. in the uc process, the luminescence center can absorb the energy of two or more photons, and then generate one emitted photon whose energy is higher than that of a single excited photon. additionally, unlike the tpa process, each process of photon absorption is like a second-order element reaction due to the existence of a real intermediate state in the uc process, which has a higher probability than the simultaneous absorption of two molecules [ , ] . upconversion nanoparticles (ucnps) based on rare earth ions provide a new method for the nir light-responsive system. for example, zou and his colleagues designed a near-infrared light-triggered nanocomposite peg-nmab-pla-ucnps (pnp-uc) to achieve photo-controllable release of dox for cancer treatment. under the irradiation of nm nir light, -nitrobenzyl group was triggered for its cleavage to release dox. compared with other ucnps-based nir responsive systems, this new nanocomposite has many advantages, such as high drug encapsulation efficiency, fast light responsiveness at low power density and less heating effect, showing potential in pharmaceutical and biomedical applications [ ] . common photoisomerization materials include azobenzene-based materials, spiropyran (sp)-based materials, and diarylethene-based materials (table ) . azobenzene is an azo group containing an aryl group, with cis and trans isomers. it shows a significant π-π transition in the uv region and a faint π-π transition in the visible region. therefore, upon irradiation of uv light, the trans structure of azobenzene changes into cis structure. additionally with heat or visible light, azobenzene can change from cis structure to trans structure [ ] . liu and colleagues assembled azobenzene-functionalized dna strands using ucnp to construct nano-pumps and load dox into nano-pumps. with nm nir irradiation, the continuous rotational inversion motion of azo molecules caused dna hybridization and de-hybridization, thereby leading to the release of dox. this novel light-responsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ring-closed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] spiropyan [ ] [ ] coumarinyl ester dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] spiropyan [ ] [ ] arylmethyl dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] spiropyan [ ] [ ] pyrenylmethyl ester dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] spiropyan [ ] [ ] o-nitrobenzyl dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] spiropyan [ ] [ dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] spiropyan [ ] [ dna hybridization and de-hybridization, thereby leading to the release of dox. this novel lightresponsive drug delivery system has shown good biocompatibility and excellent performance in both in vitro and in vivo therapies [ ] . sps are an important photoisomeric compounds. under the uv light, the spiro-c-o bond in the spiropyran structure breaks open, leading to an increase in the degree of pi-electron conjugation of the whole molecular system, forming a long conjugate chain and the colored ring-opened merocyanine (mc). mc can be reversibly transformed into a colorless ringclosed sp under specific wavelength or heating conditions. taking the advantage of this special property of sp, a novel n,n-bis(acryloyl) cystamine crosslinked poly (acrylic acid-co-spiropyran methacrylate) nanogel containing disulfide bonds was recently reported as a multi-stimulus responsive nanocarrier [ ] . upon the stimulation of low ph value or uv light, the hydrophobic sp isomerizes into hydrophilic mc, making the nanogel swollen. with the addition of reductant agent, the structure of nanogel was destroyed after disulfide bond was oxidized and cleaved, which caused the release of the loaded drug. cytotoxicity experiments showed that dox-loaded nanogels could effectively kill cancer cells, while their cytotoxicity could be enhanced by uv light irradiation. in addition, isomerized mcs in nanogels could emit strong green light after illumination, so they could also be used for fluorescent cell imaging. anthracene [ ] coumarinyl ester [ ] arylmethyl [ ] pyrenylmethyl ester [ ] o-nitrobenzyl [ ] [ ] [ ] azobenzene [ , ] diarylethene [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] diarylethenes (daes) are also popular photoisomeric molecules with many useful properties, such as easy modification, fatigue resistance, high thermal stability and significant changes in optical and electronic properties after photoisomerization [ ] ( table ). the performance and application of diaryl ethylene mainly depend on the modification of its parental structure, that is, the design and selection process of alkene bridges and aryl units. one common way is the modification of aryl units such as substituting the benzene ring of diaryl ethylene with thiophene ring [ ] (which could improve the stability of the closed loop) and modifying the alkene bridge using five-membered rings [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] diarylethenes (daes) are also popular photoisomeric molecules with many useful properties, such as easy modification, fatigue resistance, high thermal stability and significant changes in optical and electronic properties after photoisomerization [ ] (table ) . the performance and application of diaryl ethylene mainly depend on the modification of its parental structure, that is, the design and selection process of alkene bridges and aryl units. one common way is the modification of aryl units such as substituting the benzene ring of diaryl ethylene with thiophene ring [ ] (which could improve the stability of the closed loop) and modifying the alkene bridge using five-membered rings such as alkene bridges (such as cyclopentene [ ] , thiazole [ ] , imidazole [ ] , furan [ ] ) or using six-membered rings (such as benzoquinone [ ] , coumarin fluorophores [ ] , benzothiazole [ ] ). in addition, some light-sensitive molecules-vitamin b derivatives [ , ] , ruthenium complexes [ ] , etc.-have also been applied to light-sensitive systems. still, further development of light-stimulated systems is hindered by some factors. for example, for the treatment of solid tumors, penetration depth, effective area, power intensity and irradiation time are some important factors to be considered for the light-responsive therapy. in addition, many photosensitizers and photothermal agents have inherent toxicity or photo-toxicity. ongoing research is still making efforts for the application of light stimulation systems for clinical advances. temperature is a frequently studied stimuli for responsive delivery systems. inflammatory pathological sites and the hyperthermic nature of tumors can be used as internal stimuli [ , ] . another strategy is to increase the temperature by applying an external heat source. ideally, the thermal response nanocarrier should maintain its stability at body temperature and release the drug rapidly when the diseased site is heated. thermal stimulus-responsive polymers usually have lower critical solubility temperature (lcst) or upper critical solubility temperature (ucst), and the former is more widely used. for example, poly (n-isopropylacrylamide) (pnipam) and its derivatives have been used in the design of thermal stimulation response systems because their corresponding lcst is about • c, which is close to the physiological temperature of human body [ ] [ ] [ ] . a core-shell drug delivery system was designed to improve the solubility of drug by encapsulating hydrophobic drugs in nanoparticles with thermo-responsive materials as the shell. luo et al. developed a core-shell micelle that used hydrophilic thermo-responsive material pnipam as a shell for the encapsulation of the drug of paclitaxel (ptx). both drug and shell were conjugated by diselenide bond (pnipam-sese-ptx). the nanoparticles exhibited dual characteristics of temperature-responsiveness and redox-responsiveness, with high loading capacity ( . %) and encapsulation efficiency ( . %) [ ] . compared with pnipam that cannot be degraded in vivo, temperature-sensitive oligomers containing oligo (ethylene glycol) (oeg) potentially has better biocompatibility. jayakannan et al. developed a dual stimulus-responsive amphiphilic copolymer formed by copolymerization of hydrophobic polymerizable monomers with methacrylamide based on hydrophobic -pentadecylphenol (pdp) and oligopolyethylene glycol (peg) methacrylate, which thus was temperature sensitive and enzyme sensitive. the nanoparticles formed from the amphiphilic copolymer, which remained stable in normal tissues in vivo with less than % drug release, while above lcst, the nanoparticles had an up to % drug release rate in two hours due to the thermal response property of acrylamide copolymers. in the presence of esterase, the drug release rate could exceed % within h. such type of dox-polymer showed better anticancer efficacy [ ] . in addition to thermosensitive hydrogels and thermosensitive polymers, temperature-sensitive liposomes have also been investigated. since yatvin first proposed the concept of temperature-sensitive liposomes (tsl) in the late s [ ] , tsl has been attracting much attention from molecular design to clinical applications. thermodox is an example of temperature-sensitive liposomes, which was tested in phase iii clinical trials for hepatocellular carcinoma and phase ii clinical trials for breast cancer. its drug release rate is - fold higher than other liposomal drugs at elevated temperatures. porter et al. developed a novel polymer-modified temperature-sensitive liposome (ptsl) for the delivery of dox for the treatment of cancers. by reversible addition-break chain transfer (raft) polymerization of n-isopropylacrylamide (nipaam) and ph-responsive propylacrylic acid (paa) copolymers with temperature responsiveness, copolymers with dual ph/temperature phase transition properties were obtained. when attached to liposomes, these copolymers can cause membrane destruction with a ph/temperature-dependent manner. compared with traditional thermosensitive formulations, ptsl exhibited enhanced release profiles, significantly reduced thermal dose thresholds and better stability in serum with minimal drug leakage over time. therefore, these liposomes have the potential of significantly reducing damage to healthy tissue commonly associated with liposomal cancer therapies [ ] (figure ). was tested in phase iii clinical trials for hepatocellular carcinoma and phase ii clinical trials for breast cancer. its drug release rate is - fold higher than other liposomal drugs at elevated temperatures. porter et al. developed a novel polymer-modified temperature-sensitive liposome (ptsl) for the delivery of dox for the treatment of cancers. by reversible addition-break chain transfer (raft) polymerization of n-isopropylacrylamide (nipaam) and ph-responsive propylacrylic acid (paa) copolymers with temperature responsiveness, copolymers with dual ph/temperature phase transition properties were obtained. when attached to liposomes, these copolymers can cause membrane destruction with a ph/temperature-dependent manner. compared with traditional thermosensitive formulations, ptsl exhibited enhanced release profiles, significantly reduced thermal dose thresholds and better stability in serum with minimal drug leakage over time. therefore, these liposomes have the potential of significantly reducing damage to healthy tissue commonly associated with liposomal cancer therapies [ ] (figure ). for temperature-sensitive systems, thermo-specificity is one of the biggest issues because it is usually hard for chemistry to be exquisitely specific. for the future research directions, temperatureresponsive drug carriers with higher sensitivity to the minimum temperature change, better stability and enhanced safety profile in normal tissues are greatly desired. compared with other stimuli such as light radiation and ultrasonic, magnetic force is an intriguing condition for external stimuli-responsiveness because it has almost no physical interaction with the human body. additionally, owing to spatial focusing, magnetic stimulus-responsive systems can overcome some limitations of traditional delivery systems, such as difficulty of passing through physiological barrier in vivo and lack of specificity to diseased tissues. magnetic fields was first proposed as external triggers for drug release in [ ] . magnetic hyperthermia caused by the rotation of magnetic nanoparticles when exposed to alternating magnetic field, and the heat generated by magnetic loss will dissipate to the surrounding tissues. it is generally believed that the heat generated by magnetic nanoparticles is based on mechanisms of internal rotation axis and external movement, namely, thermal rotation and diffusion relaxation of magnetic moment. in terms of local drug release, the thermodynamic phase and conformation transition of polymeric for temperature-sensitive systems, thermo-specificity is one of the biggest issues because it is usually hard for chemistry to be exquisitely specific. for the future research directions, temperature-responsive drug carriers with higher sensitivity to the minimum temperature change, better stability and enhanced safety profile in normal tissues are greatly desired. compared with other stimuli such as light radiation and ultrasonic, magnetic force is an intriguing condition for external stimuli-responsiveness because it has almost no physical interaction with the human body. additionally, owing to spatial focusing, magnetic stimulus-responsive systems can overcome some limitations of traditional delivery systems, such as difficulty of passing through physiological barrier in vivo and lack of specificity to diseased tissues. magnetic fields was first proposed as external triggers for drug release in [ ] . magnetic hyperthermia caused by the rotation of magnetic nanoparticles when exposed to alternating magnetic field, and the heat generated by magnetic loss will dissipate to the surrounding tissues. it is generally believed that the heat generated by magnetic nanoparticles is based on mechanisms of internal rotation axis and external movement, namely, thermal rotation and diffusion relaxation of magnetic moment. in terms of local drug release, the thermodynamic phase and conformation transition of polymeric nanoparticles depends on their lcst/ucst and then expands/contracts, possibly leading to the drug release [ , ] . zink et al. developed a magnetic nanoparticle with superparamagnetic particles as the core and thermo-responsive peptide phe-phe-glycine-glycine (n-porcine linolenic acid a) as the nano-valve. this novel class of mesoporous silica nanoparticles (msns) could maintain stability and safety under normal environment in human body. once the magnetic core is triggered by magnetic field, the heat generated can lead to the change of its structure, giving rise to the release of drugs [ ] (figure ) . nanoparticles depends on their lcst/ucst and then expands/contracts, possibly leading to the drug release [ , ] . zink et al. developed a magnetic nanoparticle with superparamagnetic particles as the core and thermo-responsive peptide phe-phe-glycine-glycine (n-porcine linolenic acid a) as the nano-valve. this novel class of mesoporous silica nanoparticles (msns) could maintain stability and safety under normal environment in human body. once the magnetic core is triggered by magnetic field, the heat generated can lead to the change of its structure, giving rise to the release of drugs [ ] ( figure ). magnetic transfection or magnetically-aided transfection is a method to control gene delivery and improve transfection efficiency for gene therapy. the magnetic transfection method is based on the principle of magnetic targeted drug delivery proposed by widder et al. in [ ] . in , mah et al. first linked the magnetic microspheres to recombinant adeno-associated virus (aav ) gene vector through heparin for c s cells and mice in vivo [ ] . in the same year, plank et al. associated gene carriers with superparamagnetic iron oxide nanoparticles coated with polycation polyethyleneimine. it was shown that the magnetic-assisted transfection method improved the transfection efficiency of vectors by at least three orders of magnitude by either viral or non-viral vectors [ ] . ultrasound is also commonly used in stimulus responsive system. compared with other external stimuli, ultrasound is non-invasive and can improve drug release profile and drug permeability. the mechanism is based on acoustic cavitation, which means the formation and activity of gas-filled bubbles in the medium under the action of ultrasound [ ] . these gas-filled bubbles can be produced either naturally or manually (microbubbles, mbs). with microbubbles, ultrasound cavitation affects the permeability of the plasma membrane, enhances cell uptake of drugs, or allows internalization of other cell-impermeable substances. even in the absence of microbubbles, highintensity ultrasound can still induce drug uptake by cells. these characteristics enable ultrasoundmediated drug delivery systems to improve the absorption efficiency of weak or non-permeable drugs by cells [ ] . porous lipid polymer hybrid microbubbles (lipid/plga mbs) have also been developed using water/oil/water (w/o/w) double emulsification process, which addressed the limitation of low drug encapsulation efficiency of lipid-based mbs and poor ultrasound imaging ability of polymer-based mbs. compared with pure plga mbs, lipid/plga mbs had hollow microcapsule structure, which reduced the cavitation threshold intensity and enhanced the ultrasonic imaging ability of mbs. in addition, the increased surface area and porous structure of lipid/plga mbs make them have good drug encapsulation properties. using this method, controllable drug release and real-time monitoring of drug release could be achieved [ ] . another example is that zheng et al. developed ultrasound stimulus-responsive microbubbles for ultrasound imaging, which magnetic transfection or magnetically-aided transfection is a method to control gene delivery and improve transfection efficiency for gene therapy. the magnetic transfection method is based on the principle of magnetic targeted drug delivery proposed by widder et al. in [ ] . in , mah et al. first linked the magnetic microspheres to recombinant adeno-associated virus (aav ) gene vector through heparin for c s cells and mice in vivo [ ] . in the same year, plank et al. associated gene carriers with superparamagnetic iron oxide nanoparticles coated with polycation polyethyleneimine. it was shown that the magnetic-assisted transfection method improved the transfection efficiency of vectors by at least three orders of magnitude by either viral or non-viral vectors [ ] . ultrasound is also commonly used in stimulus responsive system. compared with other external stimuli, ultrasound is non-invasive and can improve drug release profile and drug permeability. the mechanism is based on acoustic cavitation, which means the formation and activity of gas-filled bubbles in the medium under the action of ultrasound [ ] . these gas-filled bubbles can be produced either naturally or manually (microbubbles, mbs). with microbubbles, ultrasound cavitation affects the permeability of the plasma membrane, enhances cell uptake of drugs, or allows internalization of other cell-impermeable substances. even in the absence of microbubbles, high-intensity ultrasound can still induce drug uptake by cells. these characteristics enable ultrasound-mediated drug delivery systems to improve the absorption efficiency of weak or non-permeable drugs by cells [ ] . porous lipid polymer hybrid microbubbles (lipid/plga mbs) have also been developed using water/oil/water (w/o/w) double emulsification process, which addressed the limitation of low drug encapsulation efficiency of lipid-based mbs and poor ultrasound imaging ability of polymer-based mbs. compared with pure plga mbs, lipid/plga mbs had hollow microcapsule structure, which reduced the cavitation threshold intensity and enhanced the ultrasonic imaging ability of mbs. in addition, the increased surface area and porous structure of lipid/plga mbs make them have good drug encapsulation properties. using this method, controllable drug release and real-time monitoring of drug release could be achieved [ ] . another example is that zheng et al. developed ultrasound stimulus-responsive microbubbles for ultrasound imaging, which can be converted into porphyrin nanomaterials with fluorescent and photoacoustic activity upon low-frequency ultrasound pulses. larger microcarriers could reach the tumor vasculature (independent of epr effect), and its principle is that the energy of ultrasound could rupture microbubbles, pushing nanomaterials into the tumor interstitium. these nanomaterials (porphyrins) could further utilize photoacoustic imaging (pai) and fluorescence for multistep multimodal imaging in tumor-bearing mice [ ] . liu et al. recently designed an ultrasound responsive photoacoustic (pa) probe based on microbubbles containing gold nanoparticles. in this design, gold nanoparticles were encapsulated in the lipid shell of mbs and filled with sulfur hexafluoride gas, thereby forming mbs with strong cavitation characteristics and low toxicity. au@lip mbs exhibited lower nir pa signal. when triggered by ultrasound, mbs was ruptured and au@lip aggregates were formed, showing enhanced nir pa signals. background-free pa imaging was achieved by subtracting the pa image before us stimulation from the pa image after us stimulation [ ] (figure ). although ultrasound-responsive nanotechnology shows some advantages, its side effects (such as skin irritations, transient pain and nerve injury) should be also considered [ , ] . the intensity, frequency and duty cycle should be carefully selected for the optimized therapeutic effects and reduced side effects. can be converted into porphyrin nanomaterials with fluorescent and photoacoustic activity upon low-frequency ultrasound pulses. larger microcarriers could reach the tumor vasculature (independent of epr effect), and its principle is that the energy of ultrasound could rupture microbubbles, pushing nanomaterials into the tumor interstitium. these nanomaterials (porphyrins) could further utilize photoacoustic imaging (pai) and fluorescence for multistep multimodal imaging in tumor-bearing mice [ ] . liu et al. recently designed an ultrasound responsive photoacoustic (pa) probe based on microbubbles containing gold nanoparticles. in this design, gold nanoparticles were encapsulated in the lipid shell of mbs and filled with sulfur hexafluoride gas, thereby forming mbs with strong cavitation characteristics and low toxicity. au@lip mbs exhibited lower nir pa signal. when triggered by ultrasound, mbs was ruptured and au@lip aggregates were formed, showing enhanced nir pa signals. background-free pa imaging was achieved by subtracting the pa image before us stimulation from the pa image after us stimulation [ ] (figure ). although ultrasound-responsive nanotechnology shows some advantages, its side effects (such as skin irritations, transient pain and nerve injury) should be also considered [ , ] . the intensity, frequency and duty cycle should be carefully selected for the optimized therapeutic effects and reduced side effects. the mechanism of self-immolation is that two chemical bonds in the inactive precursor are associated by the self-immolation spacer. the precursor usually contains a protective group (pg), self-immolation spacer (sis) and a target compound (tc). after appropriate stimulus, the protective group is removed, and then a step-by-step decomposition reaction similar to "domino" is produced, resulting in the release of target compounds (figure ). philip et al. proposed the concept of selfimmolative structures in that a pristine drug can be released without chemical modification after the bond between the carrier component and the drug component is cleaved [ ] . self-immolative linkers have subsequently been widely used in prodrug design [ , ] , sensors [ , ] , drug delivery [ , ] and other applications. the mechanism of self-immolation is that two chemical bonds in the inactive precursor are associated by the self-immolation spacer. the precursor usually contains a protective group (pg), self-immolation spacer (sis) and a target compound (tc). after appropriate stimulus, the protective group is removed, and then a step-by-step decomposition reaction similar to "domino" is produced, resulting in the release of target compounds (figure ). philip et al. proposed the concept of self-immolative structures in that a pristine drug can be released without chemical modification after the bond between the carrier component and the drug component is cleaved [ ] . self-immolative linkers have subsequently been widely used in prodrug design [ , ] , sensors [ , ] , drug delivery [ , ] and other applications. can be converted into porphyrin nanomaterials with fluorescent and photoacoustic activity upon low-frequency ultrasound pulses. larger microcarriers could reach the tumor vasculature (independent of epr effect), and its principle is that the energy of ultrasound could rupture microbubbles, pushing nanomaterials into the tumor interstitium. these nanomaterials (porphyrins) could further utilize photoacoustic imaging (pai) and fluorescence for multistep multimodal imaging in tumor-bearing mice [ ] . liu et al. recently designed an ultrasound responsive photoacoustic (pa) probe based on microbubbles containing gold nanoparticles. in this design, gold nanoparticles were encapsulated in the lipid shell of mbs and filled with sulfur hexafluoride gas, thereby forming mbs with strong cavitation characteristics and low toxicity. au@lip mbs exhibited lower nir pa signal. when triggered by ultrasound, mbs was ruptured and au@lip aggregates were formed, showing enhanced nir pa signals. background-free pa imaging was achieved by subtracting the pa image before us stimulation from the pa image after us stimulation [ ] (figure ). although ultrasound-responsive nanotechnology shows some advantages, its side effects (such as skin irritations, transient pain and nerve injury) should be also considered [ , ] . the intensity, frequency and duty cycle should be carefully selected for the optimized therapeutic effects and reduced side effects. the mechanism of self-immolation is that two chemical bonds in the inactive precursor are associated by the self-immolation spacer. the precursor usually contains a protective group (pg), self-immolation spacer (sis) and a target compound (tc). after appropriate stimulus, the protective group is removed, and then a step-by-step decomposition reaction similar to "domino" is produced, resulting in the release of target compounds (figure ). philip et al. proposed the concept of selfimmolative structures in that a pristine drug can be released without chemical modification after the bond between the carrier component and the drug component is cleaved [ ] . self-immolative linkers have subsequently been widely used in prodrug design [ , ] , sensors [ , ] , drug delivery [ , ] and other applications. the self-immolation mechanism can be categorized into electron rearrangement and intramolecular cyclization. electron rearrangement is mainly based on the structure of quinone or its derivatives (such as thioquinone methide or azaquinone methide). the mechanism of electron rearrangement includes , -elimination, , -elimination, , -elimination and β-elimination. the electron rearrangement reaction usually contain aromatic ring structure with hydroxyl [ , ] , amino [ , ] or mercaptan groups [ , ] . when they are masked by protective groups, the electron rearrangement process of these functional groups is inhibited. when the external stimulus triggers the cleavage of the protective groups, these functional groups undergo irreversible self-immolation process driven by positive entropy or the generation of stable products. common protection groups that can be triggered by various stimuli are summarized in table . self-immolative spacers based on electron rearrangement are mostly based on , -elimination or , -elimination [ , ] . additionally, , -cleavage may occur in para amino (or hydroxy) cinnamyl alcohol or coumarin alcohol. in contrast, self-immolative spacer groups based on continuous combinations (such as , -elimination of naphthalene or , -elimination of biphenyls) usually do not result in the release of drug groups because the high energy barrier destroys the aromaticity, and the repulsion of the hydrogen atom in adjacent biphenyl prevents the formation of the planar structure needed for electron rearrangement [ ] . the cyclization reaction is based on alkyl chain or aromatic spacer groups [ ] [ ] [ ] . once the cleavage is triggered, the nucleophilic attack of carbonyl or electrophilic aliphatic carbon atoms could result in the cyclization of spacer groups. as of the electron rearrangement, the self-immolation cyclization is driven by the formation of positive reaction entropy and thermodynamic stable products (such as -membered and -membered rings). additionally, the triggers of self-immolation include chemical reagents, enzymes, light and others. self-immolative linkers that can be triggered by chemical reagents; boc(tert-butoxycarbonyl), fmoc( -fluorenylmethyl) and -oxobutyl carbamate are typically ph-responsive for organic synthesis [ ] . boc and fmoc are protecting groups for protecting amino groups, and the fmoc is the only widely used amino acid protecting group of carbamates which can be dissociated in weak base condition. these functional groups undergo self-immolation under some specific triggering conditions (trifluoroacetic acid, piperidine), which in turn releases the attached cargo [ , ] . redox-triggered self-immolative linkers could be broadly divided into three categories by triggers: transition metal (e.g., zn, pd) based reagents [ ] [ ] [ ] , reducing reagents (dtt, gsh or tcep) [ ] , and oxidant (e.g., h o ) [ ] [ ] [ ] [ ] . disulfide bonds are the most widely used reductant-responsive linkers. wu et al. employed α, α-dimethyl groups with disulfide bonds, so that p-dithiobenzyl (dtb) intermediates could maintain faster self-immolation rate and improve the stability. this novel self-immolative linker is expected to promote the design of targeted drug delivery systems and achieve traceless drug release [ ] . similarly, self-immolative linkers with h o as trigger condition have also been widely used in stimulus-responsive systems in various fields. recently, clausen et al. reported the synthesis of arylboronic acid-based hydrogen peroxide-responsive methotrexate and aminopterin prodrugs. this new prodrug can deliver drugs to the lesion of chronic rheumatoid arthritis, so that the side effects of the drug on normal cells were significantly reduced [ ] . enzyme sensitive self-immolative linkers are also commonly used in enzyme responsive systems. plasmin can induce the hydrolysis of tripeptides, which in turn causes the cleavage of self-immolative linkers and release of drugs [ , ] . penicillin g amidase (pga) and bovine serum albumin (bsa) can be used to trigger the cleavage of phenylacetamide or carbamate bonds to release the linked cargo [ , ] . β-galactoside and β-glucuronide are functional groups that can be cleaved by β-galactosidase and β-glucuronidase, respectively, and these two functional groups can also be combined with other self-immolative linkers [ ] [ ] [ ] [ ] . in addition, dibenzyl phosphate derivatives are also used for enzymatic (alkaline phosphatase) self-immolative design for the delivery and release of fluorescent probes and prodrugs [ , ] . enzymatically activated self-immolation is usually slow and requires synergy with other self-immolative linkers. light-activated self-immolation does not require an additional step for synergy. a variety of similar structures based on nitrobenzyl groups can undergo self-immolation under uv light [ , ] . examples of self-immolative structure triggered by nir light are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] h + ph [ , ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] piperidine/ morpholine ph [ ] int. j. mol. sci. , , x for peer review of are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] zn/acoh redox [ ] int. j. mol. sci. , , x for peer review of are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] dithiothreitol redox [ , ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] thiols redox [ ] int. j. mol. sci. , , x for peer review of are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] plasmin enzyme [ , ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] penicillin-g-amidase (pga) enzyme [ ] int. j. mol. sci. , , x for peer review of are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] bsa enzyme [ ] int. j. mol. sci. , , x for peer review of are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] β-glucuronidase enzyme [ ] [ ] [ ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] β-galactosidase enzyme [ ] int. j. mol. sci. , , x for peer review of are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] alkaline phosphatase enzyme [ , ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] are polymers with o-nitrobenzyl group as the capping group [ , ] and polymers based on coumarin derivatives [ , ] . [ , ] piperidine/ morpholine ph [ ] zn/acoh redox [ ] pd (pph ) redox [ , ] dithiothreitol redox [ , ] thiols redox [ ] h o redox [ ] [ ] [ ] [ ] plasmin enzyme [ , ] penicillin-gamidase (pga) enzyme [ ] bsa enzyme [ ] β-glucuronidase enzyme [ ] [ ] [ ] β-galactosidase enzyme [ ] alkaline phosphatase enzyme [ , ] uv light/ nir light light [ ] [ ] [ ] [ ] light [ , ] nir light light [ , ] conventional drug delivery systems lack controlled drug release schemes whereas stimulus-responsive drug delivery systems or drug conjugates with rational design can release loaded drugs at specific sites triggered by various endogenous or exogenous stimuli. the human body is a complex collection of various microenvironments, so occasionally it may not suffice if only one stimulation condition is used for the design of stimulus-responsive systems. in this section, we mainly describe the combination of dual or multiple stimuli-responsive conditions for the design of nanoplatforms to improve the specificity and accuracy of nanotherapeutic systems. although single stimulus-responsive systems can solve the problems of specificity and side effects to some extent, the microenvironment within tumor tissues is complex. by contrast, dual or multiple stimuli-responsive nanocarriers can better detect subtle changes in diseased tissues. the traditional combination of ph and temperature has certain defects, such as possible premature drug release in blood or normal tissue species [ , ] , and the ph responsive system might be affected by various factors. to solve the problem of premature leakage, cui et al. made n-isopropylacrylamide-methacrylic acid-octadecyl acrylate (nipam-maa-oda) copolymer liposomes as nanodrug carriers that can be responsive to dual stimulates of ph and temperature [ ] . in recent years, the combination of light and ph has also become a popular choice for dual response systems. for example, researchers have conjugated ph-sensitive i-motif dna (converted from single-stranded structure to c-quadruplex structure in acidic ph environment) to gold nanostars (gns) with as used as a targeting structure. it has been discovered that a-gns/dna/dox nanocomposites have strong photothermal conversion ability, and the combination of ph and nir irradiation can effectively trigger drug release. this nanocomposite showed good stability in not-carcinogenic tissues, and therapeutic effect and biocompatibility were achieved using the construct for combined chemoand photo-therapy [ ] (figure ). in addition to the combination with photothermal therapy, ph responsive systems can also be combined with the uv light-responsive systems. host-guest interaction of β-cyclodextrin with azobenzene has also been studied, but most supramolecular polymer drug carriers are single stimulus-responsive with low delivery accuracy [ ] [ ] [ ] . chen et al. recently constructed supramolecular polymers harnessing host-guest interaction between β-cyclodextrin and azobenzene. β-cyclodextrin was combined with ph-sensitive hydrophilic poly( -(dimethylamino) ethyl methacrylate, and azobenzene was modified with hydrophobic poly(ε-caprolactone), enabling nanodrug micelles to be responsive to both ph and uv light. additionally, higher anticancer activity and stronger cancer cell inhibition than free dox could be achieved using this method [ ] . dual stimuli-responsive system using ph and redox is also a common combination. with low ph and high gsh levels in tumor cells, a strong controlled release effect can be achieved. recently, ph/redox-responsive mixed polymeric micelles formed by self-assembly of two amphiphilic diblock copolymers (poly(ethylene glycol) methyl ether-b-poly(β-amino ester)) (mpeg-b-pae) and poly(ethylene glycol) methyl ether-grafted disulfide bond-poly(β-amino ester) (pae-ss-mpeg) have been developed, which released drugs at low ph and high gsh concentration in tumor cells [ ] . in addition, there are also many combinations, such as ph/ros responsive system [ , ] , ph/enzyme responsive system [ , ] and others. as a significant characteristic stimulus condition in vivo, the combination of ph with other stimulus has shown promise for cancer treatments. redox and ros responsive systems are often used in combination with exogenous stimuli to achieve better stability and controlled release schemes. these two stimuli conditions can be combined to form a dual stimuli-responsive systems [ ] . for example, xu et al. copolymerized two camptothecin (cpt) prodrug monomers with disulfide bond and oxalate bond on β-cyclodextrin and hydrophilic poly (ethylene glycol) methyl ether methacrylate (oegma) to prepare a novel dual redox stimulus-responsive prodrug. this prodrug has small particle size, high stability, excellent biocompatibility, good permeability, improved safety and high anti-tumor efficiency. this prodrug strategy is expected to provide a feasible method for advanced chemotherapy [ ] . in addition, redox/light responsive system is also an effective strategy for cancer treatments due to the scheme of dual triggers [ , ] . in a recent report, a thioacetal-based ros-sensitive amphiphilic copolymer (ptk) was developed to load nir cyanine dye ir and dox to obtain stable nanoparticles (ir /dox@ptk). ir was used as photosensitizer, photothermal agent and imaging contrast agent simultaneously [ , ] . upon the irradiation of nm nir light, ir could not only initiate pdt and ptt, but also used for photothermal imaging. the generated singlet oxygen ( o ) led to the cleavage of thioacetal linker in ptk and the release of dox, enabling chemotherapy and phototherapy [ ] . pu et al. synthesized an organic semiconducting pro-nanoenzyme (ospe) recently, which could be activated by nir light irradiation. ospe is made by binding semiconductor polymer nanoparticles (spn) to proenzyme (based on cytotoxic ribonuclease a) via singlet oxygen ( o )-sensitive linkers. when spn released singlet oxygen, it could not only carry out photodynamic therapy, but also released proenzyme to degrade cancer-specific rna, the synergistic treatment by chemo/phototherapy for cancers [ ] (figure ). other dual/multiple stimuli-responsive systems for cancer treatments include ph/magnetic responsive systems [ , ] and other ph-based multiple stimuli-responsive systems [ ] [ ] [ ] . redox and ros responsive systems are often used in combination with exogenous stimuli to achieve better stability and controlled release schemes. these two stimuli conditions can be combined to form a dual stimuli-responsive systems [ ] . for example, xu et al. copolymerized two camptothecin (cpt) prodrug monomers with disulfide bond and oxalate bond on β-cyclodextrin and hydrophilic poly (ethylene glycol) methyl ether methacrylate (oegma) to prepare a novel dual redox stimulus-responsive prodrug. this prodrug has small particle size, high stability, excellent biocompatibility, good permeability, improved safety and high anti-tumor efficiency. this prodrug strategy is expected to provide a feasible method for advanced chemotherapy [ ] . in addition, redox/light responsive system is also an effective strategy for cancer treatments due to the scheme of dual triggers [ , ] . in a recent report, a thioacetal-based ros-sensitive amphiphilic copolymer (ptk) was developed to load nir cyanine dye ir and dox to obtain stable nanoparticles (ir /dox@ptk). ir was used as photosensitizer, photothermal agent and imaging contrast agent simultaneously [ , ] . upon the irradiation of nm nir light, ir could not only initiate pdt and ptt, but also used for photothermal imaging. the generated singlet oxygen ( o ) led to the cleavage of thioacetal linker in ptk and the release of dox, enabling chemotherapy and phototherapy [ ] . pu et al. synthesized an organic semiconducting pro-nanoenzyme (ospe) recently, which could be activated by nir light irradiation. ospe is made by binding semiconductor polymer nanoparticles (spn) to proenzyme (based on cytotoxic ribonuclease a) via singlet oxygen ( o )-sensitive linkers. when spn released singlet oxygen, it could not only carry out photodynamic therapy, but also released proenzyme to degrade cancer-specific rna, the synergistic treatment by chemo/phototherapy for cancers [ ] (figure ). other dual/multiple stimuli-responsive systems for cancer treatments include ph/magnetic responsive systems [ , ] and other ph-based multiple stimuli-responsive systems [ ] [ ] [ ] . inflammation is an immune response to infection and tissue damage so that the body can be protected from injury. however, it can also cause many diseases such as asthma, cardiovascular diseases, neurodegenerative diseases and autoimmune diseases (including rheumatoid arthritis, systemic lupus erythematosus and other diseases) [ , ] . these inflammatory chronic diseases can seriously affect health, and there are many obstacles to the treatment of the inflammatory chronic diseases. inflammation is an immune response to infection and tissue damage so that the body can be protected from injury. however, it can also cause many diseases such as asthma, cardiovascular diseases, neurodegenerative diseases and autoimmune diseases (including rheumatoid arthritis, systemic lupus erythematosus and other diseases) [ , ] . these inflammatory chronic diseases can seriously affect health, and there are many obstacles to the treatment of the inflammatory chronic diseases. the characteristic conditions within the microenvironment of inflammatory tissues include lower ph value and higher ros concentration [ , ] . therefore, ph/ros dual stimulus-responsive system is the most widely used treatment strategy for inflammation therapy [ , ] . for example, almutairi et al. designed a ros-reactive dextran-drug conjugate (nap-dex) and blended nap-dex with an acid-sensitive acetal-dextran polymer (ac-dex) to obtain ph/ros dual stimulus-responsive nanoparticles. the ros-responsive pba-modified anti-inflammatory drug naproxen was used. when the nanodrug was stimulated by h o and acidic environment, the ac-dex and pba structure of the nanodrug micelle was cleaved, thereby releasing naproxen (nap) for the treatment of inflammatory tissues. dual stimuli-responsive nanodrug micelles are more effective in scavenging ros. compared with free naproxen, dual stimuli-responsive nanoparticles reduced the levels of proinflammatory cytokines il- and tnfα by times and times respectively [ ] . in addition to lower ph and higher ros concentrations, inflammation of tissues tends to have slightly increased local temperature ( - • c above ambient temperature) [ ] . therefore, the temperature/ph dual stimulus-responsive system has also been applied in the treatment of inflammation [ , ] . a microbead system was designed with a porous poly (d, l-lactic-co-glycolic acid) (plga) shell coupled with a gelatin plug (thermal-responsive switch). additionally, n-palmitoyl chitosan (npcs) (ph-responsive switch) was employed as a core. vancomycin was loaded in the nanoparticles as the inflammatory therapeutic drug. this dual stimuli-responsive drug mbs could release the drug only in the presence of both stimuli (temperature and ph), preventing unexpected drug release due to accidental stimulus [ ] . inflammatory bowel disease (ibd) is a chronic condition of idiopathic inflammation. ibd can affect the entire gastrointestinal tract (gi) and increases the risk of colorectal cancer [ ] . the incidence of ibd worldwide has increased and effective treatment approaches are needed. therefore, drug delivery systems utilizing ibd microenvironment characteristic conditions (low ph, colonic enzymes, and high ros concentration) as responsive factors have attracted extensive attention [ , ] . li et al. reported a nano-platform of oxidatively sensitive dextran (oxidex) with its exterior modified by chitosan (cs) and then further encapsulated by ph-sensitive hydroxypropyl methylcellulose acetate succinate (hpmcas). the intestinal-specific antibiotic rifampicin was used as a model drug to form ph/ros dual stimulus-responsive nanodrug composites. this novel nanodrug composite remained stable in the upper gastrointestinal tract, but hpmcas were cleaved in the intestine, thereby releasing nanodrug particles. triggered by higher ros levels, rifampicin is released into inflamed tissues. compared with traditional enteric drug formulations, this nanodrug composite effectively reduced the permeability of drugs at the intestinal epithelium, preventing non-specific absorption and side effects [ ] (figure ). in addition, some oral drug formulations responsive to ph have been used for clinical treatments. examples include -aminosalicylic acid encapsulated in capsules made from copolymers of acrylic acid derivatives and methyl methacrylate (eudragit ® ), such as salofalk ® , calitoflak ® , claversal ® , pentasa ® and other brands [ ] [ ] [ ] . other inflammation examples include neuroinflammation, which represents an abnormal condition of central nervous system in many neurodegenerative diseases [ ] . some serious neurodegenerative diseases include alzheimer's disease, epilepsy, huntington's disease and parkinson's disease [ ] [ ] [ ] . strategies for the treatment of these diseases are desired and some ongoing efforts have been made on stimulus-responsive systems that can target excessive ros concentrations in neuroinflammatory tissues [ , ] . oral medication is the preferred route of administration because of better patient compliance. however, many drugs themselves (protein drugs/polypeptide drugs) are sensitive to the acidic and proteolytic environment of the gastrointestinal tract, so oral administration remains a challenge. factors for oral administration that should be considered include ( ) the activity of biopharmaceutical macromolecules in the gastrointestinal (git) environment, ( ) the permeability in the intestine, and ( ) drug molecules can be absorbed into the systemic circulation through the intestine [ , ] . diabetes mellitus is an endocrine disease characterized by hyperglycemia, which is difficult to cure and can lead to various serious complications. the conventional way to treat diabetes is to inject insulin using subcutaneous administration, but the development of an effective orally administered insulin formulation would be preferred. the first attempt to treat diabetes with oral insulin can date back to but the failure made researchers realize that the main obstacle to oral delivery of biomacromolecules was mainly from the human body itself [ ] . chitosan/insulin/heparin sodium (cs/ins/hs) nanoparticles were synthesized by ionic gel method, in which heparin sodium with three acidic functional groups can enhance the stability of the nanoparticle system in the stomach. mucosal affinity for cs/ins/hs in the small intestine were also improved due to the interaction of ins/hs with the positive charge on chitosan. in addition, acrylate-grafted-carboxymethyl starch (cms-g-aa) and oral medication is the preferred route of administration because of better patient compliance. however, many drugs themselves (protein drugs/polypeptide drugs) are sensitive to the acidic and proteolytic environment of the gastrointestinal tract, so oral administration remains a challenge. factors for oral administration that should be considered include ( ) the activity of biopharmaceutical macromolecules in the gastrointestinal (git) environment, ( ) the permeability in the intestine, and ( ) drug molecules can be absorbed into the systemic circulation through the intestine [ , ] . diabetes mellitus is an endocrine disease characterized by hyperglycemia, which is difficult to cure and can lead to various serious complications. the conventional way to treat diabetes is to inject insulin using subcutaneous administration, but the development of an effective orally administered insulin formulation would be preferred. the first attempt to treat diabetes with oral insulin can date back to but the failure made researchers realize that the main obstacle to oral delivery of biomacromolecules was mainly from the human body itself [ ] . chitosan/insulin/heparin sodium (cs/ins/hs) nanoparticles were synthesized by ionic gel method, in which heparin sodium with three acidic functional groups can enhance the stability of the nanoparticle system in the stomach. mucosal affinity for cs/ins/hs in the small intestine were also improved due to the interaction of ins/hs with the positive charge on chitosan. in addition, acrylate-grafted-carboxymethyl starch (cms-g-aa) and methacrylic acid (maa) were used to synthesize ph/amylase dual stimuli-responsive hydrogels (cms-g-aa/pmaa). drug-loaded hydrogels can be contracted in the stomach to ensure the stability of nanoparticles. however, in the small intestine the hydrogel can be swollen so that insulin could be released, resulting from the carrier degradation by intestinal amylase. additionally, because of chitosan and heparin sodium, the reversible opening of the pathway between adjacent intestinal epithelial cells allowed insulin molecules to pass through the intestinal epithelial cells. this approach can also be used for the design of orally administered insulin formulations [ ] . in addition to oral insulin, many stimulus-responsive systems have been designed for the treatment of other diseases such as helicobacter pylori [ ] , various cancers [ ] [ ] [ ] . for example, oupciky et al. reported a nanostructured lipid carrier (nlc)-based ptt formulation that can be orally administered. nlc showed its excellent biocompatibility, degradability and stability in the gastrointestinal tract. under nir light, nlc loaded with ir can be used for photothermal treatment on cancer cells [ ] (figure ). zhang et al. developed a supramolecular elastomer gel based on poly (acryloyl -aminocaproicacid) (pa aca) and poly (methacrylic acid-ethyl acrylate) (eudragit l - ), which remained stable in acidic gastric environment, but dissolved in the small intestine with neutral ph, allowing safe passage through the stomach and into the intestine afterwards. under acidic conditions, carboxyl groups were protonated, and the inter-chain hydrogen bonds between carboxyl groups and amide units on pa aca and l - formed a loosely cross-linked supramolecular network with water trapped inside, providing good elasticity and stability. under neutral ph conditions, the carboxyl groups in the molecule underwent deprotonation, and the disappearance of hydrogen bonds made the gel dissolve quickly. this type of gel material could be used as a good carrier for oral drugs with the advantages of strong elasticity, easy compression and folding. the use of this material for oral delivery drug was also evaluated in pigs [ ] . stimulus-responsive oral drug delivery systems are still under development and much efforts are being endeavored in order to improve delivery efficiency and targeting capability and to reduce the side effects (e.g., potential pathogenic risk of agents used for increased intestinal permeability). methacrylic acid (maa) were used to synthesize ph/amylase dual stimuli-responsive hydrogels (cms-g-aa/pmaa). drug-loaded hydrogels can be contracted in the stomach to ensure the stability of nanoparticles. however, in the small intestine the hydrogel can be swollen so that insulin could be released, resulting from the carrier degradation by intestinal amylase. additionally, because of chitosan and heparin sodium, the reversible opening of the pathway between adjacent intestinal epithelial cells allowed insulin molecules to pass through the intestinal epithelial cells. this approach can also be used for the design of orally administered insulin formulations [ ] . in addition to oral insulin, many stimulus-responsive systems have been designed for the treatment of other diseases such as helicobacter pylori [ ] , various cancers [ ] [ ] [ ] . for example, oupciky et al. reported a nanostructured lipid carrier (nlc)-based ptt formulation that can be orally administered. nlc showed its excellent biocompatibility, degradability and stability in the gastrointestinal tract. under nir light, nlc loaded with ir can be used for photothermal treatment on cancer cells [ ] (figure ). zhang et al. developed a supramolecular elastomer gel based on poly (acryloyl -aminocaproicacid) (pa aca) and poly (methacrylic acid-ethyl acrylate) (eudragit l - ), which remained stable in acidic gastric environment, but dissolved in the small intestine with neutral ph, allowing safe passage through the stomach and into the intestine afterwards. under acidic conditions, carboxyl groups were protonated, and the inter-chain hydrogen bonds between carboxyl groups and amide units on pa aca and l - formed a loosely crosslinked supramolecular network with water trapped inside, providing good elasticity and stability. under neutral ph conditions, the carboxyl groups in the molecule underwent deprotonation, and the disappearance of hydrogen bonds made the gel dissolve quickly. this type of gel material could be used as a good carrier for oral drugs with the advantages of strong elasticity, easy compression and folding. the use of this material for oral delivery drug was also evaluated in pigs [ ] . stimulusresponsive oral drug delivery systems are still under development and much efforts are being endeavored in order to improve delivery efficiency and targeting capability and to reduce the side effects (e.g., potential pathogenic risk of agents used for increased intestinal permeability). non-invasive bioimaging approaches have been used for observation of biological activities and disease diagnosis. commonly used bioimaging techniques include optical imaging, magnetic resonance imaging (mri), ultrasound (us), computed tomography (ct), positron emission tomography (pet). photoacoustic imaging is another emerging imaging modality that shows potential for future clinical use. in this section, we will mainly introduce the latest research results of stimulus responsive nano-systems used for bio-imaging. among the optical imaging methods, fluorescence imaging plays an important role because of its high resolution and low cost. for higher accuracy and specificity, optical imaging is also synergistically used with chemo-pharmacotherapy [ , ] . huang et al. recently reported a ph responsive nanocarrier using nir-ii dye-based multifunctional telechelic glycopolymer (ttq-tc-pfru). the nir-ii dyes in pfru-btz-pbob nanoparticles could be used for optical imaging and non-invasive bioimaging approaches have been used for observation of biological activities and disease diagnosis. commonly used bioimaging techniques include optical imaging, magnetic resonance imaging (mri), ultrasound (us), computed tomography (ct), positron emission tomography (pet). photoacoustic imaging is another emerging imaging modality that shows potential for future clinical use. in this section, we will mainly introduce the latest research results of stimulus responsive nano-systems used for bio-imaging. among the optical imaging methods, fluorescence imaging plays an important role because of its high resolution and low cost. for higher accuracy and specificity, optical imaging is also synergistically used with chemo-pharmacotherapy [ , ] . huang et al. recently reported a ph responsive nanocarrier using nir-ii dye-based multifunctional telechelic glycopolymer (ttq-tc-pfru). the nir-ii dyes in pfru-btz-pbob nanoparticles could be used for optical imaging and photothermal therapy. additionally, anticancer drug bortezomib (btz) was conjugated for chemotherapy. this novel nir-ii dye-based drug-loaded nanoparticles showed potential because of their precise targeting capability and the real time imaging modality to achieve better therapeutic effects [ ] . in addition, a site-selective in situ growth-induced self-assembly method was reported recently. ph-responsive micelles were synthesized based on site-specific human serum albumin-poly( -(diisopropylamino) ethyl methacrylate) (hsa-pdpa) conjugates. icg could be loaded into the core of micelles. when present in the weak acid microenvironment in tumors, the nanoparticles rapidly decomposed into protonated moieties, allowing the fluorescence enhancement by - fold compared to non-stimulus-responsive nanoprobe and icg [ ] (figure a ). photothermal therapy. additionally, anticancer drug bortezomib (btz) was conjugated for chemotherapy. this novel nir-ii dye-based drug-loaded nanoparticles showed potential because of their precise targeting capability and the real time imaging modality to achieve better therapeutic effects [ ] . in addition, a site-selective in situ growth-induced self-assembly method was reported recently. ph-responsive micelles were synthesized based on site-specific human serum albuminpoly( -(diisopropylamino) ethyl methacrylate) (hsa-pdpa) conjugates. icg could be loaded into the core of micelles. when present in the weak acid microenvironment in tumors, the nanoparticles rapidly decomposed into protonated moieties, allowing the fluorescence enhancement by - fold compared to non-stimulus-responsive nanoprobe and icg [ ] (figure a ). [ ] . (b) ros significantly enhanced the ultrasound signal [ ] . (c) mr contrast could be enhanced in the tumor microenvironment by atp/ph stimuli [ ] . (d) pa signal could be significantly enhanced in tumor microenvironment by no/ph stimulation [ ] . (e) pet imaging enabled realtime monitoring of enzyme/nir/ph stimulus-responsive drug delivery systems [ ] . (f) ph and nir responsive system resulted in significant enhancement of ct contrast [ ] . reproduced with permission from the publishers of corresponding references. us has become one of the most commonly used medical imaging techniques due to its excellent portability, noninvasiveness and low cost. however, ultrasound contrast imaging typically requires the use of diffusible microbubbles to diffuse into the surrounding media to enhance contrast. with the diffusion of gas, these microbubbles will be quickly cleared in vivo, for this reason the ultrasonic imaging has a shorter imaging time [ , , ] (figure b ). in recent reports, a ph responsive solid ultrasound nanosensor (sun) was developed that did not require the generation of bubbles. the sun consists of three parts, a multi-empty silicone shell, a solid silica core and a ph-sensitive coating on its surface. this new type of sun could expand and shrink responsive to the change of [ ] . (b) ros significantly enhanced the ultrasound signal [ ] . (c) mr contrast could be enhanced in the tumor microenvironment by atp/ph stimuli [ ] . (d) pa signal could be significantly enhanced in tumor microenvironment by no/ph stimulation [ ] . (e) pet imaging enabled real-time monitoring of enzyme/nir/ph stimulus-responsive drug delivery systems [ ] . (f) ph and nir responsive system resulted in significant enhancement of ct contrast [ ] . reproduced with permission from the publishers of corresponding references. us has become one of the most commonly used medical imaging techniques due to its excellent portability, noninvasiveness and low cost. however, ultrasound contrast imaging typically requires the use of diffusible microbubbles to diffuse into the surrounding media to enhance contrast. with the diffusion of gas, these microbubbles will be quickly cleared in vivo, for this reason the ultrasonic imaging has a shorter imaging time [ , , ] (figure b ). in recent reports, a ph responsive solid ultrasound nanosensor (sun) was developed that did not require the generation of bubbles. the sun consists of three parts, a multi-empty silicone shell, a solid silica core and a ph-sensitive coating on its surface. this new type of sun could expand and shrink responsive to the change of ph value owing to the ph-sensitive coating. when the concentration of hydrogen ions decreased, the ultrasound imaging was significantly enhanced [ ] . magnetic resonance imaging (mri) is a powerful technology for diagnosis and bioimaging of diseases because of its non-invasiveness, reasonable imaging time, deep tissue penetration and high resolution [ , ] . zhang et al. recently developed a triple stimulus (gsh/ph/nir)-responsive nanocarrier based on magnetic hollow porous carbon nanoparticles (mhpcn). the outer shell of mhpcn consists of two layers: the inner layer with iron oxide (fe o ) as contrast agent for mri and the outer layer with fluorescent carbon nanodots as the outer layer. dox was selected as a model drug and folic acid (fa) was crosslinked for targeting purpose. this new triple stimulus-responsive nanodrug carrier can achieve synergistic photothermal/chemical therapy of tumor guided by mri under the synergistic stimulation of low ph, high concentration of gsh and external nir light in tumor cells [ ] . in addition, a strategy using magnetic resonance/near-infrared fluorescence (mr/nirfl) was developed to determine the location of tumors. using ph and nir as stimuli, chemo/photothermal therapy could be achieved for efficient cancer nanotheranostics [ ] . yang et al. used polyphenols as phase transfer agents to increase the solubility of hydrophobic magnetic nanoparticles. meanwhile, polyphenols also promoted the self-assembly of nanoparticles. the core of this assembly is atp/ph responsive for the dual imaging of mr/fl and photothermal therapy. when the nano-assembly entered the tumor microenvironment with lower ph value and higher concentration of atp, they showed enhanced mr contrast (due to the strong binding affinity of atp to fe o ) and significantly altered fluorescence signal (the protonation of hydroxyl groups in tannic acid (ta). thus, functional activities in tumors could be tracked using this mr responsive nanoplatform [ ] (figure c ). photoacoustic (pa) imaging is an emerging biomedical imaging modality with deep penetration depth, high spatial resolution, and high selectivity that combines the advantages of optical imaging and ultrasound imaging [ ] . when laser irradiation is applied to biological tissues, pa signals are generated in the tissues, and the detailed information of human internal tissues could be collected and reconstructed in computer. tan et al. designed a nitric oxide (no)/ph activatable theranostic nanoprobe (datn), taking advantage of high concentration of nitric oxide and low ph in tumor microenvironment. benzo [c] [ , , ] thiadiazole- , -diamine was used for the stimulus responsive π-receptor-π-donor (d-π-a-π-d) molecules. in the tumor microenvironment, datn could turn on pa signals for tumor specific pa imaging in vivo. using these dual stimuli, the pa signal intensity of datn was . times higher than that no responsive system and times higher that of acid responsive system [ ] (figure d ). pu recently reported the synthesis of a fluoro-photoacoustic polymeric renal reporter (fprr) for noninvasive near-infrared fluorescence (nirf) and pa dual imaging of drug-induced acute kidney injury (aki) in mice. fprr consists of dextran (used to promote renal clarity), hemicyanine cyoh (used for imaging), and γ-glutamate (enzyme-responsive moiety). fprr exhibited high renal clearance efficiency in living mice, it also enhanced nirf and pa signals for real-time molecular imaging of aki after the enzyme responsive moiety is cleaved. this nanoplatform showed potential for early detection of aki in clinics [ ] . positron emission tomography (pet) can provide precise biological information on metabolic processes with whole-body penetration and excellent sensitivity at the molecular level using radiopharmaceuticals. it has been widely used in clinical disease treatment and diagnosis [ ] . when used in combination with computed tomography (ct), the clinical effect of pet can be improved for disease diagnosis such as tumors and heart diseases [ ] . for example, hyaluronic acid (ha)-functionalized mos was designed with the surface modified by pei and cu (positron emitter with a half-life of . h). the resulting mos -pei-ha multifunctional nanoplatform was endowed with the property of triple stimuli (ph/haase/nir) responsiveness. this new nanocarrier can be used as contrast agent for pet imaging, providing an effective way to monitor the treatment process and optimize disease management plan [ ] (figure e ). chen et al. developed a multimodal imaging synergistic therapy based on amphiphilic iron oxide-gold janus nanoparticles (fe o -au-jnps) in response to ph stimulus. it was shown that under the guidance of pet, mr and pa multimodal imaging, fe o -au-jnps could effectively carry out ros-mediated cancer treatment. after intravenous injection of [ cu] radiolabeled jnp vesicles, pet imaging showed that the drug vesicles remained stable in the blood, but gradually dissociated in the liver, showing reduced hepatotoxicity. pet imaging modality provided effective real-time monitoring of the whole treatment process [ ] . computed tomography (ct) collects information of anatomical structures via x-ray scans and cross-sectional images are reconstructed by computer operation. due to its penetration capability and fast scanning speed, ct is one of the most widely used clinical imaging methods. it is also usually used in combination with mri, pa and other imaging modalities to achieve better imaging results. for example, as coronavirus disease (covid- ) is spreading worldwide, ct has served as an important imaging tool for the assessment of covid- . ct scans can spot abnormalities of lungs such as multiple opacities (denser, more profuse and confluent) [ ] . in other applications such as cancer diagnosis and treatment, ct also plays a significant role and is mostly used in combination with other imaging approaches. for example, yu et al. recently developed bismuth nano-raspberries (bi-bsa nrs) modified by bsa. bi-bsa nrs have high drug loading efficiency (~ %) and can release the cargo in response to ph and nir stimuli. the efficiency of ct contrast agent is improved by infrared thermal and pa. % tumor treatment rate was achieved by chemo-photothermal therapy. in conclusion, this bi-bsa nrs have potential for the application of multimodality imaging and combination therapy [ ] (figure f ). in summary, nanotechnology-based therapeutics have developed rapidly in recent years, and a wide variety of novel strategies have been explored. particularly, stimulus-responsive nano-systems warrant attention as they show promise in targeting and controlled release in response to endogenous or exogenous stimuli. owing to their unique advantages, stimulus-responsive systems have been widely investigated as a strategy well-suited for nanotherapeutics. furthermore, dual/multiple stimulus-responsive systems have been investigated for more accurate disease diagnosis and management. self-immolative linkers with different cleavage and self-immolation rates hold potential as a versatile technology to address controlled drug release. stimulus-responsive systems have shown promise for the treatment of various cancers, ibd, neuroinflammation, gastrointestinal diseases, bio-imaging and many others. however, the translational potential, especially for the highly complex chemically modified systems, is a concern. drug-device combinations for external stimuli are more complicated than drug-alone approaches from regulatory perspectives. for example, for cancer treatment, stimulus-responsive systems treating only local tumors would require one to find advantages over the well-established modalities of surgery, radiotherapy and other ablation approaches, which do not have the complications of drugs. in addition, toxicity of not only the api, but also the carrier system, is another factor for consideration. nevertheless, with smart and rational design, stimulus-responsive 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beam radiotherapy on patients with recurrent glioblastoma multiforme feasibility study of particle-assisted laser ablation of brain tumors in orthotopic canine model ph-sensitive nano-systems for drug delivery in cancer therapy the relevance of tumour ph to the treatment of malignant disease extracellular ph distribution in human tumours causes and consequences of tumour acidity and implications for treatment biointerfacial self-assembly generates lipid membrane coated bacteria for enhanced oral delivery and treatment targeted delivery of small and macromolecular drugs stimuli-responsive nanomedicines for overcoming cancer multidrug resistance a. ph-dependent equilibrium swelling properties of hydrophobic polyelectrolyte copolymer gels ph-sensitive dissociable nanoscale coordination polymers with drug loading for synergistically enhanced chemoradiotherapy synthesis of a triple-responsive double hydrophilic block copolymer prodrug using a reducible raft-atrp double-head agent probe-inspired nano-prodrug with dual-color fluorogenic property reveals spatiotemporal drug release in living cells hollow chitosan-silica nanospheres as ph-sensitive targeted delivery carriers in breast cancer therapy micelles based on acid degradable poly(acetal urethane): preparation, ph-sensitivity, and triggered intracellular drug release acetal-functionalized pillar ( )arene: a ph-responsive and versatile nanomaterial for the delivery of chemotherapeutic agents doxorubicin-conjugated biodegradable polymeric micelles having acid-cleavable linkages synthesis of highly ph-responsive glucose poly(orthoester) facile fabrication of double-walled polymeric hollow spheres with independent temperature and ph dual-responsiveness for synergetic drug delivery incorporation and controlled release of silyl ether prodrugs from print nanoparticles acetal-linked pegylated paclitaxel prodrugs forming free-paclitaxel-loaded ph-responsive micelles with high drug loading capacity and improved drug delivery micromotors spontaneously neutralize gastric acid for ph-responsive payload release a transistor-like ph nanoprobe for tumour detection and image-guided surgery multistimuli-responsive pegylated polymeric bioconjugate-based nano-aggregate for cancer therapy intracellular release of doxorubicin from core-crosslinked polypeptide micelles triggered by both ph and reduction conditions smart drug delivery systems: from fundamentals to the clinic noninvasive imaging of tumor redox status and its modification by tissue glutathione levels intracellular microenvironment responsive pegylated polypeptide nanogels with ionizable cores for efficient doxorubicin loading and triggered release glutathione metabolism and its implications for health gradient redox-responsive and two-stage rocket-mimetic drug delivery system for improved tumor accumulation and safe chemotherapy bond energy data summarized enhanced bioreduction-responsive biodegradable diselenide-containing poly(ester urethane) nanocarriers thioether phosphatidylcholine liposomes: a novel ros-responsive platform for drug delivery self-assembled redox dual-responsive prodrug-nanosystem formed by single thioether-bridged paclitaxel-fatty acid conjugate for cancer chemotherapy synthetic applications of organotellurium chemistry γ-ray-responsive supramolecular hydrogel based on a diselenide-containing polymer and a peptide ultra-sensitive ros-responsive tellurium-containing polymers near-infrared light stimuli-responsive synergistic therapy nanoplatforms based on the coordination of tellurium-containing block polymer and cisplatin for cancer treatment hydrogen peroxide-responsive micelles self-assembled from a peroxalate ester-containing triblock copolymer in vivo imaging of hydrogen peroxide with chemiluminescent nanoparticles visible light-induced singlet oxygen-mediated intracellular disassembly of polymeric micelles co-loaded with a photosensitizer and an anticancer drug for enhanced photodynamic therapy nanoparticles formed from rgd-epothilone b conjugate for targeted cancer therapy synthesis of a phenylboronic ester-linked peg-lipid conjugate for ros-responsive drug delivery glutathione-responsive nanoscale mofs for effective intracellular delivery of the anticancer drug -mercaptopurine stimuli-responsive nanocarriers for drug delivery the smart drug delivery system and its clinical potential proline isomerization-regulated tumor microenvironment-adaptable self-assembly of peptides for enhanced therapeutic efficacy ros-cleavable proline oligomer crosslinking of polycaprolactone for pro-angiogenic host response orally delivered thioketal nanoparticles loaded with tnf-α-sirna target inflammation and inhibit gene expression in the intestines ros-responsive polymeric nanocarriers with photoinduced exposure of cell-penetrating moieties for specific intracellular drug delivery enzyme-responsive nanoparticle systems proteases: multifunctional enzymes in life and disease enzyme-responsive hydrogel microparticles for pulmonary drug delivery polymer-directed enzyme prodrug therapy. i. hpma copolymer-cathepsin b and pk as a model combination cascade-promoted photo-chemotherapy against resistant cancers by enzyme-responsive polyprodrug nanoplatforms matrix metalloprotease -responsive multifunctional liposomal nanocarrier for enhanced tumor targeting an mmp- responsive liposome integrating antifibrosis and chemotherapeutic drugs for enhanced drug perfusion and efficacy in pancreatic cancer mmp- -sensitive ha end-conjugated poly(amidoamine) dendrimers via click reaction to enhance drug penetration into solid tumor smart asymmetric vesicles with triggered availability of inner cell-penetrating shells for specific intracellular drug delivery peg-sheddable polyplex micelles as smart gene carriers based on mmp-cleavable peptide-linked block copolymers modular design and facile synthesis of enzyme-responsive peptide-linked block copolymers for efficient delivery of doxorubicin matrix metalloproteinase-responsive pegylated lipid nanoparticles for controlled drug delivery in the treatment of rheumatoid arthritis phospholipase a enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention expression of group iia secretory phospholipase a increases with prostate tumor grade overexpression of group ii phospholipase a in human breast cancer tissues is closely associated with their malignant potency presence of secretory group iia and v phospholipase a and cytosolic group ivα phospholipase a in chondrocytes from patients with rheumatoid arthritis phospholipase a s in cell injury and death phospholipidic colchicinoids as promising prodrugs incorporated into enzyme-responsive liposomes: chemical, biophysical, and enzymological aspects phospholipase a -susceptible liposomes of anticancer double lipid-prodrugs a new glucose oxidase fromaspergillus niger: characterization and regulation studies of enzyme and gene kinetics of glucose oxidase excretion by recombinant aspergillus niger glucose-responsive nanoparticles for rapid and extended self-regulated insulin delivery erythrocyte-membrane-camouflaged nanoplatform for intravenous glucose-responsive insulin delivery glucose-responsive metal-organic-framework nanoparticles act as "smart" sense-and-treat carriers nad(p)h:quinone oxidoreductase (dt-diaphorase) expression in normal and tumor tissues efficacy of beta-lapachone in pancreatic cancer treatment: exploiting the novel, therapeutic target nqo cytosolic nqo enzyme-activated near-infrared fluorescence imaging and photodynamic therapy with polymeric vesicles a unique case of breast carcinoma producing pancreatic-type isoamylase glycosidase activated release of fluorescent , -naphthalimide probes for tumor cell imaging from glycosylated 'pro-probes' factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles functional nanomaterials for phototherapies of cancer light-activated ros-responsive nanoplatform codelivering apatinib and doxorubicin for enhanced chemo-photodynamic therapy of multidrug-resistant tumors applications of light-responsive systems for cancer theranostics biologically active molecules with a "light switch porphyrin-phospholipid liposomes permeabilized by near-infrared light tunable luminescent lanthanide supramolecular assembly based on photoreaction of anthracene a self-assembled coumarin-anchored dendrimer for efficient gene delivery and light-responsive drug delivery photocontrolled nuclear-targeted drug delivery by single component photoresponsive fluorescent organic nanoparticles of acridin- -methanol photoinduced reversible worm-to-vesicle transformation of azo-containing block copolymer assemblies prepared by polymerization-induced self-assembly light-responsive and ph-responsive dna microcapsules for controlled release of loads photolabile linkers: exploiting labile bond chemistry to control mode and rate of hydrogel degradation and protein release a photochemical approach for controlled drug release in targeted drug delivery anti-stokes shift luminescent materials for bio-applications upconversion and anti-stokes processes with f and d ions in solids copolymer upconversion nanocomposites for triggered drug release in vitro and in vivo light-controlled tools a dna-azobenzene nanopump fueled by upconversion luminescence for controllable intracellular drug release photo, ph and redox multi-responsive nanogels for drug delivery and fluorescence cell imaging diarylethenes for memories and switches reversible photoswitching of triplet-triplet annihilation upconversion using dithienylethene photochromic switches synthesis and comparative photoswitching studies of unsymmetrical , -diarylcyclopent- -en- -ones photochromic properties of terarylene derivatives having a π-conjugation unit on central aromatic ring photoswitchable organocatalysis: using light to modulate the catalytic activities of n-heterocyclic carbenes reporting the release of caged species by a combination of two sequential photoreactions, a molecular switch, and one color of light a contribution to the design of molecular switches: novel acid-mediated ring-closing−photochemical ring-opening of , -bis(heteroaryl)quinones (heteroaryl = thienyl, furanyl, pyrrolyl) dual-mode fluorescence switching of photochromic bisthiazolylcoumarin synthesis and photochromism of a novel diarylethene compound containing terminal benzothiazole unit light-responsive platform for launching therapeutic agents on command drug delivery via cell-conveyed phototherapeutics photoactivation of anticancer ru complexes in deep tissue: how deep can we go? chem. a eur temperature-responsive smart nanocarriers for delivery of therapeutic agents: applications and recent advances multifunctional up-converting nanocomposites with smart polymer brushes gated mesopores for cell imaging and thermo/ph dual-responsive drug controlled release release kinetics of benzoic acid and its sodium salt from a series of poly(n-isopropylacrylamide) matrices with various percentage crosslinking the targeted behavior of thermally responsive nanohydrogel evaluated by nir system in mouse model thermoresponsive poly(n-vinyl caprolactam)-coated gold nanoparticles: sharp reversible response and easy tunability dual-stimuli-responsive paclitaxel delivery nanosystems from chemically conjugate self-assemblies for carcinoma treatment enzyme and thermal dual responsive amphiphilic polymer core-shell nanoparticle for doxorubicin delivery to cancer cells thermosensitive liposomes modified with poly(n-isopropylacrylamide-co-propylacrylic acid) copolymers for triggered release of doxorubicin magnetism in medicine thermoresponsive polymer brush-functionalized magnetic manganite nanoparticles for remotely triggered drug release controlled release of doxorubicin loaded within magnetic thermo-responsive nanocarriers under magnetic and thermal actuation in a microfluidic channel magnetically stimulated drug release using nanoparticles capped by self-assembling peptides magnetic microspheres: a model system for site specific drug delivery in vivo improved method of recombinant aav delivery for systemic targeted gene therapy magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo acoustic cavitation: a possible consequence of biomedical uses of ultrasound microbubble contrast agents: targeted ultrasound imaging and ultrasound-assisted drug-delivery applications lipid/plga hybrid microbubbles as a versatile platform for noninvasive image-guided targeted drug delivery in situ conversion of porphyrin microbubbles to nanoparticles for multimodality imaging ultrasound-responsive conversion of microbubbles to nanoparticles to enable background-free in vivo photoacoustic imaging short and long term efficacy of high intensity focused ultrasound therapy for advanced hepatocellular carcinoma extracorporeal high intensity focused ultrasound ablation in the treatment of patients with solid carcinomas in china: an overview a novel connector linkage applicable in prodrug design polymeric persulfide prodrugs: mitigating oxidative stress through controlled delivery of reactive sulfur species alkylamine-substituted perthiocarbamates: dual precursors to hydropersulfide and carbonyl sulfide with cardioprotective actions development of a cysteine-conjugatable disulfide fret probe: influence of charge on linker cleavage and payload trafficking for an anti-her antibody conjugate small molecule as fluorescent probes for monitoring intracellular enzymatic transformations engineered ph-responsive mesoporous carbon nanoparticles for drug delivery reactive oxygen species (ros)-responsive polymersomes with site-specific chemotherapeutic delivery into tumors via spacer design chemistry rational design of latent fluorophores from water-soluble hydroxyphenyltriazine dyes suitable for lipase sensing disassembly kinetics of quinone-methide-based self-immolative spacers that contain aromatic nitrogen heterocycles supramolecular hydrogels for enzymatically triggered self-immolative drug delivery quinone-methide species, a gateway to functional molecular systems: from self-immolative dendrimers to long-wavelength fluorescent dyes development of a drug-release strategy based on the reductive fragmentation of benzyl carbamate disulfides synthesis and evaluation of paclitaxel-loaded gold nanoparticles for tumor-targeted drug delivery real-time monitoring of drug release single-triggered ab self-immolative dendritic amplifiers elongated multiple electronic cascade and cyclization spacer systems in activatible anticancer prodrugs for enhanced drug release prodrug strategies based on intramolecular cyclization reactions oxidation degradable aliphatic polycarbonates with pendent phenylboronic ester syntheses and kinetic studies of cyclisation-based self-immolative spacers programmable microcapsules from self-immolative polymers self-immolative linkers as caps for the design of gated silica mesoporous supports intramolecular cyclization for stimuli-controlled depolymerization of polycaprolactone particles leading to disassembly and payload release use of dithiasuccinoyl-caged amines enables cos/h s release lacking electrophilic byproducts -bis(hydroxymethyl)aniline as a building block for oligomers with self-eliminating and multiple release properties convergent synthesis of geometrically disassembling dendrimers using cu(i)-catalyzed c−o bond formation disulfide-based self-immolative linkers and functional bioconjugates for biological applications dual-responsive nanoparticles release cargo upon exposure to matrix metalloproteinase and reactive oxygen species exponential molecular amplification by h o -mediated autocatalytic deprotection of boronic ester probes to redox cyclers effects of electronics, aromaticity, and solvent polarity on the rate of azaquinone-methide-mediated depolymerization of aromatic carbamate oligomers synthesis and evaluation of hydrogen peroxide sensitive prodrugs of methotrexate and aminopterin for the treatment of rheumatoid arthritis stabilizing p-dithiobenzyl urethane linkers without rate-limiting self-immolation for traceless drug release design and synthesis of new protease-triggered co-releasing peptide-metal-complex conjugates self-immolative polymers activity-based optical sensing enabled by self-immolative scaffolds: monitoring of release events by fluorescence or chemiluminescence output a gadolinium chelate for detection of β-glucuronidase: a self-immolative approach synthesis and antitumor properties of bqc-glucuronide, a camptothecin prodrug for selective tumor activation benzyl ether-linked glucuronide derivative of -hydroxycamptothecin designed for selective camptothecin-based anticancer therapy aryl sulfate is a useful motif for conjugating and releasing phenolic molecules: sulfur fluorine exchange click chemistry enables discovery of ortho-hydroxy-protected aryl sulfate linker an improved fluorogenic substrate for the detection of alkaline phosphatase activity synthesis, and pharmacokinetic evaluation of phosphate and amino acid ester prodrugs for improving the oral bioavailability of the hiv- protease inhibitor atazanavir light activation for the versatile and accurate kinetic analysis of disassembly of self-immolative spacers sterically-controlled self-immolation in phosphoramidate linkers triggered by light single uv or near ir triggering event leads to polymer degradation into small molecules multi-stimuli-responsive self-immolative polymer assemblies red and near-infrared light-cleavable polymers increasing materials' response to two-photon nir light via self-immolative dendritic scaffolds ph-sensitive, serum-stable and long-circulating liposomes as a new drug delivery system tumor-selective targeted delivery of genes and antisense oligodeoxyribonucleotides via the folate receptor ph and thermo dual-stimuli-responsive drug carrier based on mesoporous silica nanoparticles encapsulated in a copolymer-lipid bilayer facile construction of i-motif dna-conjugated gold nanostars as near-infrared and ph dual-responsive targeted drug delivery systems for combined cancer therapy light-triggered reversible assemblies of azobenzene-containing amphiphilic copolymer with β-cyclodextrin-modified hollow mesoporous silica nanoparticles for controlled drug release light-triggered specific cancer cell release from cyclodextrin/azobenzene and aptamer-modified substrate controlled drug release from cyclodextrin-gated mesoporous silica nanoparticles based on switchable host-guest interactions dual stimuli-responsive supramolecular self-assemblies based on the host-guest interaction between β-cyclodextrin and azobenzene for cellular drug release dual ph/redox-responsive mixed polymeric micelles for anticancer drug delivery and controlled release reactive oxygen species synergistic ph/h o -responsive poly(l-lactic acid)-block-poly(sodium -styrenesulfonate)/citrate-fe(iii)@zif- hybrid nanocomposites for controlled drug release intracellular enzyme-triggered assembly of amino acid-modified gold nanoparticles for accurate cancer therapy with multimode a smart drug delivery system responsive to ph/enzyme stimuli based on hydrophobic modified sodium alginate enzyme/ph dual-responsive polymer prodrug nanoparticles based on -hydroxycamptothecin-carboxymethylchitosan for enhanced drug stability and anticancer efficacy glutathione and reactive oxygen species dual-responsive block copolymer prodrugs for boosting tumor site-specific drug release and enhanced antitumor efficacy smart unimolecular micelle-based polyprodrug with dual-redox stimuli response for tumor microenvironment: enhanced in vivo delivery efficiency and tumor penetration gsh and light dual stimuli-responsive supramolecular polymer drug carriers for cancer therapy dual-stimuli-responsive microparticles ir- dye loaded tumor targeting theranostic nanoparticles for nir imaging and photothermal therapy sentinel lymph node mapping by a near-infrared fluorescent heptamethine dye nir-triggered multifunctional and degradable nanoplatform based on an ros-sensitive block copolymer for imaging-guided chemo-phototherapy photoactivatable organic semiconducting pro-nanoenzymes dual stimuli-guided lipid-based delivery system of cancer combination therapy non-magnetic injectable implant for magnetic field-driven thermochemotherapy and dual stimuli-responsive drug delivery: transformable liquid metal hybrid platform for cancer theranostics multi-stimuli responsive mesoporous carbon nano-platform gated by human serum albumin for cancer thermo-chemotherapy reduction/temperature/ph multi-stimuli responsive core cross-linked polypeptide hybrid micelles for triggered and intracellular drug release synthesis of temperature, ph, light and dual-redox quintuple-stimuli-responsive shell-crosslinked polymeric nanoparticles for controlled release reactive oxygen species in inflammation and tissue injury inflammation responsive logic gate nanoparticles for the delivery of proteins a dual ph/redox responsive copper-ligand nanoliposome bioactive complex for the treatment of chronic inflammation a ph/ros dual-responsive and targeting nanotherapy for vascular inflammatory diseases inflammation-responsive drug-conjugated dextran nanoparticles enhance anti-inflammatory drug efficacy responsive polyelectrolyte complexes for triggered release of nucleic acid therapeutics dual thermo-and ph-sensitive network-grafted hydrogels formed by macrocrosslinker as drug delivery system thermo and ph dual actuating smart porous anodic aluminum for controllable drug release injectable microbeads with a thermo-responsive shell and a ph-responsive core as a dual-switch-controlled release system inflammatory bowel disease bioresponsive drug delivery systems in intestinal inflammation: state-of-the-art and future perspectives reactive oxygen species responsive nanoplatforms as smart drug delivery systems for gastrointestinal tract targeting ph and reactive oxygen species-sequential responsive nano-in-micro composite for targeted therapy of inflammatory bowel disease nanoparticulate drug delivery systems targeting inflammation for treatment of inflammatory bowel disease drug delivery strategies in the therapy of inflammatory bowel disease short-term adverse effects of -aminosalicylic acid agents in the treatment of ulcerative colitis inflammation in neurological disorders: a help or a hindrance? neuroscience van der valk, p. inflammation in neurodegenerative diseases role of immunity and inflammation in the pathophysiology of neurodegenerative diseases dysregulation of mitochondrial calcium signaling and superoxide flashes cause mitochondrial genomic dna damage in huntington disease positively charged polyprodrug amphiphiles with enhanced drug loading and reactive oxygen species-responsive release ability for traceable synergistic therapy core-cross-linked nanoparticles reduce neuroinflammation and improve outcome in a mouse model of traumatic brain injury a review of advanced oral drug delivery technologies facilitating the protection and absorption of protein and peptide molecules insulin in hospital and home dual stimuli-responsive nanoparticle-incorporated hydrogels as an oral insulin carrier for intestine-targeted delivery and enhanced paracellular permeation development of ph-responsive chitosan/heparin nanoparticles for stomach-specific anti-helicobacter pylori therapy oral delivery of ph-responsive alginate microbeads incorporating folic acid-grafted solid lipid nanoparticles exhibits enhanced targeting effect against colorectal cancer: a dual-targeted approach in vitro evaluation of ph-responsive nanoscale hydrogels for the oral delivery of hydrophobic therapeutics ph-responsive lignin-based nanomicelles for oral drug delivery oral nanostructured lipid carriers loaded with near-infrared dye for image-guided photothermal therapy a ph-responsive supramolecular polymer gel as an enteric elastomer for use in gastric devices amphiphilic semiconducting polymer as multifunctional nanocarrier for fluorescence/photoacoustic imaging guided chemo-photothermal therapy dual-stimuli responsive and reversibly activatable theranostic nanoprobe for precision tumor-targeting and fluorescence-guided photothermal therapy nir-ii dye-based multifunctional telechelic glycopolymers for nir-iia fluorescence imaging-guided stimuli-responsive chemo-photothermal combination therapy site-selective in situ growth-induced self-assembly of protein-polymer conjugates into ph-responsive micelles for tumor microenvironment triggered fluorescence imaging dual-enzyme-loaded multifunctional hybrid nanogel system for pathological responsive ultrasound imaging and t -weighted magnetic resonance imaging polyphenol-inspired facile construction of smart assemblies for atp-and ph-responsive tumor mr/optical imaging and photothermal therapy nitric oxide-activated "dual-key-one-lock" nanoprobe for in vivo molecular imaging and high-specificity cancer therapy intelligent mos nanotheranostic for targeted and enzyme-/ph-/nir-responsive drug delivery to overcome cancer chemotherapy resistance guided by pet imaging dual-stimuli responsive bismuth nanoraspberries for multimodal imaging and combined cancer therapy microbubbles in medical imaging: current applications and future directions injectable microbubbles as contrast agents for diagnostic ultrasound imaging: the key role of perfluorochemicals dynamic solid-state ultrasound contrast agent for monitoring ph fluctuations in vivo molecular imaging by mri advances in high-field magnetic resonance imaging triple stimuli-responsive magnetic hollow porous carbon-based nanodrug delivery system for magnetic resonance imaging-guided synergistic photothermal/chemotherapy of cancer hp-β-cd functionalized fe o /cnps-based theranostic nanoplatform for ph/nir responsive drug release and mr/nirfl imaging-guided synergetic chemo/photothermal therapy of tumor noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain fluoro-photoacoustic polymeric renal reporter for real-time dual imaging of acute kidney injury the merging of biology and imaging into molecular imaging positron emission tomography basic science of pet and pet/ct self-assembled responsive bilayered vesicles with adjustable oxidative stress for enhanced cancer imaging and therapy correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -xta e j authors: nan title: deutsche gesellschaft für experimentelle und klinische pharmakologie und toxikologie e.v. date: - - journal: naunyn schmiedebergs arch pharmacol doi: . /s - - - sha: doc_id: cord_uid: xta e j nan nucleoside diphosphate kinases (ndpks) are multifunctional enzymes involved in a variety of cellular processes including cancer metastasis and heart diseases. the plasma membrane content of the three major ndpk isoforms ndpk a, b and c is increased in human heart failure. we have previously shown that the ndpk b isoform regulates camp levels and cardiac contractility through a receptor-independent gprotein activation involving direct g protein β subunit phosphorylation. the precise role of ndpk c in the heart is unknown and was the object of this study. ndpk c function was assessed in neonatal (nrcm) and adult (arcm) rat cardiomyocytes with real-time pcr, immunoblotting, and quantification of camp content. heart failure was induced by chronic treatment with isoproterenol (iso, . mg/kg/d days) via minipumps. chronic iso increased mrna levels of ndpk c by . ± . -fold and its protein levels by . ± . -fold. immunoprecipitation of the g protein β subunit resulted in coimmunoprecipitation of ndpk c and the stimulatory gαs subunit: iso enhanced this interaction. upon iso stimulation, ndpk c translocated from the cytosol to the plasma membrane within hours in both nrcms and arcms. adenoviral overexpression of ndpk c in nrcms caused a . -fold increase in basal and iso induced camp synthesis, whereas sirna mediated knockdown of endogenous ndpk c decreased camp levels by ~ %. our results establish ndpk c as a novel and critical regulator of camp synthesis and gs signaling in the heart. the up-regulation of ndpk c and the increased responsiveness to iso in failing hearts point to ndpk c as a potential counterregulatory factor in the onset of heart failure. uptake and metabolism of methylated myricetin derivatives: studies in cell culture and c. elegans ackermann d. , , büchter c. secondary plant compounds like flavonoids that are ubiquitary present in fruits and vegetables are believed to exert health protective effects in terms of lowering the incidence of widespread diseases such as cardiovascular diseases and cancer. besides their antioxidative effects, flavonoids may also modulate cell signaling pathways and thereby performing their disease-protective actions. though this class of dietary polyphenols has become increasingly popular as dietary supplements, only little is known about their metabolic fate in vivo. therefore, we investigated the absorption and metabolism of several flavonoids such as myricetin and its methylated derivatives laricitrin, syringetin, and myricetin- ', ', '-trimethylether in the human colon carcinoma cell line hct and the human hepatoma cell line hepg as well as the model organism caenorhabditis elegans. all flavonoids were rapidly taken up by both cell lines as shown by hplc analyses. the intracellular amount of myricetin and laricitrin did not increase with time and was only half of that of syringetin and myricetin- ', ', '-trimethylether. interestingly, no metabolites of these flavonoids could be detected which might at least in part be due to their low intracellular amounts. absorption as well as intracellular distribution was also evidenced by using the fluorescent dye "naturstoff reagent a" nsra) . fluorescence microscopy indicated a predominant cytosolic distribution of the employed flavonoids. in the model organism caenorhabditis elegans, the flavonoids were exclusively distributed in the intestine as visualized by nsra. the antioxidative capacity of the four flavonoids (measured by using the cell free teac assay and the h dcf-da assay in hct cells) decreased with increasing methyl groups in the b-ring. myricetin- ', ', '-trimethylether (three methyl groups) was the least effective radical scavenging flavonoid in both the cell free system and in hct cells compared to myricetin (no methyl groups). in conclusion, for exerting their biological effects, uptake and distribution as well as metabolism of flavonoids in certain organs such as liver and gut are important and more research in that field is warranted. munich heart alliance, münchen, germany signaling through g protein-coupled receptors is affected by receptor polymorphisms, yet the molecular basis for the functional differences of individual receptor variants is unclear. to investigate the impact of the frequent gly arg variant of the β -adrenergic receptor (β ar) on receptor conformation we used β ar-sensors capable of fluorescence resonance energy transfer (fret). these sensors retained the pharmacological and functional characteristics of the native receptors. upon stimulation of the sensors we determined the activation characteristics of the polymorphic receptors in real time and in living cells. we found the β ar variants to behave similar upon a single stimulation with an agonist, but to differentially respond with a change of their activation kinetics during subsequent stimulations. while the arg -β ar did not show altered activation kinetics after prestimulation, the gly -β ar became slower compared to the initial stimulation suggesting that β ars possess a memory of previous activation. we then permeabilized β ar-sensor-expressing cells with saponin to remove soluble cytosolic factors. upon permeabilization the β ar variants did not display receptor memory, suggesting that the β ar memory depended on the interaction of the receptors with soluble cytosolic factors upon their initial activation including the phosphorylation of agonist-bound receptors by protein kinase a or g protein-coupled receptor kinases. our findings suggest an intrinsic, polymorphism-specific property of βars that alters activation kinetics upon continued stimulation and that might account for individual drug responses. micro-rna replacement therapy: nanoparticle-mediated in vivo delivery of mirna- or mirna- a exerts antitumor effects in colon carcinoma xenograft mouse models weirauch u. micro-rnas (mirnas) control the expression of various genes, and under pathological conditions several mirnas are up-or downregulated. previous in vitro studies have established a pro-apoptotic and anti-proliferative role of mir- , which shows decreased levels in colon carcinoma. in contrast, while mir- a is only weakly expressed in several tumors as well, its role in cancer has not been analysed so far. in this study, we demonstrate the tumor-relevance of mir- a and identify the protooncogenic kinase pim- as a target of mir- a. pim- harbours a highly conserved mir- a binding site within its '-utr, and seed mutagenesis of this target sequence abolishes the mir- a-mediated downregulation of pim- . the knockdown of pim- by rnai or mirna transfection inhibits proliferation in leukemia and in colon carcinoma cells by decelerating cell cycle progression, thus establishing a tumor inhibitory function of mir- a. we furthermore introduce polyethylenimines (peis) for the therapeutic application of mirnas in vivo, which is critically dependent on the development of appropriate delivery tools. peis are able to form non-covalent complexes with mirnas, leading to mirna protection after systemic application in combination with an attractive biodistribution profile and the efficient uptake in target organs/cells. therapeutic effects of pei-mediated mirna delivery were demonstrated in subcutaneous colon carcinoma xenograft mouse models. the in vivo application of mirna- through systemic or local injection of pei/mirna complexes resulted in efficient mirna delivery and in antitumor effects, based on the concomitant repression of erk . likewise, tumor growth inhibition was observed upon treatment of tumor-bearing mice with pei-complexed mir- a. this is due to the mir- a-mediated downregulation of pim- expression and resembles the pim- knockdown through rnai / pim- sirnas. taken together, in tumor xenograft mouse models we establish mirna replacement therapy through the pei-complexation of mirnas as a novel therapeutic strategy and demonstrate that mir- and mir- a may be promising mirnas in colon carcinoma therapy. md , a hybrid of chloroquine and primaquine, is a potential drug against infectious diseases such as malaria. since one moiety of the hybrid, the known antimalarial drug chloroquine, is a known intercalator, the potential of md to intercalate into dna was determined. due to the ability of intercalators to cause frame shift mutations, the mutagenic potential of md was also investigated. the potential of md to intercalate into dna was investigated by means of fluorescence based micro plate assay using ethidium bromide (eb) and isolated calf thymus double stranded dna. as a positive control chloroquine was used. the potential of md to cause gene mutations was determined using the hypoxanthine-guanine phosphoribosyltransferase (hprt) test in chinese hamster v lung fibroblasts (v cells). v cells were treated with . µm, . µm and . µm md or the positive control, the direct mutagen -nitroquinoline-n-oxide (nqo, µm) for h. on day , mutants exhibiting loss of hprt function were selected with -thioguanine . whereas at µm chloroquine, a % decrease in fluorescence intensity of eb (indicating dna intercalation) was observed, µm md were needed to observe a similar decrease ( %) in fluorescence intensity of eb. therefore, md is fold less potent to intercalate into dna than its moiety chloroquine. the frequency of spontaneous -tg resistant mutants per colony-forming cells was ± . as expected, µm nqo caused a significant increase in the mutant frequency (mf, ± ) . in contrast, mf was not significantly affected by treatment with md at both noncytotoxic ( . µm: ± ) and cytotoxic ( . µm: ± ) concentrations. in conclusion, md is a less potent intercalator than the known antimalarial drug chloroquine. furthermore, md does not cause gene mutations in the hprt test. since current studies show that various metabolites of md are formed in vitro, their mutagenic potential is currently under investigation as well. -conducting channels but depends critically on the membrane potential. trp channels form cation entry channels thereby either contributing to ca + entry or depolarisation. recently, we showed that trpm acts as a ca + -activated non-selective cation channel and critically determines the driving force for ca + influx in mast cells following fcεri-stimulation ( ) . in addition to trpm we also identified the expression of other trp transcripts in bone marrow derived mast cells (bmmc) including those encoding trpc , trpc , trpc , trpc and trpm . in peritoneal mast cells (pmc), rt-pcr indicated expression of trpc , trpc , trpc , trpc , trpm , trpm and trpm . to identify the functional role of those trp channel proteins for mast cell activation we analysed ca + signaling using microfluorimetry in bmmcs and pmcs after stimulation with substances known to activate trpc channels in other cell systems such as the diacylglycerol analogue oag, the hyperforin analogue hyp- and flufenamic acid (ffa), but could not evoke a rise in the [ca + ]i in both pmc and bmmc. sphingosine phosphate and lysophosphatidylcholine, which were reported to activate trpc channels, induced only minor rise in [ca + ]i in bmmcs, respectively. here, we will present our analysis of ca + signaling following stimulation of the fcεri receptor and application of secretagogues that are supposed to affect ca + -dependent mast cell activation such as adenosine, endothelin- , substance p and compound / in bmmcs and pmcs derived from mouse lines with inactivation of trpc , trpc , trpc , trpc or trpc since specific antagonists are still lacking for these trp channels. the α a-ar is the main ar in the central nervous system and it plays a crucial role in regulating norepinephrine (ne) release from nerve terminals via presynaptic feedback inhibition. it is also associated with a number of physiological effects, including hypotension, pain perception, sedation and modulation of mood. ne, once released in the synaptic space, binds to α a-ars and induces a rearrangement of the receptors from the inactive state into an active conformation. this allows the binding and activation of the cognate gi-protein and, hence, the transduction of the transmembrane signal to the downstream effectors. generally, α a-ar activation has been deduced from the stimulation of a receptor-mediated biological response that could be easily followed experimentally. however, most of these approaches do not employ living cells and are normally applied under equilibrium conditions that need prolonged incubation periods incompatible with the physiological temporal dynamics of ne. here, we monitored the ne-mediated α a-ar and gi-protein activation by using a fluorescence resonance energy transfer (fret)-based approach in living cells. to examine the effects of increasing concentrations of ne on the speed and extent of α a-ar activation with very high temporal resolution, we took advantage of the previously described α a-ar flash/cfp sensor [ ] . the results indicate that in our system the efficacy of ne in eliciting α a-ar flash/cfp activation increases in a time-dependent way and reaches the maximum with a half-life of ~ ms. the ec values decrease in an exponential manner and arrive at ~ µm with a half-life of ~ ms. next, we analyzed the ability of increasing concentrations of ne to trigger a downstream intracellular response after α a-ar stimulation by monitoring the kinetics and amplitude of gi activation in living cells. we applied the previously well characterized gi cfp/yfp sensor [ ] . the results show that both the efficacy and the potency of ne in inducing gi activation reach the steady state slower compared to receptor activation (half-life ~ ms and ~ , ms respectively). in conclusion, we were able to monitor ne-mediated events occurring in the millisecond time scale and reaching the equilibrium in a time interval compatible with physiological conditions. sphingosine- -phosphate (s p) is an immune modulator produced by sphingosine kinase (sphk ) and sphingosine kinase (sphk ) and de-phosphorylated or degraded irreversibly by s p phosphatases and a lyase, respectively. we recently showed that tlr -induced il- p is selectively counter regulated by sphk , s pr and its extracellular ligand s p. on the other hand, spiegel et al. have demonstrated that specific, sphk -dependent, binding of s p to traf enhances the tnf-alpha signaling. therefore we were interested whether the tlr/tir and tnf-alpha-signaling pathways are interfered with each other and are modulated by s p. in a first approach we focused our investigations on sphk effects on both traf and traf stimulatory signals and cytokines produced downstream. experimentally, with gm-csf expanded, bone marrow-derived dcs we first desensitized the lps-tlr or cpg-tlr signal by a defined time period of costimulation with tnf-alpha. the initial results showed a partial decrease of il- p secretion in tnf-alpha-co-stimulated dcs in contrast to lps stimulation alone. this might indicate that traf activated via tnf-alpha interacted with the traf pathway to reduce il- p . further series with dcs derived from sphk -deficient mice confirmed our former results that il- p in contrast to other cytokines is specifically sensitive to sphk -s p feedback, but did not change the effects of tnf-alpha on wt dcs il- p release. in comparison, cpg-tlr -induced il- p release reached only % of lps-induced il- p levels and was less sensitive to tnf-alpha costimulation. however, sphk -deficiency strongly augmented cpg-dependent il- p production. in ongoing experiments we started to analyze the details of traf /rip and traf /tak activation by ubiquitination blots in wt, sphk -and s plyase-deficient dcs. in conclusion, we hope to unravel possible mechanisms of the observed differential effects of s p and its enzymes on inflammation and cancer-relevant cytokines. identification of the kh type splicing regulatory protein (ksrp) as a new important mediator of the anti-inflammatory effects of resveratrol art j. , besche v. , bros m. , li h. , handler n. , bauer f. , erker t. , behnke f. , mönch b. , förstermann u. , dirsch v. m. , werz o. , kleinert h. , pautz a. university of vienna department of pharmacognosy, althanstr. , wien, austria resveratrol, a polyphenol derived from different plants, possesses multiple pharmacological functions such as anti-oxidative, anti-diabetic, cardioprotective, anticancer, neuroprotective and anti-inflammatory properties. many of these effects have been attributed to its anti-oxidative activity but resveratrol also modulates signal transduction pathways like the p mapk pathway or the activity of different transcription factors like nf-κb redox-independently. moreover, the histone deacetylase sirtuin (sirt ) is an important mediator of resveratrol effects. nevertheless the direct molecular target of resveratrol remains unclear. in target fishing experiments we identified the rna-binding protein ksrp as direct resveratrol binding partner. ksrp is an rna-binding protein that controls proinflammatory gene expression on the post-transcriptional level by modulation of mrna stability. moreover, it is involved in the biogenesis of mirnas. resveratrol treatment of human dld- cells resulted in a decreased mrna expression of a number of well known ksrp target mrnas and enhanced mirna- function. downregulation of ksrp expression by sirna prevented the mrna destabilizing effect of resveratrol. as the activity of ksrp is mainly regulated on the post-translational level by phosphorylation of different serine and threonine residues we analyzed whether resveratrol changes ksrp activity by altering the phosphorylation of the protein. indeed, our immunoprecipitation experiments demonstrated that resveratrol reduces the p mapk-mediated phosphorylation of threonine residues in the ksrp protein and thus leads to an increase of ksrp activity. interestingly, resveratrol does not block p mapk activation or activity. in addition we have evidence that sirt is not involved in the resveratrol mediated activation of ksrp. so we believe that activation of ksrp by resveratrol is the major mechanism mediating the anti-inflammatory effects of resveratrol. in vitro testing of oecd reference nanomaterials (nm-series) in rat precision cut lung slices aumann a. the oecd has defined reference nanomaterials (nm) to be tested in different endpoints concerning human health and environmental safety ( ) in order to evaluate if the toxicity of nanomaterials can be linked to their physico-chemical properties. for nanomaterials, inhalation presents the major exposure route of concern and can be assessed using acute inhalation toxicity studies in rodents. however, these in vivo studies are resource intensive and animal consuming. the oecd working party on nanomaterials has named several alternative methods as being of particular interest for testing of nanomaterials; among them is the precision-cut lung slices model (pcls) to estimate respiratory toxicity. we have tested all nm in pcls measuring cytotoxicity, apoptosis, oxidative stress and inflammatory response of the tissues as well as observing them histological. for in vitro exposure of pcls the test material was dispersed in medium. since it is the nature of these materials to change their surface characteristics and agglomeration state in different environments, a standardized dispersion method (nanocare) using bovine serum albumin as a stabilizing agent, was used. particle size-distributions of the nanomaterial dispersions were characterized via analytical ultracentrifugation and found the nanomaterials well dispersed. silver and zinc oxide but none of the other nm showed cytotoxicity to the lung tissue in the tested concentrations. however, differences in cytokine profiles among the nm were observed and showed several correlations to the results obtained in in vivo inhalation or instillation studies. universität des saarlandes institut für molekulare zellbiologie, gebäude , homburg, germany tmem proteins show similarities in their primary sequence to motifs that are conserved amongst various members of the trp protein family. based on hydropathy analysis these proteins exhibit to membrane spanning domains. in contrast to trp channels there is no evidence that these proteins form ion channels in the plasma membrane following overexpression of their cdna in hek cells. tmem -/mice are viable and show no obvious signs of disease, but exhibit increased pancreatic amylase and lipase plasma levels. microfluorimetric measurements using fura- revealed that the elevation of the cytosolic ca + concentration after stimulation with carbachol and the cholecystokinin analogue caerulein is unchanged in tmem -deficient acinar cells. tmem is expressed in several cell types including pancreatic acinar cells, cardiac myocytes, cardiac fibroblasts, but their subcellular localization is still unkown. we generated several constructs encoding tmem fusion proteins with fluorescence protein tags by fusing eyfp, mcherry and tagrfp-t to the n-and c-terminus of the protein, respectively. based on western blot experiments and expression in hek cells the tmem -eyfp construct was most suitable for further colocalisation analysis and generation of viral vectors including adenovirus and semliki forrest virus. in contrast to the prediction by the psort ii algorithm tmem -eyfp could not yet be identified in the plasma membrane of fibroblasts, cardiac myocytes or acinar cells but showed a vesicular subcellular localization pattern. we localized tmem -eyfp in acidic compartments and predominantly in lysosomes (pearson coefficient (pcc) , ± , , n= using lysotracker ® dye). analysis of subcellular localization with independent tmem fusion constructs and additional vesicular markers will be presented as a framework to get insights towards the cellular function of tmem and to reveal the mechanisms underlying increased amylase release from acinar cells of tmem -/mice. the small molecule bcl- /mcl- inhibitor tw- shows single-agent cytotoxicity in neuroblastoma cell lines bachmann h. s. , akdeli n. high-risk neuroblastoma (nb) remains a therapeutic challenge in paediatric oncology. pro-survival bcl- family proteins critically regulate apoptosis, and may represent important therapeutic targets in nb. primary nb tumours heterogeneously express mcl- or bcl- , with high expression correlating to high risk phenotype. co-expression can be detected in approximately % and is correlated to reduced survival. recent studies with two inhibitors that predominantly target bcl- and other proteins, but not or to a lesser extend mcl- , elucidated the importance of mcl- inhibition for cytotoxicity in nb. tw- is a small molecule inhibitor that showed almost equal affinity to bcl- and mcl- . to explore the effect of combined bcl- /mcl- inhibition on neuroblastoma cells, four cell lines (sk-n-as, imr- , sy y and kelly) were treated with tw- and changes in growth properties were determined. furthermore, nude mice with kelly (human neuroblastoma cell line) xenografts were treated with tw- . using sirna, we investigated the functional relevance of mcl- and bcl- in kelly cells. for in vitro cell viability we observed ic values of . ± . µmol/l. on treatment with µmol/l dose of tw- , all neuroblastoma cell lines analyzed showed significantly reduced proliferation and increased apoptosis rates. bcl- as well as mcl- knockdown induced apoptosis in kelly cells. interestingly, tw- was able to reduce, but not to abrogate growth of kelly neuroblastoma xenografts in nude mice. in conclusion, combined inhibition of bcl- and mcl- using tw- exhibits strong single-agent antitumor activity on human neuroblastoma cells in vitro, but limited single-agent activity in vivo. therefore, inhibition of bcl- /mcl- may represent an interesting therapeutic strategy, most likely in combination with conventional chemotherapy and other specific inhibitors. localization and functional characterization of membrane transporters for sulfated steroid hormones in the human testis bakhaus k. , wapelhorst b. circulating sulfated steroid hormones like estrone sulfate (e s) or dehydroepiandrosterone sulfate (dheas) are delivered to the testis via membrane uptake carriers such as the sodium-dependent organic anion transporter (soat). inside the cell these sulfated steroids can be metabolized to active steroid hormones by the catalytic activity of the steroid sulfatase (sts), which shows high enzymatic activity in the testis ("sulfatase pathway"). in addition to soat, other candidate carriers like the organic solute carrier protein (oscp ) and the organic anion transporting polypeptides oatp a and oatp c are predominantly expressed in the human testis and demonstrate transport activity for sulfated steroids. we aimed to evaluate the cellular expression of soat and the other steroid sulfate carriers and their co-localization with the steroid sulfatase (sts) in human testis. furthermore we want to perform functional transport studies with the steroid sulfate carriers in stably transfected hek cells. we detected soat by rt-pcr and western blot analysis in the human testis. single cell analysis and in situ hybridization revealed pachytene primary spermatocytes to express the soat mrna. soat expression in specimens showing maturation arrest at the level of early round spermatids seems to be severely reduced or absent. sts mrna was detected by rt-pcr in testis homogenates. preliminary immunohistochemical data showed that sts may be expressed in germ cells and interstitial leydig cells. hek cells stably expressing the soat carrier protein showed significant transport activity for dheas. this was demonstrated by using a radiolabeled [ the hepato-intestinal induction of the detoxifying enzymes cyp a and cyp a by the xenosensing pregnane x receptor (pxr) constitutes a key adaptive response to oral drugs and dietary xenobiotics. in contrast to cyp a , cyp a is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven disturbances of the steroid homeostasis. using cell lines and mice transgenic for a cyp a promoter we demonstrate that the cyp a expression in these organs is noninducible and independent from pxr. instead, it is enabled by the loss of a suppressing yin yang (yy )-binding site from the cyp a promoter which occurred in haplorrhine primates. this yy site is conserved in cyp a , but its inhibitory effect can be offset by pxr acting on response elements such as xrem. taken together, the loss of yy binding site from promoters of the cyp a gene lineage during primate evolution may have enabled the utilization of cyp a both in the adaptive hepato-intestinal response to xenobiotics and as a constitutively expressed gene in other organs. our results thus constitute a first description of uncoupling induction from constitutive expression for a major detoxifying enzyme. they also suggest an explanation for the considerable tissue expression differences between cyp a and cyp a . serum albumin adducts as biomarkers for systemic bioavailability of active metabolites of various glucosinolates in animal models and humans barknowitz g. , engst w. glucosinolates (gls) are natural pesticides of brassicales, which comprise many important food and feed plants. upon physical damage to the plant, the enzyme myrosinase can convert gls to reactive metabolites (e.g. isothiocyanates). the same reaction can also be catalyzed by enzymes of the intestinal microbiota. modification of sensor proteins (e.g. keap- ) by some gls metabolites leads to adaptive responses, resulting in enhanced detoxification of reactive metabolites and other protective reactions. at least in experimental models, this mechanism can be exploited for chemoprevention of carcinogenesis induced by various chemical carcinogens. however, own research revealed that certain reactive gls metabolites can covalently bind to dna in vitro and in vivo, involving possible genotoxic and carcinogenic risks. animal models are useful for studying beneficial and adverse effects of gls. however, it has to be taken into account that the toxicokinetics of gls may differ between rodent and humans and that the active metabolites are short lived and thus difficult to detect and quantify. likewise, exposure of humans to gls and their breakdown products may enormously vary depending on (i) food preferences, (ii) cultivars, growth conditions and preparation of plants consumed and (iii) variations in human xenobiotic metabolizing system and composition of intestinal microbiota. in order to estimate individual levels of systemic exposure to reactive gls metabolites, blood protein adducts may be useful. we have developed lc-ms/ms methods for quantifying serum albumin adducts formed by glucoraphanin, glucotropaeolin and neoglucobrassicin. the method involves digestion of the protein to amino acids and the usage of isotope-labelled amino acid adducts as internal standards. serum albumin adducts were detected in mouse models after feeding broccoli and pak choi respectively, as well as after administration of purified gls and breakdown products. likewise, gls adducts were detected in human blood plasma after consumption of broccoli or cress. this work was financially supported by the bundesministerium für bildung und forschung (grant d) . barlow s. harrington house, harrington road, brighton, bn re, great britain values for cancer endpoints for the application of the ttc approach have been derived by linear extrapolation of results from animal carcinogenicity studies to calculate "virtually safe doses" (vsds). a vsd is defined as an exposure that represents an estimated upper bound increase in risk of in a million of developing cancer during a lifetime. in , from consideration of a range of vsds for carcinogens, the us food and drug administration (fda) proposed and adopted a value of . micrograms/kg of diet ( . ppb) as a threshold of regulation to be used for substances present in food contact materials. the fda considered that if exposure to a substance in the diet was below this value, consumers would be protected "with reasonable certainty of no harm" and no toxicological data on the substance need be submitted. the value of . ppb is equivalent to . micrograms/person per day, assuming that g of food and g of fluids diet might be consumed daily. this value was subsequently incorporated into the ttc approach to be used for assessment of substances without a structural alert for genotoxicity. in , kroes and colleagues further explored cancer as an endpoint and recommended a lower ttc value of . micrograms/person per day for substances with a structural alert for genotoxicity and exclusion from the ttc approach of certain groups of high potency carcinogens (with vsds below this value). in this presentation, the data underpinning these ttc values will be discussed from the perspective of their reliability for risk assessment of substances with low exposures, for which there are no toxicity data, but which may in fact be genotoxic or non-genotoxic carcinogens. stable conjugates which are recognized by the immune system. in the current investigations, the ability of chemical pre-treatment to interfere with antibody-protein binding has been investigated using ovalbumin (ova) as the model protein and naturally occurring anti-ova igg from healthy human donors. preparation of conjugates: ova ( mg/ml) was dissolved in sodium borate buffer ( . m, ph . ) . for chemical treatment various amounts ( - mg) of -fluoro- , dinitrobenzene (dnfb; sensitizer) or , -dichloro- -nitrobenzene(dcnb; non-sensitizer) was added and stirred for hrs at room temperature. unbound compound was removed by consecutive dialysis against phosphate buffered saline (pbs) and distilled water. inhibition elisa: plates were coated with µg/ml ova and blocked with % fcs in pbs. ova samples (native or conjugated; . - µg/ml) were pre-incubated with polyclonal antibodies (ab) from pooled normal human serum for min and then added to the plates. human anti-ova igg abs were detected by colorimetric analysis using ortho-phenylendiamine as a substrate. the concentration of soluble native ova or conjugate required to displace % of ab to plate-bound ova (ic ) was calculated (minimum of n= independent experiments). treatment of ova with the chemical sensitizer and protein reactive dnfb resulted in increased ic value, whereas mock treatment resulted in comparable ic values to native ova. treatment with dcnb showed that the presence of a chemical per se (and possible denaturation) was not sufficient to alter the ic value; conjugation of the compound to the protein was required. the analysis is not test chemical specific (specific ab against compound-protein conjugates are not required) and the colorimetric analysis is unaffected by absorbance of the compound itself. thus, this method may have utility for the identification of chemical sensitizers which are directly protein reactive. ´-deoxy-camp in human cell lines: another second messenger? beckert c. , hinz c. we have shown that ´-deoxy-camp (dcamp) is synthesized by recombinant human soluble adenylyl cyclase (sac). here, we report that dcamp can be detected and quantified by liquid chromatography coupled to mass spectrometry (lc-ms) in various human cell lines. in most cells a ratio of camp : dcamp of ~ was observed. as a remarkable exception, in hl- promyelocytic leukemia cells, the dcamp concentration exceeded the camp concentration more than -fold. a differential regulation of camp versus dcamp was determined upon replacement of the incubation medium (proliferating condition with serum / serum-free resting condition). for example, camp was dramatically reduced in hek cells after hours under resting conditions whereas dcamp was significantly increased. in cellular subfractions of hek cells ac assays (mn + /forskolin-or mn + /bicarbonate-stimulated) with either atp or datp as substrate revealed that comparable amounts of camp and dcamp accumulated. in addition to sac, membranous acs such as ac v were capable of forming dcamp with vmax and km values for datp comparable to those for atp. we also analyzed the substrate-specificity for several human phosphodiesterases. pde and pde hydrolyzed dcamp more effectively than camp. taken together, these data point to a putative second messenger role of dcamp in human cells. we are currently investigating the regulatory role(s) of camp and dcamp in apoptosis of hek cells. as a novel tool for these studies, we will use the cellpermeant dcamp-acetoxymethylester which penetrates the plasma membrane and releases dcamp intracellularly. the biogenic amine histamine is recognized by target cells via four different histamine receptors subtypes (h r -h r), which all belong to the family of g-protein-coupled seven-transmembrane receptors. histamine plays a crucial role in allergic reactions such as rhinitis or conjunctivitis and also in allergic asthma. previously, we showed an interaction of the effects of antagonists at the h r and h r in a mouse model of allergic asthma. however, not much is known about the signaling pathway activated by murine h r and h r. in order to analyze these signaling pathways, we established a cellular model using transfected hek cells which stably express recombinant mh r or mh r. proper expression of the receptors was verified by western blot analysis and flow cytometry. in functional assays we demonstrated that histamine stimulation results in the increase of intracellular ca + concentration ([ca + ]i) in cells expressing either of both, mh r (pec = . ) or mh r (pec = . ). as a second readout, we analyzed the modulation of forskolin-induced camp-accumulation. in mh r-expressing cells the intracellular camp concentration was increased by stimulation with histamine, while in mh r-expressing cells forskolin-induced camp accumulation was reduced. the histamine-induced effects in h r-expressing cells were blocked by the h r antagonist mepyramine ([ca + ]i: pkb = . ) and those in the h r-expressing cells by the h r antagonist jnj ([ca + ]i: pkb = . ) or by pertussis toxin, which selectively blocks receptor gi-protein coupling. jnj , which behaves as a partial mh r agonist in the steady-state gtpase assay using membranes of infected sf cells, was without effect on [ca + ]i and forskolin-induced camp-accumulation in the mh r-expressing cells. currently, we are investigating mitogen-activated protein (map)-kinase pathways activated by the h r and the h r. using a phospho-map-kinase array, histamine dependent phosphorylation of erk / , p , jnk, creb, pkb (akt), and mkk / were detected in cells expressing either of both, mh r and mh r. in summary, the hek cell lines stably expressing selective histamine receptors are very useful tools to investigate hxr signaling pathways in-vitro. enhanced fibroblast motility in the absence of the β regulatory subunit of voltage-activated calcium channels belkacemi a. cavβ subunits of voltage-activated ca + channels are required for trafficking the poreforming cavα subunit to the plasma membrane and modulate the kinetics of its current. mouse embryonic fibroblasts (mefs), acutely isolated cardiac fibroblasts (cfs) and nih t fibroblasts do express cavβ and cavβ subunits, but we could not detect any voltage-activated ca + influx. whereas in mouse cardiomyocytes or hek cells coexpressing cavβ and cavα subunits a dihydropyridin-sensitive voltage-activated ca + influx was readily detectable. apparently, cavβ subunits serve functions in fibroblasts unrelated to voltage-activated ca + influx. among the proteins potentially interacting with cavβ are the inositol , , -trisphosphate receptors (ip rs) [ , ] . we therefore coexpressed mouse cavβ and mouse ip r type , or in cos- cells and found coimmunoprecipitation of ip rs using an antibody for cavβ and vice versa. to study the release of ca + from ip -sensitive stores we performed fura- measurements on fibroblasts isolated from wild type and cavβ -deficient mice either in the presence of thapsigargin or after stimulation of gq-coupled receptors by par- , lpa or bradykinin. receptor-activated ca + release was more pronounced in β -deficient mefs and cfs, whereas thapsigargin-induced ca + release was the same in cells from both genotypes. in addition, ip production measured by a radioreceptor assay was already increased in β -deficient cells under basal conditions. fibroblasts are migrating cells and involved in various physiological and pathophysiological processes. we therefore started in vitro assays for proliferation, migration and angiogenesis as well as in vivo assays for skin wound healing. angiogenesis and proliferation were apparently not different in both genotypes but migration (measured as transwell migration and in scratch assays) and wound healing were affected in different ways. fluorescent staining of cytoskeleton and quantification of the f-actin/g-actin ratio show similar results in both genotypes, suggesting that the increased migration rates and wound repair in β knockout may result, in part, from the increased amount of ip -releasable ca + . [ ] berggren, yang, murakami, et al., removal of ca channel β subunit enhances caoscillation frequency and insulin exocytosis. cell , ( ) - . [ ] müller, haupt, bildl, et al., quantitative proteomics of the cav channel nanoenvironments in the mammalian brain. pnas , ( ) - . bender-sigel j., closs e. i. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße , mainz, germany human cationic amino acid transporters (hcat) are a family of multimembrane spanning proteins that mediate the transport of cationic amino acids through the plasma membrane. our earlier results have demonstrated that activation of either protein kinase c (pkc) by pma or cdc by egf leads to an internalization of these transporters. in addition, in a recent collaboration with the group of alexander sorkin (university of colorado denver) we found that ubiquitination and clathrin-dependent endocytosis are necessary for the down regulation of hcat- -mediated arginine transport by pma (vina-vilaseca et al, j biol chem : ) . this mechanism requires nedd e ligases, but hcats do not contain a ppxy motif to bind the ligases, suggesting that an adaptor protein takes part in this process. however, an involvement of the adaptor protein beta-arrestin in this mechanism could be excluded. using sirna against pkc alpha we now show that pkc alpha is the major isoform that induces the reduction of arginine transport in human u glioblastoma cells overexpressing hcat- a-egfp. in addition, sirna-mediated knock down of cdc prevented the decrease of hcat- amediated transport induced by pma. taken together pkc seems to negatively influence the constitutive cycling of cats by activation the ubiquitination machinery and clathrinmediated endocytosis. cdc is part of this pathway. converging of the classical mitochondria-related pathway in parkinson and nuclear dna-repair signaling? scherr a. -l. parkinson disease is the second most neurological disorder worldwide. despite the fact that most cases are idiopathic and only few can be traced back to specific genes, general progression between both tracks of the disease is comparable. the variety of clinical symptoms in motor control like tremor, rigor and postural problems all originate from loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. several proteins mutated in pd are involved in surveillance pathways, monitoring functionality and integrity of proteins and organelles either by the proteasome degradation machinery or by clearance of mitochondria via autophagy (mitophagy). disturbed calcium-and redox-homeostasis seems to play a major role in susceptibility to cell death signals in dopaminergic neurons, but if this is a preceding or successive event in cell death related to pd progression is not known. on the other hand, experimentally elicited parkinsonism by oxidative stress inducers like paraquat or rotenone and mptp (inhibitors of mitochondrial complex i) lead to damage in nuclear dna and activation of the dna repair protein poly(adp-ribose) polymerase . by consuming substantial amounts of its substrate nad + , this enzyme can drastically decrease energy levels and disturb the redox balance within a cell, also sensitizing it to stress induced cell death. inhibition of poly(adp-ribose) polymerase has been proven to be beneficial to some extent in cell culture models as well as in experimental pd in mice. our research focuses on a putative crosstalk mechanism we recently discovered between the two pathways of experimentally induced cell death in culture models, i.e. mitochondrial signaling and parp -dependent poly(adp-ribosyl)ation. both converge on two mitochondrial chaperones, mortalin and trap . whereas mutations in mortalin have been reported recently to be responsible for some parkinson disease cases in humans, trap is a specific target of the kinase pink (pten induced putative kinase), which is often mutated in autosomal-recessive forms of the disorder. pink is a central regulator of the mitophagy process, tagging mitochondria with dissipated membrane potential for destruction. we could show now that both chaperones bind to short-chain poly(adp-ribose) specifically synthesized by poly(adp-ribose) polymerase . we will present our most recent findings about regulation of these two chaperones after application of parkinson-inducing toxins. aldrich). the effect was reversible within ten minutes when cells were re-incubated in regular cell culture medium. stimulatory effects were not due to osmolarity or cell stress due to medium exchange. analysis of different components of both media (table ) revealed that bicarbonate stimulates accumulation of ccmp and cump besides cgmp and camp in a time-and concentration-dependent manner. bicarbonate is known to activate soluble adenylyl cyclase (sac) and particulate guanylyl cyclase g (pgc-g), regulating sugar metabolism, sperm motility and olfaction by synthesis of camp and cgmp, respectively. in order to identify a responsible cyclase for ccmp and cump generation after bicarbonate treatment, we are currently analyzing transiently and stably transfected hek cells overexpressing various known adenylyl and guanylyl cyclases (sac, mac , mac , mac , soluble guanylyl cyclase, pgc-g, pgc-a, and pgc-d) for their pyrimidinyl and purinyl cyclase activity in vivo and their regulation by bicarbonate. in addition, cell fractions will be analyzed for the detection of specific cyclase compartments. question: long term ventricular pacing, especially at the right ventricle (rv), results in left ventricular (lv) failure. there are several lines of evidence that disturbed ca + homeostasis is involved in the pathophysiology of human heart failure. in this study we examined if ventricular pacing affects the na + -and ca + -channels and the expression of ca + -handling proteins and investigated if there is a differential effect between right ventricular free wall (rvfw) pacing and left ventricular apex (lva) pacing. methods: after av-node ablation minipigs underwent ventricular pacing at beats/min (ddd mode) for one year. minipigs were paced from the rvfw and minipigs from the lva, respectively. minipigs with normal sinus-rhythm served as control group. patch-clamp-experiments were studied to measure na + -and ca + currents. western-blots were carried out to investigate the expression of the ca +handling proteins l-type ca + -channel, serca and phospholamban. results: both rvfw-and lva-pacing led to significant decreased ca + -currentdensities in cardiomyocytes of the lv compared to the control group. the plateau phase of the action potential was significantly shortened after ventricular pacing in relation to control minipigs. furthermore cardiomyocytes of rvfw-and lva-paced minipigs had significant lower na + -current-densities than control minipigs. the action potential amplitude was significantly decreased after rvfw-and lva-pacing whereas the diastolic potential remained unchanged. the expression of the l-type ca + -channel was significantly reduced after ventricular pacing, regardless of the pacing site. in contrast rvfw-and lva-paced minipigs showed significant increased serca -expression. the expression of phospholamban remained unchanged after rvfw-and lva-pacing compared to control minipigs. conclusion: in a chronic animal model ventricular pacing leads to remodeling of ionchannels and ca + -handling-protein-expression, regardless of the pacing site. investigation on metabolic competence of dermal systems: native human skin, in vitro skin models and keratinocytes blatz v. the implementation of reconstructed human skin equivalents (rhes) as an alternative method for dermal toxicity testing became very prominent in the last decades. their advantages are e. g. the human cell origin and an organ-like d structure. already regulatory accepted methodologies are widely in use for testing the skin corrosion (oecd ) and irritation (oecd ) within rhes. but there are still some questions open, one of them the metabolic competence of such dermal systems. in this context, enzyme activities of oxidizing (cyp; fmo; adh; aldh) and conjugating enzymes (nat; ugt) were investigated in subcellular fractions of in vitro systems such as keratinocytes and rhes (epidermis model epiderm tm (mattek), full-thickness skin models epiderm tm ft (mattek) and phenion ® ft (henkel ag)) and compared to those of native human skin. activities of cyp a, b and a isoenzymes were measured fluorometrically by oxidative desalkylation of alkoxyresorufines. fmo / activities were evaluated by hplc/fld detection of n-oxygenated product of benzydamine [ ] . adh and aldh activities were investigated by photometrical detection of nadh generation during ethanol (adh) [ ] or propanal (aldh) oxidation [ ] . nat activity was followed by hplc/uv detection of acetylated p-aminobenzoic acid. ugt activity was quantified fluorimetrically by glucuronidation of methylumbelliferone [ ] . during the course of this study the following results were observed: (loq = limit of quantification) since the metabolic competence of rhes is confirmed, these in vitro systems are estimated as suitable for further toxicity tests (e. g. genotoxicity by comet assay), where metabolic activation of substances may play a crucial role. however, for the data assessment, the determined metabolic profiles should be taken into account. we acknowledge bmbf funding this project ( d). signalling pathway indicates that in b cells the adenine receptor couples to a gqprotein followed by activation of phospholipase c pathway. these findings represent a new signalling pathway of the adenine receptor and allow the assumption that different adenine receptor subtypes exist in the rat brain. in the scope of the project lexukon ("foodborne exposure to environmental contaminants -data analysis to support and standardise exposure assessments based on nvs ii") exposure to the heavy metals cadmium (cd), lead (pb) and mercury (hg) via food consumption has been assessed for the german adult population based on the national nutrition study ii ( the updated intake assessments show that especially foods regularly consumed such as vegetables and grain contribute mainly to exposure of cd that is about . µg/kg body weight (bw) per week for average consumers over all food groups. this corresponds to % of the tolerable weekly intake (twi) of . µg/kg bw for cd defined by the european food safety authority (efsa) in . beverages and vegetables are the food groups most relevant for exposure to pb. about . µg pb/kg bw is taken up by average consumers that is below the benchmark dose for renal toxic effects ( . µg/kg bw) defined by efsa for the weekly pb intake. for hg the intake amounts for all population groups examined were significantly below the toxicological reference values. for average consumers the weekly intake of hg is . µg/kg bw that is primarily taken up by eating fish and fishery products. however, individual population groups and high consumers reach and/or exceed the toxicological reference values for the daily intake amounts for cd and pb. high consumers almost reach the twi for the cd with %. for pb a weekly intake of µg/kg bw was estimated for high consumers that exceeds the benchmark dose for renal toxic effects for the weekly pb intake. the results show that data collection should also focus on highly consumed and not only on highly contaminated foods. further, uncertainties in concentration levels should be reduced e.g. by lowering and standardizing the analytical limits. it's recommended to consider further measures in view of the reduction of contents of environmental contaminants in foods. however, other sources can also contribute to the intake of the mentioned heavy metals (e.g. smoking). major cell biological processes are regulated by rho-gtpases, actin-mediated processes in particular. amongst others, rho-gtpases are stimulated by the receptormediated activation of gα / and gαq via specific rhogefs. the p rhogef is activated by gαq and plays a major role in the acute response of vascular smooth muscle cells to angiotensin ii treatment. the aim of the present study was to establish a fret-assay between gαq-cfp and venus-p rhogef and characterize the dynamics of p rhogef-gαq-interaction in single living cells. the fusion of p rhogef with venus resulted in a functional gαq-regulated p rhogef-protein as determined by means of rho-luziferase-assays. whereas no specific fret signal was observed between the two interaction partners in the absence of receptor stimulation, a robust and rapid fret signal developed in response to stimulation of histaminergic h and cholinergic m -recptors. the onset of this signal after rapid application of agonist paralleled gαq activation kinetics. similar to the kinetics of gαq-protein deactivation the dissociation of p rhogef and gαq after withdrawal of agonist was slow (tens of seconds). the specificity of the fret signal between gαq-cfp and venus-p rhogef was verified by introducing point mutations rendering p rhogef unable to bind to active gαq. furtehrmore we observed a robust acceleration of the dissociation of p rhogef and gαq upon cotransfection of rgs , suggesting a very short lifetime of the p rhogef-gαq-complex or the ability of rgs to bind to p rhogef-associated gαq. taken together, fret-based imaging of the interactions between p rhogef and gαq revealed fast interaction kinetics closely resembling g-protein activation kinetics, both of which can be regulated by rgs . toxicity of silver nanoparticles in intestinal cells boehmert l. the rapid development of nanotechnology has been accompanied by an increased concern for the safety of nanomaterials. especially silver nanoparticles are used in many manufacturer identified consumer products including silver coated food contact materials or hydrosol silver supplements. these products lead to an intentional or unintentional oral uptake of silver nanoparticles and hence to a contact with the intestinal barrier. the human cell line caco- is a well established model system in studying effects on human enterocytes. although these cells are colon carcinoma cells and exhibit typical features of cancer cells when they are kept sub confluent, these cells have the capability to differentiate into polarized cells with morphological and biochemical properties of small enterocyte cells. we investigated the effects of silver nanoparticles on these colon carcinoma (proliferating) and small intestinal epithelium like (differentiated) caco- cells. the silver nanoparticles agpure were commercially available from rent-a-scientist gmbh. the behaviour of these silver nanoparticles in cell culture medium were characterised using asymmetric flow-fild flow fractionation (a f), small ankle x-ray scattering (saxs) and dynamic light scattering (dls). we investigated the particle toxicity on both cell states using cell titer blue assay, xcelligence impedance measurements, annexin-v and caspase measurements, diclorofluorescein assay and antioxidant pre incubated cells. the agpure stock solution is an aqueous suspension of silver particles with a metal core radius of . nm stabilised with tween- und polyoxyethylenglycerol trioleate. agpure silver nanoparticles were toxic for proliferating as well as differentiated caco- cells in a time and concentration depending manner. the presence of foetal calf serum in the incubation medium has a minor influence on the toxicity. prior to cell death, morphological abnormal adherence characteristics and morphological changes in the cells were observed using microscopy and quantified by xcelligence impedance measurement. it is concluded that cell death is caused by an oxidative stress related mechanism rather than apoptosis. the release of sphingosine- -phosphate from human platelets during acute coronary syndrome is attenuated by aspirin böhm a. , polzin a. in addition to atherosclerosis ttp knock out mice develop more cardiovascular dysfunctions. in tail vein bleeding assays we monitored a significant difference in the bleeding times of ttp deficient mice in comparison to wildtype mice, triggered by a stronger granulopoeisis. our results leed us to the assumption that the chonic inflammation seems to be more improtant for the development of cardiovascular diseases in ra patients than the traditional risk factors. differentially expressed cardiac genes in a mouse model with heart-specific overexpression of pp a bollmann p., makarova e. a., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. , halle (saale) , germany in transgenic (tg) mice with cardiac myocyte-specific overexpression of the catalytic subunit of protein phosphatase a (pp a) reduced cardiac protein phosphorylation, cardiac hypertrophy and impaired cardiac contractility were noted compared to wild type (wt) littermates. the hearts of tg mice also suffered from ventricular dilatation and a diminished response to β-adrenergic stimulation. analyses of mrnas expressed in tg and wt hearts (n= ) using affimetrix mouse genome microarray chips resulted in several candidate genes possibly differentially regulated. in this study, we focussed on verifying the mrna data of selected genes important for stress response and signal transduction on protein level in cardiac homogenates by western blotting. hearts from wt littermates were used as control. compared to wt heat shock protein (hsp ) and calcium calmodulin dependent protein kinase type ii (camkii) mrnas were upregulated in tg but only hsp protein was increased (p< . , n= - ) but not camkii (p> . ). protein phosphatase type (pp ) and superoxide dismutase (sod) were downregulated on mrna level in tg but on protein level this could be found only for sod (p< . , . in contrast, pp protein was upregulated (p< . , in tg compared to wt. for comparison the regulatory a-subunit of pp a and hsp were studied. both genes were unchanged on mrna level in tg: western blotting revealed the same results for the corresponding proteins. in summary, mrna expression data could only partially be confirmed on protein level elucidating the importance of western blotting studies. these data indicate that increased pp a activity is associated with modified gene expression in tg hearts possibly affecting stress response and regulation of cell signalling. (supported by the deutsche forschungsgemeinschaft) center for regenerative therapies, technische universtität, dresden, germany cell therapy in the form of beta cell replacement to cure diabetes has been practiced for decades without become a routine clinical therapy. more widespread clinical application is hindered by the scarcity of suitable organ donors, a dramatic loss of transplanted cells within the first days post-transplant, the requirement of long term immunosuppression to maintain graft survival, and despite this, a loss of graft function from a recurrence of autoimmunity in some patients. research is currently dedicated to overcome each of these limitations. additional beta cell sources investigated include embryonic stem cell derived insulin producing cells, human insulin producing cells lines, and xenogeneic beta cells. parallel to these efforts are the development of encapsulation devices to protect these sources from immune and inflammation mediated destruction, and transplantation into new sites such as the muscle and bone marrow to infuse beta cells. additional therapy to reduce immune suppression includes the infusion of t regulatory cells to control autoimmune and alloimmune response, and cytokine and chemokine receptor directed compounds aimed at blocking early inflammation or autoimmunity. these efforts are likely to lead to an expansion of clinical activity to replace beta cells in diabetes, and to novel pharmaceutical therapies that may be more generally applicable in patients with diabetes. pdgf-bb induces the h s producing enzyme cystathionine-γ-lyase via a rosdependent mechanism in rat renal mesangial cells boosen m. , hassan m. there is increasing evidence that hydrogen sulfide (h s) that is endogenously produced in several cell types serves as a potent gasotransmitter in a wide variety of physiological processes involving vascular homeostasis and inflammation. in the present study we investigate the expression and the regulation of the hydrogen sulfide synthesizing enzyme cystathionine γ-lyase (cse) in cultured rat renal mesangial cells. as demonstrated by qpcr and western blot experiments, mesangial cells showed a marked time-and dose-dependent upregulation of cse mrna and protein levels after treatment with platelet-derived growth factor (pdgf-bb). the cse upregulation by pdgf-bb is accompanied by a marked increase in reactive oxygen species (ros) formation. interestingly, co-administration of the ros scavenger n-acetylcysteine, the glutathione peroxidase mimetic ebselen and the nadph oxidase inhibitor diphenylen iodonium chloride (dpi) drastically reduced pdgf-induced cse expression, indicating a role for endogenously produced ros in mediating regulation of cse. as demonstrated by electrophoretic mobility shift (emsa) experiments pdgf-bb induces binding of the redox-sensitive transcription factor nf-e -related factor (nrf ) to a consensus antioxidant response element and this effect was also diminished by co-administration of antioxidants (dpi, nac, ebselen) . furthermore, lps/ifnγ-as well as pdgf-bb-induced cse upregulation was nearly completely abolished in nrf -/spleen macrophages and mesangial cells, respectively. as a consequence of the elevated cse levels we could demonstrate increased h s levels and a higher cse enzyme activity in mesangial cells after stimulation with pdgf-bb by using the colorimetric methylenblue method and a cse activity assay. importantly, in a rat model of anti-thy- -induced proliferative glomerulonephritis we observed a marked upregulation of cse protein during the course of the disease paralleled by a stabilization of nrf protein. from our data, we hypothesize that pdgf-bb-mediated regulation of cse via a redox-mediated activation of nrf may constitute a protective mechanism during glomerular inflammatory disease. rac knockout protects from acute hepatic damage following doxorubicin treatment bopp a., wartlick f., fritz g. heinrich-heine-universität institut für toxikologie, universitätsstr. , düsseldorf, germany rac belongs to the best characterized members of the ras-homologous (rho) family of small gtpases, which are key regulators of the actin cytoskeleton. furthermore, rac is part of the activation of the nadph oxidase, which produces reactive oxygen species and regulates the activity of stress kinases (e.g. sapk/jnk) and transcription factors such as nf-κb and ap . anticancer drugs cause dna damage, which in turn stimulates the dna damage response (ddr) regulating dna repair, cell cycle progression and, in case of non-repairable dna damage, triggers apoptosis. so far, a role of rac in the ddr has not been reported. based on its exceptional function as a regulator of transcription and because of its recently found ability to translocate to the nucleus, we hypothesize that rac may be involved in the ddr. to study the in vivo function of rac we used an inducible cre-based knockout mouse model (rac flox/flox/mxcre ). mice were treated with different doses of doxorubicin for different periods of time. we monitored gh ax foci formation as a marker of dna strand breaks, used the masson-goldner staining for the detection of collagen accumulation, analyzed phosphorylated histone as a marker of mitotic events and performed a tunel assay to detect apoptotic cells. in the absence of rac the basal mrna expression of pro-fibrotic ctgf was decreased. collagen levels were increased and mmp mrna expression was reduced in the liver of rac -/animals as compared with rac proficient animals. in addition we found more apoptotic cells in rac -/mice. hours after treatment with the anthracycline derivative doxorubicin the number of gh ax foci in rac -/animals was reduced in comparison to rac +/+ animals. we also found lower level of ctgf mrna expression and reduced amount of collagen in rac -/mice. none of these protective effects resulting from rac deficiency could be detected after administration of three consecutive doxorubicin injections over a time period of days. there were no significant differences in the number of gh ax foci or collagen accumulation. the mrna expression of ctgf was even higher in rac -/animals. furthermore the number of mitotic events was almost two times higher in the rac -/mice compared to the rac +/+ mice. summarizing, our findings show that impaired hepatic expression of rac protein is hepatoprotective against acute damage following doxorubicin exposure, but does not protect against doxorubicin-induced subacute toxicity. in vitro cytotoxicity of tbhq (tert-butyl-hydroquinone) braeuning a., vetter s., schwarz m. institut für experimentelle und klinische pharmakologie und toxikologie toxikologie, wilhelmstrasse , tübingen, germany at high concentrations, tert.-butyl-hydroquinone (tbhq), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. here we describe that treatment of murine t cells with tbhq in -well culture plates induces the death of untreated cells in neighboring wells on the same plate. the mechanisms underlying that effect were investigated. death of the seemingly untreated neighboring cells was caused by a more toxic and volatile tbhq oxidation product which was formed in a non-enzymatic process involving metal ions and oxygen. the unexpected perturbation of cytotoxicity testing by the volatile tbhq metabolite shows that not only metabolic processes, but also non-enzymatic mechanisms have to be considered as important parameters for in vitro assays. furthermore, our data show that even cells several wells distant from the site of treatment do not necessarily constitute proper "untreated" controls when cells are treated with tbhq, e.g. in assays aimed to analyze the activity of the tbhq-inducible nrf pathway. s angiotensin ii causes oxidative stress and dna damage in mouse kidneys via the angiotensin ii type receptor brand s. , amann k. , schupp n. universität würzburg institut für pharmakologie und toxikologie, versbacherstr. , würzburg, germany universität erlangen-nürnberg institut für pathologie, krankenhausstraße , erlangen, germany angiotensin ii (ang ii), the reactive peptide of the renin-angiotensin-system, causes vasoconstriction and, in higher levels hypertension, which is connected with an increased cancer risk in the kidney. treatment of male c bl/ mice with ang ii results in the formation of superoxide radicals and dna damage in the kidney as well as in the heart. to answer the question if the dna damage is caused by hypertension or by elevated ang ii concentrations, mice were treated with different compounds: the angiotensin-converting-enzyme blocker ramipril, the ang ii receptor blocker ramipril, the ang ii receptor candesartan, the antioxidant tempol and the vasodilator hydralazine. the effect on blood pressure and renal function of ang ii-treated c bl/ mice was examined. treatment with ang ii led to a significant increase in blood pressure. candesartan and hydralazine led to a decrease, whereas intervention with ramipril and tempol had no effect. equal conditions could be found by examining renal function regarding the excretion of urinary albumin, which was ameliorated by candesartan and hydralazine. in addition, histopathological changes were investigated. there was significant glomerular damage and tubulointerstitial damage in ang ii-treated animals compared to control animals, which was significantly improved by candesartan and tempol. hydralazine and ramipril mitigated the observed renal damage but were less effective than candesartan. furthermore, the ang ii-induced formation of superoxide radicals in the kidney and the heart was slightly affected by all interventions. genomic damage, in the form of double strand breaks was prevented by the ang ii receptor antagonist candesartan and the antioxidant tempol. to sum up, the results from this study show that ang ii induces the elevation of markers of kidney failure and dna damage, which is prevented by substances lowering blood pressure like candesartan, showing the receptor responsibility for the induction of dna damage. actually by substances not lowering blood pressure like tempol, the oxidative stress and dna damage was ameliorated, showing the involvement of reactive oxygen species. optimization of the balb/c- t cell transformation assay by coupling a drug metabolizing system brauneis m. d., steinberg p. stiftung tierärztliche hochschule hannover institut für lebensmitteltoxikologie und chemische analytik, bischofsholer damm , hannover, germany the analysis of the carcinogenic potential of chemicals plays an important role in toxicology. up to now the acquisition of such data requires a large amount of animal experiments. the aim of this study is to reduce the number of experimental animals being used by further optimizing the balb/c- t cell transformation assay, an already well-established in vitro method. this method, which is also well suited for high throughput screening applications, allows a quantitative analysis of the aforementioned carcinogenic potential. the incubation of balb/c- t cells (murine embryonic fibroblasts) with mutagenic compounds leads to a loss of contact inhibition between these cells, which results in the development of so-called foci. these foci can be distinguished by characteristic changes in cell growth behaviour, a result of the treatment with carcinogenic compounds, and their number is therefore directly related to the genotoxic potential of the latter. a major disadvantage of the "classic" balb/c- t cell transformation assay is that a number of compounds initially require a metabolic transformation to gain their full genotoxic potential. hence, without prior metabolic transformation many chemicals are not detected as carcinogenic in the abovementioned test system. to overcome this drawback the balb/c- t cell transformation assay has been coupled to a drug metabolizing system, in this case the so-called liver s . in a first step the well-known genotoxic agents benzo[a]pyrene, aflatoxin b and nnitrosodimethylamine were tested in this assay. all three compounds led to a concentration-dependent increase in the number of foci formed, whereby this concentration-dependent increase was observed in a non-cytotoxic concentration range. in a next step the balb/c- t cell transformation assay will be coupled to further drug metabolizing systems as well as to the soft agar assay. this study is being financially supported by the stiftung set and the doerenkamp-zbinden foundation. adenylyl cyclases (acs) synthesize the second messenger camp. the family of acs consists of nine membranous and one soluble isoforms with ac and ac being the predominantly expressed isoforms in the heart. in the heart, acs integrate β-adrenergic (β-ar) signaling as the main physiological mechanism to improve cardiac performance. although ac and ac share high sequence homology, opposing effects on cardioprotection have been reported, where disruption of ac , as well as overexpression of ac both exerting beneficial effects in heart failure. prospective pharmacological treatment of heart failure on the level of ac is under investigation. our study explored the impact of ac ko on ac-activities in the heart at a functional level. complementary, mrna expression studies of the β-ar-g-protein-ac signaling cascade were performed to detect possible compensatory alterations. hearts from - week old homozygote ac knockout and wild-type male littermates were examined in this study. ac activities where measured in cardiac membrane preparations from left ventricles. ac activities were assessed under β-ar and g-protein (g s) stimulation by isoproterenol, guanosine '-triphosphate (gtp) and '-o-( thiotriphosphate) (gtpγs) as well as for direct activation by forskolin. relative mrna expressions for ac - , gs-, gi-a and β -, β -ar where measured by quantitative realtime pcr. surprisingly, assessment of basal, β-ar and g-protein-mediated ac-stimulation as well as direct activation by forskolin revealed no changes in ac activities. besides from detection of the ac knockout, mrna expressions analysis of ac - , gs-, gi-a and β -, β -ar did not detect any compensatory alteration. these findings suggest that proximal adrenergic signaling in the heart does not necessarily require ac . whether physiological integration of beta adrenergic signaling in the heart is mediated by both isoforms ac and ac , or can be attributed to one main isoform remains to be elucidated. melanocortin-promoted pka activation decreases ampk activity via erk- / and lkb- in hypothalamic gt - cells breit a., ellen d., gudermann t. goethestrasse , münchen, germany α-melanocyte stimulating hormone (α-msh)-induced activation of the melanocortin- receptor (mc r) in hypothalamic neurons increases energy expenditure and inhibits food intake. intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of amp-activated protein kinase (ampk) that has recently been reported to enhance food intake in rodents. until now, it is not clear if α-msh affects ampk via direct intracellular signaling cascades or if the release of paracrine factors is involved. herein, we used a murine, hypothalamic cell line (gt - cells) and monitored ampk phosphorylation at thr which has been suggested to increase ampk activity. we found that α-msh dephosphorylated ampk at thr and consequently decreased phosphorylation of the established ampk substrate acetyl-coa-carboxylase at ser . inhibitory effects of α-msh on ampk were blocked by specific inhibitors of protein kinase a (pka) or extracellular-regulated kinases- / (erk- / ), pointing to an important role of both kinases in this process. since α-msh-induced activation of erk- / was blunted by pka inhibitors, we propose that erk- / serves as a link between pka and ampk in gt - cells. furthermore, down-regulation of liver kinase b- (lkb- ), but not inhibition of calcium-calmodulin-dependent kinase kinase-β or transforming growth-factor-beta-activated kinase- decreased basal phosphorylation of ampk and its dephosphorylation induced by α-msh. thus, we propose that α-msh inhibits ampk activity via a linear pathway including pka, erk- / and lkb- in gt - cells. given the importance of the melanocortin system in the formation of adipositas detailed knowledge about this pathway might help to develop drugs targeting obesity. autism spectrum disorder (asd) is a complex neurodevelopmental disorder with dysfunction of social interaction and communication. a hitherto unknown complexgenetic principle of origin probably underlies asd. so far, more than candidate genes were identified in literature. the patients affected with the monogenic timothy syndrome show multiorgan dysfunction including lethal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia and autism. this single gene disorder serves as a model disease for asd, giving insights in a possible pathophysiology. here, a point mutation in a highly-conserved region of the pore-forming subunit of the voltage-dependent calcium channel (ca v) cav . gene (cacna c) results in incomplete inactivation of the l-type calcium currents (splawski et al., cell ; : - ) . functionally similar biophysical effects can be induced by structural variation β -and β -subunits of the voltagedependent calcium channels (herzig et al., faseb j. ; : - ; jangsangthong et al., pflugers arch. ; : - ) . supported by findings in a meta-analysis of linkage data of asd patients (trikalinos et al., mol psychiatry. ; : - ) , we are investigating a function-based candidate gene hypothesis linking the β subunit gene (cacnb ) with asd. we performed a case control study sequencing all exons and flanking intronic regions of cacnb in patients with asd. we found three rare missense mutations in asd patients, but not in unaffected controls. all three mutations occur at highly conserved positions and might alter protein function; additionally results one amino acid substitution highly probable in a post-translational modification by phosphorylation. so far, we characterized two of these mutations and also a phosphorylation-mimicking mutant in electrophysiological studies. all variants show a decelerated and incomplete time-dependent inactivation of the co-transfected cav . subunit. furthermore, two variants exhibit a significant increased slope factor of voltage-dependent steady-state inactivation. we here present mutations in the β subunit gene of asd patients that result in a retardation of inactivation behavior, thus phenocopying the monogenic timothy syndrome mutations of cav . . β subunit mutations may influence neuronal function or development in some asd patients. jangsangthong, w., kuzmenkina, e., khan, i.f., matthes, j., hullin, r., and herzig, s. ( ) . inactivation of l-type calcium channels is determined by the length of the n terminus of mutant beta ( ) subunits. pflugers arch , - . herzig, s., khan, i.f., grundemann, d., matthes, j., ludwig, a., michels, g., hoppe, u.c., chaudhuri, d., schwartz, a., yue, d.t., et al. ( ) . mechanism of ca(v) . channel modulation by the amino terminus of cardiac beta -subunits. faseb j , - . splawski, i., timothy, k.w., sharpe, l.m., decher, n., kumar, p., bloise, r., napolitano, c., schwartz, p.j., joseph, r.m., condouris, k., et al. ( ) . ca(v) . calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. cell , - . trikalinos, t.a., karvouni, a., zintzaras, e., ylisaukko-oja, t., peltonen, l., jarvela, i., and ioannidis, j.p. ( ) . a heterogeneity-based genome search meta-analysis for autism-spectrum disorders. mol psychiatry , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] background: brain serotonin ( -ht) has been implicated in the regulation of food-intake. the ingestive effects of -ht are mediated by a number of different receptor subtypes under which the -ht a-receptor plays a central role. former in vivo studies have shown an increased intake of food, elicted by -ht-receptor agonists. the aim of this behavioural pharmacologic project was to determine if the hyperphagic effect is mediated by presynaptic -ht a autoreceptors in the raphe nuclei or by postsynaptic -ht a heteroreceptors in serotonergic terminal structures. methods: the effect of the -ht a receptor agonist -oh-dpat ( . , . or . mg/kg) was investigated on feeding behaviour in non-food-deprived young-adult and adult nmri and transgenic l mice. l mice are characterized by an overexpression of postsynaptic -ht a receptors. results: the administration of the -ht a receptor agonist induced hyperphagia in all groups of mice, except for the adult transgenic mice which showed no drug effect. conclusion: the results confirm a key role of the -ht a receptor in food intake. further, we make the assumption that the hyperphagic effect of -oh-dpat is mediated by presynaptic -ht a autoreceptors in the raphe nuclei which decreases -ht function in the central nervous system. it can be speculated that the aberrant feeding behaviour of the adult transgenic mice refers to a possible opposite role of the postsynaptic -ht a receptors. these receptors might affect the release of neuropeptides in the hypothalamus. the efflux transporter abcc (mrp ) expressed at different compartment barriers is important for the elimination of various endogenous and exogenous compounds. with some evidence inflammatory processes regulate abcc expression and cause changes of absorption, distribution and clearance of a number of xenobiotics. the investigation of the influence of interleukin (il) β on abcc mrna and protein expression in various cell lines representing specific tissues is the aim of our study. a further aim is to characterize the signaling pathways regulating abcc expression while inflammation. three different cell lines a) hepg cells (liver tissue) and b) caco (colon tissue) both without naïve il β expression and c) skhep cells representing physiological liver tissue with naive il β expression, were stimulated with different concentrations of il β (range pg/ml to ng/ml). over a period of h samples were taken at defined time points. abcc mrna and protein expression were quantified by qrt-pcr and western blot analysis, respectively. by using small molecule kinase inhibitors for signal transduction proteins (p mapk, akt, erk and jnk) we analysed the signal transduction pathways associated with il β-mediated transcriptional abcc regulation. on abcc mrna level an up-regulation in caco cells ( , fold) and hepg cells ( , fold) within the first hour after stimulation with ng/ml il β was shown. in contrast skhep cells demonstrated a decreased abcc mrna expression ( , - , fold) in comparison to unstimulated controls. the abcc protein expression exhibited a time and il β dependent regulation as well. the analysis for the signal transduction showed for p mapk a moderat time dependet down regulated phosphorylation ( %) in hepg cells whereas it showed no effect in caco cells. concluding, the expression of abcc is regulated moderately by il b in a concentration and time-dependent manner. interestingly, the effects are strongly tissue-dependent concerning abcc expression and signal transduction pathways and show partly contradictory results. the regulation of the different signaling pathways is currently subject of ongoing investigations. introduction: despite the remarkable success of imatinib treatment of chronic myeloid leukemia (cml), therapy resistance emerged as a major clinical problem. the aim of this study was to identify micrornas, which may serve as biomarkers for therapy response or predict pathways involved in pharmacoresistance of imatinib treatment. methods: blood was collected from cml-patients, ten of whom responded to imatinib therapy. after rna extraction from leukocytes, we performed a taqman low-density array screen to determine the expression of micrornas. statistical analysis using the -∆ct method was performed. micrornas showing a p-value< . and a fold change> were considered to be significantly differently expressed. in addition, by using microrna target prediction databases (targetscan , mirdb , pictar , microcosm , diana microt ), selected putative target genes were further functionally investigated by the david bioinformatics database . results: comparing treatment-naïve responders and non-responders four micrornas were identified to be deregulated that were predicted to target genes, especially transcription regulators ( %). pathway analysis showed that six of the predicted genes are relevant in cancer pathways, four of which play a role in cml (smad , nras, rb , raf ). when comparing patients' expression profiles before and under treatment, seven micrornas were identified to be deregulated in responders and five micrornas in nonresponders. ninety-nine targets of the latter include transcription regulators ( %), but also cellular transporters ( %, especially uptake transporters of the slc-family). most target genes are involved in mapk signalling or endocytosis pathways. conclusion: analysis of microrna expression profiles revealed four micrornas involved in imatinib-response and micrornas deregulated during imatinib treatment. predicted target genes code mainly for transcription factors as well as oncogenes relevant for cml and are involved in transporter expression and endocytotic processes. dissociations in the effects of beta -adrenergic receptor agonists on camp formation and superoxide production in human neutrophils brunskole i. , buschauer a. activation of the β -adrenergic receptor (β ar), a classically gs-coupled receptor, in neutrophil granulocytes results in an inhibition of inflammatory responses [ ] , which could be further therapeutically exploited. the aim of the present study was to evaluate the effects of various β ar ligands on cyclic adenosine ', '-monophosphate accumulation (camp assay) and n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced superoxide anion production (o •assay) in isolated human neutrophils, which are a physiologically relevant native test system. camp concentration in neutrophils was determined by hplc/tandem mass spectrometry, and o •formation was assessed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. (-)-isoproterenol, (-)-adrenaline, salbutamol and dobutamine were more potent in inhibiting fmlp-induced o •production than in stimulating camp accumulation. (-)-ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the camp assay, but could partially inhibit fmlp-induced o •production at higher concentrations. moreover, (-)-adrenaline and dobutamine were equi-efficacious in both assays whereas the efficacy of salbutamol was more than two fold higher in the o •assay. this suggests that salbutamol is able to stabilize a different receptor conformation than the other two ligands. thus, ligand-directed signaling via β ar can also occur in human neutrophils. in addition, differences between the data from neutrophils and recombinant test systems [ , ] were noticed, pointing to the problem of insufficient comparability of effects in recombinant and native test systems. the investigation of β ar antagonists on neutrophil granulocytes is subject of ongoing work, in order to find out whether pkb values of β ar antagonists in the camp assay and the o •assay are different. such differences were previously reported for β ar antagonists in other test systems [ ] . moreover, studies with protein kinase a inhibitors should give deeper insight into the signaling events in neutrophils that result in inhibition of fmlp-stimulated o •production and clarify how camp increase interferes with this events. agonist-selective internalization of the human -ht a receptor buchborn t., kahl e., höllt v., koch t. otto-von-guericke-universität magdeburg institut für pharmakologie und toxikologie, leipziger straße , magdeburg, germany the serotonin a ( -ht a) receptor is a g protein coupled receptor and the molecular target of lsd-like hallucinogens. downregulation of -ht a receptors is an adaptive process considered relevant for the therapeutic action of diverse serotonergic antidepressants, such as ssris. since the antidepressant targeting of -ht a receptors, however, is largely restricted to indirect agonists and/or antagonists, little is known about the mechanisms and implications of their regulation by direct agonists. in the present study we, therefore, investigated the capacity of various agonists to regulate the human ha-tagged -ht a receptor by internalization. using immunocytochemical techniques in stably transfected hek cells, we show that agonists differ in their capacity to internalize the receptor. serotonin, quipazine and doi are the agonists most efficaciously internalizing the receptor, dmt and methysergide, on the other hand, hardly internalize; other agonists like psilocin, ergotamine and lsd induce low to intermediate internalization. the specificity of the agonistic effect was demonstrated by the -ht a selective antagonist ketanserin, which blocked the agonist-induced internalization. in additional experiments, we show that the internalized -ht a receptors colocalize with a -labelled transferrin receptors, and that the internalization can be blocked by high molar sucrose; these results are indicative of a clathrin associated sequestration of -ht a receptors in recycling endosomes. also, we demonstrate that the proteinkinase c activator pma efficaciously induces -ht a internalization in the absence of an agonist, and that the doi-induced internalization can be blocked by the proteinkinase inhibitor staurosporine. we, thus, confirm previous findings that the activation of proteinkinases seems to be necessitated for the -ht a internalization to occur. overall, we conclude that the internalization of the human -ht a receptor is agonist-selective, and employs a proteinkinase (possibly pkc) dependent, clathrincoated endosome associated pathway. as there is recent evidence that the regulation of -ht (a) receptors by agonists might have antidepressant (-like) properties, knowledge about the agonist-selective processing of -ht a receptors could help to identify agonists most promising for future (pre-)clinical research. non-clinical safety assessment of homeopathic medicinal products: criteria for establishing a first safe dilution buchholzer m. -l., werner c., knoess w. bfarm bundesinstitut für arzneimittel und medizinprodukte zulassung , kurt-georg-kiesinger-allee , bonn, germany like all human medicinal products the homeopathic medicinal products for human use must demonstrate adequate safety. in general, they are regulated according to the analogue non-clinical safety principles (points to consider on non-clinical safety of homeopathic medicinal products of botanical, mineral and chemical origin, adoption by hma ). one particular approach is the recently introduced concept of a first safe dilution (fsd; introduction to the list of first safe dilutions, adoption by hma ) . this contribution summarizes the first experiences in establishing fsds of a selection of given homeopathic preparations by bfarm. for a given preparation the major toxicological concern and available data set is identified. this determines the safety assessment route: food regulation, permitted daily exposure (pde), threshold of toxicological concern (ttc) or lowest human recommended dose (lhrd/ ). finally the acceptable amount/tolerable daily intake is derived and the respective fsd is calculated. for example the draft evaluation for reserpinum (ph. eur. method . . ) and for atropine (ph. eur. method . . or . . ) based on lhrd leads in each case to a suggested fsd of d related to g of preparation. furthermore, the draft evaluation for potassium iodide (ph. eur. method . . or . . ) based on food legislation emerged a proposed fsd of d related to g of preparation. the concept of fsd combines a scientific and at the same time pragmatic approach in differentiated risk assessment of homeopathic medicinal products. impact of myricetin and its methylated derivatives laricitrin, syringetin and myricetin- `, `, `-trimethylether in c. elegans büchter c. , ackermann d. polyphenolic compounds ubiquitously present in herbal food are discussed to contribute to the health beneficial effects of a diet rich in vegetables and fruits. additional to a strong antioxidative activity of various flavonoids, most of these substances display a variety of other pharmacological properties. we investigated the flavonoid myricetin found in several species of berries, as well as the methylated derivatives laricitrin, syringetin and myricetin- `, `, `-trimethylether. in this study caenorhabditis elegans was used as a model to explore the impact of myricetin and its methylated derivatives in vivo and to investigate molecular modes of action. myricetin ( µm) caused an increase in mean and median adult lifespan of c. elegans. this longevity effect was associated with a decrease of the aging marker lipofuscin as well as a decrease in ros induction, measured by using the h dcf-da assay. however, myrictin failed to improve heat stress resistance, an attribute often associated with longevity in c. elegans. the methylated myricetin derivatives ( µm) showed a decrease in lipofuscin accumulation and ros induction and they further improved the heat stress resistance. in order to elucidate the basis of the life prolonging action of myricetin, we investigated its influence on factors known to have important functions in stress response and the regulation of aging, namely the foxo homologue daf- , the nad + -dependent protein deacetylase sir- . and the heat-shock transcription factor hsf- , respectively. lifespan extension by myricetin disappeared in daf- and sir- . loss of function mutant strains, showing the effect is at least partially dependent on these signaling molecules. by using a hsf- loss of function mutant strain of c. elegans, it was further shown that the life prolonging effect of myricetin is independent of hsf- . in conclusion, our results indicate that the life prolonging effect of myricetin is at least in part dependent on daf- and sir- . , probably due to a modified expression of target genes. stimulatory and inhibitory control of phospholipase c-gamma bühler a., walliser c., becker l., gierschik p. universität ulm institut für pharmakologie und toxikologie, albert-einstein-allee , ulm, germany activation of phospholipase c-γ (plcγ ) upon b cell antigen receptor (bcr) stimulation has been implicated to be a critical step in the bcr-mediated calcium signaling. therefore it is important to understand the mechanisms of how the activity of plcγ is stimulated and inhibited. the mammalian plcs are divided into six subfamilies, designated β, γ, δ, ε, ζ, and η. within the plcγ subfamily, the two plcγ isoforms share a number of features that are distinct from those of the other plc subfamilies. the most striking difference is the insertion of additional domains between the catalytic subdomains x and y. this specific array (sa) contains a second, split pleckstrin homology (spph) domain, consisting of two halves separated by two src homology (sh ) domains and one src homology (sh ) domain. there is abundant evidence in the literature that plcs are autoinhibited in their basal state by structural elements within their x/y linker, pointing to a conserved role of the x/y linker in autoinhibitory regulation of plc isozymes. data from our group show that plcγ is also regulated by autoinhibitory elements within its specific array (walliser et al., ; everett et al., ) . our recent data demonstrated that plcγ is negatively regulated by its sa. specifically, within the sh domain tandem, the c-terminal sh (sh c) and the sh domain in combination, but not either one alone, cause the strongest autoinhibitory control of plcγ . plcγ has been shown to be phosphorylated at tyrosine residues and upon bcr stimulation (kim et al., ) . both tyrosines are located in the linker between the sh c and the sh domain, which we have shown to be the major elements involved in autoinhibitory regulation of plcγ . interestingly, a novel phosphorylation site in plcγ was found in non-small cell lung cancer (nsclc) tissue which is located at tyrosine residue (rikova et al., ) . in this work, we demonstrate, for the first time, the activation of plcγ by phosphomimetic mutations in these three positions and the functional interplay of the three tyrosine phosphorylation targets. most interestingly, mimicking phosphorylation of tyr is critical to fully activate the enzyme. the results not only point to a crucial role of plcγ in pulmonary tumorigenesis, but also prompt and stimulate the search for the protein kinase involved in phosphorylating plcγ at tyr . molecular characterization of hepatotoxic effects of perfluorooctanoic acid (pfoa) buhrke t., scharmach e., lampen a. bundesinstitut für risikobewertung (bfr) lebensmittelsicherheit, max-dohrn-str. - , berlin, germany perfluorooctanoic acid (pfoa) is an industrial chemical that is used for the fabrication of numerous products with oil-, dirt-and water-repellent properties. pfoa is resistant to chemical, thermal and biological degradation and has become a global contaminant of soil, water, air and food in the meantime. the toxicological data of pfoa give cause for concern as the substance was shown to damage the liver of rodents and to impair embryo development. currently, the hazard potential of pfoa for humans is controversially discussed. in this study the human liver cell line hepg was employed to analyse the hepatotoxic effects of pfoa on the cellular and on the molecular level. pfoa was shown to stimulate cellular proliferation at concentrations in a range between µm and µm. at concentrations higher than µm the substance was cytotoxic to the cells (ic µm). cytotoxicity was not due to apoptotic mechanisms as no increase of caspase activity was detected up to a level of µm pfoa. on the molecular level pfoa is known to act as an agonist of the peroxisome proliferator-activated receptor alpha (pparα), and the observed hepatotoxic effects in rodents are associated with pfoa-mediated pparα activation. here we show that pfoa has the capacity also to activate the human isoform of pparα. additional human nuclear receptors were tested for activation by pfoa, and pparγ as well as the pregnane x receptor (pxr) were shown to be activated at high concentrations of pfoa whereas pparδ and the liver x receptor alpha (lxrα) were insensitive to activation by pfoa. notably, we observed a significant inhibition of the activity of the hepatocyte nuclear factor α (hnf α) by incubating the cells already with moderate concentrations of pfoa at a level of about µm. these findings indicate that additional, pparα-independent mechanisms may contribute to the observed hepatotoxicity of pfoa. the elucidation of novel modes of action of pfoa is relevant for the ongoing risk assessment of the substance. s human breast stem cells as a toxicological model for endocrine disruptors, such as soy isoflavones stempin s. , bumke scheer m. (kao et al., ) . two daughter cell lines were developed from m sv after x-ray irradiation (m sv r ) and an additional transfection with a mutated erbb oncogene (m sv r -n ), resulting in high and low tumorigenicity respectively and showing a change in estrogen response after growth in minimal media (wang et al., ) . isolated isoflavones are currently widely used in the treatment of postmenopausal symptoms of women. according to the stem cell theory of carcinogenesis, breast stem cells are the ideal target for the proposed research. however, the epidemiological data to the effect of isoflavone intake on breast cancer is contradictory. therefore, we want to develop a toxicological model using these human breast stem cell lines to test the effect of endocrine disruptors, such as soy isoflavones in a human relevant model. in the present study we analyzed the effect of the phytoestrogen genistein, the most intensively studied soy protein, on the differentiation of the hbec lines. the expression of different luminal epithelial cell markers, estrogen receptors and stem cell markers was measured on the mrna level by quantitative real time pcr. the analysis of several of these markers was also performed on the protein level using western blot. additionally, a broad number of genes related to breast cancer and estrogen receptordependent signal transduction were studied using a commercial pcr-array. in parallel we are also analyzing the changes on the protein level using d gel electrophoresis. we want to use this panel of different markers to establish a toxicological model that can be used in the future to analyze a wide range of different endocrine disruptors. kao cy, nomata k, oakley cs, welsch cw, chang cc. ( ) bronchial asthma is a common inflammatory disease of the airways whose occurrence has increased dramatically over the past decades. histamine plays an important role in mediating the inflammatory response leading to characteristic symptoms like wheezing, coughing, chest tightness, and shortness of breath. since antagonizing the histamine h -receptor (h r) shows no ameliorating effects on asthmatic symptoms, h r antagonists may be new drugs for asthma therapy. in addition to the h r-and h rselective antagonist thioperamide, the selective h r antagonist -[( -chloro- h-indol- yl)carbonyl]- -methylperazine (jnj ) is used in pharmacodynamic studies. a correct interpretation of the collected data requires the detailed knowledge of the pharmacokinetics of the applied substances. for this reason, we developed a fast and robust method based on high performance liquid chromatography coupled to tandem mass spectrometry (hplc-ms/ms) which allows the simultaneous quantitation of thioperamide and jnj as well as the selective h r antagonist -({ - [ -(piperidin- -yl) propoxy]phenyl}methyl)piperidine (jnj ) in murine plasma and lung tissue. the treatment of plasma samples based on protein precipitation performed with a mixture of methanol and . m znso using µl of plasma. analyte extraction from lung tissue was achieved by treating - mg of tissue with a mixture of ethanol and water followed by rigorous mixing using a fastprep-system. ten µl of the extracted samples were transferred to a synergy polar-rp a mercury column ( x mm; µm) connected to a polar-rp security guard. chromatographic separation was performed via a gradient using an acetate buffer (ph ) and methanol at a flow rate of . ml/min. the analytical run-time was minutes. for plasma samples the assay was linear over a concentration range of . - µg/ml for jnj and jnj , and . - µg/ml for thioperamide, respectively. in tissues, thioperamide could be quantified in a concentration range of . - µg/sample, jnj and jnj in a range of . - µg/sample. our results show that the developed hplc-ms/ms method is suitable for the quantitation of all tested histamine-receptor ligands in murine plasma and lung tissue. the functionality of the heart greatly depends on strict homeostasis and interplay of a range of signalling cascades. deregulation of either one is always harmful and eventually detrimental for life. some of the most relevant signals in the adult heart are triggered by the stimulation of g protein-coupled receptors such as adrenergic or angiotensin receptors. those in turn are modulated by a small subset of kinases, the g protein-coupled receptor kinases (grks). interestingly, grks, which for the longest time were believed to regulate only g protein-coupled receptors were shown to modulate also non-receptor-mediated signalling pathways. by now it is well documented that grk plays important roles in both the physiological as well pathological setting of the adult heart. in spite of the important functions of grks in the adult heart, it must be assumed that grk , one of the two main cardiac grks, may also be involved in signal modulation in the course of heart development. deregulation of grk -dependent pathways may very well be causal for impaired cardiac development up to congenital heart disease. in fact, grk is already expressed during embryogenesis and we can detect it in the developing heart. however, grk has not been studied yet for a potential function during embryonic development in general or heart formation in particular. we have established zebrafish as a very time and cost efficient vertebrate model to investigate the role of grk on cardiac signalling and development. tools for grk specific loss-as well as gain-of-function analyses have been developed in our lab. we revealed an unexpected role of grk in the development of left-right asymmetry in zebrafish. clinically, this has been associated with disorders such as heterotaxy and other syndromes linked to ciliary dysfunction. many of those disorders are known to affect proper heart development resulting for example in septum defects or in detrimental translocation of the outflow tract. precisely, depletion of the close homolog of human grk in zebrafish mirrors the human syndrome called heterotaxy by displaying randomized placement of inner organs, aberrant heart looping and disrupted valve formation in the heart. in addition, loss of zebrafish grk results in a lower heart rate as well as dilatation of the embryonic heart at later stages of development. therefore, we believe that grk may potentially serve as a candidate gene for congenital heart disease. identification of a hcn interacting protein in mouse brain cao-ehlker x., hammelmann v., zong x., fenske s., biel m. department of pharmacy, center for drug research ludwig-maximilians-universität, munich center for integrated protein science cipsm, butenandtstr. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] münchen, germany hyperpolarization activated cyclic nucleotide-gated cation channels (hcn) pass a depolarizing current (ih) that is involved in cardiac pacemaking and the control of numerous basic functions in neuronal circuits. the four hcn channel types (hcn - ) display specific expression pattern in brain suggesting that each channel fulfills a distinct physiological function. while hcn , hcn and hcn channels have been studied in quite some detail there is only little information on the particular role of the hcn channel. as an important step towards achieving a better understanding of hcn function we set out in this study to identify proteins that are assembled with hcn in brain tissue. to this end we performed a yeast two hybrid screen with a mouse cdna library using the hcn c-terminal domain as bait. several proteins were obtained and confirmed for interaction with hcn using heterologous coexpression in hek cells. here, we provide an in-depth analysis of the functional interaction between hcn and one of the identified interacting proteins (hip . ). we show that hip . physically binds to hcn channels in vitro and in vivo. still several open issues remain to be clarified i.e. the precise function of tpc and its tissue-specific and subcellular distribution. therefore we established a mouse model with a general deletion of tpcn and generated a series of tpc antibodies. using these tools we investigate the closer molecular and vesicular environment by different biochemical approaches i.e. affinity purification from native tissue derived from wild-type and as a control from knock-out mice, density gradient based vesicle separation, fluorescence activated organelle sorting (faos), total internal reflection fluorescence (tirf) and confocal microscopy. so far, we confirmed the tpc knock-out model by our self-generated tpc antibodies. tpc knockout mice are viable and do not show any obvious deficits. to isolate tpc containing vesicles or protein complexes, tissue or cell culture derived material was prepurified by sucrose density gradient centrifugation. for a subsequent mass spectrometric analysis this preparation was taken as a source material for coimmunoprecipitation or faos respectively. in another approach the migration pattern of tpc containing endosomes on linear density gradients was compared with a series of endolysosomal markers i.e. different rab-, er-, golgi-and lysosomal antibodies. potentially interesting markers were then in turn analyzed for their co-localization with tpc by confocal microscopy/tirf. by combining these results with that from mass spectrometric analysis of faos samples we collect data to get detailed information on the precise endolysosomal distribution pattern of tpc . foxos are involved in a wide spectrum of cellular functions, including cell proliferation, apoptosis and regulation of oxidative stress. in order to identify novel target genes of foxo transcription factors and to achieve further insight into their role in cancer cells, dna microarray analysis was performed using wild type mcf- breast cancer cells and mcf- cells overexpressing foxo. we found that several genes involved in the tnf receptor/nf-κb pathway were differentially regulated. one of the genes that was identified to be up-regulated in foxo overexpressing cells was a a negative regulator of nf-κb signaling pathway. at both mrna and protein level foxo -dependent up-regulation of this ubiquitin modifying enzyme was confirmed. to determine whether a is a direct target of foxo , a luciferase reporter containing a . kb of the a promoter was co-transfected with different amounts of foxo wild-type expression construct. foxo induced a dosedependent increase in a promoter activity, supporting the assumption that a is a direct transcriptional target of foxo . overexpression of foxo led to decreased activity of nf-kb signaling pathway as confirmed by reporter gene and nf-kb specific elisa assays. in addition, mcf- cells can be sentisized to tnf-α mediated cytotoxicity which is assocciated with a dimineshed activation of nf-κb. altogether, we identified a , an ubiquitin modifying enzyme, as a novel foxo target gene. our data implicate that sustained foxo expression may be involved in regulation of tnf receptor/nf-κb pathway and leading to reduced cell survival. trpm is a bi-functional protein consisting of a transient receptor potential ion channel segment linked to an α-type protein kinase domain. trpm is essential for motility, proliferation and cell growth. up-regulation of trpm function is involved in anoxic neuronal death, cardiac fibrosis and tumor cell proliferation. recently, we have demonstrated that the recombinant trpm channel is inhibited by the known modulators of sk - channels such as antimalarial plant alkaloid quinine, cyppa, dequalinium, ns , ska , ucl . the most potent of these compounds, ns (ic . µm), interferes with the regulation of trpm by cytosolic mg + . here we show that ns ( µm) fully and reversibly inhibits native trpm -like currents in hek cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. furthermore, we examined whether targeting of the native trpm currents by ns would impact cellular processes known to be affected by a genetic inactivation of trpm . we found that ns ( - µm) suppressed motility of hek cells without a detectable effect on cell viability. taken together, our findings indicate that ns is a potent and reversible inhibitor of endogenous trpm currents and may be a good candidate drug for pharmacological targeting of trpm . sulfur mustard ( , '-dichlorodiethylsulfide; sm) is a highly toxic and mutagenic warfare agent classified as a weapon of mass destruction. as soon as sm was first used as a warfare agent, research aimed at the development of an effective antidote was launched. early studies with first-generation inhibitors of poly(adp-ribose) polymerases (parp) have revealed promising therapeutic potential in sm-induced skin injury, but the underlying mechanism remains elusive. the current renaissance of parp inhibitors in cancer chemotherapy has revived the discussion on their use for treatment of sm injury. thus we established a comprehensive study aiming the elucidation of the role of parp in sm pathology based on model substance -chloroethyl ethyl sulfide (cees), which is not classified as warfare agent. we have recently demonstrated that parp becomes rapidly activated in living human keratinocytes (hacat) after treatment with cees. the maximal parp activity was observed minutes after treatment with mm cees. the activation was transient and dose dependent. to our knowledge this is the first demonstration of parp activation after treatment with mustards in the context of live cells. an important question is how parp- becomes activated upon treatment with mustards. parp- is a first-line protein involved in the cellular response to dna strand breaks. however, mustards do not directly induce large numbers of such lesions. one possibility is that parp- is activated by dna breaks incorporated as base excision repair (ber) or nucleotide excision repair (ner) intermediates. thus, we performed knockdown experiments of ape and ercc , i.e. endonucleases involved in ber and ner, respectively. the reduction of ape expression had no effect on parp activity. surprisingly, the knockdown of ercc almost completely abolished the cellular par production after cees treatment. the functional consequence of the errc -parp cross-talk with regards to adduct removal is under investigation. however, our present data indicate that parp activity is not obligatory for the survival of cells upon cees treatment, as revealed by the lack of effect of the potent parp inhibitor abt- . expression and activity of g protein coupled receptor kinase (grk ) are elevated in several conditions of compromised heart function. although grk inhibition has been characterized as a promising therapeutic strategy in heart failure, a specific grk inhibitor is not available. raf kinase inhibitor protein (rkip) inhibits raf but it also acts as a physiological inhibitor of grk upon phosphorylation by pkc at serine . a detailed understanding of the rkip/grk interaction may help to identify inhibitory compounds for grk . since phosphorylation often induces homo-oligomerization of proteins, we investigated whether this could be implicated in switching rkip from a raf -into a grk -inhibitor. co-immunoprecipitation assays showed that rkip self-association was substantially increased after pkc-mediated phosphorylation of rkip. rkip mutants either lacking or mimicking s phosphorylation confirmed that this phosphorylation is indeed a prerequisite for rkip/rkip association. cross-linking experiments with myc-tagged rkip in living cells or with purified rkip revealed that rkip phosphorylation by pkc promotes rkip dimers -not oligomers. to test whether dimerization is a critical step for the association of rkip with grk , we generated a peptide to inhibit rkip dimerization. intriguingly, the peptide did not only prevent rkip dimerization but also attenuated rkip/grk association. this implicates, that dimerization of rkip is essential to bind grk . to determine whether rkip dimers consequently inhibit grk activity, we established rkip mutants with high tendency to form dimers. subsequent functional analyses demonstrated that enhanced dimerization of rkip indeed translates into increased grk inhibition. we conclude that pkc-mediated phosphorylation of rkip is important for dimerization and that these dimers are essential for grk binding and inhibition. our results reveal new insights in the molecular mechanism of rkip/grk interaction and will help to develop specific grk inhibitors. expression and function of trpm ion channels in epithelial mdck cells dembla s., meiser j., philipp s. university of saarland institute for experimental and clinical pharmacology and toxicology, kirrberger str. , homburg, germany trpm proteins build ca + permeable cation channels [ ] activated by steroids [ ] and sensitive to increased temperatures [ ] . trpm channels are expressed in pancreatic ßcells as well as neurons of the dorsal root ganglion, where they act as mediators of insulin release [ ] or as nociceptors of noxious heat, respectively [ ] . however, northern blots and in situ hybridization experiments revealed that trpm is also expressed in epithelial cells of the choroid plexus and the ciliary body [ ] as well as in the kidney. pcr analysis of different epithelial cell lines indicated that trpm is also expressed in madin-darby canine kidney (mdck) cells. quantitative analysis of trpm expression by qrt-pcr revealed a ~ fold upregulation in mdck cells grown in confluency compared to well separated, proliferating cells. in contrast the level of expression of trpm , a related ion channel described as regulator of proliferation in other cell types, remained constant. hek cells overexpressing trpm channels did not proliferate in the presence of the trpm agonist pregnenolone sulphate. however, as indicated by impedance analysis, the proliferation of mdck cells in the presence pregs was only slightly affected. when we analysed the transepithelial resistance (ter) of mdck epithelial cells in transwells as a measure for the formation of tight junctions, we found that the ter of cells grown in the presence of pregs was reduced. interestingly, ca + imaging experiments using fura revealed that pregnenolone sulphate induces ca + entry in well separated mdck cells but not in cells growing in confluency. we hypothesize that trpm might act as a regulator of cell proliferation and/or the formation of tight junctions in mdck cells. inhibition of grk by rkip improves cardiac contractility and structure in a transgenic mouse model of heart failure denzinger s., schmitt j. p., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. , würzburg, germany the raf kinase inhibitor protein (rkip) has been identified as a physiological inhibitor of g-protein coupled receptor kinase (grk ). grk initiates g protein coupled receptor (gpcr) desensitization. since expression and activity of grk are upregulated in human heart failure, it has been proposed that grk inhibition may resensitize badrenergic receptor activity in heart failure patients. in this study, we evaluated chronic grk inhibition by rkip as a potential strategy to improve cardiac function in heart failure. to analyse the effect of rkip on heart failure, rkip transgenic mice were crossed with mice carrying a mutation in phospholamban (pln r c ). pln r c causes severe heart failure and premature death in humans and transgenic mice. cardiac function was significantly improved in the presence of rkip as shown by left ventricular catheterization and echocardiography. expression of heart failure marker genes anf and bnp was indistinguishable between wild-type mice and mice co-expressing rkip and pln r c . in line with these findings, the life span of double transgenic mice was significantly prolonged compared to pln r c transgenic mice. slow calcium transport into the sarcoplasmatic reticulum was characterised as cause for dilatated cardiomyopathy of pln r c transgenic mice. since western blot analyses of rkip transgenic heart lysates showed increased phosphorylation of important regulators of cardiomyocyte relaxation, we analysed calcium transients and contractility of isolated cardiomyocytes as possible mechanism of the rkip mediated rescue. in the presence of rkip, calcium reuptake into the sarcoplasmatic reticulum was accelerated and cardiomyocyte relaxation improved. furthermore, coexpression of rkip significantly attenuated pathological cardiac remodelling. interstitual fibrosis and apoptotic cells were quantified in histological sections after sirius red-and tunel-staining. this study revealed a protective function of rkip in a genetic mouse model of human dilated cardiomyopathy by improving cardiac contractility and attenuating interstitial fibrosis and apoptosis. a detailed understanding of this rescue may help to find a new therapeutic strategy to improve cardiac contractility in heart failure. gαi-proteins comprise a group of three highly related members characterized by specific expression patterns. based on previous work of gi-mediated signaling pathways in cardiomyocytes and platelets, we checked gαi expression in mouse heart and platelets. the analysis revealed the presence of gαi and gαi with gαi as the predominant isoform. gene-targeted mice lacking either gαi or gαi were analyzed to unravel the physiological role of gαi-proteins in the cardiovascular system. extraordinarily prolonged bleeding times in gαi -deficient animals were an obvious phenomenon. detailed analysis using isolated platelets gαi -deficient mice exhibited reduced platelet activation and attenuated aggregation in response to stimulation by various agonists accompanied by reduced thrombus formation and diminished stability on a collagen-coated surface. employing in vivo injury/thrombosis models revealed abrogated thrombus formation selectively in gαi -deficient mice. comparable results were obtained in experiments using mice with megakaryocyte/platelet-specific gαi deficiency. to assess the pathophysiological consequences of platelet gαi function, we challenged these mice in experimental models of myocardial and cerebral ischemia. the results clearly show that platelet-gαi -deficient mice were protected from both, myocardial and cerebral ischemia. in contrast, conventional gαi -deficient mice subjected to the heart ischemia/reperfusion model exhibited a significantly increased susceptibility to ischemic injury as compared to wild type controls. in contrast, gαi deficient mice were strongly protected from injury. thus we suggest that gαi and gαi play distinct roles in major cardiovascular disorders pointing to specific, non-redundant functions of these two highly related gαi isoforms. the cgmp signaling pathway is activated by nitric oxide (no), natriuretic peptides (anp, bnp & cnp), and cgmp-elevating drugs. it regulates several physiological functions such as smooth muscle relaxation, platelet inhibition, and cell growth and differentiation. recent studies indicate that cgmp signaling might also play a role in tumorigenesis, but the cellular and molecular mechanisms of cgmp's potential pro-and/or anti-tumor activities are not well understood. this study has examined the expression and function of components of the cgmp pathway in melanoma cells of murine and human origin. we have found that mouse b melanoma cells specifically express the alpha isoform of the cgmp-dependent protein kinase type i (cgkialpha) but not the beta isoform. treatment of intact cells with the membrane-permeable cgmp analog -br-cgmp induced the phosphorylation of cgki substrates, vasodilator stimulated phosphoprotein and phosphodiesterase . anp and cnp, ligands of the membrane-bound guanylyl cyclase gc-a and gc-b, respectively, did also activate the endogenous cgmp/cgki pathway. however, b melanoma cells did not respond to dea-no, which stimulates no-sensitive soluble guanylyl cyclases. interestingly, activation of cgmp/cgkialpha signal transduction was associated with an increase in erk / and p phosphorylation, growth and migration of b melanoma cells. similar results were obtained with wm human melanoma cells. in conclusion, we have identified a gc-a/gc-b/cgmp/cgkialpha pathway in melanoma cells, which stimulates tumor cell growth and migration in vitro. pharmacologic inhibition of cgmp signaling may offer a promising strategy for the treatment of melanoma. an increasing body of evidence supports important roles for voltage-gated calcium channels in idiopathic generalized epilepsies (iges), however which calcium channels participate in ige pathogenesis and how has yet to be fully understood. recently, it has been proposed that cav . (r-type) and t-type calcium channels jointly contribute to oscillatory bursting in the reticular thalamus (rt) , which is associated with absence epilepsy. cav . is one of the two t-type calcium channels known to be expressed in the rt . it has been demonstrated that ablation of either cav . or cav . reduces susceptibility to experimentally induced epilepsy ; and in addition that both channels share several pharmacological properties [ ] [ ] [ ] . to gain further insight into interacting mechanisms of these two channels in epilepsy, we tested cav . (-|-), cav . (-|-) and cav . (-|-)xcav . (+|-) mice side-by-side in the kainic acid model of epilepsy. we provide first in vivo data supporting a synergistic mode of action for cav . and cav . calcium channels in epileptogenesis. the deubiquitinase cyld regulates mechanisms of rip /rip -dependent necroptosis in neuronal cells diemert s. , krieg s. vivo model of cerebral ischemia, we found, that cyld -/-mice exhibit significantly reduced infarction volume compared to control littermates. overall, these data reveal a role for cyld in rip / -dependent mechanisms of necroptosis in a model of glutamate toxicity in neuronal cells and further suggests cyld-mediated mechanisms of neuronal cell death as a potential therapeutic target after acute brain injury in vivo. cyanamide-mediated inhibition of n-acetyltransferase dierolf d., bonifas j., blömeke b. university trier department of environmental toxicology, universitätsring , bldg. n, trier, germany cyanamide has been used for decades for medical purposes in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolised in vivo mainly via coenzyme a dependent n-acetylation by n-acetyltransferases. reported to be a substrate for n-acetyltransferases, cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for nacetyltransferases. therefore a more detailed investigation of its interrelations with nacetyltransferases was performed. we analysed nat enzyme activities after incubation of thp- cells with cyanamide for h, and found that the metabolic conversion of the classic substrate para-aminobenzoic acid was significantly reduced at physiologically relevant concentrations. in detail a significant dose-and time-dependent nat protein inhibition was observed for and µm cyanamide using over-expressed human recombinant nat (insect cell cytosol containing recombinant human nat * ). however, no inhibition was found in the presence of recombinant nat * . as we also provide evidence that cyanamide is not metabolised via coenzyme a dependent nacetylation in vitro by human nat or nat cytosol, by thp- cells or by human liver cytosol, we can conclude that this inhibition is not based on substrate-dependent downregulation of nat . further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of nat , leading to its rapid inhibition. since the presence of the reduction agent dithiothreitol did not reverse the results it could be that it is possibly not caused by oxidative processes. in sum these data indicate that cyanamide is able to interact with cysteine residues of human nat , which causes its inhibition but not by a substrate-dependent mode of action. taken together our results show, that cyanamide is not n-acetylated by human nats, but might modulate nat dependent detoxification and activation of arylamines. dissecting the signal transduction pathway of acute hypoxic vasoconstriction (hpv) in precapillary pulmonary arterial smooth muscle cells ( low levels of oxygen in the pulmonary airways induce acute hypoxic pulmonary arterial vasoconstriction (acute hpv) redirecting blood flow to normoxic areas of the lung to assure optimal uptake of oxygen during ventilation. acute hpv lasting several minutes occurs predominantly in the precapillary region of the pulmonary vascular tree [ ] . therefore, precapillary pulmonary arterial smooth muscle cells (pasmc) have been suggested as sensor as well as effector cells and trpc a member of the classical transient receptor potential (trpc) ion channel family was identified to be essential for the initiation of ca + influx and the subsequent contraction of pasmc [ ] . however, the underlying oxygen sensor and the exact signal transduction pathway(s) in pasmc have not been fully elucidated yet. by using gene-deficient mouse models as well as downregulation of potential candidate proteins by specific small interfering rnas (sirnas), we aim to dissect signaling cascades of trpc channel activation in acute hpv. for pasmc isolation and culture from mice we use a technique based on magnetic separation of intrapulmonary arteries originally developed in rats [ ] . trpc-expression in freshly isolated and passaged pasmc cultured in low ( %) and high fetal bovine serum ( %) was analyzed. interestingly higher passage numbers resulted in a significant down-regulation of trpc and trpc the most predominantly expressed channel monomers in pasmc, while different serum concentrations resulted in no significant changes in their expression rates. sirnas were designed, transfected and successfully tested in hek cells and pasmc. initial results of the dissection of the signal transduction pathway activating trpc and inducing acute hpv in pasmc will be presented. references [ ] staub, n. c. ( ) . site of hypoxic pulmonary vasoconstriction, chest , s- s. [ ] weissmann, n. et al. ( ) . classical transient receptor potential channel (trpc ) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange, proc. natl. acad. sci. u.s.a. , - trpv is a highly selective calcium channel expressed in various tissues amongst others in placenta. the channel may be involved in transcellular calcium transport in epithelial tissues thereby playing some role in calcium homeostasis of the body. in the placenta trpv is assumed to contribute to the maternal-fetal calcium transport. most probably trpv is part of a multiprotein channel complex but most of the components of this complex are unknown so far. our aim is to find interaction partners of the trpv protein in the placenta that might contribute to the regulation of the trpv protein function. therefore we expressed the intracellularly located n-and c-terminal parts of trpv (aa - and - ) as trpv -gst (glutathione-s-transferase)-fusion proteins and used the purified recombinant proteins for pulling down proteins from human placenta cell extracts. the proteins pulled down by this approach were analysed by mass spectrometry. we identified several potential trpv interacting proteins which were not associated with the gst protein only. one of the proteins which was highly enriched with the n-terminal part of the trpv protein is calpain . in contrast to the classical calpains (calcium activated cystein proteases), calpain is unique in that it lacks the cysteine residue in the active site. calpain is mainly expressed during embryogenesis and is reported to be involved in cytoskeleton stabilisation but with unknown function in placenta. the interaction between trpv and calpain was confirmed in reciprocal pulldown experiments and the trpv binding region for calpain was narrowed down by using overlapping n-terminal trpv -gst fusion proteins. after injection of trpv crna into xenopus laevis oocytes calcium uptake into the oocytes was measured; this uptake was largely reduced after co-injection of the calpain crna. in further experiments we want to study potential regulatory effects of the trpv protein on the calpain function in cell culture models. comparative studies on the effects of the human carcinogen inorganic arsenite and its recently identified thiolated metabolite thio-dma v on human urothelial cells ebert f., leffers l., unterberg m., schwerdtle t. universität münster institut für lebensmittelchemie, corrensstr. , münster, germany it has been demonstrated that chronic ingestion of - µg/day inorganic arsenic is associated with an increased risk for cancers of the skin, the lung and the bladder, but until now the underlying toxic modes of action are still under debate. in this context, in the last five years one main focus has been given to the role of human inorganic arsenic metabolism and nowadays it is generally accepted that human biomethylation contributes to inorganic arsenic induced carcinogenicity. due to further improvements in arsenic speciation techniques recently a new thiolated arsenite metabolite, the thio analogue of the well known metabolite dimethylarsinic acid (dma v ), the so called thiodimethylarsinic acid (thio-dma v ), has been discovered in human biological samples. after synthesizing and analytically characterizing this metabolite (bartel et al. , j toxicol. we investigated its toxic effects in direct comparison with ias iii in human urothelial cells. thereby cell cycle studies revealed a g /m-and s-phase arrest as well as subg peak formation in case of thio-dma v . moreover, thio-dma v induced apoptosis (subg , caspase activity) at lower concentrations and earlier time points as compared to ias iii . most likely this is partly due to the higher cellular bioavailability of thio-dma v (aas/icp-ms). regarding genotoxicity, a generation of dna single strand breaks (alkaline unwinding technique) as well as an increased formation of reactive oxygen species (ros, dcfh-da-fluorescence) occurred only at high cytotoxic concentrations. however, thio-dma v strongly increased h o induced ros formation at very low nanomolar concentrations, which might result in cogenotoxic effects. since our earlier studies have shown a strong inhibition of h o induced poly(adp-ribosyl)ation especially by trivalent methylated arsenic metabolites, actual studies investigate the impact of thio-dma v on cellular poly(adp-ribosyl)ation, parp- gene expression, protein level and cellular cleavage, which might hopefully give further hints regarding the mode of action behind thio-dma v induced apoptosis. mitochondrial dysfunction in models of alzheimer´s disease eckert a. universitäre psychiatrische kliniken basel neurobiology laboratory, wilhelm klein strasse , basel, switzerland the histopathological characteristics of alzheimer's disease (ad) are amyloid-ß (aß) containing plaques and neurofibrillary tangles (nfts) as well as neuronal and synaptic loss. until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. there is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several app and tau transgenic mouse models. recently, we examined mitochondrial function in a novel triple transgenic mouse model (pr /app/ps ) -triplead mice -that combines both pathologic features of the disease in brain. using comparative, quantitative proteomics (itraq) and mass spectroscopy we found a massive deregulation of proteins, of which one-third were mitochondrial proteins mainly related to complexes i and iv of the oxidative phosphorylation system (oxphos). remarkably, deregulation of complex i was related to tau, whereas deregulation of complex iv was aß dependent, both at the protein and activity levels. the triplead mice showed synergistic effects of aß and tau already at the age of months, resulting in a depolarized mitochondrial membrane potential. at months, the strongest defects on oxphos, synthesis of atp and reactive oxygen species were exhibited in the triplead mice, again emphasizing synergistic, ageassociated effects of aß and tau in impairing mitochondria. evidences from ad post-mortem brain as well as cellular and animal ad models indicate that aß and tau protein trigger mitochondrial dysfunction through a number of pathways, such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. moreover, recent reports indicate that aß may also interact directly with intracellular proteins such as the mitochondrial enzyme abad (aß binding alcohol dehydrogenase) in executing its toxic effects. mitochondrial dysfunction occurs early in ad, and aß's toxicity seems to be in part mediated by inhibition of abad. in total, a vicious cycle as well as several vicious circles within the cycle, each accelerating the other, can be drawn emphasizing the synergistic deterioration of mitochondria by tau and aß. olesoxime is a novel mitochondrial-targeted compound that is orally active and crosses the blood brain barrier. the cholesterol-oxime targets proteins of the outer mitochondrial membrane and represents a promising drug candidate for neurodegenerative diseases . we evaluated olexoxime's neuroprotective effects against mitochondrial dysfunction in an animal model for alzheimer`s disease (ad). dissociated brain cells (dbc) and mitochondria were isolated from brains of c /bj -thy -appsl (ad-mice) mice that were fed with mg olesoxime/kg feed for months. drug plasma levels reached approx. ng/ml. respiration of isolated mitochondria were significantly diminished in ad-mice due to reduced complex i, i+ii and cox activities. consequently, mitochondrial membrane potential (mmp) was significantly reduced in dbc from ad-mice. olesoxime normalized respiration chain complex activities and the mpp. to further evaluate the beneficial effects of olesoxime on complex i activity, we challenged dbc with rotenone ex vivo and observed that olesoxime treatment was protective. to further clarify the mode of action, we analyzed the ability of olesoxime to prevent opening of the mitochondrial permeability transition pore (mptp) in vitro using energized brain mitochondria by measuring ca + -and atractyloside (atr) induced swelling. the opening of mptp precedes apoptosis and can be induced by mitochondrial dysfunction due to calcium overload, oxidative stress, elevated phosphate concentrations or adenine nucleotide depletion. olesoxime prevented ca + -as well as atr induced mitochondrial swelling. atr inhibits the adenine nucleotide translocase (ant) that requires appropriate membrane properties to mediate mitochondrial permeability transition (mpt). since cholesterol (cho) and its derivates represent potent modulators of membrane viscosity, we related the effects of cho and olesoxime on mptp opening to membrane properties. both, cho and olesoxime reduced the flexibility of membrane acyl-chains in energized mitochondria and prevented atr induced mptp opening. however, cho didn`t prevent ca + -induced mptp opening, indicating a different mode of action for olesoxime. our data confirm olesoxime as drug candidate against mitochondrial dysfunction, which is considered to play a pivotal role in neurodegenerative diseases. the work was supported by the european union under the th framework program for rtd -project mitotarget -grant agreement health-f - - . several inflammatory glomerular kidney diseases are accompanied with a massive production of reactive oxygen species (ros) that may attack the glomerular filtration barrier by affecting podocyte function and may contribute to apoptotic or necrotic cell death of mesangial cells. otherwise, ros also trigger fine-tuned signaling processes that may result in cell proliferation or cell migration. to define such redox-driven signaling devices, we performed a non hypothesis-driven proteomic approach, to identify homo-or heteromeric protein complexes induced by ros. to this end, protein lysates of human podocytes were treated with or without hydrogen peroxide ( µm) for min. thereafter, the cell lysates were subjected to diagonal d gel electrophoresis and putative redox-affected proteins were analyzed by ms/ms-analysis. by this approach, we could identify a series of proteins that form interprotein-disulfide bonds in a redoxdependent manner. one of those proteins could be characterized as the regulatory subunit of protein kinase a (r-subunit of pka), which belongs to the family of serine/threonine kinases. to evaluate whether ros is capable to activate pka also in a more physiological setting, we treated rat mesangial cells with pdgf-bb to induce ros formation and we could demonstrate that pdgf-bb induces dimerization of r-subunits in a redox-dependent manner. to demonstrate whether pdgf-bb induces pkadependent pathways, we analyzed the effects of pdgf-bb on phosphorylation of serine of vasodilater stimulated protein (vasp) a classical target of pka. in fact, pdgf-bb induced vasp phosphorylation independently of intracellular camp levels. moreover, elevating camp levels via activation of adenylate cyclase with forskolin did not change the dimerization state of r-subunits. pdgf-bb-induced dimerization of the r-subunits and subsequent phosphorylation of vasp was blocked by diphenyljodonium (dpi), indicating activation of a nadph oxidase is essential for pka activity. taken together, we demonstrate a redox-dependent activation of pka by pdgf-bb and this may hint also for a probably protective role of ros in rat mesangial cells. testing the potential sensitizing capacity of chemicals is currently done by using the murine local lymph node assay (llna). animal welfare and eu cosmetics directive demands alternative methods to animal tests. the purpose of this study was to establish an in vitro assay for the prediction of skin sensitizers. based on the finding that the majority of skin sensitizers are electrophilic or have the potential to be metabolized to electrophilic substances, it is assumed that they can activate the nrf -keap -antioxidant response element (are) regulatory pathway. here, we report the results obtained from the lusens assay that detects electrophilic chemicals using the nrf pathway. the cell line lusens was derived from immortalized keratinocyte hacat cells and carries a luciferase reporter gene under the control of an are-element from the rat nadph quinone reductase nq . the lusens assay was in house validated with a panel of chemicals and cosmetic ingredients including the performance standard substances of the local lymph node assay. the predictivity of this assay was compared to the predictivity of the murine llna and to human patch test data and can be considered as reliable screening approach (accuracy of % compared to human data). however, in order to cope with the complex multi-step mechanism of skin sensitization, an integrated approach of in vitro assays mimicking several steps was designed; thereof, the are-dependent gene activation represents one module. time-resolved fluorescence ligand-binding assays for parathyroid hormone receptors emami-nemini a. ligand-binding studies represent essential tools for pharmacological research on g protein-coupled receptors. in recent years, time-resolved fluorescence gained significant relevance as readout for ligand binding studies. however, ligand-binding assays for parathyroid hormone receptors (pthrs) utilizing fluorescent parathyroid hormone (pth) were missing. therefore, we generated various fluorescent pth analogues which exhibit properties of native pth in terms of affinity, potency and internalization. for the purposes of academic and commercial research, we utilized labeled pth to set up three time-resolved fluorescence assay formats: (i) classical separation binding assay, based on time-resolved fluorescence and suitable for native receptors; (ii) homogeneous timeresolved fluorescence resonance energy transfer (htrf) based on tag-lite technology for high through-put screening; (iii) htrf based on antibodies, a synergistic approach using htrf with minimized receptor modification. this work will facilitate the development of new drugs directed to the pthr as well as fundamental research on the pthr. anandamide production in eosinophilic granulocytes is independent of il- and eotaxin stimulation engeli s., reinke j., zörner a., tsikas d., jordan j. medizinische hochschule hannover institut für klinische pharmakologie, carl-neuberg-straße , hannover, germany introduction: some animal and in vitro studies suggest that endocannabinoids exert anti-inflammatory effects. specifically, inhaled anandamide reduced the obstructive effect of leukotrien d in airways, and a specific cannabinoid receptor agonist significantly reduced pulmonary inflammation in guinea pigs. we have recently shown that segmental bronchial allergen challenge is associated with significant increases of anandamide concentrations in bronchoalveolar fluid of patients with asthma. the concomitant increase in eosinophilic counts, eotaxin and il- concentrations in bronchoalveolar fluid led us to hypothesize that anandamide is produced by eosinophilic granulocytes in response to chemotactic stimuli. peripheral eosinophilic granulocytes were isolated from whole blood by means of percoll gradient centrifugation and magnetic separation employing cd antibodies conjugated to magnetic beads. isolated cells were counted and anandamide measurements were typically performed in whole cell lysates of . x eosinophils. we stimulated eosinophils with varying concentrations of il- , eotaxin- (ccl ), eotaxin- (ccl ), and eotaxin- (ccl ). to prevent anandamide degradation, a specific fatty acid amide hydrolase (faah) inhibitor (oloxa) was employed. anandamide concentrations and faah activity were determined by stable isotope dilution using lc-ms/ms protocols. results: first, we confirmed the ability of eosinophilc granulocytes to synthesize anandamide. however, cellular anandamide content could only be measured when faah was effectively blocked with oloxa, and strong faah activity was demonstrated in eosinophils. with oloxa, typical anandamide concentrations were in the range of - pm/ . x eosinophils. neither il ( - pg/ml), nor any of the eotaxins ( ng/ml either alone or in varying combinations) did stimulate anandamide production after min of incubation. our results suggest that chemotactic molecules like eotaxin and il- are not responsible for increased anandamide formation in eosinophils during allergen challenge. in a next step, the effects of anandamide on eosinophils and bronchial epithelial cells need to be determined. the suprisingly high faah activity in eosinophils may point to an alternative pathway facilitating prostaglandin and leukotriene synthesis by production of arachidonic acid. screening methodology for estimatation of dermal absorption in vitro fabian e., goth c., guth k., mehling a., van ravenzwaay b., landsiedel r. basf se experimentelle toxikologie, carl-bosch-strasse , ludwigshafen, germany dermal absorption is used in the evaluation of the effectiveness of pharmaceutical or cosmetic formulations, but often dermal absorption is a critical parameter in risk assessment of pesticides or chemicals. therefore, knowledge of dermal absorption is e.g. helpful in formulation development. skin absorption is routinely measured in vivo or in vitro following oecd tg or . however, these tests are complex, time consuming and expensive. therefore, a method was developed to allow simple and rapid screening. the experiment uses dermatomized skin in modified franz type diffusion cells. µl of test substance preparation are applied to the skin preparation. after h, the skin is washed and the amount of penetrated substance is quantified. the receptor fluid and the washing solutions are optimized for subsequent analyses by lc-ms. we performed dermal absorption screenings in parallel to our routine guideline studies and demonstrated a good correlation of the results of both study types: the total recovery found in the screening studies is somewhat lower than in the corresponding guideline studies but is always in an acceptable range above %. the efficacy of the skin washing procedure is lower than under routine conditions, most probably due to the change to an lc-ms-compatible washing solution. overall, the dermal absorption screening is an easy, fast and cost-effective screening method for the estimation of dermal absorption of a wide variety of test substances and formulations. the p tumor suppressor protein is frequently inactivated in human cancers by diverse mechanisms. owing to its fundamental role in the maintenance of genomic stability and cancer prevention, p is an attractive target in cancer therapy and several approaches were pursued to restore p function in tumor cells. polyamidoamine (pamam) dendrons are positively charged molecules with a systematically branched structure that interact with the negatively charged cell membrane, inducing cellular uptake via endocytosis. in the present study, biotin-labeled p protein was attached to a dendronized streptavidin nanocarrier to facilitate its internalization into different tumor cell lines. first, biotin-substituted pamam dendrons were conjugated with streptavidin to allow formation of the dendronized streptavidin nanocarrier. the nanocarrier displayed uptake into hela cervix carcinoma and a lung adenocarcinoma cells without detectable cytotoxicity. biotin-labeled p was then conjugated to the dendronized streptavidin, preserving its specific dna-binding in vitro. immunoblot analysis revealed efficient internalization of biotin-p into hela cells in the presence of dendronized streptavidin. in line with this finding, specific cellular uptake of biotin-p was observed by confocal microscopy, which showed a cytoplasmic and peri-nuclear localization in hela, a and saos osteosarcoma cells. the internalized biotin-p also partially co-localized with early endosomes. importantly, the delivery of biotin-p into p -deficient saos cells resulted in impaired cell viability and upregulation of caspase / activity, demonstrating its biological functionality. this study intriguingly demonstrate the efficient delivery of functional biotin-p into different tumor cell lines using the novel streptavidin nanocarrier, which can be further modified to allow cell-type specific targeting and combined with cytotoxic drugs such as doxorubicin. identification of novel ahr target genes in rat liver oval cells faust d. , vondracek j. the aryl hydrocarbon receptor (ahr) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to , , , tetrachlorodibenzo-p-dioxin (tcdd), some polycyclic aromatic hydrocarbons (pahs) and dioxin-like polychlorinated biphenyls (pcbs), such as pcb . activation of the ahr by these ligands leads to its dimerization with arnt and transcriptional activation of several phase i and ii metabolising enzymes. while it is generally accepted that many pahs are thereby transformed to genotoxic metabolites, this classical signalling pathway so far failed to explain the tumour promoting effects of the nongenotoxic compounds tcdd and pcb . thus, there is an urgent need to define genetic programmes orchestrated by ahr to unravel its role in physiology and toxicology. we have recently shown that treatment of rat liver oval cells with tcdd or pcb leads to a release from contact-inhibition involving activation of the ahr, elevation of jund protein levels and transcriptional activation of cyclin a ( , ) . loss of contact-inhibition is one hallmark of tumour promotion. to better understand ahr-driven pathways we identified the transcriptional programme using high density microarrays in response to pcb . already h after treatment, genes were found to be upregulated and genes downregulated indicating that these are direct ahr-dependent target genes. david analysis revealed that these genes are involved, for instance, in drug and lipid metabolism, cancer pathways, tgf-b signalling and cell-cell communication. ten of the genes were selected for confirmation by semi-quantitative rt-pcr. using the ahr inhibitor ch- and sirna directed against ahr and arnt, we further demonstrated that ahr-and arnt-function is required for transcriptional activation of the selected genes. finally, we identified the transcription factor foxq as a novel ahr target protein in rat liver oval cells. although the function of foxq is poorly understood, it has been shown very recently that foxq is overexpressed in colorectal cancer and is involved in epithelial-mesenchymal transition in breast cancer cells. its function in ahrmediated tumour promotion, however, remains to be determined. the practical relevance of histamine h and h receptors in the brain can be easily deduced since h receptor antagonists of the first generation have a sedative effect and an inverse h agonist, pitolisant (close to its introduction to the market), is active against excessive daytime sleepiness associated with narcolepsy. in this context the question arises whether also h and h receptors possess a practical relevance in the brain. to this end, we examined whether the electrically evoked h-noradrenaline release in superfused human cerebral cortex slices is affected by agonists at the above receptors. the h agonist impromidine µm failed to affect noradrenaline release in human cortex slices although it facilitated noradrenaline release in guinea-pig cortex slices; the maximum extent of facilitation was %, the pec was . and the pa of the h antagonist ranitidine against impromidine was . . with respect to h receptors there is some controversy in the literature whether they occur in the brain at all. however, we were able to detect h receptor mrna in the human and mouse cortex by the reverse transcriptase polymerase chain reaction. in cortex slices of either species, noradrenaline release was not affected by the h agonist -methylhistamine - µm but inhibited by histamine µm via h receptors by and %, respectively. in mouse cortex membranes, -methylhistamine µm also failed to affect s-gtpγs binding although r-α-methylhistamine µm, acting via h receptors, increased it by %. in conclusion, h receptors facilitating noradrenaline release are detectable in the isolated guinea-pig but not human cortex. despite the presence of h mrna in the brain, functional readouts of this receptor, i.e. modulation of noradrenaline release (humans, mice) and modulation of s-gtpγs binding (mice), could not be shown. murine cx promoter activity is dependent on the transcription factor creb fels b., nunes f., schmitz w., müller f. u. westfälische wilhelms-universität münster institut für pharmakologie und toxikologie, domagkstraße , münster, germany connexin (cx ) is a gap junction protein expressed in atrial myocytes and the ventricular conduction system, mediating the electrical intercellular communication in the myocardium. alterations in cx function were linked to the pathophysiology of atrial fibrillation and heart failure. in the heart, camp dependent gene transcription is regulated by members of the creb/crem/atf family which bind to camp responsive elements (cres). similar to the human cx gene promoter, the murine promoter contains one cre. cardiomyocyte-specific overexpression of a crem-isoform (crem-ib∆c-x) led to cx down-regulation, suggesting that creb related transcription factors are involved in cx gene regulation. in order to study the functional role of creb in the regulation of the cx promoter we have generated a luciferase based murine cx promoter reporter gene construct and monitored its activity in a permanent cell line upon overexpression of constitutive-active creb (cacreb) and a non-phosphorylatable dominant-negative creb (dncreb) isoform. surprisingly, overexpression of cacreb and dncreb both lead to a reduction of cx promoter activity (cacreb % ± % vs control % ± %; p< . vs control , n= ), dncreb % ± % vs control % ± %; p< . vs control, n= ). the activity of the murine connexin promoter is modulated by creb. both cacreb and dncreb led to cx down-regulation, which could be explained by induction of inhibitory transcription factors creb/crem/atf transcription factor family, which in turn could suppress cx promoter activity. (supported by the dfg) results: fxa increased par- mrna, protein and cell-surface expression and augmented par- -mediated mitogenesis. par- expression was not influenced. the regulatory action of fxa on par- was concentration-dependent and mimicked by a par- selective activating peptide. the thrombin inhibitor argatroban or par- gene silencing did not influence fxa-stimulated par- expression. fxa increased oxidative stress and expression of the nadph oxidase subunit nox- in smc. nox- gene silencing prevented fxa-stimulated par- regulation, as did ebselen and catalase. exogenous hydrogen peroxide increased par- expression and mitogenic activity. fxa induced nuclear translocation and par- dna binding of nuclear factor kb (nfkb). inhibition of nfkb prevented fxa-stimulated par- expression. in separate studies, fxa promoted par- mrna stabilisation through increased human antigen r (hur)/par- mrna binding and cytoplasmic shuttling. hur gene silencing abolished fxa-stimulated par- expression. conclusion: expression and mitogenic activity of vascular par- , but not par- , is upregulated by fxa. this action involves transcriptional and post-transcriptional mechanisms mediated through nox- -containing nadph oxidase and its downstream effectors hydrogen peroxide, nfkb and the mrna stabilising protein hur. continued generation of fxa by the mural thrombus, and the autoregulatory feedback control of par- may maintain the inflammatory and proliferative state of the injured vessel, thereby promoting vascular remodeling. the mrna stabilising factor hur is a critical regulator of human proteaseactivated receptor aim: we recently reported that functional expression of par- thrombin receptors is induced in human saphenous vein smc exposed to high glucose. this effect could be attributed in part to transcriptional mechanisms mediated through nfkb but the contribution of post-transcriptional effects such as mrna stabilisation is not known. this study explored the potential role of the mrna stabilising factor human antigen r (hur) in the regulation of par- . methods: human saphenous vein smc were serum-deprived prior to study. gene expression was determined by realtime pcr, protein expression by western blotting. gene silencing utilized commercially available sirna. hur binding to par- mrna was determined by immunoprecipitation ("pull-down") pcr. results: high glucose ( mm vs . mm) slowed par- mrna degradation in the presence of actinomycin d. par- mrna decay was not affected. hur binding to par- mrna and nucleo-cytosolic shuttling was enhanced by high glucose, total hur protein expression was not affected. hur sirna abolished the high glucose-stimulated induction of par- mrna. hydrogen peroxide (h o ) also induced cytosolic hur shuttling and increased par- mrna and total protein expression. the role of endogenously generated h o in the regulatory effect of high glucose on par- expression was investigated with the nadph oxidase inhibitors apocynin/diphenyliodonium (to prevent h o generation) and cell-permeant catalase (to degrade cellular h o ). both approaches prevented the stimulatory effect of high glucose on par- expression. cyclic amp has been reported to suppress hur activation and in the present study, the cyclic amp stimuli forskolin and cicaprost (prostacyclin analog) suppressed basal hur shuttling and par- transcript stability. cicaprost also attenuated high glucose-induced hur binding to par- mrna and as a consequence normalised par- expression and inflammatory signalling in high glucosetreated cell. conclusion: the regulation of par- thrombin receptors in human vascular smc is critically dependent on the mrna stabilising actions of hur. through activation of hur, high glucose and other hur stimuli such as ang ii and exogenous h o , increase par- expression, while cyclic amp agonists such as prostacyclin oppose this effect. such interactions could potentially represent a fine-tuning mechanism to control par- expression and ultimately also the mitogenic and inflammatory actions of thrombin in the vessel wall. nucleoside diphosphate kinase b (ndpk b) is a member of a family of ubiquitously expressed enzymes required for nucleoside triphosphate synthesis. thus, they are involved in the regulation of a variety of cellular processes, e. g. g protein mediated signal transduction. however, whether ndpk b has a specific role in the regulation of angiogenic processes in endothelial cells is unknown. therefore, we studied the function of ndpk b in the vasculature in a developmental, an ischemia-induced and an in vitro model of angiogenesis. firstly, depletion of ndpk b expression was achieved by morpholino-mediated knockdown in zebrafish embryos in which the developing vasculature can be visualized by egfp expression in the endothelium. h post fertilization, ndpk b knockdown larvae showed a dramatic inhibition of intersegmental and dorsal longitudinal anastomatic vessel formation compared to control injected fish. this phenotype could be rescued by early re-expression of ndpk b. secondly, ischemia driven angiogenesis was studied in ndpk b-depleted mice and wildtype littermates after excision of the left femoral artery. hind limb blood flow was assessed by laser doppler perfusion imaging immediately before and after ligation (day ) and on postoperative days , , , , , and . a significant reduction of recovery was observed in the ndpk b depleted mice at days and . thirdly, in vitro-sprouting angiogenesis was analyzed in human umbilical vein endothelial cell (huvec) spheroids with and without sirna-mediated ndpk b knockdown. vascular endothelial growth factor (vegf)induced sprouting was significantly attenuated by ndpk b knock down by more than % in comparison with control transfected huvec. we conclude from these results that ndpk b is an essential regulator of angiogenesis. the loss of ndpk b may specifically interfere with the vegf-induced migration and proliferation during endothelial sprouting. ethylene oxide in blood of ethylene-exposed volunteers ethylene (et) is a commercially important high volume industrial chemical. inhaled and endogenous et is metabolized in mammals to ethylene oxide (eo), which is carcinogenic in rats and mice. until now, no data on the oxidation of et in et-exposed humans has been published. in the present study, we investigated the formation of eo in four male adult volunteers exposed for hours to constant atmospheric et concentrations of , , or ppm by means of a breathing mask. during exposure, et concentrations were measured in inhaled and exhaled air by gc/fid and eo concentrations in venous blood by gc/ms. rates of et metabolism were obtained from the product of the differences in the et concentrations with the pulmonary ventilation. in each subject, linear correlations were found between the et exposure concentration and the rate of et metabolism or the eo concentration in blood. mean rate of et metabolism was . ± . nmol/h/ppm/kg body weight. steady-state concentrations of eo in blood differed by a factor of between the volunteers. these inter-individual differences likely reflect the polymorphism of glutathione s-transferase theta, the main eo metabolizing enzyme in human liver. mean eo concentration in blood at steady state was . ± . nmol/l blood per ppm of et. the data will be used for validating a physiological toxicokinetic model which will describe the et related eo tissue burdens in rodents and humans. the model predictions will support risk evaluations of et. financially supported by the lower olefins sector group of cefic. in vitro effect of stw on human dendritic cells fink c. , bonaterra g. a. extracts of echinacea (purple coneflower) are used in the prevention and therapy of infectious diseases. the medicinal product stw contains the extract from purple coneflower, and in addition, extracts of monkshood, venom of honey bee and bushmaster snake in homeopathic dilutions. previous studies showed a stimulation of the cellular and humoral immune response. dendritic cells (dcs) are antigen presenting cells that act at the interface of the innate and adaptive branches of the immune system. during stages of dc differentiation, the ability to internalize antigens varies and decreases during maturation. in this study, we determined the influence of stw on the expression of maturation related genes (cd a, cd ), cytokines (tnfα, il- , il- ), chemokines (ccr ), major histocompatibility complex ii(mhc-ii) and toll-like receptors (tlr , tlr ). in mature (mdc) and immature dc (idc) using real-time rt-pcr. peripheral blood mononuclear cells (pbmcs) were isolated from buffy coats of human volunteers by densitygradient (ficoll ® ) and seeded in well plates. non-adherent cells were eliminated. to induce idc development, ng/ml il- and ng/ml granulocyte macrophagecolony stimulating factor (gm-csf) were added. at day , maturation was induced by addition of lipopolysaccharide (lps) at a final concentration of µg/ml and cultured for additional days. after incubation with different concentrations ( . in idc, compared to control, we found a significantly increased expression of cd ( . - . fold) and tnfα ( . - . -fold) genes after treatment with . - % stw , respectively, but no effect was found on the expression of cd a, il- , il , adam , cd c, cd ,tlr , tlr , mhc-ii and ccr . in summary, these data demonstrate a stimulatory effect of stw in idc and especially in mdc, concerning an increase of various genes related to maturation (cd ), immunomodulation (tnfα, cd ), adhesion (ccr ) and antigen presentation (mhc-ii) and are in accordance with the therapeutic use in infectious diseases. waterproofing sprays are widely used consumer products containing for example fluorinated polymers or silicon based compounds dissolved in alcohols or volatile petroleum distillates. there have been repeated reports on cases of severe acute respiratory disorders especially when using products that newly entered the market. it is hypothesized that impairment of the pulmonary surfactant by deposition of inhaled respirable particles of the active compound is one of the main causes of the acute lung injury. since the inhalation toxicity cannot be predicted a priori based on the physical and chemical properties of the formulation, proper test strategies are required to ensure consumer safety. we propose to combine screening tests addressing both, exposure and acute lung toxicity. the exposure potential of the spray product is characterized by determining the release fraction of the active compound in the respirable particle size range under conditions relevant for the product application. this is carried out by spraying defined quantities of the product into a control volume and measuring the concentration of health related size fractions. this procedure takes into account spray ageing, especially size reduction of the droplets due to solvent evaporation. the isolated perfused lung is used as a model for testing acute toxicity. ventilated rat lungs are exposed to aged aerosols with proper particle size of approximately µm mmad generated from the liquid spray formulation. lung compliance and lung resistance are continuously monitored during exposure. dose dependent deviations from the normal values (without exposure) are used as read-out parameters. using the combined procedure, different sprays could be ranked according to their realistic exposure risk and, most importantly, sprays with known lung toxicity could be uniquely distinguished from those that have been shown to be safe. in its current stage of development the simple test method is recommended for screening of substances only. induction of oxidative damage in calf thymus dna by the fusarium mycotoxin zearalenone after metabolic activation with liver microsomes fleck s. c., pfeiffer e., metzler m. kit -institute of applied biosciences chair of food chemistry, adenauerring a, karlsruhe, germany zearalenone (zen) is an estrogenic mycotoxin produced by fusarium species. the adverse effects of zen and its reductive metabolite zearalenol (zel) are often compared to those of -beta-estradiol (e ) and estrone (e ). these endogenous estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (i) hormonal activity and (ii) genotoxic effects by p -catalyzed metabolic activation to catechols (wang et al., chem res toxicol , . like e and e , zen and zel exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites (pfeiffer et al., mol nutr food res , . the aim of the present study was to examine the formation of catechol metabolites of zen by liver microsomes of various species and their potential for redox cycling. catechol metabolites are frequently associated with the generation of reactive oxygen species and subsequent oxidative damage of dna, for which -oxo- , -dihydro- 'deoxyguanosine ( -oxo-dg) is a common biomarker. the propensity of the catechol metabolites of zen and zel to cause the formation of -oxo-dg in isolated calf thymus dna was determined using a lc-esi-ms/ms method. to this end, zen was incubated with microsomes from human, rat, mouse, bovine and porcine liver as well as with human cyp a , and the incubations were extracted with ethyl acetate. the extract was analyzed with lc-ms and then added to a solution of calf thymus dna in the presence of copper(ii) ions and nadph. the formation of -oxo-dg could be demonstrated with each extract. the levels of -oxo-dg correlated directly with the extent of catechol formation, which increased from steer to swine to human to mouse to rat microsomes. -hydroxylated zen/zel, which is the major catechol, was more reactive than -hydroxylated zen/zel to form -oxo-dg. in conclusion, our study has shown that the catechol metabolites of zen are highly reactive and give rise to oxidative dna damage in vitro. in addition, recent research from our laboratory revealed that the catechols of zen are less efficiently inactivated by catechol-o-methyl transferase than the catechols of e and e . the genotoxic potential of zen may constitute another biological activity in addition to the well-known estrogenicity. supported by deutsche forschungsgemeinschaft (grant me / - ) and "food and health" of kit. thrombin regulates expression of sphingosine kinase- (sphk- ) in human vascular smooth muscle cells -inhibition by dabigatran reduces vascular sphk- expression and atherosclerotic burden in vivo flößer a. results: thrombin induced a time-and concentration-dependent ( - nmol/l) increase in sphk- mrna and protein expression in human saphenous vein smc, n= - . this was mimicked by a synthetic par- ligand. inhibition of sphk- attenuated thrombin-induced smc proliferation but not smc migration (n= ). the regulatory action of thrombin on sphk- expression was suppressed by sirna against the mrna stabilisiserhur. in thrombin-stimulated smc, hur binding to sphk- mrna and subsequent nucleo-cytosolic shuttling was enhanced. accordingly, thrombin induced sphk- mrna stabilisation in smc in the presence of actinomycin d. in apoe-deficient mice, long-term treatment with the direct thrombin inhibitor dabigatran significantly reduced aortic sphk- expression by % (n= ) and plaque size by % compared to control animals (n= ). conclusions: thrombin induces sphk- expression and s p synthesis in vascular smc via the mrna stabilising protein hur. this leads to increased smc proliferation. inhibition of thrombin by dabigatran treatment in vivo attenuates progression of plaques possibly by reducing sphk- expression. mycotoxin contamination and cytotoxicity of grain mill products typical grain mill products from north-rhine westphalia, i.e. grains, flour, wholemeal flour and bran (from wheat, rye and spelt) as well as typical by-products (outsourced fractions) from the milling process were analysed for their mycotoxin content by lc-ms/ms. the cytotoxicity of sample extracts with known mycotoxin composition was then assessed in v cell cultures by means of the neutral red uptake assay, in parallel with pure reference mycotoxin mixtures. extracts from flour and wholemeal flour samples with low levels of deoxynivalenol and enniatin b (from not detectable to . µg/g don and . µg/g ennb) induced no measurable cytotoxicity. on the other hand, although mycotoxin contamination levels were also rather low in bran, these samples induced strong cytotoxicity: extracts of bran derived from rye and spelt were more cytotoxic than those of wheat bran. the cytotoxic effects of the bran samples cannot be related to their mycotoxin content as comparable concentrations of pure mycotoxins and mycotoxin combinations tested in parallel were not cytotoxic. by-products from certain stages of the milling process (sorting and waste fractions) were found to contain mycotoxins at rather high levels: enniatin b was detected in nearly all samples, and also t- toxin, ht- toxin, ergotamine, ergocornin and deoxynivalenol were present. waste sample extracts with notable mycotoxin levels (up to µg/g don, µg/g ennb, µg/g ergotamine, ng/g ht- toxin) exert pronounced cytotoxicity in v cells. the cytotoxicity of these samples was somewhat stronger than expected when compared with mixtures of reference mycotoxins tested in parallel. in summary, the tested flour and wholemeal flour extracts contained only low levels of mycotoxins and were not cytotoxic. in contrast, bran samples showed cytotoxicity which cannot be explained solely by their mycotoxin content. this unexpected observation in real samples and combination effects of mycotoxin mixtures require further studies. the ahr is a ligand-activated transcription factor that mediates the toxicity of dioxins and related compounds. upon ligand binding the ahr translocates into the nucleus and dimerizes with arnt to modulate gene expression, e.g. of cyp a . recently, we have shown that uvb irradiation of human keratinocytes results in activation of the ahr and associated egfr signaling leading to an enhanced expression of cyp a and proinflammatory cox- , respectively. the initial step is the uvb induced intracellular formation of the tryptophan photoproduct -formylindolo [ , b] carbazole (ficz), a high affinity ahr ligand. thus, the ficz activated ahr is an important mediator of the dna damage independent part of the uvb response. our current study aims to identify further aspects of ahr mediated uvb responses. therefore, we analysed changes in protein expression, proliferation and apoptosis in ahr+/+ and ahr-/-keratinocytes (nctc ) by western blot, flow cytometry and brdu incorporation. uvb exposure of nctc cells led to a dose-dependent increase in apoptosis. compared to ahr+/+ cells, ahr-/-cultures exhibited an increased amount of apoptotic cells. this finding was confirmed in irradiated ahr+/+ cells, pretreated with the ahr antagonist 'methoxy- '-nitroflavone. moreover, the proliferation of sham as well as uvb irradiated ahr-/-cells was significantly decreased. in ahr-/-cells we found a reduced expression of checkpoint kinase (chk ), an important cell cycle regulator that arrests the cell in g /m upon dna damage. interestingly, uvb exposure led to a higher net phosphorylation of chk in ahr-/-cells, indicating that this pathway is responsible for the observed ahr-dependent differences in proliferation and apoptosis. further expression analyses of chk client proteins emphasize our hypothesis. in conclusion our study identifies the ahr as an anti-apoptotic player in uvb irradiated human nctc cells. therefore, we propose that the ahr is a suitable target to prevent uvb induced skin diseases. synthetic progestins exert divergent effects on thrombosis in a murine model of atherothrombosis background: medroxyprogesterone acetate (mpa), a synthetic progestin often used in postmenopausal hormone replacement therapy, has previously been described to be pro-thrombotic in a murine model of accelerated atherosclerosis. however, nothing is so far known about effects of progestins with receptor profiles different from mpa (i.e. agonism or antagonism of mineralocorticoid-or androgen-receptors), such as drospirenone, levonorgestrel and norethisterone acetate. methods: apo -/mice were bilaterally ovariectomized (ovx) and substituted subcutaneously with mpa, drospirenone, levonorgestrel and norethisterone acetate as well as the respective placebo pellets for days on a western-type diet. subsequently, thrombosis was induced by photochemical injury to the right carotid artery using rose bengal and a green light laser. results: compared to placebo, animals substituted with mpa showed significantly shortened times to occlusion of the right carotid artery (placebo mpa: . ± . min. vs. mpa: . ± . min., n = - , p < . ). in contrast, drospirenone, levonorgestrel or norethisterone acetate did not alter thrombotic responses. however, at least drospirenone (placebo drospirenone: . ± . min. vs. drospirenone: . ± . min., n = - ) and levonorgestrel (placebo levonorgestrel: . ± . min. vs. levonorgestrel: . ± . min., n = ) showed a trend towards shorter times to stable occlusion. furthermore, analysis of aortic gene expression revealed that in aortas of mpa-treated mice expression of matrix-metalloproteinase (mmp- ) was induced as compared to placebo-treated mice. conclusion: mpa, a progestin with glucocorticoid effects, exerts a pro-thrombotic effect that is either progesterone-or glucocorticoidreceptor-dependent while progestins with receptor profiles different from mpa do not show a significant pro-thrombotic effect. furthermore, the pro-thrombotic effect exerted by mpa may be associated with increased expression of mmp- , a metalloproteinase being known to destabilize atherosclerotic plaques and make them more prone to rupture. rapid screening for mitochondrial toxicity in vitro using an oxygen-sensitive phosphorescent probe freyberger a. bayer healthcare bph gdd ged toxikologie -p & cp, aprather weg , wuppertal, germany impaired mitochondrial function has been implicated with disease, aging, and druginduced toxicities. analyzing mitochondrial respiration (mr) rates is one of the most informative ways to assess mitochondrial function as it provides information on the the bioenergetic capacity of a tissue, however, previously measurements using polarography (clark electrode) were cumbersome with only low throughput. in this work we explored luxcel's water-soluble phosphorescent oxygen-sensitive probe mitoxpress tm for the assessment of mitochondrial toxicity in freshly isolated male rat liver mitochondria (rlm) in a -well plate format using glutamate/malate ( mm/ . mm) and succinate ( mm) as respiratory substrates. inhibition of mitochondrial complexes i to iv, adenosine triphosphate synthetase and the adenosine diphosphate (adp) / adenosine triphosphate (atp) antiporter by rotenone, thenoyltrifluoracetone (ttfa), antimycin a, potassium cyanide, oligomycin, and atractyloside was readily detected in adp-stimulated rlm. use of the two different substrates in parallel allowed to discriminate complex i inhibition by rotenone from complex ii inhibition by ttfa, whereas downstream of these complexes inhibition by the other model inhibitors occurred independent of the substrate used. decoupling of mr from oxidative phosphorylation by carbonylcyanid-p-trifluormethoxyphenylhydrazone (fccp) was detected best in the absence of adp. compared to polarographic measurement, the use of an oxygen-sensitive probe is superior with regard to assay cycle time and sample throughput and offers new opportunities to characterize and screen for mitochondrial toxicity, but also to support studies on mitochondria-mediated modes of action of new chemical entities. the murine local lymph node assay (llna) and the guinea pig maximization test (gpmt) have been used to study the sensitization potential of a series of unsaturated compounds by kreiling et al. ( ) . we have examined the same substances in the loose-fit coculture-based sensitization assay (lcsa), developed by our working group (schreiner et al., ) . eight unsaturated compounds [oleic acid (oa), linoleic acid (la), linolenic acid (lna), undecylenic acid (ua), fumaric acid (fa), maleic acid (ma), squalene (sq), -octyn- -ol (oc)] and succinic acid (sua) were investigated using a coculture of keratinocytes and dendritic cell-related cells (dcrc). sensitization potential was quantified by flow cytometry measuring the increase of cd expressed on dcrc (ec = half maximal effective concentration). a pronounced induction of cd at low concentrations was seen with la, lna and oa (ec : , and µmol/l, respectively). ua exhibited an intermediate response (ec : µmol/l). with oc and ma, we observed effects at higher concentrations only (ec : and µmol/l). no significant increase of cd was observed with fa, sua and sq. because of poor solubility, sq could not be studied adequately. induction of cd was generally observed at concentrations which did not cause a major impairment of cell viability. our results show a high degree of concordance with those obtained by the gpmt, except for oa. in comparison with the results of the llna, those compounds which showed a strong effect in the llna (oa, la, lna) also induced an increase of cd at low concentrations, whereas those with low stimulation indices in the llna induced no significant increase of cd (fa, sua) or only at higher concentrations (ua). we observed a discrepancy between the tests with ma and oc, causing a strong stimulation of the murine lymph nodes, while the expression of cd was increased at high concentrations only. we assume that ma and oc might be false-positives in the llna, because they were also negative in the gpmt. background: the first step in elimination of many cationic drugs is their uptake from the blood into hepatocytes and renal proximal tubular cells by the organic cation transporter (oct ) and oct , respectively. the pivotal role of octs in the excretion of cationic drugs raises the possibility of drug-drug interactions in which one drug reduces octmediated elimination of a second drug. although many psychoactive drugs are cationic at ph . and some of these have already been recognized as oct inhibitors, a systematic screen of this class of compounds is missing. methods: we screened a drug library of most frequently prescribed psychoactive drugs (inpatient prescriptions in germany, at least million ddd each) for their inhibitory interaction with oct and oct . human embryonic kidney (hek) cells stably overexpressing oct or oct and the prototypical oct substrate -methyl- phenylpyridinium (mpp+) were used as a test system. cells transfected with the empty vector were used as controls. results: at µm, % and %, respectively, of the tested compounds significantly decreased oct -and oct -mediated uptake of mpp+. the most potent inhibitors (inhibition > %) of oct were chlorprothixen and clomipramine, whereas olanzapine, clomipramine and doxepin were the most potent inhibitors of oct . in contrast, neither at µm nor at µm carbamazepin, haloperidol, lithium, moclobemide and valproic acid did significantly inhibit mpp+ uptake into hek-oct or hek-oct cells. there was a significant correlation between the degree of oct and oct inhibition (p conclusions: our results demonstrate that inhibition of oct function by psychoactive drugs has to be considered as a potential mechanism underlying drug-drug interactions. considering estimated peak sinusoidal concentrations e.g., of chlorprothixen and clomipramine between and µm in humans, inhibitory interactions of these compounds with hepatic oct have to be taken in account. our data will help to create a chemoinformatic model to predict potential oct-dependent interactions of psychoactive drugs with the hepatic or renal elimination of coadministered drugs. this project is supported by the german federal ministry of education and research (bmbf), project grant no. ex b. cardiac gene expression is altered during the development of hypertrophy and heart failure compared to the healthy heart. the molecular mechanisms controlling gene expression in cardiac failure are only partially known. dna methylation is one epigenetic mechanism that regulates long-term changes in gene-expression. to elucidate whether dna methylation is altered during the development and progression of chronic heart failure, genome-wide dna methylation profiles were determined in myocardial biopsies from control patients and patients with cardiac hypertrophy or failure. cardiac biopsies were obtained from patients with aortic aneurysm who served as control and did not show clinical signs of chronic heart disease (ef: ± %, n= ) and from patients with aortic stenosis. the latter group was subdivided according to the ejection fraction into hypertrophic (ef: ± %, n= ) and failing patients (ef: ± %, n= ). after bisulfite conversion of extracted dna, the methylation status of genomic dna was quantified using the infinium® humanmethylation beadchip (illumina). this microarray allows analysis of more than , methylation-sites throughout the whole genome at single-base-pair resolution. these experiments identified cpg sites in hypertrophic samples and cpg sites in failing samples which were differentially methylated compared to control specimens (delta > %; p< . ). cpg sites were significantly altered in both aortic stenosis groups compared with control hearts. from these cpgs, sites were altered concordantly in hypertrophic and failing samples. analysis of regions harbouring distinct cpg densities revealed that most changes occured in shelf regions of cpg islands whereas the methylation status in the cpg islands was more stable. further analysis showed that differences in methylation were most frequent in gene body, enhancer and `utr regions. specifically probes spanning a cpg-island at the promotor region of the muscle-specific serine kinase (srpk ) showed diminished cpg-methylation in hypertrophic (- . ± . %) and failing (- ± . %) as compared to control biopsies. remarkably, no alterations of dna-methylation were observed in loci of classic marker genes of chronic heart failure like nppa, serca, ctgf, myh or myh . these results indicate that dna methylation is specifically altered in chronic heart disease but does not affect classic marker genes of chronic heart failure. gliomas are the most abundant type of primary brain tumor in the central nervous system in adults. the current standard of glioblastoma multiforme (gbm) therapy is surgery followed by radiotherapy and chemotherapy. however the morbidity and mortality of gbm remain very high and the median survival period is only months even with treatment. therefore it is important to identify novel drugs to reduce gbm cell proliferation. purine-analogues (pa) are well known for their anti-proliferative effects on eukaryotic cells. in this study novel pa were synthesized and the library of substance-derivatives was tested using different gbm cell lines namely ln , u -mg and gl . the effect on proliferation and viability was assessed by using brdu and resazurin assays. using these in vitro methods we were able to identify several compounds with cytotoxic and anti-proliferative effects in vitro showing ic values in the deeper µm range. cytotoxicity of selected compounds was further analyzed by assessment of caspase and propidium iodide based cell cycle facs analysis to discriminate between apoptosis and cell cycle arrest. based on these data purine-derivatives might inhibit proliferation and induce apoptosis in glioma cells. as a result we hypothesize that these compounds could be potentially interesting for the drug-development of gbm therapy and therefore a clue for chemical modifications. further studies are required to identify the exact underlying mechanism of action of the tested purine-analogues. the biological role of adenosine receptors in brown adipose tissue gnad t. brown adipose tissue (bat) is responsible for basal and inducible energy expenditure in mammals. bat contains large amounts of mitochondria and is highly vascularized. bat lipolysis and thermogenesis are stimulated by sympathetic neurons. importantly, recent findings indicate that adult humans possess metabolically active bat . here, we analyzed the expression and function of adenosine receptors in bat. adenosine receptors (ador) are members of the superfamily of g protein-coupled receptors. there are four subtypes of adors in humans referred to as adora , a a, a b and a . they are widely expressed in tissues and mediate a variety of cellular functions, mostly due to their regulation of camp levels within cells. interestingly, it has been shown that adenosine can either inhibit or stimulate lipolysis in white adipocytes through adora or a a, respectively . however, the role of adenosine in the differentiation of brown preadipocytes to adipocytes and in bat function is not clear. to analyze the role of adors in bat, we use preadipocytes isolated from bat of newborn mice and subjected them to a differentiation protocol. abundance of adora , a a, a b and a mrna was measured using qpcr. all four receptor subtypes are present in preadipocytes with adora b being the most abundant. adora , adora a and adora are significantly transcriptionally upregulated -albeit at varying degree -during differentiation. adora is upregulated . fold (+/- . fold) and fold (+/- . fold) at day and at day , respectively, as compared to preadipocytes (n= ). adora a is fold (+/- . fold) upregulated at day and fold upregulated (+/- . fold) at day , respectively (n= ). adora was found upregulated . fold (+/- . fold) at day (n= ). in contrast to this, ador b was downregulated to . fold (+/- . fold) at day and to . fold (+/- . ) day compared to preadipocytes (n= ). to investigate the functional role of ador in bati cells, we analyzed lipolysis in mature cells after acute treatment with specific agonists and antagonists. we observed that adora a activation by cgs significantly increased lipolysis by % (+/- . %) compared to untreated control. moreover, adora antagonist psb increased lipolysis by % (+/- . %) (n= ). in conclusion, ador are highly regulated during brown fat cell differentiation. lipolysis of mature brown fat cells is significantly increased by adora a agonist or adora antagonist, respectively. munich heart alliance, münchen, germany activation of the sympathetic nervous system and the subsequent activation of βadrenergic receptors (βars) through catecholamines represents the strongest mechanism to increase cardiac function. however, long-term activation of cardiac βars is clearly detrimental and β-blockers have been introduced as an effective treatment modality in cardiac failure. despite their central role in cardiac physiology and disease, our knowledge about the intracellular mechanism of βar stimulation is confined to a few targets and is likely incomplete. here, we report a functional proteomics approach to directly assess the entire phosphoproteome of βar-stimulated mouse hearts in vivo. to identify proteins that are phosphorylated in response to β-adrenergic stimulation in vivo, we treated mice with isoproterenol or, as a control, with propranolol. after lysis of hearts and tryptic digest, phosphopeptides were enriched by tio or immobilized metal ion affinity chromatography (imac). subsequent analysis of eluated peptides by tandem mass spectrometry (ms/ms) mapped several phosphopeptides to cardiac proteins, among which known mediators of βar signaling such as phospholamban, troponin i and myosin binding protein c. we then employed multiple reaction monitoring (mrm) as a quantitative approach to assess changes of phosphorylation after βar stimulation. using this combination of ms approaches, we identified peptides with pka consensus phosphosites that were more abundantly detected under βar stimulation. among those, we found myozenin- (myoz ) and g protein signaling modulator (gpsm , also termed ags ) as proteins previously not related to βar signaling. we validated the βar-dependence of phosphorylation at these sites in isolated cardiomyocytes by in vivo labelling or phosphoepitope-specific antibodies. current efforts aim at the functional characterization of these novel candidate mediators of βar signaling in the heart. taken together, we report the β-adrenergic phosphoproteome of the mammalian heart in vivo. we have identified several new targets of βar signaling that may represent essential factors in cardiac physiology and disease. background: drug measurement in autopsy material is normally used to investigate the cause of death. in our study it was possible to measure concentrations of drugs that were part of a regular treatment without connection to the cause of death. metamizole is used as an analgetic and spasmolytic agent. the active metabolite maa ( -methyl-aminoantipyrin) is metabolized by the liver and eliminated by the kidney. hepatic and renal dysfunction can therefore influence maa clearance. methods: maa concentrations were measured in different samples of the autopsy material (heart blood, venous blood, urine, liver, kidney and brain) using an hplc-ms/ms method. information about the dosage and time of drug application as well as information about existing renal or hepatic disorders were taken from the corresponding patient records. because of the low number of cases an explorative single-case study was necessary. results: cases with oral intake of metamizole in a customary continuous dosage could be indentified. the maa distribution into body liquids and organs depended on the time between last oral intake and death. in two cases without renal or hepatic diseases maa blood levels were below µg/ml. five cases with combined renal and hepatic disorders showed either increased blood levels of - µg/ml or prolonged maa elimination half-life of up to hours. in one case with manifest hepatic insufficiency an maa concentration of more than µg/ml was measured in venous blood. two cases with renal insufficiency alone had maa venous blood levels of less than µg/ml. (pet) . pet detects the positron emission of neutron-deficient radioactive nuclides and allows their external localization in vivo. fet, a modified amino acid, is not incorporated in proteins but accumulates in glioblastomas. one pathway responsible for its accumulation is the preferential transport into the tumor cells, probably via amino acid transporters. we investigated in more detail (a) which individual, cloned amino acid transporters accept fet as substrate and (b) which transporter is responsible for the major fet transport into glioblastoma cells. studies with xenopus laevis oocytes, expressing individual human amino acid transporters, revealed that system l, y + l and b + amino acid transporters recognize fet as substrate (lat and , y + lat , and b + at, respectively). in contrast, y + lat and atb ,+ did not transport fet. rna expression studies using qrt/pcr revealed that lat is the dominant amino acid transporter in all glioblastoma cells investigated (ln /u /u mg/u /a /t g). a strong lat expression was also shown on the protein level. to find out whether lat is the main transporter responsible for fet accumulation, we first studied transport of the parent amino acid l-tyrosine in ln glioblastoma cells. [ h] tyr uptake was completely na + -independent and inhibited by leu, phe and trp, but not by arg, pro or ser. sirna-mediated down-regulation of lat in ln cells led to a concomitant decrease of lat mrna and tyr transport (down to % and %, respectively). these results indicate that tyr is exclusively transported by lat in ln cells. we are currently performing transport studies using [ f]fet to investigate whether fet transport is also exclusively mediated by lat in glioblastoma cells. a further question is if lat , a sodium-independent transporter, can be responsible for the accumulation of fet observed in glioblastoma cells. if true, other amino acid derivatives that are lat substrates might also proof useful in cancer diagnosis. telmisartan reduces adipose tissue inflammation and biglycan accumulation in diabetogenic ldl-receptor knockout mice grandoch m., nagy n., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße , düsseldorf, germany in addition to lowering blood pressure some of the angiotensin ii at receptor antagonists (arb) such as telmisartan have additional beneficial effects on the onset of type diabetes mellitus and obesity. this was contributed mainly to peroxisome proliferator activated receptor (ppar)γ modulating activity. hyaluronan (ha), a high molecular weight polysaccharide and the small leucine rich proteoglycans, decorin and biglycan, are known to be involved in atheroprogression. mechanistically these matrix components contribute to inflammatory processes via toll-like receptor-signalling and are supposed to modulate lipid retention. the aim of this study was to elucidate the effects of telmisartan in comparison to valsartan, an arb without pparγ activity, on extracellular matrix remodelling and inflammation in atherosclerosis and the interrelationship with adipose tissue inflammation using the ldlr-/-model of accelerated atherosclerosis. male ldlr-/-mice were fed either a diabetogenic diet alone or in combination with telmisartan ( mg/kg), valsartan ( mg/kg) or valsartan ( mg/kg) from weeks of age for weeks. all treatment groups except of the lower valsartan dose showed significant effects on reducing the aortic plaque score. the content of ha, collagen and decorin in the aortic root were not changed. however, telmisartan reduced the content of biglycan in the aortic root significantly in contrast to valsartan. in addition, a trend towards decreased mac -positive macrophages in abdominal adipose tissue was detectable after telmisartan treatment as well as a strong reduction in the adipose tissue mrna expression of biglycan. finally, telmisartan reduced the expression of hyaluronan catabolizing enzymes potentially leading to an increase of high molecular weight ha in the adipose tissue, which is thought to be homeostatic and antiinflammatory. in summary, the results of this study underline the pronounced anti-inflammatory capacity of telmisartan on atherosclerosis and adipose tissue inflammation in comparison to valsartan and strongly suggest that biglycan might be an additional target of telmisartan not only concerning matrix composition of atherosclerotic lesions but also concerning the structure of adipose tissue and metabolic effects of the compound. human primary malignant cancer cells derived from peritoneal effusions of a patient with colorectal carcinoma, as assessed by comet assay. the primary cancer cells were more efficient in dsb repair than ht- cells, and their doxorubicin ic was four times higher. comparative protein expression levels showed that the primary cells had less rad and as well as less topoiiα, while ku and levels were similar. another very interesting protein is the mrn (mre -rad -nbs ) complex that initializes the phosphorylation of atm and thereby starts the signalling cascade. the newly described mrn-atm pathway inhibitor mirin interrupts mrn activity by inhibiting the exonuclease activity of mre . the toxicity of mirin in ht- cells was measured using a luminescence-based assay detecting the amount of atp, which is correlated with cellular viability. mirin did not show any toxic effects up to a concentration of µm and incubation times of hours, indicating that mirin can be used under these conditions without detrimental effects. we are currently investigating the effect of mirin on the toxicity of topoiiα inhibitors. the inhibition of dna repair may be a valuable strategy to enhance the effect of dnadamaging anticancer drugs. since tumours (even of the same entity) are not only heterogeneous, but also polyclonal, a broad selection of response modifiers of anticancer drugs would be helpful to individually enhance chemotherapeutic effectiveness. evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. epigenetics play an important role in the control of gene expression. epigenetic mechanisms comprise modulation in dna methylation, histone modification and non-coding rna. several polyphenols have been reported to possess histondeacetylase (hdac) inhibitory properties [ ] . histone deacetylation is generally linked to transcription repression. furthermore, hdac belongs to the group of small ubiquitin-related modifier (sumo) substrate proteins. sumoylation of hdac is associated with a modulation of its biological activity [ ] . little is known so far about the mechanism by which hdac sumoylation mediates inhibition of gene transcription. we addressed the question whether sumo e and hdac expression and whether potential hdac-sumoylation will be affected by polyphenols such as chlorogenic acid, genistein and (-)epigallocatechin- -gallate (egcg). chlorogenic acid, genistein and egcg decreased sumo e protein level in the human colon carcinoma cell line ht after h of incubation measured with western blot analysis. egcg exhibited the most pronounced effect at concentrations ≥ µm. hdac expression was also affected by these polyphenols. the direct impact of polyphenols on the hdac sumoylation is detected by co-immunoprecipitation experiments with the respective antibodies against hdac- and sumo e . these experiments are still under investigation. in conclusion, chlorogenic acid, genistein and (-)-epigallocatechin- -gallate influenced the sumo and hdac expression in vitro. in further studies the direct impact on subtract-sumoylation will be investigated. these studies contribute to a better understanding of potential chemopreventive effects of dietary polyphenols on specific epigenetic alterations may provide chemopreventive strategies for reducing cancer risk. the no/cgmp cascade is thought to be essential for penile erection. within the smooth muscle of corpus cavernosum, nitric oxide activates the no-sensitive guanylyl cyclase (no-gc) which raises the intracellular concentration of cgmp. this second messenger activates the cgmp-dependent protein kinase i (pkgi) and subsequent phosphorylation of target proteins leads to relaxation of cavernosal smooth muscle. knock out of key enzymes of the no/cgmp cascade has led to discrepant results: the deletion of pkgi in the mouse has been shown to lead to erectile dysfunction whereas mice lacking neuronal no synthase are fertile. to investigate the role of the no receptor in fertility we have generated mice lacking no-gc (gcko), a bottleneck enzyme of the no/cgmp cascade. we have shown that lack of no-gc resulted in arterial hypertension concomitant with a totally abolished no responsiveness of vascular and gastrointestinal smooth muscle. in addition, we generated a mouse line in which no-gc was specifically deleted in smooth muscle cells (sm-gcko). using these ko strains we here examined the role of no/cgmp signaling with regards to the smooth muscle relaxation of corpus cavernosum. no failed to affect corpus cavernosum from gcko in organ bath experiments: neither exogenously produced no by no donors nor endogenous no release from neurons induced by electrical field stimulation led to relaxation. similar results were observed in the corpus cavernosum of sm-gcko mice. to our surprise, the gcko animals were fertile and produced offspring albeit at a reduced rate compared to wt animals. our data show that interruption of no/cgmp signaling results in complete absence of no-induced relaxation of penile corpus cavernosum in mice and reduces the ability to produce offspring but does not abolish fertility. novel modes of invasive cell motility regulated by the formin class of actin nucleators khan j., grosse r. philipps-universität marburg, pharmakologisches institut, karl-von-frisch-str. , marburg, germany pathological invasive cell migration essentially reqires actin polymerization. formins are the largest group of rho-gtpase effectors involved in actin nucleation and assembly as well as microtubule dynamics. here we studied the role of formins in cytoskeletal regulation during homotypic cancer cell invasion. we identified the actin-dependent steps and structures involved for this process. using live cell analysis we characterize the distinct actin dynamics controlled by formin-like and rho function. the specific involvement of this signaling module will be discussed. formin-driven nuclear actin assembly controls mal/srf activity baarlink c., wang h. polymerization of actin in the cytoplasm is tightly linked to transcriptional activation of the srf cofactor mal (also known as mrtf-a) through release of actin/mal interactions and subsequent nuclear accumulation of mal. formins directly promote assembly of actin filaments thereby efficiently regulating mal-dependent transcription for cell shape, adhesion and motility. here we show that formins assemble f-actin and promote mal activation inside the mammalian nucleus. the rho-gtpase effector mdia rapidly enters the nucleus in a signal-dependent fashion and an active mdia confined to the nucleus potently promotes release of g-actin from mal to specifically activate srf. live cell imaging reveals formin-mediated nuclear actin dynamics. moreover, using actin assembly assays we find that inhibition of endogenous mdia formins controls f-actin turnover in isolated nuclear extracts. thus, formin activity is dynamically compartmentalized to the mammalian nucleus to potently regulate actindependent mrtf function. in women the placenta becomes the main source of maternal estrogens during pregnancy. placental estrogen biosynthesis is located in the syncytiotrophoblast, a syncytium that builds the main part of the placental barrier and limits the transfer of substances between the fetal and maternal compartment. since the human placenta is unable to convert cholesterol into -oh-pregnenolone, the placenta tissue highly depends on the supply of c- steroids for their conversion into c- estrogens. in contrast to lipophilic unconjugated steroids that penetrate the cell membrane passively via diffusion, circulating sulfated steroid hormones are delivered to the placenta via carrier-mediated transport, followed by their reactivation via the catalytic activity of the steroid sulfatase (sts). dheas of maternal and fetal origin contributes about equally to the placental formation of estrone (e ) and estradiol (e ), while αoh-dheas supplied by the fetus contributes to over % of placental estriol (e ) synthesis. soat, a member of the slc family with highest expression in hormone-responsive tissues such as testis, placenta, and mammary gland has been shown to transport the sulfoconjugated steroid hormones dehydroepiandrosterone sulfate (dheas), estrone sulfate (e s), and pregnenolone sulfate (pregs) [ ] . aim of this project is to investigate the role of soat for placental estrogen synthesis by means of the choriocarcinoma cell line jeg- as in vitro model for the human syncytiotrophoblast. therefore, we characterized a jeg- cell line that transformed dhea into e and αoh-dhea into e . by qrt-pcr we found expression of sts and aromatase, both essential for estrogen synthesis in these cells. upon transient transfection of soat the carrier was located in the cell membrane of transfected jeg- cells. currently we investigate the transformation of dheas of these soat-jeg- cells by lc-ms-ms. we could demonstrate transport of αoh-dheas for stably transfected soat-hek cells. in situ hybridization and immunohistochemistry showed coloured syncytiotrophoblasts and vascular endothelial cells in late term placenta. in conclusion, soat-mediated transport of sulfated steroids could play a pivotal role for placental estrogen synthesis from sulfated steroid hormones. developing non-animal test systems for evaluation of toxicity was important in the past and will remain essential in the future. here we present a toxicity test using the chicken yolk sac area vasculosa (cav) of fertilized white leghorn chicken eggs [ , ] and compare it to hen's egg test on chorioallantoic membrane (het-cam) [ ] for polymer toxicity testing. fertilized chicken eggs were incubated and after h explanted shell less into sterile petri dishes. test substances were applied on the cav and the appearance of different effects (vascular lysis, haemorrhage, aggregation of blood components, lethality) was determined by light microscopy after - h (fig. ). these effects were combined to a cav test score based on the irritation score calculation used for het-cam evaluation. different polymers like poly(ethylene glycol) (peg; neutral), poly(ethylene imine) (pei; cationic) and dextran sulphate (ds; anionic), as well as guideline-conform (recommended het-cam protocol from the interagency coordination committee on the validation of alternative methods) negative ( . % nacl) and positive controls ( % sodium dodecyl sulphate (sds) and . n naoh) were investigated. additionally ld values for different cationic polymers have been determined. within the selected incubation times ( - h) , effects such as vessel lysis and blood component aggregation could be detected. additionally to het-cam, lethality as well as recovery of the cav could be observed. differences between neutral, positively and negatively charged polymers were obtained. pei showed strong vessel lysis and aggregation of blood components whereas ds and peg showed none of these effects. lethality was found to increase from peg < ds < pei and is concentration and time dependent. the results demonstrate that differences, regarding the toxicity of the used polymers, can be shown with this test. these findings in the cav test can be well correlated with already existing data. in summary, the cav test provides same data (testing control substances) and more information (recovery and lethality) compared to het-cam and could be a suitable model for toxicity testing of polymers. risk characterisation of chemicals consists of three steps ( ) hazard identification and characterisation, based on substance-specific toxicological hazard data, ( ) estimates of the level of exposure toward the substance and ( ) the comparison between the toxicologically safe level and the exposure level. in contrast to the classical risk assessment approach, the threshold of toxicological concern (ttc) approach is developed as a tool to assess the risk of substances without toxicity data. its application requires ( ) information on human exposure, for which it is essential that exposure is fully captured and ( ) knowledge of the chemical structure to assess whether the chemical is not excluded from the application of the ttc concept. instead of chemical specific no observed (adverse) effect levels (noels/noaels), the ttc approach utilises knowledge on the empirical distribution of several hundreds of noels/noaels, originally , based on toxicological testing in animals (munroe et al., ) . with the basis on noaels, the ttc concept builds on the fundamental principle of toxicology, that toxicity is a function of dose and that a dose exists, below which no adverse effects of the substance can be detected. it is assumed that exposures below this level will not result in health risks. three separate ttcs were derived (munroe et al., ) by classifying the chemicals into three toxicity classes using a decision tree based on a series of questions related to chemical structure, and on natural occurrence in food and in the body (cramer et al., ) . the ttc values are derived from empirical distribution of the noels/noaels in the class taking the th percentiles and dividing them by the default uncertainty factor of . it is assumed that the probability is very low that the unknown noael of a not tested chemical will be lower than the value of the th percentile in the distribution of the known noels/noaels. hence, at exposures below the ttc values, the probability of adverse effects on human health is considered to be very low. introduction: kibra, mainly expressed in kidney and brain tissue, is involved in brain development and memory formation as a postsynaptic scaffold protein. in podocytes, kibra is proposed to regulate cell motility as a linker between components of the cytoskeleton and polarity protein complexes (duning et al, jasn ) . furthermore, kibra has been identified as key regulator of the hippo pathway, which is involved in organ size control and tumorigenesis. in the current study, we focused on the identification of kibra gene expression regulation and functional promoter characterization. methods: serial promoter deletion constructs were generated by cloning bp of the 'flanking region of kibra into the pgl -vector system. deletion constructs were transiently transfected into human neuroblastoma cells (sh-sy y) and immortalized human kidney epithelial (ihke) cells. potential transcription factors (tfs) were investigated in cotransfection experiments. transcriptional start sites (tss) were determined by rapid amplification of 'cdna ends ( 'race) . tss utilization between cell lines was assessed by semiquantitative pcr. transcriptional activity (ta) of the kibra promoter p was separated by ~ bp into two distinct regions, promoter p a and p b. deletion constructs harbouring promoter p b were transcriptionally active only in ihke cells. 'race revealed two alternative tss in both cell lines upstream of the annotated tss (nm_ ). exclusively in ihke cells, two additional tss were detected in intron , resulting in two alternative exons. deletion constructs harbouring the putative regulatory regions (p and p ) of both exons were transcriptionally active only in ihke cells. overexpression of full length tcf l (transcription factor -like [t-cell specific, hmgbox]) resulted in a ~ -fold increase of promoter p a and intron promoter p ta. kibra gene expression is driven by a complex alternative promoter system comprising the constitutional promoter p and three alternative promoters p b, p and p . the tss utilization is cell type-specific. subsequent usage of an alternative translation start site within exon could result in truncated kibra protein isoforms. tcf l is involved in the differential kibra gene expression regulation. resulting kibra protein isoform and their cellular function will be assessed in further studies. skin absorption in vitro based on the study of human/animal skin ex vivo or reconstructed human epidermis, respectively, is an alternative method which is accepted by the oecd. guideline tg and a corresponding technical guidance document (gd ) give technical guidance how to perform valid experiments , . the requirements include integrity evaluation tests for the skin samples. different tests are proposed to ensure an exclusively use of undamaged skin. to decide which test suites best to our routine test strategy, we investigated the correlation between integrity test results and absorption profiles of various penetrants (logp range: - . - . ). finite dose experiments using rat and human skin were performed with c-labeled testosterone, caffeine, mcpa ( -chloro- -methylphenoxyacetic acid) and its -ethylhexyl-ester mcpa- ehe. for each experiment at least three of the five following integrity tests were conducted: transepidermal electrical resistance (teer), transepidermal water loss (tewl), transepidermal tritiated water flux (³h o), transepidermal absorption of methylene blue (blue) , transepidermal absorption and flux of a ³h-labeled internal standard (istd); ³h-testosterone or ³h-mannitol was used as istd. the applied radioactivity of the ³h-istd was selected to show no analytical interference with the c-penetrants. teer, tewl and ³h o represent pre-study, istd concurrent and blue post-study tests. calculated maximal permeability constants (kp) and absorbed doses (ad) of the penetrants were compared to the results of the integrity tests. individual linear regression analysis was used to evaluate the correlation the correlations varied over a wide range for all five methods and four penetrants. the best correlations in average were achieved with the istd. no inverse correlations were obtained for the istd, but partly for tewl, teer, ³h o and blue. in conclusion, the istd represents best the achieved absorption profiles of the test compounds and is based on that the most suitable integrity test for our dermal absorption studies. we will further confirm its effectiveness and generate a sufficient historical database in order to include the istd in our routine test protocol. investigation of mirna expression and dna methylation in focal and non-focal brain tissue of therapy-resistant epilepsy patients haenisch s. background: resistance to anticonvulsants affects one third of all epilepsy patients. limited bioavailability of the drug at the target site caused by increased expression of efflux transporters on the blood brain barrier or alterations of target genes are potential mechanisms for therapy resistance. however, these mechanisms alone cannot completely explain the observed resistance and it is likely that multifactorial alterations lead to pharmacoresistance. there is increasing evidence that expression of micrornas probably caused by dna modifications is deregulated in many neuronal diseases. we hypothesize that mirna regulation of target genes is involved in drug resistance in epilepsy. methods: hippocampal focal and cortical non-focal brain tissue samples from patients diagnosed with mts (mesial temporal sclerosis) who underwent neurosurgery have been screened for mirna expression using taqman low density arrays. in silico approaches for both a hypothesis-based (efflux-transporter and target gene) as well as a hypothesis-free approach were used to identify potential phenotype-relevant target genes. using the program r (bioconductor) a mann-whitney-u test was performed to compare mirna expression between brain regions. pyrosequencing was performed to investigate methylation status '-upstream of dna regions encoding for selected candidate mirnas. results: out of mirnas, were detected in both tissue types. the expression of one mirna was . fold higher (q= . ) and another was . fold lower (q= . ) in the hippocampus relative to the cortex. evidence could be found that down-regulation of the latter is possibly caused by hypermethylation of '-flanking region of its encoding dna locus. bioinformatic analysis has identified eight genes important for neuronal regulation and signal transmission (e.g. sox , mecp , bsn), as well as one abc effluxtransporter, as potential targets for these differentially regulated mirnas. conclusion: differential regulation of two mirnas could contribute to an altered function of several genes resulting in an imbalance between neuronal excitation and inhibition that is independent from mechanisms presently targeted by anticonvulsants. this work was supported by a fellowship from dfg and nih grant gm . recently, it has been reported that human b cells express and secrete the cytotoxic protease granzyme b (grb) after the combined stimulation of the il- -and the b cell receptors. grb produced by b cells is enzymatically active and b cells deliver grb to sensitive cancer cell lines, thereby inducing apoptosis. to date, there is little experimental evidence on the mechanisms involved in grb expression, or its function in b cell biology. as experimental transgenic murine systems should enable us insights into these issues, we assayed for grb in c bl/ b cells using an extensive array of physiologically relevant stimuli, but were unable to detect either grb expression or its proteolytic activity, even when antigen specific transgenic b cell receptors were cross-linked. similar results were also obtained with b cells from dba/ , cba or balb/c mice. in vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of grb in cytotoxic t lymphocytes, but not in b cell populations. we also investigated a possible role of grb on the humoral immune response to np-klh, but grb-deficient mice produced normal amounts of antibody with typical affinity maturation and heightened secondary response, demonstrating conclusively the redundancy of grb for antibody responses. our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans and demonstrate the need to develop novel in vivo systems to study human humoral immune responses. investigations of the cholinergic neurotransmitter system in dyt mice hamann m. early-onset torsion dystonia is an autosomal dominant inherited movement disorder associated with the dyt gene defect with deletion of a glutamic acid residue in the protein torsina. despite the gene defect, the pathophysiology is poorly understood. animal models can help to understand the underlying mechanisms and thereby to develop new therapeutic strategies. sharma et al. ( , j. neurosci. [ ] , - ) initially described a transgenic mouse model (dyt mice) with overexpression of mutant torsina. previous studies in these mice pointed to alterations in the cholinergic system. to investigate the functional relevance of these in-vitro findings, we carried out pharmacological in-vivo experiments and determined the density of striatal cholinergic interneurons as well as the expression of choline acetyltransferase in different brain regions. the acute intraperitoneal administration of the cholinomimetic drug pilocarpine ( , and mg/kg) as well as a long-term treatment over days ( mg/kg/d) did not induce pronounced effects in dyt mice compared to wildtype controls. the higher incidence of epileptic seizures in dyt mice compared to controls after repeated local striatal applications of pilocarpine ( and µg/ . µl/hemisphere) let presume an altered synaptic plasticity in dyt mice. the immunohistochemical investigations revealed a moderately reduced density of striatal cholinergic interneurons in the dorsomedial subregion of dyt mice compared to wildtype controls, while significant differences in other striatal subregions were not detected. western blot analysis did not show clear differences in the expression of choline acetyltransferase between dyt and wildtype control mice. these results indicate that the cholinergic system seems not to play a key role in this line of dyt mice. ongoing receptor autoradiographic analysis of binding to different muscarinic receptors subtypes have to further clarify the existence of possible alterations within the cholinergic system of these dyt mice. inhibitors direct against cell cycle-regulatory kinases are being tested in clinical trials as anti-proliferative agents. thus, the atp-competitive kinase inhibitor pd which inhibits cdk and cdk is currently tested in patients with solid tumors such as glioma. we found that pd suppressed il- -induced expression of il- suggesting that cdk or cdk may have unknown anti-inflammatory properties. to study the effects of cdk on the il- -signaling network, we established a bidirectional doxycyline-inducible system to express a constitutively active mutant of cdk , cdk s p, in asynchronized hela cells. cdk -expressing cells were identified by gfp which was expressed from the same promoter, isolated by laser-microdissection and analysed for mrna expression using a down-scaled rt-qpcr assay. compared to the uninduced state, cdk s p enhanced il- -induced il- and il- mrna expression. moreover, shrna-mediated suppression of endogenous cdk confirmed a role of this kinase in regulation of maximal il- -induced gene expression of il- . these data also revealed that the contribution of cdk to inflammatory gene expression is highest in g , when activity of endogenous cdk is activated by d-type cyclins. these findings were corroborated in hela cells expressing fluorescent ubiquitin-dependent cell cycle indicator (fucci) proteins. hela-fucci cells from g , g /s, g or mitotic states were isolated by laser-microdissection and analyzed by rt-qpcr for tnf-inducible gene expression. stable knockdown of cdk in hela fucci or inhibition by pd suppressed inducible il- expression. microarray experiments identified many additional genes that required active cdk for maximal il- -or tnf-inducible gene expression. we also found that cdk co-immunoprecipitated with p nf-κb, colocalized with p in the nucleus and was recruited together with the p subunit to the proximal il- promoter as assessed by chip and re-chip experiments. collectively, these results suggest an unexpected control of inflammatory gene expression through a classical cell cycle regulatory pathway. these results also imply that pharmacological targeting of cdks may have effects and side-effects on the immune system in addition to inhibition of cell cycle progression. trp channels form a heterogeneous family of calcium-permeable channels, which play major roles in physiological functions ranging from sensory reception to cellular signal transduction. members of the trpc subfamily (classic transient receptor potential channels) are downstream targets of hormone receptors. of particular interest is the biological role of trpc channels. they are directly activated by diacylglycerol due to phospholipase c-driven signalling pathways which are involved in smooth muscle contractility, neuronal plasticity, keratinocyte differentiation and renal function. their impact in renal function became evident from analyzing patients suffering from familial forms of focal segmental glomerolusclerosis (fsgs) which could be linked to trpc mutations. since the first descriptions at least different pathogenic mutations have been identified in humans to cause fsgs. in order to study the underlying pathophysiological mechanisms of trpc mutations, we have analysed all mutated trpc channels known to date heterologously expressed cells. one set of mutations showed a gain-of-function phenotype which has been previously suggested to cause an increased intracellular calcium load and subsequent cell death. hence, gain of function mutations fit to the current paradigm of fsgs pathophysiology. however, another set of mutations found in the patients showed a loss-of-function phenotype. our results enable a change in the current paradigm for the role of trpc in renal pathophysiology and may provide a basis for our understanding of the pathophysiology of loss-of-function mutations in familial focal segmental glomerolusclerosis. karlsruher institut für technologie (kit) institut für angewandte biowissenschaften, abteilung lebensmittelchemie und toxikologie, adenauerring a, karlsruhe, germany risk assessment for genotoxic carcinogens is an important challenge in toxicology. even though manifold attempts have been made to substitute carcinogens and to reduce exposures, their complete elimination appears to be not possible. thus, low concentrations of known or suspected genotoxic carcinogens are present at workplaces, in the environment and in food. in order to deal with this situation and to set priorities for risk management, different concepts have been established such as the alara principle (as low as reasonably achievable) and the margin of exposure (moe), based on the ratio between concentrations being carcinogenic in experimental animals and the actual exposure of humans for example via foodstuff. while usually linear doseresponse-relationships have been used as default assumption, analytical methods are now available to assess the induction and repair of dna lesions on low exposure conditions, including environmental background exposure, and to relate the extent of exposure-induced dna lesions to endogenous dna damage. this may be an important prerequisite to establish health-based limit values for selected genotoxic carcinogens. within this workshop, different examples will be discussed and research need will be identified. dendritic cells from h r-deficient mice lose their ability to properly stimulate t lymphocytes hartwig c., seifert r., neumann d. mhh pharmakologie, carl-neuberg str. , hannover, germany the incidence of allergic airway diseases is increasing throughout the world, especially in western countries. although histamine (ha) is found at high concentrations in asthmatic lungs, a role for ha in bronchial asthma is still a neglected topic in clinical research. in particular, the capacity of ha to modulate the underlying immune reaction is far from being understood. the histamine h -receptor (h r) is involved in acute inflammation and th cytokine production. consequently, we intended to analyze the role of h r in a murine th lymphocyte transfer-based model of asthma. specifically the ability of h r expressed on dendritic cells (dcs) to modulate t cell function was analyzed. ova-specific cd + t cells were polarized in vitro under th -favoring conditions with ova peptide-pulsed dcs, obtained either from wild-type or h r -/mice. analysis of the polarized t cells after in vitro restimulation revealed a marked decrease of il- production in t cells polarized in the presence of h r -/-dcs compared to those polarized in the presence of wild-type dcs. thus, on dcs, the h r is essential for proper stimulation of spleen t cells and for directing their polarization towards a th phenotype. the transfer of in vitro polarized t cells into recipient mice and subsequent provocation elicited an asthma-like disease. the h r on dcs not only affects in vitro polarization of t cells, but also the in vivo function of the obtained polarized t cells. a parameter indicating allergic inflammation is the enhanced influx of inflammatory immune cells into the lung tissue, mainly driven by eosinophils, which are virtually absent in non-asthmatics. when analyzing the number of eosinophils, a dramatic difference due to the polarizing conditions of t cells occurs. in bal fluids of mice that received t cells polarized in the presence of wild-type dcs, about % eosinophils were detected. in contrast, the transfer of t cells polarized in the presence of h r -/-dcs yielded only about - % eosinophils in bal fluids. in summery, the h r on dcs plays an important role for t cell polarization and consequently affects the allergic reaction during sensitization. since the lack of the h r on dcs reduced their ability to stimulate proper th polarization of cd + t cells, we conclude that ha via the h r significantly affects the manifestation of asthmatic inflammation. antioxidant polyphenols and their effects on nrf (skn- ) signalling in a cell culture system and the model organism c. elegans havermann s., wätjen w. heinrich-heine-universität düsseldorf institut für toxikologie, p.o. box , düsseldorf, germany oxidative stress has been connected with a variety of diseases, (e.g. alzheimer`s and parkinson´s disease), cancer and ageing over the last years. certain polyphenols were shown to have an antioxidant capacity as well as being able to activate the protective nrf signalling pathway. compared to direct radical scavengers modulators have the advantage of building up a permanent defense against oxidative insults whereas scavengers do not protect any more after consumption or may even cause stress due to redox cycling. we have employed cell culture based assays (dcf, western blot, gfp reporters) to analyse the effects of polyphenols. further we tested the coumpounds in vivo in the nematode c. elegans where skn- is the nrf homologue. baicalein and caffeic acid phenethylester (cape) protected cells and the nematode from ros accumulation after application of stress (shown by dcf assay). activation of nrf signalling is correlated with translocation of the transcription factor into the nucleus. in both systems nrf ::gfp accumulation in the nuclei could be observed after incubation with baicalein (fluorescence microscopy). but while cape is a potent activator of nrf in cells, it has no effect on skn- localisation. further the effect on the nrf protein amount was investigated by western blot analysis. the expression of target genes can be investigated by differing means: while pcr methods and western blotting are standard for in vitro studies, the vast number of available gfp reporter strains offers opportunities for research using c. elegans. we have performed congruent assays in a cell culture system and the model organism c. elegans to compare antioxidative capacity and effects of polyphenols on nrf signalling. therefore, depending on the substance tested, c. elegans is a suitable model system to investigate effects of natural compounds in an organism. being associated with adverse health effects, the human exposure to dehp is subject to concern. quantifying the population's exposure and determining the contributions of different exposure routes is a key task of environmental health risk assessment. the study presented comprises a review of the available data on dehp levels in foods, consumer products, and house dust. extensive survey data, e.g. from the current national nutrition survey ii and the eu rapex system were processed for modeling the exposure by the oral, inhalative and dermal path of the population in germany. the study also included analytical analyses of dehp levels in selected foods and consumer goods (incl. migration rates for mouthing). probabilistic techniques allowed elucidating the exposure's variation and the relevance of different routes. mean exposure estimates for german children and adults to dehp are and µg/(kg d), resp. for children, food accounts for % of the total exposure, followed by mouthing ( %) and house dust ( %). the adult exposure is almost entirely ( %) due to food. as dietary exposure is a result from concentration and consumption, foods exhibiting high contamination e.g. butter ( %) and dressings (mayonnaise) ( %) as well as highly consumed foods e.g. bread and bakery ( %) and vegetables ( , %) contributed significantly. the mean estimate of children's dehp exposure via mouthing revealed , µg/(kg d). high exposures were estimated ( th percentile) up to , µg/(kg d). on average people in germany are exposed to dehp below the current tdi of µg/(kg d). however, individual exposures exceeding the tdi still cannot be excluded. current data on dehp and other plasticizers in foods are scarce, which warrants broader monitoring. our findings highly facilitate further exposure modeling focusing on dehp substitutes and risks of combined exposure. this study was funded by the federal ministry for the environment, nature conservation and nuclear safety in the frame of the environmental research plan (umweltforschungsplan, förderkennzeichen (ufoplan) ). on the basis of the available measurements of dehp, the exposure assessment has been focused on food categories characterising a selection of the most important food groups covering all major food classes of the german population. based on an extensive literature survey, the analysis considered the available data of dehp measurements in food, as well as the official german food control data taken from the national food consumption survey. the high amount of considered data allowed the consideration of several exposure assessment tiers (deterministic and probabilistic by using monte carlo simulation). a quantitative evaluation of the uncertainties of the estimate of the food categories groups has been performed by means of a sensitivity analysis by using the methodology proposed within the who ipcs guidance document of characterising and communication uncertainty in exposure analysis. qualitative uncertainty analysis (tier ) was applied to determine the most important sources of uncertainty, i.e. concentration of dehp in all food categories. the probabilistic monte carlo simulation (tier ) was then used to rank the cumulative probability distributions of the exposure assessments of food categories on the basis of the food categories that appear to dominate. sensitivity analyses were applied to prove the impact correlation of food groups for uncertainties. by a scenario based concept, the aggregation of the food groups to groups has been evaluated, as well as the sensitivities by characterising particular scenarios. for this purpose, particular "meals" have been described as fixed combined scenarios and. the aggregation leads to a considerable higher exposure estimate which can be explained by the combination of high contaminated foods with others of high consumption. the evaluation confirms the considerable role of possibly high contaminated foods e.g. fats, or mayonnaise. the evaluation shows that quantitative probabilistic sensitivity analysis is a suitable and pragmatic tool for uncertainty analysis in exposure assessment. the transcription factor camp response element (cre)-binding protein (creb) plays a critical role in regulating gene expression in response to activation of the campdependent signaling pathway, which is implicated in the pathophysiology of heart failure. we observed creb knock-out cardiomyocytes to be larger than wildtype cardiomyocytes (cell area in µm ; mean±sem; creb-ko ± vs. wt ± ; n= / cells, n= mice, p< . vs. ctr.). the nuclear factor of activated t-cells c , nfatc , is another transcription factor involved in the development of heart failure and also a known positive regulator of hypertrophy. hence, we investigated whether inhibition of the cre-dependent transcriptional activation has an impact on the nfatc signaling pathway. we first studied the effects of an overexpression of a dominant negative creb mutant (dncreb) or of nfatc on the activity of a nfat-dependent model promoter in a permanent cell line. overexpression of dncreb evoked an . ± . fold increase of the nfat model promoter activity (n= ; n= transfections), but had no impact on kv . promoter activity which is known to be regulated by nfatc ( . ± . fold; n= ; n= ; p< . vs. ctr.). nfatc overexpression led to a . ± . fold increase of the nfat-dependent model promoter activity (n= ; n= ) and to an inhibition of kv . promoter activity ( . ± . fold; n= ; n= ; p< . vs. ctr.). we conclude that creb is a negative regulator of nfat-mediated gene transcription and that activation of nfatc might contribute to the observed hypertrophy of creb-ko cardiomyocytes. neuroleptika der perazin-klasse sind potente modulatoren des p x -rezeptors -perazine-type neuroleptic drugs are potent modulators of p x receptors hempel c., nörenberg w., urban n., sobottka h., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße - , leipzig, germany p x receptors belong to a family of atp-gated, non-selective cation channels, which play an important role in immune cell activation, inflammatory hyperalgesia and neuropathic pain. they differ from other p x family members by the low atp affinity, and by the ability to form or recruit dilated pores in the sustained presence of atp. owing to its involvement in many diseased states, p x is a promising target for pharmacological intervention. accordingly, p x blockers are currently tested in phase ii clinical trials. in an attempt to identify p x -modulating properties of approved drugs or natural compounds, we performed a medium-throughput screen, using an appropriate compound library (spectrum collection) and a stably transfected hek hp x cell line. with ic values of - µm, the tricyclic antipsychotics prochlorperazine and trifluoperazine showed a high potency and efficacy to block the atp ( mm)-triggered increases in the intracellular ca + concentration ([ca + ]i) that was mediated by human p x (hp x ). the closely related phenothiazine-class neuroleptic drugs, such as chlorpromazine or triflupromazine did not have an appreciable effect on hp x mediated ca + influx. whole-cell inward currents, measured at - mv, were blocked by more than % by - µm prochlorperazine. the inhibitory effects of perazines developed within about ms, hinting to a direct mode of action by binding to the p x protein. prochlorperazine added intracellularly via the patch pipette did not substitute for the extracellularly applied drug, indicating that its binding site is accessible from the extracellular side. in addition, both compounds blocked yo-pro- uptake when preincubated before p x stimulation with mm atp or when applied subsequent to the agonist. interestingly, when added to a hek cell line expressing the rat p x , perazines potentiated the atp-induced increase in [ca + ]i. measurements in human monocyte-derived macrophages confirmed the ability of prochlorperazine and trifluoperazine to inhibit atp-evoked increases in [ca + ]i, changes in yo-pro- permeability and whole cell currents. taken together, we conclude that perazine-type neuroleptics impede on p x activity in a species-specific manner, presumably by binding to an extracellularly accessible binding site of recombinant or natively expressed p x . similarly, pre-treatment with lov also lowered dox-induced stabilisation of p and phosphorylation of chek and sapk/jnk. while lov had no influence on ir-induced initial dna damage formation in huvec and rat cardiomyoblasts (h c ), it decreased dox-and eto-induced phosphorylation of histone h ax, which is a surrogate marker of dna-double strand breaks. this indicates that lov specifically protects against the genotoxicity of topoisomerase type ii poisons. in an acute and subacute balb/c mouse model lov protected from ir-induced toxicity. this effect rested on inhibition of pro-inflammatory and pro-fibrotic processes as measured via quantification of mrna levels of il , ctgf and tnfα. the same was true for dox-induced toxicity, i.e. heart and liver damage. similar to the in vitro experiments, dox-induced hepatic dna-damage was attenuated by lov treatment. overall, liver and heart toxicity were reduced by lov as mirrored by the serum levels of gldh/gpt and ctn-i, respectively. both in liver and in heart we observed collagen rich perivascular areas following dox treatment. under situation of lov-co-treatment these areas occurred more rarely and were less pronounced, pointing to a lowered level of fibrosis. pcr-array-based mrna analyses showed inhibitory effects of lov on dox-triggered expression of genes involved in oxidative stress response, drug transport, dna repair, cell cycle progression and cell death. for instance, up-regulation of p , wee , cjun/fos and hmox- following dox administration was attenuated by lov. altogether, we suggest that including lov in current cancer therapeutic regimen might widen the therapeutic window of anticancer therapeutics by lowering normal tissue damage. the p values. in addition, array data underwent cluster analysis for identification of substantial differences of gene regulation among the three different types of biopsies. results: of particular interest in our study was the expression of genes coding for metabolism and transport proteins. therefore genes from the significant differentially regulated genes, were selected for the qrt-pcr analysis. genes coding for abcb and abcg transport proteins showed higher expression in the jejunal tissue one year after surgery compared to the duodenal tissue (fold change . and . ). moreover, cyp a mrna involved in metabolic processes is higher expressed in postoperative jejunum than in the jejunum tissue taken during the surgery (fold change . ). in conclusion roux-en-y gastric bypass operation leeds a change of mucosal gene expression profile in the jejunum during one year. there was also a significant differential gene expression between the original duodenum and jejunum one year after surgery. these results give strong evidence that jejunum not exposed to pancreatic but only to gastric fluids may change its gene regulation. background. numerous genome-wide association studies (gwas) identified polymorphisms located in transporter genes such as slc a , abcg , npt , and urat as predicitive for the serum levels of urate . these genes encode membrane proteins expressed in the apical membrane of human kidney proximal tubule cells and are assumed to facilitate tubular exchange of urate , , , . importantly several single nucleotide polymorphisms (snp) located in vicinity of slc a have been identified as highly associated with serum urate levels. little is known about the transcriptional regulation of slc a . therefore, the aim of our study was to investigate which sequences in the slc a gene harbour ciselements and regulate its gene expression. we also asked whether intronic snps influence gene expression at the transcriptional level. methods and results. performing dual luciferase reporter gene assays we found gene regulating modules in the slc a gene. dna from human kidney samples was then genotyped for rs being part of this region. next total slc a mrna-expression levels of the samples were determined using real-time quantitative rt-pcr assay. male samples with two minor alleles of snp rs showed lower slc a mrna levels than samples with the wild type alleles. the effect was not seen in females. reporter gene constructs with either minor or major allele of rs were then used in luciferase assays, however showing no significant difference in activity. furthermore mrna-expression levels of other urate transporter genes were determined in kidney samples. after linear regression a positive correlation of mrna-expression of slc a , urat , npt , and oat , respectively was observed. conclusion. our data suggest that the slc a snps rs and rs might influence slc a mrna-level without controlling the transcriptional activity. it needs to be elucidated whether those snps alter mrna stability. however, the mrna coexpression of slc a and other urate transporter might be attributed to a common gene regulating pathway of an "transportosome" controlling urate homeostasis. sulfotransferases mediate the bioactivation of methyleugenol to a reactive sulfate ester binding to dna in vitro and in vivo herrmann k. methyleugenol (me) is a secondary metabolite occurring in many herbs and spices. although me is hepatocarcinogenic in rodents, standard genotoxicity tests were negative. this may be due to the lack of critical activating enzymes responsible for the terminal bioactivation of me to a genotoxicant. me is initially hydroxylated by cytochrome p enzymes yielding ´-hydroxymethyleugenol ( ´-ohme). this alcohol can be further activated by sulfotransferases (sults) to an electrophilic sulfate ester that can be easily attacked by dna. the dna adducts formed could lead to mutation and further carcinogenicity observed in animals. the aim of the present study was to clarify whether individual human (h) and murine sult forms are involved in the activation of me to a genotoxicant. in order to identify critical sults, mutagenicity tests including bacteria expressing different sult forms were conducted. (±)- ´-ohme (separated into its enantiomers) served as test compound. we could show that hsult a , standing out due to its high expression level in many tissues, can efficiently activate both enantiomers even at low concentrations. furthermore, dna adduct formation in hsult a -proficient and sult-deficient bacteria was examined after incubation with µm of (+)-or (-)- ´-ohme. for selective detection and quantification of me-derived ´-deoxyadenosine (da) and ´-deoxyguanosine (dg) adducts we developed a sensitive tandem mass spectrometry method including stable isotope dilution analysis. adduct formation was only observed in bacteria expressing hsult a . the concentration dependence of adduct formation in hsult a -proficient bacteria was examined for (+)- ´-ohme. both adducts turned out to be concentrationdependent. to check the extent and organ specificity of adduct formation in vivo we administered mg/kg bw (±)- ´-ohme (i.p.) to mice carrying the hsult a / a gene cluster. mice getting only the vehicle served as controls. animals were sacrificed and dna from eight organs was extracted. by means of tandem mass spectrometry adducts were measured and quantified. da and dg adduct formation was observed in all tissues studied, but not in untreated animals. furthermore, adduct levels were higher than in experiments using wild-type mice. altogether, we herein could show that sulfo conjugation leads to bioactivation of me to a dna-binding intermediate in vitro and in vivo. this work was financially supported by bundesinstitut für risikobewertung. objective: the soluble adenylyl cyclase (sac) activates the na + /k + -atpase in renal epithelial collecting duct cells. nuclear sac constitutes a functional complex with camp response element binding protein (creb), suggesting a more general role of sac in overall gene regulation. we determined the chromatin binding capacities of sac at cre sequences and its influence on genes, which play a role in aldosterone signalling. furthermore, we functionally characterised expression relevant promoter portions of sac and the influence of aldosterone and camp mediated signalling pathways on sac gene regulation. design and methods: in vascular endothelial cells (ea.hy ) and in human kidney cell lines (hek t; ihke), we performed chromation immunoprecipitation (chip) assay with antibodies against sac and creb. we conducted transfection with a cre luciferase reporter vector and sac promoter constructs, following treatment with sac inhibitors and aldosterone. total rna of ea.hy cells, which were treated with sac inhibitors and aldosterone, was isolated and subsequently analysed by real-time pcr for expression of genes involved in aldosterone signalling. in vivo binding of sac at cre motifs was shown using cre consensus sequences in chip experiments. specific pharmacological inhibition of sac led to a significant decrease of transcriptional activity of the cre control vector in endothelial and kidney cell lines. furthermore, we were able to show the different effects of sac on the expression of downstream targets of aldosterone signalling, e.g. mineralocorticoid receptor and na + /k + -atpase alpha and beta and sac itself. regulation of sac itself is mediated by two different promoter portions, which are influenced by aldosterone and inhibition of sac and differentially accessed in kidney and endothelial cells. sac has transcriptional trans-acting properties as it interacts with cre sites and potentially influences the expression of genes, which play a role in aldosterone signalling. transcription of sac is regulated via aldosterone and camp. the location of promoter ta is cell type-and stimulation-specific. the role of the sodium-calcium exchanger (ncx ) in cardiac pacemaking herrmann s., stieber j., ludwig a. friedrich-alexander-universität erlangen-nürnberg institut für experimentelle und klinische pharmakologie und toxikologie, fahrstrasse , erlangen, germany the mammalian heart is driven by the sinoatrial node, the primary cardiac pacemaker. the unique feature of sinoatrial node (sn) cells is the ability to generate a spontaneous diastolic depolarization that periodically initiates action potentials which set the heart rhythm. the molecular origin of this cardiac pacemaker activity is still a matter of debate. recent findings point to a coordinated interplay between intracellular ca + -cycling processes and plasma membrane-localized ion channels which determines the origin, periodicity and rate modulation of pacemaker potentials. in this study, we investigated the contribution of the cardiac sodium-calcium exchanger (ncx ) to pacemaking. ncx is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. it was speculated that the membrane depolarizing current incx, whose activity is dependent on intracellular ca + -fluctuations, represents a main determinant of the spontaneous diastolic depolarization. we used an inducible and sinoatrial node-specific cre-transgene to delete ncx in the murine pacemaker system. the successful creation of a cardiac pacemaking and conduction system specific ncx knockout (cpncx ko) was demonstrated by transcript quantification as well as immunofluorescence experiments. telemetric ecg recordings of cpncx ko displayed a distinct cardiac phenotype. mutant animals were deeply bradycardic and lost their capability of maintaining a stable heart beat as demonstrated by various ecg abnormalities like sn arrhythmia, sn pauses, av block and ventricular tachycardia. analysis of the spontaneous activity of isolated sn preparations showed a slower and arrhythmic contraction rate of the mutant tissues strips confirming that the bradycardia and arrhythmia induced by deletion of ncx results from a slower and arrhythmic intrinsic pacemaker activity. a battery of experiments using different heart rate lowering as well as increasing drugs revealed an altered heart rate modulation in cpncx ko animals as compared to controls. in conclusion, these initial results establish ncx as a major contributor to cardiac pacemaking. a wide variety of contaminants are ingested through food, among them the procarcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (bp) which is resorbed and partially metabolized in the enterocytes of the small intestine. previous in vitro studies revealed that bp phenols are excreted as phase ii metabolites including bp glucuronides and bp sulfates. this export is mediated by the breast cancer resistance protein (bcrp/abcg ). the ultimate carcinogenic phase i bp metabolite anti-bp- , dihydrodiol- , -epoxide (bpde) can be detoxified by glutathione conjugate formation catalyzed by various glutathione s-transferases. in the present study, differentiated human intestinal caco- cells were used as a model for the human small intestine to investigate the detoxification of bpde and the subsequent transport of the stereoisomeric glutathione conjugates in the presence of an inhibitor (acivicin) of the glutathione-cleaving enzyme gamma-glutamyl transpeptidase (ggt) at the surface of the cells. the results indicate that the glutathione conjugates of bpde are formed and excreted mainly to the apical and to a minor extent to the basolateral side of the polarized caco- monolayer. to stimulate the transport rate several inducers known to enhance gene expression of xenobiotic-metabolizing enzymes as well as transport proteins were used (quercetin, oltipraz, butyrate). however, solely oltipraz substantially increased the efflux of bpde glutathione conjugates after inhibition of ggt. inhibition studies revealed that the multidrug resistance-associated proteins (mrps/abccs) are involved in the transport of the bpde glutathione conjugates. stable abcc , abcc and abcc knockdown cell lines were generated allowing to demonstrate that abcc mediates the basolateral, abcc the apical excretion of the bpde glutathione conjugates. in conclusion, the ultimate carcinogen bpde is detoxified via glutathione conjugation and subsequently excreted by caco- cells in both apical and basolateral directions. .this finding is equivalent to a transport into the feces as well as blood system in the in vivo situation. signaling via irag regulates store operated calcium entry (soce) in aortic vsmc hieke b., hüttner j., schlossmann j. university of regensburg department of pharmacology and toxicology, universitätsstr. , regensburg, germany the mechanisms involved in the activation of store operated calcium entry (soce) through depletion of intracellular stores and their regulation are not yet fully understood. we examined the effect of inositoltriphosphate-receptor associated cgmp-kinase substrate (irag) on soce. aortic vascular smooth muscle cells (vsmc) from wild type (wt) and irag-knock-out (ko) mice were loaded with the calcium indicator fura -am and soce was measured as a change in the intracellular calcium concentration. in experiments with vsmc from wt mice soce was attenuated by the application of -br-cgmp. this effect was not observed in vsmc isolated from irag-ko mice. these differences in the strength of the soce-signal were abolished by the replacement of extracellular sodium with n-methyl-d-glucamine. the observed sodium dependence of the soce regulation via irag suggests, that an alternated sodium conductance might be responsible to some extent for the differences detected in wt and irag-ko vsmc. as a change in sodium conductance might result in a changed membrane potential we tried to track these changes with the flipr membrane potential assay kit while executing the soce protocol with and without -br-cgmp. no significant differences in membrane potential could be detected in the various stages of soce. in conclusion, our results indicate that irag exhibits a dual action on calcium regulation as it inhibits not only the intracellular calcium release but also the extracellular calcium influx through soce. induction of the icer promoter in vascular smooth muscle cells hildebrandt i. tokyo metropolitan institute of gerontology, tokyo japan several transcription factor isoforms are encoded by the crem (camp response element modulator) gene. one prominent isoform is the inducible camp early repressor (icer), which acts as a transcriptional repressor on so called camp responsive elements (cres) in its target gene promoters. the icer mrna expression is regulated by an intronic promoter of the crem gene. in vascular smooth muscle cells (vsmcs) crem/icer is involved in the regulation of cell proliferation and apoptosis with physiological consequences in vivo. for instance crem-knockout mice, in which none of the known isoforms can be expressed, exhibit an increased neointima formation after carotid ligation as well as an increased atherosclerotic plaque formation after high fat diet on an apoe background. these observations were associated with an increased proliferation rate in isolated crem deficient vsmcs. on this background we wanted to clarify the specific role of icer isoforms in the vasculature. in first experiments we examined the inducibility of icer in primary vsmcs and smooth muscle cell lines. reporter luciferase assays showed that the activity of the icer promoter is induced at the maximum of fourteen fold after hours of stimulation with forskolin (fsk) in immortalized rat vsmcs ( . ± . ; n= from isolations). in a r rat smooth muscle cells the icer promoter showed a maximum stimulation of . ± . fold after two hours of fsk stimulation (n= from isolations). these pilot experiments showed that the icer promoter is inducible in vsmcs by camp dependent pathways. further experiments have to be carried out to elucidate the role of icer in the vascular system for example by stimulation of primary vsmcs and analysis of icer knockout mice. (supported by the dfg) overexpression of transmembrane channel-like proteins (tmcs) uncouples receptor-mediated calcium mobilisation hill k., urban n., straub i., schaefer m. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. - , leipzig, germany the family of transmembrane channel-like proteins (tmcs) consist of members (tmc -tmc ) all tmc genes are predicted to encode transmembrane proteins with at least six membrane-spanning helices. mutations of tmc cause deafness in human and mice whereas tmc and tmc (also referred to as ever and ) are linked to epidermodysplasia verruciformis (ev), a skin disorder, which is characterised by an enhanced susceptibility towards cutanous infections by human papillomaviruses. the cell biological and physiological functions of tmc proteins still remain elusive. we have overexpressed tmc and tmc in hek cells to get insights into their physiological function. all tmcs were located within the endoplasmic reticulum (er) after overexpression of yfp or cfp-tagged constructs. ratiometric calcium imaging revealed that after overexpression of tmc or tmc , stimulation of gq-coupled receptors with carbachol and atp resulted in a greatly reduced amount of calcium release from the er. moreover, challenging tmc -or tmc -expressing cells with the serca pump inhibitor thapsigargin was also not followed by a release of er-based calcium within the cell. to test whether the lack of calcium release was caused by a reduced calcium content within the er, we investigated calcium dynamics within the er using an er-targeted fret-based calcium indicator (d er cameleon). the experiments revealed that the amount of calcium within the er was reduced upon overexpression of tmc or tmc . recently, it has been reported that tmc and tmc might influence intracellular zinc distribution in human keratinocytes. we could confirm the presence of tmc and tmc mrna in a human keratinocytes cell line (hacat). upon overexpression of tmc , hacat cells revealed the same phenotype as described above for the hek cells with an uncoupling of the receptor-mediated calcium mobilisation due to a depletion of the er calcium store. the mechanism by which overexpression of tmc proteins causes a reduced calcium concentration within the er remains unclear. considering that the presumed topology of the tmc proteins distantly resembles those of other ion channel superfamilies such as anoctamins, one might speculate that a conductance through the tmc protein itself leads to a calcium leak from the er. terahertz radiation is defined as radiation between . thz and thz. a number of applications are currently being developed using radiation in this frequency range. these applications will lead to exposure of the general public, making it very important to study potential effects on biological systems. historically, only a few studies on effects caused by terahertz radiation have been conducted because of the lack of suitable generators and detectors. during the last decade, a number of studies on effects caused by radiation with frequencies around ghz have been published. the present study investigated the genotoxic potential of terahertz radiation at three different frequencies, . thz, . thz and . thz. two skin cell types were used, primary human dermal fibroblasts (hdf) and a keratinocyte cell line (hacat). the cells were irradiated applying different exposure times and different power intensities. two genotoxicity tests were applied: the comet assay quantifies dna strand breaks as well as alkali-labile sites whereas the micronucleus test quantifies chromosomal damage. all experiments were performed and evaluated under blinded conditions as three independent replicate experiments. positive (mms-treated) and negative (untreated, sham-exposed) controls were included. in the comet assay no dna damage was observed as a consequence of the exposure under all experimental conditions. the same was true for the chromosomal damage investigated with the micronucleus test. the latter finding was particularly interesting for the experiments at . thz, because this type of radiation had been reported to cause mitotic disturbances. therefore these experiments were extended, applying higher power intensities and longer exposure periods. also with these modifications, no genomic damage was observed in the form of micronucleus formation. all in all, terahertz radiation did not induce genomic damage under the applied experimental conditions. this result is in line with published findings on genotoxicity of low-frequency terahertz radiation around . thz. the question, why the reported mitotic disturbances do not lead to manifest genomic damage remains open and requires further research. introduction: tea flavonoids derived from camomile and green tea such as apigenin and epigallocatechin gallate (egcg) can inhibit intestinal neoplasia. recurrences of adenomas and cancers were reduced in patients with resected colorectal cancer by treatment with tea bioflavonoids after tumor operation [ ] . to clarify the biomolecular pathway for suppression of neoplasia we investigated the anti-inflammatory effect of a nutritional supplement flavo natin® (fn) which had been used in the clinical study on tertiary tumor prevention and of egcg in a colon tumor cell line. the aim of our study was to investigate if tea flavonoids are capable to suppress the inflammatory markers produced by tumor cells after cytokine stimulation. method: we studied the cytotoxicity of fn in the colon cancer cell line t- by resazurin fluorescence and compared it with the placebo supplement. additionally, the t- cells were incubated with fn, egcg or placebo and stimulated with tnf-alpha, if-gamma and il- -beta. after the cytokine stimulation the mrna expression of ip- , il- and tnf-alpha was measured by quantitative real-time pcr (qrt-pcr). results: stimulation of t- cells increased the expression of ip- (gamma-interferon inducible protein ), tnf-alpha and il- . by preincubation with fn at µm the mrna expression of ip- was strongly reduced (log -ratio - ). the tnf-alpha mrna was also but less decreased by fn. egcg displayed an inhibition pattern similar to fn. placebo did not influence the mrna expression of the chemokines and tnf-alpha. discussion & conclusion: clinically useful dietary tea bioflavonoids inhibit the expression of inflammatory genes in a colon cancer cell line. down-regulation of inflammatory gene products could be achieved in vivo by botanicals without clinically relevant side effects. [ ] h. hoensch, b. groh, l. edler, w. kirch ( ) . prospective cohort comparison of flavonoid treatment in patients with resected colorectal cancer to prevent recurrence. world j gastroenterol, , - . the cxcr c-terminal domain mediates efficient cxcl uptake and degradation hoffmann f., müller w., schütz d., schulz s., stumm r. universitätsklinikum jena institut für pharmakologie und toxikologie, drackendorfer str. , jena, germany cxcl -signaling mediated by the g protein-coupled cxcr receptor plays a key role during embryonic development and disease states including cancer and inflammation. the second cxcl -receptor cxcr modulates cxcl /cxcr -signaling by acting as a cxcl -scavenger. given the distinct functions of cxcr and cxcr , we hypothesized that trafficking and receptor stability are differently regulated by the distinct c-terminal domains. here, we examined epitope-tagged wild type and c-terminal mutant receptors expressed in human embryonic kidney cells (hek ) with respect to trafficking, stability, i-cxcl radioligand degradation, and g protein-coupling. we found that the c-terminal residues of cxcr were sufficient for cxcr to undergo rapid spontaneous internalization. replacement of the cxcr c-terminal domain with that of cxcr (cxcr - tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization in conjunction with c-terminal phosphorylation. conversely, replacement of the cxcr c-terminal domain by that of cxcr caused ligand-independent internalization of cxcr . receptor-mediated i-cxcl -uptake, release of i-cxcl -degradation products, and degradation of the receptor protein itself were accelerated with receptors bearing the cxcr c-terminus. while the cxcr c-terminus was sufficient to abolish g protein coupling in the cxcr - tail mutant, replacement of the cxcr c-terminus, cxcr second intracellular loop or both domains with the corresponding cxcr domain did not generate a g protein-coupled cxcr chimera. taken together, we provide evidence that the cxcr c-terminal domain influences the ligand-uptake/degradation rate, g protein-coupling, and stability of the receptor. this suggests that heterologous regulatory pathways targeting the cxcr -c-terminal domain may effectively control cxcr functions. soluble guanylyl cyclase is a key mediator of brown adipocyte differentiation hoffmann l. s. brown adipose tissue (bat) uses energy to produce heat by inducible thermogenesis. recent studies show that active bat is present in adults and involved in human energy balance, suggesting that the energy consuming property of bat might be exploited to fight obesity and related diseases. the no/cgmp signaling pathway is a key player in diverse physiological processes. recently, we have shown in bat that cgmp signaling is connected with insulin signaling and abrogation of cgmp signaling leads to impaired bat differentiation and function (haas, b. et al., sci signal, ). here we investigated the role of the cgmp generating enzyme soluble guanylate cyclase (sgc) in bat differentiation in vitro. mesenchymal stem cells isolated from bat of newborn sgcβ -/mice and wt littermates were differentiated in vitro into brown adipocytes in the presence or absence of cgmp. abundance of sgc isoforms was determined by qrt-pcr and western blotting. bat differentiation was assessed by redo staining of accumulated intracellular lipids, measurement of triglyceride (tg) content, determination of expression of bat marker proteins pparγ, c/ebpα, ap and bat marker genes ucp , pgc α, cidea. the α and β isoforms of sgc were highly expressed in bat whereas α sgc could not be detected. redo staining of wt brown adipocytes showed basal differentiation which was increased upon addition of -pcpt-cgmp. in contrast, staining was lower in sgcβ -/cells compared to wt under control conditions and increased in the presence of cgmp. tg measurement showed that sgcβ -/brown adipocytes contain approximately % less lipids than wt cells under basal conditions. addition of cgmp doubled tg content in both genotypes. similar results were observed for marker protein expression. deletion of sgc resulted in - % decrease in c/ebpα, pparγ and ap expression compared to wt. again, cgmp roughly doubled protein expression in sgcβ -/and wt cells compared to control. under basal conditions, bat marker gene expression was decreased by approximately % in sgcβ -/cells compared to wt cells. this decrease was prevented by addition of cgmp. these results show that sgc deletion leads to dysfunctional bat differentiation and emphasize the central role of cgmp signaling in bat differentiation. further investigation of sgc/cgmp signaling in bat might reveal new drugable targets bringing bat-dependent pharmacological therapy to treat obesity and related disease into closer reach. comparative inhalation toxicity of carbon-nanomaterials (multi-wall carbon nanotubes, graphene and carbon black) hofmann t. carbon black is a spherical carbon anomaterial whereas multi-wall carbon nanotubes (mwcnt) are cylindrical and graphene is a laminar allotrope of carbon. processing and handling as well as abrasion processes can set free inhalable cnt particles. results of rodent studies collectively show that regardless of the process by which cnts were synthesized and the types and amounts of metals they contained, cnts were capable of producing inflammation, epithelioid granulomas, fibrosis, biochemical and or toxicological changes in the lungs (lam et al. , muller et al. , ma-hock , pauluhn . graphene possess similar physical properties as cnt but may different toxicological property. we performed short-term inhalation studies in rats to compare the toxic potency of four different cnt, two graphenes and one carbon black. the materials are characterized thoroughly according to the oecd list. the four mwcnt caused morphological changes as descriped above. several biochemical and cytological parameters in the broncho-alveolar lavage fluid were strongly increased consistent with the histological findings. two mwcnt exhibited a higher toxic potency than two other mwcnts and findings caused by one graphene typ were even less severe. the graphene with lower surface area as well as low surface area carbon black did not cause any adverse effects up to mg/m . the short-term inhalation studies were able to descriminate different toxic potencies of carbon-based nanomaterials and is hence used for the selection of less toxic materials for further product development as well as to define and prioritize higher-tier toxicological testing of nanomaterials. synthesis and analytical assessment of possible dna adducts formed after activation of the tobacco alkaloid myosmine högg c., zwickenpflug w., gudermann t. walther-straub-institut abt.: toxikologie, nußbaumstraße , münchen, germany myosmine represents one of the minor tobacco alkaloids and its effective uptake from smokeless tobacco or tobacco smoke, as well as by consumption of food is not understood in detail. myosmine can be activated by peroxidation and n-nitrosation yielding -hydroxy- -( -pyridyl)- -butanone (hpb) which is well known as reactive intermediate during activation of a variety of tobacco specific n-nitrosamines (tsna). therefore, myosmine may be a potential candidate for possible mutagenic or carcinogenic risk to human health. furthermore, myosmine n-nitrosation yields the tobacco specific nitrosamine n-nitrosonornicotine (nnn), which is classified as carcinogenic to humans. considerable efforts have been undertaken, especially in organic synthesis, to verify and elucidate the significance of the hpb precursor the pyridyloxobutyl (pob) intermediate and its dna-adducts, which were analysed only in animal experiments till now. the formation of -pyridylmethanol was observed under myosmine peroxidation and identified as a metabolite in rat urine after application of myosmine to rats. the formation of the -pyridylmethanol intermediate, might provide for the reactive electrophilic picolinium ion which could interact with dna. these possible adducts might be of special interest to elucidate the role of myosmine concerning its uptake by smokers and passive-smokers in contrast to non smokers ingesting the substance by consumption of food. dna adducts may help to obtain more information about possible risk assessment of myosmine and to differentiate between the activation from the other nicotinoids and tsna. the specific dna adducts, -methyl- , -bispyridin- -ylmethyl- h-pyrimidin- , -dion and -( ''-picolyl)thymidine have been synthesised as reference substances. the former was prepared by addition of diisopropylazodicarboxylate (diad) to a mixture of -pyridylmethanol, thymine and triphenylphosphine. the reaction product was identified using nmr ( h, c, cosy, hmqc, hmbc). for synthesis of -( ''-picolyl)thymidine the hydroxyl groups of thymidine were initially acetylated using acetic acid anhydride and dimethylaminopyridine (dmap). the existence of this thymidine adduct was confirmed using nmr. this adduct was labelled with -([ , ,-dichlorotriazin- -yl]amino)fluorescin (dtaf) to enhance the sensitivity using hplc-fluorescence chromatography and used as reference substances for analysis of dna samples from biological tissue. supported by dfg grant (ty / - ). cancer and cardiovascular diseases such as atherosclerosis are the most important causes of death in western societies. common to both diseases is a deregulation of cell death, with significant contribution of inflammatory processes. enhanced oxidative stress plays a dominant role in such events as it forms a vicious cycle with inflammation and controls multiple forms of cell demise. therefore, anti-oxidative enzyme systems gained considerable interest since control of reactive oxygen species (ros) has the capacity to regulate cell death in either direction. the human enzyme family of paraoxonases consists of three members, known as pon , pon and pon . while pon is found predominantly in the circulation, pon and pon are intracellular enzymes with established anti-oxidative functions. it has been shown that both pon and pon are protective against atherosclerosis. underlying mechanisms of their protective and antioxidative functions however remained elusive. here we demonstrate that both enzymes locate to the endoplasmic reticulum (er) and mitochondria where they fulfill vital functions in the control of ros generation. in particular, pon and pon were shown to interact with coenzyme q which diminishes mitochondrial ros formation. as a consequence, these enzymes reduce execution of mitochondrial apoptosis, such as cardiolipin peroxidation, cytochrome c release and caspase activation. moreover, pon and pon reduced er stress-triggered cell death, i.e. by diminishing jnk signaling and chop expression. while these results elucidate their protective role in cardiovascular diseases, it also establishes a relevant function in survival of tumor cells. in accordance, we demonstrate that both enzymes are frequently found overexpressed in various tumors. in cancer cell culture studies, overexpression of both enzymes granted considerable resistance against chemotherapeutics. in turn, knock-down of pon caused spontaneous apoptosis of several cancer cell lines. finally, our analyses also revealed that pon -knockout mice show severe alterations of the hematopoetic stem cell compartment, which implies a significant role in leukemias where these enzymes are frequently found overexpressed. together, our results propose pon and pon as new putative anti-tumor candidates and demonstrate the efficacy of interventions targeting cellular redox-balance. steigerwald arzneimittelwerk gmbh wissenschaftliche abteilung, havelstr. , darmstadt, germany stw (iberogast ® ), a multi-component herbal drug, is successfully used in the therapy of functional dyspepsia and irritable bowel syndrome (ibs). previous studies revealed effects of stw on disturbed motility and inflammatory processes. although the antiinflammatory properties of stw are well examined, the contribution each of the individual herbal constituents to the anti-inflammatory effect remains unclear. therefore, we studied the effects of stw and its components on inflammation-induced cell death and on the release of the pro-inflammatory cytokine tnf-α. the aim of these investigations was to analyse additive or synergistic effects of the components. the experiments were carried out on caco- cells after stimulation with lps ( ng/ml) for hours. cytotoxicity was evaluated using a commercially available ldh (lactate dehydrogenase)-assay. furthermore, the release of tnf-α after lps ( ng/ml) stimulation of differentiated thp- cells was measured using a commercially available elisa. stw ( . - . µg/ml) reduced lps ( ng/ml)-induced cell death in a concentration-dependent manner with a maximum inhibition of . %. the herbal components in equivalent concentrations contributed to the inhibitory effect of stw to different extents. the maximum inhibition differed in a wide range between the components. stw ( . µg/ml) reduced significantly the release of tnf-α by % in lps ( ng/ml)-stimulated differentiated thp- cells while having no effect in untreated cells. in concentrations equivalent to stw caraway, milk thistle, lemon balm and greater celandine had no effect on the lps-induced increase in tnf-α release. bitter candytuft, peppermint, chamomile, liquorice and angelica reduced the tnf-α release, though less pronounced as compared to stw , indicating a possible synergistic effect. our results indicate a multi-target effect of stw . the anti-inflammatory effect may be due to a reduction of the cytotoxic effect on intestinal mucosa cells and to an inhibition of the release of the pro-inflammatory cytokine tnf-α from immune cells. the individual herbal components seem to contribute by different mechanisms of action to the overall effect of stw . immune cell-induced local steroidogenesis in the lung: implications for asthmatic disease and therapeutic intervention hostettler n. , brunner t. glucocorticoids are steroid hormones with potent anti-inflammatory properties. synthetic glucocorticoids are frequently used for the therapeutic treatment of inflammatory disorders, such as asthma. endogenous glucocorticoids are predominantly produced in the adrenal glands in response to emotional, physical and immunological stress. recent years, however, revealed several alternative sources of these immunoregulatory steroid hormones. thus, we have found that the intestinal epithelium is a rich source of glucocorticoids and intestinal glucocorticoids contribute to the maintenance of local immune homeostasis ( ) . as the intestinal and the pulmonary epithelium have much in common, i.e. barrier functions and transport of nutrients, resp. gases, we wondered whether the lung mucosa is also capable of synthesizing immunoregulatory glucocorticoids in response to immune cell activation. the murine lung was found to expresses all enzymes required for the synthesis of corticosterone from cholesterol. while most enzymes where expressed in a constitutive manner, cyp a , encoding p ssc, was strongly induced in response to immunological tress. treatment of mice with t cell-activating anti-cd antibody of macrophage-activating lipopolysaccharides induced strong local glucocorticoid synthesis, which was effectively blocked by the corticosterone synthesis inhibitor metyrapone, indicating that glucocorticoids measured were produced bona fide in the lung tissue. surprisingly, allergen-induced allergic airway inflammation failed to trigger local glucocorticoid synthesis despite the massive infiltration of neutrophils, eosinophils and t cells. in contrast to that in the intestinal epithelium local glucocorticoid synthesis in the lung was found to be dependent on the presence of adrenal glands as adrenalectomy abolished pulmonary steroidogenesis. in line with the notion that the lung metabolizes steroid precursors we found that ex vivo cultured lung tissue metabolized h- during the last decade small rna molecules has been identified as important regulators for gene expression. these micrornas (mirna) are single-stranded transcripts, which are expressed in many cell types, where they modulate rna stability and translation and, therefore, controlling various cellular mechanism and tissue development. against this background, in the present study the mirna expression and potential target genes were studied in the human placenta as a tissue demonstrating various developmental changes in a limited period of time. taqman®array microrna cards for profiling of mirnas in placentas of different gestation times revealed a significant expression of mirnas by comparing placentas of early gestation (< .week), preterm (< .week) and term (> .week). when comparing the analyzed groups, of these mirnas expose a continuous up-(including mir- a, mir- ) or downregulation (including mir- , mir- a). while comparing placentas of early gestation to term placentas, % (e.g. mir- , mir- ) were up-and % (e.g. mir- , mir- ) were downregulated. by focusing on the latter group with early preterm placentas the ratio is opposed. here, % of mirnas showed higher (e.g. mir- ) and % lower expression (e.g. mir- , mir- ). with emphasize on these mirnas, a computational prediction algorithm using the mirò database predicted a potential interaction between corticotropin-releasing hormone (crh) and the mirnas mir- and mir- . specific expression analyses validate an inverse expression between mirnas and target with a reduced expression of mir- ( %) and mir- ( %) in term placentas, while crh is upregulated fold. this interaction was verified on functional level by reporter gene assay. a significantly suppressed luciferase activity of the reporter plasmid containing the ´-utr sequence complementary to crh was exhibit for the predicted mir- binding site. here, the mirna-mrna-interaction reduces the luciferase activity by ~ %, whereas the decreased luciferase activity for the predicted mir- binding site is not significant. the results demonstrate a gestation dependent expression of placental mirnas, which may help to explain gestational changes in gene expression and highlight the potential of mirnas as biomarker for pregnancy-related pathologies. since homo sapiens era wound treatment by early civilizations was based on the use of local flora. scilla indica knuth liliaceae (s.i) is a perennial herb with a pear shaped, tunicated bulb, bearing fibrous roots, white flowers and leaves on the stem resembling u. maritima. it is used as cardiac tonic, against inflammation, ulcers and sinus diseases. traditionally the powdered bulb is used topically for warts treatment, while roasted and crushed bulbs are applied to corns of the feet soles. the plant contains steroid glycosides (bufadienolides - %), glucoscillarene a, proscillaridine a, scillarene a, scillcyanoside ,scilliglaucoside ,mucilage and alkyresorcinol derivatives exerting skin healing properties similar to wheat bran products. the study is focused on the investigation of the restoration quality of a skin dorsal incision wound in wistar rats in three groups, control (a ) and experimental (a ,a ) of rats weighing - g local application of dichloromethane extract of s.i in vehicle olive oil preparations of and mg were used. animals were anaesthetized ( ether pro narcosi ), trauma cm long and mm deep was performed by lancet on depilated dorsal area skin until the muscular aponeurosis and were treated for days with λ of each preparation. group a showed increased remodelling of trauma area (limited width). granulomatous tissue was more pronounced in length, width and surface in group a . the macroscopic ulcus dimensions were better in group a while the microscopic view were similar in a and a and better in comparison to control a . a tendency to trauma remodelling was observed, without statistical significance (t = . ) in recent years, it has been suggested that nanoparticles generated from combustion processes (e.g. diesel engine exhaust particles), may contribute to the pathogenesis of neurodegenerative diseases such as alzheimer's disease (ad). the aim of our current study was to investigate the effects of subchronic exposure to diesel engine exhaust (dee) in the xfad mouse model, which is characterised by progressive behavioural deficits as well as amyloid plaque formation and neuron loss. ten weeks old female xfad mice and their nontransgenic littermates were exposed by whole body inhalation to diluted dee (~ mg particles/m ) or clean air (controls) for or weeks ( days/week and hour/day). subsequently, all animals were subjected to a series of behavioural tests. at ten days post-exposure, mice were sacrificed to investigate lungs and brain tissues for pathological and biochemical and molecular-biological changes. in line with the expectations, the xfad mice displayed typically age-dependent behavioural deficits and amyloid plaque formation in cortex and hippocampus. a significant dee exposure-related effect was observed for the string suspension test, representing a measure of motor coordination/grip strength. dee exposure was also associated with mrna expression changes of markers of inflammation and oxidative stress in specific brain regions, including the olfactory bulb. histopathology of plaqueload in cortex and hippocampus (from a limited number of animals investigated so far) did not reveal clear evidence for increased plaque formation due to the dee exposures. further research is needed to evaluate the effects of long term exposure to nanoparticles in the central nervous system. this work is supported by funds from the research committee of the medical faculty of the university of düsseldorf ( - ), the dfg graduate school grk and the rivm -centre for environmental health, bilthoven, netherlands. mono-glucosylation of (h/k/n)ras by clostridium sordellii lethal toxin (tcsl) blocks critical survival pathways, including the pi k/akt, the ralgef/ral, or the raf/erk, and results in apoptosis. in this study, ras glucosylation is presented to result in expression of the cell death-regulating small gtpase rhob based on transcriptional activitation. rhob expression depends on k-ras inhibition, as sirna-mediated knock-down of specifically k-ras (neither h-ras nor n-ras) provokes rhob expression. rhob expression further depends on inhibition of pi k/akt, as activation of pi k/akt using a pi k activator prevents rhob expression downstream of inactivated ras. newly synthesized rhob is gtp-loaded and rapidly degraded in a proteasome-and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a rho family protein. although often characterised as a pro-apoptotic protein, rhob suppresses caspase- activation in tcsl-treated fibroblasts. conclusions: . rapid and efficacious ras inactivation by tcsl turns out to be particularly useful in the characterisation of ras inactivation-induced rhob expression as an immediate-early gene response. . the finding on the cytoprotective activity of rhob in tcsl-treated cells re-enforces the concept that rhob exhibits cytoprotective rather than pro-apoptotic activity on cellular background of inactive ras. the activity of the rhob promoter is suppressed by rhoa (through a not yet identified pathway or ras (through the pi k/akt pathway. ras glucosylation by tcsl results in de-suppression of the rhob promoter and rhob expression. the calcium-binding protein annexin a (anxa ) is involved in diverse cellular processes including e.g. vesicular transport, ion channel regulation and transcriptional regulation. upon ca + binding anxa undergoes conformational changes, which lead to oligomerization of anxa to homotrimers and cause an increased affinity for membrane phospholipids, which in turn provokes the translocation from the cytosol and the nucleoplasm to plasma and nuclear membranes. in order to examine the effect of anxa on cre-and creb-(camp response element-binding protein) dependent transcription, a series of transient transfections using a luciferase reporter gene driven by the creb target promoter of the inducible camp early repressor (icer) was carried out. when the luciferase reporter construct was co-transfected with anxa , there was significant reduction of basal (dmso) luciferase activity ( the implantation of drug eluting stents (des) after coronary artery intervention was an important step treating coronary artery disease. indeed, cytotoxic compounds like sirolimus used on des are responsible for reduced in-stent restenosis in comparison with bare metal stents. pimecrolimus, a potent anti-inflammatory drug has also been investigated for its efficacy on des. preclinical studies in pigs revealed promising antiproliferative effects of pimecrolimus on neointima formation. however, in humans, pimecrolimus coated stents exerted adverse effects. we hypothesize that compared to the highly active sirolimus, pimecrolimus may influence additional cellular processes leading to the worse outcome. in order to identify those processes we conducted in vitro studies in human coronary artery endothelial cells (hcaec) and smooth muscle cells (hcasmc). brdu in vitro assays of hcaec treated with pimecrolimus examined an ic value of . µm [ . to . ] which is much higher than ic values of sirolimus described in literature [ . nm to nm]. genechip array analysis comparing gene expression in pimecrolimus and sirolimus treated hcaec and hcasmc showed significant induction of several genes involved in interferon signaling. in detail, the expression of ifnβ activated genes like irf and ifitm was up regulated in cells treated with pimecrolimus while no or oppositional effects were observed with sirolimus. gene regulatory effects were validated by real time pcr. incubation with ifnβ itself showed similar effects in up regulation of genes involved in interferon signaling. furthermore, we were able to demonstrate a significant increase of ifnβ secretion in hcasmc and hcaec treated with pimecrolimus. however, comparison of ifnβ and pimecrolimus on proliferation of hcaec and hcasmc revealed different cellular responses. while ifnβ significantly decreased hcasmc and increased hcaec proliferation, treatment with pimecrolimus lead to anti-proliferative effects on both cell types. in conclusion, pimecrolimus activates pathways involved in interferon signaling but exerts different pharmacological effects, compared to the endogenous compound suggesting that infβ secretion is not the major factor contributing to the difference in pimecrolimus function. identification of novel phospho acceptor sites of the mu opioid receptor regulating receptor internalization illing s., just s., doll c., schulz s. uniklinikum der fsu jena pharmakologie und toxikologie, drackendorfer straße , jena, germany the opioid alkaloid morphine is among the most potent clinically used analgesics. however, the clinical utility of morphine to treat chronic pain is limited by its rapid development of tolerance and dependence. the stimulation of the mu opioid receptor (mor) with damgo or morphine results in different patterns of receptor phosphorylation and trafficking. so far, the three major phosphorylation sites of the mu opioid receptor namely serine (s ) threonine (t ) and serine (s ) have been identified using phosphosite-specific antibodies. however, mutations of these three residues to alanine (s a/t a/s a) did not prevent agonist-dependent internalization. in the present study, we have constructed a series of phosphorylationdeficient mutants showing that mutation of at least six residues (s a/t a/s a/t a/t a/t a) was required to completely block agonistdriven endocytosis. consequently, we generated phosphosite-specific antibodies to t and t which enabled us to provide direct evidence for agonist-dependent phosphorylation of these sites. our analysis of time-and dose-dependent phosphorylation of mor revealed that s was the primary phosphorylation site, whereas t , t and t were secondary sites. moreover, the partial agonist morphine induced only the phosphorylation of s but not of t , t of t . our results show, that mor phosphorylation occurs in a hierarchical and agonist-selective manner directly regulating receptor sequestration. assessment of mda effects from toxicity studies with regard to endocrine modulation jäger r. methylene dianiline (mda; cas no. - - ) is on the usepa list for endocrine disruption screen testing. in a yeast androgen screening (yas) assay (in vitro assay on receptor binding or blocking) mda revealed a significant anti-androgenic activity at a concentration of . mm. to assess the biological relevance of this observation under in vivo conditions the existing data from animal toxicity studies with mda were reviewed for indications of possible endocrine modulating (em) effects (weight-of-evidence approach). in addition, literature was searched for relevant studies on mda and structural analogues using the american chemical society scifinder client-server software. structural analogues indicated several drivers for em activity, notably the diphenolic ring structure. however, in vitro receptor binding assays showed mda had no androgenic or anti-estrogenic activitiy. one report described a weak estrogenic binding at . mm mda, while another described no effect at similar concentrations and a rat estrogen receptor binding study found no effect at . mm. overall it is concluded that mda does not bind to the estrogen receptor. endocrine related effects of mda were investigated in several species and dose routes in unvalidated research-type protocols. guideline subchronic and chronic toxicity studies with rodents revealed neither adverse organ weight changes nor histopathological alterations of sex organs. the main systemic effects from the oral -week studies were body weight reduction and histopathological changes of the thyroid and bile duct (lowest loael: . mg/kg bw). mda was carcinogenic to rats and mice (thyroid and liver) after oral administration over years. non-neoplastic effects in the thyroid (rats and mice) were observed (lowest loael for systemic toxicity: mg/kg bw). mda inhibits iodide oxidation which with concomitant decreased thyroid hormone formation is known to induce thyroid tumors. in summary, in vitro screening tests revealed no consistent endocrine related effects of mda. in vivo, there was an effect on the thyroid gland, possibly by enzyme inhibition. there were no histopathological changes of gonads and accessory sex organs and no evidence of sex hormone related em. the evidence from the full dataset on mda does not indicate androgenic or estrogenic effects. overall, based on a weight of evidence assessment there are insufficient alerts for em activity to suggest further testing should be done. the gtpase arfrp controls the assembly of apoa-i to and the lipidation of chylomicrons in the golgi of intestinal epithelium jaschke a. background: the uptake and processing of dietary lipid by the small intestine is a multi-step process that involves luminal digestion, cellular uptake of fatty acids by the mucosa, and subsequent synthesis and export of chylomicrons. the gtpase adpribosylation factor related protein (arfrp ) is a member of the arf-family and controls the arf-like (arl )-mediated golgi recruitment of grip domain proteins which in turn bind several rab-gtpases. the aim of the study was to define the role of arfrp in intestinal nutrient absorption. methods: for the generation of intestine-specific null mutants arfrp flox/flox mice were crossed with mice expressing the cre recombinase under the control of the intestinespecific villin promoter (vil-cre) and arfrp expression was suppressed by sirna in caco -cells. the phenotype in respect to lipid absorption and chylomicron production in the intestinal epithelium and in caco -cells, respectively, was analyzed. results: arfrp vil-/mice were viable but showed an early postnatal growth retardation (mean body weight of arfrp vil-/was . ± % lower than that of control mice at the age of days) arfrp vil-/mice displayed reduced triglycerides, free fatty acids and glucose plasma levels indicating that the growth retardation is the result of a malabsorption. uptake of glucose and amino acids were unaffected by the deletion of arfrp . in contrast, lipid uptake as elucidated by oral fat tolerance tests was impaired in arfrp vil-/mice. arfrp vil-/enterocytes as well as arfrp mrna depleted caco- cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triglyceride content. in addition, while the release of apolipoprotein a-i (apoa-i) was dramatically decreased apoa-i accumulated in the arfrp vil-/epithelium and was predominantly colocalized with rab . our results demonstrate that the growth retardation of arfrp vil-/mice is a consequence of impaired intestinal fatty acid absorption. we suggest that arfrp is required for the assembly of aopa-i to the chylomicrons and for the further lipidation of chylomicrons in the golgi of intestinal epithelial cells. this finally leads to an secretion of chylomicrons with a markedly reduced triglyceride content. rhoa influences adhesion and spreading of cardiac fibroblasts via complex regulation of cytoskeletal proteins jatho a. the monomeric gtpase rhoa is thought to be involved in the pathology of heart diseases, however, its role in cardiac cells is not well defined. therefore we intended to analyze the effect of rhoa in cardiac fibroblasts by using a lentivirus-based knockdown approach. by doing so, we could show that the knockdown of this gtpase by about % resulted in a massive change in cell morphology, but displayed no effect on cell viability. the appearance of the cells in the d-culture changed from a fibroblast-typical stretched morphology with intracellular stress fibers to a more epithelial-like cell morphology. by morphometrical analyses we demonstrate that fibroblasts with reduced rhoa expression display an increase in cell surface by . -fold and in perimeter by . fold. moreover, the depletion of rhoa significantly influences the adhesion velocity, as within the first hour after cell detachment about % of the rhoa knockdown cells reattach, but only % of the respective control cells. based on these findings, we investigated the distribution and composition of different cytoskeletal proteins by immunofluorescence stainings and immunoblot analysis. we found, that the amount of b-actin is not reduced in rhoa knockdown cells, however, the distribution is markedly changed. in these cells internal star-shape bundles of actin could be found instead of the commonly appearing stress fibers in control cells. in contrast, the cortical actin fibers, mainly consisting of g-actin, were not affected. in addition, smooth muscle-actin, which is characteristic for myofibroblasts, was clearly reduced in rhoa knockdown cells compared to control cells by %. this reduction might be responsible for the more relaxed cell shape. in summary, the knockdown of rhoa influences cell adhesion and the morphological characteristics of cardiac fibroblasts, without obviously affecting cell proliferation and viability. using site-specific fluorescence labeling to study uptake of pasteurella multocida toxin into eucaryotic cells jehle d., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abteilung , albertstraße , freiburg, germany pasteurella multocida is an opportunistic pathogenic bacterium living in the nasal pharyngeal space of animals. p. multocida is of particular importance in the livestock management of pigs. under special conditions infection of pigs with p. multocida leads to an atrophic rhinitis, which is characterized by the atrophy of nasal turbinate bones accompanied by a shortening and twisting of the snout. the causative agent of the atrophic rhinitis was found to be the bacterial protein toxin pmt (pasteurella multocida toxin). after entering the cell the -kda toxin activates various signal transduction pathways by stimulating heterotrimeric g proteins of the gαq, gα and gαi family. the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. the uptake of pmt into cells is not comprehensively understood. therefore, we utilized a recently described technique, called "sortagging" (popp mw et al. nat chem biol. ; : ) , to specifically couple fluorescence tags to the n-or c-terminus of pmt. the enzyme sortase a (srta) from staphylococcus aureus attaches proteins to the bacterial cell wall. the substrates are recognized by an lpxtg motif. srta cleaves the peptide bond after the threonine and adds a glycine-containing nucleophile. we introduced these motifs into pmt to express srta-recognized toxin and coupled the toxin with fluorescence tags, respectively. fluorescently labeled pmt was used to study the uptake of the toxin into eucaryotic cells by laser scanning microscopy. munich heart alliance, münchen, germany cells within the myocardium communicate by secreted factors and this has been suggested to contribute to cardiac remodeling. to identify novel factors secreted by the myocardium, we have previously reported a genetic yeast screen which led to the identification of protease inhibitor (pi ). here we report the generation of a mouse line where pi can be deleted globally or conditionally using the cre/loxp system. after electroporation of the pi floxneo targeting vector in embryonic stem cells and injection into murine blastocysts we gained a mouse line that carried the targeted modification of the pi allele. global pi deficiency (pi lox/lox ) per se did not lead to a cardiac phenoytpe (hw/bw (mg/g): pi +/+ = . ± . (n = ), pi lox/lox = . ± . (n = ), p > . ; fibrosis (%): pi +/+ = . ± . (n = ), pi lox/lox = . ± . (n = ), p > . ; fractional shortening (%): pi +/+ = . ± . (n = ), pi lox/lox = . ± . (n= ), p > . ). in addition we carried out an immunohistochemical analysis of pi expression using pi -deficient mice as negative controls. pi localized to cardiac fibroblasts, to the epididymis and the trachea. in the failing heart we detected accumulation of pi preferentially in fibrotic areas. we are currently applying cardiac stress models to gain a broader understanding of the function of pi and its potential as a therapeutic target molecule. enantioselective determination of r-and s-hyoscyamine in mammalian plasma and urine samples john atropine (atr) is the racemic mixture of the tropane alkaloids s-and r-hyoscyamine (hyo). s-hyo acts as a competitive acetylcholine antagonist at the muscarinic receptors (eutomer) inducing mydriasis, excitations, hallucinations, coma and ultimately death, whereas r-hyo does not (distomer). atr is used for clinical intervention of poisoning with organophosphorus (op) pesticides or nerve agents. despite well known differences in pharmacological behavior, individual pharmacokinetics of both atr enantiomers have rarely been addressed in the literature [ , , ] . therefore, we initially developed a nonchiral liquid-chromatography-electrospray tandem mass spectrometric method (lc-esi ms/ms) that allows quantification of atr and additional natural and synthetic tropane alkaloids from plasma after simple deproteinization [ ] . to discriminate both atr enantiomers the sample preparation step was expanded by an enzymatic pretreatment. samples were incubated either with diluted human serum (not containing atropinesterase, atre, procedure a) or with diluted rabbit serum (procedure b). rabbit serum contains atre (ec . . . ) which is suitable for stereospecific hydrolysis of shyo into tropine and tropic acid while r-hyo remains unaffected. after sample precipitation, hyoscyamines were quantified by the lc-esi ms/ms method. following procedure a the concentration of total hyo and following procedure b remaining r-hyo were determined. s-hyo was calculated by the difference between these concentrations [ ] . the impact of potential matrix ingredients that may appear in samples from oppoisoned patients under atr therapy were evaluated (oximes, op agents, carbamates) [ ] . the assay was applied to diverse toxicological and pharmacological samples. i) measurement of natural s-hyo in an extract of atropa belladonna leaves as well as in plasma and urine of a female patient who was poisoned after ingestion of such leaves revealed that no biotransformation to r-hyo occurred. ii) analysis of plasma obtained from an op-poisoned female patient under atr therapy revealed faster elimination of shyo when compared to r-hyo [ ] . iii) in contrast, no enantioselective differences were obvious in healthy anaesthetized swine after intravenous injection of atr [ ] . data indicate that the enzymatic enantioselective procedure represents a useful tool to characterize in vivo behavior of r-and s-hyo allowing to reveal individual kinetics. failing hearts are unable to adequately supply the body with blood and oxygen. common therapeutic strategies interfere with cardiac remodelling, reduce cardiac preand afterload or aim at direct improvement of cardiac contractility. cardiac contractility is mainly controlled by β -adrenergic receptors. resensitization of b-adrenergic receptors by inhibition of g-protein coupled receptor kinase (grk ) is discussed as a potential strategy to treat heart failure. we characterized raf kinase inhibitor protein (rkip) as a physiological inhibitor of grk and found rkip to increase contractility of neonatal cardiomyocytes. the present study evaluated the role of rkip in heart failure. we assessed its effects on cardiac function in pressure overload induced heart failure and determined the expression patterns of rkip in failing hearts of humans and mice. transverse aortic contriction (tac) was performed on -week-old c /bl mice to induce heart failure by pressure overload of left ventricles. after three weeks of tac, echocardiography showed distinct signs of decreased cardiac function in wild-type mice. fractional shortening was reduced and left ventricular diameters were increased. histological analyses revealed increased interstitial fibrosis, caspase-and tunelassays indicated myocyte apoptosis. western blot analysis showed significant upregulation of rkip expression in failing hearts compared to non-banded control hearts. interestingly, this upregulation of rkip expression was also detected in failing human hearts and in samples of patients with aortic valve stenosis but not in healthy control samples. to assess the effects of rkip overexpression on heart failure, we analysed heart function and structure of rkip transgenic mice and wild-type mice weeks after tac. while left ventricular hypertrophy was increased to similar extents in wild-type and rkip trangenic mice, rkip mice did neither develop dilatation of the left ventricle nor a decrease in fractional shortening. in contrast to wild-type mice, the expression of the heart failure markers bnp and anp was not upregulated in banded rkip transgenic mice after weeks of tac. taken together, cardiac overexpression of rkip prevented left ventricular dilatation and loss of contractile function in a mouse model of pressure overload induced heart failure. therefore, increased rkip expression may be an interesting target to prevent detrimental effects from increased left ventricular pressure. the main focus of this study was the chromatographic separation. the most frequently prescribed antibiotic drugs clarithromycin, erythromycin, clindamycin, cefuroxim, doxycyclin, amoxicillin, levofloxacin, and ciprofloxacin were selected for the screening method. method: a relatively short operation time and a sufficient separation were reached by column, eluent, and gradient optimization with poplc (phase optimized liquid chromatography). in the first step columns with the five stationary phases c eps , c sh , phenyl , cn , and c were used to determine the retention times of the drugs in an isocratic mode. the stationary phases, the column length and the retention times were fed in the poplc software and the optimal column was calculated. this column contained different stationary phases and was compared with customary columns. results: using the optimal column a sufficient chromatographic separation of the eight antibiotic drugs was reached. that was not possible with the customary columns. with the optimal column the time of measurement was too long. using a mobile phase gradient the measuring time could be reduced. discussion: with lc-ms/ms a complete chromatographic separation of all analytes is not necessary. but when measuring many transitions in a biological matrix two problems should be excluded: ion suppression and a too small number of measurement points per peak. especially when using positive and negative ionization in the ms a good separation is mostly necessary. to determine only the eight antibiotic drugs an optimized column is not necessary, but for a screening method with more than twenty drugs the poplc system is very helpful. we have investigated the uptake mechanism of cdt and in particular the intracellular membrane translocation of cdta. our data indicate that cdt requires acidification of the endosomal lumen for translocation of cdta across endosomal membranes into the cytosol. bafilomycin a , an inhibitor of endosomal acidification protects vero (african green monkey kidney) cells from intoxication with cdt. consistently, translocation of cdta was observed when the acidic conditions of the endosomal lumen were mimicked at the cytoplasmic membrane of intact cells. next, we tested whether host cell factors are involved in membrane translocation of the toxin. radicicol, a specific pharmacological inhibitor of the chaperone heat shock protein hsp as well as cyclosporine a, an inhibitor of cyclophilins delayed the intoxication of cells with cdt but not with toxins a and b [ ] . this result was confirmed by analyzing the adp-ribosylation status of actin from such cells in the presence or absence of the inhibitors. in addition, we excluded that the inhibitors of hsp and cyclophilins have any effect on receptor binding, endocytosis or enzyme activity of cdt. the data strongly suggest that the participation of hsp and cyclophilin is crucial for translocation of cdta into the cytosol. comparable results were obtained for the related binary iota toxin of c. perfringens. in vitro purified immobilized hsp and cyclophilin a specifically bound to the enzyme components of cdt and iota toxins. in conclusion, the results imply a common hsp /cyclophilin a-dependent translocation mechanism for the family of binary actin-adp-ribosylating toxins. our current investigations focus on the participation of fk -binding proteins (fkbps), another group of peptidyl-prolyl cis/trans isomerases in the membrane translocation step of these toxins. [ ] kaiser, e., kroll, c., ernst, k., schwan, c., popoff, m. r., fischer, g., buchner, j., aktories, k. and barth, h. ( ) . complex multicellular in vitro coculture models represent a promising tool regarding e. g. cellular interactions with nanoparticles, since they more closely mimic the cellular composition of the body. therefore, we used our developed coculture model of the alveolar-capillary barrier composed of lung epithelial cells (nci h ) on top and microvascular endothelial cells (iso-has- ) on the bottom side of a filter-membrane to study nanoparticle cytotoxicity and cellular uptake. with a coculture period of about days the cells achieve a more differentiated and polarized state and develop a tight barrier, which can be measured via ter (transepithelial electrical resistance). regarding cellular uptake of fluorescently labeled amorphous silica nanoparticles (asnps, nm) the coculture took up much less asnps than conventional monocultures. besides, we could not verify a specific uptake mechanism (e. g. clathrin-, caveolae-mediated endocytosis) via immunofluorescence staining of the cells. however, we detected asnps incorporated in flotillin- and - labelled vesicles. former studies concerning cytotoxicity (lactate dehydrogenase assay) of amorphous silica nanoparticles (asnps, nm) revealed that our coculture behaved much more robustly compared to conventional monocultures. however, regarding inflammatory responses (e. g. sicam, il- increase) the coculture responded more sensitively than conventional monocultures. in a further development we added a third cell type, the alveolar macrophage (am), to our coculture. since ams embody the front-line of alveolar defense against inhaled pathogens and particles, they play a central role in regulating lung immunity. as model we applied the human acute monocytic leukemia cell line, thp- (prestimulated with nm pma for d) apically to the epithelial monolayer of the coculture. our preliminary studies concerning inflammatory responses of the tripleculture (h /iso-has- with thp- ) revealed a higher sensitivity of the triple-culture compared to the double coculture. the triple-culture responded with an increased il- release upon lps or tnf-a stimulation. in conclusion, this triple-culture model offers a promising prospect to mimic more closely realistic cell interactions with nanoparticles in the distal lung. ethanol is a component of many herbal fluid preparations [ ] , since it is an excellent extraction solvent for the phytochemical components of herbal drugs and contributes to the stability of these medicines. toxicological and pharmacokinetic evaluations [ ] have shown that the small amounts of ethanol applied with therapeutic doses are safe even in children. despite that these medicines have been used safely since many decades, they have occasionally been subject of discussion in the public, triggered by the increasing problem of recreational abuse of alcoholic beverages by children and young persons [ , ] . therefore, there is a growing need of a systematic evaluation of pharmacovigilance data on these medicines. for evaluating the experience gained from the therapeutic use of these medicines, pro-and retrospective studies with herbal medicinal products containing ethanol at doses of to mg per single dose, depending on the age group, have been analyzed, covering patients of - years of age. in these studies, altogether adverse drug effects have been described, none of which was attributable to the ethanol content of the medicines. in a survey of the worldwide use of these and other herbal medicinal products it was shown that during the past few years, more than mio daily doses have been sold, corresponding to more than mio of patients (data obtained from manufacturers; figures available partly from onwards, partly from / onwards). from the packages sold in germany in the years between and , . mio were attributable to self-medication, . mio to prescriptions reimbursed by health insurance (ims, frankfurt). as non prescription medicines are reimbursed in germany only in children, at least the latter part of the prescriptions can be attributed mainly to children. all of these medicines are registered or licensed by regulatory authorities. adverse effects are covered by the pharmacovigilance system, and no adverse effects attributable to the ethanol content have been reported. this set of data supports the conclusion drawn from the experience of a safe use over decades, i.e. that the ethanol content of herbal medicinal products does not give any causes for concern regarding their safety even in children. dedication: this contribution is dedicated to prof. dr. hilke winterhoff, institute for pharmacology and toxicology, university of münster, who died on may . she has initiated this work. prucalopride was introduced as a new selective -ht receptor agonist that is approved for treating obstipation. whereas one could expect -due to the fact that -ht receptors are functionally expressed in the human heart -in clinical trials prucalopride did not show cardiac effects. this is quite surprising because other -ht receptor agonists have been withdrawn from the market just for that reason. in this study we used prucalopride for in vitro studies with atrial preparations from transgenic (tg) mice with cardiac myocyte-specific overexpression of the human -ht a receptor. isolated electrically driven ( hz) left and spontaneous beating right atria of tg mice were compared with those of wild type (wt) littermates. moreover, we used isolated electrically driven ( hz) human right atrial preparations from patients undergoing cardiac surgery. finally gr , a -ht receptor antagonist, was added. prucalopride exhibited a dose dependent positive inotropic effect in left atria and a positive chronotropic effect in right atria of tg mice with a logec of - . and - . , respectively (p< . vs. wt, n= ). in human atrial tissue prucalopride also acts as an agonist, leading to an inotropic effect. all effects could be antagonized by µm gr . we could demonstrate that prucalopride acts as an agonist at the -ht receptor in our transgenic mice model and also in human right atrial preparations. these findings suggest that tachycardia and arrhythmias are possible side effects, which should be carefully looked for. the involvement of psychological factors, especially stress, are known to play an important role in functional gastrointestinal diseases ( , ) probably by affecting the brain-gut axis. based on the good correlation between stress & functional dyspepsia (fd), many animal models for fd have been developed where animals are subjected to various kinds of psychological stress either during the neonatal period or in adulthood. this stress was found to induce gastric motor dysfunction resembling symptoms of fd. two models for stressinduced fd were performed in order to choose the more adequate one for studying sensitivity changes of the fundus to various mediators as well as changes in some relevant hormone levels in the blood for subsequently testing the efficacy of treatment with stw (a component herbal preparation of proven clinical efficacy in fd and in irritable bowel syndrome ( , ) ). in one model, maternal separation ( ) was performed on weanling rats starting from postnatal day for h each day for weeks. rats were then allowed to mature to an adult age. the other model was that of restraint stress (rs) ( , ) . adult animals were restrained for min/ day for week. the animals of both models were eventually sacrificed, the stomach fundus was isolated and its sensitivity in vitro to carbachol, potassium chloride, serotonin and adrenaline was tested. blood samples were taken to assess levels of ghrelin, corticosterone releasing factor (crf) and corticosterone. the sensitivity of fundus strips from restrained rats towards the agents tested, partly representing autonomic responsiveness, was more depressed than those from maternally separated ones. levels of ghrelin, crf and corticosterone were also more elevated in the rs model. that model was therefore chosen to test the efficacy of stw in restoring the deranged parameters. a group of animals received stw orally once daily starting treatment week before exposing them to rs and continuing treatment for a further week during subjection to rs. stw was effective in normalizing the depressed stomach fundus responses exhibited by animals subjected to rs and to normalize to a large extent the deranged blood levels of ghrelin, crf and corticosterone. the findings lend further evidence to the role of the brain-gut axis in fd and gives supportive evidence for the first time for the clinical usefulness of stw in this condition. there is good evidence that oxidative damage to dna leads to down-regulation of transcription of affected genes and epigenetic gene silencing in a mechanism dependent on the -oxoguanine dna glycosylase (ogg ), which generates harmful repair intermediates [ , ] . we have recently shown that the magnitude of inhibition of transcription of an egfp reporter gene by single -oxo- , -dihydro-deoxyguanosine ( -oxodg) varies between the opposing dna strands of the gene [ ] . we now have addressed the question, to which extent the transcription inhibitory potential of -oxodg depends on its position in the gene and on the dna microsequence surrounding the modified nucleobase. to investigate the effect of position, we produced plasmid vectors containing single -oxodg or dg (underlined) in the second position of an agc trinucleotide. measurements of egfp expression in transfected mammalian host cells showed that a single -oxodg caused a strong inhibition of gene transcription. the magnitude of this effect for -oxodg situated in the transcribed dna strand of the gene was the same as in the non-transcribed dna strand. similarly, there was no quantitative difference between the effects of -oxodg present in the ′-versus ′-utr of the gene. the results thus indicate that inhibition of transcription by this base modification does not depend on the position in the gene. further comparisons were done between the effects of -oxodg nucleobases localised in the same dna strand but within different sequence contexts. gene expression analyses in the repair proficient host cells showed that the degree of inhibition of transcription caused by single -oxodg was dependent on the neighbouring nucleotides. among three tested sequence motifs, the minimal effect on the gene transcription was found to correlate with a significantly less efficient base excision by the purified human ogg protein. the results thus support the initiatory role of ogg in the mechanism of transcriptional repression. in addition, the finding of the effect of dna sequence on the base excision activity of ogg suggests that repair rates of single base modifications in genome could also be heterogeneous. allgayer j., kitsera n., epe b., khobta a. johannes gutenberg university of mainz institute of pharmacy and biochemistry, staudingerweg , mainz, germany interference of dna base modifications with gene transcription is an important biological consequence of genotoxic damage [ ] . an efficient method for incorporation of a single -oxo- , -dihydroguanine ( -oxog) at a defined position in the egfp gene in a plasmid vector was recently developed in our lab. the method relies on the availability of adjacent sites for a sequence-specific nicking endonuclease, which allow the insertion of a synthetic oligonucleotide containing -oxog in a chosen position. we further showed that this single lesion inhibits transcription after excision by ogg [ ] . in order to determine to which extend the observed effect depends on the position of the modified base in the gene, we constructed several new plasmid vectors which allow incorporation of the same dna oligonucleotide containing -oxog or g in different positions in different dna-strands. dna sequence cassettes were designed to contain a ′-cattgcttcgctagcacg nucleotide sequence in different orientations, which was flanked by two unidirectional bsrdi recognition sites ( ′-gcaatgnn). adapters containing the restriction sites for directional cloning into the ′-or the ′-utr of the plasmid-borne egfp gene were added. the produced plasmid vectors thus allow the insertion of a single -oxog in the same sequence context into opposing dna strands in the 'utr and in the 'utr. keratinocytes are the major cell type of epidermis and are responsible for the formation of an outer barrier, the statum corneum, to protect an organism against harmful environmental influences. for generation of this barrier, keratinocytes pass through a complex differentiation program that is accompanied by synthesis of lipids, like cholesterol and ceramides. finally, the differentiation of keratinocytes leads to apoptosis. another function of keratinocytes is to sense environmental factors, some of which are decoded by members of the transient receptor potential (trp) ion channel family. trpv , for example, is predominantly expressed in keratinocytes, and decodes different chemical and physical stimuli like the terpenoid-derived ligands camphor and thymol or temperatures above °c. less is known about the influence of cholesterol on trpv signalling. we modified the cholesterol content of hek cells stably transfected with trpv and performed flipr-based calcium measurements. these experiments revealed that cholesterol enrichment robustly potentiates trpv by sensitizing it to lower agonist concentrations. we verified these results with whole-cell patch-clamp measurements. in contrast, trpv , another heat-sensing channel, was not affected by cholesterol modification. since former studies showed a defective formation of epidermal barrier in trpv -/mice, our results imply that a cholesterol-regulated trpv signalling may contribute to the progression of differentiation or initiation of apoptosis of keratinocytes. ischemia-reperfusion injury causes severe problems in the early period after lung transplantation. since transient receptor potential (trp) channels are important regulators of vascular permeability and tone, we investigated the influence of a trpc blocker on pulmonary function after simulated transplantation, using an ex vivo model of isolated perfused and ventilated rabbit lungs. to this end, heart-lung blocks were excised and mounted in an artificial thorax chamber. negative pleural pressure ventilation was initiated, and lungs were perfused with albumin-containing tyrode-solution ( ml min - ). after equilibration in a stable perfusion and ventilation mode for min, lungs were flush-perfused with perfadex ® solution, followed by an ischemic storage for h on ice. subsequently, ventilation and perfusion were re-initiated to simulate a transplantation situation. in the oxygenated group, pulmonary artery po was adjusted to mmhg, in the deoxygenated group, the perfusate inflow was gassed with nitrogen to achieve a pulmonary artery po of mmhg. both transplantation conditions were conducted in the absence or in the presence of the trpc blocker larixol acetate ( µm). hemodynamic and ventilatory parameters were continuously monitored. the weight of deoxygenated lungs steadily increased during h of reperfusion from . ± . g to . ± . g. this weight gain was inhibited by supplementation of the trpc blocker (from . ± . g to . ± . g h after reperfusion). in contrast, oxygenated lungs showed no marked weight gain after reestablishment of perfusion (from . ± . g to . ± . g h after reperfusion), and the trpc blocker had no additional effect (initial . ± . g, h reperfusion . ± . g). we conclude that a trpc -blocking compound to the lung perfusate during simulated transplantation counteracts the endothelial permeability increase and the resulting post transplant weight gain. the results indicate a role for trpc in the regulation of pulmonary vessel permeability and support the concept that perfusion of donor lungs with trpc blockers may prevent edema formation caused by ischemiareperfusion injury shortly after lung transplantation. regulation of human inducible nitric oxide synthase (inos) expression by noncoding rnas (ncrnas) kleinert h., schmitz k., koch k., hahn s., pautz a. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher str. , mainz, germany the transcriptome analyses of human cells showed that additionally to the . protein coding sequences the human dna codes for much more ( . ?) non-coding rnas . beside the ribosomal, and transfer rnas (rrna and trna) involved in the mechanism of translation there are also short (snrna, sorna, mirna) and long noncoding rnas (ncrnas) implicated in regulation of gene expression (splicing, translation, chromatin packaging etc.). matsui et al. described regulation of il- β-induced inos expression by an antisense rna (as- -utr-rna) in rat hepatocytes . a promoter located on the antisense strand ' to the last exon (exon ) of the rat inos gene drives the expression of an as-rna complementary to the '-utr of the rat inos mrna. using different sense primers with homology to the '-utr sequences of the human inos gene for specific rt-pcr reactions (detecting only an as-rna) we were not able to detect such an as- -utr-rna in human cells. in addition, transient transfection analyses using constructs containing a . kb fragment of the '-flanking genomic sequences (as used by matsui et al. in the rat system) of the human inos gene in front of a luciferase reportergene into dld- cells revealed no promoter activity of these sequences. korneev et al. described the expression of a kb anti-inos-ncrna in different brain tumors and showed reciprocal expression to the inos mrna in human embryonic stem cells . analyzing the expression of this anti-inos-ncrna in cytokine-treated dld- cells also showed a basal expression of this as-rna and an enhancement of the expression by cytokine-treatment. downregulation of the anti-inos-ncrna by sirnas reduced whereas overexpression enhanced cm-induced inos expression in human dld- cells. this indicates that this anti-inos-ncrna regulates cytokine-induced inos expression in a positive manner. rna , rna , - rna , ( . using directed evolution to improve functional expression of class b g-protein coupled receptors klenk c., scott d. j., plückthun a. universität zürich institut für biochemie, winterthurerstrasse , zürich, switzerland the class b of g-protein coupled receptors (gpcrs) comprises peptide hormonebinding receptors which regulate important endocrine and neuroendocrine functions of the human body. several of these receptors are implicated in the pathogenesis of severe diseases such as diabetes, osteoporosis, growth disorders and depression, which makes them attractive targets for drug therapy. to develop new compounds targeting these receptors a detailed understanding of the molecular structure is required which has not been succeeded to date. structural studies of proteins by x-ray crystallography or nmr spectroscopy generally require large and homogenous quantities of protein. for gpcrs this prerequisite is difficult to achieve as the vast majority of gpcrs exhibits low endogenous expression and is very unstable in solution. therefore, improved expression conditions are necessary for the efficient characterization of new gpcr structures. here we present a method to optimize class b gpcrs for improved heterologous expression and increased thermostability by means of directed evolution. libraries of class b gpcrs were obtained by random mutagenesis and were expressed in a heterologous expression system in which functional gpcr is targeted to the inner membrane of e. coli. mutants that display increased receptor expression levels and ligand binding were selected by flow cytometry using fluorescently labeled ligands. repetitive cycles of randomization and selection allow to gradually increasing the level of protein expression and stability. with this evolutionary approach key residues within the receptor sequence can be identified rapidly that are responsible for improved biophysical properties without affecting the pharmacological features of the receptor. such gpcr mutants will become a valuable tool on the way to express high quantities of stable receptor protein for subsequent structural studies. pasireotide (som ) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, cushing's disease and carcinoid tumors. whereas octreotide acts primarily via the sst somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst activity combined with enhanced binding to other somatostatin receptor subtypes. somatostatin and octreotide stimulate the complete phosphorylation of at least six carboxyl-terminal serine and threonine residues namely s , s , t , t , t and t , which in turn leads to a robust endocytosis of the sst receptor. surprisingly, pasireotide failed to phosphorylate the four threonine residues and induced only a detectable phosphorylation of s and s . somatoprim and ke are recently developed somatostatin analogs capable of binding to four of five somatostatin receptor subtypes. here, we performed an in vitro study comparing the effects of pasireotide, somatoprim and ke on sst somatostatin receptor binding, phosphorylation, internalization and signaling. further somatostatin, octreotide, pasireotide, somatoprim and ke were tested for functional selectivity at sst receptor mutants, which possess a carboxyl-terminal sst tail. this approach allows detection of receptor activation by phospho-specific sst antibodies. compared to octreotide, somatoprim activates sst but has a higher activity on sst . ke and pasireotide are partial agonists at the sst receptor. pasireotide, ke and somatoprim show comparable effects on sst receptor. however, none of these new pan-somatostatin analogs behaves like natural somatostatin on the sst receptor. cadmium modulates ahr-associated gene expression in the rat intestine after oral exposure kluxen f. m. , , höfer n. cadmium has been shown to mimic steroid estrogen effects in vivo and in vitro. we have recently identified cross-talk of estrogen receptor (er) and aryl hydrocarbon receptor (ahr) in the rat uterus where beta-estradiol (e ) and cdcl modulate ahrassociated genes via er after i.p. injection in a similar fashion (kluxen et al., arch toxicol doi . /s - - -x). however, the predominant route of exposure to cadmium in non-smokers is via diet. moreover, uterus expresses mainly the receptor subtype eralpha, whilst small intestine and colon express mainly erbeta. thus, we now investigated by real-time rt-pcr the effects of cadmium ( mg/kg b.wt cdcl (cd )) or steroid estrogen ( . mg/kg b.wt. alpha-ethinylestradiol (ee )) on ahrassociated gene expression (i.e., ahr, cyp a , gsta , nqo ) in the small intestine of rats after oral exposure ( days gavage). the animals were also co-treated with cd and pure anti-estrogen ( . mg/kg b.wt zk (zk)) or ee , to asseess whether ermediated processes are involved. we also measured cyp a mrna expression in two estrogen receptor negative colon cancer cell lines (ht- and caco ) treated for days with cdcl ( µm) and e ( . µm). the dose-dependency of cadmium induced ahr target gene modulation was studied in a second animal experiment, with administration of cadmium in drinking water for days ( . - mg/kg b.wt cdcl equivalent to , , and ppm) and ee ( . mg/kg b.wt) as steroid reference. in summary we present two major results: the metalloestrogen cadmium modulates dose-dependently the ahr-associated gene expression in the intestine after oral exposure. yet, since the cadmium induced modulation of ahr target genes was not antagonized by anti-estrogen in the small intestine in vivo and was also found to occur in er-negative colon cells in vitro, we propose that er-independent mechanisms might play a role in this effect. meg) is the most cytotoxic lesion. if not repaired by o -methylguanine-dna methyltransferase (mgmt), o meg/t mismatch is recognized by the mismatch repair system (mmr) that performs futile repair cycles. during this process secondary lesions (i.e. dna single-strand brakes) are formed, which block dna replication in the next replication cycle, leading to dna double-strand breaks (dsbs). these dsbs eventually signal to apoptosis and other genotoxic endpoints. here, we wished to address the question whether autophagy is part of the cellular response triggered by o meg. we also assessed whether autophagy influences apoptosis induced by tmz in glioma cells. we show that tmz induces autophagy in u- mg and ln- glioma cell lines. the maximum amount of autophagy was observed several days ( h) after tmz treatment and mgmt proficient cells did not display significant autophagy. thus, the data show that mgmt protects against tmz induced autophagy, pointing to o meg as the critical lesion responsible for induction of autophagy. using colon cancer cell lines proficient and deficient in mmr, we show that mmr is required for tmz-induced autophagy. because dsbs, which emerge during the processing of o meg, are repaired preferably by homologous recombination (hr) ln- cells stably down-regulated for hr were tested for autophagy induction. the data indicate that dsbs are involved in tmz-induced autophagy. because autophagy following tmz treatment occurs earlier than apoptosis we hypothesize that autophagy protects glioma cells against apoptosis. using an early stage autophagy inhibitor -methyladenin we have shown, that autophagy inhibition sensitized glioma cells to tmz-induced apoptosis. taken together our data point out that tmz induces autophagy in glioma cells and that autophagy protects glioma cells against tmz-induced apoptosis. o meg is the lesion responsible for autophagy induction. furthermore, the data also shows that mmr and dsbs are involved in the induction of autophagy after tmz treatment. work was supported by bmbf ( nuk ). retinitis pigmentosa (rp) is a severe human retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. in most cases, rp finally leads to legal blindness. mutations in the regulatory subunit of the rod cyclic nucleotide-gated (cng) channel (cngb a) have been found in patients suffering from rp. we used cngb -deficient (cngb -/-) mice to establish a gene replacement therapy as a potential treatment for rp by means of recombinant adenoassociated viral (raav) vectors. the packaging limitations of raav vectors required a capacity-optimized vector of the large cngb a cdna (approx. kb). therefore, we replaced regulatory elements within the expression cassette and used a short mouse rhodopsin promoter element for rod-specific expression. after injection of therapeutic raavs (serotype ) into the subretinal space of -week-old cngb -/mice, we assessed the restoration of vision by analyzing i) protein expression and localization, ii) retinal function and morphology and iii) vision guided behavior. we found that treated cngb -/mice expressed full-length cngb a and cnga , which were previously downregulated. both proteins co-localized in rod outer segments and formed regular cng channel complexes in the treated area of the cngb -/retina. using electroretinography (erg) we observed a distinct rescue of rod-driven responses. moreover, cngb replacement significantly preserved outer segment morphology and delayed retinal degeneration. finally, treated cngb -/mice performed significantly better than untreated mice in a modified water maze task designed to test for rod-mediated, vision-guided behavior. in summary, this work provides a proof-of-concept for the treatment of rod channelopathyassociated retinitis pigmentosa by raav-mediated gene replacement. most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones. here, we present an alternative testing paradigm for detection of endocrine modes of actions that replace and reduce animal testing through refinement. receptor mediated endocrine effects were assessed using the yeast based receptor mediated transcriptional activation yes/yas assays and effects on steroid hormone biosynthesis were assessed using the human cell line h r in the steroidogenesis assay. in our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile. the combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. thus, this proposal constitutes a potentially attractive alternative to epa's endocrine disruptor screening program. data on reference substances for which the in vitro yes/yas and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine mode of action is presented here. the bovine corneal opacity and permeability (bcop) test has been adopted by oecd for the identification of corrosive and severe ocular irritants (ghs category ) for single component substances and multi-component formulations. eye irritation tests (eit) using human reconstructed tissue models (such as epiocular) have been described to predict ocular non-irritants (ghs no category). thus the ultimate repaltement of the draize rabbit eye irritation test (oecd tg ) by a combined or tiered testing strategy could be possible. the purpose of this study was to evaluate whether the bcop with additional corneal histology together with the eit could be used to predict eye irritancy of agrochemical formulations according to different classification schemes including un ghs and epa systems. we have performed the bcop (plus histology) and the eit of agrochemical formulations for which in vivo eye irritation data were already available (for registration purposes). using the oecd tg guideline evaluation scheme for opacity and permeability in the bcop was not predictive for the agrochemical formulations assessed here, while corneal histology grades and the epiocular tissue viabilities were useful predictors of eye irritancy potencies and could be applied for the different classification schemes. the nanomaterials offers extraordinary opportunities. the nano-structure can change the physical and chemical properties, and often also alters the biological effects. hence the toxicity of a nanomaterial can differ from its larger-scale material; but as of today, no new quality of a general nano-specific toxic effect has been observed. therefore the established testing methods are generally suitable. it is, however, difficult to apply the nanomaterials to in vitro test systems, since it is the nature of these materials to change their surface properties and agglomeration stateand the uptake and distribution in the body may differ from their larger-scale materials. while the methods for topical effects may be used for nanomaterials without further modification, the in vitro methods for genotoxicity testing require the dispersion in culture media. the use of reproducible and well-documented dispersion protocols andthe characterization of its particle size distribution is de rigueur . [ ] . for many nanomaterials published genotoxicity studies did not give a consistent picture [ ] and therefore there are rather effects of individual nanomaterials than nano-genotoxicity per se. modern toxicology is based on the insight into toxic pathways. for nanomaterials a testing strategy will include testing for their primary effects (which might be only a handful: particle effects, catalyzing the formation of reactive molecules and ion release) and their uptake, distribution and clearance. the use of dermal penetration studies in vitro for the risk assessment of sunscreen nanomaterials has been demonstrated [ ] . in vitro methods for specific effects (such as inflammation, pulmonary toxicity, sensitization) are currently awaiting validation (for both chemicals/molecules as well as nanomaterials). in the meantime alternative short-term in vivo studies with optimized biological readouts can deliver information on the toxic pathways as well as the biokinetics and dose-response relation of nanomaterials in the body. a short-term inhalation test for nanomaterials has already been used successfully [ ] . testing strategies based on those methods engage less animals and provides more significant data than classical testing. moreover data from these methods will serve as a benchmark and a validation for the in vitro models under evaluation. nanoparticles (np) are increasingly used in various field of industry which necessitates evaluation of their safety. also in the food industry, nps have gained strong interest, for example as food additives or to improve food packing. however, the potential risks of ingested np have been rarely investigated. inhalation studies have revealed that inflammation and oxidative stress may represent unifying mechanism for the induction of adverse health effects of toxic np. in the present study, a co-incubation model of human polymorphonuclear neutrohils (pmns) and caco- human intestinal epithelial cells was used as a model to address potential genotoxic effect of np during intestinal inflammation. oxidative dna damage induction (measured by the fpg-modified comet assay) was induced in the caco- cells by activated pmn and this effect increased with increasing pmn to caco- cell ratio. the crucial involvement of the phagocyte nadph oxidase complex could be demonstrated using treatment of caco- cells with bone marrow-derived pmn from nadph oxidase deficient mice. dna damage by pmn as well as h o was increased in buthionine sulphoximine (bso) pre-treated caco- cells, illustrating the importance of the cellular glutathione (gsh) status in these target cells. gsh depletion in caco- cells could also be shown upon treatment with various types of np. our data suggests that ingested np may increase the susceptibility of the colon mucosa to genetic damage during the occurrence of intestinal inflammation. the ingestion of seafood contaminated by acute toxic doses of the marine toxin okadaic acid (oa) is responsible for diarrhetic shellfish poisoning. it is recently known that both the rat and the human hepatic cytochrome p monooxygenases (cyp) are able to metabolize this toxin. currently, there is a lack of data about the toxicity and mode of action of oa after xenobiotic metabolism. the aim of our study was the measurement of the toxicity and oxidative stress status in hepg cells incubated with oa in the absence and presence of s mix. pure oa, as well as oa pre-activated with liver homogenisates (s mix) were used to treat human hepg cells that have nearly undetectable levels of functional cyp but express phase ii enzymes. the experiments were performed with both human and rat s fraction plus cofactors of phase i enzymes. the cell viability was measured after h using mtt-test and xcelligence real time cell monitoring system. furthermore, levels of intracellular reactive oxygen species (ros) were detected by ´, ´-dichlorofluorescein diacetate and additionally by measuring the intracellular glutathione content. in the presence of both human and rat s mix oa showed a higher toxicity than the parental substance. oa pre-incubated in rat s mix was toxic at nm oa. strong effects could be observed when oa was pre-activated with human s -mix at a concentration of nm oa. pure oa was non-toxic in that concentration range. we could also detect an increase of oxidative stress in hepg cells treated with oa in the presence of all investigated s -mix. these results suggest that oa is activated after oxidative xenobiotic metabolism into metabolites which possess a higher cytotoxic activity and increase the amount of intracellular ros in hepg cells. ballast water treatment -emerging health risks werschkun b., banerji s., krätke r. bundesinstitut für risikobewertung, max-dohrn-strasse , berlin, germany the introduction of invasive marine species into new environments by ships' ballast water, via ships' hulls and other vectors has severe impacts on the oceans. in the international maritime organisation (imo) launched the international convention for the control and management of ships ballast water and sediments which requires ballast water to be treated in order to eliminate alien aquatic species. ballast water treatment may include mechanical, physical or chemical measures. any ballast water management system using active substances needs imo approval. therefore identification of active substances, relevant chemicals and submission of specified datasets on their physical, chemical and toxicological properties is required in order to assess the safety for the aquatic environment and for human health. the bfr is the german federal agency responsible for health risk assessment and has been involved in more than twenty assessment and approval processes so far. the majority of imo approved systems are based on oxidative principles such as chlorination and ozonation. these methods can generate disinfection by-products (dbps), which are a mixed group mostly of halogenated organic substances like trihalomethanes, haloacetic acids and haloacetonitriles. the formation of dbps is well known from the disinfection of drinking water. some dbps are regulated under drinking water directives because of their long-term toxicity but many are unregulated and have unknown toxicological properties. the formation of dbps may vary significantly depending on the treatment system as well as on environmental parameters like temperature, ph and composition of the organic matter within the aquatic environment. in sea water, sources for dbp formation besides ballast water treatment are aquaculture and the cooling systems of coastal power plants. in order to address possible health and environmental risks from dbps formed during ballast water treatment a conference on emerging risks from ballast water treatment was held at bfr in october . here we summarise the main conference findings and identify areas for future research. two presentations corroborated that significant amounts of dbps can be formed in sea water and a presentation on the toxicological properties of dbps pointed out that many have genotoxic properties. accordingly, the determination of dbp species and generated concentrations under different ballast water treatment conditions was seen as a mayor task. different approaches for health and environmental risk assessments were also discussed. appropriate human exposure scenarios and methods for exposure assessment, taking into account common approaches used in risk assessment were presented during the conference. a suitable approach based on derived pec-values for exposure quantification was proposed in order to improve the procedure available for risk assessment of chemical agents used for ballast water treatment. agonist-selective signaling of µ-opioid receptors in t lymphocytes kraus j., börner c., lanciotti s., koch t., höllt v. inst. für pharmakologie und toxikologie, leipzigerstr. , magdeburg, germany opioids are the most potent analgesics and irreplaceable for the treatment of severe pain. in addition to their central effects, opioids modulate a great variety of immune effector cell functions, which may result in unwanted side effects during opioid treatment. the effects of most of the commonly used opioids are mediated by µ-opioid receptors, which belong to the superfamily of g protein coupled receptors. recent data support the concept that g protein coupled receptors function as dynamic entities, which may occupy multiple conformations and activate multiple signaling pathways in a ligand-dependent manner. consequently, different ligands activating the same receptor may have different cellular effects, which has been termed "agonistselective signaling". little is known about agonist-selective signaling of µ-opioid receptors in immune effector cells. in a first attempt to understand if and why such different profiles among different opioids occur we investigated effects of different opioids in human jurkat t cells. we report that the µ-opioid receptor ligands fentanyl, methadone, loperamide and betaendorphin induce internalization of a µ-opioid receptor-green fluorescent reporter construct, whereas morphine and buprenorphine did not induce internalization. the internalization was dependent on p mapk and phospholipase d . in line with this, we observed marked phosphorylation of p mapk and activation of phospholipase d induced by the internalizing opioids, but no or little such activity by morphine and buprenorphine. as a physiological result, fentanyl, methadone, loperamide and betaendorphin treatment of primary human t cells and jurkat t cells resulted in a strong, up to fold induction of il- , which was dependent on p . in contrast, morphine and buprenorphine only showed a weak, approximately one order of magnitude lower induction of il- . by inducing il- opioids significantly modulate the t helper cell balance into the type direction, which influences various immune responses, e. g. the antiviral, t helper cell type -mediated response. considering the vital necessity of opioid use in humans, it is an intriguing goal to identify analgetically feasible opioids that have little or no immunosuppressive or -modulatory effects. modulation of cgmp signals by phosphodiesterases in smooth muscle cells krawutschke c., koesling d., russwurm m. ruhr-universität bochum pharmakologie und toxikologie, universitätsstrasse , bochum, germany within the cardiovascular system, cgmp mediates smooth muscle relaxation and inhibition of platelet aggregation. cgmp is formed by particulate guanylyl cyclases and nitric oxide-sensitive guanylyl cyclases, that are activated by natriuretic peptides or nitric oxide (no), respectively. besides the cgmp-forming enzymes, the cgmp-degrading phosphodiesterases strongly determine amplitude and shape of cgmp signals. in vascular smooth muscle cells, three phosphodiesterases are considered to be responsible for cgmp degradation: pde , the cgmp-specific phosphodiesterase is activated directly by cgmp binding to its gaf domains; this activation if further stabilized by cgmp-dependent protein kinase-mediated phosphorylation. pde , the ca +/calmodulin-stimulated pde constitutes the majority of cgmp-hydrolyzing activity in smooth muscle cells at least in the presence of high intracellular ca + concentrations. and lastly pde , the cgmp-inhibited pde displays some cgmp-degrading activity, although cgmp binding to its catalytic domain is primarily thought to inhibit the campdegrading activity of pde . cgmp signals measurable in radioimmunoassays require stimulation with cgmpincreasing vasodilator concentrations that are orders of magnitudes higher than those required for relaxation. thus, we developed fluorescent sensors for real-time measurement of cgmp signals in single cells. by using these indicators, we analyzed the contribution of different cyclic nucleotide-degrading phosphodiesterases to the modulation of cgmp signals elicited by physiologically relevant vasodilator concentrations. hyaluronan (ha) is a major component of extracellular matrices and is thought to control cellular phenotypes such as proliferation and migration. therefore, ha synthesis may play an important role in the pathophysiology of atherosclerosis. there are three major ha-synthase isoenzymes (has - ). the has gene is alternatively spliced. has transcript variant (has v ) encodes the smallest has isoenzyme which has a different c-terminus and contains only two transmembrane domains compared to has transcript variant . the aim of the present study was to investigate whether has v is expressed by vascular cells, how it is regulated and where it is localized in cells and whether it indeed synthesizes ha. has v mrna expression was monitored by quantitative real-time rt-pcr. protein expression was determined by western blotting using a polyclonal antibody that was raised in rabbit. an n-terminal eyfp-has v fusion protein and a ddk-tagged has v construct were expressed for subcellular localization studies and co-immunoprecipitation (co-ip). endogenous has v mrna was expressed in both vascular smooth muscle cells (vsmc) and endothelial cells. furthermore, western blotting revealed has v protein expression in vsmc and platelets. in vsmc has v mrna expression was strongly up-regulated in response to interleukin- β (il- β, ng/ml) whereas stimulation with interleukin- ( ng/ml), platelet-derived growth factor-bb ( ng/ml), transforming growth factor β ( ng/ml), tumor necrosis factor α ( ng/ml) and interferon-γ ( ng/ml) had no effect. transfection of hek cells with eyfp-has v fusion protein revealed localization to the endoplasmic reticulum but not to the plasma membrane. furthermore, co-ip experiments showed that tagged has v proteins were precipitated together suggesting formation of multimeric has v complexes. transfection of has v did not cause increased secretion of ha into the cell culture supernatant in hek cells. in conclusion, has v is present in vascular cells and responds to inflammatory cytokines such as il- ß. because of the intracellular localization and the lack of ha secretion in has v transfected cells, has v may serve intracellular functions apart from ha synthesis. the tubulin antagonist pretubulysin shows strong vascular-disrupting properties in vitro several epidemiological studies indicate a correlation of human exposure to ultrafine particulate air pollution caused by incomplete combustion processes and an increase in the incidence of pulmonary immune diseases like asthma. as a possible mechanism behind this pathological phenomenon, the adjuvant effect of lung inflammation induced by poorly soluble environmental particles has been hypothesised. the aim of our study was to investigate the causal link between carbon nanoparticle-induced lung inflammation and modulations of immune cell populations during processes leading to sensitization, and allergic immune responses of the airways. therefore mice were treated with ovalbumin (ova) alone or in combination with carbon nanoparticles (cnp) by pharyngeal aspiration. the induction of inflammation and the immune adjuvant activity were studied in the lungs and lung draining peribronchial lymph nodes (pbln) at the level of sensitization, and at the level of the immune response. ova-specific ige antibodies were measured in blood serum, and the development of allergic airway inflammation was studied after ova challenge. results at the level of sensitization showed that cnp-induced immediate airway inflammation had immune adjuvant activity resulting in an increase of specific cell populations in pbln and in a stimulation of asthma-specific th cytokines. a specific reduction of the neutrophilic lung inflammation by application of the compatible solute ectoine significantly reduced the adjuvant effects of cnp. in ova-sensitized mice, application of cnp hours prior to allergen challenge, led to a significant increase in inflammatory cell infiltrate and respective cytokines in broncho-alveolar lavages. coapplication of mm ectoine together with cnp reduced the particle-induced effects. our data show a link between neutrophilic lung inflammation and adjuvant effects of cnp. a specific reduction of neutrophils by the application of ectoine attenuated this np induced adjuvant effect, indicating that particle-induced lung inflammation rather than the direct interaction of nanoparticles with immune cells is the critical step in environmentally modulated pulmonary immune diseases like asthma. introduction: drug-eluting stents (des) are commonly used in the treatment of acute artery occlusion. however, even if released cytotoxic drugs reduced neointimal proliferation significantly there is still the risk of in-stent thrombosis. it is presumed that this is due to reduced reendothelialization. it has been suggested that coating the stent with biomolecules may provide a new approach to circumvent the lack of healing of the endothelial layer. one approach would be the use of biomolecular signals, such as (poly)peptides and growth factors. rgd and redv are peptide motifs, known to enhance cell attachment and spreading. aim: the aim of our study was to evaluate the efficacy of proteins, derived from elastin-like proteins (elp) and artificial modified by incorporating with the amino acid motifs rgd,redv and p , in terms of endothelial healing on stents and other cardiovascular devices. results: in this work, we generated vectors encoding for different biopolymers consisting of various bioactive signal molecule sequences. the peptides, e.g. based on the elastinlike matrix (vpgig) -vpgkg-(vpgig) , were synthesized using heterologous expression. after optimizing culture conditions and extraction procedures their biological activity was assessed using human umbilical vein endothelial cells (huvec). elastin like proteins with differently incorporated bioactive signals (redv, rgd and a small p peptide) were linked covalently via carbodiimide coupling to poly(l-lactide) (plla) films. huvec growth was determined on these modified surfaces using the brdu assay (cell proliferation) and resazurin assay (cell viability). the chemically modified plla surfaces conferred higher cell viability after h adhesion ( %) and an enhanced proliferation ( %, h adhesion, h cultivation) in comparison to the unmodified plla. these results indicate that the synthesized elp incorporated with amino acid motifs promote an accelerated endothelialization of the biodegradable stent material plla. discussion: in summary, we were able to generate elastin-like proteins modified by bioactive sequences. those sequences enhanced endothelial cell proliferation and adhesion. further studies are warranted to determine the activity on smooth muscle cells of these peptides. ( ) the failing heart is characterized by excessive extracellular matrix production by myofibroblasts (myocfs) causing fibrosis and myocardial stiffening. myocfs represent phenotypically transformed cardiac fibroblasts (cfs) and are characterized by the expression of contractile proteins like a-smooth muscle actin (α-sma) and enhanced secretion of growth factors (e. g. ctgf). identification of intracellular enzymes that modulate this transformation process is desired to therapeutically modulate pro-fibrotic progression in heart failure. we show that pde a, a phosphodiesterase isoform, that is able to hydrolyse cgmp and camp, is markedly upregulated in failing hearts from patients with end-stage heart failure ( - -fold, p< . , n= ). notably, pde a protein abundance is -fold higher in myocfs compared to cardiomyocytes from neonatal rat hearts (p< . , n= ). to this end we tested whether pde a modulates the transformation of cfs isolated from neonatal rat hearts to myocfs. indeed, as assessed by immunoblotting and fluorescent microscopy (α-sma, phalloidin, dapi), adenoviral pde a overexpression induced α-sma expression ( . -fold p< . , n≥ ) and to a lower extent ctgf synthesis ( . -fold, p< . , n≥ ). mechanistically, pde a showed preferential subsarcolemmal localisation with diminished total cgmp levels (- %, p< . , n≥ ). consistently, parallel stimulation with atrial natriuretic peptide (anp), a selective activator of membrane-bound guanylyl cyclase, normalized ctgf synthesis indicating that pde a controls cgmp in a discrete subdomain near the plasma membrane. moreover pde a overexpression diminished the protein levels of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component (- %, p< . , n≥ ). these data implicate pde a-dependent subsarcolemmal cgmp regulation in myofibroblast formation and potentially cardiac fibrosis. therefore, targeting pde a may lead to regression of the fibrotic remodeling associated with heart failure. several anorganic nanoparticles (np) causedhigher inhalation toxicity than the corresponding coarse particles (oberdoerster et al. ) . we examined an organic pigment and a polymer dispersion each as nanomaterial and as the chemical identical coarse material in short-term inhalation studies in malerats. the polymer was an anionic acrylic ester copolymer containing free carboxylic groups. three different particle sizes were synthesized by varying polymerization conditions: , or nm. although polymeric acrylic ester was reported to be irritating to the respiratory tract at mg/m , all three tested polymers -including the np ( and nm) -did not cause any changes in lavage fluid and in histopathology at mg/m . the organic pigment was a poorly soluble pyrrol with an intense orange color. the np ( to nm width and to nm length) and the coarse pigments ( to nm width and . to µm length) are both needle-like. they were tested at , , and mg/m for the np and , and mg/m for the coarse materials. mild and partly reversible morphological changes were observed in lung and lymph nodes at the highest concentrations, but the more pronounced effect were found in rats exposed to the coarse material. likewise there was an increase of lavage parameters in rats exposed to thecoarse material but not to the np. these data demonstrate that inhalation of finer np is not necessarily associated with higher toxicity compared to the coarse material. the results were obtained with two organic particles of rather different size and composition but are in contrast to the more severe effects seen with several anorganic np when compared to the corresponding coarse particles. within the nanocare project a standard short-term inhalation test to examine the toxicity of inhaled aerosols from nanomaterials has been developed. the inhalation toxicity of nano-andpigmentary materials was studied: baso , zno, ceo , al-doped ceo , zro , amorphous silica, surface-coated amorphous silica, titania, carbon black and three multi-wall carbon nano tubes, all with complete phys-chem-characterization as planned for the oecd sponsorship program. quartz dust tio and zno were tested as pigmentary materials. rats were exposed nose-only to three concentrations of one of these materials, h a day for five consecutive days. positive controls were exposed to quartz dust or pigmentary zno, negative controls to clean air. a wide range of endpoints for pulmonary toxicity were evaluated immediately after the last exposure and after days and weeks after the last exposure. among these parameters, polymorphnuclear granulocyte count in bronchoalveolar lavage fluid is the most sensitive early parameter indicating inflammation process in lung, while histological examination reveals the type and localization of inflammation. among these substances, we identified baso as having the lowest toxicity. all mwcnts were most potent in producing progressive inflammation in the lung; granulomas in lung and lung associated lymph nodes were observed without indication for fibrosis. the noaecs of the substances ranged between < . and > mg/m . generally the material was only found in the lung (surface and macrophages) and in the draining lymph nodes. surface modified amorphous silica was also found in the spleen. the data demonstrate that the method is able to differentiate the toxic potential of different nanomaterials and to indicate regression or progression of the effects effects. moreover the lung burden and potential translocation to other tissues was detecable. comparing the material properties and effects of the materials, no general relationship between the toxicity and either particle size, specific surface area or aerosol particle number concentration was found. hence we must not expect to find a gerneral "nanotoxicology" or a unifying dosimetry for all nanomaterials. we must rather be prepared to test individual nanomaterials for their effects. and to develop grouping concepts not only based on material properties but also on biopersistence, biokinetics and biological effects. part of this studes has been sponsored by bmbf (nanocare). endpoint-centric search for toxicological information and data to support the information retrieval for regulatory programs landsiedel r. , wächter t. the eu reach regulation no / requires industry to ensure the safety of chemical use and manufacturing. all substances manufactured or imported in quantities above one tonne per year must be registered. information requirements for the dossiers increase with increasing tonnage or once hazards are suspected. searching for substance specific literature and the compilation of hazard data for safety assessments are highly challenging procedures. the novel web-based search engine go r, accessible free of charge at www.go r.org, has been created to allow quickly finding relevant hazard information and data. go r provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information. furthermore, go r specifically highlights information on animal testing alternatives. search results are presented automatically linked to an "intelligent table of contents" which enables the user to sort the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. retrieved documents are automatically organized in categories relating to the iuclid chapters. hereby, the user can browse directly through the entire million documents without even having to start the search with an initial query. the semantically enriched platform supports the user during query formulation, allows for bibliographic analysis, and specifically highlights information related to the replacement, reduction, and refinement of animal experiments. search results in go r are shown in an dynamic table of contents (left) making them browsable for the contained information on animal testing alternatives and toxicologogical endpoints. towards a differentiation therapy of acute myelogenous leukemia with histamine h -receptor agonists laue s., burhenne h., seifert r. hannover medical school institute of pharmacology, carl-neuberg-str. , hannover, germany acute myelogenous leukemia (aml) is a devastating malignancy characterized by a differentiation block of myeloid progenitor cells. recently, histamine dihydrochloride in combination with interleukin has been approved as orphan drug for the consolidation treatment of aml ( ) . it is assumed that histamine exerts its effects by activating the histamine h -receptor (h r) in human neutrophils, resulting in improved anti-tumor function of t killer cells by inhibiting nadph oxidase-catalyzed superoxide formation. previous studies had shown that histamine also induces myeloid differentiation ( ) . considering the fact that all-trans-retinoic acids constitutes a powerful differentiation therapy of acute promyelocytic leukaemia, a specific subtype of aml ( ), we initiated a study to explore the possibility that h r-mediated myeloid differentiation provides an alternative or complementary strategy to treat leukemias associated with differentiation blocks. as model system, we used hl- cells. in hl- cells, histamine and various h -receptor agonists induced concentrationdependent increases in camp levels. interestingly, ligands differentially increased cytosolic calcium concentration and extracellular receptor kinase (erk) pathways, indicative for ligand-specific h r conformations. h r activation resulted in myeloid differentiation as assessed by enhanced formyl peptide receptor-mediated increases in cytosolic calcium concentration. h r agonists showed no signs of cytotoxicity. intriguingly, following h r activation, the majority of the formed camp was exported into the extracellular space via multi-drug resistance protein (mrp) , indicating that export is a more important pathway for signal termination than cleavage of camp by phosphodiesterases. despite effective camp export, even a short-term exposure ( minutes) of cells was sufficient to induce expression of functionally active formyl peptide receptors. these data indicate that in contrast to previously held dogma, induction of myeloid differentiation does not require continuous presence of a camp signal. from a therapeutic point of view this is very important since "spike" therapy with campincreasing substances may be sufficient to induce a therapeutic effect in aml, thereby also reducing toxic side effects. currently, we are systematically exploring the effects of h r agonists on signal transduction pathways and differentiation in various myeloid cell types to identify highly efficacious compounds. introduction. activation of gαq/ protein-coupled receptors of postsynaptic neurons can elicit the production of endogenous cannabinoids (endocannabinoids), which in turn inhibit transmitter release from axon terminals by activating presynaptic cb receptors. the aim of the present experiments was to study the mechanism of the endocannabinoid production. specifically, we wanted to clarify the role of ca + release from intracellular stores in triggering endocannabinoid production. methods. patch-clamp-and ca + imaging experiments were performed on purkinje cells in mouse cerebellar brain slices. glutamatergic excitatory postsynaptic currents (eepscs) were elicited by electrical stimulation of parallel fibers. the gαq/ proteincoupled metabotropic glutamate receptor (mglur ) was activated by superfusion of (rs)- , -dihydroxyphenylglycine (dhpg) or -more physiologically -by burst stimulation of the parallel fibers. results. both dhpg superfusion and burst stimulation of parallel fibers elicited an increase in intracellular ca + concentration in the postsynaptic purkinje cells. dhpg superfusion and burst stimulation suppressed eepscs, and this suppression was abolished in the presence of the mglur antagonist cpccoet. the suppression of the eepscs was also sensitive to the cb receptor antagonist rimonabant, pointing to involvement of endocannabinoids and cb receptors. the suppression of the eepscs was attenuated after depletion of the endoplasmic reticulum ca + stores by thapsigargin, cyclopiazonic acid and ip . the results indicate that after activation of the gαq/ protein-coupled metabotropic glutamate receptor (mglur ) of the postsynaptic neuron ca + is released from the endoplasmic reticulum. this ca + release significantly contributes to the production of endocannabinoids. the endocannabinoids diffuse in the synaptic cleft retrogradely to the terminals of afferent axons and inhibit transmitter release there through presynaptic cb receptors. the guanine nucleotide exchange factor dock controls reelin dependent cdc effects on radial migration pichler m. the regulation of blood glucose levels is under tight control of a complex system including hormone and neurotransmitter signalling. many of these cellular signalling pathways are initiated by binding of the ligand to a g-protein coupled receptor (gpcr), e.g. noradrenaline inhibits insulin secretion upon binding to a gi-coupled receptor. upon gpcr activation the heterotrimeric g-protein is activated and both the α-subunit and βγdimers are released and interact with their specific target proteins. by the usage of bordetella pertussis toxin (ptx) as a common gαi inhibitor gαi-dependent signalling pathways are interrupted which leads to increased insulin secretion, and significantly improves glucose tolerance. since the gαi-isoform specific roles in the regulation of glucose homeostasis are still debated we studied the glycemic control in gαi -deficient mice. surprisingly and in contrast to the ptx data, glucose tolerance was unchanged in the gαi -deficient mice compared to wild type controls. however, the plasma insulin levels were significantly reduced upon glucose challenge. these findings point to disturbed islets function and improved peripheral insulin sensitivity. analysing gαi deficient islets we show that islet size and number of nuclei are reduced. nevertheless, in vitro insulin secretion is improved at low ( mm) and high ( mm) glucose concentrations and can be further stimulated upon ptx-treatment. these data indicate that gαi proteins influence islet development and inhibit insulin secretion. in addition, these findings support our hypothesis that gαi -deletion influences peripheral insulin sensitivity. therefore, we investigated glucose homeostasis and pakt-levels after two hours feeding ad libitum in gαi -deficient mice. under feeding conditions no differences in plasma insulin levels were visible although blood glucose levels were significantly reduced in gαi -targeted mice. pakt-levels of liver and skeletal muscle were unaltered, whereas akt phosphorylation in white adipose tissue was significantly increased, indicating improved glucose uptake of adipocytes. in conclusion, gαi is a negative regulator of both insulin secretion and peripheral insulin sensitivity and important for the maintenance of glucose homeostasis. , ) in a radioligand binding test and to determine their functional effects with a membrane potential test using the dye r (molecular probes, . mg/ml, excitation nm, emission nm). the affinity of compounds in radioligand binding was slightly higher in sur b than in sur a-type channels, but the enantiomeric ratio in sur a channels matched that one determined for the sur b-type indicating some conformity of the binding pockets of sur a and sur b-proteins. surprisingly, however, the membrane potential tests revealed that the ( r, s)-enantiomer acted as agonist (a) whereas the ( s, r)-enantiomer acted as antagonist (b): ( r, s)-bms- induced membrane hyperpolarisation whereas ( s, r)-bms- repolarised cells prestimulated with submaximally effective concentrations of diazoxide. concluding, bms- is not selective for sur a as compared to sur b-type k atp channels. its enantiomers activate and block sur -type katp channels in a stereospecific manner. thieno-thiadiazine derivatives with full agonistic activity at sur b-type katp channels act as partial agonists at cardiac sur a-subtypes oldenhage c., grittner d., schmidt c., lemoine h. heinrich-heine universität, inst. für lasermedizin, mol. wirkstoff-forschung, universitätsstr. , düsseldorf, germany new potassium channel openers (kco) of the thieno-thiadiazine(ttd)-type initially developed as agonists for the sur -type katp channels (nielsen et al., j med chem : , ) were characterized in sur b-type katp channels as agonists and antagonists, if r contains a quaternary (methyl-cycloalkyl) and a tertiary (r = cycloalkyl) carbon, respectively (lemoine et al., this journal , r , ) . to investigate the selectivity of ttd-derivatives for myocardial katp channels the membrane potential actions of compounds were tested in hek (kir . /sur a)-cells and compared to hek (kir . /sur b)-cells as a model for smooth muscle-type katp channels. membrane potential was measured by fluorescence (excitation nm, emission nm) using . mg/ml of the dye r (molecular probes). standard-kco induced hyperpolarisation with ~ -fold smaller potency (pec ) in sur a. ttd-compounds with ch -cycloalkyl residues not only lost potency but also intrinsic activity for channel activation (emax) in sur a. possibly, this loss of emax would be much greater in native heart cells with a normal channel density. in contrast, ttd-compounds with cycloalkyl residues acted as antagonists of cells pre-hyperpolarized with diazoxide with similar affinity in sur a and sur b-type katp channels. concluding, selectivity of kco for katp channel-subtypes cannot only be achieved by a different affinity but also by a selective stimulation of the channel of interest. small-conductance calcium activated potassium (kcnn/sk/kca ) channels maintain neuronal calcium homeostasis, shape synaptic functions and prevent excitotoxic neuronal death. so far, little is known about the function of kca channels in nonneuronal cells. the aim of this study was to investigate the expression of kca channels in microglial cells and their potential function in microglial activation and maintenance. expression of kca channel subtypes in microglial cells was assessed by mrna analysis, western blots and immunocytochemistry. lipopolysaccharide (lps)-induced microglial proliferation was evaluated by the xcelligence impedance-based system and mtt assays, and immunogenic activation of microglia was determined by measuring cytokines and nitric oxide (no) release into the cell culture medium. the kca . and kca . channel activator cyppa ( µm) and specific inhibitory peptides ( µm) were applied to distinguish effects mediated by the kca channel subtypes. all kca channel subtypes were detected on mrna and protein levels in resting and in lps-activated microglial cells. xcelligence real-time measurements and mtt assays demonstrated that lps ( ng/ml) induced microglial proliferation. the kca . /kca . channel activator cyppa reduced lps-induced microglial proliferation in a concentration-dependent manner. specific peptide inhibitors of kca . channels, but not of kca . channels, reversed the cyppa-effects on lps-induced microglial proliferation. cyppa alone did not alter the production of tnf-alpha or il- , but strongly reduced the lps-dependent cytokine production. interestingly, chelation of extracellular calcium by edta induced differential cytokine kinetics by decreasing lps-dependent il- production while tnf-a production was not affected. moreover, using inhibitory sk /kca . channel peptides, we demonstrated that sk /kca . channels modulate lps-induced cytokine il- production in a calcium-dependent manner, while the tnf-a release was independent of extracellular calcium. in summary, the present study revealed that kca . channel stimulation reversed microglial activation. thus, kca . channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in cns diseases. ( r, s)-( s, r)- intracellular amyloid beta (aß) oligomers and extracellular aß plaques are key players in the progression of sporadic alzheimer disease (ad). still, the molecular signals triggering aß production are largely unclear. we asked whether mitochondria-derived reactive oxygen species (ros) are sufficient to increase aß generation and thereby initiate a vicious cycle further impairing mitochondrial function. complex i and iii dysfunction were induced in a cell model using the respiratory inhibitors rotenone and antimycin resulting in mitochondrial dysfunction and enhanced ros levels. both treatments lead to elevated levels of aß. presence of an antioxidant rescued mitochondrial function and reduced formation of aß demonstrating that the observed effects depended on ros. conversely, cells overproducing aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. again, the capability of these cells to generate aß was partly reduced by an antioxidant indicating that aß formation was also ros-dependent. moreover, mice with a genetic defect in complex i, or ad mice treated with a complex i inhibitor, showed enhanced aß levels in vivo. several lines of evidence show that mitochondria-derived ros result in enhanced amyloidogenic amyloid precursor protein processing, and that aß itself leads to mitochondrial dysfunction and increased ros levels. we propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic ad. comparison of methods to derive health-based guidance or limit values for chemicals licht o., voss j. -u., mangelsdorf i. fraunhofer item chemikalienbewertung, nikolai-fuchs-str. , hannover, germany health-based guidance or limit values are derived for chemicals to compare measured or estimated exposure concentrations with these values. if the exposure is below the limit value, adverse effect for human health can be regarded as negligible, e.g. the exposure is expected to be tolerable. in germany such values have been derived since years for chemicals that can be found in soil, water and air as well as human blood and urine (biomonitoring). in a research project sponsored by the german umweltbundesamt several methods used by the agency are compared to the method laid in reach guidance document r. to derive a derived no effect level (dnel). the aim was to identify possibilities for standardization as well as to figure out specific elements in individual methods. in addition to extrapolation factors the public availability of guidance and specific derivations as well as procedures for consensus on the limit value were evaluated. the comparison of extrapolation factors revealed that, although they are named differently such as extrapolation, safety or assessment factors, they are used in a comparable manner. factors for interspecies and intraspecies extrapolation are presented in more detail. the standard factor for such extrapolation is in most cases. in the reach guidance this factor consists of a part for allometric scaling and remaining differences. other factors are only defined in some methods, like a factor for extrapolation from loael to noael, data quality or data gaps. a factor for data quality is not laid down in the basic scheme for setting of indoor air guidance values, but is used in some of the recent derivations of limit values. also the who guidelines for drinkingwater quality use comparable factors to account for adequacy of studies or database and nature and severity of effect. a transparent and documented derivation is necessary for acceptance of the value. the derivation methods as well as the evaluation document on a specific substance are available through publications or the internet in nearly all cases. for the dnel only the numeric value is available at the echa website, but not any information on starting point and extrapolation factors. although all guide or limit values are derived in a comparable way, differences, however, exist in some details. in most cases detailed explanation is lacking when deviating from standard or default assumptions. often such deviation is based on expert judgement. hepatocellular carcinoma (hcc) is the fifth most common cancer in the world and has a poor prognosis with limited therapeutic options. up to now, no curative systemic therapy exists emphasizing the high clinical importance of new therapies for hcc. therefore, the identification and characterization of novel drugable targets is a relevant goal. cyclin-dependent kinase (cdk ) is well characterized for its function in cns development and disease. recently, few reports indicate functions of cdk in cancer. cdk was shown to regulate tumor growth, and our group discovered that cdk regulates angiogenesis. since hcc is a highly vascularized tumor and anti-angiogenic treatment (sorafenib) has shown some therapeutic benefit, we hypothesize that cdk is an interesting target for hcc therapy. the aim of this study was to characterize the function of cdk in hcc. histology of tissue micro arrays indicates an increased expression of cdk in human hcc tissue in comparison to healthy liver tissue of the same patient. to investigate the function of cdk in hcc, we analyzed the impact of both pharmacological inhibition of cdk and specific downregulation of cdk with rna interference on hcc cells. pharmacological inhibition of cdk with the small molecule roscovitine (r-roscovitine, seliciclib) decreased proliferation and clonogenic survival, induced g /m cell cycle arrest and cell death, and reduced motility of huh and hepg cells. transient downregulation (sirna) and stable knockdown (shrna) of cdk also reduced proliferation, clonogenic survival, migration and invasion of huh cells. in a subcutaneous hcc xenograft model, treatment with roscovitine reduced tumor growth and angiogenesis, indicated by decreased tumor weight and volume, and reduced vessel density. moreover, cotreatment of hcc cells with roscovitine and tumor necrosis factor related apoptosis inducing ligand (trail) resulted in an over-additive additive effect on the induction of apoptosis. this coincided with reduced phosphorylation and activity of the anti-apoptotic transcription factor stat at ser that is directly phosphorylated by cdk , and tyr . in line with this, the expression of the antiapoptotic protein mcl- is reduced by inhibition of cdk . our results point to an important function of cdk in hcc and suggest cdk as an interesting pharmacologically druggable target for hcc therapy. delivery of mono-biotinylated rnasea into macrophages with streptavidinconjugated clostridium botulinum c toxin lillich m. , chen x. clostridium botulinum produces the adp-ribosyltransferase c , which modifies and thereby inactivates exclusively the small gtp binding proteins rho-a,-b and -c. recently, we discovered a specific endocytotic internalization of c toxin in macrophages and myeloid leukaemia cells, but not in epithelial cells [ ] . thus, c toxin provides a tool to target cells of the monocyte/macrophage lineage, which are involved in various diseases and are of great clinical interest. we used a biochemical crosslinking approach to design a delivery system based on an enzymatic inactive c bot mutant (c mut) and streptavidin. the c portion mediates uptake of the transporter into monocytes/macrophages and streptavidin allows for binding of biotinylated cargo molecules to the transporter. in vitro, the generated c mut-streptavidin bioconjugate showed specific and concentration dependent binding to biotinylated oligonucleotides as demonstrated by electrophoretic mobility shift assay. cell fractionation experiments indicated an uptake of the bioconjugate into the cytosol of j a. macrophages. in the next step, mono-biotinylated bovine pancreatic ribonuclease a (rnasea) was used as a model cargo for the delivery of macromolecules by the bioconjugate. rnasea is a highly stable, well studied protein which catalyzes the degradation of rna. mono-biotinylated rnasea interacts in a specific and concentration dependent manner with the c mut-streptavidin bioconjugate in vitro as analysed with dot blot technique. the c mut-streptavidin bioconjugate efficiently mediates the internalization of biotinylated rnasea into j a. macrophages as analyzed with laser scanning microscopy in fixed cells. this finding was also confirmed by live cell imaging. furthermore, cell fractionation showed a cytosolic delivery of biotinylated rnasea in the presence of c mut-streptavidin. as expected we could not observe a cytotoxic effect of biotinylated wild-type rnasea on j a. macrophages, which is attributable to the presence of ribonuclease inhibitor protein in mammalian cells. in summary, the c mut-streptavidin bioconjugate mediates the efficient internalization of biotinylated (macro)molecules into macrophage like cells, and therefore represents a useful tool for the transduction of exogenous molecules into macrophages. in addition, cytotoxic rnasea mutants are available and will be used in further studies. organometal compounds such as cisplatin or the second generation complexes carboplatin and oxaliplatin have become more and more important as antitumor agents. nevertheless there is still an increasing demand for novel metal-based compounds. this is necessary due to severe side effects and the occurence of resistent tumour cells. in this context we investigated the cytotoxic effects of imidazole-based phosphane gold(i) complexes as potential agents for cancer treatment. initially we have used the mtt-assay to examine the toxic potential of the gold complexes in h iie rat hepatoma cells. in this context cw (a diphosphane ligand with azoyl substituents r p(ch ) pr , r= thiazol- -yl) turned out to be the compound with the highest cytotoxic potential with an ic value of , mm ( h incubation). further investigations revealed that cw induced an apoptotic cell death in h iie demonstrated by the activation of caspase / ( h incubation with mm cw ). in addition the induction of apoptosis was confirmed by the dna ladder formation ( h incubation with mm cw ). in connection with the molecular mechanisms of apoptosis induction we used the comet assay to analyse the generation of dna strand breaks as well as the dcf-assay to detect the formation of reactive oxygen species. however neither dna strand breaks nor increased levels of reactive oxygen species were detected after h of incubation. furthermore we analysed if the compound influences intracellular signalling pathways such as the jnk pathway and the pi k/akt but after h of incubation neither pakt nor jnk were influenced. the imidazole based phosphane gold (i) complex cw shows strong toxic effects in h iie cells and turned out to be a promising compound as a potential agent for cancer treatment. the high and inappropriate intake of loop diuretics in hypertensive elderly reported in former studies has again been confirmed. remembering that inappropriate intake of loop diuretics can lead to exsiccosis and electrolyte loss especially in elderly, better medical education has to follow these alarming results to improve the pattern of diuretic prescription. furthermore, our results lead us to assume a high estimated number of unreported cases of torasemide use in uncomplicated arterial hypertension in elderly. this loop diuretic agent shows a longer duration of action compared with furosemide (elimination half-life: - hrs vs. hr) and is effective in decreasing blood pressure in subdiuretic doses. it must be pointed out that loop diuretics are still frequently inadequately prescribed because current guidelines recommend loop diuretics only in complicated arterial hypertension. the role of cgmp/cgki signaling and trpc channels in regulation of vascular tone loga f., domes k., hofmann f., wegener j. pharmakologie und toxikologie for , biedersteiner str , münchen, germany signaling by intracellular cgmp and cgmp-dependent protein kinase i (cgki) is the major pathway in vascular smooth muscle, by which endothelial no regulates vascular tone. recent evidence suggests that trpc channels are targets of cgki in smooth muscle and mediate, at least partially, the relaxant effects of cgmp. we tested this concept by investigating the role of cgmp/cgki signaling on vascular tone and peripheral resistance using cgki-, trpc -, and trpc -, and trpc /c -double knock-out mice. we found larger contractile responses to α-adrenergic stimulation in intact aorta from cgki-, trpc -, and trpc /c -double knock-out mice as compared to aorta from ctr and trpc -knock-out mice indicating a functional link between cgki and trpc channels. no differences were found if the vasodilator tone, provided by the no generation in the vascular endothelium, was inhibited by l-name. likewise, no differences were observed in the increase in peripheral resistance by α-adrenergic stimulation using the hind limb perfusion system. activation of cgki by -br-cgmp diminished aortic tone and peripheral resistance to a similar extent in control, trpc -, trpc -, and trpc /c -double knock-out mice. no effect of -br-cgmp was observed in preparations from cgki -/mice. to test the co-localization of cgki and trpc channels, we performed immunocytochemistry on isolated smooth muscle and endothelial cells from aorta of ctr, trpc -, and trpc -knockout mice. trpc could be detected in both smooth muscle and endothelial cells whereas trpc was only detected in endothelial cells. the results suggest that absence of cgki or trpc impairs the vasodilator tone induced by endothelial no production but that cgki and trpc channels are not functionally coupled in vascular smooth muscle. we thank profs birnbaumer (nih) and freichel (homburg) for providing us with trpc -/-, and trpc -/mice and prof. flockerzi (homburg) for the antibodies against the trpc channels. whole genome microarray analysis of the effects of tcdd and pcb in human hepatic cell models lohr c. , neser s. after the treatment with tcdd, however, a total of genes were more than -fold up regulated in hepg e.g. cytochrome p a (cyp a ) ( -fold) a sensitive marker for ahr activation. additional up regulated genes in hepg were; arylhydrocarbon receptor repressor (ahrr) -fold, aldehyde dehydrogenase a (aldh a ) -fold, and cytochrome p b (cyp b ) -fold. genes were more than -fold down regulated in hepg cells e.g. -proprotein convertase subtilisin/kexin type . markedly different findings were obtained in hheps, i.e., genes were up regulated, the highest up regulated gene was cyp b with a -fold increase in gene expression, followed by cyp a ( -fold) and aldh a ( -fold). only a small group of genes were significantly down regulated ( in total), e.g., solute carrier family (facilitated glucose transporter). comparing both human cell types, there was an unexpected small overlap of genes being up or down regulated. interestingly, in both cell types, only in common genes were up regulated, including cyp a , cyp a , cyp b and aldh a . only platelet-derived growth factor receptor, beta polypeptide, was down-regulated in both hepg and hheps. in conclusion, our data indicate pronounced differences in the patterns of tcdd-regulated genes between hepg cells and hheps. detection of redox modified proteins in nociceptive processing lorenz j. e. , kallenborn-gerhardt w. recent data indicate that redox modifications of proteins induced by reactive oxygen species (ros) contribute to sensitization of pain pathways during persistent pain. however, little is known about the targets of ros in pain processing, because the relatively unstable nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. here, we used the quantitative thiol trapping technique termed oxicat to identify proteins which are redox modified during nociceptive processing. we investigated spinal cords of untreated mice, after zymosan injection into a hindpaw (inflammatory pain model) and after spared nerve injury (neuropathic pain model). we identified several proteins with marked changes in their redox states after nociceptive stimulation. our results show that the oxicat method is an efficient method to detect redox modifications in proteins and that redox modifications seem to play a role in pain processing. supported by the deutsche forschungsgemeinschaft (sfb /a ). additive antinociceptive effects of a combination of vitamin c and vitamin e after peripheral nerve injury lu r., kallenborn-gerhardt w., geisslinger g., schmidtko a. pharmazentrum frankfurt/zafes institute of clinical pharmacology, goethe university, frankfurt am main, germany accumulating evidence indicates that increased generation of reactive oxygen species (ros) contributes to the development of exaggerated pain hypersensitivity during persistent pain. in the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin c and vitamin e in mouse models of inflammatory and neuropathic pain. we show that systemic administration of a combination of vitamins c and e inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by complete freund's adjuvant. in contrast, vitamin c or vitamin e given alone failed to affect the nociceptive behavior in all tested models. the attenuated neuropathic pain behavior induced by the vitamin c and e combination was paralleled by a reduced p phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. moreover, the vitamin c and e combination ameliorated the allodynia induced by an intrathecally delivered ros donor. our results suggest that administration of vitamins c and e in combination may exert synergistic antinociceptive effects, and further indicate that ros essentially contribute to nociceptive processing in special pain states. - , - and - ) replaced by leucine residues. both amino acids are comparable in terms of hydrophobicity, volume and the preference for forming α-helices, but only methionine is oxidizable to a sulfoxide, in contrast to leucine. in the present study we examined the protein-protein interaction (ppi) of recombinant ac , expressed in sf insect cell membranes, with cam, cam- , - , - and - by measuring the catalytic activity of ac . cam-mutants show a - -fold lower potency than cam, but they are more efficacious than cam. most prominently, cam- was % more efficacious than cam. such striking differences between cam and cam-mutants have not yet been observed for other mammalian effector proteins. as a result of the exchange of all methionine against leucine residues in cam- , it is more hydrophobic than cam and this leads to a better ppi with ac . in future studies we will examine the effects of cam inhibitors, antidepressants and antipsychotics on cam/ac interaction. furthermore we will analyze the effects of oxidized cam and cam-mutants on the catalytic activity of ac . because oxidative stress is of great importance in aging, it is important to know more about the abovenamed interaction in view to the demographic change. taken together, our data point to a unique cam/ac interaction that may be selectively targeted by small molecules. in particular, enhancers of these interaction could be useful to improve memory and learning. gender differences in fat distribution and diabetes prevalence in nzo mouse lubura m., scherneck s., zucker a., schürmann a. deutsches institut für ernährungsforschung experimentelle diabetologie, arthur-scheunert-allee - , potsdam, germany background: excessive fat accumulation in visceral but not subcutaneous fat depots as well as ectopic fat storage in liver, skeletal muscle and pancreas are associated with an increased risk for the development of type diabetes in humans. in this study we aimed to examine the influence of early fat distribution on onset of type diabetes in mice. methods: nzo mice are regarded as insulin resistant model in which only males become diabetic. we used male and female mice fed with high-fat and standard diet. we determined fat distribution by computed tomography for three times and conducted oral glucose tolerance tests on two different time points. besides we assessed body weight and blood glucose levels on weekly basis. results: contrary to previous findings, we observed that not only male nzo mice on high-fat diet develop diabetes. blood glucose levels at the th week of age and total pancreatic insulin content indicated diabetes prevalence of % in males and % in females these results lead to the conclusion that high-fat diet counteracts protective action of estrogens against diabetes. inversely to the findings in humans, female mice tend to store more fat in abdominal region than males. there was no relationship between early accumulation of fat in abdominal region and onset of type diabetes. however, visceral fat was associated with liver fat in males as well as in females. furthermore, at the age of ten weeks hepatic fat content correlated with blood glucose levels (r² = . ) indicating that the early hepatosteatosis is a predictor for hyperglycemia. however, there was no correlation between hepatic insulin sensitivity (indicated by quantitative insulin sensitivity index-quicki) and amounts of hepatic fat we conclude that early hepatosteatosis does not predict for glucose intolerance in nzo mice. in the nzo mouse, the amount of liver fat but not the early fat distribution predicts for the later onset of type diabetes. further experiments are needed to examine the gender dependent differences in the diabetes prevalence of this mouse strain. with a prevalence of about - % non-alcoholic fatty liver disease (nafld) represents the most common liver disorder in europe. nafld manifestation ranges from steatosis through steatohepatitis (nash) to fibrosis and cirrhosis, followed in some cases by liver failure and hepatocellular carcinoma. fatty degeneration of liver cells, increased oxidative stress with concomitant lipid peroxidation and an induction of pro-inflammatory cytokines are proposed as possible causes for developing inflammation and fibrosis, but the exact pathogenesis of the progression of nafld into nash is still unknown. thus, besides life style modifications and weight reduction interventions, no established pharmacological therapy exists so far. to gain further insights into the pathogenesis of nafld and nash and to develop new therapeutic strategies, appropriate animal models are essential. thus, in the present study three different dietary animal models for nafld were evaluated and compared to the biochemical and metabolic alterations seen with nafld and nash in man. male adult lewis rats were given standard food or one of three different diets: fatty liver diet [fld] , methionine/choline deficient diet [mcd] or methionine/choline deficient plus high fat diet [mcd+hf] . after , , , or weeks of treatment, animals were sacrificed and body and liver weights, laboratory parameters (asat, alat) as well as histopathological changes in the livers and different parameters indicating oxidative stress or representing the biotransformation capacity of the livers were analyzed. with fld and mcd+hf a normal body weight gain was observed, whereas with mcd body weight gain was strongly impaired. liver weights were mainly increased after mcd+hf. elevation of asat and alat values and hepatic steatosis were more pronounced after mcd and mcd+hf than after fld. all three diets caused an increase in the oxidative stress in liver tissue, but especially with mcd a tremendous elevation in the hepatic levels of lipid peroxidation products was seen. with regard to liver biotransformation capacity, with all three diets mainly an induction of the cytochrome p e and a isoforms expression and activity was observed, which was most pronounced after mcd and mcd+hf. in summary, the changes induced by mcd or mcd+hf most closely resemble the alterations described in literature for nafld in man and thus should be preferred over fld in future investigations on nafld and nash. ep receptors for prostaglandin e convey stimulatory and inhibitory effects. e.g., their stimulatory effect leads to vasoconstriction in the human pulmonary artery and their inhibitory activity to reduction of neurotransmitter release from neuron endings. the aim of our study was ( ) the pharmacological characterization of ep receptors in human pulmonary arteries and ( ) the examination of the involvement of these receptors in the regulation of the neurogenic tachycardia in pithed rats. l- served as the ep antagonist. experiments were performed in human pulmonary arterial rings isolated from patients undergoing lobectomy during resection of lung carcinoma and in pithed and vagotomised rats. the ep /ep agonist sulprostone ( nm - mm) concentrationdependently contracted human pulmonary artery rings (pec and emax; . ± . and . ± . %, relative to the contraction induced by kcl mm). the concentrationresponse curve of sulprostone was not affected by the ep antagonist sc ( µm) but shifted to the right by l- ( µm) (apparent pa . ). extending the exposure time to l- from . to h increased its antagonistic potency to . (schild plot-based pa ; concentrations . , and µm). in pithed rats electrical stimulation ( . hz, ms, v for s) of the preganglionic sympathetic nerve fibers or intravenous isoprenaline ( . nmol/kg) increased heart rate (hr) by beats/min. sulprostone ( - nmol/kg) did not affect the isoprenaline-induced increase in hr but inhibited the neurogenic tachycardia dose-dependently, maximally by %. l- ( µmol/kg) diminished the inhibitory effect of sulprostone nmol/kg on the neurogenic tachycardia by %. in conclusion, ep receptors ( ) located postsynaptically strongly contract human pulmonary arteries and ( ) located presynaptically on sympathetic nerve fibres supplying the heart of rats strongly inhibit the neurogenic tachycardia. - -bromo-n- [ -( - voltage-gated ca + channels of the central nervous system control a multitude of ca + dependent processes such as neurotransmitter release, neuronal excitability, neurite outgrowth, synaptogenesis, plasticity and neuronal survival. the cav . ca + channelalso known as p/q-type channel -belongs to the subfamily of high voltage activated ca + -channels. ca + influx via cav . ca + channels located at presynaptic nerve terminals triggers vesicle fusion and transmitter release at brain synapses and at the neuromuscular junction. thus, cav . ca + channels play a crucial role in synaptic transmission. the global cav . knock-out phenotype is characterized by severe ataxia, dystonia and lethality during the first postnatal weeks and is therefore an unsuitable model to analyze the importance of cav . ca + channels for learning and memory. therefore, we crossed a floxed cav . mouse line with nex-cre transgenic mice to establish a viable, forebrain specific knock-out mouse line (fbko-mice). results from western blot analysis confirmed an efficient knock out of cav . in hippocampal and cortical preparations, whereas the expression level in the cerebellum was not altered. to investigate the specific role of cav . channels in hippocampus and neocortex dependent behavior, we performed tests for motor functions and sensory abilities and in particular learning and memory tasks. mice with a forebrain specific cav . knock-out show significant deficits in spatial learning & reference memory and a significant reduced recognition memory as revealed by the morris water maze and an object recognition task. the fbko-mice exhibit no obvious locomotor deficits during behavioral tasks in the open field test and elevated plus maze. some fbko-mice demonstrate episodes of seizures in the morris water maze and during different rotarod tasks. to assess motor-function of fbko-mice in a stress reduced environment, we performed home cage based running-wheel motor-learning tasks. in summary, the diverse phenotypes of the forebrain specific knock-out mouse line emphasize the critical importance of cav . for learning and memory. helicobacter hepaticus-infected rag -/mice emulate many aspects of human inflammatory bowel disease (ibd), including the development of colitis and colon cancer [erdman et al., , pnas : - . toward the goal of elucidating mechanisms of inflammation-induced carcinogenesis and developing biomarkers of inflammation, we undertook a comprehensive analysis of macromolecular damage products during disease progression in h. hepaticus-infected rag -/mice. infected mice developed severe colitis and hepatitis, accompanied by infiltration of myeloperoxidase-positive neutrophils and f / -positive macrophages, by wks postinfection (pi), progressing into colon carcinoma by wks pi. qpcr array-based gene expression profiling revealed that pathophysiological changes were associated with characteristic alterations in the expression of genes related to inflammation, dna repair, and oxidative stress response. to study inflammation-related macromolecular damage, colon and liver tissues were analyzed by isotope-dilution chromatography-coupled mass spectrometry to quantify a battery of different dna, rna and protein damage products thought to represent the full spectrum of inflammation-related chemistries. our data revealed a significant predominance of chlorinated dna-, rna-, and protein damage products by weeks pi. in contrast, levels of damage products arising from oxidation, nitration and nitrosation changed only modestly or remained unchanged. our analyses also revealed higher levels of damage products in rna than in dna and demonstrated organ-specific differences of oxidative damage products, such as -oxo-dg and its oxidation products spiroiminodihydantoin and guanidinohydantoin. collectively, these results suggest that neutrophil and myeloperoxidase-induced chlorination chemistry may serve as a biomarker of ibd and may play important roles in the pathophysiology of ibd and colitis-associated cancer. characterization of a membrane protein expressed in mouse heart and brain mannebach s. recently, a novel membrane protein in drosophila was shown to be localized in presynaptic vesicles. it appears to mediate a ca influx after vesicle fusion with the plasma membrane. disruption of the corresponding gene leads to endocytic defects in drosophila [ ] . apparently, this protein plays a role in exo-and endocytosis and could serve as a ca channel supplying ca required for endocytosis. we have identified a protein in mouse, c rf , which shares , % amino acid sequence identity with the drosophila protein. it covers amino acid residues. using rt-pcr the full length transcripts could be identified in brain, kidney, pancreas, heart, spleen, thymus and mast cells. coexpression of c rf and the ca v . channel in xenopus oocytes reduced the amount of the α b and cavβ subunits of the ca + channel in the plasma membrane but did not affect the gating properties of the cav . channel. expression of c rf alone did not yield any channel activity. we therefore started to produce recombinant protein using the his-sumo-prokaryotic expression vector. the protein was efficiently expressed as his-sumo-c orf -fusion in e.coli (yield mg at mg/ml). we are currently preparing the c orf part of the his-sumo-c orf fusion protein by ulp -protease digestion followed by various chromatographic steps. the purified recombinant protein will be used to immunize rabbits to get antibodies. in parallel we generated antisera by immunizing rabbits with peptide fragments derived from the c orf sequence. we could not identify any homologues of c orf in the mouse genome and to analyze its function we are currently generating c orf deficient mouse lines by gene targeting. we have chosen a strategy for conditionally inactivation of the gene with the option to study the cellular localization of c orf by expression of the bgalactosidase gene under the control of the endogenous c orf promoter. by southern blot analysis we´ve already identified homologous recombinant embryonic stem cell clones out of analyzed ones and we will proceed with blastocyst injection to get chimeric mice and finally mice carrying the introduced mutations in the c rf gene. parps are involved in various biological processes such as regulation of dna repair, cell cycle progression, and cell death. consequently, several parp inhibitors are currently in clinical development as chemo-and radiosensitizers as well as monotherapeutic agents following the concept of synthetic lethality. pharmacological and toxicological studies call for an accurate analysis of parp activity in terms of a detailed knowledge of the structure of par and a reliable method for its quantification. we have developed a sensitive, precise, and accurate bioanalytical method based on liquid chromatography coupled to electrospray tandem mass spectrometry (lc/ms-ms) to characterize and quantify par with femtmol sensitivity: par is extracted from cells and hydrolysed to specific monomeric units, i.e., ribosyladenosine, which is characteristic for linear par, diribosyladenosine, which is characteristic for branching points, and adenosine, which represents the terminal part of the polymer. using this method, we are currently analyzing par levels in different cell lines and in primary human peripheral blood mononuclear cells (pbmcs) both under physiological conditions as well as upon genotoxic stress and in the presence of potent parp inhibitors. we expect that after completing method validation this assay will be useful for a wide range of applications in pharmacology and toxicology. gene mutagenic potential and metabolite profile of β-estradiol in cultured v cells expressing human cytochrome p a martínez jaramillo d., lehmann l. university of wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, wuerzburg, germany oxidative metabolism of the female sex hormone β-estradiol (e ) is considered to play a major role in the initiation of hormone-induced carcinogenesis. in extrahepatic tissues, e undergoes metabolic activation by cytochrome p -dependent monooxygenase (cyp) isozyme a to -hydroxy-( -ho) and to a lesser extent to -ho-e . if not conjugated, these catecholestrogens (ce) can further oxidize to electrophilic quinones (q), which may react with dna and induce thereby mutations. conjugation of these ce in extrahepatic tissues is mainly catalyzed by catechol-omethyltransferase. in order to identify possible mutagenic metabolites (i) the induction of gene mutations by e was determined in male chinese hamster lung fibroblasts (v cells) expressing human (h) cyp a and (ii) the metabolite profile of e in these cells was analyzed via gas chromatography/mass spectrometry after solid phase extraction of the cell suspension in the culture medium. (i) gene mutations were assessed using the hypoxanthine-guanine phosphoribosyltransferase assay. the promutagen benzo[a]pyrene (bap) served as positive control requiring metabolic activation by hcyp a and dimethylsulfoxide as solvent control. v hcyp a were treated with nm e for weeks and the resulting -thioguanine ( -tg) resistant mutants selected at weeks (w) and . the frequency of spontaneous -tg resistant mutants per colony-forming cells ranged from ± (w ) to ± (w ). as expected, . µm bap induced a significant increase in mutant frequency (mf, ± , w and ± , w ) . treatment with nm e resulted in a -fold ( ± , w ) and a -fold ( ± , w ) increase in mf, suggesting slight mutagenic activity. in culture medium of v hcyp a treated with nm e , -ho-e , -methoxy-(meo)-e , -o-methyl- ho-e and -meo-e (suggesting intracellular formation of -ho-e ) were detected. while -meo-e concentration remained constant over the exposure period, the concentration of the other metabolites increased in a timedependent manner. the maximum concentration increase was reached at w for methylcatechols and at w for -ho-e , correlating with the maximum increase in mf, observed after weeks as well. in conclusion, e possessed a slight mutagenic potential after hcyp a -mediated activation to -, -ho-e and their corresponding methylcatechols. cumulative effects of three triazole fungicides in a broad dose range in vitro rieke s., kneuer c., bumke scheer m., lampen a., hirsch-ernst k., marx-stoelting p. bundesinstitut für risikobewertung chemikaliensicherheit, max-dohrn-str., berlin, germany consumers are exposed to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. the aim of this work was to investigate potential combination effects of the three triazole fungicides epoxiconazol, tebuconazol and flusilazol for selected parameters in a broad dose range in vitro. parameters investigated were cytotoxicity, hormone synthesis ( -β estradiol, progesterone and β-hcg), expression of a panel of androgen-or estrogen-responsive genes in the human placental choriocarcinoma cell-line jeg- and transactivation via estrogen receptors α and β in stably-transfected hek cells. the ability to inhibit steroidogenesis was analysed by measuring the concentrations of β-estradiol and progesterone in cell culture supernatants of jeg- cells. additionally, the placental peptide hormone β-hcg was measured. while no change in β-hcg and β-estradiol concentrations were observed, all triazoles induced a dose-dependent decrease in progesterone concentration and a cumulative effect was observed implying dose additivity at individual doses of > . µg triazole/ml. significant activation of erβ by the three triazoles, especially by flusilazol, was observed at µg triazole/ml and combined exposure showed additive effects, while no significant activation of erα was observed. based on the data, our findings suggest dose-additivity of triazole pesticides with the same mode of action for selected parameters in vitro. no significant effects were observed at lower doses [ ng - µg triazole/ml] neither for substances applied individually nor in combination. transient receptor potential channels as mediators of catecholamine release mathar i. trp proteins form cation channels that are regulated through strikingly diverse mechanisms. recently, genetic association studies identified many trp genes including trpm as risk factors for disease states such as arrhythmias, hypertension and cardiomyopathy. the melastatin trp channels trpm and trpm have distinct properties within the trp channel family; they form non-selective cation channels activated by intracellular calcium ions and are expressed in heart, aortic endothelial cells, kidney and adrenal gland. disruption of the trpm gene in mice leads to increased basal blood pressure without evidence for impairment of endothelium-or smooth muscle-dependent regulation of contractility of peripheral resistance vessels, the renin angiotensin aldosterone system, basal cardiac output or body fluid homeostasis. instead, trpm -deficient chromaffin cells exhibit increased acetylcholine-induced exocytosis of catecholamines which is associated with elevated level of epinephrine in the plasma and its metabolites in the urine. this indicates that trpm serves as an inhibitory regulator of exocytotic catecholamine release, at least in chromaffin cells. whether catecholamine release is also regulated by trpm in other cells of the sympathetic nervous system such as perivascular neurons still needs to be clarified as well as the molecular mechanism underlying how trpm regulates catecholamine release. besides trpm we recently identified transcripts encoding additional trp channels including trpc and trpc in chromaffin cells isolated by laser capture microdissection but their functional role in these cells is still unknown. measurements of the time course of the intracellular calcium concentration before and during acetylcholine stimulation ( µm) of catecholamine release as well as the analysis of the number of released vesicles in chromaffin cells relvealed no changes in trpc /c /c triple knock out mice compared to wildtype controls. although it seems that these trpc proteins are not directly involved in catecholamine release from chromaffin cells induced by acetylcholine application in our hitherto existing experiments, their contribution to the modulation of catecholamine release by agonists of gq-coupled receptors still needs to be analysed. aims: sulfonylureas (sus) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. in addition, sus have pleiotropic effects on other tissues. regarding the effects of sus on adipocytes conflicting findings were reported. we have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. methods: primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. lipid accumulation was assessed by oil red o staining and determination of triglyceride content; gene expression was measured by real-time pcr and western blotting. results: we initially characterized the genes regulated during human preadipocyte differentiation by a global microarray analysis. treatment with glimepiride and glibenclamide caused a strong accumulation of lipid droplets and an increase in triglyceride content. genes involved in lipid metabolism were induced, chemokine expression was decreased. interestingly, the effects of sus were over all qualitatively and quantitatively similar to pioglitazone. in direct comparison glibenclamide was more potent than glimepiride in respect to the induction of fabp (ec . vs. . µm), an important adipocyte marker gene. su-induced differentiation was virtually completely blocked by the pparγ-antagonist t but not affected by diazoxide, indicating pparγ activation by sus. repaglinide, causing insulin liberation like sus but being structurally different, had no effect on adipocytes. conclusions: in primary human pre-adipocytes, glibenclamide and glimepiride strongly induced differentiation, apparently by activating pparγ . thus, sus but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation. the role of at a and at b receptors as mechanosensors in myogenic vasoconstriction blodow s. , schneider h. arterial myogenic tone denotes the intrinsic property of vascular smooth muscle cells to constrict in response to an elevated intraluminal blood pressure. this physiological reaction is more distinct in small resistance arteries than in large conduit arteries. understanding the underlying mechanisms should provide useful information for the treatment of diseases like anaphylactic shock and systemic hypertension in which this reaction is altered. whereas the underlying signaling cascade has been extensively studied, the molecular identity of the mechanosensory elements still remains elusive. recent studies at the cellular level suggest a sensory function for a subgroup of gprotein coupled receptors (gpcrs) coupling to gq/ -proteins. by determining mrnaexpression levels of selected gpcrs in consecutive pairs of resistance and conduit vessels, we could identify a subset of gq/ -coupled receptors such as angiotensin ii at b, vasopressin v a, endothelin eta and etb and α a adrenoceptor significantly enriched in resistance vessels. by pharmacological blocking of those highly expressed gpcrs by different antagonists and inverse agonists, we evaluated their influence on the formation or the intensity of myogenic tone, as measured in isolated murine mesenteric arteries ex vivo. while blocking of v a receptor and α a and α ab adrenoceptors showed no differences of myogenic tone, blocking of at a and at b receptors by losartan and candesartan, eta receptor by bq and α a adrenoceptor by prazosin caused significant reductions of the vascular response. analyzing the myogenic response of at a -/mice with and without additional blocking of at b receptors by candesartan suggested that especially at b receptors play a dominant role for mechanosensitivity in mice. this was further supported by investigating the myogenic response of at b -/mice. these findings suggest that mechanosensitive gq/ -protein coupled receptors, especially at b receptors, play a dominant role for the development of myogenic vasoconstriction. trpm ion channels are activated by steroidal compounds and noxious heat and are considered to be involved in insulin secretion and pain perception. the expression of the trpm gene generates a variety of different transcripts which arise by alternative splicing and the use of different promoters [ ] . they encode a substantial variety of isoformes and so far we have identified more than distinct trpm proteins in mouse and rat each varying in exons , , , , , and . these variants differ enormously in their biophysical properties. for example splicing within exon affects the channel pore and causes significant changes of the ionic selectivity of trpm channels [ ] , whereas splicing of nucleotides encoded by exon leads to dormant trpm proteins. however, the frequency of these different isoformes in trpm expressing tissues is completely unknown. to get insight into the significance of the different trpm isoformes we investigated the abundance of alternative trpm transcripts in different tissues and cell types by reverse transcription quantitative pcr (rt-qpcr). we found that the frequency of splicing within exon ranges from up to % in different cell types and tissues. furthermore we analyzed the trpm transcriptome in the choroid plexus of the brain and the pituitary gland, tissues in which trpm transcripts are most abundant. for that purpose we sequenced more than clones, each. corresponding to our rt-qpcr result, we found a significant number of transcripts lacking exon . in cells of the choroid plexus nearly all ( / clones) carried the short ca + permeable pore. furthermore, we identified seven variants spliced in exon encoding truncated trpm proteins. however, the composition of the trpm transcriptome in the choroid plexus and pituitary gland differed enormously, indicating the importance of alternative splicing for trpm function in different tissues. the concept of "thresholds of toxicological concern" (ttc) defines tolerable dietary intakes for chemicals without toxicity data and is widely applied to chemicals present in food in low concentrations such as flavorings. based on a statistical evaluation of the results of many toxicity studies and considerations of chemical structures, the ttc concept derives a maximum daily oral intake without concern of , or µg/person/day for non-genotoxic chemicals depending on the allocation to so-called cramer classes i, ii or iii. for substances with a structural alert for genotoxicity a ttc value of . µg/person/day might be used. recently, it has been investigated, whether the ttc values, which were derived based on mostly chronic oral dietary rodent studies would cover all relevant toxicities (neurotoxic, repeated dose, reproductive and developmental, immune effects and endocrine-related effects). several authors using different specific databases have confirmed that the ttc values derived using cramer classes are also covering immunotoxic, neurotoxic, reproductive and developmental effects. a respective decision tree is going to be presented, also considering substances or substance classes which shall be excluded from the ttc approach. there are several areas in which the ttc concept is already used, or a ttc approach is considered useful, to assess low-level human exposures, or help in prioritizing toxicological testing; as for example the assessment of plant metabolites and degradates of pesticide active substances, feed and food additives, chemicals with a low exposure profile under reach, residues, metabolites and impurities in plants, chemicals, plant protection products or pharmaceuticals. if no structural alert for genotoxicity is given or standard genotoxicity tests are negative the cramer class iii value of µg/person/day, which corresponds to a dose of . µg/kg bw is considered to represent a chronic tolerable daily intake of the test substance. examples for current and future uses of the ttc concept in regulatory toxicology are presented. objective: hyaluronan (ha), synthesized by three ha-synthases (has , - , - ), is a prominent matrix component of atherosclerotic lesions. the aim of the present study was to identify the has isoenzyme that is associated with ha-matrix remodeling in inflammatory regions of atherosclerotic plaques. furthermore the underlying regulatory pathways were determined and functional aspects of this regulation in vascular smooth muscle cell (vsmc) were addressed. methods and results: during atherosclerosis in apoe deficient mice the peak of macrophage invasion at weeks coincided with ha deposition and induction of has in aortic root plaques. in human symptomatic carotid artery plaques has was by far the most prominent has isoenzyme as determined by quantitative real time rtpcr. in vitro, in human vascular smooth muscle cell (vsmc) has was specifically induced via activation of nfkb by interleukin- β (il- β) and tumor necrosis factor alpha (tnfa) as shown by chip assay and utilization of nfkb inhibitor bay - . has was also upregulated in a co-culture system by activated macrophages via paracrine release of tnfa and il- β as verified by neutralizing antibodies. in human atherosclerotic lesions nfkb positive vsmc were frequently detected in close proximity with ha and f / positive macrophages as shown by immunohistochemistry. to study the effects of has mediated ha synthesis in human coronary vsmc, lentiviral overexpression and knockdown of human has were employed. overexpression of has resulted in increased migration and proliferation whereas knock down had the opposite effect. the effects of has were mediated by both pi k signaling and mapk signaling via hyaluronan receptors cd and rhamm. conclusion: the present results suggest that has -dependent ha synthesis is induced in human vsmc by inflammatory cytokines released from activated macrophages. moreover, has -mediated ha production induced phenotypic activation of vsmc. pulmonary inflammation and airway remodeling are major features of chronic obstructive lung disease (copd). in addition, pulmonary hypertension is a common comorbidity, which is associated with a poor prognosis of the disease. recent studies in a guinea pig model of allergic asthma have shown that increased arginase activity, which converts larginine into l-ornithine and urea and competes with nitric oxide synthases for the common substrate, contributes to allergen-induced airway inflammation, hyperresponsiveness and remodeling. there is evidence that cigarette smoke and lipopolysaccharide (lps), both involved in the pathogenesis of copd, increase the expression of arginase, however, its role in the pathogenesis of copd is currently unknown. this study aimed to investigate the role of arginase in pulmonary inflammation and remodeling, using a guinea pig model of lps-induced copd. to this aim, guinea pigs were instilled intranasally with lps or saline twice weekly for weeks and were pretreated by inhalation of the arginase inhibitor ( )s-amino-boronohexanoic acid (abh) or pbs. repeated lps exposure increased lung arginase activity, resulting in increased lornithine/l-arginine and l-ornithine/l-citrulline ratio's. both ratio's were reversed by abh treatment. repeated lps exposure also induced increased il- levels, neutrophils, goblet cells, hydroxyproline and airway collagen content in the lung, which were all abrogated by abh. moreover, repeated lps exposure increased right ventricular mass, indicative of pulmonary hypertension, which was similarly prevented by abh. in conclusion, increased arginase activity contributes to pulmonary inflammation, airway remodeling and right ventricular hypertrophy in a guinea pig model of copd, indicating that arginase inhibitors may have therapeutic potential in the treatment of this disease. (supported by msd). behavioral abnormalities in hcn -deficient mice michalakis s., schöll-weidinger m., mader r., cao-ehlker x., fenske s., wahl-schott c., biel m. center for integrated protein science munich (cipsm) department of pharmacy -center for drug research, ludwig-maximilians-universität münchen, butenandtstr. - , münchen, germany hcn encodes a hyperpolarization-activated and cyclic nucleotide-gated channel, which is expressed in various brain regions including thalamic, hypothalamic and habenular nuclei as well as brain stem and olfactory bulb. in this study we performed a comparative analysis of hcn -/and hcn +/+ mice using a battery of behavioral tests and telemetric biopotential measurements to evaluate a potential role of hcn in central nervous system function. in general, the knockout mice showed normal motor function as assessed by the rotarod and open field tests. telemetric home cage activity and core body temperature measurements confirmed a normal circadian behavior, but revealed a lower basal activity that concurred with decreased body temperature during the light phase and the light-dark transition phase. hippocampus-dependent spatial learning was normal. by contrast, hcn knockout mice showed more immobility than control mice on day two of the porsolt forced swimming test, which could reflect increased depressionlike behavior. however, center exploration in the open field test as well as performance in the light-dark transition and the elevated-plus maze tests was normal in hcn -/mice. this suggests that general anxiety was not changed in the knockout mice. in addition, hcn knockout mice were less active on the second day of the open field test, which supports a habituation phenotype. finally, hcn -/mice had higher burying scores in the marble-burying test, which is a test for certain aspects of obsessive compulsive disorder in rodents. taken together, genetic deletion of hcn in mice results in distinct behavioral abnormalities related to behavioral despair and expression of repetitive behaviors in response to mild stressors. mielke h. , gundert-remy u. alcohol consumption when breast feeding is discussed controversially. some groups recommend breast pumping before alcohol consumption and feeding the stored milk instead of breast feeding after drinking alcohol. this study was performed to simulate the blood concentration in the breastfed baby and to assess the health impact. method: we established a physiologically based kinetic model. its parameters were calculated (partition coefficients tissue/blood ; schmitt, ) silva et al., ) . we simulated . the alcohol concentration in a breastfed neonate and a -month-old suckling infant after the nursing mother had consumed alcohol, . the alcohol concentration in utero/fetal compartment during pregnancy assuming the identical alcohol consumption of the pregnant woman . the alcohol concentration during infant´s treatment of bloating by an approved herbal drug containing alcohol. results: peak maternal alcohol concentration was . ‰ after consuming . l of wine, peak concentration was . ‰ in the newborn, . ‰ in the -month-old infant and . ‰ in the utero/fetal compartment. the peak concentration after herbal drug treatment was . ‰ in the neonate and . ‰ in the -month-old infant, respectively. we discuss the results of the simulations and compare it with doses and published concentrations measured in experimental animals or in vitro studies. conclusions: we conclude that the recommendation " to glasses of wine on occasion" (agence nationale d'accréditation et d'Évaluation en santé, assante ) is in accordance with the simulation results presented here whereas stricter rules are not scientifically sound. ( ) http://www.has-sante.fr/portail/upload/docs/application/pdf/ breastfeeding_guidelines.pdf da silva et al. ( ) adenylyl cyclases (ac) mediate physiological responses in virtually all cells, where their regulation through receptors and g proteins results in the modulation of camp. in the present study we focused on the kinetics of interactions between the alpha-subunit from inhibitory g protein type (gαi ) and adenylyl cyclase type v (ac ). these proteins were labeled with cfp and yfp, respectively. the dynamics of their interactions was monitored by means of high temporal resolution fret imaging in hek cells expressing unlabeled a a-receptor and gβg subunits. to activate the signaling pathway, we applied agonist using a rapid superfusion device. application of norepinephrine resulted in the development of a fret signal, indicating interaction between gai -cfp and yfp-ac . after withdrawal of agonist the fret signal recovered with a remarkably slow time course compared to the deactivation kinetics of gi proteins reported previously (bünemann et al. ) . to further analyze the properties of the dissociation between gai and ac we measured in parallel the offset kinetics of the interaction between gai -yfp and gβg-cfp (gi -fret) after agonist withdrawal under comparable conditions. in addition we tested to what degree the coexpression of rgs accelerated the deactivation of gi proteins and the dissociation of gai -cfp from yfp-ac . these experiments revealed that in the absence of rgs the dissociation of gai from ac after agonist withdrawal takes about times longer than the deactivation of gi proteins. in the presence of rgs this difference is even larger due to the pronounced acceleration of g protein deactivation. the dissociation of gai from ac was only marginally accelerated by rgs . these observations lead us to hypothesize, that ac might trap activated g protein-subunits and thereby affect the g protein cycle by shifting the equilibrium towards activated g proteins. if this hypothesis is true, it should result in a left-shifted dose response curve compared to g protein activation dose response. in support of this hypothesis we found that the concentration response curve for gai -ac interaction was several-fold leftward-shifted compared to the concentration-response curve of gi-protein activation under very similar conditions. influencing the dynamics of the g protein cycle by effectors may represent a novel and powerful mechanism for finetuning the sensitivity of receptor evoked responses in an effector-specific manner. obesity, the excessive accumulation of white adipose tissue (wat), has reached pandemic dimensions. the factors that determine fat mass are not fully understood, but adipocyte hypertrophy and adipokine secretion are thought to be important. in present study, we investigated the role of the cyclic gmp (cgmp) signaling pathway focusing on cgmp-dependent protein kinase i (pkgi) in white adipocytes. pkgi is expressed in wat, preadipocytes and differentiated adipocytes as demonstrated by real-time pcr, western blot and immunochemistry. differentiation of pkgifl/fl preadipocytes, using an optimized protocol, resulted in an enhanced lipid accumulation as evidenced by oil red o staining. deletion of pkgi in pkgifl/fl adipocytes infected with a cre lentivirus (lv-cre, pkgi / ) exhibited reduced differentiation. analysis of the triglyceride (tg) content revealed a significant decrease of tg levels by % ± % in pkgi / as compared to pkgifl/fl adipocytes. western blot analysis of white adipocytes showed a significant decrease of c/ebpalpha ( % ± . %), ppargamma ( % ± . %) and ap ( % ± . %) expression in pkgi / cells as compared to pkgifl/fl. treatment of t -l cells with cgmp resulted in increased lipid accumulation and enhanced expression of fat marker genes. lentiviral overexpression of pkgi further increased differentiation. importantly, pkgi significantly induced mitochondrial biogenesis in t -l cells. concomitant activation of pkgi in t -l preadipocytes and treatment with the demethylating agent -aza-deoxycytidine significantly increased expression of uncoupling protein- (ucp- ) -a unique protein of brown fat cells. we found rhoa as major target of pkgi signaling with increased phosphorylation of rhoa at ser- in pkgi overexpressing cells. moreover, pkgi-dependent phosphorylation counteracts the effects of rhoa on insulin signaling as well as adipokine expression. taken together, pkgi is a key player in white adipocyte differentiation that regulates cell size and has an anti-inflammatory effect. pkgi decreases the secretion of proinflammatory adipokines via inhibition of rhoa signaling. in addition, activation of pkgi can establish a brown fat cell like phenotype during white adipocyte differentiation if the ucp- promoter is accessible. the rag gtpases, raga, ragb, ragc, and ragd form a subfamily gtpases of the ras-related superfamiliy. rag proteins are characterized by a modified ras-like gtpbinding domain and a unique c-terminal region lacking a lipid modification motif. interestingly, rag proteins have been proposed to function as heterodimeric complexes consisting of raga or ragb associated with ragc or ragd. rag gtpases have been implicated in the control of mammalian target of rapamycin (mtor) function, in particular in regulation of the nutrient-stimulated and/or hormone-regulated mtor activity. the protein kinase mtor is found as the catalytic subunit of two larger protein complexes referred to as mtor complex and , mtorc and . under amino-acidrich conditions, activated mtorc promotes protein synthesis and inhibits autophagy, while under starvation autophagy inhibition is released. increasing evidence suggests that activation of rag gtpases contributes to mtorc function. thus, rag proteins were found to be associated with a protein complex termed ragulator, a major regulatory protein of mtorc function and guanine nucleotide exchange of rag gtpases within the rag-ragulator-complex were described to promote mtorc translocation to its functional lysosomal compartment. however, the guanine nucleotide exchange properties of rag proteins are poorly characterized, and it is currently unknown, how amino acids promote rag proteins to facilitate the formation of the active, raptor-binding state of the rag heterodimers. to characterize the guanine nucleotide exchange properties of the rag gtpases as momomers or heterodimers in more detail, recombinant rag proteins were expressed in bacteria and purified from this source to near homogeneity. first, the parameters of gdp/gtp exchange of each of these proteins were compared using the non-hydrolysable gtp analogon gtpgs. the results showed that the rag isoforms are distinct in their guanine nucleotide exchange activities. in particular, nucleotide exchange on raga and ragc, but not on ragb and ragd, was only observed at low concentrations of gdp and mgcl in extraction and assay buffers, i.e. conditions favoring the gdp/gtp exchange. these findings may indicate that guanine nucleotide exchange on raga and ragc is controlled by guanine nucleotide exchange factors and suggest specific functions of the individual rag gtpases within individual rag heterodimers. in in-vitro studies on rat and canine mast cells and human mast cell leukemia cells hmc . bz at micromolar concentrations inhibited mediator release which appeared to be related to an inhibition of the intracellular camp pathway. in order to identify potential targets on/in mast cells at which bz may cause an inhibitory effect on mast cell activation, the , -bz flunitrazepam (flu), clonazepam (clo) and chlorodiazepam ( -cd) were selected because of their different affinity and selectivity to/for the gaba-a-receptor and the translocator protein (tspo): flu and clo bind with nanomolar affinity to gaba-a receptors, whereas -cd is a selective ligand at tspo with nanomolar affinity to tspo but only micromolar affinity to gaba-a receptors. flu also possesses nanomolar affinity to tspo, whereas clo has no or only micromolar affinity to tspo. after incubation of hmc . cells with -cd, flu and clo for , and hours up to genes were significantly differently expressed in a substance-specific and timedependent manner. comparison of the genes differently expressed at hours revealed that the expression of genes was regulated by both flu and clo but only genes were regulated by both -cd and flu suggesting that flu and clo induce gene expression by acting at a target site different from that of -cd. the difference between the gene regulation by flu and clo on the one hand and that of -cd on the other hand is also reflected in pathway analysis. since it was conceivable that the beneficial effects of the , -bz could be mediated by the recognition sites targeted by the , -bz, i.e. the gria -encoded ionotropic glutamate receptor ampa , we investigated by quantitative pcr whether hmc . cells express gria , tspo, the genes encoding the subunits of the gaba-a receptor and the gaba-forming enzyme glutamic acid decarboxylase. tspo, gabra , gabrb , gabre and gabrd were moderately expressed. in addition, there was a week or very week expression of gabra , gabra , gabrb , gabrg and gabrr . expression of gria was not detectable. taken together, it cannot be decided yet from our data whether the inhibitory effect of benzodiazepines on mast cell activation is due to an action at tspo or at gaba-a receptors of a novel subunit composition. monien b. h., glatt h. german institute of human nutrition (dife) department of nutritional toxicology, arthur-scheunert-allee - , nuthetal, germany -hydroxymethylfurfural (hmf) and furfuryl alcohol (ffa) are common constituents of foodstuffs in which they are formed by heat-and acid catalyzed reactions from carbohydrates. hmf and ffa have been reported to induce the formation of hepatocellular adenomas in female mice and renal tubule neoplasms in male mice, respectively. we studied whether the carcinogenic effect of these hydroxymethylsubstituted furans may originate from sulfotransferase (sult)-catalyzed formation of electrophilic esters. hmf was inactive in in vitro mutagenicity tests using standard activating systems. in contrast, it was mutagenic in v cells genetically engineered for expression of human sult a suggesting that hmf is converted into the reactive sulfooxymethylfurfural (smf). following incubation of mutagenic smf with porcine liver dna in vitro, specific methylfurfural adducts were detected using liquid chromatography tandem mass spectrometry (lc-ms/ms), i.e., n -(( -formylfuran- -yl)methyl)- 'deoxyadenosine (n -ffmda) and n -(( -formylfuran- -yl)methyl)- '-deoxyguanosie (n -ffmdg). these adducts were also detected in dna from v -sult a cells incubated with hmf. in order to determine sulfo conjugation of hmf in mice in vivo, we conducted pharmacokinetic measurements showing that about ppm of the hmf dose was converted to smf and reached the circulation. like hmf, ffa was negative in the standard ames test and various other in vitro genotoxicity tests. we showed that ffa is mutagenic in salmonella typhimurium ta engineered for expression of human sult a . the putative mutagen -sulfooxymethylfuran was synthesized and incubated with porcine liver dna, in which various nucleoside adducts were found. the main adducts, -mfda were detected in dna of ffa-exposed salmonella strain ta -sult a and in dna of liver, lung and kidney of fvb/n mice that had received about mg ffa/kg body weight per day via the drinking water for days. in summary, both furan derivatives form mutagenic sulfate esters in vitro and in vivo. in the future, we will use genetically engineered mice to characterize the role of single murine and human sult forms in the bioactivation of the furan derivatives and the contribution to tumor induction. background: micrornas are small non-coding rnas that can negatively regulate gene expression on a post-transcriptional level and have been shown to interact with epigenetic mechanisms like dna methylation. mecp (methyl cpg binding protein ) is a protein that binds methylated dna cpgs in the promoter region of genes and can thus regulate their expression. otherwise, mecp is known to be a target gene for several micrornas including the cluster mir- / in the brain. recently, our group could show that mecp expression is downregulated in human heart failure suggesting that mecp might be involved in cardiac pathogenesis. the aim of this project is to study the upstream regulation of mecp by the cluster mir- / in the heart during cardiac hypertrophy in-vitro and in-vivo. methods and results: to test whether hypertrophic stimuli can induce mir- / expression, we treated cultured nrcms with µm norepinephrine for hours. this induced cardiomyocyte hypertrophy and expression of the hypertrophy marker nppa, but also of mir- ( . ± . -fold of untreated cells, p< . ) and of mir- - p ( . ± . -fold of untreated cells, p< . ) and downregulated mecp mrna and protein levels ( . ± . -fold of untreated cells, p< . ). to check whether mecp downregulation also occurs by direct mir- / activation we increased levels of mir- and mir- in cardiac myocytes by transfecting precursor mir- and mir- - p molecules. again, we observed nrcm hypertrophy, nppa mrna upregulation and mecp mrna and protein downregulation ( . ± . -fold of control, p< . ) after mir- overexpression. similar results were obtained by overexpression of mir- - p. to test the effects of adrenoceptor activation on the mir- / -mecp axis in-vivo, wild-type mice received isoprenaline and phenylephrine via osmotic minipumps ( mg/kg/day each). after days, cardiac ventricles were analyzed. nppa gene expression ( . ± . -fold of control animals), mir- and mir- - p levels ( . ± . and . ± . -fold of control animals, p< . and p< . , respectively) were increased while mecp protein levels decreased to % (p< . ) conclusion: these results suggest that in-vitro and in-vivo adrenoceptor stimulation leads to the activation of mir- / expression and to downregulation of mecp in cardiac myocytes in-vitro and in-vivo. leopold-franzens-universität, innsbruck, austria at- receptor antagonists block the angiotensin ii-enhancing effect on noradrenaline release from sympathetic neurons. in a cell-free assay the binding affinity of the at- receptor antagonists telmisartan and valsartan to the gamma peroxisome proliferatoractivated receptor (pparγ) is close to that of the pparγ selective agonists thiazolidinediones (tzds). we tested whether the tzds rosiglitazone and pioglitazone would also modify the prejunctional facilitatory effect of angiotensin ii. left ventricular slices of rats were incubated with tritiated noradrenaline, perifused and electrically stimulated. the negative logarithm of the drug concentration that caused a % increase of control (pec %) was calculated. angiotensin ii caused a concentration-dependent increase of tritium overflow induced by electrical stimulation [pec %= . ± . (mean±sem, n= ); maximum increase= ± %]. neither rosiglitazone nor pioglitazone ( . - µm) had a direct effect. the concentrationresponse to angiotensin ii in the presence of fixed concentrations of rosiglitazone was shifted to the left with increase of the maximum (pec %= . ± . , . ± . and . ± . ; maximum increase= ± %, ± % and ± %, in the presence of . , and µm of rosiglitazone, respectively, n= - , each). in contrast, pioglitazone in concentrations up to µm had no effect on the release-enhancing effects of angiotensin ii. results show that rosiglitazone but not pioglitazone potentiates the noradrenalinerelease enhancing effect of angiotensin ii. this action might contribute to the risk for myocardial infarction from rosiglitazone use but not from pioglitazone use. deleted in liver cancer (dlc ) is a tumor suppressor whose allele is lost in % of liver, breast, lung and % of colon cancers. despite its significance, the molecular mechanisms that drive cancerous transformation upon dlc loss remain unclear. we found that the transcriptional coactivators megakaryoblastic leukemia and (mkl / ) are constitutively localized to the nucleus in hepatocellular and mammary carcinoma cells that lack dlc . moreover, dlc loss and mkl nuclear localization correlated in primary human hepatocellular carcinoma. nuclear accumulation of mkl in dlc -deficient cancer cells was accomplished by activation of the rhoa/actin signaling pathway and concomitant impairment of erk-mediated mkl phosphorylation. dlc loss led to constitutive activation of the mkl-dependent, tumor-relevant target genes ctgf, cyr , myl and myh . furthermore, we identified a novel target gene, integrin a , with a key role in cell migration and metastasis, that exhibited a dlc -and mkldependent regulation. depletion of mkl / suppressed not only cell migration, but also cell proliferation and anchorage-independent cell growth induced by dlc loss. our data provide insight into the mechanism by which dlc loss initiates tumorigenesis. as mkl and have a key role in this process, this pathway may provide promising pharmacological targets for cancer therapy. universität bonn, pharma-zentrum bonn pharmazeutisches institut, pharm. chemie i, an der immenburg, bonn, germany membrane receptors activated by purine and/or pyrimidine nucleotides ("p receptors") are widely distributed in the body and constitute novel (potential) drug targets. they are subdivided into g protein-coupled p y receptors (p y , , , , , , , ) , and homo-or heterotrimeric ligand-gated ion channel or p x receptors (subunits: p x - ). we have been interested in the identification and development of potent and subtype-selective ligands -as tool compounds and potential drugs -for the various p y and p x receptor subtypes. our strategy involves (i) establishment of a proprietary compound library consisting of synthetic small molecules and natural products; (ii) development of screening assays suitable for medium throughput screening; (iii) careful analysis of structure-activity relationships at each target and systematic optimization of the lead structures; (iv) pharmacological evaluation of selected compounds. this approach has led to new biological tools for several targets, including p y and p x receptors [ ] [ ] [ ] [ ] [ ] [ ] . fine particles in particulate matter (pm) are effective vehicles to transport toxicants into the lung; depending on their size, smaller particles may reach the bronchiolar or alveolar space. in recent years the pm fraction pm . has especially been correlated with both pulmonary and cardiovascular diseases. in order to better characterize pm emission and distribution of environmental tobacco smoke (ets) from cigarettes (reference cigarette (rc), brand cigarette (bc)) we have developed an ets emitter to simulate human smoking emission and measured pm . concentration in a telephone booth ( , m volume) as an example for small indoor spaces like cars. fine particulate matter was measured using an aerosol spectrometer with sec time resolution; laser scatter allowed a size resolution from , µm to µm. for the pm . concentration the following values were calculated: cumulative pm . concentration as auc-pm . (µg/m /sec), peak pm . concentration as cmax-pm . (µg/m ) and average pm . concentration cmean-pm (µg/m ). in closed door condition both cigarettes produced particulate auc-pm . values of ± µg/m /sec (rc) to ± µg/m /sec (bc after myocardial infarction (mi) inflammatory cells and cardiac fibroblasts (cf) determine the remodeling response. interleukin- (il- ) is induced in the ischemic myocardium and is known to stimulate the differentiation of fibroblasts to myofibroblasts. hyaluronan (ha) is an extracellular matrix component synthesized by ha-synthase isoenzymes (has - ) and is also known to control fibroblast phenotypes. however, it is presently unknown whether il- participates in the remodeling of the ha-matrix or whether the ha-matrix modulates the responses to il- . therefore, the aim of the present study was to elucidate whether il- regulates the expression and function of ha-matrix in cfs. cells were isolated from c bl/ j mice and used during passage - for experiments. cfs were stimulated with il- or hyper-il- which is a fusion protein of il- and soluble il- receptor (sil- r). after and min signal transducer and activator of transcription (stat ) was phosphorylated in response to hyper-il- but not in response to il- . rt-pcr revealed rapid upregulation of has ( . ± . fold of unstimulated control, h) in response to hyper-il- . has was induced to a lesser degree ( . ± . fold of unstimulated control, h) whereas has was not responsive ( . ± . fold of unstimulated control, h). in contrast, il- had no effect on transcript levels of has isoenzymes. in turn, expression of has and has in response to hyper-il- was inhibited by ag , which indicates the involvement of stat signaling. interestingly, despite induction of has and has the amount of secreted ha as determined by an elisa-like assay was not affected by hyper-il- . this may indicate that il- regulates the cell surface associated ha-matrix of cfs. in conclusion, the present data demonstrate that cardiac fibroblasts respond to il- trans-signaling (hyper-il- ) via the soluble il- r and subsequent stat signaling with increased ha-synthesis. the fact that il- had no significant effect suggests that the expression of the non-signaling membrane-bound il- α-receptor (il- r) in cultured murine cardiac fibroblasts is not sufficient to induce has and - gene expression. therefore, il- trans-signaling mediated by il- and the circulating sil- r might be necessary to mediate the il- -induced has expression in vivo. mrgprd receptor endogenously expressed in dorsal root ganglia: evidence for an activation by -aminoisobutyric acid müller s., hoffmann k., von kügelgen i. universität bonn institut für pharmakologie und toxikologie, sigmund-freud-straße , bonn, germany the gpcr mrgprd (mrgd) is highly expressed in small diameter dorsal root ganglion (drg) neurons and has been implicated to play a role in nociception. the receptor was previously shown to respond to β-alanine. in the present study we searched for agonistic activity of structural analogues of β-alanine. for further characterization of the receptor we used fura- fluorimetry, a nfat luciferase reportergene assay and the determination of the inhibition of forskolin-induced camp production ([ h]-camp affinity assay). first, we confirmed the activation of the receptor by β-alanine and gaba. in reportergene experiments we then identified -dlaminoisobutyric acid as an agonist, with similar potency but weaker affinity when compared to β-alanine (ec µm). fura- fluorimetry showed an increase in intracellular ca + levels by -dl-aminoisobutyric acid ( µm). moreover, -dlaminoisobutyric acid reduced the forskolin-induced camp production by up to % (ec µm). in addition to -dl-aminoisobutyric acid, we identified -dl-aminobutyric acid as a weak agonist acting at the mrgprd. other closely related substances failed to show significant responses. next to the agonists we further characterized antagonists inhibiting the response to βalanine mediated by mrgprd. chlorpromazine shifted the concentration-response curve of β-alanine to the right with an apparent pkb of . (nfat assay), thioridazine with an apparent pkb of . (nfat assay) and . (camp assay) and rimcazole with an apparent pkb of . (nfat assay) and . (camp assay). in conclusion we show for the first time that -dl-aminoisobutyric acid is an agonist at the mrgprd and that the structure-activity relationship of agonists at mrgprd is very close. the sdf- -chemokine receptor cxcr plays a key role during embryogenesis and regulates functions of immune and stem cells in adult life. furthermore, cxcr is involved in disease states including inflammation and cancer. it is well established that sdf- -stimulated cxcr receptors activate gi protein-dependent signal transduction pathways and undergo c-terminal phosphorylation and internalization. because the cxcr c-terminal domain contains serine and threonine residues, it is incompletely understood which of the potential phosphorylation sites contribute to homologous and heterologous regulation of cxcr . here, we analyzed the phosphorylation pattern of cxcr at c-terminal sites after stimulation of the receptor with sdf- and after pma-induced activation of the pkc pathway as a model for heterologous receptor phosphorylation. using phospho-specific antibodies against s / , s / and s / in immunoblot analyses, we showed that the sites were phosphorylated after stimulation with sdf- or pma. stimulation with egf or forskolin did not induce phosphorylation at these sites. sdf- -induced phosphorylation at s / , s / and s / was reversible after wash out of the ligand. time course analyses revealed that phosphorylation occurred first at s / and then at s / and s / . taken together, these results indicate that the c-terminus of cxcr is phosphorylated at multiple sites by homologous and heterologous pathways and that phosphorylation at the different sites may be hierarchically organized. human milk represents the best form of nutrition for infants early in life. however, it can also contain toxic contaminants that may adversely affect infant's development. the nephrotoxin ochratoxin a (ota) is present in human milk (tab. in [ ] ), but information on transfer from maternal blood to milk is scarce: published data [ ] indicate that levels of ota in milk are roughly one tenth ( . ) of those in blood. but, the efficiency of the ota-transfer at various stages of breastfeeding may vary since studies in animals revealed that transfer of ota is apparently time-and dose-dependent. thus, the aim of this study was to assess the ota transfer from blood to milk at different stages of breastfeeding in humans. in a small chilean cohort, lactating women were asked to provide blood and milk on the same day. these samples were collected on four different occasions within the first months after delivery and analyzed using hplc with fluorescence and/or tandem mass spectrometric detection [ ] . the transfer of ota from blood to milk was quantitatively assessed by measuring the milk to plasma ratio (m/p). the average ota level in blood plasma was ± ng/l, and no major variations were observed over time (p = . ). on the other hand, ota levels found in colostrum ( ± ng/l) were higher than in mature milk (p < . ). in line with these data, higher m/p ratios (table) were obtained with samples collected in the first six days after delivery. this study showed that the transfer of ota from blood to milk was more efficient with colostrum (m/p . ± . ) than with mature milk. thus, a higher exposure to ota can be expected for neonates than for infants at later stages of breastfeeding. moreover, the lactating women have lower average ota levels in plasma than non lactating women from chile [ ] , indicative of milk as additional excretion route. acknowledgement: this work has been supported by a stipend from conicyt/daad to km. exposure of infants to ochratoxin a (ota) deserves particular attention since ota is nephrotoxic, and one of the most potent rodent renal carcinogen studied to date [ ] . moreover, infants may be more vulnerable to the toxic effects of contaminants than adults. ota-levels in plasma of infants are indicative of an early exposure in life [ ] . but blood sampling is an invasive method not readily applicable for breastfed infants. thus, the aim of this study was to implement a non invasive biomonitoring method to assess ota-exposure in this group. to assess the exposure to ota, breast milk and infants' urine specimens were collected, from two different cohorts: chile (n= ) and turkey (n= , only urine). analysis of the samples was performed using enzymatic hydrolysis prior to extraction and hplc-ms/ms [ ] . the magnitude of infants' exposure was assessed by calculating the ota-daily intake with human milk and relating it also to urinary ota levels. calculations of the daily intake with human milk [ ] showed that infants may be exposed to ota at high levels, exceeding the tolerable daily intake (tdi) of ng/kg-bw/day set for adults [ ] . in both cohorts, most of the urine samples tested positive for ota (chile %, turkey %). ota levels observed in urine samples from the turkish infants (range: - , ng/l) were fold higher than levels found in chilean samples (range positive samples: - ng/l). further analysis of phase ii metabolites in urine confirm the excretion of ota as conjugate (glucuronide) in highly exposed infants. in conclusion, ota exposure of infants early in life was documented. given that otaintake by several infants exceeded the tdi for adults, further biomonitoring in this vulnerable group is advised including also suitable biomarkers of effect. a mixture of (e)-and (z)-clomiphene citrate is the first line therapy of female infertility. however, up to % of patients do not respond. (e)-clomiphene is structurally closely related to another selective estrogen receptor modulator, tamoxifen which is frequently used for the treatment of hormone receptor-positive breast cancer. like tamoxifen, clomiphene is extensively metabolised by the cytochrome p system. using the estrogen receptor response assay (e)- -hydroxyclomiphene and (e)- -hydroxy-n-desethylclomiphene (ec : . and . nm, respectively) turned out to be times more active at the er compared to the parent drug isomers and de-ethylated metabolites. using recombinant expressed human cyp isoforms and inhibitory antibodies, cyp d revealed to be the major isoenzyme involved in the formation of -hydroxlated metabolites. n-deethylation was catalysed by cyps a / , d , c and c . rates of -hydroxylation in microsomes from human liver donors correlated with the number of functional cyp d genes. these in vitro results were confirmed in a pharmacokinetic study with female healthy volunteers receiving a single dose of clomiphene. in carriers of two non-functional cyp d genes (poor metabolizers) cmax of (e)- -hydroxyclomiphene and -hydroxy-ndesethylclomiphene was and times lower, respectively, when compared with subjects with at least one fully functional cyp d allele. in contrast, half-life of (e)-clomiphene and (e)-n-desethylclomiphene was and -fold higher, respectively, in poor metabolizers. our data provide first evidence of a pharmacogenetic rational for the variability in the response to clomiphene treatment. among the tested compounds, compound proved to be the most active derivative, showing a significant toxicity at a concentration of , µm. compounds and showed significant toxic effects at a concentration of µm. the compound showed no toxicity up to a concentration of µm. all derivatives , and have a ec between and µm. we further proved the induction of apoptosis by apo-one assay (caspase / activity) and life/dead-assay (fluorescence microscopy). in conclusion, these gold complexes exhibit an example of interesting potential candidates for future anticancer pharmaceuticals due to relatively high cytotoxicity. gene regulating effects in mouse liver subsequent to treatment with selected dioxin-like compounds and pcb using whole genome microarray analysis neser s. , lohr c. , van ede k. i. , andresen k. interaction with the aryl hydrocarbon receptor (ahr), with , , , -tetrachlorodibenzo-pdioxin (tcdd) being the most potent congener amongst the ahr agonists. recent risk assessments have employed the toxic equivalency factor (tef) concept. the current eu-project systeq aims at developing, validating, and implementing human systemic tefs as indicators of toxicity for dl-compounds. at present, the best known parameter of ahr mediated effects is the induction of cytochrome p isoenzymes (cyps), i.e., cyp a , a , and b . one of the major objectives of the systeq project is the identification of novel quantifiable biomarkers. in a three day study, female c bl/ mice were treated with single doses of six dl-congeners (tcdd, pcb , pcb , and pcb ) , and the 'non-dioxin-like' (ndl) pcb . quality tested (agilent ® bioanalyzer) mrna isolated from livers was analyzed using the agilent ® mouse whole genome array ( x k) system. the quantity of genes affected (≥ fold) was highly heterogeneous amongst the dl-compounds. whereas tcdd-treatment upregulated genes, and down-regulated , -pncdf-treatment had impact on (up), and (down). treatment with pcb led to marginal numbers of up and down-regulated genes. with (up), and (down) genes shared, the most extensive overlap occurred between tcdd-and -pncdd-treatment. no overlap was found due to treatments with the ndl pcb ( up, down) and tcdd. when comparing the effects of all dl-congeners, minor numbers of genes of up, and down-regulated remain, most of them being related to drug metabolism. while pcb regulated only genes involved in drug metabolism, omission of pcb -regulated genes resulted in consistently (up), and (down) regulated among dl-compounds. in conclusion, our findings suggest that the pattern of gene regulation in mouse liver elicited by pcb was strictly different from tcdd, while a very limited coincidence of genes was found even among dl-compounds. comparison of these 'core' genes with data from human models is required with respect to determination of novel biomarkers. introduction: proper use of antibiotics is essential with regard to effective treatment of bacterial infections. providing adequate information for patients can contribute to achieve this aim. materials and methods: data was collected from the relational database of the drug information service at dresden university of technology. the patients, who used the service, were interviewed concerning socio-demographic characteristics, reason for enquiry, number and kind of drugs taken, and diseases. possible contact paths were phone, e-mail or letter. in the present evaluation, all enquiries from the years and were analyzed descriptively focussing primarily on systemic antibiotics as reason for the enquiry. results: in the evaluated period, enquiries were registered in total. in . % of those enquiries systemic antibiotics were named with a total number of drugs. . % of those antibiotics were found to be the direct reason for the enquiry. most common information requested by patients corresponded to adverse drug reactions ( . %), diagnosis/treatment ( . %), drug application ( . %) and drug-druginteractions ( . %). the majority of the requesting patients ( . %) was born before . a correlation between incidence of enquiries especially concerning antibiotics and quarterly statistics could not be detected. conclusion: mainly patients aged years or more seem to need or search for further information about antibiotic medication. advice is required especially regarding adverse drug reactions and diagnosis or treatment. in order to this, the advisory service can help patients to lose their insecurity and to gain more confidence in handling antibiotic drugs. colon cancer is one of the most frequent cancers in the industrialized nations. epidemiological studies show a correlation between highly processed meat and the development of colorectal tumours. it is assumed that the risk of developing colorectal cancer, among various different factors, is related to the uptake of toxic substances contained in food such as heterocyclic aromatic amines that arise during the processing of fish and meat. phip is the most abundantly formed heterocyclic compound, and therefore has the biggest impact. in a previous study, we measured the absorption of phip in different intestinal segments of the rat. in the present study we focussed on the potential mechanisms by which phip is reabsorbed. the unidirectional phip transport from the mucosal to the serosal compartment (j ms) and in the opposite direction (jsm) was examined using the ussing chamber technique and c-phip as a radiotracer. the proximal jejunum and distal colon of male fischer rats in short-circuit current chambers was clamped, so that mucosal and serosal compartments were built. the phip flux rates were determined at defined intervals over min. the experimental conditions were selected in such a way that negative net flux rates (jnet = jms-jsm) were indicative of an active secretion. both intestinal segments showed large differences. while in the jejunum jms and jsm of phip were not significantly different, there was an active secretion in the colon. in a next step the transport proteins involved in this process should be examined. introduction: human organic anion transporter , oat (slc a ), is abundantly expressed in kidney and liver and mediates the sodium-independent uptake of clinically relevant drugs like -fluorouracil, paclitaxel, bumetanide, tetracycline, and zidovudine. while immunohistochemical studies have localized human oat to the basolateral membrane of kidney proximal tubules, its hepatic localization is currently unknown. we, therefore, firstly determined oat localization in human liver. because interindividual variability of oat expression may affect hepatobiliary drug uptake and elimination, we next systematically investigated the influence of genetic and non-genetic factors on hepatic oat expression. methods: an expression profile of oat for human tissues was determined by realtime quantitative polymerase chain reaction (taqman). oat mrna expression was analyzed in well-characterized human liver samples from caucasians that were accompanied by detailed demographic and clinical data. oat was localized in human liver cryosections using a commercial rabbit polyclonal antibody and hepatic oat protein levels were determined. resequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and genome-wide single nucleotide polymorphism microarray technology served to genotype variants in the slc a gene region. results: oat mrna was expressed in several human tissues, including liver. moreover, a new alternatively spliced variant of oat was identified in human liver. hepatic expression of full-length oat mrna and oat protein varied -fold and fold, respectively. oat mrna and protein levels did not correlate with each other. oat was localized to the sinusoidal membrane of human hepatocytes. no novel variants in the exons, the '-flanking region, or the '-untranslated region of the slc a gene were identified. univariate analysis showed that oat mrna is reduced in patients diagnosed for cholestasis (p= . ) and is affected by genetic variants. whereas the influence of genetic variants on hepatic oat expression appears to be limited, cholestasis significantly contributes to the variable interindividual oat expression. this indicates consequences for hepatic drug elimination of and response to oat drug substrates such as paclitaxel or tetracycline. the life threatening toxicity of organophosphorus (op) nerve agents is caused by the inhibition of the acetylcholine esterase (ache). oximes were shown to be potent reactivators of inhibited ache, but in poisoning by some compounds, e.g. soman, they have only a small therapeutic effect. for such cases, an alternative new strategy may be the intervention at nicotinic acetylcholine receptors (nachr). previous studies with the bispyridinium non-oxime mb demonstrated therapeutic effects against soman in vitro and in vivo which was partly attributed to its direct interaction with nachrs [ ] . we investigated the interaction of mb and several structure analogous at the orthosteric binding site of human α nachr (hα nachrs), a subtype which appears to be widespread in the human body, and compared the results with data obtained from torpedo-nachrs, which show a high degree of homology with human muscle-type nachrs. interaction of compounds with the orthosteric binding site of hα nachrs were investigated with radioligand binding experiments performed as high-throughput method [ ] . membrane preparations of gh c cells stably expressed hα nachrs were incubated with the nachr agonist [³h] epibatidine and appropriate concentrations of the unlabelled competitors e.g. bispyridinium compounds. after incubation, bound and free [³h] epibatidine were separated by rapid vacuum filtration. ki values of the competing compounds were calculated with nonlinear regression. three bispyridinium compounds, mb , mb and mb exhibited ki values at micromolar concentrations while three other compounds, mb , mb and the pharmacological inactive mb (negative control) did not show any interaction with the orthosteric binding site of hα nachrs. with torpedo-nachrs, ki values were in similar orders of magnitude -except mb which indicated significant subtype selectivity. interestingly, the affinity of monomeric pyridinium derivates did not correlate with their bispyridinium structure analogues. obviously, no correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission exists, although species-related differences cannot be excluded. in this study, we analysed the cytotoxic and clastogenic effects of the anticancer drug nimustine (acnu) in cells deficient in repair proteins involved either in homologous recombination (hr) or non-homologous end-joining (nhej). we show that hr mutants are extremely sensitive to acnu as measured by the induction of apoptosis and colony formation as well as the induction of chromosomal aberrations. the nhej mutants were slightly sensitive to acnu and differed in their sensitivity, with the ku mutants being moderately sensitive and the dna-pkcs mutant resistant, comparable to the wild-type (wt). cell death was mostly executed via the caspase-dependent apoptotic pathway with involvement of caspase- and - , and necrosis was also induced. further, we investigated the kinetics of dna double-strand break (dsb) formation that resulted from the repair of acnu-induced interstrand cross-links by means of γh ax and bp foci analysis in wt and mutant cells. cells mutant in hr did not repair dsb and went into the apoptotic or necrotic pathway, whereas wt cells were able to repair most of the dsb. cells deficient in ku formed at early times after acnu treatment less γh ax and bp foci compared to the corresponding wt, which might be due to a reduced capacity of recognising dsb. at later times after treatment, ku mutant cells show foci levels similar to the wt indicating restitution of h ax phosphorylation. we also analysed whether dsb formation after acnu treatment was replication-dependent using synchronised cells. we determined the formation of γh ax and other dsb marker in wt cells that passed through the first cell cycle after demecolcine synchronization. the level of γh ax foci increased significantly in the s-phase and remained at a high level during g where a fraction of cells remained arrested. rad , atm, mdc- and rpa- foci were also formed and shown to co-localize with γh ax. these foci were ameliorated significantly in s-and g -phase, which was similar to the time course of γh ax foci formation. in western blots, we confirmed a higher phosphorylation level of atm and chk and less phosphorylation of chk in hr mutants. the data indicate that acnuinduced dna cross-links give rise to cyto-and genotoxicity via the formation of dsbs that activate the cellular dna damage response. the endocannabinoid system has been established as a mediator of numerous central and peripheral biological functions. cannabinoids have emerged as attractive alternatives or supplements to therapy with opioids for chronic pain states. however, in human the activation of cannabinoid receptors is associated with side effects. for clinical exploitation of the analgesics properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without attenuating their analgesics effect. in animal studies, the anti-nociceptive efficacy of cannabinoids has been unequivocally demonstrated in several models of inflammatory and neuropathic pain. however, there are marked inconsistencies between different reports with respect to the locus of these pain-protective effects. we are working towards establishing the contribution of cb receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia. using cb globally knock-out animal as background, we induce the expression of cb specifically in nociceptive neurons localized in the peripheral nervous system and test the analgesic effects of cannabinoid systemical delivery in these mice. our results support the development of peripherally acting cb analgesic agonist with reduced central side effects. furthermore, we are utilizing proteomics approach to identify protein complexes that interact with cb receptor which hold promise in understanding cannabinoid signaling in health and disease. most chemoattractants, including chemokines, complement c a, fmlp, and leukotriene b are signaling through heterotrimeric g proteins of the pertussis toxin (ptx)-sensitive gi family. the functional inactivation of all gαi proteins with ptx leads to a fulminant decompensation of the immune system, whereas the constitutive inactivation of a single gαi coding gene results in mild phenotypes in mice. we are mostly interested in the nonneuronally found gαi and gαi isoforms and their redundant and specific roles in immune function and infection. for this purpose cellular in vivo and ex vivo models and in vivo infection model with listeria monocytogenes are being used. macrophages were isolated from the peritoneal cavity of wild type (wt) and gαi-deficient mice days after i.p. injection of % thioglycolate that induces peritonitis in vivo. we confirmed previous observations that in gαi -deficient mice the migration of macrophages into the peritoneal cavity was reduced after induction of peritonitis. regarding the expression levels of gαi and gβ isoforms in the lavage samples, the predominant gαi isoform gαi was upregulated in gαi -deficient macrophages. vice versa gαi was upregulated in gαi -deficient macrophages. concerning gβ isoforms, both gβ and gβ were strongly reduced in the gαi -deficient macrophages which resulted in a reduced total amount of gβ. surprisingly, the gαi -deficient macrophages showed reduced gβ protein levels only which caused a change in the gβ / gβ quotient in favour of gβ . we are currently establishing an in vivo infection model with l. monocytogenes in gαi and gαi -deficient mice. our previous in vitro infection studies in mice embryonic fibroblasts provided us with information about possible distinct roles of these two isoforms as far as the uptake of l. monocytogenes in the cells is concerned. challenging the immune system of gαi-deficient mice with this pathogenic organism will give us new insights into the systemic immune response in these mice upon bacterial infection. our data indicate that we may surmount the redundancy between these two isoforms and focus on their distinct and specific roles in pathogen defense. fret-based β-arrestin biosensors reveal conformational changes upon binding to the β -adrenergic receptor in real time and living cells nuber s., zabel u., ziegler n., hoffmann c., lohse m. j. institut für pharmakologie und toxikologie pharmakologie, versbacherstr. , würzburg, germany β-arrestins are multifunctional adapter proteins that regulate seven transmembranespanning receptor ( tmr) signaling and initiate also alternative signaling pathways. studies have shown that β-arrestins undergo conformational changes upon receptor stimulation, which are thought to be necessary for its downstream actions. to investigate these conformational changes in living cells we constructed fret based biosensors of β-arrestin , in which cfp was fused to the c-terminus and the flashbinding motif (ccpgcc) was inserted to different positions within the n-or c-domain of β-arrestin . upon β -adrenergic receptor (β ar) stimulation we observed a decrease of the intramolecular fret signal between cfp and flash at the n-domain (β-arrestin flash ), indicating a conformational change moving the c-terminus and the ndomain of β-arrestin relative to each other. kinetic analysis revealed that this conformational change immediately follows β-arrestin /β ar interaction on a timescale of seconds. a β ar mutant that was previously shown not to interact with β-arrestin was utilized as control and did not induce a conformational change in the β-arrestin molecule. our data provide evidence that β-arrestin changes it`s conformation upon binding to the activated β ar in living cells. the β-arrestin flash sensor could serve as universal biosensor for gpcr activation. studies on the physiological role of annexin a in the heart nunes f. the calcium binding protein annexin a has been examined in the context of heart failure in the past. annexin a expression level was found to be elevated in ventricles of human failing heart in comparison to expression levels in non-failing ventricles. furthermore the intracellular localization pattern in atrial cardiomyocytes was found to be altered in the failing human heart (moravec and matteo, cardiovasc res ). in order to gain insight into the possible physiological significance of these findings we utilized an annexin a gene trap model (gt) in which the annexin a protein content was not detectable in ventricles and atria. measurements of sarcomere shortening and calcium transient kinetics in isolated ventricular cardiomyocytes revealed a prolonged calcium transient decay at stimulation frequencies of . hz, hz and hz as well as an increased sarcomere shortening at hz and hz in anxa gene trap animals in comparison to wild type (wt) ( the effects of the β-adrenoreceptor agonist isoprenaline (iso) on the shortening of ventricular cardiomyocytes was increased in gt as compared to wt ( , ± . vs. . ± . , *=p< . vs. wt; n= - / ). western blot analyses indicated that the expression of the sarcoplasmic reticulum (sr) ca + -atpase (serca a) and the phosphorylation status of its regulator protein phospholamban (plb) did not differ between groups (n= ). however, co-immunoprecipitation experiments suggest, that anxa is able to interact with hax , which acts as a repressor of serca a (n= ). we performed force measurements in isolated and electrically stimulated left atria in response to rising isoprenaline concentrations ( - m- - m). the positiv inotropic effect of isoprenaline was significantly increased in gt atria (rel. force at - m iso [%]: wt: ± ; gt: ± *= p< . vs wt; n= - ). in conclusion, annexin a contributes to the regulation of cardiomyocyte contractility. the anxa up-regulation might therefore contribute to diminished cardiac performance in heart failure. matteo rg, moravec cs. immunolocalization of annexins iv, v and vi in thefailing and non-failing human heart. cardiovasc res. mar; ( ) background: pregnane x receptor (pxr) is considered the most important sensor of natural and anthropogenic xenobiotics in vertebrates. in contrast, the amphibian ortholog is involved in neural development and irresponsive to xenobiotics. instead, the xenopus laevis constitutive androstane receptor (car) was recently found to possess pxr-like properties, featuring low basal activity and a pronounced ligand spectrum. thus a structural and functional characterisation of x. laevis car may provide further insights into human car basal and ligand-induced activity. methods: the time-point of origin of car genes was determined by macrosynteny analyses of car, pxr, and vdr (vitamin d receptor) gene loci, which form the nr i subfamily of nuclear receptors. based on a -dimensional protein model of xenopus laevis car, docking studies with structurally diverse agonists were conducted. proteinligand-interactions as well as sequence comparisons were performed in order to select amino acids to be mutated towards human car. the organ response to car activators was determined in xenopus laevis using rna microarrays. results: car emerged together with pxr and vdr from an ancestral nr i gene in early vertebrates via two whole-genome duplications. this was followed by losses of car from the fish lineage and of pxr from sauropsida (reptiles and birds). amino acids important for ligand binding were identified. structural features responsible for the pronounced basal activity in human constitutive androstane receptor are not present in x. laevis car. in human pxr the inter-helical loop in front of helix is part of the ligandbinding pocket and supposed to be responsible for the wide substrate spectrum. in amphibian car this inter-helical loop plays no role in ligand binding. car agonists resulted in a pronounced induction of antimicrobial peptides in the ovary. conclusions: car emerged already in early vertebrates and it is conserved in land vertebrates, whereas xenosensing pxr is found only in the fishes and mammals. we provide a comprehensive modeling and mutational analysis of this first reported amphibian xenosensor. the induction of antimicrobial peptides by car activators suggests a link between xenosensing and innate immunity. the latter one may play a previously unrecognized role in the amphibian reproduction. background: retigabine belongs to a novel class of potent anticonvulsant drugs and is currently being investigated in clinical routine. the therapeutic range of retigabine serum concentration is unknown. a therapeutic drug monitoring (tdm) is used for most other anticonvulsant drugs. the aim of this study was to develop a method for the determination of retigabine in serum of patients and to compare the effect and the side effects of retigabine with the blood levels of the drug. method: a hplc method with tandem mass spectrometric detection for the sensitive determination of retigabine was developed. solid-phase extraction (spe) of µl serum with oasis hlb cartridges allowed a reliable quantification down to ng/ml. in order to develop an assay with high sample throughput and to obtain maximum response for the analytes we required the shortest possible retention time. to implement the determination of retigabine in a second step in the routine tdm of anticonvulsant drugs the corresponding hplc method was selected: a purospher rp column ( mm x mm; µm, merck) and a mobile phase with a steep acetonitrile gradient. results: the great advantage of having analytes with different molecular masses and similar retention times in combination with ms/ms detection enabled us to aim at a minimum separation that might remove some salts or matrix components that can suppress or interfere with the analyses from the target components, while maintaining good sample throughput. the method was validated. the assay is precise, accurate, fast, sensitive, and selective. discussion: the developed method is suitable for therapeutic drug monitoring of retigabine. the correlation of the serum concentration and the effect of the drug and thus the necessity of tdm have to be tested. targeting inflammatory t lymphocytes with conditional chemokine receptor antagonist expression for a tissue-specific therapy of chronic inflammatory disorders ogrissek n., giegold o., pfeilschifter j., radeke h. h. uniklinikum der goethe-universität pharmazentrum / zafes, theodor-stern-kai , frankfurt am main, germany chemokines and their receptors are known to be involved in the pathogenesis of chronic inflammation and autoimmune diseases. several approaches tried to use chemokine receptor antagonists as therapeutics to reduce exagerrated immune response, however, due to compensation and systemic side effects clinical trials often failed. in previous experiments our group identified three promising antagonists. cxcl ( - ) has antagonistic function for cxcr , cxcl (p g ) is able to inhibit cxcr and the herpesvirus encoded protein vmip-ii interferes with ccr , - and - as well as with cxcr , - and cx cr . their expression and secretion was confirmed in pichia pastoris and antagonistic function has been proven by a reduction of t cell migration. the aim of this project is to develop a cell-based therapy for chronic inflammation with a treatment that is based on the collective effect of cxcl ( - ), cxcl (p g ) and vmip-ii. with targeting of stable transduced memory t cells these antagonists should be conditional expressed and secreted directly in the centre of inflammation, resulting in inhibition of further inflammatory t cell accumulation. to realize this project we first cloned constitutive lentiviral constructs containing these antagonists and optimized transduction of t cells, such as the ova-specific memory th- cell clone if with the potential to initiate antigen specific nephritis in scid mice. next we investigated expression and secretion of cxcl ( - ), cxcl (p g ) and vmip-ii with pcr, western blot and elisa. at the moment we want to measure the inhibition efficiency of t cell migration in vitro with chemotaxis and flow chamber assays. construction of an inducible lentiviral vector plasmid to ensure expression of the antagonists only upon t cell activation, is also part of our current work. finally we would like to test the chemokine receptor antagonists in vivo in two relevant mouse models of type- -diabetes and contact dermatitis. small heterodimer partner (shp- ) is a member of the superfamily of nuclear receptors (nrs). in contrast to other nrs this orphan receptor lacks the dna binding domain. however, shp- is known to inhibit activity of several nrs by direct proteinprotein interaction. importantly several of the interacting nrs have been shown to directly regulate shp- expression, suggesting that shp- is involved in negative feedback loops of various metabolic pathways, such as cholesterol-, bile acid-and drug metabolism and glucose homeostasis. recently binding sites for nrs were identified in the promoter region of shp- , including hnf α, lrh , lxr, fxr, srebp c and pparγ. the aim of our study was to identify single nucleotide polymorphisms (snps) in the promoter region of shp- and to determine their impact on the transactivation of shp- . dna samples from subjects of the population based cohort study of health in pomerania were analyzed by sanger sequencing, thereby we identified four snps namely - t>c (rs ), - g>c, - c>t (rs ) and del- ctga (rs ). subsequently those polymorphisms were tested for their functional consequence performing cell based reporter gene assays testing all above mentioned modulators (lrh , lxr, fxr, srebp c and pparγ) of shp- expression. only the transactivation by hnf α was decreased in the presence of the - c>t polymorphism to % and the - g>c polymorphism to %. in conclusion we described snps with impact on transactivation. it will be aim of future studies to determine the potential impact on physiological processes or disease development. autosomal recessive polycystic kidney disease (arpkd) is a rare genetic disease, afflicting about in . individuals. arpkd is characterized by cystic fusiform dilatations of the renal collecting ducts leading to massive enlargement of the kidneys and ultimately loss of renal function. in addition, the patients suffer from congenital hepatic fibrosis (chf), possibly leading to portal hypertension and liver enlargement. so far, there is no cure for arpkd. therapy is focussing on controlling the disease symptoms [ ] . mutations in the pkhd gene cause arpkd. more than different mutations in this gene have been reported, all leading to the same phenotype, though there are differences regarding the severity of the disease [ ] . in animal models of autosomal dominant polycystic kidney disease (adpkd) as well as arpkd elevated levels of camp were shown [ ] [ ] [ ] . in isolated kidney cells camp stimulates cl-secretion and activates the b-raf /mek/erk pathway. these both are important factors for cyst development and disease progression [ , ] . intracellular camp regulation is based on conversion of atp to camp by adenylyl cyclases (acs) and degradation by phosphodiesterases . referring to this, we asked the question if there are differences in the activation and expression pattern of acs in pck rats, an animal model of arpkd [ ] and in sprague dawley rats. therefore, we examined membranes in a radioactive ac activity assay using various stimulatory compounds, e.g. forskolin, a direct ac activator, or hormones like glucagon and vasopressin to characterize acs. furthermore, we examined ac isoform expression on the mrna level via rt-pcr. we observed that in pck rats ac activity was decreased in general in comparison to sprague dawley rats. in future experiments we are aiming to obtain further knowledge about the influence various hormones exhibit on pck rat acs and to biochemically characterize acs. the major pathogenicity factors tcda and tcdb from clostridium difficile monoglucosylate and thereby inactivate small gtp-binding proteins of the rho subfamily after entering host cells via receptor-mediated endocytosis. although the intracellular mode of action of the toxins is well understood, far less is known about binding structure and internalization pathway of tcda and tcdb. since antibodies directed against the c-terminal located clostridial repetitive oligopeptides (crops) are able to neutralize toxin cytotoxicity the crop domain is acknowledged to mediate receptor binding. however, we recently demonstrated that crop deletion mutants of tcda (tcda - ) and tcdb (tcdb - ) enter host cells and exhibit full cytopathic potency though lacking the proposed receptor binding domain. we therefore refute the accepted opinion of a solely crop-mediated toxin uptake and re-evaluate the role of the crops in toxin endocytosis. tcda - and tcdb - induced time and concentration dependent cell rounding and rac -glucosylation. however, depending on the cell line, truncated toxins exhibit up to -fold reduced potency towards host cells compared to the respective full length toxin. the observed difference in toxin potency might reflect the recognition of different receptor structures or the use of various endocytotic routes. interestingly, pre-incubation of cells with the isolated crop domain enhances binding as well as cytotoxicity of subsequent applied truncated tcda indicating that the crops primarily determine toxin uptake. in fact, competition experiments revealed that tcda and tcda - predominantly use different receptor structures corroborating the notion of alternative internalization processes utilized by tcda. different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of c. difficile. thus, characterization of alternative endocytotic pathways used by the c. difficile toxins might therefore be the basis to investigate the opportunity of toxin uptake inhibition as therapeutic option. in neurodegenerative diseases, such as alzheimer´s disease and parkinson´s disease, mitochondrial pathways of apoptosis are considered as major features of the underlying neuronal cell death. such mitochondrial mechanisms of apoptosis are mediated by the bh -only protein bid, a member of the bcl- family that triggers mitochondrial permeabilization and the subsequent release of death-promoting proteins into the cytosol. the pivotal role of bid in apoptotic cascades of neuronal cells has been shown in our previous studies showing a neuroprotective effect of bid sirna and small molecule bid inhibitors such as bi c in vitro. in vivo, however, the available bidinhibitors failed to protect brain tissue likely because the compounds were not bioavailable or did not cross the blood brain barrier. therefore, chemical modifications of bi- c were generated resulting in new structures and molecules with different pharmacophors. the aim of the present study is to identify novel potent bid inhibitors available for applications in model systems of brain damage in vivo. for the first screening of compounds we used a model of glutamate toxicity in immortalized mouse hippocampal neurons (ht- cells). in this model system, glutamate induces a decrease of intracellular glutathione levels resulting in lipoxygenase activity and enhanced formation of toxic reactive oxygen species (ros). to investigate the compounds' ability to prevent glutamate induced cell death, we first analyzed the cell viability by the mtt assay. in addition, we examined the cell survival by using real time monitoring of cell impedance (xcelligence system) to determine the neuroprotective potency of the new structures. using these assays, we identified novel molecules that significantly prevented glutamate-induced toxicity in ht- cells. further we were able to express and to purify recombinant bid in a high amount. in the ongoing study the purified bid protein will be used for co-crystallization with the identified neuroprotective structures for further optimization of novel bid inhibitors for therapeutic applications in experimental models of neurodegenerative diseases in vivo. polymorphic enzymes, urinary bladder cancer risk and structural change in the local industry ovsiannikov d. , selinski s. in the s, an uncommonly high percentage of glutathione s-transferase m (gstm ) negative bladder cancer cases ( %) was reported in the greater dortmund area (golka et al., ) . the question arose whether this uncommonly high percentage of gstm negative bladder cancer cases was due to environmental and occupational exposure decades ago. thus, years later, another study on bladder cancer was performed in the same area after the coal, iron and steel industries had finally closed in the s. in total bladder cancer patients from the st.-josefs-hospital dortmund-hörde and controls with benign urological diseases were investigated by a questionnaire and genotyped for gstm , gstt and the n-acetyltransferase (nat ) tag snp rs . the frequency of the gstm negative genotype was % in bladder cancer cases and thus much lower, compared to a previous study performed from - in the same area ( %). nat genotypes were distributed equally among cases and controls ( % slow acetylators). less gstt negative genotypes were present in cases ( %; controls %). apoptosis inducing factor (aif) has been identified as a key factor in intrinsic pathways of caspase-independent neuronal death in model systems of acute brain injury and neurodegenerative diseases, such as alzheimer's disease and parkinson's disease. aif is a mitochondrial intermembrane flavoprotein with the capacity to translocate to the nucleus where it induces chromatin condensation and large-scale dna fragmentation. previous studies revealed that aif deficiency leads to protective effects in different models of neuronal death in vitro and in vivo. however, aif also plays an important physiological role for the integrity and function of the mitochondrial respiratory chain. thus, aif deficiency may significantly alter mitochondrial functions and metabolic homeostasis thereby preconditioning the cells to tolerate subsequent stress stimuli. the present study addresses this hypothesis and investigates whether neuroprotection by aif depletion was attributed to a preconditioning effect, i.e. protecting mitochondrial function and integrity. as model system we use glutamate induced oxytosis in immortalized mouse hippocampal ht- neurons. silencing of aif expression by sirna ( nm) protected mitochondrial morphology and integrity against glutamate induced damage. microscopy analysis of the mitochondrial morphology revealed that aif sirna prevented mitochondrial fission. furthermore, facs analysis confirmed that mitochondrial membrane potential was stable in cells with aif silencing. this protection of mitochondrial morphology and integrity by aif depletion was associated with preserved atp levels and inhibition of cell death as detected by an mtt assay. pronounced formation of lipidperoxides as another indicator of mitochondrial damage was also attenuated in cells preconditioned by aif sirna. these protective effects of aif sirna were highly similar to effects obtained with low doses of rotenone ( nm), which was applied as an inhibitor of complex i and mediated comparable preconditioning effects in the ht- cells. overall, these findings support the conclusion that aif depletion mediates a preconditioning effect protecting neuronal cells from a subsequent glutamate toxicity. in order to link these preconditioning effects to complex i functions, protein expression and functional analysis of complex i are being analysed to identify the molecular mechanisms of aif dependent control of neuronal life and death. -dependent inactivation and display very slow voltage-dependent inactivation. both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained ca + influx through cav . channels is required to couple slowly graded changes of the membrane potential with tonic glutamate release. mutations in the gene coding for cav . cause severe impairment of retinal circuitry function and have been linked to congenital stationary night blindness type a (csnb ), aland island eye disease (aied) and cone-rod dystrophy type (cordx ). the clinical phenotypes of these eye diseases vary substantially regarding the ratio of rod to cone functional impairment. the reasons for this variability are not known. to gain more insights into the pathophysiology caused by loss of cav . function we analyzed the visual phenotype of cav . -deficient mice. to this end, we combined immunohistochemistry, electroretinography (erg) and vision-dependent behavioral testing. immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in cav . -deficient mice. heterozygous cav . mice showed mosaic synaptic defects most probably caused by random x-chromosomal inactivation of the healthy allele. electroretinography revealed a loss of scotopic and photopic photoreceptor function. this loss of retinal network function resulted in impaired performance of cav . knockout mice in a water maze-based behavioral test of rod and cone function. in conclusion, loss of cav . channels strongly impairs rod and cone retinal function and vision in mice. lsr is the host cell receptor for clostridial iota-like toxins papatheodorou p., aktories k. albert-ludwigs-universität freiburg institut für experimentelle und klinische pharmakologie und toxikologie, albertstr. , freiburg, germany the human enteric pathogen clostridium difficile is the most serious cause of antibioticassociated diarrhea and pseudomembranous colitis. hypervirulent strains of the pathogen, associated with more severe disease and increased death rates, produce the binary actin-adp-ribosylating toxin cdt (c. difficile transferase) in addition to the rho glucosylating toxins a and b. cdt is member of the family of clostridial iota-like toxins, including c. spiroforme toxin (cst) and the eponym c. perfringens iota toxin. the toxins induce depolymerization of the actin cytoskeleton and the formation of microtubulebased cell protrusions that increase adherence and colonization of clostridia. using a haploid genetic screen, we identify the lipolysis-stimulated lipoprotein receptor (lsr) as the target molecule for entry of cdt into host cells. in addition, we present evidence that lsr is shared as a cell entry point by all members of the iota-like toxin family. identification of the toxin receptor provides a most valuable basis for antitoxin strategies. bisphenol a (bpa) is a chemical of high interest due to its endocrine activity. controversy exists concerning the blood concentration due to normal exposures. some authors claimed to have measured concentrations in the ng/ml range which is in contrast to kinetic properties of bpa. bpa is excreted in the urine as glucuronide and sulfate metabolites. recently, data on the in vitro metabolism of bpa by recombinant udpglucuronyltransferase b enzymes (ugt b ) revealed that ugt b . and ugt b . had markedly lower intrinsic clearance as compared to ugt b . (hanioka et al., ) . using the in vitro metabolism data, we scaled the kmand vmaxvalues in an established human physiologically based toxicokinetic (pbtk) model (mielke and gundert-remy, , mielke et al., ) to the values of the variants. for oral doses at relevant exposure levels, the maximum blood concentration (cmax) for the ugt b . variant (v ) was fold and those of the ugt b . variant (v ) was fold higher than that of the ugt . variant. with dermal exposure at a relevant exposure level, the cmax values were . (v ) and . fold (v ) of ugt . variant. a combined exposure of oral and dermal exposure, an exposure scenario, which occurs in daily life, resulted in . fold (v ) and . fold (v ) higher cmax values as compared to ugt . variant. the values for the area under the blood concentration time curve (auc) were for a relevant oral dose . fold (v ) and . fold (v ), for relevant dermal exposure . fold (v ) and . fold (v ), and for combined exposure . fold (v ) and . fold (v ) of ugt . variant. from the results we conclude: ( ) polymorphism of udpglucuronyltransferase ( b . and b . ) has an impact on the blood concentrations which, however, is less than fold for cmax and for auc. the effect is more pronounced for oral as compared to dermal or combined exposure. ( ) polymorphism of metabolism does not explain the blood/plasma concentrations in the ng/ml range measured by some authors. hanioka n, oka h, nagaoka k, ikushiro s, narimatsu s. effect of udpglucuronosyltransferase b polymorphism on bisphenol a glucuronidation. arch toxicol. , ( ) : - . mielke h, gundert-remy u. bisphenol a levels in blood depend on age and exposure. toxicol lett. ; ( ) : - . mielke h, partosch f, gundert- the heart responds to maladaptive pro-hypertrophic stimuli by stimulating intrinsic signals that contrast and dampen the onset and development of hypertrophy. cyclic guanosine monophosphate (cgmp) and its downstream effector cgmp kinase i (cgki) have been suggested to be an important anti-hypertrophic signaling pathway ( ) . intracellular levels of cgmp can be raised by the action of nitric oxide (no) and natriuretic peptides (anf, bnf), or by inhibiting cgmp-degrading phosphodiesterases (pde). a growing body of evidence suggests that the pde specific inhibitor sildenafil (sil) prevents and reverses hypertrophy and chamber remodelling in the heart of mice subjected to thoracic aorta constriction (tac) by elevating cgmp levels and cgki activation ( ) . in contrast, using a mouse model that lacks cgki expression in every cell type except smooth muscle cells (βres mice; see ref. ), we recently showed that the absence of this kinase does not alter the onset of hypertrophy induced by tac or isoproterenol infusion ( ) . sil is believed to increase cardiac cgmp levels, although it is unclear, if its target (pde ) is expressed in cm ( ). sil may act on other pdes, such as pde c which is abundant in cms. it is also unclear if sil effects are mediated by other cardiac cell types, in particular by cardiofibroblast. to answer these questions, we are currently investigating whether sil is able to prevent hormone induced cardiac hypertrophy in the absence of cgki in cm. preliminary results on βres mice show that even in the case of chronic angii infusion, lack of cgki in cm does not alter the induction of hypertrophic response, at least in the initial phase ( days of angii infusion at mg/kg/day). interestingly, βres mice showed impaired cardiac function, as indicated by decreased fractional shortening. sil was able to partially block the onset of cardiac hypertrophy in wt animals, but not in βres mice, indicating a requirement of cgki in this process. in particular, sil was able to block the transcription of pro-fibrotic genes such as tgfβ, ctgf, collageni and fibronectin. the overexpression of the somatostatin receptors sst and sst in neuroendocrine tumors provides the molecular basis for therapeutic application of the stable somatostatin analogs octreotide and pasireotide. whereas the phosphorylation of the carboxyl-terminal region of the sst receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst receptor. here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines (t ) and (t ), which enabled us to selectively detect either the t -or the t -phosphorylated form of sst . we show that agonist-mediated phosphorylation occurs at t , whereas t is constitutively phosphorylated in the absence of agonist. we further demonstrate that the pan-somatostatin analog pasireotide and the sst -selective ligand l- , but not octreotide or ke were able to promote a clearly detectable t phosphorylation. however, none of these compounds was able to stimulate t phosphorylation and sst internalization to the same extent as the natural somatostatin. agonist-induced t phosphorylation was dose-dependent and selectively mediated by g protein-coupled receptor kinase (grk ). like that observed for the sst receptor, phosphorylation of sst occurred within seconds. however, unlike that seen for the sst receptor, dephosphorylation and recycling of sst were complete within minutes. we also identify protein phosphatase g (pp g) as sst receptor phosphatase. together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal t . in addition, we identify grk -mediated phosphorylation and pp g-mediated dephosphorylation of t as key regulators of rapid internalization and recycling of the human sst receptor. termination of signaling of activated g protein-coupled receptors (gpcrs) is essential for maintenance of cellular homeostasis. although the regulation of agonist-induced phosphorylation has been studied in detail for many gpcrs, the molecular mechanisms and functional consequences of receptor dephosphorylation are far from understood. recent studies have shown that phosphatase inhibitors, such as okadaic acid and calyculin a, can block the dephosphorylation of a number of gpcrs including the ß adrenergic receptor, d dopamine receptor, parathyroid hormone receptor , thromboxane a receptor and the vasopressin receptor . however, a specific phosphatase has not been identified so far. in present studies, we have examined the mechanism and function of receptor dephosphorylation using the sst a somatostatin receptor and the µ-opioid receptor (mor) as models. within those analyses, we have identified protein phosphatase beta (pp ß) as the phosphatase for the cluster of phosphorylated threonines ( ttetqrt ) within the sst a somatostatin receptor carboxylterminus using sirna knock down screeening. those phosphorylation sites mediate ß-arrestin binding. we have also identified protein phosphatase gamma (pp γ) as mor phosphatase that catalyzed t and s dephosphorylation at or near the plasma membrane within minutes after agonist removal. here, we show the different activated phosphatases with functional selective mutants. we examined tailswap mutants which specify the different phosphatase activities. therefore we produced a mor-rsst a chimera with the rsst a c-terminal tail and a rsst a-mor chimera with a mor c-terminal tail. detoxification by conjugation of glutathione? formations of dna adducts of patulin activated by glutathione pfenning c., lehmann l. university wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, wuerzburg, germany as a frequent contaminant in apple juice, the mycotoxin patulin (pat) has shown mutagenic potential in cultured mammalian cells at concentrations which are equivalent to those found in marketable foods. this fact is in contrast with the assumption that conjugation to the major intracellular nucleophile glutathione (gsh) leads to detoxification of the electrophile pat. although pat reacts readily with gsh, previous studies showed that co-incubation of pat with model thiols and amine compounds increased the reactivity of pat towards amines forming mixed-type adducts. thus, we hypothesise that the potential to react with dna bases after being activated by gsh might contribute to the mutagenicity of pat. adduct formation of dna bases (adenine, guanine or cytosine) with pat in the presence and absence of gsh was studied under neutral conditions. liquid chromatography coupled with electrospray ionization tandem mass spectrometry was applied for identification and structure elucidation of putative adducts. besides published as well as hitherto unknown pat-gsh adducts, several pat-dna base adducts were formed both in presence and absence of gsh. in addition, with each of the three dna bases one product exhibiting a mass to charge ratio and fragmentation pattern suggesting a mixed thiol-amine adduct was detected. based on the fragment ions of adducts formed with gsh and chemically modified derivatives, we postulate a cyclic structure of the pat-gsh-dna base adducts, resulting from the reaction of the α-amino group of the glutamic acid residue with the c -carbonyl function of pat. the exocyclic amino group of the dna base is linked to c of the pat backbone by an amid bond. thus, the present study demonstrates the reactivity of pat towards dna bases and the participation of gsh in adduct formation. the postulated structures of dna adducts could be used as biomarkers for the determination of the internal exposure to pat in humans. prescribing information detailed in the summary of product characteristics (spc) forms the officially approved basis for safe prescribing of drugs. in a project funded by the german federal ministry of education and research (bmbf) we aimed to derive an internationally valid data set for safe prescribing of psychiatric drugs and therefore analyzed and compared the content of internationally available prescribing information. a team of pharmacists and clinical pharmacologists performed an in-depth comparison of the german, swiss, british and us-american spcs of top prescribed psychiatric drugs. for drugs (of identical pharmaceutical form) the spcs from the same manufacturer were available for all countries, whereas for three drugs spcs were only available from different companies. in these cases the most recent prescribing information from each country was included in the comparison. in spcs individual data points ( . ± . per individual spc) were compared. between countries the timeliness of prescribing information for an individual drug varied by a median of . (range: - ) months. the respective spcs covered on average . ± . % (range: . - %) of all mentioned indications and . ± . % (range . - %) of all mentioned contraindications. the warnings and precautions section of an individual spc covered on average . ± . % (range: . - . %) of all mentioned warnings and precautions for that drug. the variation observed was only marginally improved when restricting the analysis to the spcs of the drugs available in all four countries from the same manufacturer. across countries, the summary of product characteristics of individual psychiatric drugs show substantial variation in crucial prescribing information. as different manufacturers are unlikely to explain much of the observed variation, these data argue for a better international cooperation and standardization of the content of summary of product characteristics. this project is supported by the german federal ministry of education and research (bmbf), project grant no. ex b. protein kinase ck (former name 'casein kinase ') is a highly conserved serine/threonine kinase, which acts as a component of regulatory networks implicated in many cellular processes but is also linked to various types of human cancer [ , ] . elevated ck activity has been associated with aggressive tumor behavior and results in growth advantage, enhanced survival and dynamic adaption to stress of cancer cells [ ] . ck is a heterotetramer consisting of two catalytic subunits (ck α) attached to a dimer of regulatory subunits (ck β) [ ] . ck β stabilizes ck α against denaturation and modulates the substrate specificity of the catalytic subunit [ ] . due to the relatively small and hydrophobic ck α-ck β interface ( Ų) [ ] , low molecular weight inhibitors are able to interfere with the ck subunit interaction and thus affect the kinase activity [ ] . such inhibitors might exhibit an increased specificity in comparison to those compounds interacting with the conserved atp binding site [ ] . we have developed an elisa-based ck α-ck β binding assay using recombinant human ck subunits. different blocking reagents were analyzed to minimize nonspecific binding. the optimized binding assay was then applied to screen for inhibitors of the ck subunit interaction. primary hits were further characterized by determination of the parameters ic and ki as well as by comparing the results from the binding assay with literature-known or recently obtained crystal structures. numerous epidemiological studies have shown associations between exposure to ultra fine particles and an increase in cardiovascular diseases such as atherosclerosis, coronary heart disease and myocardial infarction. ultra-fine particles have an aerodynamic diameter of < . µm and are highly diverse with impurities of transition metals and organic compounds (e.g. polycyclic aromatic hydrocarbons). posttranslational modification (ptm) of proteins, particularly phosphorylation, is a key element in the regulation of cell function and any disturbance can lead to multiple diseases. the present study focused on the proteomic-based identification of phosphorylated proteins to understand the mechanism behind ultrafine particle exposure and cardiovascular disease development. as one of the major sources for ufp emissions are diesel exhaust, therefore to mimic the diesel particles, carbon black (cb) and benzo[a]pyrene loaded carbon black (cb+) were used in the present study. cells of the endothelial cell line ea.hy were exposed for days to ng/ml cb and cb+. phosphoprotein extraction of whole cell lysates was carried out by the method developed in our lab . the obtained proteins were then separated by two-dimensional gel-electrophoresis followed by maldi-tof-ms (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of differently expressed proteins. to further validate the results invasive potential of cells were monitored by plating exposed cells for hrs on top of matrigel-coated inserts. differential expressions of phosphoproteins were found in cb-treated cells while an altered expression of phosphoproteins was observed in cb+-treated cells. the maldi-tof analysis revealed proteins involved in the regulation of the endothelial permeability and the cellular plasticity such as vimentin, actin and transitional endoplasmic reticulum atpase. further, the invasion assay supported these results as the cb-exposed cells showed a high invasive potential as compared to control. [ ] pink m. et. al. , precipitation by lanthanum ions: a straightforward approach to isolate phosphoproteins, j.protomics, , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] application of the ttc approach in cosmetics platzek t. bundesinstitut für risikobewertung, max-dohrn-straße - , berlin, germany regulatory toxicologists in europe have been discussing the ttc approach since more than a decade, e.g. the previous scientific committee on food in . since then, the concept was further developed and is now applied in the eu for the assessment of flavouring substances by efsa. two committees are discussing possible applications: the efsa scientific committee prepared an opinion exploring options for the application in food and feed, e.g. for impurities of food additives, thermal reaction products, food contact materials, contaminants etc. in addition, an eu non-food expert committee consisting of members of sccs, scher and schenir discussed the ttc concept in general as well as additional possible fields of application with the focus on cosmetics and an opinion was already published for public consultation. the proposal to apply the ttc approach also for cosmetic ingredients was introduced by a paper published in by a colipa (the european cosmetics association) supported working group. major aspects to be considered are the following: . applicability domain. the chemical space of the ttc dataset (> compounds) has to be compared with that of cosmetic ingredients (> compounds). route to route extrapolation. since no adequate dermal toxicity database is available both data on oral intake used in the ttc approach and on dermal exposure to cosmetic ingredients have to be transfomed to internal exposure figures. gastrointestinal and dermal bioavailability as well as route specific differences in metabolism have to be integrated. exposure. the reliability of exposure estimation is the second pillar of the ttc approach. compared to food, data on exposure to substances in cosmetics and consumer products is scarce. a pragmatic step forward is the comparison of ttcs and noael-derived safe exposure levels for cosmetic ingredients. this work was already done with substances in food contact materials and chemicals from the elincs list. for cosmetic ingredients a similar european project is ongoing. further refinement of the ttc approach is needed taking into account the up-to-date toxicological knowledge. with cosmetics specific problems may arise in praxi: according to the new eu cosmetic legislation the safety of cosmetic products available on the market has to be assessed by the manufacturer or importer and also assessors with limited toxicological experience may apply the ttc approach, e.g. by running the toxtree software. plöttner s., marczynski b., käfferlein h. u., welge p., groth h., engelhardt b., schmitz k., erkes a., brüning t. institut für prävention und arbeitsmedizin der deutschen gesetzlichen unfallversicherung -institut der ruhr-universität bochum (ipa) toxikologie, bürkle-dela-camp-platz , bochum, germany polycyclic aromatic hydrocarbons (pahs) comprise several hundred compounds with different carcinogenic potentials, typically occurring in complex mixtures. due to a lack of data risk estimations for pah mixtures are usually based on those for benzo[a]pyrene (b[a]p). the aim of our present study was to explore the suitability of a permanent human lung cell line as tool for future studies on genotoxicity of pah mixtures. in this pilot study we investigated the time-and concentration-dependent generation of specific anti-benzo[a]pyrene - , -diol- , -epoxide (anti-bpde)-dna adducts as well as cytochrome p (cyp) a / b enzyme activities after b[a]p-incubation in vitro. we used metabolically competent a lung carcinoma cells which display several characteristics of alveolar epithelial type ii cells. after h and h incubations with different b[a]p-concentrations cytotoxic effects were assessed with the neutral red assay and cyp a / b activities using luminescent tests. the formation of specific anti-bpde-dna adducts was determined by hplc with fluorescence detection. a time-and concentration-dependent formation of anti-bpde-dna adducts was observed with maximum rates of . ± . and . ± . anti-bpde/ nucleotides after incubation with µm ( h) and µm b[a]p ( h), respectively. however, the mean adduct rates decreased at higher b[a]p-concentrations. the reduction was more pronounced after h than after h. increased cyp a / b activities were observed at > . - µm ( h) and > . - µm ( h). a clear decrease of enzyme activities was observed at higher concentrations for both incubation times. in the neutral red assay no more than % cytotoxicity in relation to the negative control were found after h incubation with ≥ µm b[a]p and after h with ≥ µm b[a]p. overall, incubation of a cells with b[a]p resulted in a time-and concentration-dependent increase of cyp-activities and anti-bpde-dna adducts. this clearly shows that a cells are able to generate mutagenic dna-adducts. thus, the in vitro model used in the present work appears suitable for genotoxicity studies of individual pahs and pah mixtures, and therefore may be a useful tool for research on syncarcinogenesis. the β subunit of the cav . channel complex has been shown to play a key role in cav . channel trafficking and channel characteristics like opening probability. furthermore, the last exon of the cacnb gene coding for cavβ , exon , contains several potential phosphorylation sites, e.g. for protein kinase a or ca + /calmodulin dependent camkii. pka-dependent phosphorylation mediates β-adrenergic stimulation of cav . . potential phosphorylation sites are ser and ser (in the β subunit in rat, perez-reyes et al. a ) and ser in the c-terminus of the poreforming α c subunit (lemke et al. b ) . in cardiomyocytes camkii regulates ca + release and reuptake from and into the sr and is involved in the facilitation of the calcium channel. potential interaction sites between the cav . channel complex and camkii are thr in the β subunit (in rat, grueter et al. c ) and ser and ser in the cterminus of the α c subunit (blaich et al. d ) . however, the exact pathways remained widely unclear up to now. to clarify these mechanisms and to identify the relevant phosphorylation sites for pka and camkii we established a mouse line carrying a stop codon in exon after aa pro . this mutation prevented translation of the cavβ c-terminus containing the corresponding potential phosphorylation sites mentioned above (cavβ stop mouse). these mice were viable, showed unaltered expression of the truncated of cavβ protein and unchanged ecg and echocardiography. electophysiological analysis of isolated cardiomyocytes showed no differences in current density, the effect of isoproterenol, the time course of inactivation and the facilitation property when compared to cells isolated from littermate controls. for further investigations we bred the cavβ stop mice with s a mice (lemke et al. b ) lacking the pka phosphorylation site s in the α c subunit (s aβ stop mouse) or with sf mice (blaich et al. d ) lacking the camkii phosphorylation sites s and s in the α c c-terminus (sfβ stop). both mouse lines were viable and showed unchanged echocardiography recordings compared to their control littermates and unaltered ecg for s aβ stop mice. electrophysiological investigations on cardiomyocytes of s aβ stop mice showed unchanged β-adrenergic stimulation with isoproterenol compared to littermate controls. these results suggest that β adrenergic regulation of the cardiac cav . channel is not mediated by these phosphorylation sites. introduction. chronic atrial fibrillation (caf) is marked by increased fibrosis which contributes to the perpetuation of the disease. in addition to the role of fibrosis in structural remodeling of cardiac tissue, fibroblasts can couple with cardiomyocytes via gap junction thereby altering the electrophysiological properties of the later and potentially participating in atrial electrical dysfunction. in order to understand the importance of fibroblasts in the pathophysiology of caf, we compared the electrical properties of atrial fibroblasts isolated from patients in sinus rhythm (sr) and caf. methods. fibroblasts were isolated by outgrowth culture from right atrial biopsies and cultivated in medium containing % fetal calf serum. we used whole-cell patch clamp techniques to investigate ion currents and membrane potential. results. sr and caf fibroblasts showed similar capacitance (sr: . ± . pf, n= ; caf: . ± . pf, n = ) and membrane potential (sr: - . ± . mv, n = ; caf: - . ± . mv, n = ) . in both groups, we observed fast activating outward currents with a mean threshold at - mv. interestingly, current amplitude was significantly larger in sr than caf cells (sr: . ± . pa/pf, n = ; caf: . ± . , n = ; p < . ). when maintained in culture for - weeks, cells from both groups developed na + currents. surprisingly, the fraction of caf cells displaying such currents was larger than the sr counterpart (caf: %; sr: %). furthermore, na + current amplitude was significantly larger in caf fibroblasts (sr: . ± . pa/pf, n = ; caf: . ± . pa/pf, n = ; p < . ). na + currents were not altered by nm tetrodotoxin (ttx), but µm ttx reduced current amplitude to % of control, suggesting that the channel involved is the cardiac ttx-resistant isoform nav . . conclusion. in the context of caf, fibroblasts undergo electrophysiological changes which need to be thoroughly described. understanding whether those changes contribute to the af substrate might provide new therapeutic targets for the treatment of caf. the initial recruitment of gi to the alpha a-adrenergic receptor is affected by gprotein-coupled receptor kinases and arrestins prokopets o. s., krasel c., marburg, germany most g-protein-coupled receptors undergo homologous desensitization after agonist stimulation. in this process, agonist-activated receptors are phosphorylated by gprotein-coupled receptor kinases (grks), followed by binding of arrestins to the still agonist-occupied, phosphorylated receptors. it is assumed that arrestin competes with heterotrimeric g-proteins for the receptor molecule and thereby causes desensitization of g-protein-mediated responses. we tested this idea by investigating the effect of grks and arrestins on the recruitment of g-proteins by the alpha a-adrenergic receptor. hek t cells were transfected with yfp-tagged alpha a-adrenergic receptor and the three subunits of gi . the g(beta) subunit was cfp-tagged. upon stimulation with noradrenaline, a very rapid, robust increase in fret between the receptor and the g(beta) subunit was observed, confirming previous observations that the alpha aadrenergic receptor recruits gi with a half-life of around milliseconds. when arrestin and grk were also co-transfected, the half-life of gi recruitment was substantially delayed, increasing to around seconds. there seemed to be no effect of grk +arrestin cotransfection on a second stimulation; neither the kinetics nor the extent were altered compared to the first stimulation. interestingly, grk alone seemed to cause a similar but slightly less pronounced delay of g(beta) recruitment to the alpha a-adrenergic receptor. in corresponding experiments, we measured the recruitment of arrestin -cfp to yfp-tagged alpha a-adrenergic receptors in the presence of grk . upon stimulation with noradrenaline, we observed a robust increase in fret between the receptor and arrestin , confirming previous observations that the alpha a-adrenergic receptor recruits arrestin . co-transfection of the three subunits of gi had no effect on the kinetics of arrestin binding to the alpha a-adrenergic receptors. based on previous results with other receptors, an attenuation of gi recruiting to the alpha a-adrenergic receptor is quite unexpected. our data suggest that the relation between receptors, g-proteins, grks and arrestins may be more complex than previously postulated. introduction: human urinary bladder expresses mrna of the three known badrenoceptor (b-ar) subtypes (b , b , b ) in detrusor and urothelium. we have shown previously that only b -ar is involved in human detrusor relaxation. to investigate the urothelium-induced modulation of b-ar-mediated relaxation, we have examined systematically whether other b-ar subtypes are involved. human detrusor tissue samples were obtained from patients undergoing radical cystectomy for the treatment of bladder cancer. detrusor strips were studied with and without an intact mucosa layer. muscle strips were precontracted with µm carbachol and relaxation was studied in response to the b-ar agonist ne. a-ar mediated processes were blocked with the a-ar antagonists phentolamine and prazosin. selective b-ars antagonists were used to investigate b-ar mediated relaxation. at the end of each experiment µm forskolin was used to determine maximum camp-mediated relaxation. the presence of intact urothelium reduces potency but not effectivity of ne indicating involvement of urothelium-mediated processes not only during detrusor contraction but also during relaxation. ne-mediated detrusor relaxation is mediated through b -ar in the absence of urothelium. but in intact detrusor strips b -ars seem to have an additional inhibitory effect. affinity of the selective b -ar antagonist l , is unchanged, therefore the intact urothelium does not interact with the function of b -ars. aldosterone causes oxidative stress and dna damage independent of blood pressure in vivo queisser n., schupp n. universität würzburg institut für toxikologie, versbacherstr. , würzburg, germany background: an inappropriate increase of the mineralocorticoid aldosterone (ald) can be induced by a stimulated renin-angiotensin-aldosterone system. epidemiological studies exploring the connection between hypertension and cancer found higher cancer mortality and an increased risk to develop kidney cancer in hypertensive individuals. we recently showed that ald produces oxidative stress, activates transcription factor nf-kb and is genotoxic in kidney tubule cells. objectives: this study investigated the capacity of ald to induce oxidative/nitrosative stress, dna damage, dna repair, apoptosis, cell proliferation and the activation of nf-κb in rat kidneys. methods: mineralocorticoid-dependent hypertension was induced by ald/salt in sprague dawley rats. dna damage and oxidative/nitrosative stress markers were detected immunohistochemically. results: ald/salt treatment caused increased blood pressure compared to untreated rats. tempol, an antioxidant, and hydralazine, a vasodilator acting independent of the renin-angiotensin-aldosterone system, could lower the blood pressure, while the mineralocorticoid receptor (mr) antagonist spironolactone was administered in a subtherapeutical dose not lowering the blood pressure. ald/salt treatment caused oxidative and nitrosative stress, structural dna damage, double strand breaks, dna repair and nf-κb activation. spironolactone decreased these markers significantly. tempol was also able to reduce these markers, while hydralazine had no effect. ald/salttreated kidneys showed a tendency to lower apoptosis and to increased cell proliferation compared to control rat kidneys. discussion: this study provides a first hint of blood pressure-independent effects of ald. the mr and the production of ros seem to be crucial for the damaging effects of ald. an aberrant or long-term activation of nf-κb by persistently high ald levels could support resistance to apoptosis and the survival of cells with damaged dna, and increase cell proliferation. these actions could contribute to the increased cancer incidence in hypertension by initiating carcinogenesis. grant support by the dfg is gratefully acknowledged. creb regulating transcriptional coactivator (crtc ) is a transcriptional coactivator of the transcription factor creb. we have recently shown its expression in cardiomyocytes and its activation by beta-adrenergic signaling. beta-adrenergic signaling contributes to the pathogenesis of cardiac hypertrophy, leading to heart failure, as evidenced by the therapeutic success of the beta-adrenoceptor antagonists. in order to investigate if crtc is involved in this process, we investigated the expression of crtc in hypertrophied myocardium from mice and humans. methods: protein lysates from mouse and human samples were investigated for crtc protein expression. we distinguished between an acquired and an inherited form of cardiac hypertrophy. acquired cardiac hypertrophy is an adaptation of the heart to an increased cardiac workload and can be found in patients with an aortic valve stenosis. in mice this kind of hypertrophy can be evoked by transverse aortic constriction (tac). the inherited form of cardiac hypertrophy is caused by mutations in genes coding for proteins of the sarcomeric apparatus and is referred to hypertrophic cardiomyopathy (hcm). as a model for hcm, transgenic mice with a mutation in the mybpc gene, coding for the sarcomeric protein cardiac myosin-binding protein c (cmybp-c), were used. these transgenic mice were characterized by a hypertrophied heart. in case of human samples we distinguished between patients with an acquired form of hypertrophy due to an aortic valve stenosis, and patients with a hypertrophic obstructive cardiomyopathy (hocm), a special form of hcm. results: in the tac mice and in the myppc mutant mice the expression of crtc was significantly higher than in the controls ( . -and . -fold, respectively; n= ). in both forms of human hypertrophy, we found a significantly -fold upregulation of crtc protein level in comparison to human samples from non-failing myocardium or samples from patients with a dilatative or ischemic cardiomyopathy (n= - for each group). conclusions: crtc is upregulated in cardiac hypertrophy with acquired or geneticbased origin. the evaluation of the role of crtc in the heart may help to elucidate the role of beta-adrenergic signaling in the development of cardiac hypertrophy. microarray gene expression profiling reveals up-regulation of the cardiac lipid metabolic process at the onset of heart failure fu x., abd alla j., quitterer u. eth zürich molekulare pharmakologie, winterthurerstrasse , zürich, switzerland atherosclerosis and chronic pressure overload are major cardiovascular risk factors for the development of heart failure in patients. to mimic those risk factors in experimental models we used atherosclerosis-prone apolipoprotein e (apoe)-deficient mice, and chronic pressure overload was imposed by abdominal aortic constriction (aac). cardiac function was monitored by echocardiography. severe atherosclerosis in aged apoedeficient mice or chronic pressure overload induced signs of heart failure as evidenced by a significantly reduced cardiac ejection fraction (< %). to investigate pathomechanisms underlying the development of heart failure, microarray gene expression analysis and qt-rt-pcr were performed of failing heart tissue relative to age-matched controls. gene ontology analysis of the microarray data revealed that the onset of heart failure, in two different experimental models, was characterized by a strong up-regulation (≥ -fold) of the cardiac lipid metabolic process and lipid overload. lipid metabolism genes were involved in lipid synthesis, storage and oxidation. the major palmitate-synthesizing enzyme, fatty acid synthase, was causally related to the development of cardiac dysfunction by enhancing cardiomyocyte apoptosis. taken together the data support that the onset of experimental heart failure is characterized by a dysfunction of the cardiac lipid metabolism promoting cardiomyocyte death. at -receptor blockers (arbs) are established for the treatment of high blood pressure and new onset of diabetes is reduced by arbs. in the past years evidence increased that body weight may also be lessened particularly in rats and mice. however, less data are available whether arbs still reduce weight, when treatment was initiated not until animals became obese by diet. prior to drug treatment, spontaneously hypertensive rats were fed for months with chow but also with a cafeteria diet (cd) to develop obesity. controls received only chow (conchow). cd-fed shr were treated for months with telmisartan (tel mg/kg/d) or amlodipine (aml, mg/kg/d), whereas controls received vehicle (concd). systolic blood pressure (sbp), feeding behaviour, body weight, abdominal fat mass (by mrt) and energy expenditure (by indirect calorimetry) was monitored. leptin sensitivity was assessed by measuring energy intake and expenditure after repetitive injections (s.c.) of leptin. insulin sensitivity was functionally determined by glucose and insulin tolerance tests. due to cd feeding body weight was increased after months by more than g. tel normalized sbp whereas it remained > mmhg in concd and conchow. tel additionally reduced cd-induced increase of body weight and abdominal fat mass. food intake was diminished during the first weeks, but raised beyond control levels during the last weeks of treatment. the shift of the respiratory index to lower levels indicated improved energy expenditure. in response to exogenous leptin, the food intake of concd was higher compared to conchow, indicating a leptin resistance. this assumption is further supported by high triglyceride concentrations of concd. after tel, leptininduced food intake was reduced and energy expenditure was increased compared to concd, indicating that leptin sensitivity was at least partially restored. accordingly, triglycerides were reduced. compared to concd, the insulin sensitivity was improved by tel since maximal increases in plasma concentrations of glucose and insulin in response to glucose challenge were reduced, but glucose response to insulin challenge was diminished. even though reduction in blood pressure was almost similar between tel and aml, metabolic and antiobese efficacies of aml were markedly attenuated. we conclude that telmisartan reveals wide efficacies in improving all symptoms of the metabolic syndrome. the pleiotropic effects are not related to the hypotensive action of tel. a β -adrenoceptor -camp mediated, immediate stimulation of β -adrenoceptor gene expression in human lung fibroblasts is opposed by a delayed up-regulation of inhibitory factors racké k., lamyel f., kämpfer n., schütz i., warnken m. univ. bonn dept. pharmacol. &toxicol., bonn, germany based on their bronchodilatory effects, β -adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and copd. however, treatment with long-acting β -adrenoceptor agonists has been associated with possible worsening of airway hyper-reactivity, possibly because of loss of β-adrenoceptor function. therefore, the molecular regulation of β -adrenoceptor expression was addressed here. mrc- human lung fibroblasts were cultured for up to h in absence or presence of test substances, followed by β -adrenoceptor mrna determination by qpcr. β -adrenoceptor mrna decreased with a half-life of min after inhibition of mrna synthesis with actinomycin d ( µm), but increased by ± %, ± % and ± % (means±sem) within . , and . h, resp. after inhibition of protein synthesis by cycloheximide ( µm). the β -adrenoceptor agonists formoterol and olodaterol ( - nm) induced a rapid increase in β -adrenoceptor mrna (maximally within h by ± % and ± % at nm, resp.). however, after h exposure to nm formoterol or olodaterol a reduction in β -adrenoceptor mrna by ± % and ± %, resp., was observed. both, the stimulatory and inhibitory effects of β adrenoceptor agonists were mimicked by forskolin ( µm, increase by ± % and inhibition by ± %) and cholera toxin ( ng/ml, increase by ± % and inhibition by ± %). the formoterol-induced up-regulation of β -adrenoceptor mrna was blocked by actinomycin d, but not by cycloheximide. moreover, in presence of cycloheximide, β adrenoceptor agonist induced inhibition was converted into a marked stimulation. in conclusion, expression of β -adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. the observations with cycloheximide indicate that the β -adrenoceptor gene is under strong inhibitory control of short-living, not yet identified suppressors. although both, the time-dependent up-and down-regulation of the β adrenoceptor gene expression by β -adrenoceptor activation appears to be mediated via adenylyl cyclase -camp signalling, only the stimulatory effect appears to be a direct action on the β -adrenoceptor gene. et- appears to be involved in the pathogenesis not only of pulmonary hypertension, but also in fibrotic remodeling associated with chronic obstructive airway diseases. since human lung fibroblasts (hlf) are a source of et- and have been shown to be controlled by muscarinic receptors and β-adrenoceptors, a possible muscarinic and βadrenergic modulation of et- expression in hlf was explored. mrc- hlf were cultured for up to h in absence or presence of test substances, followed by prepro-et- (ppet- ) mrna determination by qpcr. the muscarinic agonist oxotremorine ( µm) induced an increase in ppet- mrna by %, an effect prevented by nm tiotropium. the β -adrenoceptor agonist olodaterol (up to nm) caused a reduction of ppet- mrna expression by %. the effect of nm olodaterol was prevented by ici , ( µm), but not affect by cgp ( µm) . the pka agonist -bnz-camp ( µm) caused a reduction in ppet- mrna expression by %, whereas the epac agonist -cpt- '-o-me-camp ( µm) caused only a marginal inhibition by %. olodaterol ( nm) strongly opposed the stimulatory effect of µm oxotremorine. an increase in ppet- mrna expression by % caused by . ng/ml tgf-β was effectively opposed by and nm olodaterol, resulting in an inhibition comparable to that in absence of tgf-β. however, the increase in ppet- mrna caused by a maximally effective concentration of tgf-β ( ng/ml, increase by %) was not significantly affected by or nm olodaterol. likewise, the pkaagonist -bnz-camp ( µm) opposed the increase in ppet- mrna expression caused by . ng/ml tgf-β, but not that caused by ng/ml tgf-β. tgf-β caused, with an ic of . ng/ml, a marked down-regulation of β -adrenoceptor mrna expression, maximally by % within h. et- expression in hlf is stimulated by muscarinic receptors and inhibited by β adrenoceptors. the effect of β -adrenoceptors may be mediated via pka. et- expression in hlf is markedly up-regulated by tgf-β, but only effects of sub-maximally effective concentrations of tgf-β are opposed by the β -adrenoceptor -pka pathway, in part because of tgf-β-induced down-regulation of β -adrenoceptors. since et- can promote pro-fibrotic features in hlf, inhibition of et- expression could contribute to long-term beneficial effects of long-acting β -adrenoceptor agonists such as olodaterol and long-acting muscarinic antagonists such as tiotropium. cardiac hypertrophy leads to up-regulation of dipeptidyl aminopeptidase-like proteins in human and rat radicke s., hutschenreuther a., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstr. cardiac hypertrophy is a major risk factor for heart failure and associated morbidity and mortality. functional down-regulation of k + currents is a prominent feature of cells isolated from failing ventricles. a marked decrease in the transient outward potassium current ito has been shown in various models. changes in the k + channel expression differ depending on the species, and the mechanism of induction of heart failure. to study the regulation of ito channel subunits we compared the hypertrophic responses in human ventricular tissues from failing hearts with doxorubicin-induced hypertrophy in rats and in h c embryonic rat cardiac cells. specifically, we quantified mrna expression of the pore-forming subunits kv . and kv . , the cytosolic β-subunit kchip and the transmembrane subunits dpp and dpp using rt-pcr. treatment with doxorubicin ( µm) induced hypertrophy and increased the mrna expression of the hypertrophy marker genes anf, bnp and beta-mhc in h c cardiac myoblasts. while kv . was detected in h c cells and hearts from human and rat, kv . mrna was only expressed in adult rats. during hypertrophy kv . was downregulated in human tissue as well as in doxorubicin-treated h c cells compared to the controls. in rat hearts kv . expression was increased after doxorubicin treatment. interestingly, kv . was also found to be up-regulated in rat heart tissues and h c cells after treatment with doxorubicin. as previously shown, kchip mrna expression was significantly reduced in tissues of failing hearts. in contrast, kchip mrna was upregulated in hypertrophic rat hearts and h c cells. the expression of dpp and dpp was observed only in human hearts. but mrna levels of both were significantly increased in failing tissues. dpp and dpp were not expressed in the adult rat heart or h c cells, whereas in rats with doxorubicin-induced cardiac hypertrophy and in doxorubicin-treated h c cells, the mrna of dpp and dpp was up-regulated. h c cells showed almost identical hypertrophic responses to those observed in human ventricles and rat hearts. this finding validates h c cells as a model for in vitro studies of cardiac hypertrophy. in further studies we will investigate the consequences of a knock-down of dpp and dpp in doxorubicin-induced hypertrophic h c cells. in preliminary experiments specific short-hairpin rna, targeting dpp and dpp , has been designed and tested in heterologous expression systems. the nonsynonymous c. t>c germline genetic variation in the liver-specific organic anion transporter slco b is associated with methotrexate pharmacokinetics in pediatric acute lymphoblastic leukemia radtke s. background: methotrexate (mtx) plasma concentration is related to its clinical effect. transport proteins, such as abcc , slco a , and slco b , have been implicated in the disposition of mtx. here we investigated whether common reduced-function variants in abcc , slco a , and slco b contribute to the interindividual variability in methotrexate pharmacokinetics in children with acute lymphoblastic leukemia (all). we analyzed mtx pharmacokinetics (mtx plasma concentration at the end of infusion c h, mtx auc - h, and mtx clearance cl) in an unselected population of children with all from the all-bfm trial (clinicaltrials.gov: nct ) who received courses of mtx at g/m as h infusions. the contribution of genes (genetic component, rgc) to the interindividual variability in mtx pharmacokinetics was estimated according to the method of kalow et al. ( ) . abcc c.- c>t (rs ), slco a c. g>a (p.his arg, rs ), slco b c. t>c (p.val ala, rs ) and slco b a>g (p.asn asp, rs ) genotypes were analyzed by taqman polymerase chain reaction. there was substantial interpatient variability in average (± sd) mtx c h ( . ± . µmol/l), auc - h ( . ± . h*mg/l), and cl ( . ± . ml/min/m²). the rgc values of c h, auc - h, and cl ranged from . - . suggesting that variation in mtx pharmacokinetics has a substantial genetic component. after adjustment for age and sex by multiple regression, the slco b c. t>c snp was significantly associated with c h (p< . ), auc - h (p< . ), and cl (p= . ) of mtx. compared with the wildtype genotype, in patients with the tc genotype c h and auc - h increased by % (p= . ) and % (p= . ), respectively, whereas cl significantly decreased by % (p= . ). pharmacokinetic variables significantly changed with increasing number of variant c. t>c alleles (p< . , jonckheere-terpstra), suggesting a per allele effect consistent with a co-dominant model of association. in contrast, the abcc c.- c>t, slco a c. g>a, and slco b a>g polymorphisms did not show an association with mtx pharmacokinetics. conclusions: the nonsynonymous c. t>c polymorphism in slco b contributes to the variability of mtx pharmacokinetics in this study of high-dose mtx in pediatric all. this project is supported by the johannes und frieda marohn-stiftung. the antitumorigenic mechanism of the selective cyclooxygenase- (cox- ) inhibitor celecoxib is still a matter of debate. using human lung cancer cell lines (a , h , h ), the present study investigates the contribution of cox- and peroxisome proliferator activated receptor γ (pparγ) to apoptosis elicited by celecoxib. celecoxib was found to cause apoptotic cell death in a concentration-dependent manner ( - µm), whereas structurally-related cox- inhibitors (etoricoxib, rofecoxib, valdecoxib) were inactive in this respect. apoptotic cell death by celecoxib was suppressed by preincubation of tumor cells with the selective cox- inhibitor ns- , the pparγ antagonist gw- and by sirna targeting cox- or pparγ. celecoxib-induced apoptosis was paralleled by a time-and concentration-dependent upregulation of cox- and pparγ at both mrna and protein level. using an established cox- activity assay monitoring immediate conversion of exogenously added arachidonic acid to the respective prostaglandins (pgs), ns- was shown to suppress celecoxib-induced cox- activity when added prior to arachidonic acid. among the cox- -dependent pgs analyzed, pgd and its dehydration product -deoxy-∆ , -pgj were found to induce cytosol-to-nucleus translocation of pparγ as well as pparγ-dependent apoptosis. celecoxib-elicited translocation of pparγ was inhibited by preincubation of cells with ns- which itself did not alter celecoxib-induced total pparγ protein expression. finally, a cox- -and pparγ-dependent proapoptotic mechanism of celecoxib was confirmed in primary tumor cells obtained from brain metastases of two lung cancer patients. together, our data demonstrate a proapoptotic mechanism of celecoxib involving initial upregulation of cox- and pparγ and a subsequent nuclear translocation of pparγ by cox- -dependent pgs. uncoagulated ppp contained - nm thrombin throughout. fxa was initially measured in clots at nm. this level declined over time while clot-conditioned pbs accumulated fxa. exposure of human aortic smc to clots or native unclotted ppp for h only marginally influenced smc apoptosis but increased mitogenesis over -fold. this was reduced by all inhibitors. clot-stimulated induction of tnfα and interleukin- mrna was also attenuated by the inhibitors. denatured ppp (no protease activity) increased smc mitogenesis to a level seen in smc exposed to clot and combined hirudin + dx a, reflecting the well-known mitogenic actions of serum alone. conclusion: coagulation of human plasma generates nanomolar amounts of thrombin and fxa, sufficient to stimulate the proliferative and inflammatory properties of adjacent smc. our observations validate the use of purified thrombin and fxa at nanomolar concentrations for in vitro studies, and support the individual and coagulationindependent roles of these proteases in cell proliferation and inflammation. antithrombotic therapy with argatroban or rivaroxaban may limit the cellular effects of clot-derived thrombin and fxa, while normal anti-platelet therapy would not. this aspect should be considered in the clinical use of these agents, specifically in healing processes after vessel injury. rhogef mediates cgmp/cgk induced adherence and relaxation of vascular smooth muscle cells rauch j. , stephan-schnatz k. the guanine nucleotide exchange factor rhogef is the only gef known so far to be directly activated by cgmp-dependent kinase. it is expressed in various types of smooth muscle cells and has been shown to play a role in the regulation of cell integrity. in a previous investigation we showed that the knockdown of rhogef by a shrna approach caused a loss of actin stress fibers and a subsequent change of smooth muscle cell morphology that finally resulted in cell rounding. we now provide evidence that the expression level of rhogef influences the re-attachment of cultured rat aortic smooth muscle cells (rasmc) to a surface after detachment. although rhogef depleted rasmc were still able to adhere and spread, their cell surface area remained considerably smaller than that of control cells in the first hours after seeding. cell counting revealed that to hours after seeding the percentage of adherent cells was significantly lower in the rhogef knockdown group compared to the control group. these data indicate a delay in attachment. interestingly, the knockdown of rhogef was paralleled by a loss in rhoa and cadherine expression. as rhogef mediated a cgmp-induced activation of the small gtpase rhoa in rasmc, we studied the effect of a stable cgmp analogon ( -pcpt-cgmp) on the adhesion process. in accordance with previously published data, cgmp treatment accelerated the attachment of rasmc to the surface within the first hours. in contrast, the adhesion of rasmc after rhogef knockdown was no longer stimulated by cgmp. as these data indicate that rhogef mediates cgmp-dependent signalling in a physiological process we wondered whether this protein might also play a role in the regulation of cgmpdependent relaxation of vascular smooth muscle cells. thus, we performed a collagenbased contraction assay with rhogef depleted rasmc. while the contraction in response to serum was not affected by the depletion of rhogef , we observed a slight decrease in basal contractility. interestingly, cgmp was not able to counteract the serum-driven contraction of rhogef knockdown cells. there was no cgmp-induced relaxation in these cells. we conclude that rhogef is involved in the cell adhesion of vascular smooth muscle cells and likely promotes the expression of specific proteins. its activation is required to mediate cgmp-induced signalling in terms of vascular smooth muscle cell adherence and relaxation. human eosinophil and neutrophil granulocytes are cells of the innate immune system. they both express formyl peptide receptors (fpr) und histamine h receptors (h r). activation of fpr leads to a release of reactive oxygen species (ros). h r activation results in an increase of intracellular '- '-cyclic adenosine monophosphate (camp) concentration and inhibition of fpr-mediated ros release via adenylyl cyclase activation. in this study we compared the effects of various h r ligands on camp accumulation and formyl peptide n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced ros release in isolated eosinophils and neutrophils. camp concentration was determined by hplc/tandem mass spectrometry, and ros release was assessed by monitoring superoxide dismutase-inhibitable reduction of ferricytochrome c. in eosinophils, histamine, amthamine and -methylhistamine exhibited similar potencies and efficacies with regard to camp accumulation and inhibtion of ros release. in marked contrast, in human neutrophils, we observed dissociations in potencies and efficacies of ligands at increasing camp accumulation and inhibition of ros production. our data suggest that in human eosinophils, but not neutrophils, camp mediates inhibtion of ros production. in a broader context our data provide a compelling example of the context-dependency of the pharmacological properties of g-proteincoupled-receptors. specifically, one has to be cautious when extrapolating experimental observations from one cell type to another, even when they are very closely related to each other. comonomers and monomers are used as dental restorative materials (e.g. in dental composites). unconverted compounds can be released from dental composites and can enter the body in humans. comonomers can induce various side effects in humans. this study was evaluated to qualify and to quantify eluted compounds from various dental composites. following composites were tested (producer in parentheses): els extra low shrinkage (saremco), synergy duo shade (coltène), grandio (voco), tetric evo ceram (vivadent), venus (kulzer), gradia (g.c.), and premise (kerr).polymerized composites ( mg) were incubated in gc vials with ml dest. water or ml methanol, each at °c for hours. aliquots were taken, and eluted compounds were analyzed with the method of gas chromatography/mass spectrometry (gc-ms) and liquid chromatography/mass spectrometry (lc-ms).from all composites different compounds were found. following comonomers were quantified (µg/ml; mean ± s.d.; n= )( fig. ).following range of the eluted and detected comonomers from dental composites was found (dest. water; decreasing elution): venus > gradia > synergy duo shade > tetric evo ceram premise > grandio > els extra low shrinkage. * n.d. = not detectable (below limit of detection). triphenylstibane was detected in tetric evo ceram ( ± µg/ml). reimann c., lupp a., schulz s. institut für pharmakologie und toxikologie universitätsklinikum jena, drackendorfer str. objective: cxcr is a plasma membrane chemokine receptor which is involved e.g. in organogenesis, hematopoesis and inflammation. in the adult organism cxcr is physiologically expressed on various cell types, in particular on lymphocytes. with respect to neoplastic tissues, in the current literature an over-expression of cxcr is described in different types of tumors, especially in breast and prostate cancer. additionally, an involvement of cxcr in tumor metastasis is discussed. subsequently, detection of cxcr expression in a given human tumor sample would provide a valuable predictive information on disease prognosis and possible therapeutic intervention. however, previous attempts to localize cxcr using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have yielded predominant nuclear and occasional cytoplasmic staining, but did not result in the identification of cell surface receptors. thus, the aim of the present study was to reassess the cxcr expression in a panel of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples by means of immunohistochemistry using the well characterized novel rabbit monoclonal anti-cxcr antibody umb- . methods: in comparison to negative and positive control samples (cxcr -knockout and wild-type mouse embryos) the extent of staining in the different normal and neoplastic tissue specimens was scored from zero (no expression) to three (high expression). results: cxcr was found to be expressed in all neoplastic tissue entities analyzed. in many cases, the receptor was predominantly localized at the plasma membrane of the tumor cells. however, in all cxcr -expressing tumor entities a huge interindividual variability both in the percentage of positive cells and in the intensity of staining was noted which strongly differed also between the various types of cancer. the most intense (score three) staining was found in the samples of (small cell) lung cancer, ovarian cancer and of pheochromocytoma. additionally, lymphatic organs such as lymph nodes, spleens and tonsils were cxcr positive, with mainly b cells displaying a distinct staining of the plasma membrane. the rabbit monoclonal antibody umb- may prove to be of great value in the assessment of cxcr expression in different human tumor entities and of the mechanisms underlying the formation of metastases, thus helping to find new targets and strategies in cancer therapy. link between β -adrenoceptor-mediated inhibition of formyl-peptide-induced o β -adrenoceptor (adrb )-agonists are in daily use for asthma therapy. although most cases of asthma are controlled by standard medication, a subpopulation of asthmatics remains difficult to treat. the adrb gene contains a total of polymorphisms. this variability could cause part of the ~ % genetically-determined differences in therapy response. genetic and corresponding functional data on adrb can help to understand the complex disease and, in cases of severe asthma, optimize therapy with adrb agonists for each individual. (ortega et al. ; chung et al. ) our present study connects sequence data with pharmacological data of prototypical adrb -ligands, namely, (r)-isoproterenol, (r,r)-and (s,s)-fenoterol, (r)-and (s)salbutamol and (r,r)-formoterol. as a pathophysiologically relevant cell type, we analysed human neutrophils from peripheral blood of healthy volunteers. formyl-peptide-induced o .production and its inhibition by the agonists are examined in a -well cytochrome-c assay. characteristic pharmacological values (pic , emax) are obtained for each individual. the data-set for each individual is supplemented by a differential blood cell analysis and an asthma-related questionnaire. most importantly, each volunteer's adrb -sequence variant is determined by sanger-sequencing. complete determination of a , bp sequence, including the entire adrb exon and part of the flanking '-and '-untranslated regions, allows mapping of the most common, but also of new or rare polymorphisms. first data demonstrate the inter-day and intra-individual robustness of the functional data. in the next step, we will link sequence variants and functional differences within a population of sixty volunteers with sufficient statistical power. collectively, this study represents a straightforward approach to link functional and genetic data of a clinically relevant receptor. cyclic nucleotide phosphodiesterases (pdes) are classified into eleven families and are essential for second messenger metabolism in human cells. recently, we have shown that several human pdes possess a much broader substrate-specificity than previously assumed, being capable of hydrolyzing not only the purine nucleotides cyclic adenosine ', '-monophosphate (camp) and cyclic guanosine ', '-monophosphate (cgmp), but also pyrimidine nucleotides such as cyclic uridine ', '-monophosphate (cump). these data were obtained using a highly sensitive hplc mass spectrometric assay which is quite expensive and whose technical requirements are available only in few laboratories. in our present study we developed a fluorimetric pde activity assay using '-o-(n'methylanthraniloyl) (mant)-substituted cyclic purine and pyrimidine nucleotides that can be used more broadly in the scientific community. human pde a is important for the regulation of platelet aggregation, oozyte maturation, vascular smooth muscle relaxation and contractility of cardiac myocytes. moreover, this pde shows a broad substrate-specificity, hydrolyzing camp, cgmp, cump and cyclic inosine ', '-monophosphate (cimp). using this enzyme, here, we demonstrate that various mant-substituted cyclic nucleotides are substrates of pde a and undergo a significant change in fluorescence whilst being hydrolyzed, thus allowing a quantitative analysis of catalysis via fluorescence detection. in fact, not only native cump but also mant-cump is a substrate of pde a. this finding is consistent with data published by hardman and sutherland who described a cump-degrading pde activity in homogenates from beef and dog heart, leading to the assumption that the pde activity described there could be attributed to pde a. as cump has furthermore been proven to be present in mammalian cells , to differentially activate camp-and cgmp-dependent protein kinases and to be synthesized from utp by mammalian soluble guanylyl cyclase , our present study supports the hypothesis that this cyclic nucleotide could play an important role in cell metabolism. the newly established fluorescence assay with mant-cump facilitates future studies on pde a and the assumed second messenger function of cump. rhoa is reportedly involved in stat-dependent transcription. however, the pathway connecting the gtpase and stat signaling has not been characterized. we made use of bacterial toxins, which directly activate rho gtpases to analyse this pathway. cytotoxic necrotizing factors (cnfs) are produced by pathogenic escherichia coli strains and by yersinia pseudotuberculosis. they activate small gtpases of the rho family by deamidation of a glutamine, which is crucial for gtp hydrolysis. we show that rhoa activation leads to phosphorylation and activation of stat and identify signal proteins involved in this pathway. rhoa-dependent stat stimulation requires rock and junkinase activation as well as ap -induced protein synthesis. the secretion of one or more factor/s activate/s the jak-stat pathway in an auto/paracrine manner. we identify ccl /i- as an essential cytokine, which is produced and secreted upon rhoa activation and which is able to activate stat -dependent signaling pathways. the knowledge about the connection between rhoa and stat signaling is crucial for understanding several deseases, especially cancer. acid sphingomyelinase-deficient mice are protected from the lethal cardiovascular effects in tnf-induced septic shock reiss l. k. christian-albrechts universität institut für immunologie, michealisstrasse , kiel, germany introduction: the cytokine tumor necrosis factor (tnf) is a mediator of septic shock. sepsis is a major cause of acute respiratory distress syndrome (ards), a heterogeneous lung disease with a mortality of about %. the present study was designed to investigate the effects of high systemic tnf-levels on the lung and on the systemic circulation in wildtype and acid sphingomyelinase-deficient (asm -/-) mice. the enzyme acid sphingomyelinase generates the signaling molecule ceramide that plays a critical role in edema formation and vasodilatation. material and methods: asm -/and wildtype mice were ventilated mechanically at vt= ml/kg and f= min - with fio = . and peep= cmh o while lung mechanics were followed. half of the mice received µg of murine tnf intravenously. blood pressure was stabilized by intra-arterial fluid support and body temperature was kept at °c to prevent lethal shock and to allow investigation of blood gases, lung histopathology, pro-inflammatory mediators and microvascular permeability hours after tnf application. results: tnf induced septic shock in wildtype mice, as indicated by metabolic acidosis, high serum levels of the sepsis marker procalcitonin, decreasing blood pressure and reflex tachycardia. interestingly, asm -/mice were protected from the tnf-induced cardiovascular effects and mortality. in the present study, circulating tnf failed to cause lung injury. lung mechanics stayed stable during ventilation in all groups and also pulmonary histopathology, cytokine levels and microvascular permeability were unaffected. conclusion: circulating tnf alone is not sufficient to cause acute lung injury. we conclude that the cardiovascular effects in tnf-induced septic shock are partly mediated by acid sphingomyelinase. cyclophilin a sirna provides mitoprotection and prevents aif-dependent neuronal cell death reuther c. cyclophilin a (cypa) is a peptidyl-prolyl-cis-trans isomerase which is localized in the cytosol. recent data suggested that neuronal cell death involved cytosolic cypa translocation to the nucleus, where it formed a pro-apoptotic complex with apoptosis inducing factor (aif). this cypa-aif complex induced caspase-independent chromatin condensation, dna degradation and cell death in various paradigms of apoptosis. on the basis of these data, the selective inhibition of the aif-cypa complex was proposed as a potential strategy to prevent aif-dependent cell death in neurons. therefore, the aim of this study was to determine effects of cypa silencing in a model of glutamate toxicity in immortalized hippocampal ht neurons. first, we addressed the interaction of aif and cypa by immunoprecipitation and their translocation to the nucleus by immunohistochemistry and confocal fluorescence microscopy. after exposure of ht- cells to glutamate the translocation of aif and cyp a occurred prior to cell death. cypa sirna attenuated glutamate-induced cell death as detected by the mtt-assay, impedance measurements (xcelligence system), and by facs analysis after annexin v/ propidium iodide staining. most intriguingly, cypa sirna also preserved the mitochondrial membrane potential as shown by facs analysis after tmre staining. further, confocal microscopy showed that cypa silencing prevented mitomorphology alterations and blocked the release of mitochondrial aif to the nucleus. the inhibition of the aif translocation to the nucleus was also shown by western blot analysis. in summary this study demonstrates that silencing of cypa prevents mitochondrial disruption and attenuates glutamate toxicity in vitro. thus, cypa is a promising target for mitoprotection as a basis for novel strategies of neuroprotection. up to now the question is unresolved how the ingested dose influences the absorption and metabolism of chlorogenic acids (cga) from food. so far no studies have been performed on the impact of the dose on cga absorption, circulation and excretion. recently we performed a dose-response study in a randomized, double-blinded, crossover design with five ileostomy subjects. in three trials the volunteers consumed after a two day polyphenol free diet coffee with varying cga content (high µmol; medium µmol; low µmol). the cga concentrations in plasma, ileal effluent and urine were subsequently identified and quantified by hplc-esi-ms, hplc-esi-ms/ms and hplc-dad. the results showed that the consumption of higher cga concentrations lead to a faster ileal excretion measured in the ileal effluents. this corresponded to the renal excretion of . ± . % (high), . ± . % (medium) and . ± . % (low) of total cga and metabolites. we found that cgas with a caffeic acid moiety are predominantly sulphated and those with a ferulic acid moiety are predominantly conjugated via glucuronidation prior renal excretion. furthermore, in the ileal effluents, sulphation of both structural units dominated. in plasma samples (after enzymatic deconjugation) the auc values were determined by the major cga classes in coffee, the caffeoylquinic acids: . ± . nm*h - (high); . ± . nm*h - (medium) and . ± . nm*h - (low). no major differences in the metabolic pattern were observed. additionally, we were able to identify new metabolites of cga in urine and ileal fluids. we conclude that the consumption of high concentrations of cga via coffee might influence the gastrointestinal transit time and consequently affect cga bioavailability. this study was supported by the nestlé research centre (lausanne, switzerland). interaction of antagonists with the atp binding pocket at the human p x ion channel helms n., riedel t., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße - , leipzig, germany the homomeric p x receptor (p x r) is a rapidly activating and desensitizing cation channel, gated by extracellular atp. it consists of three homomeric subunits. this representative of the p x receptor family is highly expressed on sensory afferent neurons and plays a significant role in chronic pain, bladder reflexes and taste sensation. therefore, the development of selective antagonists for p x receptors and knowledge about the binding of these antagonists are of great significance for future pain therapy and therapy of urge incontinence. to simulate the shape of the rapidly desensitizing agonist-induced current responses via p x receptors, we created a specific markov model to describe the binding of agonists and competitive antagonists. this model can be used to prove the competitive character of inhibition and to calculate the association and dissociation constants of the antagonists. furthermore we use this model to fit current responses at p x wild type receptors and their mutants to α,βmethylene atp in the presence of different antagonists. whole-cell patch-clamp recordings were performed on hek cells, heterologously expressing the human p x receptor, to determine the concentration-response relationship of different antagonists. by applying increasing concentrations, differences of antagonist potency could be observed at the wild type receptor. afterwards, we chose amino acid residues for replacement by alanine, which seem to be important for agonist binding and should be so for competitive antagonist binding as well, based on our homology model, developed from the zebrafish p x r crystal structure and previous mutagenesis studies. we intend to identify those amino acids which are important for competitive antagonist binding by monitoring the altered antagonist potency on the mutated receptor when compared with the wild-type receptor. analysis of the p x agonist binding site by double mutant cycles riedel t., wiese s., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße - , leipzig, germany purinergic p x receptors belong to the family of ligand-gated ion channels. they are non-selective cation channels, activated by extracellular atp. one of the seven members of the p x receptor family, the p x receptor, is localized at the plasma membrane of sensory neurons and is involved in pain perception. therefore, this receptor is a possible target for new drugs in pain treatment. the development of such drugs can be supported by an exact knowledge of the receptor structure and function. there are many hints to the atp binding site, but the interaction of the p x receptor with its agonists and antagonists remains still unknown. in this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed atp binding site of the homomeric human p x receptor on the effect of nucleotide analogues. the mutant receptors were expressed in hek cells and the nucleotide effects were measured by means of the whole-cell patch-clamp method. modifications in the receptor binding site changed the concentration-response dependency as well as the current kinetics during fast pulsed agonist applications. based on this fact, we were able to distinguish binding from gating, conductance, and desensitisation, using a markov model that describes the complete channel behaviour by a matrix of rate constants. the results were also checked for consistency with a structural hp x model that we developed from the known zebra fish p x crystal structure in the closed state. voltage-dependent modulation of alpha a adrenergic receptor signaling rinne a., birk a., bünemann m. philipps-universität marburg institut für pharmakologie und klinische pharmazie, karlvon-frisch str. , marburg, germany g protein-coupled receptors (gpcrs) are proteins that regulate numerous signaling pathways by activation of intracellular g proteins. gpcrs are activated by extracellular stimuli, such as light, hormones and neurotransmitters. recent evidence suggests that some gpcrs exhibit voltage-sensitivity leading to a modulation of their activity by the membrane potential (vm). we used a fret-based biosensor of the α a adrenergic receptor to analyze receptor activation at defined membrane potentials in hek cells by means of voltage-clamp recording. the biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (ne) and receptor activation was measured as decrease in the ratio of acceptor-/donor-fluorescence. receptor stimulation by ne was inhibited at depolarizing membrane potentials but enhanced by hyperpolarization. inhibition of ne activated receptors was strong at low concentrations ( nm: % inhibition) but almost absent at saturating agonist concentrations ( µm: % inhibition). both agonist-induced and hyperpolarizationinduced receptor activation exhibited a similar monoexponential time course and speed of activation was primarily dependent on agonist concentration for both activation modes. the latter indicates that depolarization lowers the apparent affinity of the ne receptor interaction and thus causes receptor deactivation by means of ne release. application of clonidine ( µm, vm=- mv) resulted in a fret response that was inhibited by % at + mv. in contrast to ne, strong receptor inhibition at + mv was present even at super-saturating concentrations of clonidine ( µm), suggesting that voltage alters the equilibrium between active and inactive conformations of the receptor. voltage-dependence of the a a adrenergic receptor also modulated downstream receptor signaling: g protein activation or the recruitment of arrestins, which we determined in fret assays that directly detect gαi protein activation or receptor-arrestin interactions, were both substantially inhibited at vm = + mv. therefore we conclude that negative membrane potentials promote active conformations of the a a adrenergic receptor, increase affinity of full agonists and enhance receptor signaling. in conclusion the present data show that scc cells extrude more ha, possibly related to increased levels of has , in comparison to keratinocytes. increased amounts of ha appear to be essential for the uvb induced tumourgenesis of sccs in mice. this effect might be related to the pro-proliferative property of high molecular weight ha. furthermore biological active ha fragments derived from ha degradation by hyaluronidases (hyal , ) are thought to be pro-angiogenetic, anti-apoptotic and proinflammatory, thus possibly also promoting tumour growth and malignancy. estradiol induced paracrine release of egf from keratinocytes protects the dermal hyaluronan/versican matrix during photoaging röck k. , meusch m. hyaluronan (ha) and versican are key components of the dermis and are responsive to uvb induced remodeling. the aim of the present study was to investigate the molecular mechanisms of estrogen (e ) mediated effects on ha-rich ecm during actinic aging. weeks of uvb irradiation ( x med ( mj/cm ), weekly) of hairless skh- mice caused a marked decline of dermal ha, which was aggravated by ovariectomy (ovx). subcutaneous substitution of estrogen (e ) by means of controlled release pellets abolished these effects confirming the stimulatory role of e . the increase of dermal ha correlated with induction of ha synthase has by e . in addition the ha-binding proteoglycan versican was induced by uvb and further increased by e . however in cultured skin fibroblasts e reduced the expression of versican and had no effect on has . therefore, direct upregulation of has and versican in fiborblasts by e was excluded. however, e increased the expression of egf in uvb irradiated skin in vivo and in keratinocytes in vitro. egf in turn upregulated the expression of has and versican in dermal fibroblasts. furthermore the supernatants of estradiol treated keratinocytes led to the same effects in dermal fibroblasts, which could be abolished by previous treatment of the supernatant with neutralizing egf antibody or treatment of the fibroblasts with egf receptor blocker erlotinib. functionally, dermal ha and versican induction by e correlated positively with proliferation and negatively with accumulation of inflammatory macrophages in the dermis. collectively these data suggest that e treatment increases the amount of dermal ha and versican via paracrine release of egf which may be implicated in the pro-proliferative and anti-inflammatory effects of e during photoaging. differential pro-and eukaryotic toxicity of silver released from nanocomposite surfaces increases the therapeutic window of silver in antibacterial treatments röhl c. , hrkac t. silver has been used since ancient times as antimicrobial agent. recently, silver gained new attention due to its higher effectiveness in its nanoform. this led to new developments of silver nanomaterials, e.g., for medical devices and consumer products. though, it is generally assumed that silver is less toxic for eukaryotes than for prokaryotes, concern is raised, if nanosilver at the same time might also increase mammalian cytotoxicity. in our study we examined the toxicity of silver released from nanocomposite surfaces with that of silver from agno solutions for adherent bacteria and mammalian cells. therefore, we established an in vitro reference system which enabled us to compare the therapeutic window between prokaryotic toxicity and eukaryotic integrity in both exposure settings. we focussed especially on the comparability of the bacterial and mammalian cell systems and the development of characterized ag/tio nanocomposite coatings with well-defined silver filling factors and silver surface release, which could be varied over a wide concentration range. as reference cells the e. coli sar strain and human dermal fibroblasts, which are of special relevance in the context of medical devices like implants or wound dressings, were chosen. bactericidal effects were determined by direct growth visualization of the gfp-producing e. coli strain by epifluorescence microscopy. mammalian cell growth and toxicity was determined by the mtt assay, protein measurements and phase contrast microscopy. the ag/tio samples were prepared by sputter co-deposition from two separate magnetron sources. the silver surface concentration release was determined by xps and the silver release by icp-ms. in solution a concentration-dependent constant silver concentration could be determined between and at least hours at the surface. while lowest bactericidal and cytotoxic concentrations of ag + from agno solutions with . and . mg/cm , respectively, differed only slightly, the therapeutic window increased significantly if ag + was released from the nanocomposite surface. while the toxicity on the fibroblasts was unchanged the bactericidal potency increased at least one order of magnitude. taken together, it can be concluded that local exposure factors i) can be modulated by silver nanocomposites and ii) play an important role for the differential toxicity of surface silver on bacteria and mammalian cells. charite -universitätsmedizin institut für integrative neuroanatomie, funktionelle zellbiologie, philippstr. , berlin, germany c exoenzyme (c bot) a clostridial adp-ribosyltransferase does not possess a cellbinding/-translocation domain. nevertheless, c is able to efficiently enter intact cells, including neuronal cells but the mechanism of uptake is not yet understood. in the present work, binding of c bot to the hippocampus-derived ht cell line was characterized by means of binding and blot overlay assays as well as mass spectrometry analysis to identify binding partners of c bot. the binding assays established that c bot bound in a concentration-dependent manner to ht cells. in the overlay assay we detected one clear band of kda. to elucidate whether glycosylation is important for the c bot-protein interaction, ht cells were incubated with glycosidase f resulting in a decreased binding of c to the kda band. to explore the involvement of phosphorylation in the binding of c to the putative binding protein, blot was pre-treated with cip (calf intestinal phosphatase) before overlay with c bot. pretreatment greatly reduced the c bot-protein interaction. moreover, inhibition of dephosphorylation by vanadate before in intact cells showed an increased level of c botprotein interaction in the following overlay. thus, interactions between c bot and ht cell proteins may require phosphorylation. to further characterize the kda band as binding target of c bot, the kda band was digested with trypsin and then subjected to lc-orbitrap mass spectrometry analysis. from this kda single gel band proteins were identified. further analysis of the identified proteins will provide a possible interaction partner of c bot. in sum, protein overlay assays revealed that phosphorylation and glycosylation are critical for efficient c bot-protein interaction. s solvent effects on enzyme kinetics in vitro rokitta d., pfeiffer k., gerwin h., streich c., fuhr u. uniklinik köln institut für pharmakologie, gleueler str. , köln, germany kinetic parameters provide essential quantitative information for characterisation of drug metabolising enzymes. such enzymes are located in an a partially queous environment, but to solve potential lipophilic substrates for in vitro measurements organic solvents are regularly needed. to preserve the enzymes from denaturation and other solvent related effects, the concentration of these solvents must be kept low. data on nature and extent of such solvent effects is sparse. in this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethylsulfoxide ( % to %) on the assessment of k m, vmax and clint with regard to the -hydroxylation of midazolam via cyp a and the cyp a catalyzed metabolism of caffeine to paraxanthine in vitro. the presence of acetonitrile showed the highest vmax value for paraxanthine formation but the lowest values for -hydroxymidazolam formation. the km value for midazolam showed no systematic effects of organic solvents, while for caffeine km was up to eightfold lower for solvent free samples compared to solvent containing samples. the present example suggests that the presence of organic solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. these effects are differing between enzyme-substrate systems and solvents. it remains to be determined to which extent such effects compromise in vitro -in vivo extrapolations, and which solvents are most appropriate. atrial remodeling and arrhythmia induced by the transcription factor er rommel c. , rösner s. introduction: the transcription factor er (ets related ) belongs to the large family of ets-transcription factors that are involved in developmental processes and in the pathogenesis of cancer. er is activated by gq-and gs-coupled receptors leading to a phosphorylation of the transcription factor by map-kinases and protein kinase a, respectively. cardiac er mrna expression is increased in failing human hearts. however mechanical unloading by a left ventricular assist device leads to normalization of er expression. thus, the aim of the present study was to investigate the cardiac function of er in genetically modified mouse models. we previously generated transgenic mice overexpressing er under control of the cardiomyocyte-specific α-myosin heavy chain gene (αmhc) promoter by pronuclear injection and established independent transgenic lines. electrocardiography (ecg) was assessed in mice at day after birth (p ) and in adult mice ( months) during isoflurane anesthesia and by ecg telemetry in awake mice, respectively. ecg analysis revealed no differences between the genotypes at day after birth. however, we found a decreased heart rate, a replacement of regular p-waves by an undulating baseline and frequent supraventricular extrasystoles in adult er αmhc transgenic mice. next, isometric contractile force measurements on isolated left atria were carried out in organ baths. while wt left atria responded to increasing concentrations of isoprenaline, nkh and calcium with an increase in contractility, the maximal positive inotropic responses to these substances were severely blunted in er αmhc atria. we performed western blots to identify potential aberrations of calcium handling and regulatory proteins. phosphorylation of serine of phospholamban (pln) was reduced in er αmhc mice. in addition, protein phosphatase (pp ) expression was significantly increased in er αmhc mice, which is consistent with the increased dephosphorylation of phospholamban. furthermore, we found a decreased expression of calsequestrin and serca a protein in er αmhc atria. electron microscopy revealed the significant structural remodeling of er αmhc atria at months of age. conclusion: increased cardiac expression of the ets-transcription factor er leads to structural and electrical remodeling of the atria. thus, er may play an important role in the pathogenesis of cardiac arrhythmias in chronic heart failure. signal transduction pathway of atp and utp in neonatal rat cardiac myocytes rothkirch d., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. , halle (saale), germany extracellular atp and utp can be released from the heart during pathological conditions such as ischemia or hypoxia. in humans, atp and utp levels are increased during myocardial infarction. atp and utp can act via p -purinoceptors which are further divided in p x - and p y - -receptors. as previously shown atp and utp can induce inotropic effects in cardiac preparations of mice and man. for rat articular chondrocytes and human intestinal cells it has been demonstrated that the mapk cascade can be activated by atp and utp. therefore, the cardiac effects of atp and utp on force of contraction probably occur via the mapk pathway. to investigate the signal transduction pathway involved, we studied the effects of atp and utp on mapk phosphorylation in isolated neonatal rat cardiac myocytes using phosphorylation-specific antibodies. µm atp as well as utp transiently increased phosphorylation of erk / and p mapk with a maximum effect at to minutes after application of atp and utp in neonatal cardiac myocytes (n= preparations each). the maximum phosphorylation of p increased with atp to % ± % (p< . ) at minutes and with utp up to % ± % (p< . ) at minutes. the phosphorylation with erk / mapk increased with atp to % ± . % (p< . ) at minutes and to % ± % (p< . ) with utp at minutes of basal values, respectively. after minutes, predrug values of mapk phosphorylation were reached again. in summary, we noted an atp-and utp-induced phosphorylation of erk / and p mapk in isolated neonatal rat cardiac myocytes. the involved receptor subtype(s) and the link between mapk phosphorylation and inotropic effect of atp and utp need to be elucidated. hameel: use of elearning in teaching pharmacology and toxicology -the halle experience rulf k. , gergs u. introduction: during the past three years, our faculty has started to integrate items of elearning into the standard curriculum of a classical medical school: the "hallesches medizinisches elearning -hameel". our hypothesis was that these new elearning tools would improve the willingness of students to spend more time into learning and this would lead to an improved outcome (in multiple choice tests). methods: hence, we offered medical students ( th or th semester) additional learning environments. the courses for students (experimental pharmacology and toxicology or clinical pharmacology) were existed of a weekly lecture and in addition tutorials (problem-based-learning style, paper cases) or classical seminars. furthermore, we offered the possibility to use an online multiple choice quiz (involving - previously used tests) and/or an online module on heart failure each week. we used the learning management system ilias software in combination with the content management system stud.ip. all students were subjected to an introductory test (to assess knowledge prior to our teaching section and allowing us to exclude a conceivable bias due to previous knowledge, involving basic items from prior teaching opportunities), a mid-term test and a final test to assess gain of knowledge. a maximum of points could be obtained as a sum of both tests. results: in the means % of students used the new elearning tools (quizzes, heart failure module). however, there was no association between the use of self-assessment quizzes and examination results. the usage of the online quizzes increased in the periods before the exams. however, usage of the heart failure module was accompanied by significantly increased scores in exams. moreover, in a formalized evaluation system, students positively commented on our elearning efforts. conclusions: while usage of our quizzes did not improve test marks, another more sophisticated clinically oriented elearning module seemed to be improving test outcomes marginally. targeting of erk thr phosphorylation attenuates cardiac hypertrophy but preserves the anti-apoptotic effects of erk / ruppert c., vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. , würzburg, germany background and aims: the extracellular regulated kinases and (erk / ) play an important role in cardiac hypertrophy and cell survival. erk / are phosphorylated at the so-called tey motif, which in turn activates erk / . hypertrophic stimuli lead to an additional autophosphorylation threonine (thr ). this autophosphorylation of erk / stimulates activation of nuclear erk targets, which are known to induce hypertrophy. the aim of this study is to investigate whether specific targeting of erk thr phosphorylation affects both erk functions -erk mediated hypertrophy and cardioprotective cell survival. methods and results: for the analysis of cardiomyocyte hypertrophy in vitro, we stimulated cardiomyocytes with phenylephrine and measured the incorporation of tritiated isoleucine. cardiac hypertrophy was assessed by echocardiography before and after transverse aortic constriction (tac). for analysis of cell survival, caspase activity and dna fragmentation was determined upon hydrogen peroxide stimulation in vitro and in response to tac in vivo. to differentiate between inhibition of erk / activity and prevention of erk thr phosphorylation, we either inhibited erk activity with pd or overexpressed a mutant of erk , which cannot be phosphorylated at thr phosphorylation. while inhibition of overall erk activity with pd attenuated cell survival and hypertrophy in vitro, specific targeting of erk thr phosphorylation by overexpression of the phosphorylation deficient mutant (erk t a ) attentuated phenylephrine induced hypertrophy, but preserved the anti-apoptotic effects of erk. cardiac overexpression of erk t a significantly reduced tac-induced hypertrophy compared to wild-type erk overexpressing mice. in line with the in vitro experiments, erk thr inhibition only prevented hypertrophy in the tac model without promoting apoptosis. conclusions: these results show that blockade of erk thr phosphorylation attenuates cardiomyocyte hypertrophy but preserves anti-apoptotic effects of erk / . therefore, specific targeting of erk thr phosphorylation might be a promising strategy for the treatment of pathological hypertrophy. intracellular camp levels are determined by interplay of camp formation by adenylyl cyclases and camp degradation by phosphodiesterases (pde). eleven families of pdes are known. one of the most recently identified pdes is pde , a pde in principle capable of hydrolysing camp as well as cgmp. pde contains a tandem of so called gaf domains in its n-terminal regulatory domain that mediate activation by camp. because current knowledge about the tissue distribution of pde was mostly based on the analysis of mrna distribution, we generated antisera against pde to analyze tissue distribution of the protein level. using these antibodies, we found a prominent occurrence of the enzyme in testis and in brain, where it was confined to the striatum. thus, pde displays a comparably restricted tissue distribution which is in contrast to that of many other pdes. low camp levels in so called medium spiny neurons of the striatum have been implicated in schizophrenia. furthermore, studies using the nonspecific pde inhibitor papaverine as well as specific pde inhibitors suggest pde as a target for the treatment of schizophrenia. here we set out to analyze the contribution of pde to camp degradation in striatum, to identify the physiological pathways pde is involved in and to clarify the functional impact of the proposed phosphorylation of the enzyme. identification of cgmp-dependent kinase i substrate complexes salb k., schlossmann j. universität regensburg lehrstuhl pharmakologie, universitätsstrasse , regensburg, germany the cgmp-dependent kinases (cgks) are components of the no/cgmp/cgk-signalling pathway and have a great physiological importance in a multitude of tissues and organs such as smooth muscles and platelets. two isoforms of the cgki and the cgkii are known. cgkiα and cgkiβ differ only in their first ~ amino acids which constitute the leucine zipper and the autoinhibitory domains. the n-terminal leucine zipper domains mediate homodimerization of the kinase and the interaction with diverse substrate proteins. since cgkiα and cgkiβ express different n-termini they interact with different substrates. the cgkiβ isoform is assembled in a macrocomplex at the endoplasmic reticulum (er) with the intracellular calcium release channel inositoltrisphosphate receptor i (insp r-i) and the inositol-trisphosphate receptor associated cgmp kinase substrate (irag). we investigated, whether irag also interacts with the insp r-ii and the insp r-iii in murine platelets and tissues. additionally, we analyzed the interaction between the amino acid peptide phospholamban (plb), which is also located at the er and regulates the er calcium reuptake by the sarco/endoplasmic reticulum ca + -atpase (serca), and the two cgki isoforms. we performed cgmp-agarose experiments with murine wt and irag-ko platelets to examine the irag-insp r interactions. the insp r-ii isoform was neither bound to cgmp-agarose nor detected in the anti-irag immunoprecipitate. on the other hand, insp r-iii from wt but not from irag-ko platelets was bound to cgmp-agarose. hence, insp r-iii interacts directly with irag but not with cgkiβ in murine platelets. however, in colon smooth muscle lysate, insp r-iii not only interacted with the irag protein but was also detected in the anti-cgkiα-immunoprecipitate. phospholamban from wt and irag-ko platelets was also bound to cgmp-agarose. subsequent immunoprecipitation experiments with the respective antibodies against the two cgki isoforms revealed that plb interacted both with cgkiβ and cgkiα. these results were supported by analysis of colon smooth muscle tissue from wt and irag-ko mice. in conclusion, irag interacts with insp r-i and insp r-iii but not with insp r-ii in murine platelets and colon smooth muscle tissue. moreover, phospholamban is an interacting partner of both the cgkiα and the cgkiβ isoform. the human immunodeficiency virus type enhancer binding protein (hivep ) is regulated by proinflammatory stimuli and statins salomon a. , , schmitz b. objective: hivep binds nf-ĸb and other proinflammatory consensus sequences, and is suggested to be involved in inflammatory processes. we recently identified two tagging snps, one positioned kb upstream (rs ) and another in exon (rs ) of the hivep gene, to be replicatively associated with venous thrombosis in gwas and follow-up studies (ajhg, ; plos one, ) . methods: total rna isolation was performed after treatment of vascular endothelial cells (ea.hy ) with proinflammatory cytokines or statins ( h). serial hivep promoter deletion constructs were cloned into the pgl -basic vector, a potential enhancer fragment, harbouring rs c/t, into the pgl -promoter vector. in ea.hy cells and thp monocytes, reporter gene assays were performed by transient transfection and overexpression of transcription factors. chip and bandshift assays were performed to identify candidate transcription factors. results: in ea.hy cells, endogenous hivep expression was increased by proinflammatory cytokines tnfα and il- β. simvastatin ( . and . µm) and atorvastatin ( µm) -but not pravastatin or aspirin -both dose-dependently decreased basal and tnfα-stimulated hivep expression. the construct harbouring rs t exerted significantly higher transcriptional activity (ta) compared to rs c (p< . ). for an intronic modulator, reporter gene assays demonstrated a regulatory effect on hivep expression in ea.hy and thp cells. cotransfection of sp and egr led to an increase in ta, while wt exclusively upregulated ta of constructs comprising the intronic modulator. chip and bandshift assays combined with specific antibody detection revealed binding of sp to the '-flanking region and the intronic modulator of hivep . conclusion: increased hivep expression during inflammatory conditions can be repressed by simvastatin and atorvastatin, and not by pravastatin or aspirin. basal hivep expression is regulated by sp combined in a transcription factor module with egr and wt under basal and/or inflammatory conditions. the rs site harbours potential activational capacity for hivep gene transcription and may communicate with the sp /egr /wt module. to date, the treatment of various movement disorders of the central nervous system is still insufficient. in most cases this is due to the sparse knowledge of the pathophysiology. l-dopa-induced dyskinesias (lid) represent a severe complication of long-time pharmacotherapy in parkinson's disease that deserves novel therapeutics. an increased activity of striatal projection neurons, which express kv . / channels, seems to be involved in the pathophysiology of these spontaneous involuntary dystonic and choreatic movements. previous studies demonstrated an antidyskinetic effect of the kv . - . channel opener retigabine after acute and chronic treatment in a rat model of lid. in order to clarify if this effect was based on the modulation of kv . / channels, we examined the acute effects of the preferred kv . / channel opener ica on lid in this animal model. four weeks post -ohda lesioning of the left forebrain bundle, dyskinesia was induced by chronic treatment with mg/kg l-dopa and mg/kg benserazide for days. three subtypes of dyskinesia (limb, axial and orolingual) were rated according to a score system from to over min. for drug testing, ica ( , and mg/kg) was administered intraperitoneal additionally to l-dopa (or vehicle). effects of drug action in comparison to vehicle controls were detected by adding up the severity scores of each observation time. additionally, effects on parkinsonian symptoms were examined min after drug administration using the block and the stepping test. ica reduced the severity of dyskinesia significantly at all doses while no negative impact on the antiparkinsonian effect of l-dopa was observed. whereas the antidyskinetic effect was restricted to the first min after the application of mg, it lasted up to min in rats treated with mg ica . a higher dose of mg did not further enhance the antidyskinetic effect. the results of our study suggest that the antidyskinetic effect of the k v channel opener retigabine was based on its action on striatal kv . / channels. in line with the results of previous studies with retigabine, this action does not seem to interfere with the antiparkinsonian effect of l-dopa. this study was supported by the micheal j. fox foundation. background: skeletal muscle toxicity is the major side effect of hmg-coa-reductase inhibitors (statins) and can be simulated in engineered skeletal muscle. statins are known to exert "pleiotropic" effects, e.g. reducing endothelial dysfunction by inducing no synthases and no production. the role of no synthases in skeletal muscle under statin treatment is largely unknown. interestingly, some skeletal muscle pathologies (e.g. duchenne muscular dystrophy) may be exacerbated by increased inos activity. here we tested whether or not statin-induced skeletal muscle toxicity would be associated with enhanced no synthesis. we generated engineered skeletal muscle (esm) from rat skeletal muscle cells, matrigel and collagen. esms displayed typical skeletal muscle properties (differentiated muscle fibres, tetanic contractions). under baseline conditions esm expressed enos most abundantly, followed by inos and nnos (n= - ). myotoxic cerivastatin ( . , . , µm for days) caused a concentration-dependent decrease of contractile force (p< , , n= - ) paralleled by an increase in inos transcript (mean±sem: . µm ± . -fold, n= p< . ; . µm . ± . -fold, n= p< . ) and protein ( . µm . ± -fold, n= p< . ; . µm . ± . -fold, n= p< . ). mevalonic acid fully prevented the inos increase suggesting that the induction is hmg-coa reductase-dependent. to test whether inos may contribute to the decrease in contractile force we co-treated esm with w, a specific inos inhibitor. we applied µm of w, a concentration found to potently reduce lipopolysaccharide (lps)induced no-production in cultured myotubes. however, we did not observe a rescue effect (n= - ). also, l-name ( mm), an unspecific nos inhibitor, did not improve contractile function, instead we observed increased myotoxictiy (n= - , p< . ). to further investigate the role of no for muscle function we treated the esms with increasing concentrations of the no-donor snp. only high concentrations of snp ( µm) caused a reduction of contractile force. combined treatment with cerivastatin and . µm snp showed a tendency towards improved force development in esm. conclusions: statins increase inos activity in our skeletal muscle model (esm). however, this does not seem to functionally contribute to myopathy in esm. increased production of no may in fact be a protective measure. esm may help to dissect clinically relevant functional changes in statin myotoxicity. characterization of primary skin fibroblasts of patients with m syndrome and mutations in the cul gene meyer k., hieber m., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. , münchen, germany m syndrome is an autosomal-recessive disorder characterized by pre-and postnatal growth retardation (< - sd), facial dysmorphism and skeletal anomalies. the majority of patients harbor missense mutations of the cul ( %) or obsl ( %) gene, respectively. cul constitutes an e ubiquitin ligase that is involved in the regulation of the insulin-like growth factor (igf- ) signaling pathway via ubiquitin mediated degradation of insulin receptor substrate (irs- ). to investigate the role of cul mediated irs- degradation in the pathogenesis of m syndrome. primary skin fibroblasts of seven m syndrome patients (six with cul mutations, one with a obsl mutation) and control fibroblasts were analyzed for proliferation rate (cell counter), cell cycle profile (facs), cell morphology and cellular senescence (histochemistry), irs- protein concentrations and activation of the igf- signaling pathway (western blot). the proliferation rate of m patient fibroblasts was significantly increased when compared to control cells. in contrast, irs- protein levels and activation of the pi k/akt and erk mapk pathway were only increased in a subset of m cells that carried cul mutations, but not in cells from a patient with the obsl mutation. no significant differences in cell cycle profile, cell morphology or cellular senescence were observed in m patient fibroblasts when compared to control cells. to determine the pathogenetic contribution of increased irs- levels to the observed phenotype, human imr fibroblasts were stably transfected with retroviral vectors encoding irs- . despite -fold overexpression of irs- compared to empty vector controls, no significant effect of igf- stimulation on proliferation rate or pi k/akt and erk mapk signaling was observed. skin fibroblasts of m patients with cul mutations displayed an increased proliferation rate and enhanced activation of the igf- signaling pathways. despite accumulation of irs- in fibroblasts from a subset of m patients with cul mutations, no pathomechanistic role for irs- could be demonstrated. collectively, our data indicate that a dysregulated igf- signaling may contribute to the pathogenesis of m syndrome, yet in an irs- independent manner. pharmacases.de -a student-centered elearning project of clinical pharmacology zollner b., berg c., gros n., muß n., oestreicher d., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. , münchen, germany pharmacases.de is a novel e-learning website of clinical pharmacology that presents clinically relevant aspects of pharmacology and toxicology in an interactive and multimedial manner. the aim of the project pharmacases.de was to develop an innovative concept for creating high quality elearning content that i) integrates and promotes the theoretical and cooperative skills of final year medical students and ii) is easily adoptable by cooperating institutes and hospitals. a peer-teaching concept was developed in which final year medical students with the elective pharmacology (pj wahlfach pharmakologie) independently researched and wrote elearning lessions ("pharmacases"). subject-specific expertise was acquired by consulting elective students of other disciplines. at present ( / ) , this "peer network" consists of elective students of nine cooperating institutions (pathology, microbiology, radiology, cardiology, psychiatry, dermatology, neurology, ophthalmology, pediatrics) at the technische universität münchen. the average time for the generation of one elearning lession by the peer network was days. to date, the website consists of pharmacases that are available to all students online (http://www.pharmacases.de). the website also contains a discussion forum and evaluation form for direct feedback. on average, pharmacases.de has visitors per month with the following evaluation results: "excellent": %, "good": % and "satisfactory": % (n= ). the didactic concept of pharmacases.de enabled the efficient generation of high quality elearning content in a student-centered and interdisciplinary manner. the peer-teaching approach supports the collaborative skills of final year medical students and facilitates the transfer of theoretical pharmacological knowledge into clinical practice. improved glucose tolerance, less chronic adipose tissue inflammation and reduced adipose tissue mass in mice with adipocyte-specific loss of tak sassmann a., offermanns s., wettschureck n. max-planck-institut für herz-und lungenforschung pharmakologie, ludwigstr. , bad nauheim, germany tgf-β activated kinase (tak ) is known to be involved in numerous inflammatory processes by linking receptors for inflammatory stimuli like lps, interleukin- or tnfa to ikk, p and jnk activation. chronic inflammation of white adipose tissue is one of the major causes for the development of insulin resistance and impaired glucose tolerance in states of obesity. to investigate the role of tak in white adipose tissue, we crossed the tamoxifen-inducible white adipocyte-specific cre mouse line adipoqcreer t with animals carrying floxed alleles of the tak gene. adipoqcreer ; tak fl/fl animals and cre negative control littermates are viable and fertile and do not show any developmental defects. after tamoxifen induction and high fat diet feeding adipocytespecific tak knockout mice show improved glucose tolerance and lower fasting insulin levels compared to control animals. in line with this, serum levels of the adipose tissuespecific hormone resistin are reduced in adipocyte-specific tak knockout mice. these findings are accompanied by a lower state of chronic inflammation of adipose tissue as indicated by a dramatic reduction of adipose tissue macrophage number and lower serum levels of tnfα and interleukin- . stimuli like tnfα, interleukins and tgf-β released from macrophages and adipocytes are known to promote obesity-related adipose tissue inflammation. when stimulated with these substances tak deficient adipocytes show reduced activation of jnk and p which both play an important role in the development of insulin resistance. interestingly, we observe a lean phenotype in adipocyte-specific tak knockout mice when fed a high fat diet which reflects a reduction of white adipose tissue mass. currently we are investigating the molecular mechanisms underlying the reduced adiposity and lower state of chronic inflammation in adipose tissue. growth of small cell lung cancer (sclc) cells is regulated via the autocrine stimulation of g protein coupled receptors (gpcrs), i. e., neuropeptide and muscarinic acetyl choline (ach) receptors. the activation of gq/ and calcium-dependent gpcr pathways results in the stimulation of erk signaling which is necessary for the mitogenic effects of neuropeptides or ach on sclc cells. in contrast, the role of calcium-independent gpcr signaling and its interplay with gq/ -regulated pathways in sclc cells are less well defined. the aim of our studies was to characterize the molecular make-up and the interaction of these pathways, and to delineate the phenotypic effects of calciumdependent and -independent signaling cascades in sclc cells. using a panel of sclc cell lines, we found that the stimulation of neuropeptide receptors led to an increase of calcium which was independent of extracellular calcium and could be prevented by depleting internal calcium stores. this calcium increase was sufficient to activate the tyrosine kinase pyk and subsequently the erk / cascade. the role of pyk for the growth of sclc cells was further supported by the fact that inhibition of pyk using a sirna approach or a novel specific inhibitor, pf , exerted pronounced cytotoxic effects on sclc cells, whereas non-sclc cells were less sensitive. interestingly, the inhibition of g / signaling by sirna-mediated g(alpha) or g(alpha) knockdown also markedly reduced the growth of sclc in vitro or in subcutaneous tumor xenografts, and increased the sensitivity of sclc cells towards certain cytostatics. to further define the role of calcium-dependent signaling via pyk versus the role of calcium-independent signaling via g / , we tested the effect of pyk inhibition in cells with impaired g / signaling. notably, pyk and g / double inhibition led to an even increased proliferation. thus, we propose that dysbalanced g protein signaling favoring either pyk activation or g / -dependent cascades inhibits the growth of sclc cells, whereas the parallel inhibition of both pathways restores again the balance and the growth capacity in this tumor entity. dendritic cells (dcs) are essential for the initial immune response and for the defence against inhalated pulmonary toxins and carcinogens in lung. to differentiate dcs, the cell line thp- were used for days and stimulated with various cytokines (il- , gm-csf, tnf-a, ionomycin). the dcs were characterized by flow cytometry with different typical dendritic cell markers (for example cd c, cd , cd ) and by immunfluorescence compared to monocytes. the bronchial tract contains up to dcs per mm² and therefore we established a triple culture model to mimic the situation in vivo. the triple culture consists out of primary human epithelial cells from small bronchi (hbec) and lung fibroblasts which are cultured under air-liquid conditions on filter membranes for weeks and dcs which were added after the differentiation phase of the bronchial cells. during the cultivation time the hbec formed an epithelial layer expressing both tight and adherens junctions. they also produced mucus, formed functional cilia with a beat frequency of between to hz and the transepithelial resistance values were stable between to Ω·cm². pathomechanisms of pulmonary toxicity in vivo are difficult to investigate, so the tripleculture model is the basis for investigations of the toxic effects at cellular level. lungtoxic substances such as organophosphates are usually absorbed through inhalation. organophosphates are dangerous nerve agents for the human organism. at high concentrations organophosphates damage in the coculture without dcs the cell-cell contacts of the epithelial layer. in the triple culture dcs firstly respond to inhaled organophosphates and seem to compensate effects on the other cells. in summary, it is very important to understand the pathogenic mechanisms of lung injury in relation to the role of dendritic cells in lung. they could play an essential role in therapy against damage of organophosphates in the lung. co-purification of arf gtpase-activating protein git and cavb schalkowsky p., wissenbach u., fecher-trost c., flockerzi v. universität des saarlandes institut für experimentelle und klinische pharmakologie und toxikologie, kirrbergerstraße, homburg, germany high-voltage activated ca channels are assembled from pore-forming α subunits and two distinct types of auxiliary subunits, cavβ -β and, maybe, α δ -δ . by a cavβ -specific antibody based affinity chromatography the cavβ protein was highly enriched from rat brain microsomal membranes. proteins associated with cavβ were identified by mass spectrometry (lc-esi-ms/ms) and include α -subunits, α δ-subunits and β-subunits. in addition to these expected interacting proteins additional proteins were co-purified with the cavβ protein, including the g protein-coupled receptor kinase-interactor (git ). the aa git is a ubiquitously expressed multidomain protein which may serve as a scaffold to bring together molecules to form signaling modules controlling, for example, vesicle trafficking, cytoskeletal organization and cell migration. in rat brain lysates the git and cavβ proteins were co-immunoprecipitated by the antibodies for cavβ and git, respectively. we cloned the git cdna from mouse brain and co-expressed it with the cavβ subunit in hek cells. like in brain lysates the git protein was retained by cavβ precipitated by the antibody for cavβ and cavβ was retained by the git protein precipitated by the antibody for git . both proteins, cavβ and git are endogenously co-expressed in mouse embryonic fibroblasts (mef). we could not observe potassium-induced voltage-activated ca influx in these acutely prepared cells. accordingly, mefs can be used as a model system to study the impact of cavβ -git interaction in the absence of functional cav channels. in addition, using mefs from cavβ -deficient mice enables us to control the impact of cavβ on git function. vice versa down-regulation of git by specific sirnas might allow to control the impact of git on cavβ function. as read-outs we use cell migration assays and monitor receptor-dependent and receptor-independent calcium signaling in these cells. effects of sphingosine- -phosphate and fty on epidermal hyperproliferation and inflammation in an imiquimod induced mouse model of psoriasis the sphingolipid sphingosine -phosphate (s p) is a mediator that modulates various physiological functions of skin cells. s p has distinct direct effects on keratinocytes as it diminishes proliferation and induces differentiation which is a classical goal of psoriasis therapy. furthermore, s p modulates the function of various immune cells, mainly to an anti-inflammatory direction. thus, the strategy of targeting immune cells with locally acting s p was explored in an experimental animal model of psoriasis vulgaris, the recently established imiquimod induced psoriasis mouse model and in the mouse tail test. topical administration of imiquimod onto back and ear skin led to a distinct inflammatory response characterized by epidermal hyperproliferation, scaling and redness which was scored with a modified pasi (psoriasis area and severity index). the positive control diflorasone diacetate and s p, but not fty reduced the epidermal hyperproliferation by topical administration onto ear skin, indicating a mode of action for s p via the s p receptor, which is not activated by fty . there was also a moderate reduction of inflammatory cell influx and edema formation in ear skin by s p treatment, which was even more pronounced by treatment with diflorasone diacetate. the pasi determined on back skin was, however, only significantly reduced by diflorasone diacetate. the discrepancy between outcome on ear and back skin remains elusive. in the mouse tail assay, the influence of s p in stratum granulosum formation (orthokeratosis) was tested compared to the positive control calcipotriol. whereas topical administration of calcipotriol led to the expected significant increase of stratum granulosum in mouse tail epidermis, s p lacked such an effect, indicating a different mode of action in epidermal differentiation. taken together, these results imply that topical administration of s p might be a new option for the treatment of mild to moderate psoriasis lesions. inhalation of toxicants such as sulphur mustard (sm), an alkylating chemical warfare agent, cause pulmonary complications like respiratory failure, pulmonary edema and secondary pneumonia. in order to investigate pathomechanisms of pulmonary toxicity, an in vitro alveolar-capillary co-culture model has been established recently by our group. in this model the human lung adenocarcinoma epithelial cell line (h ) is mimicking the epithelial site of the alveoli while the human hemangiosarcoma cell line (iso-has) represents the endothelial site. acute respiratory injuries are accompanied by disruption of the alveolar-capillary barrier that can be detected by the use of biochemical markers (e.g. ldh) and electrochemical indicators (e.g. transepithelial resistance). sm-mediated pulmonary injury is characterized by the increased secretion of proinflammatory mediators (e.g. il- ). a shortcoming of this model is the missing inflammatory component in the lung. aim of the present project is the addition of macrophages to the established co-culture model to improve the model and to investigate the relevance of inflammatory processes in toxic lung injury. the effect of sm on this triple-culture model is characterized with special regard to the interaction of epithelial cells and macrophages. the human acute monocytic leukemia cell line (thp- ) was stimulated to allow differentiation into macrophages. validation of the cellular differentiation was checked by specific clusters of differentiation (e.g. cd ) using flow cytometric analysis. after successful differentiation into macrophages, these inflammatory cells were added to the co-culture model before and after exposure with sm, respectively. the cytotoxicity of sm on the triple-culture model was evaluated by xtt assays and ter measurements. furthermore, immunohistochemical staining of tight junction proteins (e.g. zo- ) and of adherens junction proteins (e.g. e-cadherin) was conducted to enhance the knowledge of the function of the intercellular junction in injured and rejuvenated regions as well as the interaction of epithelial cells and macrophages. for the contact allergen para-phenylenediamine (ppd) we showed that concentrations above µm are accompanied with inhibition of nat activity in human keratinocytes [ ] . in the following we investigated the impact of ppd on nat activity in antigenpresenting cells using dendritic cell-like cells, namely the monocytic thp- cells. measured nat activity of thp- was comparable to those found in primary keratinocytes. a h treatment of thp- cells with physiologically relevant concentrations of ppd ( - µm) led to a % reduction of nat activity. comparable results were found for mono-acetylated ppd (mappd) whereas di-acetylated ppd demonstrated no inhibition. time-dependent studies found a significant decrease in enzyme activity already h after application of ppd or mappd while nat mrna levels were not modified. these results are indicative for a substrate-dependent inhibition. further investigations concentrated on the restoration of nat activity after treatment with ppd or mappd. here we found that n-acetylation capacities were restored after h cultivation of the treated cells in fresh medium. independent of the enzymatic activity, certain compounds are known to oxidise the catalytic cysteine or form adducts with the nat protein. therefore we studied whether ppd and/or oxidised ppd including the trimer bandrowski´s base interact additionally with recombinant nat protein itself in the absence of acetyl-coenzyme a. we found that all compounds but mappd bind to nat protein after h. the greatest inhibition was found for oxidised ppd (up to %). due to the greater inhibition by oxidized ppd we propose that oxidation products interact with the protein whereas ppd itself modulates nat enzyme activity in a substrate-dependent mode of action. overall we demonstrated that ppd can inhibit nat in two different ways. the work was partially financed by federal office of public health (foph), switzerland and stiftung zur förderung begabter studierender und des wissenschaftlichen nachwuchses objective: fabry's disease is a rare progressive multisystem disorder resulting from deficiency of the lysosomal enzyme alpha-galactosidase a (gla, ec . . . ). we hypothesize that genetic gla variants, especially those in its promoter region are of pathophysiological relevance for the development and progression of fabry's disease phenotypes. this study focuses on the characterization of the gla promoter, identification of functional genetic variants and impact of transcription factor eb (tfeb), a regulator of lysosomal genes. we screened bp of the '-flanking region of gla in patients with fabry's disease and controls for genetic variants. serial promoter deletion constructs for reporter gene assays were designed and identified genetic variants were introduced by site-directed mutagenesis. constructs were transiently transfected into immortalized human kidney epithelial (ihke) cells and human vascular endothelial cells (ea.hy ) to determine transcriptional promoter activity (ta). sequencing of patients' dna revealed five genetic variants in the 'flanking region of gla, significantly more frequent in fabry's patients compared to control group (rs ; rs ; rs ; rs ; rs ; all minor alleles p< . ). we identified two regions, a proximal one between - and - and a distal region between and - with significant ta, in both cell lines. cotransfection with tfeb activated ta of both regions significantly up to . -fold (p< . ). in ihke cells, insertion of the minor t allele (rs ) significantly enhanced basal ta of the proximal promoter region (p= . ), while insertion decreased basal ta (p< . ) of the distal promoter portion. the combined insertion of the minor c alleles (rs ; rs ), which were in complete linkage disequilibrium, significantly increased basal ta of the distal promoter region (p= . ). our results indicate that three genetic variants, overrepresented in fabry's patients, are located within transcriptionally active regions, possibly altering tf binding sites and therefore, affecting gla expression. future analysis will assess the impact of gla promoter variants and gla regulation by tfeb with respect to fabry's phenotypes. multiple sclerosis (ms) and its animal counterpart experimental autoimmune encephalomyelitis (eae) have a major inflammatory component that drives and orchestrates both diseases. ceramides (cer) are known as mediators of inflammatory processes, but until now their role in ms was not elucidated. we measured the ceramide levels in the cerebrospinal fluid of ms patients and control patients using lc-ms/ms. interestingly, the c : -cer levels were . fold increased in ms patients. this translates into the finding that c : -cer levels were also significantly elevated in the lumbar spinal cord of eae mice. the raised c : -cer levels in the lumbar spinal cord were caused by a transiently increased expression of ceramide synthase (cers) in macrophages. nitric oxide (no) and tumor necrosis factor alpha (tnf-α) secreted by interferon gamma (infγ ) induced macrophages play an essential role in the development of ms. astonishingly, rnai experiments reveal that cers and its product c : -cer are mediators of inf-γ induced no/tnf-α release in raw macrophages. moreover, treatment of eae mice with l-cycloserine prevented the increase of c : -cer and of inos/tnf-α expression and caused a remission of the disease. in summary, cers plays a critical role in the initial phase of ms, most likely by regulating the no and tnf-α synthesis. this let us speculate, that a substance designed to inhibit cers and therefore to limit the inflammatory effects of c : -cer may represent a new drug in ms therapy. role of cgmp-dependent protein kinase i for kidney fibrosis schinner e. cgmp is synthesized via nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions also in the kidney. hence, the integration of cgmp signaling via cgmp-dependent protein kinases (cgk) might play a critical role for renal physiology. both isozymes were detected in arterioles, mesangium and within the cortical interstitium. in contrast to cgkiα, the β isoform was not detected in the juxtaglomerular apparatus and medullary fibroblasts. here, we focused on the function of cgki in the renal interstitium emphasizing a functional differentiation of both isoforms. the interstitium exists mainly of fibroblasts playing a prominent role in the interstitial fibrosis. accordingly, cgki could also be involved in this pathophysiological process. therefore, we studied whether cgki influences renal fibrosis which was induced by unilateral ureter obstruction (uuo). at first we analysed the role of the no/cgmp signaling by application of cgmp increasing yc or isdn. thereby we detected antifibrotic effects of these substances. subsequently we tested whether these effects are mediated by cgki by using mutant mice. on the one hand we examined αsm-rescue mice (expressing cgkiα only in smooth muscle under the control of the sm promotor with a cgki-ko background) and cgki-ko mice (expressing no cgki). on the other hand we used tgtg mice expressing more cgkiα in smooth muscle than wt mice (transgenic cgkiα under the control of the sm promotor). due to the steeply increased use of nanomaterials for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. moreover, the continuous development of novel materials requires the implementation of hazard-predicting models to prevent potential health effects resulting from human exposure. in the present study, we studied the toxic potential of a set of nanoparticles (np) with varying physicochemical properties in human a lung epithelial cells, hepg liver epithelial cells and hk- proximal tubule epithelial cells. the used nanomaterials incorporated five tio samples, two zno samples (i.e. uncoated and coated), two multi-walled carbon nanotube (mw-cnt) samples and a nanoparticulate ag sample. cells were treated with np at doses ranging from . to µg/cm for cytotoxicity and from to µg/cm for genotoxicity. dna damage was evaluated using the alkaline comet assay while concurrent cytotoxicity was determined by the wst- assay. marked contrasts in cytotoxic and dna damaging properties were observed among the different materials. the overall strongest responses were observed with the uncoated zno-np sample and with ag-np, although effects were found to depend on the cell type. notably, the dna damaging effect of ag-np could at least partly be attributed to its dispersant. present results form part of a growing data set which are generated in the framework of the eu fp project enpra (fp -nmp) to establish dose-response relationships and a mathematical model to predict the hazard of nanoparticles. increased spontaneous hprt mutant frequency in v cells expressing human cytochrome p b schlechtweg a., esch h. , martínez jaramillo d., lehmann l. university of wuerzburg/institute of pharmacy and food chemistry section of food chemistry, am hubland, wuerzburg, germany the hypoxanthine-guanine phosphoribosyltransferase (hprt) assay in chinese hamster v lung fibroblasts (v cells) represents a widely-used mammalian test system to detect gene mutations. since v cells do not express any cytochrome-p dependent monooxygenase (cyp) isozymes, usually an activating system has to be added. therefore, v cells expressing human (h) cyp isozymes have been commercialized. to test these v cells for their use in the hprt test, v h a and h b cells were characterized regarding (i) spontaneous frequency of -thioguanineresistant clones per clonable cells (smf), (ii) the stability of which over weeks (w), and (iii) the mutational spectrum (ms) of cdna from mutant clones. ms of cdna was determined by isolation of total rna, reverse transcription/amplification of the coding region by polymerase chain reaction and sanger sequencing of the amplification product. activity of cyp isozymes was verified by ethoxyresorufin-o-deethylase (erod) assay. (i)/(ii) whereas the smf of v cells (w : ± ; w : ± ) and v h a (w : ± ; w : ± ) only varied within the range of historical controls, smf of v h b increased continuously over time (w : ± ; w : ± ). (iii) although the smf of v and v h a were similar, the mutational spectrum of v cells was characterized by as many transversions as transitions and deletions of exon or exon + , whereas the mutational spectrum of v h a was characterized exclusively by transversions and deletion of exon + . surprisingly, with out of cdnas derived from v h b mutant clones, no amplification product was detected. first results indicate that there is at least one gene mutation in the untranslated region before and behind the coding region precluding amplification with the original primers. (ii)to reduce the smf of v h b , cells with wildtype hprt activity were cloned and one clone with an erod activity which did not differ significantly from the original cell population was further characterized. initially, smf of the clone varied between . ± . and . ± . . yet its smf was unstable reaching up to ± . in conclusion, the mutational spectrum differed between the v cell lines. furthermore, h b expression seemed to enhance smf in v cells. even though a temporary reduction of the smf by cloning was possible, smf of v h b cells was unstable. we wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adp-induced aggregation of human platelets by wild garlic, allium ursinum l. method: bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. for the bioassay, blood was taken from healthy human volunteers and platelet rich plasma (prp) was prepared for turbidimetric platelet aggregation tests. prp, stimulated with µm adp, was treated with extracts of different polarities, fractions and isolated single compounds from allium ursinum. the extracts were investigated by thin layer chromatography, hplc, mass spectroscopy, esi-ms and d/ d h/ c-nmr spectroscopic techniques. for references the adt-antagonist mes-amp was used. result: fresh allium ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited adp-induced aggregation of human platelets. this ethanolic extract was subjected to liquid-liquid partition. whilst the aqueous phase containing the moiety of cysteine sulphoxide and thiosulphinate derivatives showed only weak activity on platelet aggregation, the ethyl acetate and especially the chloroform partitions showed highest aggregation inhibiting potency. thus, in our bioassay effects of alliins/allicins could be neglected. the chloroform phase, possessing the strongest activity, was separated into fractions by gradient elution open cc on silica gel. the most active fractions - were separated again yielding subfractions. this afforded , -di-o-α-linolenoyl- -o-β-d-galactopyranosyl-sn-glycerol and β-sitosterol- -o-β-dglucopyranoside, the structures of which were determined by esi-ms and d/ d h/ c-nmr spectroscopic techniques. furthermore, the diminutive amounts of volatile oil of a.ursinum leaves obtained by steam distillation according to ph.eur. could be evaluated as a third aggregation inhibiting principle. conclusions: at the first time two active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on adp-induced aggregation in human blood platelets. as a major constituent, the galactolipid , -di-o-α-linolenoyl- -o-β-d-galactopyranosyl-sn-glycerol, not yet found in allium spec., appears as a new, highly useful marker substance for a.ursinum drugs, or their pharmaceutical preparations. in recent years, public attention focused more and more on risk factors which may impair sperm quality and thereby human reproduction. in this context, for example pesticides, alcohol, cigarettes, and even mobile phones are discussed. a variety of parameters exists including sperm counts as well as sperm motility, which are considered to be two of the most important parameters to evaluate sperm quality in animal models with the final aim to assess human risk. in recent years computer assisted sperm analysis (casa) devices mostly replaced the formerly used manual counting and manual motility assessment. however, although casa offers multiple opportunities and can allow for an objective and more detailed evaluation, several pitfalls exist which can alter the results profoundly and consequently compromise the quality of the data and ultimately the validity of a study. the aim of the present study was to establish and validate the casa device tox ivos sperm analyzer from hamilton thorne and thereby to gain detailed knowledge about the practical advantages but also intricacies which may alter the obtained results. in this regard healthy adult male rats ( - weeks old) were used. ultrasonic sound resistant sperm heads were isolated from the testis and in addition, sperms were isolated from the cauda epididymis. testicular sperm head counts and sperm motility were assessed using different isolation procedures and/or instrument settings. results different instrument settings modulate both -sperm motility and testicular sperm counts. in this regard, a wide range of results including slight changes as well as false positive/negative results were obtained. in addition, the modification of the isolation procedure can lead to variable results especially for sperm motility. conclusion isolation procedures as well as instrument settings can alter the results. consequently, in an experimental setting, potential adverse effects can be confounded with methodologically mediated apparent findings exerted via inappropriate use of the device -depending on the respective conditions in the test laboratory. this study demonstrates the relevance of standardization of testing conditions adopted for computer assisted sperm analysis and the need for a robust validation prior to use in experimental settings. orai and stim proteins have been identified as central components of the highly ca + selective, store-operated current in immune cells (icrac). the molecular basis of selective orai-mediated activation of the calcineurin/nfat pathway and the crosstalk with other channel and scaffold molecules of the trpc family are still incompletely understood. using patch clamp recordings complemented by fluorescence and tirf microscopy we investigated interactions between orai and trpc in plasma membrane microdomains of rbl- h mast cells. orai -mediated crac currents, activated by passive store depletion, were found significantly reduced by over-expression of trpc . this negative impact of trpc on icrac was independent of channel function as the trpc pore dead mutant (e k) inhibited icrac to a similar extent as wild type trpc . importantly, despite a reduction in icrac, nfat translocation in trpc overexpressing rbl cells remained unchanged, or was even slightly promoted. store depletion-induced nfat translocation in rbl cells was as well unaffected by trpc e k but substantially reduced by trpc mutants with either i) eliminated fkbp /calcineurin binding (p q) or ii) deficiency in pkc phosphorylation (s a). moreover, inhibition of pkc phosphorylation by (gfx x; µm) strongly suppressed nfat signaling. we suggest trpc as a scaffold that links orai-mediated ca + -entry to nfat/calcineurin signaling within plasma membrane microdomains. neurally-induced bronchoconstriction in human and guinea pig precision-cut lung slices schlepütz m. , rieg a. d. introduction: precision-cut lung slices (pcls) are well suited to study peripheral airway responses in different species. airway tone is under close control of the autonomic nervous system and dysregulation may contribute to airway hyperresponsiveness as observed in human lung diseases such as asthma. hence, the aim of the present study was to characterize neurally induced bronchoconstriction (bc) in guinea pigs (gp) and to compare the results with those in human pcls. methods: pcls were prepared from gp or human lung tissue. nerve endings in pcls were activated by electric field stimulation (efs) or capsaicin addition. cholinergic nerve responses were proven by atropine. capsaicin was used to show excitatory nonadrenergic non-cholinergic (enanc) responses. ruthenium red or skf were used to confirm transient receptor potential (trp) channel contributions upon enanc activation. results: gp and human pcls were both sensitive to efs and airways contracted to ± % of the initial airway area (%-iaa) and ± %-iaa, respectively. in frequency response curves half maximal response was found at . ± . hz for guinea pig pcls and . ± . hz for human pcls. efs-induced bc was inhibited by atropine in both species. capsaicin contracted gp to ± %-iaa. % of human pcls were responsive to capsaicin and airways contracted to ± %-iaa, respectively. in gp ruthenium red and skf blocked capsaicin-as well as efs-induced bc. conclusion: gp and human pcls contain atropine sensitive cholinergic and capsaicin sensitive enanc nerve endings. since gp pcls were sensitive to trp channel inhibitors, the involvement of those channels can be characterized with respect to lung diseases. in conclusion, gp pcls resemble the human distal lung innervation and represent a useful model to study neural airway pharmacology. the erk / -pathway is involved in pkc-induced nox up-regulation schlufter f., xia n., förstermann u., li h. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße , mainz, germany nadph oxidases (nox) are major producers of reactive oxygen species in the vascular wall and nox is the most abundant nox isoform in human endothelial cells. we have previously shown that treatment of human ea.hy endothelial cells with phorbol myristate -acetate (pma) for h leads to an up-regulation of nox expression. this effect of pma is mediated by protein kinase cα, because it is preventable by the pkc inhibitor gö and by pkcα-sirna. the present study is aimed to investigate the signal transduction cascade downstream of pkcα. pma-induced nox up-regulation can be attenuated by pd , (an erk / inhibitor), but not by sp (a jnk inhibitor), indicating in the involvement of erk / . consistently, pma treatment leads to a sustained activation of erk / , and sirnamediated knockdown of erk / markedly reduces the pma-induced nox up-regulation. h , an inhibitor of the mitogen-and stress-activated protein kinases (msks) has no effect on the pma-stimulated nox expression, indicating that msks are not the target molecules of erk / in this scenario. on the contrary, knockdown of the transcription factor elk- by sirna significantly reduces the pma-induced nox up-regulation. in conclusion, erk / and elk- are involved in the pkcα-induced nox up-regulation. determination of spontaneous mutation frequencies in normal human mammary gland tissue using the random mutation capture technique schmalbach k., lehmann l. university of wuerzburg section of food chemistry, am hubland, wuerzburg, germany annually, over , women develop breast cancer in germany. the accumulation of genetic mutations in mammary gland tissue during lifetime may be reasonable for developing breast cancer. in particular mutations in tumor suppressor genes, e.g. p , seem to play an important role in developing cancer. up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (smf) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p gene in epidemiological studies. the only test with the potential to determine low smfs was the random mutation capture (rmc) assay, a genotype selective method which detects mutants that render the mutational sequence non-cleavable by the taqi restriction enzyme after accumulation of the target sequence. therefore, the suitability of the rmc assay to determine smf in p gene in normal human mammary gland tissue was evaluated. thus, the rmc assay was optimized concerning (i) dna isolation, (ii) pcr conditions, and (iii) amount of mammary gland tissue. (i) genomic dna from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery for cosmetic reasons, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) the target sequence in intron of p gene was captured by hybridization with a complementary uracil-containing dna-probe synthesized via polymerase chain reaction (pcr), followed by magnetic separation from the remaining genomic dna. the copy number of the target sequence was quantified by competitive pcr. the number of mutants was detected after cleavage of the target dna with taqi by means of pcr with a primer set flanking the restriction site. (iii) with g of normal mammary gland tissue a smf of . ± . x - per base pair was determined indicating the rmc assay suitable for smf determination. in conclusion, the smf in the p gene in normal human mammary gland tissue was determined for the first time, enabling the future investigation of factors influencing the smf during breast cancer development. cigarette smoke-induced release of pro-inflammatory cytokines including interleukin- (il- ) from inflammatory as well as structural cells in the airways, including airway smooth muscle (asm) cells, may contribute to the development of chronic obstructive pulmonary disease (copd). despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic amp (camp), little is known on its exact mechanism of action. we report here that next to the β -agonist fenoterol, direct and specific activation of either exchange protein directly activated by camp (epac) or protein kinase a (pka) reduced cigarette smoke extract (cse)-induced il- mrna expression and protein release by human asm cells. cse-induced iκbαdegradation and p nuclear translocation, processes that were primarily reversed by epac activation. further, cse increased extracellular signal-regulated kinase (erk) phosphorylation, which was selectively reduced by pka activation. cse decreased epac expression, but did not affect epac and pka expression. importantly, epac expression was also reduced in lung tissue from copd patients. in conclusion, epac and pka decrease cse-induced il- release by human asm cells via inhibition of nf-κb and erk, respectively, pointing at these camp effectors as potential targets for antiinflammatory therapy in copd. however, cigarette smoke exposure may reduce antiinflammatory effects of camp elevating agents via down-regulation of epac . polycyclic aromatic hydrocarbons (key marker substance benzo[a]pyrene (bap)) have been assumed to play a role in the development of bladder cancer. the objective of the present study was to unravel cellular and in particular cytoskeletal response to bap. to follow the sequential steps of chemical carcinogenesis the differential proteomic profile was analyzed at early and late time points. the study was carried out in a superficial human bladder cancer cell line (rt ) exposed to . µm bap, a subacute concentration based on results of proliferation (brdu) and dna damage (tunel) tests. cells of a human bladder cancer cell line (rt ) were exposed to . µm bap for h (n= ), wk (n= ) and wk (n= ). proteins of whole cell lysate were separated by twodimensional electrophoresis (fig. ) . differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight analysis. cortactin, actin and tubulin were immunohistochemical stained. changes in migration and colony forming ability were assessed by scratch wound-healing assay and soft-agar colony formation. results: by using several databases (uniprot, reactome, panther) the identified proteins were categorized into different functional classes such as mrna processing, translation, protein metabolic process, or several areas associated with the organization of the cytoskeleton. % of the differentially expressed proteins after h of treatment are involved in the processing of pre-mrna ( %) and protein metabolism ( %). this pattern changed after wk of treatment. then, % of the proteins affected the cytoskeleton whereas still % were categorized to protein metabolism and only % to pre-mrna processing. in the immunhistochemical staining, the treated cells appeared after wk of exposure larger and rounder predominantly due to the modifications of the actin cytoskeleton. merged images of actin and cortactin revealed that both proteins colocalized in invadopodiae. after wk, a higher number of treated cells moved toward the centre of the wound and they formed more soft-agar colonies compared to vehicle conditions, suggesting a transformation of the cells. in conclusion, bap exposure causes in an early phase an activation of the spliceosome which can led to an epithelial-tomesenchymal transition. two coordinators of a cell-type-specific splicing program, epithelial splicing regulatory proteins and , are currently being validate by pcr. fused master gel : representative -de gel of rt cells exposed to . µm bap for wk. protein spots which were differentially expressed compared to control and identified were marked. cannabinoids stimulate mesenchymal stem cell migration via a mitogen-activated protein kinase pathway schmuhl e. , ramer r. mesenchymal stem cells (mscs) are known to be involved in various regenerative processes such as cardiac, ocular, skin and bone tissue healing. however, little is known about the pharmacotherapeutical options aiming at tissue healing steps such as the mobilization and homing of mscs. here, we show that cannabidiol (cbd), a nonpsychoactive cannabinoid, stimulates the migration of human adipose-derived mscs in both boyden chamber and in vitro scratch wound assays. in boyden chambers cbd ( . - µm) was shown to promote cell migration in a time-and concentration dependent manner. this promigratory action was inhibited by am- (cb receptor antagonist) and by o- (g protein-coupled receptor [gpr] agonist). moreover, cbd activated the mitogen-activated protein kinase (mapk) pathway as evidenced by increased phosphorylation of extracellular signal-regulated kinase (erk) / . blockade of erk activation by pd prevented cbd-stimulated msc migration, whereas inhibition of p mapk by sb was inactive in this respect. furthermore, am- and o- were found to attenuate cbd-induced erk activation. an erk-dependent promigratory action was likewise demonstrated for the phytocannabinoid ∆ tetrahydrocannabinol and for the hydrolysis-stable anandamide analogue r(+)methanandamide. we conclude that cbd promotes msc migration via receptordependent erk activation, possibly contributing to tissue healing. the duffy antigen receptor for chemokines (darc) binds promiscuously many inflammatory chemokines without showing intracellular signal transduction. it is mainly expressed on endothelial cells of postcapillary venules and on red blood cells, where it acts as a transendothelial transporter of chemokines and as a chemokine sink, respectively. surprisingly, as shown for human and mouse brain, darc is also expressed at high density in the cerebellum. however, nothing is known about the function of darc in this location. we addressed this question by subjecting c bl/ wildtype and darc-deficient mice to a series of behavior experiments including morris water maze-, elevated plus maze-, rotarod-and actometer tests. while the results from the water maze experiments are ambiguous, elevated plus maze trials show a strong aversion of darc -/mice to walk to the end of the open arm, which is consistent with anxiety-like behavior. moreover, darc -/mice show greatly reduced locomotor activity, which is at least partly caused by episodes of reduced mobility occurring more frequently than in the corresponding wildtype controls (elevated plus maze, actometer). finally, darc -/mice spend a significantly reduced time on the rotating rod compared to c bl/ wildtype controls, which may indicate an impaired cerebellar function. we conclude that darc in fact modulates brain function. surprisingly, this appears to be happening under homeostatic conditions, although darc binds for the most part to inflammatory chemokines. it remains to be elucidated, how this effect can be caused by a non-signaling chemokine receptor. it may be an indirect consequence of altered brain chemokine concentrations or of as yet unknown signaling pathways activated by darc. transporter gene expression in human head-neck squamous cell carcinoma and epigenetic regulation mechanisms schnepf r. hals-nasen-ohren-klinik, kopf-und halschirurgie, friedrich-alexander-universität erlangen-nürnberg, waldstraße , erlangen, germany background: membrane transporters may affect the disposition and thereby treatment efficiency of anticancer drugs in human head-neck squamous cell carcinoma (hnscc). the gene expression profile of transporters in hnscc, however, is unknown and was evaluated in this study. moreover, we evaluated mechanisms by which transporters are regulated in hnscc. we focused on the role of the nuclear pregnane x receptors (pxr, nr i ) and epigenetic mechanisms. methods and results: real-time rt-pcr revealed a significantly increased mrna expression of slco a and slco b and a significantly decreased expression of transporters such as slco b , slco a and abcc in human hnscc tissue samples compared to adjacent normal mucosa. moreover, an association between slco b mrna levels in tumor tissues and five-year survival of hnscc patients was observed (χ = . ; p= . ; n= ). bisulfite sequencing revealed that promoter cpg islands of abcc and slco a were not methylated and thus these genes were not epigenetically silenced in hnscc tissues. in the hnscc-derived umscc- and scc- cell lines, transcript expression of transporters (e.g., abcc , slco a ; p< . ) and pxr (nr i ; p< . ) was markedly induced by the dna methyltransferase inhibitor decitabine. cotreatment with the prototypical pxr activator rifampicin significantly reversed decitabine-induced abcc and slco a expression. conclusions: transporter expression profiles significantly differed between hnscc and normal mucosa and expression levels of slco b may serve as a marker for prognosis. modulation of the epigenome with the anticancer drug decitabine substantially affects transporter expression in umscc- and scc- cells, suggesting epigenetic regulation mechanisms. moreover, interactions between epigenetic and nuclear receptor-mediated mechanisms in transporter regulation occur. this work was in part supported by the johannes und frieda marohn foundation. the role of hcn in neuropathic and inflammatory pain schnorr s. , eberhardt m. the pacemaker current ih is carried by hyperpolarization-activated cyclic nucleotidegated cation channels (hcn - ) and contributes to cellular excitability in the heart and the nervous system. hcn and hcn are the two most abundant hcn subunits in peripheral sensory neurons with hcn being the prevalent isoform in nociceptive small sized c-fibre dorsal root ganglion (drg) neurons. we examined the role of hcn for peripheral sensitization and spontaneous neuronal activity in neuropathic and inflammatory pain. we generated a conditional deletion of hcn by using a nociceptor specific cre-transgene driven by the nav . promoter. the nociceptor-specific knockout of hcn in drg neurons (nosphcn ko) was confirmed by quantitative rt-pcr and western blot. immunohistochemical staining revealed that the deletion of hcn was mainly restricted to small sized drg neurons. the conditional loss of hcn resulted in a significant reduction of ih positive small diameter drg neurons pointing to a central role of this isoform to the hcn current in nociceptive neurons. behavioral studies showed that the lack of hcn did not influence basal pain responses but led to a significant reduction in mechanosensation in both neuropathic and inflammatory pain models. however, thermosensation of the mutants was only decreased in neuropathic pain conditions. in wild-type animals, intraperitoneal, intraplantar and even intrathecal injection of the hcn channel blocker zd nearly eliminated tactile allodynia caused by inflammation in contrast to thermal hyperalgesia which remained unaffected. in contrast, pain thresholds in nosphcn ko mice did not significantly increase after pharmacological block of ih. additionally, experiments revealed that the inflammatory condition induced an upregulation of hcn protein in the spinal dorsal horn compared to saline injected mice. our results suggest that hcn might be a new target in the treatment of neuropathic and inflammatory pain. the proper functioning of the central, as well as the peripheral nervous systems is of outstanding importance to the survival and well-being of humans. yet, the integrity of neuronal systems is constantly challenged by a plethora of environmental and occupational toxins. some of these toxins preferentially target neural cells. these neurotoxins can exert their devastating effects by very different modes of action. neurotoxins may induce apoptosis or necrosis of neurons, or interfere with axon growth and elongation. these processes can be identified by specialized in vitro tests. furthermore, neurotoxins have been described to alter glial function which may compromise the viability of surrounding neurons. as another important mode of action, several neurotoxins act on neurotransmitter receptors, thereby altering signal propagation within neuronal networks. countless natural and synthetic substances have been characterized for their effects on neurotransmitter receptors and today can be used for detailed studies of receptor function. however, environmental toxins of anthropogenic origin and occupational toxins that both represent constant sources for human exposure are still poorly studied with respect to their effects on neurotransmitter receptors. thus, the need for a better understanding of the susceptibility of neurotransmitter systems for toxic effects exerted by these substances is of outstanding importance for the protection of human health. here, we introduce an imaging-based approach for the screening of the effects of potential and known neurotoxins on neurotransmitter receptors of intact cells in vitro. different neuronal cells were tested for their sensitivities for classical neurotransmitters using life-cell imaging experiments. in more detail, we examined the proportion of responding cells and determined the ec values for the most prominent neurotransmitters in cell lines widely used for in vitro neurotoxicity studies on the one hand, namely sh-sy y and lumes cells, and primary mouse neurons on the other hand. with these data at hand, we are now able to identify and characterize the effects of neurotoxins on receptor function in chronic, as well as acute exposition paradigms. the use of an in vitro imaging-based physiological test system is at the interface between non-functional in vitro approaches and in vivo toxicity tests, thus, giving mechanistic insight into neurotoxic processes without requiring animal experiments. apomorphine acts on trpa channels scholze a., schaefer m., hill k. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. - , leipzig, germany apomorphine is a non-narcotic derivative of morphine which acts as a dopamine agonist and is clinically used to treat "off-states" in patients suffering from parkinson´s disease. adverse effects of apomorphine treatment include dopaminergic effects such as nausea, but also ulceration and pain at the injection site. we wanted to test whether an activation of trp (transient receptor potential) channels in sensory neurones contributes to the perception of pain after apomorphine injection. while the warm/heat receptors trpv , trpv , trpv , and trpv and the cold receptor trpm were insensitive towards apomorphine treatment, trpa could concentration-dependently be modulated by apomorphine. low micromolar apomorphine concentrations potently activated heterologously expressed trpa channels in a stably transfected cell line (hek -trpa ), as well as natively expressed trpa in cultured dorsal root ganglion neurones. on the other hand, when using higher concentrations of apomorphine, we observed inhibition of trpa activity. previous studies have shown that subcutaneously administered apomorphine produces a biphasic dose response relationship in rats, inducing hyperalgesia at low doses whereas high doses of the substance cause antinociception. from our studies we conclude that such in vivo effects of apomorphine are presumably mediated by activation/inhibition of trpa expressed in sensory neurones agonist binding to a g protein-coupled receptor (gpcr) induces a conformational change of the receptor protein, which results in the activation of receptor-associated heterotrimeric g proteins [ ] . in radioligand binding studies, conducted to investigate ligand binding to specific gpcrs, receptors are usually probed with radioantagonists. as in other gpcrs [ ] , agonists of the muscarinic m receptor exhibit biphasic kinetics and biphasic competition curves with radioantagonists, indicating a more complex situation probably caused by g protein interactions. here, we present a detailed study of the binding of agonists to muscarinic m receptors including the novel super-high affinity agonist iperoxo and a differential chemical knockout of g proteins. in addition to membrane homogenates living cells were employed. we demonstrate that the high affinity fraction in biphasic curves does not differ between selected full agonist and is sensitive to pertussis toxin, thus indicating that this receptor population is associated with gi proteins. however, despite promiscuous signalling properties of m receptors, the low affinity fraction is not associated with any other g protein, since low affinity binding is insensitive to high concentrations of guanylnucleotides and cholera toxin. moreover, high affinity agonist binding appears solely in membrane homogenates but not in experiments conducted with living cells, probably due to their high intracellular concentration of guanylnucleotides. taken together the chemical knock-out of g proteins revealed that the high affinity binding of agonists in membrane homogenates is associated with the interaction of the muscarinic m receptor with gi proteins. the low affinity binding cannot be related to another g protein, although the muscarinic m receptor exhibits promiscuous g protein signalling properties. interestingly data obtained with living cells do not reveal any high affinity binding of agonists. prolonged stress leads to a dysregulation of the hypothalamus-pituitary-adrenal (hpa)axis and may affect the sensitivity of pain perception. however, it is not yet known whether the alterations of hpa-axis and increased pain sensitivity are related. to create a long lasting stressful situation, male wistar rats were exposed to a restraint-stress for h daily over a period of two weeks. the effect of stress on the hpa-axis was determined by adrenal morphology and stress hormone levels, the influence on mechanical pain sensitivity was evaluated by the randall-selitto paw pressure test. on day the animals exhibited a significant mechanical hyperalgesia. they also showed increased acth and corticosterone plasma levels and an enlarged zona fasciculata of the adrenal gland, indicating a dysregulation of the hpa-axis. for testing the correlation of hpa-axis dysregulation and hyperalgesia a persistent increase in plasma corticosterone in wistar rats was generated by the administration of corticosterone via the drinking water for two weeks. these animals also showed an increased mechanical nociceptive sensitivity with an accompanied decrease of the adrenal glands and reduced acth levels. the results show that chronic stress leads to a dysfunction of the hpa-axis with an accompanied mechanical hyperalgesia which can be mimicked by oral administration of corticosterone. thus, this in-vivo test system may provide a new animal-friendly pharmacological model for stress-related pain disorders. the alternaria mycotoxins aoh and ame induce cyp a and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor mycotoxins are secondary metabolites of fungi including the genus alternaria (black mold). alternaria fungi are known to infest different types of foodstuffs and produce diverse toxins amongst them the mycotoxins alternariol (aoh) and alternariol methyl ether (ame) which are potential carcinogens. as planar compounds, aoh and ame are preferentially metabolized by cytochrome p (cyp) a and a . the most prominent regulator of cyp a is the dimeric transcription factor complex ahr/arnt, which is activated by planar ligands. therefore we studied the activation of ahr/arnt by aoh and ame and monitored cyp a induction in murine hepatoma cells (hepa- c c ). indeed, aoh and ame enhanced the levels of cyp a in hepa- c c cells but not in cells with inactivated ahr (hepa- c c ) or arnt (hepa- c c ). furthermore, we studied the cytotoxicity of aoh and ame. by using a fluorescence-based microscopic readout we measured effects on cell counts, apoptosis, senescence and micronuclei formation. both aoh and ame reduce the cell number and the cell nuclei show drastic morphological changes e.g. enlargement after aoh treatment or micronuclei formation. the observed effects where, except for the induction of apoptosis, independent of ahr/arnt. in summary, aoh and ame activate the ahr/arnt pathway to induce cyp a expression and apoptosis. however, the predominant cytotoxic effect of aoh and ame in hepatoma cells is a profound reduction in cell numbers, which is independent of the ahr/arnt pathway. special purpose databases are the first place for researchers in the life sciences to obtain expert curated data. naturally, such resources are limited in terms of timeliness and comprehensiveness. the literature database pubmed alone lists more than , , scientific abstracts, and , are newly added every year. the protein sequence database uniprotkb stores over , , sequences, a hundred times more than ten years ago. turning these data into meaningful information and making it accessible to both humans and computers have become an essential part of biological discovery and biomedical research. text mining techniques have proven useful to extract the missing links in areas such as drug-target and drug-disease prediction, the extraction of mutation-phenotype associations, or the prediction of protein-protein and protein-ligand interactions. by systematically extracting information from available literature, reports or patents, text mining techniques can help to refine existing categorical knowledge stored in databases, and hence will support drug repositioning, the discovery of novel cancer biomarkers, or help to understand causes of hereditary diseases. in the area of regulatory toxicology we developed go r, the first knowledge-based search engine for alternative methods to animal experiments. the system not only helps retrieving information on the availability of alternative methods that allows for replacing, reducing or refining animal experiments, but also provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information and data. the up-to-date taxonomicstructured "table of contents" provided by go r allows for search in the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. the semantically enriched platform supports the user during the query formulation, allows for bibliographic analysis, and reveals existing relations to replacement, reduction, and refinement of animal experiments. impaired cardiac excitation-contraction-coupling in mice with complete inactivation of the crem gene schulte j. s., tekook m., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße , münster, germany the structurally related transcription factors camp response element binding protein (creb) and camp response element modulator (crem) mediate a regulation of gene transcription in response to camp and are expressed in the heart. mice with complete inactivation of crem display impaired cardiac contraction and relaxation, decreased expression of serca and down-regulation of β -adrenoceptors. to elucidate the underlying functional mechanisms on the cellular level we here investigated cellular electrophysiology and ca + -cycling in ventricular cardiomyocytes from crem ko mice (ko). adult ventricular cardiomyocytes were isolated from ko and wildtype (wt) mice (age - weeks) and subsequently used for experiments within hours after isolation. action potentials (aps) were recorded from ventricular cardiomyocytes (perforated patch, whole cell current clamp inactivation of crem seems to have no consequences for ap duration and possibly associated ion channels but leads to impairment of ca + cycling and sarcomere shortening under basal conditions well explaining the previous findings in vivo. our results show that crem is essential for a regular excitation-contraction coupling in the mouse heart. (supported by the izkf münster) new mechanistic insights in no/cgmp actions in the vasculature schulte k., koesling d., universitätsstr. , bochum, germany hypertension, a major risk factor for cardiovascular diseases, is associated with vascular changes resulting in increased vascular contractility und vascular peripheral resistance. a prominent factor in the development and maintenance of hypertension is the reninangiotensin-aldostrerone system. angiotensin ii (ang ii) is the main mediator of this system and a powerful vasoconstrictor. ang ii increases the intracellular ca + concentration thereby activating myosin light chain (mlc) kinase, which enhances mlc phosphorylation and subsequent vascular contraction. opposite to angii-induced vascular contraction, no/cgmp pathway promotes vascular relaxation by decreasing ca + concentration and lowering mlc phosphorylation. responsible for mlc dephosphorylation is the mlc phosphatase (mlcp), whose activity is regulated by different phosphorylations. phosphorylation of mlcp via rhoa-activated rho-kinase enhances phosphatase activity while phosphorylation via the cgmp-dependent protein kinase has been proposed to decrease enzymatic activity. to investigate the interplay of angii with the no/cgmp pathway, we treated wild-type and ko mice lacking the cgmp forming no receptor, no-gc , with angiotensinii ( . mg / kg bw / d) for two weeks. in addition to various cardiovascular parameters, physiological changes in vascular reactivity of aortic rings of angii-treated wt and no-gc ko mice were assessed in organ bath experiments and correlated with biochemical parameters as the phosphorylation state of mlc, mlcp and rho-kinase activities examined by immunoblot analysis. analysis of cgmp levels revealed that angii treatment decreased cgmp in wt mice to levels comparable to those of the ko mice which were unaltered by the treatment. our study will provide further mechanistic insights in the molecular interactions between constrictor and dilator stimuli in the vasculature. nanomaterials are already used today and offer even greater use and benefits in the future. the progress of nanotechnology must be accompanied by investigations of their potential harmful effects. for airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. while standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. we have investigated two genotoxic endpoints -alkaline comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow -in a combined study hours after a single instillation of µg gold nanoparticles (np) into the trachea of male adult wistar rats. the administration of three test materials differing only in their primary particle size ( -, -and -nm) did not lead to relevant dna damage in the mentioned tests. the measurement of clinical pathology parameters in bronchoalveolar lavage fluid (balf) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. minor histopathology findings occurred in the lung of the animals exposed to -nm and -nm sized nanomaterials. in conclusion, under the conditions of this study the different sized gold np tested were non-genotoxic and showed no systemic and local adverse effects at the given dose. platelet dense granule secretion mediates platelet-dependent enhancement of tumor cell transmigration and formation of metastases schumacher d., strilic b., wettschureck n., offermanns s. mpi für herz-und lungenforschung offermanns, ludwigstr. , bad nauheim, germany tumor cell metastasis to distant organs is the primary cause of mortality in cancer patients. tumor cells leave the primary tumor, intravasate, survive in the circulation and extravasate through the endothelial cell layer to grow in the target organ. it has long been known that blood platelets play an important role in tumor cell survival and dissemination, but the mechanism by which platelets promote metastasis remained unclear. given that platelets are found closely associated with tumor cells shortly after vascular arrest, we explored whether platelets can facilitate the transmigration of tumor cells through the endothelium and thereby promote extravasation of tumor cells into the organ parenchyma. the ability of various mouse and human tumor cells like lewis-lung carcinoma cells (llc ), b f melanoma cells or human neuroblastoma cells (sh-sy y) to transmigrate through an endothelial cell layer was strongly enhanced by seeding tumor cells together with mouse or human platelets onto the endothelial cell layer. this indicates that platelets facilitate tumor cell transmigration in vitro. we found that platelet granule secretion is involved in this process as supernatant from platelets incubated with tumor cells but not from resting platelets was sufficient to enhance tumor cell transmigration. additionally, no platelet-mediated increase of tumor cell transmigration was observed in dense granule secretion-defective platelets of munc - deficient mice. thus, dense granule secretion is required for platelet-dependent tumor cell extravasation in vitro. while the growth and weight of primary tumors after subcutaneous injection of llc and b cells was indistinguishable between wild-type mice and animals lacking munc - , the number of metastases in the lung was strongly reduced in munc - -deficient animals. the strong decrease in formation of metastases in munc - deficient mice was also observed after i.v. injection of llc and b f cells. thus, platelet dense granule secretion plays a critical role in tumor cell metastasis by enhancing tumor cell transmigration through the endothelial cell layer. formation of dna adducts in mouse tissues by the brassica ingredient methoxy- -indolylmethyl glucosinolate and its break-down product -methoxy- indolylmethyl alcohol schumacher f. , engst w. glucosinolates are secondary metabolites present at substantial levels in cruciferous vegetables, such as broccoli and cabbage. after injury of plant tissue they are activated by the enzyme myrosinase to form various electrophilic degradation products like isothiocyanates. we previously showed that -methoxy- -indolylmethyl ( -mim) glucosinolate (or neoglucobrassicin) is a potent genotoxicant in bacterial and mammalian cells after activation by myrosinase. the induction of mutations could be correlated with the formation of dna adducts [ ] . we have identified and synthesized the major dna adducts n -( -mim)-dg and n -( -mim)-da. moreover, we developed a highly sensitive uplc-esi-ms/ms method for selective mrm quantification of these adducts using stable-isotopic labeled adducts as internal standards. while the plant enzyme myrosinase is probably almost completely inactivated after cooking the vegetables, the glucosinolates reach the gut mostly intact due to their good heat and ph stability. enzymes of individual intestinal bacteria are able to cleave the glycosidic bond of the glucosinolates, which leads to the formation of reactive metabolites within the gut lumen. we were able to detect significant levels of n -( -mim)-dg and n -( -mim)-da in a dose-dependent manner in the large intestine of mice treated orally with isolated -mim glucosinolate. the peak levels of n -( -mim)-dg and n -( -mim)-da in the murine large intestine were reached h after a single administration of µmol -mim glucosinolate/ kg body weight. the oral application of the relatively stable metabolite -mim alcohol to mice led to the formation of identical dna adducts. this benzylic alcohol can be activated by sulfotransferases to an electrophilic sulfo conjugate. in contrast to the intact glucosinolate the orally administered -mim alcohol generated significant levels of n -( -mim)-dg and n -( -mim)-da not only in the large intestine but also in other tissues, such as the liver, of mice. [ ] h. glatt, c. baasanjav-gerber, f. schumacher, b. h. monien, m. schreiner, h. frank, a. seidel, w. engst, chem.-biol. interact., ( ) human pregnane x receptor genotype of the donor but not of the recipient is a risk factor for delayed graft function after renal transplantation schwab m. , , schaeffeler e. delayed graft function (dgf) is an important immediate complication in renal transplantation significantly contributing to decreased long-term allograft survival. in addition to donor-and recipient-related risk factors altered renal excretion of xenobiotics by membrane transporters may influence dgf as well. using recipients' and donors' dna, we assessed the impact of genetic variants on dgf for the transporter proteins, pglycoprotein (abcb ) and multidrug resistance protein (abcc ), and the nuclear pregnane x receptor (pxr/nr i ), regulating the transcription of drug metabolizing enzymes and membrane transporters. in our local cohort of transplant patients (n= ) dgf occurred in . %. logistic regression analysis using four different genetic models (i.e. co-dominant, dominant, recessive and log additive) indicates that only the donor's pxr rs tt genotype was significantly associated with dgf (recessive model: or, . ; %ci, p= . unadjusted) , even after correction for multiple testing (p= . holm-adjusted). when we performed multivariate analysis including genetic and clinical co-variates (i.e. age, gender, hla mismatches, panelreactive antibodies, immunosuppression using cni, t cell-depleting agents, anti-il- receptor antibody and steroids, cold or warm ischemia time, living vs deceased donors or graft loss) again dgf was significantly associated only with the pxr rs tt genotype of the donor (recessive model: or, . ; % ci, . - . ; p= . unadjusted) which held true after correction for multiple testing (p= . ). for abcc variants only the donor rs t>a genotype correlated with dgf by univariate (or, . ; %ci, p= . unadjusted) as well as by multivariate analysis (or, . ; %ci, p= . ; table ) but not after correction for multiple testing (p= . ). for variants in the abcb gene no significant associations with dgf were detected for both the donor's and the recipient's genotype. in summary, our findings suggest for the first time that pxr may be a risk gene for the development of dgf independently from previously identified risk factors. supported by the robert-bosch foundation, stuttgart, germany, the bmbf grant is c (berlin, germany) and by the ferdinand eisenberger grant of the german society of urology (id krs /fe- ). formation, morphology and structural requirements for formation of microtubule protrusions by clostridium difficile toxin cdt schwan c., kruppke a. s., nölke t., aktories k. institut für experimentelle und klinische pharmakologie und toxikologie i, albertstr. , freiburg, germany clostridium difficile is an anaerobe, gram-positive pathogen. it causes antibioticassociated diarrhoea and pseudomembranous colitis by production of the rho gtpaseglucosylating toxins a and b. recently emerging hypervirulent clostridium difficile strains additionally produce the binary adp-ribosyltransferase toxin cdt (clostridium difficile transferase). cdt is taken up via receptor-mediated endocytosis after binding to the lipolysis stimulated lipoprotein receptor (papatheodorou et al., pnas ) . in the cytosol, cdt adp-ribosylates actin at arg , thereby actin polymerization is blocked, resulting in disruption of the f-actin network. cdt and other binary actin-adp-ribosylating toxins induce redistribution of microtubules in the cell interior and formation of long (> µm) microtubule-based protrusions at the surface of intestinal epithelial cells which increase bacterial adherence (schwan et al., plos pathog ). the clostridial actin-adp-ribosyltransferases influence the dynamicity of microtubules and their capture at the cell cortex by indirectly affecting different microtubule regulating proteins like clasp and acf . besides the influence of cdt on microtubule regulatory proteins, the formation of protrusions depends on plasma membrane composition. depletion of cholesterol, the breakdown of sphingomyelin or inhibition of sphingolipid-synthesis reduce the formation of microtubule-based protrusions. surprisingly, most of the cdt-induced processes contain membrane-tubules derived from the endoplasmatic reticulum (er). the remodeling of the er is microtubule dependent and is mainly mediated by stim that usually functions as a calcium sensor in the er and activates the store operated orai calcium ion channels in the plasma membrane. the data suggest that toxin-induced changes of the microtubule system including alterations of the er, may affect trafficking and er-dependent signalling. bilobalide is a neuroprotective constituent of ginkgo biloba with an unknown mechanism of action. in the present study, we first used microdialysis in mice to evaluate changes in the extracellular fluid of the brain during and after stroke. microdialysis probes were implanted into the striatum of cd- mice, and dialysates were obtained while a monofilament was inserted for min via the common carotid artery (cca) to block perfusion through the middle cerebral artery (mca). while glucose levels dropped immediately upon middle cerebral artery occlusion (mcao), glutamate concentrations in the microdialysates -as measured with a cma analyzer -rose extensively during ischemia to more than % of baseline level. both glucose and glutamate levels recovered rapidly when mcao was terminated after min. when bilobalide ( µm) was perfused into the striatum through the microdialysis probe during mcao, glucose levels dropped but the neurotoxic rise of glutamate was significantly attenuated and reached only % of baseline level (p< . ). in the following experiments, we investigated the activity of mitochondria in ischemic brain. ischemia was induced by mcao, and ischemic as well as "healthy" tissue from the opposite hemisphere was obtained. mitochondria were isolated and mitochondrial respiration was monitored using the oroboros ® oxygraph. significant deficits of respiration were observed after ischemia. in the healthy hemisphere, the respiratory states (leak i+ii, complex i+ii+iv, oxidative phosphorylation (oxphos) and electron transport system (ets) capacity) showed a decrease of oxygen consumption to - % of sham-operated mice. in the ischemic hemisphere, several values were lower at % of sham-operated mice (leak i+ii, complex ii+iv,oxphos and ets) whereas complex i showed a remarkably low respiratory capacity of % of baseline. direct addition of bilobalide ( µm) to post-ischemic mitochondria caused a -fold increase of complex i activity in vitro. pretreatment of mice with bilobalide ( mg/kg i.p.) one hour before mcao caused a significant, -fold improvement of complex i respiration when measured ex vivo. these data clearly indicate that bilobalide targets mitochondrial processes within the respiratory chain, preserving complex i function during ischemia. this action likely explains its neuroprotextive activity in vivo. unreacted resin monomers such as -hydroxyethyl methacrylate (hema) are environmental stressors released from dental composites after incomplete polymerization. the production of reactive oxygen species (ros) is a major response of cells to monomer exposure. moreover, adverse effects of monomers including delayed cell differentiation or mineralization processes, dna damage or apoptosis are associated with increased ros production. the intracellular redox homeostasis is controlled by the major non-enzymatic antioxidant glutathione (gsh), and antioxidant enzymes. here, we hypothesized that cells exposed to hema responded by a differential expression of antioxidant enzymes such as superoxide dismutase (sod- ), catalase (cat) or glutathione peroxidase (gpx / ). raw . mouse macrophages were exposed to hema ( - mm) for h, and protein expression was analyzed by western blotting. to study the influence of intracellular gsh on enzyme expression, gsh synthesis was reduced by the inhibitor buthionine sulfoximine ( µm bso), or enhanced by -oxothiazolidine- -carboxylate ( mm otc) and n-acetyl cysteine ( mm nac). expression of sod- found in untreated cultures was decreased in the presence of hema and even further reduced by bso. in contrast, nac counteracted hemainduced inhibition of sod- expression. cat expression was not detected in untreated cells, however, the enhanced expression of cat in cells exposed to hema indicated the decomposition of abundant levels of hydrogen peroxide. the minor influence of bso or otc showed that expression of cat was independent of gsh levels while a decrease of hema-induced cat expression in the presence of nac indicated reduced oxidative stress. gpx / was expressed in untreated cultures, and its down-regulation by bso indicated that this enzyme was primarily responsible for h o decomposition. the inhibitory effect of hema on gpx / expression was enhanced by bso but counteracted by otc or nac. these findings indicate that h o is the predominant reactive oxygen species generated in the presence of dental resin monomers like hema. abundant h o production leads to the activation of cat expression and a feed-back inhibition of sod- expression. the hema-induced reduction of gpx / expression is most likely a consequence of reduced gsh levels because of the formation of glutathione disulfide (gssg) or by gsh-hema adducts. the life-threatening effects of certain organophosphorus compounds such as soman or fenamiphos cannot be antagonized adequately by the treatment with atropine and oximes. alternative approaches are necessary. since the adequate restoration of disturbed muscle function is considered to be life-saving, a model is needed for screening of potentially therapeutic substances. an established model for the development of such new therapies is the diaphragm preparation. however, this model requires a large number of animals and experimental available time frame is limited to some hours. here, the organotypic nerve-muscle co-culture may be an appropriate alternative, because a large number of specimens with low numbers of animals and a long period of investigation over several days is possible. in the present study, the restoration of vx paralysed muscle function with obidoxime was investigated by using both models. slices of spinal cord and muscle tissue were dissected from mice embryos (e - ) , fixed to coverslips and incubated in roller tubes for about - weeks. spontaneous muscle activity was recorded by video microscopic techniques and was quantified offline. muscle force production in mice diaphragm preparations was elicited by indirect field stimulation technique in a chamber organ bath and quantified as time-force diagrams (auc) that were expressed as relative changes of the muscle force compared to the control data. application of the nerve agent vx ( . µm) resulted in a strong reduction of muscle activity in the co-culture and of muscle force production in the diaphragm muscle. after obidoxime ( µm) was added spontaneous muscle activity in the co culture recovered from . ± . hz to . ± . hz (control . + . hz) . muscle force remained stable over the next days. the vx-blocked muscle force of diaphragm was restored to . ± . % by obidoxime compared to control. muscle force production after indirect stimulation was stable for hours only. our results suggest that the organotypic nerve-muscle co-cultures may be an appropriate tool for the screening of new therapeutic approaches in restoration of blocked neuromuscular transmission. moreover, the model offers an additional advantage as long-term experiments may be performed and pre-and postsynaptic effects may be assessed directly. additionally, the number of experimental animals could be reduced. the modulation of gene expression by the transcription factor crem (camp responsiveelement modulator) represents a fundamental mechanism of gene control in response to elevation of intracellular camp levels. in vascular smooth muscle cells (vsmcs) crem is involved in the regulation of cell proliferation and apoptosis supporting its relevance for vascular proliferative diseases. mice with a global inactivation of crem (cko) showed a significant increase in neointima formation after ligation of the carotid artery and an increase of atherosclerotic plaque formation after high fat diet on an apoe knockout background compared to wildtype controls (wt). on the cellular level a crem deficiency was associated with a . fold increased proliferation rate of primary vsmcs after stimulation with the platelet-derived growth factor (pdgf; n= from isolations). microarray analysis and subsequent realtime-rt-pcr validation revealed that the alpha-type platelet-derived growth factor receptor (pdgfra) the regulator of g-protein signaling (rgs ) and peptidylprolyl isomerase a (ppia) were . - . fold upregulated in pdgf treated cko vsmcs compared to wt vsmcs (n= ). transcripts of rgs ( . fold, ) and ppia ( . fold, were also upregulated in the carotid artery of cko mice in comparison to wt mice (n= - ). we conclude that crem deficiency is associated with transcriptional changes of rgs , pdgfra, ppia, which might explain the increased proliferation rate in cko vsmcs and the increased responsiveness to pathophysiological conditions. (supported by the izkf münster). the role of non-catalytic p and p subunits in regulating phosphoinositide kinase γ by gβγ and h-ras shymanets a. phosphoinositide -kinase γ (pi kγ) controls a plethora of cellular responses. pi kγ, a heterodimer formed by non-catalytic p or p and catalytic p γ subunits, is regulated by gβγ and ras. earlier we speculated that p binds to gβγ to translocate cytosolic pi kγ to the plasma membrane, enabling direct activation of p γ (brock et al., j. cell biol. ) . however, the p subunit does not function as gβγ adapter (kurig et al., pnas ) . since the impact of each non-catalytic subunit in regulating p γ by gβγ and ras still remains elusive, we studied their role in detail. gβ γ variants harbouring mutations in positions involved in interaction with gα subunit were purified from sf cells and tested for their ability to activate pi kγ. we observed that p , but not p , was able to rescue the stimulatory activity of gβ γ mutants incapable to activate p γ (shymanets et al., biochem. j. doi: . /bj ). to further study the functional impact of the non-catalytic subunits on pi kγ regulation, we have designed phospholipid vesicles containing similar amounts of recombinant pi kγ variants. although p /p γ exhibited stronger sensitivity to gβ γ than p γ, the activity of vesicles-associated p /p γ was significantly higher as compared to vesicles-associated p /p γ or p γ in the absence and in the presence of gβ γ . to study an effect of ras proteins on pi kγ variants, recombinantly expressed h-ras was purified from sf cells. the posttranslational processing and lipidation of the protein was verified by mass spectrometry analysis. the impact of h-ras on regulation of p /p γ and p /p γ differed, which may explain integration of pi kγ variants in different signalling pathways. taken together, p and p subunits implement discrete functions in respect to (i) membrane recruitment of pi kγ and (ii) regulation of enzymatic activity by gβγ and h-ras. preparation of consolidated exposure scenarios for mixtures under reach sica m., dorn s., mostert v. dr. knoell consult gmbh, marie-curie-str. , leverkusen, germany under reach, formulators of mixtures need to include substance-related information into extended safety data sheets (esds), if mixtures are classified as dangerous according to the dangerous preparation directive (directive / /ec). one way to add information on substances into esds of mixtures is to generate exposure scenarios (ess) for mixtures. in order to fulfil this task, two approaches have been developed for the identification of the risk-determining substances (lead substances) in the mixtures: the critical component approach (cca) relies on dnels and pnecs for all substances, their concentrations in the mixtures as well as substance and use-specific availability parameters (echa, ) . in contrast, the dpd+ method is based on the legislation for classification of preparations (directive / /ec). the dpd+ method defines a lead substance for each route of human exposure and for the aquatic environment (cefic, ) . however, each of these methods has certain limitations. the aim of the present work is to improve the preparation of consolidated ess for a number of mixtures and provide information about their safe use. to this end, we first adopted information on risk management measures (rmms) and operational conditions (ocs) of the lead substances using the dpd+ methodology. at the same time, we considered the specific conditions of use of the mixtures (e.g. spraying, brush painting). we then conducted risk assessments by deriving dnels for the mixtures and using exposure modelling tools recommended under reach (e.g., ecetoc tra, riskofderm, art). we compared the outcome of these assessments with results obtained from the application of the dpd+ methodology. the work presents the results of application of dpd+ approach and the cca and indicates possible improvements for the risk assessment of mixtures. to check for seasonal and weather dependent influences in the prescription rate of drugs used to treat cardiovascular and respiratory diseases (atc codes c and r) a survey covering all prescriptions of a specimen german general regional health insurance (aok plus, data for saxony, largest health insurance service, approx. % of all saxonian citizens are inscribed to this service) for the years to was analysed on a monthly basis. the number of prescriptions for cardiovascular drugs changed approximately +/- % around the mean for the different month without a clear seasonal pattern. for respiratory drugs only the systemic anticholinergics and drugs used to treat obstructive lung disease displayed a distinct seasonal pattern with a - % above average prescription figure during spring time (february to may) and a to % trough in late summer/autumn (july to october). the data have to be analysed for further cofounders (e.g. influenza prevalence, environmental conditions etc.) to fully understand the fluctuations observed. the prescription rate for cardiovascular drugs and respiratory drugs seems to be influenced by multiple factors aside seasonal influences. pasteurella multocida toxin prevents osteoblast differentiation by activation of heterotrimeric g proteins siegert p., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abt. i, albertstr. , freiburg i. br., germany pasteurella multocida toxin (pmt) is a major virulence factor of pasteurella multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. the toxin modulates various signaling pathways by acting on the heterotrimeric g proteins gαq, gα / and gαi. pmt activates gq to increase inositol phosphate production via phospholipase cβ and alteration of gene expression via the jak/stat pathway. the toxin also activates rhoa via gαq and gα / family proteins. we showed that the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. because pmt is the causative agent to induce progressive atrophic rhinitis in pigs, which is characterized by loss of nasal turbinate bones leading to a twisting and/or shortening of the snout, we studied the effect of pmt on bone cells. here we studied the effect of the toxin on osteoblast differentiation in st- cells and in primary osteoblasts from rat calvaria. st- cells are stromal derived cells, which can be differentiated into osteoblasts or adipocytes. the toxin inhibits the differentiation of st- cells into osteoblasts studied by determination of specific osteoblast markers. additionally, pmt represses the induction of transcription factors essential for osteoblast differentiation. moreover, the principal pathways activated by pmt to induce these effects were investigated. ventilator-induced lung injury (vili) is a serious problem in intensive care medicine. its mechanisms are only incompletely understood, although it is widely accepted that ventilation-induced inflammation (biotrauma) makes an important contribution. the isolated perfused mouse lung (ipl) is a valuable tool to investigate the mechanisms of vili. several studies have shown considerable differences between various mouse strains with respect to lung mechanics and inflammatory responses. therefore, we hypothesized that the pulmonary responses to mechanical ventilation differ between c bl/ and balb/c mice. in addition, this study introduces the novel half lung technique that allows to obtain lung tissue from the same mouse at two different time points. isolated perfused mouse lungs from c bl/ or balb/c were subjected to high ( cmh o) or low pressure ( cmh o) ventilation for minutes. after minutes the left lung was removed and used for western blot analysis. the right lung was ventilated for another minutes. by the end of experiment the right lung was removed and qrt-pcr performed. during the whole experiment perfusate sample were taken from the venous catheter and used for protein quantification by elisa. it was possible to remove half of the lung and to further ventilate the other half without acute changes in lung mechanics. in both strains high pressure ventilation elicited a significantly higher cytokine release than low pressure ventilation. c bl/ mice showed higher tnf, il- β and amphiregulin levels after high pressure ventilation, whereas balb/c exhibited increased production of cxc chemokines (cxcl- , cxcl- ) and il- . kinase activities (jnk, akt, erk / , p map kinase) were increased in high pressure ventilated animals, but were strain independent. the novel half lung technique builds on the well established ipl method. it permits to separately analyze the left and the right lung at different time points during continual ventilation. this method reduces animal numbers by % and allows statistical within subject analysis. using this method, the present study showed that inflammatory response to mechanical ventilation differ between c bl/ and balb/c. these findings show that the biotrauma response in mice depends on the strain that is studied. macrophages play an important role as an integral part of the first line of immune defense. two different macrophage populations have been described. m macrophages produce proinflammatory cytokines and are involved in inflammatory processes. by contrast, m macrophages release anti-inflammatory cytokines and extracellular matrix components. they can enhance wound repair and angiogenesis, but they can also promote tumor progression. recently, industrial nanoparticles have raised concerns because of their putative toxic effects. on the other hand, specifically designed nanoparticles can be used as clinical diagnostics and as drug carriers for pharmacotherapy. thus, investigations on the interactions of engineered nanoparticles with living cells and organisms are of great importance. macrophages as phagocytosing cells scavenge nanoparticles circulating in the bloodstream. therefore, we analyzed how nanoparticles with different surface functionalization might affect functions of human macrophages. monocytes were isolated from buffy coats and differentiated to macrophages with macrophage colony-stimulating factor. carboxy-(ps-cooh) and amino-(ps-nh ) functionalized polystyrene nanoparticles were produced by the miniemulsion polymerization process and the average particle size, the polydispersity index and their zeta potential were determined by dynamic light scattering. the macrophages were cultured in the presence or absence of different concentrations of ps-cooh and ps-nh nanoparticles for up to days. analysis of cell viability revealed that ps-nh but not ps-cooh concentration-and time-dependently reduced the macrophage viability. by annexin v/propidium iodide double staining we could show that ps-nh trigger apoptosis in macrophages. we further polarized macrophages to either m or m using ifn-γ and lps or il- , respectively. these macrophage populations were characterized by their expression of extracellular markers by flow cytometry and their production of cytokines by elisa. the effects of functionalized polysterene nanoparticles on the cytokine production and surface marker expression of m and m macrophages were analyzed. our data indicate that surface functionalization is a critical parameter in the nanoparticle-induced toxicity in human macrophages. this work was supported by the dfg spp . loos c., lunov o., syrovets t., simmet t. ulm university institute of pharmacology of natural products & clinical pharmacology, helmholtzstr. , ulm, germany nanoparticles are currently used for various medical applications including imaging, diagnosis and drug delivery. due to particle size and surface area, their fundamental properties differ significantly from those of corresponding bulk materials. nanoparticles circulating in the blood are mainly sequestrated by the reticuloendothelial system that consists predominantly of phagocytic macrophages. macrophages express a variety of cellular receptors for sensing and internalizing particular material like viruses, microorganisms, and foreign particulate matter including nanoparticles. therefore, a detailed understanding of the intracellular fate and processing of the nanoparticles by macrophages is indispensable for controlled biomedical applications of nanoparticles. introducing distinct surface modifications, one might control nanoparticle uptake by different cell types and thereby target specific tissues and cellular compartments. tumor cell lines are frequently used as models for primary cells to analyze the effect of nanoparticles on cells. here we show that carboxy-(ps-cooh) and aminofunctionalized (ps-nh ) polystyrene nanoparticles of ~ nm in diameter are internalized by human macrophages, thp- monocytic leukemia cells, and by pmadifferentiated thp- cells via different mechanisms. in buffer, macrophages and thp- rapidly internalize both types of nanoparticles, yet, the carboxy-functionalized particles were taken up to a higher extent. the uptake of both nanoparticles was drastically reduced in media containing serum. using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between cd receptors and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization by thp- cells occurred via dynamin iidependent endocytosis. by contrast, pma-differentiated thp- cells took up the particles via macropinocytosis. in line with the in vitro data, more intravenously applied ps-cooh particles accumulated in liver tissue, whereas ps-nh were preferentially targeted to tumor tissue. these data show that the amount of particle internalization, the uptake mechanisms, and kinetics differ significantly among primary cells and model tumor cells, whether differentiated or not, and that they are further critically dependent on the particle opsonisation by serum proteins. this work was supported by the dfg spp . specifically designed and functionalized nanoparticles hold great promise for a variety of biomedical applications. to ensure their safe application, such particles require a rigorous analysis of their effects on cell functions. here we demonstrate that aminofunctionalized polystyrene nanoparticles (ps-nh ) of ~ nm in diameter in contrast to carboxy-(ps-cooh) and nonfunctionalized (ps) particles induce an nlrp inflammasome activation and the subsequent release of il- β in human macrophages. amino-functionalized ps nanoparticles induced time-dependent lysosomal destabilization followed by release of lysosomal enzymes. this resulted in mitochondrial damage and formation of reactive oxygen species. accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (txnip). liberated txnip, in turn, interacted with the nlrp protein resulting in a conformational change of the pyrin domain of the nlrp protein as predicted by molecular modeling. txnip interaction with nlrp led to assembly of the nlrp inflammasome complex, to caspase- activation, and release of il- β. using an in vitro knockdown approach, we showed that ps-nh induced activation exclusively of nlrp , whereas other inflammasomes remained unaffected. treatment of macrophages with n-acetyl-l-cysteine, a scavenger of reactive oxygen species, abolished both, the caspase- activation and the subsequent release of il- β caused by ps-nh nanoparticles. these data reveal a novel mechanism of the nlrp activation induced by amino-functionalized nanoparticles and provide a strategy as to how such an effect can be functionally antagonized by supplementation with a radical scavenger. this work was supported by the dfg spp . the semi-permeable barrier of the endothelial cell lining of the blood vessels has important synthetic and metabolic functions including transport of cells and biomolecules, regulation of vascular smooth muscle tone, and control of hemostasis. plasmin is a serine protease, which is generated from its zymogen plasminogen under physiological and pathological conditions. small amounts of plasmin are produced in the context of contact activation during inflammation. consistently, increased generation of plasmin has been reported during atherosclerosis. we have shown previously that plasmin, in addition to its role in fibrinolysis, could induce proinflammatory activation of various cells including monocytes, macrophages, and dendritic cells. therefore, we analyzed how plasmin might affect the functions of endothelial cells, which could be relevant during inflammation and atherosclerosis. using flow cytometry, western immunoblotting and fluorescent microscopy, we show that endothelial cells of different origin express the plasmin receptor complex composed of annexin a and s a . addition of plasmin to human umbilical vein endothelial cells (huvec) induced timeand concentration-dependent cytotoxic effects in the cells. in addition, within min plasmin triggered a rapid and prolonged expression of free radical oxygen species (ros) in endothelial cells as analyzed by microscopy and fluorometry using the rossensitive dye carboxy-h dcfda. the ros production in endothelial cells was accompanied by cell detachment. fluorometric and western blot analysis of caspase activation in the cells treated with plasmin showed that plasmin induced apoptotic cell death in endothelial cells, which was evident already several hours after exposure to plasmin. thus, plasmin might induce production of ros in endothelial cells, their detachment and apoptosis, events which might be relevant for the development of atherosclerosis. this work was supported by the dfg. sesquiterpene lactones (stl) comprise a large group of secondary plant metabolites that constitute the active principle of a number of traditional anti-inflammatory phytomedicines. specifically helenalin and parthenolide have recently gained considerable attention as lead compounds or putative therapeutics for the treatment of inflammation and possibly cancer. both compounds have been shown to interfere with the signal transduction through inhibition of the nuclear factor κb (nf-κb). whereas the inhibitory effects of the stl on nf-κb are undisputed, their molecular mechanism of action remains a matter of debate. surface plasmon resonance (spr) analysis allows label-free measurement of molecular interactions. yet, analysis of the interaction of immobilized recombinant proteins with small molecular ligands remains a technically challenging task. in the present study we used spr technology to investigate the molecular interaction of the stl helenalin with putative intracellular target proteins such as the nf-κb protein p /rela, the catalytic subunits of the ikk complex, namely ikkα and ikkβ, and the intracellular antioxidant glutathione (gsh). at physiological ph . , helenalin interacts with rela (k d = . µm), yet it failed to bind either ikkα or ikkβ. hence, when dna with nf-κb binding consensus sequence was immobilized on sensor chips, the binding of rela was inhibited by helenalin with an ic of . µm. moreover, we provided several lines of evidence that stl may modify rela on cysteine by a michael-type addition. this interaction was confirmed by molecular docking that identified the best matching interaction between rela and helenalin with predicted hydrogen bonding interactions between helenalin and residues arg , lys , gly and ile of rela. consistent with our hypothesis that helenalin interacts with sulfhydryl groups at ph . , helenalin was also able to interact with reduced, but not oxidized, glutathione with a kd of µm, though no significant interaction was observed at ph . . thus, we showed that the sesquiterpene lactone helenalin interacts with the nf-κb protein rela but not with ikkα or ikkβ. moreover, at physiological ph, helenalin does not interact with glutathione to any significant extent. direct interaction of helenalin with rela leading to inhibition of rela-dna binding and transactivation might present the molecular mechanisms underlying the anti-inflammatory effects of stls. although nanosized materials are quickly taken up by macrophages, our understanding of the involved processes is still rather limited. therefore, we analyzed the uptake of diagnostically used carboxydextran-coated iron oxide nanoparticles of two different sizes, superparamagnetic iron oxide nanoparticles of nm (spio) and ultrasmall superparamagnetic iron oxide nanoparticles of nm (uspio), by human macrophages. by pharmacological and in vitro knockdown approaches, the principal uptake mechanism of macrophages for both particles was identified as clathrin-mediated, scavenger receptor a-dependent endocytosis. further, we created a mathematical model of the nanoparticle uptake by macrophages that permitted determination of key parameters of endocytotic process, such as the uptake rate, the mean uptake time, the number of particles taken up by a cell, and the correlation between the number of internalized particles and their extracellular concentration. the model also provided information on the individual and collective wrapping time of the nanoparticles and described the relation between biophysical parameters such as cytoskeletal forces, membrane elasticity, and the uptake time. finally, we gained information on the minimal linear spacing between simultaneously acting neighboring endocytotic pits that contain single nanoparticles and govern the collective uptake process. the calculated parameters were further confirmed experimentally using spinning disc confocal microscopy. thus, the new model provides important insights into the biophysical processes involved in endocytosis of nanoparticles by human macrophages. this work was supported by the dfg spp . prostaglandins (pg) are hormones which are formed during inflammatory processes from arachidonic acid by cyclooxygenases and prostaglandin synthases [ ] . in the subsequent metabolism, in which the five-membered ring is dehydrated, α,β-unsaturated carbonyl compounds are generated [ , ] . these come along with mercapto groups of amino acids in a michael addition reaction associated with activation of cellular enzyme cascades [ ] that potentially contribute to their possessed antiinflammatory, antineoplastic and antiviral effects [ ] . however little is known so far about possible adverse health effects.we addressed the question whether selected cyclopentenone prostaglandins (cypg) exhibit potential mutagenic and genotoxic properties in the hamster lung fibroblast cell line v . induction of dna damage was investigated by single cell gel electrophoresis assay (scge). the impact of cypg on cellular redox status was detected by total glutathione (tgsh) assay. the induction of micronuclei and apoptosis was determined by staining with ', -diamidino- -phenylindole (dapi). furthermore the hypo-xanthine-guanine phosphoribosyltransferase (hprt) assay was used for mutagenicity testing. , followed by prostaglandin a (pga ), showed the most distinctive genotoxicity, i.e., induction of micronuclei, and apoptotic effects. furthermore, the dpgj and pga -induced significant decrease in the tgsh level in v may contribute to the observed increase in oxidative dna-damage. however, none of the tested cypg exhibited mutagenic properties in the hprt assay. in conclusion, a potential in vitro genotoxicity of cypg has been observed which may be involved in carcinogenesis associated with chronic inflammation. parabens and methylisothiazolinone are used as preservatives in personal care products. sensitization to parabens and methylisothiazolinone is relatively rare considering their wide use in cosmetics, but only few quantitative or clinical data exist. therefore, we have tested methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, phenyl-and benzylparaben , and methylisothiazolinone in the loose-fit coculture-based sensitization assay (lcsa) developed by our working group. the coculture of primary human keratinocytes and allogenic dendritic cell-related cells (dc-rc) in this assay emulates the in vivo situation of the human skin. sensitization potential of the test substances was determined by flow cytometric analysis of the dc-rc maturation marker cd . determination of the concentration required to cause a half-maximal increase in cd -expression (ec ) allowed a quantitative evaluation. the irritative potential of the substances was assessed by -aad ( -amino-actinomycin d)-staining. the concentration required to devitalize % of the examined cells compared to a zero control was termed ec %. parabens exhibited weak (methyl-, ethyl-, propyl-and isopropylparaben) or strong (butyl-, isobutyl-, pentyl-and benzylparaben) sensitizing potential, phenylparaben was found to be a moderate sensitizer, with ec -values ranging from . µmol/l (pentylparaben) to . µmol/l (methylparaben). due to a pronounced cytotoxicity (ec % = . µmol/l), we could not estimate an ec -value for methylisothiazolinone. sensitization potential of parabens correlated with side chain length. parabens showed no (methyl-and ethylparaben) or weak irritative potential (propyl-, isopropyl-, butyl-, isobutyl-, phenyl-and benzylparaben) , only pentylparaben was rated to be irritative. apart from phenyl-and benzylparaben, irritative potential also correlated with side chain length but did not correlate strictly with the sensitization potential. overall, we were able to demonstrate and compare the sensitizing potential of parabens in this in vitro test. it was weak for methyl-and propylparaben, the most commonly used parabens. furthermore, we showed an irritative potential for most of the perservatives. thus the lcsa is a useful in vitro test to compare the sensitizing potential of xenobiotics. phosphorylation of the neurodegeneration-related septin by protein kinase dyrk a at serine affects protein stability soppa u. septins are gtp-binding proteins forming heterooligomeric complexes and filaments by interactions of the family members. these complexes have important functions by building scaffolds for proteins involved in cell cycle or cell polarity but their subcellular distribution as well as their regulation remain largely unclear. septin (sept ) was found in neurodegeneration related protein aggregates and is associated with migration of cortical neurons. here we first describe a potential mechanism for regulation of sept by phosphorylation via dual specificity tyrosine-phosphorylation-regulated kinase a (dyrk a). dyrk a is overexpressed in down syndrome and supposed to be involved in neurodevelopment and neurodegeneration. by site directed mutagenesis of flag tagged mouse sept and overexpression in hela cells we identified serine as the major phosphorylation site of dyrk a and generated a phosphospecific antibody. transient coexpression of sept and dyrk a in hela cells increased phosphorylation of serine by % in relation to basal phosphorylation. in contrast, cotransfection of the kinase deficient dyrk a mutants k r and d n did not increase serine phosphorylation. moreover we could show, that inhibition of kinase activity by the dyrk a inhibitor harmine reduced phosphorylation of exogenous sept at serine about % in hela cells. furthermore, down regulation of dyrk a by rna interference lead to decreased phosphorylated serine . these results indicate that endogenous dyrk a contributes to sept phosphorylation in hela cells. finally we analyzed protein stability of wild type sept compared to the phosphorylation resistant s a mutant in hela cells by inhibition of translation with cycloheximide. we found that in living cells the sept s a mutant is more stable than wild type sept . in summary, our results suggest phosphorylation at serine by dyrk a as a novel mechanism to regulate sept stability and indicate a possible link of these proteins in cellular processes. is formaldehyde a good example for a "genotoxic carcinogen" with a threshold mode of action? speit g. institut für humangenetik, universität ulm, ulm, germany formaldehyde (fa) induces toxic and genotoxic effects in directly exposed cells (site of contact). several studies in which fa was administered to rats by inhalation showed evidence of tumor induction in nasal epithelium. there is also some epidemiological evidence that fa causes nasopharyngeal cancer in humans. although fa is a known mutagen, it is still a matter of discussion whether carcinogenesis is primarily mediated via a mutagenic mode of action. there is evidence that cytotoxicity and induced proliferation are the main causes for tumor formation. however, a decisive role of mutagenesis cannot be excluded and a mutagenic mode of action has to be considered for risk estimation. the basic assumption is that mutagens have a non-threshold mode of action. a threshold mode of action for a chemical is likely when a substance with a known mutagenic potential does not induce mutations at low concentrations due to a specific type of reaction with the genetic material and / or physiological protective mechanisms. because fa is a directly acting dna-reactive substance, a threshold mode of action may only be considered because of physiological protective mechanisms. after inhalation, pre-lesion protection occurs by unspecific binding to mucus, cellular proteins and glutathione. furthermore, fa is efficiently inactivated by enzymatic pathways. if fa reaches and damages the nuclear dna, dna repair mechanisms act as efficient postlesion protection mechanisms. in vivo inhalation studies with rats indicated that fa induces primary dna damage in the nasal epithelium but increased mutation frequencies were not measured. data are now available to show the relative distribution of endogenous versus exogenous dna adducts in different locations of the nose and other organs. considering the fa concentrations present in every living cell and the background levels of endogenous dna adducts, appropriate risk assessment and the identification of practical thresholds for fa-induced genotoxicity become feasible. fraud and misconduct in clinical trials steffen c. formerly federal institute for drugs and medical devices clinical trials unit, kurt-georg-kiesinger-allee , bonn, germany plagiarism in scientific publications has been the subject of public ("guttenplag") and scientific debate. plagiarism violates, however, "only" the intellectual property of its authors. in clinical trials, misconduct may also endager the health of patients. the suppression or falsification of data in clinical trials may mislead patients and doctors to use worthless treatments. clinicians may be provoked to repeat these trials, thereby wasting time and money. in clinical trials, misconduct includes everything from suppression or their repeated publication, the "correction" of unwanted results to the complete invention of the data, their intentional or negligent misinterpretation leading to the publication of biased conclusions from otherwise correctly performed clinical studies. the peer review system cannot protect against fraud, as few reviewers will have the means and the time to reevaluate original data. they will have to rely on these data, the calculations and statistics that are presented to them. some kinds of fraudulent behavior, such as double publications, can be found in the review process, but this is also time-consuming and depends on a helpful librarian. other kind of fraud, as the suppression of patient data in clinical trials (the deletion of "non-responders", according to cinderella: the good ones go into the pot, the bad ones go into your crop) can only be detected by the national competent authorities performing an inspection according to good clinical practice. even if the results of such an inspection become public as in the case of ukrain (gansauge et al. ) , there is no institution that will further analyze and publish the misconduct or initiate the retraction of the incriminated paper. a german agency similar to the us office of scientific integrity could foster good clinical practice in germany. although it is sometimes very difficult to decide whether improper results arise from fraud or error, a bias for the source of funding is obvious. trials funded by for-profit organizations were significantly more likely to recommend the experimental drug as treatment of choice (als-nielsen et al. ) . medicinal products with disputed efficacy such as orally applied enzymes for systemic action, bacterial preparations for irritable bowel syndrome and food supplements are to be reviewed with special attention. further examples from the author's experience will be presented. ]i favors further kca opening, kca may establish a feed-forward regulation of ca + influx. in the present study we analyzed whether kca channels of sk and bk type play a role in sustaining ca + oscillations at g -to s-phase transition of primary mouse tumor cells and in human breast cancer cells, aiming towards a better understanding how kca modulate tumor cell proliferation. methods: kca expression was quantified by qpcr in human breast cancer biopsies, transgenic mmtv/c-neu + mouse mammary tumors and primary tumor cells derived thereof. the identity of the tumor cells was verified by gliolan staining. proliferation of the primary mammary tumor cells in the presence or absence of bk and sk modulators was tested using a real time cell monitoring system. the cellular dna content as a measure for cell ploidity was determined by propidium iodide staining and flow cytometry. changes in [ca + ]i oscillations and peak amplitude were determined using ca + indicator fura- am. results: sk , but not bk, expression is detectable in human and mouse breast cancer biopsies and in primary tumor cells derived from the mmtv/c-neu + mouse model. sk inhibition by tram- dose-dependently ( , to µm) inhibits the growth of primary mammary tumor cells probably by a g cell cycle arrest. ca + oscillations in proliferating mmtv/c-neu + tumor cells were ablated upon pharmacologic inhibition of sk channels. -dependent cell cycle progression is dependent on sk activity. blocking sk disrupts a feed-forward loop that coordinates ca + influx via trp or crac channels in tumor cells. the consequences of sk inhibition in mammary tumors in vivo will be discussed. synthesis of a triphenylphosphonium substituted derivative of -hydroxymethyl- -methylpyrroline n-oxide stolze k. esr combined with spin trapping is a well-known analytical approach to detect free radicals formed in various biological systems, e.g. superoxide, hydroxyl and a series of carbon-centered free radicals, which are involved in oxidative stress. our aim was to modify the established spin trap , -dimethyl-pyrroline n-oxide (dmpo) with a functional side chain, which can be used further as anchor for moieties enabling the spin trap to penetrate mitochondrial membranes, such as the positively charged triphenylphosphonium substituent. several synthetic routes were tested to introduce a -carboxybutyltriphenylphosphonium-substituent to the spin trap -hydroxymethyl- -methylpyrroline noxide (hmmpo). while the activation of the carboxy group via the corresponding chloride was not successful, the use of a mixed anhydride with acetic acid appeared to be a promising way, although the reaction is considerably slower. preliminary spin trapping experiments have been performed with model systems generating superoxide, hydroxyl-, and carbon-centered radicals. othman e. m., stopper h. universität würzburg toxikologie, versbacher str. , würzburg, germany type diabetes mellitus (dm ) is a growing health problem affecting more than million people worldwide. it is associated with severe acute and chronic complications that negatively influence both the quality of life and survival of affected individuals. epidemiological studies clearly indicate that the risk of several types of cancer (including pancreas, liver, breast, colorectal, urinary tract and female reproductive organs) is increased in diabetic patients. diabetic patients are exposed to oxidative stress which plays a pivotal role in the pathogenesis of both micro-and macro-vascular complications. this is due to a decreased antioxidant capacity and chronic exposure to increased levels of reactive oxygen species (ros). since the insulin resistance in dm leads to hyperinsulinemia we studied the cellular consequences of the elevated insulin level and showed that it generates superoxide anions (o -) and dna damage by a nadph oxidase dependent mechanism in cultured cells. in addition, we found elevated genomic damage in the lymphocytes of diabetic patients as well as oxidative stress and genomic damage in kidneys of diabetic rats. this effect of insulin may contribute to the pathogenesis and progression of dm complications including the elevated cancer risk. the classical transient receptor potential (trpc) channel subfamily is regarded as nonselective, calcium permeable cation channels involved in a wide range of physiological events that require calcium signaling. until now, the specific roles of trpc channels in neuronal function are still elusive. given that trpc is able to form receptor-operated heterotetrameric channel complexes with other trpc channel subunits, we investigated the role of trpc for receptor-operated calcium influx in the heterologous expression system as well as in neurons. for this electrophysiological whole-cell measurements, fluorimetric calcium measurements, mn + quenching and qpcr analysis were applied. furthermore, the effect of trpc knock-down on neuronal migration was monitored performing scratch assays, videomicroscopy and g-actin/f-actin assays. employing these techniques, we found that recombinant trpc was not able to function as a homomeric channel. instead, trpc subunits formed functional receptor-operated heteromeric channel complexes with trpc , , , , and . heteromers containing trpc subunits showed significantly decreased calcium permeation in heterologous cell systems. mutation of amino acids in the putative pore forming region of trpc further reduced calcium permeability. in gnrh neurons endogenously expressing trpc , , , and , downregulation of trpc by shrna resulted in increased basal cytosolic calcium concentrations and elevated calcium permeability. trpc was not involved in store-operated cation influx in gnrh neurons. moreover, trpc suppressed the migration of gnrh neurons without affecting cell proliferation. these findings suggest a novel regulatory mechanism relying on the expression of trpc and the subsequent formation of heteromeric trpc channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration. the transient receptor potential melastatin- (trpm ) is a calcium permeable nonselective cation channel that can be activated by the neurosteroid pregnenolonesulfate (pregs) or heat. trpm is expressed in various tissues, including insulin-secreting βcells and a subset of sensory neurons from dorsal root (drg) and trigeminal ganglia. the ability of pregs to evoke trpm -like currents in pancreatic β-cells and to induce insulin secretion indicated its involvement in blood glucose regulation. however, trpm -/mice show so far no metabolic deficits but further investigations are recommended to evaluate its function in insulin secretion. further studies showed that trpm is a nociceptor channel involved in sensing heat and inflammatory thermal hyperalgesia. we performed a calcium-based screening of a compound library (spectrum collection) that identified several natural compounds as trpm blockers. the most potent blockers were the citrus fruit flavonoids hesperetin and naringenin as well as ononetin, a chalcon from ononis spinosa. the ic values of the substances are in the low micromoles ranges. electrophysiological whole cell measurements as well as calcium measurements confirmed the potency of the trpm blockers. furthermore, we could show that these blockers are effective on endogenous trpm in drg neurons from mice and isolated β-cells. by drinking grapefruit juice naringenin could be consumed in concentrations that are sufficiently high enough to block trpm activity in vivo. in sensory neurons, trpm may exert similar functions as trpv . thus, trpm blocker could bear a therapeutic potential for analgesic treatment. xtt-based cell viability assay was used to determine the half-maximum effect concentration (ec ) for the investigated composite components in hgf. following concentrations of substances were used to determine the induced double strand dna breaks (dsbs): / × ec , / × ec , / × ec , and × ec . each experiment was performed at least four times. hgf were incubated with various concentrations of substances for a period of hours. induced dna double-strand breaks (dsbs) were tested by the γh ax focus assay, which is a direct marker for dsbs using anti γh ax antibodies. for quantitative γh ax analysis foci in cell nucleus were counted by eye down using a fluorescence microscope. each experiment was performed at least four times. in the xtt test following ec values of substances were found (mmol/l;mean +/-sem): tmp(eo) ta a, b . ± . ; , -hddma b, c . ± . ; etma a, c ; . ± . ; a significantly (p < . ) differently to , -hddma, b significantly (p < . ) differently to etma, c significantly (p < . ) differently to tmp(eo) ta. after six hours of exposure with tmp(eo) ta at . mm there were induced . γ-h ax foci-formations in hgfs, at . mm . foci, at . mm . foci and at . mm . foci. after exposure with , -hddma at . mm there were induced . γ-h ax foci, at . mm . foci, at . mm . foci and at . mm . foci. after exposure with etma at . mm there were induced . γ-h ax foci, at . mm . foci, at . mm . foci and at mm . foci. the negative controls dmso and medium cultures displayed . - . γ-h ax foci/cell. it was found that the induction of foci/cell were concentration-dependet for all xenobiotics in the order of: , -hddma > tmp(eo) ta > etma. these results show that dental composite components can induce dsbs in primary oral cells and therefore these substances demonstrate a genotoxic potential. effects of antioxidants on the dna-toxicity of dental (co)monomers in human gingival fibroblasts styllou p. , scherthan h. unreacted (co)monomers can be released from restorative dental materials and may show biologic activity after ingestion in the human organism. in previous studies the mutagenic/carcinogenic effect of dental monomers/co-monomers (e.g. methacrylates) on the human dna was demonstrated. in this study the effects of the antioxidants vitamin c and n-acetylcysteine on the dna toxicity of the (co)monomers triethylenglycol-dimethacrylate (tegdma) and -hydroxyethyl methacrylate (hema) was investigated. the induction of dna double-strand breaks with (co)monomers alone and in combination with antioxidants was investigated in human gingival fibroblasts (hgf). hgf were incubated with substances without or with antioxidants for a period of hours. induced dna double-strand breaks (dsbs) were tested by the γh ax focus assay, which is a direct marker for dsbs using anti γh ax antibodies. for quantitative analysis of the γ-h ax test, foci were counted by the same investigator by eye down the fluorescence microscope. each experiment was performed at least four times. the halfmaximum effect concentration ec (mmol/l) of triethylenglykol dimethacrylat (tegdma) and -hydroxyethyl methacrylat (hema) was taken from of a previous study after using xtt-based cell viability assay. tegdma induced significantly (p < . ) higher dsbs compared to hema ( . ± . vs . ± . ). the mean number of cells scored and the standard deviation (sd) were calculated. when cells were exposed to tegdma in combination with the antioxidant vitamin c an increase of dsbs was observed ( . ± . ), compared to tegdma alone. when cells were exposed to hema in combination with vitamin c an increase of dsbs was observed ( . ± . ), compared to hema alone. when cells were exposed to tegdma in combination with the antioxidant nacetylcysteine a decrease of dsbs was observed ( . ± . ), compared to tegdma alone. when cells were exposed to hema in combination with n-acetylcysteine a decrease of dsbs was observed ( . ± . ), compared to hema alone. these results show that dental (co)monomers can induce dsbs in primary oral cells. it also shows for the first time that the genotoxic potential may be reduced by the addition of the antioxidant n-acetylcysteine. purpose: we aimed to investigate the role of superoxide and peroxynitrite generated by genetically destabilized enos for the development of endothelial dysfunction and vascular remodelling. methods: a mutant of bovine enos in which cys was replaced by ala (c a) resulting in destabilization of enos has been generated (enos-c a). transgenic mice carrying c a were generated on a c bl/ background using the endotheliumspecific tie- promoter. by breeding these mice with enos knockouts (enos-ko), mice that express enos-c a (enos-ko/enos-c a-tg) exclusively in the endothelium were obtained. unilateral common carotid artery ligation experiments were performed in c bl/ , enos-ko, and enos-ko/ enos-c a-tg to study a role of destabilized enos for vascular lesion formation. results: western blot analysis confirmed the expression of enos in enos-ko/enos-c a-tg in aorta ( . ± . %, n= ), skeletal muscle ( . ± . %, n= ) and myocardium ( . ± . %, n= ) and revealed an increased phosphorylation of enos on ser / ( ± %) as compared to c bl/ (p< . , n= ). endothelium-specific overexpression of destabilized enos induced a large increase in superoxide and peroxynitrite formation in the aorta and the heart of enos-ko/enos-c a-tg (p< . , n= - ), which was abolished by nos-inhibitor l-nitroarginine (l-na) suggesting enos-c a as a source of elevated radical generation. endothelium-specific introduction of enos-c a at ~ % of c bl/ level almost completely restored aortic endotheliumdependent relaxation. experiments with l-na, soluble guanylyl cyclase inhibitor odq, peg-catalase and no-scavenger fe(detc) indicated that endothelium-dependent relaxation in enos-ko/enos-c a-tg is nos-and cgmp-dependent and nomediated. four weeks after the carotid artery ligation, neointima formation, media thickening and luminal narrowing were observed in the ligated arteries of all studied genotypes (p< . , n= - ). consistent with vasoprotective roles of enos, neointima formation was accelerated in enos-ko (n= - , p< . ). despite significantly higher vascular levels of nitrotyrosine and peroxynitrite, neointima formation in enos-ko/enos-c a-tg was substantially lower then in enos-ko and tended to be similar to c bl/ . conclusions: increased vascular superoxide and peroxynitrite formation caused by destabilization of enos does not induce endothelial dysfunction in healthy mice and has negligible effect on neointima formation. fenton reactivity as a determining parameter for the interaction of manganese oxide nanoparticles with lung epithelial cells sydlik u. , bieschke c. nanoparticles consisting of manganese oxide have been suggested for several innovative technological approaches, including the use in nanomedicine and diagnostics. therefore, the interaction of such nanoparticles with human target cells is of particular interest for the success of nanomedical approaches but also with regard to unintended side effects. to address this problem, we tested different kinds of manganese nanoparticles (mnnp) in an in vitro system which we earlier evaluated for proliferative, apoptotic, and pro-inflammatory endpoints induced by carbon nanoparticles (cnp). mnnp were synthesized by hydrothermal treatment of manganese salt solutions. the particles were subsequently characterized by scanning electron microscopy and dynamic light scattering. biological and toxic effects of the generated particles were studied in comparison to carbon nanoparticles (cnp) in experiments with rat and human lung epithelial cells (rle- tn and hbe o-). cytotoxicity was determined as measures of membrane damage (lactate dehydrogenase release) and metabolic activity (water soluble tetrazolium conversion). the oxidative capacity of the particles as well as the generation of intracellular oxidative stress was monitored using dichlorofluorescein diacetate in cell free experiments and flow cytometry assays (facs), respectively. the particle-specific phosphorylation of src family kinases (sfk) and mitogen activated protein kinases erk / were investigated using western blot techniques. after physico-chemical characterization, a set of three mnnp consisting of mn o or mno with significant differences in size and shape were selected. according to the different oxidation stages of manganese, the particles showed significant differences in fenton reactivity in the cell free system. these data did not reflect the capacity of the particles to induce intracellular oxidative stress. the characteristic to trigger membranedependent signaling processes, however, was correlated to the intrinsic oxidative capacity of mnnp than to the ability to induce intracellular ros. furthermore, the metabolic activity (wst) was negatively correlated with intracellular ros, indicating a link between mitochondrial activity and ros generation. none of the particles had effects on the membrane integrity of the cells. the data demonstrate that mnnp, unlike other poorly soluble nanoparticles (e.g. cnp), mainly trigger adverse health effects through ros production via the fenton reaction. acute ozone induced airway inflammation does not effect resting human sympathetic nerve traffic tank j. numerous mediators released in inflammatory and neuropathic pain states activate gprotein-coupled receptors (gpcrs) and modulate nociception via activation of gs, gi/o, g / , or gq/ g proteins. each of the g protein-coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of the individual signaling pathways in vivo has not yet been worked out. the gq/ signaling branch is of particular interest in pain research because it leads to the activation of phospholipase c, protein kinase c, and the release of calcium from intracellular stores. using a conditional gene-targeting approach we generated double-deficient mice lacking gaq and ga selectively in nociceptors to investigate the contribution of the entire gq/ signaling pathway in nociceptors towards the regulation of pain. we observed that mice lacking gq/ in nociceptive neurons show normal development of the nociceptive circuitry. the nociceptor-specific loss of gq/ results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in gq/ deficient mice, suggesting a novel function for gq/ in tonic modulation of acute nociception. patch-clamp recordings revealed changes in voltagedependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in nociceptors upon a loss of gq/ , whereas potassium currents remained unchanged. our results indicate that the functional role of the gq/ branch of g-protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain. provocation of arrhythmic events in single primary isolated adult mouse ventricular cardiomyocytes tekook m., fehrmann e., schulte j. s., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße , münster, germany ap duration and ca + cycling are altered in cardiomyocytes of different genetic mouse models. here, we systematically tested various protocols to study the inducibility of arrhythmic events in mouse cardiomyocytes. adult ventricular cardiomyocytes were isolated from wildtype (wt) mice by enzymatic digestion and subsequently tested to trigger arrhythmic events within hours after isolation. in patch clamp experiments (perforated patch, whole cell current clamp) aps were stimulated for second ( hz; hz; hz + s -stimulus after - ms) followed by a s rest period. the resting-membrane-potential (rmp) was observed over cycles. we observed rmp-fluctuations of different length (amplitude < mv; % of observed cardiomyocytes, mean events/cell; < s: %, . ; < s: %, . ; > s: %, . ), spontaneous depolarizations (> mv; %, . ) and spontaneous aps ( %, . ). intracellular ca + transients and sarcomere shortening were measured after loading cardiomyocytes with indo- /am. after preconditioning ( min/ hz) cells were measured under basal and continuous isoprenaline ( - m) stimulation (iso). a min pacing period was followed by a min interval of no pacing. pacing frequency was reduced after each cycle ( hz, . hz, . arrhythmic events were provocable with stimulation/rest protocols both by field stimulation and direct stimulation via patch pipette. however, low stimulation frequencies seem to lead to distinct destabilization of cardiomyocytes probably due to ca + overload. we conclude that the tested stimulation protocols are able to provoke arrhythmic events even in wt single adult mouse ventricular cardiomyocytes and may serve as a tool to test for the relevance of potential proarrhythmic substrates in mouse models. ruhr-universität bochum pharmakologie ma nord , bochum, germany cgmp is a second messenger involved in many (patho-)physiological processes such as smooth muscle relaxation, platelet inhibition, and the development and plasticity of the nervous system. however, it is not fully understood how cgmp regulates these and other processes on a mechanistic level. in particular, the existence and functional relevance of global and local cgmp signaling domains is not clear. recently, highly specific genetically-encoded optical biosensors for cgmp have been developed. these cgmp indicators are either based on fluorescence resonance energy transfer (fret), with cgmp-binding domains sandwiched between fluorescent proteins with overlapping spectra, or they consist of a single fluorescent protein fused to cgmp-binding domains. with these cgmp indicators, the spatiotemporal dynamics of cgmp signals, which result from the interplay between cgmp-producing guanylyl cyclases, cgmp-binding effectors, and cgmp-degrading phosphodiesterases (pdes), can be monitored in living cells. here, we report the generation of transgenic mice expressing the fret-based cgmp indicators cgi and cgi with apparent cgmp affinities of nm and nm, respectively. one mouse line expresses cgi driven by a cmv promoter in neural cells. fret experiments were performed with isolated cerebellar granule neurons, hippocampal neurons, and astrocytes. we observed nitric oxide (no)-induced cgmp transients and analyzed the capability of other agents (natriuretic peptides, glutamate) to induce cgmp responses. in another mouse line, the sm alpha promoter directs cgi expression specifically to smooth muscle cells (smcs). fret experiments have been performed with smcs isolated from aorta, bladder and colon, as well as with intact vessels in the retina and cremaster muscle of transgenic animals. in primary smcs we studied responses to no, atrial and c-type natriuretic peptide (anp,cnp). in different smc types we observed differences in the overall ability to react to these stimuli and in the kinetics of the induced cgmp transients. we also studied the effects of pde inhibitors on the no-, anp-, and cnp-induced cgmp signals. importantly, we were able to detect cgmp transients upon no stimulation in intact vessels of the retina and cremaster. we conclude that the cgi transgenic mouse lines are valuable tools to visualize cgmp signals in living cells in vitro and, possibly, also in vivo in the intact animal under physiological and pathophysiological conditions. current research data dealing with pharmacotherapy of α-ama intoxication shows a particularly high variability regarding the protective effect of silibinin. the aim of this study was therefore to evaluate the influence of the frequently used clinical antidotes benzylpenicillin, silibinin and their combination in human hepatocyte culture intoxicated with α-ama. cytotoxicity and apoptosis testing were performed after two and five days of simultaneously exposure to α-ama and/ or tested antidotes. to quantify apoptosis, necrosis and cell viability, we used cell death detection elisa plus®, toxilight® bioluminescence assay and cell proliferation kit ii (xtt). furthermore, we analysed the ways of apoptosis by using immunohistochemistry (differential detection of caspase , and , activated caspase , and aif). exposure of hepatocytes to α-ama at concentrations of , µm, , µm and µm resulted in disorder of cell cultures, apoptosis and reduction in cell viability compared with unexposed hepatocytes. in hepatocyte cultures treated with benzylpenicillin at concentrations of µm and mm, silibinin at µm and µm and a combination of both ( µm benzylpenicillin and µm silibinin, mm benzylpenicillin and µm silibinin), toxilight® values in the supernatant and xtt values were not significantly different from untreated cultures. simultaneous exposure to α-ama (at all tested concentrations) and benzylpenicillin, silibinin or combination of both showed higher cell viability and lower values of necrosis compared to the cultures exposed to α-ama alone (exept µm silibinin at , µm α-ama); however, in both groups dosed with benzylpenicillin the highest hepatocyte viabilitiy was observed. this protective effect was particularly revealed at high α-ama concentrations ( , µm and µm). in conclusion, our data suggest that benzylpenicillin in monotherapy is more effective than in combination with silibinin or silibinin alone. glucocorticoids (gcs) are important hormones in the regulation of metabolic homeostasis. synthetic gcs, such as dexamethasone (dex), play a fundamental role in the treatment of inflammatory diseases. there are numerous side effects of a dex therapy, e.g. the development of hypertension. in the pathogenesis of hypertension oxidative stress is a crucial factor. glucocorticoid-induced hypertension has been shown to be associated with an imbalance between nitric oxide (no) and superoxide. however, the source of this elevated superoxide production is unknown. we hypothesize that an uncoupling of the no synthase (enos), a key mediator of vascular homeostasis, may contribute to dex-induced oxidative stress. incubation of human endothelial cells (ea.hy ) with dexamethasone led to a decrease in enos expression at mrna and protein levels. this effect of dex was timeand concentration-dependent. since the major cause of enos uncoupling is a deficiency of its co-factor tetrahydrobiopterin (bh ), we analyzed the amount of bh in ea.hy by hplc. a concentration-dependent reduction of bh and also bh (dihydrobiopterin) could be demonstrated in response to treatment with dexamethasone. bh can be synthesized endogenously by two different pathways -the de novo pathway (from gtp with gtp cyclohydrolase i, gch , acting as the rate-limiting enzyme) and the salvage pathway (conversion of sepiapterin to bh involving dihydrofolate reductase, dhfr). treatment of ea.hy cells with dex decreased mrna and protein expression of both gch and dhfr. because bh is the major "coupling switch", an enos uncoupling is likely to occur in dex-treated cells. in summary, we showed that dex treatment led to a reduced availability of the important co-factor bh which could lead to enos uncoupling. the uncoupled enos may possibly contribute to glucocorticoid-induced vascular oxidative stress. the cellular oncoprotein c-fos is a major component of the heterodimeric transcription factor ap- and has been commonly found over-expressed in tumors and cancer cells of different origin. previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (uvc) light. here we demonstrate that in human telomerase-immortalized vh tert foreskin fibroblasts (behaving like primary cells) and sv -immortalized gm fibroblasts, uvc-triggered induction of c-fos protein is a delayed and long-lasting event. sustained up-regulation of c-fos went along with transcriptional stimulation of the nucleotide excision repair (ner) gene xpf, carrying an ap- binding site in the promoter. c-fos mrna was induced in a biphasic manner. an immediate c-fos mrna expression ( - min after exposure) was not translated into the protein, the second wave of transcription ( - h after uvc exposure) resulted in c-fos protein expression, - h post-uv. the stress-activated/mitogen-activated protein kinases (jnk, p k and erks) were immediately induced upon uvc exposure and stayed active for at least h. inhibitor experiments revealed that c-fos was phosphorylated by erks and jnk. the activation of c-fos preceded re-synthesis and the induction of xpf mrna, which was observed - h post-uvc, resulting in the increased expression of the xpf protein. cells over-expressing c-fos showed an accelerated induction of xpf mrna, and consequently a faster repair of cyclobutane pyrimidine dimers (cpds). sirna-mediated silencing of c-fos (transient c-fos knockdown) resulted in abrogated uvc-triggered induction of xpf, attenuated repair of cpds and increased apoptosis. finally, we observed that the removal of cpds but not of photoproducts was significantly faster when cells were pre-exposed to a low uvc dose, indicative of an adaptive response to dna damage. the work was financed by deutsche forschungsgemeinschaft (dfg ch / - ). the addition of clopidogrel to aspirin reduces ischemic events in patients with acute coronary syndrome and in those undergoing percutaneous coronary intervention (pci). however, recurrent ischemic event occurrence during dual antiplatelet therapy remains a major concern. variability in the pharmacodynamic response to clopidogrel is well recognized, and patients with higher platelet reactivity while receiving clopidogrel are at increased risk of ischemic cardiovascular events. clopidogrel is an inactive prodrug requiring biotransformation to form the platelet inhibiting metabolite. interindividual differences in clopidogrel metabolism are the major source of variability in antiplatelet response. polymorphically expressed cytochrome p (cyp) enzymes play a critical role in the metabolism of clopidogrel. these findings gave rise to the concept of personalized antiplatelet therapy -i.e. individual platelet function testing and correction of insufficient platelet inhibition to reduce ischemic events in patients with high on-clopidogrel platelet reactivity (hcpr). gravitas was the first study to test this concept by comparing double-dose clopidogrel to standard-dose clopidogrel in patients with hcpr. gravitas failed to correct hcpr consistently in the study arm, which coupled with a low overall event rate precluded demonstrating a substantial benefit from improved platelet inhibition. the trigger-pci trial tested the effectiveness of the more potent thienopyridine prasugrel versus clopidogrel in patients with hcpr after elective pci with implantation of drug-eluting stents (des). switching from clopidogrel to prasugrel in patients with hcpr afforded effective platelet inhibition. however, given the low rate of adverse ischemic effects using contemporary des after pci in stable ischemic heart disease, the clinical utility of this strategy could not be demonstrated and the study was terminated prematurely for futility. multiple studies have shown that both heterozygotes and homozygotes for loss-offunction cyp c alleles have higher rates of adverse cardiovascular events as compared with noncarriers on approved maintenance dosing of clopidogrel ( mg qd), albeit carriage of cyp c loss-of-function alleles accounted for only a minor proportion of the variability in on-clopidogrel platelet reactivity. results of ongoing studies with antiplatelet treatment stratified by cyp c genotyping are awaited to assess the clinical benefit of this approach. the organic cation transporter novel type (octn /slc a ) represents a high affinity uptake system for carnitine. besides metabolic disease like severe system carnitine deficiency, genetic variants within the slc a gene have been associated with inflammatory diseases like colitis ulcerosa. against this background, we characterized octn expression in peripheral blood cells thereby identifying its expression in all cell types. in the present work we studied octn expression in monocytes and thp- cells as an in vitro model for this cell type. in addition we examined transcriptional regulation of the carnitine transporter in lps activated thp- and investigated the effect of carnitine and its analog mildronate on the respective cytokine response. octn expression was characterized on monocytes and thp- cells on mrna and protein level. transporter mrna expression could be shown in both cell types by realtime pcr. however, the protein expression was analyzed by western blot, flow cytometry and immunofluorescence microscopy demonstrating octn specific signals as well as a localization in the plasma membrane. following thp- cells were activated using lps ( ng/ml) for up to h, thereby indicating the expected cytokine response as demonstrated by increased tnfα ( fold induction) and il- β ( fold induction) mrna levels. in addition, octn expression was analyzed identifying an initial reduction of around % compared to untreated cells. in parallel activated thp- cells were coincubated with increasing concentrations of the octn substrate carnitine or its analog resulting in reduced cytokine release as shown by elisa for tnfα. here, the tnfα effect was diminished by % in the presence of mm carnitine. this effect does not rely on a direct neutralization of lps by carnitine since it was also present in cells only preincubated with carnitine. in the present work we could show that thp- cells represent a useful model to study octn expression and function. in addition, we demonstrate immunosuppressive effects of octn substrates like carnitine. further experiments will be necessary to identify the underlying mechanism of this observation. castor oil has been used for more than years for its laxative effects and also to induce labor in pregnant women. despite its wide-spread use, the mechanism of action remained unknown. the active metabolite of castor oil is ricinoleic acid which is released from castor oil by intestinal lipases. we have found that exposure of meg- cells to ricinoleic acid caused an increase in [ca + ]i, an effect which was dose-dependent and abolished by pretreatment of cells with pertussis toxin, suggesting the involvement of a g-protein coupled receptor. to search for a putative receptor, we determined ricinoleic acid-induced [ca + ]i increases in cells transfected with a sirna library directed against human gpcrs. in this way, we identified prostaglandin e receptors ep and ep as mediators of ricinoleic acid-induced effects. to test if ep and ep receptors mediate pharmacological effects of castor oil in vivo, we analyzed laxative effects induced by castor oil in wild-type (wt) mice, ep -deficient (ep -/-) or ep -deficient mice (ep -/-). while ep -/mice responded similarly to the wt mice, ep -/animals were totally insensitive to castor oil-induced laxation. moreover, mice lacking the ep receptor only in the smooth muscle cells did not respond to castor oil, in contrast to mice which lack ep receptor only in epithelial cells of the intestinal mucosa. similarly, ricinoleic acidinduced contractions of isolated ileal segments were absent in segments lacking ep , consistent with a preferential expression of the ep receptor in the longitudinal muscle layer of the intestine. also, ricinoleic acid-induced contractions of isolated uteri were dependent on the expression of ep receptor in the myometrium. these findings identify the cellular and molecular mechanism underlying the effects of castor oil and indicate a role of the ep receptor as a pharmacological target to induce laxative effects. introduction: patients seek health information from various sources. they are facing the challenge to differentiate between reliable and untrustworthy sources and at the same time identify the best drug therapy for them. furthermore generalised health information confuses more than they benefit or rather unsettle. patients are not necessarily qualified to assess the evidence of statements properly. there is thus a need for providing competent drug information, which is offered by the independent drug information service at the institute of clinical pharmacology in dresden, germany. for the present descriptive evaluation we selected drugs (arimidex ® , cipralex ® pentalong ® , onbrez ® and pradaxa ® ), that were affected by new referenceprice formation, generic registration, warnings or directions in . in specified time frames we assessed the increase in and the cause of enquiries. deductively we draw conclusions for a perspicuous presentation of patient information. since generic registrations of the aromatase inhibitor arimidex ® enquiries on side effects of this drug were stable, but additional consultations were held on generic changeovers. the antidepressant cipralex ® as well as the long-acting β-agonist onbrez ® were assigned to reference-price groups, which resulted in an -times (cipralex ® : → ) and -times (onbrez ® : → ), respectively, increase in enquiries. main aspect was to give background information on reference prices and point out therapeutic alternatives (cipralex ® : of ; onbrez ® : of ). an additional amount of conversations were carried on the fictive registered drug pentalong ® after health insurance companies advised practitioners to avoid recourse by not prescribing this organic nitrate. notable insecurity was aroused by media reporting on lethal bleeding after taking pradaxa ® for anticoagulation. every tenth enquiry in the evaluation period was focussing on these instigative reports ( of ). patients are confronted by current changes, but often do not get enough background information from their health care providers to become acquainted with the tidings. health seekers may find eligible data from media coverage. however the individual assessment as well as the risk-benefit-relation may not be feasible for them. the drug information service for patients is a convenient helpline to reduce lack of knowledge and uncertainties and therefore support shared decision making. insulin effects on hyaluronan production -a possible link between diabetes and cancer? twarock s., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße , düsseldorf, germany background: epidemiological studies have shown an elevated incidence of certain tumor entities in diabetes type and type patients. to reveal the underlying mechanisms we focused on the effects of increased glucose uptake in cancer cells with respect to matrix production. abundant production of hyaluronic acid (ha) in the vicinity of gastrointestinal cancer cells is a hallmark in tumor development. esophageal cancer is a rare but severe kind of gastrointestinal cancer which is differentiated in adenocarcinoma and squamous cell carcinoma (scc) of the esophagus. we studied the effects of increased glucose levels on ha production in an scc cell line (osc ). in starving and full media, elevated glucose concentrations increased the production of ha secreted to the medium in h as measured by an ha-binding protein linked assay (starved, g/l glucose: ± . %; g/l: . ± . %; g/l: . ± . %; full medium ± . %; . ± . %; . ± . %). surprisingly, total ha concentrations were about . - . fold higher under starved conditions. to investigate whether this effect might be due to insulin actions, starved cells were treated with mg/l insulin for h. we observed a dosedependent decrease in ha production following insulin treatment (control vs insulin, g/l glucose: ± . % vs . ± . %; g/l: ± . % vs . ± . %). this finding might suggest that insulin directs glucose usage to the glycolytic pathway thereby diminishing ha synthesis. a premise to this assumption is the ability of osc cells for insulin independent glucose uptake. to verify this thesis, mrna expression levels of insulinindependent and insulin-dependent glucose transporters (glut , glut ) were analyzed by qrt-pcr. the relative abundance was . ± . in favor of glut indicating the presence of insulin-independent glucose transport. conclusion: in osc the absence of insulin actions caused increased ha production which might be due to diminished insulin driven glycolysis, thus leading to the use of early glucose metabolites for ha production instead of energy gain. this finding could be important in the context of diabetes type , where insulin actions are also diminished because of insulin resistance. since increased ha production is of critical importance for cancer growth and spread, the cellular shift in glucose usage from glucose catabolism to ha anabolism could therefore indicate a possible link between diabetes type and cancer progression. schwarz m., unterberger e. universtität tübingen, institut für klinische und experimentelle pharmakologie und toxikologie abteilung toxikologie, wilhelmstraße , tübingen, germany chemical hepatocarcinogenesis is a multi-stage process triggered by an intitiating mutation in a gene encoding an important cell-regulatory protein. tumour initiation may be caused by genotoxic substances which directly interact with the dna, causing mutations. cells carrying permanent mutations experience clonal expansion which may be accelerated by exposure of the experimental animals to tumour promoters during the following step of tumour promotion. it has been shown that substances which constantly activate certain nuclear receptors act as tumour promoters in rodent liver, such as the model tumour promoter phenobarbital which, amongst others, activates constitutive androstane receptor (car). since these tumour promoters do not seem to directly interact with dna causing mutations they can be regarded as non-genotoxic carcinogens. however, the molecular mechanisms of non-genotoxic carcinogenesis are still widely unknown which also poses a major problem in preclinical drug-development. the aim of the marcar (biomarkers and molecular tumour classification for nongenotoxic carcinogenesis) project is to establish early biomarkers for non-genotoxic carcinogenesis by creating a comprehensive molecular profile of tumours generated by a regimen including model tumour promoters such as phenobarbital. the ultimate aim is to differentiate spontaneous liver tumours from tumours generated by non-genotoxic carcinogens. this molecular profile includes mutational analyses, immunostaining for known tumour-specific markers, phospho-proteome analyses, genome wide and promoter-specific dna methylation analyses, as well as mirna analyses. mutation analyses were carried out with mouse and rat tissue from phenobarbital promoted liver tumours to identify mutations which phenobarbital provides a growth advantage for. furthermore, real time pcr measurements show that the expression of a particular non-coding rna and mirna precursor is up-regulated in tissue isolated from phenobarbital promoted mouse liver tumours. additional in-situ-hybridisation experiments demonstrated the localisation of this transcript in ctnnb -mutated tumours. larch-derived diterpenes are potent and selective trpc blockers urban n. , kübler w. the transient receptor potential channel trpc is a poorly ca + -selective cation channel that is activated by the membrane-resident second messenger diacylglycerol (dag). consistent with the major sites of trpc expression, its activation has been implicated in pulmonary and renal diseases, such as pulmonary hypertension, lung edema, chronic obstructive lung disease, allergic airway disease, and focal segmental glomerulosclerosis. amongst various plant extracts, conifer oils and resins are traditionally used to treat pulmonary ailments. therefore, we reasoned that they may contain constituents with a biological activity to modulate trpc activity. the true turpentines, oils and resins of various coniferous genera were tested with respect to a possible inhibition of dag-or receptor-induced activation of trpc and trpc . indeed, turpentines and resins, but not coniphere oils blocked trpc and trpc in a concentration-dependent manner. interestingly, the larch-derived turpentine exerted a trpc -prevalent inhibition. we identified larixol and its mono-and diacetates as the specific compounds that are contained in larch resin and give rise to a trpc -selective block. larixol acetates displayed an ic towards the dag-or receptor-stimulated trpc activity of about . - µm, but did not strongly inhibit a number of other trp channels, including trpv , trpm , trpm , trpm , or trpa . selectivity for trpc compared to its closest relative, trpc , was about -fold. unlike conipherous oils, which contain toxic pinenes, the resin constituent larixol ant its acetates exerted no significant cellular toxicity at concentrations that are required to block trpc . electrophysiological analysis confirmed the highly potent block, which was voltageindependent and reversible. in a murine hypoxia-induced pulmonary vasoconstriction (hpv) model, larixol acetate abrogated the euler-liljestrand mechanism and, thus, mimicked the phenotype of trpc -/mice. we conclude that trpc blockers and, more specifically, larixol-related derivatives may provide novel therapeutic strategies to treat or prevent pulmonary diseases. dioxin is an environmental contaminant, believed to affect basic biological equilibria such as calcium and iron homeostasis. however, the molecular mechanisms underlying these effects are still largely unknown. this strongly hampers the estimation of the hazard to humans associated with dioxin exposure and necessitates further studies aimed at the clarification of these mechanisms. it has been suggested that nearly all biological and biochemical processes are mediated by protein complexes. the most commonly used technology for monitoring changes in the expression of complex protein mixtures is still d gel electrophoresis, but this method suffers from poor expression of low or moderately abundant proteins. blue native page and subcellular fractionation form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. the aim of the study is to identify and characterize multiprotein complexes by blue native page to elucidate the network of protein-protein interactions that regulate protein function after dioxin exposure. sample preparation and subcellular fractionation rt cells were cultured in mccoy's a medium. cells at confluence were harvested and fractionated into cytosolic, membrane/organelle and nuclear fraction by using the proteoextract subcellular proteome extraction kit. first dimension (bn-page) mg of protein sample was mixed with % of coomassie blue g- (cbb g- ) and loaded in each lane of - % polyacrylamide native gradient gels. the lanes from the first dimension were cut into individual strips and were placed into a % sds gel. the gels were stained with coomassie and the spots were picked up for mass spectrometry. bn/sds-page combined with ms led to the identification of proteins involved in the regulation of both calcium and iron homeostasis in dioxin-exposed cells. these results demonstrate for the first time that dioxin exposure simultaneously affects calcium and iron metabolism. since important iron and calcium requirement changes occur during the regulation of cell growth, the protein expression changes observed in our study may be associated with dioxin-dependent cell-fate decisions. the murine protease inhibitor serpina n inhibits mechanical allodynia in a model of neuropathic pain vicuna l. , , simonetti m. several chronic diseases are accompanied by strong, long-lasting pain. a majority of chronic pain diseases are not well understood yet and cannot be controlled by conventional analgesics or non-pharmacological approaches. therefore, there is a major need to develop novel therapeutic principles. using a genetic screen, we identified serpina n, a serine protease inhibitor, which is homologous to human a -antichymotrypsin, to be a determinant of low neuropathic pain. we found that serpina n is expressed in the dorsal root ganglia (drg) and spinal cord and that it is upregulated in these tissues in mice developing neuropathic pain. importantly, we observed that spinal delivery of recombinant serpina n inhibits mechanical allodynia in a mouse model of neuropathic pain. we identified a novel serine protease substrate for serpina n, which is upregulated in the spinal cord in mice undergoing neuropathic pain ('enzyme e'). recombinant enzyme e delivered intrathecally to the spinal cord of mice elicited rapid and long-lasting allodynia, which was fully blocked by concomitant administration of serpina n. our results suggest that serine protease-serpin signaling modulates spinal neuronal and glial cell networks involved in processing pain and that activity-induced spinal release of serpina n constitutes an endogenous defence mechanism against establishing chronic pain hypersensitivity. these data have important implications for the pathophysiology of pathological pain and potentially hold therapeutic relevance. gβγ subunits are involved in β-adrenergic receptor induced cardiac hypertrophy vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. , würzburg, germany introduction. activated β -adrenergic receptors and their g protein gαs induce the development of cardiac hypertrophy. however, the hypertropic effects of direct activation of downstream effectors, such as adenylyl cyclase, camp or pka, are controversely discussed. recently, a hypertrophic pathway involving a g protein βγ subunit induced phosphorylation of the mitogenic kinases erk / at threonine (erk thr phosphorylation) has been described to mediate erk-induced hypertrophy. this study aims to investigate whether erk thr -phosphorylation is involved in cardiac hypertrophy triggered by β-adrenergic receptors. -phosphorylation was detected in hek cells overexpressing β -receptors, murine hearts and neonatal rat cardiomyocytes after isoprenaline treatment. we performed [ h]-isoleucine incorporation assays to assess cardiomyocyte hypertrophy in vitro. neonatal rat cardiomyocytes (nrcms) overexpressing wild-type erk showed a significant increase in [ h]-isoleucine incorporation after isoprenaline treatment. in contrast, nrcms transfected with erk thr -phosphorylation deficient mutants (erk t a and erk t s ) or pretreatment with the erk inhibitor, pd , significantly attenuated cardiomyocyte hypertrophy. for in vivo studies, isoprenaline was given subcutaneously for days to wild-type mice and transgenic mice overexpressing either wild-type erk t t or erk t s . echocardiography and histological analyses revealed that erk t s mice developed less left ventricular hypertrophy than control mice. hypertrophic target proteins of erk (e.g. elk ) are located in the nucleus. western blot and confocal microscopy analyses showed that overexpressed erk t a or erk t s are retained in the cytosol and prevented elk -phosphorylation after isoprenaline stimulation. co-immunopreciptation assays in hek cells and nrcms underlined the direct involvement of g protein βγ/erk interaction upon isoprenaline stimulation. in line with this finding, direct activation of adenylyl cyclase by forskolin did not lead to gβγ induced erk thr -phosphorylation. conclusion. taken together, gβγ-subunits participate in β -adrenergic receptor mediated hypertrophy by enhancing erk thr -phosphorylation. these findings add important insight to the molecular signaling of g proteins in cardiac hypertrophy. the protein tyrosine kinase src and its role upon alpha-toxin stimulation of human platelets vogel k. , burke m. introduction: alpha-toxin, a kda calcium pore forming exotoxin, is a major virulence factor in the pathogenesis of staphylococcus aureus infections. alpha-toxin affects human blood cells such as platelets and induces aggregation that is accompanied by multiple changes in platelet protein tyrosine phosphorylation and dephosphorylation ( ). in the present paper, we focused our interest on the protein tyrosine kinase src, the most abundant member of the src-family kinases present in platelets ( ) . by the use of various inhibitors, we studied src and its role in α-toxin-induced platelet aggregation. methods: isolated human platelets from healthy volunteers were stimulated with α-toxin in the presence or absence of the src-family member inhibitors pp , pp or su (referred as src inhibitors). src and autophosphorylation of src were analyzed by sds-page and western blotting using specific antibodies against src and tyr- -phospho-src from calbiochem and cell signaling, respectively ( ). furthermore, calpeptin, an inhibitor of the calcium-dependent protease calpain, was used. platelet aggregation was measured by the method of born. staphylococcal α-toxin induced platelet aggregation in a concentration-dependent manner ( . - . µg/ml of toxin). pre-incubation with src inhibitors (pp , pp or su ) reduced α-toxin-induced platelet aggregation by about %. similar inhibitory effects have been observed by the use of calpeptin that acts as an inhibitor of the src degrading protease calpain. with respect to src itself, a-toxin induced autophosphorylation at tyr- followed by a fast and complete dephosphorylation within min. while calpeptin modified the time course of dephosphorylation, only little effect of the src inhibitors has been seen on tyr- phosphorylation/dephosphorylation. the typical calpain-dependent degradation of src can be blocked by calpeptin ( µm), but also by depletion of extracellular calcium indicating that it is a calcium-dependent process. conclusion: taken together, our data demonstrate that α-toxin of staphylococcus aureus induces platelet aggregation accompanied by src degradation and autophosphorylation at tyr- typically observed in activated platelets. inhibition of the cellular tyrosine kinase src as well as the protease calpeptin reduces aggregation indicating an important role of src and/or other src-family members in α-toxin-induced platelet stimulation. the five subtypes of muscarinic acetylcholine receptors belong to the superfamily of gprotein coupled receptors. the even-numbered subtypes m and m prefer coupling to gi proteins, whereas the odd-numbered receptors m , m and m prefer coupling to gq proteins. with respect to ligand binding and m receptor activation, the conserved epitope trp . at the beginning of tm displays remarkable functional features. it is located at the junction between the orthosteric and the allosteric binding site of the m receptor [ ] . in the inactive m receptor, it provides subtype-independent baseline affinity for allosteric antagonists [ ] . in the active receptor, m trp . affords binding affinity for the full agonist acetylcholine and intrinsic efficacy for the partial agonist pilocarpine [ ] . to study the role of trp . for m receptor activation, agonist-induced formation of dmyo-inositol-monophosphate was measured in cho-cells transfected with the respective human receptor-cdna. surface receptor expression measured by radioligand binding was similar in hm wild-type-cells and hm trp . →ala-cells, amounting to . and . x receptors per cell, respectively. the intrinsic efficacy of acetylcholine was not influenced at m trp . →ala relative to m wild-type, whereas potency was reduced about tenfold. these findings resemble those made previously in m and the corresponding mutant. in the case of pilocarpine, replacement of trp . by alanine in m did not reduce intrinsic efficacy. this finding is in contrast to m , where the corresponding mutation induced a loss of pilocarpine's intrinsic efficacy. the potency of pilocarpine was diminished about tenfold at the m trp . →ala mutant relative to m wild-type. this finding is also in contrast to m , at which pilocarpine's potency was not sensitive to the trp . →ala mutation. taken together, the diverging sensitivity of pilocarpine to the trp . →ala mutation between the m and the m receptor suggests that the role of this epitope for receptor function may differ between even-and odd-numbered muscarinic acetylcholine receptors. in vivo experiments for inhalation toxicity are time and animal consuming. thus several in vitro methods aim to replace or reduce and refine the in vivo experiments. human dtissue models are commercially available reconstructed from different donors (normal, smokers, chronic obstructive pulmonary diseases), which show a normal human bronchiole tissue that reveals a pseudostratified epithelial structure, numerous microvilli and cilia on the apical surface of the cultures. the presence of tight junctions and mucus secretion has also been confirmed comparable to the in vivo situation. these d-models are cultured on a porous membrane as air-liquid interface. test substances can be applied apically, either as solution or with an aerosol-inducer. in our in house validation to test the strengths, handling and reproducibility of such dmodel systems as well as determining the correlation between in vivo inhalation data, we have assessed the epiairway tm model from mattek, usa. a set of substances were selected with known in vivo toxicity data and mode of action. the substances were tested in the epiairway model an in parallel, in t and a cell lines to assess putative unspecific cytotoxic effects of the test substances. a comparison of toxicity data from the d-model and the in vivo data revealed, that the model is only predictive of respiratory toxicity in vivo for a subset of substances with specific modes of action. the epiairway tm model has proven to be robust, showing high reproducibility between pre-and main-tests as well as in the concurrent controls but it will need a strict definition of its applicability domain or further development of the test protocol to achieve a wider applicability. remodeling of intracellular ca + handling and cyclic amp-dependent signaling in atrial myocytes from patients with chronic atrial fibrillation. voigt n. background: in atrial myocytes ca + entry through l-type ca + channels (ica,l) triggers a larger ca + release (ca + transient,cat) from the sarcoplasmic reticulum activating contractile myofilaments. reduced ica,l is a hallmark of atrial remodeling in chronic atrial fibrillation (caf) and is supposed to contribute to action potential shortening and contractile dysfunction. however, the coupling efficiency between ica,l and cat and its regulation by camp-dependent signaling in caf patients are unexplored. methods: ica,l (voltage-clamp) and cat (fluo- ) were measured simultaneously in rightatrial myocytes from sinus-rhythm (ctl) or caf patients. a saturating concentration of the non-selective β-adrenoceptor (ar) agonist isoprenaline (iso, µm) and the nonselective phosphodiasterase (pde) inhibitor -isobutyl- -methylxanthine (ibmx, µm) were used to increase cellular camp content. camp content was assessed with immunoassay. results: in caf amplitudes of ica,l ( . ± . pa/pf, n= / [myocytes/patients] vs . ± . pa/pf, n= / , p< . ) and cat ( . ± . nm vs . ± . nm, p< . ) were lower than in ctl myocytes, whereas diastolic [ca + ]i levels were unchanged (caf, . ± . nm; ctl, . ± . nm). the coupling efficiency between ica,l and cat was similar in ctl and caf. application of iso increased ica,l amplitude to . ± . pa/pf (n= / ) in caf and to . ± . pa/pf (n= / ) in ctl. the corresponding cats increased to . ± . nm in caf and to . ± . nm in ctl. although the amplitudes of ica,l and cat also increased after pde inhibition with ibmx, the magnitude of these increases was smaller than the iso-induced enhancements. both iso and ibmx had no effect on diastolic [ca + ]i and coupling efficiency. however, the relative iso-induced increases in ica,l (caf, + . ± . % vs ctl, + . ± . %, p< . ) and cat (caf, + . ± . % vs ctl, + . ± . %, p= . ) were significantly higher in caf compared to ctl myocytes and a similar tendency was found for ibmx. basal camp levels were higher in caf compared to ctl (caf, . ± . pmol/mg, n= vs ctl, . ± . pmol/mg, n= , p< . ), pointing to an increased camp-dependent signaling in caf patients. conclusions: these data point to remodeling of camp-dependent signaling in caf patients which likely contributes to the stronger relative increases of ica,l and cat amplitudes after β-ar stimulation and pde inhibition. remodeling of camp-dependent signaling might be a novel contributor to af pathophysiology. direct visualisation of g-protein-coupled receptors and heterotrimeric g-proteins using single-molecule microscopy wagner j. g-protein-coupled receptors (gpcrs) form the largest family of membrane-bound receptors and mediate the effects of several extracellular stimuli. although the basic mechanisms of gpcr signalling have been extensively studied, a full characterization of the involved protein-protein interaction is still missing, largely due to technical limitations. in this study, we developed new methods for labelling gpcrs and g-protein subunits based on snap-and clip-tags and visualise them with single-molecule sensitivity. the snap-tag is a mutant of the dna repair protein o -alkylguanine-dna alkyltransferase that reacts with fluorescent benzylguanine derivatives, whereas the clip-tag is reacting specifically with o -benzylcytosine derivatives. these tags allow labelling proteins directly in living cells with very high specificity and low background. snap/clip-tagged receptors and g-proteins were covalently labelled with small organic fluorophores and visualised by total internal reflection fluorescence microscopy, which allows to selectively illuminate only fluorescent molecules located on or immediately underneath the cell surface. particles were automatically analysed with previously published as well as newly developed algorithms. the results indicated that both receptors and gproteins, although diffusing with high speed on the cell surface (diffusion coefficients: receptors ~ . mm /s, g-proteins ~ . mm /s), can be visualised and correctly tracked. a variable fraction of receptors and g-proteins are immobile or show hop movements, possibly suggesting their interaction with cytoskeletal or other membranebound proteins. our data also suggest the feasibility of performing two-colour analyses with snap-and clip-tagged proteins aimed at directly visualizing transient interactions between receptors and g-proteins or among g-protein subunits. in-vitro screening systems are particularly well suited to preclinical toxicology testing at an early stage of drug development as they have the advantage of being fast and requiring only a small amount of test substance. the demands for in-vitro screening assays for systemic toxicity are multiple and include the need of organ specific cell systems, the use of optimal cell numbers, cell passages and incubation times. even minimal changes in the conditions of the test system may lead to significant changes of the biological system. therefore a reliable normalization compensating biological variability is crucial prior to any interpretation of results generated from a biological system. basf has developed an in-vitro metabolite profiling assay and a subsequently tuned normalization strategy allowing the prediction of specific organ toxicity. the in-vitro assay consist of exposing cells lines to test substances and to determine the metabolite profile using chromatography coupled to mass spectrometry systems. herein, we compare five different normalization strategies referring to their suitability in the application to in-vitro metabolite profiling data. the strategies comprise statistical approaches, approaches referring to reference values from each individual sample or samples generated in dependent batches. best results were achieved by an individual strategy using a new reference value correlating well over a large range of cell counts previously used for generating corresponding cell extracts. statistical analysis revealed the normalization based on the new reference value greatly improved the quality of the results compared to non-normalized samples as well as to all remaining strategies. generation and application of this new reference value and the corresponding normalization strategy will be presented the first time. validation will be featured on the basis of extracts of the human hepatocellular carcinoma cell line hep g . molecular mechanisms of the inhibitory function of rhoh in phospholipase cmediated signalling walliser c., löschmann y., ziegler v., kühne e., schilling p., rasonabe z., bühler a., vatter p., gierschik p. universitätsklinikum ulm institut für pharmakologie und toxikologie, albert-einstein-allee , ulm, germany rho gtpases are a subfamily of ras gtpases regulating diverse signalling pathways, for example those regulating the reorganisation of the actin cytoskeleton. among them, rac and rhoh show an expression restricted to the hematopoietic lineage. rhoh is constitutively active, because it carries mutations in two positions (s and n ) known to be important for gtp hydrolysis. hence, rhoh is controlled on the level of protein expression and, possibly, by tyrosine phosphorylation. rhoh has been implicated in human malignancies, since the gene is subject to somatic hypermutation in its noncoding regions and to translocation to the gene encoding laz /bcl or to other genes in human b-cell lymphomas. furthermore, rhoh is overexpressed in primary human chronic lymphocytic leukemia (cll) cells. these findings suggested that rhoh is involved in the initiation and/or progression of cll. we previously showed that rhoh acts as a potent inhibitor of both rac -mediated phospholipase c-β (plcβ ) and plcγ activation in intact cells. the aim of this study was to elucidate the molecular mechanisms of the inhibitory effect of rhoh on plc activity. the results showed that rhoh directly inhibited the activity of constitutively active variants of plcγ , plcβ , and plcδ , but that it had little or no effect on the activity of plcγ and plcε. the amino acid residues s and n , likely to be the cause for the gtpase-deficiency of rhoh, are not required for the inhibitory function of rhoh. furthermore, the switch-i and switch-ii regions of rhoh are not necessary for the inhibitory effect of rhoh, since rhoh mutants carrying switch-i or switch-ii regions of rac caused inhibitory effects on rac -mediated plcβ and plcγ stimulation indistinguishable from wild-type. interestingly, rhoh seems to interact with regions of plcγ distinct from those which are necessary for rac interaction, as the split pleckstrin homology domain of plcγ , which is essential for its interaction with activated rac , is dispensable for the inhibitory effect of rhoh. in summary, our results indicate, that rhoh acts as a plc-isozyme-specific negative regulator of the activity of plcβ and plcγ , both of which are specifically expressed in hematopoietic cells. these findings suggest a novel mechanism of plc isozyme regulation by rhoh. the results also suggest that rhoh plays an important role in b cell maturation, function, and leukemogenesis by modulating b-cell-receptor-mediated plcγ activation. effect of rac inhibition on doxorubicin mediated cell response wartlick f., fritz g. heinrich-heine-universität düsseldorf institut für toxikologie, universitätsstrasse , düsseldorf, germany background: the small gtpase rac is a well characterized member of the rashomologous (rho) family. rac is not only a key regulator of the actin cytoskeleton but also regulates the activity of nadph oxidase, stress kinases and transcription factors (e.g. nf-κb, ap ). furthermore, rac can translocate into the nucleus and interacts with topoisomerase type ii (topo ii). yet the general nuclear function of rac is still unclear. here, we address the question how rac influences the genotoxicity of the topo ii poisons doxorubicin and etoposide. methods: to study the function of rac , human hepatoma cells were pretreated with the rac -inhibitor eht before they were exposed to doxorubicin, etoposide or, for control, ionizing radiation (ir). to check the influence of rac inhibition on the outcome of genotoxin treatments, cell viability and cellular stress response were analyzed by the wst-assay, western blot (wb), co-immunoprecipitation experiments, facs-analysis and the alkaline comet-assay. results: as compared to the control, cells that have been pretreated with the rac inhibitor showed a higher viability, less phosphorylation of h ax (s ) and a reduced dna damage formation (measured by alkaline comet-assay) after treatment with doxorubicin and etoposide but not after treatment with ir. furthermore inhibition of rac resulted in a reduced phosphorylation of topo iiα (s ) and an increased interaction of topo iiα with hsp in doxorubicin treated cells. the data indicate that inhibition of rac protects human hepatoma cells against topo ii poisons due to interference with topo iiα function. the presence of drugs or other potential toxic substances in milk has enormous toxicological and nutritional consequences for consumers of dairy products. the atpbinding cassette (abc) transport protein breast cancer resistance protein (bcrp; abcg ) is expressed in alveolar epithelial cells of the mammary gland in cows, sheeps and goats. bcrp is known to play a major role in the active secretion of a variety of xenobiotics into human milk. so far there is little information about the transport activity and substrate specificity of dairy bcrp. therefore we aimed to establish a mdck cell in vitro model expressing bcrp of dairy animals. bcrp mrna was isolated from bovine, caprine and ovine mammary gland. full-length clones were generated using race (rapid amplification of cdna ends) pcr. the final full-length bovine, ovine and caprine abcg cdna-clone sequences were submitted to the ncbi genebank (eu , gq and gq ). stable transfection of bcrp in mdck cells was performed and the subcellular localization of bcrp at the apical plasma membrane was identified by confocal laser scanning microscopy. bcrp-mediated transport of the substrate hoechst was measured and the selectivity was determined by the bcrp inhibitor ko . inhibition studies using hoechst identified various drugs including the antibiotic enrofloxacin or anthelmintic agents like oxfendazole as substrates of bovine, caprine and ovine bcrp. to further characterize bcrp carrier activity, bidirectional transport studies were performed with transwell® filter inserts that allow studying drug transport between an apical and basolateral compartment. cell monolayer integrity was checked by measuring teer values as well as by measuring the paracellular flux marker atenolol by lc/ms. bidirectional transport studies with enrofloxacin were performed to characterize the bcrp transporter activity. our results may contribute to increase the understanding of carrier associated drug transport into the milk of dairy cattle and therefore enlarge consumer protection. acrolein and acrylamide: excretion of mercapturic acids after consumption of potato chips watzek n., scherbl d., berger f., feld j., eisenbrand g., richling e. technische universität kaiserslautern fachbereich chemie; fachrichtung lebensmittelchemie & toxikologie, erwin-schrödinger-str. , kaiserslautern, germany acrolein (ac) and acrylamide (aa) may be formed from food constituents during heating of food. ac is supposed to be generated via heat induced formation from glycerides/glycerol, aa is known to arise during the maillard reaction from asparagine and reducing carbohydrates. ac also has also been suggested to be formed by endogenous metabolism as a side product of carbohydrate and/or amino acid turnover or by oxidative desamination of polyamines [ ] . as an α,b-unsaturated aldehyde, ac forms , -michael-adducts with biomolecular nucleophiles, such as sulfhydryl and amino groups. in the organism, ac and aa are preferentially conjugated to glutathione and are excreted as mercapturic acids (ma), n-acetyl-s-( -hydroxypropyl)-cysteine , n-acetyl-s-(carboxyethyl)-cysteine (cema), (n-acetyl-s-( -carbamoylethyl)-cysteine (aama), and (n-acetyl-s-( -hydroxy- -carbamoylethyl)-cysteine (gama). data on human exposure to ac and its occurrence in the diet are scarce. in general, contents in heat treated foods are considered to be in the low ppb range (µg/kg) [ ] . nevertheless, in a pilot study in humans urinary -hpma excretion of non-smokers was reported to be about three fold higher, as compared to aama [ ] . in the present human intervention study we monitored the excretion of mas in five healthy volunteers (male) after ingestion of commercially available potato chips ( g), equivalent to an uptake of µg aa (absolute amount), together with an as yet unknown amount of acrolein [ ] . urinary ma contents were monitored by hplc-ms/ms following solid phase clean-up of urine for up to h after test meal uptake. the results demonstrated kinetics of -hpma and cema excretion in human urine to be clearly related to ingestion of the potato chip meal. on the basis of auc values, total excretion of -hpma plus cema exceeded that of aama plus gama by a factor of about four. the results confirm earlier findings on urinary mas, suggesting markedly higher human exposure to dietary ac / potential ac precursors than to aa. it is an as yet unresolved question, whether and to what extent concomitant substantial ac exposure may influence toxicology of such dietary heat-induced toxicants. [ ] stevens, j.f. and maier, c.s. ( ) molecular nutrition & food research ; [ ] osorio, v. m. and de lourdes cardeal, z., ( ) journal of chromatography a ; [ ] schettgen, t., musiol, a., and kraus, t., ( ) rapid communications in mass spectrometry ; [ ] ewert, a., granvogl, m., and schieberle, p., ( ) lebensmittelchemikertag protective effects of increased nad + levels in human peripheral blood mononuclear cells exposed to dna damaging agents weidele k., beneke s., bürkle a. university of konstanz molecular toxicology group, department of biology, jacob-burckhardt-str. , konstanz, germany the dna damage-activated enzyme poly(adp-ribose) polymerase (parp- ) acts as a nick sensor and modifies target proteins by covalent attachment of poly(adp-ribose) [par] using nad + as substrate. the intracellular levels of par and nad + are important parameters for biological responses to genotoxic stress and influence diverse cellular functions including dna repair or maintenance of genomic stability. notably, loss of genomic stability is a hallmark of both carcinogenesis and the ageing process. here we analysed the impact of elevated nad + levels in human blood peripheral mononuclear cells (pbmc) with regard to (i) poly(adp-ribose) formation, (ii) cell death, (iii) initial dna damage and subsequent repair, as well as the influence on (iv) genomic stability under genotoxic stress. after ex vivo supplementation of pbmc with low concentration of nad + precursor nicotinic acid (na) intracellular nad + level significantly increased up to fold in unstimulated [ ] and . fold in mitogen-stimulated cells. after dna damage infliction, parp activity was dramatically increased in supplemented cells, necrotic cell death was reduced and dna strand break repair was significantly affected. furthermore the frequency of micronuclei decreased significantly after irradiation damage, emphasizing the fundamental role of adequate nad + levels in maintaining genomic integrity. the cyclic purine nucleotides adenosine ': ' monophosphate (camp) and guanosine ': ' monophosphate (cgmp) are well-examined second messengers with many proven biological functions. in a recent study, using a highly sensitive and specific mass spectrometry method, we have shown that cyclic ': ' cytidine monophosphate (ccmp), a pyrimidine nucleotide, is naturally occurring in several mammalian cells [ ] . ccmp activates both camp-and cgmp-dependent protein kinases with low potency [ ] but the physiological function of ccmp is still very poorly understood. in an effort to delineate the function of ccmp, we analyzed expression of the early response gene egr . we chose this gene because it is regulated by numerous stimuli including camp [ ] . in our first study, we showed that dibutyryl-ccmp and ccmp failed to increase egr gene expression levels after stimulation of kb cells under various experimental conditions using real-time pcr (taqman®). we have now changed the experimental set-up using hela cells and the new ccmp analogue, ccmp-acetoxymethyl ester (ccmp-am), still focusing on gene expression of egr . esterification of the negatively charged cyclic phosphate of ccmp allows better transport of the nucleotide across the cell membrane, thus augmenting possible intracellular effects. hela cells were stimulated in cell culture medium with extracellularly applied ccmp-am ( , , , µm) over to min h after seeding. analysis of real time pcr (taqman®) experiments, using β-actin as a housekeeping gene, showed a significant increase of egr expression in a time and concentration dependent manner. these effects were specific for stimulation with ccmp-am but not the control phosphate trisacetoxymethyl ester. hela cells were also cultured in serum free resting medium (mcdb , sigma) that induces growth arrest, one to eight hours prior to stimulation. here, even higher egr expression levels through ccmp-am stimulation could be seen. these results suggest that ccmp could function as a second messenger just as camp and cgmp do. studies are in progress to further examine the mechanisms of the ccmp-am effects on egr expression in hela cells. methyl-cpg-binding protein (mecp ) recognizes methylated dna, it is involved in chromatin remodeling and it acts as a transcriptional repressor or activator. we have previously shown that expression of mecp is diminished in murine and human heart failure. prevention of mecp downregulation in transgenic mouse models aggravated cardiac hallmarks of heart failure. in patients with rett syndrome, which is caused by mutations in the mecp gene, mitochondrial function was found to be altered in the central nervous system. as the impact of mecp on mitochondrial function in the heart is unknown, the aim of the present study was to characterize the significance of mecp of cardiac mitochondria in mouse models with cardiac myocyte-specific expression or ablation of mecp . in order to investigate the cardiac function of mecp , two genetically modified mouse models were previously generated, including mice with inducible transgenic expression of mecp in cardiac myocytes under control of the tetracycline-system (mecp -tg) and mice with targeted ablation of mecp in myocytes (mecp mlccre ). these mice were analyzed under basal conditions and after chronic transverse aortic constriction (tac). at baseline, cardiac-specific overexpression of mecp did not cause any difference in cardiac function as compared to control mice using millar catheterization. isolated interfibrillar mitochondria showed a decrease in citrate synthase activity. after chronic pressure overload, the decrease in cardiac mecp expression could be completely prevented by the mecp transgene. cardiac contractility and relaxation were significantly decreased in mecp -tg animals. upon electron microscopical investigation, transgenic mecp expression was associated with a significant reduction of interfibrillar mitochondria, clustering of mitochondria in the perinuclear region and smaller mitochondrial cross sections as compared with control specimens. in contrast, cardiac myocyte-specific ablation of mecp caused a rightward shift in the size distribution of mitochondria as compared with mecp -tg hearts. epigenetic processes, including the recognition of dna methylation by mecp , may play an important role in the control of mitochondrial gene expression, structure, subcellular localization and function in the heart. thus, precise control of mecp expression and function is essential to prevent deterioration of metabolic function during chronic heart failure. normalisation of blood pressure does not prevent angiotensin ii-induced dna damage in kidney and heart of ren rats weissenberger s., hey v., lau d., schupp n. universität würzburg institut für pharmakologie und toxikologie, versbacher strass , würzburg, germany increased activity of the renin angiotensin system (ras) with enhanced levels of angiotensin ii (angii) leads to oxidative stress with endothelial dysfunction, hypertension and atherosclerosis. epidemiological studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. we could show in vitro and in vivo that angii causes structural dna damage dose-dependently in kidney cells and in the kidney. elevated angii levels therefore might contribute to carcinogenesis of the kidney. in a model of high angii organ levels, the transgenic ren rat, carrying an additional renin gene, dna damage in the kidney was analysed in animals of and weeks. untreated ren rats exhibit increased blood pressure from the age of weeks on. therefore, the line is kept on angiotensin i converting enzyme inhibitor therapy, which normalizes blood pressure and kidney function to values of control sprague dawley rats. despite this normalized blood pressure of the ren animals, a significant higher superoxide production could be observed in kidneys already in week old animals. also a higher frequency of structural dna damage and double strand breaks could be detected in the comet assay and with an antibody against the double strand break marker γ-h ax in kidneys. further, fittingly, an increased dna repair activity exists in kidneys of ren rats compared to control rats. as another organ affected by hypertension the heart of the ren animals was analysed for oxidative dna damage. although only a marginal increase of superoxide production could be found, also in the heart a significant higher frequency of dna double strand breaks and cells positive for dna repair activity could be observed. our data let us conclude that normalization of blood pressure in a state of activated ras is not sufficient to prevent angii-induced genotoxicity. this further implies that also patients with treated hypertension still might suffer from endorgan-damaging effects of elevated angii levels. the d a pore mutation leads to complete inactivation of trpv channels in epididymis weißgerber p. , kriebs u. replacement of aspartate residue by alanine (d a) in the pore of trpv channels in mice disrupts ca + absorption by the epididymal epithelium resulting in abnormally high ca + concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilisation capacity raising the possibility of residual activity of channels formed by trpv d a proteins (sci signal , ra , ) . it is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared to the deletion of the corresponding protein. to gain insights whether the trpv d a pore mutant still contributes to residual channel activity and/or channelindependent functions in vivo, we compared important fertility-parameters between trpv -/and trpv d a/d a mice: the fertilization rate observed in permanent matings, the in vivo fertilization rate as judged by the rate of embryos isolated from plug positive females of matings with males homozygous for either trpv mutation as well as the motility, in vitro fertilization capacity and viability of sperm were reduced to the same extent in both genotypes. also, no differences were observed in copulatory behavior between trpv -/and trpv d a/d a males. the profound reduction in ca + uptake by the epididymal epithelium was identical in trpv -/and trpv d a/d a males. this direct comparison of these parameters indicate that deleting trpv does not further aggravate the phenotype observed in trpv d a/d a mice, and -in our opinion -allows the conclusion that the d a pore mutant of the trpv protein leads to complete inactivation of the trpv channel activity or channel-independent scaffolding functions in epididymal epithelium. characterization of a naturally occurring c-terminal mutation (n i) on herg channel function in hek cells sellmaier v. , moretti a. long-qt-syndromes (lqts) are acquired or inherited disorders which predispose patients to cardiac arrhythmias and sudden death. in affected individuals, the electrocardiogram shows a prolongation in the qt interval, due to an unstable repolarization of the action potential. acquired forms of lqts are often the result of treatment with medications that block cardiac potassium channels, such as class iii antiarrhythmic drugs or antihistamines. inherited lqts are caused by mutations in cardiac ion channels. congenital long qt syndrome (lqt ) is caused by loss-offunction mutations in the human ether-á-go-go-related gene herg (also known as kcnh or kv . ). herg encodes the pore-forming α-subunit of the rapid delayed rectifier potassium current i kr, whose physiological role is to repolarize the late phase of cardiac action potentials. herg channel α-subunits exist as isoforms ( a and b) that are identical except for structurally divergent n termini. native cardiac ikr channels are tetraheteromers containing of each α-subunit types. a loss-of-function can be due to either defects in a) channel opening (gating), b) ion permeation or c) protein maturation and trafficking. we have identified a so far uncharacterized dominant missense mutation in the herg gene (n i) in a patient with lqt. both herg a and herg b subunits were cloned from a human heart cdna library and the specific n i mutation introduced by site directed mutagenesis. hek cells were transiently transfected with equal amounts of mutated herg a and wild type (wt) herg b cdnas and the resulting potassium current compared to herg a/b wt. whole-cell patch-clamp analysis showed similar current densities for wt versus mutated channels. also the voltage-dependence of activation was unchanged with a halfmaximal activation at - mv for wt and - mv for the mutated channel assembly. differences were found for the deactivation and inactivation kinetics. the deactivation was faster in the mutated channels with t fast = ms and tslow = ms versus tfast = ms and tfast = ms in wt channels, determined from the tail current at - mv after a -second pulse to + mv. the half-maximal steady-state inactivation of the tail current was shifted by mv to the depolarizing direction in the mutated channel compared to the wt. a defect in channel gating appears to be the most likely explanation. background: abcc is adjacent to p-glycoprotein the most important efflux transporter for various endogenous and exogenous compounds and is expressed at several compartment barriers. by increasing evidence it is shown that the abcc polymorphisms are of clinical significance. the aim of our study is to analyze the epigenetic regulation of distinct abcc haplotypes by the influence of micrornas. methods: abcc cdna clones containing five distinct haplotypes were generated by site-directed mutagenesis, cloned into pires-zsgreen expression vectors and transfected into different cell lines for further functional analysis. one modified vector set contained a short 'utr sequence of abcc whereas the other contained the full length (fl) sequence. mirnas potentially interacting with abcc were identified form an mirna array after rifampicin stimulation of hepg cells. results: we could demonstrate that there is no difference in the basal protein expression level comparing the two vector types (fl 'utr vs. mod. 'utr) concerning the - c/ g/ c (cgc) abcc wt in hepg cells. using the fl vector construct, the expression level of cac haplotype protein was increased ( , % ± %). transfection assays with the mir- , which was identified as a candidate mirna targeting abcc mrna, and the two different vector constructs harboring the cac or the cgc (wt) haplotype, confirmed that mir- was able to down regulate the abcc protein expression. there was no significance in downregulation for one abcc haplotype, respectively (modified 'utr: cgc - %; cac - %, fl 'utr: cgc - %; cac - % down regulation compared to mir-negative control). discussion: differences of abcc protein expression level are due to the epigenetic regulation of abcc haplotypes. to further characterize the effect of mir- on tcg, tgt and cgt abcc haplotypes, transfection assays are currently performed using cell cultures as well human primary leukocyte cultures. sex differences affect the pathophysiology and pharmacology of leukotriene biosynthesis werz o. friedrich-schiller-university jena, institute of pharmacy, philosophenweg , jena, germany inflammatory diseases affect more females than males. thus, women suffer more often from asthma, rheumatoide arthritis, alzheimer´s disease, and many autoimmune diseases than men. of interest, sex differences also exist in drug responses, with respect to both pharmacodynamics and pharmacokinetics. we have recently discovered a sex bias in the biosynthesis of pro-inflammatory leukotrienes (lts) due to testosterone, which may represent a molecular basis for gender differences in inflammation and asthma. interestingly, testosterone downregulates lt biosynthesis and also causes a sex bias in the efficiency of lt synthesis inhibitors, which demands for the clinical evaluation of a gender-tailored therapy with anti-lts. we found that certain inhibitors of lt biosynthesis were more efficiently in females than in males, and that androgens are responsible for these gender differences. in fact, the flap inhibitor mk effectively reduced ltb pleural levels in female but not in male rats treated with carrageenan, and mk increased survival only of female mice in the lt-related disease model of paf-induced lethal shock. administration of testosterone to female mice abolished the protective effects of mk . in view of the current active development of lt synthesis inhibitors as therapeutics in respiratory and cardiovascular diseases, our data prompt for consideration of gender issues in the development and use of such drugs, in order to optimize medical therapy both for men and women. considering the complexity of deposition and kinetics of air-borne nanomaterials in the lung, potential pulmonary toxicity of biopersistent nanomaterials should be evaluated by inhalation studies. those studies demand special equipment and large quantities of test material. intratracheal instillation appears as a simple and low substance-consuming alternative, although bolus dosing and the more central distribution of the particles in the lung are a well known trade-off. we compared the inflammatory response of the lung to amorphous silica (as) after instillation and inhalation. for inhalation the established short-term protocol for nanomaterials was employed (ma-hock et al. inhalation toxicology, : , ): male wistar rats were exposed to the test items for h/day on five consecutive days. the lungs were evaluated by analysis of bronchoalveolar lavage fluid (balf) and by histopathology three days and three weeks after the end of the exposure. in a parallel study, rats were intratracheally instilled and equally evaluated three days after instillation. assuming a deposition rate of %, the instilled dose corresponded to the aerosol concentration of mg/m used for inhalation. results show that inhalation and instillation of nominally equal mounts of amourphous silica elicit different results in the lung with inhalative treatment being less harmfull. this difference may be due to the bolus effect inevitable linked to instillation. instillation stuldies with amorphous silica may, therefore, be of limited value with respect to doseresponse assessment. sunscreen products containing uv filters protect consumers from the harmful effects of uv exposure. pigmentary grades of metal oxides like zno result in an opaque whiteness as a result of scattering visible light, whereasnanoparticles result in transparent products for better consumer acceptance and thus improved protection of human skin against uv-induced damage. in addition scatter uv light is most efficiently reflected at a nanosize of - nm. in the last years the toxicological properties of nanozno in comparison with pigmentary zno were examined, the results of these comprehensive studies are presented. all tests were performed according to oecd guidelines, which were modified, especially in regard of substance preparation where appropriate. nanosized zno showed no acute toxicity after dermal application, in the bcop assay as well as in the epiderm assay it showed no corrosion / irritation potential. nanosized zno does not penetrate the intact as well as the sunburned skin. a dermal absorption test in rats (oecd ) with zn-labelled test item as well as penetration tests in weanling pigs after uv radiation did not show a penetration of the zno nanoparticles through the skin. genotoxicity was tested in vitro in the ames test, in the chromosomal aberration test in v cells, both showing negative results whereas the mouse lymphoma mutation test / l y/tk+/-cells was positive. in vivo no mutagenic effect was detected in two mouse micronucleus tests, on with intraperitoneally application and another after repeated inhalation. nanosized zno was tested in -days, -days and -days inhalation studies, in all studies the predominant effects were reversible local inflammatory changes in the nasal cavity and lungs, with a noaec of . mg/m in the -day study. in a prenatal developmental toxicity study according to oecd tg , with repeated inhalation exposure to female wistar rats from gestation day through , maternal toxicity was observed by increase lung weights and inflammations in the lungs. but no substance-related effects on reproductive parameters (conception rate, corpora lutea, implantation sites, preimplantation loss, postimplantation loss, resorptions, dead fetuses) and no increase in external and soft tissue malformations and variations could be detected. the overall result of all the toxicological studies with nanosized and pigmentary zno is that the toxicological profile of both is very similar. studies were sponsored partly by cefci lri and partly by basf se. use of reach registration data for improving thresholds of toxicological concern (ttc) wieneke n., dorn s., jakupoglu c., schäfer c., sica m., wiegand c., mostert v. dr. knoell consult gmbh, marie-curie-str. , leverkusen, germany the threshold of toxicological concern (ttc) concept is utilised to identify human exposures that are so low that in-depth toxicological investigations are expendable. this is called "exposure-based waiving". exposure-based waiving serves to focus available resources on substances with relevant human exposure potential. important work into establishing ttc values has been published by munro et al. ( ) . the initial report used a database of organic substances compiled from publicly available sources. in total, noels were collected in this fashion. the munro concept used the cramer classification to categorise substances according to their hazard potential. we broadened the ttc database by including noaels published on the echa website as per november , containing data for more than registrations. only nongaseous mono-constituent substances with oral noaels were included in the ttc database. organophosphates and genotoxic substances were excluded from the database as well as noaels obtained for surrogate substances. noaels for all systemic endpoints (general toxicity, developmental toxicity, fertility, neoplasia) were taken into account. where appropriate, default assessment factors of up to were used to establish chronic noaels for each substance. for every eligible substance, we collected the published clp category for acute oral toxicity as a potential predictor of overall hazard potential. this gives rise to five categories of acute oral toxicity. a ttc is calculated from the th percentile of noaels in each of these categories using the reach rules for establishing dnels for workers and the general population. this poster presents the preliminary results for more than substances. the results indicate that the ttc concept becomes more robust when using the very broad echa database. it also suggests that acute oral toxicity categories can be used as a predictor for the overall hazard potential of a substance. comparison of different in-vitro models for inhalation toxicology with respect to the effects of cigarette smoke total particulate matter wiese j. , schumann b. b-and l-moc can be cultivated up to days without loss of viability, as determined by resazurin-assay. viability of cell cultures was determined by mtt-assay after incubation with increasing doses of tpm. exposure of h to tpm ( mg/l) reduced viability to % or % after or h, respectively. in a viability was % after h with tpm ( mg/l). the same dose of tpm lead to a decrease in viability to % ( h) or % ( h) in nhbec and to % ( h) or % ( h) in plc. as a marker of oxidative stress the level of intra-cellular glutathione (gsh) was determined by hplc. in both the tumor cell lines analysed gsh-level was increased by tpm ( mg/l). in h the induction was . and . fold after or h, respectively. while in a it was . ( h) and . fold ( h). in nhec and plc tpm ( mg/l) did not have a significant effect on gsh-levels after h. in n-moc tpm ( mg/l) also did not modulate gsh after h, but it diminished gsh-level by . fold upon prolonged contact ( h). in b-moc gsh concentrations also decreased to . or . fold the level of controls after or h incubation. the results presented show that in-vitro models of varying complexity and origin within the respiratory tract clearly differ in their response to tpm, which was used a model inhalative toxicant. the tumor cell lines used seem to be better adapted to chemical stress, while the models closer to the in-vivo situation are more vulnerable. the nongenomic effects of the mineralocorticoid receptor in transgenic mouse heart winter s. , schreier b. within the renin-angiotensin-aldosterone system (raas), the mineralocorticoid receptor (mr) is important for the regulation of fluid and electrolyte balance in the kidney, salivary glands, sweat glands and colon. however, survival of patients with severe heart failure is increased when mr blockage is combined with standard therapy suggesting aldosterone, the mr ligand, as a key factor in the development of cardiovascular diseases, but the mechanism is not yet fully understood. in recent years, evidence accumulated that besides its function as a hormone-activated transcription factor the mr also functions via nongenomic pathways. to investigate the function of the nongenomic effects of the mr in cardiovascular dysfunction, we generated a transgenic (tg) mouse model expressing a truncated human mr lacking the dna-binding site (hmr def ) under control of the cardiac specific α myosin heavy chain promoter (αmhc), a model for nongenomic effects of the mr in the heart. in this mouse model no enhanced mortality could be observed. body weight (bw), heart weight and relative heart weight were not different compared to wild type (wt) while left atrial weight/bw was increased by % (wt , ± , mg/g vs. tg , ± , mg/g, p< . , n= ). compared to wt mice neither surface electrocardiographic experiments nor echocardiographic experiments revealed modified parameters for tg mice under basal (i.e. unstimulated) conditons as well as under β-adrenergic stimulation by isoproterenol (iso, µl mm iso intraperitoneally applied). to uncover the role of aldosterone in the development of cardiovascular diseases treatment with aldosterone and high-salt diet ( %) was performed. after days cardiac function and heart dimensions were analyzed, surface electrocardiographie uncovered increased p duration ( ± . ms vs. ± . ms, p< . ) and qtc interval ( ± ms vs. ± ms, p< . ) in tg (n= ) compared to wt (n= ) animals. these findings probably indicate more sensitive conduction pathways to aldosterone in tg mice. oligomerization is important for regulation of phospholamban activity wittmann t., lohse m. j., schmitt j. p. institut für pharmakologie und toxikologie, versbacher straße , würzburg, germany phospholamban (pln) is a heart specific protein located in the membrane of the sarcoplasmic reticulum. it inhibits the ca + -atpase serca a, thereby decelerating cytosolic ca + clearance during diastole of the cardiac cycle. upon phosphorylation the inhibitory activity of pln on myocyte ca + transport is attenuated. further, it is believed that phosphorylation favors the formation of (rather inactive) pentamers and that pln pentamers itselves were an inferior substrate for phosphorylation compared to monomers. this would suggest an important role of pln oligomerization in the regulation of pln activity. to prove this hypothesis, we are investigating the patterns and kinetics of pln phosphorylation in the context of alterations in pln structure. the introduction of specific point mutations into the transmembrane region of pln yielded mutants that are purely monomeric (l a, i a and c f) or favor pentamer formation (i a, v a). transfected hek cells expressing these mutants or wildtype pln were stimulated with forskolin to induce pln phosphorylation before lysis of cells and western blot analysis using antibodies directed against phosphorylated pln. surprisingly, phosphorylation was increased for both monomeric and pentameric pln after stimulation with . µm forskolin for less than one minute. at increasing forskolin concentrations phosphorylation signals increased in parallel for monomers and pentamers. for measurement of phosphorylation kinetics stimulation of cells with . µm forskolin was stopped at different time points. we found phosphorylation of both pln monomers and pentamers within seconds of stimulation. differences in phosphorylation patterns became more pronounced when assays were performed at low temperature ( °c). intriguingly, preliminary analyses suggest that pka dependent phosphorylation occurs first in pentamers and that phosphorylation of monomers may catch up only after pentamer phosphorylation is almost complete. our data suggest that both pln pentamers and monomers are suitable substrates for pka dependent pln phosphorylation. unlike the prevalent assumption, kinetics of pentamer phosphorylation seem to be at least as fast as that of pln monomers in transfected hek cells suggesting an important role of pln pentamers in the regulation of pln activity. regulation of cardiac contractility by nucleoside diphosphate kinases in zebrafish wolf n. m. , , abu-taha i. in the heart, nucleoside diphosphate kinases (ndpks) can interact with heterotrimeric g proteins, thus regulating camp synthesis in a receptor independent manner and thereby influencing contractility in cardiomyocytes. we further investigated the interaction of ndpk isoforms with heterotrimeric g proteins in the heart in vivo and in vitro using zebrafish embryos and embryonic fibroblast from ndpk a/b double knockout mice (ndpk a/b ko mefs). in zebrafish the morpholino-mediated knockdown of ndpk a did not lead to an obvious phenotype, although the total ndpk activity was reduced. depletion of ndpk b caused a cardiac phenotype characterized by severely impaired atrial and ventricular contractility and insufficient blood flow. the depletion of ndpk b was associated with a significant decrease of protein levels of the heterotrimeric g protein subunits gβγ, gα s and gαi. the knockdown of ndpk c led to a more restricted cardiac phenotype with markedly reduced pumping function of the ventricle, while the atrium was unaffected. in accordance to the reduced cardiac pumping function, camp levels were significantly diminished in the ndpk b and ndpk c morphants. similar findings were obtained in ndpk a/b ko mefs. the absence of ndpk a and b resulted in a decrease of the plasma membrane content of gβ and gαs and a significant reduction in camp synthesis. the protein expression of the isoform ndpk c was also significantly reduced in the ndpk a/b ko mefs. the re-expression of ndpk b but not ndpk a rescued the basal camp production and the membrane content of g proteins. interestingly, the overexpression of ndpk c led to a -fold enhancement of the camp level and a significant increase of the membrane content of gβ and gα, and thus rescued the knockout phenotype. our data indicate, that the ndpk isoforms b and c are essential for cardiac contractility, most likely by forming a signaling complex at the plasma membrane including ndpk b, ndpk c and heterotrimeric g proteins. the isoform ndpk c, with its n-terminal hydrophobic region, might serve as a membrane anchor for the ndpk/g protein complex. induction of apoptosis via pka-dependent and pka-independent pathways by cyclic purine and pyrimidine nucleotides in mouse lymphoma cell lines wolter s. camp is a second messenger that plays an important role in intracellular signal transduction of various hormones and neurotransmitters. a major function of camp in eukaryotes is the activation of camp-dependent protein kinase a (pka). pka is involved in the control of a variety of cellular processes. pka exists as an inactive tetramer of a dimeric regulatory (r ) and two catalytic (c) subunits that releases the active c-subunits upon binding of camp. stimulation of the mouse t-lymphoma cell line s wild-type (wt) with dibutyryl (db)-camp induces apoptosis by an intrinsic, mitochondria-dependent mechanism. apoptosis induced by db-camp occurs via a pka-dependent mechanism, since s kincells lacking the catalytic subunit of pka are resistant to db-camp-mediated cell death. db-camp is cleaved by esterases into the biologically active compound n -mb-camp and into '-o-mb-camp. other cyclic nucleotides (cnmps) in addition to camp, like ccmp and cump can also function as second messengers and activate pka and cgmp-dependent protein kinase (pkg) . therefore, we investigated the effects of a series of membrane-permeable analogues of camp, cgmp, ccmp and cump in s wt und s kincells on apoptosis. stimulation with db-ccmp or db-cgmp induced neither apoptosis in s wt nor in s kincells. interestingly, we observed apoptosis in s wt and s kin cells after incubation with membrane-permeable nucleotide acetoxymethyl ester (am)-analogues of cgmp, ccmp, cump and also camp. induction of apoptosis occurs via pkadependent and also pka-independent pathways. a potential role of pkg and of the exchange protein activated by camp (epac) in the induction of apoptosis is unsolved and will be explored by specific pkg-and epac-activators in this system. investigations are on the way to identify the targets, the involved signal transduction pathways and the mechanisms of pro-apoptotic actions mediated by cnmp-ams. ( ) wolter s, golombek m, seifert r ( ) differential activation of camp-and cgmpdependent protein kinases by cyclic purine and pyrimidine nucleotides. biochem biophys res commun. in press apoptosis in s cells : induction of apoptosis in s wt and in s cells lacking the catalytic subunit of pka (s kin-) with a) db-cnmps and b) cnmp-ams for h. nebivolol reduces vascular inflammation in spontaneously hypertensive rats wu z., xia n., förstermann u., li h. institut für pharmakologie, obere zahlbacher str. , mainz, germany nebivolol is a third generation β receptor blocker with additional effects on endothelial nitric oxide production. the aim of the present study is to investigate the antiinflammatory effects of nebivolol in vivo. spontaneously hypertensive rats (shr) were divided into two groups: control or nebivolol treatment group. nebivolol treatment ( mg/kg/day for days) significantly reduced the blood pressure and the heart rate in shr. the drug had no effect on coagulation. aorta from nebivolol-treated rats showed significantly improved endothelial function. nebivolol did not change the expression levels of aortic nf-kb, but significantly reduced its dna binding activity. furthermore, nebivolol decreased the expression of adhesion molecules (e.g. icam- , vcam- ) and pro-inflammatory cytokines (e.g. il- ). in conclusion, nebivolol reduces vascular inflammation in experimental hypertension. it inhibits nf-kb activity, decreases the expression of adhesion molecules and pro-inflammatory cytokine, and improves endothelial function. characterization of the cellular activity of pde inhibitors using two novel pde reporter cell lines wunder f., quednau r., barg m., tersteegen a. bayer pharma ag lead discovery wuppertal, aprather weg a, wuppertal, germany cyclic nucleotide-specific phosphodiesterases (pdes) play an essential role in cellular signal transduction by regulating the intracellular levels of camp and cgmp and, therefore, are important pharmacological targets. we report here the generation and pharmacological characterization of two novel pde reporter cell lines. plasmid constructs encoding human pde b or pde d were stably co-transfected with the beta -adrenoceptor in a parental camp reporter cell line expressing a cyclic nucleotide-gated (cng) cation channel, acting as the biosensor for intracellular camp. in this reporter cell line, camp levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the cng channel. by using different pde and non-pde inhibitors, we could show that our novel pde b and pde d reporter cell lines specifically monitor pde inhibition with high sensitivity. pde -selective inhibitors alone did not increase basal luminescence levels in this experimental setting. however, these inhibitors induced concentration-dependent luminescence signals in combination with the adrenoceptor agonist isoproterenol. in contrast, in a stable beta -adrenoceptor reporter cell line with no recombinant pde expression, pde inhibitors had no effect on isoproterenol-stimulated luminescence signals. we compared the cellular activity of different pde inhibitors with the in vitro inhibition of full-length and truncated (catalytic domain) pde d from cell lysates. two different groups of pde inhibitors could be identified. the first group, including the allosteric inhibitors pmnpq and d , showed high cellular activity and much better inhibition of full-length versus truncated pde d . the second inhibitor group, including classical competitive inhibitors like roflumilast, cilomilast and piclamilast, showed comparably lower cellular activity and similar inhibitory activity on full-length and truncated pde d . the results imply that these novel pde reporter cell lines are well-suited for the characterization of the cellular activity of pde inhibitors and may also support a better understanding of the complex pde pharmacology. plexin-b is required for kidney regeneration after acute renal failure xia j. , gröne h acute renal failure is a common clinical problem with unsatisfactory therapeutic options and high mortality in humans. therefore, unraveling the mechanisms that promote kidney regeneration and repair may provide new therapeutic strategies for acute renal injury. plexin-b belongs to a family of transmembrane receptors which mediate the cellular effects of semaphorins. while plexins have first been described in the context of axon guidance, several recent studies have established them as key regulators of organogenesis, the immune system and cancer. we have recently found that plexin-b is highly expressed in the adult kidney, particularly in tubular epithelial cells which are most sensitive to acute ischemic injury. to study the role of plexin-b during kidney regeneration we generated mice lacking plexin-b specifically in tubular epithelial cells. under physiological conditions, these mice displayed normal kidney morphology and function. in contrast, following ischemia/reperfusion injury, plexin-b conditional knockout mice exhibited severely impaired kidney regeneration. while the renal function of control mice fully recovered within weeks after injury, plexin-b knockout mice had strongly elevated serum creatinine and urea levels associated with increased morbidity and mortality. this was accompanied by hyperproliferation of tubular epithelial cells and obstruction of tubular lumina. we conclude that plexin-b is required for regeneration after acute ischemic renal injury and that pharmacological interventions activating plexin-b might represent a new therapeutic strategy in acute renal failure. the nadph oxidase enzyme complex consists of two membrane-bound catalytic subunits (a nox protein and p phox) and several cytosolic regulatory components including p phox, p phox, p phox and the small gtpase rac . we have previously shown that treatment of apolipoprotein e knockout mice with resveratrol led to a downregulation of nox and nox in the heart. our recent data demonstrated that resveratrol also reduced the enzymatic activity of cardiac nadph oxidase. because activation of nadph oxidase enzyme complex is induced by translocation of the regulatory subunits, we studied whether the reduced enzymatic activity is due to an inhibition of such a translocation. indeed, resveratrol treatment prevented rac membrane translocation from cytosol. resveratrol is known as an activator and expression enhancer of the longevity gene sirtuin (sirt ). we then wanted to find out whether the effect of resveratrol on rac was mediated by sirt . sirt is a histone/protein deacetylase. in vitro incubation of rac with sirt led to a reduction of lysine acetylation. deacetylation of rac on lysine could be identified by mass spectrometry analyses. the lysine lies within the p phox-binding region of rac . consistently, in vitro incubation of rac with sirt markedly reduced its binding activity to p phox. in conclusion, we provide evidence that rac is a direct target molecule of sirt . sirt deacetylates rac on lysine and thereby inhibits its interaction with p phox. this is a novel mechanism of nadph oxidase inhibition by sirt /resveratrol. mutational analysis of the effects of the cardioprotective drug dexrazoxane on topoisomerase ii beta in vitro yan t., deng s., frensch i., gödtel-armbrust u., wojnowski l. universitÄtsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher straße , mainz, germany dexrazoxane (drz, icrf- ) is the only approved drug shown to protect against anthracycline-induced heart failure. the protection is usually ascribed to the ironchelation by drz, which is thought to reduce anthracycline-induced oxidative stress. however, similarly to anthracyclines, drz is also an inhibitor of the anthracycline target topoisomerase ii (top ). we hypothesized that the cardioprotective effects of drz are mediated by the prevention of the anthracycline-induced dna damage mediated by top b, the dominant cardiac top isoform. this was investigated using top b mutants resistant to drz, which were expressed in cells depleted of wild-type top isoforms. top b-mediated double-strand dna breaks were assessed as γ-h ax. the levesl of dsb generated by the top b mutants in response to the anthracycline doxorubicine (dox) was indistinguishable from that mediated by a wild-type top b. preincubation with drz depleted wild-type top b and this was accompanied by a decrease in the dna damage following a subsequent exposure to dox. in contrast, neither top b depletion nor the reduction of dsb by drz was seen in drz-resistant top b mutants. furthermore, the cardially ineffective drz analog icrf- , capable of iron chelation but not of top binding, affected neither the stability of top b, nor the dox-induced dna damage mediated by this enzyme. these results indicate that drz may exert its cardioprotective effects by reducing the dna damage mediated by doxpoisoned top b rather than by iron chelation. they also suggest a cardioprotective function of top b, which is currently under investigation using cardiomyocyte-specific top b mouse knockouts. aminoglycosides are important antibiotics in the treatment of life-threatening infections, especially those caused by gram-negative bacteria. their nephrotoxic and ototoxic potential is well-known, but little is known about the effects of aminoglycosides on the male reproductive system. we studied the effects of four aminoglycosides on sertolicells in vitro. rat sertoli-cells from the cell line serw were cultivated for , , and days in dmem supplemented with three different concentrations of amikacin, streptomycin ( mg/l, mg/l, mg/l), gentamicin or tobramycin ( mg/l, mg/l, mg/l). we determined the expression of two junctional proteins (connexin , ncadherin) and one protein of the cytoskeleton (vimentin) by western blot. cells were solubilized in lysis buffer. lysates were separated by sds-page and electroblotted on a pvdf-membrane. after incubation with primary antibodies overnight and horseradish peroxidase-conjugated secondary antibody the visualization was achieved by a chemiluminescence-detection system. in addition, proteins were detected by immunohistochemistry. after three days in culture amikacin caused the most pronounced effect. at the lowest concentration tested ( mg/l) connexin and n-cadherin were reduced to ± % and ± % of the controls (n= ). no change was recognized for vimentin ( ± %). effects obtained with streptomycin were less pronounced for these these proteins ( ± %, ± %, and ± %, respectively). similar, but less pronounced effects were observed with gentamicin and tobramycin at a concentration of mg/l (connexin : ± % and ± %; n-cadherin: ± % and ± %; vimentin: ± % and ± %) and mg/l (connexin- : ± % and ± %; n-cadherin: ± % and ± %; vimentin: ± % and ± %). after incubation for and days the effects occurred in the same range. the substances showed no influence on the viability of serw sertoli-cells up to mg/l in the mtt assay. by immunohistochemistry we showed that the localisation of the proteins -connexin and n-cadherin at the cell membrane and vimentin in the cytoplasm -was not influenced by the aminoglycosides. large conductance calcium-and voltage-gated potassium (bk) channels play an important role in controlling membrane potential and calcium influx, and are strongly modulated by protein kinases at multiple sites. the stress-regulated exon (strex) adds to the bk channel c terminus a cysteine-rich insert of amino acids that inverts the channel regulation by protein kinase a (pka) from excitatory to inhibitory. here we investigated the mechanisms by which the strex insert influences bk channel regulation by protein kinase c (pkc). activity of bk channels without strex insert (bk-zero), transiently expressed in hek cells, was inhibited by pkc in inside-out membrane patches (~ % inhibition). bk channels with strex insert (bk-strex), however, were insensitive to pkc. phosphomimetic mutation of a pkc phosphorylation site (s e) in bk-strex, resulted in a ~ % reduction of basal channel activity, whereas the s a mutant retained normal activity. to examine whether palmitoylation, and thus association of the strex domain with the plasma membrane, prevents pkc inhibition of bk channel gating, palmitoylation was abolished by either site-directed mutagenesis (c : a) or by pharmacological inhibition of palmitoyl transferases with -bromopalmitate ( -bp). both, mutation and pretreatment with -bp resulted in the expression of bk-strex channels which were sensitive to pkc (~ % inhibition of channel activity). no inhibitory pkc effect was observed in patches of the bk-strex s a channel mutant pretreated with -bp. in a clonal rat somatomammotroph pituitary cell line (gh b ), in which pcr products without (zero) and with the bp strex exon could be identified, the pkc activator pma blocked channel activity by ~ %. this inhibition was increased to over % when gh b cells were pretreated with -bp, indicating that both channel isoforms were functionally active. in summary, the present study demonstrates that palmitoylation of strex prevents bk channel regulation by pkc, which is mediated by phosphorylation of ser , probably by steric hindrance. our results provide further evidence for a cross-talk between palmitoylation and phosphorylation as a crucial mechanism underlying the dynamic regulation of ion channels. human pleural mesothelial met- a cells are a limited in vitro model system in determining potential asbestos-like genotoxic effects of multiwall carbon nanotubes ziemann c. , reamon-büttner s. multiwall carbon nanotubes (mwcnt) are nanomaterials with important technological impact. depending on their diameter, length, and biopersistence, however, some mwcnt seem to exhibit a fiber-like cytotoxic and genotoxic potential, similar to asbestos. thus, a project funded by the german federal ministry of education and research (bmbf contract no. x a) focuses on potential adverse biological effects of different types of mwcnt to enlarge the knowledge base about toxicity determining parameters. this project comprises both in vitro (rat) and in vivo endpoints with long amosite asbestos as a positive control. as mesothelial cells are target cells for adverse effects of asbestos, in particular mesothelioma development, the human sv transformed, non-malignant pleural mesothelial cell line met- a was chosen as the main in vitro model in this project. in the present study part, met- a cells were characterized concerning their usefulness as an in vitro model to study potential asbestos-like cytotoxic and genotoxic effects of different mwcnt varieties. using an mwcnt-optimized lactate dehydrogenase liberation assay and proliferation parameters derived from cell counts, concentration-dependent cytotoxicity of long amosite asbestos ( , , and µg/cm ) was demonstrated in met- a cells. cells also showed asbestosinduced increase in dna-strand breaks and oxidative dna-damage in the hogg modified comet assay. thus, met- a cells were responsive to asbestos treatment. owing to asbestos potential to induce aneugenic effects and spindle fiber damage, micronucleus induction, determination of numerical chromosome aberration, and altered meta-, ana-, and telophase morphology were planned as in vitro endpoints. met- a cells were thus initially characterized in this regard and were found to exhibit highly variable chromosome numbers with lower than % cells exhibiting a normal diploid chromosome set, an up to twentyfold higher spontaneous micronucleus frequency, as compared to polychromatic bone marrow erythrocytes in rodents, and a profound number of aberrant meta-, ana-and telophases with bridges, lagging chromosomes and multipolar divisions. in conclusion, met- a cells are of only limited value as an in vitro model system to study potential asbestos-like effects of mwcnt and also biopersistent fibers. the cells are indeed responsive to asbestos, but unfortunately demonstrate marked genomic instability and thus limited significance concerning genotoxic effects. waixenicin a inhibits cell proliferation through magnesium-dependent block of trpm channels zierler s. transient receptor potential melastatin (trpm ) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. they are abundantly expressed in a variety of human carcinoma cells controlling survival, growth and migration. these characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. we screened a chemical library of marine organism-derived extracts and identified waixenicin a from the soft coral sarcothelia edmondsoni as a strong inhibitor of overexpressed and native trpm . waixenicin a activity was cytosolic and potentiated by intracellular free magnesium (mg + ) concentration. mutating a mg + -sensitive site on the trpm kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of mg + . waixenicin a failed to inhibit the closely homologous trpm channel and did not significantly affect trpm , trpm , and crac (calcium release activated calcium) channels. therefore, waixenicin a represents the first potent and relatively specific inhibitor of trpm ion channels. consistent with trpm inhibition, the compound blocked cell proliferation in human jurkat t-cells and rat basophilic leukemia cells. based on the compound's ability to inhibit cell proliferation through mg + -dependent block of trpm , waixenicin a or structural analogs may have cancer-specific therapeutic potential, particularly since certain cancers accumulate cytosolic mg + . release of metals from different sections of domestic drinking water installations zietz b. p., richter k., laß j., suchenwirth r., huppmann r. governmental institute of public health of lower saxony division of environmental medicine and environmental epidemiology, roesebeckstraße - , hanover, germany different metals were used as important piping materials in the drinking water supply for a long time. due to corrosion metals can leach into the tap water. of special importance is the toxic element lead. however other heavy metals in drinking water such as copper, nickel and cadmium can also give reason for health concerns. in this study it was investigated in which amount relevant metals were released from different parts of domestic installations into the cold water. for the spatial allocation of the emission sources a sequential water sampling protocol was used after three hours of stagnation time representing the first litre of the water column. after stagnation ten sample volumes were collected in series. existing facilities of domestic installations constructed with different plumbing materials were examined predominantly from residential buildings. the elements al, as, cd, cr, cu, fe, mg, mn, ni, pb, sb, se, u and zn were detected by means of icp-ms. in total water pipe strands of domestic installation systems were examined. they comprised single water samples and single parameters. depending upon the type of plumbing different courses and concentration ranges of the elements could be measured in the tap water samples. terminal taps or installation parts were frequently responsible for a release of nickel and in several cases of cadmium. the concentration courses of the element zinc proved as a good indicator for the allocation of the emission source to a brass containing section of the installation (zinc as an alloy component of brass). one can conclude that an investigation by means of a sequential water sampling protocol and multi-element detection can be a valuable non-destructive method for drinking water-hygienic investigations of domestic installations. novel interaction partners of the murine trpc protein zimmermann j., beck a., flockerzi v. universität des saarlandes experimentelle und klinische pharmakologie und toxikologie, kirrberger str. , homburg, germany in this work novel interaction partners of the murine protein transient receptor potential canonical (mtrpc ) were identified. the trpc protein is the major subunit of a cation channel, residing in the plasma membrane. it comprises six trans-membrane domains and cytosolic amino and carboxyl termini. two major splice variants of the trpc gene exist, trpc a ( aa) and trpc b ( aa), trpc b lacks aa to of the trpc a variant. both trpc variants are co-expressed in endothelial cells, intestinal smooth muscle and brain. to identify trpc -interacting proteins a yeast two-hybrid system, cytotrap®, which allows identification of protein-protein interactions within the cytosol was used. a premade mouse brain cdna library was screened by the cytosolic amino and carboxyl terminal parts of mouse trpc a (aa to ; aa to ). for the carboxyl terminal part fourteen proteins were identified. to independently prove the interaction, the fulllength cdnas of all fourteen proteins were cloned, fused to a flag-tag and coexpressed with trpc in hek cells. co-immunoprecipitations were performed for all candidates using both the anti-flag-antibody and the antibody for trpc . in addition, changes of cytosolic calcium were monitored and trpc currents were recorded in hek cells expressing the candidate cdnas and stably expressing the trpc a or trpc b and the muscarinic receptor type cdnas. the tarbp protein, one of the candidates shown to interact with trpc , changed calcium influx when coexpressed with trpc . in order to identify the domains of trpc responsible for its interaction with the tarbp protein, six trpc -gst-fusion proteins covering the carboxyl terminal aa of trpc a were expressed in e. coli and used for pull-down experiments. by this approach two domains of trpc could be identified to interact with tarbp . one of these domains is well conserved within the trpc protein, corresponding to the result, that trpc and tarbp effectively co-immunoprecipitate, too. the tarbp protein has been shown to be a component of the risc loading complex, also known as the micro-rna loading complex which is composed of dicer , eif c /ago and tarbp (chendrimada et al. ) . by its interaction it may link trpc to pre-microrna processing. increased levels of angiotensin ii provoke dna damage and have influence on dna repair in mouse kidneys zimnol a., brand s., schupp n. universität würzburg institut für toxikologie, versbacherstr. , würzburg, germany the renin-angiotensin system (ras) plays a crucial role concerning the blood pressure, electrolyte balance and cardiovascular homeostasis. angiotensin ii (ang ii), the active hormone of the ras, in higher concentrations leads to vasoconstriction, oxidative stress and hypertension. hypertensive patients have an increased risk to develop cancer, especially kidney cancer. we have shown in vitro and in vivo, that ang ii is capable to cause an elevation of blood pressure as well as dna damage dose-dependently. to investigate whether the high blood pressure or the enhanced levels of ang ii are responsible for dna damage, male c bl/ -mice were equipped with osmotic minipumps, delivering ang ii in a concentration of ng/kg · min during days. additionally they were treated with ramipril, an angiotensin-converting-enzyme blocker, with the ang ii receptor antagonist candesartan, the vasodilator hydralazine, and the antioxidant tempol. dna damage was analysed with the comet assay. we measured the base excision repair (ber)-related dna repair in the kidney with a comet-based in vitro repair assay. furthermore, the distribution and expression of the ang ii-type (at ) receptor in the kidney was analyzed by immunohistochemistry. treatment with ang ii led to a significant increase of blood pressure, whereas the medication with candesartan decreased the systolic blood pressure. the intervention with hydralazine lowered the blood pressure only for a short time. the other substances had no effect at all on the blood pressure. genomic damage, quantified with the comet assay, was augmented by ang ii and improved by all interventions, particularly by candesartan and tempol. beyond that, ang ii showed a tendency to reduce dna repair. treatment with candesartan, hydralazine and tempol increased the repair capacity. furthermore, ang ii tended to result in a downregulation of the at receptor in kidney tubule cells. candesartan and ramipril, especially were able to augment the expression of the at receptor, whereas hydralazine achieved the opposite. these results demonstrate that ang ii leads to dna damage in the kidney independent of blood pressure. apparently elevated levels of ang ii affect dna repair and expression of at receptor. to confirm these findings we are going to examine more precisely the manifestation of other enzymes, which are implicated in dna repair. regulation of hcn channel activity by cyclic cytidine ´, ´-monophosphate zong x., krause s., chen c. -c., gruner c., cao-ehlker x., fenske s., wahl-schott c., biel m. lmu münchen, department pharmazie, pharmakologie für naturwissenschaften center for integrated protein science munich (cipsm), butenandtstr. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] münchen, germany hyperpolarization-activated cyclic nucleotide-gated (hcn) channels play a key role in controlling cardiac pacemaker activity and are essential for normal function of neuronal circuits. hcn channels are principally gated by voltage but are coactivated by the cyclic nucleotides camp and cgmp which directly bind to a c-terminal binding domain. recently, cyclic cmp (ccmp) was shown to be present in various cell lines and tissues at concentrations that are comparable to cellular cgmp levels. moreover, there is recent evidence that ccmp can activate camp-and cgmp-dependent protein kinases in vivo. here, we examined whether ccmp exerts effects on hcn channels. to this end, we recorded hcn channel-mediated currents (i h) in hek cells that stably express hcn , hcn , hcn or hcn , respectively. currents were measured either with a standard patch-clamp setup or by employing the planar patch-clamp technology. in hcn and hcn channels, ccmp shifted the membrane potential for half maximal activation (v . ) to more positive values. in addition, ccmp accelerated activation while it slowed down deactivation kinetics. the ec for ccmp was µm which is about times higher than the ec of cgmp. cyclic cmp is a partial agonist of hcn channels since it activates only % of the maximal current obtained with camp or cgmp. to identify in vivo effects of ccmp we recorded ih of murine sinoatrial node (san) cells in the presence and absence of mm ccmp. like for heterologously expressed hcn channels, ccmp shifted v . of ih by about + mv. importantly, the steepness of the diastolic depolarization of san pacemaker potentials which is mainly determined by the amplitude of ih was profoundly increased by ccmp, compared to control conditions. as a consequence of the upregulation of ih the frequency of san pacemaker potentials was increased by about % in the presence of ccmp. our results suggest that ccmp is a physiological regulator of hcn channel activity. a d actinic keratosis like construct for assessment of innovative tumour therapeutics zoschke c. the incidence of actinic keratosis (ak) has increased dramatically in the last decades and it is considered the most frequent carcinoma in situ today. especially immunosuppressed patients are at high risk to develop invasive squamous cell carcinoma (scc) [ ] which asks for most efficient and well-tolerated ak therapy. yet, current measures do not fit with these demands. nucleotide analogues, recently identified by molecular modelling [ , ] , outperformed the current standard for the therapy of actinic keratosis, -fluoruracil, when tested in the tumour cell line scc , in normal human keratinocytes and fibroblasts [ ] . as next step in pre-clinical drug assessment, we aimed to characterise the effect of the most selective nucleotide analogue oxbu in reconstructed human tumour skin. based on the d construct of scc developed by höller and co-workers [ ] for start we introduced several adaptations with respect to keratinocyte, fibroblast and scc seeding to grow an aklike construct with scc cells forming nests in particular in the epidermis. in addition, first experiments with the oxbu on the ak like constructs showed promising results for an efficient treatment of actinic keratosis. efficacy was derived from immunohistology (marker for proliferation: ki- , marker for scc: cytokeratin- , axl, marker for invasion: mmp , marker for apoptosis: caspase- , nuclei were stained with dapi) as well as the effects on the secretion of cytokeratin- and its caspase-induced cleavage product into the culture medium following drug exposure for up to days. efficacy of a . % oxbu solution proved close to or even better when compared to both a . % fluorouracil and . % aphidicolin solution. the former being the gold standard of current ak therapy, the latter is a frequently used inhibitor of human polymerase alpha and delta, however, failed to be introduced into clinical use. -in fact, in monolayer cultures aphidicolin proved most toxic for normal human keratinocytes which was not true with oxbu [ ] . -therefore, these d tumour constructs offer a new approach to pre-clinical drug assessment and may be added to other d models of skin diseases currently gaining increased interest as test platforms. ima , a multi-peptide cancer vaccine for advanced colorectal cancer, induces multiple cd + and cd + t-cell responses associated with improved survival walter s. , kuttruff s. ima is a novel peptide-based vaccine consisting of hla-a* and hla-dr binding synthetic peptides that were identified based on natural presentation on human colorectal cancer (crc) samples. ima was characterized in a phase i/ii trial in advanced/metastatic crc patients being at least clinically stable after weeks of first-line oxaliplatin-based therapy. all patients received a single application of low-dose cyclophosphamide for immunomodulation. this was followed by repeated ima vaccinations in combination with low-dose gm-csf (cohort ; n= ) or ima / gm-csf plus topically applied imiquimod (cohort ; n= ). before and post vaccination, patients were analyzed by hla-multimer assay and intracellular cytokine (ics) assay for cd + t-cell responses and by ics assay for cd + tcell responses. as immune status biomarkers, phenotypically defined myeloid derived suppressor cell populations (mdsc - ) were analyzed prior to immunotherapy. tumor status of patients was monitored repeatedly by ct/mri according to recist and corresponding tumor scans were reviewed centrally. clinical assessment included disease control rate (dcr), time to progression (ttp), progression-free survival (pfs) and overall survival (os). ima overall was immunogenic in / ( %) evaluable patients, with % and % of patients mounting multiple cd + and cd + t-cell responses, respectively. patients that received the immunomodulator imiquimod presented with significantly more multiple cd + cell responses as detected by ics (p= . ). multiple cd + and multiple cd + responses were individually associated with significantly better clinical outcome. the association was most pronounced for patients with both multiple cd + and multiple cd + responses. these patients had significantly higher dcr at months (p= . ), improved ttp (p= . ) and improved pfs (p= . ) than other patients. most importantly, a trend for prolonged os was also observed in these patients (p= . , hazard ratio . ). in the study population, levels of different mdsc phenotypes were significantly increased as compared to age/gender matched controls. high mdsc levels were associated with fewer immune responses and for mdsc and mdsc high frequencies were associated with shorter os (p= . and p= . , respectively). to summarize, both hla-a* and hla-dr restricted peptides in ima were immunogenic. a significantly better clinical outcome of multi-tumap responders in comparison to patients with one/no tumap response strongly indicates clinical activity of ima . acrylamide (aa), a genotoxic carcinogen (iarc class a) is formed in food by thermal treatment from different precursors. after oral ingestion, aa is metabolically epoxidized in the liver by cyp e into glycidamide (ga). ga binds to dna, forming covalent adducts, primarily at n of guanine (n -ga-gua). both, aa and ga undergo conjugation to glutathione (gsh) to be excreted via urine as mercapturic acids (ma), namely nacetyl-s-( -carbamoylethyl)-cysteine (aama), and n-acetyl-s-( -hydroxy- carbamoylethyl)-cysteine (gama). in a dose response study, encompassing the dosage range from human dietary exposure levels up to mg/kg bw, female sprague dawley (sd) rats on a diet devoid of detectable aa content were gavaged with single doses of aa. formation of urinary mas and of n -ga-gua dna adducts in liver, kidney and lung was measured h after application, a time point where cmax of n -ga-gua was reached. the untreated control group was found to excrete about . nmol (aama plus gama) in the urine ( h), indicating a background of endogenous aa formation. compared to untreated control, the lowest dosage of . µg aa/kg bw neither resulted in significantly enhanced ma excretion, nor in a detectable n -ga-gua adduct levels in any organ tested (limit of detection, lod, . adducts/ nucleotides). at the tenfold higher dose ( µg/kg bw), adducts were found in kidney (about adduct/ nucleotides) and lung (< adduct/ nucleotides), but not in liver. at and µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at µg aa/kg bw (about - adducts/ nucleotides). the results of this in vivo study and of further recent research on aa toxicology will be discussed with respect to risk assessment. exposure of rats to single doses of aa in the range of human dietary exposure ( . - µg/kg bw ) leads to n -ga-gua adduct levels in the tissues monitored obviously not exceeding the range of steady state background dna lesions associated with endogenous/exogenous exposure to various genotoxic electrophiles. thus, the question of significant impact on human background dna damage resulting from exposure to a given genotoxic carcinogen, and on potentially ensuing biological consequences may become a highly relevant issue in risk assessment. pharmaco-economic impact of price, volume and demographic development böcking w., kirch w. institut für klinische pharmakologie, medizinische fakultät der tu dresden, fiedlerstr. , dresden health insurance costs in germany have grown by % p.a. over the last ten years and amount to approx. bn eur in . while costs for stationary treatment as the largest cost category have been intensely analyzed over the past years, pharmaceutical expenses have been analyzed in less detail, mostly focusing on the statutory health insurance side, even though pharmaceutical expenses have grown almost twice as much as costs for ambulant treatments. therefore, the question was asked how pharmaceutical expenses in a large german private health insurance company are allocated with respect to age and indication groups, and how those have developed from to . the data of a private health insurance company with more than . customers was split into price and volume effects per age group to understand if price or volume drives the cost development. additionally, the two largest indication groups are analyzed in detail. as a result, both price and volume effects drive an overall cost increase. these effects are even stronger in older age groups. this cost increase is not sustainable for the german health insurance system over a longer period of time and will even further increase due to the ageing of the german population. a novel animal replacement system for the detection of endocrine disruptive capabilities in sexual development scheider j. , , winter p. alternatives to animal testing for prediction of local toxicity and genotoxicity have been recently established. however, currently these methods are not suitable for measuring endocrine effects in developing organs such as e.g. embryonic gonads. here we present a phenotypic anchoring of a comprehensive study on sex-specific gene expression analysis accompanied by histological analysis of endocrine disruption in chicken embryo gonads, having the potential for an animal replacing system for endocrine disruptive toxicologic and ecotoxicologic examinations of chemicals. chicken embryos were inoculated with different amounts of tributyltin (tbt) and bisphenol-a (bpa). embryos were incubated and their gonads analyzed histologically d prior to hatching. from identically treated embryos right and left testes and ovaries were separated and genome-wide transcription profiles generated using supertag digital gene expression (st-dge, supersage) profiling. male and female gonadal tissues both revealed histological aberrations in response to tbt and bpa. female gonads became masculinized in response to tbt and, viceversa, bpa-treated male gonads underwent feminization whereas in female gonads clearly visible structural aberrations occurred. in both chemicals mortality increased especially in the most affected sex (tbt: females, bpa: males). the expression profiles of more than million mrnas revealed massive effects of both chemicals, tbt and bpa, on important cellular signaling pathways. gene expression differences were most pronounced in the phenotypically most affected sex. our results demonstrate that endocrine disruptive chemicals exert their effects on several levels including but not restricted to known hormone-based pathways. together with an ongoing study of gene expression differences in very young life stages and different chemicals these data will form the base for a blow-by-blow analysis of sexspecific gene expression of embryonic development. the project builds on already existing and further to generate data with the aim of the development of an in vitro method for testing chemicals at chicken eggs for ) replacement of tests on juveniles and (sub-) adult rodents, ) stages with impossibility of sensation of pain in the individuals, ) highly sensitive prospects of modes of action of chemicals, which ) might show consequences in the next generations. channels are critical for oscillatory burst discharges in the reticular thalamus and absence epilepsy differential distribution of three members of a gene family encoding low voltage-activated (t-type) calcium channels hippocampal seizure resistance and reduced neuronal excitotoxicity in mice lacking the cav . e/r-type voltage-gated calcium channel transcriptional upregulation of cav . mediates epileptogenesis in the pilocarpine model of epilepsy structure and functional expression of a member of the low voltage-activated calcium channel family a molecular determinant of nickel inhibition in cav . t-type calcium channels histidine residues in the is -is loop are critical for nickel-sensitive inhibition of the cav . calcium channel substrate recognition and translocation by polyspecific organic cation transporters proton pump inhibitors inhibit metformin uptake by organic cation transporters (octs) structural determinants of inhibitor interaction with the human organic cation transporter oct (slc a ) functional characterization of the human organic cation transporter variant p. ala>ser extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean? local glucocorticoid production in the mouse lung is induced by immune cell stimulation biomimetic materials in tissue engineering biomaterials offer cancer research the third dimension synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells directed growth of fibroblasts into three dimensional micropatterned geometries via selfassembling scaffolds novel pcl-based honeycomb scaffolds as drug delivery systems for rhbmp- tissue engineering spatio-temporal vegf and pdgf delivery patterns blood vessel formation and maturation presentation of rgds epitopes on self-assembled nanofibers of branched peptide amphiphiles controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces langmuir avintegrins as receptors for tumor targeting by circulating ligands heparin binding nanostructures to promote growth of blood vessels tirrell endothelial cell adhesion to the fibronectin cs domain in artificial extracellular matrix proteins design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes stimuli-responsive thin coatings using elastin-like polymers for epac as a novel effector of airway smooth muscle relaxation ) camp inhibits airway smooth muscle phenotype modulation functional roles of epac and pka in human airway smooth muscle phenotype plasticity assessment of the sensitizing and irritative potential of preservatives by loose-fit coculture-based sensitization assay (lcsa) sonnenburg a nsc- (ukrain) in the palliative treatment of pancreatic cancer. results of a phase ii trial association of funding and conclusions in randomized drug trials: a reflection of treatment effect or adverse events a general method for the covalent labeling of fusion proteins with small molecules in vivo robust single-particle tracking in live-cell time-lapse sequences correlation of structural class with no-observed-effect levels: a proposal for establishing a threshold of concern trbp recruits the dicer complex to ago for microrna processing and gene silencing index a , , , , objective: hypertension and arterial stiffness is influenced by environmental and genetic factors. high plasma sodium concentration leads to mechanical stiffening of endothelial cells resulting in endothelial dysfunction and elevated blood pressure. here we investigated whether endothelial cell stiffness of ex vivo preparations of human arteries is linked to plasma sodium concentrations and functional genetic variants of the mineralocorticoid receptor (nr c ), rs modulating blood pressure, renin, and aldosterone levels, and rs , which alters a mirna binding site. design and methods: twenty patients were enrolled after a vein stripping procedure and collateral arterial blood vessels were prepared for atomic force microscopy (afm). plasma sodium concentration was routinely determined and dna for genotyping was extracted from edta blood samples. sodium levels > mmol/l were defined as 'high'. after application of µm amiloride, a specific blocker of the endothelial sodium channel (enac) changes in endothelial cell stiffness, were defined as 'weak' (≤ %), or 'strong' (> %). statistical analyzes were performed by anova. results: in ex vivo artery preparations of patients with high sodium levels (n= ), mechanical stiffness of endothelial cells was tend to increase (∆ amiloride) (p= . ). both nr c variants were associated with a change > % in endothelial stiffness after amiloride treatment. the rs c allele was significantly associated with a strong amiloride response (p= . ), while the rs a allele only showed a trend towards stronger amiloride effects (p= . ). conclusion: our findings indicate that high plasma sodium concentration results in an increased endothelial amiloride response and thus influencing mechanical stiffness, modulated by functional nr c variants. our novel approach linking patients' sodium levels and genetic status to endothelial stiffness by afm will be further evaluated in larger clinical settings. protein expression changes in bap-exposed human bladder cancer cells from spliceosome activation towards redistribution of the cytoskeleton after long-term exposure to subacute concentration schmitz-spanke s., pink m., jeske e., stempelmann k., rehn s., verma n., rettenmeier a. w. universitätsklinikum essen institut für hygiene und arbeitsmedizin, hufelandstr. , essen, germany deregulation of the β-catenin signaling pathway plays an important role in the development of hepatocellular tumors. activating mutations in ctnnb (encoding β-catenin) are frequently observed in murine and human liver tumors (e.g. human hepatoblastomas). activation of β-catenin signaling induces an overexpression of several cytochrome p (cyp) enzymes, including cyp e . cytotoxicity of acetaminophen (aap) is based on its cyp e -catalyzed metabolism to the electrophilic compound n-acetyl-p-benzo-quinone imine, which forms covalent adducts with cellular macromolecules if depletion of glutathione occurs. treatment with aap should therefore lead to a selective damage of cyp e -overexpressing ctnnb mutated hepatoma cells. mice were injected with a single dose of the liver carcinogen n-nitrosodiethylamine (den) and subsequently treated with the tumor promoter phenobarbital to select for ctnnb -mutated tumors. administration of a single dose of aap ( mg/kg of body weight) followed the tumor promotion protocol. two days after treatment immunohistological analysis of the livers showed about % necrotic tissue in the larger tumors which were positive for glutamine synthetase (gs), a marker for ctnnb -mutated tumor cells. by contrast, gs-negative tumors remained unaffected. at later time points we observed regeneration processes with infiltration of the necrotic tissue by inflammatory cells followed by fibrotic cells. proliferation of normal hepatocytes surrounding the damaged areas could also be observed. however, repopulation of parts of the former tumor areas by remaining gs-positive tumor cells was also detected. these results suggest that treatment with aap might serve as a future therapeutic possibility to selectively poison cyp e -overexpressing hepatoma. release of , -epoxyeicosatrienoic acid ( , -eet) upon neuronal activity induces trpa -dependent mechanical pain hypersensitivity sisignano m. , epoxyeicosatrienoic acids (eets) are cyp-epoxygenase (cyp ) derived metabolites of arachidonic acid (aa) which act as endogenous signaling molecules in multiple biological systems. we investigated the specific contribution of , -eet to transient-receptor potential-(trp)-channel activation in nociceptor neurons, and its consequence for nociceptive processing. we found that during capsaicin-induced nociception , -eet-levels increased in the drg and it is released from activated sensory neurons in vitro. , -eet potently induced a calcium flux [ nm] in cultured drg-neurons which was completely abolished when trpa was deleted or inhibited. in spinal cord slices , -eet dose-dependently enhanced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sepsc) in lamina ii neurons that also respond to mustard oil (aitc), indicating a presynaptic mechanism. furthermore, , -eet-induced enhancement of sepsc frequency was abolished in trpa null mice, suggesting that , -eet pre-synaptically facilitates spinal cord synaptic transmission via trpa . finally, intrathecal injection of , -eet caused mechanical hyperagesia in wild type but not trpa null mice. we conclude that , -eet is synthesized upon acute activation of nociceptors and leads to mechanical hypersensitivity via trpa at central afferent terminals in the spinal cord. sisnaiske j. , hardelauf h. introduction: neurite outgrowth and plasticity of neuronal networks are essential processes e.g. during brain development and learning. thus, morphological readouts of neuronal connectivity are thought to be important endpoints to assess neurotoxic effects of environmental chemicals as well as when discovering new drugs. to analyze neurite outgrowth and connectivity level rapidly and easily in vitro we developed the network formation assay (nfa) (pct/ep / ). this platform requires a spatially standardized hexagonal array for culturing neuronal networks with no need to fix or stain the cells to visualize neuritic processes. methods: to demonstrate the feasibility of the nfa we performed experiments in which we disrupted mature neurite networks or inhibited generating networks of human sh-sy y cells with different concentrations of acrylamide (acr). we also observed the counteracting effects of brain-derived neurotrophic factor (bdnf) and calpeptin in these systems. to create the hexagonal array we used a poly(dimethylsulfoxide) bilayer stencil comprising through holes for adhesion spots and interconnecting tracks. plasma stencilling a peg-coated glass substrate produces adhesive nodes for the neurons and micron-scale-tracks for guiding neurite outgrowth and connectivity. results: in both systems, the developing and mature network, we found not only a concentration dependant effect of acr and bdnf but also a time dependant effect with a limited capability of the developing system to regenerate, even in the presence of acr. the co-treatment of the cells showed that inhibition of calpains by calpeptin might reduce the effect of intracellular elevated ca +, a known neurotoxic mechanism of acr. moreover, the neurothrophin bdnf acts via trkb receptors on pathways stimulating neurite outgrowth and thereby counteracting the adverse effect of acr. conclusion: with the nfa we provide a rapid and simple way to analyze neurite outgrowth and connection formation in real time. by spatially standardizing the array we provide assay coordinates to streamline the analysis process and bring it towards high throughput testing. furthermore preliminary data showed that modification of the surface with biomolecules allows cell adhesion of other neuronal celltypes (e.g. primary mouse neurons) and extracellular matrix proteins (e.g. laminin) stimulate neurite outgrowth via integrins. transcriptional regulation of nox by histone deacetylases siuda d. , , zechner u. , prawitt d. nox is a member of the nadph oxidase family, which represents a major source of reactive oxygen species (ros) in the vascular wall. nox -mediated ros production mainly depends on the expression levels of the enzyme. the present study is aimed to investigate the regulation mechanisms of nox transcription by histone deacetylase (hdac). in cultured human ea.hy endothelial cells, treatment with the pan-hdac inhibitors (scriptaid, trichostatin a, tsa, and suberoylanilide hydroxamic acid, saha) leads to a drastic decrease in nox mrna expression. a similar down-regulation of nox mrna expression can be achieved with sirna-mediated knockdown of hdac . hdac inhibition in endothelial cells is associated with enhanced histone acetylation in the human nox promoter region, with no significant changes in dna methylation. consistently, scriptaid-treated cells show increased chromatin accessibility in nox promoter. in addition, we provide evidence that c-jun plays an important role in controlling nox transcription. knockdown of c-jun with sirna leads to a marked downregulation of nox mrna expression. in response to scriptaid treatment, the binding to c-jun to the nox promoter region is reduced despite the open chromatin structure. in parallel, the binding of polymerase iia to the nox promoter is significantly inhibited as well, which may explain the reduction in nox transcription. in conclusion, hdac inhibition decreases nox transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the nox promoter. this is very likely because of a hyperacetylation-mediated steric inhibition. cyclopentenone prostaglandins induce oxidative dna-damage in hamster lung fibroblast v cells solecki g. m. key: cord- -atbjwpo authors: nan title: poster sessions date: - - journal: febs j doi: . /febs. sha: doc_id: cord_uid: atbjwpo nan ** each poster has been given a unique number beginning with the letter p; the next part relates to the session in which the poster will be presented. moreover, klf is also acting on cellular processes such as cell migration, apoptosis, inflammation, angiogenesis and differentiation. previous studies showed a novel role for klf as a regulator of proliferation and differentiation in skeletal muscle stem cells. detecting klf at the protein level harbored technical obstacles. commercially available antibodies exhibited low affinity, low specificity and failed to recognize post-translationally modified forms that are directly relevant to the function. thus, these obstacles prevent further functional protein studies such as western blots, protein co-immunoprecipitation and chromatin immunoprecipitation (chip) assays. therefore, we used crispr/cas system to establish a stable cell line which carry v epitope tag into the n-terminal of klf gene. insertion into the target side of klf gene via crispr-cas system provided an opportunity to overwhelm the above mentioned obstacles. v epitope tag would not interfere with the function of the klf and also enable us to recognize endogenous klf via anti-v antibody in the mouse myoblast cell lines (c c ). we confirmed the targeted insertion into the exon of the klf gene both at the dna and protein levels. the conformational dynamics of structural domains plays an important role in functioning of many proteins. the reca proteins from e. coli are known to be the central catalyst of homologous recombination and repair in bacteria. it forms a helical filament on ssdna capable to bind homologous dsdna and catalysis of the exchange of the complementary strand. significant mobility if its c-terminal domain has been observed experimentally by cryo-electron microscopy. however its potential significance for reca protein activities still remains unclear. in this work we investigated this question by construction of a mutant reca protein with artificial disulfide bridge between central and c-terminal domains. the wild type protein has no disulfide bonds, and therefore its native mobility can be restored in vitro, by addition of b-mercaptoethanol. our data suggest that the s-s bridge decreases both the rate of atp hydrolysis in vitro and the e. coli resistance to uv in vivo. thus, our experimental results indicate that the flexibility of the c-terminal domain significantly affects recombination activity of reca protein in vivo and in vitro. hydroxiurea (hu) is an inhibitor of ribonucleotide reductasethe enzyme that catalyzes the process of free dntps synthesis in living cells. treating cells with hu causes diminishment of the nucleotide pool. as a result, single-stranded dna regions are generated, which leads to s-phase checkpoint activation. the progression of replication forks is blocked and the completion of dna replication is prevented. this results in s-phase cell arrest. nevertheless, our results demonstrate that after prolonged hu treatment, the saccharomyces cerevisiae cells seam to escape the arrest and continue the progression of their cell cycle. we show that when cells re-enter the cell cycle, mrc , but not ctf is detached from chromatin. our data also shows that meanwhile, rad checkpoint activity is diminished in order to allow s-phase checkpoint escape and completion of the cell cycle. moreover, cells not only continue the cell cycle, but steadily surmount in the presence of hu. all this data indicates that cells have made the decision to compromise s-phase checkpoint and to adapt to the novel environmental conditions in order to survive. as both mrc and ctf are known to be responsible for polymerase and helicase harmonization during replicative arrest, our data indicates that mrc has a more specific role in the process of adaptation. our data demonstrates that mrc is a leading protein to regulate the stability of s-phase checkpoint arrested replication forks. zinc finger domain is the most common dna binding domain in metazoa. almost drosophila proteins with c h zinc fingers also have zinc finger associated domain (zad). several proteins with zad (zw , pita and zipic) were found to interact with cp and act as insulator proteins. for some of the zad-containing proteins (for example, weekle and grauzone) it was shown that their zad domains can form dimers with each other. the ability of these proteins to dimerize appears to be especially important in the light of the model suggesting that dna-binding insulator proteins can support genome looping and organization of chromatin structure via interaction with each other. in this work we aimed to understand the role that zadmediated protein-protein interactions play in maintenance of dna loops, focusing on proteins: zw , pita, zipic and cg . first, we performed co-precipitation and yeast two hybrid assays to confirm dimerization of isolated zads in vitro. we observed that only zads from the same protein can specifically interact with each other (homodimerization) and they are unable to interact with zads from different proteins (heterodimerization). we confirmed homo-but not heterodimerization of zads in vivo with coimmunoprecipitation experiments in s cells. furthermore, we found that zad proteins can support longdistance interactions in transgenic constructs in flies. using model system with cg protein, we demonstrated that zad is essential for these interactions. proteins without zad cannot maintain loop formation. finally, analysis of chip-seq experiments for zw , pita, zipic and cg revealed that binding sites of zad proteins often overlap with regions of inter-chromosomal contacts known from hi-c experiments. we conclude that zad-containing proteins can support longdistance genomic interactions and dimerization of zads is necessary for these interactions. this study was supported by the russian science foundation (project № - - ). over the years a large body of clinical knowledge has accumulated on pharmacological effects of drugs on thyroid function. antipsychotics are administered over long periods in humans; therefore their possible adverse side effects should be taken into consideration. the aim of this study is to evaluate the effects of haloperidol and clozapine on plasma t and t concentrations in adult male wistar rats. fifty rats aged between and weeks ( ae g) were divided into five groups (n = in each group), and drugs were administered each day intraperitoneally (ip) for days. the first group was a sham group. the other four groups were considered as low and high treatment doses of the drugs. after a one-week habituation period, animals was administered haloperidol ( . mg/kg, n = and mg/kg, n = ) and clozapine ( . mg/kg, n = and mg/kg, n = ). the rats were anesthetized with ether, and bloods were collected by direct cardiac puncture hours after the last injection. the t and t plasma concentration levels were analyzed with chemiluminescent immunoassay. statistical analysis was performed with ibm spss v . . kruskal-wallis and bonferroni tests were used. t plasma concentration levels significantly differ between sham (median= . mg/kg) and haloperidol ( mg/kg) (median= . mg/kg), haloperidol ( . mg/kg) (me-dian= . mg/kg) and clozapine ( mg/kg) (median= . mg/ kg), haloperidol ( mg/kg) (median= . mg/kg) and clozapine ( mg/kg) (median= . mg/kg) groups (p < . ). however, no significant differences between the groups regarding to t plasma levels were observed. in conclusion, haloperidol and clozapine increased the t plasma concentrations, but didn't have any significant effect on t plasma concentrations. p- . . - isolation of lipase producing strains of bacillus obtained from olive wastewater and screening for substrate specificity in this work, wastewater samples of an olive factory from yusufeli (artvin, turkey) were collected carefully. after a centrifugation period of samples, supernatants were applied to a . lm filter and incubated on lb agar medium for hours. based on differencies of colony morphologies, isolates were selected and purified for identification. s lipase activity assay was carried out by rhodamine b. all of the strains exhibited lipase activity. for determining the substrat specificity of isolates, different substrates were used; nitrophenyl-butyrate, -nitrophenyl-caprylate, -nitrophenyl-laurate, -nitrophenyl-myristate, -nitrophenyl-palmitate. results were measured spectrophotometrically at nm. all of the strains hydrolyzed -nitrophenyl-butirat, while there was no activity with -nitrophenyl-palmitate. bacillus sp. l was the most efficient strain that hydrolyzed all of the substrates. the gene encoding for lipase of bacillus sp. l will be cloned and expressed for more analyses and industrial applications. p- . . - some quantitative aspects of hair follicle layers differentiation e. vsevolodov , , v. golichenkov , a. mussayeva , , i. latypov llc "kazcytogen", almaty, kazakhstan, "institute of general genetics and cytology" sc mes, almaty, kazakhstan, lomonosov's moscow state university, moscow, russia in the course of stable hair growth the differentiation of hair bulb cambium cells to several layers with dissimilar cytochemistry and morphology takes place. this means the activation of different genes in the cells of different layers. depending upon the hair diameter some layers may be absent (medulla in the thin hairs). the hair diameters of the carpet sheep breeds vary widely even within the same square mm of the skin. we compared the different layers thicknesses proportions for the follicles with varying hair diameters. the follicle layers were measured on microphotos of transverse histological sections of the follicles made under the standard magnification. all follicles belonged to the same skin biopsy. the measurements were made at the levels just below the fissure separating the hair and inner root sheath appeared. the empirical regressions of the layers thicknesses and of ratios of different layers against hair diameters were counted. the computer model was made on the basis of these regressions which allowed to obtain the absolute parameters of the layers as well as ratios of these parameters for every chosen hair diameter. using this model we found an essential trend in changing the proportions in relative layers dimensions as we choose the follicles with more and more thick hair. when we change the follicles with mcm hair diameter for those with the hair diameter mcm the ratio of hair medulla diameter to hair diameter increases from . to . . the ratio of hair diameter to the diameter of inner root sheath increased from . to . . it means that the thicker is the hair the higher proportion of cells produced by cambium are spent to build innermost layers (medulla layer within the hair or hair within the complexinner root sheath + hair). these data may throw some light on positional information mechanism of layers differentiation. lignin is a heterogeneous polymer that constitutes % of woody plant cell walls. microorganisms that degrade lignin are fungi, actinomycetes and to a lesser extent, bacteria. in case of industrial applications, the use of fungi is not feasible due to the structural hindrance caused by fungal filaments, requirement of particular culture conditions such as humidity, aeration which are not compatible with industrial processing environments. bacteria are worthy of being studied for their ligninolytic potential due to their immense environmental adaptability. environmental concerns and increasingly stringent emissions standards have led the pulp and paper industry to devise ways to decrease the level of chlorinated lignin residues in its effluents through both production process changes and improved treatment technologies. bleaching with the enzymes is the most promising because the enzymes may be very efficient, and can be used under industrial conditions. the main objective of this study was to investigate the adequency of klebsiella pnuemonia gst (glutathione-s-transferase) pretreatment for bleaching of calabrian pine kraft pulp. for this purpose the following conditions were investigated: enzyme loadings from to u/g pulp basis and the consistecy of the pulp was between and %. enzyme at the desired concentration was added to the pulp and the mixture was incubated at °c for hours. after the enzymatic pretreatment to determine the optimum conditions the kappa number of all reactions were analyzed according to tappi standarts. as a result of this study we determine the optimum conditons as % pulp consistecy, u/g enzyme for pulp treatment. after the enzymatic treatment carried out under optimum conditions we are planning to submit a short bleaching sequence and analyze for physical properties such as viscosity and brightness. owing to this bleacing sequence we are going to able to compare the enzymatic and chemical treatments of pulp in bleaching prosess. p- . . - biochemical characterization of lipase from bacillus subtilis strain a from olive waste water f. ay sal, m. kac ßagan, s. c ß anakc ßi, a. o. beld€ uz karadeniz technical university, trabzon, turkey lipases (triacylglycerol acyl hydrolases, ec . . . ) are regarded as mild and environment-friendly biocatalysts for triacylglycerols hydrolysis. in addition to this hydrolytic reaction, they also catalyze reverse reactions of esterification, transesterification, and interesterification in non-aqueous environments. substrate, stereo-, regio-and enantio-specificities, and chiral selectivity are certain unique attributes of lipases that make them industrially attractive. these properties are often exploited in the manufacturing of detergent formulations, synthesis of fine chemicals, useful esters and peptides, food processing, paper manufacturing, degreasing of leather as well as in bioremediation. in this study, lipase from bacillus subtilis strain a is partially purified and characterized. bacillus subtilis strain a is isolated from olive factory from soke (aydin, turkey) and identified with s rrna analysis. lipase activity is screened on petri supplemented with rhodamine b. bacteria was grown in lb medium supplemented with % olive oil (vol/vol) for hour at °c. after incubation, cells were harvested by centrifugation at , rpm for minutes, resuspended in mm tris-hcl (ph . ) buffer, followed by sonication with sartorius labsonic m to release intracellular proteins. q-sepharose is used as ionexchange column chromatography for lipase purification. effects of temperature on activity and stability were determined spectrophotometrically using p-nitrophenyl laurate as the substrate. effects of ph on activity and stability were also determined. the effects of various metal ions and other reagents on the hydrolytic activity were assayed at °c. the enzyme was active and stable in the broad ph range of . - . and temperature range of - °c. bacillus subtilis strain a have high lipolytic activity. after cloning this enzyme to an expression vector and detailed characterization, this may suggests its usefulness in industrial applications. p- . . - investigation of pin as a nuclear factor one binding partner s. saritas, a. e. yilmaz, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey the nuclear factor one (nfi) proteins are important regulators of gene expression in the developing embryo and in adult stem cell niches. this transcription factor family has four members: nfia, nfib, nfic, and nfix. nfi proteins bind a consensus sequence on gene regulatory regions as homo or heterodimers. each member of nfi family has a highly conserved n-terminal dna binding and dimerization domain and a diverse proline rich c-terminal transcriptional activation/repression domain. as knockouts of nfi genes display distinct developmental phenotypes, we hypothesized that specificity of nfi protein function may arise from their interactions with binding partners. a yeast-two hybrid screen identified protein interacting with never in mitosis a (pin ) as a potential nfib interactor. pin is a ubiquitously expressed protein that specifically recognizes and binds to a phospho-serine or a phospho-threonine followed by a proline (ps/pt-p motif), and catalyzes isomerization of peptidyl-prolyl bonds. interestingly, both n-terminal and c-terminal domains of four nfi isoforms contain several conserved putative ps/pt-p motifs and some of these are reportedly phosphorylated. we looked for nfi pin interactions in vitro by gst-pulldown and co-immunoprecipitation assays. while gst-pin fusion protein interacts with all of four nfi isoforms, it binds nfib most strongly, nfia and nfic moderately, nfix most weakly. moreover, deletion of the cterminal domain leads to loss of nfi affinity for pin implicating this domain in nfi-pin interactions. co-immunoprecipitation assays where we co-expressed various epitope tagged nfi and pin proteins in hek t cells showed that pin precipitates nfib, as well as other nfi isoforms and nfib can, in turn, precipitate pin . we are currently carrying out site-directed mutagenesis on nfib to identify the specific residues that pin recognizes. we will further explore if this interaction regulates nfi function during embryonic development. that pre-adapt migrating fish to the life in seawater. among others, smoltification induces intense growth of fish that enter the ocean at a size where risk of predation is significantly reduced. skeletal muscle growth depends on a tightly controlled balance between protein synthesis and degradation. protein synthesis driven by hormone regulation is well studied in smoltified atlantic salmon; while less is known on protein degradation occurring via a number of pathways including cytosolic ubiquitin-proteasome system and calcium dependent calpains. the aim of this study was to compare calpain and proteasome enzymatic activities in the skeletal muscles of s. salar parr, pre-smolts and smolts. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from indera river (kola peninsula, russia). our results demonstrated the significant differences in studied protease activity levels between parr and smolts. calpain and proteasome activities in s. salar smolt muscles showed a significant drop compared with that of parr. the negative correlation between proteases activity levels in the muscle tissue and overall fish growth rate was shown. so, our data indicated life stage specificity in skeletal muscle protein degradation capacity in migrating fish. we suppose that intense muscle growth in s. salar pre-smolts is supported by various mechanisms including accelerated muscle protein accretion through the reduction of protease activities. obtained results enhance our knowledge in the mechanisms of atlantic salmon smoltification. the work was supported by the russian scientific foundation, project no. - - . p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the sociodemographic characteristics of the pregnant women who double and triple prenatal screening test h. d€ ulger, s. yabanci€ un meram medical faculty, n.e.university, konya, turkey double and triple prenatal screening tests which are applicable during first and second trimesters of pregnancy predict existent abnormalities at early stage. the aim of this study is to investigate the relationship between positive results of double and triple tests, further confirmatory tests during prenatal phase, postnatal status of babies and maternal age. in this study, double and triple test results of pregnant women who were admitted to meram faculty of medicine during - period were scanned from archive and test results indicating risk were detected. from these results, those which were above cut-off values for down syndrome, trisomy , open spina bifida were determined. a questionnaire was carried out with voluntary participants by reaching to these individuals. positive-negative result ratio of all double and triple test results and sociodemographic features such as age, occupation, presence of consanguineous marriage were investigated. all data from archive and answers from survey questions were assessed statistically. participants of the study were to years old and their average age was . ae . . ofthem ( . %) were under years of age whereas of them ( . %) were above years of age. number of pregnancies were scaling between to with an average of . ae . . of mothers ( . %) were not undergone amniocentesis, whereas babies with chromosomal abnormalities were detected among mothers who were undergone amniocentesis. in conclusion, there may be regional, sociological and such that reasons for those who were not undergone amniocentesis despite positive double and triple test results. ( . %) chromosomal abnormalities were detected among pregnancies with increased risk assessment with positive double and triple results. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the effects of oil on the growth and development of amphibians l. sutuyeva, t. shalakhmetova, a. ondassynova al-farabi kazakh national university, almaty, kazakhstan currently, the pollution of ecosystems by oil and oil products is increasing everywhere. the oil gets into water and ground during oil production, transportation and accidents. as a result, terrestrial animals and hydrobionts are exposed to oil contamination. thus, populations of animals decline. it can be assumed that the most sensitive to the effects of pollutants are animals in early stages of development. amphibians have established themselves as the most convenient bioindicator species. since lake frog (rana ridibunda) and green toad (bufo viridis) are the bioindicator species in kazakhstan, the study of the effects of oil on their larvae was carried out. we used water-soluble fraction of the oil from zhanazhol field (aktobe region) in our test. the larvae of control group were kept in pure water, and larvae of test groupsin aquariums with . , . and % concentrations of the oil fractions. the concentrations were chosen in accordance with the level of pollution of kazakhstan's water bodies with oil. mortality of larvae, morphometric parameters and morphogenesis were studied. it was found that high mortality of larvae is the most visible reaction when exposed to oil. this indicator rose noticeably depending on the doses ( . , . % and %) in both species with percentages %, % and % in r. ridibunda and %, % and % in b. viridis, respectively, while in the control group it was about %. furthermore, delayed larval development was detected. thus, the larvae from the control and . % oil group reached gosner stage (gs) , tadpoles from . % and % groups were at gs- and gs- , respectively, by the th day of life. moreover, behavioral abnormalities (sluggish movements) and decreased sensitivity to mechanical stress (touch) were observed under the influence of high concentrations of oil fractions. thus, oil in low concentrations alters the growth and development of tadpoles of anurans, and causes their increased mortality in high concentrations. p- . . - effect of catechin loaded plga nanoparticles on glioma cell line histone h t is a linker histone which binds to dna and contribute in chromatin condensation as well as regulation of specific genes through spermatogenesis. replacement of this histone h subtype and hyperacetylation of histone h tail, facilitate the replacement of histones with sperm chromatin condensing proteins of tnps and prms. ethical approval and informed patient consent was gained from infertile men referred to royan institute. testicular biopsies were collected from patients through assisted reproductive techniques (art) procedure. based on pathological results samples were classified into the following three subgroups: obstructive azoospermia (as positive control), complete maturation arrest and sertoli cell only syndrome (negative control). chromatin of tissues evaluated for presence/absence of histone h t protein in regulatory regions of tnps and prms genes using chip-real time pcr. results showed lower incorporation of h t protein on regulatory regions of tnps and prms genes in two spermatogenic failure group versus positive control. in this study, it can be concluded that the decreased levels of h t histone variant in testis tissues and failure in chromatin condensation have significant association with male infertility. p- . . - serum dickkopf- levels in obese children and adolescents that obesity is detrimental to bone health despite potential positive effects of mechanical loading conferred by increased body mass on bones. the wnt/b-catenin pathway is essential for normal osteogenesis. serum dickkopf- (dkk- ) is one of the most important inhibitors of the wnt//b-catenin pathway. the aim of this study was to investigate the serum dkk- levels in obese and non-obese children and adolescents. materials and methods: the study included obese children and adolescents ( males and females) aged from to years and healthy normal-weight controls ( males and females) aged from to years. serum dkk- levels were measured by elisa method using commercially available kit. results: body mass index of the obese children was significantly higher than that of non-obese children (p = . ). however, there was no significant difference between dkk- levels of the groups. (these results are preliminary and the study is continuing). discussion and conclusion: our result showed that serum dkk- levels were not changed in obese children and adolescents. p- . . - transcriptional regulation of cdo by nuclear factor one proteins b. kutay, c. lektemur, v. g€ uler, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey nuclear factor one (nfi) transcription factors play important roles in regulation of central nervous system development. three of the four members of nfi family, nfia, nfib, and nfix are expressed in neural progenitors, as well as neurons and glia in the embryo. inactivation of these genes in mice show that they function in development of neocortex and hippocampus in the forebrain, cerebellum, spinal cord and precerebellar nuclei of the hindbrain, regulating neurogenesis, gliogenesis, as well as neuronal migration, axonal outgrowth and guidance. all three neural specific nfis are expressed in precerebellar neuroprogenitors, however, only deletion of nfib leads to a delay in development of precerebellar neurons. investigation of misregulated genes in nfib À/À precerebellar neuroprogenitors identified cell adhesion associated, oncogene regulated (cdo) as a potential downstream target of nfib. interestingly, this gene has been reported to be upregulated in nfia À/À hippocampus as well. cdo, a cell surface glycoprotein of the ig superfamily, has been found to regulate neurogenesis in vivo, is highly expressed in the developing brain and can induce neural differentiation by promoting heterodimerization of basic helix loop helix transcription factors with e proteins. bioinformatic analysis of the kb human cdo promoter region identified five nfi binding sites: one cluster in the first kb region, another in the . kb upstream region. electrophoretic mobility shift and supershift assays showed that nfib binds to all five sites. furthermore, nfib, along with the other neural nfis, inhibits the proximal cdo promoter driven luciferase activity by up to % in hek t cells. preliminary data indicate that nfis bind to sites in both clusters in human neural stem cells (hnscs) suggesting that these sites are functional in vivo. we are currently investigating this possibility through nfi overexpression and silencing experiments that will examine regulation of cdo in hnscs. differentiation. the aim of this study is to investigate bdnf and drd /ankk gene variants in eos development. in this study, eos patients and healthy controls were used. genomic dna extraction was performed from peripheral blood leukocytes. drd /ankk taq a (rs ) and bdnf val met (rs ) polymorphisms were determined by real-time polymerase chain reaction (rt-pcr). positive and negative syndrome scale (panss) was used to determine eos severity. for drd /ankk rs polymorphism, there was a significant difference in the genotype frequencies between patients and controls for the co-dominant model (p = . , or = . ; % ci: . - . ). however, no significant relationship was observed in the genotype frequencies of bdnf val met polymorphism between eos patients and controls (p = . ). these results indicate that, drd /ankk rs genotypes may affect eos development. however, bdnf val met polymorphism may not be associated with eos. lack of association of bdnf val met polymorphism may be due to limited number of patients. our findings need to be confirmed by further studies. various dyes used in the textile industry are discharged in large quantities to the receiving environment in the manufacturing process. this is the beginning of a process that is difficult to compensate for environmental and human health. therefore, contaminated areas should be cleaned. in addition, technologies with high polluting potential should be integrated with biological approach and thereby the impurities consisting of dyes should be reduced. in this experiment; burdirect black meta konz (c.i. direct black ) was intended to decolorization using laccase. firstly, enzymatic decolorization of the dye was determined using spectrophotometry. the wavelengths of maximum absorption of burdirect black meta konz (c.i. direct black ) was determined between and nm. then, optimization studies have been done. for optimization studies; dye concentration, laccase activity, ph, buffer concentration, temperature, mediator effect, mediator concentration and time parameters were determined. lastly, in optimal conditions, atr-ftir and gc-ms analyzes of ensuring decolorization of dye were analyzed. decolorization of burdirect black meta konz (c.i. direct black ) was performed successfully and the absence of any metobolite in the decolorization medium has been provided by atr-ftir and gc-ms analyzes. assessing in terms of application, it can be easily applied by provided the reaction conditions in textile factories. laccase is a tool of decolorization of dyes in environmental friendly process. thus for the development of spermatids into mature sperm able to fertilize the oocyte.one of the causes of male infertility is in fact impaired sperm fertilization capacity due to sperm chromatin abnormalities and aberrant protamine replacement.recent research has focused on protamine biology,including protamine gene and protein structure,mechanisms of protamine expression regulation and involvement of the protamines in male fertility.various studies reported abnormal expressions of protamine (prm) genes in sperm of infertile men.the aim of the study is to investigate the gene expression of prm , prm and their relationship with defective spermatogenesis. materials and methods: this study has been performed on infertile and fertile turkish men.total rna was extracted from the sperm pellet using trizol reagent.after rna extraction and cdna synthesis,real-time quantitative polymerase chain reaction (rt-qpcr) was used to determine the expression of prm and prm . results: distinct levels of spermatozoal prm and prm mrna were found in infertile patients compared to fertile control groups.we found that the mrna levels of prm was reduced in (% ), and the mrna levels of prm was reduced in (% ) out of infertile patients.in the current study,we found statistical significant association between the prm expression and infertility (p < . ).although prm gene expression was decreased in most of infertile patients compared to fertile control groups,the differences between the groups were statistically insignificant (p > . ). discussion: the results of the study suggested that, the protamine expressions which were associated with spermatogenesis may be important in infertility treatment. further studies are required in a large series of different populations to clarify the role both prm and prm themselves and their mrna expression on male fertility. the study was conducted to characterize the processes of muscle growth in atlantic salmon (salmo salar l.) of different ages inhabited rivers indera and varzuga (kola peninsula, russia) in summer and autumn. the expression levels of genes myosin heavy chain myhc, myostatin (mstn), and myogenic regulatory factors myf , myogenin) in white muscle were studied in salmon parr of age groups +, +, + in june and october. the changes in expression levels of mrfs, myhc and mstn indicating the extent of hyperplasia, hypertrophy, and restriction of muscle growth at different ages of parr were revealed. the pattern of age-related changes differed between seasons. especially, the expression of genes myod, myogenin and myhc peaked in yearling parr ( +) in summer, that indicated the high rate of hyperplastic and hypertrophic muscle growth in yearlings ( +). at the same time, the mstn expression level, the negative regulator of muscle growth, was highest in parr at age +. possibly, it is the necessary regulation mechanism to attenuate hyperplasia and hypertrophy and control muscle growth. in autumn, the expression level of myhc and myogenin were higher in salmon of age + and + then in +, indicating the higher intensity of hypertrophy in parr at both first ages in comparison to +. there was no differences in expression level of myod, myf and mstn between age groups in autumn. moreover, the expression levels of genes studied were lower in autumn than in summer. thus, it indicated the decrease of muscle protein synthesis and muscle growth rate in autumn. these findings expand knowledge on age-and season-related features of muscle development in young atlantic salmon in their natural habitat. the study was supported by the grant of the russian science foundation no. - - . p- . . - lmp and lmp gene polymorphisms in the southeastern anatolia population of turkey d. mihc ßioglu , f. ozbas gerceker sanko university, gaziantep, turkey, gaziantep € universitesi, gaziantep, turkey introduction: the low molecular weight polypeptide (lmp ) and low molecular weight polypeptide (lmp ) genes are located in the class ii region of the major histocompatability complex (mhc) locus on chromosome . these genes encode peptides forming the large components of the proteosome complex which degrades short-lived cytoplasmic proteins. due to the significant role of lmp products antigen presentation, these genes can be accepted as strong candidates of susceptibility factors for different diseases. population genetic studies can also contribute to understanding of the possible role of lmp gene polymorphisms. the aim of this study was to determine the allele and genotype frequencies of the lmp and lmp gene polymorphisms in southeastern anatolia population and to compare these with the frequencies in other populations previously reported. material and methods: a total of healthy and unrelated individuals participated in this study. polymorphism analyses were done by polymerase chain reaction (pcr)-restriction fragment length polymorphism (rflp) method and allele/ genotype frequencies of lmp and lmp genes were determined. results: a deviation from the hardy-weinberg equilibrium (v = . ,p < . ) was found for the genotype distribution of lmp gene polymorphism, while the lmp genotypes found to be distributed (v = . ,p > . ). discussion: available allele frequency data for different populations were used to calculate genetic distances and to construct a neighbor-joining tree. among the included populations, nahuas (mexico) population was found to have the lowest genetic distance from the southeastern anatolia-turkey population. conclusion: it can be concluded that, more studies using different types of genetic markers are needed to clarify the filogenetic relationships of southeastern anatolia population with other populations and also the number of population studies on lmp and lmp genes should be increased to understand their effects as a genetic marker. p- . . - investigation of in vitro antioxidant activity of quercetin loaded plga nanoparticles pharmacological effects. but its usage is restricted because of low aqueous solubility, poor bioavailability, poor permeability and instability in physiological medium. these problems can be overcome with encapsulation of quercetin into nanocarriers such as biodegradable plga based nanoparticles. polymeric nanoparticles which have - nm particle size and providing controlled released of biological active agent are prepared by using biodegradable and biocompatible polymers. in this study, encapsulation of quercetin molecules into plga nanoparticles was carried with using the single emulsion (w/o) solvent evaporation method. size measurements of the obtained nanoparticles were performed by zetasizer and their size were found . ; . ; . nm respectively. the morphological features were examined by sem images. antioxidant activities of q , q ve q nanoformulations have been investigated by dpph and no (nitric oxide) methods. it is thought that the nanoparticular formulations that is developed in this study can be useful model for the other antioxidant molecules and will provide a significant contribution to the food and pharmaceutical industry. "this research has been supported by yıldız technical university scientific research projects coordination department. project number: - - -gep ". p- . . the effect of environmental enrichment on spatial memory and certain nmdars, and ht a expressions in rat pups introduction: the aim of the study was to investigate the effect of environmental enrichment exposed during whole childhood on spatial learning and memory and certain nmdars, and ht a in the hippocampi of pups. materials and methods: four-weeks old, male, weaning rats were randomised into groups as enviromental enrichment (ee, n = ) and standard cage control (scc,n = ) groups. eeg housed in an enriched environment and sccg were kept in standard cages for weeks. following the experiment the rats were trained and tested in the morris water maze (mwm) , open field test (oft) and forced swim test (fst) in order to assess the neurobehavioural effects of ee. nr a, nr b, ht a protein levels were analyzed by western blotting from hippocampi of rats. results: the positive effect of ee was seen at the learning phase in the mwm as 'latency to locate the hidden platform' between groups thoughout the training days showed that eeg located the hidden platform significantly earlier than sccg on days , (p = . , p < . ). also eeg significantly spent lower time in the outer zone of the maze on days , which was the sign of low anxiety level (p = . , p = . ). the parameters of oft which indicated increased locomotion, exploration and low anxiety were significantly higher in eeg (p < . ), in fst comparison of groups showed no difference (p > . ). the levels of nr b and ht a were significantly increased as compared to sccg as well (p < . , p = . ). discussion & conclusion: these findings showed that exposure to ee throughout the whole childhood causes several neurobehavioural effects like increased exploration and low anxiety. these effects may lead to improvement in speed of learning. increase in the nr b and ht a concentrations which are the receptors that are related to learning and memory in the hippocampi accompanied these changes which may be basis of the neurobehavioural improvements or may provide contribution to positive neurobehavioural effects. p- . . - effects of monosodium glutamate exposure during prepubertal term on several biochemical parameters in rats h. i. b€ uy€ ukbayram, d. kumbul doguc ß, i. ilhan, a. y. ismail s€ uleyman demirel university, isparta, turkey monosodium glutamate, which is commonly used in processed foods as flavor enhancer, is considered 'generally recognised as safe' by fda; however many studies have revealed the negative effects of msg.we aimed to evaluate the effects of msg in childhood on several serum parameters. sixty-six rats, ( weeks old) were divided into groups as control (cg, n = ; + , male+female) , experiment (msg-low dose, e g, n = ; + , male+female) and experiment (msg-high dose, e g, n = ; + ) groups. msg was administered at mg/kg/d to e g, . g/kg/d to e g for weeks by oral gavage. the rats were sacrified and blood samples were collected from aorta. the blood samples were centrifuged, the serum samples were separated and glucose, alt, total protein, albumin, creatinine, cholesterole and triglyceride levels were analysed by beckmann au autoanalyser. level of total protein was significantly increased in e g and e g groups when compared to cg (p < . ). level of alb€ umine was also increased in both egs but significant difference was seen in e g as compared to cg. creatinine levels were significantly increased in egs when compared to cg (p < . ). although the glucose levels in both egs were increased, the increase in e g was statistically significant (p < . ). the alt levels of in egs were also increased but the significant increase was seen in e g (p < . ). the effect of msg seem to be dose dependent and especially effect on carbonhydrate metabolism. increasing doses caused increase in glucose level, and tendency to glucose intolerance. increasing doses of msg also caused increase in creatinine and urea. another apparent effect of msg was detected on alt activity. in conclusion the negative effect of msg on glucose level, liver and kidney functions depends on daily dose intake. consumption of msg seem to be inevitable it has to be restrained in children otherwise early metabolic problems may be future problems for these children. ( mg/kg) + tartrazine ( mg/kg) + brilliant blue fcf ( mg/kg) + ponceau r ( mg/kg) + azorubine ( mg/kg) + indigotine ( mg/kg) + erythrosine ( mg/kg). artificial food color mixture were administered to g and g and drinking water was applied simultaneously to g by oral gavage per day for weeks. after application all rats were sacrificed, the total oxidant (tos)/antioxidant (tas) capacity were analyzed in rats' brain, liver, kidney homogenate and serum with rel tos-tas diagnostics assay kit.the statistical analysis was carried out by using kruskal wallis test. tas and tos levels in liver homogenate were not found significantly different between all groups (p > . ). in serum and kidney and brain homogenate, tas levels were not significantly different between all groups. tos levels in g were higher than g and g in serum and kidney and brain homogenate (p < . ). exposure to synthetic food colors may increase oxidative stres in vitale organs such as brain, kidney in female rats. these alterations differ according to organ and dose. parallel with increasing trends on healthy eating habits, consumption of prebiotics and probiotic microorganisms have been popular due to their benefits on human health. functional dairy foods such as probiotic yoghurt and cheese are the most common foods including probiotic microorganisms. due to some considerations such as standardization and quality in bulk production, starter cultures are used in industrialised fermentative food production to start fermentation. starter culture basically refers the microorganisms which induce and maintain fermentation of the fermentative foods and starter cultures including probiotic microorganisms are called as probiotic starter cultures. in this study, probiotic cheese starter cultures as a microbial community were investigated using computational systems biology tools. a metabolic network model of probiotic cheese starter culture was reconstructed using microbial community network modeling approach. literature-based genome-scale metabolic models of commonly used lactic acid bacteria were used for the microbial community metabolic model. the microbial community metabolic model simulated metabolic interactions of the microorganisms in the probiotic starter culture. metabolic flux values computed by the metabolic network model also predicted the metabolic pattern of the glycolysis (conversion of lactose), lipolysis (conversion of fat) and especially amino acid catabolism which are associated cheese flavor metabolism. simulations obtained by metabolic network-based analysis of cheese starter cultures can also be used for other fields like genetic engineering, upstream processing of the functional cheese production. p- . . - er quality control protein network in cf to modulate f del-cftr rescued phase ii xenobiotic metabolizing enzymes convert parent compounds into more hydrophilic metabolite by catalyzing conjugation reactions including glutathione and amino acid conjugation, glucuronidation, sulfation and acetylation. this study was aimed to describe the best cell line model for studying phase ii xenobiotic metabolizing nqo and gst-pi enzymes. for this purpose, mrna and protein expression of nqo and gst-pi enzymes were analyzed in ht and sw (colon); hepg and huh (liver); pnt a and pc (prostate) cell lines by qrt-pcr and western blotting techniques, respectively. protein expression analysis revealed that nqo protein was expressed in all cell lines and relative protein expression is highest in the hepg ( %) and pnt a ( %) while huh ( . %) showed relatively low expression of nqo . in addition, nqo mrna expression was relatively high in ht ( . fold) and pnt a ( . fold) when compared with liver cell line hepg ( . fold). gst-pi protein expression was found very high in huh ( %) while there was no expression in hepg . gst-pi mrna expression was relatively higher in pnt a ( . fold) and ht ( . fold) when compared with huh ( . fold). according to these results, choosing the best cell line as model depends on the purpose of the research. for studying metabolism of a chemical by nqo and gst-pi or effect of a chemical on translational regulation of these enzymes, it is better to consider protein expression of the cell lines for choosing best model. however, if the aim is to study effect of a chemical on transcriptional regulation of these enzymes, it is better to choose a cell line that expressing highest mrna of gene of interest. in conclusion, considering both mrna and protein expression levels together, the best model cell lines for studying phase ii xenobiotic metabolizing nqo and gst-pi are ht and huh , respectively. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the studies on the pancreatic cells' surface glycoconjugates profiles in rats fed with high fat with lectin labelling methods by flouresans microscopy y. mater, s. beyhan ozdas gebze technical university, kocaeli, turkey in this study, the backbone of the cellular adhesion-recognition mechanism, located in the cell membrane. the study material selected pancreas tissue, has a privileged structures. the pancreas is one of the main organs to aid in digestion. the pancreas functions as an exocrine gland and role in digestion. in addition, the pancreas also functions as an endocrine gland, secreting several hormones into the blood that control the blood levels of glucose and other nutrients. due to the pancreas have been selected for this unique feature. thus, different types of cells in the same sample will be able to study the structure of the surface glycoconjugates. generally researches about determination of carbohydrates in the cell, glycoproteins or/and glycolipids are cut with enzymes. next step, the oligosaccharide mixture obtained, than establishing the complete structure of oligosaccharides and polysaccharides requires determination of branching positions, the sequence in each branch, the configuration of each monosaccharide unit, and the positions of the glycosidic links. this is a more complex problem than protein and nucleic acid analysis. these processes are indispensable for the understanding of the chemical structure of the sugar. whereas in cells using labeled lectins specific sugars, it is possible to accurately determine. in this study, was used triticum vulgaris (wga) labeled with fluorescein (fitc). thus, the cells located on the cell surface and neu ac (sialic acid) for wga sugar residues were investigated. according to preliminary results of this study, wga labeled with fitc is specifically binding of these sugars. when this study is completed, the differences of sugar on the surface of different type of cells in the pancreas can be distinguished in micrographs. thus, in the cells of the pancreas, the sugar units involved in adhesion-recognition can be possible to determine specifically. large scale gene networks could be topologically analyzed in order to obtain possible global system-level structure cancer gene co-expression networks can have lower connectivity as compared to normal samples. using colorectal tissue gene expression datasets, we observed that tumor specific networks are less connected than normal networks. functional enrichment analysis suggested that cell cycle genes and methylation-associated cell adhesion genes can specifically play a role in the connectivity loss of carcinoma samples. literature confirmation provided a gene network including significant genes playing roles in the intersections between cell cycle, cell adhesion, and cell skeleton dynamics. this network can provide novel insight to our understanding of the molecular mechanisms of colorectal cancer. p- . . - tf-mirna circuits specific to epithelial cancers y. oztemur, a. aydos, b. gur dedeoglu ankara university biotechnology institute, ankara, turkey cancer is the most common cause of death in the world but there are still a lot of uncertainties about the exact mechanism taking roles in regulation of it. cancers can be classified according to cell type; in which they start. carcinomas are the most prevalent types of cancer and start in epithelial tissues. they are also named as epithelial cancers (ecs) and make up about out of every cancers. over the past few years, many studies are concentrated on mirnas, which have emerged as important regulators of gene expression like transcription factors (tfs). tfs are regulators at transcriptional level while mirnas are post-transcriptional regulatory key-elements. otherwise the transcription of mrnas and mirnas are known to be regulated by tfs and tfs are the targets of mirnas. therefore, it is crucial to characterize the relation of tfs, mirnas and their targets by building circuits in diseases such as in ecs. for this study, mirna and mrna expression studies including epithelial tumors and normal samples searched in geo and array express microarray databases. mrna studies and mirna study, which were designed for different ecs (breast, lung, ovary and colorectal) were selected to be analyzed. differentially expressed (de) mrnas and mirnas between epithelial tumors and normal samples were extracted (p ≤ . , fold change). among de genes, transcription factors and mirnas were identified and listed for epithelial tumor vs. normal comparison. circuit analysis resulted with remarkable circuit, which was common for all the types of ecs that includes klf transcription factor and hsa-mir- . in the literature hsa-mir- and klf are known as important regulators in different types of cancer, which indicated that the motifs involving tfs and mirnas might be useful for understanding the regulation of ecs. as a conclusion finding out new and common circuits may aid us in predicting new or alternative diagnostic and/or prognostic biomarkers for ecs. mesenchymal stem cells (mscs) are multipotent stromal cells that can differentiate into a variety of cell types which are used in cell therapy. although they are the most attractive cell type for cell therapy studies, primary mscs lose their differentiation potential with increasing time in culture and passage so they are of limited use. due to this disadvantage, msc lines are more suitable for in vitro researches owing to their immortality. in this study we compared primary bone marrow-derived msc (bm-msc) with bone marrow derived msc line (rcb ) in terms of cell characteristics and gene expression profiles to determine the functional differences among mscs types. firstly, mscs were identified by using cd , cd , cd and cd as positive markers and cd as a negative marker. gene expression profilling was investigated using affymetrix hg-u -plus arrays. the significant go biological process terms and kegg pathway enrichment analyses of the identified degs were performed using david (p < . , fold change≥ ). the analysis showed similar pathway clustering in both cell types. the resulting quantitave transcriptome of genes were identified that differentially expressed in msc line versus primer mscs ( upregulated and down-regulated). functional classification of changed genes was mainly clustered in cell cycle, cell death and mismatch repair. kegg pathway analysis revealed that the genes were significantly enriched in pathways including "cell cycle, dna replication and focal adhesion" pathways. in conclusion, our results indicate that msc lines can be used instead of primary mscs. these quatitative results provide an important basis to adapt cell lines to more closely resemble physiological conditions as oppossed to animal experimentation. this could help to minimize the use of animals in research. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] association between loss of q , gain of q . and progression in sporadic colorectal cancer n. belder , b. savas , m. a. kuzu , i. pak , h. s€ umer c ß elebi , a. ensari , h. € ozdag biotechnology institute, ankara university, ankara, turkey, school of medicine, ankara university, ankara, turkey, ankara oncology training and research hospital, ankara, turkey colorectal cancer (crc) is one of the most diagnosed cancer and the third leading cause of cancer deaths throughout the world. identifying of copy number variation (cnv) profiles between early and late stage cancers can be useful to understand the progression and aggressiveness of cancer. the main goal of this study was to construct a comprehensive insight of association between cnv and sporadic crc stages in order to identify novel candidate targets which may contribute to tumor progression. affymetrix . genechip snp arrays were used for characterization of cnvs in tumor and matched normal formalin-fixed, paraffin-embedded (ffpe) tissues from stage i, stage ii and stage iii samples. paired cnv analyses were performed using partek genomic suite . and genomic segmentation algorithm was performed using a minimum of markers per segment, a signal-to-noise ratio of . and the cut-off value for the gain and loss was set of ae . . the adjusted p-value ≤ . were considered to be significant. whole genome cnv analysis revealed that amplification of q . with genes was found the most frequent ( . %) in stage ii tumors. the most frequent ( %) amplifications were q . and p . in stage iii tumors. while deletion of chromosome q . in stage iii with a frequency of % was found the most frequent loss, deletion of q . was seen the most frequent ( . %) in stage ii tumors. two tumor suppressor genes smad and smad which are found in these deletion regions were common genes between stage ii and stage iii. our results showed that gain of q . might have a significant role in the progression of cancer. loss of q comprising two tumor suppressor genes is also another important finding. q loss can be a significant prognostic value in colorectal cancer even though validation of target genes requires additional study and larger sample size. this work was supported by tubi-tak project no: s . p- . . - meta-analysis based mirna signature discriminates cervical cancer from normal samples a. yucel polat, y. oztemur, a. aydos, b . gur dedeoglu biotechnology institute, ankara university, ankara, turkey gynaecological cancers are common problems in female health. squamous cell carcinoma (scc) is a type of these malignancies. this tumor type is derived from pre-cancerous lesions, which is called cervical intraepithelial neoplasia (cin). cin is classified as cin , cin and cin according to their dysplasia grade in the cervical tissues. mirnas are small non-coding rnas that were shown to have important roles in the development and progression of various cancers. the aim of this study is identifying mir-nas, which are playing a part in progression of cervical lesions by a ranking based meta-analysis approach. two mrna and three mirna expression studies, which include normal, cin , cin and scc samples were selected from arrayexpress and gene expression omnibus (geo) databases. three mirna studies were combined with anova dependent ranking based meta-analysis program which was developed in our laboratory to find out a mirna signature that can discriminate cin , cin and scc samples from normal samples. the top five mirnas with the highest ranks in meta-list were selected for further analysis. predicted targets of these mirnas were identified by mirdb target prediction tool. additionally two mrna datasets were selected for mirna-target validation studies. common genes, which were obtained from meta-mirna targets and differentially expressed genes between normal and cin , cin and scc groups from two independent studies, were identified and they were subjected to pathway enrichment analysis. pathway enrichment analysis that was performed with common genes showed that these targets were significantly enriched (p < . ) in especially cell proliferation, cell survival and cell cycle pathways, which are the key players of cancer development and progression. the meta-analysis results together with validation analysis of their targets may point out the potential roles of mirnas as biomarkers for the diagnosis and the treatment of cervical cancer. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the hypoglycaemic and regenerative activity of thymbra spicata in alloxanized-diabetic rats thymbra spicata (labiatae), a carvacrol and thymol containing plant, is one of the medicinal herbs used by diabetic individuals to reduce blood glucose in turkey. we investigated the hypoglycaemic and anti-lipemic effects of the aqueous extract prepared from dried leaves and flowers of this plant in alloxanized-diabetic rat model. rats were divided as: diabetic control (group ), dia-betic+glibenclamide (group ), diabetic+plant extract (group ), untreated control (group ) and control+plant extract (group ) groups (n = for each group). serum glucose, lipid levels and body weight changes were mesasured and pancreas and liver histology of the rats were examined. each rat in all groups were administered the plant extract ( mg), and the reference drug glibenclamide ( mg/kg) by gastric gavage every day for weeks. in group , blood glucose, serum alt, ast, triglyceride, cholesterol and ldl cholesterol levels increased while body weights decreased. in group , serum glucose, alt, ast, triglyseride and hba c levels decreased compared to group while cholesterol and ldl levels were high. in group , serum alt, ast, trigliserit, cholesterol, ldl levels decreased significantly but serum glucose and hba c were higher compared to group . body weights increased except group and hdl levels were not altered. histologically degenerative changes observed on pancreas of group were decreased in groups and . there was no difference on liver histology of the groups. in conclusion, thymbra spicata showed a protective and regenerative effect on diabetic pancreas. the hypo-lipidemic effect of the plant extract was also more effective than glibenclamide possibly due to the flavonoids, saponins and triterpenoids contents in the extract. its hypoglycaemic and protective activity should be tested for different doses and extract preparations and for longer periods. our study suggests that thymbra spicata is an excellent candidate for future studies on diabetes mellitus. with three different transcriptome data sets from the public gene expression omnibus database: time dependent data of dphop mutant, dargr mutant and wild type strain. the dynamic data spanned both primary and secondary phases of the metabolism. statistical results of transcriptome data were used for reporter metabolite analysis and reporter pathway analysis, which identify the metabolites (or pathways) with a significant coordinated transcriptional change in response to gene deletion perturbation in phosphate and nitrogen metabolisms. further, the production of actinorhodin, a pharmaceutically important compound, was modeled in the two deletion strains by calculating the metabolic fluxes subject to transcriptional level constraints on enzyme-coding genes. the metabolic switch from primary to secondary metabolism was highlighted in terms of the activity of pathways and fluxes as a result of the computational analyses in this work, leading to a better understanding of the role of phosphate and nitrogen metabolisms in increasing production levels. introduction: as a member of legume family licorice (glycyrrhiza glabra l.) has been widely used by human kind for many years as food constituent. especially by folks in rural sites licorice consumed widely. beside food constituent licorice has been used for medical purposes as well. licorice found effective with scientific datas on peptic skin infections, ulcers, inflammation, eczema, alzheimer disease, liver disease, and cancers. it also has been used as natural sweetener and food additive for preparing candies, chewing gum and beverage since ancient times. like all other medicine it has not been free of adverse event or toxicological effects. material and methods: alcoholic extracts of plant obtained by maceration process. for in vitro examination of anti-oxidant profile of licorice dpph free radical scavenging, abts cation radical scavenging and cupric ion reducing antioxidant capacity assay applied. application of extract made by oral route to rats for a week. anti-oxidant profile has been evaluated by myeloperoxidase (mpo), arylesterase (ares), total oxidative stress (tos) and total antioxidant status (tas) of serum levels. determination of toxicological effects alt, ast, ldh and alp values studied. histological investigation applied on liver and kidney tissues. results and discussion: results compared with control and standarts. antioxidant potential of licorice has been observed by in-vitro assays. serum mpo and ares values also compared with in-vitro results and correlation between them has evaluated. toxicological investigations made after evaluation of ast, alt, ldh and alp values. conclusion: in vitro assays has showed that licorice has potential anti-oxidant effect. investigation revealed that a mild toxic effect of licorice by biochemical tests. toxicological profile compared with control group and alt, ast values found slightly decreased and a mild elevation has been seen in ldh and alp values. for further and detailed investigation is needed. p- . . - on the applications of a metabolic network model of mesenchymal stem cells h. fouladiha, s. a. marashi, m. a. shokrgozar, m. farokhi mesenchymal stem cells (mscs) have several applications in tissue engineering and regenerative medicine. mscs can be very useful in stem cell therapy, because they can be isolated bone marrow or adipose of an adult. these cells have also been used as gene or protein carriers. therefore, maintaining them in a desire metabolic state has been the subject of several studies. here, we have used a genome scale metabolic network model of bone marrow derived mscs for exploring the metabolism of these cells. then, we try to validate the computational results by experimenal tests. we analyzed metabolic fluxes in order to increase stem cell proliferation using the metabolic model. consequently, the experimental results were in consistency with computational results. in the present work, the applicability of the metabolic model was successfully approved. therefore, this metabolic model can be useful in biomedical researches of stem cells. p- . . - qtl analysis for body weight and fatness in bxd recombinant inbred mouse strains a. dogan , c. neuschl , r. alberts , g. a. brockmann school of medicine, istanbul kemerburgaz university, istanbul, turkey, department for crop and animal sciences, humboldt-universit€ at zu berlin, berlin, germany, helmholtz-zentrum f€ ur infektionsforschung, braunschweig, germany genetic variation in body weight and composition is under the influence of many genes and have different genetic architectures. in the present study, the genetic factors contributing to body weight and fatness were examined under energy rich feeding conditions. growth traits, lean and fat weight, fat mass gain were analyzed to map qtls in a set of bxd ri strains. genome-wide analyses were revealed several genomic loci that control body weight and associated bodily changes in a sex and age-specific manner. the genetic data provided evidence for significant qtls on chromosome (chr) , , , and . most likely candidate genes within or near the regions with the highest significance levels were identified. the genes f rik, gbe , a n , and four genes cenpc , stap , uba , gnrhr for example, are suggested as most likely positional candidates accounting for the qtl effects on chr for fat mass, on chr for fat mass gain and on chr for lean weight, and chr for body weight, respectively. our results showed that body composition and fatness are highly complex that many genetic factors regulating and suggested candidate genes, which may help for studies of human fatness. related to serotonergic and gaba systems in response to hormonal changes. the nutrients involved in neurotransmitter synthesis may be the cause of relationship between diet and premenstrual syndrome. therefore, the aim of this study was to investigate the effect of various nutrients and premenstrual syndrome. this study was conducted to healthy women aged - years. participants were asked to fill in premenstrual assessment form. dietary intakes (three days in each phases) were recorded during premenstrual, menstrual and postmenstrual phases. energy, protein, amino acids, iron, calcium, and magnesium intakes were estimated. statistical analyses were performed using the spss software. friedman tests were conducted and differences were considered to be statistically significant for p-values lower than . . . % of the participants reported premenstrual symptoms and premenstrual symptoms related nutrient intake were increased in these women. it was determined that energy (p = . ) and protein (p = . ) intakes were higher in the premenstrual phase. during premenstrual phase; tyrosine (p = . ), isoleucine (p = . ), leucine (p = . ), lysine (p = . ), methionine (p = . ), cysteine (p = . ), tryptophan (p = . ), and glutamic acid (p = . ) intakes were higher than other phases. likewise, iron intake was higher on premenstrual phase (p = . ). on the other hand, intake of other potential premenstrual syndrome related nutrients like fat, cholesterol, calcium, magnesium, and vitamin b were not significantly different within the menstrual phases. amino acids including tyrosine, tryptophan, glutamine, and vitamin b are involved in neurotransmitter synthesis and might be related to premenstrual symptoms. consequently, elevated intakes of dietary protein and some amino acids during premenstrual phase may be related to premenstrual syndrome symptoms. until now far uv cd spectra of only two potexviruses were published. the papaya mosaic virus (papmv) spectrum, measured by leclerc and co-authors contained no obvious anomalies and was similar to the spectrum of isolated papmv coat protein (cp). but measured years earlier by homer and goodmanfar uv cd spectrum of potato virus x (pvx) itself had anomalous character and differed strongly from the spectrum of isolated pvx cp. in the present work we measured far uv cd spectra for two more members of potexvirusgenus: alternanthera mosaic virus (altmv) and potato aucuba mosaic virus (pamv) and their free cps. the altmv virion and altmv cp spectra were similar to each other and to the spectra of papmv and its cp. the pamv spectrum resembled the pvx spectrum in anomalously low ellipticity of the negative band at nm, but in contrast to pvx, did not have additional peak at nm. homologous modeling showed that cp of the three viruses is very similar in the core structure, and the observed difference may be explained by differences in disordered parts of proteins. possible reasons of potexvirus structural variability are discussed and it is suggested that the intravirus potexvirus cps may assume different conformations in different virions of the same preparation or even along the length of one virus particle. this work was supported by the russian science foundation (grant - - ). the antimicrobial potential of different phenolics was tested on pectobacterium in search of possible mode of action. in this respect, biofilm formation, exoenzyme activity, gene expression and virulence on its natural host (potato, cabbage, calla lily) were performed. also computational approach to show interaction between phenolic compounds and target protein was carried out using docking tools. the virulence determinants of pectobacterium were significantly impaired, at compound concentrations that did not affect bacterial cell growth. these observations suggested a mechanism which specifically interferes with bacterial virulence. since, these virulence determinants in pectobacterium are controlled by quorum sensing (qs), we focused on the effect of phenolics on the qs system in pectobacteria. the study revealed an inhibiting effect of the tested compounds on the expression level of central qs system and controlled genes, using qrt-pcr. also, there was a prominent reduction in the level of qs signal molecules n-acyl-homoserine lactone (ahl) accumulation. in addition infection capability was also practically blocked, which was completely recovered by application of exogenous-ahl. these results were supported by a potential interaction of plant phenolics with qs targets, as shown by molecular docking tool. collectively, results suggest the potential interference of phenolic compounds with qs central components (expi/expr proteins). moreover, it holds potential for future development of control measures against pectobacterium, and possibly other pathogens with similar mode of virulence. saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including double-stranded rna (dsrna) viruses. the l-a dsrna virus family of s. cerevisiae is widely distributed in nature. several versions of l-a virus are described and new ones continue to be discovered. some s. cerevisiae strains along with l-a dsrna possess smaller dsrnas, called m satellites. these dsrnas encode a sole secretable protein, known as k , k , k and k-lus toxin. l-a genome encodes the gag major structural protein and gag-pol fusion protein, formed by ribosomal frameshifting. gag-pol has transcriptase and replicase activities are necessary for maintenance of both l-a and m satellite dsrnas. so far, it's not known whether certain l-a virus has evolved to maintain a distinct type of satellite dsrna or this phenomenon lacks inherent specificity. we developed universal strategy to obtain full length l-a and m dsrna genomes from s. cerevisiae. complete viral dsrna genomes can now be cloned, as evidenced by l-a- dsrna, analyzed and sequenced directly from any yeast strain by means of enzymatic manipulations on total or fractioned rna content. we have identified previously undescribed l-a variant from different yeast strains specifically associated with certain type of m satellites. moreover, we identified for the first time full -utr and -utr sequences of m satellite. highly conserved sequence regions along with variable fragments were discovered at protein level, revealing clear trend to form clusters among different l-a gag-pol proteins. the obtained data suggest that each l-a virus variant can specifically maintain a distinct type of satellite dsrna. p- . . - physic-chemical characterization of plga adjuvants for immunization per os t. chudina, d. kolybo palladin institute of biochemisry of the national academy of sciences of ukraine (nasu), kyiv, ukraine antibodies against diphtheria toxin play the most important role in the immunity against corynebacterium diphtheriae. all current diphtheria vaccines have parenteral route of administration. undoubtedly, oral administration of antigens would be the most patient-friendly way of immunization. however, the efficacy of free antigens oral administration is limited by their degradation in the gastrointestinal tract and poor absorption by m-cells. biodegradable and biocompatible polymers, like poly (d,l lactide-co-glycolide) (plga), are widely used for the design of mucosal immunizing agents. importantly, that the way of particle preparation plays an important role in plga biodegradation and antigen release. the aim of this work was to characterize the main physic-chemical properties of two types of plga particles: with immobilized antigen (plga ) and with encapsulated antigen (plga ) . we have prepared two types of plga particles containing egfp-sbb proteins (non-toxic recombinant fragment b of dt fused with egfp). the antigen loading efficiency of particles was determined based on the ratio of protein concentration in solution before and after loading and shown better results for plga particles (plga - . %, plga - . %). the flow cytometry results demonstrated that % of plga particles conjugated with egfp-sbb, and only . % of plga particles conjugated with protein.the particle sizes had the slight difference by the results of two different techniques (ntanumber based, the software tracks individual particles; dls -scattering intensity weighted), however demonstrate similar patterns. dls data showed that the mean plga particles size was . nm and plga - . nm. nta data also showed that mean plga particles size a little smaller than plga ( . nm and . nm respectively) . demonstrated differences in the properties of synthesized particles may have an influence on the immunogenicity of the used for oral immunization antigen. p- . . - a suitable system for studying the functionality of a plasmodial protein in mammalian cell lines cho-mt , a mutant cell line was proved to be an appropriate tool for investigating intracellular function of cct. in this cell line, the endogenous cct activity decreases dramatically at °c, blocking membrane synthesis and ultimately leading to apoptosis. we have studied the rescuing potential of pfcct in cho-mt cells with the isogenic cho-k cells as a control. cells after transient transfection were incubated at °c and then analysed by facs using the fluorescence of egfp fused to pfcct. the proportion of cells undergoing apoptosis was determined by propidium-iodide staining. we have demonstrated for the first time that heterologously expressed pfcct is able to complement endogenous cct activity in mammalian cells. thus, a suitable system has been established for functional investigation of structural elements of pfcct. in order to reveal the role of different protein sequences in enzymatic function, we redesigned the structural gene of pfcct obtaining a modular system where different domains are easy to be removed or exchanged. here we designed a series of different truncation and deletion constructs to reveal the role of plasmodium specific sequences. in parallel, heterologous expression experiments of different constructs in the mutant cho-mt and the wild type control cell lines are performed to validate the reported model system. p- . . - host-pathogen interactions: is there a relationship between tlr polymorphisms and tuberculosis in a group of turkish patients? introduction: tuberculosis (tb) is a global health problem and according to world health organization (who) each year more than million individuals die from tb and each year , cases of tb are notified in turkey. malatya is the third largest city in east anatolian region of turkey and tb incidence rate is higher ( . / , ) comparing to the general population of the country. for this reason it is important to determine the factors that lead to tb in this population. disease agent can stay in the latent phase for long periods of time after infecting the individuals. while some infected individuals show the symptoms some others never do and even % of these never develop clinical disease. various mechanisms take place during the host response to infectious agents. toll-like receptor (tlr) genes are shown to be candidate genes in these responses. materials and methods: in this study tb patients and healthy controls were included. tlr genotyping for rs , rs was performed by using a commercial taqman snp genotyping assay kit. data were summarized by count and percent. hardy-weinberg equilibrium was tested by chi-square distribution with df. differences between groups due to allelic and genotypic distributions were analyzed by pearson's exact or fisher's exact tests. in all comparisons significance level was considered to be . . results: the single nucleotide polymorphisms (snps) which were subject of this study haven't been screened in turkish population earlier. no significant association was found between tb and the snps we screened in our group of patients. discussion and conclusion: unlike other populations results we couldn't find a significant association between the disease and the genotypes of our patients. the study should be performed in bigger populations in order to confirm the results. p- . . - lytic action of bacteriophages as a tool for the obtaining of images p. boltovets , r. radutny , t. shevchenko institute of semiconductor physics nas of ukraine, kyiv, ukraine, scientific and technical center of advanced technologies nas of ukraine, kyiv, ukraine, taras shevchenko national univercity of kyiv, kyiv, ukraine obtaining of images by different types of bacteria now became a very special branch of skill at the interface between science and art. however the authors did not found any scientific article, where bacterial lawn was used as the background and the image was formed by the lytic action of the virus (bacteriophage). whereas the mentioned approach could be used not only with artistic aims but for the practical use. the aim of this work was to demonstrate a possibility to obtain the image on the bacterial lawn by the lytic action of the bacteriophage. the bacterial lawn was obtained by the standard metod using the . % agar with the nutrient medium and the . % agar containing escherichia coli culture. stencils with the preparation of the bacteriophage t were applied. samples were incubated during the twenty-four hours at + °c. after that stencils were removed and the samples were stained by coomassie blue r- or fuchsine (with further fixation by the % acetic acid). several approaches to obtain the image by the lytic action of the virus were applied. first of all stencils made from printing paper and filter paper were compared. it was demonstrated, that the use of filter paper stensil allows to obtain more accurate and controllable images, than the use of the printing paper stensil. in the next series of the experiment the possibility of the reversed stencil use (where the image is formed not by the lytic zone but by the zone of bacterial growth) was demonstrated. also the possibility of the partial staining of the obtained image was explored. it gives an opportunity to obtain polychrome images using available colorants. summarizing the above it should be noted, that it was the first time when the graphical image was obtained by the lytic action of the virus on bacteria. this approach could be used not only for the artistic aims but as well for the practical use, for example, for the restriction of the action of microorganisms in out-of-theway places. burgdorferi the identification and characterization of possible antigens is essential for the improvement of current laboratory diagnostics for lyme disease and vaccine development. in this study, several recombinant b. burgdorferi outer surface proteins have been obtained and their antigenic properties have been evaluated in an effort to characterize novel immunodominant antigens. because b. afzelii and b. garinii are the most prevalent species in latvian ticks, proteins with conserved domains were included in this study. a panel of serum samples of lyme disease patients with early and disseminate disease stage was used. the controls were matched by age and sex to the patients and represented the same geographic area. the results show that proteins of several b. burgdorferi gene families have properties with respect to their candidacy as a subunit assay for a novel lyme disease immunodiagnostic. especially, the difference in their size in a range on the western blot assay may provide good discrimination between protein bands. however, they have potential for diagnosis if used in combination with other antigens but not as a "stand alone" test. in conclusions, this study showed the existing challenges in serological testing of early lyme disease. the conservation of the sequence of antigen between species of b. burgdorferi complex is essential for the most successful serodiagnostic marker candidate. the presence of homologous proteins in treponema species could lead to the cross reactivity in syphilis patients, and should be carefully evaluated. antimicrobial resistance is one of the greatest challenges in modern medicine. there is a pressing need for better understanding of the specific mechanisms that contribute to resistance to optimize existing therapies. in in georgia extended-spectrum beta-lactamase (esbl)-producing e. coli strain was isolated from the post-surgical sample obtained from gallbladder of the patients with chronic calculous cholecystitis which belongs to the sequence type (st ) complexes with ctx-m gene. is this strain characterized by other differences on a proteome level? are antibiotics against which the strain is resistant inducing the changes in bacterial proteome? the present work was aimed (i) to study the differences on a proteome level (i) between e. coli - / -g and attc e. coli-reference strain and (ii) to compare the proteomes of strain at two conditions: with and without antibiotics. strain was grown in the presence of three antibiotics: rocephin (ceftriaxone), fortum (ceftazydym) and claforan (cefotaxime sodium) together. proteomic expression was analyzed using two-dimensional gel electrophoresis and mass spectrometry. significant differences were found for several proteins, including putative abc trnsporter arginine protein , cystine-binding periplasmic protein, fkbp-type peptidyl-prolyl cis-trans isomerase, outer membrane protein a, d-galactose binding periplasmic protein and some others. the importance of these differences for anti-microbial resistance will be discussed. p- . . - molecular characterization of resistance and virulence features in staphylococcus aureus clinical strains isolated from cutanaeus lesions in patients with drug adverse reactions i. lupu , i. gheorghe , , m. popa , , a. ion , m. mihai , v. lazar , , m. c. chifiriuc , carol davila" university of medicine and pharmacy, bucharest, romania, research institute of the university of bucharest-icub, bucharest, romania, faculty of biology, university of bucharest, bucharest, romania patients treated with epidermal growth factor inhibitors often experience cutaneous adverse reactions. however, the infectious complications of these toxic effects and the contribution of specific pathogens, such as the community emergent methicillin resistant staphylococcus aureus strains. the present study was aimed to identify the types of sccmec and virulence genes profile in clinical s. aureus isolated from cutaneous lesions of different severity degrees in patients with dermatologic toxic effects. this study was conducted on a total of s. aureus clinical strains isolated in from acneiform reactions pustulae and periungual lesions in patients with drug cutaneous adverse reactions. multiplex pcr was performed on genomic dna from isolates in order to identify the sccmeccentral elements and the virulence genes: bbp (bone bound sialoprotein), ebps (elastinbinding protein), fnbb, fnba (fibronectin-binding proteins), fib, clfa, clfb (clumping factors a and b), cna (collagen-binding protein), luk-pv (panton-valentine leucocidin), hlg (haemolysin), tst (toxic shock toxin). the mrsa phenotype was genetically confirmed by the presence of meci gene in case of . %, meca in . %, sscmec type ivd element in . %, ccrb in . % and sccmec types i, iii, iv in . % of the studied s. aureus strains. regarding the virulence genes encountered in s. aureus strains, the most frequent was clfa ( . % of the isolates), followed by clfb ( . %), fib ( . %), hlg ( . %) and bbp ( . %). these results confirm the high prevalence of mec i and sscmec type iv elements, usually encountered in communityacquired mrsa strains, in cutaneous isolates from patients with dermatologic toxic effects. more data on the virulence and genetic background of these local strains are needed to appropriately assess the risk of such infections and avoid the inappropriate administration of beta-lactams. p- . . - analysis of toxicogenic properties of staphylococcus aureus strains isolated from cows with subclinical form of mastitis in the central area of russian federation. pore-forming toxins and enterotoxins), which are present in s. aureus strains isolated from clinically healthy cows. staphylococcus strains were isolated from cow's milk. disk diffusion method was used to determine the sensitivity to antibiotics. pcr analysis was used for detection of meca, mecc genes and genes of toxins. investigated strains were resistant to oxacillin ( %), vancomycin ( %) . it was found that all strains, which contain meca and mecc genes, showed resistant to more than antibiotics. it was determined that among the investigated strains % contained meca, % -mecc, % contained both meca and mecc. some strains contained genes of panton-valentine leukocidin (pvl) or alpha-hemolysin and several strains contained both types of genes. enterotoxin a (sea) gene was detected in . % of cases, sed - %, seg - %, sei - %. genes of staphylococcal toxins b, c, e, h were not found. the presence of phenol soluble modulin biosynthesis genes was determined: genes of alpha peptide synthesis were found in % of strains, beta peptide toxin genes in %, delta toxin gene in %. it was determined, that clinically healthy animals are carriers of s. aureus strains that cause mastitis. high level of antibiotic resistance was found in strains containing meca and mecc genes. the major part of the strains carried genes of phenol soluble modulin biosynthesis. the role of phenol soluble modulins as well as of pvl and alpha-hemolyzin in the development of mastitis is not completely clear. we conclude that pore-forming toxins have dominant role in the latent form of mastitis. p- . . - impact of lactoferrin on the hydrophobicity and adherence to the inert substratum of staphylococcus aureus strains isolated from patients with cutaneous drug reaction skin healing is a complex biological process that requires the involvement of different cell types and humoral effectors. one of the main factors are aggravating and delaying the healing process is represented by the supra-infection with pathogenic or opportunistic microorganisms that grow in specialized consortia embedded in a self-produced extracellular polymeric matrix, called biofilms, which are extremely resistant to any antimicrobials and host immune response. lactoferrin (lf) is an ironbinding glycoprotein which promotes skin healing by enhancing the initial inflammatory phase, but also by inducing an overabundant immune response. the aim of this study was to investigate the influence of lf, one of the main components of innate, humoral anti-infectious immunity on some microbial features, involved in the first steps of the infectious process, such as hydrophobicity and adherence of staphylococcus aureus strains isolated from maculo-pustular lesions in patients with adverse reactions to epidermal growth factor inhibitors. for hydrophobicity measurement the bacterial suspensions were grown in the presence or absence of lf, and then, the "microbial adherence to hydrocarbons test" (math) was performed. the capacity to develop biofilms on inert substrata and the influence of lf on this feature was spectrophotometrically quantified using an adapted microtiter method, after crystal violet staining. our results showed that lf decreased the hydrophobicity and limited the biofilm development of all s. aureus tested strains, in a dose and time dependent manner. the decreasing effect on the microbial hydrophobicity was accompanied by a lowering effect on the adhesion of microbial strains to the inert substratum. in conclusion these observations indicate that lf exhibits a wound pro-healing effect, by limiting the microbial colonization and biofilm formation and thus, the occurrence of infectious complications of skin lesion. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] host-specificity determinants of bacteriophage vb_ecom_fv considered vehicles of s.aureus intoxication in humans throughout the world. the objective of the present study was to assess the presence of enterotoxigenic and methicillin-resistant s. aureus in water buffalo milk and dairy products. a total of water buffalo milk and dairy products ( water buffalo cream and water buffalo cheese) were collected from different dairy farms, smallholders and local bazaars in samsun, turkey. all samples were analyzed using the standard procedure en iso - and isolates were confirmed for the presence of the s rrna and nuc gene by polymerase chain reaction. s. aureus was identified in of water buffalo milk ( %), of water buffalo cream ( %), and of water buffalo cheese ( %). a total of isolates were confirmed as s. aureus by pcr. genotypic methicillin resistance was evaluated using pcr for the meca gene. out of isolates, ( %) were found to be methicillin resistant (meca gene positive) by pcr. the enterotoxigenic s. aureus was identified in out of ( %) isolates by the mpcr technique. five isolates produced staphylococcal enterotoxins sea ( / ; . %), two isolates produced sec ( / ; . %), one isolate produced ( / ; . %) sed, one isolate produced ( / ; . %) see and three isolates produced sec+sed ( / ; %) . none of samples were positive for seb. in conclusion, the presence of enterotoxigenic and methicillin-resistant s. aureus in milk and dairy products is of significant for public health concern and also these enterotoxin genes sea and sed are predominant toxins that can cause staphylococcus intoxication in humans. this study was funded by ondokuz mayıs university, samsun, turkey, scientific research project programs (project no: pyo. vet - . . ) and this article was part of a phd thesis. p- . . - identification and biochemical characterization of an immune modulating protein from helicobacter pylori b. kaplan t€ urk€ oz faculty of engineering, department of food engineering, ege university, izmir, turkey helicobacter pylori is able to achieve persistent infection with minimal immune response. the first line of defence during h.pylori infection is through gastric epithelial cells which present toll like receptors (tlr). a family of bacterial proteins which share homology with the toll/il- receptor (tir) domain were identified. the structure of btpa from brucella showed that bacterial tir proteins (btp) mimick human tir domain proteins and act on myd signaling pathways to suppress tlr signaling. h.pylori might also produce a similar protein. a putative h. pylori tir protein was found based on sequence homology and the corresponding gene; hp ; was cloned in fusion with an n terminal cleavable his-tag. the recombinant protein, his- was purified using nickel affinity chromatography. was subjected to limited proteolysis and the bands were analyzed by peptide mass fingerprinting (pmf). oligomerization of was investigated by in vitro pull-down and size-exclusion chromatography. , a amino acid protein, has a predicted c terminal tir domain similar to other btps and sequence alignments verified the presence of tir domain signature regions. recombinant his- was produced with a yield of mg/l culture. a structurally stable kda fragment was obtained from limited proteolysis which contained the tir domain as verified by pmf. in vitro pull down assays showed interacts with itself forming dimers as shown by size-exclusion chromatography. tir domain proteins function by interacting with themselves and other tir domains. our results showed that also form dimers, supporting that it is a btp. current research is focused on solving the structure of and investigating its interaction with myd . might play a direct role in reduced immune response against h.pylori by binding to myd analogous to other btps. further characterization of will provide the first solid evidence of presence of a tir domain protein in h.pylori. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] lipopolysaccharides with different lipid a acylation status from vibrio cholerae and campylobacter jejuni contribute differently to il production by bone marrow-derived macrophages k. korneev , , e. sviriaeva , lipid a is a biologically active part of lipopolysaccharide (lps) from gram-negative bacteria that is responsible for the activation of the innate immunity through interaction with toll-like receptor (tlr ) and subsequent production of proinflammatory cytokines. bacteria frequently transform their lipid a so that its recognition by tlr is not sufficient for induction of effective antibacterial immune response. we compared biological activity of various lps from pathogenic bacteria vibrio cholerae and campylobacter jejuni. we purified r-form lps for each strain by hydrophobic chromatography. the biological activity of lps preparations was evaluated by their ability to activate production of proinflammatory cytokine il by bone marrow-derived macrophages from c bl/ mice, using tlr -deficient macrophages to control for specificity of tlr signaling. lps from e. coli and inactive lps from f. tularensis were used as positive and negative controls. lps from v. cholerae demonstrated biological activity similar to that of lps from e. coli, consistent with the presence of highly acylated lipid a in both strains. however, the former was a slightly weaker activator than the latter, because lipid a from v. cholerae had on average shorter acyl chains. lipid a from c. jejuni had on average longer acyl groups than in e. coli, while degree of acylation was lower, and as a result its lipid a displayed significantly lower biological activity. our study demonstrates importance of functional groups of lipid a in the ability of lps to activate production of il by macrophages. in line with our previous reports, we confirmed a direct correlation between biological activity of various lps species with their lipid a acylation status: the biological activity increases with increase in the length and in the number of the acyl chains. excess proinflammatory cytokine production through tlr activation can cause sepsis, while inefficient activation may result in the failure to clear bacteria. clostridium perfringens phospholipase c (cpplc) is the most toxic extracellular enzyme produced by this bacterium and it is an essential virulence factor in the pathogenesis of gas gangrene. cpplc may lead to cell lysis at concentrations that causes extensive degradation of plasma membrane phospholipids. however, at sublytic concentrations it induces cytotoxicity without causing evident membrane damage. the results of this work demonstrated that the cytotoxic effect of cpplc requires its internalization and the activation of the mek-erk pathway. cpplc internalizartion occurs through a dynamin-dependent mechanism and in a time progressive process: first, cpplc colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. the results also showed that cpplc requires endocytosis in order to activate mek-erk, because treatment with the dynamin inhibitor, dynasore, prevents cpplc endocytosis, erk / activation and cytotoxcity. cholesterol sequestration as well as inhibition of actin polymerization also prevents cpplc internalization and cytotoxocity, involving endocytosis in the signaling events required for cpplc cytotoxic effect. once internalized, cpplc induces reactive oxygen species production through the activation of pkc, mek/erk and nfjb dependent pathways. inhibition of either of these signaling pathways prevents cpplc's cytotoxic effect. in addition, it was demonstrated that nfjb inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of cpplc in mice. these data provide new insights about the mode of action of this bacterial phospholipase c, previously considered to act only locally on cell membrane. understanding the role of these signaling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridial myonecrosis. p- . . - apoptosis induced by clostridium perfringens phospholipase c is mediated by reactive oxygen species m. flores-d ıaz , l. monturiol-gross , m. j. pineda padilla , c. araya-castillo , a. alape-gir on bacterial phospholipases are lipolytic esterases surface associated or secreted by a wide variety of bacterial pathogens. clostridium perfringens, the most broadly distributed pathogen in nature, secretes a prototype phospholipase c (plc), also called a-toxin, which plays a key role in the pathogenesis of gas gangrene. this toxin causes death to cultured cells and extensive myonecrosis when injected intramuscularly in experimental animals. the results of the present study showed that c. perfringens plc ( - ng/ml) induces morphological and biochemical changes characteristic of apoptosis in cultured cells, as determined by scanning electron microscopy. nuclei condensation and fragmentation were observed by fluorescence microscopy and a typical ladder fragmentation pattern of genomic dna was detected by dna in agarose gels. cell death was prevented by the caspases inhibitors z-devd-fmk and z-vad-fmk. c. perfringens plc induces oxidative stress in cultured cells as determined by fluorescence microscopy and flow cytometry using the membrane permeable probe dcfda. different antioxidants including the gluthation precursor nac, several iron chelators and the free radical scavengers tiron and edaravone prevent cell death induced by c. perfringens plc in cultured cells or in mice challenged intramuscularly with . lg of that toxin. thus, this work provides compelling evidence that superoxide, hydrogen peroxide, and the hydroxyl radical are involved in the cytotoxic and myotoxic effects of c. perfringens plc. furthermore, the data demonstrated that edaravone, a clinically used hydroxyl radical trap, reduced the myonecrosis and the mortality caused by c. perfringens in a murine model of gas gangrene, induced by intramuscular bacterial injection of bacteria. this knowledge provides new insights for the development of novel therapies to reduce tissue damage during clostridial myonecrosis. lectins are ubiquitous proteins able to recognize mono-and oligosaccharides with high specificity and low affinity. lectins do not have any catalytic activity, unlike enzymes, and they are not products of the immune system in contrast to antibodies. lectins play a crucial role in cell interactions on molecular level showing their importance in various physiological and pathophysiological processes as well as both mutualistic and parasitic interactions between microorganism and hosts. photorhabdus luminescens is a gram-negative bacterium from the family enterobacteriaceae. the bacteria have a complex life cycle that involves mutualistic and pathogenic interaction with two different invertebrate hosts. it is highly pathogenic towards insect larvae. in addition, p. luminescens lives in the intestine of infective juveniles of nematode heterorhabditis bacteriophora, together forming an effective entomopathogenic complex. we have identified several soluble lectins produced by p.luminescens. in this study, we focus on proteins from p. luminescens, which show a high sequence homology with each other. a wide range of methods was used for structural and functional studies of photorhabdus lectins, e.g. surface plasmon resonance, isothermal titration calorimetry, analytical ultracentrifugation and x-ray crystallography. all lectins from p.luminescens recognize l-fucose and d-mannose. despite being closely related, they differ in fine binding specificities. to determine their biological function, knock-out mutants of p. luminescens are being prepared to study its interaction with axenic nematodes and insect larvae. breast cancer is the major disease of women in developed countries occuring predominantly after the age of . triple negative breast cancer (tnbc) is a typical subtype of epithelial breast cancer which lacks estrogen receptor (er), progesterone receptor (pr) and human epidermal growth factor receptor (her ) all together. although various researches have been focused on characterizing tnbc and enlightening different molecular markers with the aim of improving the overall outcome, currently the sole affective therapy action for tnbc is chemotherapy. thus chemoresistance is the main clinical challange and accounts for % of failures in terms of treating the disease. multidrug resistance (mdr) is defined as simultaneous resistance towards the drugs which do or do not demonstrate structural resemblance and have different effects on their molecular targets. p-glycoprotein (p-gp) is a membrane protein coded by abcb (mdr- ) gene. p-gp is an atp-dependent pump which pumps a wide range of drugs out of the cells including chemotherapeutic agents such as doxorubicin (dox) and pactilaxel. in the present study, tnbc cell line mda-mb- was treated with increasing doses of dox, cell viability was examined with srb assay and development of mdr was investigated through mdr assay and rt-pcr. results demonstrated that cell viabiliy decreased significantly with the treatment of higher doses. mdr was shown to be increased when cells were treated with , and nm of the drug respectively along with lm of p-gp inhibitor verapamil. rt-pcr results were obtained to be consistent with mdr assay results and indicated increased mdr- gene expression with the treatment of dox. especially after nm of dox treatment, mdr- was overexpressed to be fold when compared to control. in conclusion, it was demonstrated that mda-mb- cells have shown to display elevated resistance to higher doses of dox. p- . . - targeting dna damage response pathway in cancer cells under heat stress and the mechanical effect of ultrasound y. furusawa , t. kondo toyama prefectural university, imizu-shi, japan, university of toyama, toyama, japan ultrasound (us) has been widely utilized for diagnosis and therapy in many medical fields. the biophysical modes of us are divided into three classes, thermal, cavitation and non-thermal non-cavitation effects. in clinical use for cancer therapy, the thermal effect was utilized for hyperthermia therapy with focusing us on cancer to rise the temperature from °c to °c, or further which could induce thermal ablation of cancers. cavitation leads to a variety of mechanical stress such as shear stress, shock wave, high pressure, and chemical stress such as free radical formation, both of which have been inferred to act simultaneously on all biological materials. it has been indicated that us induces cell killing, cell lysis, loss of viability, and loss of clonogenicity. recently, we found that heat stress as well as us without thermal effect induce not only dna single-strand breaks but also dna double-strand breaks, a most cytotoxic region of dna, in chromatin dna detected by both gammah ax staining and neutral comet assay. in response to the stresses which induce dna damage, the dna damage sensor protein kinase, ataxia telangiectasia mutated (atm), atm and rad related (atr), and dna-dependent protein kinase (dna-pk) become activated form to initiate signal transduction pathways activating cell-cycle checkpoints, dna repair, and apoptosis. the molecules consisting of dna damage response pathway were expected as therapeutic targets because defects in the response to dna damage agents can be lethal. this work was designed to explore the possible therapeutic targets of the molecules in dna damage response pathways for future us-aided therapy. finally, several kinases (e.g., checkpoint kinase) on dna damage response pathway seems to be the targets for hyperthermia and us therapy. (ural branch) , ekaterinburg, russia, shemyakin and ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia based on the recently synthesized (s)-( -aminopurin- -yl) amino acids (gly, ala, val, phe, pro), we obtained a series of novel modified nucleosides using the transglycosylation reaction. for the first time, it has been demonstrated that the corresponding nucleobases are good substrates for the genetically engineered recombinant e. coli purine nucleoside phosphorylase (conversion to nucleosides reached - %). nucleosides, such as ribosides, -deoxyribosides, and arabinosides were obtained in high yields ( - %). it has been found that yield in the transglycosylation reaction does not depend on the structure of the amino acid fragment. the nucleosides synthesized are considered as potential inhibitors of intracellular adenosine deaminase (ad), the increasing activity of which is observed in hepatitis, cirrhosis, hemochromatosis, obstructive jaundice, prostate and bladder cancer, hemolytic anemia, rheumatic and typhoid fever, gout, and cooley's anemia. cytotoxicity of the synthesized nucleosides was tested in the jurkat (model of human t-lymphoblastic leukemia) and el- (model of mice t-lymphoblastic leukemia) cell lines. the compounds studied did not exhibit cytotoxic activity compared to the activity of the known antitumor agent nelarabin. the work was financially supported by the russian science foundation (grant - - ). p- . . - dna binding, dna cleavage, antimicrobial activities, antimutagenic and anticancer studies of a schiff base and its complexes n. yildirim , n. demir , m. yildiz health services vocational school, c ß anakkale onsekiz mart university, c ß anakkale, turkey, department of biology, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, department of chemistry, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. the rational design and synthesis of new schiff bases and their metal complexes have been drawing great interest because of their diverse biological and pharmaceutical activities. so, exploring and designing novel molecules that have biological activities and capable of interacting with nucleic acids has a great significance for disease defence and to discover new dna-targeted anticancer drugs for chemotherapy. in this study, we report the synthesis and characterization of a novel schiff base and its ni(ii) and cu(ii) complexes. the minimal inhibitory concentration (mic) of the compounds was screened in vitro against bacteria and yeast cultures using broth micro dilution test. dna binding and dna cleavage activity of the compounds were investigated by uv-vis spectroscopy and agarose gel electrophoresis. antimutagenic activity of compounds were tested in the absence of microsomal enzymes (s -). also, cytotoxicity of the compounds against hepg cell lines was assayed by the mtt ( -( , -dimethylthyazolyl- )- , -diphenyltetrazolium bromide) method. consequently, uv-vis spectroscopy studies indicated that the compounds interact with calf thymus dna (ct-dna) via intercalative binding mode. dna cleavage activity studies showed that the cu(ii) complex can effectively cleave pbr plasmid dna. compounds inhibited the base pair mutation with high inhibition rate in the absence of s . also, schiff base complex had cytotoxic activity towards hepg cell line, that it was found to be more potent than the control cisplatin. p- . . - single particle electron tomography of rnap elongation complex, stalled at position + genome in vivo is constantly exposed to the damaging effects of the environment. single-strand breaks (ssbs) are the most frequently occurring dna lesions. accumulation of unrepaired ssbs can interfere with the cells metabolism and increase genomic instability. in vivo, ssbs are repaired in specific pathway, but, in eukaryotic nuclei, dna is organized in chromatin that could affect the accessibility of lesions to sensor proteins. breaks in a template strand induce arrest of rna polymerase ii (polii) in vitro and in vivo and can be revealed in a transcription-dependent manner. our recent biochemical studies identified two key intermediates formed during transcription through a nucleosome by rnap that are nearly homogeneous, active and stable by biochemical criteria (complexes stalled after entering or bp into the nucleosome; ec+ or ec+ , respectively). hear we produced two complexes, both stalled in the + position, one without break in the dna, and the other with introduced ssb at position + of a non-template dna strand. complexes were purified using affinity chromatography and applied to a carbon-coated, glow-discharged em grid. tomographic studies were performed at ae °in a jeol microscope at kv accelerated voltage. images were recorded using a gatan ccd camera. image analysis was performed using the imod software. the resulting structure of the ec+ complex with no break in dna consist of two domains, connected by a single dna string. the complex with a break introduced into the dna has a more compact appearance and its two domains were connected by two dna strings, thus forming an intranucleosomal dna loop. our data suggest that ssbs in a non-template strand can induce the formation of stable non-productive transcription intermediate. the inhibitory effect of ssbs onto transcription may suggest a possible mechanism for their recognition in vivo with a transcription-dependent pathway. this work has been supported by the rsf grant # - - . colorectal cancer (crc) is one of the leading causes of cancerrelated deaths in the developed countries. according to who report new incidence rate of crc in turkey is . % among other cancer types. owing to difficulty of the low allele frequency variations detection, genetic association profiles of crc have not been entirely identified. low allele frequency variations mlh À g>a (rs ) promotor substitution, mlh g>c (rs ) exonic substitution, mthfr c t (rs ) and apc t>a (rs ) were investigated in this study. these snps "rs , rs , rs , rs " are located on p . , q , p respectively. colonoscopic investigations were performed on both cancer and control group. the snps were genotyped using kompetitive allele specific pcr technology in cases and healthy controls. statistical analysis was carried out with cochran-armitage chi-square test. in this study these of the snps in mlh , mthfr genes were examined for the first time in turkish sporadic crc cases. statistical analysis showed no significant association within our turkish sporadic crc population. percentage of mlh À aa genotype in group aged ≥ was found to be . % in cancer versus % in control group. moreover apc a, mlh c alleles were detected only and allele respectively. previously, apc a allele was determined in . % of a turkish cohort. however in the present study apc a allele was detected on allele only. studies showed mlh À promoter variation as a risk factor for microsatellite instabile crc but for the current study this data is not available. in spite of literature mthfr c t and mlh g>c snps were not found to be associated with sporadic crc in turkish population. this research demonstrates that importance of population based studies in multifactorial disease. p- . . - excision of damaged bases from transcription intermediates by fpg/nei superfamily dna glycosylases k. makasheva, d. zharkov sb ras institute of chemical biology and fundamental medicine, novosibirsk, russia oxidative lesions are abundant due to constant presence of reactive oxygen species in living cells. repair of oxidative base lesions is initiated by dna glycosylases. for example, bacterial fpg and nei dna glycosylases excise oxidized purines and pyrimidines, respectively, from dna. their human homologs, neil and neil , have been reported to show preference towards oxidized lesions in dna bubbles. from these observations, it had been hypothesized that neil proteins may be involved in the repair of lesions in dna bubbles generated during transcription. however, it is not presently clear how neils would behave on bubbles more closely resembling transcription intermediates (e. g., containing the rna strand), and bacterial homologs fpg and nei had never been investigated with bubble substrates. we have studied excision of either -oxoguanine ( -oxog) or , -dihydrouracil (dhu) by e. coli fpg and nei and human neil and neil from single-strand oligonucleotides, perfect duplexes, bubbles with different number of unpaired bases ( to ), d-loops with dna or rna and from complexes with rna polymerase. fpg, neil and neil efficiently excised dhu located inside a bubble. fpg and neil was generally more active than neil in excision of -oxog from ssdna and bubbles. nei, on the other hand, was active only on dhu located in dsdna (either perfect duplex or dna/dna d-loop). fpg and neil also have shown activity in d-loops with rna. the presence of an additional unpaired -tail of the third strand of d-loops didn't affect the glycosylases activity. the activity of fpg was observed in pre-assembled transcriptional complexes with e. coli rna polymerase and depended on the position of the lesion in the transcription bubble, possibly reflecting local accessibility of the lesion within the elongation complex. this work was supported by rsf ( - - ). nucleotide excision repair (ner) is a multistep process that eliminates a wide range of lesions in dna, including uv photoproducts and base modifications by many carcinogenic and chemotherapeutic agents. one of the advanced approaches to ner process investigation is based on reproducing the repair reaction by mixing protein extracts from mammalian cells with model linear dnas, bearing lesions. long linear dnas ( bp) containing efficiently recognized and processed by ner system lesions (fluoro-azidobenzoyl photoactive lesion fab-dc, nonnucleoside lesions nflu and nant) in both strands have been synthesized. we have demonstrated that dnas containing closely positioned lesions in the both strands represent difficult-to-repair (fab-dc/nflu(+ ), fab-dc/nflu(À )) or unrepairable (nflu/nflu (+ ), nflu/nflu(À ), nant/nflu(+ ), nant/nflu(À )) structures. besides, it has been shown that model dnas bearing bulky lesions in opposite positions (fab-dc/nflu( ), nflu/nflu( )) represent unrepairable structure as well. the model substrates with increasing distance between lesions in the duplex demonstrated the full recovery of substrate properties in ner process (fab-dc/nflu(+ ), fab-dc/nflu(À ), fab-dc/nflu(À ), nflu/nflu (+ ), and nant/nflu(+ )), whereas the level of specific excision from nflu/nflu(À ), nflu/nflu(À ) and nant/nflu(À ), nant/nflu(À ) was approximately % of the nflu/dg or nant/dg dna respectively. it has been shown that modified dna-duplex ( bp) with fab-dc has decreased structurally dependent affinity for xpc-hr b compared to duplexes containing lesions in both strands being analyzed (fab-dc/dg, fab-dc/nflu(+ ), fab-dc/nflu (À ), fab-dc/nflu(+ ), fab-dc/nflu(À ), fab-dc/nflu(- )) and increased compared to umdna. the data provide an argument that the ner system of higher eukaryotes recognizes and eliminates injured dna fragments on a multi-criteria basis. it is well known that dna plays crucial role in the biological system because of including all the genetic information for cellular function. therefore, the interaction of molecules with dna has gained interest in the medicinal chemistry to explore new anticancer agent. photodynamic therapy which is alternative cancer treatment method depends on free radicals and singlet oxygen to destroy tumor tissue via necrosis and apoptosis. phthalocyanines (pcs) are used for photodynamic therapy because of their absorption of high wavelength light ability and they have high triplet quantum state yields and long lifetimes in triplet states. also they do not have any toxic effect without light. in this study the novel synthesized - [ -( morpholin- -ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanines were used. the potential properties of phthalocyanine compounds for photodynamic therapy were purposed to reveal by the preliminary work. for this aim, the mode of dna binding, photocleavage and topoisomerase i inhibition of these compounds were investigated. - [ -( -morpholin- -ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanine compounds have been synthesized. the interaction of novel pcs compounds with calf thymus (ct) dna was investigated by using uv-vis spectroscopy, thermal denaturation studies and viscosity measurements. additionally, dna photocleavage and topoisomerase i inhibition studies were performed to pbr dna by using agarose gel electrophoresis. the interaction studies indicated that pcs compounds powerfully bound via an intercalation mechanism with ct-dna. these compounds showed efficiently dna photocleavage under irradiation at nm. the all of pcs inhibited topoisomerase i in a dose-dependent manner. all the experimental studies showed that pc compounds might be used agents for photodynamic therapy. p- . . - target search by base excision repair dna glycosylases e. dyatlova, g. mechetin, d. zharkov institute of chemical biology and fundamental medicine, novosibirsk, russia the problem of rapid target search in dna is faced by transcription factors, restriction endonucleases, dna repair enzymes and other sequence-or structure-specific dna-binding proteins. theoretically, the fastest target search in dna can be achieved by combining one-dimensional diffusion along the dna contour (processive search) and three-dimensional diffusion (distributive search). the balance between these search modes depends on many factors affecting dna-protein interactions, such as the presence of mono-and divalent cations, competing proteins, crowding effect, etc. presently, the mechanisms of target search are understood only for a handful of enzymes. we have recently developed an assay to study target search by dna repair enzymes, based on cleavage of oligonucleotide substrate containing two targets. thus, the distance between the targets can be precisely controlled, and any modification can be introduced into dna. subsequently, the probability of correlated cleavage (p cc ) is estimated, reflecting the efficiency of enzyme transfer between the specific sites. in this work, we have investigated five repair enzymes: e. coli endonuclease viii (nei), its human homologs neil and neil , and uracil-dna-glycosylases (ung) from e. coli and vaccinia virus. as expected, p cc of all enzymes depended on the ionic strength of the solution and the presence of mg + . ung from vaccinia virus was the most sensitive to these factors, raising questions about its proficiency as a suggested processivity factor of viral dna polymerase. nei, neil and neil showed a peak of p cc at low but non-zero ionic strength indicating that nonpolar interactions contribute to binding of these proteins to nonspecific dna. this conclusion was also supported by analyzing amino acid conservation in the catalytic core of nei. introduction of bulky fluorescent group between two specific sites greatly reduced the ability of glycosylases to slide along dna. this work was supported by rsf ( - - ). p- . . - does causes mhz magnetic field application kras and p mutations in colon?: occurences histopatologically and microbiologically changes in colon determination of kirsten rat sarcoma (kras) and p gene mutations in colon. materials and methods: in this study, three groups were prepared as control,sham and electromagnetic field (emf) group. mhz radiofrequency (rf) radiation was produced by using an electromagnetic energy generator.the emf group rats were exposed to electromagnetic field for weeks as minutes per day.at the end of experiments, rats were sacrificed under ethyl ether anesthesia and the rat colons were dissected.fecal speciments were collected.fecal dna (for detection of fusobacterium and bacteroides) and colonic dna (for detection of kras and p mutations) were isolated.rt-pcr tchnique was used for detection of bacterias and mutations. results: no any differences was observed histopathologically between control and sham groups.erosions and partial losses were observed at mucosal epitelium in the emf group.the corrupted gland structure, the mucosal edema and the inflammatory cell infiltration were observed.the amout of collagen was increased and fibrosis was detected in emf group.goblet cell number decreased statistically significant when compared to control and sham groups (p < . ).the amount of fusobacterium increased significantly in emf group compared to controls.the difference was not detected between groups in the amount of bacteroides.all the samples analysed for kras and tp mutations in the colon tissue were found to be wild type.no significant difference was observed between the control group and the emf applied group. discussion and conclusion: in conclusions,for weeks minute/day exposure to mhz emf caused histopatological damage in rat colon.the amount of fusobacterium is increased.emf exposure did not caused to kras and p mutations in colon tissue. p- . . - synthesis, antimicrobial activity, genotoxicity, dna binding and dna cleavage studies of new glycine methyl ester derivative schiff base there has been an increasing focus on the binding study of small molecules to dna during the last decades, since dna is an important genetic substance in organisms. therefore, the current growing interest in small molecules that are capable of binding and cleaving dna is related to their utility in the design and development of synthetic restriction enzymes, new drugs, dna agents, and also to their ability to probe the structure of dna itself. in recent years, schiff bases have found increased application in pharmaceutical research, organic synthesis, and bio-processes. schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. in this study, we report the synthesis and characterization of a novel glycine methyl ester derivative schiff base. the minimal inhibitory concentration (mic) of the compound was screened in vitro against bacteria and yeast cultures using broth micro dilution tests. antimutagenic activity of compound was tested in the absence of metabolic activation. also, dna binding and dna cleavage were investigated of compound by uv-vis spectroscopy and agarose gel electrophoresis respectively. consequently, this compound differs significantly in its activity against tested microorganisms. this difference may be attributed to the fact that the cell wall in gram-positive bacteria is a single layer, whereas the gram-negative bacteria cell wall is a multilayered structure, and the yeast cell wall is quite complex. the compound inhibited the base pair mutation in the absence of s with high inhibition rate. uv-vis spectroscopy studies of the interactions between the compound and calf thymus dna (ct-dna) showed that the compound interacts with dna via intercalative binding. to date a large number of the sequences in the human genome (g motifs) with the potential to form a spatial structure, gquadruplexes is known. g motifs were found in the promoter regions of most of the known oncogenes. recent experimental studies have shown that genome instability directly related to the non-canonical dna structures, including g-quadruplexes. in this work we study the distribution of somatic snvs within the g motifs in tumor samples with the aim to identify involvement of the motifs in the process of mutagenesis in pancreatic cancer. using the access kindly provided by the international icgc consortium to the database, we analyzed samples of pancreatic ductal adenocarcinoma and samples of pancreatic endocrine neoplasms. we considered only the promoter regions as the richest with g-quadruplex motifs. we found that quadruplex sequences have the ability to focus somatic snvs. this could be explained by the errors of polymerase during replication through secondary dna structures. furthermore, the snvs occur much more often in loops of g motifs than in g blocks, without changing the motive. in addition, t>g(a>c) and t>c(a>g) substitutions occur significantly more likely in loops which in turn stabilize the g-quadruplex structure. the cancer-related mutations tend to increasing the length of g blocks. the conservation of g motifs may indicate an important functional significance of g-quadruplex structures in human genome. supported by project no. - - of the russian science foundation. background: multiple myeloma (mm) is a rare, leading to bone destruction and marrow failure, largely incurable malignant disease of plasma cells. anemia (mostly normocytic normochromic) is seen in most patients. mean platelet volume (mpv) is a laboratory marker of platelet function and activity, the most accurate measure of platelet size. the aim of this study was to investigate the mean platelet volume (mpv) values in this disease. materials and methods: whole blood samples were collected from healthy controls and patients with mm. the mean age for controls and patients were . ae . and . ae . years, respectively. mpv levels were calculated with cancer is a chronic disease in the world which is the second leading cause of death, after cardiovascular diseases. benzimidazoles have been known to act as antiproliferative or anticancer agents in chemotherapeutic drug research area. in this regard we aimed to investigate the cytotoxic and apoptotic properties of novel benzimidazole derivatives bearing pyridyl/pyrimidinyl piperazine moiety against a lung adenocarcinoma cells. a lung adenocarcinoma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by mtt assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic values of the compounds were determined for a cell line. compounds , and which were including -chlorophenyl, -nitrophenyl on pyridine ring; -fluorophenyl on pyrimidine moiety, had significant cytotoxic activity with ic values lower than . ae . lg/ml. compound showed the highest cytotoxic activity with a ic value of . ae . lg/ml, whereas cisplatin ic values were . ae . lg/ml lg/ml against a cells. cytotoxic activity of compound and with a ic value were . ae . and . ae . lg/ml, respectively. also, compound showed the highest population of early apoptotic cells ( . %) of the tested compounds which was . -fold higher than for cisplatin. compound produced a comparable population of apoptotic cells with a percentage of . %, respectively according to cisplatin's percentage of . %. it was determined that synthesized compounds , and had considerable anticancer activity against a cell lines compared to cisplatin. compound including -florophenyl on pyrimidine ring was the most cytotoxic compound against the a cell line. our study results demonstrated that compound , also induced apopototic pathway on a cells. p- . . in vitro/in vivo antimitotic activity and structure-activity relationships of new glaziovianin a isoflavone series glaziovianin a (gva), isolated from the leaves of the astelia glazioviana, demonstrated cytotoxicity, disrupting microtubule structure and dynamics of hl- cells. the aim of the present work was to devise a concise synthetic route toward gva and its derivatives in order to expand structure-activity relationship studies and to investigate their anti-mitotic effect. a concise six-step protocol for the synthesis of gva and its alkoxyphenyl derivatives starting with readily available plant metabolites from dill and parsley seeds was developed. the sea urchin embryo tests confirmed that gva directly affects tubulin/ microtubule dynamics and structure. the b-ring substitution pattern of gva derivatives exhibited strong effects on activity. according to the assay results, the anti-mitotic activity decreased in the following order: gva > myristicin ≥ , , -trimethoxyphenyl = -methoxyphenyl > dillapiol > -methoxyphenyl> , dimethoxyphenyl > , , , -tetramethoxyphenyl derivatives. a methylenedioxy moiety was essential for the activity of compounds substituted with four b-ring alkoxy groups. the mts assay of the limited panel of cancer cell lines shows that gva displayed the highest inhibitory activity, with ic values ranging from . (a cells) to . lm (mda-mb- cells). compounds, containing , , -trimethoxy and apiol-derived b-rings, respectively, were less active. other isoflavones did not affect cancer cell growth up to lm. anti-proliferative effects of isoflavones observed in both the sea urchin embryo model and human cancer cell lines correlated well. importantly, none of the synthesized isoflavones demonstrated cytotoxicity in human pbmcs, up to lm. in summary, gva and its analogues were synthesized via a scalable six-step reaction sequence. the gva and its analogues containing , , -trimethoxy and apiol-derived b-rings were found to be promising anti-mitotic microtubule destabilizing agents with low toxicity against human pbmcs. bag- is a multifunctional protein which has interactions with a number of cellular proteins; nuclear hormone receptors, bcl- , hsp /hsc family, growth hormone receptors, raf- , ubiquitin machinery and dna to regulate cell survival. for this reason, bag- is a critical molecular player in the regulation of cell survival signaling and apoptosis mechanism. elevated expression levels of bag- are associated with progression of cancer. in the treatment of breast cancer, silencing tools as a promising combined therapy strategies in the presence of classical chemotherapeutics gain importance to investigate interaction networks of cell death and survival signaling pathways. therefore, we aim to understand potential role of bag- silencing in the treatment of breast cancer cells with apoptotic agents; cisplatin or paclitaxel. our results showed that, silencing of bag- enhanced cisplatin or paclitaxel-induced apoptosis in mcf- cells by down-regulating antiapoptotic and upregulating proapoptotic bcl- family proteins, changes on cell cycle, upregulation on subg phase, activating caspases and cleavage of parp. in addition, knockdown of antiapoptotic bag- has a suppressive role in pi k and akt signaling pathway in mcf- breast cancer cells through inhibition of akt phosphorylation and downregulation on pi k. investigation targets of akt pathway showed that mtor cell survival pathway also affected through bag- silencing. bag- silencing inhibited mtor signaling via downregulating both rictor and raptor proteins which are the members of rapamycininsensitive mtorc and rapamycin-sensitive mtorc complexes, respectively. knockdown strategies of bag- is important to enlighten the network interactions of bag- and clarify its interaction partners in the cells. therefore utilization of bag- targeted strategies might further increase therapeutic efficiency of drugs through inhibiting cell survival machinery in the treatment of metastatic breast cancer. p- . . - biological activity evaluation of new , , trisubstituted triazine derivatives bearing different heterocyclic rings against lung cancer cell lines l. yurttas, g. akalin c ß iftc ßi, h. e. temel, b. demir anadolu university, eskisehir, turkey cancer is one of the major death causing disease worldwide. among the various cell types occurs on different organs, lung cancer is one leading cause of cancer death accounting for approximately % of all female and % of all male cancer deaths in . the resistance development, cytotoxicity and inadequacy are the main encountered problems by the treatment with existing chemotherapeutic agents. therefore, there is continuous need to discover new active and non-toxic molecules. -[ -( , -bis( -substituted phenyl)- , , -triazin- -yl)piperazin- -yl]- -[benzimidazole/benzoxazole/benzothiazole- -yl)thio]ethanone ( - ) derivatives were synthesized with a four-step synthetic procedure using toluil, anisil and -chlorobenzil as starting materials. the anticancer activity of the compounds was evaluated using the methods mtt ( -( , -dimethylthiazol- -yl )- , -diphenyltetrazolium bromide), brdu (bromodeoxyuridine) assays and flow cytometric analysis against lung cancer cell lines. the lipoxygenase enzyme inhibition activity of the compounds were also investigated using the method described by baylac and racine. compounds was found to have (inhibition concentration) ic values between - lg/ml. the early and late apoptotic cell percentage was determined as . for compound by flow cytometric analysis. the lox inhibition activity was found . ae . for compound . compound bearing -chlorobenzil and benzoxazole moieties was found as the most active compound when we evaluate anticancer potential of all compounds. the lox enzyme inhibition was indicated for the compound including methyl substituent on phenyl rings. the dna synthesis inhibition of the compounds has been still studied at the concentrations ic / , ic and ic x . p- . . - single amino acid substitutions and deletions modulate the drp-lyase activity of human dna polymerase iota n. miropolskaya, i. petushkov, a. kulbachinskiy, a. makarova institute of molecular genetics, moscow, russia dna polymerase iota (pol ι) is a y-family dna polymerase that possesses an unusual combination of properties. due to the special organization of the active site pol ι has a very low accuracy of dna synthesis but possesses an ability to bypass a variety of dna lesions. in addition to the dna polymerization activity, human pol ι also possesses an intrinsic -deoxyribose phosphate (drp)-lyase activity. removal of the drp group is a pivotal step in base excision repair (ber) in vivo. although pol b plays a key role in the drp group cleavage and dna synthesis during ber, pol ι was shown to complement the in vitro single-nucleotide ber deficiency of pol b null cell extracts and was suggested to be involved in ber under oxidative stress. the drp-lyase active site in pol ι is still not known. to address the mechanism of the drp-lyase activity of pol ι we obtained a series of pol ι mutant variants including point mutations of conserved lysine residues and deletions in different locations. we purified human pol ι variants from yeast saccharomyces cerevisiae and tested the effect of mutations on the cleavage of an internal -drp group in oligonucleotide dna substrates in the presence or absence for me + ions. the experiments revealed several point amino acids substitutions that significantly affected the drp-lyase activity of pol ι, thus suggesting a possible location of the drp-lyase active site. furthermore, we showed that deletions in the n-terminus of pol ι and metal ions modulate its drp-lyase activity, which may play an important role in the regulation of pol ι activities in vivo. this work was supported by russian foundation for basic research grants - - -a and - - -mol-a-mos and by the russian academy of sciences presidium program in molecular and cellular biology. rosmarinus officinalis, commonly known as rosemary, is an aromatic plant belongs to lamiaceae family. from past to now, rosemary have been used as a traditional medicine to cure for various illnesses such as diabetes, rheumatism and cancer. recent studies have shown that rosemary is effective for various cancer types. in this study we aimed to investigate the effect of rosemary in glioblastoma cells (gbm) by comparison with etoposide and the effect of rosemary by concurrent application with the etoposide. gbm cells (u mg) were seeded into the well plates and cultured with dmem supplemented with % fetal bovine serum. rosmarinus officinalis tea was prepared just as traditional usage and filter sterilized. at the second day of the culture rosemary in / (v/v) dilution ratio was given to first group, lm etoposide was given to second group, / (v/v) diluted rosemary and lm etoposide together were given to third group. after one day incubation cell viability was measured by neutral red assay. it was observed that rosemary reduced the viability of gbm cells by nearly % , etoposide reduced the viability by nearly % and rosemary with the etoposide reduced the viability by nearly % . the results showed that rosemary was able to reduce the viability of gbm cells but hadn't got an increasing or inhibiting potential over the etoposide's cytotoxic effect. from our previous studies we know that rosemary increases the proliferation of mouse embryonic fibroblasts. it is considered that rosemary might have a protection potential from dna damages and when rosemary is used with etoposide during the cancer treatment, it might reduce the side effects on healthy cells. in conclusion rosemary promises hope for developing new cancer treatment strategies and reducing the side effects of chemotherapeutics. for further studies it is aimed to examine the effects of rosemary with other chemotherapeutics and if rosemary has got a protection potential from the genotoxic stress. morpholine moiety has been found to be an excellent pharmacophore in medicinal chemistry and a number of molecules possessing morpholine skeleton are the clinically approved drugs. in this present study, we aimed to investigate the possible underlying apoptotic mechanism for the cytotoxicity of new morpholine dithiocarbamate derivatives bearing -( -aryl- -oxoethyl)- -substituted benzimidazole moiety on c glioma. c glioma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by cell proliferation analysis using standard ( -( , -dimethylthiazol- -yl)- , diphenyltetrazolium bromide (mtt) assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic values of the compounds were determined for c cell line. compounds , , , , and , which were including hydrogen, -methyl, -methoxy, -chloro and -floro substituents on phenyl acetyl moiety, had significant cytotoxic activity with ic values lower than lg/ml. compound showed the highest cytotoxic activity with a ic value of lg/ml, whereas cisplatin ic values were lg/ml against c cells. cytotoxic activity of compound , , , and with a ic value were , , and lg/ml, respectively. compound , and showed the highest population of early apoptotic cells as . , . , and . % respectively compared to cisplatin ( . %). also, compounds caused dna synthesis inhibition depend on their ic values by brdu assay. conclusions: it was concluded that synthesized compounds had considerable anticancer activity against c cell lines. however, compound , and including -methyl, -chloro and -floro substituents were the most active compounds against the c cell line. also our study results showed that compound , , induced apoptosis in c glioma cells. rutin is a glycosided flavonoid and known to have antioxidant and anti-inflammatory properties.trail induces the apoptosis of tumor cells and has no significant toxic effect on normal cells. although trail is a promising anticancer agent, trail resistance is a major barrier to effective cancer therapy. this study was conducted to examine the utility of the combined use of rutin and trail in prostate cancer cells. pc- and du prostate cancer cells were treated with rutin ( - um) and/or trail ( ng/ml), cell viability and migration were examined. cell viability was determined by trypan blue exclusion and mtt assay. cell migration was determined by wound healing assay. furthermore, lactate dehydrogenases (ldh) levels of medium were determined as biochemical markers of cell viability. pc- and du- prostate cancer cells were treated with rutin for and hours incubation and ic doses for hours incubation were determined um and um respectively. treatment with rutin, pc- cells is more sensitive than du cells. rutin and rutin plus trail inhibit prostate cancer cell growth in a dose-dependent manner. treatment with trail has no effect at inhibiting growth of pc- and du prostate cancer cells. the combination of rutin and trail elicit a synergistic antitumor effect on pc- and du prostate cancer cells. there is a significant increased in rutin and rutin+trail treatments group of ldh activities with respect to control and trail group. conclusion: present data show that rutin efficiently enhanced trail effects in prostate cancer cells. combined treatment with rutin and trail is more effective than the individual treatments of trail at inhibiting growth of prostate cancer cells. p- . . - determination of antigenotoxic, proliferative and cytotoxic properties of ellagic acid since ancient time, people use plant for traditional treatment. plants or fruits are produced different type of secondary metabolites. particularly phenolic phytochemicals from plants play an important role in the prevention and treatment of radical damage by inactivating the reactive oxygen compounds due to their antioxidant properties. however, the structure and the activities of many herbal products are not fully elucidated yet and there are several studies about the toxicity of herbal antioxidants and their possible risks to human health. ellagic acid, phenolic compounds, is an important substance. ellagic acid is a naturally occurring plant phenol found in numerous fruits, including blackberries, raspberries, strawberries, cranberries, walnuts, pecans, pomegranates and wolfberries. different researchers give some information about the biological activities of ellagic acid. in this study, we aimed to determine the cytotoxic, proliferative and antigenotoxic effects of ellagic acid, which is phenolic compounds found in natural products. cytotoxic effects of ellagic acid on huvec is investigated by lactate dehydrogenase (ldh) and cell proliferation (wst- ) methods; and antigenotoxic effects against ccl on human lymphocytes is investigated by single cell gel electrophoresis (comet) methods. the rusults showed that high concentration ( and lm) of ellagic acid has cytotoxic and mutagenic effects, but showed antiproliferative effects. on the contrary, low concentrations ( , , . lm) of ellagic acid has anticytotoxic and antimutagenic effects. as a conclusion, low concentrations of ellagic acid might be use treatment of some disease. but high concentrations of ellagic acid constitute a risk factors for people. keywords: cytotoxicity, antiproliferation, wst- , ldh, rtca-sp the constitutive nuclear factor kappa b (nf-kb) activation is widely found in diverse types of hematologic malignancies such as acute myeloid leukemia (aml) and chronic myeloid leukemia (cml) as well as solid tumors. inhibition of nf-kb signaling via proteasome inhibitors such as bortezomib can induce apoptosis in myeloid leukemia cell lines. however it is not clear whether the cytotoxic effects of bortezomib on myeloid leukemia cell lines is due to direct inhibition of nf-kb or another pathway, such as dna damage. in this study, cml cell line k and aml cell line hl- were treated with bortezomib (bor) , etoposide (eto) and camptothecin (cpt) alone or in dual combination with these drugs, following by measuring the effects on cell viability, apoptosis and signal pathways. the effect on cell viability was determined using the mtt assay. the data were used in combination index and isobologram analysis. the expression levels of apoptototic genes (bcl , bax and caspase ), the related dna damage genes (atm and atr) and the involved genes in nf-kb signaling (rela and p ) were determined by real time rt-pcr. we showed that combinations of bor with topoisomerase inhibitors (cpt and eto) exhibited synergistic cytotoxic effect in k cell line but not in hl- cell line. the combination treatment increased apoptosis and dna damage response. dnadamage-sensing kinases were detected in k and hl- cells following treatment with bor as similar as topoisomerase inhibitors. bor increased the mrna levels of atm and atr dramatically, which indicated active dna damage in the myeloid cell lines. furthermore, bor induced apoptotic cell death by decreasing bcl and increasing bax and caspase levels. these effects of bor were observed to correlated with increasing the p expression levels. this study on the mechanism of action of bor indicates that this compound affects several pathways involved in the control of cell cycle progression, apoptosis and dna damage. p- . . - analysis of molecular cytogenetic alterations in gastric and colon carcinoma by array-based comparative genomic hybridization (array cgh) introduction: genomic dna regions are frequently lost or gained during tumor progression. we aimed to evaluate tumor samples of patients with gastric cancer and colorectal carcinoma to show these genetic alterations by array-based comparative genomic hybridization (array cgh) method. materials and methods: dna isolation was performed from the tumor samples obtained from sixteen patients with primary gastric adenocarcinoma and twelve patients with colon adenocarcinoma. then, agarose gel electrophoresis was performed in those dna samples. following electrophoresis of dna, array cgh procedure was performed to four patients with gastric adenocarcinoma and three patients with colon adenocarcinoma who had dna breaks with - kb. results: after array-cgh study, many common genetic changes in gastric and colon cancer genome were determined. in gastric cancer dna samples, common losses were detected in chromosome p . , p . , q , q , p . , q . , q . , q . , q . , p , q . , q . , q . , q . , q . , q . , and q . , and also common gains were detected in chromosome p . , q , q . , q . and xq . in colon cancer dna samples, common losses were detected in chromosome p . , q , p . , p . , q . , q . , q . , q . , p . , p . , q . , and q . , and also common gains were detected.in chromosome q . , xp . , xp . , xp . , xp . and xq . both in gastric and colon cancer dna samples, common losses were detected in chromosome q . , q . , and p . , and common gains were detected in xq . discussion and conclusion: we think that these common changes, generally in dna loss areas harboring tumor suppressor genes and dna gain areas harboring oncogenes, may important in gastrointestinal tumorigenesis. the dna of every cell is under a constant attack by various mutagenic factors which damage the dna and can cause cell cycle arrest and even cell death. accumulation of dna damage is the basis for cancer development and one of the reasons for aging of the organisms. in order to preserve the integrity of its dna cells have evolved an impressive array of dna repair pathways, which are precisely coordinated with the progression of the cell cycle. one of the first events at the site of dna damage is poly(adp-ribose) polymerase (parp ) recruitment which is a sensor for single strand breaks in dna. parp catalyzes the synthesis of poly(adp-ribose) or par which is needed for the recruitment of many other dna repair proteins by means of par-binding domains. we used high speed confocal spinning-disk microscopy of living cells to obtain precise kinetics of recruitment of par-dependent proteins to the sites of laser induced dna damage. our results show that the investigated par-dependent proteins are recruited to dna damage sites in the matter of seconds, they reach peak intensities for to seconds after damage infliction and start dissociating. the recruitment of the proteins is entirely dependent on par because addition of parp inhibitor abbrogated their recruitment. the use of spinning-disk microscopy of living cells allowed us to obtain the kinetics of recruitment of the studied proteins to the sites of dna damage. the results are consistent with the fact that parp and par-dependent proteins are quickly recruited to damage sites and generation of par is essential for other dna repair protein recruitment. the precise kinetic curves may serve as a basis for investigating how they will change or if they will change at all when cells are put in different conditions or treated with various chemical substances affecting dna metabolism and repair. introduction: chronic myeloid leukemia (cml) is a myeloproliferative disease associated with reciprocal translocation between chromosomes and . bcr-abl fusion gene which exhibits constitutively active tyrosine kinase activity has a main role in cml. the tyrosine kinase inhibitor imatinib is used as a first line treatment in cml patients, but imatinib resistance leads to failure in therapy. the application of imatinib in combination with other anticancer agents may be a strategy to increase the antileukemic effect of imatinib. in this study, we have investigated the antiproliferative effect two novel agents: a benzamide derivative xt and a benzoxazole derivative xt b in combination with imatinib. these molecules were investigated in imatinib-sensitive (k s) and imatinib-resistant (k r) cml cell lines. materials and methods: antiproliferative and apoptotic effects were assessed by mtt assays and flow-cytometry, respectively. we also evaluated the effects of these compounds on the expression of apoptosis-related genes bax, bcl- , bad, bim, bcl-xl and mcl by real-time quantitative pcr. results: treatment of k cells with xt increased the expression levels of the pro-apoptotic genes bax, bad and bim in both sensitive and resistant cells. however, xt b was not found to have similar effects on k r and k s cells. combined application of xt increased cell death in the mtt assay. mtt assay demonstrated that ic for xt treated cells in k r with imatinib (ic = . ) is lower than k r without imatinib (ic = . ). discussion and conclusion: our results showed that combining xt with imatinib has more antiproliferative and apoptotic effect on a cml cell line. as a result combination of xt with imatinib can be an alternative approach to overcome imatinib resistance. introduction: the mmr(mismatche repair) system recognizes base-base mismatches and insertion or deletion loops in doublestranded dna, and it degrades the error-containing region of the newly synthesized strand, allowing the polymerase to correctly resynthesize the second strand according to the template sequence. the human mmr system includes the mlh and msh . alteration in expression or a defect in mlh or msh can cause resistance to anti-cancer drugs used in chemotherapy. the attempt of the mmr system to detect drug induced dna damage, triggers the activation of apoptosis, a mechanism which may enhance the cytotoxicity of chemotherapy. loss of the mmr system would make the neoplastic cell less able to initiate apoptosis. inability to initiate apoptosis could be a mechanism of resistance to drugs. chronic myeloid leukemia (cml) is a clonal disease originating from aberrations in hematopoietic stem cell. imatinib, a tyrosine kinase inhibitor has significantly improved clinical outcome for cml patients. however, patients develop resistance when the disease progresses to the blast phase (bp) and there are several mechanisms involved in imatinib resistance. in this study we investigated the role of mmr system in imatinib resistance. materials and methods: k s (sensitive) and k r (resistance) were grown in rpmi- . k r cells were maintained in rpmi- medium supplemented with lm imatinib rna isolation, cdna synthesis, rt-pcr was performed respectively. results: the results demonstrated that expression of mlh in k r cells is dramatically lower than equal amount of imatinib treated k s cells, whereas msh expression level did not change in both cell lines. conclusion: it can be suggested that alteration and down-regulation of mlh genes leads to imatinib resistance. p- . . - characterization of interaction between rad inhibitor dids and human serum albumin d. velic, s. henry, c. charlier, m. popova, p. weigel, j. masson, i. nabiev, f. fleury cnrs/university of nantes, nantes, france -diisothiocyanostilbene- , -disulfonic acid (dids) has been largely used during the last years for its inhibitory effect on anion transporters and channels. more recently, ishida and colleagues have described a possible mechanism by which dids inhibits rad -mediated homologous pairing and strand exchange, key processes in dna repair by homologous recombination. thus, dids could act as a potential revertant of radioand chemo-resistance in cancer cells, which is the major cause of failure during therapeutic protocols. new drugs targeting rad protein have since been developed with potential use for medical applications. in this context, we attempted to determine the behaviour of dids towards blood and plasma proteins such as serum albumins. firstly, we analysed the effects of several environmental factors such as solvent polarity, which may affect the stability of the molecule. secondly, we analysed the spectroscopic properties of dids in the presence of human or bovine serum albumin proteins. uv-visible absorption, circular dichroism, fluorescence spectroscopy and isothermal calorimetry were used. here we show for the first time that dids can interact with both serum albumins. we have also determined the characteristics of these interactions. the comparison of several dids derivatives led us to identify the essential chemical moiety of this compound involved in the interaction. moreover, by using site competition approaches we show that the main binding site for this molecule is in subdomain ib of the protein. these findings show that the binding of dids to serum albumin proteins may change the equilibrium between the free and bound dids forms, thereby affecting its bioavailability and efficiency against the rad recombinase protein. p- . . - mechanism of tap beta-mdm autoregulation p is a transcription factor which is the member of a p family. it regulates many cellular processes, such as apoptosis, cell cycle, and senescence. in contrast to p , p is rarely mutated in tumors and elevated p expression is observed in many types of cancers including hepatocellular carcinoma, neuroblastoma, and lung. defining regulatory mechanisms which control p protein abundance and activity will be crucial for the development of new therapeutic strategies for cancers. mdm is known as the key player in regulation stability and activity of p . in addition, p induces mdm transcriptional activity, and caspase- , activations which cleave mdm n-terminal at asp . cleaved form of mdm binds p and promotes its stabilization. mdm suggested as a candidate to modulate p activity and stability too. however, an interaction between p and mdm has not defined well. in this study, we aimed to analyze the role of mdm in p stability. to define this relationship, firstly, we overexpressed the tap beta isoform using trex system in hep b. tap beta and mdm protein levels were determined by western blot. to examine whether mdm mediate tap beta protein degradation by the proteasomes, cells were treated with proteasome inhibitor, mg for hours prior to analysis. previous studies showed that p -induced caspase- and caspase- activation cleaves mdm . considering this, we firstly examined caspase- activation by western blot in hep b tap beta cells. then we analyzed expression of cleaved mdm and tap beta levels following caspase inhibitor, z-vad-fmk treatment. as a conclusion, tap beta-induced full-length mdm- expression. furthermore, tap beta enhanced cleavage of mdm via increased caspase- activation. in addition, inhibition of caspase- activation caused a decrease in cleaved-mdm levels in parallel with tap beta expression repression. our results suggested positive regulation between mdm -tap beta. hepatocellular carcinoma (hcc) is one of the most common type of liver cancer and third leading cause of cancer related deaths in worldwide. discovery of new targets is important in survival of hcc patients. p is a transcription factor which is the member of p family. it has two promoters; while p promoter expresses apoptotic ta isoforms, p promoter expresses anti-apoptotic dn isoforms. in addition, alternative splicing in c terminal creates many isoforms of ta and dn p . it has been shown that both tap and dnp isoforms are expressed in hcc patient tissue and cell lines. the ratio between tap and dnp affects the apoptotic response, drug response and prognosis. accordingly, identification of the role of p and its targets are important in discovery of new treatment strategies in hcc. to understand the role of p isoforms in hcc, firstly we performed mtt assays following dna-damaging drugs and multikinase inhibitor, sorafenib treatment to categorize hcc cell lines as resistant or sensitive. after that, we analyzed the expression levels of tap isoforms via western blot in all hcc cell lines. then we overexpressed the tap beta isoform using trex system in hep b and snu cells. these two clones were analyzed for dna damaging drug response by mtt, cell cycle and apoptosis by flow cytometry, and tumor formation by in vitro and in vivo experiments. in scope of our study; . only tap alpha isoform is expressed in a few hcc cell lines. . there is no correlation between basal expression of p isoforms and drug responses in hcc cell lines. . there is no change in expression of p isoforms after treatment of drugs. . we showed that the ectopic expression of tap beta in hep b arrested the cell cycle in g / s and decreased the colony formation. therefore, the capacity of tumor formation of the cells dramatically decreased in scid mice. as a result, we revealed that tap beta play role in tumor formation, cell cycle arrest, dna damage responses in hcc. p- . . - biochemical characterization of exonuclease iii-family ap endonuclease point mutants reveals role of conserved amino acid residues in the nir-specific enzymes a. mursalimov, z. koshenov, t. yeleussizov, m. redrejo-rodriguez, a. ishchenko, b. t. matkarimov, m. saparbaev national laboratory astana, astana, kazakhstan oxidative dna damage caused by reactive oxygen species is believed to be a major type of endogenous cellular damage. oxidatively damaged dna bases are substrates for two overlapping repair pathways: dna glycosylase-initiated base excision (ber) and apurinic/apyrimidinic (ap) endonuclease-initiated nucleotide incision repair (nir). in the ber pathway, an ap endonuclease cleaves dna at ap sites and -blocking moieties generated by dna glycosylases, whereas in the nir pathway, the same ap endonuclease incises dna to a number of oxidized bases. majority of characterized ap endonucleases possess classic ber activities and about half of them are able to catalyze nir activity. at present, the molecular basis of dna substrate specificities of various ap endonucleases remains unclear. here, we examined amino-acid sequence requirement of the nir activity of human major ap endonuclease (ape ). amino acid sequence alignment of various ap endonucleases including e coli exonuclease iii (xth), human ape and archaeal mth revealed conserved amino acid residues in the nir-specific ap endonucleases ape , mth and exoa that are absent in xth. based on these data, we constructed four ape point mutants y h, n q, g s and t d and examined their dna substrate specificities. results obtained from biochemical characterization of ape mutants are discussed in the light of the evolutionary conserved dna repair functions of ap endonucleases and whether these functions can be mutationally separated from. since its discovery some years ago, cisplatin has evolved for its efficacy in one of the most used drugs in treatment of various cancer types. huge effort was invested in understanding the action of cisplatin and development of more potent drugs. they target mainly neighboring purine bases of nuclear dna forming covalent intra-or inter-strand cross-links that affect inhibition of replication and transcription, cell cycle arrest, and attempted repair of the damaged nucleotides. if such damage cannot be removed the cell dies. we have studied the details of the binding site of the short oligonucleotide modified by a platinum compound using complementary solution techniques used in modern structural biology, including raman spectroscopy with dft calculations aided interpretation of the obtained vibrational spectra. moreover, the calculated structure of the dna duplex was verified using saxs (small angle x-ray scattering) curve. in our contribution, we will present an nmr structure of a dna cross-linked with a cisplatin derivative containing a cyclohexane ring. at this atomic level resolution, structural features probably influencing cytostatic effects are described and compared with previously published structures. common structural features of previously determined structures are: a significant roll ( - °) of the guanine bases involved in the cross-link, bending and unwinding of the double helix at the site of cross-link and orientation towards the major groove. also, the platinum-guanine plane angle varies between and °. although the experimental structures were often used as the starting models for molecular dynamics (md) simulations, results of these md still leave many questions unresolved. the results of this research have been acquired within ceitec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. p- . . - ercc /xpd polymorphisms and colorectal cancer risk: a case control study in a north eastern iranian population j. mehrzad islamic azad university, neyshabur, iran excision repair cross-complimentary group (ercc ) is one of the important dna repair genes.ercc codon and polymorphisms has been shown to modulate cancer risk. we therefore assessed the relationship between the ercc polymorphisms and the susceptibility to colorectal cancer in a case-control study. there were lung cancer cases and matched healthy controls in this study. information concerning demographic and risk factors was obtained, each person donated ml blood for biomarker testing. ercc genotypes were determined by t-arms-pcr method. all of the statistical analyses were performed with spss (v . ). there was significant difference between the frequencies of ercc polymorphism in cancer cases and controls (p < . ). the frequencies of ercc gln allele were . % in controls and . % in cancer cases. the individuals with lys/gln+gln/gln combined genotype were at an increased risk for lung cancer as compared with those carrying the lys/lys genotype (adjusted or= . , %=ci . À . ). the above findings indicate that the genetic polymorphism in the ercc codon is associated with the risk of colorectal cancer in an iranian population (neyshabur citizenship). peptide pore blockers are potent tools to study structure and function of potassium voltage-gated channels (kv). kcsa-kv .x chimeras, in which a ligand-binding site of eukaryotic kv-channel is inserted into bacterial kcsa channel, mimic properly the pore domain of kv-channels. a fluorescence-based approach to study the binding of peptide blockers with kcsa-kv . -kcsa-kv . chimeras was developed by us. this approach rested on high-level expression of kcsa-kv .x chimeras in e.coli inner membrane, binding of fluorescently-labeled toxin at the surface of the spheroplast and analysis of competitive binding of studied ligands by laser scanning confocal microscopy (lscm). here we report on a new analytical system for search and study of kv . -channel blockers that combines bl (de ) cells expressing kcsa-kv . and rhodamine-labelled agitoxin (rh-agtx ) as a fluorescent probe. by tuning cultivation conditions, the high-level of membrane expression of kcsa-kv . was achieved. it was found that lowering both the growth temperature and the concentration of inducer resulted in significant increase in membrane-embedded kcsa-kv . . for system validation, wellknown kv channel blockers were studied by the method of competitive binding, and equilibrium dissociation constants were estimated for agtx , osk , and kaliotoxin. a new system was applied to study molecular determinants of peptide-kv . channel binding using a number of agtx mutants constructed by us, whose affinities to kcsakv . were measured. a new bioengineering fluorescent system is a robust and sensitive assay for assessing the binding activity of kv . channel blockers. it can be used to study interaction interfaces of toxinchannel complexes, to search for novel peptide blockers and to develop new potent and selective kv . -blockers for scientific and medical purposes. the work was supported by the grant - - from russian science foundation. asparagus racemosus root extracts (ar) have been exhibited to show a wide range of pharmacological benefits. in this study, liposomes of ar were developed and assessed their physicochemical properties and anti-inflammatory activity in monocytic leukemia cell line (thp- ). liposomes containing ratios of ar to lipid and phosphatidylcholine to cholesterol ratio were synthesized by thin-film hydration (tf), reverse-phase evaporation (rev), and polyol dilution (pd). the in vitro anti-inflammatory activity was assessed in terms of inhibition of tumor necrosis factor alpha (tnf-a) in lipopolysaccharide activated thp- by elisa. the size of ar liposomes prepared by tf were larger, whereas those prepared by rev and pd were smaller. ar to lipid ratio was shown to have no influence on particle size, whereas zeta potential enhanced with increasing ar to lipid ratio. ar liposomes with lipid ratio of : achieved the highest value of entrapment efficiency and were at the highest with polyol dilution method. ar was found to have no toxic effects on thp- cells. the anti-inflammatory activities of ar and ar liposomes in terms of tnf-a in thp- cells were was exhibited to possess the highest values of around % at ar concentration of lg/ml and % tnf-a inhibition tended to decline with the increasing amount of ar. this result may be attributed to the increased amount of liposomal particles being uptaken into the cells as a result of the increasing ar concentrations. it can be suggested that ar liposomes could be an alternative choice of topical/transdermal drug delivery for anti-inflammatory activity. p-mis- inhibition of ire signaling enzyme increases the expression of tumor suppressor genes and modifies their hypoxic regulation in u glioma cells d. tsymbal, o. minchenko palladin institute of biochemistry of the national academy of sciences of ukraine (nasu), kyiv, ukraine gliomas constitute one of the most aggressive groups of malignant neoplasms with poor survival prognosis and scarce therapeutic options. plentiful studies have proven the connection between endoplasmic reticulum stress and malignant growth. we have studied the effect of inhibition of ire (inositol requiring enzyme ), which is a central mediator of endoplasmic reticulum stress and controls cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in u glioma cells. it was shown that inhibition of ire leads to up-regulation of the expression of krt , cd , mest, cenpu, myl , ing , ing , mybl , and mybl genes at the mrna level in u glioma cells, with more profound changes for mest, mybl , and cd genes. hypoxia leads to up-regulation of the expression of cd , ing , and ing genes and to down-regulationof krt gene in glioma cells. at the same time, inhibition of ire modifies the effect of hypoxia on the expression of all studied genes: suppresses effect of hypoxia on ing gene, eliminates hypoxic regulation of krt , cd , and ing genes in glioma cells. the present study demonstrates that inhibition of ire enhances the expression of all studied genes and modifies the hypoxic regulation of these gene expressions in gene specific manner and thus possibly contributes to slower glioma cell proliferation, but several aspects of this regulation remain to be further clarified. amplification and clonig of dna polymerase (pol ) of thermus scotoductus k isolated from an armenian goethermal spring a. saghatelyan, h. panosyan, a. trchounian, n. birkeland yerevan state university, yerevan, armenia the most important enzyme ''mined'' from thermophilic microorganisms is dna polymerase, which widely used in molecular biological studies. although dna polymerase produced by thermus aquaticus (taq polymerase) was launched into the market long back, isolation of more processive, reliable and stable dna polymerases from other species is a demand. the purpose of this work was to amplify and clone the pol gene of t. scotoductus strain k recently isolated from an armenian geothermal spring. the draft genome sequence of strain k was deposited under accession number ljjr . . genomic dna was isolated using genelute bacterial genomic dna kit. primers for the pol gene were designed manually. the gene was amplified using pfu polymerase, and amplicons (~ . kb) were ligated into the pet- b(+) vector (novagen) and transformed into chemically competent top escherichia coli. inserts were sequenced with t prom and t term primers, which showed that the gene sequence was correct and in the right reading frame and could be expressed in mesophilic e.coli. dna polymerases patented form different species of thermus are mostly comparable, suggesting that only limited natural variations in taq-like dna polymerase may be discovered. the pol gene from k shares % and % similarity with pol of t. scotoductus sa- ( . kb) and t. aquaticus, respectively. although the difference is not huge at sequence level, possible functional differences (e.g. stability, proofreading activity, resistance to different pcr inhibitors etc.) may occur. therefore, it is important to express and purify dna polymerase from strain k for further investigations. peptide ligands of the immunoglobulin g fc region identified by screening phage libraries and site-directed mutagenesis n. kruljec, p. molek, t. bratkovic young researcher, ljubljana, slovenia affinity chromatography based on immunoglobulin (ig)-binding proteins, such as staphylococcal protein a and streptococcal protein g, typically represents the initial step in therapeutic antibody purification process. however, this approach suffers from high cost, poor ligand stability and the requirement for relatively harsh elution conditions that can negatively impact activity and immunogenicity of antibodies. compared to protein ligands, peptides represent an interesting alternative due to higher stability and less expensive production. furthermore, the expected lower affinity for immunoglobulins should allow for elution under milder conditions. the aim of our research was to identify short peptide ligands for the fc region of human iggs. we have screened three commercially available phage display libraries of random cyclic and linear peptides for binding to human fc region in solution using an optimized biopanning approach. five non-homologous linear peptides were shown to specifically interact with the fc portion of immunoglobulins as verified by a set of phage elisa assays. individual phage-displayed peptides were able to recognize specific subclasses of igg. the highest-affinity peptide ( l- fc), which competed for fc binding with protein a, was subjected to mutagenesis studies. we displayed on phage several variants of l- fc with individual amino acid residues exchanged for alanine as well fragments of the parent peptide of different lengths and evaluated binding to fc with phage elisa to identify the minimal binding motif. binding characteristics of the minimized peptide were further analyzed using spr biosensor. the details will be disclosed at the meeting. diverse effects of ganoderma lucidum in combination with tamoxifen citrate and doxorubicin in mcf- breast cancer cells ganoderma lucidum, an edible medicinal fungus, has been known with its anti-metastatic, anti-carcinogenic bioactivities and widely used in asian countries in complementary and alternative medicine. however, there is no information regarding its combined usage with tamoxifen and doxorubicin in breast cancer treatment. we investigated the interactions between ganoderma lucidum and tamoxifen or doxorubicin in mcf- human estrogen receptor positive breast cancer cell line. anti-proliferative properties of six extracts were assessed by wst- method. the most effective extract in inhibition of mcf- cell viability was then evaluated in terms of its anti-metastatic activity by boyden chamber assay. apoptosis and cell cycle assays were performed by flow cytometry. ganoderma lucidum ether extract (g.ether) was the most effective extract on inhibition of cell viability among others with ic ( ) values of lg/ml and . lg/ml at h. and h. respectively. we found that g.ether is capable of inducing apoptosis and changing cell cycle dynamics. however, incubation with g.ether did not affect mcf- cell motility significantly. we then assessed the interactions between g.ether and tamoxifen or doxorubicin in mcf- cells. the interactions between g.ether and cancer therapeutics were examined by combination index analysis and macsynergy ii software. interestingly, g.ether increased the anti-proliferative effect of tamoxifen although exhibited strong antagonism with doxorubicin in mcf- cell line. testing the best matrix/analyte combination for maldi tof mass spectrometric detection of steroid hormones, amino acids, vitamins and carbohydrates in spite of numerous advantages, there are serious drawbacks of the application of matrix assisted laser desorption/ionization time-of-flight mass spectrometry (maldi tof ms) for smallmolecule analyses (below da) and quantification. the main problem is the background interference from commonly used maldi matrix materials. the aim of this work is to evaluate maldi tof mass spectra of physiologically relevant small molecules: steroid hormones, vitamins, amino acids and carbohydrates, acquired with several organic, traditional matrices. small volume, . ll, of each sample solution (testosterone, progesterone, estradiol, l-cysteine, l-alanine, dl-methionine, glutathione, d-(+)-glucose, d-(+)-maltose, vitamin a, vitamin e) was mixed on the sample plate with the same volume of organic matrix solutions (dhb, thap, chca, -aa). for each molecule/matrix pair, we determined quantitative and qualitative parameters of ms analysis. to calculate within day and day-today variation we used excel tools (anova tests). in addition, homogeneity of the sample/matrix distribution on the target was also calculated and expressed as the coefficient of variation of a series of measurements. our results show selectivity of the detection of individual molecules related with the matrix applied. the statistical analysis of certain molecule/matrix pairs gave within and day-to-day variations less than %. additionally, homogeneity of the sample/ matrix mixture distribution on the target plate was with some matrices, also less than %. some of the used matrices have a great potential for the analysis of small molecules with good analytical parameters, with low variations and high homogeneity of samples on the maldi target plate. these results hold potential for quantification of metabolically-significant small molecules and are very promising for future applications of maldi tof ms analyses. stress causes different expression of mitochondrial biogenesis markers in rat steroid-producing cells of adrenal gland and testes i. starovlah, s. radovic, t. kostic, s. andric faculty of science univeristy of novi sad, novi sad, serbia functional mitochondria of steroid producing cells of adrenal cortex and leydig cells of testes are essential for steroid hormones biosynthesis and regulation. the aim of this study was to determine transcriptional profile of mitochondrial biogenesis markers in adrenal cortex and leydig cells by applying in vivo and in vitro studies. immobilization stress (imo), was performed for hours daily for one ( ximo), two ( ximo) or ten ( ximo) consecutive days. in in vitro studies, primary cultures of purified leydig cells from undisturbed rats were stimulated with stress hormone adrenaline, propranolol (nonselective b-adrs-blocker) and prazosin (the selective a -adrs antagonist). rq-pcr results showed that the transcription of the main regulator of mitochondrial biogenesis, ppargc a and ppargc b, significantly decreased in adrenal cortex of ximo rats. oppositely, the significant increase of the same transcript was registered in leydig cells from the same rats. in parallel, transcription of ucp , the mediator of regulated proton leak, decreased in adrenal cortex, but increased in leydig cells of the same group of rats. incubation of leydig cells with adrenaline, increased transcription of the main markers of mitochondrial biogenesis (ppargc a, ppargc b, nrf and nrf a). nonselective b-adrsblocker attenuated this effect. the selective a -adrs antagonist did not change adrenaline-induced stimulation of ppargc a, ppargc b, nrf and nrf a transcription in leydig cells, indicating that the most of the effects are probably mediated by b-adrenergic receptors, not by a -adrs of leydig cells. in summary, the results suggest that reduction of transcription of mitochondrial biogenesis markers could be a possible mechanism that protects body from excessive glucocorticoid production from adrenal glands in stress conditions, while at the same time stimulation of mitochondrial biogenesis markers transcription in leydig cells could serve as mechanism to preserve testosterone production. p-mis- generation of new mitochondria is possible protection mechanism of basal steroidogenesis in leydig cells s. radovic, i. gak, t. kostic, s. andric faculty of science, university of novi sad, novi sad, serbia mitochondria are the most important component of stress response in all cells and for steroid-hormones-producing cells they are the starting point for steroid biosynthesis. here we investigated the parameters of mitochondrial biogenesis in these cells from rats exposed to the psychophysical stress by immobilization (imo). imo stress was applied for hours daily for one ( ximo), two ( ximo) or ten ( ximo) days.hormone levels were measured employing eia, elisa kit or ria. mitochondrial membrane potential (Δwm) was measured by tmre fluorescence, mitochondrial mass was detected by quantitative analysis of mitotracker-green fluorescence as well as relative intensity of fluorescence, since number of mitochondria and mitochondrial architecture were defined using transmission electron microscopy. relative gene expression and proteins analyses were performed by rq-pcr and western blot. there was positive correlation between Δw m of leydig cells and androgens production of leydig cells. both of them were reduced in all stressed rats but partially recovered in ximo group. the mitochondrial mass in leydig cells from ximo group was increased. transmission electron microscopy analyses showed that acute and two times repeated stress altered architecture of mitochondrial cristae, while ximo increased number of mitochondria and recovered mitochondrial architecture. there was significant increase in the expression of the all markers of mitochondrial biogenesis in leydig cells from ximo rats compared with other groups. accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. supporting the evidence that stress, a constant factor in life of humans, induces mitochondrial biogenesis in leydig cells, our results indicate this mechanism probably protects the basal steroid production in stress conditions. targeting survival pathways in leukemic cells through synergism of metformin and thymoquinone u. glamoclija, m. suljagic international university of sarajevo, sarajevo, bosnia and herzegovina generation of resistance to current treatment options is common problem in the therapy of many hematological malignancies. combined therapies utilizing compounds with low toxicity that act synergistically, are proposed to overcome this problem. metformin and thymoquinone (tq) are two molecules which have proven safety profile and represent potential candidates for treatment of hematological malignancies. there are more than clinical trials, at different stages, exploring metformin anticancer activity. metformin activates amp activated protein kinase (ampk) leading to inhibition of the mammalian target of rapamycin (mtor) and induction of apoptosis in different cancers. however, human leukemic cells with increased basal protein kinase b (akt) phosphorylation were shown to be resistant to metformin-induced apoptosis. it was found that activity of metformin can be enhanced by combination with akt and/or nuclear factor 'kappa-lightchain-enhancer' of activated b-cells (nf-jb) inhibitors. tq is phytochemical compound that has shown inhibitory capacity on both of these targets. wst- assay was used to evaluate the effects of metformin and tq in dhl (b cell lymphoma) and k (chronic myelogenous leukemia) cell lines. compusyn software was used in order to calculate the combination index (ci). the ci value indicates whether two drugs have synergistic (ci< ), additive (ci= ) or antagonistic effects (ci> ). we have shown that separately, metformin and tq, exhibit dose dependent inhibition of dhl and k cells. in combinatorial study with fixed constant ratio and simultaneous drug exposure, in dhl and k cell lines, ci values were . and . , respectively. to our knowledge, this is the first report showing synergistic effects of metformin and tq in lymphoma and chronic myelogenous leukemia derived cell lines. these promising data are currently being investigated in order to obtain the insight into their molecular mechanisms. for the last decade many methods of calculating and analysing the physical characteristics of dna has been developed. these methods allow to estimate distributions of free energy, propensity to bend, stress-induced duplex destabilization (sidd), electrostatic potential (ep) etc. and most of them have been used for prediction of genomic regulatory site positions. the main idea of such approach is that proteins recognize genome regulatory sites by these physical and chemical properties, so the physical characteristics are used to predict the location of regulatory sites. most of the characteristics mentioned above describe properties of dna at equilibrium or steady state, but we propose to use characteristics of internal dna dynamics. in this work we used the coarse-grained model of dna, developed recently, to simulate dynamics of the dna open states. with this model we were able to calculate trajectories of the open states moving along the molecule and their dynamical characteristics, such as: open state activation energy, size, half decay time and sound velocity in dna. we use distribution of four dynamical characteristics around transcription start site of experimentally found e.coli promoters taken from regulon db to organise them in stable clusters. clusterization was made with ward method and consensus clustering technique was applied to clusterization results for analysis of its consistency. the same procedure was applied to equilibrium dna characteristics for comparison. distribution of go functions among clusters was also analysed. stable promoter clusters obtained with different physical properties share some similarity. it was not surprise that clusters obtained with dynamical characteristics of dna more similar to sidd clusters then to ep clusters. the data highlights the possible role of dna dynamical properties in transcription initiation and its applicability to promoter identification together with other physical and textual properties of dna. chromium complex with -hydroxyflavone acts on metabolic pathways the development of novel therapeutic strategies for obesity treatment are urgently required as obesity is currently the main leading cause in type ii diabetes and insulin resistance. among natural compounds, flavonoids have recently gained interest due to their positive role in maintaining blood glucose levels and insulin secretion. their association with trace elements, wellknown for their capacity in increasing the efficiency of insulin, might potentiate flavonoids biological effects. in this context, the aim of our study was to investigate the in vitro changes in energetic metabolism related genes expression profile in the presence of a chromium complex with -hydroxyflavone. dna microarray technology was used for a large scale screening of differentially expressed genes in human adipose stem cells (hascs) after weeks of adipogenic induction in the presence of the chromium complex with -hydroxyflavone. moreover, perilipin expression was assessed by flowcytometry. the chromium complex with primuletin negatively regulates the expression of key genes involved in adipogenesis and also modulates the expression of the genes associated with triglyceride synthesis and subsequent fat storage in mature adipocytes. consequently, the chromium complex with -hydroxyflavone can be further employed in studies on animal models to investigate the possible improvement of metabolic disorders. deinococcus radiodurans is a highly radioresistant and stress-resistant bacterium. despite extensive studies, the mechanisms of transcription regulation that contribute to the stress-resistance are still poorly understood. d. radiodurans encodes multipe stress-related proteins including three members of the gre-family of transcription factors: grea, gfh and gfh . while grea is a universal bacterial factor that stimulates rna cleavage by rna polymerase (rnap), the functions of lineage-specific gfh proteins remain unknown. we cloned, expressed and purified d. radiodurans rnap and gfh factors and their mutant variants and analyzed their properties using various in vitro transcription approaches. we tested gfh effects on rnap activity in promoter, elongation and termination complexes assembled on natural and synthetic dna templates under different conditions. we found that the gfh factors strongly enhance site-specific pausing and intrinsic transcription termination by d. radiodurans rnap but do not act on active transcription complexes and do not compete with the grea factor. uniquely, the pause-stimulatory activity of gfh is greatly enhanced by manganese ions, which are accumulated in d. radiodurans cells under stress conditions, and is modulated by the secondary rna structure. we revealed functionally important regions in the gfh factors and the rnap active site involved in transcriptional pausing. we propose that gfh factors inhibit rna extension in paused complexes through binding within the secondary rnap channel, coordinating metal ions in the rnap active site and stabilizing an inactive enzyme conformation. this may serve as a sensitive mechanism to regulate transcription under stress conditions and coordinate it with dna repair and replication. our data suggest that gre and gfh proteins target different structural states of the transcription elongation complex and reveal functional diversity of the factors that bind within the secondary channel of rnap. from planktonic to biofilm state of growth, flagella formation is turned off, and the production of fimbriae and extracellular polysaccharides is activated. bola protein is widespread in nature and has been associated with several cellular processes. using high-troughput techniques we showed that bola protein is a new bacterial transcription factor, which regulates the switch between motile and sessile lifestyle. it negatively modulates flagellar biosynthesis and swimming capacity in escherichia coli. moreover, bola overexpression favors biofilm development, involving fimbriae-like adhesins and curli production. our recent results show that bola action in these pathways is related with cdi-gmp a relevant intracellular signaling molecule involved in biofilm formation. we demonstrate that bola contributes to a fine-tuned expression of different diguanylate cyclases and phosphodiesterases and c-di-gmp has a negative influence in the bola mrna transcription. herein we propose that bola is a key player in motile/adhesive transcriptional switch, contributing to a fine-tuned regulation of these important pathways. background: deep venous thrombosis (dvt) is an important health problem worldwide. its pathophysiology is multicausal and involves environmental, genetic and acquired factors. factor v leiden (fvl), prothrombin g a (pt g a), and methylenetetrahydrofolate reductase (mthfr) gene mutations are to predispose to venous thrombosis. the aim of this study was to compare the frequency of fvl, pt g a and mthfr polymorphisms between patients with dvt and healthy controls. methods: this study was conducted at the bozok university hospital. total participants were included in this study, patients with dvt and healthy blood donors. in order to identify fvl, pt g a, mthfr c t and mthfr a c, the polymerase chain reaction (pcr) method was utilized combined with the amplification refractory mutation system. results: in patients fvl was present in ( . %) patients while in controls fvl was present in only ( . %). frequency of fvl was significantly higher in cases as compared to controls (p < . ). pt g a mutation was present in patients ( . %) and in healthy participants ( . %). mthfr c t and mthfr a c polymorphisms were almost equally distributed among patients and healthy participants. however, the concomitant presence of fvl and double heterozygous polymorphisms of mthfr c t/a c was found in patients ( . %) and in healthy controls ( . %), showing significant association with deep venous thrombosis. conclusion: in this study, the frequencies of fvl and pt g a polymorphisms were found significantly higher in patients with dvt than those in healthy participants. thus, fvl and pt g a polymorphisms have a contributory role on the development of dvt in contrast, mthfr c t and mthfr a c genotypes were not associated with a predisposition to development of dvt. but, a combination of double heterozygous polymorphisms of mthfr c t/a c with fvl may be associated with increased risk of dvt. p-mis- self-assembling micellar clusters comprising drugs, nanoparticles and fluorescent compounds for bilogical applications when designing drug carriers, the drug-carrier ratio is an important consideration, because the use of wrong drug-carriers relation can result in toxicity as a consequence of poor metabolism and elimination of the carriers. solubility problem of various substances also plays an important role in many aspects of fundamental science and practical field. specifically, it is an important parameter as well as bioavailability, which determines the required concentration of drug in the body needed to achieve a pharmacological response. among the variety of solubilization methods micellar solubilization is widely used as an alternative to the dissolution of poorly soluble drugs. here, we show a specific approach based on sequential selfassembly of nonionoc detergent micelles (t , tx ) followed by enacpsulation of various nanoparticles (noble metals, magnet etc.), drugs, fluorescent compounds leads to the formation of stable micellar nano-amd microcomplexes. we propose ways of micellar clusterisation. in the first one micelles are modified by semi-hydrophobic chelator followed by addition of metal ion to make cross-linking. the second way is similar to the first one and suggests application of the metal complex with incresed denticity instead of naked metal ion, and the third one involves micelles clusterisation by semi-hydrophobyc metal complex directly. therefore, one can stabilize micellar network by means of 'interactions on interface': semi-hydrophobyc metal complexes are embedded inside micelle due to hydrophobyc interactions. hydrophobic fluorescent compounds-loaded micellar complexes demonstrates better optical response in aqueous media without crystallization. such obtaining clusters are also very flexible and can be modified by nanoparticles to obtain various nanocomposites, such as fluoromagnetic clusters. this work was supported by russian foundation of basic research grants no. - - r_center_a ( no. - - r_center_a ( - no. - - r_center_a ( ) and - - mol_a_ved ( no. - - r_center_a ( - . lamellipodia and membrane blebs utilize different signalling pathways to induce directional movement of walker carcinosarcoma wc cells in a physiological electric field clear if those reactions are mediated by similar mechanisms. to establish that, we performed proteomic analysis and subsequent investigation of the role of differential signalling pathways in electrotaxis of cells representing various strategies of movement. cells were exposed to ef in galvanotaxis apparatus and their reaction was recorded. in some experiments cells were pre-incubated with erk / or btk- inhibitors. the phosphorylation of erk / and btk- was determined by western blot analysis. proteomic analysis was performed by ultimate rs lc nanosystem coupled with a q-exactive mass spectrometer. both blebbing (bc) and lamellipodial (lc) cells show cathodal migration in a physiological ef ( v/cm). comparative analysis of bc and lc cells proteomes revealed about differential proteins. functional analysis in ingenuity analysis pathway allowed to determine the statistically significant signalling pathways in which these proteins are engaged. among the most distinctively regulated pathways are tec kinase and erk/ mapk signalling activated in lc but not bc. it was found that btk- is required for directional movement of lc but not for bc cells. moreover, ef induced stronger and faster btk- phosphorylation in lc than bc cells. in contrast erk / activity was not necessary for electrotaxis of lc cells and ef did not induce erk / phosphorylation. our results reveal that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of wc cells but it is mediated by different signalling pathways. this work was supported by a grant from the national science centre / /b/nz / , poland. newborn screening for congenital hypothyroidism in turkey: a regional evaluation € o. demirelce , n. y. saral , f. b. aksungar , , a. coskun , , m. serteser , , i. unsal , acibadem labmed, istanbul, turkey, acibadem university, istanbul, turkey congenital hypothroidism (ch) is the most common congenital endocrine disorder and the most important cause of preventable mental retardation. it is important to begin the treatment within weeks before the development of brain damage. tsh based newborn screening programs are shown to be useful for implementing early treatment of ch. in this study, regional results of ch screening program in turkey between and were assessed retrospectively. we have evaluated the results of marmara, central anatolia, aegean and mediterranean regions in which our laboratories are located. screening was based on tsh determination in dried blood spot specimens. tsh limits determined to be lu/ml for cut off point and lu/ml for clinical decision point. tsh was measured using enzyme immune assay (eia). blood spot tsh data for newborns during this time period were evaluated. permanent or transient ch was determined according to the results of thyroid function tests. confirmed ch cases were based on local endocrinologists' report and initiation of thyroxine treatment. the frequency of neonatal tsh levels were found to be under the cut off level of lu/ml in ( . %), between and lu/ml in ( . %) and above the level of lu/ml in ( . %) babies, respectively. recall rate was . %. ch cases of neonatal tsh levels greater than lu/ml were . the incidence of ch of this group was : . there were no significant differences in the number of congenital hypothyroidism between males and females (p > . ). the preliminary results of our study indicate that the incidence of ch in our region is higher than the worldwide reports as has been proved by preceding studies. iodine deficiency, dyshormonogenesis, highly consanguineous population, may contribute to the high incidence of ch in turkey. newborn screening of ch must be developed for detecting true cases and tsh cut off point must be reviewed for decreasing redundant recall rate. in silico analysis of the first complete genome sequence of lactobacillus acidipiscis species k. papadimitriou , m. kazou , v. alexandraki , b. pot , e. tsakalidou agricultural university of athens, athens, greece, institut pasteur de lille, lille, france introduction: lactic acid bacteria (lab) constitute a significant group of microorganisms for the food industry, as they play a key role in food fermentation and consequently in human health. lactobacillus acidipiscis aca-dc is a gram-positive, motile, rod-shaped lab isolated from traditional greek kopanisti cheese. here we present the in silico analysis of the first complete genome sequence of l. acidipiscis in order to explore the biology of the species. materials and methods: sequencing of l. acidipiscis genome was performed using the hiseq and pacbio rsii sequencing platform technologies and the genome assembly was validated against an nhei optical map of the l. acidipiscis genome. protein-coding sequences were predicted by glimmer, rrna genes by rnammer and trna genes by the trnascan-se server. potential genomic islands were detected using the island-viewer software tool, prophage regions by phast and the subsystem-based annotation by rast server. finally, the circular representation of l. acidipiscis genome keyed to the cog groups was constructed by cgview server. results: the sequencing analysis resulted in one continuous genomic scaffold of , , bp with a g+c content of . %. the genome contains , protein-coding genes on the chromosome covering up to . % of the genome sequence, trna and rrna. according to the subsystem-based annotation, , protein-coding genes were assigned to metabolic subsystems. the most abundant of the subsystems are related to carbohydrates (n = , . % of total protein-coding genes) and protein metabolism (n = , . % of total protein-coding genes). furthermore, three prophage regions were detected; one intact ( . kb), one incomplete ( . kb) and one questionable ( . kb). discussion and conclusion: the whole genome analysis of l. acidipiscis aca-dc provided interesting information about a not well-studied species. investigation of serum irisin levels of patients with metformin taking new onset type diabetes mellitus increases glucose tolerance and energy expenditure and improves carbohydrate homeostasis. metformin is a biguanidine class antidiabetic drug which inhibits liver gluconeogenesis and decreases insulin resistance and is frequently recommended in treatment of new onset type diabetes mellitus (t dm). irisin has a role in the regulation of energy metabolism pathways and its level in blood of persons with t dm has been reported to decrease. regarding this relationship, it was aimed to reveal the effect of metformin on serum irisin levels. patients with impaired oral glucose tolerance test were included to this investigation. they were recommended to take metformin and to change their life style, such as exercise and diet. their blood were taken at the beginning and after month. also, a healthy control group (n = ) was formed from persons with similar age and sexual distribution as the patient group. irisin levels of their sera were measured by enzyme-linked immunosorbent assay (elisa) method. statistical evaluation of the measurements showed no significant difference (p = . ) between the irisin levels of the patients at the beginning and after month treatment. a similar result was found between the control and the treated groups (p = . ), while a significant difference (p = . ) was observed between the control and untreated patients groups. the results obtained from this study do not show a clear and significant change in the blood irisin levels of the patients with new onset t dm taking metformin together with life style change. a longer period of treatment and a higher number of patients may be needed for more reliable results. thermodynamics of dna ligands binding at specific sites of telomeric g-quadruplex dna g-quadruplexes are a perspective target for anticancer therapy. for example stabilization of the telomeric g-quadruplex dna formed by single-stranded ends of the chromosomes leads to inhibition of telomerase, which is active in % of cancer cells. similarly, small molecules targeted to a specific g-quadruplex would inhibit various cellular processes. stoichiometry and affinity of interaction of these compounds to dna is determined by specific structural motifs within a g-quadruplex. rational design of novel chemical compounds requires an in depth knowledge of interactions between known ligands and g-quadruplex structures. experimental methods that are used for determination of thermodynamic binding parameters, such as isothermal titration calorimetry, differential scanning calorimetry, ultraviolet absorption and circular dichroism spectroscopy provide a collective characteristic for all of the ligand molecules bound to dna, while the information on ligand affinity to individual dna binding sites is lost. we propose a complimentary method for detailed analysis of thermodynamic parameters of ligand binding based on the introduction of fluorescent probes in the structure of g-quadruplex. monitoring fluorescence quenching of the fluorescent labels allows to derive binding constants of the dna-ligand interaction at a specific binding site. temperature dependence of the fluorescence quenching determines the thermodynamics of the dnaligand complex formation. since only a proximal ligand is able to quench the fluorescence, this method allows characterization of the ligand binding to a particular site the g-quadruplex structure. the study was supported by project no. - - of the russian science foundation. the correlation between biochemical and dynamic surface tension parameters of calves blood serum during the animal ontogenesis, as well as by various pathologies or poor diet, the imbalance of protein, mineral, lipid components is observed (the changes in all parameters of biological liquids are accompanied of these metabolism peculiarities). the dynamic surface tension (dst) of serum essentially depends on these factors and (in combination with the biochemical parameters) can provide the valuable information for evaluation of the physiological and biochemical status of the organism (can be used as an express test for animal diagnostics in future). the aim of the work was to study dst and biochemical parameters of calve serum, as well as their correlations, as the main indicators of the animals. ) of calve serum were in the range of the normal values for healthy animals and can be considered as reference data for animal science and practice. the obtained results enable us to establish correlations between the dst and biochemical parameters of calves serum. this work was supported by the russian scientific foundation (grant - - ). the middle strong correlations of dst values of calves serum with the level of total protein, albumin, billirubin, some enzymes and cholesterol, whereas only weak correlations with the other biochemical parameters (urea, calcium, magnesium, phosphorus, etc.) were found. in the veterinary science and practice such correlations are important for the estimation of the organism physiologicaland biochemical status, for general inspections of cattle before vaccination (immunization) or slaughter, for "quick separation" of healthy and ill animals in the case of infection, etc. role of protein kinase c in the regulation of astrocytic glutamine transporter sn in ammonia-exposed mouse cortical astrocytes (bisi; lm). total pkc activity was analyzed by a direct pkc assay and phosphoserine detection by western blot (wb) analysis. protein level of sn and sn , second astrocytic gln transporter belonging to system n, in a membrane fraction was also analyzed. the total uptake and system n-mediated (l-ala and l-leu-inhibitable) gln uptake was tested. treatment of astrocytes with ammonia resulted in a decrease of pkc activity, whereas pma treatment increased pkc activity in ammonia-independent way. bisi treatment reversed fully, and ammonia partially, the pma-induced pkc activity. pma treatment resulted in only a slight decrease in sn protein level in both control and ammonia-treated astrocytes, while a decrease of total and system n-mediated gln uptake were noted in control astrocytes, an effect not exacerbated by ammonia. in turn, cotreatment with pma and bisi reversed the decrease of total gln uptake and showed tendency towards increase in system nmediated gln transport. the results suggest that: a) ammonia changes the dominating direction of system n transport from release to uptake, which may be related to decreased phosphorylation or to alterations in relative phosphorylation by different pkc isoforms. this inference remains to be verified in further studies; b) changes in system n transporter function induced by ammonia appear to involve mechanisms other than changes in transporter expression. evidence for human ghrelin ghs-r a and orexin ox heteroreceptor complex formation in a heterologous system ghrelin and orexin are two peptides implicated in the regulation of energy balance and modulation of food-related motivation at the level of the midbrain dopamine reward system. their function in the hypothalamic arcuate nucleus and the ventral tegmental area (vta) has already been described, but the modulation at the level of receptors remains unclear. the action of these peptides is mediated by g-protein-coupled receptors (gpcrs): ghrelin a and b (ghs-r a , ghs-r b ) for ghrelin, and orexin and (ox , ox ) for orexin. traditional approaches to know the mechanism of neurotransmission of dopaminergic neurons in the mesolimbic system have focused on targeting neuronal receptors as single entities. from the discovery that gpcrs for neuromodulators may form heteroreceptor complexes, our hypothesis is that ghrelin and orexin receptors may interact and form novel functional units that may specifically participate in the central regulation of food intake and energy balance. as a proof of concept we have investigated the potential of human ghs-r a and ox receptors to form heterocomplexes. formation of ghs-r a -ox receptor heteromers in transfected hek t cells was detected by bioluminescence resonance energy transfer (bret) and proximity ligation (pla) assays. furthermore, a negative crosstalk was identified in cells co-expressing both receptors by assessing mitogen-activated protein kinase (mapk) and adenylyl cyclase (camp) pathways, and by a label-free dynamic mass redistribution assay. experiments in sources endogenously expressing ghs-r a and ox receptors are needed to know the functional relevance of the heteromer. from the negative crosstalk here identified, it is tempting to speculate that ghs-r a -ox receptor heteromers are important players in mediating the response to the combination of different orexigenic signals. lysosomal storage diseases which are related to deficiency of specific lysosomal hydrolases resulted to clinical aspects due to accumulation of substrates in different tissues. since dried blood spot (dbs) is non-invasive, low-cost, easy transportable, acceptable enzyme stability compared to leucocyte and/or fibroblast culture, it's recommended as a first screening test. however the false positive rate with dbs sample is higher compared to other samples. we aimed to investigate any possible effect of leucocyte number on enzyme activity in dried blood samples in a retrospective study. we re-evaluated the lysosomal enzyme activity results in regard to leucocyte number among data within last year. enzyme activities had measured by using fluorometric and lc msms method. we determined the correlations between the lysosomal enzyme activities of alpha glycosidase, glycocerebrosidase, alpha galactosidase, sphingomyelinase, galactocerebrosidase and alpha-l-iduronidase in healthy population (n = ). while glycocerebrosidase and galactocerebrosidase positively correlated with the number of neutrophils, alpha galactosidase, sphingomyelinase and alpha-l-iduronidase positively correlated with the number of lymphocytes. alpha glycosidase activity showed a correlation both lymphocytes and neutrophils. the patients having the glycocerebrosidase enzyme activity which was lower than . nmol/ml/hour (which is accepted as the cut off value to recall the patients) existed significantly lower number of leukocyte, lymphocyte and neutrophil compared to those of patients having higher enzyme activity than . . our data indicated that the enzyme activity in dried blood samples including low leucocyte number might be found lower than reference intervals resulting in false positive diagnosis. therefore we suggest that the laboratory scientists should evaluate the number of leucocyte levels while they were interpreting data. using dna-markers for estimation of genetical variability of two kazakh sheep breeds a. mussayeva , , b. bekmanov , , a. amirgalieva , k. dosybaev , , z. orazymbetova , , r. zhapbasov , a. zhomartov , n. zumadillaev , n. zumadillaev llp "kazcytogen", almaty, kazakhstan, "institute of general genetics and cytology" sc mes, almaty, kazakhstan, branch "scientific research institute of sheep" llp "kazakh research institute of animal husbandry and feed", almaty, kazakhstan to compare the frequencies of different microsatellite loci in sheep breeds subpopulations genomic structure of edilbay and kazakh archaromerinos was investigated. different methods for homogeneity testing of two breeds were elaborated. inter simple sequence repeats (issr) pcr analysis of the breeds studied displayed species and breed specific fragments with different frequencies (population frequency more than . ) there were found rarely met fragments (frequency lower than . ). the combinations of these fragments present the specific issr-spectra which arrange genofond profiles of breeds. using panels of microsatellits (recommended by isag) breeds ( populations) were characterized. informative value and resolving capacity of the sum of str-loci were estimated. wide polymorphism of alleles length was demonstrated both when the breeds were compared and within the breeds. informative markers were chosen for both two breeds, markers being used for both breeds, while other markers were informative for one of the breed only. when the animals of one breed were compared unique alleles which were met only within one of populations were of much interest. for example the allele of bm was met in birlik population of edilbay breed as often as in % of animals while in two other populations there were no this allele. in kumtekey population one can meet % animals having particular locus (dyms ), while in the other population (cf ablay) this locus was not met at all. basing on genetical distances obtained using fragment analysis phylogenetic relationships between populations were estimated. so for example edilbay population of cf ajar has the larger distance from two other populations (birlik and bayserke-agro) than each of them from one another. two subpopulations of kazakh arkharomerinos breed (cf kumtekei and cf ablay) also have the genetical difference. how preeclampsia affects oxidant status and antiinflammatory potential of breast milk? preeclampsia is a pregnancy syndrome associated with hypertension, proteinuria and edema, leading to maternal morbidity/mortality and preterm delivery. in this study we aimed to investigate if the breast milk of preeclamptic mothers is effected in oxidative status and anti-inflammatory activity in comparison to the breast milk of mothers with healthy pregnancies. for the aim of the study, hyaluronidase and myeloperoxidase activities (mpo), total oxidant status (tos), total antioxidant status (tas), oxidative stress index (osi) and tbars levels were measured in breast milks of preeclamptic mothers and mothers with healthy pregnancies as control group. when the control group and preeclamptic group were compared, hyaluronidase activity, tas, tos and osi levels showed statistically significant differences in the preeclamptic group. hyaluronidase activity was significantly higher in the preeclamptic mothers' breast milk ( vs u/ml, p = . ). while tos levels were significantly higher in the preeclampsia group ( . vs . lmol/l, p = . ), the tas levels were significantly higher in the control breast milks ( . vs . mmol/l, p = . ). as expected osi levels (tos/tas ratio) were significantly higher in the preeclampsia group. even though the mean levels were higher in preeclamptic group, the difference in mpo activities and tbars levels did not show statistic significance. oxidant status parameters also suggest that preeclampsia effects in both ways by increasing oxidant status and also decreasing antioxidant capacity shifting the balance to the increased oxidant stress side. as the results showed that the preeclampsia group had higher hyaluronidase activity, this can be interpreted as preeclamptic mothers' milk have higher inflammatory potential as this enzyme enhances inflammation by catalyzing the depolymerization of certain acidic glycosaminoglcans. p-mis- investigation of relationship between postprandial lipemia and erythrocyte membrane cholesterol level postprandial lipemia is a metabolic condition related to an increase in plasma triglycerides. remnant-like lipoprotein particles are predominant in postprandial phase and they play an important role in development of atherosclerosis. cholesterol is a prominent component of erythrocyte membranes and regulates the membrane functions such as viscosity and permeability. free cholesterol derived from erythrocytes is thought to participate in the atherosclerotic plaque formation. in the current study, it was aimed to investigate the relationship between postprandial lipemia and erythrocyte membrane cholesterol level in healthy subjects. study group included subjects ( female and male with age range of - years). then these individuals were divided into three groups according to the values of area under curve (auc) calculated by using triglyceride levels at the fasting state and at nd, th and th hours after the high fat diet (ottt). lipid and erythrocyte membrane cholesterol (emc) values were compared between groups with low and high ottt response. while tc, tg, ldl-c and emc were significantly higher, hdl-c was significantly lower in high ottt response group than low ottt response group. it was not observed any statistically significant difference when compared emc values between women and men study groups. on the other part, it was seen positive correlation between emc and auc (r = . , p = . ), tg (r = . , p = . ), tc (r = . , p = . ), ldl-c (r = . , p = . ) in the total study group. it was concluded that, postprandial lipemia may show atherosclerotic tendency not only with atherogenic lipid profile but also with increasing emc. p-mis- eu-openscreen: the european infrastructure for chemical biology b. stechmann, p. gribbon eu-openscreen, fmp leibniz institute for molecular pharmacology, berlin, germany small molecules that can be applied as chemical 'tool' compounds (or 'probes') have become indispensable in basic research for the elucidation of fundamental biological mechanisms. they act directly with the protein-of-interest and often allow for the interrogation of biological processes that cannot be properly studied with traditional genetic or rna interference approaches. eu-openscreen (www.eu-openscreen.eu) is the largest emerging academic chemical biology research infrastructure initiative in europe and will provide access for molecular and cell biologists to screening infrastructure, well-characterized highquality chemical libraries, and facilities for medicinal chemistry services for compound optimization. molecular biologists who have a robust and suitable biological assay and are interested in collaboratively developing chemical tool compounds to validate their targets-of-interest are welcome to work with eu-openscreen. selected assays are screened against a collection of more than , compounds, incl. confirmatory and counter screening, ic/ec determination, sar (structure-activity relationships) and qc of confirmed hit compounds. eu-openscreen will start operations in , but it can already look back on a growing number of transnational activities: joint screening projects, exchange of local compound libraries, development of new design principles for its compound collection; exchange of experimental data through its pilot database etc. steps towards an arthrobacter nicotinovorans based biotechnology for production of hidroxy-nicotine as the archetypal agonist of nachr, nicotine stands up as a powerful scaffold for developing new alzheimer disease therapeutic agents in form of nicotine derivatives. in this context, arthrobacter nicotinovorans pao and its wide range of nicotinederivatives produced when grown on nicotine have a huge biotechnological potential. indeed, the metabolic intermediate -hydroxy-nicotine ( hnic) produced by a. nicotinovorans pao was shown to bind to the nachrs, and by modulating their function, to sustain spatial memory formation in a rat model of ad. the current work presents the first attempts to produce and isolate hnic by using a genetically engineered a. nicotinovorans strain. the growth and the hnic accumulation were compared for two strains: . a. nicotinovorans pao wild type strain and . a genetically engineered a. nicotinovorans pao strain (part ndh) containing the nicotine-dehydrogenase (ndh) genes cloned in the nicotine inducible part vector. the growth curves were followed spectrophotometrically. the consumption of nicotine and accumulation of hnic were monitored by hplc using a mn nucleodur - c ec column and . m sulfuric acid at a flow rate of ml/minute. the growth curve of the part ndh strain shows that the bacteria grow slower when compared with the wt. as a result, in the wt strain, the nicotine is quickly depleted from the medium and only low amounts of hnic are observed. although the sds-page analysis of the total protein extracts from the part ndh strain did not show clear signs of ndh overexpression, the enzyme is produced and is active, allowing a fold accumulation of hnic in the growth medium. the first attempts to purify ndh from the part ndh strain using imac were nevertheless unsuccessful. in conclusion, using the part ndh strain for hnic production is feasible. further improvements of the growth condition and strain are envisioned (i.e. knocking the ndh downstream genes; adding inhibitors for the downstream enzymes). studies on the impact of butyrylcholinesterase (bche) on the symptoms and progression of cognitive impairments like alzheimer's disease (ad) or other neurodegenerative disruptions speak in favour of selective bche inhibitors as a new approach in future ad pharmacotherapy. some derivatives of quinine and quinidine, present in the cinchona species bark, have already been identified as selective bche inhibitors with respect to acetylcholinesterase (ache); therefore, further investigation of these compounds might result in promising leads for enhanced anti-ad drugs. we synthesised ten quaternary derivatives of cinchonines and their corresponding pseudo-enantiomeric cinchonidines. quaternization of quinuclidine moiety was carried out with groups diverse in size: methyl and differently meta and para substituted benzyl groups. all of the compounds were prepared in good yields, characterized by standard analytical spectroscopy methods, and were tested for their bche and ache inhibition potency. the inhibition potency of the compounds was defined by the dissociation constants of the enzymeÀinhibitor complex (ki). all of the tested compounds reversibly inhibited both human bche and ache. the compounds inhibited bche with ki constants in the range of . - lm, and ache in the range . - lm. five cinchonidines displayed a - times higher inhibition selectivity to bche over ache, and four of them were potent bche inhibitors with ki constants up to nm. bche affinity toward the studied compounds depended on the size of the substituent on the nitrogen of the quinuclidinium part of the molecule and on the resonance stabilization of the substituent at the quaternized nitrogen. based on the presented results, cinchonidine cd-(pbr) can be pointed out as a potent and selective bche inhibitor that could be considered for further research in alzheimer disease pharmacotherapy. exposure to nmda ( lm) for h increased the expression of kir . mrna and decreased that aqp -and gs mrna. the expression of kir . was decreased by h exposure to glu ( mm) and tnfa ( ng/ml). at h incubation, nmda induced a decrease of kir . expression in the presence but not in the absence of calcium in the medium. nmda did not alter the expression of nmda receptor subunits. tnfa increased the expression of the nr subunit, and decreased that of nr b mrna. glu decreased the expression of out of subunits. the study demonstrates, to our knowledge for the first time, that prolonged exposure of astrocytes to nmda alters the expression of mrna coding for critical astrocytic proteins. the dependence of the decrease of kir . mrna expression on extracellular calcium suggests the ionotropic nature of nmda receptor stimulation. the effects of nmda receptor stimulation occurred by a mechanism bypassing changes in subunit composition of the nmda receptor. experiments are under way to establish whether the tnfa-induced changes in the expression of nmda receptor subunits contribute to modulation of nmda receptor stimulation by inflammation. the importance of education in reducing preanalytical errors the preanalytical phase includes the request of test, the preparation of patient, the obtaining of sample from the patient, the transport of the sample to the laboratory, and the pretreatment of sample. the preparation of patient and the obtaining of sample are considered as the most common error sources. in order to reduce preanalytical errors, we aimed to provide training for phlebotomists and to also determine their knowledge level about the preanalytical phase before and after these training. it was given the training related with preanalytic phases to pediatric nurses and adult nurses, other phlebotomists in march. the surveys which are made before and after the training were consisted of questions that are related with demographyic features and preanalytic phases. in order to determine the effects of training to the preanalytic phase, the preanalytic error rates before (in february) and after (in april) traning was calculated with the formula of: (the number of rejected samples/the number of total samples)x . the average age of participants was ae years. it was not found significant difference between their correct answers rate before the training and the education degree of the participants. the correct answer rate before the training was % and after the training it was %, which showed an increase of %. the preanalytic error rates which were . % in february were decreased to . % in april. in our study, the positive results were obtained through the training aimed to reduce the preanalytical errors. by providing regular training to the phlebotomists and also providing pretraining to the beginners, the updating of their information about preanalytic phase can be achieved. in this way, the loss of labor and economic related to preanalytical errors can be avoided and the accurate results can be obtained in short time. curcumin, the active compound of turmeric (curcuma longa) has antiinflammatory, antioxidative and antitumour effects. unfortunately, curcumin has a poor absorption and low stability. both can be solved by encapsulation of curcumin using a proper technique like electrospray. it was reported that piperin, the active compound of black pepper, enhances the intestinal absorption of curcumin and thus its bioavailability. due to these facts it was aimed in this study to nanoencapsulate turmeric extract in order to enhance its absorption and stability. for that purpose, it was encapsulated with the maize protein zein, chitosane and black pepper extract by varying the voltage and flow rate of electrospray and the concentration of the compounds. the nanocapsules were characterised by measuring their particle size and with help of sem photographs. the particle size of the final nanocapsule formulation was nm and had a sufficient stability over a period of months, visually determined. by encapsulating turmeric extract into double layer nanocapsules with help of black pepper extract, zein and chitosane, the turmeric extract could be protected from degradation, which was observed for the pure turmeric extract in form of clearing its yellow colour. analysis of the human genome reveals that potential g-quadruplex sequences are enriched in promoters of the oncogenes. growing body of evidence suggests that g-quadruplexes (g ) may play putative roles in various biological processes, such as the regulation of gene expression. consequently, targeting the oncogenic g-quadruplexes using small molecules is an alternative strategy for the potential treatment of cancers. porphyrin derivatives are promising class of drug in this respect, being nucleic acids binders and generators of reactive oxygen species under visual light irradiation. interaction between porphyrin derivatives and g dna from oncogene promoter region has been studied in vitro. we applied chemical probing, circular dichroism spectroscopy and uv melting techniques in order to map the oxidized bases, monitor structural rearrangements and evaluate stability of the resulting dna structures. specifically, we observed that g dna is considerably more susceptible to lightinduced modification than duplex dna; -terminal tetrads of the g dna are preferably oxidized; structural changes induced by oxidation result in decrease of the thermodynamic stability of the g dna. irreversibility of these effects on dna make porphyrin derivatives perspective lead compounds for rational design of ligands targeting human oncogenes. the study was financially supported by project no. - - from the russian science foundation. resistin levels in denervated obese rats n. saglam , t. ahmedi rendi , c. kahraman , a. alver department of medical biochemistry, faculty of medicine, karadeniz technical university, trabzon, turkey, school of health, d€ uzce university, d€ uzce, turkey the sympathetic nervous system is an important factor affecting the metabolic and secretory function of the white adipose tissue. resistin is mainly expressed by mononuclear cells, also it is expressed by adipocytes, pancreatic cells, and muscle. resistin induces insulin resistance and glucose intolerance in mice. resistin plasma levels depend on fat depots size and sex. resistin levels decrease in short-term fasting in mice, then it increase refeeding. also, it increase as a response to fed with the high fat diet. in our study we aimed to determination of the effect of high-fat diet and denervation on serum resistin levels in rats. in this study experimental groups were formed each consisted of rats. during weeks, first two groups are fed with high-fat diet and other two groups are fed with standart diet which they purchased from research diets company. at the beginning of the feeding periods, retroperitoneal fat tissiues of animals assigned to the first and the third groups were denervated. second and fourth groups were not denervated. at the end of the week feeding periods, blood collected from rats and blood resistin concentration was determinated by elisa. in denervated and fed with high fat diet groups serum resistin levels higher than control groups (p < . ). according to our literature research, there are no studies demonstrating the relationship between resistin and the sympathetic nervous system. also, denervation may lead to increase in serum resistin levels. the amount of resistin is possibly reduced by b -adrenergic activation. in conclusion, it was concluded that there is differences on serum resistin levels depending on diet in bilateral denervation of retroperitoneal fat tissues of rats. stress activated protein kinases regulates the ribosomal frameshift rate in est gene, encoding subunit of telomerase s. t€ urkel, s. sarica uludag university, bursa, turkey est gene (ever shorter telomere) of s. cerevisiae encodes one of the essential subunits of telomerase enzyme. expression of est gene is regulated at the translation level by + programmed ribosomal frameshift (prf). it is known that the physiological stresses affect telomere length. in this study, we have investigated the effects of stress activated protein kinases snf p (ampk) and gcn p (eif kinase) on the prf rate in est gene. prf rate of est gene was quantified in plasmid based expression system. expression vectors were transformed in to the wild type and mutant yeast strains that deleted for snf , or gcn genes. yeast cells were grown in normal conditions or subjected to acid stress, osmotic stress, or glucose limitations to activate protein kinases gcn p, and snf p, respectively. prf rate of est gene was measured as % in the normal growth conditions in the wild type cells. but, the prf rate of the wild type strain grown in glucose limited conditions decreased more than -fold, giving less than % prf rate. contrary to glucose limitation, osmotic or acid stress activated frameshift rate by -fold in the wild type cells and prf rate increased to %. when the prf rate was analyzed in gcn and snf mutants, frame shift rate of est was - % in normal growth conditions. when these mutants were subjected to acidic or osmotic stress, prf rate activated slightly. we have also shown that gcn p and gcn p, positive regulator of gcn p, is also essential for the regulation of prf in est in response to stress conditions. it is clear that the basal level expression of est is highly dependent on the gcn p kinase complex. gcn p is also associates with ribosomes, indicating that gcn p might have a significant function in connecting the stress signals to biosynthesis of the full length est peptide. this regulation might also link the biosynthesis of functional telomerase and telomere replications to cell physiology through protein kinases such as snf p and gcn p. inflammation might have a role in erosive esophagitis but not in non-erosive reflux disease the relationship between inflammatory activation mechanisms and acid-peptic injured esophageal tissue is not clear. we evaluated whether there are differences between inflammation and tight junctional proteins such as e-cadherine among subtypes of gastroesophageal reflux disease. the aim of this study was to investigate any possible role of inflammation in pathologic mechanism of reflux disease by determining the inflammatory markers in injured esophageal tissue as well as serum of patients. three groups (erosive-ee, n = ; nonerosive-nerd, n = ; healthy controls-hc, n = ) were evaluated with upper gastrointestinal endoscopy. the esophageal biopsies and blood samples were collected. serum e-cadherine levels, nfkb, chitotirosidase (chit), myeloperoxidase (mpo) activities in serum and homogenized tissues were determined. nkfb levels in tissue was significantly higher in subjects with ee ( . ae . ng/mg.prt) versus hc ( . ae . ng/mg.prt, p = . ). mpo tissue activities in ee group were significantly lower ( . ae . u/mg.prt) than hc ( . ae . u/mg.prt, p = . ) while mpo serum levels were higher in ee ( . ae . ul) versus hc ( . ae . ul, p = . ). tissue chit levels were three fold increased in ee versus hc (p = . ). none of these measurements showed any differences in nerd group. nfkb and mpo levels had a negative correlation (r=À . , p = . ) in tissue. nfkb and ecad levels had a positive correlation in serum (r = . , p < . ). inflammatory process might play a pivotal role in injured mechanism only in erosive esophagitis but not in nerd. noninflammatory mechanisms might be responsible such as hypersensitivity in patients with non-erosive reflux disease. d-dimer (a fibrin degradation product) test is used to aid in the diagnosis of intravascular coagulation. the aim of this study is to investigate the correlation between d-dimer levels and other inflammatory markers including procalcitonin. anonymized data on d-dimer, fibrinogen, hscrp, wbc, neutrophil% (neut%) and procalcitonin levels from , patients (mean age ae sd, . ae . ) were used for the correlation (excel analyze-it v . . ) and linear regression (pasw statistics v . ) analysis between the measured parameters. there was a significant (p < . ) age-dependent increase in d-dimer levels between different age groups. patients with the highest d-dimer levels were also found to have an increased frequency of hscrp levels. d-dimer levels showed a significant correlation with hscrp, wbc and neut%. a model describing the positive association between these parameters were built. the resulting equation is as follows: d-dimer = (hscrp* . ) + ( . *age) + ( . *wbc) + ( . *neut%)À . . correlation analysis between procalcitonin and d-dimer levels gave pearson's correlation coefficient of . . our results suggest that the age-dependent variations should be taken into account while interpreting d-dimer test results. in addition, neut% ratio was found to be the most important parameter for estimating d-dimer levels. our equation can be used when the d-dimer test is not available or for control purposes only. in the field of cancer research great hope lies in finding more powerful and selective way for the direct elimination of cancer cells. this task can be solved by means of nanobiotechnology. recent progress in this field has arisen interest in a carbon nanostructurefullerene c . fullerene exhibits not only unique physico-chemical properties and biological activity but also a significant potential to serve as a nanocarrier for selective drug delivery into cancer cells. the aim of this study is to analyze a unique tool for cancer therapy. the main idea is realized by the non-covalent conjugation of c with the well-known anticancer drug -doxorubicin (dox). two types of conjugate with different c -dox ratio ( : and : ) were studied. conjugates absorbance and fluorescence, size distribution as well as a mass data were recorded utilizing optical and analytical equipment (microplate reader, zetasizer, lc-ms/ms and maldi-tof). in vitro studies were performed including evaluation of c -dox conjugate effects on human leukemic cells (jurkat, ccrf-cem, thp ad molt- ) viability. conjugates accumulation and distribution within cancer cells was monitored using fluorescent microscopy accompanied with fluorescence-activated cell sorting. it was evidently proven that both c -dox conjugates were stable and could be used as reliable candidates for biological application. cellular accumulation and distribution studies showed that conjugation of dox with fullerene promoted its entry into leukemic cells. accumulation of dox in the form of conjugates within cancer cells was intensified compared to the free drug. the results show that conjugated dox is more cytotoxic and the value of its ic are lower compared with the free dox. obtained results confirm nanocarrier function of fullerene c and the perspective of its application for optimization of doxorubicin efficiency against leukemic cells. comperative investigation of protective effects of tea and tea-related wastes on reducing potaential of h o -induced erythrocytes tea processing waste (tpw) formed during the tea production process in tea factories is up to , tones/year in turkey. tpw is one of the abundant available phenolic biomass among plantal wastes. in this study, black and green teas and their wastes were used. the aim of the study is to determinate the phenolic content and the radical scavenging activities of the samples, and to measure their effects on hydrogen peroxide-induced erythrocyte damage due to analyzing the reducing potential of erythrocyte involving glutathione reductase (gr), glutathione peroxidase (gpx) activities and reduced glutathione (gsh) content. total polyphenol content of samples was determined as mg catechine per dry mass by using folin-ciocalteau reactive and dpph radical scavenging activity was estimated by cuendet method as equivalent catechine standard. in erythrocyte, gsh level was measured by method of sedlak and lindsay while gr and gpx activities were assayed by the methods of bergmeyer and beutler, respectively. the highest phenolic content was observed in green tea and its wastes (p < . ) whereas black fiber waste had the lowest phenolic content. therefore, the highest radical scavenging activity and gsh level were detected in green tea and its wastes (p < . ). erythrocyte with the extracts of the teas and their wastes had the similar enzyme activities for both gpx and gr. in sum, the teas and wastes have antioxidant activity but, green tea and its leaf waste hade higher antioxidant activity than other samples. the tea wastes might be evaluated as many of protective health products, particularly in cosmetic fields thus, these by-products no application for any area is expected to become an economical value. fluorouracil ( -fu) is a chemotherapeutic drug classified as an "antimetabolite". it works through irreversible inhibition of thymidylate synthase. chemical derivatization of -fu with carbohydrtates is being investigated widely in order to enhance its bioavailability, therapeutic efficiency and to reduce its toxicity. however, water solubility of the newly derived compounds is usually very low. so, in order to obtain a pharmaceutically relevant formulation they need to be formulated appropriately. in this study, we prepared micellar delivery system for the new tetra-o-acetylglycose derivative of -fu synthesized via "click reaction", namely f -[{ -( ″, ″, ″, ″-tetra-o-acetyl-b-dglycopyronosyl)- h- , , -triazole- -yl}methyl] -fluorouracil. since the water solubility of this compound is very limited, we tested its solubility in several pharmaceutically relevant solvents by visual estimation after stiring increasing amount of the compound in ml of solvent for h. to estimate the carcinogenic potential of this compound, salmonella/microsome mutagenicity assay (ames test) was performed in four histidine-requiring strains of s. typhimurium, tester strains ta , ta (for the detection of frameshift mutations) ta and ta (for detection of base pair substitutions) according to the oecd guideline . the drug was solubilized ( lg/ml) with no precipitation in lutrol Ò -f /ethanol/water ( . : . : . , wt/wt) micelles ( . ae . nm). the results of ames test were negative so the compound neither produced frame shift mutations nor base pair mutations in s. typhimurium strains. the results imply that the new compound can be dissolved in aqueous micellar delivery system in order to be used for further studies, and that it was not mutagenic in the tested s. typhimurium strains. in conclusion, the formulation of the newly synthesized compound is not carcinogenic, and can be evaluated for anticancer activity in vitro and in vivo. integral metabolism parameters of dairy goats during reproductive cycle periods d. solovyeva, e. zarudnaya, s. zaitsev moscow savmb, moscow, russia study of the goat metabolism at different periods of the reproductive cycle allows to correct feeding ration, to increase the age of the productive use of animals and to receive high-quality products. the aim of the work was to determine the metabolic parameters of blood serum of goats, expressed in terms of biochemical parameters and interfacial tensiometry and study their relationship to metabolic processes in the body goats depending on the age and the period of the reproductive cycle. the healthy goats were divided into groups. the dynamic surface tension (dst) parameters were obtained from dependences of a surface tension (r) vs. time (t): at t? (r ), at t = . s (r ), t = s (r ) and t?∞ (r ). this work was supported by the russian scientific foundation ( - - ). all animals had - % fat content. the contents of total protein ( . %), albumin ( . %) and urea ( . %) are higher for the lactating animals as compared to the normal goat values. the levels of total cholesterol ( . %) and creatinine ( . %) are higher for the lactating animals. in lactating animals have the highest level of, which along with high phosphorus level talks about the intensification of energy processes during lactation. the correlations were found between the biochemical and dst parameters of the goat blood: lipids or cholesterol levels with r (r = À . ), r (r = À . ), r (r = À . ); total protein or albumin levels with r (r = À . ), r (r = À . ), r (r = À . ); aminotransferase activity with r (r = À . ), r (r = À . ). the correlations were found between the total protein and albumin levels with k (r = . ), k (r = . ); glucose levels and r (r = . ), r (r = . ). thus, the dst and biochemical parameters of goats have strong correlation relationships that are important for biomedical and veterinary applications. the relation of the severity of atherosclerotic disease with oxidative stress in patients with stable coronary artery disease h. sezen harran university, sanliurfa, turkey introduction: because, to the best of our knowledge, the relationship of total oxidant status (tos) and total antioxidant status (tas) with the severity of stable coronary artery disease (cad) has not been investigated in the literature so far, the present study was conducted to address this issue. materials and methods: this study consisted of consecutive patients and controls who underwent coronary angiography. for each patient, the total gensiniscore (gs) was calculated andthose with a gs of > were classified as the high gs group (hgg), and those with a gs less than were defined as the low gs group (lgg). the total oxidant status (tos) and total antioxidant status (tas) levels were measured using the erelmethod. the osi, which is an indicator of the oxidative balance, was calculated as the percentage ratio of tos to tas. results: the tas was lower in the hgg than lgg. the tos and osi were higher in the hgg than lgg. the correlation analysis showed that gs was negatively associated with the tas and positively with the tos and the osi. the multivariate analysis showed that age, tos, and hdl-c were independent variables for a high gs. the cut-off level of . lmol h o equiv./ l for serum tos levels predicted high gs with a sensitivity of % and a specificity of %. discussion: information on the severity of atherosclerosis is requiredtopredicttheprognosis of an individualpatientandtodetermine the proper treatment modality. the gs system has beenproventodemonstratethe severity of atherosclerotic disease. inthepresentstudy, thepatientswith a high gs had increasedlevels of oxidants. inaddition, tos was an independentindicator of theseverity of atherosclerosis. the optimal cut-offvaluefor tos topredict high-gens score was . (sensitivity % and specificity %). conclusions: the results suggest that the severity of atherosclerosis in stable cad is associated with increased oxidative status. evaluation of roemerine as a multidrug resistance pump inhibitor f. g. avci , c. velioglu , e. recber , c. unsal , g. gulsoy , b. sariyar akbulut marmara university, istanbul, turkey, istanbul university, istanbul, turkey efflux by multidrug resistance (mdr) pumps is a common defense mechanism used against antimicrobials. by pumping the drugs out, these pumps significantly reduce the efficacy of drugs. one approach to overcome this limitation is offered by the combinatorial therapies where drugs are co-administered with together with pump inhibitors. by simply preventing the efflux of the drug, the presence of inhibitors enhance drug efficacy. (-)-roemerine is an aporphine type alkaloid with significant antibacterial (against bacillus cereus, escherichia coli) and antifungal (against candida strains) activities. interestingly, (-)-roemerine was also found to enhance the cytotoxic response mediated by vinblastine in multidrug-resistant kb-v cells. in the same study, this finding was linked to its possible interaction with p-glycoprotein, a eukaryotic mdr pump. taking this finding as the starting point, the current study investigates the potential of roemerine as an inhibitor of the p-glycoprotein homologue pump, bmra, in bacillus subtilis . the antimicrobial agent berberine was used as the model agent since its efficacy is reduced by efflux through mdr pumps. to this end, bacillus subtilis cells were subjected to lg/ml berberine, a value well below the mic. this concentration only slightly retarded growth for hours but then cells resumed their regular growth. upon addition of lg/ml (-)-roemerine to the bacillus subtilis cells treated with berberine, growth pattern changed, indicating possible interaction with bmra. further investigation for the change in the expression of bmra was achieved with real time pcr analysis. glucose oxidase is an enzyme that catalyzes the oxidation of glucose to d-glucono- , -lactone and hydrogen peroxide. we hypothesized that enzyme would cause a double negative effect on cancer cells, by reducing the presence of glucose in cancer microenvironment and producing reactive oxygen species. to increase enzyme stability and enhance cellular uptake we encapsulated the enzyme with a thin acrylamide layer. the purpose of this work was to optimize the synthesis of these glucose oxidase nanoparticles and investigate their effect on cancer cells. nanoparticles containing single glucose oxidase were synthesized in two steps; first by introducing the vinyl groups onto the surface of enzyme by acyloylation followed by polymerization step with acrylamide monomers. encapsulated enzymes are approximately nm in size and retain most of their activity. after the optimization of nanoparticles, the anticancer potency of these nanoparticles was in vitro tested in mcf- breast cancer cell line. according to results, both nanoparticles and free enzyme are capable of inhibiting viability of cancer cells in a similar manner at very low concentrations. currently we are investigating mechanisms involved in this viability inhibition. initial results demonstrated that glucose supplement does not rescue cells from death induced by the activity of glucose oxidase, suggesting an oxidative stress related cause of inhibition. further studies are required to elucidate the exact mechanism. until now there is no determined advantage of glucose oxidase encapsulation against proteolysis. however, encapsulation may induce the accumulation of enzyme in cancer microenvironment. furthermore results suggest that glucose oxidase has a high effect on the viability of mcf- breast cancer cells indicating that this enzyme may have a potential use in cancer treatment. studies on the interaction of human phospholipid scramblase with c-terminal domain of topoisomerase iia u. sivagnanam, s. n. gummadi applied and industrial microbiology lab, bhupat and jyoti mehta school of biosciences, indian institute of technology madras, chennai, india human phospholipid scramblase (hplscr ) is a multifunctional protein that plays key roles in several cellular processes including apoptosis, tumorigenesis, anti-viral defense, cell signalling and several protein-protein interactions. it has been shown that hplscr interacts with the c-terminal domain of topoisomerase iia (topo iia) and enhances its decatenation activity in vitro. the interacting region in topo iia was identified but till date, no reports exist on the binding region in hplscr . this study aims to identify the region of hplscr that interacts with topo iia. to identify the topo iia interacting sites in hplscr , nterminal deletion constructs of hplscr viz Δ -hplscr , Δ -hplscr , Δ -hplscr , Δ -hplscr and Δ -hplscr were generated by pcr, cloned, overexpressed and purified to homogeneity using ni + -nta purification. the cterminal domain (ctd) of topo iia was cloned in pgex p- and was expressed as a gst fusion protein. gst pull down assays will be performed with the deletion constructs of hplscr and the gst-ctd-topo iia. the binding region in hplscr will be confirmed by peptide competition assays. our initial results show that the decatenation activity of topo iia was enhanced when the topo ii was pretreated with hplscr . Δ -hplscr did not show any enhancement of the decatenation activity compared to full length hplscr . hence, the binding region could be in the - region of hplscr . further deletions were done in the - region of hplscr as described earlier. gst-pull down assays and decatenation assays will be performed for the deletion constructs to narrow down the region of hplscr that binds to topo iia. we conclude that hplscr interacts with and enhances the activity of topo iia and the - region of hplscr is critical for enhancement of decatenation activity. further work is under progress to identify the exact topo iia binding region of hplscr and the physiological relevance of this interaction in the cell. a. ugur kurtoglu , v. aslan , e. kurtoglu department of biochemistry, antalya education and research hospital, antalya, turkey, department of hematology, antalya education and research hospital, antalya, turkey beta-thalassemia is a common autosomal recessive disorder resulting from over different mutations of the beta-globin genes. our aim was to creat a mutation map of beta thalassemia in province of antalya, turkey. in this study, mutation analysis of a total of beta-thalassemia patients followed up at the thalassemia center of the antalya education and research hospital, antalya, turkey, were included. according to our results, the ivs . is the most frequent mutation type in our province same as other geographical regions of turkey. the most frequent mutations in heterozygous or homozygous patients are ivs . , ivs . , ivs . and ivs . . our results indicate the importance of micromaping and epidemiology studies of thalassemia, which will assist in establishing the national prevention and control program in turkey. keywords: beta-thalassemia, beta-globin gene, mutation p-mis- investigation of the in vitro effects of some antibiotics on the purified beta-glucosidases from the rat liver n. t€ urkmen , h. kara karadeniz technical university medical biochemistry department, trabzon, turkey, balikesir university veterinary faculty biochemistry department, balikesir, turkey beta-glucosidases catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in b-d-glucosides and oligosaccharides.b-glucosidases are widely distributed in the living world.b-glucosidases which in mammals, primarily found in the liver and kidneys;lysosomal b-glucosidase (gba ),non-lysosomal b-glucosidase (gba ), cytosolic b-glucosidase (gba ),intestinal lactase-phloriz the hydrolase (lph). liver tissues of wistar-albino rats were homogenized with homogenizer in the extraction buffer and crude extract was obtained after centrifugation.ammoniumsulfate precipitation range designated crude extract was purified by sepharose b-ltyrosine- -naphthylamine hydrophobic gel.commercially availabled antibiotics were prepared with substrate buffer.it was investigated inhbition effects of cefuroximesodium, ampicillin-sulbactam, amoxicillin trihydrate/potassium clavulanate, cefazolin sodium, gentamicin sulfate and ceftriaxone disodium antibiotics onto gba .inhibition types and k i values of related antibiotics were determined with p-npg substrate.lineweaver-burk plot was used for that purpose. rat liver gba was purified at . -fold with . % yield.gba was illustrated and kda at sds-page.ic value of ampicillin/sulbactam antibiotic for gba was found . mg/ml with competitive type inhibition and other antibiotics didn't inhibit. purfication methods are being used in the literature for the purified b-glucosidase from different sources.purified gba was illustrated and kda at sds-page.about molecular weight of bglucosidases is presented different information in the literat€ ure. this has been reported because of acid beta glucosidases are abnormal migration at the acrylamide or agarose gels.it was investigated inhbition effects of various antibiotics onto purified gba .ampicillin/sulbactam antibiotic inhibited to purfied gba at the competitive type.similiar antibiotics studies have been made in the literature for different enzymes. effect of glutamine on insulin resistance and endoplasmic reticulum stress g. aydogdu , p. b. sermikli , a. abbasi taghidizaj , e. yilmaz ankara university, institute of science, ankara, turkey, ankara university, biotechnology institute, ankara, turkey obesity and diseases are one of the most important public health problems of the world.excess fat storage in adipocytes leads to the release of increased amounts of non-esterified fatty acids, glycerol, hormones, cytokines, which are factors involved in the development of insulin resistance that cause type diabetes. one of the major differences between obese and lean individuals is the amino acid concentration in the circulation. although there are many studies about the amino acid metabolism associated with insulin resistance in obese individuals, the effect of glutamine metabolism in insulin resistance mechanisms are not well understood yet. glutamine can be used as fuel and its levels in tissues and circulation can regulate cell responsiveness to insulin and cellular metabolism. therefore, glutamine is a potentially important factor that might help us better understand insulin resistance and type diabetes. to determine whether glutamine effect on insulin resistance and endoplasmic reticulum stress, t -l cell is treated with different concentration of glutamine and analyzed by western blot for er stress markers. our results indicated that glutamine reduced endoplasmic reticulum stress and related with that attenuated insulin resistance. in case of transport of amino acids, insulin resistance, how it is affected when we have the information about the important tips on energy requirements and metabolism reach insulin resistance and type- diabetes treatment is likely to reveal a possible new targets. how does different lead levels affect tsh, ft , ft , vitamin b and folate? e. ozkan ankara occupational disease hospital, ankara, turkey exposure to heavy metals is increasing with the industrialization of society. one of the most intense exposure to heavy metals is pb on this issue. this study was aimed to determine the relationship between different blood pb levels and serum thyroid hormones (th), vit b , folate. the cases were - years old, male individuals who admitted to our hospital between april -march for periodic inspections because of occupational exposure to pb. the parameters of the cases were retrospectively retrieved. according to their pb levels, exposed workers (n: ) were divided into four subgroups; group (g) : - . lg/dl, g : - . , g : - . , g : ≥ . from these, the number of cases whose th levels were measured (n: ) given respectively; g : , g : , g : , g : cases. also the number of cases whose vit b and folate levels were measured (n: ) given respectively; g : , g : , g : ,g : cases. levels of pb were determined by icp-ms. th, vit b , folate were determined by cmia. between the groups formed according to pb levels, there was no significant difference in terms of average t , tsh and vitamin b (p > . ). on the other hand there was statistically significant difference between t and group , , (p < . ) but there was no difference between group (p > . ). the average folate belongs to the first group was about % higher than the other groups, and found that the difference was statistically significant (p < . ). there are many publications which have various results between pb levels and t ,t , tsh. but this study is important to compare the effect of different levels of pb. up to day there was no publication about the relation between different pb levels and vit b , folate. it was seen that there was no significant clinical relation between different pb levels and thyroid parameters, vit b . but the low levels of folate in the high pb levels groups shows us that we need further studies about this relationship. fluorescent study of in meso crystallization of membrane proteins with the introduction of membrane protein in meso crystallization years ago by landau and rosenbusch, a new era of membrane protein structural research has emerged ( ). since that time this method became associated with a number of major breakthroughs in the field ( ) including exceptional successes in structural studies of microbial rhodopsins and g-protein coupled receptors ( ) . here we used fluorescence microscopy to study in meso crystallization process of bacteriorhodopsin. several observations provide new insights into the in meso crystallization process. the crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. these observations demonstrate an ostwald ripening mechanism of the in meso crystal growth. the depletion zone formed around the growing crystal is consistent with the previously proposed analogy relating in meso crystallization with the crystallization in a microgravity convection-free environment. this work is supported by rsf - - . ( ) landau, e. m.; rosenbusch, j. p. proc. natl. acad. sci. u. s. a. , , À . ( ) caffrey, m. acta crystallogr., sect. f: struct. biol. commun. , , - . ( ) katritch v., cherezov v., stevens r.c. ( ) . annu rev pharmacol toxicol , - . p-mis- stamp is critical for both ar and mtor signaling in prostate cancer cells x. sheng, y. jin, f. saatcioglu university of oslo, oslo, norway androgen receptor (ar) signaling plays a central role in the initiation and progression of prostate cancer (pca), including when the disease progresses to castration-resistant pca (crpc). the second central signaling pathway in pca, similar to various other cancers, is the pi k-akt-mtor signaling. importantly, these two oncogenic pathways cross-regulate each other in pca cells by reciprocal feedback, thereby maintaining tumor cell survival even when one is suppressed. we have previously identified that the six transmembrane protein of prostate (stamp ) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk mapk signaling. human pca cell lines lncap and vcap were used in the study. colony formation, soft-agar growth, prostatosphere formation assays were performed. for in vivo xenograft experiment, the cells were implanted subcutaneously into the flanks of nude mice. here, we show that stamp knockdown caused defects in colony formation, anchorage-independent growth and prostatosphere formation in lncap and vcap cells both in vitro, as well as tumor formation and growth in vivo. this may be due to the impaired ar and mtor signaling in these cells upon stamp knockdown. interestingly, in the crpc cell line rv , where-stamp knockdown did not affect mtor signaling, there was a remarkable repression of tumor take rate and growth. these results clearly indicate that stamp is essential for both ar and mtor signaling, and is crucial for pca growth in vitro and in vivo. however, the detailed molecular mechanism requires further investigation. taken together, these data unveil a critical role for stamp in coordinating the ar and mtor signaling pathways in pca cells, solidifying the basis for its pro-survival effects in pca, including in advanced disease. quantification of d thin layer chromatograms using d gel analysis software and gel documentation system o. kaynar, m. ileriturk, d. kaynar, s. kurt ataturk university, erzurum, turkey introduction: thin layer chromatography (tlc) is an important chromatographic technique that is widely used as a cost-effective method for rapid-sensitive analysis of compounds in plants, animals, and humans. however, one dimentional ( d) tlc is not sufficient for the separation of complex compounds. therefore, two-dimentional ( d) tlc was developed. the quantitative evaluation of plates are performed with tlc scanners or documentation systems. however, these systems specific for d plates, and cannot be adapted to quantitative evaluations of d plates. in this study, the applicability of the gel documentation systems and d analysis software for the analysis of d tlc plates were examined. material and method: d tlc of lipids: st dimension: methyl acetate/n-propanol/chloroform/methanol/ . % kcl ( / / / / v/v); nd dimension: chloroform/methanol/acetic acid/ water ( / / / v/v); detection: charring. d tlc of aminoacids: st dimension: . % (v/v) formic acid; nd dimension: toluene/glacial acetic acid ( : v/v); detection: uv. phospholipid and aminoacid standards, each include different classes were developed by d tlc. plates visualized with biorad geldoc xr, and band volumes on plates were calculated with biorad pdquest d gel analysis software. for the method validationa) plates containing same lipid classes were developed in the same day, and results were used for the calculation of intra-assay cv;cv% = average of each sample standard deviation/mean of sample b) plates containing same lipid classes were developed in different days, and results were used for the calculation of interassay cv; cv% = standard deviation of each sample average/mean of the plates results: volume of each phospholipid and aminoacid had less than % intra and inter-assay cv. conclusion: gel documentation system with d gel analysis software can be used for the quantitative analysis of the d tl plates both at uv and visible light. the role of na + k + atpase activity in the vasodilatory effect of n-acetylcysteine introduction: spasm occurred at the stage of and after the preparation of arterial grafts used in coronary artery bypass surgery (cabg) is effective on morbidity and mortality in the first hours of postoperative patients. n-acetyl cysteine (nac) that vasodilatory effect is known,may be considered as a suppressor agent for vasospasm developing during cabg. however, for the prevention of complications that may arise during or after cabg mechanism of these vasodilatory effects should be described. this study was aimed to investigate the role of atpase enzyme on the vasodilatory effect of nac. materials and methods: in this study, adult male wistar albino rats were used. rats were separated into four groups as control rats (g ), mm nac (g ), mm nac (g ) and mm nac (g ). a portion of the thoracic aorta isolated from rats was used for the relaxation response recording, and the other portion was used for measurement of nakatpase activity. isolated smooth muscle rings are suspended in the ml organ bath containing krebs solution for relaxation responses. in all groups, level of smooth muscle contraction were allowed to reach a plateau by adding mm kcl to the organ bath. then, in the first minutes of application relaxation responses which created by adding nac to the medium were recorded and the maximum relaxation responses were measured. nak atpase activity was determined using the mazzanti method. groups means were compared by one-way analysis of variance (anova). the threshold for statistical significance was set at . . results: the contraction force decreased in all nac dose groups compared to control group and this reduction was statistically significant (p < . ). similarly, nak atpase activity is also decreased in a dose dependent manner (p < . ). the findings obtained in this study suggest that vasodilator effect of nac formed in thoracic aortic smooth muscle was associated with the activity of the enzyme na k atpase. in the presented study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic activity. a bacillus uk , which was isolated from the soil samples taken from farmland on kahramanmaras sutcu imam university campus, showed high keratinolytic activity when cultured on feather meal medium. the enzyme activity was studied in the ph range of . - . . the optimum temperature for keratinase activity was investigated by varying the incubation temperature between °c and °c. optimum keratinolytic activity was observed at °c and ph . . the enzyme was stable at °c. the activity was investigated in the presence of some chemicals, including sds, tween , dmso, triton x- , edta, nacl, zncl , cacl , glucose. the keratinolytic activity was inhibited by all chemicals tested to some degree. the molecular weight of keratinase was determined by polyacrylamide gel ( %) using standard molecular weight marker and estimated about kda by sds-page. the keratinase isolated from bacillus uk could be used in biotechnological processes i.e. feather degradation, wastewater treatment and in industrial processes, such as detergent, food and leather industries. introduction: hemoglobinopathies, including thalassemia, abnormal hemoglobins, constitutes a major group of inherited disorders of hemoglobin synthesis. the reduced or absent of the beta (b) or alpha (a) globin chains of the adult human hemoglobin molecule results beta or alpha thalassemias, leading to imbalanced a-globin/non a-globin chains. the aim of this study was to give a quik desicion with a/b-globin mrna ratio for sequencing of a or b gene, when the anemia is not detectable. materials and methods: mrna and cdna extraction of bthalassemia and a-(including two of . kb del./hbs) thalassemia subjects and normal controls were accomplished using the high pure rna isolation kit and transcriptor first strand cdna synthesis kit, respectively, following the manufacturer's instructions. we used cdna as a template in the real-time pcr amplification using primers specific for a, b globin genes. amplification was performed in a lightcycler Ò instrument. the a/b-globin mrna ratio of each sample was calculated based on the Àddct method. results: a/b-globin mrna ratios calculated in a-thalassemia subjects relative to normal control as a result of numbers of defective a-globin genes. the a/b-globin mrna ratio was found higher in b-thalassemia subjects. coinheritance of a-thalassemia in hb s subjects concluded a stabile a/b-globin mrna ratio as per a-thalassemia or b-thalassemia subjects. discussion and conclusion: instability in a/b-globin chains is a significant factor of thalassemia disease severity and can be used before deciding type of gene sequencing when the anemia is not detectable. this study indicates that imbalance in globin gene expression could be demonstrated by measuring a/b-globin mrna ratio, which was conveniently and accurately determined by qrt-pcr and give an information about globin gene function which gene should be correct to investigate an individual for globin gene mutation. p-mis- self-assembling peptides mimic supramolecular biochirality r. garifullin , , m. o. guler bilkent university unam, institute of materials science and nanotechnology, ankara, turkey, kazan federal university, institute of fundamental medicine and biology, kazan, russia supramolecular chirality is rooted in asymmetric spatial arrangement of structural elements (e.g. molecules or units with higher hierarchy). self-assembled systems giving rise to this kind of chirality are of great importance because they closely resemble natural biological systems and potentially can lead to new advanced functional materials. in the process of self-assembly, both molecularly chiral and achiral structural units can organize into chiral nanostructures. chiral arrangement of chromophore molecules in space is known to result in emergence of chiroptical properties of a chromophore. organization of pigment-protein complexes into macrodomains in green plants gives rise to biochirality emanating from long-range chiral order of complexes. owing to this order, macrodomains start to absorb circularly polarized light intensively and thus exhibit huge circular dichroism (cd) signal. in our study, a simple approach which was aimed at mimicking the biochirality phenomenon makes use of self-assembling peptide amphiphiles and their interactions with pyrene chromophore. designed peptide amphiphiles are capable of self-assembly into nanofibers with chiral interior, which in principle gives an opportunity to achieve long-range chiral order. two modes of interactioncovalent and noncovalentwere utilized in order to induce supramolecular chirality. covalent interaction mode included direct covalent attachment of pyrene to peptide sequence. upon self-assembly of peptide amphiphile into nanofibers intense circular dichroism phenomenon was observed. noncovalent interaction mode envisioned encapsulation of pyrene molecules in the hydrophobic core of nanofibers of another peptide amphiphile. co-assembly of peptide amphiphile and pyrene molecules led to chiral order and intense cd signal. in addition, it was possible to control the sign of cd signals by using either of peptide isomers, l or d. p-mis- pon activity in hdl subgroups of obese, overweight and normal weight subjects objective: the aims of this study were isolation of hdl-c subgroups by using precipitation method, determination of pon- activity in both total and hdl subgroups, and evaluation of performance characteristics of pon- activity measurement method in newly diagnosed obese, overweight and normal subjects. material and methods: the study population consists of newly diagnosed obese, overweight and normal subjects. fasting morning blood samples were taken from all study groups. hdl subgroup was obtained by heparin-mn-dextran sulphate precipitation method and cholesterol was measured with direct (homegenous) hdl-c method. hdl -c concentrations were calculated with the subtraction of hdl -c from total hdl-c. hdl -c and total pon- activity were determined by using eckerson method. non-hdl pon- activitiy was calculated with subtraction of hdl pon- activity from total pon- activity. results: total hdl-c, hdl -c and hdl -c concentrations and the activity of total pon- and hdl pon- were found lower in obesity according to overweight and normal subjects (p < . ). negative correlations were found between body mass index and hdl -c, total pon- and hdl pon- (r = À . , p < . ; r = À . , p < . ; r = À . , p < . , respectively). conclusion: our findings indicated that hdl-c metabolism and lipoprotein associated antioxidant defense mechanisms were adversely affected with obesity. in conclusion we think that precipitation method using for separating hdl subgroup, is simple and cost effective for routine applications in clinical laboratories. besides hdl -c measurements, pon activity, measurement of total and hdl -c subgroup might be helpful to evaluate the atherosclerotic process in obese subjects. keywords: obesity, body mass index, paraoxonase, hdl subgroup, cholesterol p-mis- hepatitis e virus antibody prevelance among persons who work with animals in north cyprus introductions: hepatitis e infection is a major cause of viral hepatitis in many developing countries. the objectives of the present study was to determine the seroprevelance of hev infection in peoples who work with animals in northern cyprus. materials and methods: prevelance of hev infection were determined in study group population: persons without occupational exposure to animals; persons who work with animals; veterinarian and butcher. a total of blood samples were collected. all serum samples were tested elisa using a commercially available kit according to the manufacturer's instructions. ti-test were used for istatistical analyses. p > . was accepted as significant value. results: in a study of blood donors ( male, female), the overall prevelance of anti-hev igg antibodies were . %. the blood samples were collected different areas. the prevelance of anti-hev igm antibodies was . % and he was years and acting a butcher during years. the prevelance of anti-hev igg of women were approximately two fold higher than men. no significant difference in anti-hev prevelance was observed between the age of the blood donors. according to the anti-hev igg prevelance, the without occupation expose to animal animal were %, the animal husbandry were % and the veterians and the butcher were % were found. discussion: the prevelance of anti-hev in the north cyprus ( %) was found low such as the prevelance of the turkey ( %). the prevelance of anti-hev igg in animal husbandry were higher that the other groups because of they may be more spend of time and contact with animals. the prevelance of igm results suggested that the possibility of outbreaks may be low in north cyprus. conclusion: this study was the first seroprevelance analysis of north cyprus according to the population number.the further studies could be included the seroprevelance of anti-hev from the animals. most errors in the clinical laboratory occur in the preanalytical phase the aim of this study was to investigate the causes and rates of rejected samples, regarding to certain test groups in our laboratory. this study was designed on the rejected samples between january and january . clinical chemistry, coagulation, hormone, cardiac markers, total urine evaluation and other (ethanol level, hba c, hb electrophoresis, neonatal bilirubin, drug level, blood gas, fecal occult blood) test groups were included. the total number of specimen and rejected samples was obtained from the hospital information system retrospectively. types of inappropriateness were evaluated as follows: erroneous coding, clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen. it was determined that blood samples were sent to our laboratory in one-year period. . % of them were rejected because of preanalytical errors. erroneous coding was found as the most common rejection cause ( %). rejection rates of clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen were found to be %, %, %, %, % and % respectively. in our study, erroneous coding was the most common cause of preanalytical errors. education of medical secretaries is relevant and important as can be seen in the decrease of sample errors and the resulting quality improvement. glycosylated hemoglobin test (hba c) is important for screening, diagnosing, and monitoring diabetes and prediabetes. however, hba c levels may dependent on patient ethnicity suggesting that the diagnostic cut-offs should be evaluated for specific populations. therefore, our aim in this study was to evaluate the efficiency of hba c for predicting diabetes in comparison to oral glucose tolerance test (ogtt) results for turkish population. the study included anonymous lab results (acibadem labmed laboratories in turkey) of patients ( female, male) aging . ae . years ( - ) who had an initial diagnosis of diabetes. glucose and insulin levels during ogtt were measured after the initial administration of g sugar ( hour), -hour and -hour. these parameters were statistically analyzed in comparison to simultaneous hba c results. glucose measurements at hour had better distinction power (p < . ) between these individual groups than initial and -hour glucose measurements. the average hba c (%) levels for healthy, pre-diabetic and diabetic individuals were . ae . , . ae . and . ae . , respectively. roc curve analysis showed . % sensitivity and . % specificity for the clinically accepted hba c cut-off value of . %. hba c cut-off value of . % had a higher sensitivity of . % and comparable specificity of . %. the highest discrimination power between healthy, pre-diabetic and diabetic individuals was observed at glucose concentration at -hour after sugar administration in ogtt test as opposed -hours generally used for diagnosis. low sensitivity was observed for the clinically adapted . % cut-off value of hba c. the cut-off value of . % for hba c was found to be more sensitive with comparable specificity than the . % cut-off values for diabetes screening in our population. our results suggest that . % for hba c should be considered for diabetes cutoff value for turkish population. induction of the glutathione-dependent detoxification capacity is involved in the hepatoprotective effect of silymarin against acetaminophen-induced hepatotoxicity y. kim, d. kwon, c. ahn seoul national university, seoul, south korea recent findings in this laboratory showed that silymarin was capable of promoting hepatic glutathione (gsh) synthesis via a modification of the transsulfuration reactions in the liver. to investigate its pharmacological significance, we examined the hepatoprotective effect of silymarin against liver injury induced by acetaminophen (apap). adult male mice were treated with silymarin ( mg/kg, po) every hours for a total of doses prior to an apap challenge ( mg/kg, ip). the apap-induced liver injury was assessed by histopathological examination and measurement of changes in plasma enzyme activities, lipid peroxidation and formation of nitrotyrosine protein adducts in the liver. plasma levels of apap and its major metabolites were monitored for hours to estimate the metabolic transformation of apap. also protein and activity of the major cyp subtypes involved in the metabolic activation of apap into a toxic metabolite were determined in liver of the mice treated with silymarin only. silymarin pretreatment attenuated the apap-induced liver injury significantly when determined hours later. plasma concentrations of apap, apap-glucuronide or apap-sulfate in plasma were not changed, but thiol conjugates of apap, such as apap-glutathione, apap-cysteine and apap-n-acetylcysteine, were elevated significantly in the mice pretreated with silymarin. however, silymarin treatment did not affect protein expression of cyp e , cyp a , or cyp a in the liver. also hepatic microsomal enzyme activities measured using p-nitrophenol, ethoxyresorufin and erythromycin as substrates, were not increased by silymarin, indicating that the elevation of apap-thiol conjugates should be attributed to an augmentation of the gsh conjugation capacity. it is suggested that silymarin may protect the liver against an electrophilic substance-induced toxicity by increasing gsh availability which would enhance the detoxifying capacity of liver cells. prostate cancer (pca) is the second leading cause of death among men in western countries. we have previously found that the six transmembrane protein of prostate (stamp ) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk/mapk signaling. we also found that stamp is highly mobile in pca cells and shuttles between the plasma membrane and the golgi, often found in vesiculotubular structures in the cytosol. using advanced imaging techniques, we have now characterized the trafficking of stamp from the plasma membrane to early endosomes in lncap cells, by analysing its dynamic targeting to the three main endocytosis pathways: clathrin-mediated endocytosis, caveolae/lipid rafts, and the arf -dependent pathway. we found that stamp fused to cyan fluorescent protein (cfp-stamp ) is present at the plasma membrane where it accumulates in punctate structures. live cell confocal imaging showed that these puncta were dynamic over time indicating that stamp may be constitutively delivered to the plasma membrane and removed from it by endocytosis. co-expression of cfp-stamp with various fluorescent protein markers revealed that cfp-stamp puncta corresponded to lipid rafts that were labelled with caveolin- -rfp or antibodies against flotillin. live cell imaging showed that cfp-stamp and caveolin- -rfp disappeared at the same time from the same region of the plasma membrane suggesting that lipid rafts are likely to be responsible for stamp internalization. notably, stamp was absent from other endocytosis structures such as clathrin-coated pits/vesicles. further work is needed to determine whether stamp internalization is required for its function, such as its link to erk signaling, and whether interference with lipid rafts influences stamp effects on pca cell proliferation and survival. antithrombin-iii, mpv and plasma total homocysteine levels in behcet's disease introduction: behcet's disease is a multi-systemic and chronic inflammatory vasculitis of unknown etiology characterized by recurrent oral and genital ulcers, uveitis, arthritis, arterial aneurysms, venous thrombosis and skin lesions. platelet indices such as mean platelet volume (mpv) is a standart indicator of platelet function in disease pathophysiology. antithrombin, a glycoprotein synthesized in the liver, is the major plasma inhibitor of thrombin thus modulating blood coagulation. antithrombi-iii (at-iii) is a enzyme even moderate deficiency significantly increases the risk of thrombosis. homocysteine (hcy), that is formed during the metabolism of methionine. several clinical studies have clarified that elevated blood hcy levels are related to atherosclerotic disease. in our study, we investigated ovocystatin is one of the best characterized members of cystatin superfamily of protease inhibitors, and it has been frequently used for pathophysiological studies as the model protein, representative for this superfamily. its application has been supported by high structural similarity to human cystatin c as well as several common biological activities. as regard to biological activity, cystatins, including ovocystatin, are best characterized as inhibitors of cysteine proteases of papain family (c ), such as cathepsins b, h, l and s. these inhibitors participate in intra-and extracellular control of proteolytic events, both in physiological and pathological states. in the recent decade also new activities of cystatins, not assigned to inhibition of papain-like cysteine cathepsins, were found. these activities are associated with an alternative active center for legumain-type proteases in the molecule. here we report a chemical modification of ovocystatin that disables the anti-papain activity of the inhibitor but does not affect its anti-legumain activity. the chemical knockout has been obtained by reaction with -hydroxy- -nitrobenzyl bromide (hnbb) that covalently modifies the trp residue in the molecule. the reaction has been monitored by uv-vis and fluorescence spectroscopy. the anti-papain activity of the inhibitor has been measured colorimetrically against bana as a substrate. the anti-legumain activity was assessed fluorometrically using z-ala-ala-asn-amc. the reacted inhibitor exhibited an additional, characteristic for hnbb, band at nm in uv-vis scan. accordingly, an ablation of trp fluorescence was also observed. the molecule fully retained the anti-legumain activity, while only residual antipapain activity ( %) was observed. the modified ovocystatin can be a useful molecular tool for studying the physiological and pathological processes specifically associated with legumain activity. departments of medicine (hematology/oncology) and biochemistry and molecular biology, university of louisville, james graham brown cancer center, louisville, ky, united states -phosphofructo- -kinase/fructose- , -bisphospatase (pfkfb) family of enzymes are responsible for the conversion of fructose- -phosphate (f p) to fructose- , -bisphosphate (f , bp) and vice versa, and f , bp is an allosteric activator of phosphofructokinase- (pfk ), a rate-limiting enzyme of glycolysis. among the four identified pfkfb isozymes (pfkfb - ), pfkfb is the least studied isozyme in human cancers. there exists two different splice variants of pfkfb , variant- and variant- , coding two different isoforms, isoform a and b, respectively. in this study, we first analyzed the effect of k-ras(g d)induced oncogenic transformation on pfkfb expression in pancreatic duct cells. we found that oncogenic k-ras induction in immortalized pancreatic duct cells (ipde) was associated with decreases in total pfkfb mrna and protein expressions (mrna; ipde: ae . ; ipde+kras: . ae . and protein; ipde: ipde+kras: . ). we then, checked individual expressions of splice variants and observed that while pfkfb splice variant- (p -v ) expression was reduced by k-ras induction (ipde: ; ipde+kras: . ), pfkfb splice variant- (p -v ) expression was increased (ipde: ; ipde+kras: . ). then, we checked effects of p -v and p -v on glycolytic phenotype of ipde and ipde+kras cells. over-expression of pfkfb variants increased f , bp concentration (p -v : . ; p -v : . fold; compared to empty vec), glucose uptake (p -v : %; p -v : %) and glycolysis (p -v : %; p -v : %) in ipde+k-ras cells. we next analyzed the subcellular localizations of pfkfb isoforms and observed that both pfkfb isozymes localize to the nucleus, with more prominent nuclear localization of p -v compared to p -v . also, nuclear localization ratio of p -v increases after oncogenic transformation with mutant k-ras. taken together, these results suggest that pfkfb may have a role in the glycolytic phenotype of pancreatic cancers characterized with hyperactive k-ras signaling. effects of p map kinase inhibitors on mda-mb- cell line introduction: p mapk phosphorylates serine and/or threonine residues of the target proteins. the activation of p mapk leads to cell growth, differentiation, survival or apoptosis. in this study, we tested the effect p mapk sb and sb on mda-mb- cells to further elucidate the controversial role of p mapk on cell proliferation or cell migration. materials and methods: mda-mb- cancer line was cultured in rpmi- supplemented with % fbs. the cytotoxic and cell migration effects of sb and sb inhibitors were tested by mtt assay and wound assay, respectively. the effects of both inhibitors on proliferation and adhesion of md-mb- cells were determined by icelligence system. results: it was found that sb p map kinase inhibitor was more effective than sb . however, no significant effects of low doses of lm and lm of both inhibitors were seen on cell proliferation as compared to the dmso-treated control cells for up to hours as determined by icelligence system. on the other hand, both sb and sb significantly prevented cell proliferation at a concentration of lm. both sb and sb significantly reduced cell migration in a time-dependent manner at a concentration of lm. then, we tested whether each p mapk inhibitors have any effect on cell adhesion during a treatment period of hours using icelligence system. only lm concentration of sb reduced cell adhesion for about . hour (p < . ). conclusion: p mapk inhibitors sb and sb differentially affect cell proliferation, survival and migration. acknowledgements: this study is financially supported by dumlupınar university, scientific research project no - . mutagenicity of a series efficacious benzoxazine derivativesa new approach to evaluate ames test data e. foto , f. zilifdar , s. yilmaz , t. sarac ßbasi , i. yalc ßin , n. diril hacettepe university, ankara, turkey, ankara university, ankara, turkey testing safety of drug candidates is as crucial as evaluating their efficacy in early drug development. we previously synthesized a series of , -benzoxazine- -one derivatives showing significant antimicrobial, in vitro anticancer, topoisomerase i inhibitory activities and studied their several mechanisms of action. in this present study, we have evaluated mutagenic activities of these compounds and their potential metabolites. moreover, we aimed to develop a new statistical algorithm available for structureactivity relationship analysis to identify the regions responsible for the activity. to evaluate mutagenicity of the compounds, ames salmonella/microsome test was used. salmonella typhimurium ta and ta strains were used to detect for frameshift and basepair substitution mutagens, respectively. additionally, mutagenicity of potential metabolites of them were evaluated by adding metabolic activation system (s ) which was prepared from a pool of male sprague dawley rats. results were evaluated with student's-t test. following regression model estimation analysis, we detected minimum mutagenic doses of all tested compounds for generating a d-common features pharmacophore model with hiphop method. according to the results, only bs , bs , bs and bs exhibited strong mutagenic effects on both strains in the presence and absence of s . additionally bs , bs , bs and bs (in the absence of the s ), bs , bs and bs (in the presence of the s ) showed weak mutagenic effects on ta . hiphop analysis results revealed that mutagenicity was increased in the presence of aromatic desactivating groups which might form hydrogen bonds at the position of r and hydrophobic groups at the position of r of the benzene ring in the structure of benzoxazine. the new statistical approach developed in this study can be useful for assessing the ames test data available for structure activity relationship analyses. background: recently more than thirty different diseases can screen simultaneously with expanded newborn screening (nbs) programs by tandem ms.expanded nbs with tandem ms is performed routinely at akdeniz university hospital central laboratory since .the aim of this study was to evaluate our nbs results with some second-tier and confirmatory tests. materials and methods: nbs results (n = ) were evaluated in dried blood samples which sent to our laboratory for the study between august and august . electrospray ionisation (esi)triple quadrupole mass spectrometer (shimadzu lc-ms/ms ,japan) was used for nbs analysis,acylcarnitine and amino acid profile were screened with mrm (multiple reaction monitoring) spectrum within minutes.second-tier tests were performed as urine organic acid analysis by gas chromatography-mass spectrometry (gc-ms),plasma and urine quantitative amino acid analysis by high pressure liquid chromatography (hplc).pathological nbs results were assessed in three separate groups as amino acid metabolism disorders, fatty acid oxidation defects and organic acidemias. results: metabolic diseases were found in ( . %) patients by the second-tier tests performed.there were detected amino acid metabolism disorders in ,organic acidemia in ,fatty acid oxidation defects in patients. conclusions: the reason of high positive results in our laboratory could explain that our study includes both screening and monitoring of previously diagnosed metabolic patients.nbs is performed in only a few centers in turkey although there were the national screening programs included nbs in many foreign countries.more expanded nbs programmes in our country is required to start treatment of patients before irreversible damage is not occured. although many reports indicate the involvement of calpain in several human pathologies, it is not yet clarified how the protease can recognize the substrates to digest and how can escape to its natural inhibitor calpastatin. answers to these questions have been obtained by identifying specific intracellular localizations of calpain and its substrates and analyzing the interactions of the protease with calpastatin. these studies were carried out using human sknbe neuroblastoma cells. protein-protein interactions and intracellular localization of calpain and the related proteins were determined by immunoprecipitation and isolation of membrane microdomains. we have observed that small amounts of calpain- are localized in lipid rafts microdomains together with n-methyl-d-aspartate receptor (nmdar) containing nr /nr b subunits. immunoprecipitation experiments have demonstrated that nmdar containing nr /nr b subunits, calpain- , hsp and neuronal nitric oxide synthase (nnos) but not calpastatin and calpain- are present in specific protein complexes. thus, in this localization calpain activity is regulated by hsp that reduces the affinity for ca + of the protease. cell stimulation with nmdar agonists induces calpain activation that specifically cleaves the subunits nr b of the receptor promoting changes in lipid rafts organization and internalization of nmdar without affecting cell viability. moreover, in these conditions, also nnos is digested and converted in the active form by calpain- . our data suggest a physiological role of calpain- at specific cell sites. the protease inserted in lipid rafts microdomains is in strict contact with its targets and escapes to calpastatin which is not inserted in these structures. following an increase in ca + influx, the activated protease regulated by hsp , promotes the removal of nmdar from the plasma membranes, decreasing ca + entrance through this receptor-channel and protecting cells from ca + overloading. tissue transglutaminase (tg ) is a multifunctional protein complex that can act as a crosslinking enzyme, gtpase/atpase, protein kinase and protein disulfide isomerase. at the cell surface, tg was shown to be involved in adhesion, migration, invasion, growth, epithelial mesenchymal transition and hence implied in the metastatic development of many different tumor types. renal cell cancer (rcc) is one of the most common type of cancer in adult males that generally grows as a single tumor within a kidney. our previous findings indicate that the increased expression of tg in rcc results in tumor metastasis with a significant decrease in disease-and cancer-specific survival outcome. herewith, the role of tg in cell migration of rcc was investigated in this study by transducing the model rcc mouse cell line renca with a series of tg mutant constructs. renca cells were transduced by lentiviral particles encoding wttg , transaminase-defective tg -c s form with low gtpbinding affinity, gtp-binding deficient form tg -r a, and transaminase-inactive tg -w a. in order to investigate the role of tg transamidating and gtpase activity in cell migration, scattering assay was used where colonies for each mutant clone was followed for a time interval of hours. our results showed that non-transduced control and tg -c s mutant renca cells demonstrated a similar migration pattern with a % of scatter activity. on the other hand, % colonies formed by renca cells overexpressing wttg and tg -w a mutant scattered away from each other. a small insignificant increase in scattering was seen in % of the total number of colonies for renca cells overexpressing tg-r a construct. data from this study supports that gtp-binding activity of tg is the drive force in migration driven scattering of renca cells, suggesting that inhibitors targeting the gtp-binding activity of tg may serve as a new therapeutic approach in the treatment of rcc. background: in this study, we aimed to investigate the relationship between level of vitamin d with subclinical hypothyroidism and subclinical hyperthyroidism. material and metod: study groups planned as three groups such as euthyroid (n = ), subclinical hypothyroid (n = ), subclinical hyperthyroid (n = ). serum tsh, free t (ft ) and free t (ft ) levels were determined by chemiluminescence immunoassay and serum -hydroxy (oh) vitamin d (oh) d level were determined by liquid chromatography-tandem mass spectrometry. euthyroidism was defined as a normal level of tsh (range, . to . miu/l), ft (range, . to . ng/dl) and ft (range, . to . ng/dl). subclinical hypothyroidism is defined as an elevated serum tsh level associated with normal total or free t and t levels. subclinical hyperthyroidism is defined as low serum tsh levels associated with normal free t and free t levels. results: subclinical hyperthyroid group had significantly higher (oh) vitamin d levels compared to the euthyroid and subclinical hypothyroid groups (p < . ). (oh) vitamin d levels in subclinical hypothyroid group was not statistically significant when compared with the euthyroid group. food processing wastes provide carbon sources in high amounts for fermentative microorganisms to produce energy. converting carbon-rich biomass into bioethanol through fermentation by microorganisms both provides energy requirement for humankind and also decrease pollutant gases like co , no x and so x (ghorbani et al., ) . fermentation processes for bio-ethanol production could be achieved by saccharomyces cerevisiae, zymomonas mobilis, and escherichia coli. bacterial hemoglobin (vitreoscilla hemoglobin, vhb) is the first and best characterized prokaryotic hemoglobin molecule. the function of vhb is supporting the cellular respiration through binding to oxygen at microaerobic environment, transferring it to the terminal respiration oxidases (geckil et al., ) and thus improving growth and productivity of the microorganisms. in this study, e.coli strains fbr , ts and ts were used as ethanologenic microorganisms. expression of vhb in ts is lower than in ts strain. for the efficient ethanol production effect of different inoculum sizes, sugar species and sugar concentrations in the growth medium were investigated. vhb expression increased effectively the viability of ts strain by up to . x cfu per ml of fructose ( %, w/v) supplemented lb medium starting with small inoculum for fermentation. this indicates that vgb expression should be at the certain level to maintain sufficient the cell growth for ethanol production. geckil h, gencer s ( ) . production of l-asparaginase in enterobacter aerogenes expressing vitreoscilla hemoglobin for efficient oxygen uptake. applied microbiology and biotechnology : - . ghorbani, f., younesi, h., sari, a. e., najafpour, g. ( ) . cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by saccharomyces cerevisiae. ethanol production from dairy industry by product using bacterial hemoglobin t. sar, g. seker, a. g. erman, m. yesilcimen akbas gebze technical university, depertment of molecular biology and genetics, kocaeli, turkey bioethanol production from biomass has a great potential to reduce greenhouse gases emissions. ethanol has several applications in industries (chemical, medical, pharmaceutical, food etc.) in the form of raw material, solvent and fuel. one of the most abundant liquid wastes is cheese whey generated from dairy industries. whey powder is concentrated form of whey and contains lactose and also protein, lipid, minerals and vitamins. vitreoscilla hemoglobin (vhb) is the first bacterial hemoglobin. the main function of this molecule is to improve oxygen transfer to cellular oxidases and thus supporting cellular growth and productivity at low oxygen levels (kallio et al. ) . in this work, e. coli strains fbr , ts (low level vhb expressing) and ts (high level vhb expressing) were used as ethanol producing microorganisms. fermentation medium containing whey powder supplemented with lb material was inoculated with these strains and incubated for hours at °c and rpm in a ml erlenmayer flask. the ethanol production was improved over % by using lower vhb expressing strain. the ethanol levels (v/v, %) were determined as . , . and . for fbr , ts and ts strains respectively. it is shown that the certain levels of vhb could be useful tool to increase the growth and productivity of ethanol from dairy industry wastes. kallio p.t., kim d.j., tsai p.s. and bailey j.e. ( ) . bioethanol is usually produced from cellulose, hemicellulose and lignin. the lignocellulosic wastes should be hydrolysed into fermentable sugars by using enzymes or dilute acids before microbial fermentation. acidic hydrolysis methodology is cheaper than enzymatic hydrolysis but it can cause production of some inhibitors like aliphatic acids, which affect the growth of microorganisms. vitreoscilla hemoglobin (vhb) is the first described prokaryotic hemoglobin. the recombinant strains carrying vgb gene (e. coli, p. aureginosa) which encodes vhb showed increased bacterial growth, productivity of metabolites compared to untransformant counterparts under low oxygen concentrations [nasr et al., ; geckil et al., ] . in this study, ethanologic e. coli strains fbr , its derivative strains ts (vgb+) and ts (vgb+) were used. ts was constructed in such that it could express more vhb than ts . bioethanol production by these strains in presence of lignocellulosic hydrolysates derived inhibitors was investigated. different acetic acid concentrations ( . - mm) were used as inhibitors from lignocellulose hydrolysate. . mm acetic acid was used as an inhibitor. the growth of vhb expressing ts and ts strains was inhibited about % after hours fermentation time. strain fbr was inhibited as high as % by using the same inhibitor including growth medium. it was shown that the expression of vhb could improve growth and productivity in presence of lignocellulosic inhibitors. differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. however, excessive adipogenesis is closely linked to the development of obesity. thus, any drug or chemical that can inhibit adipogenesis may have preventive and/or therapeutic potential against obesity and related diseases. azd , an inhibitor of the family of pim kinases, is known for anti-cancer activity. here we investigated the effect of azd on adipogenesis in t -l preadipocytes. notably, azd treatment led to a concentration-dependent inhibition of both lipid accumulation and triglyceride (tg) synthesis during the differentiation of t -l preadipocytes into adipocytes with no cytotoxicity. on mechanistic levels, azd strongly reduced not only the expression levels of ccaat/enhancer-binding protein-a (c/ebp-a), peroxisome proliferator-activated receptor-c (ppar-c), fatty acid synthase (fas), and perilipin a but also the phosphorylation levels of signal transducer and activator of transcription- (stat- ) during adipocyte differentiation. furthermore, azd largely decreased leptin, but not adiponectin, mrna expressions during adipocyte differentiation. collectively, these results demonstrate that azd inhibits adipogenesis in t -l preadipocytes and the inhibition is largely attributable to the reduced expression and/or phosphorylation levels of c/ebp-a, ppar-c, fas, perilipin a, and stat- . effect of intrauterin exposure to artificial food colourings on dna damage in rats in many research genotoxic potential of food additives has been investigated. however there are few findings about the effect of artificial food colourings (afc) on dna. in this experimental study, we aimed to analyze whether in utero exposed artificial food colourings would have effect on dna and cause damage.thirteen female rats were included to the study which were equally divided into two groups as control (cg, n = ) and food colouring (fcg, n = ) groups. a mixture of nine food colours were given daily to fcg by oral gavage from preconception to birth. no adverse effect level (noael) of artificial food colourings for each colouring was administered to fcg. three months after the birth, offspring from each group were selected randomly as control (cg) and experiment (eg) groups. then they were sacrified under anesthesia. for performing the alkaline comet comet assay leukocytes were seperated from whole blood samples. the alkaline comet assay was performed. the extent of dna damage was assessed from the length of dna migration derived by subtracting the diameter of the nucleus from the total length of the image and graded into categories and these grades were converted into arbitrary unit (au). differences between the means of data were compared by independent samples t test. the results were given as the meanaesd, p values of less than . were considered as statistically significant. although the extent of dna damage was higher in eg, the comparison of experiment ( . ae . ) and control ( . ae . ) groups showed no statistical difference (p = . ). relationship between glucocorticoid receptor gene polymorphisms and recurrent depression l. aydogan, i. benli, z. c. ozmen, i. butun gaziosmanpasa university medical faculty, department of biochemistry, tokat, turkey objective: sensitivity to glucocorticoids varies between individuals and these differences have been implicated in the etiology of psychiatric diseases such as depression. recent studies have found relationship between common glucocorticoid receptor (gr) gene (nr c ) polymorphisms and unipolar or bipolar depression. the nr c gene is a candidate gene affecting depressive disorder risk and management. the aim of the present study was to evaluate the relative distribution of specific polymorphisms of nr c (bcl and rs ) in recurrent depressive disorder (rdd) patients. methods: our study included volunteers with recurrent depressive disorder and healthy individuals without any mental illness. depression was assessed by hamilton and madrs depression scale. nr c gene polymorphisms were detected by real-time pcr, with hybridization probe method. allele and genotype frequencies at two loci (bcli and rs ) were investigated in rdd patients and controls. results: genotype distribution among rdd patients and the control group for bcl- (g/c) were as follows: cc % and %, gc % and %, gg % and %, respectively. there was not a significant difference when the frequency of the allele (p = . ) and genotype frequency (p = . ) were compared between the patients and the control. genotype distribution in the rs region (a/t) of the patients and controls were tt % and %, ta % and %, aa % and %, respectively. allele frequency (p = . ) and the genotype frequencies (p = . ) were not significantly different among the groups. conclusion: numerous nr c gene polymorphisms were previously reported in association with modification of depressive disorders. the results of our study showed no association between gr genotype and recurrent depressive disorder. nr c polymorphism does not play a role in the development of recurrent depressive disorder. thymoquinone (tq) has been shown to supress the proliferation of various tumor cells, while it is minimally toxic to normal cells. the aim of this study is to investigate the potential therapeutic effects of tq on cell proliferation, apoptosis, invasion, migration, colony formation and wound-healing in sh-sy y human neuroblastoma cell line. sh-sy y cell line treated with - lm tq by solving medium for , and h considering a time-and dose-dependent manner. the cytotoxic effect of tq was determined by mtt method. total rna was isolated by trizol reagent. cdna synthesis was performed by using commercial kit. mdm , p , p , akt, pten, cdk , cyclin d , caspase- , - , - , - , bcl- , bax, parp, bcl-xl, bid, dr , dr , puma, noxa, mmp- , - , timp- , - and gapdh gene expression profiles were analysed by real-time pcr method. effects of tq in sh-sy y cells on invasion, colony formation and cell migration were detected by matrigel-chamber, colony formation assay and woundhealing assay, respectively. statistical analysis were performed with rt profiles array data analysis by using student's t test. ic value of tq in sh-sy y cells was detected as lm at th hours. by rt-pcr results, it was determined that tq caused a decrease in the expression of mdm , akt, cdk , cyclin d , bcl- and mmp- . it is also observed that tq caused a significant increase in the expression of p , pten, caspase- , - , bid, dr , puma, noxa and timp- . it was also found that tq in sh-sy y cells suppressed invasion, migration and colony formation by using matrigel invasion chamber, wound healing and colony formation assay, respectively. in conclusion, we demonstrate that tq significantly effect cell cycle, apoptosis, invasion, migration and colony formation of sh-sy y cells. tq may be a potential candidate as chemotherapeutic agent for the treatment of neuroblastoma. more studies have to be performed to profile the mechanisms and genome wide effects of tq to prove its therapeutic potential. dna aptamers can achieve a very high affinity to the target due to the potential of developing broad target-binding interface. however, classic strategy selection of aptamer binders is a challenging task requiring multiple rounds of panning and post-selection optimization. we have developed fast and convenient technique for the selection of dna aptamers based on the offrate selection and tandem affinity purification (tap). we constructed and produced in e.coli recombinant chimeric protein, comprising two affinity tags (his and gst) separated from each other and from the target protein (anthrax protective antigen domain iv, padiv) by sumo protease recognition polypeptide and synthetic cleavage site for the anthrax lethal factor (lf). the protein bound to aptamer library is first captured by imac resin, cleaved by sumo protease, captured by gst resin and eluted by lf following the lines of the tap method. the gst-captured aptamer-target complexes were subjected to the off-rate selection using soluble padiv as the competitor. multiple selection rounds are cumbersome and can result in carryover. high abundance of moderate affinity aptamers in the resulting pools obtained by classic selection approaches suggests that the procedure to counter-select them at the beginning of panning is needed. reduction of the contact duration between the aptamer library and the target was crucial for efficient selection of high-affinity binders. on the other hand, tap prevents contamination, and bundled with the off-rate selection, allows for clean isolation of high-affinity binders with affinity in the low nanomolar range. the developed technique is applicable for efficient selection of high affinity dna binders to soluble recombinant proteins and their fragments. dna aptamers obtained will be further used for the development of diagnostic and therapeutic tools for the detection and treatment of anthrax. the work was supported by russian science foundation research grant no. - - . the role of macab efflux pump in protection of serratia marcescens against antibiotics and oxidative stress the emergence of bacterial multi-drug resistance is a growing problem of public health worldwide. bacterial drug efflux pumps are membrane protein complexes that function to expulse drugs from the cell. they play a crucial role in the rising rates of antibiotic therapy failures. the homolog of macrolide-specific pump macab was identified in opportunistic pathogen serratia marcescens and was used in this study to characterize its role in protection against antimicrobials and other processes beyond the active efflux of antibiotics. here we used method of serial dilutions to determine minimum inhibitory concentration (mic) for s. marcescens sm wild type (wt) and its isogenic Δmacab mutant strains. we also used h o survival assay to evaluate the ability of wt and the mutant strain to withstand an oxidative stress. finally, we used b-galactosidase assay to evaluate macab promotor activation in the reporter strain and followed macab expression by western blotting analysis using macab- xhis strain. we show that in contrary to its e. coli homolog, macab pump in s. marcescens is not involved in the protection against macrolides but instead it is required for protection against aminoglycosides. we further show that similar to its salmonella typhimurium homolog, s. marcescens macab is essential for protection of bacteria against h o . transcriptional analyses demonstrate that while low level of macab promotor activity can be detected after hours of growth in lb-broth there is at least -fold increase in expression in response to the presence of h o . on the protein level macab can be detected starting from hours of growth in lb-broth and it reaches maximum expression on hour of growth. our data suggest that macab pump in s. marcescens is involved in protection of bacteria against aminoglycoside antibiotics and is crucial for protection against reactive oxygen species. we are currently working on identification of macab substrate with anti-h o properties. antiproliferative and apoptotic effects of noscapine on mcf- and mda-mb- human breast cancer cell lines approximately - % of breast cancers are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor . these are most aggressive tumor and a clinical problem because of lack of targeted therapies. noscapine is an alkaloid from opium. noscapine is a microtubule-interfering agent. it causes mitotic arrest, induces apoptosis. in this study, we aimed to investigate the effects of noscapine in mcf- and mda-mb- human breast cancer cell lines. the cytotoxic effects of docetaxel, tamoxifen, and noscapine on the mcf- and mda-mb- cell lines were analyzed by roche xcelligence system. the cells were cultured in % fetal bovine serum containing dulbecco's modified eagle medium at °c in a humidified atmosphere containing % co . h after seeding, the cells were treated with different doses of docetaxel ( . to nm), tamoxifen ( . to lm), and noscapine ( . to lm). cultured cells were harvested, fixed with % formalin, and centrifuged. pellet was blocked, fixed, and embedded in paraffin. paraffin-embedded cells blocks were sectioned at lm thickness and stained with h&e, ki- , bcl- , cyclin-d , and bax. sides were assessed under a light microscope. quantification of the analyzed proteins were evaluated by the percentage of positive cells. all drugs showed cytotoxic effects on both cell lines. all drugs inhibited the proliferation of breast cancer cells, but effects were dependent on time and dose. all drugs were especially more effective on mcf- cells. immunohistochemical examinations revealed that tamoxifen was more effective on mcf- cells, hovewer docetaxel and noscapine were more effective on mda-mb- cells. tamoxifen has more apoptotic and antiproliferative effects on mcf- cells. docetaxel and noscapine showed more apoptotic and antiproliferative effects on mda-mb- cells. noscapine may be an effective anticancer agent due to antiproliferative and apoptotic effects on breast cancer cells. negative selection of dna aptamers to reduce non-specific binding in solid-phase-based selection procedures carryover by binders specific to the components of the selection system can be a serious issue in hampering the aptamer selection campaign. solution-or "mass"-based techniques still cannot substitute classic phase-separation strategies. one approach to prevent selection of "passenger" phase-specific (plastic, beads) or blocking agent specific aptamer species is their depletion from the initial library pool. our aim was to develop the universal technique for removal of such aptamers exemplified by bsa-and casein-specific binders, while preserving the initial library complexity. the dna aptamer library was subjected to three rounds of depletion using magnetic beads with covalently attached casein and bsa. to ensure high depletion efficiency, beads were pelleted in a -ml centrifuge tube by a neodymium magnet through a -cm cushion of % sucrose, thus preventing weakly bound aptamers from re-populating the library. high complexity of the input library helped to avoid pcr amplification after depletion rounds preventing the library bias introduced by dna amplification. the depletion effciency was confirmed by real-time pcr. resulting oligonucleotide sub-library was analyzed for binding to the targets using solid-phase real-time pcr assay. we have shown that three rounds of panning under the conditions employed provided full depletion of the initial dna pool from nucleic acid structures capable of binding to protein competitors and hampering the process of aptamer selection. we compared selection efficiency of aptamers specific to type a botulinum neurotoxin light chain in depleted vs undepleted library. the yield of the target-specific aptamers was -fold higher in the library subjected to the depletion procedure. removal of undesired binders from aptamer libraries appears an important step of solid-phase selex procedure. it can become a useful approach in optimizing solid-phase selex. the work was supported by russian science foundation research grant no. - - . epithelial mesenchymal transition (emt) is a critical trans-differentiation program driving cancer metastasis. patients showing signs of emt or presence of distant metastasis have poor prognosis. another well-known feature of decreased cancer-associated survival is the lack of anti-cancer immune responses. thus we hypothesized that the emt and anti-tumor response should be linked via altered secretion of soluble factors by metastatic cells. all cell lines were grown in dmem. emt status of crc cell lines were assessed by investigating canonical markers of emt. cytokine/chemokine expression of crc cells was performed using r&d systems antibody arrays and validated using ccl sandwich elisa and rt-pcr. the mechanism of action of zeb / on ccl promoter has been studied by luciferace assay and chip. ccl coding region was cloned into pcdna . and stably transfected into dld- cells. ccl deficient ct cells were generated using lentivirual shrna transduction. cells overexpressing or knock/down ccl were injected orthotopically into mice. t lymphocyte (til) infiltration in respect to ccl and sip expression was studied using ihc or flow cytometry. emt status catagorised crc cell lines into epithelial, intermediate epithelial, intermediate mesechymal and mesenchymal. cytokine/chemokine antibody arrays showed a significant increase in ccl in induced dld-sip cells. elisa, multiplex assays and rt-pcr confirmed a significant increase of secreted ccl in the induced dld-sip cells as well as mesenchymal crc cells as compared to epithelial ones (p = . ). promoter studies showed that zeb / bind to ccl promoter and and activate ccl gene expression. no metastasis was observed for dld- cells overexpressing ccl but significant alterations of tumour associated lymphocytes were identified in syngeneic orthotopic crc models. our data shows that ccl is up-regulated by emt inducing transcription factor sip , and mesenchymal (metastatic) crc cells secrete significantly more ccl compared to epithelial (non-metastatic) ones. ccl did not induce emt per se but abundant secretion of ccl by metastatic crc cells was a crucial regulator of immune infiltrate in crc. inhibiting ccl in metastatic crc may have a therapeutic potential. barley (hordeum vulgare l.) belongs to the grass family, poaceae (gramineae). it is the fourth most important cereal crop after wheat, maize and rice and is among the top ten crop plants in the world. talbina was used to be recommended for the sick and for one who is grieving over a dead person. talbina is made by adding one or two tablespoon of barley flour (must be percent wholegrain barley flour) to one-and-a-half cups of water and placed on low heat for - minutes (optional: add milk or yoghurt and sweeten with honey). the main objectives of this investigation were determine the a-tocopherol contents and antimutagenicity activity of talbina (hordeum vulgare l.). our results showed that the total tocopherol content was in the range of . to . lmol/g fw. talbina extract was shown to have greater antimutagenic activity observed in the lg/plate concentration s. typhimurium ta . at all the doses antimutagenic response was significant at (p < . ) against both the strains with a percent mutagenicity decrease from to for ta followed by ta with percent antimutagenicity from to . the results of the study concluded that talbina is a better antimutagenic agent than vitamin e and combination of vitamins did not produce any synergistic activity. the compounds containing thiadiazoles have diverse applications as antifungals, anticancer agents, antibacterial, antiinflammatory drugs, antidepressants and carbonic anhydrase inhibitors according to literature. in this study some novel thiadiazole compounds [( , , , )-tetrathia[ . ] ( , )- , , -thiadiazolophane; ( , )dioxo- , , , )-tetrathia[ . ]( , )- , , -thiadiazolophane; ( , , , )-tetraoxo- ( , , , )-tetrathia[ . ]( , )- , , -thiadiazolophane and ( , , , , , )-hexaoxo- ( , , , )-tetrathia [ . ] ( , )- , , -thiadiazolophane] were used to evaluate the cytotoxicity on healthy human lymphocytes and the antibacterial activities. cytotoxicity tests were perfomed using mts assay and the trypan blue test. cells were incubated with the compounds for hours. at the end of the each hour, cell vitality was assessed by measuring the absorbance ( nm) of each well using a microplate reader for mts assay. in addition, viability percents of the cells were determined after trypan blue test. as a result, the compounds showed cytotoxicity in a dose dependent manner. for the concentrations of : of . mg/ml, the cytotoxic effect was eliminated. also, antioxidant capacity was determined using , -diphenyl- -picrylhydrazyl (dpph) reagent. moreover, the antibacterial activities of the compounds were analyzed using a microdilution test against e. coli and s.aureus. compounds having various concentrations showed different antibacterial effects against these two bacteria. arabidopsis thaliana ecotypes vary in their ability to utilize organic p substrates insufficient quantity of inorganic phosphorus in soil is an evergrowing problem that affects many fields of agriculture. unlike inorganic phosphates, organic phosphorus compounds are very common in many soil types, but plants are often unable to efficiently utilize them. to better characterize the extent of natural variation in the ability of the model plant arabidopsis thaliana to grow on organic phosphorus compounds, we grew arabidopsis ecotypes on several organic and inorganic sources of phosphorus. plants were grown in liquid or solid media containing naphosphate, phytate and atp as the sole supply of phosphorus or in absence of any phosphorus source. after several weeks of growth, plants were assayed for changes in their morphological and physiological characteristics. phytate was shown to be the least preferred source of phosphorus compared to inorganic phosphate and atp. the rate of biomass accumulation in all ecotypes decreased in the following order from inorganic phosphate to atp to phytate. lateral root formation was markedly reduced in the absence of any phosphorus source or in the presence of phytate. we also showed that phosphomonoesterase activity in intact roots increased when plants were grown on atp and phytate. overall phosphorus content in leaves and roots was similar when plants were grown on atp or inorganic phosphate, but it was markedly reduced on phytate. substantial differences between ecotypes were also observed in root length, p content in ash and phosphomonoesterase activity in intact roots. our analysis of the ability of arabidopsis ecotypes to grow on several different phosphorus sources provides a unique opportunity to investigate the degree of natural variation in this plant's ability to adapt to different nutritional environments. analysis of many important morphological and physiological changes observed in these plants can further extend our understanding of the full range of plant responses to phosphorus availability. laboratory tests are important in terms of confirmation of diagnosis given by clinics and implementation of appropriate treatment protocols for patients. laboratory tests used by the clinics have been increased in parallel with time.there are many reasons for increased use of the test such as increase of elderly population, increase in standard of care, lack of information and shortening of turn around time. unnecessary laboratory testing also constitute one of the reasons for increased use of laboratory tests. in our study we aimed to investigate the unnecessary laboratory testing for fpsa test. fpsa tests which are ordered with total psa tests that values of less than ng/ml or greater than ng/ml were accepted as inappropriate initial testing. fpsa tests were evaluated as unnecessary laboratory testing. the clinic which ordered the maximum unnecessary laboratory testing with was urology within all the clinics. although to the restrictions about the ordering of total psa and fpsa tests there were no decrease in the number of unnecessary laboratory testing. unnecessary usage of laboratory testing may cause increase of false positive results, increase in the use of invasive testing, unnecessary drug consumption and increase of healtcare costs. some precautions may be effective in reducing unnecessary tests such as to inform clinicians about the cost of laboratory tests, to increase the clinician education programs and to develop usage of disease specific diagnostic algorithms about test ordering. local clinical validation of blood collection tubes although the tubes with gel and clot activator are widely used due to the advantages, there are ongoing discussions about the effects of the blood collection tube on clinical outcomes in the analysis of biochemical parameters. therefore, we aimed to prove the local clinical validation of the new produced blood collection tubes with low-volume. the blood samples of patients who referred to the hospital phlebotomy unit were collected using holder into the different tubes. first tube was ml glass tube and with no additive, second was ml tube with gel separator, third was ml tube with gel separator. serum was separated and immediadiately analysed for biochemical parameters. the difference between the analyte amounts in the different tubes was evaluated using paired t-test. the clinical significance was evaluated using significant change limit. bias (%) between the other tubes with the reference tube was also evaluated according to the ''allowable total error". when we compared the other test tubes to a glass tube which was assumed reference tube, total protein, albumin, amylase, calcium, triglyceride, cholesterol, hdl-cholesterol, total and direct bilirubin, iron, gamma glutamyl transferase, magnesium, phosphorus results were statistically significant. but the results of all the analytes were within the significant changes limit and the allowable total error was not significant. while a biochemical parameters have analysed, it may be absorbed into the gel and this may caused from factors such as the chemical structure of the gel, analyte itself, the residence time in the gel, storage temperature and volume of the sample e.g. as well as the leaking of gel material to the sample was reported to be another factor for affecting the analysis. despite these factors, we observed that neither gel-clot activator tube with low nor high volume affect the clinical results. the research of the frequency of interference in thyroid function tests interference is defined as the effect of substance in the sample which changes the correct value of laboratory results. the frequency of interference in immune techniques is varied. the frequency of interference depends on population of the study, technique for detecting the reaction and researcher's method. unexpected or inconsistent results with clinical findings should suggest the possibility of interference. in this study it is aimed to investigate the frequency of interference in thyroid function tests (tsh, ft , ft ) which are the most common requested laboratory tests. thyroid function tests of patients are analyzed in ankara numune education and research hospital in october -may . five samples which had the incompatible results with clinical findings are re-evaluated just because of the suspicion of interference. the detection of interference included; repetition of test via different immune techniques, serial dilution, polyethylene glycol (peg) precipitation and incubation with heterophilic blocking tubes (hbt). the results of two different immune techniques and before/ after incubation with hbt showed no significant difference. linear curves had observed in serial dilution. after peg precipitation; below % of recovery had obtained in one sample, therefore it is interpreted as macro-tsh. the frequency of interference in thyroid function tests for -month study period was . %. no information is found about the best test for defining the cross reaction. it is also aforethought that interference should not be excluded by using any single procedure. p-mis- development of polyclonal and monoclonal antibodies against fatty acid binding protein (fabp /ap ) a. abbasi taghidizaj, g. aydogdu, b. p. sermikli, e. yilmaz ankara university, ankara, turkey recombinant proteins and antibodies can be use for therapeutic or diagnostic purposes which produced in many different host organisms. the technique for the production of immortal cell making single antibody, fusing target antibody-forming b lymphocyte precursor with a suitable myeloma cells. the fused hybrid cells (called hybridomas), as a cancer cell will reproduce rapidly and will produce large amounts of the desired antibodies. fatty acid binding protein (fabp ) is a well characterized intracellular lipid transport protein and plays a key role in the intracellular fatty acid transport and adipose tissue metabolism. fabp as a adipokine that regulates glucose homeostasis and has various features for metabolic syndrome associated with obesity. in this study, production of monoclonal antibodies against immunogenic fabp protein made by recombinant dna technology. recombinant his-fabp was expressed in e.coli and purified. balb/c mice used for immunization and serum anti-fabp antibodies determined by enzyme-linked immunosorbent assay (elisa). hybridoma cells created by fusion of splenocytes and myeloma partner cells. after selection of antibody producing cell clones, injecting hybridomas into the peritoneal cavity in balb/c mice ascites fluids was obtained. we have selected fifteen hybridoma clones that produced antibodies specific for fabp , as shown by western blotting and immunocytochemistry. as a result we produced mabs that will be useful for the scientific community working on fatty acid binding proteins and lipid metabolism. in near future, therapeutic approach for this antibody maybe a possibility in metabolic syndrome. thioridazine, an anti-psychotic drug, inhibits migration, invasion and epithelial mesenchymal transition in breast cancer cell lines thioridazine (thz), an antipsychotic drug, exhibits anti-angiogenic effects on breast cancer cell lines. however the mechanistic insight in exerting antiangiogenic effect is not clearly understood. the objective was to investigate the role of thz in epithelialmesenchymal transition (emt) by using cell migration assay, scratch assay, western blot (wb) and immunocytochemistry. thz treatment reduced cell viability on mda-mb- , mcf- and cd + /cd -cells and ic values of thz were found to be lm, . lm and lm respectively, at hours. invasion potency of mcf- , cd + /cd -and mda-mb- cells were determined as %, %, . % when compared to relevant treatment controls. migration potency of mcf- , cd + /cd -and mda-mb- cells was determined as . %, . %, % respectively. among the three cell lines mda-mb- cells display enhanced invasive and migration ability when compared to other cell lines. western blotting results demonstrate that thz significantly increases e-cadherin, cytokeratin- , b-catenin, while inhibiting n-cadherin, vimentin, fibronectin. immunocytochemistry studies revealed decrease in e cadherin and a concomitant increase in vimentin level for all three cell lines upon treatment with thz. moreover thz significantly inhibited the cell migration, invasion and emt in mda-mb- , mcf- and cd + /cd cell lines by suppressing mesenchymal markers. in conclusion, these data suggest that thz might be a novel anti-proliferative and anti-metastatic agent for treatment of breast cancer. effect of seasonal temperature and humidity on urine density in children environmental heat and humidity are important factors affecting hydration status in childhood. hereby, we aimed to investigate the effects of seasonal climate changes on urine density of children living in mediterranean climate, cyprus. healthy - year children's ( girls, boys) age, sex and urine density results were collected retrospectively for three consecutive years. the correlation of urine density with each seasonal and months' average temperature and humidity has been analysed. the urine density results had a positive correlation with temperature (r = . , p = . ) and a negative correlation with humidity (r= À . , p = . ). mean urine density in spring was higher than that of autumn (p = . ) and winter (p = . ). mean value of summer was higher than autumn (p = . ) and winter (p = . ). - months age group had lower urine density. evaluation of urine density based on gender and puberty revealed no statistically significant difference. seasonal mediterranean climate changes have an impact on urine density in children which may affect hydration status especially in infants < yrs of age. during high temperature seasons ensuring adequate water intake is essential in this age group in mediterranean climate. p-mis- implementation related to the use of antibiotics and data sources by community pharmacists in north cyprus as the resistance to antibiotics is gaining importance in today's world;the solution to this problem is possible through a common consciousness of the doctor who prescribes antibiotics,the pharmacist who sells and the patient who consumes antibiotics. irrational use of drugs is an economic and medical problem in many developed and developing countries around the world.the aim of this study is to determine the sales ratio of non-prescription antibiotics in pharmacies which is the biggest category of the antibiotic group sold as well as the indications that lead to its' prescription. eighty-four pharmacies out of pharmacies located in north cyprus were involved in the study with %stratified systematic sampling, questionnaires were filled and a consent form was signed by the participating pharmacists. the pharmacists involved in the study stated that non-prescribed antibiotics were demanded from the pharmacists and all except two ( . %),responded positively to this demand. it has also been identified in the study that . % of the daily sale of antibiotics in the first half of the year was non-prescribed. the most purchased antibiotics either with or without prescription was found to be the penicillin and its derivatives with . % and upper respiratory tract with . %. when the level of selfawareness of the pharmacists was examined, the rate is found in north cyprus to be ( . %),compared with the studies conducted in greece,italy,malta and spain % and egypt . %that designated the non prescribed antibiotics purchased from the public pharmacies. the rate of sale of non-prescribed antibiotics in north cyprus has been found to be at a higher level compared to the rates in many developed and developing countries. furthermore, the upper respiratory tract infections are amongst the most common viral causes which lead to a high consumption of both prescribed and non-prescribed antibiotics. this study was supported by turkish viral hepatitis prevention society. acrylamide has cytotoxic, antiproliferative and apoptotic effects on human lung adeno carcinoma cell line a acrylamide (aa), a widespread substance in many fields, forms in foods during high temperature processing such as baking, roasting, frying. aa is a potent neurotoxic, genotoxic and clastogenic agent being a strong electrophile and forming adduct with biological molecules or potent nucleophiles. up to now, several studies confirmed the toxicity of acrylamide to several organs. on the other hand, aa is reported to have inhibition effects both on proliferation and differentiation of different cancer cells in a time and dose-dependent manner. in addition, natural and synthetic acrylamide derivatives are also used as potent anti-cancer agents. moreover, inhibition concentration (ic ) values of aa against these cancer cells have not been investigated in detail yet. thus, the goal of this study is to investigate the cytotoxicity of aa on a cells including with ultrastructural and morphological effects. ic value of aa on a cells for h was detected with mtt ( -( , -dimethyl- -thiazolyl)- , -diphenyl- h-tetrazolium bromide) colorimetric assay. we evaluated morphological changes under confocal microscopy and ultrastructural changes under transmission electron microscopy (tem). our results demonstrate that aa inhibits the proliferation of a cells in dose-dependent manner and ic on a cells was found to be . mm for hours. confocal microscopy evaluations showed that aa caused nuclear condensations, fragmentations, cytoskeleton lacerations and membrane blebbing. tem results revealed membrane blebbing, chromatin condensations and cell shrinkage. although aa is a probable carcinogen substance, it drastically inhibited cell viability in dose-dependent manner. from microscopic assessments, aa is suggested to induce apoptosis in a cells. in conclusion, the present study confirms the high potential of aa for cytotoxic, antiproliferative and apoptotic activity on a cells. however, appropriate aa dose is critical to prevent its possible adverse effects. effect of hemolysis and lipemia on some immunochemical tests in beckman coulter unicell dxi immunoassay analyzer c. yilmaz, s. yildiz, m. senes, v. fidanci, d. y€ ucel ankara training and research hospital, ankara, turkey the aim of the study was to investigate the effects of in vitro hemolysis and lipemia on immunoassays studied by the beckman coulter unicell dxi immunoassay analyzer. we prepared a serum pool without hemolysis, lipemia and icterus. baseline serum pool concentrations of tests were measured by the beckman coulter unicell dxi . d _ ifferent serum pools, six for hemolysis and five for lipemia, were spiked with increasing concentrations of hemoglobin ( . , . , . , . , . and . g/l hemoglobin) and intralipid ( . , . , . , and g/l intralipid). the hemolysate was prepared by osmotic shock method. intralipid ( %, baxter, deerfield, il) was used to mimic the effect of lipemia. the hemolysis (h), lipemia (l) and icterus (i) indices were measured on beckman coulter au . after spiking the pools, the tests were measured again in duplicate on beckman-coulter dxi analyzer. a change of % from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. we observed a positive interference due to hemolysis for folat, vitamin b , testosterone and by lipemia for cortisol. there was a negative interference of hemolysis for ca . , ca , ca . , insulin, pth and e , and of lipemia for progesterone, ca . , vitamin b and pth. we found clinically significant effect (>total analytical error) of hemolysis on folate and insulin, and lipemia on cortisol. investigation of the effect of two different p mapk inhibitors in rats subjected to isoproterenol-induced acute myocardial injury: an experimental study objective: acute myocardial infarction is a serious acute condition. in the current study, we aimed to investigate the possible effect of two different mitogen-activated protein kinase (p mapk) inhibitors in rats subjected to isoproterenol (iso)induced myocardial injury. materials and methods: a total of male wistar-albino rats were equally and randomly seperated into four groups as follows: control, iso, iso plus sb andiso plus tak- . treatment agents were orally administered and myocardial injury was induced by subcutaneous injection of iso. serum cardiac troponin-i (ctni), ischemia modified albumin (ima), heart fatty acid binding protein (hfabp) levels and paraoxonase- (pon- ) activity, tissue tos (total oxidant status), tas (total antioxidant status), tt (total thiol), tumor necrosis factor-a (tnf-a) levels, superoxide dismutase (sod) and glutathione peroxidase (gsh-px) activity levels were measured. tissue mrna levels of nf-jb, p mapk and nuclear factor erythroid -related factor (nrf ) were analyzed. heart tissues were also immunohistochemically and histopathologically evaluated. results: both compounds have led to a decrement in myocardial damage, apoptosis, ctni, ima, hfabp, tos, and tnf-a levels, nf-jb, p mapk, phosphorylated c-jun n-terminal protein kinase (pjnk / ) expressions. on the other hand, the applied treatment increased sod, gsh-px, tas and tt levels, as well as phosphorylated extracellular signal-regulated kinase (perk / ) and nrf expressions. conclusion: data established from the current study suggest that administered agents have protective effect against cardiac injury induced by iso, which was more prominent in rats received sb treatment. p mapk inhibitors may constitute a useful choice as cardioprotective agents due to their antiinflammatory, antioxidant and anti-apoptotic effects. keywords: _ isoproterenol, myocardial infarction, myocardial ischemia, p mitogen-activated protein kinases, sb , tak- . silicosis composes the vast majority of occupational lung diseases. silicosis, caused by inhalation of crystalline silica, is a chronic lung disease characterized by parenchymal nodules and pulmonary fibrosis. the susceptibility of patients with silicosis to infection is thought to be due to toxic effects of silica on pulmonary macrophages. ada activity is considered as a nonspecific marker of t cell activation and cellular immunity. this study aimed to compare the serum ada activity in silicosis patients with spirometric values. in this study there were males in each groups which contained patients with silicosis (group ), individuals having similar symptoms with silicosis from same occupational area (group ) and healthy subjects (group ). routine hematological and biochemical parameters were also measured. the serum ada activity and spirometric values (fev , fev %, fev /fvc, fev / fvc%, fef - and fef - %) were compared. the average age of group , and are . ae . , . ae and ae . years, respectively. there was a significant difference between group and in terms of the ada level (p < . ). there was a negative correlation between ada activity and fev , fev %, fev /fvc, fev /fvc%, fef - values. elevated serum ada activity has been shown in many diseases with induced cellular immunity. despite initially toxic effects were lead to a little immunological reaction in patients with silicosis, continuation of this immunological response is important in some chronic manifestations of silicosis. the release of chemotactic factors and inflammatory mediators cause the migration of polymorphonuclear leukocytes, t lymphocytes and macrophages. in this study, the ada activity was significantly higher in patients with silicosis than others. increased immunity in patients with silicosis is being considered, increasing ada activity might be help of earlier recognition of these patients and to take better quality of life. atlantic salmon (salmo salar l.) is an important model system in evolutionary and conservation biology that provides fundamental knowledge into population persistence, adaptive response and the effects of anthropogenic change. the role of behavioral and body size variation in environmental adaptation of atlantic salmon is well known, by contrast, the underlying biochemical mechanisms are largely unknown. intracellular proteases, such as cathepsins b and d in lysosomes and calpains and proteasome in cytosol, due to their metabolic and regulatory role may contribute to phenotyping speciation of salmon young. we examined the activity of intracellular proteolytic enzymes in skeletal muscles of atlantic salmon parr from two local habitats of the varzuga river (the main channel and small tributaries) differing in hydrological and feeding parameters. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from varzuga river (kola peninsula, russia). it is known that salmon parr originated from a common hatch became phenotypically divergent during the settle in the biotopes. reliable difference in studied enzyme activities in the salmon parr from two local habitats was found; furthermore, calpain and cathepsin b proteolytic activities were found to negatively correlate with parr body size. muscle proteolytic activity data support an idea on protease contribution to environmentally-driven adaptation and speciation process in fish. the work was supported by the russian scientific foundation, project no. - - . the phylogenetic analyses of anthriscus (apiacea) species from turkey based on non-coding "trn" regions of chloroplast genome p. yilmaz sancar , m. tekin , s. civelek firat university, elazig, turkey, cumhuriyet university, sivas, turkey anthriscus pers. (apiaceae) species belongs to apiaceae family and is represented by genus on the world and by genus in turkey. anhriscus species are used extensively for treatment various disease such as asthma, alzheimer and show anti-tumoral, anti-microbial, antioxidant features. for determining exact species which treat disease it is necessary sorting species correctly with molecular markers to support morphological features. anthriscus species were defined by examining insufficient quantity of samples in turkey flora. besides, no detailed study was found in our country after flora study. for this reason a revision study was made with the aim of solving some systematical problems in by tekin. the result of the study provided important contribution to the systematic of the species in turkey. however a molecular study was also required for building the obtained results on a more solid ground. in this study, the aim to reveal systematic and phylogenetic relationship among species of anthriscus in turkey, by using trnl-f region in chloroplast genome. dna was isolated by ctab method and amplified in pcr by using e-f primaries. the obtained data was evaluated by mega . program and phylogenetic tree was prepared by using maximum likelihood method. according to the phylogenetic tree that we prepared by using the sequence line up of trnl-f section, it was observed that a. cerefolium, a. caucalis and a. tenerrima species completed their speciation and an isolation with other species in terms of speciation was provided. it was also observed that a. kotchi, a. sylvestris subs. sylvestris, a. sylvestris subs. nemarosa and a. lamprocarpa'nın provided hybridization among themselves but they did not complete their speciation. it was determined that a.lamprocarpa var. chelikhii which is one of the two different varieties of a. lamprocarpa is actually a new sub-species. this fact was supported by molecular data obtained from the study we made after morphologic data. introduction: excessive production of androstenedione can becaused by defects of adrenal steroid biosynthesis, tumors of ovarian and adrenal origin, polycystic ovarian syndrome, increased peripheral sensitivity to androgens, and increased peripheral production of androgens. most epidemiologic studies use enzyme-linked immunosorbentassay (elisa) to measure sex steroid hormones because they have acceptable turnaround times and arerelatively inexpensive. mass spectrometry-based methods are currently the most specific quantitative analytical methods for steroid determination. mass spectrometry methods are independent of matrix effects or cross-reactivity. in this study, a new liquid chromatography-tandem mass spectrometry (lc-ms/ms) method was developed. materials and methods: for serum androstenedione measurement, ll of internal standard (d - deoxycortizol) in methanol was added to ll standart or serum and centrifuged at . rpm for minutes to remove the precipitated proteins. supernatant was transferred to clean tubes and this procedure was performed twice. the supernatant was collected and dried under a nitrogen gas flow at • c and dissolved in mobile phase. ll was injected in to the ultra performance liquid chromatography analytical column for chromatography. elisa study was conducted with drg (lot. no. k ) brand kit. results: method comporison between lc-ms/ms and elisa was found slope value , , intercept value À . and r² value . . the regressione quation was elisa= À . + . lc-ms/ms. discussion and conclusion: method comparison study presented higher results in elisa compared to lc-ms/ms. in our opinion, this might due to the interference in elisa systems. our lc-ms/ms method allows rapid, sensitive and specific determination of androgens in plasma and serum.the specificity of liquid chromatography-tandem mass spectrometry (lc-ms/ ms) offers advantages over immunoassays. heparins play an important role in cell growth, differentiation, migration and invasion. however, the molecular mechanisms of heparin mediated cellular behaviors are not well defined. to determine the effect of heparin on gene expression, we performed a cdna microarray in a hepatocellular carcinoma cell line and found that heparin regulates transcription of genes involved in glucose metabolism. in this study, we showed a new role of heparin in the regulation of thioredoxin interacting protein, which is a major regulator of glucose metabolism, in hepatocellular carcinoma cell lines. we determined the importance of a unique carbohydrate response element located on its promoter for the heparin-induced activation of thioredoxin-interacting protein and the modulatory role of heparin on nuclear accumulation of carbohydrate response element associated proteins. we showed the importance of heparin mediated histone modifications and downregulation of enhancer of zeste polycomb repressive complex expression for heparin mediated overexpression of thioredoxininteracting protein. when we tested biological significance of these data; we observed that cells overexpressing thioredoxininteracting protein are less adhesive and proliferative, however they have a higher migration and invasion ability. interestingly, heparin treatment increased thioredoxin-interacting protein expression in liver of diabetic rats. in conclusion, our results show that heparin activates thioredoxin-interacting protein expression in liver and hepatocellular carcinoma cells and provide the first evidences of regulatory roles of heparin on carbohydrate response element associated factors. this study will contribute future understanding of the effect of heparin on glucose metabolism and glucose independent overexpression of thioredoxin-interacting protein during hepatocarcinogenesis. prolidase activity in chronic obstructive pulmonary disease and asthma t. g€ uc ßl€ u , h. s€ urer , g. bilgin , d. y€ ucel ankara training and research hospital, medical biochemistry department, ankara, turkey, ankara training and research hospital, chest diseases department, ankara, turkey chronic obstructive pulmonary disease (copd) is a consequence of an underlying chronic inflammatory disorder of the airways that is usually progressive and causes dysregulation in the metabolism of collagen. and asthma is a disease where there is an accumulation of collagen in the reticular basal membrane of the airway leading to chronic inflammation. prolidase has an important role in the recycling of proline for collagen synthesis and cell growth. we measured and compared prolidase activity in healthy individuals with copd and asthma patients to find out that whether its activity might reflect disturbances of collagen metabolism in the patients. patients with copd, patients with asthma and healthy control subjects with similar age range and sex were included in our study. the patient and control groups do not have any other chronic disease. serum prolidase activity was measured in the patient and control groups. ferritin and alpha- antitrypsin concentrations were also compared. there was no significant difference between serum prolidaz activities of asthma and copd patients. serum prolidase activities of both copd and asthma patients were significantly lower than those of the control subjects (p < . ). there was no significant difference for ferritine and alpha- antityripsin levels between the groups. the prolidase activity is significantly lower in asthma and copd patients comparing with control subjects. the collagen metabolism may be undergone to a change in these patients. hence, there may be an effect on the accumulation of collagen in the reticular basal membrane. the results suggest that collagen turnover are altered by the development of copd and asthma in human lungs, and prolidase activity may reflect disturbances of collagen metabolism in these pulmonary diseases. monitoring serum prolidase activity may be useful in evaluating fibrotic processes and in the chronic inflammatory lung diseases in human. acyclovir molecule in the active site of e. coli purine nucleoside phosphorylase (on the basis of x-ray study) i. kuranova , , v. timofeev , , n. zhukhlistova , y. abramchik , t. muravieva , r. esipov shubnikov institute of crystallography of fsrc "crystallography and photonics" ras, moscow, russia, national research centre "kurchatov institute", moscow, russia, shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia e. coli purine nucleoside phosphorylase (pnp), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family i of hexameric pnps. due to key role in the purine sulvage pathway pnps are attractive targets for drug design against some pathogens. they also used widely in biotechnology for the synthesis of nucleoside analogues as well as for the activation of the prodrugs in anti-cancer gene therapies. the acyclovir (acv), acyclic derivative of guanosine, is antiviral drug for the treatment of some human viral infections. the crystalline complex of e. coli pnp with acyclovir was prepared by co-crystallization using counter diffusion in capillary through the gel layer. the set of x-ray data at k from single crystal grown in space (sp. group p ) was collected on the spring- synchrotron-radiation facility (japan) and the structure was solved at . a resolution, using the molecular replacement method (pdb id i c). acv molecule was located in the nucleoside binding pocket of the enzyme in two conformations. the phosphate binding site was occupied by so ion. the hydrogen bonds network and hydrophobic interactions stabilising acv molecule in the active site as well as the conformational changes upon ligand binding were described. the comparison of e. coli pnp/acyclovir complex and the similar complexes of bacillus subtilis pnp (pdb id da ) and human pnp (pdb id pwy) allowed to establish the peculiarities of acv binding of in the e. coli enzyme. gonadotropins are glycoprotein hormones that regulate normal growth, sexual development, and reproductive function. these are large, up to kda proteins, which are synthesized and secreted by the gonadotropic cells of the anterior pituitary gland. these hormones may vary in the level of glycosylation depending on the tissue and the metabolism cycles. follicle-stimulating hormone (fsh) and upon binding to fsh receptor, a g-protein coupled receptor (gpcr), regulates the development, growth, pubertal maturation, and reproductive processes of the body. human chorionic gonadotropin (hcg) and luteinizing hormone (lh) act via a shared gpcr (lh receptor) and regulate mechanisms essential for ovulation, early pregnancy and placental function in females as well as spermatogenesis and testosterone production in males. activation of gpcrs by these hormones can be measured by monitoring formation of cellular cyclic adenosine monophosphate (camp). the level on camp was measured using a f€ orster resonance energy transfer (fret)-based biosensor tepacvv (h ) kindly provided by dr, kees jalink. the biosensor was expressed using the developed bacmam gene delivery system (recombinant baculoviruses carrying the transgene under a strong mammalian promoter). kgn cells expressing the fsh receptor and cos cells expressing the lh receptor served as study objects. monitoring of specific gpcr activation in living cells, allows detection of only the biologically active agonists, which has real impact in quantification of large hormones. differences in levels of hormone glycosylation may affect their biological function. investigation of this phenomena is planned for near future. detection of biological activity of gonadotropins is of importance for pharmaceutical industry, where today the concentration of recombinant proteins is mostly estimated using immunological assays only. development of a colorimetric aptasensor for the detection of peanut allergen protein ara h in food samples b. bora ege university, izmir, turkey food allergy, especially peanut allergy is a life-threatening problem, and severe reactions against these foods can be observed. since unintnded consumption of non-labeled foods is the most dangerous risk, any residual allergen protein should be tested and labeled by the manufacturers. an aptamer based colorimetric test is a powerful alternative to commercially available rt-pcr and elisa test kits. the main objective of this study is to develop an aptamer based colorimetric test fort he detection of major peanut allergen protein ara h . ara h aptamer was used to recognize any residual peanut major allergen protein ara h in food samples. recombinant ara h protein was produced and puirifed to be used as a target. ara h aptamer was used in combination with a blocking sequence, to prevent non-specific binding event, a biotinylated complementary strand to the blocking sequence, and finally strp-hrp interaction in order to facilitate colorimetric reaction. optimal blocking sequence length was optimized and introduced to the site of aptamer sequence to construct an aptamer-hairpin structure. liberation of the blocking sequence allows biotinylated complementary strand to bind to the blocking sequence and consequently str-hrp conjugate to achieve color development that is proportional to the target concentration. since, the aptasensor will be used for the detection of ara h in food samples, total protein extraction from chocolate samples was also optimized. in order to lower the detection limit of aptasensor, aptamer coupled magnetic bead based pre-enrichment assay was aslo optimized for the total protein extraction. as a result, a sensitive, fast and reliable aptamer based colorimetric assay was developed for the detection of peanut allergen protein from food samples. moreover, the assay has the advantages like ease of application and low cost which makes the assay a promising and a powerful alternative to commercially available rt-pcr and elisa tests. the association between lipid parameters and waist circumference in female university students in turkey s. ozen, a. cort sanko university, department of nutrition and dietetics, gaziantep, turkey a high waist circumference is associated with an increased risk for type diabetes, dyslipidemia, hypertension, and cvd in patients with a bmi in a range between and . kg/m . monitoring changes in waist circumference may be helpful, in addition to measuring bmi, since it can provide an estimate of increased abdominal fat even in the absence of a change in bmi. objective of the study was to find an association between plasma lipid profile and anthropometric parameters (waist circumference percentage of body fat and body mass index (bmi)) in abdominal obesity in turkish university students. lipid profile and anthropometric parameters of obesity were studied in a sample of women. students with high bmi (> ) had higher values of low-density lipoprotein (ldl), triglycerides (tg) and cholesterol (c) than students with low bmi (< ) but these differences were not significant. high-density lipoprotein (hdl) levels were non-significantly higher in low bmi (< ) student group. waist circumference, percentage of body fat was higher in high bmi (> ) group than low bmi (< ) group. waist circumference, percentage of body fat was positively correlated with bmi in both samples (bmi (> ) and bmi (< )). students were grouped depend on their waist circumference. healty individuals who had lower than cm waist circumference had decreased tg levels compared to cardiovascular risk group who had higher waist circumference than cm. this study shows an association between waist circumference, percentage of body fat, body mass index and lipid parameters in young female university students. with regard to the relationship, the screening females for central obesity to prevention of cardiovascular disease are recommended. a new biotechnological product from propolis with low allergen: anti-inflammatory effect propolis is extensively used in food industry due to its special medical properies (antioxidant, antimicrobial, antiseptic, antibacterial, anti-inflammatory and antimutagenic effects). even these positive properties it may cause some allergic reactions in consumers with allergic predispositons. previously, we demonstrated that biotransformation of propolis by some special strains of lactobacillus plantarum ( , , aatc strains) might decrease the allergenic molecules in propolis. in this study, we aimed to investigate the effect of biotransformation of popolis on it's antiinflammatory activities. before biotransformation, propolis samples were treated with different solutions ( % ethanol and polyethylene glycol -peg %) and different method (ultrasonic treatment w/ o c/ minutes) in order to facilitate solvation of solid samples which are very dense and not suitable for fermentation. fermantations were performed at o c/ hours under constant agitation conditions. the anti-inflammatory activity was determined in-vitro conditions using hyaluronidase's analysis and the xanthine oxidase activity. the highest inhibition (%) of radicals produced by xanthine oxidase was determined in solid samples treated by peg prior to biotransformation and using of l.plantarum strain during fermentation ( . %), followed by liquid samples treated by ultrasonic method prior to transformation ( . %). concernig the results of hyaluronidase activity (%) inhibitions, the best value were determined in the solid sample treated by peg prior to biotransformation and using of l.plantarum strain during fermentation ( . %). results indicated that the anti-inflammatory activities of analysed samples are quite high and depending of used extraction methods prior the biotransformation and used specific strain of l.plantarum could be optimized in terms of other required parameters. faceanti-mullerian hormone is not predictive for poor neonatal outcome aim: anti-mullerian hormone (amh) is a growth factor specific to ovaries. it is commonly used to predict ovarian reserve and outcomes of fertility treatments. recently, low levels of amh have been shown to be related to hypertensive diseases of the pregnancy and the risk of preterm labor. the aim of this study was to investigate the diagnostic performance of amh levels of mothers to predict poor neonatal outcome in term pregnancies and the relationship between amh and birthweights of the newborns. materials and methods: patients, having delivery beyond weeks, and who did not have any other medical problems were included in the study. the patients had normal g. oral glucose tolerance test results. they were divided as groups, based on their newborns' birthweight as " g. and g.". level of amh was determined by elisa method. results: there was not any relation with the amh of the mothers and the poor neonatal outcome of the newborns, in all groups. also no siginificant difference was observed in amh levels of the patients having delivery in early term and late term periods. when the patients of the same group were evaluated; amh levels were irrelevant to age, gravidy, delivery week, body mass index, the weight gain during pregnancy, and poor neonatal outcome. conclusion: amh is not a predictive factor for poor neonatal outcome and it is not a determinant of the weight of the newborn. objectives: the aim of the study was to investigate the effects of differing amounts of hemolysis on serum high sensitvity troponin i (hs-tni), ck-mb mass and myoglobin measurements. materials and methods: we prepared serum pools having troponin i, ck-mb and myoglobulin concentrations at low ( . ng/l, . ng/ml and . ng/ml respectively), normal ( . ng/l, ng/ml, . ng/l respectively) and high ( ng/l, ng/ml, g/ml respectively) values. the osmotic shock method was utilized to prepare a hemolysate. hemolysate was added into serum pools increasing concentrations of hemoglobin ( . , . , . , , . and . g/l hemoglobin). troponin i, ck-mb (mass) and myoglobin concentrations were measured in duplicate by beckman coulter access analyzer. the hemolysis indices were measured on beckman coulter au . a change of % from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. results: we found a positive interference due to hemolysis for ck-mb (mass) at low concentrations ( . ng/ml), and a negative interference for myoglobin at low concentrations ( . ng/ml) and high concentrations ( ng/ml). conclusions: ck-mb increase and myoglobin decrease in hemolyzed samples with hemoglobin ≥ . g/l, but the bias might not be clinically significant (< total analytical error) in samples. a retrospective study to determine a reliable marker for selective screening of pompe disease lysosomal storage diseases (lsd) are rare inherited metabolic disorders caused as consequence of a deficiency in a specific enzyme required for lysosomal function. pompe disease is one of these disorders with deficiency of a- , glycosidase enzyme with an incidence of : , - : , . as enzyme replacement therapies are available nowadays, early diagnosis is crucial and selective screening is a rational method to reach pompe patients among people who administer to healthcare with lsd suspected symptoms. this study aims to examine the relationship between basic biochemistry parameters and a- , -glycosidase activities retrospectively, in order to find a key parameter for selective screening of pompe disease. for this reason a- , glycosidase, creatine kinase (ck), creatine kinase-mb (ck-mb) activities calcium, phosphate levels of those who had been suspected to be lsd patients and administered to our laboratory for analysis are examined retrospectively. out of patients's examined, of them were diagnosed with pompe disease depending on clinical findings & low a- , glycosidase activity. enzyme activities of pompe patients were . nmol/ml/hour as lsd suspected patients'activities had a mean of . nmol/ml/hour (p = . ).comparison of ck activity was compared results showed significant difference between pompe patients and lsd suspected patients. even though ck activity levels of the lsd suspected patients were much higher ( vs - u/l) than reference interval, the levels of the pompe disease patients' were still more than twice of the lsd suspected group ( vs u/l, p = . ). ck-mb, ca, p levels didn't show a significant difference. a strong (-) correlation (p = . r=À . ) was observed between a- , -glycosidase and ck activities (n: ). selective screening is a rational way to diagnose rare diseases. this study's results show that ck activity can be used as a key parameter to determine patients for selective screening of pompe disease within lsd suspected population. the functional effect of stem cells on the reproductive organs infertitility is considered as a major health problem of recent century. importance of stem cell is increasing so it is searched new features and supposed to be involved in the infertitility treatment where oxidative stress and apoptosis play importany role. we aimed to investigate the beneficial effect of the stem cells related to free radicals and cell death on testis and ovary. biopcy model of wound healing was created in the rat testis and ovary with ppd syringe where stem cells were delivered by injection. rats were divided into four groups including controls, sham, wound healing and wound healing with stem cell. after the creation of the wound, bone marrow-derived mesenchymal stem cells from the tibia of the mature rats and medium were administered to ovaries and testes. following the applications, ovary and testis samples were investigated for oxidative stress and apoptosis by immunohistochemistry. in comparison with the medium and stem cell applications without a medium support, it was meaningfully determined that healing effect in testicles and ovaries were spotted specifically on the seven day. tissues were analysed for these staining by h-score and h-score results were determined using one-way anova test statistically. our results show the positive effects which clinic applications can bring by displaying the great contribution of the stem cell application in the treatment of testicle and ovary damage. these findings suggest that transplantation of the mesenchymal stem cells may help to promote better enviroment for the reproductive organs by the effect on oxidative stress and apoptosis. the further studies of these results in the molecular level can lead the way to solve the problem of infertility, to increase the percentage of success in the ivf and icsi techniques and more importantly to perform a differentiation from a somatic cell to a germ cell. the antimicrobial activity of ( h)-furanone derivative on staphylococcus aureus nosocomial infections caused by methicillin-resistant staphylococcus aureus strains are known to be a reason of many infectious diseases like osteomyelitis, endocarditis, sepsis etc. being organized in biofilms these bacteria become extremely resistant to antimicrobials and host immune system leading to difficulties in treatments. here we report the effect of ( h)-furanone derivative possessing sulfonyl group and l-menthol moiety (f ) on biofilms formed by s. aureus atcc and mrsa cells. while exhibiting relatively high minimal inhibiting concentration -mic ( mg/l), clear synergy with a number of antibiotics was found in the checkerboard assay. thus, in the presence of mg/l of f the mic of kanamycin was decreased -fold, and the mics of both erythromycin and ampicillin were lowered -fold. at the concentration of mg/l f also completely inhibited the biofilm formation by s. aureus; the cell growth was suppressed by two orders of magnitude as judged by differential fluorescent staining with syto and propidium iodide. the addition of f to preformed h-old biofilms increased the fraction of red-stained (dead) cells of both s. aureus atcc and mrsa strains uniformly throughout the whole profile of the biofilm. the quantitative analysis of clsm microphotographs revealed that f at concentration of mg/l led to death of up to % of biofilm-embedded cells. this fact suggests that f efficiently penetrates into the biofilm matrix and kills the cells without visible damage of biofilm structure. in summary, furanone f seems to be a promising compound for drugs design to treat biofilm-embedded s. aureus. this work is supported by the russian science foundation, project № - - and the german academic exchange service (№ ). pneumonia is an inflammatory lung disease which can be associated with inadequacy of host defense system and the proliferation of various pathogenic microorganisms into the lower respiratory tract. community acquired pneumonia (cap) is one of the leading causes of death in elderly. the incidence of pneumonia in people aged and over is - times more than young adults. creactive protein (crp) is an acute-phase protein of hepatic origin that increases following interleukin- secretion by t cells and macrophages. procalcitonin (pct) is a peptide precursor of the hormone calcitonin, the latter being involved with calcium homeostasis. it is composed of amino acids and is produced by parafollicular cells (c cells) of the thyroid and by the neuroendocrine cells of the lung and the intestine. the level of pct rises in a response to a proinflammatory stimulus, especially of bacterial origin. the aim of this study was to compare crp and pct levels in young and elderly patients with pneumonia. recently diagnosed young and elderly patients with pneumonia and their respective aged matched controls (n = , n = ) were enrolled this study. crp and pct levels were by immunoturbidometric and by elisa methods respectively. crp and pct levels for young control and patients and elderly control and patients respectively are . ae . mg/l, . ae . ng/ml, . ae . mg/l, . ae . ng/ml, . ae . mg/l, . ae . ng/ml and . ae . mg/l, . ae . ng/ml. young patients with pneumonia have significantly higher crp and pct levels than their controls (p < . and p < . ). elderly patients with pneumonia have significantly higher crp levels than their controls (p < . ). crp and pct are important markers in the diagnosis of pneumonia. effect of serum albumin concentration on total and ionized calcium z. adiyaman, c. yilmaz, s. a. peker, d. y€ ucel ankara training and research hospital, ankara, turkey objective: the aim of the study is to investigate in vitro effect of albumin concentration on total and ionized calcium concentrations. materials and methods: a serum pool with low albumin ( . g/dl) and normal calcium ( . mg/dl) concentrations was prepared from leftover sera. from this serum pool, two parts, each of ml were aliquoted. purified albumin, . g, was added to one of these pools and albumin concentration was determined as . g/dl. the low and high albumin pools were mixed at different ratios and pools with . , . , and . g/dl albumin concentrations. total calcium and albumin concentrations of these pools were measured at a beckman-coulter au analyzer and ionized calcium was measured at a radiometer abl blood gas analyzer in triplicate. total and ionized calcium concentrations were evaluated as compared to those of the original pool with an albumin concentration of . g/dl. results: total calcium concentrations are increased with the increasing albumin concentrations: . %, . %, . %, and . %, respectively. whereas, ionized calcium concentrations were decreased with increasing albumin: . %, . %, . %, and . %, respectively. conclusions: when total allowable error limits based on biological variation were considered, total calcium concentrations are significantly increased at > g/dl albumin concentrations. ionized calcium is significantly affected by . g/dl and over albumin concentrations. a regression equation based on albumin concentration may be useful for corrected ionized calcium concentrations. relationship between lipoprotein (a) and hba c in patients with type ii diabetes , is a complex lipoprotein consisting of ldl and apolipoprotein(a). lp(a) is a risk factor for coronary artery disease and stroke. the relationship between lp(a) and diabetes mellitus is not clear. in this study, the relationship between lp(a) and glycemic parameters such as hba c and fasting glucose concentration was investigated. lp(a), hba c, fasting glucose, triglyceride, total cholesterol, ldl-and hdl-cholesterol concentrations were screened retrospectively from july to july . there were patients with these test results at the same time. the patients were grouped according to hba c values: group i < . % (n = ), group ii . - . % (n = ), and group iii > . % (n = ). the relationship between these parameters were statistically within each group and all groups. there was not a statistically significant difference between the lp(a) concentrations of group i and group ii. lp(a) concentrations of group i and ii were significantly higher than those of group iii.. _ in total, lp (a) was negatively correlated with hba c (r = . ; p < . ), but there was not a significant correlation with fasting blood glucose. _ in groups, there was a significant and negative correlation between lp(a) and fasting glucose in only group i. the negative correlation between lp(a) and glycemic parameters is interesting in patients with diabetes. despite lp(a) is an independent risk factor for cardiovascular diseases, on the contrary to expectations, lp(a) concentrations are decreased in diabetes. effect of blood collection through intravenous lines on hemolysis erroneous results are one of the most important causes of medical errors and may lead to unnecessary investigations or inappropriate interventions. total testing process consists of preanalytical, analytical and postanalytical phases. hemolyzed specimens that one of the most common source of preanalytical errors are frequently observed in laboratory practice and associated with incorrect laboratory results. blood collection through intravenous lines frequently results in hemolysis especially at eds and icus. in this study, we aimed to compare the effect of blood drawing by using bd luer-lock adapters and injector on the hemolysis rates at the ed. patients who has been admitted to the ed were included in this study. all samples were drawn from newly inserted iv lines. the first blood sample was drawn with injector and the second one was drawn with luer-lock adapters to vacuum tubes. after the centrifugation routine chemistry tests and hemolysis indices were analysed on a beckman coulter au analyzer for each serum tube. the statistical significance of differences between two tubes was calculated with paired samples t test and statistical significance was accepted as p < . . there were statistically significant differences between the two groups of tubes for the following parameters: ldh, ck, ast, k + , total bilirubin, protein, albumin, alp, calcium and hemolysis index (p < . ). the use of luer-lock adapters instead of injector could reduce the hemolysis rate. because of it reduces false results and unnecessary investigations, this approach will be more appropriate and cost-effective in ed. hemolysis and test rejection: are we following a reliable process? introduction: in laboratories, some blood samples are rejected due to hemolysis. we usually cancel only some of the tests that are affected by hemolysis. however, the frequency of the test cancellation may be relative. each test is affected in different degrees of hemolysis; some of them are not even affected at all. in this study, we aim to investigate unnecessary cancellations and explain the relationship between hemolysis and test results according to their kit inserts. materials and methods: we measured hemoglobin levels of hemolyzed serum using drabkin method (abbott). interference studies are conducted using clsi protocol nccls ep -p is written in kit inserts. target values ( %) and their change due to different degree of hemolysis have been defined. results: hb concentration ranges of hemolyzed sera were found from to mg/dl. according to kit inserts, aspartate aminotransferase (ast) test results deviate . % from the target when the degrees of hb are mg/dl. when the degree of hemoglobin is mg/dl, the test strays about . %. potassium levels increase ( %) at mg/dl hb while this increase reaches to . % at mg/dl hb. sodium, calcium, ck, crea, total bil, lipase are not significantly affected even at mg/dl. in lactate dehydrogenase (ldh) tests, test reporting is not allowed at any hemolysis level. alt increases %, at the mg/dl hb. ast and potassium results were excluded from patients' reports even though those samples had low hb. some of them were reported despite of excess hemolysis. some tests are even blocked without ever being studied. discussion: prior to the approval of the lab specialist, technicians decide whether to cancel the tests affected by the hemolysis according to the visible hemolysis based on their personal knowledge. conclusion: we should use the hemolysis index, in which standards would be defined via guidelines. this way, all technicians and specialists could know which results are false. the dna-binding hu-proteins are present in all bacteria and belong to the family of nucleoid-associated proteins. these proteins can be considered precursors to eukaryotic histones. gene knockout of hu-proteins partially inhibits the growth of bacteria, their ability to resist various stressing factors and in some cases leads to their death. since the spatial structure of hu-proteins is highly conserved it is possible to create inhibitors that will affect them in a broad spectrum of pathogenic bacteria. in the present work the preparation of the recombinant hu protein from mycoplasma gallisepticum, crystallization of this protein, and x-ray diffraction study of this protein has been reported. the crystallization conditions for studying protein were found by the hanging-drop vapor-diffusion method. found conditions have been adapted to the counther-diffusion method in the capillary. the x-ray diffaction dataset from grown crystals have been collected using synchrotron radiation. d-structure of the hu protein from mycoplasma gallisepticum have been determined with a resolution. structural features of the investigated protein are described. this work is supported by russian scientific fund ( - - ). a novel sensitive disposable indium tin oxide (ito)-based electrochemical immunosensor was developed for simple, rapid and sensitive biomonitoring of sox . sox is a cancer biomarker and used for detecting of small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, skin cancer, prostate cancer, and breast cancer. in this study, indium thin oxide (ito) thin film was used as working electrode. carboxyethylsilanetriol was also used for electrode modifying so as to obtain self-assembled monolayers. the formed self-assembled monolayers were activated with -ethyl- -( -dimethylaminopropyl) carbodiimide (edc)/n-hydroxysuccinimide (nhs) chemistry. edc was used as a heterobifunctional crosslinker. nhs was used in conjunction with the crosslinker edc. anti-sox antibody was used as a biorecognition element and it was covalently immobilized onto the ito electrode modified with carboxyethylsilanetriol. immobiliztion steps were characterized by cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis), and scanning electron microscopy (sem). the optimal immobilization conditions for the best sensitivity of the new immunosensor were investigated. under optimal conditions, this immunosensor demonstrated a wide linear range ( . - pg/ml) with a detection limit as low as . ng/ml sox . furthermore, the developed sox immunosensor had good storage stability, repeatability and reproducibility. in this work, we successfully fabricated disposable ito thin film based electrodes for sensing the interaction between sox antigen and anti-sox antibody by electrochemical impedance spectroscopy and cyclic voltammetry. and our developed immunosensor has an acceptable performances for the detection of sox antigen, exhibits low detection limit, has selective and reproducible results in immunoreaction analysis. we are thankful for the support from t € ub _ itak (the scientific and technological research council of turkey, project number: z ). applying multiple linear regression model to determine the relationship between anti mullerian hormone with age, luteinizing hormone, follicle stimulating hormone and estradiol: a data mining study introduction: anti mullerian hormone (amh) has a widely used in our life because it is a good indicator of reproductive age to estimate the time of menopause. the purpose of this retrospective data mining study is the estimate of ovarian reserve by using amh and determines relationship between other indicators which are luteinizing hormone (lh), follicle stimulating hormone (fsh), estradiol and age. materials and methods: . women members were included this retrospective data mining study who were applying to acıbadem labmed laboratory. multiple regression analysis of age related changes of amh ( - ) and lh, fsh and estradiol were investigated. beckman gen ii elisa kit was used for amh and the technique of electrochemiluminescence and roche elecsys cobas analyzer were used for the measure of other hormones. results: amh shows meaningful correlation between lh, fsh, estradiol and age but also seen there is no correlation between progesterone. after the multiple linear regression analysiz amh= . -( . age)À( . fsh) + ( . lh)À( . estradiol) is detected and the model's r = . is also detected. conclusion: nowadays there are lots of methodology were developed the estimate the function of ovary and biological age of ovarian. age, fsh, lh and estradiol show ovarian reserve by indirectly. this study shows the mathematical relationship between amh and the other indicators and results are thought to lead to future developments. antioxidant and anticancer effect of artemisia absinthium extract on colon and endometrium adenocarcinoma cells plants have always been among the common sources of medicines that have many phytochemicals with various bioactivities, including antioxidant and anticancer activities artemisia absinthium (ar) has been used as an antipyretic, antiseptic, anthelmintic, tonic, diuretic, and for the treatment of stomachaches in turkish folk medicine. this study aimed to investigate antioxidant, cytotoxic, genotoxic and apoptotic effect of methanol extracts of ar activities on the human colon (dld- ) and endometrium (ecc- ) adenocarcinoma cell line. total phenolic, flavonoid content, and antioxidant activities were determined using suitable methods (abts, cuprac i.e). cytotoxic effects of ar on cells were determined by mtt and neutral red uptake assays. genotoxicity was evaluated by comet assay and, apoptosis induction were detected by apoptosis elisa and acridine orange staining methods at the half maximal inhibitory concentrations (ic ) levels. it was determined that extract have shown antioxidant activity in all tests and that they could be considered as a source of natural antioxidants. cytotoxic effects were concentration-time dependent. specifically, apoptotic and genotoxic effect increased at and lg/ml concentrations by hours. we found that ar extract had antiproliferative, genotoxic and apoptotic effects on the human cancer cell lines dld- and ecc- . however, further studies at molecular level are required to support our findings and to elucidate chemopreventive and chemotherapeutic effects of ar on colon and endometrium cancers. keywords: artemisia absinthium, antioxidant, anticancer, apoptosis, genotoxicity introduction: colorectal cancer is considered as a major gastrointestinal. this cancer is the second cancer related cause of death after lung cancer in worldwide. we designed a vaccine chimeric including cea and ca - against colorectal cancer (ce-ca). materials and methods: the construct were analyzed by bioinformatics softwares. in this study, the ce-ca gene was optimized using the codon bias of e.coli and synthesized by biomatik company. then construct (ce-ca) was cloned into an expression vector and recombinant constructs transferred to e.coli bl de bacterium and desired recombinant protein was expressed. recombinant protein was purified using ni-nta affinity chromatography. the content of secondary structures was obtained by circular dichroism (cd) spectrum. then recombinant protein was confirmed using western blot analysis and indirect elisa method. results: sds-page analysis showed that the recombinant protein was highly expressed and purified. western blot analysis confirmed recombinant protein. also cd spectrum confirmed predicted structures by bioinformatics tools. the elisa results showed significantly high affinity toward recombinant ce-ca protein. discussion: based on many studies, cea as potential immunogenic candidate could be considered in vaccine studies. also ca - is a cell-surface antigen that has significant increase of expression in colorectal cancer, thus as marker of colorectal cancer. based in available data, these two antigens, in combination can provide specificity for production of colorectal cancer vaccine. conclusion: these findings suggest that ce-ca as potential immunogenic candidate which could be considered in future vaccine studies and detection of colorectal cancer. flow cytometric cell cycle and apoptosis analyses of some wild animal species a. tas, e. koban bostanlar tubitak, marmara research center (mrc), genetic engineering and biotechnology institute (gebi), animal genetic and reproductive biology laboratory, kocaeli, turkey cell biobanking; more specifically cryopreservation of biological diversity, is promising as a tool to preserve wild animals as well as domestic ones via nuclear transfer. in this study, we investigated the viability and cell cycle characteristics of wild animal species (fallow deer, red deer, wild sheep, wolf, wild goat). auricular tissue samples were maintained in pbs+ %psa. tissues were seeded on mm petri dishes containing dmem/high glucose supplemented with % (v/v) fcs and incubated %co in air at % relative humidity and at °c. after seeding, the medium was unchanged for days and then it was changed in every days for days at maximum. once the cells were obtained; flow cytometric cell cycle and apoptosis analyses were done. in terms of apoptosis, all the groups showed high viability rates (over %) in culture when compared with the negative control ( %). the cell cycle comparisons were made between serum-starved cells and roscovitine treated cells, both for which untreated cells were used as control, which revealed different results for different species. there was no difference found between serum-starved cells and roscovitine treated cells for red deer and wolf. the serum-starved cells resulted in higher g /g phase for fallow deer and wild goat. on the contrary, roscovitine treated cells resulted in higher g /g phase for wild sheep. as a result; the cells obtained from wild animals had high viability and g /g phase rates. therefore, they may serve as a donor cell source for nuclear transfer studies.(grant: tubitak kamag, turkey, g ). the interaction of different types of antibiotics with endothelial cells in the presence of nanoparticles the interaction of nanomaterials with cells and lipid bilayers is critical in many applications such as phototherapy, imaging, and drug/gene delivery. the aim of this study was to investigate the interaction of nanoparticles (fe o ) or nanoparticles fused with different antibiotics with cell membranes in order to reveal changes in the membrane organization. endothelial cells were used to determine the effect of different antibiotics (gentamicin, kanamycin, amikacin, penicillin, polymyxin, neomycin, cefotaxime, bacitracin, moxicillin, erythromycin, streptomycin and vancomycin) on the membrane organization. for recording the anisotropy of cell suspensions treated with antibiotics or nanoparticles fused with antibiotics we used - -trimethyl- -phenyl , , hexatrien p-toluenesulfonate (tma-dph). we decided to use nanoparticles fused with antibiotics because they contain small amounts of antibiotics which makes them less toxic than simple antibiotics,which is very important in patients with genetic diseases such as cystic fibrosis, that should be treated with antibiotics for a long time. our results showed that at temperatures between and °c simple nanoparticles decreased the membrane fluidity. at physiological temperatures ( - °c) nanoparticles fused with antibiotics (gentamicin, vancomicin, cefotaxim, bacitracin, amoxicillin) increase more the membrane rigidity compare with simple antibiotics or nanoparticles.erythromycin, polymyxin and penicillin increase the membrane rigidity at °c, and at °c the same effect was obtained in the presence of nanoparticles fused with these antibiotics,suggesting that the nanoparticles are dependent to temperature for penetrating the membrane. in conclusion the membrane fluidity does not depend on antibiotics types, the modification are present in many antibiotics irrespective of class type.the presence of nanoparticles fused with antibiotics is very important for long term treatment. objectives: hypertension is an important cardiovascular risk factor for the development of atrial fibrillation (af). increased atrial electromechanical coupling time interval measured by tissue doppler is accepted as an important factor for prediction of af development in hypertensive patients. monoamine oxidases (maos), are enzymes which catalyze the oxidation of monoamines. -isoprostane is considered as an indicator of oxidative stress. mao activity and -isoprostane levels were measured in some diseases. however, there are no information on -isoprostane levels and mao activity in newly diagnosed patients with stage hypertension has not been observed in a study of literature. aim: this is the first study, we aimed to evaluate the levels of mao and -isoprostane in newly diagnosed patients with stage hypertension. the study included newly diagnosed stage hypertensive patients with no other systemic disease. patients were selected as randomized ( women, men; range of age - years) and healthy individuals as control ( women, men; range of age - years). all the underwent tissue doppler echocardiographic examination. blood samples were taken from patients and controls and, the levels of mao and -isoprostane in serum samples were measured by elisa. results: baseline blood pressures, electrocardiographic and echocardiographic findings, and atrial electromechanical coupling were similar in both groups (p > . ). compared to the control group, the activity of mao and -isoprostane levels were found significantly higher in patients (p < . ). conclusion: increased -isoprostane level indicate that there is oxidative stress in newly diagnosed patients with stage hypertension. also, increased mao activity may be biochemical biomarkers for the diagnosis of hypertension. keywords: hypertension, monoamine oxidase, -isoprostane p-mis- determining the indirect reference intervals for complete blood count parameters in bursa, turkey reference intervals (ris) for laboratory test results are defined as the most commonly used diagnostic tool in medicine. therefore, careful determination of ris by the laboratory for use is a very important task. although c -a guideline recommends the direct ris (dris) calculated from healthy subjects, ris can be calculated from laboratory data which are called as indirect ris (iris). the study was carried out at the central laboratory for clinical chemistry, teaching and research (uludag university, bursa, turkey) . the results of the laboratory analyses from , males, , females, stored for approximately one year, were used for statistical analysis. data for hospitalized patients and for ambulatory patients from the intensive care unit were eliminated. furthermore, we used evidence based criteria to enrich the health-related values. a modified bhattacharya procedure was used to estimate the iris from hospital patient data. the nested anova was used to evaluate variations among genders and ages. cell dyn analyzer (abbott diagnostics, il, us) was used for the measurements of complete blood count. the obtained iris were also compared the dris determined in our previous ri study and the ris suggested by the manufacturer. we found that the ris of rbc, hb and hct required strong gender partition and calculated the ris of rbc, hb and hct separately. the observed iris for wbc, sub-fractions of wbc and plt in both genders are in good accordance with the dris reported in previous study. age-related changes were noted for rbc, hb, and hct. the calculated iris for rbc, mcv and rdw are different from the ris suggested by the manufacturer. we believe that, using this relatively easy technique, every laboratory can produce its own iris, divided, where possible, according to sex and age and according to local conditions. these ranges can be complementary to dris obtained for reference individuals according to the ifcc recommendations. the principal sigma subunit, involved in transcription of most house-keeping genes in escherichia coli, was also shown to induce rnap pausing during transcription elongation, by interacting with promoter-like motifs in the transcribed dna. such pauses were proposed to play important roles in the regulation of phage and cellular genes. e. coli contains six alternative s subunits but little is known about their ability to induce transcriptional pausing. we expressed and purified alternative s subunits of the sigma family and tested their effects on transcription elongation in vitro on natural and synthetic dna templates containing consensus promoter motifs. the structure of the paused complexes was analyzed by dna footprinting methods. in vivo analysis of transcription was performed using reporter genes placed under the control of corresponding promoters. we demonstrated that the stationary phase sigma subunit induced efficient rnap pausing on both synthetic and natural dna templates containing promoter-like motifs in initially transcribed regions. in contrast, the sigma and sigma subunits did not affect rna elongation. we showed that the sigma -induced pausing depends on sigma contacts with both nontemplate dna strand and rnap core. the pausing results in formation of backtracked transcription elongation complexes which can be reactivated by gre factors that stimulate rna cleavage by rnap. our results for the first time reveal transcriptional pausing induced by an alternative s subunit. analysis of sigma -dependent promoters shows that a substantial fraction of them contains potential pause-inducing motifs suggesting that such pausing may be a widespread phenomenon. we propose that sigma -dependent pauses may play important roles in genetic regulation and modulate the binding of transcription repressors or activators to promoter regions. the crosstalk between streptococcus pneumoniae rnase r, ribosomes and translation c. b arria, s. domingues, c. arraiano instituto de tecnologia qu ımica e biol ogica, lisbon, portugal ribonucleases (rnases) are enzymes that ensure maturation, degradation and quality control of rna thus, contributing to the maintenance of the optimal amount of each transcript in the cells. escherichia coli rnb family of enzymes is present in all domains of life and includes rnase r, rnase ii and the eukaryotic rrp /dis , dis l and dis l proteins. in streptococcus pneumoniae only rnase r was identified. rnase r, encoded by the rnr gene, hydrolyzes rnas starting from the end. rnase r level is increased in several stress conditions such as heat shock, stationary phase or cold shock, conditions in which most of the proteins translation is blocked. moreover, rnase r is the only exoribonuclease able to degrade highly structured rnas without the help of a helicase which is critical at low temperatures. here, we investigated the role of this enzyme by comparing the wild type strain with an rnr mutant strain. for that purpose we performed northern blots analysis of transcripts involved in translation. also, we investigated rnase r connection to the ribosome and polysome fractions using sucrose gradient polysome separation and western blots. in this study, we highlight the importance of s. pneumoniae rnase r in translation. we show that this enzyme interacts with ribosomes mostly with the s subunit at °c. moreover, in the absence of this enzyme we have observed a decrease in the amount of the s ribosomal subunit, concomitantly with the increase of s and s subunits. rnase r seems also to modulate the amount of the elongation factors ef-tu and ef-g transcripts. nevertheless, preliminary results further suggest other roles of rnase r in translation. modified nucleotides are present in many rna species in all domains of life. the biosynthetic pathways of such nucleotides are well studied. however, much less is known about the degradation of rnas and the salvage of modified nucleotides, their respective nucleosides or heterocyclic bases. using an e. coli uracil auxotrophic strain, we screened the metagenomic libraries for genes, which would allow the conversion of -thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. we show that a novel gene encoding previously uncharacterized domain of unknown function (duf) is responsible for such phenotype. we have purified this recombinant protein and demonstrated that it contains a fe-s cluster. the substitution of cysteines, which have been predicted to bind such clusters, with alanines abolished the growth phenotype. we conclude that this domain is required for conversion of -thiouracil into uracil in vivo. this work is supported by the research council of lithuania (lmt, mip- / modified nucleotides are present in almost all classes of rna. they have great chemical diversity and are critical for rna folding, stability, interaction with cellular proteins and thereby for various cellular processes such as translation, stress response, and signaling pathways. biosynthesis of pyrimidine nucleotides and their modified derivatives in rna is well studied. nonetheless, not much is known about the cellular degradation of these compounds and the enzymes catalyzing such processes. using an e. coli uracil auxotrophic strain, we screened metagenomic libraries for genes encoding isocytosine deaminases. three novel genes were obtained, one of which encodes a protein similar to oxoguanine deaminases. the other two encode proteins resembling hydroxydechloroatrazine ethylaminohydrolases. we confirmed that these proteins are functional in vivo, allowing growth of e.coli on minimal medium with isocytosine. we also demonstrated that such purified recombinant enzymes catalyze the conversion of isocytosine, but not cytosine, into uracil in vitro. natural products display special attributes in the treatment and prevention of various human diseases, including cancer. a significant number of organic compounds from plants exhibit anticancer properties as attested by in vitro and in vivo studies. emerging evidence supporting the antineoplastic activity of natural compounds has rendered them promising agents in the fight against cancer. in this study, skin from limnio grape, a red greek grape variety that is indigenous to the greek island of lemnos, was extracted using mixtures of methanol, water and acetone; the apoptosis-inducing properties of these extracts were studied in the human ovarian malignant adenocarcinoma cell lines tov- g and tov- d. for this purpose, tov- g and tov- d cells were treated with limnio grape skin extracts at a range of concentrations, at °c, for , and hours. untreated cells incubated for the same time intervals served as controls. cell viability was determined by measuring metabolic activity (colorimetric mtt assay) and observing cell membrane integrity (cell staining with trypan blue). after the determination of the optimal concentration of the extract, total rna was extracted from treated and untreated (control) tov- g and tov- d cells. after determination of rna concentration and subsequent first-strand cdna synthesis, mrna expression analysis of apoptosis-related genes was performed with rt-pcr using gene-specific primers. an increasing percentage of non-viable cells was observed by increasing cell exposure time and extract concentration. distinct modulations of the expression of apoptosis-related genes at the mrna level were also observed, mainly concerning bcl , bclx, bax, bak and bcl l , along apoptosis induction. in conclusion, the cytotoxic properties of limnio grape skin extracts against ovarian malignant adenocarcinoma cells merit further investigation. the intrinsic apoptotic pathway seems to be the major mechanism of action induced by these plant extracts. almost all eukaryotic mrnas are polyadenylated by a complex machinery that recognizes the poly (a) signal, cleaves the mrna and adds the poly (a) tail. % of human genes harbor multiple poly (a) signals. alternative polyadenylation (apa) generates transcript isoforms with different utr (untranslated region) lengths due to the use of proximal or distal poly (a) signals. hence, tightly regulated apa has been observed in normal physiological settings as well as in diseases. considering that utr shortening cases have been linked to increased protein levels, we hypothesized deregulated apa to be one of the potential cancer related mechanisms. we investigated the utr alterations in er(+) breast cancer patients and cell models compared to normal breast tissue, using gene expression data and a probe-based quantification tool, apadetect. based on means of proximal to distal probe sets, slr (short-long ratio) were calculated as an indication apa. significance analysis of microarrays (sam) determined significant genes. the gse numbers of the datasets are gse , gse and gse . we analyzed two datasets of er(+) breast cancer patient samples (n = , n = ) compared to normal breast tissue (n = ) using apadetect and sam. a total of utr shortening and utr lengthening events were detected in breast cancer samples compared to normal breast tissue. ontology analysis suggested almost all the utr shortening genes were proliferation related and were indeed reported to be upregulated in breast cancer. to further investigate the connection between apa and era status, we used data from a cell line model; wild type or era transfected mda-mb- cells that are otherwise of triple negative nature. our results suggested that most of the genes are utr shortened or lengthened via direct binding of era to dna. our results suggest involvement of apa mechanisms in era action mechanisms. possible link between era regulated transcription and apa remains to be elucidated. contamination of nucleic acids (na) as a result of na extraction protocols may result an inaccurate measurement of dna copy number. agarose gel electrophoresis and spectrophotometric methods are commonly used to check dna purity. however, the resolution of these methods may not be good enough for special applications such as determination of dna copy number and separation of base pairs (bp) that are close in their bp number. in this study, we have developed a new method for separating na's ranging between - bp also detecting the impurities in dna solution in %, % and % ratios to the dna of interest. the developed method was validated using the in-house dna fragments of , and bp. the dna mixture analyzed using analytical hitachi elite lachrom hplc using the guard and analytical columns tskgel dna-npr, . lm, . mm id . cm and tskgel dna-npr, . lm, . mm id . cm, respectively. the validation of the analysis was performed by running each sample five times on three different days. the linearity of the detector response was established by plotting a graph to quantity versus area of bp dna. the lod and loq were then measured by calculating the minimum level at which analyte can be readily detected and quantified. the ratios calculated with hplc were compared to the ratios calculated by quant-it kit. recovery values were calculated for each measurement and the uncertainty were calculated for each ratio. the method was found linear for bp in the range of . ng to ng dna with the regression coefficient of r = . . lod and loq for the bp dna was found to be . ng and . ng, respectively. the recovery values for the %, % and % impurity ratios were found . . . and . , respectively. the purity of the synthetic dna was determined by hplc and related uncertainty was calculated. the developed method is a simple alternative to electrophoresis and spectrophotometric methods with higher resolution and separation range. physical and chemical factors can disturb the conformation of proteins maturing within the cellular secretory pathway. in response to unfolded proteins the cell activates several stress signaling and adaptive response mechanisms. the aim of our study was to investigate small non-coding rnas as the potential regulators of cellular response to unfolded proteins (upr). for this, we conduct the next generation sequencing of small rna and transcriptome analysis of mrna from jurkat cells exposed to dithiothreitol (dtt), which reduces protein disulfide bounds. analysis of mirnas reveals the differential expression of mirnas. we observe a decrease in the normalized amount of reads aligned to mirna loci in stressed cells. affymetrix analysis with subsequent gsea reveals downregulation of reactome mirna biogenesis pathway (fdr = . ). the length distribution of small rnas revealed nt-peak corresponding to trna-derived fragments, amount of which was increased by . -fold under dtt treatment. the trna isotypes that gave rise to almost % and % of all nt rna fragments in stressed and control cells, respectively, include glycine, glutamic acid, aspartic acid and valine. the vast majority of nt fragments produced from these trnas are precisely phased halves with the characteristic cleavage patterns generated by rnase a angiogenin (ang). observed upregulation of tirna in stressed cells is accompanied with upregulation of ang mrna and down-regulation of angiogenin inhibitor (rnh ). we speculate that translational repression, associated with observed tirna, is an additional mechanism of reducing global protein synthesis in response to dtt-induced stress. collectively, our findings reveal the increase in tirna, the differential regulation of mirna expression together with the global mirna downregulation as the most prominent small rnome reprogramming events and possible fine-tuned levels of post-transcriptional regulation upon dtt-induced cellular stress response. global gene expression changes after spinal cord injury j. k. hyun , , , j. kim , , j. y. hong dankook university, cheonan, south korea, institute of tissue regeneration engineering (itren), cheonan, south korea, the neuronal regeneration is hardly achieved spontaneously after spinal cord injury (sci), and the restoration of somatic and autonomic functions after sci is also challenging in the clinical field. the pathophysiology of sci is extremely complex and many in vitro and in vivo studies continued to report opposite results each other in spite of the same treatments, therefore a fundamental analysis such as an extensive assay of global gene expression is required to find a way for spinal cord regeneration. in this study, we aimed to detect the changes of global gene expression after spinal cord contusion in rats according to the time sequence. the spinal cord tissues at contusion site were sequenced after spinal cord contusion in rats using rna-sequencing technology. for time sequence analysis, five time points was determined; hour, day, week, month and months after spinal cord contusion, and sham operated rats at each time point were used as controls. quantitative rt-pcr analysis was also performed to validate expression changes of candidate genes in each category. we found that the pattern of changes in gene expression at acute and subacute stages was quite different from that at chronic stage, especially genes associated with with neurotrophin signaling and apoptosis pathways. most of gene expression levels of inflammatory cell markers were increased and peak during acute stage ( hour to week) and maintained until chronic stage. some of regeneration-associated genes (rags) including brain derived neurotrophic factor, glial cell derived neurotrophic factor and ciliary neurotrophic factor were increased at hour or day after sci. we concluded that the information of gene expression level according to the time sequence after sci might be useful to determine treatment strategies for spinal cord regeneration especially in chronic stage. p- . . - utr length isoform generation profile in a differentiation model alternative polyadenylation (apa) is the regulated selection of a specific poly(a) signal among other proximal and/or distal signals on the utrs (untranslated region) for the endolytic cleavage and addition of a poly(a) tail to form the mature mrna. consequently, position of the poly(a) site determines the length of the utr which is known to harbor microrna and rna binding protein sites. such apa isoforms have already been linked to altered protein levels and even functions. therefore we hypothesized apa to be one of the mechanisms to generate isoform diversity in proliferating and differentiated cells to better understand the molecular basis of cancer. we used a combinatorial in silico and in vitro approach to analyze a well known enterocyte differentiation model; caco- cells. initially we analyzed gene expression datasets for the proliferative and differentiated caco- cells using a probe based apa detection tool. to better understand the significance and to validate these results, we used proliferating and differentiated (day ) caco- cells and tested sample apa events by rt-qpcr. utr isoforms were identified by using race pcr. we identified genes ( % of all apa events) to undergo utr shortening in differentiated cells compared to proliferating cells. on the contrary genes ( % of all apa events) went through utr lengthening events. several genes have been validated to follow the pattern that was seen in apa detection tool so far. to begin understanding the mechanism behind these observations, we are investigating potential inducers of apa during the complex events of differentiation. our next aim will be to further validate and investigate the consequence of such isoform generation events both in the context of differentiation in colon cancer cells. recognition of phosphorylated threonine- of rna polymerase ii c-terminal domain by end processing apparatus o. jasnovidova, m. krejcikova, k. kubicek, r. stefl central european institute of technology, masaryk university, brno, czech republic rna polymerase ii has evolved an array of heptad repeats with the consensus sequence y -s -p -t -s -p -s at the c-terminal domain (ctd) of its largest subunit, rpb . phosphorylation of serines (s , s , and s ) and tyrosine- orchestrate the binding of rna processing and transcription factors in the site of transcription. several recent studies showed that also threonine- site can be phosphorylated which has a number of functional consequences. to reveal the structural basis for the recognition of threonine- phosphorylated ctd, we set out to investigate several proteins factors that were implicated with a high levels of threonine- ctd phosphomarks using integrative structural biology. one of them, a factor involved in the -end processing and transcription termination, showed a high affinity to the phosphothreonine ctd. using nuclear magnetic resonance spectroscopy (nmr), we determined its structure bound to the ctd phosphorylated at threonine- that reveals a direct read-out of the phosphothreonine. altogether, our data provides the first insights into the recognition of this poorly understood ctd mark that plays important role in the ctd code of rna polymerase ii. the results of this research have been acquired within ceitec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. introduction: the treatment of brain tumor glioblastoma (gbm) is still one of the greatest challenge. anti-inflammatory drug indomethacin (ind) mainly acting through the inhibition of cyclooxygenase (cox) has also anti-cancer activity including brain tumors. the aim was to investigate how ind effects an immortality enzyme telomerases' activity. materials and methods: monolayer and spheroid cultures of t g human gbm cell line were used to evaluate the effects of ind ( lm) on cell proliferation, viability, apoptosis, cell cycle, camp levels, the levels of apoptotic and anti-apoptotic proteins, morphology (sem) and ultrastructure (tem) for hours. results were analyzed using the student's t-test. results: ind decreased cell proliferation (p < . ), cell viability (p < . ), cell rate at s phase (p < . ) and g + m phase (p < . ), camp levels (p < . ), the levels of pdgfr-a (p < . ), mrp- (p < . ), nf-jb (p < . ) and cox- (p < . ) in comparison to control group. ind mildly increased apoptosis (p < . ) and caspase- levels (p < . ). interestingly, ind increased htert levels ( %, p < . ; %, p < . ). sem evaluation showed that ind led to decreased and shortened microvilli, the lost of cell interactions and the conversion of many cell shapes from spindle to oval. many cell remnants in the intercellular area, intact cell membranes, many dense lipid droplets and few autophagic vacuols in the cytoplasm were observed under tem. discussion and conclusions: the effect of ind on telomerase activity can only be found in publications at pubmed research that they only showed its' inhibitory effect in colon, gastric, head and neck cancers. in contrast to previous studies, it was shown for the first time that ind increased telomerase activity in gbm cells and this increase was independent from cox- and other tested factors. p- . . - interaction between fibrinogen and insulin-like growth factor binding protein- under physiologic conditions and influence of diabetes mellitus type on this interaction n. gligorijevic, o. nedic institute for the application of nuclear energy, university of belgrade, belgrade, serbia fibrinogen is plasma glycoprotein and principle participant in blood coagulation. it interacts with many proteins, including insulin-like growth factor binding proteins (igfbps). one of them, igfbp- , is controlled by insulin. metabolic changes due to diabetes mellitus (dm) affect igfbp- . besides glucose regulation, igfbp- stimulates wound healing. we have investigated complexes formed between fibrinogen and igfbp- , their change in dm type (dm ) patients and involvement in fibrin clot. samples from adult healthy persons and dm patients were studied: plasma, isolated fibrinogen and fibrin. the amount of igfbp- /fibrinogen complexes was determined using immunoblotting. immunoprecipitation and lectin affinity chromatography were used to confirm interaction between fibrinogen and igfbp- . in vitro incubation of fibrinogen with excess glucose or methylgyoxal (mgo) was employed to demonstrate influence of glyco-oxidation on complexes. results have shown that igfbp- /fibrinogen complexes can be differentiated from igfbp- oligomers and igfbp- /alpha- macroglobulin complexes. the amount of igfbp- /fibrinogen complexes was lower in patients with dm . complexes participated in fibrin clot formation, the amount being significantly lower in patients' samples. the quantity of igfbp- monomer in fibrin clot was greater in patients' samples. in vitro experiments revealed that complexes undergo glyco-oxidative modifications leading to their reduced formation, cross-linking and increased acidity (faster electrophoretic movement). isolated fibrinogen from patients with dm was additionally able to bind exogenous igfbp- . since igfbp- stimulates wound healing, directly and by delivering igfs, igfbp- /fibrinogen complexes may be seen as igfbp- storage instrument, ready to participate in fibrin formation and to assist in damage repair. reduction of complexes due to glyco-oxidative stress in patients with dm may be part of the mechanism responsible for impaired coagulation process. human interferon gamma (hifnc) is a proinflammatory cytokine involved in the regulation of nearly all phases of immune and inflammatory responses. its abnormal expression is associated with the aetiology of many inflammatory and autoimmune diseases. recently we have been exploring the idea to counteract the over-expression of the endogenous hifnc by competitive inhibition with inactive hifnc mutants. they are designed to have preserved affinity to the hifnc receptor, but to be deprived in their capability to trigger the intracellular signal transduction. to this end a library of mutants was created and two potential hifnc antagonists were selected for further investigations: a single point mutant k q (q substitution for k in position ) and a double mutant with additional substitution in the n-terminus. both mutants and the wild type hifnc were expressed in e. coli employing the established by us methodology for large scale production of aggregation-prone proteins in soluble native form. the purified mutants were screened for interferon activity (antiproliferative assay), binding affinity (isothermal titration calorimetry) and ability to compete with the wild type for the hifnc receptor (competition assay on wish cells). the selected mutants demonstrated (single mutant) and (double mutant) times lower antiproliferative activity than the wild type. measuring the binding thermodynamic parameters, we proved that the receptor binding affinity of both mutants was preserved, which is an indication for their potential to compete with the wild type hifnc for its receptor. finally, the biological assay performed on wish cells showed a distinct dose-dependent competition between the wild type hifnc and the mutants. based on the results presented in this study we conclude that the two hifnc mutants are potential candidates for autoimmune therapy based on selective suppression of the endogenous hifnc activity. mesencephalic astrocyte-derived neurotrophic factor (manf) is an er (endoplasmic reticulum) stress-inducible protein and widely expressed in mammalian tissues. it has been identified as a secretory protein that protects cells against er stress-induced damage. er-stress is one of the main mechanisms that play a role in ischemia/reperfusion (i/r)-induced renal injury. recent studies demonstrated that manf can protect cardiac myocytes and cortical neurons against i/r-induced injury. moreover, it has been suggested that it has a restorative effect in ischemic injury. nevertheless, the function of manf in i/r-induced renal injury is still not known. in the present study, we investigated the function of manf by manipulation its expression level in ischemic acute renal failure model established in proximal tubular kidney cells (hk- cells). for this purpose, the cells were transfected with either manf sirna or manf encoding plasmids for silencing or over-expression of manf, respectively. then, the cells were exposed to hypoxia-reperfusion (h/r) induction for indicated times. evaluations of cell viability were determined with wst- reagent. the changes in protein levels of h/r-induced stress markers were analyzed byimmunoblotting. the results showed that the overexpression of manf has provided a significant resistance to h/r-induced cell death, whereas silencing of manf has rendered the cells more susceptible to death. it was also determined that the pretreatment of cells with manf conditioned medium caused a decrease in cell death. additionally, oxidative/nitrosative stress (os/ns) and er stress levels were decreased with over-expression of manf and increased by silencing of manf in hk- cells. taken together, our study suggests that manf may have a protective role against h/r-induced renal cell injury, possibly through the reducing effects on os/ns and er stress. p- . . - his-flag tag as a fusion partner in insect expression systemgain or loss? e. krachmarova , m. tileva , k. maskos , i. ivanov , g. nacheva institute of molecular biology "roumen tsanev", sofia, bulgaria, proteros biostructures, martinsried, germany human interferon gamma (hifnc) is a glycoprotein playing major role in the regulation of innate and adaptive immunity. glycosylation is not essential for hifnc activity but is important for its stability, half-life and protease resistance in blood. the commonly used hifnc in therapy and research is produced in e. coli and therefore is not glycosylated. bearing in mind the above mentioned shortcomings of the non-glycosylated hifnc we expressed it in mammalian cells and transgenic mice, however very low yields were achieved. to obtain glycosylated hifnc, here we employed a secretory expression of n-terminal his-flag fusion protein in baculovirus-infected insect high five Ò cells. this small hydrophilic tag is designed to not affect the proper folding of the target protein and to facilitate the detection and purification procedures. in parallel the same fusion was expressed in e. coli cells. the fusion proteins were purified to high degree of purity by affinity and size-exclusion chromatography. bioassay carried out on wish cells showed that the antiproliferative activity of both fusion proteins was times lower than that of the native hifnc. this result shows that, in contrast to the generally hold view, the n-terminal his-flag tag interferes with the biological activity of hifnc despite of the protein glycosylation. in order to restore the biological activity we attempted to remove the his-flag tag enzymatically. surprisingly, we found that the fusion protein obtained from insect cells was resistant to enterokinase, independently of the enzyme source and experimental conditions, whereas the protein isolated from e. coli was susceptible and the tag-free protein showed fully restored biological activity. we are prone to explain the enterokinase resistance of the fusion protein from insect cells with either the specific conformation of the glycosylated protein or with the interaction of the carbohydrate residues with the enzymatic activity of the enterokinase. p- . . - development of fluorescence assay for highthroughput screening system based on flow cytometry for directed evolution of cellobiose dehydrogenase cellobiose dehydrogenase (cdh) is an enzyme produced by phanerochaete chrysosporium and it has been already successfully cloned in other organisms. one of the most important roles of cdh is removing products of cellulose degradation. cdh is very important for biofuel and biosensor industry. for improvements of enzyme properties we have used directed evolution. the most important step is to develop screening system that reflects properties of interest. screening in microtiter plates (mtp) is expensive, time-consuming and has low throughput with a small number of variants detected ( - in months). the aim of this work was the development of screening system for mutant libraries of cdh expressed on surface of yeast cells based on fluorescent enzymatic assay and flow cytometry. the screening method should be capable of screening cellobiose dehydrogenase variants mutated for higher activity and higher thermostability by error prone pcr. the fluorescent assay was beta-galactosidase (ec . . . ) also known as lactase is the enzyme that typically catalyzes hydrolysis of beta- , -d-galactosidic linkages in beta-d-galactosides, including disaccharide lactose, with glucose and galactose as end reaction products. this enzyme is able to catalyze synthesis of oligosaccharides, in particular galactooligosaccharides via galactosyl transfer reaction. arthrobacter sulfonivorans beta-galactosidase of unique for prokaryotes extracellular localization may find application in food industry for manufacturing lactose-free dairy products and in pharmacology as bioactive principle of medicines prescribed for patients suffering from lactase deficiency. the study was aimed at cloning of the gene encoding a. sulfonivorans beta-galactosidase, purification and characterization of the enzyme. a novel extracellular beta-galactosidase from a. sulfonivorans was recovered with an overall -fold purification, a . % yield and specific activity uÁmg À protein. the subunit molecular mass of the enzyme determined by sds-page analysis equalled kda. it was found that the enzyme displays pi . , prefers ortho-nitrophenyl-beta-galactoside as substrate (km mm) and shows maximum activity at °c and at ph . - . . the beta-galactosidase gene was isolated from the genomic dna library of a. sulfonivorans, sequenced, cloned and deposited in the genbank database under accession number km . . it was established that the gene carries an open reading frame consisting of bp ( amino acids) and encodes beta-galactosidase referred to glycosyl hydrolase family (cazy database). p- . . - different splice-forms of tdrd protein mutated in cataract's and glaucoma's interacts with s k / o. skorokhod, v. filonenko department of cellular signalling, institute of molecular biology and genetics nas of ukraine, kyiv, ukraine ribosomal s kinases (s k) are important players in cellular pi k/mtor signalling network, deregulation of which has been associated with methabolic disorders, inflammation and cancer. previously we had identified a novel binding partner of s k -tdrd (trap). tdrd is a scaffold protein detected in complexes involved in the regulation of cytoskeleton dynamics, mrna transport, protein translation, non-coding pirnas processing, transposons silensing. it was reported recently that mutations in human tdrd result in cataract and glaucoma formation, defined by elevated intraocular pressure (iop) and optic nerve damage. the aim of our study was to confirm s k-tdrd interaplay and study its role in cells. bioinformatical analysis of tdrd sequence revealed the presence of potential phosphorylation sites of s k . using in vitro kinase assay, we have demonstrated that recombinant s k phosphorylate from fragments of tdrd . formation of s k -tdrd complexes in vivo was further confirmed by coimmunoprecipitation using anti-s k and anti-tdrd antibodies generated previously in rat brain lysates. this interaction was further confirmed by confocal microscopy, oleksandr had shown that tdrd co-localize with s k in hepg cells, predominantly in perinuclear region, enreached for one of the tdrd isoforms identified previously. moreover, we have detected that c-terminal synthetic peptides of s k with methylated arg interfere with tdrd from hepg lysates. the physiological characteristics of s k -tdrd interaction and the role of this complex formation in neuropathology's development need further investigation. many biological function of placenta are performed not just a set of individual proteins, but also different oligomeric structures and complexes. herewith, activities of complexes may considerably differ from activities of individual proteins. therefore, identification and characterization of placental multi-protein complexes is an important step to understanding the placenta function. the aim of the present work was to investigate a composition and biological functions of the very stable high molecular mass multi-protein complexes (spc) from placenta of healthy mother. we isolated spcs (~ kda) from the soluble fraction of three human placentas. light scattering measurements and gel filtration showed that the spc is stable in presence salts, acetonitrile and triton x- in high concentrations, but efficiently dissociates in the presence of m urea and mm edta. such a stable complex is unlikely to be a random associate of different proteins. it was shown the spc includes a number of proteins with molecular weights of to kda. several protein components of the spc were identified, including serum albumin, transferrin, iggs, annexin a and other proteins. serum albumin, transferrin and protein with molecular weight , kda are the main proteins of the complex. it was shown high the spcs from three placentas possesses dnsase and catalase activities. an addition, investigation of cytotoxic effect on human cancerous cell lines has shown that the spcs reveal high cytotoxicity. antibody-cytochrome b fusion protein, characterization and applications for antibody development process antibodies have recently become an essential tool being a part of immunodiagnostics, therapeutics and as a valuable instrument in life science research. an enormous number of options utilizing a various tags were used to create a universal antigen-binding domain, which can be easily detectable, highly soluble and might be produced in high yields with low costs, but no multipurpose solution exists yet. we addressed the question whether a single tag could be found for enhancing solubility of recombinant fab antibody fragment and providing its detection and accurate quantification by rather simple method. a new application for hemeproteincytochrome b as the antibodies fusion partner were proposed. we have constructed of recombinant fab antibody fragment cytochrome b fusion protein. we have shown that cytochrome b enhance expression of fab antibodies fragments in bacterial system, and could be a versatile tool for recombinant proteins folding, redox (oxidation) state studies and for their precise concentration determination in the turbid solutions. fusion fab-b protein has a stable red color and characteristic absorbance spectrum with the maximum absorbance at nm in oxidized environment. cytochrome b change its spectrum maximum depending on environmental redox potential and its folded state, so one can track these events in real time spectrophotometrically. binding activities of fab-b fusion protein and hybridoma secreted immunoglobulin were measured by biolayer interferometry and elisa. no significant difference between them was revealed. due to this feature we can distinguish the chimeric protein of interest in complex mixtures and control the process of recombinant proteins expression and purification in real-time. besides, cytochrome b fusion tags multiples recombinant antibody yield (from to times) and doesn't affect antigen-binding properties. the bb - site of fibrin molecule is the site of fibrin protofibrils lateral association l. urvant palladin institute of biochemistry nas of ukraine, kyiv, ukraine previously we showed that fibrin-specific monoclonal antibody i- c (monab i- c) inhibited the fibrin protofibrils lateral association. we suggested that the epitope of monab i- c in bb - of coiled-coil region of fibrin molecule coincides with the site involved in fibrin protofibrils lateral association. the aim of this study was to localize the site of protofibrils lateral association in fibrin molecule using the synthetic peptides bb - , bb - and both their scrambled version, and bb - peptide. monab i- c was isolated from hybridoma culture medium by affinity chromatography on fibrin-sepharose. turbidity analysis was used to study the effect of synthetic peptides on fibrin polymerization. the interaction between peptides and monab i- c was investigated by spr method using plasmon- device. we investigated the effect of synthetic peptides which corresponded to amino acide sequences of fibrin molecule bb - , bb - , bb - , and the scrambled versions of bb - and bb - peptides on a binding to monab i- c and on the fibrin polymerization process. in spr analysis was showed that bb - and bb - peptides, but not their scrambled version, binds to monab i- c, immobilized to a chip. turbidity data showed that only bb - and bb - peptides caused the -fold decrease of the rate of the lateral association of protofibryls at the concentration . À m and . À m, respectively. both of them decreased the final clot turbidity. our data let us to suggest that the bb - site is the site that involved in protofibryls lateral association. it has been recently shown that irisin immunoreactivity was altered in gastrointestinal cancers. as known hematological malignancies was one of the most common malignancies through world, but no study was present how irisin was changed in this type of cancers. therefore, purpose of this was to investigate how immunoreactivity to irisin was altered in hematological malignancies (blood cancers). we used an antibody from phoenix to demonstrate how a kda band after deglycosylation of irisin altered in hematological malignancies. here we first time showed that irisin tissue immunoreactivity from acute lymphoblastic leukemia (all) and acute myelogenous leukemia (aml) patients was increased when compared with unaffected biological tissue parts. from the immune-histochemical (ihc) investigations it is concluded that hematological tissue and blood cells may be another source of irisin and increased with cancer, thus this finding might help to enlighten pathophysiology of hematological malignancies. the value of urine neutrophil gelatinaseassociated lipocalin (ngal) in acute heart failure n. serdarevic clinical centre, sarajevo, bosnia and herzegovina introduction: renal dysfunction is very common in heart failure (hf) and neutrophil gelatinase-associated lipocalin (ngal) is used as an early marker of acute renal tubular injury. recent studies have been reporting that ngal is inhibitor of inactivation of matrix metalloproteinases (mmp- ) which results in enhanced proteolytic activity with prolonged effects on collagen degradation. due to its relation to extracellular matrix degradation in myocardium and infammation, we hypothesized possible increased ngal expression in hf besides it renal dysfunction etiology. patients and methods: in study were included patients hospitalised with signs and symtoms of ahf. urine samples for ngal analysis were collected at admission and analysed by the chemiluminescent microparticle immunoassay (cmia) for the quantitative determination of neutrophil gelatinase-associated lipocalin in human urine (abbott, architect analyzer). refferent range for urine ngal is - ng/ml. on admission blood samples for bnp (brain natriuretic peptide) analysis were drawn and tested by architect bnp chemiluminescent microparticle immunoassay (cmia), abbott laboratory. results: the mean age of the patients (male= , female= ) was . years (sd . years). among them ( %) patients was diagnosed as a hf-pef (hf with preserved ejection fraction) while ( %) as a hf-ref (hf with reduced ejection fraction). mean bnp values was . pg/ml (sd . pg/ml) and mean lvef was . % (sd . %). mean urine ngal was . ng/ml (sd . ng/ml). we found significantly positive, but weak correlation among ngal and bnp only by pearson correlation test (r = . , p = . , wilcoxon signed rank test z = À . p < . ). conclusion: bnp levels are elevated in hf with reduced and preserved ejection fraction. urine ngal is not elevated in acute heart failure, but it is slightly positively correlated with serum bnp values. converging evidence implicates the intermediate and medial mesopallium (imm) of the domestic chick forebrain in memory for a visual imprinting stimulus. a number of learning-related changes have been found in plasma membrane and mitochondrial proteins of imm. for broader analysis of these changes we employed two-dimensional gel electrophoresis/mass spectrometry approach and identified differentially expressed proteins in membrane-mitochondrial fraction of the imm across chicks with different estimated levels of imprinting h after training. we further inquired whether the amounts of those proteins in the imm and a control region (posterior pole of the nidopallium, ppn) are correlated with memory for the imprinting stimulus. learning-related increase in the amounts of the following proteins was demonstrated in the left imm, but not in the right imm or left and right ppn: (i) membrane cognin;(ii) a protein resembling the p subunit of splicing factor sf ;(iii) voltage dependent anionic channel- ;(iv) dynamin- ; (v) heterogeneous nuclear ribonucleoprotein a /b . obtained results indicate that the molecular processes involved in learning and memory of imprinting cover a wide range of cellular activities, including stabilization of protein structures, increased mrna trafficking, synaptic vesicle recycling and specific changes in the mitochondrial proteome. the aim of this work is to study the substrate and inhibitory properties of uridine derivatives in the reactions catalyzed by e.coli up in order to shed some light on the substrate's conformation in the productive complex with the enzyme. we studied the e.coli up-catalyzed phosphorolysis of uridine and its derivatives modified in the heterocyclic base and the sugar moiety. the kinetic constants (km, ki, kcat ) of the phosphorolysis reaction of near uridine derivatives were determined. the combined kinetic (nnna, , ) and structural data (acta crystallogr., d , , ) provide clear evidence that up binds uridine in the most energetically unfavorable conformation, which, to the best of our knowledge, has no precedents in the enzymes of nucleic acid metabolism. this is possible due to multiple interactions between the substrate and the protein environment (active site residues) mainly through hydrogen bonds. these results are important for understanding the mechanism of action of this class of enzymes. an analysis of the conformations of nucleosides in solution and rotational barriers suggests that the energy difference between the ground state of uridine and uridine complexed with up may be high as - kj/mol. the binding in a high-energy conformation results in the weakening of the glycosidic bond. the observed conformation of uridine complexed with sulfate (mimetic of phosphate) may be very similar to its conformation in the transient state. until now, foxp (forkhead box p ) has been identified as a tumor suppressor in several correlation studies in breast cancer. although, foxp is defined as a transcriptional repressor that interacts with other transcription factors in various mechanistic studies, there is no study that explains its repressor functions in breast cancer biology. here we demonstrate the repressor function of foxp on nfat (nuclear factor of activated t cells) and the migratory effect of this repression in mda mb breast cancer cells. we performed co-immunoprecipitation experiments for the investigation of protein-protein interaction between two transcription factors. protein-protein interaction on dna was investigated with emsa and transcriptional effects of foxp on nfat, lusiferase reporter assay was performed. wound healing assay was used to analyse the effects of overexpression of foxp on tumor cell migration. our results showed that foxp has protein-protein interaction with nfat on dna and enhances breast cancer cell migration by repressing nfat transcriptional activity and foxp shows oncogenic function by regulating breast cancer cell motility. introduction: phosphodiesterase (pde ) is one of phosphodiesterase lead to hydrolyzing cgmp.the cgmp signaling pathway has an important role in proliferation of cells. previous studies showed pde was increased in cell lines cancers thus pde inhibitors can used as efficacious therapeutic option for treatment of cancers. the current study was to investigation the effect of hydroalcoholic achillea.wilhelmsii extract (hawe) on the pde gene expression and cgmp signaling in the mcf- er + and mda-mb- er À . methods and materials: the ed of the hawe on both cell lines were examined by using mtt viability test then the expression of pde and cgmp concentration were measured in timedependent manner (in the ed ) by real-time rt-pcr and colorimetric assay respectively. results: treatment with the hawe showed, lg/ml is ed for both cell lines and the hawe lead to reduction in pde mrna expression and evaluation of intracellular cgmp showed an increase pattern in the time-dependent manner. conclusion: our results showed that the hawe has anti-proliferative property in the mcf- and mda-mb- , cell lines of breast cancer through the cgmp pathway, these data suggested that the hawe can be potential source for the isolation of effective anti-proliferative molecules. keywords: achillea.wilhelmsii, breast cancer, anti-proliferative, phosphodiesterase, cgmp signaling pathway. outer membrane protein g (ompg) is a stable monomeric porin having -stranded beta barrel form from e.coli. its exact function is not fully understood; however, it allows the passage of molecules up to da in neutral ph but the pore is closed by going through a conformational change under ph . . as being monomeric and having ph-dependent gating characters, it is suitable for biosensor and targeted drug delivery applications. an attempt on ompg is to create a larger pore while its stability is undisturbed. ompg- s is obtained by adding amino acids to the primary chain in order to have a -stranded beta barrel porin. ompg- sl is formed by further adding amino acids to loop l and by replacing lysines with arginines. ompg- s and ompg- sl mutants are investigated by fourier transform infrared spectroscopy (ftir) and compared with ompg-wild type (wt) in terms of ph-dependent conformational changes and thermal stability. each mutant is prepared in na-phosphate buffer pd . / . and infrared spectra are recorded. further, temperature profiling are recorded for the range between to °c. results show that both mutants are responsive to ph changes. while turning the ph from acidic to neutral, beta sheet signals shift to lower wavenumbers showing difference in secondary structure, implying the existence of closed and open states. on the other hand, mutant proteins show structural differences compared with the wt protein. porins are known for their remarkable thermal stability. the mutans retain this character by having transition temperature of $ °c, although this is less than the wt transition at $ °c. in conclusion, two mutants show signs of open and closed states as ompg-wt and even if the mutants are less stable than ompg-wt. this study shows that the attempted alterations in ompg structure are successful in terms of ph-response but it needs improvement in terms of stability when necessary. nad is a key factor in the regulation of mitochondrial metabolism. besides its vital role as redox carrier, nad serves as substrate for protein adp-ribosylation and deacetylation, modifications which modulate enzyme activities in mitochondria. these functions depend on how nad levels are maintained in this organelle. in human cells, mitochondrial nad is segregated from the cytosolic pool and can be synthesized from nmn, which is probably imported into the matrix. here, we tested whether the nudix pyrophosphatase nudt participates in the regulation of the mitochondrial nad pool. this enzyme has a predicted nadh pyrophosphatase zinc ribbon domain and a mitochondrial targeting sequence at its n-terminus. however, it has not yet been functionally characterized. we overexpressed nudt endowed with a c-terminal flag-epitope in human cells. to evaluate changes in the mitochondrial nad concentration, we used a reporter system which includes the overexpression of the catalytic domain of poly(adpribose) polymerase (parp ) within the organelles (mitoparp). thereby mitochondrial nad is converted into protein-bound poly(adp-ribose) (par). the extent of par formation correlates with the mitochondrial nad availability and is detected by western blotting. our results established that nudt is indeed a mitochondrial protein, as it was localized exclusively to these organelles. moreover, when nudt was overexpressed along with the mitoparp detector system, a dramatic decrease of par was observed. the obtained results indicate that nudt is enzymatically active upon overexpression in the mitochondrial matrix and that it might cleave nad, thereby modulating its organellar level. however, at this point we cannot exclude the possibility of direct par cleavage by nudt . further characterization of nudt will define its substrate specificity and clarify its role in mitochondrial metabolism. the incidence of increase in colorectal cancer (crc) worldwide has become a major health problem. early diagnosis and treatment of crcs are of importance for improving survival. in the present study, it was aimed to investigate chemopreventive effect of rosmarinic acid and evaluate the angiogenesis process in azoxymethane (aom)-induced crc model. male sprague-dawley rats were randomly divided into a control group, aom-induced rat colorectal cancer group ( mg/kg body weight aom; ip, weekly for four weeks), and rosmarinic acid ( mg/kg body weight; oral, daily for four weeks)-treated group. in addition to the standart diet of the all groups . % peanut oil was added throughout the experiment. the all rats were sacrificed at the end of weeks. biochemical examinations were performed in rat plasma. histopathological adenocarcinoma rates were observed in . % of aom group. the incidence of adenocarcinoma was showed a reduction in the treatment group. significant increases in plasma tos and mcp- levels were found in the aom group compared to controls. these increases were reduced in the treatment groups but no significant. a significant increase was detected in tas levels in the treatment group when compared to the aom group. significant decreases in plasma adiponectin levels were found in the aom and the treatment groups compared to controls. in conclusion, treatment with rosmarinic acid reduced the occurrence of inflammation and was helped to maintain the oxidant-antioxidant balance in the model of aom-induced rat colon cancer. mitochondrial genome, while being strongly reduced in course of evolution, still codes for several proteins. the vast majority of them are components of the respiratory chain complexes. to produce these proteins, the system of mitochondrial translation is presented in the organelles, which is in common close to that in bacteria. translation initiation in bacterial cells is orchestrated by three protein factors called if , if and if . the orthologs of the two latter proteins are commonly found in mitochondria. however, mitochondrial if could not been identified in several groups of organisms, including s.cerevisiae, for a long time. recently we have shown that baker's yeast protein aim p possesses a function of mtif . however, the mitochondrial translation has not been stopped in the yeast strain without aim p which is surprising taking into account the fact that if is obligatory for the translation in bacterial systems. instead of blocking of mitochondrial protein synthesis in absence of aim p, we observed the translational imbalance: the synthesis rate of the complex v subunits was increased while the synthesis rate of the complex iv subunits was repressed. thus, in addition to its general role in translation initiation, aim p might specifically affect the biosynthesis of individual mitochondrial-encoded protein species. our genetic experiments have revealed that, indeed, aim p is almost indispensable for cox p synthesis, and that it affects the translation of cox mrna through its -utr, like classical mitochondrial translational activators. this is in accordance with our measurements of complex iv activity which is several times less in yeast lacking aim gene than in the wild-type. taken together, our results point on the multiple role of aim p in mitochondrial translation: in addition to its function as mitochondrial if , it specifically regulates the amount of complex iv subunits and its activity. p- . . - the circulating betatrophin and irisin levels in polycystic ovary syndrome patients with and without insulin resistance introduction: polycystic ovary syndrome (pcos) is the most common endocrine/metabolic disease in women around the world, characterized by oligo-or anovulation, polycystic ovary, and/or hyper-androgenism. insulin resistance (ir) and obesity are common findings in patients with pcos. irisin is a recently identified myokine secreted from skeletal muscle in response to physical activity. irisin has been postulated to induce the differentiation of white fat tissue into brown fat tissue. betatrophin is a currently discovered new hormone proposed to stimulate b-cell proliferation. in this study we investigated the levels of irisin and betatrophin in pcos patients. materials and methods: our study group was consisted of patients with pcos and healthy volunteers. patients group was divided into two subgroups according to presence of ir. (pcos+ir and pcos-ir). the oral glucose tolerance test (ogtt) and the homeostatic model assessment (homa-ir) were performed to assess glucose tolerance and insulin sensitivity. irisin and betatrophin levels were measured by elisa method. results: circulating irisin was significantly higher in the pco-s+ir subgroup than the control group (p < . ). circulating betatrophin was significantly lower in both patients subgroups than the control group (p < . ). there was no negative or positive correlation between irisin and betatrophin levels. discussion: these data suggest that irisin and betatrophin may act a role together in the ir mechanism in pcos patients. butyrylcholinesterase (bche) synthesized in liver has long been associated with hyperlipidemia, type diabetes and obesity. there are also reports on bche knockout mice becoming obese. the exact involvement of how bche interacts with lipids is still not clear. previously we displayed a correlation between leptin, waist circumference, fat mass and bche levels. recently, we have also shown that bche overexpression in hepg cells is regulated by alpha linoleic acid. as the next approach on the analysis of lipid metabolism and bche interaction, we considered the capability of bche to hydrolyze lipids. human serum bche was purified by subsequent deae-tris-acryl m and procainamide chromatography. the purified bche was utilized in a modified acid lipase assay with the acid lipase substrate -methylumbelliferyl palmitate ( -mu-palmitate). as the second alternative substrate trioleic acid was utilized. the triolein hydrolysis was measured by the nefa kit. verification that bche hydrolysis of these lipid substrates was not due to another esterase was done by iso-ompa inhibition studies. also, lectin binding studies with bche and rca were carried out to rule out non-specific esterase activity. using purified human serum bche and hepatic lipase as control enzyme we found that bche is able to hydrolyze the acid lipase substrate -methylumbelliferyl palmitate ( -mu-palmitate). we found that bche hydrolyses this molecule at ph rather better than at ph . . at ph values, purified human bche has a km value that was times bigger than that of human pancreatic lipase. with the bigger molecule the triolein, the difference between the km values of bche and pancreatic lipase was smaller. bche seems to hydrolyze triolein with an efficacy comparable to approximately % that of human pancreatic lipase. our results display that another function of bche may be its lipid hydrolyzing activity. p- . . - determination of regional reference ranges for erythropoietin with laboratory data mining serum erythropoietin (epo) levels are the main regulator factor of erythrocyte production and increase in response to hypoxia. our region is a location dominated by hypoxic conditions due to the high attitude. in this study we aimed to investigate the mean serum epo levels in the living conditions of our region. two hundred and eighty epo results from our laboratory data whose hemoglobin levels were normal were evaluated in the study. mean serum epo levels were analyzed via chemiluminescence method in beckman coulter dxi auto analyzer. the epo levels of samples was . ae . mul/ml (ranged between . and . ) mul/ml. when we performed ae sd for the studies population we determined normal serum epo levels were as . - . mul/ml. the upper limit determined by our results was % higher than that of determined by the manufacturer as . mul/ml and the lower limit determined by our results was % higher than that of determined by the manufacturer as . mul/ml. normal serum epo levels were considerable for our region and the upper and lower limits were higher than those of determined by the manufacturer. more detailed studies considering the physical properties of participants including a higher number participants are necessary. subclinical hypothyroidism is the precursor to hypothyroidism because it has a tendency to transform into hypothyroidism. subclinical hypothyroidism is considered one of the risk factors causing metabolic syndrome. metabolic syndrome can be characterized by plasma level of apelin released from adipocytes. in the present study, we aimed to measure serum apelin level of patients with subclinical hypothyroidism and compare them with serum apelin level from healthy individuals. our study group included patients diagnosed with subclinical hypothyroidism and healthy volunteers. serum samples were obtained from each participant for the measurement of apelin. these were then stored at À •c until the time of analysis. serum apelin concentrations were determined using an enzymelinked immunosorbent assay. the mean serum apelin levels of subclinical hypothyroidism and control groups were ng/l, control group ng/l respectively. there was no statistically significant difference in terms of the mean apelin levels between the groups (p > . ). apelin levels didn't show significant correlation with bmi (p > . ). in the present study, no significant difference of serum apelin level was observed between patients with subclinical hypothyroidism and healthy control subjects. however, the apelin levels were higher in the patients with subclinical hypothyroidism than in the control group. the possible relationship between thyroid hormones and apelins is critical to understanding the etiopathogenesis of metabolic disorders. the mitochondrial erv /mia import system does not impact cytosolic fe-s cluster protein maturation and iron regulation erv is a sulfhydryl oxidase that partners with the import receptor mia to import small cysteine-rich proteins into the mitochondrial intermembrane space. it has also been suggested that erv has an additional role in maturation of cytosolic fe-s cluster proteins and regulation of iron homeostasis in s. cerevisiae. however, these studies were performed on one particular erv mutant strain (erv - ) that we discovered has additional defects in glutathione (gsh) metabolism. since gsh is required for iron regulation and cytosolic fe-s cluster assembly, this complicates our understanding of erv s role in these processes. we discovered that the erv - strain originally tested for fe-s cluster defects was the only strain to exhibit defects in the cytosolic fe-s enzymes. mitochondrial and cytosolic fe-s protein activities in the other erv and mia mutants tested were similar to the wt control. in addition, while all the erv and mia mutants tested exhibit temperature-dependent defects in mia oxidation, only the erv - strain has significantly reduced gsh levels and more oxidized gsh: gssg redox state. we determined that the cause of gsh depletion in the erv - strain is an additional mutation in the gene encoding the glutathione biosynthesis enzyme (gsh ) that compromises gsh protein folding and/or stability. to address whether gsh deficiency in the erv - mutant is the underlying cause for the cytosolic fe-s cluster defects and iron misregulation for this strain, we measured fe-s protein activity, iron-regulated gene expression, and iron accumulation in erv and mia mutant strains. only the erv - strain exhibited iron misregulation and accumulation of mitochondrial iron, while exogenous gsh rescued these defects. these results demonstrate that the defects in cytosolic fe-s enzymes and iron homeostasis in erv - are due to gsh depletion and neither erv nor mia play significant roles in cytosolic fe-s cluster assembly and iron homeostasis. human c-peptide is a amino acid polypeptide, which is secreted into blood from b-cells in the pancreas where pro-insulin undergoes a post translational modification and cleaved into insulin and c-peptide. human c-peptide concentrations in blood plasma and urine reflect the level of insulin resistance associated b-cell function and can point out insulin secretory failure. the reference intervals in blood plasma and urine are . - . ng/ml and - ng/ml respectively. c-peptide measurement in urine and plasma provides a guide for therapy in diabetes. this study describes a method for the development and validation of picaa (peptide impurity corrected amino acid analysis) method for the determination of the purity of the human c-peptide which could be used as a reference material to measure cpeptide concentrations in plasma. two different methods were performed for the picaa; aaa-id-ms/ms for quantification of constituent amino acids following hydrolysis of the material and rp-hplc-esi-tof ms for determination of the peptide related impurities. the result of the aaa id ms/ms method was corrected for the amino acids originating from the impurities. id ms/ms-aaa was performed with zivak Ò hplc and zivak Ò tandem gold triple quadrupole ms equipped with a phenomenex ez:faast l aaa column ( mm i.d). the mobile phase was composed of, a: mm ammonium formate (af) in water, b: mm af in acetonitrile (acn). the intact peptide analysis was performed by a hitachi lachrome elite hplc and bruker microtof-q mass spectrometer equipped with a capcell pak mg-ii c column ( mm i.d., mm particle size). the purity of the synthetic c-peptide was determined by picaa analysis and related uncertainty was calculated. traceability to si was established using the amino acid standards of which the purity was determined by tub _ itak ume using qnmr analysis. picaa is a simpler alternative to the full mass balance approach which requires large quantities of the peptide material. p- . . - heat shock proteins: complementary therapies in brain tumors with viscum album e. onay-ucar, s. n. biltekin istanbul university, faculty of science, department of molecular biology and genetics, vezneciler, istanbul, turkey cancer is one of the lethal diseases in the world. different cancer types possess overexpressed hsps levels. viscum album extracts with their anticancer and antioxidant properties are being used in cancer therapies. biochemical composition of this plant is known to vary its features depending on the host trees and time of harvest. in our previous study, it has been found that v.album inhibited hsp expression and induced caspase-dependent apoptosis in c rat gliomas. the aim of the current study is to find out whether different v.album extracts have different effects on hsps expression level and apoptosis in c glioma cell line or not. in this study, three different extracts of v.album were compared for their potential inhibition effects on hsps. the cytotoxic effects of extracts have been determined via mtt test. different experiment groups were set up subjected to heat shock and/or incubated without any heat shock application. overexpression of hsps was induced by heat shock at °c for h in c cells. expression levels of hsps were determined by western blot analysis. the apoptosis inducing effect was also evaluated via caspase- activation in c glioma cells. pretreatment of the cells with non-toxic dose ( lg/ml) of v.album extracts prior to heat shock, reduced significantly the expression levels of hsps. similarly, pretreatment with the extracts prior to heat shock increased apoptosis via caspase- activation in c glioma cells. these results will be utilized in the determination of the relation between extract composition and stress protein expressions. these results suggest that different extracts of v.album are able to down regulate expression of hsps, and induce apoptosis. this warrants further exploration as a potential resource of bioactive compounds that can be used in cancer therapy. future studies targeting hsps for the development of chemosensitizers may help improve the treatment of cancer in combinational therapy. biological drugs (biologics) are the fastest-growing category of therapeutics among those approved by the agencies for drugs regulation. most biologics are proteins designed for parenteral use. however, proteins are characterized by poor pharmacokinetic and safety profiles. peg-coating (poly-ethylene glycol coating) of biologics provides several benefits, including an increased half-life related to reduced renal clearance, an increased stability to degradation, and a reduced immunogenic/antigenic response. preservation of the three-dimensional structure and activity of the pegylated form is a strict requirement for human use. the recombinant proteins used for this studies (as-sod, superoxide dismutase; mmp , matrix metalloproteases ; ansii, l-asparaginase ii) were cloned and then over-expressed in escherichia coli. pegylation reactions were performed using commercial reagents. all the protein samples were purified and analyzed by solution and solid-state nmr (fields from mhz to mhz). we developed new protocols to prepare samples of pegylated proteins, demonstrating that solid-state nmr spectra of exceptionally good quality can be obtained for pegylated proteins in the sedimented state (obtained by either ultracentrifugation or rehydration of freeze-dried samples); surprisingly, sedimentation of pegylated proteins to this end has never been attempted. the spectral quality is comparable toor better thanthat of the corresponding crystalline samples. the excellent quality of the solid-state nmr spectra would make it possible to perform extensive resonance assignment and even a conventional full structure determination of biologics. the proposed method is based on the comparison of a standard twodimensional solid-state nmr spectrum of the sedimented pegylated protein with that of the crystalline state of the native proteinfor which the x-ray structure is available. all eukaryotic creatures hereditarily have natural defense mechanisms and are protected from the infections with this defense mechanism. antimicrobial peptides (amp) contain - amino acid content, are positively charged with amphipathic feature. the antimicrobial activities of amps are thought to be depended on the microbiocidal effects by binding to the surface of microorganisms and creating pores in their membranes. defensins are both effector and mediator small antimicrobial peptides of the immune system. these peptides in cationic and amphipathic structure have broad spectrum antibacterial, antifungal and antiviral features. defensins regulate the innate and acquired immune systems by suppressing proinflammatory responses during infection. mammals have three structural subfamilies of defensins. these show differences according to the trisulfide arrays in their structure and are classified as a,b, h defensins. human beings have tissue-specific six functional a defensins. human hnp- and hnp- encoded by defa , defa and defa genes are firstly expressed in neutrophils. human hdp and hdp encoded by defa and defa are firstly expressed in paneth cells in the intestines and play important role in the defense and homeostasis. human beings have many pseudogenes such as defap and deftp in addition to these functional genes. according to literature data, defensins play an important role in defense against microbial placements on mucosal surfaces. in addition, the antimicrobial spectra of defensins include gram negative and gram positive bacteria, fungi and viruses. in addition to their antimicrobial efficiency, they can accelerate the wound healing due to their mitogenic effects on epithelium cells and fibroblasts. bile salt hydrolase (bsh) enzyme catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts into amino acid residues and the free bile acids that reduce cholesterol. however, some intestinal bacteria have an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid having potential harmful side effects to the host. the catalytic mechanism and substrate preference of such bsh enzyme is not clear. in this study, bsh gene from lactobacillus plantarum gd strain was cloned, expressed, characterized in escherichia coli blr(de ) strain, and then val- and phe- amino acids, supposed to be responsible for substrate preference, were substituted for met- and ile- amino acids respectively by site directed mutagenesis. the hydrolysis activities and stability of the mutant recombinant bsh (mrbsh) enzymes were examined along with six different bile acids by ninhydrin assay and sds-page respectively. ninhydrin test results indicated that wild-type recombinant bsh (wrbsh) hydrolyzed six major human bile salts with an apparent preference towards glycine-conjugated to tauro-conjugated bile salts. however, the activities of mrbsh/phe ile enzyme are %, %, %, %, % and % of the activity of wrbsh against to glycocholic acid (gca), glycodeoxycholic acid (gdca), glycochenodeoxycholic acid (gcdca), taurocholic acid (tca), taurodeoxycholic acid (tdca) and taurochenodexycholic acid (tcdca) respectively. the activities of val met mrbsh enzyme are %, %, %, %, % and % of wrbsh against to gca, gdca, gcdca, tca, tdca and tcdca respectively. our findings support the suggestion that bsh enzymes recognize their substrates predominantly at the amino acid moieties and not at the cholate moieties. however, further pcr-based site-directed mutagenesis and structure-driven computational and theoretical approaches are required for the precise determination of their substrate specificities and the selection of probiotic bacteria. we deposited bacteriorhodopsin in purple membranes under applied electrical field onto ito (indium tin oxide) support. purple membranes film, highly oriented in one direction, was placed between two ito electrodes. we studied dependence of electrical properties of these films on light illumination. we argue that this setup can be used for functional studies of microbial rhodopsins. in opposite to already published results where this system was used as a photocondensor for studying functional properties of bacteriorhodopsin, we studied electric properties of such systems and we found strong light dependence of resistivity of bacteriorhodopsin in purple membranes films. optogenetics is already used in study of neuronal cells cooperation in vitro and in vivo by means of microbial rhodopsinsion pumps and channels incorporated in membranes of neurons changing their electrical potential while receiving a light quantum by laser or led source. best perspectives optogenetics will give after successful transfer to medical applications, such as the treatment of blindness, treatment of disorders like parkinson's disease etc. but to achieve these we need a broad set of tools, optogenetics tools, highly specialized to solve specific problems of neurophysiology. to the creation of such tools our work is dedicated. new optogenetic tools can be made by mutations in existing ones altering their properties (mainly spectral characteristics, selectivity and conductivity) or some promising mutations in conserved residues can be found in existing organisms. a halophilic archaeon halosimplex carlsbadens is a host of protein of our interest. according to the theoretical data based on the alignment with br and the d structure model of this novel protein, we suppose this protein functions like the light-driven h+ pump: all the key residues are the same or at worst have the similar properties, except one in the position leucine instead of the aspartic acid. a gram-positive bacteria deinococcus-thermus phylum syntheses rhodopsin with substitution of this aspartic acid to alanine. sphingomonas paucimobilis has rhodopsin where aspardic acid in position is changed to serine residue. and one yet uncharacterised guillardia theta rhodopsin even has the same as br motif (d , t , and d ) but according to alignment is closer to chr even the last one motif is e , t , n . it is expected that all of them will show us new properties. though the further experimental data are essential. the work is supported by rsf - - . evaluation of some thymus proteins in patients with crimean congo hemorrhagic fever i. b€ ut€ un , s. sahin , f. duygu university of gaziosmanpasa, department of biochemistry, tokat, turkey, oncology education and reasearch center, ankara, turkey crimean congo hemorrhagic fever (cchf) is a tick-borne viral zoonotic disease. it has a high fatal rate (% - ). tokat is one of the cities having the most reported cchf cases, in turkey. clinical presentation of the disease varies widely among patients. thymic peptides are small molecules synthesized by thymic epithelial cells. they play role in the immune response, as well as anti-inflammatory process. fourty patients referring to the hospital with tick-contact history and/or presenting clinical manifestations consistent withcchf and with positive pcr results for cchf virus in blood samples were included to the study. the wbc and platelet values at application and before the patients were discharged were recorded. the healthy control group consisted of age and gender matched healthy volunteer adults free of any chronic disease. thymosin alpha (ta ), thymuline and thymosin beta (tb ) were studied by the elisa method in this study. biochemical parameters were also analysed. ast and alt values were significantly higher (p < . ) and plt and wbc levels were significantly lower in the cchf group (p < . ). levels of tf, ta and tb were found to be significantly higher in cchf (p < . ). there was no mortality during the study period. duration of hospitalization was . ae . days. levels of tb were significantly correlated with duration of hospitalization (r= À . , p = . ). alt levels were significantly correlated with tf levels (r = . , p = . ). patients received ffp and apheresis for the supportive treatment, while patients received only ffp and patients got only apheresis. patients did not get any of these blood products. there was not a statistically significant differences in thymus peptides among these treatment groups (p > . ). we report survived cchf patients with elevated thymic peptides. pathogenesis of cchf has many points to be highlighted. thymic peptides may play role in the clinical situation of the patients with the disease. the effect of methocarbamol on the peroxiadse activity of human erythrocyte hemoglobin hemoglobin is released to blood circulation, after red blood cells lysis. it is carried in circulation by binding to haptoglobin. in normal persons, no free hemoglobin is observed in the blood, because most of hemoglobin is in the form of haptoglobin complex. in some diseases that are accompanied by hemolysis, the amount of released hemoglobin is higher than its complementary haptoglobin. as a result, free hemoglobin appears in the blood, which is a toxic compound for these patients. free hemoglobin has been showed to have peroxidase activity and considered a pseudoenzyme. in this research, the effect of methocarbamol on the peroxidase activity of human hemoglobin was studied. our results showed that the drug inhibited the pseudoenzyme by un-competitive inhibition. both k m and v max decreased by increasing the drug concentration. k i and ic values were determined as and mm, respectively. molecular docking results showed that methocarbamol did not attach to heme group directly. a hydrogen bond connected nh of carbamate group of methocarbamol to the carboxyl group of asp side chain. two other hydrogen bonds could be also observed between hydroxyl group of the drug and ser and ser residues of the pseudoenzyme. p- . . - dca reduces viability and down regulates mapk protein activations in human malignant mesothelioma cells and pericardium. microarray analyze results performed in mm patients revealed that one of the most prominent changes is upregulation of many genes involved in glycolysis and the krebs cycle. dichloroacetate (dca) is an inhibitor of pyruvate dehydrogenase kinase (pdk) that enhances the oxidative activity of cells by activating pyruvate dehydrogenase (pdh) in mitochondria. dca has shown as a promising anti-neoplastic agent that re-sensitizes cancer cells to apoptosis. the aim of this study is to elucidate the coupling between pdk inhibition and mm cell proliferation and cell cycle. human malignant mesothelioma (spc ) cell line was used as a model for dca treatments. cell viability was measured by mts assay; mapk protein activations and expressions were assessed by western blotting; cell cycle profile was analyzed by flow cytometry. statistical analysis was performed by utilizing one-way anova test. results showed that dca reduced viability of spc cells in a concentration and time dependent manner. protein analysis indicated that mapk pathway was down regulated at concentrations greater than mm. moreover, primary cell cycle analysis has indicated arrestment at g /m phase in hours. our findings corroborate with recent reports where dca treatments resulted in reduction of viability and g /g and g / m arrest in other cell lines. abnormalities in mapk signalling play a critical role in the progression of cancer. here, we showed for the first time that dca decreased mapk activation in h. our results suggest that dca is an anti-prolifertive agents for mm cells in vitro. however, it requres extra analysis with other mesothelial cells. future study will focus on investigating relation between mapk and mitochondrial apoptosis. adrenomedullin (adm) is a vasodilator peptide consisting of amino acids. adm is synthesized in many tissues. and is a biologically active peptide that has various effects including vasodilatation, the regulation of vascular endothelial function and adjusting adipogenesis. hypoxia inducible factor alpha (hif a) is a subunit of a heterodimeric transcription factor hypoxia inducible factor . it is the master transcriptional regulator of cellular and developmental response to hypoxia. the dysregulation and overexpression of hif a by either hypoxia or genetic alternations have been heavily implicated in cancer biology as well as a number of other pathophysiologies. in our research, the adm and hif -a levels in heart, kidney and lung tissues of rats were investigated in control, hypoxia, control+adm and hypoxia+adm groups. rats in hypoxia groups were provided hypoxic environment containing of - % oxygen and - % nitrogen for week. rats in adm groups were injected intraperitoneally in a dose of . nmol/kg for four days before the collection of the tissues. the control group was oxygenated with normal air. the control and treatment groups were formed from - animals and adm, hif -a levels were measured in taken tissues with immunoassay method. the aim of this study was to investigate the reaction of the organism when exposed to hypoxic conditions and the effect of adm over hif -a level. adm levels and hif -a in heart tissue were found decreased in hypoxia group, and adm levels increased in hipoxia+adm group. hif -a levels decreased in hypoxi+adm group. adm levels in liver tissue were found decreased in hipoxia and control+adm groups than control group. hif -a levels were higher in control+adm group. adm has a role in angiogenic process, and our experiment showed that adm reacts earlier than hif -a, and affects its synthesis. organism increases its vascularization as a reaction to hypoxic condition, and adm treatment may provide a rapid adjustment. p- . . - covalent conjugation and characterization of immunogenic protein of toxoplasma gondii and polyacryclic acid as vaccine candidate r. c ß akir koc ß yildiz technical university, department of bioengineering, istanbul, turkey toxoplasmosis is a major medical and veterinary disease caused by toxoplasma gondii which infect approximately half of the world's population. this infectious disease especially gains importance in pregnant women and immunodeficient individuals. also t. gondii infection has economic importance. however, there are only one attenuated-live t. gondii vaccine for veterinary uses and no vaccine against t. gondii is available for humans. therefore development of an effective vaccine would be extremely valuable for preventing disease in human and veterinary medicine. subunit vaccines are very attractive vaccine candidates but there is low antigenicity problem when they are used alone. polymers themselves don't stimulate immune response while they used with antigenic structure of various infective agents enhance immune response because when proteins are covalently conjugated with hydrophilic polymers, ( ) their circulatory-lives and stability (in different ph and temperature values) enhance ( ) binding to proteases and clearance by the reticuloendothelial-system decreases. in this study, immunogenic protein of t.gondii and polyacrylic acid with immune stimulant properties was covalently conjugated and conjugation was demonstrated by size-exclusion chromatography (sec) and fluorescence spectroscopy. it is significant to detect time of death in case of a sudden death for medical and legal concerns. there is no known method that can be used for post mortem time detection. based on this deficiency pmi detection in narrow time frame is a big problem. in this study, we aimed to investigate and determine timedependent expressional changes of apoptotic markers by western blot technique. postmortem skeletal muscle were analyzed hour periods in first -hour after death. nd and rd -hour periods were statistically significant (p < . ). keywords: post mortem interval, time of death, apoptosis. hyaluronidases are excessively found in nature and involved in numerous biological functions. hyaluronidases primarily degrade hyaluronic acid (ha) and have significant role in fertilization during acrosomal reactions. therefore, the measurement of hyaluronidase enzyme activity may provide valuable information about acrosomal function and the fertilizing ability of the sperm. the aim of this study was to investigate the semen hyaluronidase enzyme activity changes among four different sheep breeds (akkaraman, suffolk, merino, and kıvırcık). in this research, ten ram testis tissues from each sheep breed, a total of , were cut and collected on ice. ovine testicular hyaluronidase of four different sheep breeds was purified from a crude ammonium sulfate-precipitated fraction of an extract of ram testis. the semen hyaluronidase enzyme activity differences between the sheep breeds were examined by spectrophotometrically monitoring the appearance of ha at nm. analysis of variance test was used to examine the possible mean differences among the four different sheep breeds. the observed mean differences in enzyme units for kıvırcık, suffolk, akkaraman, and merino were as follows . , . , . , and . , respectively. the observed mean differences in absorbance values for kıvırcık, suffolk, akkaraman, and merino were as follows . , . , . , and . , respectively. the results showed that the observed mean differences in enzyme units and absorbance values among the four different sheep breeds were not statistically significant. despite that, in average kıvırcık had higher values for the activity of each sample and yet it had the smallest values for standard deviation. therefore, in order to achieve higher enzyme activity and more homogenous samples kıvırcık breed should be preferred. what is extra to learn from protein drying measurements? hydrations of soluble proteins are crucial for their functionality. therefore elucidating the details of protein hydration is still of interest in the proteins' action mechanisms. this is the motivation of the present study. in order to study protein hydration, changing concentrations of the well-studied serum albumin protein was measured with the spectroscopic techniques like uv-vis and ft-ir spectroscopy. spectral data is analysed and calculations were performed on the data to extract the relevant changes in the protein. experimental parameters' variation in association with the spectral changes implies the involvement of protein structure and hydrogen bonding in the drying process. the protein's reactions may not be merely a feature of the protein structure in the common sense but it could be related directly to the protein hydration states as well. this is understandable since it is already known that enzymatic proteins lose their functionality when they are dried while this drying may or may not involve dramatic structural changes. on the other hand, here it is claimed that the role of water in gaining the functionality that was lost in the dried state is not just about enhanced diffusion processes and the dynamicity but could be related to the functionality of water in the energy transfer processes as well. investigating the cellular effects of the aldoketo reductases akr b and akr b in hct- colon cancer cells b. taskoparan, e. g. seza, m. s. ceyhan, s. banerjee middle east technical university, ankara, turkey aldo-keto reductases (akr) are nad(p)h dependent oxidoreductases are best characterized as glucose reducing agents, and have been implicated in diabetic pathophysiology. increased expression of akr has been associated with tumors of lung, breast, prostate, cervix, ovarian and colon. two members of the akr superfamily that have been associated with different cancer types are akr b ; aldo-keto reductase family , member b , and akr b ; aldo-keto reductase family , member b . both are -kda cytosolic reductases that are similar in both amino acid sequence identity ( %) and tertiary structure with the (a/b) barrel topology. while hct- , a colorectal cancer cell line, cells expresses akr b robustly, there is no expression of akr b . in this study, we have stably knocked down akr b through shrna technology and overexpressed akr b in hct- cells. comparisons were made with a known akr inhibitor sorbinil. with the knock down of akr b , we have observed reduced cellular proliferation, enhanced apoptosis, delay in cell cycle progression, reduced expression of mitogenic proteins and a decrease in activation of the inflammatory transcription factor nuclear factor kappa b (nf-kappab). interestingly, although akr b overexpression did not affect cell proliferation, apoptosis or cell cycle, some effect was observed with nf-kb signaling. our data indicate that, although closely related, akr b and akr b have very different contributions towards signaling pathways in colorectal cancer. comparison of different nisin quantification methods and optimization of nisin production by lactococcus lactis z. girgin ersoy, g. demir, m. f. cesur, s. tunca gedik gebze technical university, kocaeli, turkey nisin, which is produced by certain strains of lactococcus lactis, is the only bacteriocin approved by world health organization (who) as a food additive. it prevents the growth of foodborne bacteria which cause food spoilage. nisin research and applications necessitates developing an accurate and reproducible method for its quantification. the agar diffusion bioassay is the most widely used method for quantifying nisin, although it has limitations especially diffusion-related difficulties of the active substance. in the present study, "agar diffusion bioassay", "enumeration of colony forming units", "colorimetric assay" and "flow cytometry" methods were compared with each other to determine antibacterial activity of nisin on micrococcus luteus. moreover, this study also covers the results about the effect of different cultural conditions to optimize nisin production by l. lactis. galactose, lactose and their combination in m medium (ph ) boosted nisin production at °c, as the addition of . lg/ml hemin into the fermentation broth. to our knowledge, this is the first study showing the usage of "flow cytometry" method to determine nisin activity of fermentation broth filtrates. p- . . - coronaviral nucleocapsid protein is an antiviral target for drug development institute of genomics and bioinformatics, national chung hsing university, taichung, taiwan between and , the severe acute respiratory syndrome (sars)-cov caused a worldwide epidemic and had a significant economic impact in the countries affected by the outbreak. recently, the middle east respiratory syndrome human coronavirus (mers-cov) was found in patients with severe acute respiratory tract infections in the middle east and south korea. as is true for all coronavirus infections, there are no efficacious therapies currently available against coronaviral diseases, making the development of anti-coronavirus compounds a priority. the cov genome consists of positive-sense, single-stranded rna approximately kb, and it contains several genes encoding several structural and non-structural proteins that are required for progeny virion production with a conserved order. the n proteins exist in the center of the viral particle and represent a helical structure complex. nucleocapsid protein is most abundant structural protein of covs, binds the viral rna genome to form the virion core, leading to the formation of a ribonucleoprotein (rnp) complex or to a long helical nucleocapsid structure, that is important for maintaining the rna in an ordered conformation for replication and transcription. the cov n protein is also involved in the regulation of cellular processes, such as gene transcription, interferon inhibition, actin reorganization, host cell cycle progression, and apoptosis. two strategies to inhibit oligomeric n protein function have been reported. the first strategy is to discover antiviral agents that target the rna-binding site. the second one is to impair normal n protein function by interfering with monomer-oligomer equilibrium. our recent studies suggest that n proteins in infections caused by coronaviruses will be useful antiviral drug targets because they serve many critical functions during the viral life cycle. post-translational modification of vascular endothelial growth factor (vegf) in colon cancer cells s. tunc ßer, e. solel, s. banerjee middle east technical university, ankara, turkey vascular endothelial growth factor a (vegf-a), commonly referred as vegf, is a potent secreted mitogen crucial for tumor initiation and progression. the gene for vegf is translated into a number of splice isoforms that lead to , , and amino acid proteins, with different receptor-binding and matrixbinding properties. in the present study, we discuss the functional significance of post-translational modification/processing of vegf isoform in hct- colon cancer cells. we also focus on the role of calcium in the post-translational modification of vegf . we show that vegf undergoes n-linked glycosylation in hct- cells. perturbation of cellular calcium may affect vegf driven malignant phenotypes. p- . . - novel methods for modulating the activity of bcl family proteins in apoptosis p. rowell, j. miles, a. wilson, t. edwards apoptosis, also known as programmed cell death, is an essential cellular process, but is implicated in several human diseases, including diabetes and cancer, when it is up-or down-regulated respectively. bcl- family proteins are major players in the control of intrinsic, or mitochondrial apoptosis; they respond to intracellular stress signals, function through protein-protein interactions and converge on the mitochondrial outer membrane to cause membrane permeabilisation, release of cytotoxic molecules, and initiation of an apoptotic cascade that leads to cellular demise. our work aims to identify molecules able to bind and modulate the activity of several key players in the bcl- family, including the pro-survival members bcl- , bcl xl and mcl , and the death promoting family member bax. adhirons, novel non-antibody peptide display scaffolds developed at the university of leeds, have been used to construct a phage display library containing over clones, and form a key part of the strategy to identify such molecular modulators. adhirons able to selectively bind individual bcl- family members have been identified, in vitro assays carried out to test for modulatory activity, and xray crystallography used to elucidate details of how they interact with their target proteins. more recently, studies have been carried out to identify adhirons able to target multiple bcl- family members, with the aim of selectively inhibiting defined groups of proteins in cells. this work provides opportunities to differentiate the activities carried out by different bcl- family proteins in apoptosis, enabling us to better understand how their dysregulation contributes to human disease. biophysical and evolutionary study of the structural flexibility of adp-dependent sugar kinases from mesophilic and psychrophilic archaea r. zamora , v. castro-fern andez , c. a. ramirez-sarmiento , e. a. komives , v. guixe facultad de ciencias, universidad de chile, santiago, chile, department of chemistry and biochemistry, university of california, san diego, united states of america the capability of extremophiles microorganisms to live at low temperatures is mainly attributed to the high structural flexibility of its enzymes. several sequence and structure features have been associated to a high structural flexibility that enables metabolic processes to occur at low temperatures. during evolution, the general mechanism adopted by these enzymes has been to reduce the free energy of the transition state rather than the michaelis constant, k m . increased structural flexibility and decreased affinity for its substrates in psychrophilic enzymes is compensated by an increase in the catalytic rate, k cat . few psychrophilic enzymes have been reported to performance the optimization of their catalytic efficiency (k cat /k m ) by decreasing k m values. we use the adp-dependent kinase sugar family of archaea as a model, to identify particular structure and sequence features of a psychrophilic enzyme that would make this enzyme more flexible than their thermostable homologues. we characterize the bifunctional psychrophilic enzyme phosphofructokinase/glucokinase from methanococcoides burtonii (mbpfk-gk) and the bifunctional mesophilic enzyme phosphofructokinase/glucokinase from methanococcus maripaludis (mmpfk-gk) by spectroscopic, biophysical and computational techniques. the comparison showed that the absence of two ion pairs is primarily responsible for the increased structural flexibility accounted in the psychrophilic model. this increase in structural flexibility is reflected in the exponential increase in the k m values with temperature. additionally, we reconstruct the sequences of all ancestral enzymes between the current enzymes and their last common ancestor, which was used to trace the occurrence of these electrostatic interactions during evolution in the adp-dependent sugar kinase family. our results suggest that electrostatic interactions are a dominant feature in the transition from psychrophilic to thermophilic environments. fondecyt . elucidating the domain swapping mechanism of the forkhead domain of human foxp fox transcription factors control gene transcription during key processes, such as embryogenesis and immunity, and feature a conserved dna-binding domain known as forkhead. while most forkhead domains are monomeric, solved structures of members from the p subfamily (foxp) show that they can oligomerize by domain swapping (ds), a mechanism where protein segments are exchanged between subunits leading to an intertwined dimer. the biological relevance of ds in foxp has been stressed by disease-causing mutations that impair this process. however, for many proteins ds takes days to occur and requires global protein unfolding. thus, the current mechanism impedes a conciliation of the occurrence of ds of foxp in a biological context. here, we elucidate the biological feasibility of this process by biophysically characterizing the ds mechanism of the forkhead domain of foxp using size exclusion chromatography (sec), circular dichroism, and hydrogen-deuterium exchange mass spectrometry (hdxms). our results show that ds of foxp occurs at micromolar protein concentrations, within hours and is energetically favored. remarkably, dimeric foxp follows a threestate n « i « u folding mechanism, where dimer dissociation into a monomeric intermediate (i) precedes protein unfolding, in contrast to other ds proteins. using sec and hdxms, we show that the i state of foxp largely resembles the native state, but has a larger hydrodynamic radius and a higher deuterium uptake in regions that maintain the compact monomer, suggesting that the i state is an 'open' conformation en route of ds. finally, we compared the local flexibility of the dimer and monomer of foxp , showing that only the hinge region connecting ds segments exhibits different deuteration rates. our results show that ds in foxp follows an unusual threestate folding mechanism that proceeds through transient structural changes rather than needing protein unfolding as in most ds proteins. (fondecyt , ). p- . . - the sustained release of growth factor proteins following their implantation in tissue engineering j. jang bone tissue engineering has become a promising approach for bone regeneration. however, insufficient vascularization during bone regeneration, particularly with large bone defects, results in poor and unsustainable bone formation due to central necrosis. therefore, vascularization following implantation in vivo is essential to the successful formation of new bone tissue. we evaluated the release profile of vegf from bgs using a novel fluorescence-based retention assay, which revealed that vegf loaded on bgs can be released in a sustained manner without an initial burst (near zero-order cumulative release) with a controlled release rate of . % per week for up to weeks. in contrast, an elisa-based release assay showed vegf to have an early burst-release profile for the first week. however, the biological activity of vegf released from the bgs was preserved over the -week release period, which is consistent with the sustainedrelease profile observed in the fluorescence-based retention assay. we developed a novel fluorescence-based retention assay to evaluate the release of vegf from bgs. this fluorescence-based retention assay, which detects the vegf that remains on bgs, reveals that vegf loaded on bgs can be released in a sustained manner, with a minimal initial burst, for up to weeks. these results indicate that the sustained biological activity of the vegf released from bgs over the full -week period promotes bone regeneration, and suggest its potential use for bone tissue engineering. irisin is a recently discovered myokine which regulates energy metabolism and is associated overweight. we aimed to evaluate serum irisin levels in the patients with morbid obesity. a total of sixty patients with morbidly obese and thirty healthy control subjects were included in this study. all participants were measured body weight and height, the lipid profile, and plasma glucose, hba c, insulin and irisin levels. irisin levels were measured by elisa method. serum irisin levels were significantly lower in morbidly obese patients than healthy controls (p < . ). there was no statistically significant difference in terms of age or gender. serum irisin was negatively correlated with bmi, insulin levels, and homa-ir (p = . , p = . , p = . , respectively). our study revealed lower irisin levels in morbidly obese patients with respect to control subjects. the lower irisin levels observed in morbidly obese patients might suggest a loss of browning of subcutaneous adipose tissues. p- . . - pka inhibition restores adenosine uptake in renal tubular epithelial cells under high d-glucose conditions w. garrido, j. catal an, s. alarc on, r. san mart ın universidad austral de chile, valdivia, chile introduction: diabetic nephropathy (dn) is the leading a cause of end-stage renal failure whose pathogenesis must to be elucidated. the progression of dn has been associated with elevated levels of adenosine. extracellular adenosine availability is regulated by the activity of the equilibrative nucleoside transporters (ents). due to the ents have putative sites of phosphorylation our objective was to evaluate the role of pka and ck kinases on ents activity. methods: adenosine uptake ( lm adenosine, s, °c) was assayed in hk cells preincubated with mm or mm d-glucose for h and exposed to lm -br camp, lmh or lm tbb for h. plasma membrane and intracellular proteins were fractionated by the biotinylation method and ent and ent contents were quantified by western blot. results: high d-glucose in hk cells inhibited the uptake of adenosine. this effect was reversed using a pka inhibitor (h ) through an increased ent uptake activity. we noticed this pka inhibitor did not regulate the plasma membrane localization of ent or ent under normal d-glucose ( mm) or high d-glucose conditions ( mm). also, we saw that tbb (ck inhibitor) decreased the activity of ent and ent under normal glucose conditions, decreasing the localization of ent at cell surface, while the membrane localization of ent decreases under the effect of tbb and high d-glucose conditions. conclusions: pka inhibition reversed the effect of high d-glucose, increasing the uptake of adenosine mediated by ent . this could be a new target for the restoration of adenosine levels in dn. relation between serum lipo (a), plasma fibrinogen, red cell distribution width and mean platelet volume in healthy adult men the aim of this study was to investigate the relationship between the serum lipo (a) and plasma fibrinogen, red cell distribution width (rdw) and mean platelet volume (mpv) in healthy adult men. for this purpose, healthy adult men have normal physical examination and laboratory findings and not use any drug were included to the study. serum lipo (a) levels and hematologic parameters (fibrinogen, rdw-sd and mpv) were measured by autoanalyzer and commercial kits. the mean of the age of the persons was . ; body mass index was . ; serum lipo (a) level was . and plasma fibrinogen level was . . there was significant positive correlation between the serum lipo (a) and plasma fibrinogen levels (r = . ; p = . ), significant positive correlation between the serum lipo (a) and rdw-sd (r = . ; p = . ) and significant negative correlation between lipo (a) and mpv (r= À . ; p = . ). the plasma fibrinogen and the serum lipo (a) levels have been known as the risk factors for cad (coronary artery disease) increase together in healthy adult men. similar findings have been reported in cad patients. it has reported that elevated rdw is associated with intracoronary thrombotic burden and may be associated with the severity and instability of acute myocardial infarction. in addition, mpv is predictor of severe atherosclerosis and may be used for the prediction and identification of cardiac risks in cad patients. our findings show that elevated rdw and decreased mpv may predict to increased risk of cad in the future, in healthy adult men. follicular fluid is rich in peptides, which significantly influence the growing oocyte. due to existence of a link between kisspeptin (metastin) cells and gonadal steroids kisspeptin might manipulate the gonadotropin axis and folliculogenesis. in this context, the study was planned to investigate for the first time that the follicular fluid (ff) and serum concentration of kisspeptin in high and poor responders undergoing in vitro fertilization (ivf)/intracytoplasmic sperm injection (icsi). biological samples were collected from twenty infertile women with polycystic ovary syndrome (pcos) and poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone (gnrh) antagonist protocol for ivf/icsi treatment. kisspeptin concentrations were measured in serum and follicular fluid by using elisa, whereas fsh and lh levels were detected by routine laboratory methods. it was found that kisspeptin levels were significantly lower in serum and follicular fluid of infertile women with pcos. kisspeptin levels were correlated with fsh and lh level in infertile women with pcos. it can be concluded that low level of kisspeptin might inhibit gnrh release that might cause to the inhibition of fsh and lh release and might disrupt folliculogenesis. decline in serum and ff levels of kisspeptin might be possible cause of anovulation and subfertility in pcos subjects. cryptococcus albidus d is a newly identified yeast isolates from petroleum area in _ izmir as a lipase producer. the molecular weight of the enzyme is . kda as found. optimum temperature was °c and half-life times were , , and . min at , , and °c, respectively. optimum ph value was . . however, it shows significant ph stability at ph values . and lower. the existence of acetone in the solution as a solvent enhanced lipase activity. cryptococcus albidus d lipase was able to catalyze the esterification reaction between fructose and palmitic acid to produce fructose palmitate using acetone as the solvent. due to its stability in organic solvents, we propose that in order to increase the yield of fructose palmitate, we could immobilize d lipase. therefore, the effect of immobilization on kinetic parameters of d lipase was investigated. different immobilization materials and methods were used to find efficient support materials for d lipase immobilization. additionally, fructose palmitate production processes will be optimized with immobilized lipase. introduction: the diagnosis of osteoarthritis (oa) is based on clinical symptoms and radiographic findings. it is known that the pathologic changes at the molecular level in the joint cartilage tissue start before symptoms appear in oa. c q tumor necrosis factor-related protein (ctrp- ), c q tumor necrosis factor-related protein (ctrp- ) and kallistatin are related to many different cellular processes including bone and cartilage tissue metabolism. the aim of this study was to investigate any probable association between the serum ctrp- , ctrp- and kallistatin levels and diagnosis and radiologic staging of knee oa patients. materials and methods: this study included patients with knee oa and healthy individuals for control purposes. the patient group was divided into four stages radiologically. ctrp- , ctrp- and kallistatin levels were measured in serum samples of patient and control groups with elisa method, and the differences between the groups were analyzed with statistical methods. results: the levels of serum ctrp- in the patient group were significantly higher than in the control group (p = . ), serum ctrp- and kallistatin levels were not statistically different (in order of p = . , p = . ). in the patient group, there was not a significant difference between serum ctrp- , ctrp- and kallistatin levels and radiologic stages (respectively p = . , p = . , p = . ). there was a significant positive correlation between the radiologic stage and patient's age, body mass index, western ontario and mcmaster universities arthritis index and visual analogue scale values (respectively p = . , r = . ; p = . , r = . ; p = . , r = . ; p = . , r = ). discussion and conclusion: serum ctrp- levels were detected significantly increased in patients with knee oa, but there was no significant difference in ctrp- and kallistatin levels. there was not a significant association between the radiologic stage and levels of ctrp- , ctrp- and kallistatin. enzymes in microorganisms, especially termophilic bacteria are more attractive in biotechnology and molecular biology due to the higher catalytic activity. turkey is rich in geothermal resources and it is important to determine unknown microbial content of geothermal sources. in this study, water and sludge samples were taken from ayder, kizilcahamam and gonen hotsprings. firstly, ph, temperature, salt concentration, gram reaction, mobility, endospore formation, oxidase and catalase tests were carried out as conventional characterization. molecular characterizations of isolates were achieved by fames, rep-pcr and s rrna sequencing. finally, test isolates were evaluated according to enzyme production capability by petri dish. as result of conventional tests, isolates were determined as gram positive, mobile-rod shaped, aerobic, oxidase, catalase and endospore positive. the growth range for ph and temperature of the isolates were determined as - and - °c. in consequence of the salt test, the test isolates were grown at - % nacl. of thermophilic isolates were selected by rep-pcr and according to s rrna sequencing analysis test isolates were belonging to bacillus, geobacillus, anoxybacillus and brevibacillus genus at a range of - %. enzyme tests showed that, some of the isolates were able to produce protease (f , f , f , f , f and f ), amylase (f , f , f , f and f ), cellulase (f , f , f , f , f and f ), xylanase (f , f , f , f and f ) and lipase (f , f , f , f and f ). it can be concluded that, geothermal regions are rich in bacillus and related genera. fame analysis was particularly insufficient for diagnosis of thermophilic microorganisms, but rep-pcr was successful in separation of organisms at species and even subspecies levels. most of our bacterial isolates have industrially important enzyme production capacities. it is a pioneer result to use bacteria for industrial applications which need higher temperatures. p- . . - warburg effect was investigated by studying various metabolic molecules and assays in mammalian cell lines z. i. kanbagli, b. kiratli, h. cimen yeditepe university, istanbul, turkey majority of the different cancer cells switch their metabolism from oxidative phosphorylation pathway to glycolytic pathway; in order to meet excessive energy requirement, which is also called warburg effect. acetylation is one of the most crucial post-translational modifications playing key roles in metabolic reprograming. in this study, the relationship between acetylation dependent changes in energy metabolism and apoptotic pathways were investigated in pnt a, du , hela, hep b, hek t, shsy y. immunoblotting experiments were applied by using antibodies against acetylated-lysine to examine the changes in overall acetylome. candidate proteins displaying elevated acetylation was identified with mass spectrometry based-proteomic analysis. glucose transporter (glut ) was used to detect insulin-stimulated glucose transport, total oxphos rodent antibody cocktail to identify variations in complexes which are responsible for most of the atp production. caspases (casp- , - ) to unreveal different activation levels of apoptotic pathway among the cell lines. mitochondrial membrane potential was measured by using rho-damine by employing confocal microscopy. the expression level of respiratory chain complex iv subunit mtco and casp- was higher in hek t compared to other cell lines. casp- was upregulated in cancer cell lines, mostly in hep b. glut- levels were downregulated in cancer cell lines in contrast to healthy cell lines. findings imply that these proteins might have significant roles leading to variable metabolic and apoptotic activity of each cell line during energy production. due to the results, mtco might be important in adaptation of different cell lines to regulate the overall respiratory chain complex activity. reduction in glut level demonstrates insulin desensitization in cancer cell lines, which might lead to metabolic defects in these cells. besides, since p has a repressive effect on glut , it also can lead us to study about p levels. the effect of inhibition of pi k/akt/mtor signaling pathway on receptor tyrosine kinase expression in breast cancer cells g. tunali, a. l. dogan department of basic oncology, hacettepe university cancer institute, ankara, turkey the increased expression and activation of receptor tyrosine kinases (rtk) (egfr, her , her ) play important roles in breast cancer pathogenesis. her /her interaction is the most potent heterodimer and it causes oncogenic pi k/akt activation. inhibitors of pi k/akt pathway (akt inhibitor and pi k/mtor dual inhibitors) lead to increase in rtk levels and activities while blocking signaling pathway. in this study, the time dependent effect of dual pi k/akt inhibitor pi- on receptor tyrosine kinase expressions' in breast cancer cells was investigated. two breast cancer cell lines, mda-mb- cells (which has egfr overexpression and pten deficiency) and skbr- cells (which has her overexpression) were evaluated for the effect of dual inhibitor. these cells were treated with dual pi k/akt inhibitor pi- for different time periods ( - h) . egfr, her her total rtks expression and pi k/akt pathway inhibition (p-akt and p-p s k expression) were evaluated by western blot. in mda-mb- cells, there were significant decrease in p-akt and p-p s k proteins' expression during the first h. this inhibition was followed by reactivation of the signaling pathway after h. in skbr- cells, p-akt and p-p s k proteins' expression were significantly decreased during the first h. the pi k/ akt signaling pathway in these cells were reactivated after h. basal expression of egfr and her in mda-mb- cells and basal expression of her and her in skbr- cells were found to be very high. transient inhibition of akt and mtor protein kinase activation in tumor cells followed by reactivation of signaling pathway did not result in a time-dependent difference on egfr, her and her expression levels. these results suggest that dual pi k/mtor inhibiton by pi- may trigger receptor tyrosine kinase reactivation due to the signal distruption without affecting total protein expression level. site-specific bioorthogonal reactions are one of the significant tools for discovering different aspects of biological systems in live cells. the reactions should be highly stable and rapid in physiological conditions. various chemical tools can be used in bioorthogonal reactions to monitor biological systems, therapeutics, microscopy and diagnostic applications in live cells. synthetic covalent chemistry in the study of biological systems has been used to label biomolecules selectively in their native environment. for example, small synthetic fluorophores can be added to biomolecules without disturbing other molecular biological pathways. aldehydes or ketone-based functional handles can be attached onto protein at specific sites via chemoenzymatic reactions. labeling of carboxy terminus of a-tubulin has been successfully studied in our previous studies by replacement of wild type tyrosine with unnatural amino acid -formyltyrosine in the presence of tubulin tyrosine ligase enzyme (ttl)-as its role can suggest whether certain cancer cells might grow more aggressively than others. in this work, we highlight the synthesis and spectroscopic properties of azacoumarin chemical probes to study tubulin-tubulin tyrosine ligase (ttl) system in live cells. significant increase in fluorescence quantum yield or a red shift on absorption and emission maxima is observed when the conjugated product is formed. bioorthogonal fluorescent labeling is such a favorable reaction to perform rapid kinetics, localization and high site-specificity in cell environment. newly synthesized azacoumarin fluorophores should therefore not only be useful for studying ttlbased biological systems, but also would enable broad range of high-yielding and fast diagnostics for future biolabeling applications in biochemistry, cell biology and beyond. binase is an extracellular ribonuclease from bacillus pumilus which shows antiviral and antitumor activities in cell cultures. however, the action of binase on intracellular functions and processes has not yet been identified. here, for the first time we report the whole transcriptome analysis of binase-treated human lung adenocarcinoma epithelial a cells. a plasmid-based reverse genetics system and colorimetric cell viability assay were used to identify the binase internalization and binase cytotoxicity towards a cell line, respectively. for the whole cell transcriptome analysis a cells were treated with lg/ml of binase for h followed by mrna extraction and library preparation. sequencing was performed on solid xl wildfire next-generation sequencer. we found that binase internalized into a cells after h of incubation. the binase at lg/ml was absolutely non-cytotoxic towards a cell line and was active in the cell culture medium during h incubation. the analysis of rna-seq data showed that among thousands of protein coding transcripts transcripts were up and down regulated by binase, among them transcripts were induced and repressed. binase repressed the production of s a and tnxb which act cancer biomarkers, scn a and drd which play a crucial role in cancer metastasis and responsible for pediatric tumors, respectively. the induction of transmembrane protein transcript abcb by binase can help binase to internalize into the cell as abc transporters are often account for transporting drugs across the cellular membrane. binase induced the production of nlrp , rasgrp and alpk transcripts which can activate apoptosis, cytokine or t cell activation in cancer cells. thus, binase exerts different effects in cancer cells. the rnaseq data obtained will help to understand the mechanism of binase anticancer action. . ) is a specific group of phosphatases capable of hydrolyzing myo-inositol , , , , , -hexakisphosphate (phytate) with the formation of less phosphorylated inositol derivatives (from mono-to pentaphosphate). three major types of phytases are recognized on the basis of the first phosphate group hydrolyzed by the enzymes: -phytase, / -phytase, and -phytase. due to the stereospecific way of phosphate release from the phytate molecule by the action of phytases, these enzymes by themselves and their composition may serve as a potential alternative for production of myo-inositol phosphate isomers with therapeutic properties. chemical synthesis of these compounds is inefficient and costly. pantoea sp. strain . . showing high phytase activity was isolated from the forest soil sample of the republic of tatarstan, russia. in this study the main objective was the cloning and expression of pantoea sp. . . phytase gene in e. coli. first, we amplified the phytase gene (agpp) from the genomic dna of the bacteria using specific primers phmh_dir and phmh_rev. size of phytase gene corresponded to base pairs. during the optimization of amplification conditions it was found that the optimum temperature for primer annealing was °c. this temperature increases the specificity and efficiency of annealing. then the pcr-product of agpp gene was cloned into the pbad myc/ his vector first. on the next step we carried out subcloning of the agpp into a pet a expression vector. multicopy plasmid pet a/agpp contained the sequence of the phytase gene of pantoea sp. . . under t promoter. the corresponding recombinant protein was expressed in e. coli as a fusion with a his-tag and was detected by western blotting. recombinant phytase was purified via affine chromotography on the ni-nta column and displayed high phosphomonoesterase and phytase activities. bag- (bcl- associated athanogene) is a multifunctional protein that interacts with diverse array of cellular targets and modulates a wide range of cellular processes, including proliferation, cell survival, transcription, apoptosis, metastasis and motility. in human cells bag- exists as three major isoforms (bag- s, bag- m and bag- l) derived by alternative translation initiation from a single mrna, which allows interactions with various molecular targets such as hsp /hsc molecular chaperones, components of the ubiquitylation/proteasome machinery, bcl- , raf- kinase, nuclear hormone receptors and dna. our work aims to investigate how altered bag- expression levels affect cell survival in mda-mb- (er, pr and her /neu negative) breast cancer cell lines. we first cloned bag- l gene to a cloning vector, later we transfected mda-mb- cells for overexpression of bag- . we also used bag- sirna to silence bag- gene. western blot analysis was applied to demonstrate relative expression levels of bag- , its interacting partners and certain proteins which are important for apoptosis pathway. we performed xtt cell viability assay for bag- overexpressed cells to checkbag- 's impact on cell survival, and observed enhanced survival rates on cells compared to that of the untreated cells with bag- overexpression. in addition, our study revealed that once bag- forms a complex with c-raf/b-raf/hsp /akt/bcl- , modulation of cell survival was observed. we believe that once the exact localization and involved molecular mechanisms of bag- and its isoforms are found, the role of each bag- isoform in cell survival can be understood better. this can further provide routes to study tumor development. the aim of this study is testing the recombinant glp encapsulation into a biocompatible material. we also tested if it can be a therapeutic candidate drug for type diabetes. the incretin hormones, which are also named as endogenic peptide hormones have become a more attractive therapy for type diabetes because of different physiological effects. in circulation, glp is cleaved by ddp in a very short time. if glp can be protected from cleaving, the effective time of glp would be increased and by this way it can replace the therapy of insulin. chemical synthesis methods of peptides are limited because of low efficiency and high cost. the production of peptides by recombinant e. coli is an alternative way because of effective production, simplicity and low cost. however, the major disadvantage derived from the recombinant e. coli is the frequent formation of inclusion body. for that reason, extra methods are needed for obtaining soluble recombinant peptides. glutathione s-transferase (gst) tag is commonly used as affinity and solubility tag to improve the solubility of recombinant peptides. in this study, we cloned and heterologously produced glp using the gst fusion system in e. coli. affinity purification of recombinant protein was achieved by using glutathione immobilized columns. characterization of the gst-tagged glp was performed by sds-page. the purity of fusion protein was found to be %, as confirmed by glp elisa kit. then, the fusion protein was encapsulated in a chitosan coated polygalacturonic acid. the different ph stability and in vitro release tests also in different phs was studied. morganella morganii is an opportunistic pathogen capable of causing a wide range of clinical infections. it is known that microbial metalloproteases play an important role in the development of bacterial infections. thus, investigation of m. morganii metalloproteases has a particular interest. bacteria were grown in lb medium at °c. as a bioinformatics tool blast was used. for molecular biological experiments, thermo scientific kits and sibenzyme enzymes were used. pbad/myc-his plasmid was used as an expression vector. bacterial transformation was carried out by heat shock method. bacterial cells were disrupted by sonication. gene expression products were analyzed by western blotting. to analyze the actinolytic activity of bacterial extracts sds-page electrophoresis was used. the putative metalloprotease gene (an cp . ) has been found in the genome of annotated strain of m. morganii kt. its amino acid sequence has partial homology ( %) with actin specific metalloproteases grimelysin from s. grimesii and protealysin from s. proteamaculans. using specific primers the gene with % homology was identified in the genome of clinical isolate of m. morganii . rt-pcr analysis showed that this gene had the maximum expression at h of growth. in addition, the cellular extract of m. morganii had small actinolytic activity. cloning of the gene into e. coli dh a cells led to the synthesis of the kda protein. it is known that the highest expression of serratia proteases is observed at h of growth, and the molecular weight of the mature proteins is kda. it was shown that metalloprotease gene of m. morganii expressed at the same time of growth. moreover, the recombinant e. coli cells synthesized protein with the similar weight ( kda) which perhaps is a mature form of the metalloprotease from m. morganii. as a result, in the genome of m. morganii the metalloprotease with similar properties to grimelysin and protealysin proteases was identified. the preliminary characterization of p-ii like protein glnk from lactobacillus brevis z. iskhakova , d. zhuravleva , a. laykov , k. forchhammer , a. kayumov kazan federal university, kazan, russia, eberhard-karls university tuebingen, tuebingen, germany the p-ii proteins in bacteria, archaea and plants regulate the activity of a variety of proteins in response to specific metabolic signals which affect their structural state and interaction ability. among various bacteria belonging to lactobacillus only some species have genes encoding pii protein in the genome. here we report the preliminary characterization of pii-like protein lbrglnk from lactobacillus brevis. while the amino acid sequence alignment revealed only - % homology of lbrglnk with other well studied pii proteins, lbrglnk also has the atp binding motive gdgk. trimeric structure of lbrglnk was confirmed in vitro by size exclusion chromatography, suggesting possible similarities of lbrglnk properties with pii proteins. the isothermal titration calorimetry revealed a preferential binding of adp (kd = lm) over atp (kd = lm) suggesting that they compete for binding to lbrglnk. neither -oxoglutaric acid nor other nucleotides were interacting with lbrglnk in itc measurements. the mutation gly ala in the atp binding motive completely abolished the interaction with both adp and atp. the pull down of lbrglnk with l. brevis cell extract allowed identification of chaperonin grol, transketolase and glnr-like transcriptional regulator from merr family as most probable partner proteins for interactions with lbrglnk. this work was supported by the russian foundation for basic research (project no. - - a background: hemophilia is a bleeding disorder due to the deficiency in coagulation factors viii (hemophilia a) or ix (hemophilia b). hemophilia patients are essentially treated with intravenous replacement of the missing or dysfunctional factors fviii or fix by recombinant proteins. these therapies often induce the generation of acquired antibodies, and thus, novel approaches are needed. most recent hemophilia strategies target the tissue factor pathway inhibitor (tfpi), which is the major inhibitor of the coagulation cascade, particularly of the extrinsic tenase complex. anti-tfpi agents have been empirically developed such as aptamers, peptides, monoclonal antibodies. we have followed a structure-based strategy, to design a mutated fxa that would show more affinity for tfpi, and thus trap this inhibitor. tfpi exists as two isoforms are folded as multi-kunitz domains related by linkers. the second kunitz type domain of tfpi (tfpi-k ) is known to bind the catalytic site of fxa. methods: the molecular complex of tfpi-k -fxa was modeled and submitted to molecular dynamics (md), allowing the identification of low-spots interaction. modified fxa with theoretically stronger interaction with tfpi-k were predicted using md. the mutants and wild type proteins were expressed in hek cells, and their processing status was checked. they were tested by western blotting, by chromogenic activity using a specific substrate of fxa, by thrombin generation assay in fviii depleted plasma. finally, their binding to a tfpi-k peptide array was compared. results: the mutants showed better efficiency to restore thrombin generation in plasmas from hemophiliacs and displayed stronger binding to tfpi-k than the wild type fxa. conclusions: the proof of concept of the synergistic approaches of md and mutagenesis was obtained and an efficient tfpi trap was designed. the mutated fxa is a candidate for a new hemophilia therapy. organophosphorus acid anhydride (op) nerve agents are potent inhibitors which disrupt the mechanisms of neural transmission. organophosphorus acid anhydrolase (opaa; e.c. . . . ) is a class of enzyme that potentially acts on phosphorus anhydride bonds, reported intracellularly in diverse organisms, albeit notably the enzyme belongs to alteromonas species are more extensively studied. whereas mass-transfer problem is a major issue in native whole cell biocatalysis, new anchor system derived from the n-terminal domain of ice-nucleation protein from pseudomonas syringe inav (inav-n) was used for the first time to display opaa onto the cell surface. tracing of the recombinant protein and its activity assay showed a successful presentation of opaa and its significant ability for biodegradation of organophosphorus compounds. further studies on bacterial fractions confirmed that opaa is remarkably located on the outer membrane. the specific activity of recombinant bacteria to degrade diisopropylfluorophosphate (dfp) was measured at u/mg of cell wet weight, which almost all was observed in the outer membrane fraction. recombinant cells could also degrade chlorpyrifos (cp) compound in . u/mg activity. it can be concluded that inav-n anchor is efficient for targeting opaa on the cell surface and can effectively eliminate the masstransfer problem in native whole cell bioconversion system. proper spatial and temporal organization of proteins involved in cell signal transduction is crucial for the specific and efficient information transfer. scaffold proteins coordinate the action of signaling molecules by their physical binding and organization in multiprotein complex assemblies. multiple protein binding is often mediated through intrinsically disordered regions of the scaffold, where the interaction epitopes are defined by linear peptide motifs. using a hub scaffolding protein axin as a paradigm, we have employed peptide microarray technique to identify the binding epitopes for axin interaction partners at high resolution. this enabled us to design axin-derived peptides corresponding to the respective binding epitopes that compete for the interaction in vitro. by transfection of chemically stabilized competitive peptides directly into the cells, we have shown the effect of specific interaction blocking on axin-mediated signaling in vivo. our data demonstrate a proof of concept for a rational design of inhibitors of protein-protein interactions that allow specific intervention with single function of the targeted protein (i.e. recruitment to the axin complex). contrary to the inhibitors that completely disrupt the protein function (e.g. inhibition of a kinase catalytic site), this approach provides a tool for investigating specific action within the axin complex, while the other cellular functions of the targeted protein remain preserved. the results of this research have been acquired within cei-tec (lq ) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. de novo design of an artificial biocatalyst using immunoglobulin template became rather routine procedure due to the achievements of molecular biology and crystallography. recently the 'reactibody' approach was developed based on the chemical selection of catalytic repertoires from immunoglobulin library followed by expression of these biocatalysts in eukaryotic system. in this study we structurally characterize the a antibody, its kappa and lambda variants, in order to understand the difference on the active site between a and a which although there are two antibodies sharing very high homology and sequence identity their active residues are located in a different region of the antibody. the structures of the a antibody kappa and lambda variants have been already determined, there was no structural information though about the a antibody. the structural analysis revealed dramatically different angle in position of nucleophilic residue tyr and area of solvent accessible surface. the structural difference of active center reflects on kinetics of the a organophosphate modification. both variants of antibody bind with organophosphate throw induce-fit mechanism, but rate of the step of induce fitting is different (k obs are s À and s À for a kappa and a lambda respectively). this observation may hint at novel means of regulation of velocity and specificity of artificial biocatalysts. this study was supported by grant #rfmefi x . translation elongation factor ba (eef ba) is a component of a heavy form of translation elongation factor (eef h). it functions as a catalyst of gdp/gtp exchange in translation elongation factor a (eef a) restoring its active conformation appropriate for aminoacylated trna binding. eef ba forms a tight complex with translation elongation factor bc (eef bc) via the n-terminal domain, while its c-terminal domain executes the catalytic activity. eef bc has been shown to enhance the attributed to the c-terminal domain catalytic activity of eef ba. this suggests that the eef ba n-terminal domain may influence the guanine nucleotide exchange process. to test this hypothesis we prepared a set of n-terminal truncated forms of human eef ba and checked their activity in the guanine nucleotide exchange assay on both isoforms of eef a, eef a and eef a . we showed that recombinant eef ba is a non-globular monomeric protein in solution with an elongated shape by analytical ultracentrifugation approach. the truncation of the dispensable for the catalytic activity n-terminal domain of eef ba resulted in significant acceleration of the rate of guanine nucleotide exchange in eef a comparing to full-length eef ba. similar effect on the catalytic activity of eef ba was observed after its interaction with eef bc. in contrast, the effect of full-length eef ba and its truncated forms on the rate of guanine nucleotide exchange in eef a was similar but relatively modest compared to eef a . this can be explained by higher rate of spontaneous gdp dissociation from eef a comparing to eef a and lower affinity of eef a to eef ba. thus, we propose that the n-terminal domain of eef ba via flexible linker region may interfere with eef a binding to the cterminal catalytic domain that results in a decrease of the overall rate of guanine nucleotide exchange reaction. the formation of a tight complex between eef bc and eef ba n-terminal domains abolishes this inhibitory effect. p- . . - assessment of quantitative proteomics results in large-scale data-independent with fragmentation spectra reproducibility measure reduces variation and allows to use lowintensity signals organisms with reduced genomes that lack the vast majority of transcriptional or translational regulation systems tend to adapt to changing environment with a variety of subtle changes in protein abundances. as soon as relative changes for most proteins fall below %, the power of traditional label-free proteomic analysis rapidly becomes insufficient for robust profiling of hundreds of samples. intoduction of frament-by-fragment and sample-by-sample signal quality assesment in mrm and dia experiments helps to increase accuracy of methods and at the same time reintroduce cases which could have been excluded during bulk quality assessment due to lower signal-to-noise ratio for several fragments. samples of mycoplasma gallisepticum were acquired in data-independent manner on sciex tripletof + mass-spectrometer (swath acquisition) during the year. the samples were produced from mycoplasma gallisepticum culture cultivated at different temperatures. the signals for each fragment were extracted with vendor-supplied software with the theoretical fragment ions for each peptides instead of spectral library. the results were used for relative protein quantitation in two manners the first conventional method included direct use of peptides with top most intense signals. the second included selection of peptides and ions for quantification for each pair of samples based on the reproducibility of fragmentation patterns after computing the areas of chromatographic peaks for each ion. spectral angle was used as a distance measure for fragmentation patterns for clustering. further, a base set of detected ions was selected for each peptide and a subset for comparison of each pair of runs. the first method resulted in quantitation of proteins across all samples with variation across lc-ms replicates was % on average, and the second approach led to quantitation of proteins in total, of them across % of samples, all with the variation about % on average. p- . . - interaction of plasminogen fragments k - and k with fibrin fragment dd t. yatsenko, v. rybachuk, l. kapustianenko, s. kharchenko, o. yusova, t. grinenko palladin institute of biochemistry of nas of ukraine, kyiv, ukraine introduction: plasminogen interaction with specific binding sites in c-terminal d-domains of fibrin molecule initiates the activation process of proenzyme and subsequent fibrin clot lysis. the sites are exposed under fibrin polymerization. plasminogen kringle domains ensure the proenzyme interactions with fibrin clot. in this study, we investigated the binding of human plasminogen kringle fragments k - and k with human fibrin fragment dd and their effect on glu-plasminogen interaction with dd. results: kringle-containing fragments k - and k reduce plasminogen activation by tissue-type activator on fibrin fragment dd. the level of glu-plasminogen binding to dd is decreased by - % in the presence of k - and k . fragments k - and k have high affinity to fibrin fragment dd (dissociation constant is . lm for k - and . lm for k ). analysis of k - and k binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with c-c-chains of fragment dd. k - interacts with complex of fragment dd-immobilized k as well as k with complex of fragment dd-immobilized k - . the plasminogen fragments do not displace each other from binding sites located in fibrin fragment dd, but can compete for the interaction. analysis of k - and k binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with aand c-chains of fragment dd. conclusions: widely known specific plasminogen-binding site located in aa - region of fibrin molecule is not a single binding sequence of fibrin peripheral domains or plasminogenbinding site is not linear and contains amino acid residues from other polypeptide chains of fibrin d-domains. fibrin fragment dd contains different binding sites for plasminogen kringle fragments k - and k , which can be located close to each other. possible plasminogen kringle-binding sites are located in aand c-chains. p- . . - implementation of budded baculovirus particles for characterization of ligand binding to g protein-coupled receptors a. allikalt, a. rinken university of tartu, tartu, estonia g protein-coupled receptors (gpcrs) constitute the largest class of membrane receptors involved in regulation of signal transduction into the cell in response to various extracellular stimuli. for that reason, gpcrs have become important targets for variety of drugs. as these receptors are present in native tissues at very low concentrations, efficient recombinant expression systems are needed to produce sufficient amounts of protein. we have shown that budded baculovirus particles, which display gpcrs on their surfaces can be used as a source of receptors for the investigation of ligand-receptor interactions. this expression system can be used for radioligand binding assay as well as for fluorescence anisotropy-based assay (fa). we have validated the system with budded baculovirus particles produced in spodoptera frugiperda (sf ) cells expressing human dopamine d receptors using [ h]sch- and bodipy-fl-skf- as reporter ligands for corresponding assays. this system has many advantages, for example good signal to noise ratio, homogeneity of the receptor, high expression levels and long-term stability of the receptor preparation. fa method allowed real-time monitoring of reporter ligand binding in the absence and presence of different dopaminergic ligands, giving information about their kinetic properties. association, as well as dissociation of the bodipy-fl-skf- itself were rapid with an apparent half-life of t / = . ae . s for association ( nm) and t / = . ae . s for dissociation. we determined the pharmacological profiles of different dopaminergic ligands in displacement binding assays with membranes of sf cells or budded baculovirus particles. the data were in good agreement for both membrane preparations tested in radioligand binding as well as in fa assay. obtained results indicate that budded baculovirus particles can be proposed as a source of gpcrs for performing fluorescence anisotropy as well as radioligand binding assays. gastrointestinal (gi) cancer includes a variety of cancer types affecting the structures and functions of the gi system, encompassing the gi tract and the accessory organs of digestion, from the esophagus, stomach, biliary system and pancreas to the intestine, rectum and anus. despite the significant advances however, much remains to be learned in the spectrum of gi cancer. several investigators have shown that both gas and its receptors, axl, sky, and mer are expressed in various types of cancers. however, the expression level of gas in gi cancer remains unclear. the aim of the study was to determine and compare plasma gas levels in gi cancer patients. female and male patients were included in the study (n = ): colorectal, gastric, pancreatic, liver, ampullary, gall bladder and esophageal. from all gi cancer patients, ml venous blood was collected in citrate tubes before surgery. blood samples were centrifuged at g for min, and plasma samples were carefully removed and stored in À °c prior to use. the level of plasma gas was measured using a commercial developmental elisa kit (r&d systems, minneapolis, mn) which is validated by our laboratory. plasma gas levels in cancer patients were determined as follows: . ae . ng/ml in colorectal; . ae . ng/ml in gastric; . ae . ng/ml in pancreatic; . ae . ng/ml in liver; . ae . ng/ml in ampullary; . ae . ng/ml in gall bladder and . ae . ng/ml in esophageal cancer. preliminary findings indicate that there is a relation between gi cancers and plasma gas levels. taken together, these results suggest that gas may be a candidate biomarker for diagnostic use in gi cancer. inhibition of gas would be an attractive therapeutic target for slow down the progression of gi cancer. monday september : - : computational biology p- . . - computational approaches as an assay for blactam hydrolysis in class a b-lactamases c. tooke university of bristol, bristol, united kingdom b-lactam hydrolysing enzymes, in particular carbapenem-hydrolysing enzymes, are an increasing clinical threat. herein we show that molecular dynamics (md) and combined quantum mechanics/molecular mechanics (qm/mm) approaches are a predictive tool of carbepenemase activity in class a b-lactamases. b-lactam drugs are the most prescribed class of antibiotics worldwide, especially in the treatment of gram-negative pathogens such as klebsiella pneumoniae and escherichia coli. these organisms produce b-lactamases, enzymes which hydrolyse the b-lactam ring, a key resistance mechanism. class a b-lactamases have the ability to hydrolyse carbapenems, termed 'last resort' antibiotics. in particular, the kpc (klebsiella pneumoniae carbapenemase) family are the most clinically important, and recently identified natural kpc variants show increased hydrolytic activity against ceftazidime, a third generation cephalosporin. here we use computational simulations of b-lactam hydrolysis by b-lactamases. in particular, molecular dynamics (md) combined with qm/mm approaches have been used to model the deacylation of the carbapenem meropenem across class a b-lactamases. this method has been extended to model cephalosporin hydrolysis across class a b-lactamases, including kpc variants. these approaches calculated the free energy barriers and correctly distinguished carbapenemases from carbapenem-inhibited enzymes. preliminary results suggest this protocol is also a predictive tool for ceftazidime hydrolysis. further, md simulations of kpc variants (single and double amino acid changes) were analysed to identify structural changes in the active site, highlighting that variants differ in the size of the active site opening, corresponding with experimentally derived kcat values. these computational assays provide a predictive tool of b-lactam hydrolysis and has potential to provide insights into important mechanistic differences both across class a b-lactamases and within the same families. p- . . - computational design of a novel polyglutamic dendrimer-based platform as an anticancer therapeutic approach poly (glutamic acid) (pg)-dendrimer as potential nanocarriers for cancer therapies, to specifically deliver tumor associated antigens (taa)mannosamine and melanato target cells and to modulate cancer antigen intracellular trafficking within the cytoplasm to promote an efficient and selective antitumor immunotherapeutic effect. the theoretical structures were obtained using x-plor software. the molecular dynamics simulation of pg-g -dendrimer and taas was performed using desmond. the electronic properties of the structures were determined by semi-empirical methods using mopac. docking studies of taa to pg-g -dendrimer to mannose receptor (mr ) were performed using hex . . software. taa lumo atoms were conjugated to homo atoms of pg-g dendrimer using maestro software. results showed that pg-g -dendrimer displays carboxylic end groups available for covalent interaction with taas. the homo molecular orbitals of the dendrimer was located on the a-carbon of the carboxylic acid groups from backbone chain and it preferentially interacts with lumo molecular orbitals of amine group from taas. no differences in the gap energy of homo/lumo of all pg-g -conjugates. taas bind preferentially to a-carbon of cooh of backbone chain instead of cooh from side chain. docking results showed that majority of taa conjugated pg-g -dendrimer binds to the core of the mr receptor. increasing of the number of mannosamine conjugated to pg-g -dendrimer more close and stable is the conjugated to the receptor. this system shows promising results as a novel functionalized pg-dendrimers for cancer therapy. theileria parva is one of the the economically important protozoan of the theileria genus belong to apicomplexa phylum which include plasmodium spp. and toxoplasma gondii, causative agents of malaria and toxoplasmosis respectively. this parasite is the disease agent of tick-borne east coast fever (ecf) ranks first among the tick-borne diseases of cattle in sub-saharan africa. the disease caused by the parasite affects a large proportion of domestic and wild animals and leads serious economic losses in the world. major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. thus, it is important to develop an efficient and affordable antitheilerial agent. for this aim, -deoxy-d-xylulose- phosphate reductoisomerase (dxr) which subjected to identify novel drug aganist malaria and toxoplasmosis, of theileria parva was selected as potential target for improving novel inhibitors aganist ecf. a computational molecular modeling approach was conducted to determine the d structure of tpdxr by phyre . energy minimisation and root mean square deviation (rmsd) was performed by drefine and superpose servers. to ensure the quality of modelling, stereochemistry, energy profile and residue environment of modelled structure were checked by different servers and possible ligand binding pockets were identified by metapocket . server a reliable d model for dxr from t. parva was modeled by using au as a template. the ca rmsd and the backbone rmsd deviations for the model and the template crystal structure were found to be . and . a, respectively. the ramachandran plot for the predicted model by rampage reveals that model shows an acceptable stereochemistry. top three considered possible binding pockets have been identified. these results have important implications for future screens aimed at finding new and safe molecular entities active against tpdxr through docking studies. p- . . - molecular binding profile of protoberberine alkoloids on amyloid precursor proteincleaving enzyme (bace ) as a drug candidate for alzheimer's diseases g. yalcin , i. yildiz biotechnology institute, ankara university, ankara, turkey, department of pharmaceutical chemistry, faculty of pharmacy, ankara university, ankara, turkey alzheimer's disease (ad) is the most prevalent neurodegenerative disorder that leads to dementia and nowadays over million people live with dementia worldwide. because of the prevalence and economic burden of the disease, drug development studies have picked up speed and scientists especially focused on natural products. ad is basically characterized with tau hyperphosphorylation and accumulation of amyloid b (ab) proteins. ab proteins are generated from sequential cleavages of amyloid precursor protein (app) by b and c secretases, and b-site app cleaving enzyme (bace ) is a b secretase essential for ab production. the alkaloids represent a very extensive group of secondary metabolites, with diverse structures, distribution in nature and important pharmacological activities. protoberberine alkaloids, which belongs to isoquinoline alkaloid class, are widely arranged in many species of the berberidaceae, annonaceae, fumariaceae, papaveraceae, and other plant families. recent searches showed that some of the protoberberine alkaloids such as berberine, palmatine, jatrorrhizine, columbamine, magnoflorine prevents the progress of neurodegenerative disorder. however, the mechanisms of them are not absolutely clear. therefore, we have aimed to elucidate the binding and affect mechanism of these alkaloids on the bace open and closed forms in here. for this purpose, molecular docking studies were applied for these natural products to the both forms of bace by using autodock vina and it was subjected to explicit solvent simulations by amber molecular dynamic package. our preliminary studies indicate that gly , thr , gln , phe , tyr , lys , thr , arg , thr residues of binding pocket have affiliations with all of the mentioned alkaloids and the binding of them generates alterations on closed form of bace . the complexity of animal milk needs to apply numerous approaches and methods for its investigations. an understanding of the processes occurring in the milk can be used, for example, for quality control of the products. fat and protein are main components of milk which have a significant influence at its colloid properties, such as dynamic surface tension (dst). the application of regression-correlation analysis to milk data enables to develop a reliable quantitative model. the aim of our investigation was to perform the regression analysis to establish the relationship between above-mentioned parameters. for this purpose, we used a statistical software packages r version . . . dst was determined by bpa - p tensiometer. milk fat (f) and protein (p) contents were measured by analyzer bentley . this work was supported by the russian scientific foundation (grant - - ). obtained formulas characterized the degree of influence of fat and protein contents of a milk sample for each of the dst parameters (r , r , r , r , k , k ): r = . + . * p À . * f r = . + . * p À . * f r = . + . * p À . * f r = . + . * p + . * f k = . + . * p + . * f k = . À . * p À . * f these formulas show that the maximum total effect of fat and protein contents influences at r and r . a significant coefficient (> ) before the fat is observed in the formula, which describes the value of the tilt of final part of the tensiogram (k ). the resulting regression equations have fundamental importance. with their help it is possible to calculate the dst parameters without their experimental determination, positioning fat and protein contents data. obtained dst parameters promote more complete characterization of the properties of the milk that may be used for dairy products. p- . . - molecular studies of scorpion toxin and its mutants interactions with voltage-gated potassium channels the voltage-gated potassium kv . channel is mostly expressed in neurons and immune cells. its blockage has a high therapeutic potential, for example, specific inhibitor shk toxin is undergoing clinical trials on psoriasis. goal of the current study was an interface analysis in complexes of hybrid channel kcsa-kv . with peptide blockers agitoxin and its mutant forms. d structure was generated by homology modeling method using complex of mutated kcsa channel with charybdotoxin (pdb-code a h) as a template and equilibrated by molecular dynamic simulation in gromacs software. analysis of hydrophobic and stacking interactions, hydrogen and ionic bonds of the toxin and potassium channels was performed for representative frames with optimal toxin orientations using program platinum and apbs software package. we performed contacts energy characteristics estimation to predict key toxin residues for binding process and possible mutation sites for changing selectivity against kv .x channels. the results of investigation are in good agreement with the experimental values of binding constants, obtained by competitive binding assays. results of the conducted investigation may find an application in fundamental science and drug design. the research was supported by the russian science foundation grant no. - - . simulations were performed using the supercomputing center of lomonosov moscow state university. p- . . - homology modeling and molecular docking study of the paraoxonase- and its polymorphic variants q/r and m/l for non-statin lipid lowering drugs paraxonase- (pon ) enzyme is an hdl associated ester hydrolase exhibiting paraoxonase, arylesterase and lactonase activity, and reduces the formation of atherosclerosis blocking the ldl oxidation and reducing levels of oxidized lipids. in this study, molecular docking approach and molecular dynamics simulation were applied for finding the affinity of non-statin lipid-lowering drugs to pon and its polymorphic structures pon q/r and m/l . fibrates (bezafibrate, ciprofibrate, clofibrate, fenofibrate, gemfibrozil), phytosterols (beta-sitosterol, brassicasterol, campesterol, stigmasterol) and other lipid lowering drugs (ezetimibe, niacin, orlistat, probucol, and sibutramine) was obtained from pubchem database. x-ray crystallographic structure of human pon and its polymorphic variants pon q/r and m/l was generated via 'modeller', homology modelling software, from human-rabbit hybrid x-ray crystal structure of pon (pdb code: sre). ns molecular dynamic simulation analysis was performed using gromacs . . . affinity of lipid lowering drugs to pon and its polymorphic variants was predicted by molecular docking approach using autodock . suite. unlike other lipid lowering drugs that they have negative Δg values for affinity, probucol, orlistat and betasterol was calculated by positive Δg values ( . , . and . kcal/mol). these values suggest that they may have no affinity to pon q/r polymorphic structure. in all drug groups, brassicasterol and stigmasterol to pon -m/l and sibutramine to pon q/r were calculated as the highest affinity. in generally, phytosterols predicted by high affinity to pon and m/l polymorphic structures in comparison to other lipid lowering drugs. our study demonstrated that phytosterols predicted as high affinity compounds on pon structures may reduce the activity of antioxidant pon enzyme. this study need to be supported by in vitro and in vivo detailed studies. prolactin and its cognate receptor, prolactin receptor (prlr), are involved in over distinct functions in mammalians. the mammalian prlr gene consists of - exons and several and regulatory sequences. in this study, gaps and annotation errors in the rat prlr gene were corrected by comparing the genomes of mammals and rodents and new putative exons were identified. the rat prlr gene sequences from two different sources (rnor_ . , nc_ . and rn_celera, ac_ . ) were used and primary analysis showed that both sequences contain several gaps (varying from . to kbp), corresponding to about . % ( - kbp) of the gene. using the rat known prlr mrna exon sequences, it was found that the rnor_ . prlr gene has two exon- (one is about kbp long and the other immediately after this). comparisons of mammalian and rodent prlr gene structures showed that the kbp stretch is an assembly artifact. by comparing both gene sequences (and also other available rodent prlr genes), the gaps in the rat prlr gene were reduced from . % to . % (from kbp to kbp). functional annotation of the gene revealed that r. norvegicus prlr gene could have two more additional exons, exon- and - , similar to mus musculus prlr gene. in mammals, prlr mrnas contain non-protein coding exons in the utr (exon- and - ). in rats, there are exon- variants, resulting from alternative promotor usages. studies on the rat and mouse prlr genes revealed that both rodents share common non-protein coding exon- variants. in conclusion, it is found that the rnor_ . version of the prlr gene has the highest number of unidentified base pairs (corresponding to . % of the gene) and the second exon- is the assembly artifact. the rat prlr genes in both databases have several gaps and our corrected version is the best available and characterized form of the rat prlr gene. in silico affinities of some statins to paraoxonase- enzyme the structure of the statins (atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin) was obtained from pub chem database, and x-ray crystal structure of pon (pdb id: sre) from protein data bank. modeller software was used for homology modeling of pon and its polymorphic variants that's called as pon q/ r and m/l . amino acid sequence of human serum pon (uniprot: p ) was used as the modeller template. all molecular dynamics simulations were carried out with gro-macs . . software. molecular docking calculations on each of the polymorphic structure of the pon was performed with auto dock . . suite. for each substrate, y residue showed open conformation in pon and m/l polymorphic structures while q/r polymorphic structure showed closed conformation. in comparison between structures of pon variants, in most cases statins had lower affinity to q/r polymorphic structure than to the other variant. in this study, among statins, atorvastatin showed lowest but simvastatin highest affinities to pon . by considering that the high affinity drugs can have reducing effect of pon activities, it may be more appropriate to use the low affinity statins in hyperlipidemia treatment. however, these findings need to be supported with in vivo and in vitro studies. p- . . - self-assembly of lipidoids for sirna uptake and release mechanisms studied by molecular dynamics simulations o. acar , d. alpay , a. r. atilgan , c. atilgan sabanci university, istanbul, turkey, northwestern university, evanston, united states small interference rna (sirna) has the ability to bind a specific mrna which provides silencing of selected genes. nanocarriers made out of self-assembled lipidoids encapsulate sirna and deliver them into target cells effectively. in this study, a library of lipidoid structures is constructed and studied by molecular dynamics (md) simulations in different solvents, including sodium acetate, to ferret out their self-assembling mechanisms. the effect of the protonation state of the head group of lipidoids on the final shape of the self-assembled carrier is also studied. we further examine the role of the size of hydrocarbon tails in the packing. we study the final topology and the geometry of the self-assembled lipidoids both in the presence and in the absence of sirna. we find that stable clusters form with as few as chains. for lipidoids having neutral head groups, clusters are in the form of dense bundles, while those with charged head groups form spherical capsids which are depleted of the salt on the inside and having a salt rich phase on the outside. in the self-assembled structure, lipidoids are arranged so as to expose the nitrogen and oxygen atoms to the solvent. while partial capsids with these properties also form at lower lipidoid numbers, chains are necessary to form a fully closed capsid. in the presence of the sirna, the capsid assembles around the nucleotide. the free energy to remove the sirna from the assembly is calculated via repeated steered md calculations utilizing jarzynski's equality relating it to the irreversible work along and ensemble of trajectories. we therefore determine an optimal tail length for the most stable nanostructure, paving the way for designing nanocarriers with high efficacy. milk is one of the most valuable products for humans and attracts a lot of interest of researchers in various fields such as biochemistry, biology, food science and technology. the methods of milk study are quite varied. we chose the combination of the ultrasonic and dynamic surface tension (dst) measurements with the possible correlations among the obtained parameters. the aim of this work is to study the correlation between the parameters of milk, such as a content of fat, protein, lactose, minerals, dry milk solids and dst parameters. for this purpose we used milk analyzer 'klever- m' and tensiometer 'bpa- p'. three groups of animals were formed from clinically healthy holstein cows at the age of - years according to the fat content in the milk sample. group i - cows (milk fat content . ae . %), group ii - cows (milk fat content . ae . %), group iii - cows (milk fat content . ae . %). this work was supported by the russian scientific foundation (grant - - ). the biochemical parameters of the milk samples of all three groups are in the range of the 'normal' values for healthy holstein cows: protein content varies from . % to . %, lactose and mineral content varies from . % to . %, respectively. the dst parameters (r , r and k ) for the group i have strong positive correlations with the fat content in the studied milk samples. at the same time for the groups ii and iii the fat content in the milk indicates only medium positive and weak positive correlations with the r , r and k . obtained absolute values of the dst parameters of the milk samples showed some differences between all three groups. thus, the dst parameters are changing in direct proportion to fat content in the milk sample that can be explain by the primarily role of the milk lipids in the formation of the water/fat surfaces (such as fat globules, lipid-protein particles, etc.). p- . . - exploration of allosteric paths in caspase molecules using energy dissipation e. n. bingol, o. sercinoglu, p. ozbek sarica marmara university, istanbul, turkey caspases are highly regulated aspartate-specific cysteine proteases that have major roles in programmed cell death; apoptosis. effector caspases are at the terminal step of the pathway, hence they are considered as death switches. with the discovery of the presence of allosteric sites, these molecules attracted the attention of the pharmaceutical studies and became drug targets. as a result of the binding of small molecules to the dimeric interface, active site loops are shifted to an unfavorable position. this is associated with a network between distal allosteric sites and the active site loop. an energy dissipation model was applied in order to analyze this matter in further detail. perturbation of specific residues enable us to determine a possible signaling network in proteins using external energy as an input, while focusing on the dispersion of this energy between residues throughout the structure. molecular dynamics simulations were performed with and without energy perturbation using namd software with charmm force field. energy perturbation was applied by increasing the velocity of a chosen residue at the desired time step of the initial md simulation. energy change of each residue was calculated upon the application of perturbation. as a result, residue response times, corresponding to the time of the response of a residue after the perturbation of another chosen residue, are obtained. combining reponse time data with a residue interaction network, it is possible to construct a final network that shows the communication started by perturbation within the molecule. it is shown that perturbation of allosteric sites result in the disruption of the catalytic sites given in literature. our findings support this and also gives a little detail of the possible communication between distal allosteric site and the active site loops. this finding enables the usage of this methodology for similar structures where the exact allosteric mechanism is yet not known. p- . . - effect of complex mammalian membrane models with multiple membrane components on ras protein nanoclustering a. farcas , , l. buimaga-iarinca , c. floare , l. janosi faculty of physics, babes-bolyai university, cluj-napoca, romania, national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania ras proteins are essential for the cellular signal transduction that regulates cell proliferation and differentiation and act as binary switches between gdp and gtp forms. a wide range of human tumors are associated with defective ras protein signaling. the production of permanently activated ras proteins is correlated with mutations in ras genes. experiments and computer simulations have shown that membrane-bound ras proteins form nonoverlapping dynamic nanosized subdomains (nanoclusters) in activation state-/isoform-dependent manner. we performed coarse-grained molecular dynamics simulations to investigate the effect of complex mammalian membrane models on formation and evolution of ras nanoclusters. a fundamental part of the plasma membrane is the phospholipids bilayer, which contains phosphatidyl-choline (pc), phosphatidylethanolamine (pe), phosphatidyl-serine (ps), sphingomyelin (sm) and cholesterol (chol). the nature of lipid-lipid and protein-lipid interactions was studied in binary (pc:chol) and quinary mixtures (pc:pe:ps:sm:chol). because the polar lipids are not uniformly distributed between the two leaflets of the membrane, the construction of the plasma membrane with five-component lipid mixtures took into account the asymmetry between the outer and inner mono-layers. the phospholipids chain saturation (combined with the presence of cholesterol) constitute the dominant factor in phase separation and was, therefore, modeled in different lipid tail combinations for various headgroups. using microsecond timescale simulations of membraneembedded ras proteins, we have shown that the nanoclusters are spontaneously forming dynamic structures whose behavioral characteristics is modulated not only by the ras isoform, but also by the complexity of the membrane model. furthermore, we showed that variations in the plasma membrane lipid composition have important implications in the localization of ras protein nanoclusters. optogenetics comprises biological methods to achieve gain or loss of function of well-defined events in specific cells of living tissue by means of targetable control tools that respond to light and deliver the effector function. microbial rhodopsins (mrs) have been established as powerful light-sensitive tools for optogenetics. acting as ion pumps or channels, mrs are used to induce cell (de)polarization to control neuronal activity in a wide range of living organisms. mrs are membrane proteins found in a large clade of organisms, including eukaryotes, bacteria, and archaea. they share a common architecture of transmembrane a-helices and a covalently linked retinal, which is employed to absorb photons for energy conversion or the initiation of cellular signaling. major efforts are put into screening of natural and generating of synthetic mrs with desirable properties for optogenetics, e.g. ion selectivity. however, experimental study of mrs is difficult and resource consuming owing to, among other factors, low expression levels and protein stability. thus, there is a need in developing of computational tools for identification of mrs with desirable properties. we used non-redundant atomic structures of mrs taken from protein data bank to develop a set of numerical descriptors that reflects functional properties of mrs. then, we calculated the descriptors for non-redundant sequences of mrs with known function taken from the uniprot database, resulting in the feature matrix. we applied the support vector machine and the fold cross-validation procedure, using the feature matrix as the training set. as a result, we obtained the classifier that discriminates mrs in terms of the ion selectivity, e.g. na + , h + , or cl À pumps, with high precision. finally, we used the derived classifier on a test set of proteins and identified mrs for the further experiment in vivo. rational design of peptides with required stability and functional activity properties becomes a real instrument for the new generation drug development. the reca bacterial protein (and human rad homolog) is considered to be the central catalyst of homologous recombination, a mechanism essential for the accurate repair of double-strand dna breaks. dna repair via homologous recombination requires reca nucleoprotein filaments assembly. using seqopt (http://mml.spbstu.ru/seqopt/), a novel method for a-helix sequence optimization we present the successful design of peptide sequences capable to maintain a very stable a-helix structure and to inhibit reca activity. novel a-helical amino acids peptide is constructed based on reca-dna complex structure. we observed in vitro inhibition of reca atp hydrolysis, dna strand exchange reaction and reca filament formation. also, we observed lower e. coli resistance to uv and sos-response suppression in vivo. computational identification of promiscous enzyme activity for the morita-baylis-hillman reaction k. ozturk, s. sayin, n. celebi olcum yeditepe university, istanbul, turkey enzyme promiscuity attracts considerable attention in terms of enzyme evolution, protein engineering and biocatalysis. especially, development of highly efficient novel biocatalysts starting from promiscuous enzymes that have the catalytic machinery to perform desired chemistry is an intense area of research in recent years. in this work, we computationally explored the catalytic promiscuity of natural enzymes for the synthesis of morita-baylis-hillman (mbh) adducts, which display antitumoral activity against human cervical cancer cells, by mining structural protein databases using quantum mechanically optimized theoretical active site models (theozymes). catalytic interactions in the active sites of selected hit proteins with potential mbh activity were evaluated in solvated dynamic environment using molecular dynamics simulations. computational screening of the protein data bank for the quantum mechanically determined optimal arrangement of catalytic functional groups for the target mbh reaction successfully identified an enzyme with experimentally determined promiscuous mbh activity. ras proteins mediate a wide variety of signal transduction pathways that regulate cell growth, proliferation and differentiation. these proteins are small gtpases that act as binary switches between gdp-bound 'off' and gtp-bound 'on' states. oncogenic point mutations of ras are associated with~ % of all cancers and up to % in specific tumors and many developmental disorders. both experimental and in silico results showed that the membrane-bound ras proteins form non-overlapping dynamic nanosized subdomains called nanoclusters in an activation state-/ isoform-dependent manner. we performed coarse-grained molecular dynamics simulations in order to investigate the formation and evolution of ras nanoclusters in mammalian model membranes. ras proteins were inserted into the cytoplasmic side of the plasma membrane model (di-c : -phosphatidyl-choline: di- : -phosphatidyl-choline: cholesterol : : ) where they formed highly dynamic nanoclusters, both in size and in composition. furhermore, we found that the presence of ras protein nanoclusters has a significant impact on the model membrane behavior. properties such as phase behavior, diffusion coefficient, surface tension and lipid tails order parameter are also influenced by the temperature variation of the model membrane. we have investigated dynamics in three different crystal forms of ubiquitin, as well as ubiquitin in solution, with particular emphasis on (i) conformational exchange between b turn type i and ii in the region - and (ii) rocking dynamics where protein molecules as a whole undergo subtle reorientational motion within the confines of the crystal lattice. experimentally, both motional processes have been probed using relaxation dispersion techniques, including recently developed near-rotary-resonance dipolar relaxation dispersion experiments. thereby it has been determined that rocking motion in one of the crystal forms (pdb id n ) occurs on the time scale of tens of microseconds, whereas the conformational exchange has characteristic time constant of ca. ls. using molecular dynamics simulations, we have shown that the similarity of motional time scales is not accidental: bi↔bii exchange and rocking motion appear to be coupled. we have investigated the mechanisms of this coupling and predicted a number of point mutations that are expected to abrogate (or enhance) rocking. the crystals of ubiquitin containing these mutations have been modeled in silico. we have also investigated the interactions (in particular, crystal contacts) that control the balance between bi and bii conformations in different crystal forms. finally, we have used md simulations as a basis for chemical shift calculations and illustrated how relaxation dispersion effects can emerge as a function of bi↔bii exchange in conjunction with the rocking motion. the md simulation study was supported by rsf grant - - . serine/threonine kinases are attractive targets in targeted cancer therapy due to their overexpression in several forms of cancer. flavonoids are highly bioactive plant secondary metabolites that are important in human health due to their antioxidant property. quercetin, a natural flavonoid derivative, has been shown to regulate several signal transduction pathways and is in phase i clinical trial as an anticancer drug. this study explored the inhibitory potential of quercetin and its derivatives using in silico methods like molecular docking and molecular dynamics simulations. quercetin and its derivatives were observed to bind to several serine/threonine kinase family proteins with binding energy significantly better than other known inhibitors and commercially available drugs. this study thus sheds light on the atomic level interactions that define the polypharmacological nature of quercetin and its ability to interfere with a number of cancer pathways. introduction: noninvasive prenatal diagnosis (nipd) of the fetal rhd status by rhd genotyping of the maternal plasma was initially applied in alloimmunized pregnant women. fetal rhesus d status detection for management of rhd incompatibility using circulating cell-free fetal dna from maternal plasma or serum is now accepted by many obstetricians in europe as reliable and useful. the aim of the study was to detect fetal rhd specific antibodies in maternal plasma using a nanopolimer based electrochemical biosensor. materials and methods: a three-electrode system, consisting of a gold electrode, an ag/agcl reference electrode and a pt counter electrode, was accommodated in a -ml electrochemical cell. anilin and jelatin were used for immobilization of rhd antibody. the polimerization was occured at nm uv light. antibodies of rhd antigen were detected using differential pulse method at between . and . v potentials by observing the differentiations in the current values. results: the rhd status of the fetus was predicted in rhdnegative pregnant women ( - th week of pregnancy). rhd antibody were detected in maternal bood using biosensor in of the fetuses. the results were confirmed with real-time pcr. the fetuses found rhd (+) for exon and of rhd gene by multiplex real-time pcr. discussion and conclusion: biosensors based studies might be useful, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses. we found that more advantages in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, specific, economic, practical and less time-consuming. fetal rhd detection at low concentrations and in the early week of pregnancy is possible with this method. p- . . - investigation of phylogeography of cricotopus sylvestris (diptera: chironomidae) using mitochondrial and nuclear molecular markers the family chironomidae is one of the most widely distributed insect families of diptera, and this family is distributed in all continents and all habitats from the tropics to the arctic in lakes, streams and puddles. in this study, we aimed to determine the dispersal of c. sylvestris using molecular phylogenetic markers not only in turkey but in the world and to reveal from where this species may have entered to turkey in the past. c. sylvestris larvae were collected from lakes across turkey. after total genomic dna extraction from body of larvae, fragments of two mitochondrial genes, cytochrome c oxidase subunit i (coi) and cytochrome b (cytb), and one nuclear gene, carbomyl phosphate synthase domain (cps) of cad, were amplified and sequenced. in addition, several sequences of these three genes of c. sylvestris from different countries of different continents such as south corea, japan, canada, denmark, and sweden were obtained from genbank. all sequences were aligned using mega and bioedit version . . . and were used for phylogenetic analyses. neighbour-joining (nj) tree was created in mega and paup . b with bootstrap replicates. maximum likelihood (ml) analysis was performed in raxmlgui . using gtrgamma model with bootstrap replicates. beast v . . was used for bayesian analysis. our phylogenetic analyses indicated that the japanese, south corean and american c. sylvestris were different from european and turkish members. turkish members of c. sylvestris were closely related to european ones according to our bayesian, nj and ml analyses. in turkish members, c. sylvestris collected from hazar and c ß ıldır lake was more ancient than those from marmara, sapanca, c ß ıldır, aygır, beys ßehir, e girdir and sıhke lakes. in conclusion, our results clearly suggest that several transoceanic dispersal events among the continents may have occurred and that the entrance of turkish c. sylvestris to turkey may have been from southeast and northeast of the country. metagenomics is providing great help to explore world of unculturable microorganisms in the natural samples to enhance our knowledge about microbial diversity. here, we have performed metagenomic analysis of fresh water lake bacterial community using pyrosequencing techniques. we have observed a wide array of bacteria from phylum proteobacteria and family enterobacteriaceae as well as very few viruses from podoviridae, siphoviridae and unclassified phages. we have conducted a metagenomics analysis with the primary focus on the examination of the community of bacteria in a fresh water lake ecosystem. roche gs flx software gave us total reads (with an average read length of . bp). there were contigs having > bp sequence length whereas contigs with > bp sequence length. for further analysis we have taken contigs with > bp only. further, we have analyzed the microbial community composition using blastn/blastx against nt/nr databases with e-value cutoff of À . ≥ % of total contigs were mapped to the reference with ≥ % contig match coverage. the community analysis revealed that domain bacteria is predominantly present ( . %) in the water sample, followed by eukaryota ( . %), viruses ( . %) and other sequences ( . %). most abundant phyla was proteobacteria ( . %) and the most dominant family was enterobacteriaceae ( %) followed by xanthomonadaceae ( %), vibrionaceae ( . %), pasteurellaceae ( . %), shewanellaceae ( . %). we performed functional analysis of all contigs using rapid annotation using subsystems technology (rast) which detected coding sequences and rnas in subsystems. among the classified cds from rast showed major cds hits for enzymes involved in the subsystems amino acids and derivatives and the carbohydrate metabolism. the great diversity of microorganisms present in the lake may reflect the human activity in the area. maldi-tof mass spectrometry is a ubiquitous and widespread tool for protein identification. once the protein sequence is unavailable, unambiguous identification cannot be performed, and predictability is limited by the presence of sequenced homologous proteins. we present a statistical approach to predict a number of structural, localization and functional properties of unknown proteins by direct analysis of mass distribution shapes of their post-cleavage fragments obtained from maldi-tof mass spectrometry data. secondary structure of proteins is best predicted by their specific cleavage at the inertial hydropathy group amino acid residues (filmv), with thermolysin (afilmv) being the closest commercially available reagent, leading to distinguishing between proteins with presumably ahelixes or b-sheets with % accuracy. cellular localization of proteins is best predicted by their specific cleavage at the external hydropathy group amino acid residues (dehknqr), exemplified by gluc(phosphate)+lysc(dek) cleavage. protein location in the cell membrane and its localization character (monotopic/ transmembrane, single-pass/multi-pass transmembrane) are predictable with~ % accuracy by this single cleavage, with optimal combination of - cleavages improving the accuracy tõ %. functional prediction of proteins is the best among membrane-associated proteins with characteristic structural conformations. we attribute the differences in the mass distribution shapes to the characteristic clustering of amino acids residues with respective hydropathy properties that are involved in the formation of d structural conformations of proteins. the suggested approach allows for a non-parametric statistical prediction of uncharacterized proteins from their maldi-tof mass spectrometry data without knowledge or reconstruction of their primary sequence. potential applications include proteomic studies of organisms with unavailable genomic sequences and highly variable proteins analysis. the cancer genome atlas (tcga) represents a comprehensive database of genomic, transcriptomic and epigenetic alterations across more than tumor types. earlier we developed cross-hub tool aimed at multi-way analysis of tcga data in the context of gene expression regulation. in the present work, the software was updated with new features that are described below. crosshub is a python-based application. one of the features of crosshub is the combining tcga rna-seq co-expression analysis to encode chip-seq data in order to reveal most possible transcription factor (tf) targets and coupling mirna-mrna co-expression to several algorithms of mirna target prediction in order to enhance its efficacy. the key feature of the updated crosshub version is the analysis of the associations between expression ratio of tf to its targets and tf mutation status. this allows identification of tfs that are functionally (in)activated with driver mutations in a particular cancer type. the second novel feature of crosshub is the analysis of associations between 'tf-to-targets' expression ratio and tumor characteristics (tnm classification, pathological stage), patient follow-up, etc. in turn, this analysis may result in the identification of 'tf-targets' functional relations that are important for disease progression, tumor invasion, response to chemotherapy. thus, crosshub was supplemented with new features that can be useful for comprehensive tcga data analysis. the updated version of crosshub is freely available at http://sourceforge.net/ projects/crosshub/. this work was financially supported by the russian foundation for basic research (grants - - , - - and - - ) and ras presidium program 'molecular and cellular biology'. p- . . - mutations leading to increased rnase production and streptomycin resistance in bacillus pumilus bacillus pumilus strain - which was derived from soil-isolated b. pumilus p using chemical mutagenesis is characterized by resistance to streptomycin (str, up to lg/ml) and ability to produce extracellular enzymes in quantities almost -fold higher than the parent strain. these features make the - strain suitable for industrial production of rnase (binase) which is known for its antitumour and antiviral properties and can be used as an rna-degrading tool in molecular biology. the whole genomes of both mutant and wild-type b. pumilus strains were sequenced recently. to reveal the exact genetic features responsible for rnase overproduction and str resistance we have fulfilled comparative genome analysis of b. pumilus p and - strains. facilities of rast server, edgar platform and additional bioinformatics tools (plasmid finder, prophinder, bl seq) were used. it is found that both b. pumilus genomes under study contain an intact prophage, while only wild-type strain bears a kb cryptic plasmid. none of the systems is inactivated in mutant strain according to the results of metabolic reconstruction. . % of total cdss differ in - strain in comparison to p one, % of them are hypothetical. the altered genes are involved in membrane transport, cell wall composition, chemotaxis, spore formation, carbohydrate metabolism, dna metabolism, translation and transcription regulation. mutation (k n) leading to str resistance is identified in s ribosomal protein s p. regulatory and coding regions of binase gene have no modifications. candidate genes which can account for binase overproduction are selected. mutation k n is classical in str resistance and leads to enhancement of decoding accuracy while decreasing elongation speed. rnase overproduction is brought about by non-specialized mechanism since other hydrolases are also overproduced in mutant strain. genes encoding extracellular serine protease, sporulation initiation phosphotransferase f, gnat-family acetyltransferase and cell wall modifying enzymes are reported previously to increase production of degradative enzymes. the action of encoded by them proteins lead to increase of stability and release of secreted proteins to environment and to derepression of their transcription from negative regulators bacillus pumilus strain p has been identified on its ability to produce ribonuclease and different extracellular proteases. in order to increase inherent biosynthesis of proteases the p strain was screened on culture medium supplemented by streptomycin. derivative b. pumilus strain - gains the resistance to streptomycin and also shows the increased ribonuclease activity. we used genomes of both these strains to explore streptomycin susceptibility and increased activity of hydrolytic enzymes. whole-genome shotgun sequencing was performed using a combination of pyrosequencing and ion semiconductor sequencing, which provided x ( p) and x ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) overall genome coverage. assembled genome sequences of p and - strains included and scaffolds > bp with a calculated genome size of bp and bp, respectively. the gc content was %. both draft genomes have been deposited at genbank (jojx . for p and jhud . for [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . detailed comparative genomic analyses of strains have been performed. we calculated average nucleotide identity (ani) values between the genomes of our strains and completed b. pumilus genomes deposited in ncbi database. two b. pumilus strains (sh-b and safr- ) revealed the max. ani value ( . % and . %, respectively). b. pumilus sh-b strain has been used as a reference for snp calling in strains p and - . snps for the p strain and for the - strain were classified as nonsynonymous variants. radical nucleotide substitutions from the - genome were not found in p genome. among them, the mutation in the codon of rpsl gene (coding s ribosomal protein s ) is probably associated with resistance to streptomycin. also, two mutations in rpob and nusa genes (coding rna polymerase and transcription termination factor rho, respectively) may be related to increased enzymes activity. both our strains contain protease-coding genes. twelve of them are encoding extracellular proteases. here we propose an algorithm that can predict an antibodies mutant forms with desired specificity. this algorithm allows to determine the position and type of amino acid residue for mutagenesis. approach is based on a hybrid method of quantum and molecular mechanics (qm/mm) that allows to understand the reaction mechanism and the role of active center amino acids. catalytic antibody a , that is able to hydrolyze pesticide paraoxon, was selected as a model. however, the hydrolysis efficiency of paraoxon by a antibody is only m À min À , that is insufficient for using this antibody as antidote. the main fundamental goal of our study is to determine the necessary conditions for improving the binding reaction of paraoxon by catalytic antibody a . the hybrid qm/mm method allows to study the reaction mechanism of interaction of a with paraoxon. it was shown that the reaction proceeds via the classical s n mechanism. the key step of the reaction is the proton transfer from the catalytic residue tyr- to paraoxon. qm/mm approach determines position for mutagenesis -leu- in light chain. for one of the mutant in this position -leu argwere predicted (i) increased probability of formation of a hydrogen bond between the catalytic moiety and paraoxon compared to the wild type antibody and (ii) smaller value of the diffusion coefficient, which reflects the best positioning of paraoxon in the active center. steady-state kinetic analysis shows that leu arg exhibits a -fold increase in k /k d compared to a ( m À min À vs. . m À min À ). double mutant leu arg/ser ala also has improved constants of interaction with paraoxon in comparison with the wild type antibody, however, a single mutant leu arg still binds paraoxon three times better, that may be due to the fact that the serine in position increase the nucleophilicity of tyr . thus, our results are in line with our computed predictions. this work was supported by rfmefi x . due to high prices of meat and meat products, low quality raw materials like offal tissues are commonly used in turkey. in the retrospect of the studies for evaluating and detection of unwanted tissues in the sample is basic histological examination. the light microscopy techniques are very strong method if a researcher qualification is enough. a new researcher-independent method must be developed. therefore, different tissues and organs constitute of unique mrna and protein. our method is based on this event, so the antigenic sites of the tissues can be detected by selected antibodies. the first set of the antibodies are for detecting muscle and adipose, consist on muscle actin and adipose triglyceride lipase. this set is used for calibration on standard meat sample. the second set of the antibodies are detecting of offal tissues, consist on trrap and casein. anti-casein antibody is selected because the mammary gland usage in grinded-meat is very common. immuno-staining started with hier (heat mediated epitope retrieval), then classical ihc method applied to slides with dab-chromogen. after all process completed the slides were photographed by las (leica application suite) on microscope. the capture settings were remained same on both sets. image capture size is x pixels and field of view (fov) is lm. all the image files were converted to binary for threshold operation. the threshold values of first set and second set were calculated and their ratios were compared. the formula is based on the distribution (dst) of pixel intensity (int) over threshold (thrs) values on all fov (axis: the results are good enough to detect the unwanted micro-structures on % raw meat and % offal tissue. calculations proofed with imagejÒ. future application of this method and opencv-based software algorithm is to port the source code to a single board computer (sbc) with a digital microscope connected. monday september : - : mechanisms of pro-inflammatory diseases p- . . - the effects of raas inhibition in rate limiting step by aliskiren on testicular torsion injury in rats testis torsion is a urological emergency condition that results in necrosis of the testis if the condition is not treated. unfortunately treatment of testis torsion is not fully understood, therefore clinical and experimental studies are performed continuously. reninangiotensin-aldosterone system (raas) contributed to pathophysiology of several diseases. aliskiren (als) inhibits the renin on the first step of this system. our aim is to investigate the protective effect of aliskiren on unilateral testis damage caused by experimental testis torsion and detorsion. the forty-eight rats were separated into eight groups of six animals: sham, sham+als mg/kg (oral) group, torsion group (tor), torsion/detorsion group (tor/det), tor+als mg/kg (oral) group, tor+als mg/kg (oral) group, tor/det+als mg/kg (oral) group, tor/det+als mg/kg (oral) group. in the tor and tor/det groups, the left testes were rotated °clockwise together with the spermatic cord and tunica vaginalis in the scrotal space. the left testes of the rats were subjected to torsion and detorsion during h. after experimental procedures, testicular tissues were examined by histopathologic and molecular analyses. the il- b and inos mrna expressions were increased in tor and tor/det groups when compared with sham group. both doses of aliskiren administration decreased these expressions in tor/det groups. the stereological results revealed that aliskiren administration promote the numerical density of mature spermatids in tor and tor/det groups. the numerical densities of tor/det+als and tor/det+als groups were similar and these two groups have significant difference when compared to the tor and tor/det groups. the administration of als may be useful for preventing ischemic damage on unilateral testes injury in rats. this study supported by a tubitak project, coded s . ) has recently been recognized as a potent immunomodulator which acts through regulation of gene expression involved in immunity response thus affecting various inflammatory and autoimmune diseases. the study was aimed at investigating hepatoprotective role of d in vdr-mediated regulation of pro-inflammatory factors in diabetic liver. materials and methods: type diabetes was induced in male c bl/j mice by i.p. injection of high-dose stz ( mg/kg b.w.). after weeks of stz-induced diabetes animals were treated with/without d ( iu/mouse per os) for weeks. blood serum ohd was assessed by elisa. rel-a, vpf, inos and vdr expression was measured by qrt-pcr and/or western-blot. results and discussion: diabetes caused two-fold reduction of serum ohd level, indicative of d deficiency. significant alterations in d -endocrine system were found as is evident from reduced expression of cyp a , cyp r , vdbp and vdr at transcriptional and translational levels. these changes were accompanied by a marked increase in mrna and protein levels of inflammation markers rel-a, vpf and inos in hepatic tissue of diabetic mice. diabetes also led to structural lesions in liver tissue. complete restoration of ohd content and partial normalization of liver tissue structure were achieved after d treatment. d administration partially normalized expression of cytochromes involved in d metabolism and hepatic pro-inflammatory factors. d treatment prevented overexpression of rel-a and phosphorylated p /rel-a translocation to hepatocellular nuclei that is most likely mediated through , (oh) d and vdr. conclusion: study confirmed that diabetes-induced liver abnormalities are associated with chronic inflammation that can be linked to impaired d metabolism and deficiency. our findings demonstrate protective vdr-mediated effect of vitamin d against diabetes-induced liver injury. p- . . - lavandula stoechas extract increased glucose uptake and protein levels of key signaling molecules in insulin resistant c c muscle cells s. savranoglu , h. ipek , s. arslan , h. delig€ oz , a. r. t€ ufekc ßi , i. demirtas , t. boyunegmez t€ umer graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, deparment of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, department of chemical engineering, faculty of engineering, pamukkale university, denizli, turkey, department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, turkey, department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: the aim of this is to identify remedial effects of lavandula stoechas, anatolian traditional medicine, against metabolic disorders developed on the ground of insulin resistance. ethyl acetate extract (eae) of l. stoechas was investigated in c c myotubes which were made insulin resistant by free fatty acid (ffa) treatment, for its effects on glucose uptake and as well as on the activation of akt- (by pakt/ akt ratio) molecule which plays a central role in insulin signaling through serine ( ) phosphorylation. in addition, the protein level of lipoprotein lipase (lpl) enzyme was also evaluated. material and methods: c c cells were made insulin resistant by palmitic acid (ffa) and effects of eae on p-akt (ser )/akt ratio and lpl level were determined by sds-page/western blot. the effect of eae on glucose uptake in insulin resistant cells were determined by the -deoxyglucose uptake assay. results: eae at and lg/ml significantly increased the glucose uptake and % compared to insulin resistant control cells. metformin at mm increased this parameter up to %. eae increased pakt ser /akt level - % and lpl expression - % for and lg/ml, in insulin resistant myotubes, respectively (p < . ). discussion: eae of l. stoechas improved impaired insulin sensitivity through both enhancing glucose uptake and activation of akt molecule through ser phosphorylation. in addition, it also considerably increased lpl level which has very important function in lipid metabolism. conclusion: overall, results demonstrated that l. stoechas contain phytochemicals which can be effective for the prevention and also treatment of insulin resistance and associated conditions. our research group is on the way for the identification of these 'bioactive' molecules with bioassay guided fractionation studies. tubitak (projectid: t ) supports this study. achievement of complete pain control is very difficult task, which requires a search for new molecular targets during the analgesic substances development. considering the importance of glial cells and their signaling molecules, development of new gliotropic therapeutic methods is a promising direction in pain treatment. polyunsaturated fatty acids, including docosahexaenoic acid demonstrating anti-inflammatory and antioxidant activity are of considerable interest. docosahexaenoic acid (dha, : n À ) analgesic activity was studied using a chronic constriction injury (cci) rat model. animals were subcutaneously injected with dha emulsion at a dose of . mg/kg ( mm/kg) daily during weeks after surgery. collection of material for subsequent immunohistochemistry investigation was performed on day . we clearly demonstrated that the activation of neurokinin neurotransmission and nnos synthesis are coincided with the astroglial activation in the spinal cord dorsal horn (scdh) superficial lamina during neuropathic pain development. however, the detailed mechanisms of interaction between substance p (sp)-, no-ergic systems and astrocytes in the spinal cord remain to be elucidated. systemic administration of dha to cci animals reduced neurogenic pain intensity and duration, leading to an earlier stabilization of paw weight distribution and preventing the development of degenerative changes in denervated limb. this drug treatment reduced the level of the sp-and no-ergic neurotransmission and decreased astrocytosis in the scdh superficial lamina. thus, the ability of dha to affect nociception is a promising and safe alternative to current pharmaceutical therapeutics. immunohistochemistry studies carried out with the russian science foundation financial support (agreement no. - - ), obtaining dha and all manipulations with animals of the material was funded by rfbr according to the research project no. - - mol_a. p- . . - circulating endothelial-derived apoptotic microparticles and aopps are related to highsensitive troponin t in patients with chronic hepatitis c infection the aim of this study was to evaluate non-standard risk factors for cardiovascular events, such as endothelial dysfunction assessed by endothelial-derived microparticles (emps) (cd +/ cd + ), advanced oxidation protein products (aopps), and low-grade inflammation, that are potentially associated with elevated levels of high-sensitivity troponin t (hs-tnt) and n-terminal pro-brain natriuretic peptide (nt-probnp) in patients with chronic hepatitis c (chc). methods and results: eighty-six chc patients and healthy control subjects were enrolled in the study. circulating levels of hs-tnt, nt-probnp, aopps-albumin (the ratio of aopps to albumin content), emps (cd +/ cd + ), hs-crp, and tnf-a were assessed. compared with chc patients, the chc patients with diabetes mellitus (dm) had higher levels of emps (cd +/ cd + ) and aopps-alb, which were associated with elevated hs-tnt levels (≥ . pg/ml). nt-probnp positively correlated with tnf-a level in all chc patients and this correlation was stronger in diabetic patients. in multivariate logistic regression analysis, the independent factors associated with the presence of elevated hs-tnt levels were the presence of dm (p < . ) as well as high levels of aopps-alb, apoptotic emps (cd + /cd + /an-v + ), and nt-probnp (p = . , p = . , p = . respectively). conclusion: the prevalence of elevated hs-tnt were increased significantly in the diabetic patients with chronic hepatitis c. hs-tnt was related to non-standard risk factors for cardiovascular events, and circulating endothelial-derived apoptotic microparticles (cd + /cd + /an-v + ) level was an independent predictor for elevated hs-tnt levels, potentially indicating some abnormalities in the myocardium. apnea; and healthy individuals; and assessing the connection between the pain and the dimension of the sleep disorder. material and methods: patients who were diagnosed with obstructive sleep apnea and healthy individuals who were similar in terms of age and gender were included in this study. the patients, who were diagnosed with obstructive sleep apnea with the examination and sleep tests, were assessed according to the american college of rheumatology (acr) criteria in terms of fms. serum d vitamin level was measured by using the ultra performance liquid chromatography method. findings: when the fibromyalgia syndrome and obstructive sleep apnea and pure obstructive sleep apnea patient groups are compared with the control group, the vitamin d level was found to be low at a significant level (p = . , p = . , respectively). no significant difference was found between the vitamin d levels in fibromyalgia syndrome, obstructive sleep apnea and pure obstructive sleep apnea patient groups. a negative correlation was found between the number of the sensitive points and vitamin d levels in fibromyalgia syndrome patients (p = . ). results: it has been concluded that the obstructive sleep apnea and fibromyalgia syndrome patients have low vitamin d levels, and this situation must be considered in treatment modalities. on the other hand, the results obtained in the study make us consider that vitamin d metabolism is not influential in the pathogenesis of the fibromyalgia syndrome and obstructive sleep apnea togetherness. p- . . - decreased chitotriosidase activity and levels in familial mediterranean fever discussion: familial mediterranean fever is an inflammatory disease. several cytokines and inflammatory mediators are playing role on pathogenesis of the disease. although ıt has been demonstrated that the increased concentrations of cht in patients with fmf. we found lower cht activity and concentrations in patients with fmf. conclusion: serum cht enzyme activity and concentrations may not be considered as a biomarker in fmf patients taking colchicine. new studies are needed to evaluate the changes of the enzyme activity, concentration and the role of cht in patients with colchicines negative patients. chronic hyperglycemic state leads to an increase in subclinical systemic inflammatory response in diabetes mellitus type (dmt ) patients. inflammation-based scores, neutrophil to lymphocyte ratio (nlr), platelet to lymphocyte ratio (plr) and red blood cell distribution width to platelet ratio (rpr) are biomarkers able to quantify systemic inflammation. the aim of the study was to investigate association of the inflammation-based scores with short-and long-term glycemic control markers, and whether they could be used as indicators of glucoregulation in dmt patients. the cross-sectional study included dmt patients, treated at the primary health care centre zenica from december to april , distributed into groups according to glycated hemoglobin (hba c) values: a (n = , hba c ≤ . %) and b (n = , hba c > . %). complete blood cell count, fasting blood glucose (fbg) and hba c measurements were determined at the primary health care centre zenica and at the department of laboratory diagnostics, cantonal hospital zenica by standard laboratory methods. all statistical tests were performed using spss . . p values fasting blood glucose and hba c were significantly higher in the group b compared to the group a (p < . ). there was no significant difference of nlr, plr and rpr between the groups (p = . ; p = . ; p = . , respectively). significant correlation of inflammation-based scores with fbg and hba c was found only between plr and hba c in the group a of dmt patients (r = . , p = . ). inflammation-based scores could gather meaningful clinical information, either diagnostic or prognostic, on a variety of hyperglycemic, inflammatory, cardiovascular and thrombotic disorders. since there was no statistically significant difference of nlr, plr and rpr between dmt patients with good and poor glycemic control, we conclude that these scores could not be used as indicators of glucoregulation in dmt patients. chronic inflamation plays a central role in the development and progression of diabetes and in the pathogenesis of its comlications. the neutrophil-lymphocyte ratio (nlr) and platelet-lymphocyte ratio (plr) are indicators of subclinical inflamation. mean platelet volume (mpv) is one of the platelet function indices that reflects the platelet production rate and stimulation. we investigated the association of nlr, plr and mpv with prediabetes and type diabetes mellitus (t dm) and determine whether or not these are reliable markers for diagnosis. we evalueted people's results who were carried out oral glucose tolerance test (ogtt). acording to -h values of plasma glucose in the ogtt; . group (normal glucose tolerance: ngt): under mg/dl (n = ), . group (prediabetic: impared glucose tolerance (igt)): ranging from mg/dl to mg/dl (n = ), . group (firstly diagnosed diabetic by ogtt): above mg/dl (n = ). . group is clear diabetic without complication (taking treatment) group (n = ). we compered nlr, plr, mpv and some biochemical markers between four groups. there are significantly differences between all groups in nlr (p = . ) and plr (p = . ) values. nlr values are significantly higher in prediabetic ( . it is recognized that a chronic low-grade inflammation and an activation of the immune system are involved in the pathogenesis of insulin resistance and type diabetes mellitus (t d). this study aimed to analyze the long-term impact of altered metabolism in female t d patients at the level of mediators of inflammatory response. this study included femalet d patients and control subjects, which were recruited at the clinical center university of sarajevo and the general hospital tesanj. in this study the effects of glycemic control on markers of the inflammatory response crp, fibrinogen, leukocytes, sedimentation, and cytokine il- , were analyzed. all subjects included in this study were free of evidence infections, surgery, thyroid disease, polycystic ovarian syndrome, active liver and kidney damage. all biochemical analyses were performed by employing standard ifcc protocols. results from this study demonstrated significant increase of fibrinogen (p = . ), crp (p = . ), il- (p = . ), leukocytes (p = . ) and sedimentation rate (p = . ) in female t d population compared to control subjects. interestingly, a significant correlation was shown between crp and hba c (p = . ), il- and glucose ( . ), il- and bmi ( . ). in our study, female t d compared to the healthy population had significantly higher levels of fibrinogen, leukocytes, il , crp and sedimentation. other studies conducted in female population associated elevated levels of il- and crp with t d independent of other risk factors for diabetes. crp being most robust predictor of diabetes. studies have shown that crp is an important predictor of t d for the female but not the male population. thus, our data suggest that inflammation play an important role in the pathogenesis in female diabetic population. a more detailed study on a far larger number of subjects should point out fact if they can effectively be used as biomarkers in the primary prevention of t d in this population. objectives: bone and mineral metabolism disorders hold an important place among the complications after renal transplantation. the purpose of this study was to demonstrate the relationship between vitamin d, calcium, phosphorus metabolism with graft function and to measure , (oh) d levels with lc-ms/ ms in renal transplant recipients. design and methods: this study included renal transplant recipients ( female, male; mean age: . ae . ) from living related donors were transplanted. blood samples were collected immediately before and after transplantation at month . serum creatinine, bun, calcium, phosphorus, alkaline phosphatase, glucose, albumin, pth, (oh)d and , (oh) d levels were measured. gfr values were estimated by ckd-epi. plasma , (oh) d levels were determined in a lcms- triple quadrupole tandem mass spectrometer (shimadzu corporation, japan) by mrm. spss . software was used for statistical analysis. results: although plasma , (oh) d levels significantly increased (p = . ), we did not find any significant differences for serum (oh)d levels after transplantation. when posttransplant levels of serum phosphorus, pth, creatinin, bun and alp levels were found to be significantly decreased (p = . , p = . for alp), we observed significantly higher calcium and gfr values (p = . ). vitamin d insufficiency was present . %, deficiency . %, severe deficiency % before transplantation, insufficiency was also seen . %, deficiency %, severe deficiency . % after transplantation at month . conclusions: in our study, all patients were found to vitamin d deficiency and insufficiency. determination of vitamin d deficiency and consequently treatment with vitamin d supplements could lead to better graft surveys. free fatty acids (ffa) represent important link between obesity, insulin resistance, type diabetes (t d), and dyslipidemia. increased adiposity, as approximated by body mass index (bmi), correlates well with increased serum levels of leptin-adipocyte derived hormone implicated in the regulation of adipose mass and alterations in insulin action and secretion. the main objective of the present study was to investigate the potential association of serum ffas with leptin levels in healthy and newly diagnosed type diabetic subjects. this study involved newly diagnosed type diabetics and healthy subjects. all participants in the study were free of evidence of hepatitis, viral infection or active liver and kidney injury. for biochemical analyses of glucose, glycosylated hemoglobin (hba c), and lipid profile, standard ifcc protocols were used. analysis of free fatty acids (ffas) was done by gas chromatography, while serum leptin levels were determined by the elisa kit. in addition to the expected differences in glucose, hba c, and bmi, our results also showed significant differences in leptin, myristoleic, palmitic, linolenic, arachidic, and arachidonic acids between t d and control subjects. in healthy subjects, a significant correlation was demonstrated between glucose and lauric, arachidic, arachidonic acid levels, body weight, and bmi. newly diagnosed diabetics showed significant association between glucose and lauric, myristoleic and linolenic acid levels; with leptin being associated with myristic and palmitoleic acid levels. interestingly, in all participants, significant association was found between glucose and hba c, glucose and leptin, myristoleic, arachidic, and bmi as well as between leptin, arachidic acid, and bmi. thus, our data point out association of different types of ffas with leptin levels in newly diagnosed type diabetics. however, further studies should be done in larger number of patients to confirm our results. rheumatoid arthritis (ra) and ankylosing spondylitis (as) are chronic inflammatory diseases with distinct clinical manifestations in many ways. the aim of this study is to evaluate the serum levels of molecules which may be used as markers for angiogenesis and vascular leakage in the processes of two clinically different pictures, ra and as. ra patients, as patients and healthy volunteers with mean age of - were included in the study. serum levels of vegf, angiopoetin- and tie- were measured by enzymelinked immuno-sorbentassay (elisa) using a commercially available kit. serum nitricoxide levels were evaluated by the griessreaction. serum vegf, ang- and no levels were significantly higher in the as group ml, p < . ; p < . ; p < . ). no differences were found between as and ra for tie- (p > . ). vegf, ang- , tie- and no levels were positively correlated in both as and ra patients (p < . ), but no correlation was detected between clinically activation index das- , basda _ i scores and laboratory measurements such as sedimentation, crp and anti-ccp (p > . ). when the diagnostic performance of the parameters were evaluated with the roc analysisonly the performance of the ang- in as patients was sufficient (auc ( % cl): . , p < . ). elevation of angiogenic factors in the serums of as and ra patients supports the role of angiogenesis in the etiopathogenesis of these diseases. however, lack of relationship between disease activity leads to not to use these factors as a marker for clinical follow-up. only ang- measurements may be useful for the differantial diagnosis. the evaluation of ischemia modified albumin as an early biomarker of acute myocardial infarction introduction: acute myocardial infarction (ami), remains a leading cause of morbidity and mortality worldwide. early diagnosis of ami is very important because early treatment may reduce the extent of injury to the myocardium. currently, biomarkers of myocardial necrosis such as myoglobin, ck-mb and troponins are highly sensitive and exhibit good specificity. however, these biomarkers increase after tissue injury, approximately - h after the cardiac event and detect only the consequences of prolonged ischemia. recently, ischemia modified albumin (ima) has been assessed and found to be very useful for the diagnosis of myocardial ischemia and it is considered as a serum biomarker. the aim of the present study was to evaluate the serum level of ima and determine the relation between patients with ami and control group, in order to verify its potential as a novel marker for early detection of mi. materials and methods: the study was performed with patients and healthy controls. blood samples from all subjects were collected by venipuncture in plain tubes, and immediately centrifuged at g for min at °c. the serum samples were stored at À °c until analysis. the serum levels of ima were determined using the cusabio biotech human ischemia modified albumin, elisa kit according to manufacturer's instructions. the results are given as international units/milliliter (iu/ml). results: our findings revealed that ima showed no significant difference between the groups. conclusion: our results suggest that ima assay is not a sensitive marker for early detection of ischemic hearth disease and cannot be used alone for the diagnosis of ami. prospective studies are needed to identify ima's potential as a biomarker for ami. p- . . - neutrophil-to-lymphocyte ratio and platelet-tolymphocyte ratio in polycystic ovary syndrome polycystic ovary syndrome is a complex and multifactorial disease with metabolic dysfunction and the etiopathogenesis is not well established. emerging data suggest that adiposity and chronic low-grade inflammation are involved in the development of the metabolic dysfunction. neutrophil-to-lymphocyte ratio (nlr) and platelet-to-lymphocyte ratio (plr) have recently been investigated as two new inflammatory markers used in the assessment of systemic inflammation in many diseases. the purpose of the study was to investigate their relation with pcos patients. the study population consisted of patients with polycystic ovary syndrome and healthy women controls. nlr and plr obtained by dividing absolute neutrophil to absolute lymphocyte count and absolute platelet count to absolute lymphocyte count, respectively. the neutrophil count ( . ae . vs. . ae . , p < . ) and platelet count ( . ae . vs. . ae . , p < . ) were higher in patients with pcos compared to the control group. lymphocyte count was . ae . in pcos patient and . ae . in control group. the nlr and plr of pcos patients were significantly higher compared to those of the controls ( . ae . , . ae . p < . , . ae . , . ae . p < . , respectively). in this study we found nlr and plr were significantly increased in patients with pcos compared to healty control. nlr and plr were two useful inflammatory markers for assessment of patients with pcos. imbalance in neurotransmission in conjunction with neuroinflammation contribute to neurological dysfunction observed during acute liver failure (alf). own observations indicate that alf in a mouse model is associated with altered expression and/or intracellular distribution of synaptic proteins. since neutralization of tgf-b appears to improve the neurological score in alf mice, we examined the possibility that increased levels of tgf-b , caused by liver damage, may affect the expression of selected synaptic proteins. expression and/or cytoplasmic vs. membrane distribution of a number of functionally critical synaptic proteins in cerebral cortex and blood tgf-b were measured in c bl mice with alf induced by single i.p. injection of aom ( mg/kg of b.w.) and after neutralization of tgf-b induced by single i.p. injection of ab-tgf-b ( mg/kg) h before aom injection. in alf mice, blood tgf-b was increased, and the expression of presynaptic proteins: synaptophysin and synaptotagmin was increased in the cytosolic (s ) fraction by~ % and %, respectively, but was slightly depressed in the membrane (p ) fraction by~ % and~ %. aom induced an increase of postsynaptic proteins: psd- and nnos by~ % in p fraction. tgf-b neutralization resulted in a reduction in the expression of presynaptic proteins by~ % in s fraction and~ % in p fraction, in control animals and normalized their amount in the cytosolic fraction after aom injection, but was ineffective with regard to psd- and nnos. the results indicate that in alf mouse, neutralization of cytokine tgf-b normalizes synaptophysin and synaptotagmin expression in the synaptoplasm, without affecting their synaptic membrane content. effect of tgf-b neutralization appear to be confined to the presynapse. % of acute pancreatitis (ap) patients develop severe acute pancreatitis (sap), which is is resulted in multiple organ dysfunction syndrome. an extensive inflammatory response occurs due to inflammatory mediators synthesized and secreted during sap. since preventing the inflammation in sap is important in the prognosis of the disease, new drug candidates having strong antiinflammatory effects will provide a new concept for therapeutic strategies against acute pancreatitis. non-steroidal anti-inflammatory drugs (nsaids) show their effects by inhibiting cyclooxygenases (cox- and cox- ) and they play an important role in the pathogenesis of acute pancreatitis. since conventional nsaids inhibit both cox- and cox- , they have serious side effects on gastrointestinal system. therefore, new highly selective cox- inhibitors having fewer side effects are needed. in the present study, selective cox- inhibitory activities and cytotoxic effects of new series of -benzoxazolinone and thiazolo [ , -b ]- , , -triazole derivatives previously synthesized as specific cox- inhibitors with no side effects on gastrointestinal system were investigated. permeability of the compounds was tested by pampa using caco- cells. compounds were found highly selective, non-toxic and permeable. ap was induced in rats via retrograde injection of stc into the pancreatic duct system. rats were pre-treated with saline or celecoxib or the new compounds before stc injection and sacrificed h later. the severity of ap was evaluated using biochemical and histopathological analyses. edema, inflammation, hemorrhage and acinar cell necrosis were detected in the pancreatic tissue of sap group. sap was remarkably increased serum lactate dehydrogenase, ast, alt, lipase and amylase activities and serum tnf-a, il- b, il- , il- and il- levels. tissue myeloperoxidse activity was also increased. pretreatment with the novel compounds reserved all these biochemical and histopathological parameters. alopecia areata (aa) is an inflammatory disease which is affects hair follicles, and sometimes nails. it is suggested that cytokinemediated immunity plays an important role in etiopathogenesis of aa. this study was planned to evaluate the serum ykl- and tgf-b levels of patients with aa. patients with aa and healthy volunteers were recruited into the study. fasting venous blood samples were collected from the participants and serum was obtained by centrifugation. serum ykl- and tgf-b levels were measured by enzyme linked immunosorbent assay (elisa). serum tgf-b levels in the patient group were significantly lower compared to the control group whereas serum ykl- levels were significantly higher in patient group. tgf-b levels of men and women with aa were found to be significantly lower than that of controls. while serum ykl- level of male control group is higher than the male patients, there were no significant differences between women groups. the increased serum ykl- levels in aa patients suggests that ykl- plays a crucial role in the pathogenesis of aa. arterial immune mediated inflammation participates centrally in all stages of the development of atherosclerosis, from the initial lesion to the end-stage thrombotic complications. although emerging evidence supports augmented cardiovascular morbidity and mortality in cutaneous psoriasis (psc) and psoriatic arthritis (psa) as compared to the general population its underlying mechanism is poorly understood. here we analyzed the inflammatory burden in recent onset of psa patients without traditional cardiovascular risk factors (cvrf) in a transversal study measuring carotid intima media thickness (cimt) (measured with ecodoppler), and proatherogenic inflammatory molecular markers like c-reactive protein (crp), interleukin (il- ), and soluble intercellular adhesion molecule- (sicam- ) in comparison with control patients. cimt values are similar in psa ( , ae . *) and psc ( , ae . ) patients. however, both of them were significant increase compared with control ( . ae , ). regarding inflammatory markers il- serum levels in patients with aps was higher than pcs ( ae . ) and healthy controls ( , ae . ) but the difference did not achieve statistical significance (*p > . ). on other hand mean of sicam- , value from patients with recent onset of psa is significant higher than controls. psc remain without significant changes compared to control (*p > . ). in addition mean value from patients with recent onset of psa is significantly higher than in controls (*p < . ) and psc group. overall, preliminary findings suggest for the first time that patients with early psa, without evident traditional cvrf have significant increased values of cimt, sicam- crp against the general population control group. this data strongly supports that early cv molecular markers are increased after the first symptoms and signs of this disease even in the absence of traditional cardiovascular risk factors. furthermore, this give new windows for a proper treatment. p- . . - protective effect of trail against proinflammatory cytokines on pancreatic beta cells correlated with decrease in dr and increase in dcr expressions universitesi, antalya, turkey introduction: proinflammatory cytokines are known to have destructive effects on beta cells, which contribute to type diabetes (t d) development. the combinatory effects of three of these cytokines in particular, namely tnf-alpha (tnf-a), ifngamma (ifn-c), and il- beta (il- b), are claimed to render beta cells prone to t cell-mediated destruction. the recently identified anti-inflammatory feature of tnf-related apoptosis-inducing ligand (trail), its possible protective role in this process. in this study, the effects of applications of trail with tnf-a, ifn-ᵞ, and il- b on beta cell viability and correlation of these effects with trail receptor expression patterns were investigated. methods: glucose-responsive insulin-secreting nit- mouse beta cell lines were treated with tnf-a, ifn-ᵞ, il- b, and soluble trail (strail) individually and in various combinations. cell viabilities were determined at and h by mtt assay. trail ligand and receptor expression profiles on nit- cells, and alterations in receptor expression levels following cytokine applications were determined by western blotting analysis. results: trail treatment did not have any cytotoxic effects on nit- beta cells at h, while increasing cell viability following il- /ifn/trail and il- /tnf/trail combined applications. substantial levels of death receptor (dr ) expression were detected on nit- cells before applications, yet it displayed decreased levels at h of trail treatment. lower levels of decoy receptor (dcr ) expression detected prior to treatments increased significantly in contrast. discussion: the fact that trail co-treatment with tnf-a, ifn-ᵞ and il- b increased cell viability in nit- beta cell lines along with reduction in dr death receptor expression and an increase in the decoy receptor dcr expression, points out to a possible protective effect of trail in insulitis, and strengthens its potential as a putative therapeutic molecule in prevention of beta cell loss. behc ßet's syndrome (bs) is a multisystemic inflammatory disorder with a strong and complex genetic background. being a prevalent disorder both in turkey and also in the ancient trade road 'silk road' countries, bs is an important cause of impairment and disability owing to its chronic and relapsing nature. besides, bs is reported to be an important cause of mortality among the young male patients. while the epidemiology of bs is substantially well documented, currently, the etiology, the molecular mechanisms underlying its pathogenesis, and the classification of the disorder remain to be elucidated. our aim was to disclose the disease mechanisms at molecular level in turkish bs patients by obtaining, comparing, and analysing the transcriptome data of bs patients with age and gender matched healthy controls. for this purpose, by using the affymetrix hg u plus . microarrays, peripheral blood cell mrna profiles of bs patients (b) and matched healthy controls (c) were obtained. following bioinformatics, gene ontology, and pathway analysis, validation experiments of the identified prominent mrnas were performed by qrt-pcr methodology. the comparison of b vs. c yielded differentially expressed gene numbers of and for the chosen fold changes of . and . respectively (p ≤ . for both). during gene ontology and pathway analysis, immune system process, immune system diseases, systemic lupus erythematosus, arthritis, and intestinal immune network for iga production categories/pathways were significantly enriched. clustering analysis revealed a molecular signature which accurately distinguished b and c samples, while the qrt-pcr analysis successfully validated the chosen mrna transcripts. this study documented differential expression of a large number of immune system and immune disease related genes in bs patients. the uncovering of the molecular disease mechanisms of bs will point to novel candidate molecules to be targeted for the treatment of the disorder. obesity is a public health problem in developed countries and worldwide with increasing prevalence through a relationship primarily with atherosclerotic cardiovascular diseases as well as several metabolic disturbances such as increased insulin resistance and diabetes. although several studies identified obesity as an independent risk factor for atherosclerotic cardiovascular diseases, the mechanism underlying the increased cardiovascular risk in obese patients has not been clearly delineated. adma, no, endothelin- and homocysteine are an indicator of endothel disfunction that plays an important role in the pathophysiology of atherosclerosis. in our study, obese children and the control group were compared in terms of adma, no, endothelin- and homocysteine, we also investigated whether there is a correlation between these parameters. obese and healthy children, participated in the study. when the obese group was compared to the healthy controls, the adma level of the obese group were significantly higher than those of the control group but there was no statistically significant difference in no, endothelin- and homocysteine. increased adma level might trigger the pathogenesis of atherosclerosis starting from the childhood years onward. that is why controlling obesity in this age group with diet and other treatment modalities will prevent the mortality and morbidities that will be seen in adult years. inh deficiency leads to the formation of bradykinin causing to dilation of blood vessels. furthermore, the study conducted by shagdarsuren, on the damage done by c -esterase, demonsrates that the complement system and triglyceride levels are affected. we investigated lipid oxidation and fetuin a levels in patients with c _ inh deficiency. materials and methods: people with c _ inh and people without any illnesses were taken into the study. fetuin a was studied using an el _ isa kit from raybio (usa). ferrous ion oxidation-xylenol orange test was used to find looh serum levels. sh (free thiol groups) test was studied with regards to ellmans method modified by hu. _ ibm spss . was used for statistical results. results: in assessments made between the healthy and the illness groups, there was significant differences in the levels of fetuin (p = . ), looh (p = . ) and sh (p = . ). when pearson correlation analysis was performed, we detected a significant positive correlation between fetuin a and looh levels (r: + . ) discussion and conclusion: in these patients, lipids is secreted from the adipose tissue. in response, anti-atherosclerotic fetuin a levels were risen. patients also possessed increased lipid peroxidation, this increase shows positive correlation with fetuin a levels. in conclusion, we identified that sh with antioxidant properties have increased levels. aim: high fructose corn syrups are found in soft drinks, juice beverages, breakfast cereals, most of the processed foods. it has been shown that high dose of fructose intake may lead to a reduction in the number of hepatocytes, deterioration of liver function, increasing reactive oxygen species and liver steatosis. the aim of this study was to explore whether caffeic acid phenethyl ester (cape) has any potential protective effect on high fructose diet-induced fatty liver model. materials and method: totally fifty rats were divided into five groups. control group, % fructose administered group, cape group, % fructose + cape administered group and ethanol group. after weeks, liver oxidant and antioxidant status, and blood tnf alpha, il- , and il- , tissue nfkb levels were quantified. protein levels were investigated against, nfkb and p-nfkb antibodies and normalized and analyzed against b-actin antibody by western blotting. results: serum tnf-alpha, il- , il- levels were found to be increased in fructose group compared with the control group (p < . ). in liver tissue of % fructose administered group, mda, protein carbonyls and no levels were higher than control group. however sod activity did not show any difference among the groups. in the fructose administered group, caspase showed liver apoptosis and was considered as positive. acquired data revealed that nfkb protein level was decreased in the presence of cape while increment in nfkb protein level was observed in the fructose administered group compared with control group. in case of pnfkb antibody, increment observed in fructose only and both cape and fructose administered groups, respectively. in cape only administered group, there was a decrement in the level of pnfkb protein. conclusion: depending on further analysis, experimental findings are expected to implicate the role of cape as a protective agent on high fructose diet-induced fatty liver model in relation of inos, nfkb and p-nfkb pathways. the investigating association of hepcidin levels with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction a. g. agg€ ul , n. uzun , e. c ß inar tanriverdi , h. € un agri ibrahim cecen university, agri, turkey, nenehatun maternity hospital, erzurum, turkey this study was designed to investigate hepcidin levels and their associations with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction (iugr). a total of pregnant women were included in this study. pregnant volunteers were divided into two groups ( healthy pregnant women and pregnant women with iugr). serum hepcidin, total free iron, ferritin, transferrin, transferrin receptor and interleukin- (il- ) levels were measured by elisa. also, hemoglobin (hb) and c-reactive protein (crp) levels were determined in serum samples from the healthy pregnant women and the pregnant women with iugr. there were significant differences in hepcidin, ferritin, transferrin receptor, crp and il- levels between healthy pregnant women and pregnant women with iugr. hepcidin, ferritin, crp and il- levels in pregnant women with iugr were significantly higher than healthy pregnant women (p). the mediators of systemic inflammation in lipopolysaccharide-induced neonatal sepsis rat model sepsis is an excessive inflammatory response that causes shock, multi-organ failure and high mortality. foreign bacterias and lipopolysaccharides lead to stimulation of endothelial cells to produce biologically active mediators such as proinflammatory cytokines and chemokines, cell adhesion molecules, and growth factors. then these mediators could be act on targets, which were involved in the initiation of systemic inflammation in neonatal sepsis. our aim was to indicate a protective role of thalidomide and etanercept, which have anti-tnf-a activity on systemic inflammatory response in lipopolysaccharide (lps)-induced neonatal sepsis rat samples. thirty -day-old wistar rats were randomly divided into five groups: a control group that received normal saline, a sepsis group that received lps, thalidomide, etanercept and both thalidomide and etanercept treatment group that were administered with therapeutic agents hrs after lps injection. the rats were sacrificed at hrs after lps or normal saline injection (n = ). hepatic tissue tnf-a, il- , icam- and pdgf levels were determined by enzyme-linked immuno sorbent assay (elisa) method in all groups. in sepsis group, tissue tnf-a, il- , icam- and pdgf levels were statistically significantly higher than in controls (p < . ). at same time, pretreatment with both thalidomide and etanercept were found statistically dramatically decreases the levels of tnf-a, il- , and pdgf when compared to sepsis group (p < . ). there were no significant differences in the icam- levels between the all treatment groups and the sepsis group. higher liver tissue tnf-a, il- , icam- and pdgf levels are associated with severe bacterial infection. these proinflammatory cytokines and angiogenic factors may be important in the endothelial dysregulation seen in sepsis. therapeutic agents used in the present study can be help to avoid devastating effects of neonatal sepsis. n-stearoylethanolamine (nse)is saturated minor compound of natural origin that represents the large family of signaling lipids n-acylethanolamines, which belong to endocannabinoid system. considering the crosstalk between obesity-induced inflammatory response and its key role in synaptic dysfunction and neurodegeneration, our current study aimed to investigate the biological effect of nse on brain tissue under high fat diet-induced insulin resistance. previously we found that nse administration to insulin resistant rats caused normalization of liver and pancreas lipid composition followed by the improvement of glucose tolerance and insulin sensitivity (decline in serum insulin level and homa-ir value). moreover, this effect of nse correlated with inhibition of nf-kb translocation into the nucleus of peritoneal macrophages and decreased pool of serum tnfa level in obesity-induced insulin resistant rats. further experiments showed that fat overload triggered significant reduction in the level of main phospholipids (phosphatidylethanolamine, phosphatidylcholine and sphingomyelin), while there were no changes in cholesterol content. nse at a dose of mg/kg during weeks of administration to insulin resistant rats showed a tendency to restore the phospholipid level that was accompanied by increased neural cell survival ( %) compared to rats without treatment ( %). neuroinflammation accompanied by intensive reactive oxygen species (ros) production impairs neurotransmission in a wide range of neurodegenerative pathologies. flow cytometry is used for quantitative analysis of global dna methylation, but fluorescence microscopy is mostly preferred to qualitatively reveal intranuclear localisation of dna methylation and its copattern with other markers. both methods use a similar immunostaining protocol. in this study, we aimed to compare these methods concerning the detection of the global amount of dna methylation. for this, mouse embryonic fibroblasts were cultured either with or without phenol red and then stained for dna methylation or positive controls (histone, betaactin, phosphoakt) by specific antibodies, or nonspecific control antibodies. some cells were incubated with trypan blue before or after the addition of antibodies. fluorescence intensities were measured by the green fluorescence channel ( / nm). autofluorescence spectrum of cells was analysed, and fluorescence channel used for dna methylation detection was changed to red ( nm lp). a poor discrimination between signal and noise was detected due to cellular autofluorescence interfering with specific detection of dna methylation by flow cytometry but not by microscopy. it was also the case for the other markers examined. conventional advances to reduce autofluorescence such using phenol red free culture media or trypan blue quenching were not effective, but using the red channel regarding autofluorescence spectra allows detecting specific staining of dna methylation by flow cytometry. but, green channel did work well for microscopy analysis. findings show that flow cytometry detection of dna methylation requires much attention to quench cellular autofluorescence compared to detection by fluorescence microscopy. one reason could be that flow cytometry detects all cellular content, but manual image-based analysis can exclude cytosolic components. these results suggest the usability of flow cytometry and microscopy as complimentary methods for dna methylation detection, but optimisation to reduce autofluorescence is crucial for flow cytometry. objectives: lung cancers are divided in two main groups as small cell lung cancer (sclc) and non-small cell lung cancer (nsclc) . docetaxel (dtx) and cisplatine are chemotherapeutic that has an anti-tumor activity against various solid tumors. the growing resistance against dtx and cisplatine (cis) still continues to be the biggest obstacle for the treatment success of nsclc patients. deguelin (deg.) is a natural plant derivative and has an encouraging activity against a lot of human cancers. the comparison of the treatment activity of the separate and combined usage of deg., which is a potential chemotherapeutic agent, and dtx, cis which are used in standard treatment, is aimed in this study. material-method: the ic doses of dtx, cis and deg. on the a and h nsclccell lines were determined via the cell vitality tests in our study. the active concentrations determined were applied to nsclc cell lines as deg., dtx, cis and their combinations. the impacts of the medicine are studied by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, colony formation, migration and angiogenesis analyzes on the treated cells and measuring the oxidative stress index (osi). statistical analyse program, rstudio (v. . . ) and the r-script language were used to examine the differences between the agents. the states in which the pvalue was lower than . were accepted as statistically meaningful. results: we found that deg. has pro-apoptotic, anti-migratory and cytotoxic potential on lung cancer cells. deg. amplified cis and dtx-related anti-cancer efficacy (increased apoptotic cell content and cytotoxicity, reduced migration). also, deguelin pretreatment sensitized the cells dtx-treatment (reduced ic values). these effects were remarkable in p -mutant cells. conclusion: deguelin, solely, has anti-cancer potential on nsclccells. both deguelin pre-treatment and combinantion with standart chemotherapeutics result in enhanced anticancer efficacy. the % of the lung cancers are non-small cell lung cancers (nsclc). despite docetaxel (dtx) and cisplatine (cddp) are agents used in the standard treatments of these patients and the recent improvements in the treatments, the response and remission rates observed on the patients are relatively nominal. selenium (se) is an essential diet component and is introduced to have a preventive impact on different levels of cancer. the aim of our study is to investigate the impacts of selenium addition on anticancer feature and tumor prevention before or/and during nsclc standard treatment. the ic doses of dtx, cddp and selenium on the a and h (p mutant) nsclc cell lines were determined via the cell vitality tests in our study. the active concentrations determined and the stipulated available concentrations were applied to cell lines as dtx, cddp, se combinations. the impacts were compared by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, western blot analyzes on the treated cells and measuring the oxidative stress index (osi) and thioredoksin reductase activity. selenium pre-treatments reduced dtx-related ic concentrations at lower doses in both nsclc cells. however, cddprelated ic concentrations reduced dose-dependent manner. selenium supplementation also altered cell-cycle charactheristics at several concentrations and combination regimens. the remarkably higher osi values were observed after dtx treatment and osi levels were found to be lower in selenium pre-treated nsclc cells. selenium sensitizes nsclc cells to dtx treatment at lower concentrations. however, this effect is obtained dose-dependent fashion for cddp regimen. breast cancer is the most common female malignancy worldwide. human epidermal growth factor receptor (her ) is overexpressed in % of breast cancers in association with aggressive phenotypes. the prognosis of metastatic breast cancer remains poor in spite of advances in therapy. as such, her has long been studied as a potential target for anticancer drugs. the modulation of intracellular signaling pathways leads to altered cell metabolism that triggers tumorigenesis and adapts cells to cancer cell metabolism. this characteristic hallmark of cancer metabolism is known as warburg effect meaning energy production via enhanced glycolysis. despite of several studies in breast cancer metabolism, little detail exists on the link between her overexpression and warburg effect. we have committed examining the nature of aerobic glycolysis in her overexpression. in breast cancer cell line mcf , her overexpression (mcf-her ) results in mitochondrial dysfunction with low mitochondrial membrane potential (Δᴪm) and ros accumulation. intracellular iron levels are also higher in mcf -her cells than vector control (mcf -vec). additionally, mcf -her cells show enhanced levels of atp and lactate in association with increase in glucose levels. we have found that complex i activity increases in mcf -her and decreases in knockdown of her in hcc cells that is her positive breast tumor cell line. based on these results, we conclude that there is a link between her overexpression and metabolic indicators of warburg effect. expression and methylation analysis revealed microrna genes deregulated by methylation and new potential target genes of mir- and mir- - p in breast cancer micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to assess the contribution of methylation to expression level alterations of mirna genes and to search for novel potential targets of these mirnas. to analyze alterations in expression we used qpcr technique with references (rnu , rnu ) and paired (tumor/normal) breast cancer (bc) samples. for methylation analysis a methylation specific pcr and the same set of bc samples were used. significant downregulation was shown for mir- b- p, - - p, - - p, - a- p, - b- p, - - p, - - p, and - - p (p ≤ . , fisher's exact test) in bc. we observed mirna genes to be hypermethylated and mir- hypomethylated. hypermethylation for of these mirna genes was shown for the first time: mir- , - , and - ( - % of bc cases). a significant correlation between methylation and expression alterations was revealed for mirnas with downregulation: mir- b- p, - - p, - - p, - a- p, and - b- p (spearman's correlation coefficient (rs) was in the range À . to À . , p ≤ . ), and for mirnas with both scene (down-and upregulation) as well: mir- a- p, - a, and - (rs = À . to À . , p ≤ . ). comparative analysis of the data on expression alterations of mirna genes and protein-coding genes, which were predicted as targets by mirwalk . , revealed the negative correlation between expression levels for some potential mirna-mrna interaction pairs. for example, for pairs mir- /rhoa, mir- /rassf (a), mir- - p/dapk (rs = À to À . , p ≤ . ). thus, both mirnas and methylation affect regulatory networks in bc. novel potential mirna-mrna interaction pairs could be useful in the development of bc therapy approach. this work was financially supported by grant - - from the russian science foundation. the authors thank the n.n. blokhin cancer research center for tissue samples. clear cell renal cell cancer (ccrcc) with metastases has pour prognosis: -year survival is about %. micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to find out mirnas which methylation contributed to ccrcc progression, including metastasis, and to reveal potential target genes of these mirnas. to analyze methylation status, we used a methylation specific pcr as a method and a representative set of paired (tumor/ normal) ccrcc samples. we also used post-mortal renal tissues from individuals without cancer history as additional control. for expression analysis we used qpcr method and paired ccrcc samples. we observed mirna genes (mir- a- /- /- , - - , - - , - b/c, - - , - a, - ) to be hypermethylated, (p ≤ . , fisher's exact test), mirna genes to be hypomethylated and mir- a with both scene (hyper-and hypomethylation was detected). methylation of mirna genes (mir- a- /- , - b/c, - - , - , - a, - a) correlated with advanced stage and/or tumor size and/or dedifferentiation. hypermethylation of mir- - , mir- a, and mir- significantly correlated with metastasis presence (p < . , fisher's exact test). besides, preliminary data revealed the positive correlation between hypermethylation of mir- - and up-regulation of p protein-coding genes: rarb( ), rhoa, nkiras , and chl , which were predicted as targets by mirwalk . (spearman's correlation coefficients (r s ) was in the range . - . , p ≤ . ). in conclusion, novel supposed interactions of mir- - with target genes could be useful as missing chains in signaling pathways. tests for hypermethylation of mir- - , mir- a, and mir- could be suggested as markers of metastasis and pour prognosis of ccrcc. because of difficulty in diagnosis and treatment hc is a clinical problem: early symptoms of hc are often non-specific and surgical resection is the only curative treatment for hc. it is well known that epigenetic alterations are linked to cancer development. the purpose of this study was to determine potential mechanisms of epigenetic regulation of genes related to energy metabolism in hc. we have performed bioinformatics analysis of the cancer genome atlas (tcga) project rna-seq data with crosshub software and found a number of genes involved in glycolysis and differentially expressed in cholangiocarcinoma. qpcr analysis revealed significantly decreased expression of pgm and eno genes in a majority of hc samples which were known as up-regulated in other human cancers according to the literature date. on the basis of tcga methylation dataset ( k illumina microarrays) we supposed that cpg methylation of pgm and eno promoters may play a role in their inactivation. using bisulfite sequencing study we identified several regions within the gene promoters (pgm :~ bp and bp upstream tss; eno :~ bp downstream tss) that are frequently methylated in hc samples (up to %, / ) with down-regulated pgm and eno expression. thus, we demonstrated frequent and significant pgm and eno down-regulation associated with hypermethylation of the specific regions within the gene promoters in hc. the pattern of pgm and eno gene promoter methylation suggests a possibility of ones to be used for the hc diagnosis and development new strategies for therapy. this work was financially supported by grant mК- . . from the president of the russian federation. the work was performed using the equipment of eimb ras 'genome' center. introduction: the development of stomach cancer is a multifactorial and complex process and includes multiple epigenetic, genetic alterations and dietary/non-dietary factors. iodine as an antioxidant may play a protective role against gastric cancer. the aim of this study was to investigate the changes in iodine level in rat with stomach cancer induced by n-methyl-n -nitro-n-nitrosoguanidine (mnng). materials and methods: a total of sprague dawley rats were randomly divided into six groups. rats were administered with mnng ( lg/ml) by oral gavage on days , and to initiate stomach cancer. during the experiment, rats died and those surviving were sacrificed in the rd, th, th, th and th months of the experimental period (group i, ii, iii, iv, v, respectively). the control group (group vi) contains rat which are given only food and water for months. the stomach tissue was examined histopathologically. and also, iodine levels in stomach tissue was determined using the foss method. results: a decrease in iodine level was determined in stomach cancer tissue of rats in group i-v compared with normal healthy stomach tissue in group vi. when the control (group vi) iodine level was taken as % baseline, the % iodine levels of all groups were determined as follows . , . , . , . and . for groups i-v, respectively. the pathological diagnosis of gastric cancer was adenocarcinoma. discussion and conclusion: the iodine levels of group i were higher than those of group ii (p < . ) and of groups iii, iv and v (p < . ) and also were lower than in the control group (p < . ). iodine deficiency as one of the risk factors of stomach cancer strongly supports the necessity for the application of effective iodine prophylaxis in the areas with iodine deficiency. iodine supplementation might be useful in stomach cancer therapy and therefore, further research is warranted. this study was supported by ataturk university (project number: / ). effect of water extract of turkish propolis on mitochondrial membrane potential in human laryngeal epidermoid carcinoma cell lines propolis is the generic name for the resinous substance collected by honeybees from the buds of various plant sources and it is used by bees to seal holes in their honeycombs, smooth out the internal walls, and protect the entrance of bee hive against intruders. the aim of this study is to investigate what kind of changes the turkish propolis cause on mitochondrial membrane potential (mmp) of human laryngeal epidermoid cell lines (hep- ), by considering its anticancer features. water extract of turkish propolis (wep, - mg/ml) and ethanolic extract of turkish propolis (eep, . - mg/ml) were prepared and incubated with hep- cell lines ( , , and h). mmp was investigated with a flourometric method by using dioc ( , -dihexyloxacarbocyanine iodide). the most significant mmp decrease was seen on rd hour. both wep and eep extracts at all concentrations decrease mmp according to that of control. the recent studies have shown that propolis extracts have induced apoptotic cell death by decreasing mitochondrial membrane potential in various cancer cells. it was concluded that both wep and eep decreased mitochondrial membrane potentials on hep- cell series according to control ( concentration) depending concentration and time. there are numerous transcription factors involved in the regulation of the inducible gene expression. thus, transcription of proinflammatory genes, steroid hormone receptors, etc. is controlled by the group of factors triggering gene expression which includes nf-kb. another group of factors is involved in the formation of the structure of the chromatin of the inducible genes regulatory regions, providing competence for gene expression. it is expected that this group of factors includes the proteins of nf (nuclear factor ) family. there are few data suggesting that the nf factors maintain potentially active state of the chromatin of the hormone-dependent gene promoter regions. these findings initiated studies of the correlation between presence of the nf transcription factors on the chromatin of a gene regulatory region and the functional state of the gene in vivo. as a model we chose the rat tryptophan dioxygenase (tdo) gene which is expressed tissue-specifically in the liver under control of glucocorticoid hormones. three constitutive dnase i-hypersensitive regions are identified in the regulatory region of this gene. to conduct the study we used rat liver and kidney. the basic methods were electrophoretic mobility shift assay (emsa), immunoblotting assay and chromatin immunoprecipitation combined with real-time pcr (chip-qpcr). using emsa we found that the proteins of nf family interact with the constitutive dnase i-hypersensitive regions in vitro. immunoblotting assay of the protein fraction from rat liver used in emsa experiments showed the presence of the nf -b isoform. chip-qpcr revealed statistically significant differences in the level of the factor nf enrichment of the tdo gene regulatory region between the rat liver and kidneys at p < . . these data suggest the involvement of the nf proteins in the formation of the chromatin structure of the rat tdo gene promoter region. reciprocal ( ; ) translocation and bcr-abl fusion protein that is responsible for developing leukemia are observed in more than % of chronic myeloid leukemia (cml) cases. epigallocathecin- -gallate (egcg) is a green-tea flavonoid and egcg is proposed as a natural anti-cancer agent. histone modifications which contain histone deacetylases (hdac) and histone acetyltransferases (hat) are parts of epigenetic regulations. hdacs play important roles in different human malignancies including leukemia via activation of abnormal signaling pathways. hdac inhibitors have become remarkable therapeutic molecules for malignancies. the aim of this study is to determine the expression changes of leukemia-related hdacs with the treatment of egcg in k- cells. the cytotoxic effect of egcg on k- cells was determined in time and dose dependent manner by wst- analysis. total rna was isolated from k- cells. reverse transcription procedure was performed for cdna synthesis and gene expressions were detected by rt-qpcr. the expression level of hdac , hdac , hdac gene that supports cell proliferation was down-regulated . , . , . folds in k- cells treated with ic dose of egcg, according to control, respectively. our current findings suggest that is a polyphenol egcg may be a hopeful agent in treatment of cml by hdac inhibitory effect. chronic lymphocytic leukemia (cll) is a disorder of morphologically mature but immunologically less mature lymphocytes and is manifested by progressive accumulation of these cells in the blood, bone marrow, and lymphatic tissues. carbonic anhydrase (ca) is a metalloenzyme which is widely distributed in the living world, and it is essential for the regulation of acid-base balance. anti-ca antibodies have been reported in many disorders, such as systemic lupus erythematosus, sj€ ogren's syndrome, rheumatoid arthritis, endometriosis, idiopathic chronic pancreatitis, type diabetes and graves' disease. the goal of this study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in cll. patients with cll and healthy controls were included in the study and ca i and ii autoantibody levels were investigated by elisa. the ca i autoantibody levels of cll group were significantly higher than the healthy group (p = . ) while there was no statistical difference between serum ca ii autoantibody levels of the groups (p = . ). we found a significant positive correlation between hemoglobin and hemotocrit levels in patients with cll (r = . , p = . ). cut-off value of . absu for anti-ca i was associated with % sensitivity and % specificity and a cut-off value of . absu for anti-ca ii was associated with % sensitivity and % specificity for predicting cll. the ca i autoantibody levels in patients with cll were found higher compared to control group and the results suggest that ca i autoantibody may be involved in the pathogenesis of cll. genetic and epigenetic aberrations can lead to the activation of oncogenes and inactivation of tumor-suppressor genes (tsgs) followed by the development of malignant tumors. in the present work we evaluated the frequency of alterations of cpg island methylation and dna copy number in paired (tumor/normal) breast cancer (bc) samples using comparative dna hybridization on noti-microarrays and original niman software. the microarrays contained noti-clones associated with chromosome genes. expression alterations were assessed with the use of qpcr technique, ddct method and original atg software. in total, noti-sites with high ( - % of cases) hypermethylation/deletion (hm/d) frequency were revealed in bc. among genes associated with these sites, there are both known tsgs and tsg-candidates (aldh l , vhl, ctdspl, etc.) as well as genes, which involvement in breast oncogenesis was shown for the first time (lrrn , foxp , prickle , etc.). noti-microarray data were verified selectively using bisulfite sequencing for vhl, nkiras , itga , lrrc b, and ctdspl genes. several genes with high hm/d frequency (aldh l , ephb , itga , and ropn ) were tested for expression alterations using qpcr. frequent ( - % of cases) and significant (> -fold) down-regulation was shown for all of them in bc. the most significant expression loss was observed for aldh l geneon the average -fold mrna level decrease in % of samples. the involvement of the majority of genes with high hm/d frequency in breast oncogenesis was shown for the first time. these genes are novel tsg-candidates in bc. functional hypermethylation associated with expression loss was shown for aldh l , ephb , itga , and ropn genes thereby strengthening the speculation on tumor suppressor abilities of these genes. methylation and expression analyses of genes, that were revealed by noti-microarrays, were financially supported by grant - - from the russian science foundation. functional hypermethylation of a number of chromosome genes was revealed in colon cancer using noti-microarrays cancer is a disease of genome caused by genetic and epigenetic aberrations. noti-microarrays, that were developed by prof. e.r. zabarovsky, is a unique tool that allows us to simultaneously detect hypermethylation of cpg islands and dna deletionstwo major reasons of inactivation of tumor suppressor genes (tsgs). in the present work, the frequency of chromosome genetic and epigenetic alterations in colon cancer (cc) was evaluated. noti-microarrays, that contained noti-clones associated with chromosome genes, were used for comparative (tumor/normal) hybridization of dna from paired cc samples. data analysis was performed using original niman software. expression alterations were evaluated using qpcr technique and original atg software. in total, noti-sites with % and above hypermethylation/ deletion (hm/d) frequency were revealed in cc. among genes associated with these sites, there are several known tsgs and tsg-candidates (for example, vhl, ctdspl, and itga ), but for the majority of genes, involvement in colon oncogenesis was shown for the first time (for example, lrrn , nbeal , and ube e ). the highest hm/d frequency was observed for ankrd , nkiras /rpl , itga , cmtm , and gor-asp /ttc a genes - - %. expression alterations were evaluated for genes with high hm/d frequency (plcl , prickle , and ppp r a) and significant mrna level decrease (> -fold) associated with hypermethylation was shown for all of them in the majority of samples. a number of novel potential tsg-candidates was revealed in cc. functional hypermethylation associated with expression decrease was shown for plcl , prickle , and ppp r a genes thereby enhancing the suggestion on tumor suppressor function of these genes. this work was financially supported by in many countries, radon is the second leading cause of lung cancer, which accounts from % to % of cases. it is obvious that the population of all the developed and industrial countries in the world spend most f their time, almost %. therefore it is necessary to explore the obtained radiation dose, because of the presence of radon in a room due to the radon emanation from the soil and exhalation from a variety of building materials. the developed countries solve this problem of radon pollution as well as create a special monitoring services. the paper presents some data of genes molecular-genetic analysis from patients with lung cancer who live in almaty located in a foothill area of tectonic faults. the object of research were blood samples obtained from patients diagnosed with lung cancer who are receiving a treatment at the almaty oncology center and living in the city of almaty, where the level of radon activity exceeds the norm approved by the international commission on radiation safety. as a control group relatively people living in the plains, characterized by a lower radon emanation have been considered. to determine mutations in the genes polymerase chain reaction with a subsequent analysis of restriction fragment length polymorphism has been conducted. the pcr products were subjected to hydrolysis by bstni restriction endonucleases haeiii, ras i. disturbances in the genes under consideration to variour types of cancer development. the analysis showed that examinees do not have mutations in the kras gene codons - , which corresponds to a control group consisting of people living in the city of balkhash. on the whole, molecular genetic studies have shown that examined patients do not have mutations in the kras gene. one mutation was been found in the egfr gene. aim: polyps are abnormal growths of tissue that can be found in gastro intestinal system. they are most often found in the colon and rectum. most polyps are noncancerous (benign) however, because of abnormal cell growth, they can eventually become cancerous. the aim of this study is to determine the concentrations of trace element contents in colon and rectum polyp tissues and whether there is any relationship between polyp tissue element levels and the disease. material and method: the present study was conducted on total of individuals including patients and healthy subjects. while receiving normal intestinal tissue from healthy control group; from the patient group both normal tissue and polyp samples were taken during colonoscopy procedure. the concentrations of the elements (al, cr, mn, fe, co, ni, cu, zn, as, se, ag, cd, hg and pb) were determined with induced coupled plasma-mass spectrometer. results: the mean concentrations of cr, mn, ni, se and ag in colorectal polyp tissues of patients were significantly higher than in colorectal tissues of control subjects (p is less than . ). on the other hand the mean concentration of cd and pb in colorectal polyp tissues of patients were significantly lower than in control colorectal tissues of control subjects (p is less than . ). there was no any significant difference between the groups in terms of concentrations of al, fe, co, zn, as and hg (p is more than . ). conclusion: the differences found in some elements between polyps and a control tissues may provide an indication about the role of trace elements in the early stage (polyps) in the colon carcinogenic process and encourages further studies to confirm the involvement of such elements in neoplastic processes. the use of herbal medicines is steadily growing, with approximately % of the population use herbs to treat various illnesses in the western world. vitex agnus-castus has been used since ancient times as a remedy. the aim of this study was to investigate the in vitro anticancer activities of vitexagnus-castus oil. for this purpose, the cytotoxicity of vitexagnus-castus oil in sh-sy y cells was investigated by crystal violet staining. ec was found to be . %(w/w) vitexagnus-castus oil for this cell line. this dose was applied to the cell for h, and the cells were harvested for further studies. vitex agnus-castus oil treatment increased bax and p mrna levels. on the other hand, bcl- , bcl l , erk- , jnk, caspase and mrna expression levels were reduced significantly withvitexagnus-castus oil treatment while p and pten remained unchanged. these results indicate that another effector caspase such as caspase or may be involved apoptosis process which remains to be elucidated. moreover, mapk pathways, p and erk, may be involved in vitexagnus-castus oil induced apoptosis in sh-sy y cells. these initial observations suggest that this agent might not be useful in treating cancers. further detailed studies should be carried out to elucidate the exact mechanism of vitexagnus-castus oil in neuroblastoma cell lines. melanoma is a skin cancer with a melanocyte origin that can occur in any part of the body that contain melanocytes. while melanoma is less common than other skin cancers, it causes the majority of deaths related to skin cancer. several gene expression databases have shown that interferon regulatory factor (irf ) is upregulated in melanomas, and genome wide association studies linked variation at irf locus with skin cancers. irf was first identified to have roles in lymphocyte development and function. studies have identified a 'non-oncogene addiction' of malignant cells to irf in various hematopoietic cancer types. the aim of this study is to investigate the role of irf in melanoma cell lines. lentiviral vectors were used to reduce irf levels in melanoma cell lines. a gfp competition assay was performed to study the competitive fitness of melanoma cells with irf knockdown (gfp positive cells) over melanoma cells with normal irf levels (gfp negative cells). cell cycle profiles were investigated in melanoma cells with irf knockdown by propidium iodide staining. migration potential was assessed as well by wound healing assay. our preliminary data showed a decreased competitive fitness for cells with decreased irf levels. cell cycle profiling showed increased g /g and decreased g /m levels in irf knockdown cells compared to controls. wound healing assay results showed no difference between controls for cells with reduced irf levels. taken together, these results indicate that irf knockdown affects the melanoma cell lines' survival and cell cycle profile, suggesting a non-oncogene addiction of melanomas to irf . these observations are largely similar to previous observations in hematopoietic cancers. unravelling the role of irf in melanoma will increase our knowledge about melanoma development and progression and thereby may lead to targeted therapy in melanoma treatment. humans are exposed to various chemicals having beneficial or toxic effects at a time in their daily lives. , -dimethylbenz[a] anthracene (dmba) is a carcinogenic compound produced during the incomplete combustion of carbon-containing compounds. endosulfan is an organochlorine pesticide used against insects on food. morin is an antioxidant, antiinflammatory and chemoprotective flavonoid. this study is aimed to determine the effect of morin in the presence of dmba and endosulfan. for this purpose, adult wistar male rats weighing - g were randomly selected and divided into eight groups. mg/kg body weight (b.wt.) morin and . mg/kg b.wt. endosulfan were given to morin and endosulfan treated groups three times in a week. the rats in dmba treated groups were gavaged with . mg/kg b.wt. dmba three times during the administration period ( days). cytochrome p a (cyp a) associated -ethoxyresorufin o-deethylase (erod) and glutathione s-transferase (gst) activities were measured in rat liver cytosols and microsomes. in addition, liver tissues were evaluated by histopathological analysis. erod activities of control, morin, endosulfan, dmba, morin+endosulfan, morin+dmba, dmba+endosulfan and morin+dmba+endosulfan groups were ae , ae , ae , ae , ae , ae , ae and ae pmol/min/mg protein, respectively. all treatments increased erod activities. gst activities of these groups were ae , ae , ae , ae , ae , ae , ae and ae nmol/min/mg protein, respectively. histopathological studies showed that endosulfan and dmba induced inflammation in the liver tissues and morin reduced their effects. in conclusion, morin treatment increased the metabolism of dmba and endosulfan by inducing cyp a activity. gst activities of morin+dmba+endosulfan group were not significantly different from those of dmba group. histopathological studies indicated that morin administration reduced the toxic effect of endosulfan and dmba in the liver cells. hepatocellular carcinoma (hcc) is the sixth most common cancer and third most frequent cause of cancer-related death worldwide. molecular mechanisms of hepatocarcinogenesis is still unclear. the impairment of epigenetic mechanisms is implicated in the development of multiple cancers, including hcc. transforming growth factor-beta has been shown to play both tumorsuppressive and tumor promoting roles. transforming growth factor-beta signaling pathway involves activation of smad and smad by the type i receptor and formation of smad / / heteromeric complexes that enter the nucleus to regulate transcription. -deazaneplanocin a is an inhibitor of the histone methyltransferase ezh . we aimed to reveal the effect of -deazaneplanocin a on transforming growth factor-beta /smad pathway in hepg cell line. hepg , a human liver cancer cell line cultured in dulbecco's minimal essential medium supplemented with % fbs. the cells were seeded the day before -deazaneplanocin a administration and then the cells were treated with lm -deazaneplanocin a for days. expression levels of genes were analyzed by roche lightcyclerÒ . gapdh was used as housekeeping gene. apoptosis assay was performed by the muse annexin v and dead cell assay kit. the unpaired t-test was used to compare variables and p < . was accepted as statistically significant. -deazaneplanocin treatment was significantly reduced transforming growth factor-beta, smads - in hepg cells (p < . ). we also found that -deazaneplanocin induces apoptosis in treated cell line (p < . ). as a result, -deazaneplanocin a may take place in treatment of hepatocellular cancer by its inhibitory effect on transforming growth factor-beta /smad pathway and inducing apoptosis in liver cancer cells. brefeldin a (bfa) is a lactone antibiotic first isolated from the fungus eupenicillium brefeldianium. bfa inhibits the transport of secreted proteins from endoplasmic reticulum (er) to golgi apparatus, leading to disruption of golgi function, accumulation of unfolded and not fully incompletely processed proteins in er. bfa also inhibits cell proliferation, phosphorylation and migration of cancer cells. therefore in this study, we investigated the effects of bfa on breast cancer cell proliferation of various phenotypes. in we observed that bfa inhibited the proliferation of all three phenotypes of breast cancer cells, but the effects of bfa were seen at different times and doses. according to time and dose, bfa was observed more effective to mcf- compared to other cell lines. physiological, pathological and physical factors. moreover, nlr may represent the two opposing inflammatory and immune pathways that exist together in cancer patients. we aimed to investigate nlr in breast cancer in our population. methods: using data retrieved from the medical records, women diagnosed primary breast cancer met our study inclusion criteria as they had a complete blood count with leukocyte differential performed before any anti-cancer therapy. and women with benign mammary neoplasm/disease, followed up in the outpatient clinics of mammary disease and confirmed with sonographical/histopathological examination, made up our controls. exclusion criteria included laboratory evidence of white blood cells count (wbc) > . /l. differential leukocyte counts were obtained by bc (mindray medical international ltd., china), we examined wbc, neutrophil, lymphocyte, platelet counts, and hematocrite, nlr, mean platelet volume values. results: although there is lack of evaluation of tumor-associated neutrophils and lymphocytes, higher nlr median values and lower lymphocyte mean counts (lymphopenia) were shown in women with breast cancer (p < . ). there was a weak negative correlation in breast cancer between nlr values and platelet counts (r s = À . ; p = . ). holmboe] is distributed throughout southern mediterranean europe from spain to the eastern mediterranean on anatolian peninsula of turkey. present study was designed to investigate the in vitro anti-cancer activities of turkish black pine essential oil. the essential oil was extracted by steam-hydrodistillation and its chemical composition analyzed by gc-ms. the major components of the essential oil were a-pinene, b-pinene and trans-b-caryophyllene, respectively. the crystal violet staining method was used to investigate the cytotoxicity of essential oil in sh-sy y cells. ec was found to be . % (w/w) essential oil for sh-sy y cells. neuroblastoma cells were incubated at °c for h. after h, cells were harvested for further studies. bax and p mrna levels were significantly elevated in essential oiltreated cells. on the other hand, bcl- , bcl l , casp- , casp- , erk- and jnk expression were significantly downregulated. unlike these proteins, p and pten mrnas were not changed. in this study, apoptosis was enhanced by turkish black pine essential oil treatment which was activated by the involvement of another effector caspase subfamily, like casp- and casp- . additionally, erk and p mapks may be associated with upregulation of the level of bax. based on these results, we suggest that p. nigra subsp. pallasiana essential oil might not be well-suited in cancer treatment. however, further detailed research is necessary to establish the exact role of p. nigra subsp. pallasiana essential oil in sh-sy y cells. p- . . - the protective effect of newly derivatized compound naringenin-oxime and relative to naringenin against cisplatin-induced nephrotoxicity and genotoxicity in rat background: the aim of this study was to evaluate the possible protective effect potentials of newly derivatized compound naringenin-oxime (ng-ox) relative to efficacy of free naringenin (ng) on cisplatin (cis) induced nephrotoxicity and genotoxiticity in rat. methods: totally, fifty six male wistar albino rats were equally divided into eight groups as follows: control; cis treatment ( mg/kg b.w., i.p.), ng and ng-ox ( mg/kg b.w., i.p daily for days) alone treatment; cis + ng ( or mg/kg b.w., i.p daily for days) and cis+ng-ox ( or mg/kg b.w., i.p daily for days) combination treatment. at the end of the study total antioxidant capacity (tac) levels, total oxidant status (tos), lipid peroxidation (lpo), total thiol, catalase (cat) were studied in homogenate kidney. peripheral lymphocyte cell dna damage was investigated with comet assay results: the results suggest that cis induces oxidative stress resulting in increased tos and lpo reduction thiol, tac and cat in kidney and increased peripheral lymphocyte cell dna damage. the treatment with naringenin and naringenin oxime alone or with cis treatment showed a protective effect against the toxic influence of cp on peroxidation of the membrane lipids and an altering of the total thiol status in the kidney of rats. from our results we conclude that naringenin and naringenin oxime functions as a potent antioxidant and suggest that it can control cp-induced nephrotoxicity and genototoxicity and ng-ox was found more protective than that of ng on cisplatin induced toxicity in rats. keywords: naringenin, naringenin-oxime, antinephrotoxic, antigenotoxic, comet assay. introduction: oxidative damage is considered to play a pivotal role in ageing, several degenerative diseases, and carcinogenesis. lung cancer is the most common type of cancer, resulting in over . million deaths each year worldwide. accurate and reliable determination of superoxide radicals has been widely investigated using spectrophotometric, electrochemical, amperometric, polarimetric, piezoelectric technologies. among these methods, electrochemical detection is a most promising approach to achieve accurate, separate and rapid superoxide radicals monitoring with using biosensor system. materials and methods: we used a new technic for detecting superoxide radicals in samples. superoxide dismutase (sod) enzyme immobilized on the surface of gold electrode with the help of gelatin, bovin serum albumin (bsa) and glutaraldehyde (ga) crosslinker. for the biosensor preparing benzoquinone selected as a mediator in working buffer and measurements were carried out at À . v. result: for the optimization studies, effect of the bsa, gelatin, glutaraldehyde, ph, buffer concentration on biosensor response. characterization of the biosensor commitment to the work process and answer reproducibility were evaluated. the analytical characteristic of the biosensor were evaluated by measuring the steady state current response to superoxide radical concentrations. the electrochemical response of the enzyme electrode was linearity gradually leveled of at higher concentration. we found that crosslinking of the sod (e.c. . . . ) with glutaraldehyde could be achieved over a wide range of relative mole ratios in mm phosphate buffer at ph . , glutaraldehyde concentration of % . . discuss and conclusion: in this study, a new technique for developed sod biosensors has been developed, which features effective combination of sod/gelatin/bsa/ga modified electrode, trapping of sod and glutaraldehyde cross-linking. this technique is reliable and cost effective. the effect of astaxanthin on apoptosis and cell arrest in u brain cancer cell line f. s€ og€ utl€ u, b. € ozmen yelken, c ß . kayabasi, a. asik, s. gonca, r. gasimli, s. yilmaz s€ usl€ uer, c ß . biray avci, c. g€ und€ uz department of medical biology, izmir, turkey a brain tumor is a collection, or mass, of abnormal cells in your brain. brain tumors can be cancerous (malignant) or non-cancerous (benign). the brain is one of the least accessible organ that active pharmacological compounds cannot be delivered. the two physiological barriers control and block the entry and exit of endogenous, exogenous compounds. one of these is the bloodbrain barrier and the other is the blood-cerebrospinal fluid barrier. this structures maintain protection of the brain. when there is a cancer case, it can lead to problem. astaxanthin with potent antioxidant properties can cross blood-brain barrier. in our study, we aimed to evaluate the effects of astaxanthin on apoptosis, cell cycle and also migration in brain cancer cell line. in present study, xcelligence real-time cell analyzer was used so as to determine cytotoxic effect of astaxanthin in u cell line. changes of apoptosis and cell cycle in u cell line exposured to ic dose of astaxanthin ( . nm- lm) are detected with annexin v-egfp apoptosis detection kit and cycle test plus dna reagent kit with facs, respectively. the result of apoptosis and cell cycle test was analysed in flow cytometry. the group to which active substance was not treated was used as controlled. the wound healing assay performed in order to measure migration ability of u cell line to which astaxanthin was treated or not. ic dose of astaxanthin was calculated as . lm at h by xcelligence rtca sp based on time and dose. astaxanthin decreased the migration ability at rate of % in u cells treated by ic dose of astaxanthin. astaxanthin had no apoptotic effect on viability in u cell line and astaxanthin caused an increase of g /m phase arrest ( . fold) and s phase arrest ( . fold). astaxanthin has cytotoxic effects in brain cancer. it determined that astaxantin decreases cell cycle potential at g /m even a little. the effect of anticancer of astaxanthin should be researched further. interferon regulatory factor (irf ) is a critical transcription factor in development and survival of different cell types including immune cells and melanocytes. furthermore, it has been demonstrated that irf expression levels are elevated in several lymphoid cancers, and irf is one of the key transcription factors for the survival of these cancers. several genome-wide association studies identified irf -linked genetic variants to increased melanoma incidence. in addition results from our lab and elsewhere have shown high levels of irf expression in melanoma cell lines. furthermore our preliminary results suggest melanoma cells are sensitive to irf expression levels. however, there are no published studies about irf target genes in melanoma cells. in this study, we are investigating the genome-wide target genes of irf in melanoma cell lines via high-throughput sequencing of immunoprecipitated chromatin (chip-seq). we have identified possible irf binding regions in loci with known key roles in development of melanocytes from neural-crest cells. one such key factor is mitf, which is the master regulator in melanocyte development and also plays critical roles in melanoma. integrating chip-seq and rna-seq data suggests irf as a transcriptional regulator of genes related to progression of melanoma. objectives: aim of this study was to evaluate prognostic importance of selected laboratory parameters (c-reactive protein (crp), gama glutamiltransferaz, ferritin (fer), potassium, chloride, calcium, phosphorus, magnesium, total protein, aspartat aminotransferaz, alanin aminotransferaz (alt), ifn-c, il- , tnf-a) in non-small cell lung cancer (nsclc). material and methods: patients with nsclc who were treated with chemoradiotherapy (crt) prospectively evaluated. all patients were newly diagnosed tumour. heparinized blood samples were taken from the patients before and after the completion of crt. fer analyzed by chemiluminescence method on beckman coulter dxi ; ifn-c, il- , tnf-a were analyzed with elisa kits (boster biological technology) and other biochemical parameters analyzed on abbott architect c . post-crt and pre-crt levels compared with survival. results: the lr cox regression analysis revealed that pre-crt ferritin was significantly associated with survival of patients with nsclc (hazard ratio (hr) = . , p = . , %ci; . - . ). it was also demonstrated by lr cox regression analysis, high levels of pre-crt crp was associated with worse outcome of patients (hr = . , %ci; . - . , p = . ). after crt, mean alt level was determined as . . there was survival difference in nsclc patients with high post-crt alt (hr = . , %ci; . - . , p = . ). conclusions: there exists a clinically relevant relationship between pre-crt fer concentration and the prognosis of survival in patients with nsclc. elevated fer is the result of inflammation rather than body iron overload. ferritin showed negative correlation with survival so it could be a useful biomarker to indicate bad prognosis of the patients with nsclc. additionally, crp which is easy to detect and feasible for the use in the routine clinical practice should be considered in the prognosis of nsclc patients. keywords: ferritin, nonsmall cell lung cancer, survival, c-reactive protein. epigenetic therapy tries to reverse the aberrations followed to the disruption of the balance of the epigenetic signaling ways through the use of natural and synthetic compounds, active on specific targets, such as dna methyltransferases (dnmts). we previously synthesized some benzoxazole and benzamide derivatives which might have anticancer activities on account of their heterocyclic structure. our studies showed that not only these compounds caused selective cytotoxicity towards cancer cells (hela) with little or no toxicity on normal cells (l ) but also were not genotoxic. in this study, we aimed to test whether these compounds changed global demethylation profile of normal and cancer cells. we used methylation specific comet assay (msc assay) to determine global methylation levels of cells. cells were treated with the tested compounds at ic concentrations for h. slides were prepared as did in alkaline comet assay, then they were incubated with methylation specific restriction enzymes (mspi, hpaii) before electrophoresis. differences in global methylation levels between nontreated control cells and cells treated with compounds were compared by using tail moment data. -aza-c, a demethylating agent, was used as reference drug. msc assay results revealed that none of the tested compounds caused hypermethylation on both cell lines. however, global methylation levels decreased statistically (p < . ) through both cells treated with c- and c- . only c- decreased methylation level on l but not on hela. consequently, c- and c- caused demethylation on hela cells similarly with -aza-c at low concentrations. for the reason that dna methylation is regulated mainly dnmt enzymes in the cell, c- and c- might cause global demethylation in the cell by inhibiting dnmt activity. further studies will be done to support this prediction. overall, macrophages and some subtypes of lymphoid cells are found in tumour stroma. these cells secrete a variety of growth factors, proinflammatory cytokines and chemokines, esp. tnf-a, il- b and il- , causing the formation of inflammatory microenvironment around tumour cells. tnf-a and il- b signaling increases activity of nf-kb pathway. at the same time, il- , triggers jak-stat signaling pathway, which effector is stat . nf-kb and stat activity facilitates hyperexpression of mir-nas mir- , mir- and mir- as well as down-regulates expression of mirnas mir- / , mir- and let- . this investigation aims to identify in what way these shifts in mirnaome can lead to epigenome reorganization supporting the cell transformation. mirna targets within gene transcripts were predicted in silico using targetscan software. transcripts of hdac / / / and sirt / genes encoding histone deacetylases carry targets for at least one of up-regulated mirnas mir- , mir- or mir- . also, these mirnas can silence ezh , mll, mll , nsd , setd / / , smyd , suv h genes encoding histone methyltransferases. mirna mir- suppresses gene encoding de novo dna methyltransferase dnmt b. at the same time, down-regulation of mirna mir- / can allow hyperexpression of gene encoding acetyltransferase elp . these shifts impair dna and histone methylation, cause the increase of overall level of chromatin acetylation and expression and, therefore, create epigenetic background for reactivation of silent transposons, oncogenes as well as other genes important for cell transformation. immune system can paradoxically facilitate the tumour growth instead of healing. cancer-related inflammation leads to the mir-naome and epigenome shifts contributing to the tumour promotion and progression. lysine acetylation is one of the key mechanisms to regulate chromatin structure and transcriptional activation. acetyl-lysine modifications are recognized by bromodomains, which are small interaction modules found on diverse proteins including histones. among these acetyl-lysine reader proteins is the family of the bet (bromodomain and extra-terminal) proteins which contain tandem bromodomains (bd and bd ). the recent discovery of potent and specific inhibitors for the bet family proteins has stimulated intensive research activity in diverse therapeutic areas, especially in oncology, where bet proteins regulate the expression of key oncogenes and anti-apoptootic proteins. several bet inhibitors are currently in clinical trials and reported to exhibit promising clinical activities. however, pleiotropic nature of bet proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. here, we report the recent progress in the development of bet inhibitors in korea research institute of chemical technology (krict). we have identified the bet inhibitors with a novel scaffold different from the previously reported diazepine and azepine scaffolds and specific for first bromodomains (bd s). a medicinal chemistry effort is currently made to optimize the pharmacokinetic properties of these lead compounds for further drug development. the experimental data from the biochemical and cell-based assays for these bd -selective bet inhibitors will be presented. family of small c-terminal domain serine phosphatases (scp), which includes ctdspl, ctdsp , and ctdsp , plays a regulatory role in a number of vital processes. in particular, it is shown that ctdspl is capable to activate the retinoblastoma protein (rb) which is well-known tumor suppressor and one of the key cell cycle regulators. although the question on whether ctdsp and ctdsp dephosphorylate rb is open, high similarity of sequences and three-dimensional structures of phosphatases may indicate the similar function of these enzymes. in the current study expression of scp genes was evaluated by quantitative pcr in non-small cell lung cancer (nsclc) samples. using original crosshub software, that combines an analysis of high-throughput sequencing data of the cancer genome atlas project (tcga) and databases of mrna-mirna interactions (targetscan, mirtarbase, etc), the involvement of mir- - - microrna cluster in co-regulation of ctdspl/ / genes in nsclc was predicted. the significant ( -fold on the average) and simultaneous decrease of mrna levels of ctdspl/ / genes was revealed in the majority of nsclc samples ( %, / ). such unidirectional expression change and strong positive correlation between phosphatase expression levels (r s = . - . , p ≤ . ) allowed us to suggest a common mechanism of their inactivation. we evaluated the expression of predicted co-regulators of scp gene expression, mir- - - family, in examined nsclc samples. as a result, the simultaneously increased levels of all three mir-nas in most nsclc samples ( %, / ) and negative correlation with phosphatase gene expression was shown. the results suggest the ability of investigated phosphatases to exhibit tumor-suppressive activity and the involvement of mir- - - micrornas in the regulation of rb protein activity via inactivation of ctdspl/ / in nsclc. cancer is one of the leading causes of death in all around the world. cancer is defined as a disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. tumor markers are substances that are produced either directly by the tumor or as an effect of the tumor on healthy tissue. tumor markers can be used for screening, determining prognosis and monitoring effectiveness of therapy and disease recurrence. the aim of this study is to investigate the frequency of tumor markers orders and the appropriateness of these requests. laboratory information systems data for were reviewed. for , a total of patients and tumor marker requests were included. carbohydrate antigen - , cancer antigen , cancer antigen - , prostate specific antigen, alphafetoprotein and carcinoembryonic antigen were measured by chemiluminescence method. according to the data from the year of , both positive tumor markertest resultsratio and the positive patient ratio were %. in the patients group with increased marker levels, % of the patients had no history of cancer. in the patients group with tumor marker levels in referenceranges, % patients with diagnosed cancer history in remission. the ratios of positive tumor markers were % forca - , % for ca , . % for psa, %for ca - , . %for afp, and . % for cea. in conclusion; unnecessary test requests increase laboratory work load and health expenses. laboratory and clinical staff collaboration is crucial to increase the appropriate use of tumor markers. dna methylation is an epigenetic modification that is involved in both normal biological and disease states. hypermethylation of promoter regions of tumor suppressor genes have a role in tumor development. therefore, the measurement of promoter methylation of genes can be used for diagnosis and prognosis purposes of cancer. to detect dna methylation alterations in a sample (biopsy, blood, saliva, etc.), sensitive detection systems and optimization of the methods are needed. as a part of a collaboration project between national metrology institute of korea (kriss) and national metrology institute of turkiye (tubitak ume). dna methylation status of apc and gstp genes were studied. dna methylation measurements were performed using stepone real-time pcr system and results were analyzed using hrm (high resolution melting) software. the parameters effecting the quantification of dna methylation were found as primers, annealing temperature, pcr cycle number, fluorescence dye and the commercial dna methylation standards used for quantification of dna methylation. since, the accurate measurement of dna methylation is very critical in early diagnosis of cancer and choosing the right therapy, optimization of the method is required. cancer is a disease that includes heterogenic and complex molecular changes. anti-carcinogenic effects of resveratrol, a natural polyphenol, have been proved in a variety of cancer cells. considering the effects of resveratrol, the influence of the signal transduction pathways in the presence or absence of p of colon cancer cells is gaining importance. our aim was to investigate the effects of resveratrol in the presence or absence of p on cell viability, apoptotic cell death ratio and fold changes of proliferative or anti-proliferative gene expressions, which may have important effects on colon cancer, in hct colon carcinoma cells. ic doses of resveratrol were determined by wst- assay. the apoptotic cell death ratios in treatments of resveratrol were determined by annexin-v-fitc/pi assay for flow cytomety . the changes of ccnd , fra , ppard, egfr, birc , pcna, mcl , stat , fos, jun, p , atf , trail, puma, gadd a, rb , faslg, tnf, socs , stat gene expressions were evaluated by real time pcr. all data were statistically analyzed by student's t test. our research has revealed that resveratrol ( lm) causes decrease in cell viability and increase in apoptotic cell death in hct p (+/+) and hct p (À/À) cells significantly (p < . ). the fold changes of the gene expressions have shown that resveratrol has significant (p < . ) and different effects on the expressions of the genes related with the existence of p in hct cell lines. therefore we proposed that resveratrol might show proliferative or apoptotic effects related with p mutation of colon cancer cells and we predicted that unconscious consumption of resveratrol in colon cancer patient might cause adverse effects. introduction: colorectal cancer (crc) is the third most common cancer worldwide. alterations in methylation profiles of tumor suppressor genes (tsgs) have been recognized as a key mechanism in colorectal cancers. in the current study, we investigated the hypermethylation status of tsgs in colorectal cancer tissues. materials and methods: formalin-fixed paraffin-embedded (ffpe) tissue samples obtained from patients with crc. methylation specific-multiplex ligation dependent probe amplification (ms-mlpa) technique was used to assess the methylation status of tsgs. the findings were evaluated in terms of age, mortality, survival, positive lymph node status, lymphovascular invasion, and perineural invasion. results: hypermethylation-detected patients and hypermethylation-undetected patients were called as group and group , respectively. hypermethylation was detected in atm, cdkn a, and gata genes. mortality rate was ( . %) in group and group (p > . ). mean -years survival rate in group was ae months and mean -years survival in group was ae months (p > . ). positive median lymph node count was ae for group and ae for group and the difference was statistically significant (p < . ). frequencies of perineural invasion and lymphovascular invasion rate in two groups were % (p > . ). discussion and conclusion: our findings suggest that tsg hypermethylation found in crc patients may increase the lymph node metastasis. further investigations with larger sample size are required to support our results. boron (b) is known to be important for cell replication and development, but the underlying mechanism remains obscure. recently b has also become important in some specific anticancer processes. some recent reports advise using of some boron compounds for the treatment of specific forms of cancer. for instance, boron-based drugs (bortezomib) are now being developed for use as therapeutic agents with anticancer activities and several other boron-based compounds are in various phases of clinical trials. it has been shown that bortezomib disrupts the regulation of cell cycle and induces apoptosis in both hematologic and solid tumor malignancies except for colon carcinoma. colorectal cancer (crc) is the third most common cancer in men and the second in women, accounting for % of all tumour types worldwide. cytotoxic effects of boron compounds on crc cells and changing of its effects related with p mutation, which is mutated % of cancer cases, have not take part in literature yet. for this purpose; the aim of the study was designed to investigate the effects of borax pentahydrate and disodium pentaborate decahydrate compounds on cell viability, apoptotic cell death ratio and parp protein expressions in p (+/+) and p (À/À) hct colon carcinoma cells lines. the effects of the boron compounds on cell viability were assessed by xtt assay and apoptotic effects and parp protein expression of the compounds were evaluated by flow cytometry and western blot analysis respectively. our results showed that borax pentahydrate ( mm) and disodium pentaborate decahydrate ( mm) significantly causes nearly % reduction of cell viability at h (p < . ). apoptotic cell death ratios and parp expressions revealed that both of the compounds might have a potential for a candidate of anticancer agent. epithelial-mesenchymal transition (emt) is a significant event for metastasis, and could be mediated by several pathways such as pi k/akt, map kinases and many epigenetic regulators. satb is an epigenetic regulator involved in emt and osteoblastic differentiation. since preliminary results indicate that there is a crosstalk between p and akt pathways in nsclc cells, we aimed to determine whether this crosstalk has a regulatory effects on emt and satb expression in nsclc cells. we used a and h cells as a model to evaluate the effects of the crosstalk between p and akt on emt of nsclc cells. therefore, cell culture, inhibition of p activation via sb , transient expression assay for (ca-akt), western blot analysis, sirna transfection for satb , wound healing and invasion assay were performed in this study. firstly, the expression statues of e-cadherin, satb , p-p , p , p-akt and akt was examined in a and h cells by western blot analysis. we observed that e-cadherin and satb are downregulated in a cells (highly active p , lowly active akt) compared to h cells (lowly active p , highly active akt), suggesting that e-cadherin and satb are associated with the crosstalk between p and akt pathways. our results demonstrated that p inhibition in a cells leads to decreased pten expression and subsequently increased akt activation. then, we found that p inhibition upregulated satb expression, and reversed emt in a cells. furthermore, alone satb knockdown is sufficient to induce emt, and prevented the effects of p inhibition on emt. all these results strongly indicate that the crosstalk between p and akt pathways might determine satb expression and epithelial characters of nsclc cells, and satb is a critical epigenetic regulator for emt in nsclc cells. therefore, it is also need to explore how p and akt signalling pathways could regulate satb expression. this work was supported by tubitak ( s , z ). introduction: lung cancer is a disease characterized by uncontrolled cell growth in the lung tissues. the most common causes of lung cancer are tobacco smoke, radon gas, asbestos, air pollution, and genetic factors. nitric oxide (no) has potential mutagenic and carcinogenic activity and may play important roles in lung cancer. endothelial no, synthesized from l-arginine by endothelial no synthase (enos), inhibits apoptosis and promotes angiogenesis and tumor cell proliferation. the aim of the present study was to examine the possible relationship between enos gene intron vntr and exon -g t (glu asp) the stressful ecosystems exert strong adaptive pressure and proteins that facilitate these adaptation processes are candidate drug targets. nucleotides are the core of biochemical pathway required for cancer cell growth and replication and genetic changes will lead in oscillation in their pools. although it is questionable whether the warburg effect actually causes cancer, impairing dglucose uptake and metabolism induces oxidative metabolism. lproline (lproline) homeostasis is critical in a constellation of human diseases, in parametabolic linkage between cancer, epigenetics (phang et al. ) and bioenergetics (pallotta ) where degradation and biosynthesis are robustly affected by oncogenes or suppressor genes that can modulate intermediates involved in epigenetic regulation. lproline-fueled mitochondrial metabolism involves the oxidative conversion to l-glutamate by a flavin dependent lproline dehydrogenase/oxidase and a nad +dependent l-d -pyrroline- -carboxylate dehydrogenase. in saccharomyces cerevisiae an important test tube, put p and put p respectively help cells to respond to changes in the nutritional microenvironment by initiating lproline breakdown after mitochondrial uptake (pallotta ) . in this preclinical study, low molecular weight compounds were tested for inhibiting lproline mitochondrial transport and put p/put p catalytic activities. thus, in seeking for natural bioactive compounds targeting lproline pathway and its substrate channeling (becker's group ), we report data using in silico screening and in vitro researches in saccharomyces cerevisiae with genetic background atcc but different phenotypic landscape induced by nutritional stress/ ph changes. cells vitality, dΨ measurements, nad(p) + /nad(p) h pool and flavine turnover were determined in spectrofluorimeter microplater reader and via hplc (pallotta et al. (pallotta et al. , (pallotta et al. , pallotta ; di martino pallotta ) thus in supporting of future cancer therapies with decreasing side effects. evaluation of lymphocyte to monocyte ratio (lmr) in patients with colorectal cancer introduction: inflammation may play an important role in cancer progression and a high neutrophil to lymphocyte ratio (nlr) has been reported to be a poor prognostic indicator in several malignancies. the aim of this retrospective study was to evaluate the prognostic value of nlr, lymphocyte to monocyte ratio (lmr) and platelet to lymphocyte ratio (plr) in patients with colorectal cancer (crc). : patients who were diagnosed with colorectal cancer between january and january ; were evaluated retrospectively. the cutoff value was determined using receiver operating characteristics curve analysis. survival analysis was performed using the kaplan-meier method and log-rank test. the cox proportional hazard model was used to identify the influence of factors related to survival. (tnm stage, tumor differentiation, age, tumour size and lmr) results: receiver operating characteristic curves showed that lmr was superior to plr and nlr as a predictive factor in patients with colorectal cancer. the cutoff value for lmr was . . cancer-specific survival was not significantly different between the high-and low-lmr groups (p = . ). age was identified as independent prognostic factor in colorectal cancer (hazard ratio: . ; % confidence interval: . - . ; p = . ). discussion and conclusion: our preliminary study showed that the lmr was not an independent prognostic factor in crc patients, but additional large sample sized prospective studies will be needed to confirm these findings. the aim of this study is to investigate the effects of luteolin treatment on enzymatic activity of arginase, and ornithine and polyamine levels (putrescine, spermidine spermine) in serum and cancer tissues of ehrlich ascites breast cancer model. balb/c female mice were divided randomly into following groups: healthy control, healthy treatment, cancer control, treatment and treatment . . ml ehrlich ascites tumor cells was inoculated subcutaneously to medial part of left hind leg. healthy treatment and treatment groups, and the treatment group were given mg/kg and mg/kg dose of luteolin, intraperitoneally, for a days period, respectively. luteolin has a hydroxylated flavonoid structure and shows potent antioxidant, anti-inflammatory, and anticarcinogenic properties. luteolin not only leads to cell death in various tumors by suppressing cell survival pathways and stimulating apoptotic pathways, but also sensitize them to cytotoxic therapy. supporting various previous studies, tumor implantation to healthy mice resulted in statistically significant elevation of serum arginase and polyamine levels (p < . ) indicating the tumor cells as the main source of this production. furthermore, luteolin treatment abolished this increase in serum arginase and polyamine levels (p < . ). tissue measurements of arginase and polyamine levels indicated that luteolin treatment resulted with an increase in these parameters of tumor tissue while the serum levels of them showed a significant decrease. our results revealed that increased tissue arginase and polyamine levels might be related with estrogenic agonistic effect of luteolin on utilized tumor model in this experiment; and decreased serum levels of these parameters while there is a significant increase of them in tissue levels might be a result of a suppression of polyamine efflux from the tumor tissue by inhibitory effect of luteolin on plasma membrane polyamine transporters. hepatocellular carcinoma (hcc) is the third most common cause of cancer-related deaths. around - % of hcc patients are diagnosed at an early stage of the disease. hepatic resection, liver transplantation are common strategies in hcc treatment. even if, most of the patients present advanced-stage tumors and have a restricted survival rate. for the reason, resistance against existing tumor stress conditions have been demonstrated in hcc. hypoxia, hyperglycemia are general stress sources in hcc and result in aggressive cell phenotype, resistance to apoptosis and therapeutic drugs. thioredoxin interacting protein (txnip) regulates cellular responses under stress conditions. over-expression of txnip results activation of oxidative stress and apoptosis. in cancer models txnip is considered as a tumor suppressor gene. however, its role in the development, progression of hcc and mechanisms behind it warrant further investigation. in this study expression levels of txnip were examined in hcc cell lines by rt-pcr and western blotting. txnip expression was significantly high in poorly-differentiated snu- , snu- and snu- than the well-differentiated hcc cell lines such as huh- , hepg and plc/prf/ . besides, expression of txnip was examined in non-hcc and hcc tissue samples by immunohistochemical staining. txnip positivity was observed in % of well and % of poor differantiated hcc tissues. however, no txnip positivity was observed in non-hcc tissues. to investigate whether txnip might be involved in biological responses such as cell proliferation, motility and invasion, we used overexpression and silencing strategies. overexpression of txnip minimally inhibited adhesion and proliferation, whereas boyden-chamber motility and invasion assay showed that invasiveness of cells were increased. our findings suggest that txnip expression is increased in hcc and txnip over-expression is important for invasive phenotype during hepatocarcinogenesis. cardiovascular diseases are the leading cause of morbidity and mortality in the western world. it was shown that ischemic tolerance of the heart can be enhanced not only by ischemic or pharmacological conditioning (pre-and postconditioning), but also by adaptation to chronic hypoxia. different studies have indicated that these cardioprotective phenomena may at least partly share the same signaling pathways. the jak/stat signaling pathway has been demonstrated to participate in the development of cardioprotection by conditiong apparently through the inhibition of gsk- b. the aim of our present study was to determine whether this pathway also takes part in cardioprotection induced by adaptation to chronic hypoxia. we investigated the effect of inhibitor of jak kinase (ag- ) on myocardial infarct size and the jak /stat signalling pathway and other effector molecules that may participate in cardioprotection conferred by adaptation to hypoxia. adult male rats were adapted to intermittent normobaric hypoxia ( % o , weeks, h/day) and part of them recieved ag- ( mg/kg) min before ischemia. control rats were kept under normoxia. infarct size was assessed in isolated perfused hearts. relative expression of the key components of the jak /stat signalling system and other proteins was detected using western blotting. preliminary data indicate that administration of the jak inhibitor ag- caused a significant increase in infarct size in hypoxic rats. western blot analysis revealed changes in phosphorylation of jak , stat and some other proteins involved in cardioprotection (akt, erk / , gsk b). these results suggest that the jak/stat signaling pathway could participate in the development of a cardioprotective phenotype in rats exposed to chronic hypoxia. however, further research will be needed to clarify in more detail the role of this signalling pathway in the cardioprotective mechanism. p- . . - detrimental effect of hypertension on myocardium was reversed by liver x receptor agonist gw hypertension is a cardiovascular disease that causes functional and structural changes in the heart. nuclear liver x receptors (lxrs) are involved in the control of cholesterol and lipid metabolism. however, effect of lxr activation on the hypertensive heart is not well characterized. in this study, the effects of lxr agonist gw on hypertension-induced damage of myocardium were investigated. hypertension was induced by deoxycorticosterone acetate (doca) injection ( mg/kg, twice a week) following the unilateral nephrectomy in male -week-old wistar albino rats for weeks. blood pressure was measured by using tail-cuff method. gw ( mg/kg/day, i.p.) was administered last week. expression of various markers (grp , perk, p-perk, ikb-a, nf-kb p , tnf-a, bax, bcl- , mmp- ) in the ventricular tissue were examined by western blotting. inflammation and fibrosis were evaluated in histopathological examination. gw treatment reduced systolic blood pressure of hypertensive animals. expressions of endoplasmic reticulum stress markers grp and p-perk were increased by hypertension and gw treatment reversed them. hypertension-induced increase in nuclear nf-kb p expression and decrease in ikb-a expression were reversed by gw treatment. while bcl- expression was lower, bax level was higher than control in the hypertensive animals. in hypertensive group, fibrosis marker mmp- expression was augmented and gw treatment reversed this elevation. hypertension-induced increase in interstitial and perivascular collagen deposition and inflammatory cell infiltration in left ventricle were prevented by gw treatment. these results suggest that lxr activation by gw restored the hypertension-induced structural changes of heart in the doca-salt hypertension. methylphenidate (mph) is a psychostimulant prescribed for the treatment of attention deficit hyperactivity disorder (adhd), one of the most common neurobehavioral disorders of childhood and adolescence. in fact, despite the widespread use of mph the full comprehension of its cellular/molecular mechanisms is still elusive, including its effect on blood-brain barrier (bbb). this barrier is a key structure in the central nervous system since it protects the brain and its dysfunction has been described as a critical event in several brain diseases. thus, the aim of the present study was to clarify the effects of mph on the bbb function in both physiological and adhd conditions. for that, we used a rat model of adhd, the spontaneously hypertensive (shr) rats, and wistar kyoto (wky) animals as inbred comparator strain. also, to mimic a clinical dosing schedule for adhd treatment, rats were administered for monday-friday with vehicle or mph ( . mg/kg/day or mg/ kg/day, per os) from p -p . chronic mph treatment ( mg/kg/day) promoted cortical bbb permeability in both wky and shr animals; however, more prominent in wky rats. this effect can be explained by the downregulation of claudin- and collagen-iv, tight junction and basal lamina protein, respectively. noteworthy, wky animals also showed an increase in the expression of caveolin- and in both vascular cell and intercelular adhesion molecules. these bbb alterations led to subsequent infiltration of peripheral immune cells, including cd + macrophages. furthermore, hippocampal bbb disruption was only observed in wky rats with mg/kg of mph. here, mph decreased collagen iv expression and upregulated caveolin- , with no alterations in claudin- . overall, our results show that chronic exposure to mph can led to brain vascular alterations particularly under physiological conditions. this highlights the importance of an appropriate mph dose regimen for adhd, and also that mph misuse can have a negative effect. regulators of g proteins signaling (rgs) serve several cellular functions varying from tolerance, dependence, neuroprotection, transcription and tumorgenesis. despite their initial role as gtpase activating proteins, evidence suggests that rgs proteins are localized in the nucleus, interact with transcription factors thus regulating transcriptional responses. it was shown that rgs directly interacts with and interferes in opioid receptor (or) signaling. rgs is mostly expressed in brain and is implicated with brain structural alterations; however, the molecular mechanisms of how rgs could be involved in cellular differential functions remains unclear. based on these observations we examined whether rgs can regulate transcriptional responses mediated by the stat b transcription factor. isolated neural stem cells from rgs À/À mice were immunostained for the mitosis marker ph and the mrna levels of antiapoptotic genes were determined. proliferation assays were performed with brdu staining in neuro a cells stably expressing rgs . the functional assays of stat b transcriptional activation were performed in hek expressing either the erythropoietin receptor (epo-r) or the delta opioid receptor (d-or). the present data demonstrate that rgs blocks stat b phosphorylation and transcriptional activation by interfering in stat b heterodimerization upon epo-r or d-or activation triggered by cytokines or opioids administration. lack of functional rgs results in increased mrna levels of stat b target genes such as the members of the bcl anti-apoptotic family bcl- , bcl- and bcl-xl. this upregulation of stat b inducible gene transcription results in an increased proliferation rate of neural stem cells. this study demonstrates for the first time a non-canonical function of rgs in stat b mediated transcriptional responses and a novel selective role of rgs in transcription. role of the pre-molten globule structure in amyloid fibril formation a. eshaghi department of biology, faculty of science, islamic azad university, mashhad branch, mashhad, iran the major factor that caused extensive research on the protein fibrillation is their crucial roles in important diseases known as the amyloidosis diseases. neurodegenerative diseases, including alzheimer's, parkinson's, diabetes and huntington are the most important types of this disease. understanding the mechanisms of fibril formation and ways of treatment can be useful in reducing this type of disorder. in this project, the fibrillation of carbonic anhydrase protein was investigated as a model. carbonic anhydrase creates two stable intermediated known as pre-molten and molten globule, in different ph solution. this protein at ph between ph - molten globule structures was formed while the pre-molten form took place under ph . in our tests at ph . when the protein in molten globule structures only the amorphous aggregates were formed. instead, at ph . in pre-molten globule structure amyloid fibrils formed in the protein. there some reports, indicated the protein from pre-molten globule structure go toward amyloid assembly. even intrinsically unstructured proteins such as alpha-synuclein first took a structure similar to pre-molten globule and then made amyloid fibrils. it seems pre-molten globule structure have the major role in promoting to amyloid fibrils. perhaps drugs that prevent the formation of premolten globule structure have an important role in inhibiting amyloid fibrils. identification of compounds preventing the biochemical changes that underlie the epileptogenesis process and understanding the mechanism of their action is of great importance. we have previously shown that myo-inositol (mi) daily treatment for days prevents certain biochemical changes that are triggered by kainic acid (ka)-induced status epilepticus (se), [ , ] . however in these studies we have not detected any effects of mi on the first day after se. in the presented study we broadened our research and focused on ka induced other early molecular and morphological changes and influence of mi treatment on these changes. the increase in the amount of voltage-dependent anionic channel- (vdac- ), mitochondrial-plasma membrane cofilin and caspase- activity was observed in the hippocampus of ka treated rats. administration of mi h later after ka treatment abolishes these changes, whereas under the same time schedule diazepam treatment has no significant influence. the number of neuronal cells in ca and ca subfields of hippocampus is decreased after ka induced se and mi post-treatment significantly lessens this reduction. no significant changes are observed in the neocortex. obtained results indicate that mi post-treatment after ka induced se could successfully target the biochemical processes involved in apoptosis, reduces cell loss and can be successfully used in the future for translational research. references . r. . neuroscience letters, vol. , no. , pp. - . . r. solomonia, et al; . cell. mol. neurobiol. vol. , no extracellular deposits of amyloid-b peptide (ab) in brain parenchyma via proteolytic processing of amyloid precursor protein (app) are one of the typical characteristics of alzheimer's disease (ad). these aggregates mainly occur as a result of an increase in ab production or a decrease in its degradation. it was found that the neurotoxicity of ab aggregates is accelerated by acetylcholinesterase (ache). besides, ab-ache complex has a prominent neurodegenerative effect in brain. thus, cholinesterase inhibition and preventing ab production are current treatment strategies for ad. recent studies have shown that methylene blue (metb), a cholinesterase inhibitor with phenothiazine structure, inhibits the formation of amyloid plaques and neurofibrillary tangles. azure b, the major metabolite of metb, has been shown to inhibit ache and bche with ic values of . lm and . lm, respectively. in the present study, we tested whether azure b, may effectively lower the levels of ab / . we treated chinese hamster ovary cells, which stably express human wild type app and presenilin- (ps ) with - mm azure b or vehicle for h. to determine the effect of azure b treatment on ab / levels, we used separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. azure b treated ps cells were also assessed by propidium iodide in flow cytometry for cellular toxicity. we observed a significant decrease in both extracellular ab and ab levels with a dose range treatment of azure b in ps cells. ab levels were reduced by . % in lm and . % in lm azure b-treated cells when compared to control. additionally, ab levels were decreased by % in lm and . % in lm azure b-treated cells when compared to control. overall, these preliminary results suggest that azure b may have beneficial effects for the treatment of ad. the effect of green silver nanoparticles (agnps) on the amyloid formation in alphalactalbumin and chaperone action of alphacasein a. ghahghaei, m. dehvari, j. valizadeh formation and deposition of protein fibrillar aggregates in the tissues is associated with several neurodegenerative diseases such as alzheimer's and parkinson's disorders. molecular chaperones are a family of proteins that are believed to have ability for inhibiting protein aggregation. in the present study the effect of different concentrations of green synthesis silver nanoparticles (agnps) from pulicaria undulate l. on the amyloid formation of a-lactalbumin (a-la) and chaperone action of a-casein have been investigated. the effects of the agnps were determined using light scattering absorption, tht binding assay, intrinsic fluorescence assay, ans binding assay, cd spectroscopy and sds-page. light scattering and tht assay results showed that agnps have the ability in preventing aggregation of a-la in a concentration-dependent manner. consistent with these results, sds-page results represented that by increasing the concentration of agnps the adsorption and interaction between agnps and protein have increased. light scattering and tht assay results, also, revealed that the amyloid fibrilation decreased in the presence of both agnps and a s -casein compared to presence of a s -casein alone. fluorescence results, however, show that agnps have no effect on the chaperone ability of a-casein and in fact they perform their protection of protein aggregation action independently. consistent with the above experiments, cd spectroscopy also revealed that agnps have decreased structural changes in reduced a-la in absence and presence of a-casein, both the tertiary and the secondary structure of the proteins. our finding represented that agnps have preventing effects on protein aggregation and have no effect on the chaperone ability of a s -casein. in the main, results of this study show that biosynthesized agnps mediated by >pulicaria undulate l. maybe could be affective as a therapeutic agent for inhibiting aggregation in treatment of amyloidosis disorders. pink is a mitochondrial kinase with multiple cellular functions. while loss-of-function mutations of pink gene lead to early onset parkinson's disease, its over-expresion is associated with cancer development. parkinson is a multifactorial neurogenerative disease, with a complex aethiology including various cellular stressors. it is now known that genotoxic stress also triggers the release of soluble factors able to induce changes in neighboring cells enhancing the initial lesions, process known as bystander phenomena. althrough the mechanisms are still unclear, recent studies point towards a role for mitochondria in this process. our work investigates pink role in intracellular and intercellular stress response, comparatively in various models: fibroblasts (mefs) and neuroblastoma (sh-sy y) used as a tumoral model or differentiated to a neuronal phenotype. pink role in this process was analyzed using genetically engineered pink deficient cells exposed to a genotoxic agent, bleomycin. the modified cell lines showed a reduced level of basal atp production. pink proved to be involved in cellular vulnerability to stress. despite differences in cellular sensibility between our models, genotoxic treatment of pink deficient cells induced consistently higher lesions compared to corresponding wild type variant. pink deficient cells showed altered intercellular signaling of stress, impairing bystander phenomena induction, by suppression of signal formation in treated cells, but also by altering the capacity to respond to the signals in neighboring cells. our hypothesis is that pink contributes to the management of cellular stress being involved in bystander transmission of detrimental effects through intercellular communication. this is determined mainly by its role in maintaining mitochondrial homeostasy and atp levels, pink deficient cells lacking the amount of energy required for rapid dna repair and stress signaling transmission. p- . . - intranasal administration of synthetic fragments from receptor for advanced glycation end products prevents memory loss in olfactory bulbectomized mice the receptor for advanced glycation end products (rage) is a member of the immunoglobulin protein superfamily. activation of rage causes brain inflammation, oxidative stress and secretion of beta-amyloid that has been recognized as an essential phase in the development of alzheimer's disease. it is known that the receptor soluble isoform (srage) which lacks the transmembrane and cytosolic domains binds to ligands and prevents negative effects of the receptor activation in in vivo and in vitro experiments. we proposed that potential ligand-binding peptide fragments from srage would demonstrate the same biological activity. we have selected and synthesized peptide fragments from unstructured surface-exposed regions of rage. synthetic peptides were intranasally administrated into olfactory bulbectomized (obe) mice with neurodegeneration of alzheimer's type. we have found that only administration of rage fragment ( - ) effectively prevents the obe murine memory from impairment, leads to decrease of beta-amyloid level and blocks the development of neuronal pathology in the brain of experimental mice. six overlapping fragments of rage ( - ) peptide were synthesized in order to find a site, responding for the therapeutic effect. tests in obe mice carried out with these fragments showed that only the n-terminal part of the molecule is responsible for preventing obe mice memory from impairment. all fragments which do not include n-terminal - dipeptide have been fully inactive in these experiments. we have proposed that active peptides can interact with beta-amyloid or s b protein preventing these ligands from binding with rage. this interaction can inhibit the development of neurodegeneration. the aim of this study was to examine effects of social isolation, enriched environment and exercise on learning in rats. the study included female day old wistar rats. the rats were randomly divided into four different groups; control, exercise, social isolation and the enriched environment groups. the social isolation group and the enriched environment group were housed under their specific conditions and the exercise group and the control group were housed in standard conditions during weeks. the rats in the exercise group swam for weeks. after weeks, the rats were evaluated in the morris water maze. brain and blood samples were taken and the hippocampus tissue was dissected. bdnf and ngf levels were measured in these samples. in conlusion, while enriched environment was a positive effect on spatial learning, social isolation was a negative effect on spatial learning and increase thigmotactic behaviors. according to the analysis results ngf and bdnf levels in the hippocampus and plasma did not change with environmental conditions and exercise. time of exposure to social isolation, procedures of the enriched environment, time of exposure to the environment, type and duration of exercise and gender may affect the results. alzheimer's disease (ad) was characterized by dementia that typically begins with subtle recognition failure and poor memory. it slowly becomes more severe and, eventually, incapacitating. the cholinergic system seemed particularly susceptible to synapse loss, especially in cortical regions associated with memory and executive function ( ) . recent studies showed that the main cause of the loss of cognitive functions in ad patients was a continuous decline of the cholinergic neurotransmission in cortical and other regions of the human brain ( ) . acetylcholinesterase (ache) and butyrylcholinesterase (bche) are hydrolytic enzymes that act on acetylcholine (ach) to terminate its actions in the synaptic cleft by cleaving the neurotransmitter to choline and acetate. both enzymes are present in the brain and detected in neurofibrillary tangles and neuritic plaques. it was suggested that ache predominates in the healthy brain, with bche considered to play aminor role in regulating brain ach levels. however, bche activity progressively increases in patients with ad, while ache activity remains unchanged or declines. both enzymes therefore represent legitimate therapeutic targets for ameliorating the cholinergic deficit considered to be responsible for the declines in cognitive, behavioral, and global functioning characteristics of ad ( ). we initiated a study to screen their acetylcholinesterase (ache, ec . . . ) inhibitory activities, which are the key enzymes taking place in pathogenesis of ad. newly synthesized chiral benzimidazole derivatives with thioure structure showed ic values in the range of . - . nm for ache. this study was financed by turkish research council-tubi-tak (kbag z ). p- . . - f -a h, novel fingolimod derivative, activates camp-dependent signalling pathway in sk-n-sh cell line g. celik turgut , a. sen , d. doyduk , y. yildirir department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, department of chemistry, faculty of sciences, gazi university, ankara, turkey fty , a sphingosine -phosphate (s p) receptor modulator, is the first oral disease-modifying therapy to be approved for the treatment of relapsing-remitting multiple sclerosis. in this study, we have synthesized and characterized novel derivative of fty , namely f -a h, and have determined its underlying camp regulation in sk-n-sh cell lines. for this purpose, we first determined the regulation of the camp response element (cre) activity and camp concentration by f -a h along with fty using pgl . luciferase reporter assay and camp immunoassay, respectively. then, we have determined their effect on camp/pka-related gene expression profiles using custom arrays along with fty treatment at non-toxic doses. it was found that f -a h significantly activate cre and increase camp concentration in the sk-n-sh cell line, indicating that it activates camp pathway through cell surface receptors as fty does. furthermore, f -a h modulates the expression of the pathway related genes that are important in camp signaling pathway. in summary, our data demonstrate that the novel fty derivative act as a modulator of camp ultimately by influencing the gene expression via the camp and downstream transcription factor cre pathway. in conclusion, f -a h might contribute future therapies for multiple sclerosis. alzheimer disease (ad) results in memory impairment and accompanied by neuroinflammation, cholinergic deficit and amyloid-beta (ab - ) accumulation in brain. we found that bacterial lipopolysaccharide (lps) injections or mice immunizations with extracellular a nicotinic acetylcholine receptor (a - nachr) domain resulted in astrogliosis, decrease of a nachr density, accumulation of ab - in brain and episodic memory impairment. the aim was to reveal main event triggering ad-like symptoms development. c bl/ mice were injected with lps, immunized with recombinant a - or a - endoglycosidase treated to remove carbohydrates. two immunizations with week interval were performed. control mice obtained complete freund's adjuvant injections. mice were tested for memory performance, blood sera were examined for presence and fine specificity of a - -specific antibodies and brain preparations were studied for a nachr, ab - and il- levels. the original a - ('glyc') was more immunogenic than 'deglyc', and their epitopes were recognized with different efficiency. in contrast to lps and 'glyc' a - immunization with 'deglyc' a - did not stimulate il- elevation in brain and had no proinflammatory effect. immunizations with 'glyc' or 'deglyc' a resulted in similar a nachrs decrease and ab - accumulation in brain and significant episodic memory decline comparable to those after lps injections. a nachr interacts directly with amyloid-beta precursor protein and facilitates its proper processing and metabolism. our data indicate that decrease of a nachr density caused by a - -specific antibody is critical for ab - accumulation and episodic memory impairment while pro-inflammatory capacity of a - -specific antibody plays secondary role in ad-like symptoms development. in vitro antioxidant and antiacetylcholinesterase activity of achillea millefolium alzheimer diseases (ad) is a neurodegenerative condition without a current effective treatment. increase in reactive oxygen species and lipid peroxidation or decrease in total antioxidant capacity causes oxidative stress-induced tissue damage. it has been suggested that decrease in oxidative stress and inhibition of acetylcholinesterase (ache) activity play a major role in the prevention and slowing of cognitive symptoms of ad. recently, studies have been directed for the discovery of medicinal plants and natural substances that are known to have natural antioxidants. achillea millefolium (a. millefolium) is a traditional herbal medicine that contains natural compounds with antioxidant activity and has been used as a carminative, diuretic, menstrual regulator and wound healer, however the mechanism of its actions are unclear. the aim of our study was to investigate the effects of a. millefolium extracts on free radical production, acetylcholinesterase (ache) activity and lipid peroxidation in vitro. methanol (me) and ethanol (ee) extracts of a. millefolium were prepared to determine (a) in vitro antioxidant capacity (by using , -diphenyl- -picrylhydrazyl assay, radical scavenging activity, phosphomolibdenum-reducing antioxidant power, ferricreducing antioxidant power, and total phenolic-total flavonoid contents), (b) effects on ache kinetics (by using a colorimetric assay) and (c) effects on sodium nitroprusside-induced lipid peroxidation in mice brain homogenates. me showed a higher antioxidant activity compared with ee in the biochemical assays tested. similarly, me demonstrated significant inhibition of ache activity that was potent than ee. both extracts dose-dependently decreased malondialdehyde content in mice brain homogenates suggesting a strong inhibition of lipid peroxidation. these results showed that a. millefolium has a high antioxidant capacity and antiache activity, indicating a potential use as an adjuvant therapy in ad. b-cells are known to play a key role in multiple sclerosis (ms) progression and autoimmune response. cxcr is the main b-cell chemokine receptor that under normal conditions directs their migration to specific areas of secondary lymphoid organs. in ms, areas of demyelinating lesions have been reported to attract bcells due to overexpression of cxcl , the cxcr ligand. we aimed to determine whether snp rs located in the promoter of cxcr gene and associated with high risk of multiple sclerosis could have a direct effect on of cxcr promoter activity. mef c binding to dna was assessed using pull-down assay. b-cell stimulation was performed using lps, pma and ionomycin. activities of variants of cxcr promoters containing different rs alleles were estimated using luciferase reporter assay. we determined that minor rs allele creates functional mef c-binding site within one of the regions required for the basal activity of the cxcr promoter. cxcr promoter containing minor rs variant that is statistically associated with low risk of ms showed significantly decreased activity in stimulated human b-lymphoblastoid cell lines. mef c has been reported to play an essential role in b-cell survival and b-cell responses. we determined mef c as the main regulator of rs -dependent modulation of cxcr promoter activity in b-lymphoblastoid cell lines. this link may be directly related to pathogenic b-cell activities in multiple sclerosis. introduction: parkinson's disease (pd) is the second most common neurodegenerative progressive brain disease with increasing prevalence in aging population. the etiopathogenesis involves many cellular procesess, but is not fully elucidated yet. treatment of pd is based on levodopa and dopamine agonists, but mao-b inhibitors, comt inhibitors, amantadine or anticholinergics may be used as initial monotherapy or as adjuvant therapy. treatment related adverse drug reactions (adrs) are frequent, but cannot be predicted and/or prevented. non-motor adrs, such as nausea, somnolence, hallucinations and hypotension are frequent in dopamine agonist therapy, while dyskinesias along with motor fluctuations are the most common late adrs with levodopa. the aim of our study is to combine clinical data with genetic and epigenetic biomarkers in the algorithm for personalized approach to pd management. materials and methods: we are planning a clinical study to assess the combined impact of selected clinical, genetic and epigenetic factors on the progression of pd, adrs and treatment response. our study will have a retrospective and prospective arm. we will collect peripheral blood samples of pd patients and clinical data. single nucleotide polymorphisms (snps) in the genes involved in dopamine, neurotransmitter and drug metabolism and transport, receptors and signalling pathways will be genotyped. snps within inflammatory, neurodevelopmental, antioxidative defense, synaptic transmission and immune response pathways will also be analysed. in the prospective arm we will isolate the exosomes and check their mirna content at the time of diagnosis and after the treatment initiation. the combined effects of clinical, genetic and epigenetic factors will be analyzed using lasso penalized regression analysis. conclusions: we hope to identify genetic and/or epigenetic biomarkers that may predict progression of pd, adrs and treatment response and may support personalized tratment of pd. most evidence indicates that g protein-coupled receptors form heteromers between them and with other receptors. by allosteric mechanisms, them acquire a multiplicity of unique pharmacological and functional properties. recently, we discovered that dopamine d receptors (d r) and histamine h receptors (h r) form heteromers through which h r ligands can inhibit d r function. d rs also physically interact and modulate ionotropic glutamate nmda receptors (nmdar). in the present work, we investigated if nmdar, d r and h r form a heterotrimeric complex in brain. the heteromer expression was studied in slices from both rat and mouse brain cortex by co-immunoprecipitation (co-ip) and proximity ligation assays (pla). the ability of d r and h r to interact with nmdar in transfected hek cells was analyzed by bioluminescence resonance energy transfer (bret) with bimolecular fluorescence complementation (bifc) experiments. heteromer properties were studied by analyzing erk / phosphorylation and cell death in cortical slices. endogenous d r-h r heteromers were detected in rat and mouse cortical slices, where h r ligands decreased d r signaling (erk / pathway) and were also able to block the cell death induced by overstimulation of either d r or nmdar. by bret experiments in transfected hek cells, we demonstrated that both d r and h r form heteromers with nmdar subunit a in the presence of subunit b. d r-h r-nmdar heteromers were detected by bret with bifc. endogenous d r-h r-nmdar heteromers were observed in rat and mouse cortex by pla. many systems, including the glutamatergic and dopaminergic, are involved in neurodegeneration. our innovative finding is that d r, h r and nmdar form heteromers that may be a point of intervention for cognitive disorders in neurodegeneration. d r-h r-nmdar heteromers are expressed in brain cortex and a complex interaction exists between protomers in the heteromer, where h r ligands act as a 'molecular brake' for d r and nmdar signaling. studies conducted on obesity and hfd (high fat diet) revealed hypothalamus have crucial roles on development of metabolic diseases. after chronic over nutrition or high fat diet, as a neurodegenerative condition, premise inflammation, neural stress and development of functional impairments are observed. these studies generally focused on changes in neurons, but it's effects on brain vessels are still unknown. in this study, as a neuronal damage infrastructure, changes in hypothalamic vascularity investigated. experiment initiated with weeks old total male wistar rats. in order to acquire obese phenotype, the rats were fed either cafeteria diet as hfd, or normal/chow (standard diet, sd) for months. intravenous glucose tolerance tests performed before sacrification. animals were exposed for s to co and then decapitation was performed with guillotine. isolated brains were directly immersed into liquid nitrogen and stored at À °c. the hypothalamic sections were acquired with the cryostat instrument at different. immunofluorescence was performed on serial sections through the hypothalamus using the antibody smi- and cd . changes in tight junction (tj) proteins (occluding and zone occludin- ) are evaluated via western blot (wb) analysis. the hfd-treated consumed significantly more food than did control animals, when examining average food consumption per day and rats that received the hfd diet weighted significantly more at the end of month diet treatment. there were no differences acquired for glucose tolerance tests. however, after hfd treatment, wb analysis have shown that tj proteins decreased even if hypothalamic micro vessel number increased and smi- staining have shown that increased. our primary results have shown that hfd diet can affect hypothalamic vascularity and such changes might initiate neurodegenerations and functional impairments as observed in neuroretinal degeneration in relation to vasculopathy in diabetic patients. defects of mitochondrial trafficking are common problem in many neurodegenerative diseases. its dysregulation can contribute to changes in bioenergetics profile of the cell and can lead to cell death. in our study we investigate distribution of mitochondria and their transport in primary fibroblasts derived from patients with sporadic form of alzheimer's (ad) and parkinson's (pd) diseases. our data revealed that in the most cases the velocity of mitochondrial movement is lower in ad and pd cells in comparison to the control. the most intense differences between ad, pd patients and control group are observed in the case of movement of large mitochondria. owing to the fact that mitochondrial trafficking depends on mitochondrial state, we investigated the 'age' of mitochondria. we observed a diminished mitochondrial turnover in ad and pd fibroblast. evaluation of the mitochondrial distribution within the cell in all groups (ad, pd and control) showed that in the perinuclear area are accumulated 'old' and 'worn out' mitochondria, probably dedicated to remove from the cell. because mitochondrial biogenesis, shape and size depends on fusion/fission proteins we assessed their level within the cell. to summarise, our results revealed alterations in mitochondrial trafficking in fibroblasts derived from patients with alzheimer's and parkinson's diseases in comparison to the healthy control cells. carbonic anhydrases (cas, ec . . . ) is a zinc metalloenzyme that catalyzes the reversible reactions of co and water. carbonic anhydrases (cas, ec . . . ) form a family of metalloenzymes that play an important function in various physiological and pathological processes. therefore, many researchers work in this field in order to design and synthesize new drugs. carbonic anhydrase activators are important as much as inhibitors. caas have polar groups to make hydrogen bond in the main body and the activation property of enzyme increaase in this way. caas are have polar groups to make hydrogen bond in the main body and the activation property increaase in this way. furthermore, recent studies suggest that ca activation may provide a novel therapy for alzheimer's disease. in this study ca activators are determined. human carbonic anhydrase isozymes ca i and ca ii are isolated from human blood erythrocyte. hca-i and hca-ii isoenzymes were purified using sepharose- b-l tyrosine-sulfanilamide affinity colum. finally, hca-i and hca-ii isoenzymes were eluted with appropriate elution buffers. enzyme purity was checked by sds-page. the enzyme activity system contained . m tris-so ph . , r-nitro phenol in ml total volume. effects of some macrocyclic thiacrown ethers derivates were investigated. enzyme activities were measured at constant substrate and different activator concentrations to find ac value. these compounds are thought to be useful for treating alzheimer's disease. introduction: gender differences in stress models are not studied in detail. we compared different stress conditions on brain bdnf levels, in social isolation (sit) and predator scent tests (pst) in rats. bdnf levels in cortex, hippocampus and amygdala were compared, effects of chronic fluoxetine (flu) treatment were evaluated. methods: rats were used. for sit, animals were kept individually for month and for pst, rats were exposed to dirty cat litter for min at the first day of month stress. flu was given ( mg/kg, ip) through stress. controls, stress and treated groups were evaluated in elevated plus maze (epm), anxiety scores were calculated. brain bdnf levels were determined in cortex, hippocampus and amygdala by western blot. p < . were considered significant. results: sit and pst induced anxiety in both male and female rats, females having greater anxiety scores than males (p < . ). flu restored anxiety scores in both sexes (p < . ) in two settings. male and female rats exhibited reduced cortical bdnf levels in sit (p < . ). pst reduced cortical bdnf in females, but increased in males. hippocampal bdnf expression was lowered in sit (p < . ) and pst (p < . ) in both sexes. female rats had % lower bdnf expression than males in amygdala in sit. flu did not restore cortical bdnf in females in both tests, but reduced incresed bdnf levels in males (p < . ). flu did not restore reduced brain bdnf in males in hippocampus and amygdala, but restored in hippocampus, in females. discussion: our findings indicate that sex differences must be considered in studies related to mood disorders of animal models, and suggest that bdnf expression in different brain regions are altered differentially in a gender-dependent manner in rats. antianxiety effect of flu is not mediated through increasing bdnf activity in cortex in both genders. increased bdnf in hippocampus and amygdala may reflect its antidepressant effect in female rats, but not in males. perineuronal nets (pnns) are special forms of neural extracellular matrix found around neuron bodies and neurites. hyaluronan and proteoglycan link protein (hapln ) is one of the major elements of pnns. hapln interacts with tenascins and aggrecan which are other essential pnns components. in most of neurodegenerative disorders caused by neuritogenesis defects, disrupted pnns structure and decreased expression of hapln were observed. however, the role of hapln in neural differentiation is unknown. the aim of this study is to determine mrna and protein levels of hapln during differentiation using pc cell line as a neural differentation model, derived from rat pheochromocytoma. after pc cells were stimulated to differentiate into neurons by nerve growth factor on days , and ; cells were collected, qrt-pcr and western blot were performed. also, in order to find out whether there is a physical interaction between hapln and proteins related to neuritogenesis defects, spinal muscular atrophy (sma) was used as a neurodegenerative disease model. therefore, a detailed hapln and survival motor protein (smn ) network analysis were performed in-silico. as a result, we analyzed fold increase in hapln mrna level compared to undifferentiated state. on the other hand, a decrease in protein level was detected. this decrease in cellular hapln level suggests that, hapln is required for formation of pnns structure, thus secreted to extracellular environment at early stage of differentiation. in addition, according to in-silico analysis, an indirect path between hapln and smn through fibulin (fbln ) was detected. fbln was also found to be an interaction partner between different matrix molecules such as aggrecan and hapln which form a macromolecular meshwork. the results of this study will pave the way for investigating the role of hapln and fbln in neurodegenerative disease models. also it will help us to understand the mechanism of neuritogenesis defects. determination of properties of bone marrow and tissue-derived mesenchymal stromal/stem cell population in neurofibromatosis type patients neurofibromas, complex tumors deriving from schwann cells and containing fibroblasts, vascular structures and mast cells, are part of the clinical picture in nf . the risk of malignancy is increased in nf , wound healing is delayed and keloid formation is frequent. because multiple tissues are involved in malignant and non-malignant manifestations of nf , we considered the mesenchymal stem/stromal cells (msc) carrying the nf mutation might play a role in the microenvironment. mscs affect the biological behaviour of other cells: they alter their proliferation, apoptosis and migration through various secreted growth factors, cytokines, chemokines, or by direct contact. we examined the msc of nf patients. methods: the adipogenic and osteogenic differentiation potential of mscs from nf and healthy subjects was examined in vitro and by rt-pcr. msc's migration potential was measured in the scracth assay. mscs' interaction with schwann cells and their effect on tumorigenesis was examined in co-culture by apoptosis markers on flow cytometry. results: nf -mscs' adipogenic and osteogenic differentiation potential was lower than healthy controls as assessed by staining aizerin red s and oil red o and rt-pcr for osteopontin and collagen . mscs cultured from dermal neurofibroma showed faster closing of the scratch compared to the same patient's normal and caf e au lait skin. on the other hand, mscs obtained from plexiform neurofibroma healed late, while mscs derived from the same patient's caf e au lait skin showed the fastest healing. hepatic encephalopathy with ammonium ions accumulation is accompanied by some disorder in the brain due to toxic material concentration being usually detoxified in the liver. one of the reasons for hyperammoniemia could be some imbalance in brain glutamine metabolisation induced by the key enzymes glutamine transferases (gts), which catalyze the reaction of glutamine transamination resulting in neurotoxic product of a-ketoglutaramate (akgm). akgm is hydrolyzed to a-ketoglutarate and ammonia by x-amidases. in the study, the dynamics of the enzymes activity in the tissues and biological liquids of experimental animals with hepatic dysfunction induced by thioacetamide (taa) was under investigation. white laboratory rats of wistar line (female, weight of g) chronically intoxicated with hepatotrophic toxine of taa. every weeks, some biological samples were collected to assess gt-k and x-amidase activities. x-amidase activity was the highest in the kidney tissue in the control and decreased by % in the experimental group. in the experimental hepatic x-amidase activity decreased by % compared to those in the control. the average x-amidase activity in the blood serum ( . nmols/ mg/min) and in the brain ( . nmols/mg/min) differed faintly. maximal gt-k activities were revealed in the kidneys; in the controls, it was about % higher than those in the experimental animals. the difference between average enzyme activities in the liver of the control and experimental animals reached %. the average gt-k activities in the blood serum and brain of the control and experimental animals were rather similar. the decrease in x-amidase and gt-k activities obtained in the study during hepatic pathology development could testify to imbalance of glutamine metabolism, possibly aimed at declining the level of akgm neurotoxicant under the hepatic dysfunction. acknowledgments: supported by the russian federation ministry of science and education (grant no rfme-fi x ). wilson disease is an autosomal recessive disorder of copper metabolism characterized as neurodegeneration and liver abnormalities. it is caused by defects in the atp b gene. atp b is responsible for the sequestration of cu into secretory vesicles, and this function is exhibited by the orthologous ccc p in the yeast. we aimed to characterize clinically-relevant novel mutations of p.t i, p.v i and p.r g-fsx in yeast lacking the ccc gene. the patients with these mutations have copper storage abnormalities in different parts of their bodies; p.t i mutation mainly affects the liver and the nervous system, p.v i mutation affects the nervous system, and p.r g-fsx mutation causes damages to the liver. to better understand the effects of these mutations on normal functions of atp b, we cloned human atp b gene onto a yeast expression vector and created the same mutations by site directed mutagenesis. then, wild type and mutated forms of atp b genes were transformed into yeast cells lacking the homologous ccc gene for functional comparison. first, we analyzed the expression of atp b and its variants in yeast cells by a real time pcr approach and western blot to make sure that transformed cells express the plasmids. expression of human wild type atp b gene in ccc d mutant yeast restored the growth deficiency and copper transport activity, however, expression of the mutant forms did not restore the copper transport functions and only partially supported the cell growth. our data support that p.t i, p.v i and p.r g-fsx mutations cause functional deficiency in atp b functions and suggest that these residues are important for normal atp b function. in recent years, attempts were made to develop miniaturized potentiometric biosensors which is particularly important to reduce the amount of enzyme and reagents needed. the miniaturization of a biosensor is possible by using an all solid-state polymer membrane ion selective electrode which is cheap, easy to prepare and allow micro-sized construction. the use of all solidstate polymer membrane ion selective electrode as the basic sensing element also has the advantage of providing biosensors that are easy to fabricate, exhibit rapid response and have long life-times. they are also mechanically stable and allow flow-through configuration. genetical and chemical modifications for the alteration of enzyme molecule characteristics are gained considerable importance. enzymes can be modified chemically by using water-soluble polymers or some chemicals. conjugates of natural and synthetic macromolecules with enzymes provides wide usage in medicine and in many fields of biotechnology. in this study, enzyme-polymer conjugates with different molar ratios were synthesized using urease enzyme. in this study micro sized potentiometric urea sensitive biosensor has been developed in which urease-polymer conjugates were immobilized on polymeric membrane ammonium ion selective electrodes whether pvc or derivatized-pvc via glutaraldehyde cross linking reaction. biosensor is not include inner reference electrode and inner reference solution. potentiometric performance of biosensor will be examined with a computer-controlled measurement system designated. the most important features of the obtained micro sized urea biosensor by using enzyme-polymer conjugations were being highly sensitive, having long life-time, easily built at a low cost, and having short response time when compared with conventional potentiometric urea biosensor. also, these biosensors were easily built at micro-construction. this study was supported by grant from the tub _ itak research fund (project number: z ). creative drama technique as a new tool to increase enthusiasm and to achieve learning objective for medical students e. y. sozmen , , e. erem faculty of medicine, ege university, izmir, turkey, center for continuing education, ege university, izmir, turkey, recently drama in education techniques have been implemented successfully in education program of primary and secondary high school and positive effects of these techniques on learning ability and attitude of students have been shown. the aim of this study was to organize an education program based on drama in education techniques in a special module of ege university medical faculty and to test any effect of this technique on achievement of learning objectives and student's perspectives on drama. the special module program was on the oxidative stress and antioxidants. the program covered the drama in education sessions (improvisation, role play, game) linked with learning objectives (understanding of free radical generation and free radical reactions in body, evaluation of the effect of free radical reactions in diseases as well as increase the ability usage of scientific information), laboratory work (antioxidant activity determination) and searching a special scientific topic on literature. students (in rd year of faculty) who had taken theoretical lecture on this subject a year ago, participated in this special module. the opinions of the students on the program were obtained through a questionnaire form and the increase in knowledge was evaluated via pre/posttest. the mean of pretest point was . / , that increased to . / in the posttest evaluation. % of students pointed that they enjoyed participating in drama activities in the pre-questionnaire, this rate was % in the final questionnaire. they all remarked that implementation of drama in education was beneficial for their communication skill, helping them to learn more about science and increased their enthusiasm to learn and discuss the scientific information. although the preparation process might take more time and need to hard work for teachers, we concluded that the drama methods as a new tool to increase of participant's interest might be proposed for students in higher education. laboratory-based performance assessment in medical education: an opportunity for connection between scientists and medical students h. tuncel cerrahpasa medical faculty, istanbul university, istanbul, turkey number of medical students who interested in basic medical sciences is declining and medical sciences literacy is falling, it is crucial to develop ways for students and scientists to connect. students need to know that science is an intensely human endeavor, and scientists need mechanisms to bring that truth to the community at large. based on continued interest and experience on the part of faculty, and on student feedback, the development of a more effective and stimulating interactive learning tool was undertaken. an in-depth knowledge of laboratory medicine principles is vital to all practicing physicians. great variation exists in the ways that medical students learn the principles of laboratory medicine. there are a number of programs for electronic media that emphasize laboratory-related skills. some of these are appropriate for medical students in the clinical years. programs that teach skills in common laboratory procedures, such as interpretation of peripheral blood smears and microscopic examination of urine sediment, have been shown to improve student performance. to ensure that important principles are addressed, medical schools should establish goals and objectives specifically related to laboratory medicine and experiment with optimal teaching and assessment methods. we also hope that this study will inspire dialogue among primary care and specialist physicians as to the proper degree of education in this area. ideally, it will encourage scientific studies that address evidence-based possibilities for improving critical laboratory medicine educational outcomes, that is, the training of physicians who optimally use laboratory diagnostics and therapeutics. engaging medical students in scholarly scientific activities and producing clinically competent and research-oriented medical workforces are essential demands, particularly in developing countries. an experimental special study module for medical undergraduate students: learning western blot analysis and detection of b-actin protein expression in tissue and cell culture samples learning, introduce basic principles of laboratory research and to present the results.b-actin is one of six actin isoforms which is mainly expressed in all eukaryotic cells. western blotting is a widely used laboratory technique to determine specific proteins and to evaluate protein expression in tissues and cells. in our study, different concentrations of rat spleen homogenates ( , , lg/well) and lg protein/well of human lung and ovary cancer cell lysates were used. the proteins were seperated by % sds-page, transferred to pvdf membrane, incubated with specific b-actin antibody and then with hrp-conjugated antibody. protein bands were detected with ecl and densitometric analysis of proteins was quantified by imagej software. differences in protein band intensities were compared using one way anova.a value of p < . was considered statistically significant. we detected b-actin expression in rat spleen homogenates, human lung and ovary cancer cell lysates, as a kd protein. the protein band intensities were in correlation with protein concentrations. the highest concentration resulted in the highest signal intensity in rat spleen homogenates.b-actin level was higher in ovary cancer cells than in lung cancer cells, although both proteins were loaded equally. the feedback showed that students were very satisfied with this laboratory ssm. they developed their independent study skills, planned a research, worked in a laboratory, learned and performed a technique, western blotting. the hands-on experience is very important for medical undergraduate students for their future scientific career. three-dimensional structure of truncated human kv . voltage-gated potassium channel kv . belongs to the ether-ago-go family. it has been proved that its mutants are involved in development of neurological diseases and some types of tumor. directed drug design needs knowledge of details of the threedimensional channel structure. the members of the kv - subfamilies are characterized by extremely long n-and c-terminal intracellular tails, which possess a number of structural domains. the n-terminal pas domain in kv plays an important role in activation, and is thought to alter the rate of deactivation, possibly by binding at or near the s -s linker at the inner mouth of the pore. here we present an improved d structure of the truncated human kv . channel, obtained by single particle em. this channel lacks its cytoplasmic pas domain but it still forms tetrameric particles. earlier we showed that the full length kv . channel is build according to the 'hanging gondola' type, and its cytoplasmic and transmembrane parts are connected by linkers. the cytoplasmic part includes the interconnecting pas and cnbd domains. deletion of the pas domain leads to the conformational change in the cytoplasmic part of the channel. for interpretation of the d structures we used homology modeling and molecular dynamics simulation. there are several templates available to the moment including eag domain-cnbhd complex of the mouse eag channel, full-length shaker potassium channel kv . , c-linker/cnbhd of zelk channels and others. but there are still no templates for many fragments that led to necessity of partial de novo modeling. analysis of molecular trajectory allowed estimating dynamical characteristics of channel, supposing interdomain interactions. results of the conducted investigation have a great interest at both the academic and the industrial levels. the objective of this task program is to enable students to gain scientific attitude and skills for them to be able to deal with the problems they'll confront in future research careers. it's been emphasized in various studies that medical students are keen on conducting scientific research, and it's been stated that they need to be supported in this respect, as they lack the adequate fund of knowledge. this study aims to share information throughout a -year performance of the research training program (rtp) conducted at ege university, faculty of medicine. rtp is an educational program applied in addition/parallel to the bachelor's degrees for establishing scientific thought, attitude, proceeding and knowledge of the willing medical students, and enabling them to adopt study skills. the dynamic program produced by the rtp committee in has been developing each and every year via feedback received from the students. an operating principles, a manual for advisor and an introductory booklet have been laid out. applications are being accepted at the end of first academic year, making announcement to the freshmen in advance. students are being admitted to the program, taking the assessment criterias into consideration. second and third year medical students attend the didactic part of the program during the terms devoted to special study modules. thereafter, they go through the project management phase, and accomplish their projects under supervision of a faculty member until their graduation. first graduates of the program, accomplishing their projects, received their certificates at the graduation ceremony of graduation. currently, there are students in total from all classes who perpetuate their studies within the program. an inventive training pattern of ege university faculty of medicine, rtp experience is being maintained as a dynamic process and successfully keeps the students advised of conducting scientific researches, cultivating scientific awareness. objectives: objectives selection and verification of blood collection tubes is an important preanalytical issue in clinical laboratories. in this study comparison with the reference glass tube of ayset plastic tubes containing separator gel and assessment for routine clinical chemistry laboratory testing in samples were aimed. materials and methods: thirty-four volunteers were included in the study. samples were taken into two different tubes by two experienced technologists according to the clsi protocol [tube : z (becton dickinson and company, franklin lakes, nj, usa); tube : ayset (lot , turkey)]. glucose, urea, creatinine, ast, alt, total-cholesterol, triglycerides and high density lipoprotein-cholesterol were analyzed subsequently (olympus au ) and randomised samples stored at - °c for and h. th hour sample was analyzed immediately after collection and accepted as the reference for the comparison of the other samples. a paired t-test and wilcoxon signed rank sum test were used to test the significance of differences between the reference tube and test tubes. results: the difference between the results of reference tube and test tubes for glukose, urea, creatinine, ast, alt, total cholesterol, tg and hdl-cholesterol at , and h were statistically no significant (p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , p = . , respectively). conclusions: no significant difference was observed between ayset tubes' results and the reference tube's results. e. akin c ß akir d€ uzen laboratuvarlar grubu, istanbul, turkey insulin resistance underlies the development of obesity which is a global health problem. obesity is a main concern of scientists because it's associated with type diabetes, hypertension and some cancers. recently, inflammation centered mechanisms are deeply investigated as well as the effects of anti-inflammatory diets which are highly rich in vitamins, minerals, fibers and healthy oils. these diets are proposed to inhibit or supress the secretion of the inflammatory mediators and also improve the intestinal microflora. the aim in this study is to highlight the increasing trend of publications in regard to insulin resistance and inflammation based obesity along with the effects of antiinflammatory diets used for its treatment in the last decade. we performed a pubmed search with key words of 'obesity and insulin resistance and inflammation' ( / / - / / ) (search # ). besides, we performed another search with key words of '(anti-inflammatory diet or dietary supplement) and (obesity or insulin resistance)' (search # ) to highlight the value of anti-inflammatory diets and dietary supplements in combating inflammation based obesity and insulin resistance. search # revealed articles; of these were clinical trials, were observational studies. human studies were while animal studies were . overall, there were review articles and meta-analysis in the field. search # revealed articles of which were clinical trials, were review articles, were meta-analysis. human studies were and other animal studies were . the relationship of metabolic diseases with a low grade inflammatory state has opened a new area of research to understand the consequent causes of inflammation in the human body. the increasing scientific data in the field indicates that antiinflammatory diets may serve as powerful tools to solve the inflammation and the consequent insulin resistance and obesity. medical and biological illustrations for life sciences education: is 'a picture worth a thousand words' in visualizing medicine and science? medical and biological illustrations (mbi) convey complex ideas with just an image and they are powerful intersections of science and art. the clarification of complex pathways via illustrations can be effective means in education as they help the student to visualize the biomolecular world and understand the mechanisms. our aim is to illustrate how a mbi is developed over the example of mechanism of insulin action, via the phosphoinositide (pi) kinase-protein kinase b (akt) pathway. organising one's thoughts and clarifying relationships and then using the optimal complexity level to illustrate the pathway most clearly are the basics of mbi. thus, we made a thorough investigation of insulin mechanism on glucose uptake in skeletal muscle and adipose tissue; a biochemical process that includes insulin receptor (ir), ir substrate, pi kinase, pi-dependent kinase and akt. then, we found the d structures of molecules via protein databanks and accordingly created drawings and diagrams of each component in both molecular and macrolevels by adobe photoshopÒ software. graphics tablets and a compatible pc were also used in the production phase. the use of computer hardware/software enables unlimited detail in images and provides the flexibility that classical drawing techniques can not. thus, the final diagram clarifies the underlying molecular mechanisms of a biochemical pathway along with the physiologic actions. recent improvements of computer technology have resulted in the creation, and reproduction of high-quality lower cost medical art. mbi's can be used in education to explain concepts/pathways to students to enhance learning. similarly, mbi's are great tools to show mechanism/procedures to enhance patients' understanding of their medical condition. considering their unquestionable contribution to education, research and patient care, the creation of mbi's should be promoted as a graduate level course and the discipline should be represented at academic level. biochemistry is a compelling field with broad applications in many disciplines like medicine, dentistry, pharmacy and bioengineering. biochemical research increasingly combines ideas from genetics, molecular biology, etc. and collaborates with many disciplines. our objective is to conduct a scientometric analysis of the last decade's postgraduate theses in the field of biochemistry (ptb) in regard to number, collaborations, subject area distribution, etc. to discover the characteristics and trends in turkey. we searched the turkish higher education council's theses database ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) which includes master of science (msc.), doctorate (ph.d.) and specialization (s) theses in all disciplines. an electronic search with the keyword of 'biochemistry' (in the thesis subject area) was conducted, thus it brought all theses either in the biochemistry discipline or theses in other disciplines but have a biochemistry component. we performed data cleaning and further quantitative analyses in excel. we also executed word count analysis on the titles of theses to derive the main subject areas in ptb. of the total of ptb ( s, msc, phd theses) . % was in natural sciences while . % was in health sciences. the theses output-growth measured by the compund annual growth rate was % over the -years. the top clinical disciplines in collaboration with biochemistry were pediatrics, surgery and cardiology, and the top science disciplines were biotechnology, bioengineering and biology. oxidants-stress and antioxidants ( ), endocrine-metabolism ( ) and enzymology ( ) were the top research areas in ptb, followed by genetics ( ) and cancer ( ). scientometrics is a powerful tool to understand the direction of science and research. our ptb analysis indicated that prominent areas like stem cell, biosensors, geriatrics are somewhat lagging in turkish biochemistry research while postgraduate education and research in total is growing fast with sound collaborations. the st turkish in vitro diagnostic symposium evaluation objective: in vitro diagnostic (ivd) medical laboratory tests and the equipment are closely related the public health, patient safety and the safety of all who utilize these tests. it depends on auditing of the process from the production to the consumption of these materials, that they do not pose a risk to individuals and society. upon these basic requirements; 'turkish in vitro diagnostic symposium: medical laboratory tests' was held in february , organized by the cooperation of turkish biochemical society branch of izmir, and dokuz eylul university health sciences institute. it was intended to shed light on some questions such as, what is the place and importance of ivd in turkey? what are the responsibilities of educational institutions?, what is the role of ministry of health? to put across the conditions of preparing a substructure that may provide achieving success in producing ivd medical devices and in this sector, in our country. material-method: invited speakers attended the symposium, along with the participation of both as lecturers and attendees; ministry of health, turkish standards institution; representatives of manufacturer enterprises; representatives of enterprises manufacturing in turkey; scientists conducting considerable researches on health technology; students and representatives from some of the non-governmental organizations. in addition to the presentations, gathering up the written opinions of the participants, a report was prepared. results: the symposium that lasted for days was realized with participants in total, of which from universities; representatives of their companies; from chamber-institute-public establishments and of which from public hospitals. of the participants were from izmir, of them were coming from out of izmir. conclusion: at the end of the symposium, % of the participants gave feedback. among the feedback selected; . % of the participants evaluated the symposium overall, as successful. . % of them found the symposium successful with regard to its scientific content. their feedback were . % positive in terms of the symposium's contribution to the situation assessment on ivd in turkey, and . % of them stated they would consider participating in the second of the ivd symposium if it is to be organized. perceptions of molecular life science master's students on their scientific and academic competencies and prospective plans for professional development the master's education (me) in molecular life sciences (mls) is aimed at strengthening the knowledge and skills base of the young scientist, preparing him for the competitive academia/industry positions. the rapidly developing pace of science and research forces the master's student (ms) to play a central role in monitoring and guiding his scientific education and professional development (pd). thus, the aim of our study was to examine the perceptions of ms of mls, regarding their scientific and academic competencies. with this data, we planned to analyse if this awareness channels ms to take action and/or matches with their prospective plans. we developed a item online survey with sections (demographic data, current data-contributions of me, competencies and prospective data-action for pd, future plans) and distributed it via e-mail to various postgraduate institutes. at the end of the -day period, ms students (in the thesis phase) answered the questionnaire (female: . %, male: . %). the most highly rated activities that contributed to their scientific knowledge and skills gained in the me were laboratory work ( %), visits with their mentors ( %) and theoretical lessons ( %). ms expressed low levels of sufficiency both in theoretical scientific knowledge and laboratory skills (only % and % sufficiency, respectively). communication skills ( %) and team work ( %) were rated as the highest professional competencies followed by literature search and research planning (both %). it was striking that ms perceived themselves as quite insufficient in scientific writing ( %), data analysis ( %) and project writing ( %) while proficiency in english ( %) was the first area they wanted to take action. despite their perceptions of insufficiencies in many areas, a majority ( %) wanted to continue to phd education. these and similar surveys may lead to an increase in selfawareness in ms and the data may contribute to the revision of me. the report of the st turkey in vitro diagnostic symposium results the following aspects and suggestions took place in the results report prepared after the symposium: about the national ivd-tc r&d, production, forming quality assurance and innovation strategy and policy the cooperation of the university and the industry is not sufficient most of the industrialists cannot take enough advantage of the support provided by the institutions like tubitak, the ministry of science, technology and industry and the ministry of development the statistics on the ivd-tc in turkey should be carried out as soon as possible national standards should be determined in parallel with the international standards the vat rate of the exported raw materials that would be used in ivd production should be decreased. about the education and training, the job titles and positions the related graduate programs, which would focus on all steps of the whole life cycle related to the ivd-tcs one by one, are not widespread there is no 'postdoc' application in turkey. 'postdoc' staff is needed for insufficient component human resources the lecturers should not be restricted to one discipline only graduate programs on laboratory medicine are needed to be established and spread, in order to train component labor specified on the ivd-tc about research and development there are almost no researches related to product development. this should be associated with the education and training institutions about the research centers currently, there is a real infrastructure on health technology in turkey, but there are difficulties in its instituonalism the insufficient cooperation of the university and the industry does not allow the inventions to turn into products the cooperation supports of r&d, being restricted to tech-nopark and r&d centers, are open fields for improvement p-edu- phd training in medical education: career profiles and satisfaction levels of graduates from dokuz eylul university graduate school of health sciences this survey was carried out within the scope of special study modules that is entitled 'phd training in medical sciences' by a group of medical students in deu. the purpose of the survey to investigate the members of health sciences that have successfully completed their phd training in terms of the levels of satisfaction and the status of their career. from this scope we generated two hypothesis: we expected that graduate phd graduates are mostly involved in academy and find their satisfaction levels at moderate level as to phd education. the study was designed as cross-sectional. we reached phd graduates who had graduated from deu graduate school of health sciences between and from different departments via e-mail. the survey was included questions, which were prepared in the light of the existing literature. among the phd graduates, ( %) participated in the study. through this survey, perception of phd students on supervisors' scientific and educational abilities, opinions on phd training, productivity of phd training, number of articles published, their position and related satisfaction levels after graduation were investigated. according to the results, more than half of the graduates (% . ) are well satisficed from the education they had taken. beside this, interestingly we found that % . of graduates prefers staying in the academic positions and % . of them sustains their communication with their supervisors. in conclusion, most of phd graduates were contented with phd training and their career profiles. as a result of this survey, we produced a novel and precise contribution to the literature. in a further study, this survey may extend to other parts of turkey and compile the results in order to get more accurate and inclusive data. phd is an international degree that is reached by conducting an original research after finishing bachelor or master's degree. doctoral degree (phd) can open the door of academic career; on the other hand, a person with a doctoral degree is equipped to carry out important work in research, industry, or public sector. today, gradually increasing number of phd students have brought some problems in phd training. the purpose of this study is to investigate and review activities that have been done by the following international organizations: orpheus: (organization for phd education in biomedicine and health sciences) eua-cde: european university association-council on doctoral education. febs education committee: federation of european biochemical societies these three organizations have done workshops on phd training to pay attention to the following points: *a phd student must take some courses and trainings outside her/his institute, should not be limited to the institute. *the phd training programme should include transferable skills courses. *clinicians, if involved in phd training, should be given free time from their clinic duties. *with regards to potential problems with the supervisor, the institute should provide the student an advisory system. *students should be encouraged to participate in the management of doctoral programmes in the institute. *the students should be given be opportunity to select their own supervisor (thus their thesis area). the phd training has gained quality thanks to these organizations. it is advised that graduate school of health sciences should follow the recommendations and report from these organizations. itsn and itsn are genes encoded adaptor proteins with multiple isoforms participating in clathrin-mediated endocytosis (cme), mapk signaling and reorganization of actin cytoskeleton. changes in itsns expression can lead to different neurodegenerative disorders and cancers. to date little is known about regulation of itsn genes on posttranscriptional level. the aim of our work was to predict and experimentally confirm target sites for micrornas that could potentially regulate itsns expression. using web servers we analyzed utrs of short and long isoforms of human itsn mrnas and found conservative target sites for mir- , mir- , mir- , mir- / , mir- , mir- and mir- in utr of itsn -s, predicted by servers, mir- predicted by servers for itsn -l, and mir- , mir- / , mir- , mir- and mir- predicted by - servers for itsn -l. to elucidate potential impact on cme, mapk signaling and actin cytoskeleton regulation by these mirnas we performed enrichment analysis by diana-mirpath server and found that mir- , mir- , mir- / , mir- , mir- and mir- were highly enriched for all analyzed pathways. using regrna . and scan for motifs services we predicted types of different regulatory elements in utr of itsn and itsn : k-box and brd-box, musashi binding element for rbps musashi and musashi , gu-rich element (gre) and au-rich elements (are) that regulate mrna decay. to confirm itsn -s regulation by micrornas we cloned utr of itsn -s into luciferase reporter vector and transfect hek cells by this construction and mir- a, mir- a and mir- a. for mir- transfected cells, we found - % decrease of expression of itsn -s utr-bearing construction. for other mirs we did not obtain strong reproducible effect in luciferase assay. these data may confirm mir- target site in utr of itsn -s mrna but needed additional research. objective: stroke is an acute neurological disorder that is mostly caused by ischemia in central neural system. % of stroked patients lose their life in year, and remaining / of the living patients continue to their lives as dependent to others. nihss and mrs are two scales which are used in prognosis studies because they can show stroke intensity and after stroke functional recovery. microrna's which have effects on transcription and posttranscription gene regulations are small rna molecules. their roles have been investigated on pathophysiology and treatment of diseases. in this study, it was aimed to detect changes in blood serum levels of mir- a, - , - , b, - , , - a and let- f of ischemic stroke patients and to investigate role in predicting prognosis methods: patients diagnosed by acute ischemic stroke admitted to neurology service of g€ oztepe hospital and healthy individual were included in the study. after stroke patients' blood samples were taken periodically in st day, st week, and rd month, and at the same time nihss and mrs scores were determined. set mirna blood serum levels were measured by rt pcr results: when compared to the control group, we found that after stroke st day peripheric blood levels of mir- a,- ,- a and let- f were significantly low; when st week and st day serum records were compared there was a significant increase in mir- level; and when st week and rd month records were compared we noted that there was a significant increase in mir- a,- and let- f levels. from prognosis point of view; after ischemic stroke measurements showed that mir- b in the st day, mir- a and mir- in the st week showed positive significant correlation with rd month mrs scores (p = . , p = . , p = . , respectively) conclusion: according to the outcomes of this study, after stroke st day mir- b, st week mir- a and st week mir- levels can be stipulated to use in predicting patients' rd month prognosis p- . . - inhibitory rna aptamer against lambda ci repressor showed the transcriptional activator activity s. ohuchi, b. suess tu darmstadt, darmstadt, germany because of the variety of functionalities on gene regulation and the easiness of molecular engineering, functional rnas are promising parts for the construction of genetic circuits. artificial affinity rna, or rna aptamer, is one of such genetic parts. in the previous study, an inhibitory rna aptamer against a repressor protein, tetr, was developed as a transcriptional activator [ ] . the expression of this aptamer abolishes the repressor activity of tetr, resulting in the elevated gene expression under the control of tetr. because of simplicity of the mechanism, similar transcriptional activators can be generated by using rna aptamers against other repressor proteins. here, we examined the generation of an activator based on an rna aptamer against one of the most frequently applied repressor proteins, lambda phage ci. in vitro selection (selex) was performed targeting a recombinant ci protein employing an rna pool containing -nucleotides of a random region. after rounds of selex, the pool rna showed the affinity, as well as the inhibitory activity, against ci in vitro. then, rna aptamers with the transcriptional activator activity were screened from the enriched pool in vivo employing a reporter plasmid on which the expression of a reporter gene, lacz, is repressed by ci. when the variants of the rna pool were transformed to e. coli cells harboring the reporter plasmid, about half of the transformants showed the elevated reporter expression. interestingly, all of these desired rna clones shared the same sequence. quantitative analysis indicated that -fold induction of the reporter expression was achieved upon the aptamer expression. our results suggested that diversity of artificial transcriptional activators can be extended by employing rna aptamers against repressor proteins to broaden parts for the construction of genetic circuits. [ ] hunsicker, et al. ( ) an rna aptamer that induces transcription. chem. biol., : - . p- . . - microrna expression signatures between non-atherosclerotic plaque and atherosclerotic plaque in cad with humans, and parallels whole blood rnas and represent a new important class of gene regulators. the present study was designed (i) to investigate the mirna expression profile in human atherosclerotic plaques from peripheral arteries aorta as compared to non-atherosclerotic left internal mamary artery (lima); (ii) to examine the expression levels of mirna in whole with correlation mirnas of aorta tissue. material and methods: thirty-one patients with cad were enrolled in study. lima and aorta tissue samples were obtained during coronary artery bypass surgery. whole blood samples were collected before cabg surgery. each patient with cad was obtained from whole blood, aorta and limas tissues. these tissue samples were immediately soaked in rnalater solution and homogenized using a magnalyser. the rna was extracted using the trizol reagent and the mirneasyÒ mini-kit. the expression profiles of mirnas were evaluated using highthroughput qrt-pcr. results: we found that mir- - p was expressed only in aorta. mir- - p and were expressed both aorta and whole blood. mirnas were significantly up-regulated in aorta when compared to lima tissue (fc > ). mirnas were significantly down-regulated in aorta compared to lima. conclusion: in conclusion, our study suggests that mir- - p, mir- - p and might be a potential for cardiovascular disease development. also mir- - p and might serve as novel non-invasive biomarkers for cad p- . . - mir b regulates cell proliferation and colony formation in pancreatic ductal adenocarcinoma n. gurbuz, e. isildar due to the strong metastatic potential, pancreatic cancer has the highest mortality rate of all major cancers. therefore, the investigators are in urgent need of developing the new alternative therapeutic approaches for the prevention of pancreatic cancer. mirnas, small noncoding rnas, regulates as an inducer or inhibitor in expression of key mediators related molecular mechanisms in cancer promotion. to investigate the effect of mir b on pancreatic ductal adenocarcinoma cells, we performed the cell viability and clonogenic assays by mts and crystal violet dye, respectively, in panc- and miapaca- cells transfected with mir b mimic. our data revealed that mir b led to decrease the cell viability depending on enhanced mir b doses, which are , , and nm, as the ratio of % , , and , respectively, in miapaca- cells and as the ratio of % , , and , respectively, in panc- cells compared with control condition of each cell. this inhibition mediated by mir b was also obtained in colony formation both of pancreatic cancer cells. when the induced effect of mir b on the death of pancreatic cancer cells was compared with gemcitabine, which is currently used as a clinical drug for pancreatic cancer patients, we determined that mir b was more effective than gemcitabine. based on our findings, it is clearly shown that mir b serves as a tumor suppressor in pancreatic ductal adenocarcinoma cells. we strongly believed that mir b gene therapy might be more effective and targeted approach than classical gemcitabine therapy for pancreatic cancer patients. *this work was supported by tubitak (the scientific and technological research council of turkey) grant s . breast cancer is the most common cause of cancer death in women. trastuzumab is a therapeutic agent frequently used against her + breast cancers, which has role in approximately % of invasive breast cancers. with the discovery of their activity in cancerogenesis, micrornas (mirnas) have become potential candidates to mediate therapeutic actions by targeting genes that are effective in drug response. recent studies have showed that mirnas are induced by targeted therapies. in this study, we aim to find out mirna-mediated mechanism, which is driven by common trastuzumab responsive micro-rnas in her + breast cancer. for this purpose, the common trastuzumab responsive mirnas were determined in treated bt and skbr cells by microarray profiling. two datasets were intersected to find out common mirnas for both cell lines. the overlapping predicted targets of common mirnas were provided by two different mirna-target prediction databases and then a mirna-gene network was built in cytoscape by using networkanalyzer plugin. the most shared target genes were chosen to be analyzed in the ebi-embl gene expression atlas for their expression patterns in breast cancer. common mirnas were found to have overlapped targets in two target prediction algorithms that were used to build the mirna-gene regulatory network. overlapped targets were determined as the most shared genes in the mirna-gene network. expression pattern of each shared gene in the gene expression atlas showed that out of the most shared target genes were strongly dysregulated in several breast cancer types. our results suggest that mirnas might show a common response to the targeted therapies and network analysis can be profitable to have a better explanation of the regulatory mechanisms between drug responsive mirnas and their target genes. revealing the mirna-potential target interactions might provide novel key players that mediate the mechanisms of action in drug treatment. chronic myeloid leukemia (cml) is a clonal disease of primitive pluripotent stem cells that identified with a specific t( ; ) reciprocal translocation that encoding bcr-abl oncoprotein. resveratrol (res) is a natural phytoalexin found in grapes and induces apoptosis, erythroid differentiation and autophagy in leukemic cells. micrornas are small, single strand, non-coding rna molecules that regulate post-transcriptional gene expression via disrupting the stabilization of target transcripts or inhibiting protein translation. in our study we aimed to determine cytotoxic effect of res in k human cml cell line and to evaluate the expressions of mirnas that are associated with genetics of leukemia after treatment with res; to investigate target genes of mirnas which show significant expression alterations and molecular mechanisms of res treatment. k cells were treated with lm (ic dose) res during h and cytotoxicity was evaluated by using wst- assay. the rt-qpcr is used for mirna and gene expression analysis. results showed that; res up-regulated tumor suppressor mir- - p level . fold and significantly downregulated hdac gene expression (p = . ) and upregulated p / sqsmt gene expression (p = . ), according to the control cells.p /sqstm interacts with lc and plays role as a critical player in the autophagic degradation of the bcr-abl fusion protein. our findings showed that resveratrol acts as a hdac inhibitor targeting hdac and p /sqsmt gene expression level. treatment with hdac inhibitors results apoptosis, cellcycle arrest, cell differentiation, anti-angiogenesis and autophagy. downregulation of hdac provides post-translational modification for expression of tumor supressor genes and leads to cell cycle arrest and increases apoptosis. these results could be linked to hdac dependent induction of gene associated with autophagy like p /sqsmt and resveratrol could be a therapeutic candidate as a hdac inhibitor for cml treatment. the mirna used in this study are hsa-mir- , hsa-let- a, hsa-mir- b and hsa-mir- . thereafter, we bought pre-mirnas and their mirnas commercially. we apply them to the a cell line in different combination and different concentrations. these mirnas applied solely onto cells or in combination as; four of them, let + , let + b, let + , + b, + , + let + b, + let + b. the cell viability was detected by wst- kit in a well plate elisa reader. cells were seeded as per well, mirnas incubated with cells for h in an appropriate atmosphere. according to our results some combinations and mirnas didn't alter viability, however + b and + combination increased the cell viability dramatically. on the other hand let + b and only applications decreased the cell viability. the other applications' viability results are not different from the control cells significantly. in this study, we used a cell line also called non-small lung cancer (nsclc) cell line and possibly effective mirnas on lung cancer. it is important to exhibit the mirna combinations should be effective on cancer cells' viability. the prospect combinations were determined which is crucial to develop new strategies for cancer treatment. competing endogenous rnas (cernas) act as molecular sponges for the same pool of micrornas through their mirna response elements (mre), titrate mirna levels and thereby regulate gene expression post-transcriptionally. smad interacting protein (sip ), a member of the zeb family is a regulator of epithelial-to-mesenchymal transition (emt) program, which is active during embryogenesis and tumor invasion and metastasis. hence, we investigated the regulation of sip by cernas in hepatocellular carcinoma (hcc) cells. among hundreds of sip cernas listed at competive endogenous mrna database (cerdb), transcripts (pten, zeb , ptch , creb , acvr b, enah, robo , erbb , e f , foxo , rictor, klf , ets , cdk ) sharing at least common mre sites with sip were selected and their expression in hcc cell lines were determined by qrt-pcr. ets was found to be highly correlated with sip in hcc. furthermore, repressing sip expression by shrna in hcc cells resulted in decreased expression of ets , pten and zeb . our results suggest a possible bidirectional and post-transcriptional regulation of sip and its cer-nas in hcc. a meta-analysis for the identification of common microrna signatures in colorectal cancer n. sahar , , n. belder, h. ozdag biotechnology institute, ankara university, ankara, turkey, comsats institute of information technology, islamabad, pakistan colorectal carcinogenesis (crc) has quite frequent incidence and mortality rates worldwide, despite being studied for decades now. new biomarkers are needed to be identified in addition to the existing ones, due to heterogeneous nature of this disease. the regulatory molecular machinery of a cell, including micrornas (mirnas), contributes to this heterogeneity upon aberrant expression. herein, for a mechanistic understanding of differential gene expression in crc tissue we analyzed mirna expression profiles of crc tumors against normal colorectal mucosa samples, using raw data from e-mtab- and e-geod- (affymetrix microarrays), and gse and e-mtab- (agilent microarrays) datasets obtained from gene expression omnibus and arrayexpress. raw samples were normalized (different platforms separately) using quartile normalization in brb-array-tools. differential expression of mirnas was identified using cut-off values of p ≤ . , fold change ≥ . and stringent false discovery rates. mirtarbase and mirwalk . databases were explored to identify validated targets. we found thirty (including mir- and mir- ) and thirteen (mir- , mir- , mir- , etc.) mirnas commonly upregulated and downregulated respectively, in both affymetrix and agilent microarray results. predicted targets of these mirnas frequently belong to pathways related to cancer like b-catenin, phosphoinositol- kinase, and transforming growth factor-b, to name few. moreover, the target genes were significantly enriched in clusters related to cell cycle, cell differentiation and regulation of apoptosis. these promising results will further be compared with differentially expressed gene profiles from a cohort of turkish crc patients. hence the integrated study will refine the panel of diagnostic and prognostic crc markers. hsa-mir-x modulates motility and invasion in triple breast cancer cell line s. noyan , h. g€ urdal , b. g€ ur dedeoglu biotechnology institute, ankara university, ankara, turkey, department of pharmacology, ankara university, ankara, turkey breast cancer is a heterogeneous disease and expression levels of certain receptors have demonstrated subtypes which characterize clinically distinct breast tumors. a triple-negative phenotype lacks expression of er, her and pr and is known as basallike carcinoma. micrornas are a class of small non-coding rnas that participate in the gene expression in many biological processes. e-cadherin is an important mediator of adhesion in epithelial tissues, and loss of e-cadherin can play a critical role in tumor invasive behavior. another key player of cell integrity pip ( , , ) triphosphate is generated at the leading edge of the cell and leads to cell polarization. pip is generated by hydrolysis of pip ( , ) bisphosphate, which is synthesized by pip k . any dysregulation in these molecules may support the invasive behavior of the cells. the aim of this study is to find out the role of mirna precursor (hsa-mir-x) in invasion and motility in triple negative breast cancer cells. in this study a triple-negative breast cancer cell line bt- was transfected with hsa-mir-x or scrambled control sirna. to check its role in motility and invasion, wound healing and invasion assays were performed respectively. cell invasion was monitored over a period of h by xcelligence real-time cell analyzer using a double-plate and measuring impedance-based signals. additionally emt markers were analyzed by qrt-pcr to explain the molecular mechanisms beneath motility and invasion. we observed that cell motility and cell invasion diminished after transfection of bt- cells with mimic for hsa-mir-x. furthermore, qrt-pcr experiments indicated that transfection of hsa-mir-x decreased the expression level of pip k c while increasing the e-cadherin expression level. wound healing and invasion assays together with qrt-pcr results support the role of hsa-mir-x in cell motility and invasion. this process might be explained via e-cadherin mediated met or gsk- -beta related inhibition of invasion. expression level of five micrornas as diagnostic markers in glioblastoma situated in the main brain lobes, but can also be found in other brain regions. while microrna (mirna) as non-coding rnas, play a crucial function in the post-transcriptional regulation of gene expression by mrna degradation or translational repression. in the present study, we aimed to investigate the contribution of gene expression of the five mirnas and to unravel their role in brain tumor cell lines, the mirnas to the risk of gbm tumor. the five gbm cell lines (crl- , crl- , crl- , crl- and htb- ) were evaluated with non-malignant (normal) brain cell line (hcn- ) . determinations of expression level of five mirnas (mir- , mir- , mir- , mir- , and mir- ) were evaluated by monitoring quantitative rt-pcr (qrt-pcr) technique. the expression levels of four mirnas (mir- , mir- , mir- , and mir- ) were significantly decreased while the expression level of mir- was increased in gbm cell lines according to hcn- cell line. consequently, these five mirnas can potentially be used as biomarkers for gbm tumor; further studies are mandatory to a better understand and confirm our preliminary findings. background: noncoding rna are known to be crucial molecules with diverse regulatory roles in neural oncology and neurodegenerative disease. the recent study suggested that lncrna anril play role in the development of neuroblastoma and alzheimer disease via binding disease-specific micrornas. material and methods: we used lncrnadisease, hmdd v . , mir disease to predict lncrna-and mir-associated disease in our study. in addition, we utilize tardetscan to search lncrna-mirna interaction. results: disruptions of lncrna anril expression (also named as cdkn b-as, locus cdkn a/b (ink /arf), chromosome p ) have been associated with the development of neuroblastoma and alzheimer disease. here, we predicted interactions between noncoding transcripts encoded by locus cdkn a/b and their molecular partners -microrna. anril can act as decoy while containing sequences that mimic mirna target sites to titer these mirs away from their primary targets thereby act as molecular sponge. using targetscan . , we predicted target sites for hsa-mir- -p/ -p/ -p/ -p/ -p/ -p and hsa-mir- - p/ in anril utr. then, we used hmdd v . and mir disease databases to define if any of these mirs participate in alzheimer disease and neuroblastoma. according to both databases, mir- is implicated to alzheimer disease and mir- to neuroblastoma. as soon as anril participate in the development of both abovementioned disorders and can have microrna sponge activity, it could potentially positively regulate mir- and mir- targets by competing with them for micro-rna binding sites thus restoring the expression of target genes. in our further research we plan to experimentally validate predicted microrna sites in anril utr. conclusions: we predicted sites for mir- and mir- in utr of anril lncrna that could uncover its possible sponge activity in the development of neuroblastoma and alzheimer disease. aim: matrine excracted from saphora flavescens root and demonsrated that indicates pro-apoptotic and anti-proliferative effect in many types of cancer. acute lymphoblastic leukemia (all) is an acute form of leukemia, or cancer of the white blood cells which characterized by the overproduction and accumulation of lymphoblasts. mirnas play important roles in deregulated cell death mechanisms. we aimed to investigate the effects of critical mirnas during the development of matrine resistance on all cell line ccrf-cem. material-method: ccrf-cem cells were treated with different ( . - mg/ml) concentrations for h and cell viability measurements were carried out with xcelligence device to determine the cytotoxic effects of matrine. mirnas were extracted from treated and untreated ccrf-cem cells using the mirna isolation kit. cdna was synthesised using allin-one first strand cdna synthesis kit. expressions of selected mirnas were analysed with miprofiletm custom mirna qpcr array using the applied biosystem fast real-time pcr system. results: according to the cytotoxicity assay, it was determined that 'treatment with increasing concentrations of matrine, decreased the viability of the ccrf-cem cell line. expression levels of different mirnas were studied for indicated passages in two replicates. our results showed that hsa-mir- b- p (- , fold, p = . ), hsamir- - p (- , fold, p = . ), hsa-mir- a- p (- , fold p = . ), hsa-mir- a- p (- , fold, p = . ), hsa-mir- - p (- , fold, p = . ), hsamir- b- p (- , fold, p = . ), hsa-mir- b- p (- , fold, p = . ), hsamir- b- p (- , fold, p = . ), hsa-mir- - p (- , fold, p = . ), hsamir- a- p (- , fold, p = . ) expression were decreased during the development of matrine resistance. conclusion: these data suggested that especially hsa-mir- b- p plays a critical role in the matrine response. ericd (e f -regulated inhibitor of cell death) is a newly found lncrna. it is located at q . . it has two exons and its transcript size is bp. ericd is regulated by e f (transcription factor ) and modulates the cellular response to dna damage. arid a is a family member of the at rich interaction domain (arid) dna-binding proteins that participate in diverse biological processes like development, cell cycle control, chromatin remodeling and epigenetic regulation. both, ericd and arid a have just opposite roles in apoptosis in case of dna damage indicating a probability of reciprocal interaction between each other. till now, there is no work related to the interaction between lncrna and arid a in cancers. herein we try to find a probable interactive role between these in cancers. in this study, different cancer cell lines, osteoblast cell line and different types of normal human tissues rnas were selected for expression analysis of ericd and arid a. after rna isolation, cdna was converted from their rnas. expression profile analysis of ericd and arid a in different cancer cell lines and normal tissues was done using imagej program for semiquantitative and (-ddct) method for quantitative rt-pcr. among used cancer cell lines, ericd was highly expressed and arid a had lower expression in u- os (osteosarcoma), a- (glioblastoma) and a (lung cancer). at the same time, ericd expression was lower and arid a had high expression in hfob . (osteoblast cell line) and normal tissues like brain and lung . both ericd and arid a are cell cycle regulated and are commonly regulated by e f. they have just opposite roles in apoptosis during dna damage. these two genes have a probability of reciprocal interaction between each other in cancer. our results indicate that both ericd and arid a might have opposite roles in lung cancer, glioblastoma and osteosarcoma. ericd and arid a might act as cancer promoting and tumor suppressor genes respectively in these cancers. the importance of mirna expressions in infertilty implantation process is controlled with endometrium, factors secreted by the embryos and in accordance with these factors embryo and/or endometrium via receptors on. more than human microrna (mirnas) that are small noncoding rnas were shown to play an important role in intracelluler cycle regulation in both normal and pathological conditions. in this study we aim to identify mirnas and controlling molecules expressions in different time period of endometrium in fertile and infertile cases. the endometrial samples were taken from fertile and infertile patients in proliferation and early secretion periods. the samples are fixed and stained either with hematoxylen-eosin for morphological analysis or with immunohistochemistry for distributions of anti-dicer, anti-drosha, anti-eif a and anti-eif c. mir- - p, mir- a, mir- b, mir- - p, mir- , mir- a*, mir- , mir- a, mir- a, mir- b, mir- a/b/c were analyzed with qrt-pcr. while dicer immunoreactivity was detected weakly only proliferation phase of fertile group, this immunoreactivity were detected strongly in both proliferation and early secretory phases of infertile group. drosha immunoreactivitiy was also weakly detected in the proliferation phase of fertile group, it was moderately detected in both proliferation and early secretory phases of infertile group. eif a immunoreactivitiy was similar in each groups but there were a few differences between fertile and infertile group. eif c immunoreactivitiy was negative in all groups. mir- , mir- a* and mir- a were highly expressed in proliferation phase of fertile group, mir- a and mir- b were highly expressed in early secretion phase of infertile group. in conclusion, dicer and drosha immunoreactivities and different expression of mirna's were detected in all groups. implantation problems may be reason for different mirna expression which controlling with dicer and drosha in the infertile endometrium in both proliferation and early secretory phases. wheat is an important agricultural crop with an over . million metric tons harvesting capacity annually. drought and salinity are environmental stress factors that affect yield and quality of wheat, dramatically. there are different defense mechanisms against these stress conditions in plants. altering gene expression profiles by micrornas at post-transcriptional level is one of the most conserved mechanisms among plants. micrornas are an extensive class of noncoding rnas, approximately nucleotide length which regulates the expression of genes by binding to the -untranslated regions of specific mrnas. micrornas implicated under salt and drought stress have widely been reported in numerous plant species and wheat genomes in the last years; however, studies on einkorn wheat (triticum monococcum spp. monococcum) are not yet available. the goal of this study is identification of conserved micrornas from einkorn wheat using next generation sequencing technology and bioinformatic analysis. in this study, small rna molecules were extracted from pooled plant samples grown under normal, drought and salinity conditions. sequencing analysis revealed unique small rna sequences obtained from raw reads. after bioinformatic analysis based on comparative genomics approaches, we identified putative mature microrna sequences belonging to distinct microrna families. since chromosomal sequence data is not available for triticum monococcum spp. monococcum, we used available sequences from triticum urartu, a close relative, as template to extract precursor microrna sequences. of precursor sequences showing % homology to triticum urartu genome were analyzed for secondary structure prediction using mfold software. data provided in this study is critical to investigate transcriptional regulation of genes involved in stress metabolism in einkorn wheat. the role of vim-as , a natural antisense transcript, in cancer coding or non-coding transcript. in this regard, vim-as is nats located antisense to vimentin gene. in the present study, we aimed to determine tissue and cell line distribution of vim-as . materials and method: for the tissue expressions analysis, human total rna master panel ii (containing different human tissue samples) was used. total number cancer cell lines and normal cell lines included in the study. for the expression analysis rt-pcr and qpcr methods were used. results: as a result, expression levels of vim-as were found to be tissue specific. particularly, while vim-as was highly expressed in lung and thyroid gland tissues, its expression was not observed brain, stomach and adrenal gland tissues. also, vim-as was also found to be differentially expressed in cancer cell lines. vim-as expression was found to be lost in cal , pc , and hct and highly diminished a cancer cells. also, it is found to be highly expressed in bcpap, panc and beas b cells. discussion: results of the current study suggest that vim-as may have significant role in the regulation of vim gene in thyroid gland tissues, as it is highly expressed in both thyroid gland tissues and bcpap thyroid cancer cells. moreover, vim-as and vim axis can be involved in the formation of lung tumors because vim-as was highly expressed in normal lung tissues and beas b cells and expressed very low levels in a lung cancer cells. lung cancer is the leading cause of cancer related deaths in the world and approximately % patients with lung cancer ultimately die from metastatic disease. metastasis is the most dangerous step of cancer. in our recently published work showed that akt/nf-kb pathway is continuously active and induces cellular invasion and pten suppresses cellular invasion via inhibition of akt/nf-kb pathway. in this study we aimed to show nf-kb mediated induction of mirna expression can responsible for inducing nsclc invasion. we used chromatin immunoprecipitation (chip) assay kit for detection of tnf-a induced nf-kb mediated mirnas. therefore, h and pc cells treated by tnf-a ( ng/ml) for chip assay. chromatin regions, reading with chip-seq, were analyzed using bioinformatics tools. we also performed additional bioinformatics search to find nf-kb related mirnas which potentially take a role in nsclc invasion. we investigated the effects of mirna which determined at the bioinformatics analysis results on invasion using invasion chamber method. we found mirnas which potentially induced by nf-kb and related with nsclc invasion. our invasion results indicate that mir- a- p, mir- as- p, mir- , mir- , mir- - p, mir- q mimics can induce cellular invasion on h , mir- v, mir- h- p, mir- - p, mir- a- p, mir- as- p, mir- mimics can induce cellular invasion on pc . we also verified our results by qrt-pcr, because we want to sure that mirnas which can induce invasion, can also transcriptionally regulated by nf-kb or not. we found that mir- q, mir- a- p, mir- as- p, mir- , mir- in h , mir- - p, mir- a- p, mir- as- p, mir- in pc can induce cellular invasion by nf-kb. as a conclusion, our investigation indicate that nf-kb can induce nsclc invasion via mir- a- p, mir- as- p and mir- . (this study is supported by tub _ itak grand number s ). to understand of the molecular mechanisms of cellular invasion is very important for fight against cancer mechanisms and first step of invasion is emt. cells change phenotypical and genotypical in emt progress. in this study, we focussed on the explore molecular mechanism of tnf-alpha induced cellular invasion of nsclc. we use western blot, qrt pcr and mirna transfect methods for this purpose. we find that tnf-alfa treatment reduce the expression of pten and induce e cadherin expression in a cells. when we inhibit nf-kb activity by p targeted sirna tnf-alpha can not reduce pten expression means that tnf-alpha inhibits pten expression through on nf-kb. because tnf/nf-kb pathway change the transcriptional level of mir- as and pten untranslated region has recognition site for mir- as. therefore; we transfected a cells by mir- as. mir- as transfection leads to inhibition of pten expression. our results indicate that tnf-alpha mediated activation of nf-kb can inhibit pten expression on induction of emt through on mir- as. (this study is supported by tubitak grand nummber s ). introduction: the corpus luteum (cl) is an ephemeral tissue whose regulated secretion of progesterone is essential for maintenance and/or timely termination of pregnancy in rodents. our previous finding that cl of pregnant rats does not possess fas/ fasl system suggests that this tissue may undergo autophagic, but not apoptotic, cell death during regression. we here investigated the presence of autophagic system in cl and its any implications in rodent pregnancy and parturition. materials and methods: lc (-i and -ii) expression in cl was estimated by western blot analysis in comparison with progesterone secretion and luteal mass throughout pregnancy. lc was also tested by immunocytochemistry. functional implication of autophagy in this tissue was examined by local treatment with bafilomycin a (autophagy and v-atpase inhibitor, . pg/ . ml/ovary) on day of pregnancy. reproductive, biochemical, and morphological outcomes were evaluated. results: while the expression of cytosol-associated lc -i was not significantly altered throughout pregnancy, that of autophagosome-associated lc -ii increased significantly from day , showed a peak on day , and decreased on day (day of normal parturition). lc -ii / i ratio had positive correlations with steroidogenic activity and cell size. immunoreactive lc was found to be present in the cytosol of steroidogenic cells and showed marked aggregation in cells on day . in vivo treatment with bafilomycin a resulted in unaltered delivery, but in significant reductions in steroidogenic cell size and neutrophil infiltration compared to vehicle-treated control groups. discussion and conclusion: the ratio of lc -ii / i in cl was markedly up-regulated in the late phase of pregnancy. manifestation of this autophagy parameter was temporally matched with further structural and functional activation of cl. autophagy may contribute to activation, but not regression, of rodent cl and thus their female reproduction. apoptotic and necrotic effects of low dose bisphenol a in shsy y neuroblastoma cells b. ayazg€ ok, t. t€ uyl€ u k€ uc ߀ ukkilinc ß faculty of pharmacy, hacettepe university, ankara, turkey bisphenol a (bpa) is a commonly used chemical in industry to make plastics. 'low-dose' term has been expressed for the first time in studies with bpa in . the value of low dose bpa was received as < lm. in this study, matrix metalloproteinases (mmps) together with their tissue inhibitors (timps), involved in tissue remodeling after i- therapy, have been examined in patients ( m/ f) with ptc and ( m/ f) with ptc+ht. peripheral blood samples were collected just before and, subsequently, at days after i- administration ( . gbq). ptc+ht patients had positive titers of anti-thyroglobulin autoantibodies (tgab). the serum levels of tgab, mmp- , mmp- , timp- and timp- were measured by elisa. there were no significant changes in serum concentrations of mmp- , timp- and mmp- /timp- ratio after i- in the two groups. in ptc patients, i- administration resulted in an increase with % in timp- level (p = . ) and a reduction with % in mmp- /timp- ratio (p = . ). in ptc+ht patients it has been observed an increase with % in tgab level (p = . ), % in mmp- /timp- ratio (p = . ) and unchanged timp- serum concentration. tgab titers were positively correlated with mmp- /timp- ratio (r = . , p < . ). our data suggest that radioiodine therapy for ptc patients, but not for ptc+ht, modulates the balance of mmp- /timp- for anti-invasion and anti-migration by augmenting timp- . in criminal cases, the determination of the time of death of the bodies step in the analysis of events is making a big contribution. today, forensic and molecular methods utilized time of death rather than provide clear information offers potential death time interval. principally, 'time of death' is a term that should be avoided. in forensic science, the postmortem 'interval' is the term to be considered. nowadays, there is no precise molecular method that can be used alone in the determination of pmi. aim of this study is to analyze the usability of ecm components, adamts , and and mmp , and to determine pmi. for this purpose, with iliopsoas muscle tissues provided by ethical board of the ankara institute of forensic medicine, cases have been included in this study, divided into groups according to their pmis: ' - h', ' - h' and ' - h'. from these tissues, western blotting technique is used to analyse the expression of adamts , and and mmp , and . it is determined that when pmi increases; adamts- and amounts decrease. on the other hand adamts- amounts are examined to increased related to the interval., especially on the ' - h' dataset. additionally, considering metalloprotease levels, mmp- and amounts decrease as pmi increases. unlike mmp- and ; mmp- levels increase proportional to the interval, especially on the ' - h' dataset. these results are the first data on pmi determination from iliopsoas muscle tissue. in this study, it is suggested that adamts- and mmp- , proteases responsible for ecm degradation, can be used to determine pmi as markers. here we present the first observations of postmortem variation of mmp and adamts activities in skeletal muscle. in recently, popular mmps and adamts s pathways of the relationship between time-dependent changes to investigate the post mortem time interval determination to shed light on the future. the role of functional polymorphisms of matrix metalloproteinases and in spontaneous abortion samples capillaries, which are responsible for maintenance of implantation and placental nutrition, have major effects on mechanisms underlying abortion. matrix metalloproteinases (mmps) function in various cellular pathways. they play a role in patholohical conditions, metastasis; as well as normal physiological functions like tissue and bone regeneration, physiologic function of uterus, ovulation, embryogenesis and embryo implantation. mmp and mmp (gelatinases) have key roles at organisation of extracellular matrix and affect endometrial implantation. previous studies have shown that mmp - c>t and - c>t polymorphisms cause loss of gene function, and mmp - c>t polymorphism causes a decrease in gene expression, and also gene expression levels are lower in abortion specimens, compared to control specimens. the goal of this study was to investigate the potential effects of functional mmp - c>t and - c>t polymorphisms, and mmp - c>t polymorphism on etiology of abortion. restriction fragment length polymorphism (rflp) method was used to analyze the polymorphisms those evaluated in the study. study group consisted of samples collected from spontaneous abortion specimens, and control group consisted of peripheral blood blood samples collected from healthy subjects. the risk of abortion was . -fold higher in patients with heterozygous - c>t polymorphism (p = . ). combined genotype analysis showed that the risk of abortion was . -fold higher for patients with normal - c>t polymorphism, and heterozygous - c>t polymorphism (p = . ). functional polymorphisms of mmp and mmp may have a role in etiology of abortion. p- . . - changes in the specific extracellular matrix proteins and expression of adamts proteinases and effects of hypoxia in the adriamycin-induced renal fibrosis model renal fibrosis is a common cause of renal dysfunction with chronic kidney diseases. this process is characterized by excessive production of extracellular matrix (ecm) or inhibition of ecm degradation. adamts proteinases, which are widely presented in mammals, have very critical roles in ecm remodeling. we aimed to study the role of adamts proteinases in the pathogenesis of renal fibrosis and the effets of hypoxia by studying adamts expressions in rat kidney after adriamycin (adr) administration. adr was administered intravenously in consequtive two doses ( . and mg/kg) to the rats. in addition to control and adr groups, another rats were assigned into three groups as sham, min and min ischemia-reperfusion. after months following the first dose, the expression of adamtss were determined in the renal tissues using western blot analysis. additionally, renal nephropathy and fibrosis were assessed pathological and immuno-histochemical staining methods. in the adr group rats developed renal dysfuntion and tissue damage in favor of renal fibrosis pathologically. it is observed that occurred remarkable changes in the expression of adamtss. it is showed that hypoxia and hypoxia time enhanced tubulointerstitial fibrosis in the rat kidney tissues. expression differences were absorved also in the hypoxia groups, and especially the expression of adamts- and - genes were showed an increase in various rates. the restricted and different expression pattern of adamts protein profiles in the adr-induced renal fibrosis suggest that adamts family members are related with development and progression of fibrosis. moreover, our findings raise the possibility that the hypoxia promotes renal fibrogenesis. the monitoring of adamts proteinases might contribute to the early diagnosis of renal fibrosis and chronic kidney disease. adams (a disintegrin and metalloproteas) are transmembrane proteins that contain a pro-domain, a metalloprotease, a disintegrin, a cysteine-rich, an epidermal-growth factor like and a transmembrane domain, as well as a c-terminal cytoplasmic tail. they are involved in cell adhesion and they have protease activities. previous studies showed that some adam proteins act in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis, embryonic tgf-a release and the inflammatory response. although there are more researches about adam proteins, still the function of all adam proteins remain unclear. we aimed to investigate the potential link of infertilty with adam , - , and - . in this study twenty four patients were included. the patients were classified as normozoospermia (ns; n = ), oligozoospermia (os; n = ), azoospermia (as; n = ) groups. adam , - and - protein levels in infertility indviduals were analysed by western blot. adam protein level was . fold lower in the os and as groups than in the ns group. adam protein level was . fold higher in the as group than in the ns group. adam protein level was fold lower in the as group accourding to ns group. we observed no change between protein level of adam and adam of os and ns groups . in conclusion, adam proteins may have a potential role in male infertility. our study is a preliminary and first study on this issue. keywords: adam, infertility. the role of tissue metalloproteinase inhibitors thymus chemokine and thrombospondin- on prognosis of crimean-congo hemorrhagic fever s. bakir, m. bakir, s. ersan, a. engin cumhuriyet university, sivas, turkey crimean-congo hemorrhagic fever (cchf) is a disease which is caused by an arbovirus carried by ticks and characterized by the sudden onset of high fever, severe headache, dizziness, back and abdominal pain. cchf pathogenesis is still not resolved today to fully open. therefore, in this study, to determine the level of tck- , timp- and tsp serum samples obtained from cchf patients and the control group is intended to be examined for the pathogenesis and prognosis of the disease. the study sample was created patients with diagnosis of cchf. healthy volunteers were chosen control group matched for gender and similar to in terms of age. tsp, tpc- and timp- levels of patients and a control group were analyzed using the human elisa kits. serum timp- tck- and tsp levels in cchf patients were measured significantly higher than the in the control group. cchf pathogenesis of today still have not provided fully open. therefore, it reveals the importance of this work. in our study, presence of high timp- , tck- and tsp levels indicate important direction for pathogenesis and prognosis of cchf disease. p- . . - activation of calpain and protein kinase ca promotes a constitutive expression and release of matrix metalloproteinase in peripheral blood mononuclear cells from cystic fibrosis patients matrix metalloproteinase (mmp ) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies, including cystic fibrosis. we have studied if the alteration in intracellular ca + homeostasis, observed in peripheral blood mononuclear cells (pbmcs), isolated from cystic fibrosis (cf) patients homozygous for deletion of phenylalanine in gene of cystic fibrosis transmembrane conductance regulator (f del-cftr), could be involved in the abnormal presence of mmp in the extracellular fluids of cf patients. pbmcs were isolated from healthy donors and cf patients homozygous for f del-cftr. mmp levels were evaluated following h of cell incubation. our investigations show that all cf pbmcs analyzed constitutively express and release high levels of mmp ; conversely, in pbmcs from healthy donors, expression and secretion of mmp are undetectable but both events can be evoked, after h of cell culture, by a possible paracrine stimulation. we have demonstrated that in cf and h-cultured control pbmcs mmp secretion is prevented by chelation of intracellular ca + and mediated by the concomitant activation of calpain and protein kinase ca (pkca) and also that mmp expression is mediated by the sequential activation of pkc and extracellular signal-regulated protein kinases and (erk / ). moreover, the rescue of active f del-cftr reduces the extent of mmp secretion, correlating the channel defect to the [ca + ] i dysregulation of cf pbmcs. our results indicate that the high level of intracellular ca + concentration in cf pbmcs, promoting the aberrant activation of both calpain and pkca, induces a constitutive release of mmp . these data characterize new alterations in mononuclear leukocytes of cf patients that may be of primary importance in the progression of the disease and indicate that pbmcs may contribute to the accumulation of mmp in fluids of cf patients. p- . . - aebp /aclp is upregulated in differentiation, injury repair and fibrotic degeneration of skeletal muscle c. € ozdemir , , u. akpulat , i. onbasilar , c ß . kocaefe department of medical biology, faculty of medicine, hacettepe university, ankara, turkey, department of stem cell, institute of health, hacettepe university, ankara, turkey, laboratory animal breeding and research unit, faculty of medicine, hacettepe university, ankara, turkey aebp /aclp is an ambiguous gene with several attributed functions and cellular events, adipogenic differentiation, cell adhesion, pattern development and fibrosis are the well-understood. aebp is the short isoform that acts as a transcriptional repressor by targeting the ap promoter and aclp, which is the long isoform that harbors a leader sequence that directs the peptide to the extracellular compartment. the latter is known to be associated with development of the connective tissue, injury repair and fibrosis in certain pathological conditions. aebp /aclp displays a moderate expression in skeletal muscle where the role is not known. we have investigated the spatial and temporal expression of aebp /aclp in defined models of skeletal muscle differentiation, injury repair and fibrosis. aebp /aclp expression is present in steady state dividing myoblasts. this basal expression is upregulated folds upon the induction of differentiation in c c cells. considering that differentiation and post-natal injury repair share several common aspects, we also investigated the expression of aebp /aclp in acute injury-repair model. in the course of cardiotoxin induced injury, aebp /aclp expression peaks up to folds in the th day of regeneration. this time point concomitantly corresponds to tissue remodelling. since aebp /aclp is also known to be associated with fibrotic events in chronic pathological conditions, we also have investigated its expression in tenotomy induced skeletal muscle degeneration which mimics endomysial and perimysial fibrosis. aebp /aclp expression is upregulated up to folds in early time-point samples. these results depict a novel role for aebp /aclp in extracellular remodeling of the skeletal muscle during injury repair as well as fibrotic degeneration. the source of this expression might come from fibroadipogenic precursers which reside in endomisial area of muscle. our current efforts are focused on presenting of this endomysial expression. the mprbp gene from b. pumilus - encoding a novel secreted metalloproteinase was identified. based on the primary structure the enzyme has been classified as metzincin metalloproteinase that combines the features of two families: astacin and adamalysin. representatives of the adamalysin family previously have not been described for bacilli. the aim of the work was to elucidate the mechanisms of the gene expression regulation of a new bacillus pumilus - extracellular metalloendopeptidase. promoter region analysis revealed the presence of potential binding sites for transcription factors spo a (sporulation) and degu (biodegradation). study of mprbp expression in strain defective in regulatory proteins degs and degu shows that the productivity of metalloproteinase biosynthesis decline in average % compared with the strain with a complete degs-degu system. we also studied mprbp expression in strains with a mutation in the gene degu, leading to stabilization of degu~p protein. it is known, that this mutation leads to a multiple increase in the gene expression level, positively regulated by degs-degu system. our data shows a -fold increase in metalloproteinase productivity in the recombinant strain. thus, deg-system participates in control of the proteinase synthesis but not only in the regulation of mprbp gene expression. the mprbp expression in the strain deficient in regulatory protein spo a remained at the level with expression in the strain with the complete spo a. a similar pattern we observed in the study of mprbp gene expression in strains defective in other spore-specific regulatory proteins (spo b, spo f, spo k, spo j, sigf, sigh, sigk). these data indicate that mprbp gene expression is free of spo-regulatory proteins. on this basis, we concluded that the expression of metalloproteinase gene is not correlated with the sporulation. p- . . - paricalcitol attenuate activity and expression of matrix metalloproteinases in a rat model of renal ischemia-reperfusion injury matrix metalloproteinases (mmps) are endopeptidases involved in the degradation of extracellular matrix. they have been postulated to have a role in the pathogenesis of ischemia-reperfusion injury (iri). in the present study, we investigated the effect of paricalcitol, a synthetic vitamin d analog, on mmps in renal iri. wistar albino rats were divided into three groups: sham operated, ischemia-reperfusion, and paricalcitol-pretreated. iri model was induced by bilateral clamping of renal arteries for min followed by h of reperfusion. the analysis of serum creatinine levels and activities/expressions of mmp- and - were performed after h of iri. the effects of paricalcitol on activities and expressions of mmp- and mmp- levels were investigated by gelatin zymography and immunohistochemistry, respectively. the pathological examinations were performed to score tubular damage by light microscopy. creatinine levels increased significantly in the iri group. rats in the paricalcitolpretreated group showed significant decrease in expressions and activities of mmp- and mmp- during iri. moreover, pathological examinations displayed significantly lower score of tubular damage in paricalcitol-pretreated group. in conclusion, paricalcitol attenuated iri by downregulating the expressions and activities of mmp- and - . p- . . - the changes of matrix metalloproteinase , activity and hyaluronic acid level in rat's heart and serum under cadmium influence o. fomenko , o. shaulska , y. kot , g. ushakova , a. shevtsova dnipropetrovsk medical academy, dnipropetrovsk, ukraine, kharkiv national university, kharkiv, ukraine, dnipropetrovsk national university, dnipropetrovsk, ukraine the changes in the molecular mechanisms of the extracellular matrix degradation under toxic factors are not well known. the main goal of work was the investigation of the mmp and mmp activity and hyaluronic acid level in the heart and blood serum under cadmium influence at different doses. the wister rats divided to groups were used for the experiment. cdcl x . h o in doses . lg/kg and lg/kg was given to rats intragastrically in drinking water during days. the rats were decapitated under isoflurane anesthesia according to ethical rules; the heart was quickly removed. the relative activity [in arbitrary units (au)] of pro-and active forms of mmp and mmp , total protein (tp) and hyaluronic acid levels were calculated. it was shown that low doses of exogenous cadmium ( . lg/ kg) lead to reduced activity of pro-and active forms of mmp in myocardium ( . ae . au/mgtp and . ae . au/mgtp compare to the . ae . au/mgtp and . ae . au/mgtp in the control rats accordingly) and in serum ( . ae . au/mgtp and . ae . au/mgtp compare to the . ae . au/mgtp and . ae . au/mgtp in the control rats accordingly), but pro-mmp activity in heart was increased ( . ae . au/mgtp compare to the . ae . au/mgtp in the control rats); level of ha was decreased in both tissues ( . ae . lg/ml and . ae . lg/ml compare to the . ae . lg/ml and . ae . lg/ml in the control rats accordingly). high doses of cadmium ( lg/kg) caused a reliable increase of both gelatinase activity in the myocardium: mmp increased from . ae . au/mgtp to . ae . au/mgtp, prommp to . ae . au/mgtp, mmp to . ae . au/mgtp. ha level was increased in serum ( . ae . lg/ml) and decreased in heart ( . ae . lg/ml). the results indicate the dose-dependent and tissue-specific effect of cadmium on mmp-depended protein degradation and level of hyaluronic acid. a disintegrin-like and metalloproteinase domain with thrompospondin- repeats (adamts) are a large family of proteoglycanase that show proteolytic activity towards proteoglycans like aggrecan, brevican, neurocan, and versican. interleukin- (il- ) is an il- cytokine family member that uniquely plays a role as a cytokine and nuclear factor. it is released by necrotic epithelial cells and activated innate immune cells as an alarming danger signal. adamts and il- implicated in brain cancer pathogenesis. we aimed to seek the amount of adamts in u proteolytic enzymes are able to speed up wound healing by removal of the necrotic tissues and fibrin. several investigations have reported that proteases damage also the microbial biofilms formed by opportunistic bacteria including staphylococci on surfaces of chronic and acute dermal wounds. therefore, proteases are seemingly perspective enzymes for biofilm eradication by hydrolysis of both matrix proteins and adhesins, proteins providing cells attachment onto solid surface and other bacteria, as well as by the cleavage of signalling peptides of intercellular communication of gram-positive bacteria. here we report that ficin, a plant protease, efficiently degrades the structural components of biofilm matrix formed by s. aureus and s. epidermidis at concentrations of lg/ml while trypsin and chimotrypsin are used as - mg/ml solution. the spatial structure of the biofilm was analyzed by atomic force microscopy. after ficin treatment, the biofilm structure became porous, with reduced viscosity. the congo red staining of the treated biofilms confirmed the hydrolysis of the protein component of the matrix. moreover, the biofilm treatment with ficin increased the antimicrobial efficiency of ciprofloxacin against biofilm-embedded cells of s. aureus and s. epidermidis. while h antibiotic treatment did not lead to the increase of dead cells of neither s. aureus nor s. epidermidis embedded into the biofilm matrix, in the presence of ficin the fraction of viable cells decreased significantly. accordingly, soluble ficin appears beneficial for outer wound treatment biofilm eradication and reduces the reinfection risk. the wound-healing activity of ficin requires further investigations. this work was supported by the russian science foundation (project no - - ). resveratrol (resv) is an antioxidant that belongs to the group of plant compounds, called polyphenols. resv is defined as an antimicrobial substance that is produced by several plants (red grape skin, peanuts and berries) to protect them from rough environments like excessive ultraviolet light, infections and climate changes. as an antioxidant, this polyphenol protects the body against cardio-vascular and cancer diseases. besides, resv has anti-inflammatory, neuro-protective, anti-diabetic and other pharmacological effects. although the positive pleiotropic effects of this polyphenol are well documented, there is a huge need to understand its influence on the biophysical properties of lipid bilayer. in the present work, the interaction of resv with membranes composed of palmitoyl-docosahexaenoyl phosphatidylcholine (pdpc) or palmitoyl-oleoyl phosphatidylcholine (popc), sphingomyelin (sm) and cholesterol (ch) was investigated by means of fluorescence spectroscopy. generalized polarization of the fluorescent probe laurdan (gp) as a function of temperature was used to probe the changes in the fluidity of lipid bilayer induced by different resv concentration. the obtained results showed decreased lipid ordering from to lmol resv and opposite effect from to lmol in pdpc/sm/ch mixtures as compared to the control without resv. the interaction of resv with popc/sm/ch mixtures caused only an increase in the lipid ordering as a function of resv amount. popc and pdpc have the same head group but different fatty acid chains at sn- . since resv changes the physicochemical properties of lipid bilayer by different ways one might suggest that the interaction of polyphenol with the membrane depends on the level of fatty acid unsaturation. this specific effect of resv on lipid organization could be related to its unique properties to prevent the cell from oxidative stress. neurodegeneration is the umbrella term for the deseases including progressive loss of structure or function up to death of neurons. beta-amyliod peptide is proteolytic fragment of the amyloid protein. the spontaneously formation of selective, voltage-dependent, ion-permeable channels in the lipid bilayers was reported as one of amyliod peptide toxicity mechanisms. the aim of our study was the investigation of the influence of the flavonoids (phloretin, phlorizin, quercetin and genistein) on the membrane activity of amyliod peptides. virtually solvent-free bilayer lipid membranes were prepared from mixtures of phospholipids in . m kcl (ph . ) using monolayer-opposition technique. using spectrofluorimetry we estimated prepared from phospholipids by extrusion the liposomal membrane permeability for calcein. we found that the addition of phloretin into membrane bathing solution led to an signicant increase in the channel forming activity of fragments - of amyloid peptide, fragment - of [gly ]-amyloid peptide and - of human prion protein. addition of other flavonoids did not cause any changes in the steady-state amyloid-induced current. it was found that the effect was caused by electrostatic interaction with the peptide. we found that time course of amyloid induced leakage calcein from liposome's is characterized by two components: the fast one is related to sorption of b-amyloid peptide on the membrane and the slow one is related to the oligomerization of the peptides on the surface of the lipid bilayer. addition of the phloretin simultaneously with b-amyloid peptide to the suspension of liposomes caused significant reduction in time parameters characterizing fast and slow components. from this results we can proposed that phloretin compensates the positive charge of the b-amyloid peptides and leads to the changes in their oligomerization status. the study was supported in part by rfs ( - - ) and sp- . . . ferritin nanocarriers: a focus on a metal-based drug encapsulated inside the protein cavity s. ciambellotti , c. bernacchioni , f. scaletti , p. turano cerm (magnetic resonance centre), florence, italy, department of biochemical sciences, university of florence, florence, italy, chemistry department 'ugo schiff', florence, italy ferritin is one of the main player involved in the iron metabolism. the biological role of ferritin is the storage of iron atoms inside the cavity preventing the uncontrolled accumulation of toxic species inside cells. ferritins are polymers constituted by subunits that self-assemble giving rise to an almost spherical nanocage. in vertebrates, ferritins are formed by two distinct subunits, the heavy chain (h), with oxidoreductase activity, and the light chain (l) without catalytic activity. ferritins are promising nanocarriers for the delivery of contrast agents for diagnosis and of drugs for therapeutic purpose. their endogenous origin and the possibility to stabilize and protect the enclosed cargo inside the cavity, make ferritin a biocompatible vehicle. moreover, there are specific receptors on cells that recognize and incorporate ferritin by endocytosis, prospecting a kind of targeted-delivery. following the increasing interest in nanotechnology, we studied the interaction between different isoforms of ferritin with an antimetastatic drug, called nami-a, which is the first ruthenium derived anti-cancer drug to have entered clinical evaluation. this molecule is a metal-based prodrug that can release the metal ion ligands. here, we describe nami-a hydrolysis in the presence of recombinant homopolymers of ferritin followed spectrophotometrically. thanks to characteristic time dependent changes of spectral profile in the uv-visible region, we could detect differences in the hydrolysis process. the formation of a ru-adduct with hferritin was established by uv-visible and circular dichroism spectroscopies, as well as by kinetics measurements that showed inhibition of the ferroxidase activity of h-ferritin. crystallization trials are in progress to identify the binding site of ru by solving the x-ray structure of the complex. finally, we planned to test the cytotoxicity of ferritins pre-incubated with nami-a, using different cancer cell lines. a. cort , t. ozben , a. sansone , s. barata-vallejo , c. chatgilialoglu , c. ferreri sanko university, gaziantep, turkey, akdeniz university, antalya, turkey, cnr, bologna, italy, universidad de buenos aires, buenos aires, argentina, national center for scientific research 'demokritos', athens, greece liposomes as biomimetic models of cell membranes were used for examining some novel aspects of drug-metal induced reactivity with unsaturated lipids under oxidative conditions. the chemical basis of cis to trans transformation was ascertained by liposome experiments, using bleomycin-iron complexes in the presence of thiol as a reducing agent that by incubation at °c gave rise to the thiyl radical-catalysed double bond isomerisation of membrane phospholipids. the effect of oxygen and reagent concentrations on the reaction outcome was studied. as a chemical biology model for antitumoral strategies, liposomes highlight the role of cell membranes, which are not spectators but important targets of the drug effect, with synergic roles for chemotherapeutic effects. indeed, fatty acid recruitment and membrane formation are attracting a lot of interest in cancer, and in this context the loss of the natural cis geometry and the oxidationinduced lipid remodelling are worthy of deeper studies in antitumoral strategies. furthermore, the interaction between drugs and lipids can be suggestive of novel aspects of chemical reactivity for liposome carriers when circulating in vivo. gpr is a g-protein coupled receptor (gpcr), expressed in cells of brain, heart and kidney. it is related to the leukotriene and purinergic subfamilies of the rhodopsin-like class a gpcrs. gpr plays controversial role in the brain and spinal cord cells recovery after injuries. it is assumed that gpr is one of the cell death regulators immediately following an injury but at later stages it also takes part in tissue regeneration. there are also data implying some role of gpr in glucose homeostasis. drugs targeting gpr may help with treatments of multiple sclerosis and ischemia. the damage of rat's brain in artificially induced ischemia disease decreased after gpr inhibition. in addition, gpr takes part in myelin sheath formation, the lack of which is known to be the reason of multiple sclerosis. to better understand functional role of gpr and enable design of more efficient ligands we initiated structural studies of this receptor. to improve receptor stability and facilitate crystallization we created a series of chimeric constructs using different fusion partnerssmall soluble proteins inserted into the native amino acid sequence. preliminary experiments were carried out to evaluate the influence of different ligands on the receptor stability and cell surface expression in insect sf cells. this work was supported by russian federal target sterols are significant for the structural and dynamical features of cell membranes. among them, cholesterol is the major sterol in mammalian cell membranes whereas stigmasterol is the predominant sterol in plant membranes. stigmasterol varies structurally from cholesterol in having both an ethyl group at carbon and an additional trans double bond between carbons and . dimyristoylphosphatidylcholine (dmpc) is a widely studied synthetic lipid, which has a neutral (zwitterionic) pc headgroup and two symmetrical -carbon fatty acyl chains. the studies on the interactions of cholesterol and stigmasterol with dmpc membranes at molecular level are very limited. in the present study, a calorimetric comparison of the effects of the animal sterol cholesterol and the plant sterol stigmasterol on zwitterionic dimyristoylphosphatidylcholine (dmpc) multilamellar vesicles (mlvs) was investigated for the first time by using differential scanning calorimetry (dsc). our dsc studies indicate that with the inclusion of increasing cholesterol and stigmasterol concentrations from to mol% into pure dmpc mlvs, the pretransition disappears, the main phase transition shifts to lower temperatures and then disappears at cholesterol and stigmasterol concentration above mol%. the main phase transition enthalpy (dh) is progressively reduced whereas full width at half maximum (dt ½ ) gradually increases with increasing cholesterol and stigmasterol concentrations. moreover, the main phase transition peak consists of overlapping sharp and broad components, indicating the hydrocarbon chain melting of sterol-poor and sterol-rich dmpc domains, respectively. in conclusion, this study shows clearly that both cholesterol and stigmasterol interact effectively with dmpc membranes and cause changes in their structural and functional properties. p- . . - trh receptor mobility in the plasma membrane is affected by its interaction with its cognate signaling molecules and by cholesterol depletion r. moravcova, b. melkes, j. novotny department of physiology, faculty of science, charles university in prague, prague, czech republic g protein-coupled receptors (gpcrs) play a fundamental role in transferring information from extracellular environment to the cell interior. some gpcrs are supposed to form signaling complexes with their cognate g proteins and possibly other accessory proteins, which may facilitate the activation of g proteins and thus accelerate signal transmission. here we investigated the role of some components of thyrotropin-releasing hormone (trh) receptor signaling in hek cells stably expressing yfp-tagged trh receptor using fluorescence recovery after photobleaching (frap). we observed significant changes in values of the diffusion coefficients if expression of b -arrestin or gb subunit were suppressed by sirna. results of our frap experiments indicated significant difference between control and trh-treated cells as the diffusion coefficient markedly decreased after agonist stimulation. on the other hand, the same decline of the diffusion coefficient value was found after silencing with sirna and subsequent treatment with trh for most of the screened proteins. treatment of cells with À m trh led to fast internalization of trh receptor, which was revealed by real-time confocal microscopy. it is known that cholesterol is an essential component of the cell membranes and it exerts modulatory effects on the functioning of various membrane proteins. disruption of plasma membrane integrity by cholesterol depletion using b-cyclodextrin significantly reduced the apparent diffusion coefficient values. interestingly, addition of trh to cells treated with b-cyclodextrin did not further reduced trh receptor mobility. it can be concluded that stimulation with agonist and/or sirna silencing of some components of the trh receptor signaling cascade significantly affects the mobility of trh receptor in the plasma membrane. p- . . - l-opioid receptor mobility in the plasma membrane is diversely affected by biased ligands b. melkes, l. hejnova, j. novotny opioid receptors belong to the large family of g protein-coupled receptors (gpcrs), which are currently considered among the most important targets for pharmacological manipulations. during the past few years, a great deal of attention has been devoted to biased agonism. this phenomenon reflects the ability of different ligands to selectively affect the functioning of some gpcrs so they can display marked differences in their efficacies for g protein-or b-arrestin-mediated signaling. here we decided to investigate the effect of different agonists of the l-opioid receptor (l-or) on the lateral mobility of this receptor in the plasma membrane of hek cells which were stably transfected with l-or tagged with yellow fluorescent protein (l-or-yfp). it has been found previously that damgo stimulates g-protein-dependent signaling, endomorphine stimulates arrestin-dependent signaling and morphine does not show any significant bias towards these two signaling pathways. in our experiments, we used the fluorescence recovery after photobleaching (frap) method to estimate the diffusion coefficients of l-or-yfp in the resting state and after addition of the above mentioned specific agonists. we observed that addition of damgo increased the value of the diffusion coefficient and addition of endomorphin decreased the value of diffusion coefficient of l-or-yfp. addition of morphine or the l-or antagonist naloxone did not change the value of the diffusion coefficient compared to the resting state. these results indicate that different biased agonists of l-or may differently affect the mobility of this receptor in the plasma membrane. these findings provide new insights into the dynamics of l-or in the plasma membrane and contribute to better understanding of the mechanism of biased agonism at gpcrs, which is of central importance for receptor homeostasis and fine regulation of receptor activity. color tuning and adding potassium selectivity to a light-driven sodium pump k. kovalev , , v. polovinkin , , , v. shevchenko , , v. gordeliy , , moscow institute of physics and technology, dolgoprudniy, russia, research centre j€ ulich, j€ ulich, germany, institut de biologie structurale, universit e grenoble alpes, cnrs, and cea, grenoble, france recently a light-driven sodium pump has been discovered, characterized and tested as an inhibitory optogenetic tool. sodium pumping rhodopsin from dokdonia eikasta kr has an absorption maximum at nm at ph . and to create more redshifted variants we analyzed available structures of the kr (pdb codes: xtl, xtn) and did the rational mutagenesis of residues in the retinal proximity region (i.e. m , g and s ). the mutants of kr under investigation were: m a, g v, m a/g v, s a, s f, s g, s l, s m, s n, s t, s v, s y. the protein mutants were expressed in escherichia coli c strain, expression was induced by the addition of mm isopropyl b-d- -thiogalactopyranoside. the cells were then washed trice with unbuffered mm nacl or kcl solution. finally, the ph changes in cell suspensions (od = . ) were monitored. ph changes upon the addition of lm of protonophore carbonylcyanide m-chlorophenylhydrazone were also measured. the following mutants completely abolished the protein function and were not used for further characterization: s f, s l, s m, s n, s v. the remaining mutants have shown sodium pumping activity and s a mutant has gained an additional potassium pumping activity. all functionally active mutants were purified using ni-affinity chromatography and the absorption spectra were collected for them at ph . ( mm na/na-pi, mm nacl). m a mutant absorption maximum is blue-shifted to nm. g v and m a/g vblue-shift to nm. s a, a potassium pumping variant,red-shift to nm. s g, s yred-shift to nm. s tno change in absorption maximum position. based on structural analysis of kr we discovered another potassium pumping variant and provided the variants with absorption maximum blue-shift up to nm and red-shift up to nm. human endothelin receptors belong to the g-protein coupled receptors (gpcrs) superfamily. this class is pharmacologically important, with more than % drugs targeting it. the human endothelin system, which includes endothelin receptors types a and b (etb and eta), plays a highly important role in the blood pressure regulation. endothelium cells produce peptides, known as endothelins - , which activate endothelin receptors and launch cascades of reactions that lead to vasoconstriction or vasodilatation depending on the receptor subtype and the tissue. additionally, endothelin receptors take part in such processes as transmission of nerve impulses, development of neural crest, and regulation of acid-base-salt balance in kidneys. in order to stabilize etb receptor for crystallization trials we introduced a compact soluble protein, apocytochrome b ril (bril), is the third extracellular loop of the receptor. bril is known to be an effective crystallization driver for gpcrs. the engineered protein was expressed using baculovirus system in sf insect cells, purified and subject to variety of pre-crystallization assays. localization of the overexpressed protein in insect cells was visualized via the confocal microscopy. thermal stability of the protein in the presence and absence of ligands was measured by the thermal shift assay. finally, the mobility of the receptor in lipidic cubic phase (lcp) at many different conditions was probed by the lcp-frap (fluorescence recovery after photobleaching) assay. these tests showed that the obtained protein is thermally stable, functionally active and diffuses well in lcp at certain conditions, making it a suitable candidate for proceeding to in meso crystallization trials. this work was supported by the russian science foundation (project no. - - ). mitochondrial oxidative phosphorylation is the key metabolic pathway for the production of atp. mitochondrial respiratory chain (rc) defects are some of the most commonly diagnosed inborn errors of metabolism with a diverse spectrum of clinical phenotypes. the aim of the study is to evaluate the rc enzyme activities and histopathological findings in muscle biopsies of patients with suspected mitochondrial disease. muscle biopsy samples were collected from pediatric patients. the samples were homogenized in seth buffer using a glass/glass homogenizer. the activities of citrate synthase (cs) and rc enzymes (complex i, ii-iii, and iv) were measured in tissue homogenates by kinetic spectrophotometric assays by schimadzu uv spectrophotometer (uv- ). non-collagen protein was determined by the modified lowry assay. activities of complex (c) i, ii-iii and iv (cox) were normalized by cs. histopathological investigations included h&e, modified gomori trichrome, periodic acid schiff, oil-red-o, nadh, sdh, cox and atpase stains. deficiency of rc complexes was detected in biopsies ( %). c iv deficiency was most common ( %), followed by c i ( %) and c ii-iii ( %). multiple complex deficiency was present in % and isolated deficiency was present in % of the biopsies ( % c i, % c ii-iii, % c iv). cs activity was elevated in % of the biopsies. decreased c i/cs, c ii-iii/cs and c iv/cs ratio was observed in %, % and %, respectively. comparing with histological data, biochemical analysis revealed additional findings in % of biopsies. complex iv deficiency, either isolated or accompanied by other complex deficiencies, is most common in our cohort. rc analysis may reveal additional findings to histopathological results and careful clinical investigation with correlation of clinical, biochemical and histopathological data is mandatory for the challenging diagnosis of mitochondrial disorders in childhood. investigation of adipocytokines, activity of glut and na + /k + -atpase in rats fed glucose, fructose, starch-based sugars objective: all over the world, shows a significant increase in obesity and diabetes. intake of foods that contain fructose, glucose and starch-based sugar is a potential risk for metabolic syndrome. obesity and diabetes are important effects of high fructose corn syrup (hfcs). we aimed to research, activity of na + /k + -atpase in addition to glucose transporter (glut) , resistin, adiponectin and other biochemical markers in rats fed glucose, fructose and starch-based sugars. materials and methods: study was performed on rats and groups were included in the study. rats were fed with chows that were given either normal diet for control group ( % carbohydrate, % protein and % fat), high fructose ( % carbohydrate ( % fructose and % starch), % protein and % fat), or high sucrose ( % carbohydrate ( % sucrose and % starch), % protein and % fat). rats were fed with chows for weeks. in this process, the weight of the rats were followed. at the end of the experiment, blood is taken in all groups. level of hba c, glucose, resistin and adiponectin were studied. glut and na + /k + -atpase activity were studied in the liver tissue. results: a significant increase in adiponectin levels were determined in rats fed both hfcs and sucrose (p < . ). a significant decrease in level of na + /k + -atpase activity were determined in rats fed both hfcs and sucrose (p < . ). there was no significant differance level of hba c, glucose, resistin and glut in rats fed sucrose or hfcs (p > . ). conclusions: fructose-rich diet has an effect on changes in the atpase activity and is a major risk factor for obesity. keywords: adiponectin, fructose, glucose, high-fructose corn syrup, na + /k + -atpase, resistin. p- . . - nucleic acid-biomembrane lipid selfassemblies: from primordial soup to novel genome organization model and cellular nonviral nanotherapies f. k. demirsoy , n. eruygur , e. s€ uleymanoglu biotechnology central laboratory, biotechnology institute, ankara university, ankara, turkey, department of pharmacognosy, faculty of pharmacy, cumhuriyet university, sivas, turkey, department of pharmaceutical chemistry, faculty of pharmacy, gazi university, ankara, turkey turkey nucleic acid-cell membrane complexes has attracted research interest as models in gene regulation, cell cycle and division, as biosensors designs, as well as in molecular evolution. zwitterionic phospholipids, complexed with polyribonucleotides by divalent metal cations (mg +) are considered as genosome vehicles. their formations are studied by spectroscopic, thermodynamic, interfacial and microscopic approaches to build thermodynamic and kinetic models of their structural transitions. dna forms a mg +-driven ternary complexes with neutral liposomes both at the airwater interfaces and at vesicle surfaces. the described self-assemblies form relevant models for nuclear pore complexes and their further implications in gene expression and functions. such membrane contacts could be considered also in prokaryotic nucleoids important in bacterial segregation, whereas in eukaryotes these complexes can be regarded as focal points for transcription-translationtranslocation processes. the effects of ozone/oxygen mixture on citrate synthase and mitochondrial complex activities of striated muscle tissue of healthy mice we investigated the effects of ozone/oxygen mixture and oxygen on citrate synthase (cs) and succinate dehydrogenase (sdh) activities of striated muscle tissue of healthy mice. thirty-six mice were included the study. firstly muscle samples were taken from all mice's left thigh muscle in under anesthesia (group ). secondly mice were randomly divided in four groups as: group : oxygen was given once at days for days, group : ozone/oxygen was given once at days for days, group : oxygen was given once at days for days, group : ozone/oxygen was given once at days for days. ozone/oxygen mixture and oxygen were given at a dose of mg/ kg groups ( ) ( ) ( ) ( ) . after treatment animals were sacrificed, and muscle samples were taken and stored in À °c for until the analyses. muscle tissues were homogenized in . m tris-hci and . m kci. cs and sdh activities were measured with spectrophotometer. cs and sdh activities were expressed as lmol/min/g tissue. cs and sdh activity results were given as mean ae sd. cs activity has been found in group ( . ae . ), group ( . ae . ), group ( . ae . ), group ( . ae . ) and group ( . ae . ). sdh activity has been found in group ( . ae . ), group ( . ae . ), group ( . ae . ), group ( . ae . ) and group ( . ae . ). there was no statistically significant difference among all groups in terms of cs (p > . ) and sdh activities (p > . ). there was no difference between all groups in terms of inflammation, muscle fiber size, regeneration or necrosis. vascular structures, connective tissues, lipid and glycogen content of fibers were normal. cytochrome oxidase activity was also normal. ratio of ragged blue fibers of all groups were less than . %, so they were scored as . there was no difference among groups for ragged red fiber content. we have not found a significant effect of ozone/oxygen mixture and oxygen on cs and sdh activities of striated muscle tissue of healthy mice. lipidic cubic phases (lcps) consist of bicontinuous lipid bilayers and water channels. lpcs are widely used for membrane proteins crystallization and further determination of their spatial structures by means of x-ray crystallography. this approach was successfully used for studying g-protein coupled receptors structures. usually crystallization initiates by adding the precipitants (buffers with high salt concentrations). here we propose to use photo-switchable analogs of -monoolein to change lattice parameter of lpc. we synthetized a number of novel diazo-analogs of -monoolein. their structures were confirmed by nmr-spectroscopy and mass-spectrometry. being incorporated in phospholipid vesicles or detergent micelles they subjected to trans-to cis-isomerization under the light exposure at nm. also we characterized the lcp's structures and properties by small-angle x-ray scattering on the rigaku instrument. one of the synthetized compounds, -( -{-[ -(octyloxy) phenyl] diazenyl} phenoxy) propane- , -diol ( % mol), was incorporated into the -monoolein cubic phase. according to small-angle x-ray scattering data the structure of the monoolein cubic-pn m phase with lattice parameter . angstrem was not affected by insertion of this photo-switchable monoolein's analog. after the light exposure at nm we observed trans-cis-isomerization. in the same time the cubic-pn m phase was not changed but the lattice parameter reduced to . angstrem. this effect on monoolein lpc is similar to the addition of a precipitant to initiate protein crystallization process. the spontaneous return to the initial lattice parameter was completed after days in dark. thus, we demonstrated the possible controlling of the monoolein cubic phase lattice parameters by adding the photo-switchable diazo-derivatives of monoglyceride analogs, which can be used for crystallization of membrane proteins. evaluation of certain protein and phosphoprotein expression levels by using western blot technique in head and neck squamous cell carcinoma a. kalayci yigin , t. cora cerrahpasa faculty of medicine, istanbul university, istanbul, turkey, faculty of medicine, selcuk university, konya, turkey introduction: head and neck squamous cell carcinoma (hnscc) is a primer tumor type in head and neck cancers, characterized by aggressiveness, early recurrence and metastasis. while alcohol and smoking play an important role at pathogenesis of disease, deregulation of some signaling pathways, genes and protein levels related to these pathways are considered as important at contribution of development of hnscc. materials and methods: in this study, protein and phosphoprotein expression levels of the frequently phosphorylated sites (egfr, pegfr, igf-ir, pigf-ir, pdgfrb, ppgdfrb, pten, ppten, akt and pakt) were investigated by using a western blot to confirm the expression of mrna in the context of protein levels at normal-tumor tissues of hnscc and sccl-mt that is a hnscc tumor cell line and hek- that is a normal cell line. results: as a result of western blot analysis egfr, pdgfrb and igf- r were detected as highly overexpressed cell surface receptors in tumor tissues of hnscc. discussion and conclusion: the findings of this study revealed the overexpression of other cell surface receptors as well as egfr in hnscc. in conclution, potential pathways were identified by determining the cell surface receptors overexpressed in hnscc, these data support each other and may be important in pathogenesis of hnscc. introduction: the investigation of final products of lipid peroxidation is considered as the main mechanism involved in development of pathogenesis, diagnostics and prognosis of various parasitic illnesses. materials and methods: the concentration of lp-ap in the blood was determined in the study group considered of women ( %), and men ( %). results: before antiparasitic treatment, women infected with g. intestinalis showed a statistically significant . times increase of gpx activity levels; and . times ada level increase compared to the control group. after the treatment, the cat activity showed a sharp increase, whereas the ada activity decreased by . times, compared to the average level before the treatment. the results of the blood samples of the infected men with giardiasis, show the statistically significant increase in the level of all the studied parameters of lipid peroxidation, except the total primary production (tpp). the exception was the mda level, remaining significantly increased, in contrast to the control group and to the condition after antiparasitic treatment. in infected men, the level of cat activity was more than . times higher than that noted in control group. after treatment the levels of ada activity and gpx returned to the values of the control group, while the level of cat activity remained elevated. conclusion: an accumulation of primary and secondary metabolites in the course of giardiasis seems to confirm its involvement in the induction of oxidative-antioxidative homeostasis. antiparasitic treatment in giardiasis leads to normalization of the ap parameters studied in women and men, except the mda content in the blood of men. therefore, additional antioxidant treatment is advised for the antiparasitic therapy of men. in vitro effects of ethanol on rat brain synaptosome and dose-dependent antioxidative role of boric acid ethanol is a psychoactive drug that is very large and used frequently today. it has suppressive effects brain's comminication pathway. depending on its acute or chronic use and the dose, ethanol increase membrane fluidity and it causes oxidative stress. this study deals with the in vitro effects of ethanol toxicity ( mm) and potential protective effect of different doses of boric acid (ba) ( , and mm) on rat brain synaptosomes. with this aim, five male spraque dawley rats are killed by decapitation under anesthesia. after the frontal cortexes of the rats are taken out, each of them is divided into four pieces. these pieces were used as a sample in five groups (control, ethanol, ethanol+ mm ba, ethanol+ mm ba, ethanol+ mm ba) which include six samples. the synaptosomal fractions are prepared by the homogenization of the frontal cortex pieces and centrifugation for each samples. as markers of ethanol-induced oxidative stress in the synaptosome of the rats, malondialdehyde (mda), nitric oxide (no) and catalase (cat) levels were measured. mda levels in the ethahol group were a quantity increased compared with those in the control group but it unchanged significantly as statistically (p < . ). however the mda level in the ethanol+ boric acid ( mm) group was shown to be significantly decreased compared with that in the ethanol group (p < . ). the cat activity of the ethanol group was significantly higher than that in the control group and cat activity of the ba ( mm, mm) groups were close compared with control groups (p < . ). no levels in ethanol groups were decreased but unchanged compared with control groups as statistically. neverthless, no levels in ethanol+ boric acid ( mm) groups were increased (p < . ). these results demonstrate that ethanol ( mm) is capable of triggering damage to rat brain synaptosome and ba could be influential in antioxidant mechanisms against oxidative stress resulting from ethanol exposure. acute myeloid leukemia (aml) is the most common form of acute leukemia in adults and its incidence increases with age. carbonic anhydrases (cas) are zinc-containing enzymes. these enzymes catalyze a very simple physiological reaction, the inter conversion between carbon dioxide and the bicarbonate ion, and are thus involved in crucial physiological processes connected with respiration and transport of co /bicarbonate between metabolizing tissues and lungs, ph and co homeostasis, electrolyte secretion in a variety of tissues/organs, and biosynthetic reactions and many other physiologic or pathologic processes including reproductive tract. investigation of autoantibodies in aml patients have been popular research area in recent years. the aim of the current study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in the serum of subjects with aml based on the information and considerations of autoimmune relation of acute myeloid leukemia. anti-ca i and ii antibody levels were investigated by enzyme-linked immunosorbent assay method (elisa) in serum samples of thirty patients with aml and thirty healthy peers. anti-ca i and ii antibody titers of aml group were significantly higher compared with the control group (p = . ), (p = . ), respectively. we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in patients with aml (r = . , p = . ). we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in women and the men (r = . , p = . ), (r = . , p = . ), respectively. at an anti-ca i cut-off point of . absu, sensitivity was % and specificity %. at an anti-ca ii cut-off point of . absu, sensitivity was % and specificity %. the ca i and ca ii autoantibody levels in patients with aml were found higher compared to control group and the results suggest that ca i and ca ii autoantibodies may be involved in the pathogenesis of aml. aim: behc ßet's disease (bd) is a systemic autoimmune disease. recurrent oral and genital mucosal ulcers, uveitis, and skin lesions are characteristic findings for bd. platelet-lymphocyte ratio reflects a novel marker for romatological diseases. the aim of this study was to investigate the platelet/lymphocyte ratio in behc ßet's disease. methods: whole blood samples were collected from healthy control and patients with behc ßet's disease. the mean age for controls and patients were ae . and ae , respectively (p = . ). patients with chronic disease and inflammatory disorders were excluded. thrombocyte and lymphocyte counts were analyzed with abbott cell dyne heamotolgy analyzer. statistical analysis was performed with ibm spss v . results: platelet counts were higher but not statistically significant in patients with behc ßet's disease compared to control group ( ae vs. ae ) (p = . ). lymphocyte counts were lower in patients with behc ßet's disease compared to control group ( . ae . vs. . ae . ) (p = . ). platelet/lymphocyte ratio was higher but not statistically elevated in patients with behc ßet's disease compared to control group ( ae vs. ae ) (p = . ) conclusions: platelet/lymphocyte ratio (nlr) and mean platelet volume (mpv) as inflammatory markers recently became popular because of their simplicity, cost effectivity and their advantages to predict clinical prognosis of specific diseases. according to this study's results, platelet/lymphocyte ratio must be analyzed in vast scale patient populations to identify the disease. objectives: systemic lupus erythematosus (sle) is a chronic relapsing autoimmune disease characterized by production of autoantibodies against a series of nuclear antigens and by chronic inflammation. in recent years, neutrophil-lymphocyte ratio (nlr) was determined to be a good indicator of inflammatory status. nlr can be easily calculated from a whole blood count. introduction: neuroblastoma, an embryonal malignancy, is the most common extracranial solid tumor of childhood. untreated neuroblastoma tumors and cell lines are reported to have reduced hla class i expression, rendering them potentially susceptible to natural killer cell killing due to lack of engagement of hla class i-specific inhibitory killer cell immunoglobulin-like receptors (kirs). the aim of this study was to investigate whether kir genes could influence the risk of neuroblastoma and prognosis of the patients. materials and methods: study group consisted of neuroblastoma patients ( male, female, median age: months) followed at the pediatric oncology clinic of c ß ukurova university medical faculty. control group consisted of healthy children. patients had stage , , or s disease, patients had stage disease. different kir genes were analysed by sequence specific oligonucleotide probe (ssop) analyses. statistical analysis were done using fisher's exact test. results: compared to the control group, neuroblastoma patients had lower expression of activating kir gene, kir ds (p = . ), and higher expression of inhibitory kir gene dl (p = . ). additionally kir ds genes were more common in patients with early stages (stage , , or s) (p = . ) and kir dl genes were more common in patients with stage (p = . ). furthermore, there were no statistically significant differences between the rate of aa and bx genotypes and their centromeric/ telomeric regions of patients and controls. discussion: kir dl gene can have a triggering effect in neuroblastoma pathogenesis; whereas kir ds can have a protective role. investigating nk cell infiltration and kir receptors in neuroblastoma tissue samples will give more insight to the pathogenesis p- . . - neuroprotective and immunomodulatory effects of urtica urens s. arslan , g. terzioglu , b. kabalay , a. r. tufekci , a. sen , i. demirtas department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, deparment of chemistry, faculty of arts and sciences, c ß ankiri karatekin university, c ß ankiri, turkey urtica urens (small stinging nettle) has been used for medical purposes in turkey as an alternative therapy. it has been used in the treatment of inflammation that is early, non-specific immune reaction to tissue damage or pathogen invasion, plays an important role in the initiation of neurodegenerative disorders such as multiple sclerosis. however, there are limited studies that investigate anti-inflammatory activity of urtica. therefore, aim of this study is to find out theanti-inflammatory effect of chloroform extract in caco- cell line. for this purpose, firstly, chloroform extract of urtica leaves was prepared. chemical composition of extract was determined by lc-ms. the effect of chloroform extract on selected pro-inflammatory and inflammatory proteins such as tumor necrosis factor-a (tnfa), nuclear factor kappa b (nf-jb), c-x-c motif chemokine (cxcl ), and (cxcl ) were determined. whole genome transcriptome analysis was performed by using human ht- v beadchip. extract treatment caused % and % increases in protein and mrna levels of nf-jb, respectively. on the other hand, tnf-a protein and mrna levels decreased significantly ( % and %, respectively). similarly, cxcl and cxcl mrna levels decreased % and %. transcriptome analysis showed that probes were significantly changed (p < . ). pathway analysis revealed that the extract altered a group of genes involved in immune response, calcium ion homeostasis and transport, potassium channel complex, g-protein coupled receptor protein signalling pathway, etc. it is well established that calcium is very critical for brain cell death and formation of many brain disease including multiple sclerosis. these observations suggests that urtica maybe used in neurodegenerative diseases. in order to further test this hypothesis experimental autoimmune encephalomyelitis experimentsand activity guided fractionations have been still continuing. this work is supported by tubitak t . p- . . - linear low molecular weight a- , -glucan from bifidobacterium bifidum bim b- d is implicated in pathogenesis of celiac disease the bifidobacteria are recognized as human commensals and widely used as probiotics. earlier, we have found (kiseleva et al., benef. microbes, , ( ) : - ) that bifidobacterium bifidum bim b- d contains low molecular mass ( - kda) a- , glucans (g anti-tpo and g anti-tg ) that interact selectively with human autoantibodies to thyroid peroxidase (anti-tpo) and thyroglobulin (anti-tg), recognized markers of autoimmune thyroid disease (atd). the aim of the study was isolation and identification of b. bifidum bim b- d biopolymers (bps) interacting selectively with autoantibodies to tissue transglutaminase (anti-ttg) and antibodies to gliadins (anti-gl), recognized markers of celiac disease (cd). we used affinity chromatography with anti-gl, size exclusion chromatography, h and c nmr spectroscopy, elisa with immobilized bps, tissue transglutaminase (ttg) and gliadins (gl) as positive controls. the bp isolated by affinity chromatography with anti-gl (as more available marker of cd) and size exclusion chromatography was identified by two-dimensional nmr spectroscopy as - kda linear a- , -glucan identical to g anti-tpo and g anti-tg . the functional activity of the bp named g anti-gl , viz., ability to interact selectively with anti-ttg and/or anti-gl was proven by elisa with (i) serum samples of cd patients containing either both anti-ttg and anti-gl without anti-tpo and anti-tg or anti-gl without anti-ttg, anti-tpo and anti-tg vs. serum samples of healthy donors without four types of antibodies and (ii) pure anti-gl vs. pure total igg (without anti-ttg, anti-gl, anti-tpo, anti-tg). since (i) serum samples of cd patients do not contain anti-ttg without anti-gl and (ii) pure anti-gl isolated by affinity chromatography with gliadins (gl) cross reacts with tissue transglutaminase (ttg), additional studies with pure anti-ttg are necessary to find out which of the two antibodies, anti-ttg and anti-gl, bind g anti-gl . in conclusion, we proved that b. bifidum bim b- d cells contain linear low molecular mass a- , -glucan, g anti-gl , that interacts selectively with anti-ttg and/or anti-gl. since g anti-gl is identical to earlier found g anti-tpo and g anti-tg , we hypothesize that the a- , -glucan is implicated in pathogenesis of both autoimmune diseases, cd and atd. influences of elevated serum ferritin levels on insulin resistance and non-insulin-dependent diabetes mellitus (niddm) have predicted either because of increased body iron stores or influenced by several inflammatory diseases. low serum hydroxyvitamin d is known to perturb cellular function in many tissues, including the endocrine pancreas, which are involved in obesity and niddm. we planned to investigate the association between hydroxyvitamin d with hematologic parameteres and iron status in obesity vs. diabetic patients. study groups consist of control, non-diabetic obese, obese-diabetic and lean-diabetic groups. serum triglycerides, total cholesterol, ldl-c, hdl-c, fasting glucose, hba c, uric acid, creatinine, ggt, -hydroxyvitamin d, insulin, crp, esr, total blood count and iron status. apart from the three parameters, there were no significant difference (p > . ) between groups. serum ferritin and mchc levels were significantly higher in lean-diabetic patients (p < . ). on the other hand, rdw are determined to be significantly lower (p < . ) in the non-diabetic obese group. no difference was detected in -hydroxyvitamin d levels between the control and the study groups (p > . ). non-diabetic obese patients had significantly (p < . ) higher levels of tg and lower levels of hdl compared to obese-diabetics. insulin levels were higher in nondiabetic obese and obese-diabetics than lean-diabetics (p < . ). this study provides evidence that lean diabetic patients show higher ferritin and mchc levels than obese patients. the increase in serum ferritin and mchc levels is related with altered iron metabolism at cellular level. at late mitosis, the mother cell divides, leaving two daughter cells connected by a thin intercellular bridge (icb). during abscission of the icb, the ingression of the cleavage furrow is formed, and the central spindle microtubules are compacted into the structure known as midbody (mb). the mb is situated within the icb, with the abscission usually occurring at one side of the mb. as a result, only one daughter cell inherits the post-mitotic mb. these mbs can then either accumulate in the cytoplasm or be degraded. recent studies have identified mbs as novel signaling platforms regulating stem cell fate and proliferation. indeed, stem cells as well as cancer cells were shown to accumulate post-mitotic mbs, resulting in reprogramming of the cell fate and conversion to highly-proliferative, stem cell-like phenotypes. it has been proposed that regulated macroautophagy may be playing a key role in mediating pots-mitotic mb degradation. therefore, the experimental approach involved studying the dynamics and function of a protein known as fyco , which associates with postmitotic mbs and may regulate their degradation. in this study we identified fyco as a protein, which associates with post-mitotic mbs and may regulate their degradation. interestingly, fyco is also known to be present on autophagosomes, and overexpression of fyco can induce the formation of enlarged lc -containing autophagocytic structures. here we demonstrate that fyco knock-down leads to defects in autophagic mb degradation, and that fyco functions by targeting endocytic membranes to the autophagic phagophore during early stages of mb degradation. additionally, we showed that fyco depletion leads to increased proliferation and cell growth in soft agar. based on all these data, we hypothesize that fyco mediates selective mbs degradation via endosome-dependent extension of the phagophore around the post-mitotic mbs, and that mbs may be the regulators of cancer proliferation and progression. p- . . - proliferative effect of hypericine on human skin fibroblast cells and identification of the mechanism of action in molecular level to drawbacks associated with efficiency and viral genome integration. in order to improve reprogramming efficiency and compensate for viral transduction, new chemicals have been explored through ipsc research. the aim of this study was to investigate the proliferative effect of hypericine on human skin fibroblast cells (sf) in-vitro, and to identify the mechanism of action in molecular level. the proliferation was measured using the clonogenic and dimethylthiazol diphenyltetrazolium bromide (mtt) assays. real-time quantitative polymerase chain reaction (qrt-pcr) was performed to detect the mrna levels of cyclins (d and b ) and cell cycle controller genes (p and p ). sf cells were treated with different doses ( nm- lm) of hypericine for h and h. a significant cell proliferation was observed in moderate concentrations ( . - lm; % -% ), but at high concentrations ( - lm) cytotoxic effects emerged in sf cells (ic = . m, r = . ). qrt-pcr results revealed that the most proliferative dose of hypericine ( lm) stimulates cyclin d . the anti-proliferative activity of hypericine was accompanied by inhibition of cyclin b mrna, whereas it induced expression of p and p genes, and thus apoptosis was observed by dna laddering at the same dose ( lm). overall results suggested that hypericine can compensate for viral transduction and improve reprogramming efficiency of ipscs by enforcing them in g phase. hence we report that hypericine can be a good candidate component for cocktails produced to trigger ipsc proliferation. glioblastoma (gbm) is the deadliest brain tumor. the mean survival time of gbm patients is approximately months, increasing to months after treatment with temozolomide, which is the gold standard chemotherapy. the resistance of gbm to chemotherapy seems to be associated with the blood-brain barrier (bbb) that limits the delivery of chemotherapeutics, and the presence of a population of cells that expresses stem cell-like properties, which are known to be chemo-and radioresistant, the glioblastoma stem cells (gscs). the difficulties imposed by these two factors could be reduced by the use of a targeted drugdelivery liposome-based strategy that allows bbb passage and reduces the side effects of chemotherapeutics. the present study evaluated the ability of the f peptide-targeted ph-sensitive lipid-based nanoparticle containing doxorubicin (dxr) to target gscs and non-gscs. we evaluated the expression of cell-surface nucleolin by flow cytometry, as well as of stem cell-like markers, in two gbm cell lines. we also determined the ability of gbm cell lines to specifically uptake the f peptide-targeted ph-sensitive lipid-based nanoparticles, by flow cytometry, and correlated it with the expression of stem cell-like markers. moreover, to ascertain the impact of intracellular delivery of chemotherapeutic drugs into gbm cell lines, cytotoxicity was further assessed by the mtt assay. our results showed that the f peptide-targeted ph-sensitive lipid-based nanoparticles successfully targeted glioblastoma cells and particularly gscs. in addition, the results also provided evidence of the nucleolin overexpression-dependency of this strategy, emphasizing the need to adapt the therapeutic strategy to the individual patient. this study showed that f -targeted ph-sensitive liposomes may constitute an appropriate strategy to overcome the chemoresistance associated with glioblastoma cells. p- . . - leukemic cell plasticiy as a resistance mechanism towards tyrosine kinase inhibitors chronic myelogenous leukemia (cml) is a hematopoietic stem cell disease characterized by the t( ; )(q ;q ) translocation, which encodes the chimeric tyrosine kinase onco-protein, bcr-abl. the tyrosine kinase inhibitor (tki) imatinib is the first-line treatment for patients with cml. unfortunately drug resistance is one of the main problems observed. while secondary resistance is associated with bcr-abl kinase domain mutations, oncogene amplification and mechanisms interfering with intra-cellular drug concentrations; primary resistance mechanisms haven't been elucidated. we generated high dose imatinib-resistant k subclones (k -ir) by clonal selection to study primary resistance mechanisms in vitro. drug resistance was shown by caspase and annexin v/pi assays. we also showed cellular uptake and function of imatinib with western blot technics. k -ir cells are not only resistant to imatinib but also to nd, rd generation tyrosine kinase inhibitors. we demonstrated that k -ir cells have a highly adherent character, proliferate slowly and are resistant to drug-induced senescence. microarray analysis revealed that k -ir cells differentially express tissue/organ developement and differentiation genes at high levels. we showed that k -ir cells forms intact tumor spheroids in d cell culture conditions which is a marker of tumor initiating potential. cell surface maker analyses and protein analyses of k -ir cell population, points towards an epithelial-mesenchymal plastic cell capable of adopting different morphologies. we hypotizied that imatinib and other tyrosine kinase inhibitors may cause the gain of phenotypic plasticity potential in leukemic cells, by interfering with signalling pathways; which in itself may lead to therapy resistance. hypoxia has multiple effects on cancer cells, which are critically involved in tumor progression. hypoxia leads to changes in tumor cell metabolism and can promote cancer cell survival, invasion and metastasis by its critically important role on maintenance of cancer stem cell (csc) phenotype. in this research, human cd + cscs isolated from human osteosarcoma cell line saos- using macs magnetic separation technique were characterized, and their stemness properties under hypoxic ( % o ) and normoxic ( % o ) conditions were compared in two and three dimensional culture conditions. two different d culture techniques (nanofibrous bacterial cellulose scaffolds and scaffold free microtissues) were used to evaluate effects of hypoxia on csc behavior, and the results were compared with the cell behavior in classical d culture systems. the morphologies of cells were examined by scanning electron microscopy (sem); rt-pcr and immunocytochemistry staining were used to examine the cancer stem cell phenotype maintenance under hypoxic and normoxic conditions. it is shown that hypoxia supports the expression of stemness markers such as oct / , nanog and sox compared to normoxic conditions in d cultures. although similar effects of hypoxia were observed in d cultured cscs, the expression levels of stem cell phenotypeindicative markers were significantly lower on d compared to d culture systems. this study is seen as an introduction to develop a more relevant d hypoxic cancer stem cell based tumor model to study csc behavior and tumor genesis in vitro for testing of novel cancer stem cell therapeutics and to understand signal transduction in cancer stem cells. prostate cancer (pca) is the second most frequent cause of cancer-specific mortality in the world. cancer stem cells (cscs) are a subpopulation of cells that involved in drug resistance, metastasis and recurrence of cancers. the efficacy of natural flavanone apigenin on cell survival, apoptosis and migration of cscs were evaluated. cd + cscs were isolated from human pca pc cells using a magnetic-activated cell sorting system. pc and cscs were treated with different concentrations of apigenin, docetaxel and combinations of the two agents for h. apigenin dose dependently inhibited cscs and pc cell viability, and this was accompanied with a significant increase of the cell cycle inhibitors p and p (kip ). the flavonoid significantly induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mrna expressions of caspases- , - and tnfa, but failed to regulate the intrinsic pathway as determined by the bax, cytochrome c and apaf- in cscs. in contrast to cscs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of bax, cytochrome c and caspase- while caspase- , tnf-a and bcl- levels remained unchanged in pc cells. the ability of apigenin to inhibit the proliferation of cscs through apoptosis was confirmed by tali image-based cytometer. the flavanone strongly suppressed the migration rate of cscs compared to untreated cells. significant downregulation of mmp- and - exhibits the ability of apigenin treatment to suppress invasion. the expressions of pi k/akt and nf-kb p / p were significantly decreased after h apigenin treatment. taken together, these data demonstrated that flavonoid apigenin is an invaluable chemopreventive compound that inhibits proliferation, invasion and the stemness properties of cscs. this study was funded by the scientific and technological research council of turkey (tubitak, grant no. s ). (pi k), are frequently found in patients with severe early-onset segmental overgrowth. whilst differences in timing and location of the founder mutation are likely to explain part of the observed disease heterogeneity, it is less clear whether and how quantitative differences in the strength and timing of pi k activity contribute to phenotypic variability. our aim is to characterise pik ca mutant-specific signalling as well as to explore the effects of varying the strength and/or temporal pattern of pi k activation on downstream output specificity in the cell. we are currently employing crispr/cas mediated gene editing in human induced pluripotent stem cells to generate isogenic disease models of three such activating pik ca mutations. these cells will be used for signalome profiling by reverse-phase protein arrays (rppa) to compare and contrast mutant-dependent alterations to candidate signalling networks. in parallel, ongoing efforts focus on developing an endogenously expressed optogenetic p a, allowing precise spatiotemporal control over pi k signaling to unravel the extent to which pi kdependent phenotypes are determined by strength of activation and/or dynamic encoding. ultimately, the outcome of this research will yield novel insight into fundamental aspects of pi k signalling and potentially aid the development of targeted therapies for human diseases of pi k hyperactivation. e. gov, n. kaya, k. y. arga cancer stem cells (csc) have been proposed to be the cancer initiating cells. because of their highly tumorigenic and drug resistant properties, cscs offer significant potential for developing novel anticancer drugs and therapeutic strategies. in the present study we analysed eight gene expression datasets for breast, ovarian, lung cancer and glioblastoma by comparing gene expression levels between stem cells and tumor cells and integrating them with genome scale biological networks. consequently, mutual molecular signatures (i.e: differential expressed genes, transcription factor, mirna) and biological characteristics were determined via integrative analyses, which might be feasible to uncover the mutual biological mechanism insights behind the cscs. it was identified twenty mutual differential expressed genes in four cancer types; jun and klf as transcription factors, egfr and cdk as receptors come into prominence as mutual signatures. molecules and pathways that were related to mapk, wnt, p signaling and pathways in cancer were the common indicators in csc types. our results provided similarities in gene expression profiles of various cscs and gave clues about the seed of tumorigenesis. this study proposed signatures and pathways that could be considered as effective therapeutic approaches in further experimental and clinical applications to eliminate subpopulation of csc. colorectal cancer (crc) is one of the leading causes of mortality worldwide. metastasis is associated with the presence of circulating tumor cells (ctcs) in the peripheral blood of cancer patients. ctc cut-off values have been shown to predict for poorer overall survival in metastatic breast (≥ ), prostate (≥ ), and colorectal (≥ ) cancer based on assessment of . ml of blood. in our study, ctcs were detected in blood samples of colorectal cancer patients, using with our modified convenient method for the strategies of ctc enrichment and detection. . ml peripheral blood samples were firstly collected and peripheral blood mononuclear cells (pbmcs) were isolated from the fresh blood samples by ficoll gradient separation. next, the leukocytes in pbmcs were removed by magnetic microbeads conjugated with cd for a negative selection. finally, the retained cells were labeled with anti-epithelial cell adhesion molecule (anti-epcam), cytokeratins (ck , ck ) and the leukocyte-specific marker as anti-cd . all samples were analyzed by bd facs aria iii flow cytometry. in total, patients and healthy people were included in this study. the results showed that ctcs were not detected in the blood samples of healhty volunteers, but - ctcs were detected with ck , , , -based gating strategy in the blood samples of colorectal cancer patients. it is accepted that the cut off value is ctcs for colorectal cancer and ctc is negative if it is below this value or ctc is considered as a positive, if it is equal to or above this value, which might be an indication for poor prognosis. thus ctc's detection may serve a representative surrogate tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. cells were grown in culture flasks in a humidified incubator at °c with % co and were used at the proliferation and confluent stages. cultured cells were exposed to the pemf and prfe. the proliferations of the cells are measured by mtt assay for the effect of emf on the cancer cells. on the other hand the wound healing was investigated by closure of the wound by the cell proliferation with cell morphology using inverted microscope images. the proliferation decreased significantly by the effect of pemf on the semi confluent mcf- and mda-mb- cells. this effect was observed more prominent on mcf- . considering prfe therapy this effect is much more pronounced especially for mda-mb- comparing with pemf. the phase contrast observations of these results were consistent with mtt analyses. similarly, this effect was seen less for pemf but the proliferation was more suppressed with prfe on the wound models. it was considered that the emf applications could be effective in cancer cells, but this effect has not been studied how it occurs in invasive cancers. in our cell culture study, the appropriate emf applications were found to be effective though the inhibition of proliferation of cancer cells even in invasive cancer but with lower effect. this means that emf applications may support the existing treatment methods of cancer patients and even people who suffer from invasive cancer. metastasis is the one of the most known causes of death in patients diagnosed with cancer. circulating tumor cells (ctcs) are shed from primary tumors and circulating in the bloodstream, and thought to play a key role in metastasis. a hypothesis that ctcs may contribute to metastasis was first introduced in the mid th century by thomas ashworth, an austrilian pathologist. in today's research, identification and molecular characterization of ctcs are thought to be a novel target for treatment of cancer and a key factor to understand the metastatic process. existing methods of ctc capture based on the cell search system, flow cytometers, laser scanning cytometers instruments, fiber-optic array scanning technology (fast), isolation by size of epithelial tumor cells (iset), and definition fluorescence scanning microscopy. ctcs are increasingly considered as a 'liquid biopsy' and when liquid biopsy is compared to tumor tissue biopsy, liquid biopsy for ctcs detection can be carried out routinely in patients due to accessibility and ease of blood collection. also, primary tumor sampling may not reflect the actual metastatic conditions, ctcs are thought to be a novel tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. with futher works, ctcs may be used as liquid biopsies and it might provide better understanding metastatic process, new approaches in cancer diagnostics and treatment. mesenchymal stem cells (mscs) are distributed all over the organism as a source of tissue formation and regeneration. glucose is vital for the proliferation and differentiation of mscs. glucose uptake is mediated by specific glucose transporters of two families, the na-coupled glucose transporters (sglt) and glucose transporter facilitators (glut). the presence and function of glut proteins in human placental amnion derived mscs (hamscs) is unknown. we aimed to investigate the presence of glut , glut , glut proteins and genes in hamscs isolated from term placentas. mscs were isolated from human term placenta amniotic membrane, the characterization of cells were provided by flow cytometry. mscs were used to assess their chondrogenic, osteogenic and adipogenic differentiation potential. the expression of glut , glut and glut proteins was detected in hamscs by immunofluorescence. glut , glut , glut protein and gene expression in these cells were investigated by western blot and real-time pcr, respectively. flow cytometry analysis results of isolated cells showed that they were positive for cd , cd , cd , cd (mesenchymal stem cell markers) and hematopoietic markers cd , cd b, cd , cd and hla-dr were negative. the presence of glut , glut , glut proteins and genes were identified in hamscs. in this study, for the first time in literature, glut , glut and glut gene and protein presence was determined in hamscs. therefore, gluts could mediate glucose transport in human amniotic membrane mscs. proliferation and differentiation of mscs in vitro are still not optimized. further studies are required to clarify the complex mechanisms regulating the relationship between glucose and mesenchymal stem cells. disclosure of this relationship may provide a better understanding of glucose-related pathologies such as diabetes. tumors have hierarchically organized heterogeneous cell populations and a small subpopulation of cells, termed cancer stem cells (cscs), is responsible for tumor initiation, maintenance as well as drug resistance. therefore, killing the cscs along with the other cancer cells is gaining an importance. in the present study, it was aimed to evaluate the cytotoxic and apoptotic activity of a novel platin (pt) (ii) complex [pt(hepy) cl ] on mammospheres obtained from mcf- human breast cancer line. elevated expression of stemness markers were determined by western blotting. cytotoxicity was assessed using the atp viability assay. effect of the pt (ii) complex on the formation and development of mammospheres was analyzed with sphere formation (sfa) assay. apoptosis was determined via cytofluorimetric analysis (caspase / activity, annexin-v-fitc and bcl- activity) as well as gene expression analysis. cytotoxicity was confirmed with the atp viability assay after the treatment with zvad-fmk (an apoptosis inhibitor) and necrostatin (a necroptosis inhibitor). in addition, alterations in mitochondrial membrane potential were evaluated by jc- staining. mammospheres exhibited increased oct- and sox (stemness markers) expressions compared to parental mcf- cells. cytotoxicity by pt (ii) complex was evident in a dose-dependent fashion ( . - lm) . pt (ii) complex significantly prevented mammosphere formation and disrupted mammosphere structure in a dose-dependent manner. pt (ii)-induced apoptosis was determined based on the presence of caspase / activity, annexin-v-fitc positivity and bcl- inactivation. apoptosis was also confirmed with increased tnfrsf a and hrk gene expressions. in addition to apoptosis, necroptosis was also present as evidenced with increased mlkl expression. mitochondrial membrane was depolarized. in conclusion, the pt (ii) complex seems to be a powerful apoptosis-inducing compound on cancer stem cells, thereby warrants further in vivo experiments. cancer is a disease which arises from destruction of growth and proliferation mechanisms in cells and is the second leading cause of death worldwide [ ] . in the development of primary cancers, the head and neck cancer is accounting for approximately . new cases annually around the world [ ] . laryngeal cancer is a type of head and neck cancer in which malignant cells arise from the mucosal tissues of the larynx [ ] . cancer might spread from primary tumor by getting into the lymph and blood vessel system and forms secondary tumor. greater than % of deaths in cancer patients are attributed to metastasis [ ] . circulating tumor cells (ctc's) provide an opportunity to understand the metastatic process of cancer patients. identification and molecular characterization of ctc's in the peripheral blood of cancer patients is a promising research area in the field of biomarker development and novel treatment targeting in today's cancer research [ ] . the detection of ctc methods include cell search system, flow cytometry, high-definition fluorescence scanning microscopy, fiber-optic array scanning technology, isolation by size of epithelial tumor cells, and laser scanning cytometers [ ] . in our study, . ml of peripheral blood samples were collected from larynx cancer patients and healthy volunteers and the samples were analyzed by bd facs aria iii flow cytometry via biomarkers epcam, ck , ck for positive selection and cd for negative selection [ ] . according to the results of our study; ctcs were detected in larynx cancer patients by our newly modified method whereas there was no ctc's detection in the samples of controls. thus, this study may provide us monitoring of the treatment process of larynx cancer and this method might be used as diagnostic, prognostic, and predictive biomarkers in cancer therapy as a liquid biopsy. prostate cancer is the second most common cancer and the fifth leading cause of death from cancer in men . circulating tumor cells (ctcs) present in the peripheral circulation of cancer patients with different solid malignancies including prostate cancer and have a potential as a liquid biopsy to monitor disease progression and response to therapies at cell and molecular level . one of the general methods in ctc detection is flow cytometry . radical prostatectomy is the most frequently applied procedure in the surgical management of localized prostate cancer. in this surgical operation, the surgeon removes the entire prostate gland with the seminal vesicles. a radical prostatectomy procedure can be done using the da vinci robotic system (intuitive surgical, sunnyvale, ca, usa) . robotic surgery has been suggested to have fewer complications, lower risk of infections and shorter recovery period following robotic radical prostatectomy , . in this study, our aim was to detect ctcs before and after robotic radical prostatectomy in clinical localized prostate cancer patients. the ctc detection study was performed with our modified method in which . ml of peripheral blood samples were collected from each prostate cancer patient and healthy individual; the samples, using with biomarkers epcam, ck , ck for positive selection and cd for negative selection, were analyzed by bd facs aria iii flow cytometry . according to our results, we detected ctcs in the peripheral blood samples of prostate cancer patients before robotic radical prostatectomy. however, following this surgical procedure no ctc or decreased number of ctss was detected. our study might contribute to understand disease progression after robotic radical prostatectomy in clinically localized prostate cancer patients that warrants further research. keywords: circulating tumor cells, prostate cancer, flow cytometry, robotic radical prostatectomy. p- . . - determination of effect cytotoxic, apoptotic, caspace- activity and mrna expression levels of apoptototic related genes of vulpinic acid on breast cancer cell lines n. kilic ß, s. aras, d. cansaran-duman ankara university, ankara, turkey breast cancer is the most common cancer types in women. several drugs used to treat breast cancer patients are developing resistance to the treatment for this reason success rate falls. therefore the discovery of alternative therapeutic agent and molecular detection of anticarcinogen effect because of treatment for cancer patients may be a source of hope for the contributions. in this study, different concentrations ( . , . , . , . , , , lm) vulpinic acid (va) lichen seconder metabolite was determined to cytotoxic, apoptotic effect and caspase- activity in breast cancer cells (mda-mb- , mcf , bt- , sk-br ) and normal cell (mcf a). in addition to the quantitative real-time pcr (qrt-pcr) using apoptose specific primers (tp- , bcl- , bax, birc- , gapdh, caspase- , caspase- , caspase- , caspase- ) and sybr green dye were performed to determine expression patterns of transcript level in cancer cell lines, using gapdh as a reference gene. the antiproliferative characterization of va effects identification of the gene set at molecular level and we determination role of va on apoptotic pathway. according to our study, va is demonstrated significantly (p < . ) effect cytotoxic, apoptotic, caspase- activity. beside this, dose dependent expression patterns decreased apoptose spesific genes (except of bcl- ) mrna levels from six to eleven fold change more than untreated va cell lines. va will be used as candidate molecule for effective treatment on breast cancer in the future. glycosylation largely determines the variety and functions of proteins. paucimannose, a mannosidic n-glycoepitope has long been thought to be specific for plants and invertebrates. recently, it has also been detected in mammalsin physiological conditions (stem cells) and in pathophysiological conditions (inflammation and cancer). in glioblastoma cells, paucimannose also seems to play a role in cell proliferation. glioblastoma is the most frequent brain tumor in adults with poor prognosis due to a lack of suitable treatments. we hypothesize that paucimannose could be a promising new biomarker as it is otherwise rarely found in mammals. therefore, paucimannose levels were investigated in different glioblastoma cell lines differing in their proliferation rate and tumorigenicity. the highest paucimannose levels were detected in low proliferating, nontumorigenic cells. furthermore, we found that modulation of paucimannose function by application of a specific antibody regulated cell proliferation and the capability of cells to form colonies in soft agar. these data support a functional role of paucimannosidic epitopes in tumorigenic processes. glioblastoma multiforme (gbm) is the most lethal type of malignant brain tumors. recently, gbm stem cells (gscs) have been studied in great deal and accepted that they have a legitimate role in tumor formation, development, chemo-resistance and recurrence. in this study, it is aimed to investigate new therapeutic targets within apoptosis related molecules to select and eliminate cd + gscs effectively. ten primary gbm cells were isolated from gbm tissue samples and they were cultured among with the gbm cell lines (u , u , u and t ). cd + and cd À cells were seperated by macs method via anti-cd (ac ) antibody from cultured cells and cell lines. rna isolation from cd + and (À) cells, cdna synthesis was performed. finally, by performing pcr array, mrna expression levels of genes were detected. proper results were collected and analysed statistically. according to the results of pcr array; it has been found that cd + cells express approximately fold tnfrsf and fold tnfsf when they are compared with control cells. tnfsf binds to cd that is expressed on the surface of tcells. cd does not have a death domain, instead it has a cytoplasmic tail which binds to trafs. trafs act as adaptor molecules that are related with jnk and nf-jb signalling pathways. tnfrsf (dr ) is a death receptor which are known for transmitting the pro-apoptotic signals from outside to the inside of the cell. it negatively regulates t-cell activation and the release of few cytokines. as a conclusion, tnfsf and tnfrsf both are found on immune system cells, mostly on t-cells, which may mean that gbm stem cells act as a immune system cells to avoid the elimination by the immune system. to conclude, acting as an immune system cell and promoting survival via tnfsf and tnfrsf , these molecules may be essential markers to target cd + gbm stem cells. the effect of docetaxel on p , sin a and mdm gene expression in mcf- breast cell line docetaxel is a cytotoxin effective in treating breast cancer. it stabilizes microtubules and causes catastrophic cell cycle arrest in g /m. it also initiate signaling through cell death pathways that result in programmed cell death. in this study, it was aimed to investigate apoptotic and cytotoxic effects of docetaxel has on the mcf- breast cancer cells line. in this study, mcf- breast cell line was applied different doses docetaxel ( nm, nm, lm, lm, lm) as h and h. mtt analysis was performed to the mcf- breast cancer line in control group and groups of docetaxel. afterwards, evaluation of apoptosis by tunel and levels of p , sin a and mdm gene expression by real-time pcr were determined in an order. it was observed cell variable was significant lower in docetaxel groups compared to control group (p < . ) in h as mtt analysis. the lowest cell viabilty was determinated in group applied lm docetaxel. while the lowest positive cell density was determinated in control group, it was observed apoptotic cell density gradually increased with increasing docetaxel concentration in groups treated docetaxel (p < . ). the highest p , si a and mdm expressions were apperared in nm docetaxel group compared to control group. human alpha-fetoprotein (afp) and afp receptor binding domain (afprbd) are able to bind and internalize effectively by wide range of human tumor cells and tissues. as other vector molecules afprbd has insufficient quantity of chemical groups which can be conjugated with drugs or diagnostic agents. conjugation of vector molecules with macromolecular polymer carriers like dendrimers aims to solve this problem. our study describes influence of afprbd-dendrimer-doxorubicin conjugate surface charge on intracellular trafficking routes and toxicity. the amineterminated (g ) and acetyl-terminated (g ) nd generation pamam dendrimers carrying doxorubicin (dox) were used to synthesize conjugates with afprbd. unmodified by afprbd g and g dendrimer derivates labeled with dox were absorbed by the cells at °c with different efficiency. g -dox derivate characterized much slower internalization rate than nonacetylated g -dox. only g -dox shown partial colocalization with lysosomal marker lamp after h of incubation. internalization of afprbd-g -dox and afprbd-g -dox did not show significant difference. at the same time, both conjugates contained afprbd wyкy almost fully associated with lamp already after min of incubation. cytotoxicity results revealed that ic levels of g -dox and afprbd-g -dox coincided and demonstrated a bit higher activity against sensitive to dox skov and resistant skvlb cells than afprbd-g -dox conjugate after h of incubation. at the same time, after h of incubation afprbd-g -dox and afprbd-g -dox were much more than g -dox and g -dox. we may conclude that there is significant difference in ways of dendrimers internalization by tumor cells depending on nature of surface chemical groups. on the other hand, chemical modification of dendrimer conjugated with does not afprbd influence dramatically on the protein trafficking and resulting cytotoxic effect. russian scientific foundation supported this study (no. - - . ) , a key enzyme in glycolysis, catalyzes conversion of phosphoenolpyruvate (pep) into pyruvate with regeneration of adenosine triphosphate (atp). the key regulator of the metabolic alterations found in tumor cells is the glycolytic isoenzyme pyruvate kinase type m that is generally expressed in all proliferating cells and overexpressed in all tumor cells investigated to date. during carcinogenesis a shift in the pyruvate kinase isoenzyme equipment always takes place, such that the tissue-specific isoenzymes disappear, and m -pk is expressed. breast carcinoma, the third most common cancer worldwide, accounts for the highest morbidity and mortality. breast cancer tissue analysis confirmed the upregulation of m -pk in breast cancer, and high m -pk levels were associated with poor prognosis of breast cancer patients. materials and methods: poly hema (mac) nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. pyruvate immobilization was actualysed with cross linking reagent glutaraldehyde. biosensor was developed by preparing pottasiumferrociyanide, selected as a mediator. results: cyclic voltammograms have been carried out at between~ . and . v potentials vs. ag/agci. m -pk activty was detected by using differential pulse method at between . and À . v potentials by observing the differentiations in the current values. in the optimization studies, some parameters such as optimum ph, temperature, concentration of glutaraldehyde and p-hema-mac, were investigated. discussion and conclusion: the method developed for the measurement of the tumor m -pk activity by using biosensor. we found that more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. piruvat kinase tumor m -pk activity determination at low concentrations is possible with this method. p- . . - tie /tek: a potential biomarker for targeting glioblastoma stem cells role in angiogenesis, endothelial cell survival, proliferation, migration and adhesion. therefore, tie /tek could be a potential target for therapeutic strategies directed against glioblastoma stem cells and their microenvironment. in this study, we investigated the gene expression levels of tie /tek in both cd + gscs and cd À gbm cells. gbm primary cells were freshly isolated from glioblastoma tissue samples and cultured in dmem supplemented with % fetal calf serum and % penicillin-streptomycin at °c in % co -humidified incubator. we isolated cd + and cd À cells from gbm primary cells using macs system. following rna isolation from healthy brain tissues, cd À and cd + cells, cdna synthesis was performed. finally, according to microarray protocol, cell surface marker panel array was applied. expression levels were analyzed using the delta delta ct method. statistical analysis was performed using spss software for windows version . . tie /tek gene expression was determined as . fold higher in cd + gscs than normal brain tissue (p < . ). morever it was determined . fold higher compared to normal brain tissue in cd À (p < . ). according to our results tie /tek expression was higher in gscs, indicating that tie / tek may be a potential marker for targeting cancer stem cells in gbm. this research has been supported by the scientific and technological research council of turkey (no: s ). adenosine inhibited the breast cancer stemlike cell population through erk / pathway s. m. jafari, m. aghaie cancer stem cells (cscs) are immortal tumor-initiating cells that can self-renew and drive tumorigenesis in various cancers, including breast cancer and others solid cancers. in a study indicated that extracellular atp reduces tumor sphere growth and cancer stem cell population. but at present, there are no reports available in literature on the effect of adenosine on breast cancer stem cells. in this study we evaluated the effect of adenosine inhibition and its mechanism of action in breast cancer stem cells isolated from breast cancer cell lines. our result showed that adenosine significant reduces breast cancer stem cell population. reduction of erk / protein levels was also observed after treatment cancer cells with adenosine. in conclusion, our results indicate that adenosine decreases the breast cancer stem-like cell population through erk / pathway. taxanes are commonly used for the treatment of many cancers as chemotherapeutic drugs that resistance to these agents has become a major clinical obstacle. taxane based chemotherapy drugs such as paclitaxel, docetaxel and cabazitaxel bind microtubules and inhibit to microtubule polymerization appear to stimulate programmed cell death. taxane-resistance to cancer has not been clearly in progression and development of drug resistance. multiple mechanisms are involved in the drug efflux proteins as multidrug resistance protein, differences in amino acid sequences among the b-tubulin isotypes. we investigated taxane resistance with different doses of paclitaxel, docetaxel and cabazitaxel in prostate cancer stem cells. we compared the expression level of apoptotic proteins, and its functional role in resistance mechanisms in cd + /cd + prostate cancer cell lines. taxane drugs were categorized as concentration-dependent or time-dependent. cabazitaxel caused a time-dependent and dose-dependent reduction in cell viability in all tested cell lines. resistance activity was consistently higher in docetaxel in prostate cancer cells compared with the other drugs. there are many different response of clonogenic formation cd + /cd + cells with resistance to docetaxel, paclitaxel and cabazitaxel in prostate cancer stem cells. the innate of prostate cancer resistances are important characterization steps and critically limits treatment outcomes therefore novel drugs must be focus on antiresistance and molecular based combinations. mesenchymal stem cells (mscs) are self-renewing cells with ability to differentiate into organized, functional network of cells. mscs isolated from various tissues including adipose tissues, bone marrow, umbilical cord, placenta and pancreas have different differentiation and proliferation potential. good knowledge of the metabolism and proliferation mechanisms of stem cells is required for stem cell therapies. glucose is an important molecule in the culture of stem cells. glucose concentration affects the differentiation and proliferation potential of stem cells. the aim of the study was to investigate the proliferation status by identifying the proliferating cell nuclear antigen (pcna) expression under normoglycemic and hyperglycemic conditions in mscs. mscs were isolated from human term placenta amniotic membrane. characterization of the isolated cells was performed using flow cytometry. chondrogenic, osteogenic and adipogenic differentiation potential of these cells were investigated. characterized cells were cultured in normoglycemic and hyperglycemic conditions for and h and the expression of pcna protein expression in these cells were investigated by western blot. flow cytometry analysis showed that isolated cells were positive with mesenchymal stem cell markers cd , cd , cd , cd and negative with hematopoietic markers cd , cd b, cd , cd and hla-dr. western blot result of pcna protein expression statistically significantly increased in human amniotic membrane mscs under hyperglycemic conditions for and h culture. the glucose content of stem cell medium is important because glucose is an effective molecule of the proliferation of stem cells. proliferation of mscs in vitro are still not optimized. when the relationship between glucose and stem cells be understood, it will provide a better understanding for the glucose-related pathologies such as diabetes during pregnancy. prostate cancer (pca) is the second most common type of cancer among men in the world. it is revealed that some gene, protein and metabolite sets control the pca, however the whole metabolomics changes are not completely understood yet. pca is common among older men, and this is an important health problem in developed countries. sarcosine is the n-methyl derivative of the glycine amino acid. glycine n-methyl transferase produces sarcosine from glycine. besides, it is metabolized to glycine by sarcosine dehydrogenase. in , high level of sarcosine in urine was associated with pca by sreekumar et al. they identified sarcosine as a pca biomarker that was significantly increased in urine during prostate cancer progression to metastasis. following this study, several studies have been published indicating sarcosine as a pca biomarker. in our study, a preliminary biosensor system was fabricated for determination of sarcosine in urine by using sarcosine oxidase. sarcosine oxidase was immobilized on au electrode surface using gelatin as an immobilization matrix. glutaraldehyde was used as a cross-linking agent to avoid the loss of the enzymegelatin mixture. optimization and characterization studies were carried out. sarcosine concentrations were detected carefully with the developed biosensor system. the fabricated preliminary biosensor is a promising system that can allow lower detection limits after surface modifications. activation of the epithelial-mesenchymal transition (emt) program in tumor cells is associated with invasiveness and stemness. recent studies implicate emt-inducing molecules in reprogramming energy metabolism. the -phosphofructo- -kinase/fructose- , -bisphosphatase- (pfkfb ) regulates glycolysis by producing fructose , -bisphosphate (f , bp). given that pfkfb is induced by several established emt-inducers in tumor cells, e.g. hif- a and ras, we hypothesized that pfkfb may be involved in regulation of the emt in tumor cells. silencing of pfkfb in pancreatic adenocarcinoma cell lines panc and s vp was achieved using specific sirna molecules. mrna and protein expressions of the cdh gene (encoding e-cadherin, an established epithelial marker), as well as zeb and snai genes, by real-time quantitative (q)-pcr and western blot, respectively. immunfluorescence analysis was performed to visualize e-cadherin protein expression on plasma membrane. in order to test the effect of pfkfb on the invasive ability of the cells, a matrigel invasion assay was performed. ectopic expression of zeb was achived by transfecting cells with a plasmid carrying zeb cdna. cells that were depleted of pfkfb exhibited markedly increased cdh mrna and e-cadherin protein expressions and reduced snai and zeb mrna expressions. immunfluorescence analysis confirmed the upregulation of the e-cadherin protein on plasma membrane. silencing of pfkfb caused approximately % reduction in matrigel invasion, compared to non-targeting sirna. inhibition of the matrigel invasion caused by pfkfb depletion does not appear to be associated with reduced zeb expression, as ectopic expression of zeb did not reverse the effect of pfkfb silencing on invasion. taken together, these data suggest that pfkfb may be required for the maintenance of the mesenchymal phenotype and associated traits in pancreatic adenocarcinoma cell lines. introduction: leukemias are neoplasms that arise from hematopoietic cells initially proliferate in the bone marrow, and then disseminated in the peripheral blood, spleen, lymph nodes and eventually to other tissues. lymphomas occur primarily in the lymph nodes, but can be extended in peripheral blood and bone marrow infiltrate. aim: to determine the values of haematological parameters the control and test groups. to determine the prevalence of types of chronic leukemia in relation to the experimental group. compare haematological parameters in relation to the type of chronic leukemia. materials and methods: a prospective-retrospective study included subjects who were made laboratory hematology in oj clinical chemistry and biochemistry ukcs. blood tests conducted on the hematology analyzer siemens advia hematology system and abbott cell dyn and microscopic analysis of the peripheral blood smear. results and discussion: according to the age of respondents test group was established mild form of anemia, a red blood cell count is totaled . ae . x , which is signifycantly lower compared to the control group. the average number of leukocytes was significantly higher in subjects studied groups and amounted to . x , with a maximum value of x . in the peripheral blood of patients with chronic leucosis has established a significantly higher number of cells compared to the control group (p = . ), while the number of monocytes was a significantly smaller. in the group of patients with chronic leukosis largest number had chronic lymphocytic leukemia ( %), and chronic myeloid leukemia had % of respondents. conclusion: subjects with cll were statistically older than patients with cml, and as regards the gender structure, men have dominated in cll and cml in women. white bloodline was found that the number of leukocytes in both forms of chronic leukemia high above the reference value. p- . . - effect of enzymatic and non-enzymatic isolation methods of endometrial stem cells on their cell proliferative potential and mesenchymal stem cell characteristics human endometrial stem cells (hescs) are responsible for the monthly renewal of the basal layer of the human endometrium by facilitating stromal and vascular regeneration. in this study, hescs were isolated with three different isolation methods including non-enzymatic and enzymatic digestion using trypsin and collagenase type . the effect of these three isolation methods on the acquisition of mesenchymal stem cells (msc) and on hesc proliferative potential was evaluated through flow cytometric analysis of cd surface markers and wst- tetrazolium salt assay. our findings indicate that hescs isolated with these three methods have statistically similar cell proliferation rate at h time point. however, at h time point, hescs isolated with the non-enzymatic and collagenase type method displayed a higher expansion in cell number when compared to the hescs isolated with trypsin. the late passage of hescs isolated with non-enzymatic and trypsin methods showed the highest proliferation rate in comparison to the hescs obtained via collagenase type isolation method at h, h and h. the three isolation methods for the early passages of hescs had a resemblance in their msc profile with no significant difference. on the other hand, late passage hescs isolated using trypsin non-enzymatic method showed a higher cd and lower cd profile. moreover, late passage of hescs isolated with non-enzymatic method displayed a significant reduction in their cell surface cd , cd , and cd surface expression levels. only hescs isolated with collagenase type did not present a significant shift in their mesenchymal cd marker profile from early to late passages, taken together results from this study suggest that the longterm maintenance of mesenchymal markers can only be achieved in cell isolation with collagenase type , while non-enzymatic method is more suitable to obtain higher msc cell yield for immediate use. hepatocellular carcinoma (hcc) abundantly arises on the viral and/or chemical-induced cirrhosis in liver. cirrhosis is defined as one of the premalignant stage hcc in which microenvironmental changes occurred such as uncontrolled production of collagen type i and activation of hepatocyte growth factor (hgf)/c-met signaling. it has been shown that epcam+/cd + subpopulation of cells isolated from hcc tissue can initiate tumor at very low concentration in xenograft model and behaves as hepatic cancer stem cells. however, the molecular mechanisms supporting hepatic stem cell activation are not well understood and knowledge about the role of hgf/c-met pathway in this process is not clear. in this study, we aimed to define effect of collagen type i and hgf induction on the cell behaviours of epcam+/ cd . epcam+/cd + cells were sorted by magnetic separation from huh- cells. then proliferation and invasion of cells were analyzed under the hgf induction as well as branching morphogenesis in vitro. after hgf stimulation, phosphorylation level of c-met increased in epcam+/cd + subpopulation. moreover, presence of collagen type i enhanced significantly effect of hgf stimulation in the invasion of epcam+/cd + cells. we also have showed that hgf stimulation increased branching tubulogenesis capacity of epcam+/cd + subpopulation while it did not effect proliferation of cells. these effects of hgf reverted by c-met inhibitor, su , in vitro. all these findings showed that hgf and collagen type i regulates aggressive phenotype as microenvironmental changes via induction of invasiveness of epcam+/cd + subpopulation of huh- . in conclusion, we showed that hgf/c-met signaling causes to get more metastatic phenotype based on invasion and tubulogenesis in epcam+/cd + hepatic cancer stem cells in hcc and it might be possible to use c-met inhibitors to target hepatic cancer stem cells during hepatocarcinogenesis. endometriosis is defined by the migration of endometrial mesenchymal stem cells (emscs) into the peritoneal cavity or other site of body rather than uterus in a retrograde fashion. its previously known intracellular crosslinking enzyme called tissue transglutaminase (tg ) was shown to play important roles in the extracellular matrix (ecm) modelling, fibrosis, cell adhesion and migration. we have hypothesized that tg might be expressed in emscs and take part in the formation of endometriosis. the difference in the proliferation capacity of emsc isolated from endometrial tissue with/without endometriosis was determined using wst- assay and tg activity and expression levels were analysed by btc assay and rt-pcr. the biosynthesis and activity for mmp- and - were investigated with zymography and rt-pcr, respectively. although tg activity was found to be % less in emscs isolated from endometriotic tissue, these cells showed times higher tg protein expression than those isolated from the control tissue without endometriosis. emscs from endometriotic tissue have . times higher tg and . fold higher itgb mrna levels when compared to the cells of healthy group. similar results were observed in sdc- gene expression with a . fold increase. endometriotic emscs demonstrated an average of . -fold increase in the mmp- activity while a onefold increase was evident in mmp- activity when compared to the healthy emscs. emscs from patient group possessed a higher proliferative ability in comparison to that of healthy subjects within h. the fact that emscs from the control tissue showed lower tg protein levels with a high enzyme activity suggested that tg might be important in the development of endometriosis not only by destabilizing ecm but also enhancing the cell migration. in this context, the upregulation of tg along with itgb and sdc was evident in emscs of endometriosis which was possibly associated with the increase in the activity of mmp- and - . recent studies have indicated that pluripotent stem cells and some stromal stem cells such as mesenchymal stem cells (msc) are metabolically different from their differentiated counterparts. in this study, the cellular mechanisms controlling metabolic changes in stem cells was investigated using wharton jelly mesenchymal/stromal stem cells (wj-mssc). wj-msscs were isolated by the explant method and cultured in dmem-f with % fbs. endothelial differentiation was induced by the addition of vegf, egf, insulin and hydrocortisone for days. neuronal differentiation was achieved by using commercial neuronal differentiation medium (millipore) for days. in parallel experiments, cellular metabolic activity such as lactate production was measured. the msc characterization was performed by flow cytometry using antibodies against cd , cd , cd and cd (bd human msc analysis kit). the differentiation process was followed by measuring the expression of cd , cd for endothelial and gfap, neu and tyrozine hydroxylase proteins for neuronal cells by immunofluorescence. for gene expression, nanog, cd and gapdh genes were analyzed by rt-pcr. differentiation stimuli to endothelial or neuronal cells resulted in a significant decrease in msc marker proteins. expression of stem cell markers other than cd were decreased to - %. differentiation induced the expression of cd , cd for endothelial and gfap and neu proteins for neuronal cells. in vitro lactate production was decreased following differentiation in both lineages. neuronal differentiation increased glucose consumption by~ % and the extracellular calcium concentration of these cells was significantly lower than synchronous undifferentiated cells. glycolytic activity is decreased during in vitro differentiation of wj-msscs. metabolic reprogramming and glucose uptake of cells may be an early indicator of the differentiation process in wj-msscs, supporting the view on their metabolic plasticity. store-operated ca + entry (soce) activated by depletion of intracellular ca + stores has been shown to control intracellular ca + homeostasis in many physiological and pathological events. stromal interactive protein, stim , as endoplasmic reticulum (er) ca + sensor and orai protein as pore-forming subunit of soc channels play crucial roles in the activation of soce channels. stim and orai were reported to have pathophysiological roles especially in hepatocellular carcinoma (hcc). anticancer chemotherapy frequently falls back because of these tumor-initiating subpopulations, tentatively called 'cancer stem cells'. the purpose of this study was to investigate the roles of stim and orai on soce in differentiation of huh- hccs expressing epcam and cd surface adhesion molecules (epcam + cd + ). epcam + cd + subpopulations in huh- cells were separated via flow cytometry and transfected with stim and orai- over-expressing (oe) plasmids. expression levels were confirmed by rt-pcr. changes in intracellular ca + concentration were monitored via dual wavelength spectrofluorimeter in fura -loaded cells. in epcam + cd + cells, er ca + release increased without any change in soce compared to that of epcam À cd À cells. similar results were observed in stim -oe epcam + cd + cells. on the other hand, increase in orai has no effect on either parameter. cancer is globally one of the most death causes. recently, huge improvements occurred in the cancer diagnosis and treatment due to advanced technology, however recurrence occurs almost - % of patients and their survival times decreases. in this study, we aimed to investigate of relationship between the cancer stem cells which are strongly associated with chemotherapy and radiotherapy resistance and recurrence with the non-classical mhc i antigens which have immunosuppressive properties. for this purpose, we immunohistochemically evaluated the expression patterns of cd , cd , nanog, oct / , hla-g and hla-e in the advanced stage colorectal, gastric and breast cancer and also non malign biopsy samples. we detected that the cancer stem cell markers cd , cd , nanog and oct / significantly increased in the advanced stage cancer tissues. however, the immunosuppressive hla-g and hla-e expressions increased only in the colorectal and gastric tumor tissue. in addition to the presence of cancer stem cell like cells in the tumor tissues, increased expressions of hla-g and hla-e may indicate an immune evasive adaptation of tumor cells. according to our findings, the hla-g and hla-e may be potential therapy targets to elimination of cancer stem cells of colorectal and gastric cancers. however, more detailed studies are needed to support our findings and also to determinate of clinical values of these markers. endocannabinoids increase sdf- release from human mesenchymal stem cells s. k€ ose , f. aerts kaya , d. uc ßkan c ß etinkaya , p. korkusuz stem cell research and application center, hacettepe university, ankara, turkey, department of histology and embryology, hacettepe university, ankara, turkey lipid-structured endocannabinoids are endogenous morphine ligands and present widespread receptor-mediated effects at physiological and pathological levels on the nervous system as well as many other systems. these effects are partially realized through mechanisms affecting cell growth, differentiation, apoptosis and migration at the molecular level. the hematopoietic progenitor cells (hpcs) and mesenchymal stem cells (mscs) form a distinct niche in bone marrow where they interact with each other in harmony. the stromal cell-derived factor (sdf- /cxcl ) is a chemotactic factor in bone marrow and is released from mscs and their receptor cxcr is found in hpcs. with these rationale in mind, we asked if hpcs and msc interaction mediates sdf- release via endocannabinoidal system. bone marrow mscs obtained from healthy donors and passage mscs were induced with ng/ml lipopolysaccaride (lps) for h. antagonists for cb (am ) and cb (am ) receptors were added to cultures for days. after incubation with antagonists msc culture supernatants collected and processed with human sdf- beta in elisa medium. analyses demonstrated direct decreasing effect of endocannabinoid receptor antagonists on sdf- beta release from bone marrow mscs. in conclusion, endocannabinoidal system regulates sdf- release on mscs and directly act on hpcs mobilization in bone marrow microenvironment (niche). this may have a clinical implication on therapeutic mobilization strategies for hscs in hematology clinical applications. implantation is invasion of the embryo into the endometrium and occurs in three stages apposition, adhesion and invasion, via the complex cellular and molecular mechanisms. during these stages, both of maternal endometrium and embryo should be appropriate for the implantation which is the beginning of pregnancy. receptivity of uterine consists in the existence of growth factors such as tgfbeta- , igfr , vegf. it is indicated that damages of factors relesead from endometrium and blastocyst prevent implantation. recently, stem cells can be obtained from many sources to use for therapeutic purposes and mesenchymal stem cells derived from bone marrow are the most studied. in our study, it was aimed to investigate molecules play a role in blastocyst implantation after bone marrow derived mesenchymal stem cell application into the rat endometriyum. female rats were divided into three groups which were saline (sf, n: ), media (m, n: ), stem cell in media (m+bmsc, n: ). after vaginal smear technique, female rats in estrous cylcle were injected into the uterine and periton ll saline, ll culture media and cell/ ll culture media. the pregnant female rats on the day were sacrified and uterine samples removed and were stained with heamatoxylin-eosin histochemically and anti-tgfbeta- , anti-igfr , anti-vegf and anti-pcna immunohistochemically and obseved under light microscope. h-score results were determined using one-way anova test statistically. it was found that intraperitoneal administration of stem cells with media, was increased tgfbeta- , igfr , vegf and pcna parameters when compared with the intrauterine administration of stem cells. in this study, it was revealed that distribution of molecules play role in implantation were changed due to stem cell application. it is supposed that stem cell treatments can be cured the molecules caused infertility. many unconventional biochemical factors remain to be investigated for their potential effects on stem cells. among others, endogenous gasotransmitter h s, generated from l-cysteine and organosulfur-compounds (oscs) metabolisms, plays very important roles in the central nervous, respiratory and cardiovascular system. slow-releasing h s donors are viewed as powerful tools for basic studies and innovative pharmaco-therapeutic agents for cardiovascular and neurodegenerative diseases. exogenous h s administration is able to promptly scavenge ros, activate myocardial k atp channels and increase pro-cell survival signaling, very likely activating erk and phosphatidylinositol -kinase (pi k)/akt pathways. the effects of h s-releasing agents on the growth of stem cells are not yet widely investigated. therefore, stem cell therapy combined with h s may have great clinical relevance in cell-based therapy for regenerative medicine. the effects of slow-releasing h s agents on the in vitro cell growth and differentiation of human lin À sca + cardiac progenitor cells (hcpc) were here studied. in particular, the effects of h s-releasing agents, such as na s, gyy and water-soluble gsh-garlic conjugates (gsgaws), on the cell viability and differentiation of hcpc were here investigated by colorimetric assay, immune-fluorescence microscopy and western-blotting analysis. the treatment with slow-releasing h s donors increased the cell proliferation in a concentration dependent manner respect to the control. moreover, the treatment with gsgaws led to an up-regulation of the expression of proteins involved in the cell adhesion and differentiation processes. these preliminary results highlight on the effects of this gasotransmitter on the stem/progenitor cells and on the possibility to develop functional d-systems for cardiac tissue repair, that take into account the relevant biological role of h s in the cardiovascular system. p- . . - investigation of the protective effect of boric acid and omega- fatty acid in model of acute myocardial infarction changes in myocardial rats ischemic heart disease being the most common cause of the mortality and morbidity in worldwide commonly results from the occlusion or narrowing of the coronary arteries by atheromatous plaque and thus is named as coronary artery disease. male sprague dawley rats were used in the present study. rats were divided into groups with rats in each: control, mi, mi+boric acid, mi+omega- and mi+boric acid+omega- groups. control rats were treated with ml/day saline, boric acid-treated rats received mg/kg/day boric acid and omega- -treated rats received mg/kg/day for days by oral gavage. for the experimental mi model, mg/kg izoproterenol-hcl (iso) was administered subcutaneously two times with a -h interval in the last days of the boric acid and/or omega- treatments. twelve hours after the second dose of iso, general anesthesia was induced. under general anesthesia and spontaneous respiration, ecg recordings were obtained by using a computerized data recording and analysis system (mp , biopar) and d-ii recordings were used in the analysis. compared to the control group, serum ck-mb, bnp and tnf-a levels were higher in mi group (p < . , p < . and p < . respectively). in the heart tissue homogenate, biochemically measured calpain activation and mda were increased (p < . and p < . , respectively) and pon levels were decreased (p < . ). according to the ecg recordings, st wave and heart rate were found to be decreased (p < . and p < . , respectively). on the other hand, all above mentioned parameters were found to be improved in rats treated with boric acid and/or omega- after induction of mi. moreover, histological analysis including light microscopy and tem revealed a significant histological improvement in rats treated with boric acid and/or omega- after induction of mi. results of the present study suggest that omega- and/or boric acid treatment significantly decreases the cellular damage in mi. this is study is aimed at measuring the level of serum heart-type fatty acid binding protein (h-fabp) in patients presenting with diabetic ketoacidosis (dka) and diabetic ketosis (dk) and to determine its role in identifying early period cardiac ischemia by comparing this level with the level of a control group at a comparable age this study was planned to be a prospective study and it included patients diagnosed with dka, patients diagnosed with dk and voluntary pediatric and adolescent healthy control subjects. the h-fabp, creatine kinase-mb (ck-mb) and troponin-i levels were studied in patients with dka and dk as well as in the control group at the time of presentation. for dka patients, their h-fabp values were measured once again after acidosis correction and compared with the values they had at the time of presentation there were no differences among groups in terms of sex, age, height and weight. no statistically significant differences were found among groups with respect to troponin-i values ( . ae . , . ae . . ae . ; p = . ). no statistically significant differences were found among groups with respect to ck-mb values ( . ae . , . ae . , . ae . ; p = . ). the h-fabp values of dka patients at the time of presentation were found to be statistically significantly higher than those of dk patients and control group ( . ae . ; . ae . . ae . ; p = . ). the h-fabp value of the dka group at the time of presentation was found to be statistically significantly higher than the value at hour after acidosis correction ( . ae . ; . ae . ; p = . ) the fact that h-fabp levels were found to be high in pediatric patients diagnosed with dka at the time of presentation suggested that myocardial ischemia had been triggered. in diabetic patients, every ketoacidosis attack may lead to cardiac ischemia, thereby accelerating progress to necrosis. in conclusion, we would like to propose h-fabp as a potential marker for indicating myocardial ischemia. p- . . - genome-wide analysis of hypoxic stress response in human cardiomyocytes stress in human cardiomyocytes on a genome-wide scale remains poorly understood. this study aimed to identify the gene expression patterns of adaptive response of the human cardiomyocytes (hcm) to hypoxic stress. in vitro experimental models of hypoxia mimicking in-vivo coronary ischemia, are useful tools to identify molecular pathways involved in myocardial ischemia. in the current study, we cultured ac -hcms in dmem/f with %fbs. to simulate hypoxia model, cardiomyocytes were plated in hypoxia chamber ( %o , %co , %n ) for , , , h and the control group was incubated in normal conditions ( %co , %o ). cell viability was determined using mttassay. annexin-v assay was used to monitor apoptosis. gene expression profiling was analysed with affymetrix-hg-u -plus- arrays. following bioinformatic and statistical analyses differentially expressed genes (deg) were classified according to gene ontology using david and kegg pathway analysis tools. according to mtt, annexin-v and hif gene expression results, hypoxia time was determined as h. we identified genes ( down-regulated and up-regulated) (p < . , fold change ≥ . ) were differentially expressed in hypoxic-ac vs. ac . degs were mainly clustered in cell proliferation, regulation of cell death, cell adhesion and response to stress. furthermore, transcriptome analyses revealed that 'metabolic, cytokinecytokine receptor interaction, hif- signaling, tgf-beta, cell cycle and apoptosis' pathways were involved in the hypoxic stress response of human cardiomyocytes. this study provides molecular information regarding gene expression reprogramming of human myocardial hypoxia. the pathways identified in this study may pave the road for translational medicine. this study was supported by tub _ itak project number s . autologous ips cells after reprogrammed into endothelial progenitor cells (epcs) may offer several advantages in the treatment of cardiovascular disorders because of their cardiogenic and vasculogenic differentiation potential. to reach that purpose, we differentiated and characterized mouse ips cells into flk + , a well-recognized epc marker. further maturation of epc was characterized by the expression of cd and cd markers. purified ips cells were differentiated into flk + cells with the use of differentiation medium on type iv collagen-coated dishes in the absence of lif. we then analyzed flk gene expression and protein levels with qrt-pcr, western blot and immunocytochemical methods on days . to . . flk + cells isolated with macs system and then recultured these cells in differentiation medium with vegf to induce epc cells. following induction, cd and cd gene expression and protein levels were analyzed with genomic and proteomic methods. after isolating these cells and aggregate overnight, we cultured cells in three-dimensional condition in collagen type i and used differentiated medium including vegf and egf. we found that flk expressing cell number reached to a peak level ( %) on day . followed by a progressive decline subsequently. in the second step, cd and cd positive cells were generated and enriched during day of induction. we showed optimal time for harvesting flk + cells is day . of initial differentiation. following isolation of flk + progenitor cells they were further matured into functional epcs by vegf within days of induction. additionally for evaluation of angiogenic potantial differentiated cells, we monitored epcs behavior along vascular formation in d culture. our work demonstrates that epcs could be successfully derived from ips cells and these cells have vascular formation and angiogenic potential in d culture. epc drived ips cells play important role in the treatment of cardiovascular disease. p- . . - electrophysiological, biochemical and genotoxic effects of luna experience on heart tissue in rat model pesticides are widely used for the control of agricultural, industrial and domestic pests. however, the uncontrolled use of pesticides has diverse effects on ecological system and public health. fungicides are one of the pesticide type used to kill fungi or fungal spores. in this study, the effect of different doses of luna experience, a fungicide, on the cardiac electrophysiology and genotoxicity in rats were investigated. among five groups ( mg, mg, mg, control and positive control for comet assay) treatment groups received by gavage doses of luna experience for days. electrical activity of heart were recorded using electrophysiological recording techniques. tissue activities of paraoxanase (pon) and arylesterase (are) and level of malondialdehyde (mda) were measured using biochemical methods. comet assay was performed on heart tissue. we calculated genetic damage index (gdi) and damaged cell percent (dcp) from comet assay. it was observed that there is a significant decrease in heart rate in all treated groups as compared with control group (p < . ). amplitude of p wave and qrs complex did not change (p > . ). in all treated groups, statistically significant differences were found for values of pon, are, mda, gdi and dcp when compared to control group (p < . ). according to our results, exposure to different doses luna experience have a probable hazard potential for the cardiac system. the macrocyclic cage complexes iron (ii) clathrochelates are of the interest due to their bioactivity; they are able to inhibit t- rna polymerase, possess toxicity to leukemia cells hl- and suppress amyloid fibril formation. their binding to serum albumins was reported; the extreme binding affinity to albumins is observed for the compounds bearing carboxy groups. upon this interaction, clathrochelates quench protein intrinsic fluorescence and gain optical activity inducing circular dichroism (cd) signal in - nm region. here we examine the effect of spatial arrangement (isomery of substituents) of clathrochelates on their binding to globular proteins. we study bis-substituted clathrochelates bearing two same or different isomers (ortho-/meta-/para-) of carboxyphenylsulfid groups. their interaction with bovine (bsa) and human serum albumins, b-lactoglobulin and lysozyme are explored by cd and protein fluorescence quenching method. the binding of compounds to albumins evoked the cd bands of the same shape, but their intensities vary up to times depending on substituents isomery. in the presence of b-lactoglobulin, the intensities, shape, and positions of the induced cdbands differ for the compounds with different isomer groups. the cd bands induced by the lysozyme in the case of di-para substituted clathrochelate are shifted relatively to the bands of other isomeric compounds. the pronounced quenching of protein fluorescence by clathrochelates was observed only in the case of bsa, its intensity depends on the geometry of substituents ( - times). the different spatial arrangement (isomery) of carboxyphenylsulfid substituents in clathrochelates causes the distinctions in both their cd-signal induced by interaction with proteins and their effect on the protein fluorescence. the geometry of ribbed substituents is important for their binding to biomolecules (particularly proteins) and is suggested to determine the structure of the formed guest-host complex. d bioprinting is a new technology that revolutionized the field of tissue engineering and regenerative medicine, allowing reconstruction of living tissue and organs preferably using the patient's own cells. using a d printer we can design biological structures by controlling exact deposition of cells, growth factors and extracellular molecules in a spatially-controlled manner. the aim of this study was to evaluate the differentiation of human amniotic fluid stem cells (afsc) into endothelial progenitor cells using a bioinkÒ hydrogel photopolymerized in a d network resembling vascular tissue. characterization of afsc was performed by flow cytometry, followed by sorting of the cd + stem cell subpopulation. cd + stem cells were stained with cell tracker red cmtpx and then mixed with bioinkÒ hydrogel. printing was done using a lm diameter needle, under bar pressure, and mm/min speed. the network models with define distance apart were printed and analyzed by fluorescent microscopy. mtt test was used to evaluate the viability of the cd + stem cells. our results showed that afsc remained viable as shown by mtt assay. the fluorescent microscopy images confirm the viability biochemical test showing that the cd + cells viability is maintained after days of cultured in bioinkÒ hydrogel. furthermore, histological section of hydrogel showed that cells have a relatively uniform distribution forming network interactions between cells. flow cytometry assay showed that cd + cells expressed endothelial markers such as cd , cd , cd , cd and vegfr . in conclusion d printers are useful tools for creating three-dimensional scaffolds that mimics the cell microenvironment where different types of cells could proliferate, differentiate and crosslink with each other forming tissue-like structures. this study aims to reveal the biocompatibility, biodistribution and immunomodulatory impact on the production of inflammatory citokines of magnetite (fe o ) nanoparticles functionalized with natural compounds with proved antimicrobial and immunomodulatory effects. co-precipitation synthesized fe o were functionalized with plant-derived compounds: eucalyptol, carvone, limonene and b-pinene. characterization was done by ir, sem and hr tem, while in vitro biocompatibility was tested using endothelial human cells (fluorescence microscopy and proliferation assay). in vivo biodistribution was tested in a balbc mouse model at and days post-intraperitoneal injection, followed by experimental organ removal. tissue sections obtained from vital organs were stained with hematoxylin-eosin. production of inflammatory cytokines was assessed by elisa. results demonstrated that, at concentrations of lg/ml, all prepared nanosystems have a good biocompatibility in vitro and in vivo, allowing the development of cultured cells and also not affecting any visible behavior and organ morphology of the mice. microscopy evaluation of the organs sections revealed that nanoparticles are not present in vital organs such as brain, heart, kidney and liver, but aggregates were visible in the lungs and spleen. at days post-injection no visible aggregated were found in the lungs, few dark-brown nanoparticles clusters being visible in the red pulp of spleen. elisa results revealed that fe o functionalized with carvone and limonene significantly stimulated the production of il- , il- and il- , while reducing the production of tnfa. other nanosystems din not impact significantly on the cytokine production. functional fe o nanoparticles are efficient drug delivery shuttles, able to stabilize pharmacological compounds, such as plant-derived bioactives, and their biocompatibility, specific biodistribution and limited immunomodulatory effects recommend their use in pharmacological formulations. p- . . - new approach for cell imaging with fluorescent carbon nanoparticles m. dekaliuk, k. pyrshev, o. demchenko palladin institute of biochemistry, kiev, ukraine in the nanotechnology field, much interest was focused on the new carbon nanomaterials for cell imaging. recently discovered inorganic carbon nanoparticles ('c-dots') due to their excellent fluorescence characteristics and biocompatibility have ample opportunities for their use in imaging and functional transformations in living cells. their distinctive features, such as high brightness, small sizes, high biocompatibility, small negative charge on the surface and very easy methods of their preparation present a good alternative to other nanoscale materials. the focus of our research was to determine the possibility of using c-dots as the easily available probes for apoptotic cells detection. the carbon nanoparticles were prepared from alanine, citric acid, urea, etc by hydrothermal treatment at c. the studies were performed with adherent epithelial vero and hela cell lines (atcc). with these tools we demonstrate that both native and apoptotic cells can be easily visualized. the cdots uptake occurs probably by endocytosis, which allows for much larger their number to accumulate in apoptotic cells. using the different methods of sample preparation, they show the ability for labeling various structural compartments of the cell. for living cells there are the intracellular vesicles and lysosomes. in contrast, in fixed cells the nucleus is labeled preferentially. the fact that apoptotic cells accumulate strongly increased amount of cdots can be efficiently used in flow cytometry for characterizing the cell populations regarding the relative amount of apoptotic cells in different experimental conditions. the application of such cheap and easily accessible nanoparticles provides more opportunities to simplify the popular methods of cell labeling and detection. previously, our studies showed the possibility of using these nanoscale fluorophores for super resolution method sofi. a new electrochemical microbial biosensor for the fast detecting of dopamine and epinephrine based on candida tropicalis immobilized in a carbon paste electrode (cpe) modified with single wall carbon nanotube (swcnt) was described in this paper. the immobilized cells were used as a source of polyphenol oxidase (ppo) to develop voltammetric epinephrine and dopamine biosensor. voltammetric determination of phenolic compounds like epinephrine and dopamine is a simple technique available. direct oxidation of phenols can be used, but the oxidation potentials of this compounds are similar and they can not be detected distinctively. another possibility is the use of biosensors based on the polyphenol oxidase (tyrosinase) enzyme that oxidises the phenolic compounds into their corresponding quinones. by this way phenolic compounds that epinephrine and dopamine that used in this study were detected at the different potential. the effect of varying the amounts of swcnt and microorganism on the response to epinephrine was investigated to find the optimum composition of the sensor. the effects of ph and temperature were also examined. increases in biosensor responses were linearly related to dopamine concentrations between . and . mm and epinephrine concentrations between . and . mm. limits of detection of the biosensor for dopamine and epinephrine were calculated to be . and . mm, respectively. finally, proposed systems were applied to epinephrine and dopamine analysis in pharmaceutical drugs. objective: it has started a long time ago to search for a material that can replace blood. this material does not require special storage conditions, independently of the recipient's blood group and can be applied to all individual. milk, casein derivatives, starch, saline and ringer were used for this aim in the past. the determination of toxic effect of natural hemoglobin (hb) on human, researchers have focused on development modified blood. in this work, the development of an artificial biomaterial alternative of blood for using as preoperative and operative aims was aimed. material and methods: in our study, ultrapure hb molecules are immobilized on triethanolamine coated magnetic nanoparticles using various techniques. prepared nanoparticles were characterized by ft-ir, ctem, xrd and cyclic voltammetry (cv). the cytotoxic effects of artificial blood were tried on mtt cell proliferation. results: the characteristic peaks of hemoglobin were obtained from ft-ir spectra differently from support. particles size is concluded by using debye-scherrer equation as > nm from xrd spectra. sem and ctem images supported xrd result. cv results showed that hb molecule has À . v cathodic potential against ag / agcl standard electrode. significant differences were not observed in the mtt results (p < . ). conclusion: the nanoparticles were obtained in accordance with the intended desired method. it is determined that the hemoglobin molecules give the same potential with natural blood even after weeks of immobilization and carrying oxygen as natural blood. there are statistical differences between results of mtt tests due to used concentration. but, it is considered that decantation advantage of the artificial blood minimized cytotoxic effects. proteoglycans are among the most abundant molecules of the inter-cellular structure and they are present in extracellular matrices of connective tissues. these glycosylated proteins contain one or more (gag) chains that are covalently attached to the core protein and their hydrodynamic function is mainly due to the physicochemical characteristics of this gag component which provides hydration and swelling pressure to the tissue. gag levels excreted via urine are used as a marker to monitor different diseases (chronic renal disease, renal fibrosis, glomerular filtration abnormalities, bladder stones, breast and lung cancers, hypertension and diabetes, etc.) besides the well known mucopolysaccharidoses. however, their detection by using chromatographic methods is hard, because of the high polarity of negative charges and different functional groups such as acetyl sulfates that generate microheterogenity. in this study, we developed molecularly imprinted chromatographic hplc columns for specific heparan sulfate (hsa), chondroitin sulfate (cs) and dermatan sulfate (ds) detection in urine. positively charged acrylamide monomers were first polymerized by precipitation polymerization, to produce polymers which will show specific recognition for gag's via electrostatic interactions and hydrogen bond formation. these gag selective polymers were then filled in the steel hplc columns and columns eluents were chemically degraded. degradation products of gag's were examined offline column coupled with tandem mass spectrometry. the results showed that our imprinted columns separated gag's specifically and sensitively. thus, urine gag's can be specifically determined by using a gag specific molecularly imprinted column. in this study internal standart weren't used because the matrix effect was lower than % for each urine samples. %cv of ds, cs and hsa was calculated as; supported lipid bilayers (slb) were started to be used for cell culture studies to focus on cell adhesion, cell signaling etc. testing the stability of slbs is essential to utilize them as cell culture platforms. in this study, the stability of phosphatidylcholine (pc) lipid bilayers on glass was investigated under milli-q water, phosphate buffer saline (pbs) and dulbecco's modified eagle medium culture (dme) medium supplemented with/without serum. the stability was also checked by enriching slb with different lipids. pc-liposomes were prepared by hydrating the dried thin lipid film with pbs and then by extruding the suspension through a polycarbonate membrane. a negatively charged phospholipid, phosphatidylserine (ps, %); a positively charged phospholipid, dotap ( %) and cholesterol ( %) were also used for liposome preparation. liposomes were fluorescently labelled and series of slb imaging were taken for a week. in all experiments in milli-q water and pbs, the stability was conserved for days. pc bilayers in medium supplemented with serum showed hole formations on the second day and their number and size increased rapidly in time. when the bilayers were prepared in medium without serum, disruption was lowered but not completely removed as a result of other factors in medium. cholesterol providing an increased rigidity to the membrane caused higher stability. positively charged bilayer structures also showed increased stability. this can be explained by decreased mobility of bilayer as a result of electrostatic interaction between positively charged molecules and negatively charged glass surfaces. decreased mobility decreases the interactions within the medium. lastly, negatively charged bilayers did not show high stability. strong repulsive forces between the negatively charged surface and bilayer probably prevented the integrity of the bilayer and increased the deformation. in recent years the use of biopolymers has gained priority in tissue engineering and biotechnology, both as dressing material and for enhancing treatment efficiency. there is a demand for new biopolymers designed with protease inhibitors and antimicrobials. ll- is an important antimicrobial peptide in human skin and exhibits a broad spectrum of antimicrobial activity against bacteria, fungi, and viral pathogens. using lignin which is an abundant carbohydrate polymer in nature and a polyacrylic acid, we prepared a polymer film by plastifying caprolactone and polyacyrlic acid. films were actified to immobilize ll- . the structure was elucidated in terms of its functional groups by fourier transform infrared spectroscopy (ftir), and the morphology of the film was characterized by scanning electron microscopy (sem) before and after the immobilization process. the amount of ll- immobilized was determined by elisa method. . % of ll- peptide was successfully immobilized onto the films. antimicrobial activity was determined in the film samples by quantitative antimicrobial activity method. according to the results, ll- immobilized film samples were effective on test organisms; gram-positive staphylococcus aureus and gram-negative escherichia coli. in bio-compatibility assays, the ability to support tissue cell integration was detected by using t mouse fibroblasts. samples were examined under transverse microscope, non-immobilized sample showed a huge cellular death, whereas ll- immobilized film had identical cellular growth with the control group. this dual functional film with enhanced antibacterial properties and increased tissue cell compatibility may be used to design new materials for various types of biological applications. p- . . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in vitro modulation of the cross-talk between macrophages and osteoblasts by titania nanotube-modified ti surfaces p. neacsu, a. mazare, p. schmuki, a. cimpean bone remodeling is a dynamic process that maintains a fine balance between bone formation and resorption, and is highly influenced by the inflammatory state of the local microenvironment. therefore, a proper modulation in the cellular interactions and cytokine expression is a promising approach to achieve enhanced bone healing. as the biomaterials surface has a major impact on cellular behavior, the goal of the current study was to investigate the influence of tio nanotube-modified ti surfaces (ti/tio ) on the cross-talk between raw . macrophages (mf) and mc t -e preosteoblasts (ob) in mono-and co-culture systems in comparison with flat ti (cpti). raw . and mc t -e cells were seeded on the test surfaces and grown in standard culture conditions for various periods of time. for co-culture studies, the cells were cultivated using a transwell system. inflammatory mediators released by raw . cells were measured using elisa technique, while the ob capacity to produce calcified bone matrix was evaluated by alizarin red staining. in co-cultures, lps-stimulated tnf-a, il- and mcp- release was significantly increased at h, while after days only il- exhibited higher amounts when compared with mf cultures alone. moreover, the secretion of these mediators by cells exposed to ti/tio was diminished, especially in lps evoked conditions. also, alizarin red staining demonstrated the presence of calcium deposits when ob were co-cultured with mf for h and days, whereas the presence of the mf for weeks significantly inhibited mineralization. on ti/tio surface elevated calcified matrix was observed, as compared with cpti. this study reveals that the overall effect of inflammation suppression induced by ti/tio may contribute to the enhanced mineralization. also, chronic inflammation may inhibit or delay the regeneration process. therefore, an adequate modulation of mf and ob interactions is vital for the biomaterials success in stimulating bone regeneration. p- . . - synthesis and characterization of the branched magnetic polymer for drug delivery systems t. tarhan , , b. tural , s. tural mardin artuklu university, mardin, turkey, dicle university, diyarbakir, turkey magnetic nanoparticles (mnp) have gained a lot of attention in biomedical and industrial applications due to their biocompatibility, easy of surface modification and magnetic properties. magnetic nanoparticles can be utilized in versatile ways, very similar to those of nanoparticles in general. however, the magnetic properties of these particles add a new dimension where they can be manipulated upon application of an external magnetic field. this property opens up new applications where drugs that are attached to a magnetic particle to be targeted in the body using a magnetic field. often, targeting is achieved by attaching a molecule that recognizes another molecule that is specific to the desired target area. in recent years, the development of the systems in which drug is delivered magnetically to the target is drawing considerable attention since it is a current issue. it is possible to eliminate the most of the problems caused by high doses of chemotherapy by using the magnetic drug delivery systems. therefore, it is important to design delivery systems with high drug loading capacity. it is necessary to increase the number of reactive groups on the surface of nanoparticles in order to increase drug loading capacity. in this study, we synthesized a novel magnetic surface for drug delivery systems. magnetic dextran-nta (md-nta) was synthesized by using magnetic o-carboxymethyl dextran (ocmd) and nana-bis (carboxymethyl) -l-lysine hydrate (nta) in order to increase the number of reactive carboxyl groups on the surface of biocompatible and biodegradable magnetic dextran. magnetic material (md-nta) which was prepared and characterized by the analysis of transmission electron microscopy (tem), scanning electron microscope (sem), vibrating sample magnetometer (vsm), fourier transform infrared spectroscopy (ftir) and x-ray photoelectron spectroscopy (xps). there are three subtypes of the tgf-b protein that has been reported to be involved in tissue repair process; scar tissue formation has been reported on tissues that has been affected by tgf-b and due to high collagen synthesis. on the other hand the other isoform tgf-b , suppresses the dense collagen production caused by tgf-b and prevents the scar formation. to be able to use these growth factors local or iv route, new drug transport systems are needed to protect the bioactivity during the treatment and controlled release. for this purpose poly(lactic-co-glycolic) acid polymer which is widely used in controlled release systems was chosen as the matrix material. aim of the project was to design, formulate, prepare and optimize tgf-b loaded plga nanoparticular and/or plga polymeric film drug delivery systems and to test their effect on cell proliferation. tgf-b loaded nanoparticles was prepared with emulsion-solvent evaporation method; whereas polymeric film systems was prepared with film castingsolvent evaporation method. following the preparation tgf-b loaded drug delivery systems was characterized. quantification and in vitro release of the growth factor tgf-b was studied with elisa. hepg cell line was used on mtt cell proliferation assay for both tgf-b loaded nanoparticles and films on a time course study. nanoparticles and films were prepared and loading efficiency of the nanoparticles were found to be . %. particle size, zeta index and polydispersity index for this formulation were determined as . ae . nm, . mw and . , respectively. thickness of the prepared films were ae . nm. additionaly prepared nanoparticles and films were found non-toxic. tgf-b nanoparticles and films which were prepared in this study are planned to be used as an effective treatment strategy for wound healing after injury. this project was supported by grand s from the scientific and technological research council of turkey (tubitak). polyvinylpyrrolidone (pvp) is a biodegradable material and natural polymeric biomaterial in such studies. ganoderma lucidum is a natural material containing triterpenes, polysaccharides, adenosine, polypeptides, and amino acids. these constituents have been shown to exhibit anti-cancer properties, enhance and regulate immunity, resist oxidation and ageing, and promote metabolism and cell proliferation. composites of polyvinylpyrrolidone (pvp) have been prepared by solution intercalation method using ganoderma lucidum at different loading amounts. the characterization of pvp/ ganoderma lucidum composites was made by x-ray diffraction (xrd) and scanning electron microscopy (sem); the interactions between ganoderma lucidum and pvp was determined by ftir-atr; the thermal stability was determined by simultaneous tg/dta. hemocompatibility of the prepared composite samples were investigated by a -well plate spectrophotometer. in addition, contact angles and antimicrobial activity of biomaterials were also determined. ftir-atr confirms interactions formed between ganoderma lucidum and pvp. xrd and sem results give evidence that ganoderma lucidum was well dispersed and homogenously in the pvp matrix. thermogravimetric analysis indicated that introduction of clay to the polymer network resulted in an increase in thermal stability. the results of in vitro hemocompatibility test were showed that pvp/ ganoderma lucidum composites are used as biomaterial. the development of synthetic materials, textured polymers and metals and their increasing use in medicine make research of biomaterials' hemocompatibility very relevant. composite material is a multi-phase system consisted of matrix material and reinforcing material. matrix material is a continuous phase and reinforcing material is a dispersed phase. the main two bioactive components of ganoderma lucidum can be broadly grouped into triterpenes and polysaccharides. despite triterpenes and polysaccharides being widely known as the major active ingredients at anti-cancer effect. this study describes the synthesis and characterisation of biocomposites of different molecular weight of peg (polyethylene glycol) as matrix with ganoderma lucidum as a filling material at different loading (% , % . , % wt). the composites have been prepared by solution intercalation method using ground and sieved ganoderma lucidum at micron scale. the characterization of composites was made by x-ray diffraction (xrd), scanning electron microscopy (sem) and fourier transform infrared attenuated total reflectance (ftir-atr) also in this study the hemocompatibility and antibactarial properties of composite investigated. when xrd and ftir-atr results discussed, all of the composites using the different loading amunt of ganoderma lucidum (% , % . and % wt) were shown a homogen distribution in the matrix (peg). and an interaction have occured between matrix and filling material. the sem photos have confirmed these results. peg and composites have been detected as hemocompatible. these results showed that they can be used as biomaterials. p- . . - evaluation of the genotoxic potential of some nanocomposites by comet assay b. yilmaz , s. dogan , s. celikler kasimogullari department of molecular biology and genetics, balikesir university, balikesir, turkey, department of biology, uludag university, bursa, turkey due to its similar nature to the bone, nanohydroxyapatite is a biocompatible particle and poly(methyl methacrylate) (pmma) is a polymer that has been used in dentistry and orthopedic applications for years. in this study, genotoxic potential of pmma/nanohydroxyapatite nanocomposite films composed of polymers having different molecular weights and nanohydroxyapatite fillers in different concentrations ( , . and %) were investigated by comet assay which is a kind of gel electrophoresis that can be used to measure dna damage in individual cells. if the dna is damaged we expect broken ends to migrate apart from the head. at the end of the assay performed after incubation with lymphocytes of healthy humans, we measured the dna damage index (ddi) and percentage of damaged cells (pdc). in addition, to prove the morphological properties of the nanocomposites scanning electron microscope was used and an interaction between the matrix and nanoparticles with a homogeneous dispersion was observed. protein adsorption on stimuli-responsive mixed pdmaema/peo polymer brushes a. bratek-skicki , , c. dupont-gillain universit e catholique de louvain, louvain-la-neuve, belgium, j. haber institute of catalysis and surface chemistry, polish academy of sciences, krakow, poland smart polymer brushes are made of macromolecules that are sensitive to stimuli from the external environment, including ph, ionic strength, temperature, etc. when stimuli-responsive polymer brushes are introduced onto material surfaces, their properties can be adjusted by tuning the environmental stimuli. these brushes can find promising applications across many areas of research, including surface science, nanotechnology, and biotechnology. in our work, the adsorption of human serum albumin (hsa, molecular weight of . kda, isoelectric point ip at ph . ) and lysozyme (lys, molecular weight of . kda, ip~ ) was studied on polymer brushes composed of poly(ethylene oxide) (peo) and poly ( -(dimethylamine) ethyl methacrylate) (pdmaema). peo is a protein-repellent polymer and pdmaema is a polyelectrolyte bearing a variable density of positive charges depending on ph. a gold substrate was modified by these thiolated polymers according to the 'grafting to' method. the obtained polymer brushes were characterized by xray photoelectron spectroscopy, static contact angle measurements and atomic force microscopy. polymer brush formation and protein adsorption were monitored by quartz crystal microbalance. surface characterization of the mixed brushes revealed the presence of both polymers at the surface. conformational changes of pdmaema/peo brushes were experimentally evidenced, and the results indicated that the brushes collapse at ph . (pdmaema is neutral in such conditions) and were swollen at ph . (pdmaema is positively charged). protein adsorption was performed at different ph values ( . - . ) and salt concentrations ( . - . m). it was shown that pdmaema has a high affinity to hsa at ph above its isoelectric point. however, the adsorption of positively charged lysozyme in a wide range of ph was not observed. these results indicate that pdmaema/peo brushes are promising candidates for selective adsorption from a mixture of proteins. clay-polymer nanocomposites (cpn) developed in recent years as a new type of inorganic-organic hybrid materials that were conceived for medical uses such as tissue engineering or drug delivery [ ] , [ ] . the understanding of the structure and physico-chemical properties of cpn is a first step in the investigation of biomaterials, but their potential in this respect is determined by their interaction with living tissue components. in this study, pure kaolinite was intercalated with dimethyl sulfoxide (dmso) and then intercalated kaolinite was modified pyridine, -amino pyridine and , -diamino pyridine to expand the interlayer basal spacing. modified kaolinite samples as filler and poly(vinyl chloride) (pvc) polymer as matrix were used in the nanocomposite synthesis. nanocomposites of pvc have been prepared by solvent blending method using thf as a solvent. the material characterizations were carried out by xrd, afm, ftir-atr, dta/tg and dsc. the xrd results reveal the formation of intercalation/exfoliation of modified kaolinite in the pvc matrix. ftir and afm results confirm the presence of nanomaterial in kaolinite/pvc nanocomposites. tga data show that the modified kaolinit/pvc nanocomposites have significant enhanced thermal stability. the glass transition temperature (tg) of pvc nanocomposites is higher than that of pure pvc. in addition, the antimicrobial activity of clay-polymer composites were also determined. introduction: polyhydroxyalkanoates (phas) are biocompatible and biodegradable materials obtained from microorganisms. they are produced in the cytoplasm of several bacteria as energy reserve. the physical properties of poly( -hydroxybutyrate) (phb), which is from the group of phas, make it a competitive source to petrochemical plastics. phb has potential in order to be used in a variety of application fields such as packaging industry, printing materials, agriculture and food industry. furthermore, phb meets expectations for tissue engineering applications, since it is biocompatible, biodegradable, non-toxic and has good mechanical properties. although its many advantages, blending approach could be needed in order to fulfill all expectations of a material. due to its flexibility, polycaprolactone (pcl) is a promising candidate to be blended with phb. the aim of this study is to construct a scaffold by using phb produced by extreme alkaliphilic b. marmarensis gmbe t (dsm ) and commercial pcl as components and investigate its properties. materials and methods: electrospinning method was used in order to construct scaffolds from blend polymer solution containing phb from b. marmarensis and commercial pcl. results: nanofiber structures were observed on scanning electron microscope (sem) images and fourier transform infrared resonance (ftir) analyses have shown characteristic peaks for both phb and pcl. discussion and conclusion: phb could be blended with other polymers in order to enrich its properties. in addition, nanofiber structure of electrospun phb-pcl blend makes it a rewarding material as scaffold for several tissue engineering applications. q fever is a zoonotic disease that is encountered widely around the world, the most common acute form of q fever shows the following symptoms; a sudden fever, shivering, lassitude, headache, anorexia. because this disease does not show specific symptoms its diagnosis is possible with laboratory tests. current diagnostic kits lack effectiveness; this is why the main goal of our studies is to come up with a new diagnostic kit that does not have disadvantages that current diagnostic kits show. with this goal, nine mile i strain (rsa ), s serologic virulent phase i, were obtained from slovak science academy, virology institute for rickettsia reference and research from who co-operation centre. these cells were purified and lipopolysaccharide (lps) isolation from coxiella burnetii was performed. the polymeric carrier, poly (n-vinyl- -pyrrolidone-co-acrylic acid) [p(vp-co-aa)] was synthesized and characterized. physical complexes of obtained lps and p(vp-co-aa) with varying ratios. ternary complexes of lps-cu + -p(vp-co-aa) were also synthesized with copper metal mediation. structure and interaction of lipopolysaccharide-p(vp-co-aa) complexes were investigated with zeta-sizer device using zeta potential analysis and ftir spectrophotometry according to the ratios of components, reaction environment conditions and chemical structure of the polymer. the best complex ratio according to analysis results will be used in the future studies for obtaining monoclonal antibodies which will be an important step for obtaining more effective and stable diagnostic kits that can be used for q fever. this in this study; new types of water soluble polymer-biomolecule conjugates were synthesized using covalent bonding techniques between polymers and co-polymers (varying monomers of polyacrylic acid and acrylic acid) with peptides. different compositions of polymers, varying ratios of biomolecule/polymer and different molecular weight of polymer has yielded new types of bioconjugates. conjugation mechanism, composition and structure were investigated with various physicochemical methods (uv, ftir, hplc, gpc, etc.). the peptide used in this study was the antigenic peptide epitope of sheep-goat pox disease (eakssiakhfslwksyadadiknsenk). whether this peptide series was bound to polymers or whether it was bound to polymer-protein carrier; peptide-specific immunogens that were capable of producing antibodies were synthesised. it is thought that using polymer-peptide bioconjugates that contain just peptide will yield a higher peptide-specific immunogenicity compared to traditional adjuvants. in vitro and in vivo investigations of bioconjugates effectivity is planned to be done in the future studies. p- . . - bioinert fluorinated ethylene-propylene copolymer modified for keratinocyte adhesion surface properties are crucial when adhesion of a cell to a polymeric material is required for a biomedical application. one of the methods for polymer surface tailoring is argon plasma treatment. this simple and reproducible method alters the surface properties such as roughness and wettability without affecting the bulk properties of the material. for the modification of the bioinert fluorinated ethylene-propylene (fep), related to teflonÒ, we employed argon plasma treatment with the powers of and w for - s. the human keratinocytes of the hacat cell line served as a model cell line for biocompatibility testing. we studied adhesion, proliferation and morphology of the cells on modified fep matrices as well as controls (pristine fep and standard polystyrene tissue culture dish) by means of fluorescence microscopy. further morphological details were acquired by scanning electron microscopy. furthermore, fluorescence microscopy with immunochemical labelling was used to determine the size and distribution of focal adhesions in cells grown on the modified matrices. the overall effect of the matrices on metabolic activity of cultured hacat cells was also evaluated using the wst- reagent. the ar plasma treatment of fep matrices improved significantly cell adhesion and proliferation and promoted spreading of the hacat cells compared to the pristine fep, on which cells were not able to spread properly. also, increased metabolic activity rates for cells cultured on modified matrices were found in comparison to the pristine fep. altogether, we found that ar plasma treatment improved the surface properties of fep to such extent, that it allows cultivation of adherent cells on its surface. we therefore propose that ar plasma treatment is a useful method for fep surface modification. p- . . - graphene oxide enriched biomaterials display potential for tissue engineering applications tissue engineering (te) requires more efficient systems that favor local tissue regeneration with minimum cytotoxicity. materials based on natural compounds ensure biocompatibility and have better effects for regeneration. graphene oxide (go) has been shown to enhance cell adhesion and to improve the rate of cell differentiation. in this context, the aim of this study was to evaluate if the addition of different concentrations ( . - wt.%) of graphene oxide (go) improves the properties of cellulose acetate (ca) materials for biocompatibility and cell differentiation, in the prospective of using these films for te applications. go-containing ca films were tested for cytocompatibility by quantitative and fluorescence microscopy assays, and compared to the ca control. cell cytoskeleton architecture in contact with biomaterials was revealed by confocal microscopy. furthermore, bioconstructs were exposed to in vitro osteogenic and adipogenic induction and monitored for days. histological stainings were performed to validate differentiation. osteogenic and adipogenic markers gene expressions were assessed via qpcr. ca/go wt.% revealed best biocompatibility among all tested scaffolds. adhesion proved to be dependent on the percentage of go in material's composition. cells cultivated on ca/ go wt.% expressed adipogenic and osteogenic markers earlier than cells cultivated on materials with lower go content. differentiation markers displayed an increasing profile of gene expression from to days post induction, with higher levels registered for materials with high go content as compared to films with low go content and to the control. go added to ca materials positively influenced cell survival, proliferation and cell differentiation. ca/go films represent potential candidates for te applications. the design of appropriate scaffolds remains one of the most important challenges for te. current idea is that the cell-scaffold interaction could drive cell differentiation and be linked to gene expression and protein organization. therefore, their quality is essential and should favour cell attachment, growth, migration, in situ vascularization and release of biochemical and physical factors able to address the cell fate. moreover, for an ideal scaffolding material an adequate and interconnected porosity is relevant for facilitating cell spreading and colonization of the inner layers. a combination of optimal mechanical and biochemical properties were here utilized to design a d composite hydrogel scaffold ( d-chs) in order to favour cell-scaffold interactions and promote a functional differentiation of human lin À sca + cardiac progenitor cells (hcpc). the biocompatible peg-diacrylate (pegda) was used to prepare hybrid protein-pegda hydrogels with embedded albumin-microspheres (ms) as protein component. ms were able to modulate the mechanical and biological behavior of the scaffold acting as air-reservoir, porogen agents and potential carriers of biomolecules. an increase of the hcpc viability in the ms-concentration dependent manner was observed. moreover, the microarchitecture of the d scaffold also plays a key role in the stability and functionality of cellularcomposite systems. therefore, pegda-honeycomb structures were fabricated using microstereolithography process and the hcpc viability and adhesion to the microstructures were assessed. d-chss were synthesized embedding honeycombstructures into ms-pegda hydrogel and the effects on cell proliferation, cell-cell interactions and cellular alignment were investigated. these results could be of relevant interest for expanding the knowledge on cell-scaffold interaction processes and to promote the development and the application of d-chs for tissue regeneration using the emerging bioprinting technologies p- . . - gene expressions of mesenchymal stem cells after osteogenic induction on ceraform bone substitute a. kilic s€ uloglu, e. karacaoglu, h. akel, c ß . karaaslan hacettepe university, ankara, turkey ceraformÒ, is a synthetic calcium phosphate ceramic with the chemical composition of hydroxyapatite % and tricalcium phosphate %, with - % pore volume and - lm pore diameter. in this study adipose tissue derived mesenchymal stem cells were differentiated into osteoblast cells and loaded on cer-aformÒ. in order to improve cell adherence, ceraformÒ was covered with fibronectin. the cells were cultivated for -day period by osteogenic induction medium. days , , , and were selected as specific intervals for incubations. total rna was isolated and cdna was synthesized. differences in the expression of runt-related transcription factor (runx ), bone morphogenetic protein- (bmp- ), and osteocalcin (ocn) were determined by qpcr. the peptidylprolyl isomerase a (ppia) gene was used as an internal control. according to the qpcr results, ocn gene expression was observed on the day th, continued to increase in day . bmp- gene expression was increased in , , day compared to day. on the other hand, runx gene expression was increased only on days and . these findings pointed out that the osteogenic induction was successfully activated on fn coated bone material. therefore, these results can be used in bone injury treatment and related disorders. p- . . - on the in vitro cytotoxicity of graphene oxide nanomaterials v. grumezescu , i. negut , f. sima , e. axente , l. e. sima national institute for lasers, plasma and radiation physics, magurele, romania, institute of biochemistry, romanian academy, bucharest, romania during the last decade, graphene and its derivates have proven unique physicochemical properties, several applications being continuously developed. among them, electronic, catalytic, mechanical, optical, and magnetic properties have attracted huge interests. however, the merging of graphene and graphene oxide (go) with biotechnology is still in its infancy, many challenges remaining unexplored. potential applications are related to biosensors, drug delivery or gene therapy and cells imaging. in order to use gos as drug release matrices for cancer cells targeting, it is necessary to ensure that these molecules do not affect normal cells within tissues. it was shown that the cytotoxicity of graphene nanomaterials is highly dependent on surface functionalization. studies suggest that pristine and reduced go with fewer surface functional groups tend to be more toxic than go. in striking contrast, it has been reported that functionalized graphenes, can significantly reduce the cytotoxicity even at relatively high concentrations. in this study, we report on the comparison between go and protein functionalized go when submitted to in vitro cytotoxicity tests. bovine serum albumin (bsa) was used for the noncovalent go surface conjugation. three case-studies were investigated: aqueous nano-colloids consisting of serial dilutions of both go and go-bsa conjugates, dropcasted thin films and laser-assembled thin films on glass substrates. safe concentration windows were identified by live/dead staining and mts assays for different human melanoma cell lines, while melanocytes and human dermal fibroblasts were used as normality controls. the predominant melanoma subtype is represented by cells bearing braf (v e) activating mutation. with a view to target this specific melanoma subpopulation, we embedded braf inhibitors into go laser-deposited scaffolds and tested their anti-tumoral effect. our results evidence the high potential of these nanomaterials for biomedical applications. osteoporosis is a skeletal disorder associated with low bone mass and increase in bone fragility due to increased osteoclastic activity. binding of rank ligand to its receptor on osteoclast precursor cells results in the osteoclast differentiation. sirna is a dsrna, used to inhibit the translation of the target gene. the aim of the study is to develop an injectable sirna-delivery system targeted to the bone for osteoporosis treatment. pei (polyethyleneimine) and rank complex was loaded into poly(lactic acid-co-glycolic acid) (plga) nanocapsules which are bound to hydroxyapatite (hap)-specific elastin-like protein (elp) for targeting to bone tissue. plga nanocapsules were prepared by w/o/w double emulsion technique. affinity of elp to hap was determined by ftir. elp was coated on the nanocapsules by using the transition temperature of elp. elp on plga nanocapsules were crosslinked by genipin and binding of elp on plga nanocapsules were studied by xps and tem. the optimum ratio of n (pei) to p (sirna) in the complexes to be loaded into plga nanocapsules were studied by etbr staining and zeta potential measurements with varying n/p ratios and finally pei-dnaoligo encapsulation efficiency of the capsules was determined by picogreen reagent. xps results of elp treated plga (elp-plga) nanoparticles indicated the presence of nitrogen atom ( . %) in the sample which appeared as a fuzzy halo in tem micrographs. n/p ratios up to show negatively charged particles. when n/p was , the zeta potential of complex was neutralized which also resulted in larger particles compared to the others. zeta potential moved to positive values when n/p was higher than . the migration of polyplexes with different n/p ratios ( - ) was analyzed by gel electrophoresis. dnaoligo complexes show the same patterns of complexation wih that of sirna and thus were used in the encapsulation efficiency studies instead of sirna. the encapsulation efficiency of pei-dnaoligo in plga nanocapsules was %. tuesday september : - : aging p- . . - novel benzenesulfonamides exhibit low toxicity on zebrafish embryonic development and selectively inhibit carbonic anhydrase ix with nanomolar affinity in xenopus oocytes introduction: the toxic effects of two recently discovered inhibitors (vd - and vd - - ) that selectively and with extraordinary strong, picomolar affinity bind to human carbonic anhydrase (ca) ix, an anticancer target, were investigated on zebrafish embryonic development and in xenopus laevis oocytes. zebrafish has emerged as a promising animal model to evaluate the toxicity of the drug candidates. xenopus oocytes do not natively possess any ca activity and thus became a convenient in vivo model system to study the ph effects and the selectivity of synthetic ca inhibitors. materials and methods: morphological changes in zebrafish treated with the compounds were studied by light-field microscopy and histological analysis. ca activity in xenopus oocytes was monitored by measuring ph in the cytosol and at the outer membrane surface and confirmed by mass spectrometry of lysed oocytes. the toxicity studies showed lc values to be lm for vd - , lm for vd - - and lm for ethoxzolamide (eza), a non-selective ca inhibitor commonly used in clinic. the zebrafish exposed to lc doses of vd - and vd - - showed fewer phenotypic abnormalities and less morphological changes compared to the zebrafish treated with the corresponding dose of eza. vd - - exhibited - nm ic for both intracellularly and extracellularly expressed ca ix in xenopus oocytes while exhibiting strong selectivity over ca ii, ca iv and ca xii. discussion: interestingly, the compounds exhibited -fold lower toxicity, induced fewer side effects in zebrafish than eza and the amount of vd - - needed to cause complete inhibition of ca ix enzymatic activity in xenopus oocytes was -fold lower than eza. conclusions: vd compounds did not lead to deleterious effects on the zebrafish embryonic development and reached the ic of nm for ca ix in xenopus oocytes. the compounds could be further developed as anticancer drugs. cacybp/sip is present in various cells and tissues, both normal and pathological. in normal tissues, e.g. stomach or colon, cacybp/sip is weakly or barely detected whereas in gastric or colon cancer this protein is expressed at a high level. there are also data indicating that the level of cacybp/sip expression correlates with tumor metastatic potency and multidrug resistance. taking into consideration data that suggest association of cacybp/sip with many vital cellular processes, in this work we decided to investigate the possible mechanism involved in regulation of cacybp/sip gene expression, mainly by transcription factors and, on the other hand, the influence of cacybp/sip on the expression of other genes. we have shown that nfat (nuclear factor of activated t cells) influences the cacybp/sip gene expression and that overexpression of cacybp/sip has an effect on the level of ap- and on the activity of nfat and ap- transcription factors. by analyzing the cacybp/sip gene promoter sequence we also found potential binding sites for transcription factors from the stat family, which are involved in interferon signaling. microarray data indicate that indeed overexpression of cacybp/sip affects levels of the stat proteins as well as of some interferons and interleukins. based on functional analysis we have found many genes the products of which are involved in immune response. to analyze in more detail the influence of an altered level of cacybp/sip on interferon signaling pathways as well as on factors involved in expression of interleukins, including nfkappab, we plan to apply methods such as luciferase assay, real-time pcr or immunocytochemistry. one of the pathological hallmarks of alzheimer's disease (ad) is the neuritic plaques occurred as a result of the extracellular accumulation of aß peptides formed from amyloid precursor protein (app) via the ß-amyloidogenic pathway. aß is more prone to aggregation to form plaques and more toxic to neurons than aß . in addition to change in app metabolism, the decline in levels of neurotransmitter acetylcholine and cholinergic dysfunction are also observed in ad. thus, current strategies for ad treatment focus on compounds with inhibitory effect on cholinesterases as well as preventive effect on aß aggregation. in our earlier studies, toluidine blue o (tbo), a phenothiazine dye, was shown to be a highly effective inhibitor of cholinesterases with k i values in nm range. we also found that intracellular app and aß levels are reduced in human neuroblastoma cells after treatment with tbo. additionally, an earlier study revealed that tbo has a selective inhibitory effect on tau aggregation, the other pathological characteristic of ad. the aim of this study was to investigate whether tbo may effectively lower the level of extracellular aß / in an ad-like cellular model. chinese hamster ovary cells that express human wild type app and presenilin , namely ps , were treated with a dose range of tbo ( - lm) or vehicle control for h. after treatment, aß / levels in cell culture media were assayed by separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. besides, biocompatibility of tbo was evaluated in the ps cells using cell viability assay for flow cytometry. strikingly, all dose ranges of tbo inhibited both aß and aß secreted into the cell culture media. significant reduction for both aß species was evident at lm (p < . ), lm (p < . ), and lm (p < . ) of tbo vs. vehicle control. in conclusion, these results support the idea that tbo may be used as a therapeutic in ad. monitoring the changes of key molecules participating in the osmo-regulatory response of nucleus pulposus intervertebral disc cells during stress-induced senescence e. mavrogonatou, d. kletsas ncsr 'demokritos', athens, greece introduction: intervertebral disc cells are faced with a harsh extracellular milieu characterized by hyperosmotic conditions, nutrient and oxygen deficiency because of the absence of vascularization and oxidative stress due to the accumulation of their metabolism's by-products. we have previously shown that high osmolality is anti-proliferative for disc cells through the activation of the g and g cell cycle checkpoints by p and p mapk, respectively. in addition, we have shown the participation of nine solute transporters, with the a subunit of na + /k + -atpase being central in this response. here we assessed the changes in the expression of these key osmo-regulatory molecules during in vitro stress-induced senescence. materials and methods: changes in cell cycle progression were assessed using flow cytometry; overall transcriptional alterations were assessed by whole-genome arrays; differences in expression at the mrna and protein level were revealed by quantitative rt-pcr and western blotting, respectively; knocking-down of selected proteins was performed by sirna. results: high osmolality led to the differential expression of > genes, including nine genes encoding transporters. p mapk and p were demonstrated to differently participate in the regulation of the aforementioned transporters, while knocking-down of three selected transporters had a distinct outcome on the overall cellular response towards hyperosmotic stress. these molecules were found to show differences in their expression in senescent cells. discussion: given that the presence of senescent cells has been demonstrated in the intervertebral disc in vivo and could most probably attributed to the prevailing stressful conditions, here we showed differences in the expression profile of known key molecules for osmo-adaptation during senescence. conclusion: understanding disc cells' physiology is of outmost importance when designing cell-based therapies for disc degenerative disorders. p- . . - smad specific e ubiquitin protein ligase (smurf ) and its potential effects on inhibitory transmission in aging adams , , , interdisciplinary program in neuroscience, bilkent university, ankara, turkey, national nanotechnology research center (unam), bilkent university, ankara, turkey, department of molecular biology and genetics, bilkent university, ankara, turkey, molecular biology and genetics zebrafish facility, bilkent university, ankara, turkey, department of psychology, bilkent university, ankara, turkey smad specific e ubiquitin protein ligase (smurf ) is part of the tgf-b signaling pathway associated with cellular proliferation, differentiation, genomic stability and senescence. moreover, smurf , via its downstream partners, may regulate inhibitory synaptic transmission. our research group previously found that the smurf transcript is significantly higher in old zebrafish brains. thus, smurf may alter inhibitory synaptic transmission in aged animals. the focus of this study was to examine age-related changes in smurf protein levels and related key inhibitory synaptic proteins; gephyrin (gep), a scaffolding protein for gaba receptors, and gaba a , an ionotropic gaba receptor subtype. additionally, the levels of those proteins were studied in a mutant zebrafish line, which lacks acetylcholinesterase (ache) and is suggested to be a delayed aging model. whole brain tissues were isolated from young, middle-aged and old male and female zebrafish brains (ab/wildtype strain), as well as from old male and female ache mutant zebrafish (ache sb /+ ). animals were maintained and raised in standard conditions. the extracted brain tissue was homogenized in ripa buffer and subjected to western blot analysis to determine differences in the relative protein expression levels. our preliminary data indicated that smurf and gep levels remain stable in the aging brain (p = . , p = . ), and in the ache mutants gep levels are increased compared to the wildtype controls (p = . ). further analysis of the relationships between smurf and gaba a levels and brain aging is ongoing. we predicted that alterations in smurf levels would parallel changes in key synaptic inhibitory proteins during the aging process, which was the case for the gep levels. while smurf may regulate inhibitory synaptic transmission, the exact roles of those synaptic proteins in the context of normal and delayed brain aging are not known well-understood and the subject of continuing study. in recent years, express the hypothesis that aged individuals are vulnerable to infectious and other inflammatory agents and they become more prone to develop majority of severe age pathologies, including cardiovascular and oncology diseases, neurodegenerative diseases, type diabetes mellitus and inflammatory diseases, etc. one of the central components of immune response is the family of toll like receptors (tlr). there are several opinions that single nucleotide polymorphisms (snp) leading to a loss of function of the respective tlrs can be associated with age and increase the risk of age related diseases, especially cardiovascular diseases (cvd). however, many available studies focusing on tlr snps and cvd are with conflicting results. the aim of this study was to assess the potential interaction between genetic variants of tlr and tlr and ischemic heart disease (ihd) in kazakhstan population over years old. we evaluated patients with ihd and healthy subjects aged years and over (ethnical kazakhs and russians living in republic of kazakhstan). polymorphic loci of the genes tlr rs and tlr rs were genotyped by pcr with subsequent restriction analysis. our results indicated that the genotype and allele frequencies of tlr (arg gln) and tlr (asp gly) were not significantly different between the groups (p ≥ . ). statistical analysis didn't elicit any association between studied gene polymorphisms and predisposition to ihd in individuals over years old (p ≥ . ). for these polymorphisms, age, fasting blood sugar and serum lipid levels were not also significantly different among different genotypes in the ihd and control groups. in conclusion, the data shows that there is no interaction between tlr and tlr and ischemic heart disease (ihd) in kazakhstan population over years old. we plan to include other types of polymorphisms in tlr and tlr genes and increase the volume of patient cohort in our future studies. p- . . - evaluation of prognosis with total oxidant/ antioxidant status and some oxidative stress parameters in patients with acute ischemic stroke stroke is the third most common cause of death after coronary heart disease and cancer. strokes are classified into two groups according to their pathology: ischemic stroke and hemorrhagic stroke. ischemic strokes make up % and hemorrhagic strokes % of all strokes. during ischemic stroke, oxidative stress has been shown to play a major role in the occurrence and progression, formed oxidants also affect cell membranes and genetic material such asdna, rna, and various enzymatic events, and they lead to cell damage. some studies have shown oxidant-antioxidant status but have not shown the relationship with prognosis. this study has investigated the relationship between prognosis and total oxidant/antioxidant status and biochemical parameters in patients with acute ischemic stroke patients, with acute ischemic stroke and healthy controls we reenrolled in the study. blood samples were taken within st and th days, and after rd months in the patient group for analysing serum total oxidant status (tos), antioxidant status (tas), catalase, arylesterase, and thiol. prognosis was evaluated with national institutes of health stroke scale (nihss) and-modifiedrankinscale (mrs) scores. there was no significantly difference between groups by means of serum tas, tos and catalase levels. but arylesterase (p: . ) and thiol (p: . ) levels were significantly higher in first h blood samplingthancontrolgroup. statistically significant negative correlation was observed between the rd month values of tos and nihss score (r = . , p = . ). but there was no correlation between mrs scores and serum tas, tos, catalase, thiol and arylesterase. similarly, our findings suggested some serum oxidant levels were increased in acute ischemic stroke patients and total oxidant status might be used in evaluation of prognosis but larger studies are needed. p- . . - amylin and preptin regulate glucose homeostasis in infertile women with polycystic ovary syndrome and poor responders undergoing ivf/icsi disrupted glucose homeostasis leads not only metabolic disturbance such as polycystic ovary syndrome (pcos), but also influences oocyte growing. this study was designed to evaluate follicular fluid (ff) and serum levels of glucoregulatory hormones, amylin and preptin, in infertile women with pcos and poor responders undergoing ivf/icsi. human follicular and serum were obtained from infertile women with pcos and poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone antagonist protocol for ivf/icsi treatment. ff and serum amylin and preptin levels were measured by elisa. it was found that ff and serum amylin and preptin were lower in infertile women with pcos when compared with poor responder participants. ff amylin and preptin concentrations were lower than that of the serum amylin and preptin concentrations. decreased follicular fluid amylin and preptin levels suggest that amylin and preptin may have a physiological role in follicular maturation via controlling local glucose homeostasis. despite high serum levels of amylin and preptin in pcos their low concentration within the follicle may be main culprit of defective folliculogenesis seen in pcos subjects. similar to insulin resistance in pcos subjects existence of amylin and preptin resistance support the critical role of both peptides in follicular maturation in pcos. keywords: follicular fluid; amylin; preptin; polycystic ovary syndrome; infertility. the transcription initiation on p promoter of xp bacteriophage in presence of p protein, a modulator of rna-polymerase activity a. shadrin g.k. skryabin institute of biochemistry and physiology of microorganisms, ras pushchino, russia many bacteriophages are able to manage the transcription system of their bacterial host for their own needs. for example, bacteriophage xp , in the early stages of infection of xanthomonas oryzae inhibits transcriptional activity of bacterial rna-polymerase on majority of promoters via p protein, except of bacteriophage p promoter responsible for expression of the bacteriophage 'middle' class genes. the focus of this work is to study the mechanism of action of p protein in the transcription initiation and identification of the role of the individual elements of p promoter of xp bacteriophage, enabling x. oryzae rna-polymerase escapes inhibition by p protein. we have designed a set of promoter probes representing the combination of sequences of p -resistant p promoter and p sensitive t n promoter. using fret-based assay it was shown that the truncated probes corresponding to promoter dna downstream À position, relative to the transcription initiation start site, did not lead to dissociation of the sigma-factor. longer probes, containing À promoter element, induce dissociation sigma-factor. the in vitro transcription experiments show that the deletion of region , a sigma-factor domain responsible for interaction with À promoter element during the transcription initiation, is not critical for inhibition of rna-polymerase by p protein. promoter probe with up-element of p promoter had affinity to x. oryzae rna-polymerase a several times higher than a probe containing the consensus up-element for e. coli rna-polymerase . summing up the results, it seems like the transcription initiation on p promoter of bacteriophage xp can escape inhibition by p protein through a high affinity interaction between the up-element and c-terminal domains of the alpha subunit of rna-polymerase x. oryzae. p- . . - distribution of soluble form of glial fibrillar acidic protein in the different areas of gerbils brain during development and aging y. kovalchuk, g. ushakova oles honchar dniepropetrovsk national university, dniepropetrovsk, ukraine astrocytes are the most abundant cell type within the cns and play an important role in cns homeostasis and function. glial fibrillary acidic protein (gfap) forms the main astrocytic intermediate filament (if). the overall level gfap in different parts of the brain uneven and depends on the number of astrocyte cells. gfap is very sensitive to any kind of neurodegenerative diseases and aging. during aging, a glial reaction is observed in the human brain, as well as in rat and mouse brains. the aim of our study was to investigate the quantitative astrocytes-specific protein gfap in different areas of the gerbils brain at the first stages of postnatal development and aging. for the study gerbils brains were used and divided into groups (n = ): : newborn animals ( day), - : , and days respectively, : animals aged years. the animals were decapitated under mild anesthesia (thiopental), with isolated brain three divisions: the cerebellum, thalamus and hippocampus, which are then used to produce cytosolic protein fractions. the level of gfap in the obtained fractions were determined according to the method of competitive elisa. newborn gerbils found no significant content of soluble form of glial fibrillar acidic protein in all investigated parts of the brain, and a sharp increase of amount within days (in cerebellumamounted to . ae . lg/ mg tissue; to - days increased to . ae . lg/ mg tissue, and began to grow again in older individuals aged years). unlike the cerebellum, the level of sgfap in hippocampus and thalamus reached the maximum at days p.d. ( . - . lg/ mg tissue), and unchanged for days. these results revealed that the most intensive development of astrocytes in the cerebellum to p.d. of gerbils, and in the thalamus and hippocampus are formed within the first month of life. the plastid-nucleus located protein whiry acts as an upstream regulator of leaf senescence binding to the promoter of senescence associated genes (sags) like senescence marker gene hvs . in order to investigate the impact of whirly on drought stress-induced senescence, transgenic barley plants with a knockdown of whirly (hvwhy kd) were grown under untreated and drought stress conditions. the leaf senescence evolution was monitored by physiological parameters and gene expression studies of senescence and drought stress related genes. to reveal the epigenetic indexing at hvs at onset of drought-induced senescence in wild type (wt) and hvwhy kd lines, stress-responsive loading with histone modifications at gene regions of hvs ( regions in the promoter, one region around translation start site and regions located in the gene body) was analysed by chip and quantified by rtq-pcr. in barley, drought treatment caused acceleration of leaf senescence in wildtype (wt) plants, whereas why kd lines showed a staygreen phenotype. expression of senescence-associated and drought stress responsive genes expression was delayed in hvwhy kd indicating that whirly protein acts as an upstream regulator of drought stress-induced senescence. the chip results showed that drought treatment is causing in wt a significant increase in the levels of h k ac all over the analyzed gene regions, correlating with a massive induction of hvs expression, while drought stress caused no substantial increase of h k ac in why kd plants. the results suggest that drought induced expression of hvs is under epigenetic control, and furthermore that why is involved in this epigenetic control level. oncolytic viral therapy is based on the capabilities of selective lysis of tumor cells and is a prospective trend in cancer disease treatment. in vitro experiments showed that plant rhabdoviruses does not have any direct cytotoxic effect upon sarcoma cells, causes induction of apoptosis in these cells and does not pose any threat to somatic cells of warm-blooded animals, which makes it possible to use this virus for therapy of malignant neoplasms. buckwheat burn virus (bbv), the prototypic member of the family rhabdoviridae, contains surface glycoprotein and which is lectin-active. its carbohydrate branch can aid adhesion of lymphocytes to tumor cells. the present study has addressed the effect of bbv on cancer cell viability. all studies were carried out after week of inoculated with erlich cancernome ( cells/animal, i. p.) in months male balb/c mice treated at once with or without plant extract with bbv ( mg/kg, i. p.). by fluorescent microscopy and using two due staining by acredine orange and propidium iodide it was found that in the rd day of administration of bbv lead to increasing of necrotic and apoptotic cells on % and % respectively versus to untreated group. at the same time the viability of investigated cells was impaired too and according to flow cytometry analysis using propidium iodide the amount of dead cells was elevated by fivefold ( . % versus . % in untreated group). also as was shown in previously reports bbv decreased activity of macrophages in the early stages after injection and it may have a positive effect when using this drug in tumor therapy. when using this drug appears to slow down the possibility of a sharp activation of macrophages, and as a consequence of the development of cytotoxic effect will be prolonged. key words: rhabdoviruses, buckwheat burn virus, cancer, cell viability. plants are considered as one of most promising sources for new antimicrobials, based on the evidence of their use in folk medicine to treat various infectious diseases since ancient times. despite relatively small area size, armenia has large diversity of flora with many endemic species. the main goal of this study was the screening of various parts of herbs (widely being used in armenian folk medicine) for their antimicrobial activities in order to select most prospective plants for further comprehensive studies. plant crude extracts were obtained with maceration technique using five solvents: water, methanol, chloroform, acetone and hexane. agar well diffusion assay was used to evaluate antimicrobial properties of plant crude extracts at lg/ml concentration against escherichia coli vkpm-m , pseudomonas aeruginosa grp , bacillus subtilis wt-a , salmonella typhimurium mdc and staphylococcus aureus mdc , candida albicans wt- and candida guilliermondii hp- . statistical analysis was done using graphpad prism . . crude extracts of all tested plant materials expressed antimicrobial activity against at least one test strain. most of the tested extracts inhibited growth of both gram-negative and gram-positive bacteria. in contrast, only some plant materials exhibited inhibitory activity against yeast strains. according to obtained data sanguisorba officinalis, rumex confertus, hypericum alpestre, lilium armenum and agrimonia eupatoria possessed the highest and broadest antimicrobial activity. moreover, the results showed that acetone was the most effective solvent for solubilizing antimicrobial compounds from plant materials followed by methanol, chloroform, hexane and water. the results demonstrated high antimicrobial activity of medicinal plants used in armenian traditional medicine. five plant species were selected for further comprehensive studies. besides, acetone was proposed as efficient solvent in antimicrobial screening protocols. p- . . - effects of aluminum stress on photosystem-i apoprotein a gene (psab) transcription level in lichen xanthoria parietina (l.) th. fr. unal € ozakc ßa ege university, izmir, turkey in this study the effects of shortrerm aluminium (al) toxicity on the lichen xanthoria parietina (l.) th.fr. were investigated at physiological and transcriptional level. lichen thalli were treated with alcl in different doses ( . , . , and mm). lipid peroxidation and chlorophyll integrity were determined by spectrophotometer. expression level of psab gene was also investigated. chlorophyll a content was significantly (p ˂ . ) decreased after hours treatment with mm and mm of al, while chlorophyll b content was increased significantly due to treatment with increased concentration of aluminum. also treatment with . and . mm al for hours increased the gene expression level of psab by . % and . % respectively. our results indicated that aluminum treatment has decreased the chlorophyll biosynthesis and increased the lipid peroxidation depending on time and concentration. this study also demonstrates that the psi can be readily photo-inhibited by aluminum stress. in conclusion, mm al exposure for hours could damage the electron transport in photosystem i. p- . . - nigella sativa reduces paracetamol-induced nephrotoxicity and oxidative stress in rats: biochemical evaluation background: nigella sativa l. (ranunculaceae) (ns) is traditionally used to treat many conditions such as inflammation. this study evaluates the effects of ns seeds ethanol extract in paracetamol-induced acute nephrotoxicity in rats. material method: forty-eight female wistar albino rats were divided into eight groups: i = sham; ii = sham + mg/kg ns; iii = sham + mg/kg (n-acetyl cysteine) nac; iv = g/ kg paracetamol; v = g/kg paracetamol + mg/kg nac; vi, vii and viii = g/kg paracetamol + , and mg/kg ns, respectively. paracetamol administration (oral) was carried out h after ns and nac administrations (oral), and all animals were sacrificed h later. result: urea and creatinine levels were determined in serum, while glutathione, malondialdehyde levels and superoxide dismutase activity were determined in the kidney tissues. there were significant increases in the serum levels of urea and creatinine in the paracetamol-administered group. serum levels of urea and creatinine were decreased in all groups administered ns with paracetamol. ns administration dramatically restored sod, gsh, and mda levels in the kidneys. conclusion: the results suggest ns has a significant nephroprotective activity on paracetamol-induced nephrotoxicity. it may be suggested that the antiinflammatory and antioxidant effects of ns ethanolic extract originated from different compounds of its black seeds. p- . . - the study of problems of preservation of the birches e. shadenova , , e. zhumabekov , m. sembekov , m. burchaeva institute of general genetic and cytology, almaty, laboratory of genetics and reproduction of forest culture, institute of general genetics and cytology, almaty, kazakhstan nature of deciduous trees have a whole range of various medicinal properties. instead of synthetic hormone substitutes, you can use medicinal infusions and decoctions of natural phytohormones are widely used in both folk and professional medicine. one of these plants is birch, its young leaves and buds. however, they also must be used with caution because overdose of these compounds is very dangerous, not only can you not get the desired effect, but also face the opposite of his action. in our research to mass replication of plants (different types of birches (betula ajanensis, yarmolenko, jacguemontii, maximowiczii, ulmifolia, middendorffii, kelleriana, tianschanica)) we use nutritional medium excluding the application of phyto promoters in order to prevent mutation. the object of research serve as the old, the sick, being on the verge of extinction, mature trees as explant meristema. since from the moment of calling experience and most cultivation occurs at nutritional medium without hormones. as a result of molecular analysis we get without virus, genetically identical plants. molecular certification of different types of birches of interest, both in terms of organizing, and in terms of selection and genetic improvement of valuable forms, identification of lines selected from natural populations and clones obtained in vitro. relationship between clones and installed parent form by comparing profiles amplific pcr products using issr-marking. according to the results of carried out works really recovered clones obtained from one source tree, indicating the potential for certification of clones studied forms of birches pcr. a study performed in the framework of the state grant project "conservation of breeding valuable species of birches". p- . . - fractionated triterpenoid glycosides from sea cucumber inhibit invasion and metastasis in human cancer cells sea cucumbers are slow-motioned invertebrates. holothuria polii delle chiaje, is widely distributed sea cucumber in _ izmir coastline (turkey). it secretes saponins i.e. triterpenoid glycosides (ttg) as secondary metabolites. the aim of this study is to evaluate anti-invasive and anti-migrative effects of fractionated ttgs obtained from h. polii on ht- , t and upci-scc- cancer cell lines. the semi-purified ttgs was extracted from h. polii collected from coast of _ izmir-dikili. the four different fractions (fraction a-d) were collected by using hplc (high-performance liquid chromatography) and characterizated with maldi-ms/ ms. the fractions obtained from h. polii extract include holothurin a ( . m/z) and -dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer ( . m/z). anti-invasive and anti-migrative effects of the fractions on the cancer cell lines were detected with xcelligence rtca dp system. the results showed that fraction a-d inhibited migration and invasion of human cancer cell lines at th and th hours compared to control group. this study shows that holothurin a, dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer could be evaluated as promising anti-cancer agents for human cancers. acknowledgement: the authors acknowledged the scientific and technological research council of turkey (t € ub _ itak) for financial support ( z ). p- . . - alternative splicing regulation of sr proteins in response to environmental stress in chinese cabbage serine/arginine-rich protein (sr protein) family, which acts as rna-binding protein, plays a major role in post-transcriptional regulation of pre-mrna, such as alternative splicing (as). these proteins cause pleiotropic effect by regulating as of pre-mrna in a tissue and developmental stage-specific and stress-responsive manner in arabidopsis. here, we identified genes encoding sr proteins in chinese cabbage (brassica rapa chiifu- ) from brassica database and analyzed their phylogenetic relationship. b. rapa has types of sr protein that are classified into common (sr, rsz and sc) and plant specific (scl, rs z, rs and sr-like) subfamily similar with arabidopsis. interestingly, the as pattern of most sr genes changed at the late stage ( and days after germination). to verify the correlation between sr genes and environmental stress, we screened the as pattern of sr genes to various abiotic stress using rt-pcr and a microarray analysis. in particular, the expression level and the as pattern of bra and bra were affected significantly by heat stress. these results suggest that the as regulation by sr protein correlates with adaptation mechanism to the environmental stress in chinese cabbage. p- . . - characterization of recombinant prolyl oligopeptidase from myxococcus xanthus and potential use in gluten hydrolysis e. k. kocaazorbaz, f. zihnioglu faculty of science, biochemistry department, ege university, izmir, turkey a recombinant prolyl oligopeptidase from myxococcus xanthus was purified with a specific activity of u mg(- ) by using nickel-metal-chelate affinity chromatography and gel permeation chromatography. the recombinant enzyme had a monomeric molecular weight of kda. its isoelectric point, determined by two dimension polyacryl-amide gel electrophoresis, was close to . . the optimum ph and temperature was estimated as . and °c, respectively. the purified enzyme was stable from ph . - . and able to thermal stability up to °c. the k(m) and v(max) values were . mm and . micromol/ min per mg. the enzyme exhibited hydrolytic activity for suc-gly-ala-pna, suc-gly-pro-pna, z-gly-pro-pna, igf- , substance p, whereas no activity for h-gly-pro-pna, h-val-ala-pna, h-arg-pro-pna, h-ala-pro-pna, glu-ala-pna, pro-pna, leu-pna. its proteolytic activity was inhibited by activesite inhibitors of serine protease, z-pro-prolinal pmsf, and metal ions, cd + and hg +. the potential use of the enzyme was tested by the hydrolysis of the wheat gluten. the resulting gluten hydrolysate were characterized by means of their antioxidant, antibacterial, trypsin inhibition and prolyl oligopeptidase inhibition activities. keywords: serine protease, prolyl oligopeptidase, bioactive peptides, , , -trinitrobenzene sulfonic acid. proteomic analysis is probably the best approach to analyze seed germination. however, it is difficult to analyze complex samples and there are many obstacles that must be faced in order to achieve a reasonable proteome coverage. for example, the barley (hordeum vulgare) genome was fully sequenced in , but the uniprot database contains less than reviewed sequences, which is approximately -fold less than for arabidopsis thaliana. here, to improve the barley proteome coverage, we employed several fractionation methods including polyethylene glycol precipitation, strong cation exchange chromatography, off-gel separation, sds-page and acetonitrile elution gradient. proteomic analyses were performed using an lc-ms-based analyses and an uhr q-tof mass spectrometer. the candidate peptides were targeted via selected reaction monitoring (srm) and triplestage quadrupole (tsq) mass spectrometer. in total, proteins were identified, which represents a three-to four-fold increase compared with the standard shotgun analysis of the same sample. out of these, were only accessible by one of the techniques and, besides, the detection limits were not similar. we hypothesized that an srm-based targeted analysis will allow detection and quantitation of most of these proteins, even without the application of proteome fractionation. we can conclude that all peptides from the library with ms/ms spectra of the total intensity above , are easily detectable in the total protein extracts. p- . . - transcriptome sequencing based identification of alternative oxidase genes in white waterlily, nymphaea alba alternative oxidases (aoxs) are the terminal oxidases in the respiratory electron transport chain of plants. they reduce molecular oxygen to water with low proton translocation across the inner mitochondrial membrane. in plants, aoxs increase local tissue temperature to release volatile compounds thereby attracting pollinator insects and regulation of mitochondrial retrograde signaling pathway. regulation of retrograde signaling pathway is currently under investigation to improve cultivation studies in many plants. water lilies are aquatic ornamental and economically valuable plants classified under nymphaea family. nymphaea alba, white water-lily, has a special focus since its applications in landscaping of parks and gardens, farming as vegetable and medical applications. however, cultivation of n. alba is a challenging process. we hypothesized that by controlling alternative oxidases, success rate can be increased for n. alba cultivation. to identify alternative oxidase encoding genes in n. alba, we performed transcriptome analysis. by using transcriptome analysis data, aox gene sequences, subcellular localization of aox proteins and structural modelling of aox proteins were predicted. in transcripts, database search with trinotate tool revealed transcripts with aox domains characterized in known alternative oxidases. blast analysis of these sequences with known aox proteins revealed three distinct aox genes (nalba-aox , nalba-aox and nalba-aox ). after subcellular localization analysis of three identified aox proteins by using targetp server tool, nalba-aox , nalba-aox are predicted as mitochondrial while nalba-aox is localized in chloroplasts. template based structural modelling results showed that all identifed proteins are statistically similar to known structure models of corresponding aoxs. most environmental contaminants have toxic and mutagenic effects on living organisms as a result of the activation of free radical formation and inhibition of reparation activity. it is becoming relevant to search for protectors of natural origin from the effects of xenobiotics. many biologically active substances (bas) of inartificial origin are found to be antioxidants and can increase the body's resistance to the toxic and mutagenic effects of a wide range of pollutants. the aim of the study was to investigate the antioxidant and antimutagenic properties of bas from medicinal plants limonium gmelinii (plumbaginaceae) and inula britannica (compositae). the antioxidant potential of plant extracts was determined by the activity of superoxide dismutase (sod), catalase, and the content of malonic dialdehyde. mutagenic and anti-mutagenic properties of the extracts were determined in the test by counting chromosomal aberrations in root meristem of barley seeds. barley seeds were treated with an aqueous solution of unsymmetrical dimethyl hydrazine (udmh), which is highly toxic i class hazardous material, well known pro-oxidant. the results showed that udmh enhanced the process of lipid peroxidation and decreased the mitotic activity. treatment of barley seeds with extracts from i. britannica and l. gmelinii and their germination in the presence of stress factors stimulated antioxidant defenses in the primary roots of barley seeds. increase of the activity of sod and catalase, and reduction of peroxidation level of lipids were observed. cytogenetic study showed no mutagenic activity in plant extracts. when effects of plant extracts and udmh were combined there was a significant reduction in the frequency of structural mutations, induced by the toxicant. conclusion about the presence of the antioxidant and antimutagenic activity in the studied plant extracts is made. the work done within the framework of the mes project (no. gr rk ). p- . . - comparative analysis of cytokinin dehydrogenase inhibition and trans-zeatin treatment in arabidopsis seedlings j. nov ak , v. koukalov a , z. medvedov a , c. martin , j. hradilov a , l. sp ıchal , b. brzobohat y mendel university in brno, brno, palack y university in olomouc and centre of the region han a, olomouc, czech republic cytokinins are plant hormones regulating many processes during plant life ranging from germination to senescence. manipulation of cytokinin levels and their impact on plant vitality, production and ability to defend against stresses is in great interest of agriculture. in this work we focused on comparison of inhibitor of the cytokinin degradation incyde ( -chloro- -( -methoxyphenyl)aminopurine) and exogenous application of trans-zeatin on arabidopsis thaliana seedlings. transcripts of genes regulating cytokinin metabolism were analysed by rt-qpcr analysis. classical cytokinin root essay revealed that incyde effect is comparable to that of trans-zeatin in a similar concentration-dependent manner. besides a negative effect on the primary root length, both substances induce flavonoid accumulation and an increase in the root hairs formation. histochemical staining of transgenic plants expressing glucuronidase (gus) under cytokinin-responsive promoter of arr gene revealed increased gus activity in cotyledons following incyde treatment suggesting diverse localization of cytokinin modulation upon trans-zeatin and incyde treatment, respectively. possible molecular differences originating in different cytokinin population and distribution following trans-zeatin or incyde treatments were monitored on the level of gene expression and via an lc-ms proteome analysis in roots and shoots of -day-old plantlets. rt-qpcr analysis revealed an alteration in cytokinin metabolism that could explain observed differences on the proteome level between incyde and trans-zeatin treated seedlings. pharmacologically inhibited cytokinin degradation could be very efficient tool for modulation of cytokinin levels. interestingly, the application of incyde and trans-zeatin shows a contrasting spatial and temporal pattern on molecular levels. incyde represents potent growth regulator with interesting properties useful for agriculture. p- . . - the expression yield of prokaryotic alphaamylase is significantly magnified by molecular cloning techniques randomly hydrolyzing glycosidic bond alpha-amylase has been traditionally employed in bread and similar industries. in that regard, increasing the overall expression level of the enzyme is a crucial concern in biotechnology. to reach the goal, appropriate alpha-amylase producing species and expression vector were carefully selected. therefore, genome of bacillus subtilis was extracted and amplified by polymerase chain reaction (pcr) using specifically designed primers. subsequently, the extracted gene was inserted in expression vector pht and transferred to e. coli as intermediate host followed by bacillus subtilis host replacement. the recombinant vector was expressed in bacillus subtilis and the expression was evaluated by agarose gel electrophoresis. relative purification of the recombinant enzyme was performed by kda filtration to remove impurities. to identify the biochemical characteristics, starch was used as specific substrate to measure enzyme activity and the enzyme was exposed to various ph and temperatures. the extra-cellular expression of alpha-amylase enzyme was successfully elevated by folds in comparison to the native enzyme. the optimum temperature and ph for the enzyme was carefully determined as °c and , respectively. the enzyme was stable at °c, but thermal stability was dramatically decreased at higher temperatures up to °c. kinetic parameters were also measured; vmax was . u/ml min and km was . mg/ml. it is concluded that the elevated expression extent of recombinant alpha-amylase together with appropriate qualifications could make the clone a good choice for various industrial applications. flax seedlings of cultivars tmp , lira and lines g- / _o, g- / _k were treated for and hours with lm alcl solution or distilled water (control). twelve small rna libraries were constructed and sequenced using illumina gaiix. to identify known mirnas, obtained sequences were aligned with mirnas from mirbase (http://www.mirbase.org/). fold change value was calculated to identify up-and down-regulated mirnas under al stress. in total, about million raw reads were obtained and conserved mirnas from families were identified. significant expression alterations in flax plants under al treatment were shown for mir and mir . expression level of mir was varied in similar way in resistant and susceptible to al genotypes: mir was up-regulated after hours of alcl exposure and down-regulated after hours. mir expression was increased after hours of alcl exposure and decreased after hours in susceptible to al flax genotypes (lira, g- / _o), while in resistant genotypes (tmp , g- / _k) mir level was decreased after both and hours of al treatment. in other plant species, mir and mir were identified as al-responsive. mir targets mrna of tcp (teosinte branched/cycloidea/pcf) transcription factors, which control plant growth. mir targets mrna of tas protein, which regulates lateral root growth via degradation of arfs (auxin response factors). in flax, the involvement of mir and mir in response to al stress was shown for the first time. moreover, we revealed diverse expression alterations of mir in susceptible and resistant to al genotypes. this work was financially supported by grant - - from the russian science foundation. p- . . - association genetics of phenylalanine ammonia lyase (pal) and cinnamyl alcohol dehydrogenase (cad) enzymes involved in lignin biosynthesis of european black poplar (populus nigra) b. taskiran, z. kaya middle east technical university, ankara, turkey populus nigra l. are considered as one of the most economically significant forest trees with respect to production of wood, biomass, and other wood-based products. while wood quality and biomass are directly associated with high cellulose content, lignin emerges as an undesirable polymer for both pulp and biofuel manufacturing industries. the aim of the study is by choosing the superior and eliminating the inferior clones to make a contribution to woody feedstock development and to improve wood quality of populus nigra. to estimate association genetics of pal and cad enzymes which have important functions in lignin biosynthesis, the important germplasms of populus nigra has been sampled from year old poplar trees ( clones x replicates x ramets) which were grown in behic ßbey plantation clone bank in ankara. additionally, five commercially registered clones and six foreign clones were included to the study to make comparison. the average mean values of cellulose, lignin and glucose content were calculated as . ae . lg/ml, ae . lg/ml, and ae . lg/ml, respectively. even though for pal and cad enzymes, data gathering process have been still resuming, particular clones have been separated from all in terms of pal and cad activities as expected. key words: populus, poplar, lignin, pal, cad, genetic variation, feedstock p- . . - proteomic analysis of the molecular mechanisms of the response of plant seeds to pre-sowing treatment by stressors seed treatment with non-ionizing low-level radiation (nr), such as cold plasma (cp) or electromagnetic field (ef), is a modern eco-agricultural technology for stimulation of plant germination and performance. the molecular determinants of seed response to these treatments are not established and no genomic studies of plant seed response to nr have been reported. we studied the effects of pre-sowing seed treatment, using vacuum ( min), radio-frequency ef ( - min) and cp ( - min), on germination and growth of non-oilseed helianthus annuus. to gain an insight into the molecular mechanisms underlying effect of nr on sunflower seed germination and dormancy, we estimated changes induced in the balance of plant hormones and differential protein expression. the results of the germination tests and estimation of seedling morphology showed that response develops in time and is stronger when sowing is performed in days in comparison to days after seed treatment. the d dige analysis revealed differentially expressed proteoforms in kernels of seeds treated with cp or ef. proteins involved in biological processes of seed maturation, response to stress, response to abscisic acid stimulus, processes of organonitrogen compound metabolism and glucose catabolism were identified. while expression patterns for majority of the proteins were highly specific to cp and ef treated seed kernels, accumulation of several proteoforms of seed storage proteins (ssp), including vincilin-like, miraculin-like protein and albumin- were common for both experimental groups. this suggested that response to nr treatment could be at least partially associated to function of ssps in response to oxidative stress that protects proteins required for seed germination and seedling formation. variation of abundance of distinct proteoforms of helianthinin, vicilin-like and s globulin-like ssps suggested that post-translational modifications are involved in regulation of the function of ssps. p- . . - suppression of lipopolysaccharide-induced inflammatory responses in raw . macrophages by tuber extract of cyclamen l. turkey is a prominent centre of plant diversity, being the meeting point of three main floristic zones. geophytes which have underground storage organs such as, tubers, bulbs and rhizomes. cyclamen l. is a tuberous geophyte traditionally used by some people for treating whooping cough, headaches or sinusitis, and confirmed to have antioxidant, analgesic and anti-inflammatory properties by several reports. a prolonged inflammatory response is often associated with chronic diseases such as cancer, arthritis and autoimmune disorders. recently, plant based products are used as an alternative and complementary treatment of these diseases. in this respect, the present study was aimed to determine the effects of three cyclamen tuber extracts on lps-induced inflammatory responses of murine raw . macrophages. firstly, c. cilicium (endemic), c. pseudibericum (endemic) and c. graecum subsp. anatolicum were collected from different localities of turkey. the tubers of plants were air-dried and grounded to fine powder and then extracted with ethanol. cell viability assay was performed to evaluate the nontoxic concentration in cell line by mtt assay. several measurements were performed including tnf-a, no and il- concentration assay by elisa after treatment compared to non treated cells to determine the anti-inflammatory activity. also, tnf-a and inos mrna levels were evaluated by quantitative rt-pcr. the cytotoxic activity which is considered safe on raw. . cell were found as . - lg/ml. studied cyclamen taxa inhibited tnf-a and il- release on lps stimulated-raw. . in a concentration-dependent manner. among the three cyclamen tuber extracts evaluated, the highest nitrite-associated no inhibitory activity was obtained from c. pseudibericum compared to other two cyclamen l. taxa. collectively, these results suggest that cyclamen tuber extracts possess anti-inflammatory properties. p- . . - in vitro hypoglycemic activity of ziziphus jujuba recent reports have indicated that continuous treatment with nutritional jujuba (ziziphus jujuba miller) fruit extracts in diabetic rats improved glucose utilization and produced a significant decrease in the blood glucose. in the present study, hypoglycemic activity of z. jujuba was investigated using various in vitro techniques. the hypoglycemic effect of z. jujuba in phosphate buffered saline which grown in balıkesir was studied by measuring glucose adsorption, glucose diffusion and glucose uptake by yeast cells. the glucose content in the solution measured by spectrophotometrically with commercially kits. the adsorption capacity of the z. jujuba was found to be directly proportional to the molar concentration of glucose. the glucose binding capacity of extract increased in higher glucose concentratrations. there was significant differences were observed between the adsorption capacities of z. jujuba and control samples (p < . ). the rate of glucose diffusion was directly proportional to the time. diffusion rate was significantly lower in the solution containing z. jujuba compared to control (p < . ). the extract demonstrated significant inhibitory effects on movement of glucose into external solution across dialysis membrane compared to control. the rate of glucose transport across cell membrane in yeast cells was observed to be inversely proportional to the molar glucose concentration. z. jujuba inhibited glucose transport across the yeast cells. the results showed that z. jujuba reduced glucose levels at least by three mechanisms. first by increasing glucose adsorption capacity during postprandial hyperglycemia; second by retarding glucose diffusion rate and third, at the cellular level by inhibiting glucose transport across the cell membrane. all of these decreased the absorption of glucose in the intestinal cells and the concentration of postprandial serum glucose. p- . . - cucurbitacin b increased the anticancer effect of imatinib mesylate through inhibiton of matrix metalloproteinase- expression in colorectal cancer cells f. bakar ankara university, ankara, turkey several natural products have been investigated for their anticancer effects. among these, cucurbitacin b (cub) has been reported as its inhibitory effects on cancer cell proliferation. matrix metalloproteinases (mmps) belong to endopeptidase family and they are received as potential biomarkers for several types of cancer. the aim of this study is to investigate the effect of cub in combination with imatinib mesylate (im) on mmp- mrna expression of human sw colorectal carcinoma cells. the cytotoxicity analysis was performed via mtt assay. muse cytofluorimetric analysis system was performed to evaluate apoptotic cell population. the mmp- mrna expression was determined by quantitative real-time pcr. this study was supported by scientific and technological research council of turkey grant, sbag- s . data obtained from the cell culture experiments were expressed as mean ae sd and one-way anova test was applied for multiple comparisons. cub alone significantly inhibited cell growth at lm and higher concentrations. the most potent effect was observed in cub-im combination treatment with . lm ic value. in cub-im treated group, the apoptotic effect was higher than cub and im treated groups. cub-im induced apoptosis significantly at lm concentration when compared to control and lm (p < . ). cub alone showed inhibitory effects on mmp- mrna expression at lm and higher doses significantly (p < . ). the results showed that the combination treatment of cub with imatinib synergistically inhibited human sw cell growth and induced apoptosis by increasing the anti-histone antibodybound nucleosom levels and annexin v binding. although cub could inhibit mmp- expression alone at higher treatment doses, it enhanced the inhibitory effect of im on mmp- synergistically in a dose dependent manner. in conclusion, this study suggests that cub combined with imatinib mesylate may enhance the effects of chemotherapy in patients with colorectal cancer. plants are most important parts of natural resources that alternatively referred to synthetic drugs for reasons such as being less side effects and lower costs. ziziphus jujuba miller (z. jujuba), a plant used in traditional medicine, is one of the most important ziziphus species belonging to rhamnacea family. the fruit and seeds of this plant are used different purposes such as antiinflammatory, antioxidant, immune-stimulant and wound healer. in this study, we investigated the antibacterial effects of z. jujuba. the aims of this study were to screen the antibacterial activity of z. jujuba. the extract was obtained from z. jujuba fruits pulverized with the aid of ball mill using % aqueous-ethanol solution. extracts were screened for antimicrobial activity against six different standard strains of bacteria by determining minimum inhibitory concentration (mic) according to clsi criteria. serial dilutions are made between mg/ml and . mg/ml concentration range. the lowest concentration of wells that no visible growth has been accepted as mic value. materials in the mic and lower concentrated wells were transferred to % sheep blood agar petri dishes for calculation of minimal bactericidal concentration (mbc). the lowest concentration that no colony formation has been accepted as mbc value. jujuba showed the most potent effect on strain of s. aureus atcc is gram-positive cocci (mic: mg/ml). the mic values of other gram-positive bacteria s. aureus atcc , e. faecalis atcc , l. monocytogenes f and m. smegmatis cmm were detected as , , and mg/ml respectively. mic values of gram-negative bacilli were detected as > mg/ml. consequently, z. jujuba was found to be effective on grampositive cocci bacteria (s. aureus atcc , s. aureus atcc and e. faecalis atcc ). the strongest effect was observed on s. aureus atcc strain. in contrast, extract showed less effect on gram-negative bacilli. p- . . - selective cytotoxic effect of morus rubra extract in human lung cancer cells through enhancing apoptosis and cell cycle arrest cancer is a disease that develops as a result of unlimited proliferation of abnormal cells that occurs due to loss of control over the mechanisms of normal growth and differentiation of cells. morus rubra, known as "red mulberry" belongs to family of moraceae. for many years, the fruits of morus species have been used to treat many diseases in traditional medicine. biological effects of morus species is predominantly attributed to its content of polyphenolic compounds. many studies have evaluated the cytotoxic effects of different morus species, but there is no study about cytotoxic effect of m. rubra. in this study, we aimed to evaluate the cytotoxic effect of m. rubra extract in human lung cancer cells (a ) with regard to apoptosis, cell cycle and mitochondrial membrane potential. cytotoxic effect of m. rubra extract on human lung cancer cells was determined using mtt assay. then, mechanisms of cytotoxic activity of m. rubra extract on a cells were examined in terms of cell cycle, apoptosis and mitochondrial membrane potential using flow cytometric methods. m. rubra extract exhibited selective toxicity against a cells compared to normal foreskin fibroblast cells. we determined that m. rubra extract increased cell cycle arrest at g phase, the level of apoptotic cells and decreased mitochondrial membrane potential in a cells. our results showed that m. rubra extract has pro-apoptotic and antiproliferative effect in a cells. further studies are also needed to fully mechanisms underlying this effect of m. rubra extract. p- . . - dipeptidyl peptidase iv inhibitory activity of arctium tomentosum l. a. zeytunluoglu , f. zihnioglu denizli vocational school of technical sciences, pamukkale university, denizli, department of biochemistry, faculty of science, aegean university, izmir, turkey type diabetes mellitus (t dm) is rapidly growing metabolic syndrome of multiple aetiologies causing hyperglycaemia with insulin resistance at cellular level. a novel approach in the treatment of t dm is based on preventing of rapid inactivation of the incretin hormone glucagon-like peptide- (glp- ) and glucose-dependent insulinotropic polypeptide (gip) by dipeptidyl peptidase-iv enzyme. in this study; dipeptidyl peptidase iv (dpp-iv; ec . . . ) inhibitory activity of the aqueous and methanolic extracts of arctium tomentosum leaves were successfully tested in vitro conditions. our study revealed that both aqueous and methanolic extracts obtained from test material had a significant dppiv enzyme inhibitory activity in changing ratio. the ic values were also determined by nonlinear regression curve fit using graph pad prism . with appropriately diluted of lyophilized arctium tomentosum. diprotein-a (ile-pro-ile) was used as reference inhibitor. a. tomentosum aqueous extracts showed ic . lg/ml while the standard (positive control) diprotin a displayed the ic value of . lg/ml. this study demonstrates that a. tomentosum aqueous extracts could be a good lead for further development as a new antidiabetic agent. p- . . - dna recognition determinants of arabidopsis thaliana b transcription factors g. sasnauskas, k. kauneckaite, k. lapenas, v. siksnys institute of biotechnology, vilnius university, vilnius, lithuania transcription, one of the most important cellular processes, is regulated by transcription factors (tf), proteins that often directly interact with gene promoter sequences. tf binding to dna is mediated by various dna binding domains. the b tfs constitute a large, plant-specific protein family (approx. % of all tf proteins in the flowering plants), which is characterized by the presence of one or several small (approx. amino acids) b dna binding domains. currently the b tfs are divided into four groups (lec -abi /val, arf, rav and rem). the preferred recognition sites were identified for representatives of all groups except the rem family. currently, only a single structure of a dna-bound b domain (arf , arf family) is available, thus the mechanism of site-specific dna recognition by the lec -abi /val and rav b domains remains poorly understood. based on the arf -dna structure (pdb ldx) we have built homology models of dna-bound b domains from a. thaliana abi (lec -abi /val family) and nga (rav family) transcription factors, mutated putative dna-interacting amino acid residues and characterized the dna binding ability of the purified mutants using electrophoretic mobility shift assay and a set of radiolabelled dna substrates carrying various variants of the optimal recognition site. we confirm the importance of several positively charged amino acid residues, which are conserved between the abi / nga b domains and structurally related dna-binding domains of bacterial restriction endonucleases ecorii, bfii and ngoavii; furthermore, we identify residues in the 'n-arm' and 'c-arm' loops that may be involved in specific interactions with the dna bases. our results therefore help us refine the homology models of the dna-bound b domains and in the future may help us predict the dna binding properties of currently uncharacterized b domains. p- . . - immunohistochemical analysis of inhibitory effects of origanum hypericifolium oil on dipeptidyl peptidase iv in streptozotocininduced diabetic rats p. ili , a. zeytunluoglu denizli health services vocational high school, pamukkale university, denizli, denizli vocational school of technical sciences, pamukkale university, denizli, turkey diabetes mellitus (dm) is a serious metabolic disorder with micro-and macro-vascular complications that result in a significant morbidity and mortality. glp- and gip have significant role in pancreatic beta cells and prevention of inactivation of them by dipeptidyl peptidase iv (dpp iv) inhibition is a novel approach to treatment of dm. origanum hypericifolium (lamiaceae) is an endemic turkish plant and its essential oil is mainly composed of monoterpenes including carvacrol and thymol. streptozotocin (stz) is used to induce diabetes in rats. the aim of this study is to investigate the inhibitory effects of o. hypericifolium essential oil on the dpp iv in stz-induced diabetic rats. the animals (female spraque-dawley rats) were assigned to four groups (group : control, group : stzinduced diabetic, group : o. hypericifolium injected, group : stz-induced diabetic and o. hypericifolium injected). dm was experimentally induced in groups and by a single intraperitoneal injection of stz at a dose of mg/kg body weight. in groups and , rats were intraperitoneally injected with o. hypericifolium oil at a daily dose of ml/kg body weight for consecutive days. at the end of the experimental period, all animals were sacrificed by cervical dislocation under ether anesthesia and liver and kidney tissues of each animal were rapidly excised. tissues were fixed in sainte-marie fixative. after routine histological processes, samples were embedded in paraffin, immunohistochemical staining for dpp iv was performed on sections and then they were photographed. the immunohistochemical reaction intensity differences were observed between the groups. in conclusion, the immunohistochemical distribution of dpp iv in the tissues that the test oil was applied in the diabetic rats may be important for the investigation of the inhibitory effects of oil on the enzyme. moreover, our findings suggest that o. hypericifolium oil may be used for prevention of diabetic diseases. introduction: all eukaryotic cells need microtubules for purposes of nuclear and cell division, organization of intracellular structure, and intracellular transportation, as well as ciliary and flagellar motility. microtubules are made of polymerized a/btubulin subunits. mec is important for microtubules, because it encodes the enzyme that adds acetyl groups to lysine (k ) of tubulin. k is largely conserved in a-tubulins of many eukaryotes, and acetylation is thought to stabilize microtubule structure. in algae, the effect of acetylation by mec on flagellar motility and phototaxis has not been tested previously. materials and methods: in this study, mec mutant chlamydomonas reinhardtii cells were compared to wild-type cells to see the effect on flagellar motility and phototaxis. we tested phototaxis, eyespot size and quantity under the microscopy. in addition to this, we fixed cells and examined them by immunofluorescence microscopy using antibodies to tubulin, acetylated tubulin, and photoreceptor. results: we observed that some mec mutant cells contain more than one eyespot. we detected no acetylated-tubulin (ac-tub) by immunofluorescence. the cells still phototax and have normal motility discussion and conclusion: interestingly, mec cells still have the ability to phototax and they have normal flagellar motility, even though they contain occasional additional eyespots and no ac-tub. chlorella vulgaris as a model system for screening of plant growth modulators p. volynchuk , e. marusich , r. chuprov-netochin , j. neskorodov , y. mishutkina , s. leonov life sciences center, moscow institute of physics and technology, dolgoprudny, center "bioengineering", russian academy of sciences, moscow, russia the discovery of new plant growth modulators became extremely important task as an alternative approach to overcome plants resistance to herbicides and pesticides, which leads to harmful action on plants and land rising, environmental and ecological problems. small molecules provide agricultural biotechnology with valuable tools, which help to circumvent the need for genetic engineering and offer unique benefits to modulate plant growth and development. we developed a system to explore molecular modes of action of plant growth modulators using chlorella vulgaris model. our model allows applying high content screening approach in well plate format for fast and robust effect assessment of large number of tested modulators. chlorella v. was grown in climate chamber under optimized constant temperature ( °c) and light conditions ( : hours/light:dark). modulating effect of tested compounds was estimated by spectrophotometric measurement of microalgae density at the beginning of the experiment (start point- . od) and hours later. to validate our system we used known cytokinins and auxins ( mm) as positive controls of growth stimulation. we showed that in presence of each compound the density of chlorella v. was increased in - times range, compared with only times increase in control group. eight new chemicals ( lm), which demonstrated modulation effect on nicotianatabacum l. pollen and arabidopsis thaliana models, were tested on developed chlorella v. model. positive controls showed no stimulating effect at this concentration, while tested compounds were confirmed as hits and increased the density up to %. we demonstrated that developed model system, based on chlorella v., is an effective system for primary screening of plant growth modulators. the main advantages of this system are short time of assay, simplicity of performance, possibility of automation and low cost. selected hits can be recommended as perspective candidates for future test on crop field. sunflower is under a big threat of downy mildew which is a fungal disease caused by plasmopara halstedii. the disease can cause up to an % yield loss in sunflower production. downy mildew resistance genes (r) denoted as pl has been discovered to date in sunflower. in recent years, single nucleotide polymorphism (snp) markers have become widely used in plant breeding programs. in this study snp markers have been currently developing for pl , pl , pl , pl arg genes by competitive allele specific pcr (kasp) assay which enables bi-allelic scoring of snps and insertions/deletions (indels) at specific loci. in total sequence tagged site (sts) sequences from ncbi were aligned for pl , pl , pl , pl arg genes to identify conserved regions for each gene. based on the conserved regions, specific pcr primers were designed in order to make sequencing of these genes in five crosses and their f progenies. sequence data will be used to design an allele specific primer maches the target snp and amplifies the target region with the common reverse primer provided by kasp genotyping assay. snp markers linked to pl genes which are being developed in this study, have the potential to be used in marker assisted selection (mas) for sunflower breeding programs. p- . . - investigation of the antidiabetic effects of hibiscus sabdariffa, teucrium polium and myrtus communis in hepg cells line s. altundag , pamukkale university, denizli, istanbul medeniyet university, istanbul, turkey some antidiabetic plants currently are used in alternative treatment of type ii diabetes. hibiscus sabdariffa, myrtus communis and teucirum polium plants are also known for their antidiabetic properties. hibiscus sabdariffa, myrtus communis and teucrium polium; depending on the impact on hepg - cells to investigate the possible mechanisms of type ii diabetes with researches on glucolysis and glucogenesis pathways gene expressions (pk m , glut- ,pepck). plants were obtained in dried state from reliable herbalists in denizli and mersin. plants treated with the extractor device. plants obtained aqueous extract was subjected to lyophilization process. human cancer cells have been used throughout the study. the cytotoxicity of the cells was measured by elisa plate reader . total rna was isolated using trızol Ò solution was carried out according to the instructions of the manufacturer's (thermo scientific) recommended procedures were performed, but we have to optimize our own laboratory conditions. pk m , glut- ,pepck genes were synthesized by b _ ioneer. during our study the activation of certain genes (pk m , glut- ,pepck) were examined by real time pcr. in our study hibiscus sabdariffa, mrytus communis and teucrium polium plant to extract applied hepg - cell line in the gluconeogenesis and glycolysis pathways in involved in some important genes (pepck, pk m , glut- ) analyzed the expression levels . teucrum polium plant extract is applied in hepg - cells the glycolysis pathway genes (pk m glut- ) an increased expression also genes of gluconeogenesis pathway (pepck) were not decreased. however hibiscus sabdariffa and the expression of genes involved in glycolysis and gluconeogenesis pathway mrytus communis plants were observed to have a full effect as diabetic or hypoglycemic . in this context, it is considered that the plant teucrum polium on the line hepg - cells showed antidiabetic effect. p- . . - purification of b-glucosidase from malatya apricot (prunus armeniaca l.) seeds and some of its biochemical properties h. kara , s. sinan , z. ekmekci , y. turan university of balikesir faculty of veterinary, balikesir, university of balikesir faculty of arts and sciences, balikesir, turkey introduction: b-glucosidases are one of the key enzymes in carbohydrate metabolism and located in glycosyl hydrolases (ec . . ) family. plant b-glucosidases have biotechnological significance as they are effective on glycosidic bonds of flavor and aroma precursors in plants. b-glucosidases that located in fruit seeds are important because they affect the amygdalin. aim of this study is purification and partially characterization of b-glucosidase from malatya apricot seeds. materials and methods: apricot seeds were homogenized with extraction buffer to prepare of crude extract. the enzyme protein was precipitated with % ammonium sulfate then purified by hydrophobic interaction chromatography using sepharose b-ltyrosine- -naptylamine gel. para-nitrophenyl b-d-glucopyranoside (p-npglc) was used as substrate to determine biochemical properties of the enzyme. the optimum ph was determined using buffers between ph - and thermal optima was determined using - °c temperature range. inhibitory effects was determined with mm substances. results: the enzyme was . -fold purified with yield of %. purified b-glucosidase from apricot seed was visualized about kda molecular weight on sds-page. the kinetic parameters were determined against p-npglc substrate as km and vmax values of . mm and . eu, respectively. the optimum ph and temperature were determined . and °c respectively. effects of cacl , kcl, nacl, mgcl , k so , na so , cuso , fecl , pb(ii) acetate, agno , zncl and glucose on purified enzyme activity were investigated. kcl, na so , pb(ii) acetat and cuso reduced the enzyme activity. discussion and conclusion: in this study, b-glucosidase was purified from malatya apricot seed and some of its biochemical properties were determined. because this enzyme has pharmaceutical importance hydrolyzing amygdalin. the results showed that immobilized almond b-glucosidase was used to break amygdalin and release -cn compound that effective to shrink cancer mass. p- . . - a new affinity gel for purifing polyphenol oxidase enzyme a. erg€ un , , o. arslan balikesir university, science and technology application and research center, balikesir, department of medical chemistry, faculty of science, balikesir university, balikesir, turkey polyphenol oxidase (ppo) enzyme, sometimes called as phenol oxidase, catecholase, phenolase, catechol oxidase or tyrosinase, is considered to be an o-dipenol. ppo (ec . . . ), a multifunctional copper containing metalloenzyme, is widely distributed in nature. ppo exists in many kinds of plants and fungi, such as banana, mushroom, butter lettuce, napoleon grape, potato, coffee, marula fruit, artichoke heads, longan fruit, tobacco, wheat flour. in this study, a novel affinity chromatography gel was synthesized for purifing ppo enzyme. the affinity chromatography gel was synthesized by coupling aniline as a specer arm to cnbr activeted sepharose- b. then, p-amino benzoic acid was coupled to aniline as a ligand. ppo was purified from musa sapientum var. cavendishii (banana) by using sepharose- b-aniline p-amino benzoic acid affinity chromatography gel. % . yield and . fold purification were achieved. sds-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent mw of kda. the v max and k m of the purified enzyme were determined , u/ml min and . mm, respectively. p- . . - phenolic content and antioxidant capacity of gamma irradiated olive leaves m. e. diken , b. kocat€ urk , s. dogan , h. tuner balikesir university, balikesir, kavram vocational school, istanbul, turkey in this study, dried in diffirent ways (such as microwave, infrared, convection heaters and under normal atmospheric conditions) olive leaves has been used as experimental material. radiation has been applied to dried olive leaves in three diffirent dosages in room temparature. the amount of radiation to be implemented to the samples have , , kgy/min. in this study, how gamma rays (radiation) effects phenolic components, total phenolic content and antioxidant capacity of dried olive leaves has been determined. the phenolic components, total phenolic content and antioxidant capacity were analysed with hplc, folin ciocalteu and dpph radical scavenging method, respectivelty. gamma rays is well known as a decontamination method for many foodstuffs and plant materials, being an environment friendly and effective technology to resolve technical problems in trade and commercialization. for this reason, nowadays it is utilized as an alternative method of sterilization. gamma rays are of ionizing radiation. when ionizing radiation interacts with matter, generally it causes a break in the molecular bonds and/or breaks the bonds between the molecules. these intermediates have unpaired electron and called free radical. gamma radiation or the radiation-induced free radicals would break or cause damage to the dna molecules of living organisms. gamma irradiation is predict to change phenolic content and antioxidant capacity in living tissues. phenolic compounds are secondary plant metabolites naturally present in fruits and vegetables. in recent years there has been a growing interest in food phenolics because of their potential health benefits mainly due to their antioxidant and free radical scavenging activity. despite the controversy about potential risks posed by genetically modified plants on human health, environment and microorganisms, cultivation area of these crops increases day by day. this increment has revealed concerns especially related to hgt. hgt studies indicated that antibiotic resistance genes in gm plants have a potential to transfer to soil microorganisms. in this study, hgt of widely used genetic elements such as regulatory sequences, from transgenic plants to bacteria was investigated. three soybean feed and four seed examples from turkish feed manufacturers' association were used for genetic analysis based on foreign gene determination. gmo analysis were conducted by camv s promoter-specific pcr in genomic dnas. in gm positive samples, genomic dnas sheared into appropriate fragment size by ultrasonication for the purpose of bacterial transformation. presence of s promotor region in fragmented dnas was proved by pcr. escherichia coli dh a strain was transformed by fragmented dna samples according to cacl method. s promotor sequence screened by using pcr in bacterial genomic dnas. as a result of gm screening, all feed and three of the seed samples were found to be transgenic. ultrasonication conditions were optimized for shearing dna's to - bp for bacterial transformation. fragmented dnas confirmed for carrying intact s promotor sequence. none of bacterial genomic dnas were found to be s-positive. according to the transformation results, absence of s promotor sequence in all tested bacterial genomic dnas, proved the dna samples belonging to gm plants can not transfer into compotent e. coli dh under laboratory conditions. for verification of this finding, transformation studies will continue with acinetobacter baylyi bd strain which is naturally competent soil bacterium for natural transformation. we believe that all of our findings will contribute to constitute transformation system which can be used as model in hgt studies. p- . . - preliminary studies on differential methylation in a and d sub-genomes of upland cotton (gossypium hirsutum l.) four species of cotton (gossypium l.) provide raw materials for the textile industry. among the four species, two have diploid genome and another two have tetraploid genome. tetraploid genome consists of a and d sub-genomes. a sub-genome belongs to asian cotton while d-sub genome belongs to american cotton. previous studies revealed that d sub-genome of gossypium species contributes to the superior yield and quality of tetraploid gossypium l. species (atdt). dna cytosine methylation of four regions of dna sequences located on a and d sub-genomes of gossypium hirsutum l. texas marker (tm- ) was investigated using bisulfite sequencing technique. among the regions studied two could not be located on subgenomes due to sequence identity match between a and d subgenomes. on the other hand two dna regions could be located on a and d sub-genomes using the blast searches. some of the dna sequences located on different sub-genomes showed polymorphic nucleotides including c/t and g/a polymorphisms. in silico analysis indicated that some alleles located on different sub-genomes of cotton have c/t and g/a polymorphisms. c/t polymorphisms between the sub-genomes could be thought as unmethylated cytosine using the bisulfite sequencing technique. this indicated that an extra attention needs to be paid in dna total cytosine methylation studies in polyploid species such as cotton using bisulfite sequencing, methylation sensitive amplification polymorphism (msap), whole-genome bisulfite sequencing (wgbs). otherwise, t in c/t polymorphism between the subgenomes could be thought as unmethylated cytosine. based on the two genomic regions we could conclude that a sub-genome may be more methylated than d sub-genome. differential methylation of genes located on different sub-genomes may provide another mechanism responsible for differential gene expression of genes located on different sub-genomes. p- . . - cleaved minisatellite locus (cml) markers for fingerprinting of cotton cultivars grown in turkey e. u. gocer, m. karaca akdeniz university, antalya, turkey after their discovery by alec jeffreys in , minisatellites have been used in genetic studies of many organisms. minisatellites, also called variable number tandem repeats (vntrs), are composed of arrays of longer repeats mostly dispersed throughout heterochromatin (centromeres and telomeres). direct amplification of minisatellite regions of dna using a single core primer is a powerful method to amplify minisatellites (damd-pcr). although the damd-pcr technique has been applied to many plant species, the level of polymorphisms in cotton (gossypium l.) is very low due to very narrow turkish cotton genetic base. the objective of this study was to improve the level of polymorphisms by cleaving minisatellite loci by restriction enzyme digestion. genomic dna samples of twenty-one turkish cultivars, pima - , tm- and ps- were extracted. twenty-one minisatellite primers were screened using the damd-pcr technique. monomorphic amplicons were digested using several restriction enzymes. three to five micro liters of amplified products were digested with various restriction enzymes. digested products of minisatellite loci were separated in % high resolution agarose gels. comparison studies of digested and undigested markers revealed that cleaved minisatellite markers showed polymorphisms in those cotton lines that could not be differentiated by microsatellite and minisatellite markers. this approach was called cleaved minisatellite locus markers (cml). the amplification reactions of minisatellites used touch-down (td) cycling conditions. the use of td offered a simple and rapid means of optimizing polymerase chain reaction (pcr), increased specificity, sensitivity, and efficiency without the need for lengthy optimizations of minisatellite primers. the cml markers were obtained at a °c annealing temperature, which is a temperature higher than those used in random amplified polymorphic dna (rapd) inter-simple sequence repeat (issr) markers. p- . . - association between cytosine methylation and tissue specific expression of microsatellites a. g. ince, m. karaca akdeniz university, antalya, turkey heritable covalent modification of dna, rna or protein without altering their primary sequences is defined epigenetics. because all biological events are influenced by epigenetics, it is one of the most important fields in science. dna methylation is one of the most important epigenetic mechanisms. dna cytosine methylation is a process by which methyl groups are added to cytosine bases of dna. microsatellites, also knows simple sequence repeats are dna sequences consisting of - nucleotide repeats. there is a large body of information regarding the relationship between microsatellite instability and abnormal gene expression, and between dna methylation and altered gene expression. however, there is limited information on cytosine methylation of microsatellites. in the present study, we investigated whether there is any association between cytosine methylation and tissue specific expression of microsatellites. genomic dna samples of various tissues and developmental stages of pepper line demre sivrisi (capsicum annuum l.) and cotton line tm- (gossypium hirsutum l.) were extracted. cdna samples were synthesized using mrna expressed in pepper tissues. cytosine methylation levels were investigated using bisulfite sequencing methods. screening studies of microsatellites revealed that some genes containing microsatellites were differentially expressed in tissues and developmental stages of pepper. microsatellite containing genes that expressed differently among tissues had also showed different methylation levels in cg, chg and chh (where h refers to a, c or t) contexts. methylation level differences between microsatellites were also observed. as far as our knowledge, it is the first report on differential expression of genes containing methylated microsatellites. p- . . - pcr-lg: an alternative way to assign the chromosome location of genes/markers in cotton a. aydin, m. karaca akdeniz university, antalya, turkey assignment of genes and dna markers on chromosomes is very important in life sciences, especially for plant breeding and medicine. there are several methods for the assignment of a gene or dna sequence to a specific location on a chromosome. for example, the most widely used technique is the assignment of fluorescently-labeled gene or dna sequences (markers) on chromosomes using the fluorescently-labeled gene (for instance, fish technique). another example is the construction of a genetic map (linkage map) which orders the targeted genes along the dna strand based on recombination frequency. sequencing is the most precise technique in which coding (gene-containing) and noncoding dna region of genes could be located on a chromosome molecule. aneuploid lines could also be used to locate genes in a specific chromosome, but maintenance of these lines is difficult. here we report the use of chromosome substitution lines to indirectly locate genes/markers on chromosomes. we used chromosome substitution lines (csls) that carry a chromosome pair or chromosome arms from gossypium barbadense l. while the rest of chromosomes belong to g. hirsutum l. a total of chls, a homozygous cotton line tm- and a double haploid line pima - were used as plant materials. twenty microsatellite primer pairs were utilized in touch-down polymerase chain reactions. we developed a method, called polymerase chain reaction to locate gene (pcr-lg), to assign genes/markers on chromosome or chromosome arm. with the use of pcr-lg approach any polymorphic genes/markers between tm- and pima - (g. hirsutum and g. barbadense) could be assigned to a chromosome or chromosome arm. results indicated that if csls were used any polymorphic markers (genes) with known primer pairs could be assigned to cotton chromosomes. although we used cotton chromosome substitution lines to validate the proposed technique, it could be applicable all the species that have chromosome substitution lines. p- . . - pecularities of genome variability of antarctic hairgrass deschampsia antarctica desv. from the maritime antarctic deschampsia antarctica desv. (poaceae) is the only grass species native to the antarctic region, adapted to harsh environmental conditions. however, reasons for its unique success remain unexplored. stressful environmental factors can influence a plant genome and cause changes in the chromosome number and morphology and increase genetic variation. therefore, the purpose of our research was to explore alterations in the d. antarctica genome both at the chromosomal and molecular levels and to investigate species genome stability using in vitro tissue cultures. plants used for the study were grown in vitro from seeds collected in the argentine islands region during - . chromosome number was determined in plant root apical meristems and specimens prepared from tissue cultures. rrna genes localization were determined using the fish technique. moleculargenetic analysis was performed using pcr with polymorphic issr-primers. new forms of chromosome polymorphism, associated with aneuploidy ( n = - ), polyploidy ( n = - ) and the occurrence of additional b-chromosomes ( n = + - b), were observed. fish analysis also confirmed that genotypes with a different chromosome numbers varied in the number of s rdna and s rdna sites. assessment of genetic variation demonstrated a low level of diversity: differences between the plants with different chromosome numbers do not exceed the level of within-population variation. cytology analysis of d. antarctica cultured tissues revealed aneuploidy (up to . %) with predominance cells with diploid and near-diploid chromosome number irrespective of plant's initial karyotype (diploid, mixoploid or polyploid). we assume that discovered intraspecies chromosomal polymorphism is a manifestation of quick genome reaction to harsh antarctic conditions. whereas the results of molecular-genetic analysis and study of cell cultures of this species suggests on the relative stability of d. antarctica genome. p- . . - angiotensin converting enzyme inhibitory activity of morchella esculenta (l.) pers a. zeytunluoglu , i. arslan biomedical equipment technology, pamukkale university, denizli, turkey, denizli, biomedical engineering, pamukkale university, denizli, denizli, turkey hypertension is a multi-aetiological, chronic pathophysiology that leads to multi-organ dysfunctions like cardiovascular diseases, strokes, and renal complications. natural extracts play an important role in traditional medicines for the treatment of hypertension and are also an essential resource for new drug discovery. mushrooms are use as therapeutics in alternative and complementary medicine as functional food because of contain a large number of biologically active components that offer health benefits and protection against many degenerative diseases. morchella esculenta is one of the most highly priced edible mushrooms worldwide. it contains a wide range of active constituents which include tocopherols, carotenoids, organic acids, polysaccarides and phenolic compounds which exhibit a wide range of medicinal and pharmacological properties including anti-microbial, anti-inflammatory, immunustimulatory, antitumor and antioxidant. in this study; the in vitro angiotensin converting enzyme-i (ace-i) inhibitory activity of m. esculenta peptides were generated by alcalase hydrolysis were studied. the kda < peptides < kda in the ultrafiltration fractions displayed highest ace inhibition ( . ae . % at lg/ml). the results indicate that m. esculenta derived peptides may have potential as functional food ingredients in the prevention and management of hypertension. p- . . - modulations of antioxidant enzymes, gsts and catalase, by salvia absconditiflora in hepg cell line d. irtem kartal , a. altay yuzuncuyil university, van, erzincan € university, erzincan, turkey oxidative stress is considered to play a important role in the pathogenesis of aging and several degenerative diseases, such as cancer. in order to cope with an excess of free radicals produced upon oxidative stress, humans have developed several mechanisms for maintain redox homeostasis. these protective mechanisms either scavenge or detoxify ros, block their production, and include enzymatic and nonenzymatic antioxidant defenses. in enzymatic defenses include glutathione s-transferases and catalase enzymes. many epidemiological studies have revealed that there is a strong correlation between consumption of polyphenol-rich foods and the prevention of certain diseases like cancer, cardiovascular diseases and aging. phenolic compounds are abundant in all plants. so, they form an integral part of the human diet. salvia species, commonly known as sage, have been used since ancient times for more than different ailments ranging from aches to epilepsy. there are around species of salvia, of which are represented in turkey including salvia absconditiflora. in this study, s. absconditiflora collected from metu campus (ankara, turkey) is extracted with methanol and water. effects of the water and methanol extracts on the mrna expressions of antioxidant enzymes gstm and catalase in hepg cells were investigated by q-rt-pcr technique. it was also monitored the effects of the extracts on the enzyme activities of gsts and catalase by spectrophotometrically. water and methanol extracts decreased gsts mrna expression in hepg cells for hours and hours incubation and methanol extract decrease catalase mrna expression for only hours incubation. on the other hand, extracts highly increased the gsts and catalase activities in both hour incubation. overall, these results indicate that s. absconditiflora and/or its components have regulatory activities on antioxidant enzymes and they may have a potential as a therapeutic agent in the treatment of cancer. p- . . - transcriptomics and proteomics approach to drought stress mechanism in wheat b. cevher keskin tubitak marmara research center genetic engineering & biotech. institute, kocaeli, turkey birsen cevher keskin, yasemin yıldızhan, oktay kulen, bayram y€ uksel, selma onarıcı, _ ismail t€ urkan, as ßkım hediye sekmen, u gur sezerman, bu gra € ozer identification of novel stress-responsive genes and their role in drought response is an important area for the improvement of the crops. drought-related genes were investigated in leaves and roots of three wheat genotypes after different drought stress treatments by rna sequencing (rna seq) technology and de novo assembly was performed before comparative transcriptome analysis. analyzing gigabases of bp paired end illumina reads from a hexaploid wheat poly(a) rna library, we identified common and new differentially expressed transcripts. selected differentially expressed genes were confirmed by qrt-pcr. we also performed root proteome analysis with nano electrospray ionization source coupled to a high-performance liquid chromatography system (nanouplcÀesiÀqtofÀms) to identify drought-related proteins. totally proteins were differentially expressed in root tissues of tolerant and non-tolerant wheat genotypes. responses of antioxidative defense system to drought stress were comparatively studied in the same wheat cultivars. similarities between protein and rna levels help increase our confidence in novel biomarkers, differences may also reveal other post-transcriptional regulatory junctures. all these analyses will allow us to get a better idea about the possible role of these genes in the drought-response mechanism. the drought-related genes that are functionally characterized could be introduced into agronomically important wheat cultivars. this work offers a resource for accelerating drought-related gene discovery and improving this important crop. p- . . - isolation and characterization of a hexose converter from olive s. altunok , e. d€ undar department of biology, university of balikesir, balikesir, department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey introduction: hexose sugars are key components of glycolysis and photosynthesis. the genes regulating their conversion into one another, is therefore, of great importance for the control of carbon metabolism. in this study, we report isolation and characterization of a cdna associated with conversion of hexoses in olive. the cdna putatively named aldolase based on bioinformatic and experimental analyses. material-methods: characterization based on nucleotide and amino acids were conducted using bioinformatic tools such as nucleotide and protein blast, bioedit, primer , finchtv, clc genomic workbench, expasy, targetp, sosui and web promoter scan. comparison of the genomic and cdna sequence of the gene and detailed bioinformatic analyses including cellular location, hydropathy analysis, amino acid-nucleotide composition and predicted d structure were also conducted using the bioinformatics tools mentioned above. temporal expression pattern of the putative aldolase were conducted using real-time pcr experiments. sds and western blot analyses were completed while biochemical analyses are ongoing. oleocanthal is an important secondary metabolite that has been reported to be useful against important human diseases including cancer. the aim of this study was to identify and characterize the key biochemical and genetic components of oleocanthal biosynthesis. to determine the biochemical components of the pathway, multiple olive cultivars along with their spatial and temporal points were determined. the expression levels of multiple candidate genes were also aimed via real-time pcr. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes with that of other organisms) were conducted on ncbi web page. phylogenetic tree construction, amino acid composition analysis, nucleotide composition analysis, hydropathy analysis and translations through expasy were conducted. primer was used to design forward and reverse primers to amplify the target genes from different olive tissues at different times. analysis of the first candiate gene with bioedit program revealed that a+t ratio was more than g+c according to the nucleotide composition analysis. according to amino acid composition analysis isoleucine, lysine and leucine were more than other amino acids while kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. abundance of hydrophobic amino acids (leucine and isolecine) along with an abundant hydrophilic amino acid (lysine) suggest the existance of hydrophobic pockets in the protein which may mean a membrane bound protein or a sitoplasmic protein with a strong hydrophobic core. the molecular weight of the protein was kda with a pi of . . the protein was found to have a signal peptide. according to the sosui gramn , intracellular localization was found to be in the inner membrane. analysis of other candidate genes contiunes. acknowledgements: this study was supported by tubitak with grant number o . key words: olive, olea europaea l., secologanin, polymorphism, allele diversity p- . . - antioxidant potentials of propolis and its bioactive components, and their effects on cyp e gene expression in ht- adenocarcinoma cell line a. altay , d. irtem kartal erzincan € university, erzincan, y€ uz€ unc€ u yil university, van, turkey propolis is a resinous mixture that is collected by honeybees from plants, and is combined with beeswax and secretions from the bee's salivary glands plus some pollen. it is a rich mixture of polyphenols, flavonoid aglycones, phenolic acids and their esters. it has been used in traditional medicine for thousands of years because of these ingredients. propolis, just like honey, has been the subject of many studies due to its antimicrobial, antifungal, antiviral and hepatoprotective activities. the cytochrome p enzymes are involved in phase i xenobiotic and drug metabolism. cyp e is present in high levels in human tumors demonstrated by qrt-pcr and immunohistochemical studies. in this study, propolis collected from datc ßa (mugla, turkey) is extracted with % ethanol. phenolic contents of the propolis were measured by lc-ms/ms. cytotoxic effects of the propolis on ht- human colon adenocarcinoma cell lines were examined via xtt colorimetric cell proliferation assay. effects of propolis extract and its main bioactive component caffeic acid on the expression of phase i detoxification enzyme cyp e in ht- cells were investigated bu using q-rt-pcr technique. ic values for dpph radicals scavenging activities of the extract was calculated as . mg/ml. tpc and tfc were determined as . mg gae/g extract and . mg qe/g extract, respectively. caffeic acid content of the extract was measured as . lg/g extract. ic values for xtt assay in hours incubation with the extract and caffeic acid were evaluated as . mg/ ml and . mg/ml, respectively. propolis extract and its main phenolic content caffeic acid significantly dicreased cyp e mrna expression with . and . fold, respectively in ht- human colorectal adenocarcinoma cells for hours incubation. overall, these results indicate that propolis and/or its components have regulatory activities on cyp e expression and they may have a potential as a therapeutic agent in the treatment of cancer. p- . . - effects of histone h lysine inhibition on gene expression profile in tobacco (nicotiana tabacum l.) differentiation is the characteristic of multicellular organism. cellular dedifferentiation underlies topical issues in biology such as reprogramming in stem cell research, regeneration and nuclear cloning, and has common features in plants and animals. the state of dedifferentiation is evidenced by changes in cell morphology, genome organization and the pattern of gene expression as well as by the capability of plant tissues to differentiate into multiple types of cells depending on the type of stimulus applied. chromatin reorganization appears to be a fundamental theme in cellular dedifferentiation and reentry into the cell cycle both in plants and animals. the chemical modifications of histone proteins which are structural units of the nucleosome, generate 'codes' for the recruitment of proteins or protein complexes that affect chromatin structure and gene expression according to 'histone code' hypothesis. methylation of histone h at lysine residue by specific methyltransferases suv h in humans and kyp/suvh in arabidopsis generates a'code' for the recruitment of hp proteins. in this study, to enhance the dedifferentiation efficiency, chaetocin which inhibits suv h has been used at the germination stage of tobacco seeds in in vitro conditions. our results showed that chaetocin induced callus formation from leaf discs of tobacco in the early stage of the inhibitor application. chaetocin can enhance reprogramming of plant cells in seed development treatment as callus induction. it is known that the formation of callus which is the non-differentiated cell community in plants, is a consequence of the changes in the gene expression profile. it has been found that epigenetic modifications play a crucial role in some of these changes. the definition of the genes related to callus formation by the inhibitation of an epigenetic modification -h k methylation-in tobacco might be used to bear on various dedifferentiation-driven cellular processes. induction of tumor cell death by the use of some phytochemicals that consumed through diet, and derived from medicinal plants opens up new horizons for cancer treatment researches. rheum ribes species, which is studied in this research, is one of the commonly used herbs in pharmacological researches. the high content of phenolic compounds in r. ribes extracts were known to be responsible for the high antioxidant and antibacterial activities. our aim in this study is to assess cytotoxic and apoptotic changes by way of implementing methanol extract of the rheum ribes (root) to the mcf- breast cancer cell line. cytotoxic effect of rheum ribes extract was evaluated by using the xtt ( , -bis( -metoksi- -nitro- -sulfofenil)- h-tetrazolyum) test. in order to determine the dose of ic , plant extracts were applied as time and dose dependent in the range of - ug. in nd hour, the ic value is determined as ug. to examine the apoptotic effects of the extract, total rnas were isolated from dose group and the control cells firstly, then cdnas were synthesized. expression profile of the target genes(caspase- , caspase- , caspase- , caspase- , bax, bcl- , fas) are determined by qpcr. according to the results, when the control group compared with the cells, it was determined that, while . and . -fold respectively increase in the gene expressions of caspase- and caspase- of dose group cells, . -fold decrease in the gene expressions of bcl- . no significant difference was observed in the other genes examined. based on the obtained data, we believe that methanol extract of the rheum ribes induces apoptosis by activating intrinsic pathway. as a result, this plant species can be a new and effective therapeutic candidate for the medical world in search of alternative anti-cancer approaches, and could shed light on the work to be done in this area. p- . . - the effect of ferulic acid and -fluorouracil combination on apoptosis in pc- human prostate cancer cells prostate cancer is quite often seen in industrialized countries and has the second most common death rate due to cancer after lung cancer in men. -fluorouracil ( -fu) is a pyrimidine analog and cell cycle-targeting drug by inhibiting dna synthesis. it has been widely used for treatment of several cancers such as gastric, colorectal, and breast cancers. phenolic compounds found in foods are potential antioxidants of harmful oxidative processes related to cancer and also important due to induction of different mechanism such as apoptosis. ferulic acid (fa; -hydroxy- -methoxycinnamic acid), a phenolic compound, is abundant in fruits and vegetables. the purpose of this study was to investigate combination effect of fa and -fu on apoptosis in pc- human prostate cancer cell line. the effects of -fu, fa, and combination of both of them on cell viability were determined by xtt method. total rna isolation was conducted using trizol reagent. expressions of important genes in apoptosis including casp , casp , casp , casp , bcl , bax, fas and cycs were investigated in four groups by qpcr. the ic doses of fa and -fu were found to be lm and lm for hours in pc- cells, respectively. in order to determine combination effect, pc- cells were treated with <ic doses ( lm fa and lm -fu). according to qpcr results, a significant increase in the expressions of bax and cycs genes and a decrease in the expression of bcl were observed in the fa group, compared with the control group cells. after the treatment with -fu, the expression of casp was significantly increased compared with the control group. furthermore, combination of fa and -fu significantly increased expression of casp , casp , fas and cycs genes. in conclusion, comparing with the single treatments, it was observed that combination of fa and -fu affected expression of different genes in apoptosis in pc- cell line. p- . . - combination of ferulic acid and gemcitabine affects expression of some apoptotic genes in pc- human prostate cancer cells prostate cancer, the second causing of cancer-related death in men, is an important cancer type due to the late age of onset, slow the progression, high incidence. gemcitabine is an effective agent in several cancer types including non-small cell lung cancer, pancreatic, bladder and breast cancer. it is known that gemcitabine is used single agent or as a part of combination treatment in prostate cancer therapy. nowadays, new treatment methods have been tested for cancer therapy including natural compounds with low toxicity. ferulic acid (fa) one of these agents, an abundant phenolic compound found in various fruit and vegetables. in this study, objective was to investigate combination effect of ferulic acid and gemcitabine on apoptosis in pc- human prostate cancer cell line. cell viability was determined by using xtt method after the treatment with gemcitabine, fa and combination of both of them. total rna was isolated with trizol reagent. expressions of casp , casp , casp , casp , bcl , bax, fas and cycs genes are important in apoptosis, were evaluated in four groups by qpcr. the ic doses of fa and gemcitabine were found to be lm and lm for hours in pc- cells, respectively. for determination of combination effect, pc- cells were treated with <ic doses ( lm fa and lm gemcitabine). when compared with the control group, qpcr results illustrated, a significant increase in the expressions of bax and cycs genes whereas a decrease in the expression of bcl gene in the fa treatment group. after the treatment with gemcitabine, the expression of casp and fas genes were significantly increased compared with the control group. furthermore, combination of fa and gemcitabine significantly increased expression of casp , casp , casp , fas and cycs genes. in conclusion, it was observed that combination of fa and gemcitabine affected different genes in apoptosis compared with the single treatments in pc- human prostate cancer cell line. pistacia lentiscus linn. is widely distributed in mediterranean europe, morocco and turkey. the resin part of its known as mastic gum and plant called as mastic tree. it has been referred to over centuries as having medicinal properties to treat a variety of diseases. the aim of the present study are to evaluate antioxidant, cytotoxic and antimicrobial activities of heptane, chloroform and methanol extracts from p. lentiscus leaves and mastic gum. the antioxidant activities of chloroform and methanol extracts were assayed with various methods, including tpc, dpph free radical and abts radical cation scavenging activity. also, cytotoxicity of extracts was evaluated and screened against human mcf- , hela, a , and ht- cancer cell lines and nih- t cells. the viability of cells in lg/ml concentration of extracts was evaluated using mtt assay. antimicrobial activity of extracts were investigated against s aureus, s epidermidis, e coli, p aeruginosa, p vulgaris, k pneumoniae, c albicans, c glabrata, c guilliermondii, c tropicalis, c parapsilosis test organims by the agar well diffusion and broth microdilution methods . according to our results, the methanol extract of p. lentiscus leaves showed the highest dpph free radical scavenging activity (ic = . ae . mg/ml) and abts radical cation scavenging activity ( . ae . mg/ml). this study also exhibited that methanol extract of p. lentiscus leaves had the highest tpc ( . ae . mg gallic acid equivalents/g extract). the hexane extract of mastic gum showed antifungal activity against all of the tested candida species ( - mm / < . - mg/ml). the methanol extracts of p. lentiscus leaves showed the highest antimicrobial activity against all of the tested bacteria except k pneumoniae strain ( - mm/> mg/ml). on the other hand, p. lentiscus showed no cytotoxicity against cancer and normal cells. the results suggested that p. lentiscus may be natural source of antioxidant and antimicrobial activities. p- . . - antibacterial activity of and chemical composition of alcoholic extract of marjoram against some human pathogens m. ahanjan, m. rahbar mazandaran university of medical sciences, sari, iran herbs enjoy a unique value and importance in sustaining healthy communities in terms of disease prevention ( ) . in this regard, marjoram is a plant of the mint family which has antibacterial properties on microorganisms ( ) . the current study aims to investigate the anti-microbial activity of the alcohol extracts (i.e., methanol or ethanol) of marjoram plants on the bacteria of staphylococcus (atcc: ), aureus e. coli (atcc: ), and salmonella enterica (atcc: ) and p. aeroginosa through utilizing disk diffusion method. also, the minimum inhibitory concentration and the minimum bactericidal concentrationwere measured through tube. the measurement of minimum inhibitory concentration and minimum bactericidal concentration of ethanol and methanol extracts on e.coli were equal with and milligrams per milliliter, subsequently. moreover, the measurement of the minimum inhibitory concentration and of the minimum inhibitory concentration of marjoram ethanol extraction on staphylococcus aurous was reported to be milligrams and milligrams per milliliter, subsequently. in addition, the amount of ethanol and methanol extracts on salmonella enteric and p. aeroginosa was equal with and milligrams per milliliter, subsequently. the results showed that marjoram alcoholic extract enjoy antibacterial properties. also, among the alcoholic extracts, the ethanol extract has demonstrated to be the most effective extract on salmonella enterica and e. coli and p. aeroginosa. p- . . - molecular analysis of serine/arginine rich sc splicing factor from olive b. bas , e. d€ undar depertmant of biology, university of balikesir, balikesir, department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey olive (olea europaea l.) is an evergreen fruit tree adapted to mediterranean climate and rich in tannins, essential oils and organic acids. serine/arginine rich splicing factors are essential in seqeunce specific splicing of pre-mrnas. in this study we report molecular characterization of an serine/arginine rich sc splicing factor (oesarsc sf) that was isolated from a cdna library constructed from olive pedicels. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes from other organisms) were conducted on ncbi web page. amino acid composition analysis, nucleotide composition analysis, hydropathy analysis, open reading frame determiantion, through, molecular weight and the isoelectric points calculations were conducted using online expasy software. primer was used to design forward and reverse primers to amplify the target gene from different olive tissues at different times. analysis with bioedit program revealed that a+t ratio was more than that of g+c. oesarsc sf was a protein consisting of amino acids. as expected, amino acid composition analysis revealed that serines and arginines were more than other amino acids. kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. the molecular weight of the protein was kda with an isoelectric point (pi) of . . the protein was found to have a signal peptide. according to the predotar analysis results, intracellular localization was found to be in the mitochondrial. the combined results suggest oesarsc sf might function as splicing factor as its homologs from the other plants. confirming this hypothesis with futher experimental characterization including biochemical function analysis continues. acknowledgements: this study was supported by tub _ itak with grant number o . keywords: olea europaea l., oesarsc sf, alternative splcing, pre-mrna splicing, bioedit, pedicel specific cdnas. p- . . - application of three-phase partitioning for the purification of peroxidase from kiwano (cucumis metuliferus) z. denli , g. arabaci kto karatay university faculty of medicine, konya, faculty of arts and sciences, sakarya university, sakarya, turkey peroxidases are enzymes able to catalyze reduction of h o and oxidize various substrates. the kiwano is an oval shaped fruit which has an orange skin with lots of tiny horns. in this study, peroxidase isolated from kiwano is purified with three-phase partitioning (tpp). kiwano fruit was homogenized and obtained crude enzyme extract. the extract was saturated with % (w/v) ammonium sulfate ((nh ) so ) and added t-butanol with the ratio of : . (v/v). the lower and interfacial layer was collected. the influence of percent saturations of (nh ) so ( , , , , %) and tbutanol ratios ( : . , : , : . , : ) to the partitioning behaviour of peroxidase were analyzed. after dialyzed, the interfacial and lower phases were measured for peroxidase activity and protein content. the protein pattern of the peroxidases was evaluated by using gel electrophoresis. peroxidase activity recovery and the purification fold of interfacial and lower phases were . , . % and . , . . therefore, other experiments were continued with interfacial phase. at constant t-butanol with the ratio of : . , the enzyme activity recovery and purification fold of interfacial phase for saturations of (nh ) so ( , , , %) were . , . , . , % and . , . , . , . . the interfacial phase was not dissolved in % (nh ) so . at constant % (nh ) so , the enzyme activity recovery and purification fold of interfacial phase for t-butanol ratio ( : . , : , : . , : ) were . , . , . , . % and . , . , . , . . finally, at optimum conditions (% (nh ) so , t-butanol : . ) after dialyzed interfacial phase, the enzyme activity recovery and the purification fold were . % and . . the results of gel electrophoresis showed that the molecular weight of enzyme was between - kda. the applications of tpp gave the maximum recovery of . % and . -fold purification. as a result, for purfication of peroxidases, tpp is a rapid, simple and economical technique. accumulating the most robust genes and proteins in elite genotypes without any harmful effect on the potential plant yield is an urgent need to enhance productivity under various stressors. among the stressors, drought is a major challenge for agricultural productivity. brachypodium distachyon, with its close relationship to agriculturally and economically important crops, is an important model plant species. although ongoing transcriptomic analyses in brachypodium distachyon available, proteomic analyses are required to obtain an integrated picture of drought response. in the current study, a comprehensive proteome analysis was conducted on brachypodium leaves under increasing levels of drought stress. to screen gradual changes upon drought stress, brachypodium leaves subjected to drought treatment for , and days were collected for each treatment day. the cellular responses were investigated through a proteomic approach involving two dimensional difference gel electrophoresis and subsequent combined tandem mass spectrometry. for the validation of transcriptional expression, the genes encoding selected proteins were examined through quantitative real-time pcr. spot detection on cye-dyed gels revealed a total of distinct spots in brachypodium protein repertoire. a total of differentially expressed proteins (deps), with at least -fold changes in abundance, were identified by mass spectrometry and classified according to their functions. the biological functions of deps included roles in photosynthesis, protein folding, antioxidant mechanism and metabolic processes, highlighting the significant degree of overlapping between metabolic alterations induced by drought stress. identified proteins in this study and understanding the molecular mechanisms of drought response and defense mechanisms in plants will contribute to the researches on development of drought tolerant crop species. p- . . - immunohistochemical and electron microscopy investigation of tmv-based chimeric virus particles carrying conserved influenza antigen in nicotiana benthamiana recently we obtained and partially characterized set of viral vectors based on tobacco mosaic virus (tmv) genome displaying conserved influenza a m e epitope from matrix m protein. purified chimeric virus particles (cvp) conferred protection of mice against lethal homologous and heterologous influenza virus challenge. we revealed significant difference in symptoms of infections caused by tmv-m e recombinant viruses containing cysteine (cys) to serine (ser) or alanine (ala) substitutions in the human consensus m e sequence. accumulation level of m e-ala recombinant coat protein was significantly higher than m e-cys/ ser (ratio : ). tmv-m e-ser infection, in contrast to ala mutant, suspended growth and development of nicotiana benthamiana. non-inoculated leaves ( d.p.i.) were fixed with ethanol and histological sections were incubated with mouse serum to m e and secondary antibodies conjugated with either hrpo or fitc. cvps of all three mutants were detected in epidermal and stomata cells as well as in sieve elements and minor veins. electron microscopy analysis of mesophyll cells revealed typical rigid helical particles. cys/ser mutants mostly accumulated within ground cytoplasm as aggregates of discrete tubules in parallel arrangement, which were not delimited by lipid membranes. we discovered huge amount of cvps in the cytoplasm and lesser amount diffused in the central vacuole. essential part of ala particles was located in the cytoplasm, but mentioned aggregates were not found and only insignificant number of virions was revealed in vacuole. unlike wild-type tmv, none of the mutants was revealed in chloroplasts. diameters of cvps were as follows: sersingle particles in cytoplasm ae . cyanidin is a natural anthocyanidin found in a variety of fruits (grapes, blackberry, blueberry, cherry and cranberry etc.) and vegetables (red cabbage, red onion). this polyphenolic compound is a flavonoid with significant antioxidant activity. cyanidin and its glycosides have vasoprotective effects and can interfere with inflammation, carcinogenesis, obesity, and diabetes. one important role of the macrophages is the release of pro-inflammatory mediators, such as nitric oxide, various cytokines, in response to activation signals, including chemical mediators, cytokines, and bacterial lipopolysaccharide (lps). in this study, we investigated the role of cyanidin chloride in inflammation. anti-inflammatory effects of cyanidin chloride were examined in lps-stimulated murine raw . macrophages. we observed the level of various inflammation markers such as nitric oxide (no), inducible no synthase (inos), cyclooxygenase- (cox- ), tumor necrosis factor-a (tnf-a) and interleukin- (il- ) under lps treatment with or without cyanidin chloride. cyanidin chloride inhibited not only no production but also the expression of cox- and inos, without any cytotoxicity. cyanidin chloride also attenuated pro-inflammatory cytokines and other inflammation-related markers such as il- in a dosedependent manner. in conclusion, cyanidin chloride may be beneficial for the prevention and treatment of anti-inflammatory diseases. p- . . - the investigation of centranthus longiflorus plant extacts effects on cell proliferation and apoptosis activity in the cell lines of mcf- breast cancer h. askin ataturk university, erzurum, turkey introduction: in the u.s., breast cancer is the second most common cancer in women after skin cancer. current treatment of cancer can be done by surgery, chemotherapy, and radiation therapy. in addition, there is widespread use of complementary and alternative medicine in developed countries. plants and plant extracts play a critical role in the research into new anticancerogenic agents. centranthus longiflorus (cl) is used in alternative medicine for sedative and antispasmodic purposes. a plant of turkish origin, centranthus longiflorus used as traditional turkish medicine have remained uninvestigated for familial hypercholesterolemia, diabetes, coronary artery disease and cancer for their in vitro biological activity despite their use for sleep disorders. in this study, growth-inhibiting and pro-apoptotic effects of hexane, ethyl acetate and ethanol extracts of cl in mcf- breast cancer cell line were investigated. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from cl was analyzed using a gc-ms system. dose-and time-dependent cytotoxic and apoptotic effects of cl were evaluated by mtt cell proliferation kit and cell death detection elisa kit, respectively. manufecturer's protocol was followed for analyses. then, apoptotic genes; caspase , bax and p and antiapoptotic genes; bcl- and pi expression levels were determined by rt pcr. results: according to our results, cytotoxic effect on mcf- cell was only observed in and lg/ml doses of cl. however, any of the application doses showed an apoptotic effect on mcf- cells. they exhibited a necrotic effect rather than the apoptotic effect. although alterations in expression levels of these genes were determined, this alterations was statistically insignificant. discussion and conclusion: consequently, we can say that cl have a cytotoxic effect on mcf- breast cancer cell lines. p- . . - reduction of the chloroplast genome and the loss of photosynthetic pathways in the mycoheterotrophic plant monotropa hypopitys, as revealed by genome and transcriptome sequencing e. gruzdev, a. mardanov, a. beletsky, v. kadnikov, e. kochieva, n. ravin, k. skryabin institute of bioengineering, research center of biotechnology of the russian academy of sciences, moscow, russia genomes of parasitic plants represent interesting model systems to study effects of relaxed selective pressure on photosynthetic function. previous genomic studies of nonphotosynthetic plants revealed reduction of their chloroplast genomes, but the corresponding changes in their nuclear genomes are less known. here we present the data on the transcriptome and the chloroplast genome of the non-photosynthetic mycoheterotrophic plant monotropa hypopitys. the chloroplast genomes were sequenced for two specimens of m. hypopitys, collected in different regions of russia. the cpdnas are , bp (mon- kalr) and , bp (mon- volr) long and rearranged with respect to each other. both genomes contains genes encoding ribosomal proteins, infa, matk, and ribosomal rna genes. and trna genes were predicted in two cpdna. genes encoding nadh dehydrogenase, plastid rna polymerase, all genes related to photosynthetic apparatus, clpp, ycf , ycf , accd, and some genes for ribosomal proteins are missing or became pseudogenes. the reduction of gene content is associated with extensive gene order rearrangement and the lack of inverted repeats. overall, the size and gene content of m. hypopitys cpdna indicates that it is close to the end of plastid genome degradation process. in order to get insights into the changes in the nuclear genome associated with the transition to nonphotosynthetic lifestyle, we sequenced and assembled the transcriptome of m. hypopitys. as expected for holoparasites, we did not found transcripts for the nuclear genes encoding the components of photosynthetic machinery, including photosystem i and ii, cytochrome b f complex, and ribulose bisphosphate carboxylase. contrary to the holoparasitic plant phelipanche aegyptiaca, almost all genes of chlorophyll biosynthesis pathway from protoporphyrin ix were not found in the m. hypopitys transcriptome. this work was supported by the rsf grant - - and rfbr grant - - (mon- volr cpdna sequencing). introduction: beta-sitosterol is a substance found in plants. chemists call it a plant sterol ester. it is found in fruits, vegetables, nuts, and seeds. it is used to make medicine. beta-sitosterol is used for heart disease and high cholesterol. the federal food and drug administration (fda) allows manufacturers to claim that foods containing plant sterol esters such as beta-sitosterol are for reducing the risk of coronary heart disease (chd). centranthus longiflorus (cl) is used in alternative medicine for sleep disorders. a plant of turkish origin, cl used as folk medicine have remained uninvestigated for familial hypercholesterolemia, coronary artery disease and preventing colon cancer for their in vitro biological activity despite their use for sleep disorders. we investigated of the chemical constituents from dried aerial parts of centranthus longiflorus. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from the aerial parts of cl was analyzed using a perkin-elmer gc-ms system. results: ten compounds were obtained and identified as butanoic acid, hexadecanoic acid (palmitic acid), -methyl-z-tetradecen- -ol acetate, octadecanoic acid (stearic acid), diisobutyl phthalate, -octadecenamide, octacosane, nonacosane, alfa amyrin and beta sitosterol. the latter two were obtained in all extraction (hexane, ethyl acetate and ethanol). discussion and conclusion: all of these compounds are isolated from centranthus longiflorus for the first time. these findings may shed light on the design of new drugs, the cholesterollowering effect. p- . . - role of lutein for the high light-induced inhibition of photosystem ii related reactions in thylakoid membranes of arabidopsis thaliana, wt and lut k. dobrev, d. stanoeva, m. velichkova, a. v. popova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthetic reactions taking place in thylakoid membranes of higher plants are extremely sensitive towards different environmental stress conditions such as high and low temperature, high light intensity, uv radiation etc. carotenoids are intrinsic component of photosynthetic pigment-protein complexes and are involved in performing multiple important functions. their role of accessory pigments in absorbing sun light, participation in photoprotection via dissipation of excess absorbed light, deactivating of stress-induced reactive oxygen species and structural role are well documented and recognized. the role of lack of lutein in high light-induced alterations in structural organization and functional activity of the main pigment-protein complexes was evaluated using isolated thylakoid membranes of arabidopsis thaliana, wt and mutant lut , deficient in lutein, subjected to photoinhibitory treatment for different periods of time. alterations in photochemical activity of photosystem i and photosystem ii were determined by a clark-type electrode in the presence of exogenous electron donors and acceptors. activity of oxygenevolving complex and of the grana and stroma situated photosystem ii reaction centers was evaluated by determination of flash oxygen yields and initial oxygen burst under constant light without donors and acceptors. low-temperature ( k) fluorescence was applied for unraveling of light-induced alterations in energy transfer and interaction between the main pigment-protein complexes. maximal quantum efficiency of psii was registered by pulse amplitude modulated fluorescence method. results obtained are discussed in respect to the importance of lutein for the organization and sensitivity of photosynthetic apparatus towards high light intensity treatment. modern agriculture relies heavily on phosphate rock fertilizer to improve phosphorus availability in many soils, but this approach is not sustainable long-term. phytate (myo-inositol hexakisphosphate) is an organic phosphorus compound often present in many soils. however, phytate can not be utilized by most plants, and its accumulation in soil leads to substantial ecological problems. phytases are enzymes that hydrolyze phytate and release inorganic phosphate. many microorganisms such as bacteria and fungi synthesize highly diverse phytases which are suitable for plant biotechnology. generation of transgenic plants expressing phytases of bacterial origin has been proposed as one option to improve plant phosphorus nutrition. in this study, we generated and characterized transgenic arabidopsis thaliana plants expressing a modified phytase gene paphyc from pantoea sp. under strong camv s promoter. three individual transgenic a. thaliana lines expressing the bacterial phytase gene, as well as negative control plants harboring the camv s promoter alone were identified. expression of phytase in plants was verified at both transcription and translation levels. phytase-expressing plants grown on media with phytate as the sole source of phosphorus demonstrated better than wild-type growth rates, shoot dry mass, shoot phosphorus content, as well as higher phytase activity in cell-wall extracts. overall, we show that plants expressing bacterial phytase are capable of better growth on phytate as the only source of phosphorus in laboratory conditions. further research investigating the applicability of using bacterial phytase expression to improve plant growth in soil is necessary to evaluate the different routes of solving the phosphorus deficiency problem in agriculture. p- . . - the role of elevated temperature in photoinhibition and recovery of photosystem ii in thylakoid membranes from arabidopsis thaliana a. faik, m. gerganova, m. velitchkova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthesis of higher plants is the principle process to transform light energy into biochemical usable energy. in nature, plants are exposed to the environment where light, temperature, uv-b radiation varied and very often their extreme values that are unfavorable for effective performance of photosynthetic reactions. plant are developed various strategies to cope with stress including radical scavenging enzyme system, accumulation of protective compounds, etc. pigment-protein complexes of photosystem i and photosystem ii and their light harvesting antenna, situated within thylakoid membranes, are involved in the primary reactions of photosynthesis -absorption of light, charge separation and electron transport. photosynthetic process is sensitive towards higher than optimal temperatures, the photosystem ii and oxygen evolving complexes being extremely sensitive to elevation of temperature. in present work pam fluorescence was applied to evaluate the effect of long term action of elevated temperature ( / °c) on the quantum yield of photosystem ii, non-photochemical quenching and rdf, the latter quantifying the photosynthetic process. in addition, the activity of oxygen evolving complex was determined polarographically in the presence of exogenous electron acceptor , -benzoquinone. sds-page electrophoresis and western blot were applied to determine the damage of d -reaction center protein of photosystem ii. alterations of mutual organization within photosystem ii complex and its antenna and of energy interaction between them were followed by analysis of k steady state chlorophyll fluorescence spectra. the simultaneous application of high temperature and high light intensity resulted in a well pronounced reduction of non-photochemical quenching that restore to the initial values after recovery for days at optimal conditions. d was also restored while quantum efficiency of photosystem ii did not recuperate to initial values. p- . . - reorganization of the main pigment-protein complexes in thylakoid membranes from tomato (solanum lycopersicum) during long term exposure to elevated temperature the changes of earth climate resulted in unfavorable environment for plants. depending on the duration of influence of stress factors, the response of plants includes short and long term acclimation. the population, structure and organization of pigmentprotein complexes within thylakoid membranes are dynamic and flexible, thus providing for the acclimation of the photosynthetic apparatus to the changed environment. the main pigment-protein complexes, involved in energy transduction, are photosystem i, photosystem ii and light harvesting complexes. they are separated in grana and stroma regions of thylakoid membranes but it is well established that they can rearrange as a result of alterations of light intensity, temperature increase and decrease in order to balance the perception and utilization of excitation energy. in present work the effect of long term action of elevated temperature on organization and stoichiometry of main pigmentprotein complexes in the thylakoid membranes from tomato plants (solanum lycopersicum cv. m ) was investigated. three weeks old tomato grown at optimal conditions ( / °c day/ night temperature and light intensity lmol/m /s) plants were exposed for and days to elevated temperature at / °c. by means of blue-native electrophoresis the effect elevated temperature on the populations of psii (dimmer and monomers) and lhcii (monomers and trimers) was estimated and compared with the same parameters for control plants. the ability of plants to recover from this treatment was checked after days under optimal conditions. the changes of content of chlorophylls and carotenoids were determined at every stage of treatment. based on the results obtained it can be concluded that one of the mechanisms for regulating the energy balance and maintenance of efficient photosynthetic process involves a change in the organization and stoichiometry of the photosystem ii and oligomer state of light harvesting complex ii. aim of the study, was to evaluate the anti-tumor effects of silymarin, curcumin and propolis on leptin-induced mcf- cells. mcf- cells were incubated various concentrations leptin (physiological, obesity and pharmacologically doses; respectively , and ng/ml) . then different doses of silymarin ( , , , lm), curcumin ( , , , lm) and propolis ( . , . , . , . mg/ml) were added. after , , and hours incubation periods different area images were taken digital camera. then using dye release reagent we determined the intensity of apoptosis via colorimetric determination by elisa reader. absorbance was directly proportional the number of apoptotic cells (biocolor cell-apo percentageapoptosisassay). also, we examined the effect of these natural products on proliferation rate of leptin-induced mcf- cells for , , and hours (biovision cell proliferation kit) all experiments were carried out different days, at least times. all of three compounds were stimulate the apoptosis at all time points and all different doses of leptin. the differences was statistically significant at the level of p < . between and hours. it was found that there were not seen much cells at and hours time points. we thought that most of the cells were gone necrosis instead of apoptosis. the best effective doses on apoptosis of propolis was . mg/ml, silymarin and curcumin were lm. also, we evaluated the effects of on proliferation rate the mcf- cells, we found that only propolis was effective of inhibiting proliferation at all doses of leptin induced mcf- cells in hour. we hope this study will be a guide for the further studies in anti-cancer agent development field and show that the natural origin substances cause cancer cells apoptosis and provide targeted treatment for cancer therapy. p- . . - investigation of some lichen-derived substances' cosmetic potential for skin protection against ultraviolet b due to the depletion of the stratospheric ozone layer and chronic exposure, the occurrence of various skin diseases have been increased in recent decades. thence, people and cosmetic companies have progressively given more importance natural sunscreen products for the protection from harmful sun rays, especially ultraviolet b rays. we, therefore, isolated some lichen-derived substances; -hydroxyphysodic acid and protolichesterinic acid from hypogymnia tubulosa and cetraria aculeate, respectively. chemical characterization and identification of the isolated lichen substances were accomplished by using ftir, h-nmr and melting point analyses. the theoretical uv-vis spectra and d conformations of the isolated compounds were determined by using the gaussian software with hf theory at the b lyp/ - g level. the dark toxicities and ultraviolet b protection capacities of the substances were lighted up as previously described [ , ] on hacat human keratinocyte cell line by using mtt cell viability and ldh cellular membrane degradation assays. the obtained results from the assays showed that protolichesterinic acid has a more dark toxic activity on keratinocyte cells than hydroxyphysodic acid, and the toxic activities were found sufficient as much as % at the highest doses of the substances; lm. however, it was observed that the cytotoxicity of the substances were reduced at the rate of approximately % by the irradiation. consequently, we think that the substances block the ultraviolet b rays but their cytotoxic feature is an important limitation to their usage in cosmetic industry. references [ ] varol, m., t€ urk, a., candan, m., tay, t., koparal, a. t. ( ) photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes, phytotheraphy research : - . [ ] varol, m., tay, t., candan, m., t€ urk, a., koparal, a. t. ( ) a great effort and financial supports have been consumed to explore and design novel sun protection factors due to the unhindered increase of malignant and non-malignant skin diseases caused by the chronic exposure and depletion of the stratospheric ozone layer. the testing of naturally produced compounds seems to be the best and inexpensive way to search for potentially photoprotective substances. on the other hand, as photo-resistant species, lichens are still poorly exploited. norstictic acid was, therefore, isolated from the acetone extract of pleurosticta acetabulum. ftir, h-nmr and melting point analyses were performed to identify the chemical features of norstictic acid. gaussian software with hf theory at the b lyp/ - g level was also performed to determine the theoretical uv-vis spectrum and d conformation of the isolated compound. the dark cytotoxicity and ultraviolet b-protection capacity of norstictic acid were comparatively tested as previously described [ , ] by using mtt cell viability and ldh cellular membrane degradation assays. as a result of the experiments, it is observed that norstictic acid has a dark-cytotoxicity as less as % at the highest dose of the substance; lm. however, ultraviolet b-induced damage on human keratinocytes was blocked by the lower concentrations of norstictic acid as , and -lm, and % of cells were protected according to the control experiments of irradiated cells. consequently, we think that norstictic acid might be employed as a sun protection factor at the low concentrations, and further studies should be performed. years, researchers have focused on the lichen acids because of their biological activities. it is also suggested that lichens can be used as anticancer agents. vulpinic acid, an important lichen seconder metabolite, has antimicrobial activity and strong antimutagenic, anticancer and antioxidant capacity. nanotechnology has the potential to offer solutions to current obstacles in cancer therapies, because of its unique size ( - nm) and large surfaceto-volume ratios. so, in this study we aimed to determine the cytotoxic and proliferative effects of vulpinic acid and magnetic nanoparticles loaded with vulpinic acid (fe there are four species of wild rice known around the world. zizania aquatica l., zizania palustris l. and zizania texana hitche are found in north america, whereas zizania latifolia (griseb) turcz) is native to east asia. cwr mainly grows in the areas along the yangzi river and the huai river in china without any cultivation and domestication. cwr was an ancient grain that has been used in chinese herbal medicine to treat a variety of ailments associated with nutrition, including gastrointestinal disorders and diabetes. our previous studies have demonstrated that consuming chinese wild rice can significantly improve blood lipid profiles and ameliorate high-fat/cholesterol diet-induced insulin resistance. however, compared to the well studied common dietary white rice, active composition and the associated proteomic information of chinese wild rice have yet to be investigated. in this study, we compared and analysed the different proteins between chinese wild rice and white rice by proteomics method. our study provides insights and experimental evidence for further exploration of this ancient medical food in disease prevention and therapy. the homology between cwr and n is %, but significant differences also exist between the two. we gained new insight by analyzing the biological function of the high reliability (credibility score or higher, p < . ) peptide mass fingerprint of cwr -de electrophoresis revealed differences in protein composition between cwr and n . information obtained from the pmf indicates that glutelin precursor, caffeoyl coenzyme a (coa) omethyltransferase and putative bithoraxoid-like protein can provide gene evidences for its biological function. p- . . - mir and growth-regulating factors interaction during maize leaf growth under low-temperature stress g. aktug, f. aydinoglu gebze technical university, kocaeli, turkey microrna (mirna) genes are a class of non-coding small rnas about nucleotide-long which are revealed as regulators of plant growth and stress responses. the mirna mir targets and regulates growth-regulating factors (grfs) which are plant specific transcription factors family and this regulation machinery is conserved among plant species. plant growth is a result of cell division and expansion which took place as spatial gradient zones throughout maize leaf which are meristem, elongation and mature zones. cells proliferate in meristem, migrate to elongation zone and finally reach to mature zone to get its final size. it has been shown that mir affects cell division by regulating grfs and changes leaf size which are determined by cell number and cell size of leaf. this study aims to investigate the role of mir and grfs interaction during maize leaf growth under low-temperature stress. maize seedlings were grown under low-night temperature for stress treatment to generate growth retardation and control conditions as well to make comparative analysis. length of the third and fourth leaves of seedlings was measured every day and leaf elongation rate was calculated to observe stress effects on the leaves. growth zones of fourth leaves were harvested during steady-state growth phase for determining expression level of mir s and their target by q-rt-pcr. we mined mir genes sharing sequence similarity and grf targets. the expression analyses of mir s and grf are proceeding for different growth zones. in conclusion, this is the first study investigating the regulation network between mir s and grf in different developmental stages of maize leaf under low-temperature stress. oeigpd cdna was isolated from a cdna library we constructed from olive pedicels. homology searches for nucleotide, amino acids and alternative open reading frames were conducted utilizing blastn, blastp, and blastx, respectively. nucleotide sequences of homologous genes from other plants were aligned using bioedit and the number of snps were detected. the alignment was then used to generate a phylogenetic tree using mega program. another alignment with amino acid sequences of the homologues proteins was also generated to construct a phylogenetic tree displaying oeigpd's position among other plants. various aspects of oeigpd including amino acid composition, hydropathy analysis, isoelectric point (pi) and three dimentional structure of the protein were determined using online software at expasy. multiple primer pairs to amplify the full length open reading frame of the gene, to clone the gene into the expressing vector plate , and to detect expression through real-time pcr were designed using primer . amino acid composition analysis revealed that oeigpd contained serine, arginine and isoleucine predominantly while hydropathy analysis suggested it was an hydrophilic protein. isoelectric point (pi) of the protein was calculated as . . the molecular weight of the protein was calculated as kda. analyses continue to determine the polymorphism of oeigpd among olive cultivars, and biochemical function of the gene in olive. p- . . - cytotoxic effect of fractionated triterpenoid glycosides from holothuria polii in human cancer cells sea cucumbers are the members of class holothuroidea and they have more than described living species all around the world. sea cucumbers secrete special secondary metabolites from their body walls and they are called triterpene glycosides (tggs). in this study, cytotoxic activity of fractionated ttgs from h. polii on different cancer cell lines were carried out. h. polii delle chiaje, samples were collected from dikili-_ izmir. the semipurified extracts were fractioned by using hplc. four different fractions (fraction a-d) were obtained. in order to characterize the fractions, maldi-tof/ms was used. the cytotoxic activity of the fraction a-d were tested on ht- , t and upci-scc- cell lines by using xcelligence rtca sp system. the cells were treated with three different concentrations of the fractions for hours. the cell index data were compared with the control group. ic values of the fractions for three cell lines were calculated. according to the results, the fractions have holothurin a ( . m/z), -dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer ( . m/z). the fraction d was the most effective on all cell lines with ic value of . ae . mg/l, . ae . mg/l and . ae . mg/l for ht- , upci-scc- and t , respectively. in conclusion, sea cucumber ttgs are promising agents for colon adenocarcinoma, oral squamous cell carcinoma and colorectal carcinoma (metastatic) treatment. p- . . - effect of horse-chestnut (aesculus hippocastanum) seed extract on matrix metalloproteinases during diabetic wound healing impaired wound-healing in diabetics is a major public health problem. the expression and activation of matrix metalloproteinases (mmps) are also impaired in diabetic wounds according to previous studies. their main function is to degrade the various components of the extracellular matrix. also, they participate physiological processes such as inflammation, angiogenesis, tissue remodeling. horse-chestnut seeds (hc) are rich in saponins and flavonoids. it has been shown that hc has antiinflammatory, antioedema, vessel protective, and free radical scavenging properties. the aim of this study is to determine with molecular signs on cutaneous wound healing effects of the ethanol ( %) extract of hc (hce) seed in rats by excision wound model. this study was conducted on diabetic wistar albino rats, which were injected by a single dose ( mg/kg i.p.) streptozotocin. diabetic treatment rats were applied topically % (w/w) ointment with hce and control rats were applied topically simple ointment, once a day during the experimental period. the gene expression levels of mmp- , mmp- by qpcr and levels of nitric oxide (no), hydroxyproline and malondialdehyde in wound tissue investigated at the end of rd, th, and th days. wound closure was also measured. the hydroxyproline and no levels were significantly increased in the hce treated group versus control after the rd and th days. the malondialdehyde levels were significantly lower in the treatment group. mmp gene (associated with collagen processing and reepithelialization) expression levels in hce treated rats were increased in the th day while it was reduced in th day. mmp gene (associated with inflammation and gelatinase) expression levels in hce treated rats were decreased in th, and th days compared to the control. these findings indicate that hce accelerated the cutaneous wound healing process in diabetic rats via mmp and mmp regulation. p- . . - isolation and molecular molecular characterization of vps /vam from olive b. celikkaya, e. dundar molecular biology and genetic at balikesir university, balikesir, turkey vps /vam promotes aggregating and fusing of endosomes and lysosomes. it is a component of a protein complex that is found in vacuole membranes. this gene has been studied from various organisms including humans, drosophila, arabidopsis and rice. no studies on olive vps /vam , however, have been reported. the aim of this study is to report information of olive vps /vam including expression pattern and biochemical characterization. vps /vam was isolated from a cdna library we constructed from fruited olive leaves in july. to determine the putative name of the cdna, blast analyses were conducted for nucleotide, open reading frame and amino acid sequence comparisons. bioedit program was used to determine the nucleotide and amino acid composition along with its molecular weight and isoelectric point (pi). hydropathy analysis was conducted using kyte and doolittle program. phylogenetic analysis was done using mega . cellular localization of the product was predicted using sosui gramn. the three dimentional structure of the protein was calculated using i-tasser and compared to previously known structures using cn d. the blast and bioedit analyses revealed vps /vam had base pairs coding amino acids with a molecular weight of . kda, and pi of . . the at/gc ratio was very high ( . ) comparing to its homologs from other plants suggesting to expect significant differences of this gene's function from the others. amino acid composition analysis revealed high rates of serine, leusine and isoleucine indicating a hydrophobic property of the protein. the hydrophobic feature was confirmed by kyte and doolittle analysis while the cellular location was revealed to be extracellular. the hydrophobic nature despite extracellular location suggests it is a membrane associated protein which was confirmed by transmembrane domain analysis. as expected no signal peptide was detected. the d structure of the protein was similar to its previously reported homologs. p- . . - isolation and characterization of the ribosomal l protein from olive s. cinarli, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey despite as a ribosomal protein, l is known as the inhibitor of the cellular aging gene and it has been reported to have roles in apoptosis. the ribosomal l protein is larger than the lsu of ribosome and contains domains as -layered alpha/beta domain and -layered alpha/beta domain. in ribosomes, it functions in translocation and orientation of trnas. although the ribosomal l (rl ) gene has been studied in many plants, reports on olive rl (oerl ) are very rare. this study presents molecular characterization of rl gene from olive. oerl was isolated from a cdna library we constructed from unfruited olive leaves in july. homology analyses were conducted using blast programs. nucleotide and amino acid compositions, molecular weight, isoelectric point (pi) and at/gc ratio were determined using bioedit and expasy programs. cellular location of the l protein was determined using sosui-gramn program. signal peptide detection, transmembrane domain detection, three dimensional ( d) structure analysis, and phylogenetic analysis were conducted using signalp . , thhmm, i-tasser/cn d and mega , respectively. oel was found to have an open reading frame of base pairs coding amino acids that constitutes a molecular weight of . kda and a high pi of . . lysine, leucine and valine had higher rates. the hydrophilic nature suggested by kyte and doolittle analysis despite high rates of leucine and valine suggests an amphipathic nature of the protein that can bind to both hydrophilic and hydrophobic proteins and / or function in both media. a . at/gc rate is significant comparing to that of its homologs from other plants. sitoplasmic location predicted by sosui-grann is in agreement with the hypothesis suggesting an amphyphatic nature for oerl . likewise, no signal peptide was detected and it was predicted to have at least one transmembrane domain. further characterization of oerl with respect to expression pattern and biochemical function continues. p- . . - isolation and characterization of an olive splicing factor b subunit m. nurcin, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey splicing factor b subunit (sf b ) functions in the regulation of translation and gene expression. sf b forms u small nuclear ribonucleoprotein complex (u snrnp). splicing factors a and b binds pre-mrna at the site of the intron branching point. this binding joins u snrnp to pre-mrna. although sf b from various plants have been widely studied, no studies on olive sf b (oesf b ) have been reported. this study reports information on various aspects of oesf b . oesf b was obtained from a cdna library we constructed from fruited olive leaves in december. it was putatively identified as a splicing factor using blastn, blastp and blastx. to determine wheter oesfb was a sitoplasmic protein, sosui gramn was used. tmhmm was used to detect any transmembrane domains while signal peptide analysis was conducted by signalp. i-tasser and cn d were used to generate the calculated d structure and to compare it with experimentally generated models, respectively. nucleotide and amino acid compositions along with the calculated molecular weight and isoelectric point (pi) were analyzed using bioedit and online expasy software. the phylogenetic trees revealed genetic relationship of olive among other plants based on oesfb . the orf contained nucleotides coding amino acids that produce a . kda peptide with a pi of . . alanine, valine and leucine were found at high ratios suggesting a hydrophobicity which was also predicted by kyte and doolittle analysis. the at rich property of oesf b is not unusual comparing to most plant genes. cellular localization of the gene was suggested to be in mitochondria with no signal peptide indicating oesf b could be synthesizing in mitochondria. the predicted d structure of oesf b was similar to experimentally produced structures while some hydrophobic pockets were predicted. further characterization of the gene with respect to temporal and spatial expression pattern and biochemical function continues. p- . . - kafirin profile of turkish originated sorghum populations r. temizg€ ul, s. yilmaz, m. kaplan, t. akar department of biology, faculty of science, erciyes university, kayseri, turkey sorghum bicolor l. is the fifth important crop in the world with its high photosynthetic activity and resistance to unfavourable conditions as high temperature, drought, salt, and ph changes. sorghum has attracted great interest due to its intensive usage both as human and animal nutrition, and contribution to resistance against many diseases. some proteins of sorghum exerts reducing effect on nutrient digestion through making connetions with other proteins and/or carbohydrates. kafirin proteins have the highest proportion in grain with a range of - %. they are grouped into a ( - kda), b ( kda), c ( kda) and d ( kda) subunits depending on molecular weight, solubility and structure. in the current study, kafirin proteins from turkey originated sorghum populations were acquired through sequential extraction; first, non-prolamines were removed through application of % naci concentration, and second, kafirins were obtained using tertiary butanol ( %) and reducing agents. sds-page was conducted for seperating and visualising the subunits of kafirins. the a, b, c, and d subunits of populations were respectively estimated as , , , and %. of the total proteins, % was identified as a, % b, % c, % d, and % non-prolamines. non-prolamin group of proteins were visualised as different bands ranging from . to kda. c and b group of proteins were only viewed when treated with reducing agents as -me and dtt suggesting that they are connected with complex cross-links. however, a group of proteins visualized without using these agents due to not having intra molecular disulphide bridges and inter molecular cross-links. non prolamins, except for . , . , . , . and . kda, were able visualised in the presence of reducing agents. transcriptomic analysis of the genes encoding analysed proteins needs to be elucidated for better understanding of the genetic diversity and biochemical characteristics of sorghum. p- . . - untargeted metabolomic profiling of romanian and uk tomatoes varieties by high performance liquid chromatography coupled with mass spectrometry c. socaciu , university of agricultural sciences and veterinary medicine, cluj-napoca, center for applied biotechnology ccd-biodiatech at proplanta ltd, cluj-napoca, romania tomato flesh is a rich source of many phytochemicals of high nutritional value, including a large variety of carotenoid derivatives with health promoting properties. metabolomics became the most adequate technology for an accurate chemotaxonomic classification and discriminations between different varieties, based on untargeted profiling or targeted, quantitative analysis. different varieties of tomatoes (b-carotene-rich, lycopene-rich, ketocarotenoid-rich) cultivated in romania and uk were comparatively studied using enriched fractions obtained by a preliminary fractionation of the whole pulp homogenate. two methods were applied for carotenoid extraction: a mixture of hexane/ethanol ( ) and chloroform/methanol ( ) . the dried extracts were dissolved in ethyl acetate and analyzed by uv-vis spectrometry and hplc-esi(+)qtof-ms (bruker gmbh). the base-peak chromatograms were processed by specific biostatistics software (data analysis and profile analysis) and the molecular identification were determined by comparison with the data base lipidomics gateway (www.lipidmaps.org). the content of carotenoids were significantly higher using extraction ( ) , ranging from . to . mg/ g. the major carotenoid derivatives, were represented by lycopene, hidroxy-lycopene, all-trans or cis-beta-carotene, echinenone, all-trans retinyl palmitate, but also sterols, phospholipids, di/tri glycerides and ceramides. the romanian varieties were more rich in polar carotenoids and lipids, in general, while the uk tomatoes proved to be enriched in non-polar derivatives, especially esterified carotenoids, keto-carotenoids and glycerides. new molecules were identified, as good discriminatory markers of each tomato variety. acknowledgements. this work was supported by a grant of the romanian national authority for scientific research and innovation, cccdi -uefiscdi, project nr. / , pncdi . glycogen is a multi-branched polysaccharide that serves as the main form of glucose storage in the body, where the main reserves are in the liver and muscle. it has been observed that glycogen metabolism is altered in many tumor types, and that glycogen content is inversely correlated with proliferation rate. in addition, it has recently been described that when glycogen accumulation is forced in glioblastoma u cells in hypoxia, senescence is induced and tumor growth is inhibited in vivo. our laboratory has various animal models with different parts of the glycogen metabolism pathway affected. most notably, we have two animal models lacking glycogen: muscle glycogen synthase (gys ko) and liver glycogen synthase (gys ko) knockout animals. we isolated mouse embryonic fibroblasts (mefs) from gys ko to perform replicative senescence assays. in addition, we induced hepatocellular carcinomas in gys ko animals via n-nitrosodiethylamine (den) injections in order to track tumorigenesis in animals lacking hepatic glycogen. lastly, we performed partial hepatectomies (phx), which involves the resection of two thirds of the liver, on gys kos to evaluate the effect of the lack of glycogen on hepatocyte proliferation. interestingly, we have observed that glycogen levels are increased in human and mouse fibroblasts under replicative senescence, and that mefs depleted of glycogen bypass senescence and immortalize faster than wts. we have also demonstrated that senescence pathways are down regulated in mefs lacking glycogen. furthermore, gys kos treated with den show higher tumor burden and mortality than controls. we also evaluated the effect of glycogen on hepatocyte proliferation after phx. gys ko mice present faster proliferation and liver regeneration rates, when compared to wt counterparts. collectively, our preliminary data suggest that glycogen metabolism plays a crucial role in the regulation of cell cycle in both physiological and pathological states. it is established that pineal is involved in circadian regulation of testosterone secretion from leydig cells. however, the precise routes of this regulatory involvement are still unknown. as cgmp has been also regarded as modulator of steroidogenesis we sought to study the effects of pineal removal on the circadian pattern of cgmp variations and expression of the genes that encode elements of no-cgmp signaling pathway in adult rat leydig cells. the analysis was performed on testicular leydig cells obtained from pinealectomised and shame pinealectomized rats, in six time point during hours. the pinealectomy was confirmed by serum melatonin eia measurement. the androgen levels were measured by ria; cgmp by eia and gene expression was quantified by rq-pcr. all results were analyzed by cosinor method. data revealed circadian transcriptional pattern of nos , nos (genes encoded no producers) and pde a (gene for cgmp remover) in leydig cells from adult rats. pinealectomy significantly increased expression of nos which lost rhythm and increased and delayed amplitude of nos expression. further, pinealectomy initiated cyclic transcription of gucy b and noncyclic transcription of gucy a (genes encoded cgmp producers) and increased mesor and amplitude of pde transcription. the transcription of prkg , the main effector in this signaling pathway was not affected with pineal abolition. additionally, pinealectomy did not influence the circadian transcription profile of coxi or other investigated genes (coxi , nrf , nrf a, pgc a) related to mitochondrial function and biogenesis. finally pinealectomy reversed phase of circadian cgmp oscillation in leydig cells, increased amplitude and slightly advanced peak of serum testosterone oscillation. results suggested pineal influence on circadian rhythm of no-cgmp signaling in leydig cells. further studies based on these data are needed to better understand the relationship between pineal and circadian rhythm of testosterone production. influenza is a contagious respiratory infection caused by a variety of influenza viruses. neuraminidase inhibitors is a new class of antiviral drugs that inhibit influenza viruses. the most popular antiviral agents is oseltamivir, having a commercial name of tamiflu, within anti-influenza antivirals. as well as tamiflu is a member of neuraminidase inhibitor group drug. therefore, this study was performed to determine the effect of tamiflu on cultured human peripheral blood lymphocytes. material and methods: for examining the presence of the indirect mutagenic effect of oseltamivir in iver s fraction mix was used. cells were treated with . , and lg/ml oseltamivir, the tamiflu capsule ingradient, for or hours in the absence or presence of an exogenous metabolic activation system. the test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. on the other hand, some concentrations of tamiflu induced sce and also decreased significantly the proliferation index (p ˂ . ) in the absence of s mix. result: tamiflu did not induce significant increases of ca or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but week sce induction was observed. on the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the s mix. discuss and conclusion: tamiflu weakly induced sce at the highest concentration with/without added s mix in cultured human peripheral lympocytes. it could be assumed to be a sce inducer. sces can be increased by several agents that attack dna. tamiflu decreased the proliferation index and nuclear division index at some concentrations thus interferring it as being weakly cytotoxic, though this effect disappeared in the presence of s mix applications. this finding is important for showing the inefficiency of tamiflu metabolites on the cell cycle. introduction: chronic renal failure as a result of the progression of diabetic nephropathy is the main cause of mortality in patients with type diabetes. chronic hemodialysis is a life-saving therapy for patients with strong renal disorders. the main goal of hemodialysis is toxins removal from the patient. the monitoring of hemodialysis is the best way for biomedical evaluation of correctness and efficiency of this clinical treatment. according to the published data, the markers of development of diabetes complicated with renal failure are increased levels of glucose, urea and creatinine in the patient blood. today colorimetric and spectrometric methods are most commonly used for determination of the above metabolites in biological samples. however, these methods are complex in application, have low selectivity, and require pretreatment of samples. materials and methods: we propose for levels of glucose, urea and creatinine detection the potentiometric multibiosensor based on ph-sensitive field-effect transistors and immobilized enzymes developed in our laboratory. results: we developed a potentiometric multibiosensor and studied its main analytical characteristics. linear dynamic ranges of determination of substrates were following: . - mm of glucose, . - mm of urea, and . - mm of creatinine. it was shown that the potentiometric multibiosensor had good reproducibility, and its bioselective elements were working independently from each other, because test of substrates cross-selectivity was negative. discussion and conclusion: very sensitive, fast and selective multibiosensor for simultaneous measurement of three metabolites in a single cycle based on ph-sensitive field-effect transistors and immobilized enzymes is developed. the developed potentiometric multibiosensor was verified by quantitative analysis of glucose, urea and creatinine in blood serum of patients with diabetic nephropathy. p- . . - ph-dependent interaction of asymmetrically charged peptides with a protein nanopore over the past two decades, the ability to use natural or artificial nanopores to probe at uni-molecular level the structural and kinetic features of various bio-molecules (peptides, dna, rna) was successfully achieved. the operating principles of the nanopore-based single-molecule technique are simple: the single macromolecule capture, entry and subsequent translocations through a free-standing, voltage-biased nanopore, depend upon the physico-chemical and topological features of the analyte. the concentration, identity volume and charge of the analyte are then deduced from the analysis of the stochastic current blockade events caused by the trafficked analyte across the nanopore. herein, we used the a-hemolysin (a-hl) nanopore and set up an experimental model providing efficient control of a-hl-peptide interactions, in the presence of a ph gradient across the nanopore. for this, we engineered a amino acids long peptide containing a neutral asparagines-containing sequence, flanked by oppositely charged aminoacid patches at the n-(glutamic acids) and c-termini (arginines), whose length was set as to span a single a-hl protein. when the ph of the solution in contact to the a-hl's b-barrel opening is changed from neutral to acidic values, the electrostatic interactions between the protein's mouth and either the n-or c-terminus end of the peptide occurs, and this influences strongly the dynamics of a peptide translocating the nanopore. we further proved that during the same experiment, peptide entry into the nanopore can be set to occur with either n-or c-terminus end head on, by simply changing the sign of the transmembrane potential across the nanopore. nanopores are emerging as a powerful and broadly applicable tool in biophysics, which allows one to study the features of charged macromolecules under confinement. a few noteworthy examples are: determining the electrophoretic mobility, effective charge and diffusion coefficients of charged molecules; exploring the folding and unfolding of peptides and proteins; analyzing biopolymers trafficking, protein transport, dna translocation, rna and dna sensing and sequencing. herein, we employ single molecule analysis techniques using a wild-type ahemolysin (a-hl) protein nanopore to study the capture and translocation behavior of a short cationic peptide ( amino acids in length) at an extremely low ph value. our experiments revealed that an effective absorbing field is created by the electroosmotic flow, against the electrophoretic force, which enables the peptide capture inside the nanopore. furthermore, our findings show that the trajectory of a single peptide can be experimentally visualized and the main steps determined: the peptide capture, reversible translocation across the pore's vestibule and lumen regions, and the peptide release from the nanopore. also, the kinetic analysis of the main steps observed allowed us to describe the free energy profile of the peptide interactions with the protein nanopore. the presented work provides evidence for the ability of controlling the dynamics of a single-peptide, its capture and passage inside a a-hl nanopore, that underlie the processes naturally occurring in cells, thus proving a powerful approach for probing single molecule biophysics phenomena, in general. changes in the physical conditions of the cancer microenvironment driven by elevated tissue growth and angiogenesis, may introduce exposure of laminar fluid flow, which effect the key factors of cancer, such as progression, immune-escaping and metastasis. conventional experimental models fail to mimic the physical cues on tumor microenvironment. microfluidic culture techniques allow precise control of fluids, simultaneous manipulation and analysis of cultured cancer cells. here, we present a platform that can be used for the investigation of the role of flow mediated mechanical stimuli on cancer cells. microfluidic cell culture platform was fabricated using polymethyl methacrylate and double-sided adhesive films with mm dimensions. ovary adenocarcinoma cells (efo- and onco-dg- ) were used for the optimization of the platform. to understand the fluid and gas distribution patterns, specific modeling was performed. dynamic microfluidic cell culture and static conventional cell culture conditions were compared for the differences of cancer cell phenotype, such as proliferation, viability, epithelial-mesenchymal transition. we confirmed that, the proliferation and viability of cancer cells are increasing under dynamic fluid flow. the proliferation rate of ovary adenocarcinoma cells was correlated with the increase of fluid flow rate. immunocytochemical analysis showed that fluid flow causes decrease in e-cadherin expression, and increase in n-cadherin and vimentin expressions, which indicate mesenchymal phenotype of cancer cells. our results showed that, cancer cells present different characteristics due to fluid flow of tumor microenvironment. to understand the role of physical dynamics by using microfluidic culture techniques, is a key to elucidate the mechanisms underlying disease progression, and may lead to new diagnostics and therapeutic approaches. (this study was funded by turkish scientific and technical research council (tubitak- s ). high-sensitive detection of low-affinity antibodies by immuno-pcr with supramolecular olygonucleotide-streptavidin complex detection of low affinity antibodies in blood sera and cell surface outwashes is important both in the study of molecules that bind to cellular receptors (circulating tumor cell masking antibody, for example) and medicine (diagnosis of allergy). low affinity igm and ige antibodies can not sometimes be determined by conventional methods. we using supramolecular oligonucleotide-streptavidin complex formed from single-stranded synthetic oligonucleotide ( n) contains biotin on '-and '-ends, and sterptavidin in molar ratio : . this complex represents a structure with equivalent electrophoretic mobility of bp dna and preferred "valency" of streptavidin is . this universal immuno-pcr approach make it possible to increase a signal by using several oligonucleotides per one antibody. after the method optimization we achieved - times highter sensitivity than elisa. to reduce the matrix effect we used - fold dilutions of sera samples. this approach achieved a significant advantage, because it allows working with small-volume samples (need only mkl of serum sample). antibodies to the disaccharide galb - glcnac (le c ) are typical of the natural antibodies. the igm anti-le c antibodies are found in almost healthy people without the epitope specificity variation. we have shown that the concentration of igm anti-le c antibodies was higher (p ≤ . ) for health donor sera (n = ; . ae . pg/ml) compared with sera from patients with breast cancer (n = ; . ae . pg/ml). sensitivity of igm anti-le c antibodies detection was pg/sample ( mkl) ie . molecules. thus for the immuno-pcr detection of antibodies the - tumor cells are sufficient. such amount of cells seems to be a realistic one for detection of antibodies masking circulating tumor cells. this study was supported by a grant from russian science foundation (# - - ) and by russian federation president scholarships donated to d. yu. riazantsev (# sp . . bacterial pathogen detection and identification is of crucial importance for disease diagnosis, bacterial contamination surveys and water quality assessment. we propose herein a novel method for bacterial detection based on the interaction of single gram-negative bacterial cells (i.e.: escherichia coli and pseudomonas aeruginosa) with an ahemolysin (a-hl) protein nanopore embedded in a reconstituted lipid bilayer, at neutral ph. as a consequence of an applied voltage, the negatively charged bacteria suspended in saline buffer solution are electrophoretically driven towards the pore opening, inducing reversible blockages in the ionic current through a-hl. experiments were also performed in the presence of an antimicrobial peptide, cma , as well as in acidic environment. statistical analysis of the frequency and duration of blockage events allowed us to discriminate between the two types of bacteria. the frequency of interactions was higher for escherichia coli with respect to pseudomonas aeruginosa. adsorption of cma peptides on the membrane of bacteria increased the frequency of interactions with the pore, contrary to the expected effect induced by lowering the net surface charge of the cells. in experiments performed at ph = , the frequency of blockage events was found to be two orders of magnitude higher, with longer interaction life-times. the net negative charge ( uncompensated aspartate residues) localized at the entrance of the pore contributes an additional electrostatic repulsion interaction between negatively charged bacterial cells and a-hl. thus, adsorption of cationic peptides at the interface will reduce this repulsive interaction. the same effect was recorded at ph = , when the aspartate residues are partially protonated, confirming our understanding of the previously observed results. this method could be further developed and integrated with other techniques, making nanopore-based systems a fast and reliable bacterial detection and identification tool. this study was performed to analyze the effects of tunicamycin (tm) and taurohyodeoxycholic acid (tudca) on thle- cells. cells were treated with tm to induce endoplasmic reticulum (er) stress and tudca was administered as an er stress inhibitor. cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations of either tudca, tm or both. thle cells were cultured in fibronectin, bovine collagen i and bovine serum albumin coated plates. cell lines were grown in begm media supplemented with epidermal growth factor, phosphoethanolamine, fetal bovine serum, u of penicillinstreptomycin and maintained in a humidified incubator at °c and a % co atmosphere. cell viability was measured using the colorimetric -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay kit. cells were grown to confluence in well plates and incubated with ll/ml dmso, - lg/ml tm, . - mm tudca, or lg/ml tm + . - mm tudca for - hours. control cells were prepared in plates containing only medium. at the end of the incubation period, mtt was added to each well and incubation was carried out for hours at °c. formazan production was expressed as a percentage of the values obtained from control cells. at all hours of incubation neither dmso or mm tudca was cytotoxic. at and hours incubations mm tudca and lg/ml tm + mm tudca were significantly cytotoxic compared to control, dmso and mm tudca groups. treatment of cells with . mm tudca hours before administrating ug/ml tm significantly decreased the cytotoxic effect of tm. we conclude that tudca may show cytotoxic effects at mm concentration when treated with tm. therefore . mm of tudca, administered hours before tm treatment should be applied to protect against er stress. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; s ). recent studies reveals that history of preeclampsia is an independent risk factor for cardiac events and stroke. lipoprotein-associated phospholipase a (lp-pla ) is a vascular inflammatory marker associated with cardiovascular diseases (cvd). we hypothesize that vascular inflammation (lp-pla mass, activity, index) related genetic variations (pla g ) increase the risk for developing future cardiovascular disease in women with pe. a group of preeclamptic patients and normal pregnant women were recruited from university of istanbul, cerrahpasa medical school, department of gynecology and obstetrics included into the study. the control group was matched for maternal and gestational age at time point of sampling. preeclamptic patients were starified into two groups; early-onset and late-onset according to the gestational weeks. enzyme-linked immunosorbent assay procedure was used to determine the serum lp-pla mass level. lp-pla activity were determined by kinetic method. plag snp genotyping performed by using the sequenom massarray iplex. the rs tt genotype had a higher lp-pla index (p = . ) for early onset preeclampsia, cc genotype had a higher lp-pla mass and lp-pla index for late onset preeclampsia. no difference were found for control. the rs gg genotype had higher lp-pla mass and index for late onset preeclampsia (p = . , p = . respectively). stepwise logistic regression analysis performed to identify cardiovascular disease related variables that independently and significantly contributed to the presence of alleles of rs and rs snps in early, late onset preeclampsia and control group. only lp-pla mass was independently and significantly associated with both snps in early onset preeclampsia. the association between lp-pla mass, index and rs , rs snps might be useful genetic markers to adress future cvd risk in patients with preeclampsia. introduction: b-thalassemia is one of the most monogenic autosomal recessive disorder characterized by defective production of the b-chain of hemoglobin. definition of the b-globin genotype is necessary for genetic counselling in the carriers, and for predicting prognosis and management options in the patients with thalassemia. dna-based diagnosis of b-thalassemias routinely relies on polymerase chain reaction (pcr) and gel electrophoresis. the aim of this study is to develop a new procedure, a dna-based piezoelectric biosensor, for the detection of b-thalassemia ivsi- mutation, the most common b-thalassemia mutation in turkey. materials and methods: b-globin gene of genomic dna isolated from whole blood, was amplified by pcr. bioactive layer was constituted by binding -hidroxymetacrilate metacriloamidoscystein (hema-mac) nanoploymers on the gold electrode's surface. single oligonucleotide probes specific for ivsi- mutation of b-thalassemia were attached to the nanopolymer via reactive cross-linker glutaraldehyde. the measurements were executed by piezoelectric resonance frequency which is caused by binding of pcr products in media with single oligonucleotide probe on the electrode surface. the results were confirmed by the conventional molecular method as arms. results: the piezoelectric resonance frequencies obtained by hybridization of the pcr products on bioactive layer were found ae , ae , and ae hz for the samples of normal b-globin, heterozygote, and homozygote of ivsi- mutation, respectively. discuss and conclusion: the developed biosensor serves as a specific result to ivsi- mutation. it could accurately discriminate between normal and ivsi- mutation samples. because of low costs, fast results, specificity and high detection/information effectiveness as compared with conventional methods, we can be offered this techique as an alternative to conventional molecular methods. the increasing use of nano-sized materials in the last several years has compelled the scientific community to investigate the potential hazards of these unique and useful materials. one of the most widely used nanoparticles is titanium dioxide. the objective of the research is to investigate the alterations in molecular and cellular responses in culture of primary lymphocytes to tio nps. human lymphocytes isolated from heparinized blood of healthy individuals were exposed to tio nanoparticles. viability, ros generation, the changes in the expression of genes encoding proinflammatory mediators tnf-a, il- b and il- and dna damage were assessed. human lymphocytes were incubated with nanoparticles of different concentrations and viability was determined in and hours after treatment, respectively. cell viability was decreased by a treatment with nanoparticles in both a time-and concentration-dependent manners. the ability of tio to induce ros formation in lymphocytes was evaluated using dcf fluorescence as a reporter of oxidant production. the fluorescence intensity of oxidized dcf was increased in cells treated with nps. this means that ros generation occurred in response to the treatment with tio . to investigate the expression level of mrna related to the inflammation responses in human lymphocytes real-time pcr was performed. the expression of il- b, il- and tnf-a genes were increased by the exposure to nanoparticles of , and lg/ml for - hours. tio nanoparticles were shown to induce the dose-dependent fragmentation of dna strands. much evidence of hazardous health effects of nps has been reported. in this study, viability was reduced under the exposure to tio . oxidative stress was elevated by the treatment with tio nps. oxidative stress can also trigger inflammation signals. induced by exposure to nanoparticles they may cause the translocation to the nucleus of transcription factors, which regulate proinflammatory genes, such as tnf-a, il- b, il- . background: endothelial cells (ec) represent one of the primary targets of the major pro-inflammatory cytokinetumor necrosis factor (tnf). development of the new approaches for the treatment of acute and chronic inflammatory conditions, including the strategies aimed to tnf neutralization, requires the usage of the adequate cellular models closely resembling the properties of the endothelium. the endothelium-derived ea.hy cell line expresses several inflammation and neoangiogenesis markers in response to activation factors however their expression can differ from the patterns demonstrated by primary ec. the aim of the current study was to compare the expression of the known endothelial cellular markers including receptor of vascular endothelial growth factor- (vegfr ) and a v b -integrin on d and d cultures (spheroids) of ea.hy . methods: the ea.hy cell line was used with permission from dr. edgell. the cells were cultivated in the presence of tnf ( ng/ml) or vegf a ( ng/ml) for hours. mrna was isolated using rneasy kit from qiagen and reverse-transcribed with revertaid kit (fermentas). rt-pcr was performed with specific primers. expression of vegfr and a v b -integrin was visualized by confocal microscopy using specific monoclonal antibodies and previously developed fluorescent hybrid proteins. results: the expression of a v b -integrin and vegfr- increased on the d culture compared to d according to confocal microscopy and rt-pcr. the aforecited methods revealed elevated expression of a v b -integrin in the d culture of the ea.hy cell line activated with tnf. also increased expression of vegfr in the d culture activated with vegf a. then by confocal microscopy, we analyzed our fluorescent hybrid proteins that bind a v b -integrin and vegfr on the surface of d and d cultures as well as antibodies with fluorescent label. conclusions: d cultures of the ea.hy cell line represent a promising model for the inflammation studies. tumor necrosis factor (tnf) is a trimeric cytokine associated with the inflammatory response to tissue injury and found to possess a key role in rheumatoid arthritis pathogenesis. spd is a highly toxic recently discovered tnf inhibitor that promotes trimer dissociation and lead to the inactivation of the protein. according to the traditional anti-tnf treatment of ra, we aim at extracellular inhibition of this pro inflammatory cytokine as an effective therapy. the project plan comprises design, synthesis and validation of candidate inhibitors (measurement of dissociation constant and aqueous solubility). because of the elevated percentage of insoluble compounds a solubility enhancement protocol has been developed. the experimental procedure was the following:: a. drug design. identification of novel drug compounds are based on two approaches: i) structure based drug design using the d structure of tnf and ii) design of more potent and less toxic spd analogues. b. drug synthesis. a series of spd analogues were in house synthesized while novel candidates discovered by in silico approaches were commercially available. the purity of the majority of the compounds exceeded %. c. solubility measurement and enhancement. samples were incubated under specific conditions that can enhance aqueous solubility and solubility measurement with a direct uv method pursued. d. measurement of the dissociation constant. a fluorescence binding assay was used in order to evaluate the inhibitory activity of the compounds. from our results it can be concluded that dmso, peg and b-cyclodextrin can be used for solubility enhancement without interfering with fluorescence assay. however peg -in contrast to dmso-is not suitable for isothermic titration calorimetry measurements. dissolution procedure also plays a crucial role in the levels of solubility reached. finally, it has been shown that some of the studied spd derivatives have better dissociation constants than spd . the effect of exercises on serum bmp- levels of knee osteoarthritis cytokines. more complicated approaches are expected to focus on molecular proteins as bone morphogenetic proteins (bmps) of the transforming growth factor (tgf)-beta superfamily. bmps associated with many cellular functions, such as proliferation, differentiation, and apoptosis. bmp- is significantly important for the endochondral bone formation. inflamation can induced serum bmp levels in oa patients. the aim of this study is to evaluate the clinical findings of oa patients after the isokinetic exercise together with the serum levels of bmp- to sustain the molecular approaches for treatments. a total of patients were included in this study. the groups are formed as follows: group , oa patients before the exercise; group , oa patients after the exercise; group , oa patients before the isokinetic exercise; group , oa patients after the isokinetic exercise clinical and biochemical findings were evaluated before and after weeks of the exercise programme. self reported severity of pain was measured using the mm visual analog scale (vas), womac scores were calculated and isokinetic knee muscle strength testing was measured using cybex dynamometer that a standardized protocol previously described was applied in a subject-specific range of motion. serum bmp- levels of all patients were studied by elisa method. results represented a better vas and womac scores for all exercise groups after treatment. the serum bmp- levels were significantly decreased in group compared to group ( . ae . ; . ae . respectively, p < . ) and in group compared to group ( . ae . ; . ae . respectively, p < . ). there is not any statistically differences between group and group (p > . ). as a conclusion, the decreased serum levels of bmp- may be suggested as a biochemical marker for oa patients during exercise programmes. p- . . - tnf-a blokade efficiently reduced severe intestinal damage in necrotizing enterocolitis c. tayman, s. aydemir, i. yakut, u. serkant, a. c ß iftc ßi g€ olbasi devlet hospital, ankara, turkey objectives: to ascertain the beneficial effects of infliximab an inhibitor of tumor necrosis factor alpha (tnf-a) on the development of nec in an experimental nec rat model. material and methods: thirty newborn sprague-dawley rats were randomly divided into three groups as nec, nec+ infliximab, and control. nec was induced by enteral formula feeding, exposure to hypoxia-hyperoxia and cold stress. pups in the nec+ infliximab group were administered infliximab at a dose of mg/kg daily by intraperitoneal route from the first day until the end of the study. all pups were sacrificed on the th day. proximal colon and ileum were excised for histopathologic, immunohistochemical (tunel and caspase- ), and biochemical evaluation, including, total antioxidant status (tas), total oxidant status (tos), malonaldehyde (mda), and myeloperoxdase (mpo) and tnf-a activities. results: we observed better clinical sickness scores, weight gain, and survival rate in the nec+ infliximab group compared to the nec group (p < . ). histopathological and apoptosis examination (tunel and immunohistochemical evaluation for caspase- ) revealed lower damage in the nec+ infliximab group compared to the damage in the nec group (p < . ). tissue mda, mpo, tnf-a levels, and tos were significantly decreased in the nec+infliximab group, whereas tas was significantly increased in the nec + infliximab group (p < . ). conclusion: tnf-a blockade with infliximab efficiently reduced the intestinal injury and preserve the intestinal tissues from severe intestinal damage by its complex mechanisms on nec. therefore, it may be an alternative option for the treatment of nec.keywords: tnf-a; infliximab; necrotizing enterocolitis; newborn; protection; rat; treatment p- . . - short-term diabetes causes cardiovascular inflammation: anti-inflammatory effect of resveratrol introduction: diabetes is a metabolic dysfunction and has been associated with various disorders including inflammation, cardiomyopathy and coronary artery disease. inflammation is a protective mechanism elicited by the host in response to infection, injury, and tissue damage. the aim of this study was to investigate the effect of intraperitoneally resveratrol administration on cardiac and vascular function in diabetic rats. materials and methods: diabetes was induced in sprague-dawley rats by using injection of streptozotocin ( mg/kg, i.p.). rats were divided into group i: control, ii: control/ mg/kg resveratrol; iii: diabetic/vehicle; and iv: diabetic/ mg/kg resveratrol. histopathological examinations with masson's trichrome and verhoeff-van gieson staining were carried out to reveal cardiac and vascular tissue damage and inflammation. in addition to plasma glucose and cardiac & vascular mda levels were measured by standard enzymatic kits while tnf-a, il- b, il- (mbl) were analyzed by elisa kit. results: final body weight decreased in all groups compared to control. in the diabetic rats, plasma glucose and vascular mda levels were enhanced while cardiac mda was unchanged compared to control. vascular tnf-a, il- b and mbl and cardiac mbl were increased in the diabetic groups compared to control. discussion and conclusion: it has been found that resveratrol has greatly normalized altered parameters. taken together, resveratrol partly improved cardiac and vascular inflammation induced by diabetes. this may be due to the healing activity of resveratrol on pro-inflammatory markers. p- . . - cytokine network is critical in growth hormone-induced resistance mechanism against curcumin which modulates jak/stat/ socs pathway in mda-mb- and mcf- breast cancer cells m. c ß elik, a. c ß oker g€ urkan, e. damla arisan, p. obakan yerlikaya, n. palavan unsal t.c. istanbul k€ ult€ ur university, istanbul, turkey curcumin (diferuloylmethane), a polyphenolic compound that triggers apoptotic cell death in various cancer cells such as prostate, colon, melanoma and breast cancer. a pituitary-derived hormone, growth hormone (gh) play role in elongation and differentiation of ductal epithelia into the breast terminal and buds. in this study, our aim is to determine the role of inflammation in curcumin induced apoptotic cell death via acting on jak/stat/socs pathway in wt and gh+ mda-mb- and mcf- breast cancer cell lines. according to mtt cell viability assay curcumin triggers cell viability loss in time and dose dependent manner in mda-mb- wt and mda-mb- gh+ breast cancer cell lines, respectively. selected concentrations of curcumin as lm (for mcf- ) and lm (for mda-mb- ) decreased cell proliferation and induced apoptosis through causing jak dephoshorylation, stat , , dimerization and acting on socs proteins expression in each cell lines. in addition, activated jak/stat/socs pathway, via forced gh expression has been suppressed following curcumin treatment for hours. lm curcumin-induced apoptotic cell death via dephosphorylating jak at tyr / residues and decreased phospho-stat , level in both breast cancer cell lines. although curcumin dephosphorylated stat in both mda-mb- and mcf- wt cells, no significant effect has been observed in mda-mb- gh+ and mcf- gh+ cell lines. in consequence, although forced gh expression induced cell proliferation in mcf- and mda-mb- breast cancer cells, curcumin overcame gh-mediated resistance mechanism via acting on jak/ stat/socs signaling, which is related to pparg-induced inflammation. acknowledgment breast cancer is one of the highest cancer type among women worldwide. various enviromental and genetic factors such as age, gender, family history, metabolic diseases and gene mutations are involved in the breast cancer pathogenesis. growth hormone (gh), a pituitary derived hormone, has essential role on postnatal growth and development. it is also established that signalling route of gh and its receptor (ghr) activity is increased in different cancer types. curcumin, a nutraceutical deriatives from rhizomes of turmeric (curcuma longa), has potential therapeutic activity against cancer cells, including breast cancer. curcumin inhibits proliferation of cancer cells such as prostate, colon, melanoma, cervical and breast cancer via induction of apoptosis and inflammation. stat , a major downstream target of gh/ghr signalling, is related to survival, proliferation and differentiation. in this study, our aim was to investigate curcumin-induced apoptotic cell death in gh overexpressed mda-mb- breast cancer cells via jak-stat/socs signalling and inflammatory response profile. according to mtt cell viability assay, curcumin decreased cell viability in time and dose dependent manner in wt and gh+ mda-mb- breast cancer cell lines. we found that lm curcumin-decreased in apoptotic cell death through inactivity at jak which led to dimerization of stat , stat , stat . concomitantly, curcumin affected stat regulating socs proteins in mda-mb- breast cancer cell line. in addition, we demonstrated that lm curcumin induced pparg expression and altered inflammatory cytokine signalling cascade. consequently, although gh overexpression led to agressive profile in mda-mb- breast cancer cells, curcumin overcame this resistance. inflammation is involved in many systemic disturbances, including osteoarthicular or skin diseases, coordinating the signaling network that contributes to tissue injuries. the aim of our study is to reveal pro-inflammatory messengers at the cutaneous barrier (keratinocytes, fibroblasts, endothelial cells), simulating the dermal impact of active principles, especially polyphenols and flavones from vegetal sources: salvia officinalis, asculum hippocastanum and calendula officinalis. we focused on il and il cytokines as main mediators of inflammation progression, correlated in keratinocytes with il a as skin irritation indicator and vegf as pro-angiogenic factor, as well as in endothelial cells with icam- and vcam- adhesion molecules expression. in order to in vitro mimic the inflammatory conditions, we used targeted stimuli for each type of cells: for fibroblasts and endothelial cells -tnfa, a systemic stimulus, single or combined with pma that activates protein kinase c and up regulates nadph oxidase, which lead to superoxide anion production; for keratinocytescontrolled uv-a and uv-b radiation, simulating the solar damages or potential uv interactions with active principles in light exposed skin. the main analysis technique was flow cytometry: beads bases assay for soluble factors and fluorescent antibodies staining. our results prove the different involvement of polyphenols and flavones in the anti-inflammatory mechanisms, depending of the vegetal source: active principles from salvia officinalis induce a strong inhibition of il and il in tnfa stimulated keratinocytes, fibroblasts and endothelial cells, reduce the icam- over-expression but have no effects on irradiated keratinocytes; biocomplexes from asculum hippocastanum inhibit only il release in stimulated fibroblasts, but protect keratinocytes from uv-a and uv-b radiation; compounds from calendula officinalis are active on il signaling in fibroblasts and counteracts only uv-b inflammation. ischemia and/or reperfusion injury is one of the most common causes of acute renal failure. ischemia-reperfusion associated with thrombolytic therapy, organ transplantation, coronary angioplasty, aortic cross-clamping, or cardiopulmonary bypass results in local and systemic inflammation. within the endothelium, ischemia produces expression of proinflammatory gene products (e.g. cytokines) and bioactive agents (e.g., endothelin), while preventing other "protective" gene products (e.g., thrombomodulin) and bioactive agents (e.g. nitric oxide). therefore, ischemia induces a proinflammatory state that increases tissue vulnerability to further injury on reperfusion. this experimental study was designed to investigate the protective effect of salvia l. extracts on kidneys from i/r injury. salvia lamiaceae have been used for treatment of some illnesses in turkish folk medicine. forty spraque dawley rats were divided into groups (n = ). right nephrectomy was performed to all groups. group i: control group; group ii: i/r group; group iii: i/r + mg/kg salvia l.group; group iv: i/r + mg/kg salvia l. group; group v: i/r + mg/kg rosmarinic acid. group. salvia l. and rosmarinic acid for days was given single dose as a gavage. minutes ischemia, minutes reperfusion were applied to groups except control. intracardiac blood samples were taken, high sensitive crp (hscrp), tumor necrosis factor-a (tnf-a), interleukin (il)- and interleukin ib (il- b) levels were detected. serum hscrp levels were also determined in our clinical laboratory using routine standard methods. serum tnf-a, il- and il-ib levels were evaluated using an enzyme-linked immunosorbent assay technique. mean values were evaluated by statistical analysis. serum hscrp, tnf-a, il- , and il- b concentrations were significantly increased after renal i/r as compared to the control group. our treatment group mg/kg salvia l. and mg/kg rosmarinic acid especially mg/kg of salvia l. were found to show a protective effect against renal structure and function. we concluded that salvia l. extracts could be beneficial in the treatment of renal ischemic injury. but mg/kg salvia l. extract were more effective than mg/kg salvia l. extract and used as synthetic mg/kg rosmarinic acid. acne vulgaris is a common chronic inflammatory skin disease of unknown etiology. excess levels of secretory phospholipase a (spla ) contributes to inflammatory diseases and studies indicate that lipoprotein lipase (lpl) has differential effects on several inflammatory pathways. the aim of the present study was to assess serum activity of spla , lpl and evaluate changes in circulating protein levels of angiopoietin-like protein (angptl ), angptl , cyclooxygenase (cox) and prostaglandin e (pge ). serum from control subjects and acne vulgaris patients with moderate and severe disease was evaluated for levels of spla , cox, pge , lpl, angptl and angptl . disease activity was determined according to the national health service (nhs) lambeth and southwark clinical commissioning group guidelines for the management of acne. lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods using autoanalyzers (beckman coulter au clinical chemistry and unicel dxi immunoassay systems). serum levels of spla and lpl were significantly increased in acne vulgaris patients compared to age and gender matched controls. no significant differences were found for cox, pge , angptl and angptl levels between acne vulgaris patients and controls. the results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly increased serum spla activity. increased lpl activity in serum of acne vulgaris can be protective in patients through its anti-dyslipidemic actions. to our best knowledge, this is the first study investigating spla , lpl, angptl and angptl levels in acne vulgaris. future studies are aimed to understand the regulation of spla and lpl expression in acne vulgaris patients. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; # s ). p- . . - -ohdg and hogg levels are as an oxidative dna damage markers in acne vulgaris treated with isotretinoin h. ecevit, m. izmirli, b. gogebakan, e. rifaioglu, d. sonmez, b. bulbul sen, t. sen, h. m. okuyan mustafa kemal university, hatay, turkey acne vulgaris is a skin disease that characterized by comedones, papules, pustules, nodules and cysts at face, back and body skin. isotretinoin is one of the treatment agents in acne vulgaris. about weeks after drug treatment, the amount of sebum which is produced by sebaceous gland reduces keratinization disorder and the number of propionibacterium acnes normalizes. however, isotretinoin is known that has a wide range of side effects. in recent studies, isotretinoin treatment has been shown to increase the oxidative stress. -hydroxy- -deoxyguanosine ( -ohdg), an important indicator of oxidative dna damage, hydroxyl ion is bound at the th carbon of guanine. this structure is repaired through a base excision repair mechanism and the human -oxoguanine dna glycosylase (hogg ) plays a key role in this processes. in this study we aimed to evaluate the dna damage and it's repair in acne vulgaris before and after months of isotretinoin treatment by measuring -ohdg and hogg levels. the current study includes acne vulgaris patients who are diagnosed in mustafa kemal university, department of dermatology. -ohdg and hogg levels were measured by enzymelinked immunosorbent assay (elisa) method for before and after months of isotretinoin treatment. the commercial elisa kits (cloud-clone corp; usa and cell biolabs; usa) were used for the assessment of hogg and -ohdg, respectively.both -ohdg (p as a conclusion, isotretinoin increases dna damage and high serum -ohdg and hogg levels as a result of isotretinoin treatment may effect on the amount of reactive oxygen species. the pineal gland is a circumventricular organ which serves as a major neuroendocrine gland in the brain. its primary function is the production of melatonin which is controlled by signals from the suprachiasmatic nucleus. melatonin codes the length of the night and it is well recognized for its anti-inflammatory effects. lipopolysaccharide (lps) is the essential component in the outer surface membrane of gram-negative bacteria and act as a strong stimulator of natural and innate immunity in all eukaryotic species. furthermore, lps reduces melatonin synthesis and induces the expression of the serine protease inhibitor (spi- ) in the stat -mediated manner in pinealocytes. however, the precise function of stat in the cell signaling in the pineal gland is not yet known. here we investigated the effect of inhibition of stat on lps-induced changes in melatonin levels, expression of arylalkylamine n-acetyltransferase (aa-nat) and spi- in the pineal gland. experiments were performed in vitro using organotypic and primary cultures prepared from the rat pineal glands. levels of melatonin and spi- were determined from tissue homogenate by enzyme-linked immunosorbent assay (elisa). the pinealocytes were used to carry out sirna stat transfection. the successful transfection and subsequent decline in stat expression levels were proved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the changes in synthesis of aa-nat and spi- were studied by rt-pcr. in conclusion, lipopolysaccharide can affect the immunomodulators secreted by the pineal gland. the clarification of the effect of inhibition of stat on those immunomodulators is important from the clinical point of view because inhibitors of stat are nowadays used as tumour suppressors. silica nanoparticles have a great potential for a variety of industrial, diagnostic and therapeutic applications. in this study, we have evaluated the in vitro effects of amorphous silica nanoparticles ( nm) using human lung mrc- fibroblast as model. cells were exposed to . lg/ml silica nanoparticles for , and hours. the cytotoxic and inflammatory response, and matrix metalloproteinase expression were examined. the pro-inflammatory cytokine il- b, il- , il- , tumor necrosis factor (tnf-a), matrix metalloproteinases (mmp- , mmp- , mmp- ) and tissue inhibitor of metalloproteinase- (timp- ) were analyzed by western blot method. cytotoxicity was evaluated by lactate dehydrogenase (ldh) released into the culture medium by damaged cells. the level of ldh activity was increased after exposure to silica nanoparticles, in a time-dependent manner compared to control. the protein expression of il- , il- , il- and tnf-a as well as of mmp- and timp- , was up-regulated whereas those of mmp- , mmp- was down-regulated after and hours respectively. in conclusion, our data indicate that amorphous silica nanoparticles generate a cytotoxic and inflammatory response, as well as an imbalance in extracellular matrix due to the differential regulation of mmps and tissue inhibitor of metalloproteinase- in mrc-cells after and hours. p- . . - association of fto gene variant (rs ) with markers of t dm and obesity in population from bosnia and herzegovina and kosovo fto (fat mass and obesity-associated gene), recently discovered in a genome-wide association study for type diabetes (t d) encodes a -oxoglutarate-dependent nucleic acid demethylase and is mainly expressed in the hypothalamus. this gene may play important role in the management of energy homeostasis, nucleic acid demethylation, and regulation of body fat masse by lipolysis. the aim of this study was to analyze the association of this single nucleotide polymorphisms (snps) with clinical and biochemical parameters of obesity, t d, prediabetes and at the level of healthy population from bosnia and herzegovina (bh). the study included patients with t d and prediabetes and healthy controls both sexes, aged from up to years. patients were recruited at the clinical centre university of sarajevo, university hospital of clinical centre in banja luka, general hospital in te sanj and health centre in prizren. genotyping of analyzed polymorphism was performed by rt-pcr method in cooperation with the department of clinical chemistry, faculty of pharmacy, university of ljubljana (ljubljana, slovenia) and university hospital of charles university (hradec kralove, czech republic). our results did not show significant differences in genotype frequencies of the analyzed polymorphisms between patients with t d, pre-diabetes and healthy population also, results of logistic regression analyses did not show significant association of risk a allele of fto gene polymorphism -rs with increased risk of t d (or = . , % ci . - . , p = . ). a allele was significantly associated with higher values of hba c, insulin, homa ir index, diastolic blood pressure and higher levels of inflammatory markers (fibrinogen and leukocytes). interestingly, a tendency of association of a allele with higher values of obesity markers (bmi, waist and hip circumference) was noted. further studies are needed on a larger population in order to confirm these results. the water extract of capparis ovata (cowe) has been shown to be used as an alternative medicine for the treatment of multiple sclerosis (ms). cowe was further fractionated and studied for additional anti-neuroinflammatory effects in sh-sy y cells. for this purpose, the dichloromethane sub-fraction of the cowe extract was tested for its anti-inflammatory effects on selected anti-inflammatory genes believed to be important in ms pathophysiology using sh-sy y cells. cell viability was assessed using lactate dehydrogenase (ldh) activity in the media conditioned by the crystal violet cell staining. in these cells, levels of the tumor necrosis factor-a (tnfa), nuclear factor kappa-lightchain-enhancer of activated b cells (nf-jb ), glial fibrillary acidic protein (gfap), c-x-c motif chemokine and (cxcl , cxcl ), matrix metalloproteinase (mmp ), chemokine (c-c) motif (ccl ) and tyrosine-protein phosphatase non-receptor type (ptpn ) were determined by quantitative reverse transcriptase-pcr assay (qrt-pcr). we have found out that the dichloromethane sub-fraction of cowe effectively inhibited the expression of all of the genes given above in sh-sy y cells. thus, phytochemicals present in the dichloromethane sub-fraction of the cowe extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology. studies are underway to identify the individual compound(s) in this subextract of the cowe extract contributing to these effects. this work is supported by tubitak s and pamukkale university paubap fbe . p- . . - apigenin and luteoline were identified as active anti-inflammatory constitutents of lavandula stoeachas by bioassay guided fractionation h. ipek , s. savranoglu , a. r. t€ ufekc ßi , f. g€ ul , i. demirtas , t. boyunegmez t€ umer graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: lavandula stoechas, in the genus of lavender, has distinct therapeutic uses among anatolian people. rather than worldwide use of its essential oil in aromatherapy, specifically the aqeous portion as decoction has been traditionally used in anatolia against the components of metabolic syndrome, all of which share a state of chronic inflammation as an underlying cause. the anti-inflammatory constiutents of l. stoechas were isolated using a bioassay guided fractionation in lipopolysaccharide (lps) inflammed raw . macrophages. materials and methods: an aqeous extract was partitioned into ethyl acetate (eae) and n-butanol fractions. the eae, determined as bioactive extract was seperated into subfractions by column chromatography. e was identified as active subfraction subjected to sephadex column to get pure compounds which were then applied to nmr, ir, and uv analyses for structure determination. in raw . cells, the effects of extracts/fractions/subfractions/compounds on lps induced no production was determined by using griess method. the potential inhibitory effects of each compound on lps induced inos expression were determined by qpcr and western blot. results: p-coumaric acid, apigenin and luteoline were found in the e , and the first two compounds appeared to be primarily responsible for the anti-inflammatory activity. apigenin and luteoline at lm decreased no production and %-ic : and lm-by inhibiting inos gene expression and % as well as protein expression and %, respectively (p < . ). conclusion: this is the first time that luteoline and apigenin have been found in eae of l. stoechas, and the anti-inflammatory properties of the eae can be attributed, at least in part, to the presence of these two compounds. we are on the way to gain further insight for the action mechanism of these two active principles as anti-inflammatory agent. tubitak (project id: t ) support this work. the role of tip in the inflammation process immun response generates the first line of host defense during inflammation and plays an important role inducing pro-inflammatory response by generating early response against pathogens. il- (interleukin ) is one of the pro-inflamatory cytokines and its expression increases during the infection to activate the jak/ stat pathway. jak/stat pathway is regulated by hamp (hepcidin antimicrobial peptide). our previous study, we reported that hamp gene expression was decreased in liver-specific tip conditional knockout mice, so we thought that tip may have a direct or indirect role on inflammation mechanism. tip (tat interacting protein, kda) is a member of the myst enzyme family of histone acetyltransferases (hats) and plays an important role in multiple function including cellular signaling, dna repair, cell cycle and apoptosis. in this study, the quantitative gene and protein expression of il- were investigated by using taqman real time pcr, western blot and immunohistochemistry analysis in control group, lpsinduced inflammation group and liver-specific tip conditional knockout group mouse liver. according to our preliminary results, the gene and protein expression of il- was increased in lps-induced inflammation group (p < . , p < . ) and liver-specific tip conditional knockout group mouse liver (p < . , p < . ). our initial data suggest that tip may be essential for the inflammation process. this work was funded by grants from the scientific and technological research council of turkey (tubi-tak) (grant number: z ). although intracellular reactive oxygen species (ros) level is necessary to maintain cellular homeostasis, elevated intracellular ros level with the impact of unfavorable environmental conditions leads to oxidative stress that may cause damage to dna, proteins and lipids. in case of inflammation, organism seeks to provide cellular homeostatis by increasing ros levels via antioxidant molecules and enzymes. therefore, it was thought that there can be a direct or indirect relation between inflammation and oxidative stress. in this study, inflammation was performed by intraperitoneal injection of lipopolysaccharide (lps). the gene expression and activity of antioxidant enzyme including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione s-transferase (gst), glutathione reductase (gr) and glucose -phosphate dehydrogenase (g pd). additionally, any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h o ) level are accepted as an indication for the accumulation of ros, the relative levels of them were also studied. to show our inflammation model was performed in mouse kidney with lps treatment or not, the expression of interleukin (il ), which is accepted as a inflammation marker, was investigated by real time pcr. the expression of il was significantly increased in lps treated group. while the level of mda and h o was elevated in lps treated group, gssg was decreased. no changes was seen for gsh level. the correlation was observed between enzymatic and molecular levels. while the gen expression and the enzyme activity of sod, cat, gst, gr, and g pd were decreased, gpx was increased with inflammation. in conclusion, increasing ros level was observed in the inflammation process and, the antioxidant system was affected at the molecular and protein level. this work was funded by grants from the scientific and technological research council of turkey (tubitak) (grant number: z ). the aim of our study is to evaluate effect of vitamin d levels on hemogram parameters including neutrophil %, lymphocyte %, neutrophil % / lymphocyte % ratio (nlr) and mean platelet volume (mpv) in behcet's patients. fifty eight patients with diagnosis of behcet that applied to selcuk university faculty of medicine department of dermatology are recruited to the study. clinical and laboratory characteristics of the patients were obtained from hospital automation. t test was used to examine the differences between the parameters. p < . was taken to be statistically significant. there was a statistically significant difference between vitamin d values and age (p = . ) whereas difference was not significant between vitamin d and neutrophil %, lymphocyte %, nlr, mpv values. according to the literature, there are a lot of studies that show the relationship between vitamin d and hemogram parameters. however, contrary to the previous studies, we were unable to find any significant relationship between vitamin d and these hemogram parameters. these results serve the idea that the effects of vitamin d on the hematopoietic system should be further investigated experimentally and clinically. crimean-congo hemorrhagic fever is a tick-borne disease caused by the arbovirus and characterized by a sudden onset of high fever, severe headache, dizziness, back and abdominal pains. the exact pathogenesis of cchf has not been clarified yet. the aim of this study, clinical cases of cchf in cu, se and zn is to examine the relationship between the concentration of trace elements. the study sample consisted of patients which have been diagnosed with cchf. matched for gender, healthy volunteers were similar to the control group according to age. the patients and control groups, serum cu, zn and se levels were analyzed using atomic absorption spectrophotometer. cchf patients in the group, cu zn and se serum levels were significantly lower compared with the control group. in our study, the cofactor of the antioxidant enzyme cu, zn and se elements were lower. this shows us in cchf disease, a decrease in antioxidant enzyme activity, and suggest that they contribute to the immune system's degradation. p- . . - inhibitors of mdm ubiquitin ligase as prospective modulators of autoimmunity e. bulatov, a. valiullina, r. sayarova, a. rizvanov kazan federal university, kazan, russia ubiquitin-proteasome system is seen as a pool of promising protein targets for therapeutic impact in many human diseases. mdm is an e ubiquitin ligase widely studied due to its wellknown role in cancerit negatively regulates p oncosuppressor that mediates apoptosis in tumour cells. inhibitors of p / mdm interaction have long been known as potential anticancer therapeutics. however, recent advances in the field suggest that both mdm and p might be playing a substantial role in autoimmune processes. we used a small molecule p /mdm inhibitor nutlin- a to test the effect of p activation on peripheral blood mononuclear cells (pbmcs) from both healthy volunteers and patients diagnosed with multiple sclerosis. in our study we employed a variety of molecular biology methods, such as immunoblotting, real-time pcr, mts cell proliferation assay, fluorescence flow cytometry and confocal microscopy. we demonstrated that disruption of p /mdm interaction by nutlin- a alters the p levels and also affects the lymphocyte subpopulations within pbmcs. our findings suggest that p /mdm interaction inhibitors can potentially be used as prospective modulators of immune response in autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus and other. the study was funded by rfbr research grant - - mol_a_dk. can ykl- be an inflammatory biomarker in vitamin d deficiency? object: the tumor necrosis factor (tnf) was found to be cytotoxic to tumor cells and to induce tumor regression in mice. except for one member, all receptors to the tnf superfamily bind tnf-related ligands and act mostly on inflammatory system. there are currently tnf superfamily ligands. tnf superfamily ligands share several common features. tnf ligands are generally type ii transmembrane proteins whose extracellular domains are be divided by enzyme to create cytokines. the tnf superfamily currently consists of receptors. tnf family receptors are type i or type iii transmembrane proteins that contain multiple extracellular domains. in this study, we investigated to presence, differences and effects of tnf superfamily and receptors genes in human and mice by using bioinformatics techniques. methods: the nucleotide and amino acid sequence of each protein in human and mice was determined using t-blast-n for homologous sequences. homologous sequences of human tnf family genes found an automated procedure by using psi-blast. the secondary structure of and three-dimensional of the protein were analyzed by psipred and ffas server. netcglyc . and netphos . program were used for post-translocation modifications. the web apoptosis database was also used for the lists of domains, proteins containing these domains and their associated homologs. results and conclusion: humans tnf ligands have genes encoding proteins that contain a conserved carboxy-terminal domain. this family of proteins is highly conserved between humans and mice. humans contain genes encoding tnffamily receptors. sequence data from the ncbi databases demonstrated the presence of mouse tnf-family receptors with orthologs in humans and one additional receptor found only in mice. the differences and similarities in the tnfs genes in humans and mice will provide information for understanding the utility and limitations of the mouse models of disease and comparing of immunology outcomes. the development of left ventricular remodeling after acute myocardial infarction is a predictor of shock. the genetic influence on cardiac remodeling, and shock in the early period after acute myocardial infarction are unclear. the aim of the present study was to investigate the relationship between angiotensin converting enzyme (ace) gene polymorphism and modified shock index (msi) in the early period in patients with acute anterior myocardial infarction. overall patients with a first acute ami were included in this study. dna was isolated from peripheral leukocytes. the id status was determined by pcr. based on the polymorphisms of the ace gene, they were classified into groups: deletion/deletion (dd) genotype (group , n = ), insertion/deletion (id), insertion/insertion (ii) genotypes (group , n = ). blood pressure and pulse measurements were performed in all patients within minutes admitted to coronary care unit. msi was defined as heart rate (hr) divided by mean arterial pressure (map). echocardiographic examinations were performed in accordance with the recommendations of the american echocardiography committee. one-way analysis of variance (anova) and chi-square analyses were used to compare differences among subjects with different genotypes. the study was approved by the local ethics committee, and each patient gave a written consent. there were no significant differences among clinical parameters of patients. msi was significantly higher in patients who have ace dd genotype than in patients who have ace id / ii genotypes ( . ae . and, . ae . , p < . ). presentation time hypotension or developing hypotension during admission was reported to be an important predictor of intensive care unit admission besides other vital sign measurements. our results suggested that, ace gene i/d polymorphisms d allele may affect modified shock index in patients with a first acute anterior mi. glucocorticoids (gcs) are widely used in medicine, despite their side effects, e.g. osteoporosis. however, precise molecular mechanisms of gc action, especially on bone marrow (bm) cells, remain controversial. given the osteoprotective role of vitamin d , the aim of our study was to examine prednisolone-induced changes in the rank (receptor activator of nuclear factor kappa-b)/rankl (rank ligand)/opg (osteoprotegerin) pathway of rat bm depending on the state of vitamin d endocrine system. female wistar rats received prednisolone ( mg/kg b.w.) with and without iu of d (for days). the levels of rank, rankl, opg, a-hydroxylase (cyp b ) in bm were determined by western blotting. vitamin d receptor (vdr) and rankl mrnas were measured by quantitative rt-pcr. ohd content in the serum was assayed by elisa. rankand vdr-positive bm cells were quantified using flow cytometry and visualized by confocal microscopy. prednisolone induced a marked increase in rankl and rank levels, while opg level was shown to decrease. this reflects disturbances in cytokine-mediated regulation of bm progenitor cell function. data from flow cytometry indicated a significant growth in the number of rank-positive cells (hematopoietic osteoclast precursors) compared to control. these changes were accompanied by a decrease in the levels of vdr and cyp b , which is responsible for , (oh) d synthesis, in bm and ohd content in serum. co-localization of vdr and rank in mono-and multinuclear bm cells was observed, indicating a close relation between vitamin d and rank/ rankl/opg pathway. vitamin d co-administration prevented prednisolone-induced changes in bm cells through restoration of vitamin d bioavailability and vdr signaling that resulted in a reduction of the osteoclast progenitor pool in bm. thus, prednisolone-induced imbalance in rank/rankl/ opg system components is associated with impairments of vitamin d endocrine system in bm and can be ameliorated by vitamin d treatment. p- . . - heat shock pathway in response to different stress factors the heat shock response is an emergency pathway of the cell, which mediates repair and protection from cellular stress and therefore guarantees the survival of the cell. this stress can range from heat or hypoxia to chemicals and heavy metals. it is highly conserved in all eukaryotic cells and plays an important role during atypical conditions. due to its high complexity, the pathway is not yet completely understood. most important, after activation of the pathway, is the refolding of proteins or, in case of severe misfolding, the depletion of proteins to maintain proteostasis. heat shock factor encoded by the hsf gene is known as the main switch point in heat shock regulation. after activation it trimerizes and binds to heat shock elements in target gene promoters. one of these promoters is the hspa a promoter (hsp promoter). the promoter was analyzed by dismantling it to its functional parts. especially three elements, the heat shock elements, were in the focus of this work. in first place parts of the promoter were multimerized and combined with different reporters, like luciferase, by cloning. also mutations in the natural promoter were designed by cloning. the focus now is on the heat shock elements, where hsf can bind as a trimer. the idea is that these different elements have various effects on different stressors like heat, chemicals (geldanamycine as hsp inhibitor, mg as proteasome inhibitor) or heavy metals (cadmium, arsenic, zinc) . this was tested on cells transiently transfected with those promoter variants. for promising variants stable cell lines were created. in these stable cell lines further experiments on mrna level can be conducted. in the last months experiments with the crispr/cas system were started. furthermore, experiments on transcriptional (qpcr) and translational (dual-luciferase assay) levels were done as well. in the end we hope to get a clear picture on the regulation of the hspa a promoter by different stress factors. invasive cancer cells form membrane protrusions, invadopodia, that facilitate cell invasion and metastasis. key players invadopodia include the adaptor proteins tks and tks , the actin regulators cortactin, wip and n-wasp, the kinase src and others. in spite that in the last two decades significant advances in our knowledge of the structure and development of invadopodia have been made, detailed mechanisms they are functioning is not yet available. we have identified a series of new tks binding partners including adaptor proteins itsn , itsn , crk and grb , kinase src, amph , bin , plcg and also another member of the tks family -tks . it may indicate the possible role of tks in transport and sorting of cell vesicles. current data are supported by interaction with the proteins of amph and bin , as their main functions are membrane trafficking and remodeling. adaptor proteins crk, grb and itsns are important for the actin cytoskeleton rearrangements, endocytosis and signal transduction. moreover, we have identified and characterized new tks isoform -tks -beta. we suggested that an active state of tks is regulated via intramolecular interactions between its proline-rich motifs and own sh -domains. we have shown the interaction between itsns and other prominent component of invadopodia wip. data from immunofluorescent analysis revealed co-localization of itsn and wip at the sites of invadopodia formation and in clathrin-coated pits. we have also demonstrated that the key protein itsn and wip and n-wasp can form a complex in cells. together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify itsns as scaffold proteins involved in this process. we have shown the interaction between itsns and other verprolin family members cr and wire which play an important role in the reorganization of the actin cytoskeleton. we have demonstrated that cr and wire interact with sh domains of itsns in complex with actin. p- . . - correlation between proteomic and phenazine profile of pseudomonas sp. phenazines are widely known compounds with huge variativity of biological activities which are produced by pseudomonas sp. and some other bacteria species. the results of our work shows the correlation between the changes of proteomic profile of pseudomonas aeruginosa caused by a mutagenesis and the secondary metabolism of antibiotics (phenazine) profile. different strains of pseudomonas aeruginosa were obtained using mutagenesis, after that bacterial cells were destroyed by ultrasound. protein-containing fractions were isolated using methanol-chloroform method as well as phenazines compounds were extracted from culture media using liquid phase extraction. obtained proteome was analysed by shotgun-proteomics technique. as the result of the liquid phase extraction phenazine compounds were mainly extracted to the organic phase. this phase was evaporated and re-dissolved in % methanol. after sample preparation obtained solutions were analyzed by hplc-agilent with quadrupole tof mass-detector. results of the analysis were compared with the library of known phenazine compounds mass-spectras generated by cfm-id online resource. obtained phenazine profiles were compared with each other and correlation with the changes in proteome was analyzed. received results promote better understanding of mechanisms of phenazine production. this data opens possibilities for targeted changes in the methabolic pathway in order to obtain phenazine compound with required biological activity. insulators are genomic elements which block enhancer-promoter interaction and prevent spreading of heterochromatin. cp protein is an integral component of most known drosophila insulators, it interacts directly with ctcf and pita dna-binding insulator proteins using dimeric btb-domain, but function of cp within insulators still remains to be elucidated. recently we described an interaction between cp btb-domain and cterminal domain of ctcf insulator dna-binding protein, subsequent deletion analysis allowed us to isolate aa fragment within ctcf c-terminal domain sufficient for interaction with cp btb, but deletions of flanking regions also lead to the loss of interaction with cp in vivo. at the same time crosslinking experiments suggest that a dimer of btb interacts with one molecule of ctcf, presuming that it could recognize two peptide fragments within ctcf c-terminal domain. we solved crystal structure of btb-domain from cp insulator protein at . a resolution. overall structure is similar to other btb-domains. cp btb-domain has peptide-binding groove similar to that previously found in bcl btb domain. inspection of btb-domain surface revealed several possible binding sites for polypeptide fragments from ctcf protein. based on these observations a set of point mutations within peptide-binding groove of btb-domain has been designed and we tested ctcf-interaction abilities of these mutants using gst pull-down assay and yeast two-hybrid assay. the most significant impact was found with alanine-substitutions of hydrophobic residues whereas substitutions of hydrophilic amino acids were less effective. therefore our results support that cp btb-domain recognizes ctcf protein using peptide-binding groove. this study was supported by the russian science foundation (project № - - ). p- . . - comparative study of the fatty acid composition of lipids in the raw meat samples obtained from hybrid sheep one of the most important tasks in the animal biology and husbandry is to clarify the role of animal genetic diversity in providing nutrients to the diversity of animal products. the objective of our work was to study the chemical compositions of raw meat samples obtained from domestic (group i -purebred romanov sheep) and hybrid sheep (group ii -f hybrids of romanov sheep with . % of argali blood). the significant changes in fatty acid composition of the lipid fraction from the fat and muscle tissue of the hybrid sheep as compared to the control were found. the content of saturated fatty acids (sfas) in the fat samples of the hybrid animals was by . % lower ( . ae . %, p < . ), but polyunsaturated (pufas) or monounsaturated fatty acids (mufas) contents were by . % and . % higher ( . ae . and . ae . (p < . ), respectively) as compared to purebred romanov sheep. the most pronounced changes were found for palmitic acid (decreased from . % to . %) and for oleic, linoleic, arachidonic acids (increased from . %, . %, . % to . %, . %, . %, respectively). the last two acids together with the linolenic acids belong to the so-called essential acids and very important for the animal metabolism. a similar trend was observed on the composition of the lipid fraction of muscle tissue. sfas, pufas and mufas content in muscle tissue of hybrid sheep was . ae . , . ae . and . ae . %, that was . % lower (p < . ), and . % and . % higher (p < . ) compared to purebred romanov sheep. these results emphasized the difference of the pufas/sfas ratios in fat and muscle tissues, respectively) and characterized the biological value of the lipid fraction of fat and muscle tissue. the obtained data gave evidence of the positive changes in the fatty acid compositions of the lipid fractions for the hybrid animals as compared to the purebred sheep. supported by the russian scientific foundation, no. - - . foodborne illnesses resulting from the consumption of agricultural commodities contaminated with enteric pathogens are an increasing problem around the world. while various possibilities of produce contamination with pathogens exist, the global warming combined with a widespread use of animal manure in agriculture will likely contribute to an increased number of such outbreaks. thus, phages isolated from different agroecosystems may prove to be useful in detection/biocontrol of enterobacteria in produce. during the investigation of the impact of global warming on the diversity and co-evolutionary dynamics between microorganisms and viruses in lithuanian agroecosystems, a novel enterobacteria phage vb_ecos_nbd (nbd ) was isolated from agricultural soil using e. coli novablue for phage propagation. nbd genomic dna was isolated from cscl-purified phage particles, and was subjected to illumina dna sequencing. nbd is a virulent siphovirus that has a low-temperature plating profile (fails to form plaques at a temperature > °c). the genome of nbd is $ kb long, and has a total of probable protein-encoding genes as well as gene for trna ser . the genome analysis revealed that nbd orfs encode unique proteins that have no reliable identity to database entries. among the orfs that encode proteins with matches to those in other sequenced genomes, are similar to proteins from phages that infect different members of enterobacteriaceae, while nbd orfs are most similar to those from bacteria. based on the similarity to biologically defined proteins, nbd orfs were given a putative functional annotation, including genes coding for morphogenesis-related proteins, as well as associated with dna replication, recombination, and repair. phylogenetic analysis revealed that enterobacteria phage nbd is distantly related to phages belonging to the subfamily tunavirinae. this research was funded by a grant (no. sit- / ) from the research council of lithuania. p- . . - the antibiotic novobiocin affects the composition of the escherichia coli proteome n. e. arenas , j. williamson , v. schw€ ammle , s. douthwaite universidad de cundinamarca, cundinamarca, colombia, university of southern denmark, odense, denmark novobiocin (nov) is an aminocoumarin which competitively inhibits the atp binding site in the gyrase-b subunit of prokaryotic topoisomerase ii. nov remains a therapeutic choice for treating infections with bacterial pathogens that are resistant to more commonly used drugs. the aim of this study is assess the proteomic response of e. coli strain upon nov treatment. minimum inhibitory concentrations of nov were measured by standard assays. three different e. coli strains (as , as -rlma::aph and b) were grown aerobically in nutrient rich lb media at °c during one hour. the whole cell proteome (five biological replicates in each sample,) was assessed by lc-ms by using tmt labelling protocol. raw files were imported to proteome discoverer (thermo fisher scientific) and searched together with mascot against the uniprot e. coli reference proteome. mics for nov were determined to be > -fold higher the wild-type b-strain of e. coli than for the hypersusceptible as strains ( lg/ml). whole genome comparison of the b and as strains were characterized by an increase in proteasome components ( proteins), chaperones ( ), error-prone dna polymerase components ( ), ribosomal hibernation factors ( ), heat shock response ( ), electron transport coupled proton transport ( ), pentose phosphate pathway ( ), flagellar assembly ( ), oxidative phosphorylation ( ) and tca cycle ( ). whereas ribosomal proteins ( ), aminoacyl-trna synthetases ( ), rnases ( ), abc transporters ( ), mismatch repair ( ) and sec secretion pathway ( ) were significantly down-regulated upon nov treatment. the three e. coli strains respond similarly upon nov treatment and their proteomes showed upregulation of heat shock response with changes in the components of translation and transcription, the proteasome and atp biosynthesis. the changes observed can be used to define the processes that are required for antibiotic tolerance and survival of e. coli against aminocoumarin antibiotics. postnatal growth is under control of pituitary derived hormone, growth hormone (gh) that triggers bone, fat tissue growth and development via acting on protein, carbohydrate and fat metabolism. gh functions on postnatal development by jak /stat signaling following gh:gh receptor (ghr) dimerization. isolated growth hormone deficiency (ighd) is a medical condition of insufficient production of growth hormone (gh) that is caused by mutations on gh-n gene in different ethnic origin children. various mutations within gh has been determined in different populations so far, and glutamic acid to glycine (e g), asparagine to aspartic acid (n d), threonine to alanin (t- a) missense mutations, alanine to serine (a s) substitution, tryptophan to stop codon (w- x), gaaa insertion in intron of gh-n gene and both intron (+ c) and deletion of . amino acid of gh protein phenylalanine (f del) mutations were detected in turkish ighd children. the potential role of these mutations on cell growth, proliferation, emt via acting on gh signaling pathway has not been observed yet. all these mutations were performed on wild type gh-n gene inserted pc . vector by site-direct mutagenesis and stable cell line of each gh gene mutations were generated by neomycin selection. although w- x, e g, f del, a s and n d mutations suppresses gh signaling via acting on either jak dephosphorylation or stat downregulation, t- a, gaaa insertion and deletion of + c mutations have no significant effect on gh signaling. in addition, each mutation lead different growth suppression effect and colony formation potential and intracellular polyamine levels and odc expression profiles were essential role in emt potential of hek cell lines. as a result, w- x, e g, f del, a s and n d mutations prevented gh signaling and cell growth and differentiation via polyamine metabolism. pulmonary embolism (pe) is a common cardiovascular emergency and affects a large number of patients. acute pe-induced oxidative stress can lead to the accumulation of specific nitroproteins that may play a role in disease progression. the impact of nitration of a single tyrosine residue often has broad implications on the activity of biologically critical proteins, which has become increasingly related to pathological conditions. in this study, we used a proteomic approach to analyze nitrated serum proteins in patients diagnosed with acute pe and healthy controls. nitrotyrosine (no tyr)-containing proteins were immunoprecipitated from serum with a no tyr affinity sorbent. precipitated proteins were separated by sds-page and visualized by coomassie blue staining and western blotting with mouse monoclonal anti-no tyr antibody. among the numerous immunoreactive bands observed in disease patients, the kda protein band was in-gel digested and analyzed by maldi-tof mass spectrometry (ms). mass fingerprint data sets obtained from the peptide fragment ions matched human collagen alpha- (iii) chain (co a _hu-man) with mascot algorithm analysis giving a score of (p < . ). collagen alpha- (iii) chain is a fibrillar collagen that is found in extensible connective tissues such as skin, lung, and the vascular system. altered metabolism of collagen and its excessive deposition in the matrix of the connective tissue is a hallmark of chronic interstitial lung diseases. collagen can be measured in serum and bronchoalveolar lavage fluid from patients with numerous chronic interstitial lung diseases. given these considerations, future studies are aimed understand the relevance of no tyr modifications in co a relating to changes in protein structure and function. recent studies have shown that the genes involved in dislipidemia represent potential loci to be associated with diabetes as a disease. recent genome wide association (gwa) studies have associated rs in gckr gene and rs in galnt gene with parameters of t d and diabetic dyslipidemia. in this study, the association of these single nucleotide polymorphisms (snps) with t d and dyslipidemia was tested in the population from bosnia and herzegovina (bh). our study involved patients with t d and healthy subjects. biochemical and anthropometric parameters were measured in all participants. after dna extraction, sequenom iplex platform was used for the analysis of galnt polymorphism (rs ), while polymorphism in gckr (rs ) gene was analyzed by using real time pcr. our results demonstrated significant association of gckr rs variant with waist circumference (p = . ) and fasting glucose levels (p = . ) in the control group. no such association was demonstrated for rs galnt gene. in the group of diabetic patients, significant association of gckr rs variant with levels of bilirubin (p = . ) and rs galnt variant with hba c (p = . ) and triglyceride levels (p = . ) was also demonstrated. our results suggest an association of variations of gckr and galnt genes with specific markers of t d and dyslipidemia. further studies would be needed in order to confirm these genetic effects in other ethnic groups as well. osteoporosis is the most common metabolic bone disorder affecting the normal bone turnover with low bone mineral density (bmd) and risk of fragility fractures. polymorphisms at the sp binding site of the collagen type a (col a ) gene is associated with low bmd. we examined the distribution of col a gene polymorphism in young osteoporotic women and in control group in turkish population. patients had low bmd with t score ≤ . sd and controls was healthy women ( - years). mean age ( . ae . ) and ( . ae . ) respectively. the bmd, as g/cm , was measured in the hip and the lumbar spine (l -l ) with (dexa). dna was isolated from blood. col a gene was analysed with genomica clinical array system. the x test was used to compare allele and genotype frequences between patients and controls. mean of t score in patients was À . ae . . mean bmd (as g/cm ) was . ae . , and ( . ae . ) genotype distribution were ( %) ss, (% )ss, (% )ss for patients, and ( )ss, ( )ss, ( )ss for control . patients had (% )s allele, ( %) s allele, controls had ( %)s allele, ( %)s allele. when genotypes and bmd were compared in patients, there was no significant correlation between osteoporosis and genotypes. the allelic distribution was not significant between patients and controls p > . . genotypic distribution in patients were significantly different. patients had a higher frequency of the ss(% ) than controls (ss % ) p < . . this study shows that high prevalences of the ss genotype at the col a locus, in osteoporosis . _ it is possible that the presence of the s allele causes variation col a and col a mrna's producing abnormal collagene protein. since collagen protein is major protein of bone, it is to be expected that a defect in this protein will produce bone fragility. col a gene should be detected early to initiate preventative therapy for bone health. the biological activity of nigella sativa seeds is mainly attributed to its essential oil component which is pre-dominantly ( - %) thymoquinone (tq). therapeutic effect of tq was exhibited in many diseases including inflammation, cancer, sepsis, atherosclerosis and diabetes. tq has been reported to exhibit antiproliferative effects on cell lines derived from breast, colon, ovary, larynx, lung, myeloblastic leukemia, and osteosarcoma and inhibited hormone refractory prostate cancer. tq induces apoptosis in tumor cells by suppressing nf-jb, akt activation, and extracellular signal-regulated kinase signaling pathways and also inhibits tumor angiogenesis. the aim of this study was to evaluate the anti tumor effects of tq on hepatoma cells. these antitumor assays include cell viability assay, clonogenic assay, scratch assay and molecular expression studies of death related genes. cells were treated with different concentration of tq in hep b for cell proliferation by mtt and clonogenic assay. in addition, the metastatic character of tq was investigated by scratch assay in hep b at - and hours. the effect of tq was also evaluated at mrna level by real-time-pcr. tq was treated on the hep b cells in three different concentration, namely - and . lm. tq showed the cell cytotoxicity in concentration and time dependent manner. the scratch assay revealed no healing in the scratched area due to the decreased cell viability. maximum permissible dose was lm. proapoptotic genes, bax and bad, and autophagy genes, beclin- and lc , were upregulated in hep b cells after hours treatment in contrast, antiapoptotic gene, bcl- , expression level was decreased for hep b cells after hours. p- . . - association of irs genetic variation with type diabetes and insulin resistance in patients from bosnia and herzegovina insulin receptor substrate- (irs ) encodes the irs protein, a substrate for the insulin receptor tyrosine kinase and has a critical role in insulin-stimulated signaling pathways. previous studies showed that irs single nucleotide polymorphisms (snps) were associated with type diabetes mellitus (t d). this is the first study performed in a population from bosnia and herzegovina (bh) in which we examined the association of rs (g>a), rs (t>c) and rs (a>t) with t d risk and related traits. our study involved t d patients and healthy subjects. biochemical parameters, including but not limited to insulin, homa-ir, hba c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was done by spss . our results demonstrated a significant difference in frequency of rs (p < . ) and rs (p = . ) snps between t d patients and control subjects. interestingly, here we showed a significant association of irs rs risk t allele with increased insulin levels (p < . ) and homa-ir (p < . ) in t d patients. similarly, rs variant was also associated with the same markers of insulin resistance in diabetic patients, i.e. insulin levels (p = . ) and homa-ir (p = . ). no such association was demonstrated for rs . however, this irs variant was associated with changes in lipoprotein levels, where risk c allele increased vldl (p = . ) and decreased hdl levels. our results suggest that irs variants are associated with t d susceptibility in bh population, thus confirming similar findings in other population cohorts. furthermore, the associations of these variants with markers of insulin resistance and dyslipidemic metabolic changes point to their role as potential t d biomarkers. the adra a gene encodes alpha- a adrenergic receptor which mediates adrenergic suppression of insulin. a genetic variant in adra a was recently associated with defective b-cell function. the objective of this study was to analyze association of two adra a polymorphisms (rs a>g and rs g>t) with type diabetes (t d) and its related traits. in this study we have included t d patients and healthy subjects from bosnia and herzegovina (bh). biochemical parameters, including but not limited to insulin, homa-ir, hba c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was performed by ibm spss statistics software. our data showed that frequencies of both, rs and rs , variants were not significantly different between t d and control subjects. however, rs risk a allele appear to increase insulin levels (p = . ) and homa-ir index (p = . ). furthermore, this variant also seems to affect vldl levels (p = . ) and waist circumference (p = . ) in diabetic patients. the genotype analysis of rs variant demonstrated that risk g allele decreased hdl (p = . ) and increased ldl levels (p = . ), as well as affected the waist circumference (p = . ) in diabetic patients. interestingly, haplotype analysis demonstrated the association of rs a / rs g with higher homa-ir index. here we demonstrated that although both, rs and rs , adra a polymorphisms were not associated with t d risk in our cohort, they were associated with markers of dyslipidemic perturbations and insulin resistance in diabetic patients. further studies in larger cohorts are needed in order to explore these possible interactions and confirm our findings. smad-interacting protein (sip ), also known as zeb is a member of zeb family transcription factors and was shown to regulate epithelial-to-mesenchymal transition, cell cycle, cellular senescence and cancer stemness. bipartite zing finger motifs at amino and carboxyl termini of the sip mediate its binding to ebox sequences in the genome. however, there are only limited data about sip target genes. by using a home-made anti-sip monoclonal antibody, clone e , we conducted chip-seq in three hepatocellular carcinoma cell lines, namely snu , plc/prf/ and sk-hep- and found receptor tyrosine kinase-like orphan receptor (ror ) as one of the targets. sip dnabinding consensus motif cacctg was found at + kb, + kb and + kb from ror transcription start site. chip experiments validated sip binding to all consensus motifs in the ror gene region. interestingly, the strongest enrichment was at + kb suggesting that long-range interactions play an important role in the regulation of ror by sip . sip knockdown by shrna in high-sip expressing snu cells resulted in the repression of ror expression. ror is expressed in embryogenesis and fetal life, and is absent within most of adult normal tissues. however, overexpression of ror was observed in many human cancers, from hematological malignancies to solid epithelial tumors. ror -positive cancer cells have enhanced proliferation, invasion and metastasis capacities, show resistance to apoptotic stimuli and display cancer stem cell characteristics. therefore, sip and ror act in similar pathophysiological processes. our finding that ror is regulated by sip at least in hepatocellular carcinoma cells adds another level of complexity to the molecular mechanisms of proliferation, invasion and stemness of cancer cells. hepatocellular carcinoma (hcc) is the most prevalent primary liver cancer and is one of the leading causes of cancer related deaths. smad interacting protein (sip ), a member of the zeb family of emt inducers, is involved in cellular proliferation, senescence, invasion and metastasis in human tumors. however, genes regulated by sip in hcc are yet to be identified. we conducted a chip-seq study in high-sip expressing hcc cell line snu by using a home-made anti-sip antibody, clone e . among annotated genes, we selected six for further studies because of its increased expression in multiple cancers and its association with poor prognosis. sip dna-binding motif cacctg was found at - kb from transcription start site of six gene. chip qpcr experiment validated sip binding to this region with , fold enrichment. compared to healthy liver, six transcripts were upregulated in of hcc cell lines included in this study. knockdown of sip by shrna in snu cells caused upregulation of six . immunohistochemistry studies in hcc tissue arrays showed increased expression of six in tumors and inverse association with sip expression in a tumor grade dependent manner. therefore, our results strongly suggest an inverse correlation of sip and six in hcc bone mineral density (bmd) and bone turnover are under genetic control and variations in the vitamin d receptor (vdr) are related to bmd. bmd is known to be affected by -hydroxy vitamin d ( (oh)d) and intact parathyroid hormon (ipth) levels. we aimed to determine correlation blood levels of vitamin d (vitd), ipth, and vdr gene effect in healthy turkish women. the subjects were healthy women in age - years. the bmd was measured as a t score in the lumbar spine (l -l ) with dexa. all subjects had normal t score between (- . to . ) sd. vitd was measured by lc- -at shimadzu. ipth was measured by chemiluminescence method, dna was isolated from blood. the fok i (vdrf-foki) and bsmi (vdrb-bsmi) polymorphisms of vdr gene was analysed with genomica clinical array system. the mean vitd level was ( . ae . ) lg/l, mean plasma ipth level was ( . ae . ) pg/ml. pearson correlation test showed no relation of vit d with bmd. there was moderately negative correlation between ipth and bmd (r = À . ). genotype distribution and allele frequency of subjects were as follows: ( %) ff), ( %) ff, ( %) ff genotype in vdrf -fok gene, ( %) bb, ( %) bb, ( %) bb in vdrb-bsmi gene. allele frequencies were f: %, f: %; b: %, b: %. when fok and bsmi were combined, %(ff-bb) and % (ff-bb) were found as the most frequent genotypes. bsmi frequency was in hardy weinberg equilibrium (p > . ). but foki was not (p = ). it was found that vit d, ipth levels and bmd were in normal levels in all carriers of ff genotype and in combined (ff-bb) type carrying healthy women ( %). the association vdr genotype and bmd may be different in various ethnic and geographical groups. therefore it is worthwhile to assess vdr polymorphism among turkish population. these type of distribution studies of vdr in healthy and in osteoporotic women may enlighten to earlier diagnosis and treatment planning. p- . . - determination of hb a c values in beta thalassemia f. g€ uzelg€ ul , g. s. seydel , a. e. yalin , e. s€ onmez , k. aksoy c ß ukurova university, adana, nigde university, nigde, mersin university, mersin, turkey introduction: hemoglobinopathies are most commonly seen hereditary blood diseases worldwide. our aim was to compare the hba c values measured on cation-exchange high performance liquid chromatography (hplc) in beta thalassemia cases. materials and methods: we collected ml of whole blood k edta containing tubes from forty-nine cases. arms, rflp and dna sequence analysis methodologies were carried out for determination of beta thalassemia mutations. hb a c values were measured using the agilent hplc system. results: forty-nine diabetic and non-diabetic patients were diagnosed with beta thalassemias: twenty-one ivs - /ivs - , one ivs - /ivs - , one ivs - /ivs - , two ivs - /ivs - , two ivs - /ivs - , one fsc /fsc , one fsc /fsc , two - /- , two cd /cd , one cd - /cd - , one cd - /cd - , two cd /cd -g/ cd /cd -g, two ivs - / ivs - , one ivs - / ivs - , one ivs - /fsc , one ivs - /cd , one ivs - /cd , one ivs - /cd - , one ivs - /ivs - , one ivs - / ivs - , one ivs - / ivs - , one ivs - /cd and one fsc /cd . cases were classified as diabetic ( ), prediabetic ( ) and non-diabetic ( ) introduction: members of aurora kinase family aurora a, b and c are conservative kinases of cell cycle which are encoded by genes aura, aurb and aurc respectively. overexpression of aura and aurb was found in human cancers, especially in prostate cancer. moreover, there is the evidence that aurb interacts with one of the major oncogenic kinases -braf. little is known about implication of aurc in cancer, but it was demonstrated, that it can overlap aurb function and shares its location. we studied expression of genes of these kinases in urine of prostate cancer patients aiming to evaluate their involvement in this disease and their potential as tumor markers. materials and methods: urine samples from patients with prostate cancer were gathered after prostate massage before surgical invasion. we used urine samples from healthy men as control. we obtained cells from each urine sample by centrifugation and isolated rna using standard approach with phenol and guanidine thiocyanate. cdna was synthesized and taken to qpcr reactions. data was statistically analysed. results: expression of all studied genes was detected in urine of patients with prostate cancer and of healthy men. expression of aurb and aurc in cancer samples each was higher than expression of aura. the cumulative expression aurb and aurc was higher than expression of aura in samples from . we observed positive correlation between expression of aurc and braf (rs = . , p = . ). discussion and conclusion: previous investigation showed, that for normal prostate tissue % of aurora family expression was presented by aura. we suppose that presence of aurb and aurc cumulative overexpression means presence of cell cycle deviations in prostate tissue of these patients and might be further studied as prognostic marker. in this study we first showed the correlation between aurc and other carcinogenic kinase braf expression, which opens the perspective for investigation of role of aurc in carcinogenesis. bacillus marmarensis sp. nov. is an extreme obligate alkaliphile isolated from mushroom compost near marmara region of turkey. it can survive at extreme ph values up to . . based on its genome sequence, metabolic pathways for proteases, amylases, cellulases, lipase, n-butanol and a biodegradable plastic poly-b-hydroxybutyrate were annotated. in addition to being a potential extracellular hydrolase producer, its ability to survive in the high ph range of . to . makes it an attractive microorganism for different industrial applications. in the current study, the adaptation strategy of b. marmarensis sp. nov. to alkaline conditions was investigated using proteomic tools. the organism was grown at two different ph values, . and ph . . for extraction of whole cell proteins, cells were disrupted with mp bio fast prep device. protein extracts were treated with protease inhibitors and a nuclease mix. salts were removed using a cleanup kit. obtained proteins were separated based of their isoelectric points in the first dimension and then based on their molecular weights in the second dimension. proteins maps of cells grown at these two extreme ph values showed significant differences in protein expression for alkaline adaptation. p- . . - biochemical and proteomic analyses of normal human astrocytes and glioblastoma exposed to dichloroacetate treatment f. c. atilgan, h. cimen yeditepe university, istanbul, turkey glioblastoma (gbm) is an aggressive malignant tumor composed of astrocytes in brain tissue. gbm cells utilize glycolysis rather than oxidative phosphorylation to support rapid growth rate which is called warburg effect. dichloroacetate (dca) is an antiglycolytic agent that inhibits pyruvate dehydrogenase kinase (pdk) activity and induces apoptosis via normalizing the mitochondrial activity. this study aimed to demonstrate the metabolic alterations between the normal human astrocytes (nha) and gbm cell lines which are exposed to dca, and to identify the differentially expressed proteins by ms-based proteomic analyses. nha cell line, u mg and u as gbm human cell lines were examined through analyzing the alterations in the glycolysis metabolism upon dca treatment by measuring the variations in the pyruvate levels, lactate dehydrogenase a, pdk . mts was performed to investigate the effect of dca treatment on cell viability. immunoblotting of pgc -a, oxphos complexes, and mitotracker green staining was employed to reveal the mitochondrial differences between normal and the cancer cells, and upon dca treatment of these cells. proteomic analyses were utilized for the identification of candidate proteins depending on the acetylation status. in this study, compared to nha, the pyruvate and ldha levels were elevated and pdk levels in u mg were reduced by %. due to mts results, ≤ mm dca treatment showed significant decrease in gbm cells compared to nha cells. immunoblotting and mitotracker green staining results showed increase in mitochondrial mass. elevation in the pyruvate and ldha levels and reduction in pdk level in u mg and u cells indicates glycolysis dependent metabolic switch in energy metabolism. proteomic analyses demonstrate that most of the differentially expressed proteins comprised of metabolic enzymes. this study provides novel information about metabolic alterations existing between nha and gbm, which can inspire further studies for therapeutic applications. kidney stone is a complex disease resulting from environmental as well as hereditary factors and principally composes of approximately % calcium oxalate (caox) crystals, which are formed through a multi-step process. vitamin d receptor (vdr) gene encodes the nuclear hormone receptor for vitamin d ; downstream targets of this gene are chiefly contributed in mineral metabolism though the receptor regulates a variety of other metabolic pathways. calcium sensing receptor casr plays an important role in sustaining mineral ion homeostasis. the aim of this study is to profile the expression level of vdr and calcium sensing receptor (casr) genes and to unravel their role in rat kidney stone induced by ethylene glycol, in order to explain the underlying molecular mechanisms. total rna were extracted from paired sample before and after ethylene glycol treated of rats. the mrna expression level of vdr and casr gene were measured employing quantitative rt-pcr (qrt-pcr). the mrna expression levels of both genes were significantly down-regulated according to before treated. in conclusion, our data suggest reduced mrna expression in vdr and casr genes might be a risk factor for kidney stone formation. further studies are necessary to verify these findings in different ethnic groups. p- . . - apj receptor a c gene polymorphism in turkish patients with coronary artery disease against different models of expected frequencies/counts to understand the evolutionary dynamics of saars in proteins. we obtained from ensembl the assemblies of genomes/proteomes of human and nonhuman primates (chimpanzee, gorilla, and rhesus monkey), rodents (mouse and rat), and birds (chicken and zebrafinch). the expected probabilities for the occurrence of saars based on their nucleotide frequencies in coding regions and amino acid frequencies in individual protein sequences or across the whole proteome were compared with the observed repeat occurrences. we found that with all three methods and in all eight species the correlation between observed and expected repeat counts decreased above a saar length threshold. the percentage of saar proteins for each amino acid also exhibited variability among species when both the repeat length and counts were taken into account. however, clustering based on saar characteristics generally reflected the known phylogenetic relationships between species. our comprehensive bioinformatics analyses reveal that saars show amino acid-specific occurrence patterns with respect to species as well as saar length. tissue proteins play important roles in biological metabolic processes. the qualitative and quantitative analysis of tissue proteins facilitates the understanding of molecular mechanisms that differentiate between physiologic and pathologic states. health and research institutions routinely prepare formalin-fixed paraffinembedded (ffpe) tissue blocks for histopathology. proteomics on ffpe tissue still requires standardization of tissue solubilization processes to overcome variability in protein extraction results. our aim is to compare the proteomic studies of fresh frozen and ffpe rat renal tissues. fresh frozen and ffpe preparations from renal tissues were included in this study. an adult rat was sacrificed and the dissected kidneys were divided two equal section. one immediately frozen in phosphate buffer, and the other tissue specimen not thicker than mm to allow rapid penetration of the fixative put in % buffered formalin for hours. the fresh frozen tissue was dissolved and homogenised in the cold phosphate buffer solution containing protease inhibitors. paraffin blocks were performed from formalin fixed tissue specimens. we have extracted the protein from the ffpe tissues using our previously verified method. we have utilized electrophoresis three times to compare protein yield, number, intracellular and intercellular of homogenised samples obtained from ffpe and fresh frozen kidney samples. the number of proteins identified from fresh frozen kidney tissue has generally been shown to be increased compared with ffpe tissue. decrease of the qualitative results in electrophoretic bands was found similar in all replicative studies. ffpe tissues undergo extensive cross linking between protein/ dna/rna molecules during formalin fixation, which creates inter-molecular crosslinks. on the other hand, ffpe tissues represent a valuable resource to carry out retrospective studies aimed to biomarker discovery in kidney cancer as well as other kidney diseases. background: development of atrial fibrillation (af) during the course of chronic primary mitral regurgitation (mr) is common and represents complex molecular mechanisms. however, the gene expression profile of human atrial fibrillation (af) in the setting of chronic primary mr remains uncharacterized. in the current study, we aimed to compare the gene expression profiles of patients with severe degenerative mr in sinus rhythm (sr) and af. methods: left and right atrial tissue samples were obtained from patients with chronic primary severe mr in permanent af (n = ) and sinus rhythm (n = ). we performed a novel micro-dissection technique for thin sections of atrial tissue samples and immediately fresh froze intra-operatively. transcriptomes of left and right atrial appendages of degenerative mitral regurgitation patients with sr versus af were compared by microarray analysis on affymetrix hgu- plus platform. bioinformatics, data mining and pathway analyses were conducted on partek gs and webgestalt. genome-wide gene expression profiles were compared between af and sr groups among . transcripts representing . well-characterized human genes. differentially regulated genes were evaluated according to fold change (fc ≥ . ) with a p-value ≤ . . results: most remarkable pathways altered in af atrial tissues compared to sr group, were extracellular matrix-receptor interaction; mapk, adipocytokine, and calcium signaling; apoptosis and cardiac muscle contraction pathways. conclusions: this is the first human study of comparative transcriptomics in left and right atrial tissues of patients with af versus sr associated with severe degenerative mr. the main findings of this multidisciplinary translational research provide novel candidate targets for the treatment and prevention of af. in order to acquire iron under iron-limiting growth conditions, bacteria employ specific mechanisms such as production and secretion of siderophores. siderophores are low molecular metalchelating compounds that contribute not only to iron scavenging, but also participate in other important processes including oxidative stress response and cell signaling. serratia marcescens, gramnegative bacterium, could be found in various environments, including wastewater, plant rhizosphere and hospital setting where s. marcescens can cause serious life-threatening infections. in this study, we performed a detailed characterization of the siderophores of the clinically important pigment-free s. marcescens strain sr - and environmental pigment-producing s. marcescens strain sm . bioinformatic analysis of these genomes by antismash software revealed the presence of several clusters involved in non-ribosomal peptides synthesis (nrps). we found four nrps clusters in genome of s. marcescens sm . cluster has a low level of identity to enterobactin gene cluster typical for bacteria producing catechol-like siderophores. second cluster has only % of identity to xantholipin biosynthetic gene cluster. clusters and of nrps genes of s. marcescens sm did not show any homology to known nrps clusters. in contrast, the genome of s. marcescens sr - contains only one genetic cluster of nrps genes. this cluster does not have similarity to any of the known bacterial nrps genes. thus, genetic analysis of two isolates of s. marcescens allowed us to identify nrps genetic clusters and showed that the repertoire of these genes is different between strains. we hypothesized that the strain isolated from environment has competitive advantage over clinical isolate due to genetic diversity of siderophores. on the other side, clinical isolate has specific genetic cluster of siderophores which may promote s. marcescens growth and adaptation to the extreme niches present in medical facilities. p- . . - the first glance on the genome's structure and activity in hibernator edible dormouse hibernation is a unique adaptive way of survival in extreme environmental conditions where mammals decrease their metabolic rate and demonstrate physical inactivity for prolonged periods of time (up to - months). remarkably, some hibernating animals have a long average lifespan and the ability to avoid muscle atrophy caused by disuse or immobilization. to identify main molecular pathways behind the protective musculoskeletal adaptation and genome structure in hibernator edible dormouse (glis glis), whole-genome analysis of mrna expression in muscles (m. soleus and m. edl) and lumbar spinal cord samples was conducted. three groups of the dormice: ) active animals ) hibernated animals and ) animals immobilized for weeks in laboratory, were examined. rna libraries have been sequenced using hiseq illumina platform. coupled with genome dna sequencing provided x coverage of the estimated genome, we have assembled de novo transcriptome of the dormice. differentially expressed genes in response to immobilization and hibernation were determined. transcriptional program of these phenotypes was similar. pathways enriched by differentially expressed genes were identified. gene expression of the key muscle proteins and muscle atrophy markers was analyzed. muscle-specific e -ubiquitin ligases murf and mafbx revealed no changes in mrna expression. our study represents the first attempt to elucidate changes in transcription profiles of skeletal muscles and spinal cord during hibernation and hypokinesia in edible dormice. in corroboration to the gene expression data, they demonstrated minimal morphological evidence for muscle disuse atrophy during physical inactivity. edible dormice, thus, can be considered as a novel model organisms in investigation of the genetic mechanisms of hibernation and prevention of muscle atrophy. the work is performed according to the russian government program of competitive growth of kfu and supported by rfbr jsps_a no. - - . in response to diverse environmental cues bacteria form complex structured communities called biofilms. the metabolic pathways activated by these cues are remarkably different depending on the species studied. however, they all lead to the formation of an extracellular matrix that holds the cells together. non-pathogenic gram-positive spore-forming soil b. subtilis strain is recognized as a model system for the study of biofilms. to discover the pathways regulating biofilm formation in b. subtilis, we studied the natural isolate of b. subtilis strain , and constructed the recombinant strains with knocked out genes of following regulatory proteins: abrb (global transcriptional regulator), degu (two-component response regulator of signal transduction system degs-degu), ccpa (regulator of carbon catabolism) and spooa (regulator of sporulation). in the minimal medium broth b. subtilis wild-type strain forms biofilm with its maximum on th hour of culture growth. ph-optimum for biofilms formation by the wild-type strain is in the range of . - . . the temperature optimum is in the range from °c to °c. this corresponds to the natural conditions of the b. subtilis habitat in rhizosphere. the level of biofilm formation by regulatory mutant strains with deleted abrb, degu, ccpa, spooa genes is on average % lower than by the wild-type strain. this indicates that global regulatory system controlls biofilm formation process. statistically significant differences in the levels of biofilm formation between regulatory mutants haven't been identified. ph and temperature optima of mutant strains are the same as for the wildtype strain - , - and °c - °c respectively. the crataegus genus which is a member of rosaceae family, has approximately species worldwide and species in turkey. all plant species in this genus have the common name "hawthorn". crataegus microphylla (c. microphylla) c. koch which is characterised by having erect sepals in fruit and smaller leaves in comparison with the other species, is one of the wild edible fruits in turkey. crataegus species have been used as food and also in folk medicine for the treatment of various diseases. for this purpose, the potential biological properties of crataegus microphylla were aimed to reveal by the preliminary work. in this study, prevention of oxidative dna damage using supercoiled pbr plasmid dna, acetylcholinesterase, tyrosinase, a-glucosidase inhibition and antioxidant effects: , diphenyl- -picrylhydrazyl radical scavenging effect, phosphomolibdenum-reducing antioxidant power, ferric-reducing antioxidant power with total phenolic and total flavonoid contents of the c. microphylla leaves, stem barks and fruits that extracted with ethanol, methanol and water were investigated. the experiments of oxidative dna damage studies and antioxidant activities of c. microphylla extracts showed that methanol and ethanol extracts possessed a strong ability to prevent dna damage and significantly antioxidant activities. methanol extracts of stem barks from c. microphylla exhibited the highest acetylcholinesterase and tyrosinase activities ( . ae . % and . ae . %, respectively), at lg/ml. in addition, ethanol extract of leaves from c. microphylla inhibited the a-glucosidase activity significantly when compared to acarbose. this study explained significant antioxidant, enzyme inhibitory, hypoglycemic, and neuroprotective activities of methanolic or ethanolic extracts prepared with stem bark and leaf from c. microphylla and also strong ability to prevent dna damage that corresponded to antioxidant potential of methanol extracts of leaf and stem bark. the yarrowia lipolytica species (yl) is nonconventional yeast widely used for recombinant protein expression due to its system of post-translation protein modification, which is the most similar to that of higher eukaryotes. yl appears the promising producer of recombinant proteins with much more complicated molecules compared to those of prokaryotic producers. however, an important feature for a producer strain of recombinant proteins is the genes, the expression of which undertakes under controlled conditions, and consequently, search of new effective inducible promoters in the yl genome is of great interest. proteome analysis of the yl cells grown at different ph values ( . , . , . ) showed that under alkaline conditions the amount of mitochondrial porine vdac (voltage dependent anion channel), one of the most abundant protein of the mitochondrial outer membrane, increased significantly. vdac is supposed to let reactive oxygen species out of mitochondria protecting the cell against oxidative stress. therefore, the por gene expression, encoding vdac should increase in the stress conditions. the promoter of the por gene was used to construct some new expression systems based on yl w . a new genetic construct bearing a reporter bgalactosidase gene under control of the por promoter. b-galactosidase activity was assayed in the cells grown in various ph conditions and exposed to exogenous oxidants such as hydrogen peroxide, menadione, and methyl viologen. it was shown, that in h o and methyl viologen treated cells b-galactosidase activity increased . - . -fold reaching its maximum in the cells, grown at ph of . . thus, we demonstrated high inducibility of the por promoter, which is essential for effective action of the expression system based on it and potency of application for transformed lines of producers. acknowledgments: supported by the russian foundation for basic research (grant no - - mol_a). aspergillus nidulans is able to detoxify and catabolize the toxic proline analogue, lazetidine- -carboxylic acid in nature, azc serves as a plant protectant against infections and consumption. we have obtained evidence that azc is not only non-toxic for the model ascomycete aspergillus nidulans, but it can be utilized as a poor nitrogen source. in order to elucidate the molecular mechanism underlying azc detoxification, we have constructed and studied a. nidulans strains deleted in the cognate genes involved in azc detoxification in pseudomonas and saccharomyces cerevisiae. these genes, found by in silico analysis, encode a putative hydrolase, acha, and an azc acetyltransferase, ngn , respectively. gene deletion was accomplished through double crossover. a cassette containing the~ bp ' and ' flanking sequences of each gene, with the afpyrg gene as a selection marker, was contructed. crossing the achad and the ngn d strains isolated the achad ngn d double mutant strain. rt-pcr was used for gene expression analysis in the wild type strain, area-loss of function and crea-derepressed mutant strains. our results clearly show that azc can be used as a poor nitrogen source by a. nidulans. this utilization requires a) acha, a putative azc hydrolase, and b) a fully active gaba catabolic pathway, as lack of either amdr or gata abolishes azc utilization. most importantly, the double mutant, achad ngn d, shows azc toxicity, suggesting that ngn is a true orthologue of mpr , able to detoxify azc, a phenotype that can be observed only in the absence of acha. as a final point, ngn was shown to be induced by the presence of azc and and to be under nitro the spatial genome organization plays a great role in the maintenance of the nuclear architecture and regulation of all processes occurring in the nucleus. this system is controlled by a set of special proteins having an architectural function. however, the mechanisms of their action remain unknown. among these proteins are, in particular, zad-domain-containing proteins. zinc finger-associated domain (zad) is a ubiquitous motif of c h zinc finger proteins of drosophila. genes that encode zad proteins are specific for and expanded in the genomes of insects. only a few zad-encoding genes have known functions, and the role of zad is being discussed. up to date there was only one known crystal structure of zad-domain from drosophila transcription factor grauzone (grauzad). here, we present for the first time the crystal structure of the zad-domain of serendipity-d transcriptional activator of the egg-polarity gene bicoid. zad-domain was cloned, overexpressed in e. coli, purified and the structure was solved at . a by mad technique. detailed analysis of the structure proved that the protein exists in dimeric form and revealed unique spatial organization of the protein, different from those for grauzad. this work is supported in part by russian ministry of education and science grant ( . . . ). mycoplasma gallisepticum is a convenient model object for studying the regulation of transcription because it has a reduced genome, lack of cell wall and many metabolic pathways and also easy to culture and non-pathogenic to humans. due to the nature of the genomic organization and the loss of many of the known regulators, the effect of disrupting the function of some proteins may be a useful tool for studying the regulation of transcription. the gene expression study was performed on agilent onecolor microarray with custom design and random-t polymerase primer for cdna synthesis. microarray represents probes for orf including genes and ncrna. in this work, we have investigated the effect of changes in the level of gene expression of m. gallisepticum for two different types of conditions: a genetic knock-out mutants and the cell response to treatment with sub-lethal concentrations of antibiotics. we characterized transcription of m. gallisepticum when the cell responses to dysfunction of proteins with metabolic potential, possible regulators of expression, in violation of permeability of membrane by cccp, inhibition of ribosomal synthesis by tetracycline, dna gyrase by novobiocin and atp synthase by oligomycin. the data obtained allow to characterize the transcriptional response under different conditions and to identify groups of genes that change expression together. major transcriptional changes were observed in the response of cells under cccp treatment due to uncoupling of the proton gradient and further reducing the membrane potential, as well as under novobiocin treatment due to changing the topology of dna. global problem of oil pollution forces scientists to search for a new safe remediation technologies constantly. careful attention is paid to bacteria, some of which possess additional biotechnologically valuable properties, such as utilization of hydrocarbons and production of biofurfactants. in this regard, we carried out proteogenomic characterization of tsukamurella tyrosinosolvens strain ps , which was isolated from chemical sludge and capable for alkane degradation and biosurfactant production. whole genome of the strain was sequenced on the miseq (illumina) platform, assembled and annotated. proteome on mineral medium with glucose, sucrose and hexadecane as a sole carbon and energy source was studied. shotgun proteomics approach was performed on hybrid chromatography-mass spectrometry machine (maxis impact). alkane oxidation genes (alkane- -monooxygenase, rubredoxin and rubredoxin-reductase) under genome sequence, as well as two pathways of trehalose synthesis and genes for mycolic acids production were found. emulsification activity of cell-free culture liquid was about four times higher on hexadecane in comparison with sugars. proteomic profile was different at various culture conditions. all glycolysis genes, beginning with glucose- -phosphate isomerase to pyruvate kinase, were found on the media with sugar. the medium with hexadecane helped to reveal enzymes involved in the beta-oxidation of fatty acids, for example , -dienoyl-coa reductase, -ketoacyl-coa thiolase and enzymes of the initial mycolic acid synthesis pathways. thus we have established that the strain t. tyrosinosolvens ps utilizes sugar by glycolysis. also, the bacterium is capable for alkane oxidation followed by beta-oxidation of fatty acids. based on the proteogenomic data, we assume that the bacterium is able to synthesize trehalose lipids, namely, trehalose mycolates. obtained results could be useful to create conditions for increased biosurfactants production. gestational diabetes mellitus (gdm) is a glucose intolerance firstly diagnosed during pregnancy. in this study, we aimed to investigate the association between serum adiponectin, resistin levels and insulin resistance in gestational diabetic patients. a total of patients; healthy pregnant women (control group) and pregnant women diagnosed with gdm (gdm group) were included in this study. serum adiponectin, resistin, glucose, insulin, hba c levels and lipid parameters were measured. insulin resistance index homa-ir values were calculated. in this study, serum glucose, insulin, hba c levels and homa-ir were significantly higher in gdm group compared to the control group (p = . , p = . , p = . , p = . , respectively). serum adiponectin levels were significantly lower (p < . ); whereas serum resistin levels were significantly higher (p = . ) in gdm group than in the control group. it can be concluded that resistin contributes to the formation of insulin resistance, adiponectin plays an important role in the regulation of this resistance and they also have effects on gdm pathophysiology. hematological cancers including acute myeloblastic leukemia (aml) and acute lymphoblastic leukemia (all) in terms of incidence and mortality, are the second most important cancer type in turkey. numerous studies show that cancer patients respond differently to treatment thus supporting the idea of personalized therapy need for individuals. renin angiotensin system (ras) have key roles in aml and all progression and it has been shown by many studies suggests that these system's genes might be good biomarkers for aml and all personalized therapy. we aimed to identify ras gene based homogeneous subgroups of acute leukemia and determine the most effective chemotherapoetic agent for each subgroup. after validation and verification of the results, more effective drugs can be recommended for the use in clinics for chemotherapy of aml and all. results of our preliminary studies showed that we are able to identify subgroups of aml and all as well as correlating each existing subgroup with fda approved drugs. considering the long and highly cost process of developing new drugs for cancer treatment makes the present study all the more valuable. in addition, there is a serious need for change in aml and all therapy since there is no highly effective chemotherapy protocol available for their treatment. welcome trust sanger (wts) and cancer cell line encyclopedia (ccle) databases will be used to determine subgroups of aml and all based on ras genes or whole genome expression using standard deviation and hierarchical clustering analysis. the most effective drugs for each subgroup will be identified using pearson's r correlation analysis with drug sensitivity data (ic , ic , amax, aare, etc.) available in same databases. further validation tests will be performed by in vitro validation using aml and all cell lines: drug sensitivity profiles will be determined and gene expression will be shown by q-rt-pcr. p- . . - functional polymorphisms of ephx in a turkish population h. pinarbasi, i. sari cumhuriyet university, sivas, turkey soluble epoxide hydrolase (seh; ec . . . ) is encoded by ephx and catalyses the degradation of endogenous fatty acid epoxides generated by cyp epoxygenases. these fatty acid epoxides such as epoxyeicosatrienoic acids (eets) have been shown to posses vasodilator, anti-inflammatory, anti-platelet, anti-hypertensive, anti-apoptotic, anti-thrombotic and natriuretic effects. it has been reported that eet levels are associated with hypertension, stroke and cardiovascular diseases. individual differences in the ephx gene that affect the seh activity may alter the circulating levels of eets. k r and r q polymorphisms have been known to cause increased and decreased seh activity, respectively. therefore we aimed to determine the genotype frequencies of these two polymorphisms in a turkish population. k r and r q polymorphisms were determined by the real time pcr using double-dye hydrolysis probes or pcr-rflp method. the observed genotype frequencies for k r polymorphism were . % wild type (aa) and . % polymorphic genotype (ag+gg) and for r q polymorphism . % wild type and . % polymorphic genotype (ga+aa). the genotype distributions for both polymorphisms were in hardy-weinberg equilibrium. pregnancy is one of manifestations for thrombophilia factors, which in its turn leads to various complications of its course. one of the markers of hereditary thrombophilia is mutations in the folate cycle mtr, mtrr and mthfr genes. insufficient intake of folate during pregnancy disrupts the functioning of the genome, leading to miscarriage, violation of embryogenesis and various fetal malformations. however, results of studies on the role of hereditary thrombophilia in the occurrence of complications during pregnancy are rather contradictory. aim of this study was to determine the frequency of alleles and polymorphic variants of folate cycle genes mtr a g, mtrr a g and mthfr c t in women of kazakh ethnic group with pregnancy complications. we used real-time pcr. blood samples for dna isolation were obtained from pregnant women. the main group consisted of women (n = ) which had a history of two or more pregnancy complications in the form of pre-eclampsia, eclampsia, missed abortion, miscarriage, and etc. control group consisted of women (n = ) with two or more normal pregnancy outcomes, and had no complications during pregnancy in history. average age of women in experimental group was . ae . years compared with control of the age . ae . . the analysis of the frequency distribution of alleles of genes in experimental group of women with complications of pregnancy revealed no significant differences relative to the control group. analysis of the distribution of polymorphic variants of folate cycle genes showed significant difference between the study and control groups in the occurrence frequency of heterozygotes for the mutant allele g in the gene mtrr a g (or = . , ci % = . - . ; v = . , p < , ). no significant differences in alleles between homozygous wild-type and homozygous mutant alleles were observed. this work was funded by the mes kazakhstan (gr rk project number). p- . . - a study on the association between rs polymorphism in connective tissue growth factor gene and pseudoexfoliation syndrome pseudoexfoliation syndrome (pes) is a disorder of the extracellular matrix characterized by the production and progressive accumulation of an abnormal fibrillary material in many ocular tissues. pes prevalence is . % above the age in turkey. since pes is characterized by excessive synthesis of elastic microfibrillar components throughout the body, growth factors can have important roles in the pathophysiology of pes. human connective tissue growth factor (ctgf) is a protein expressed in a variety of tissues, including the anterior chamber of the eye. ctgf coding gene has several genetic polymorphisms. rs g/c single nucleotide polymorphism (snp) is found at position À , in promoter region. the presence of a c allele for rs is critical for transcriptional suppression of the ctgf gene which would reduce ctgf production. aim of this study was to investigate if there is any association between pes and rs polymorphism of the ctgf gene. study population consisted of patients with pes and controls. blood samples were collected by g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. genomic dnas were isolated from whole blood samples using manual dna isolation. the frequency of ctgf rs polymorphic allele g was . in patients, and . in controls ( . , p = . ). distribution of genotypes was gg: . %, gc: . % and cc: . % among patients, while gg: %, gc: . % and cc: . % (or = . , p = . ) in controls. statistical analysis showed that there is no significant relationship between ctgf rs snp and pes. these are the preliminary findings of a research project which is the first study analyzing the relationship between ctgf rs snp and pes. this work did not point out a role for ctgf rs in the risk for pes. a significant relationship might be found when the study population is enlarged. p- . . - evaluation of rs single nucleotide polymorphism of clusterin gene in pseuodoexfoliation syndrome risk pseuodoexfoliation syndrome (pes), an age-related systemic disorder, is characterized by production and accumulation of abnormal fibrillar extracellular material in anterior structures of the eye. clusterin (clu) is a multifunctional glycoprotein produced and secreted by almost all cell types and is found in all body fluids and in accumulated pes material. under cellular stress conditions, clu provides inhibition of stress-induced precipitation and aggregation of misfolded proteins. clu expression level in pes patients is unexpectedly low and this could be due to single nucleotide polymorphisms (snp) on the gene coding for clu. rs c/t polymorphism has been found to be associated with alzheimer's disease and pathophysiology of alzheimer and pes are similar. this study aimed to determine whether rs snp of clu gene have a role in the development of pes. study population consisted of patients with pes and controls. blood samples were obtained from g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genomic dnas were isolated from whole blood of subjects using manual dna isolation. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. t allele frequency of pes patients was . and that of controls was . ( . , p = . ). the distribution of genotypes was cc: . %, tc: . % and tt: . % among patients while cc: . %, tc: . % and tt: . % ( . , p = . ) in controls. there was no statistically significant difference between pes patients and controls in terms of tt genotype and t allele frequency. these are the preliminary findings of a research project which is the first study analyzing the relationship between clu rs snp and pes in turkish population. this work did not point out a relation for polymorphic genotype in the risk for pes. however, a relationship between clu rs polymorphism and pes can be found when we enlarge the study population. the tumor suppressor tp is the most frequently mutated gene in head neck squamous cell carcinoma cancer and represents a known transcription factor and tumor suppressor gene that regulates different microrna and target genes. the aim of our work is to construct the transcriptional and post-transcriptional network regulated by tp and to evaluate the difference at mrna and protein expression levels of the tp target genes in hpv negative head and neck squamous cell carcinoma (hnscc) patients with distinct tp mutation states and to elucidate the molecular mechanism that underlie the poor prognosis of tp mutation. to show the tp mutation landscape and its prognostic relevance for survival, we used cbioportal for cancer genomic analysis. we downloaded mutational profiles of hpv negative hnscc patients. employing different databases we constructed the tp regulatory network. and then, to evaluate the effect on mrna, protein and microrna regulated by tp we used the mrna and protein expression profiles of patients from tcga. our results show that hotspot, truncating and missense mutations have statistical significance in the univariate analysis. the tp regulatory network show the involvement of important target involved in the progression of hnscc and the deregulation of protein expression of an important key epigenetic modifier ezh was significantly associated with tp mutational state. ezh is a member of the polycomb group protein enhancer zeste homolog which is known to be directly repressed by tp and indirectly by the activation of mir- a and mir- b. we found a significant up-regulation of ezh that depend from tp mutation. it is important to understand the difference in mrna and protein expression of tp regulatory network that could depend from its mutational state. this finding suggest that ezh might be a potential therapeutic target for hnscc. p- . . - next generation sequencing based approach for monitoring of minimal residual disease in acute lymphoblastic leukemia minimal residual disease (mrd) monitoring is widely used to evaluate efficiency of chemotherapy and to choose a strategy for further treatment in acute lymphoblastic leukemia (all). the most commonly used approaches for mrd detection are based on flow cytometry and qpcr. these methods have several important limitations including insufficient sensitivity, complicated experimental setup and false positivity. the newly developed next generation sequencing (ngs) approaches could overcome the existing limitations in mrd monitoring. here we describe a new mrd monitoring approach based on targeted deep sequencing of malignant rearrangements. first, we identified bcr/tcr rearrangements specific for the leukemic clones in initial bone marrow samples of all patients. for this, we used sanger sequencing of the products of multiplex pcr, performed with bcr/tcr specific primers combined according to the optimal frequency distribution of v/j-genes in healthy donors. second, we analyzed concentration of malignant clone rearrangements, identified at the first step, in dna samples obtained from bone marrow after days of treatment. for this purpose, we performed ngs of libraries for each identified leukemic rearrangement. four libraries were amplicons of bcr or tcr gene rearrangements generated using characteristic v and j segment specific primer combination. six additional libraries were amplicons of the same primer combination from the same dna sample which contained initial leukemic dna spike-in (in concentrations corresponding to per , and , cells) for a calibration curve generation. using this approach, we analyzed all clone specific rearrangements for three patients and calculated concentration of the leukemic clones by using the calibration curve. for one patient we didn't find any leukemic cells and for two patients we found leukemic cell per , analyzed cells. znf is considered as a transcriptional target for p and plays an important role in the homologous recombination, mitosis, centrosome dynamics. as was shown by gwas some snps in znf have strong association with the risk of breast cancer (bc). however, it was unclear whether the same snps are associated with risk of bc in kazakhstan. therefore two polymorphisms (rs , rs ) of znf were investigated in kazakh population in this study. the present case-control study was carried out with participation of kazakh females with bc and cancer-free donors. additionally, subtypes of bc, stratified by estrogen receptor (er+/À), progesterone receptor (pr+/À) and human epidermal growth factor receptor (her +/À) status were estimated. pearson p-value, odds ratio, % confidence interval tests were applied to data analysis. significant differences were found in allele frequency and genotype distribution at rs locus in znf between the patients and control groups (p = . for allele; p = . for genotype). moreover, significant association with bc was revealed for rs after dividing patients according to er+/ À, pr+/À and her +/À status of the tumor. the g allele was associated with er+ (p = . , or = . , %ci: . - . ), pr+ (p = . , or = . , %ci: . - . ) and gg genotype with her -bc carriers (p = . , or = . , %ci: . - . ). also, g allele can be considered as a risk factors in er+/ pr+/her -luminal type of tumor (p = . , or = . , % ci: . - . ). our findings correlate with the data of several gwas where the association of the rs polymorphism with higher mammographic density and the risk of breast cancer have been shown. the obtained results allow us to consider g allele and gg genotype of rs as a marker of bc risk with predictive value, restricted to er, pr and her status of the tumor in the kazakh population. breast cancer (bc) is the most common cancer among women in most of countries. alternative variants of low-penetrance genes such as fgfr (rs ), tox (rs ), map k (rs ), lsp (rs ) are shown to have high frequency in north america, south-east asia, australia, europe populations and a multiplicative effect on the development of bc. in this study was investigated assosiasion between alleles/genotypes combinations of these genes with increase/decrease of bc risk. the case-control study included bc patients and healthy women from kazakh and russian populations. genotyping was performed by pcr-rflp methods. combined effect of allele and genotype variations in four different genes on bc risk was assessed by apsampler algorithm. the fisher exact test, odds ratio (or) with % confidence intervals ( % ci) were applied to data analysis. according to obtained results combinations of allele c of tox rs and a of map k rs (p = . , or = . ), also allele c of tox rs and c of lsp rs (p = . , or = . ) associated with increased bc risk in the russian population. consequently, combinations with c allele of tox rs contribute significantly to bc risk with p-value = . , or = . . on the contrary, tt genotype of tox rs with p = . , or = . and its combination with allele t of lsp rs with p = . , or = . determine a bc risk reduction in russian population. in addition, a risk combination of allele c of lsp rs and a of map k rs was found in kazakh population (p = . , or = . ). studies have shown that a genetic predisposition to bc can be determined by the cumulative effect of individual alleles and genotypes and possible epistatic interactions of studied genes. obtained combinations of alleles and genotypes can be considered as complex genetic markers of bc and may be used as predictive. cancer that is caused by excessive proliferation of cells and reduced apoptosis is a pathological condition. currently, studies that are comitted with breast cancer are great important early detection and diagnosis of breast cancer. after the discovery of cisplatin as chemoterapy drug, new transition metal based complexes have been developed for treatment of cancer. in this study, anti-cancer activity of azo-azomethide ligand and its mononuclear metal complexes is studied on human cancer cell lines (mcf- ) and mouse fibroblast (l ) cell lines. cells were studied four different concentrations ( , ; ; ; lm). xtt ( , -bis-( -methoxy- -nitro- -sulfophenyl)- h-tetrazolium- -carboxanilide) protocol was applied after and hours. in our study % fetal bovine serum (fbs), % l-glutamine, iu/ml penicillin and mg/ml streptomycin in dmem (low glucose) were used. cancer cell lines in dmem medium is produced in % humidity and % co incubator at °c. anti-cancer activity of synthesized complexes were determined on mcf- and l . in the biological activity studies, synthesized compounds showed higher anticancer activity than positive control ( -fu). finally, our new synthesized complexes can be suggested that potent ajan for anti-tumuour for breast cancer drugs. large interindividual differences in response to chemotherapy present an important issue in cancer treatment. malignant mesothelioma (mm) is an aggressive tumor with poor prognosis, treated mostly with gemcitabine/cisplatin (gem/cis) or pemetrexed/cisplatin (pmx/cis) chemotherapy. as both clinical characteristics and genetic variability may affect treatment outcome, our aim was to construct and validate clinical-pharmacogenetic prediction models of treatment outcome in mm for both chemotherapy regimens and to develop an algorithm for genotype-based treatment recommendations. clinical-pharmacogenetic models were built on gem/cistreated and pmx/cis-treated mm patients. pharmacogenetic scores were assigned by rounding the regression coefficients. gem/cis model was validated on independent mm patients. model predicting outcome of gem/cis chemotherapy included crp level, histological type, performance status, rrm rs , ercc rs , ercc rs , and xrcc rs . values ranged between and . ; cutoff value of . had sensitivity of . and specificity of . . patients with higher score had shorter progression-free and overall survival (p < . ). in the validation group, positive predictive value was . and negative predictive value was . . model predicting outcome of pmx/cis chemotherapy included crp level, mthfd rs , and abcc rs with scores ranging between and . . cutoff value of . had sensitivity of . and specificity of . . patients with higher score had lower probability of good response and shorter progression-free survival (p < . ). clinical-pharmacogenetic models could enable stratification of mm patients based on their probability of response to gem/cis or pmx/cis and improve treatment outcome. this approach could be used for translation of pharmacogenetic testing to clinical practice as it would facilitate the selection of the best treatment option for each patient. p- . . - evaluation of anti-diabetic potential of circiliol and circilineol using caco cell line diabetes mellitus is a metabolic disorder, and many people are suffering from this disease in the worldwide. oral hypoglycemic agents such as sulfonylureas and biguanides are currently used for the treatment. however, studies searching for a more effective anti-diabetic agents are being carried out continıously. based on that we aim to investigate the potential anti-diabetic effects of circilineol and circiliol isolated from teucrium alyssifolium extract, using in vitro cell culture models. for this purpose, the anti-diabetic actions were investigated by applying model caco (colorectal adenocarcinoma) cell line. we determined the level of ag (alpha-glucosidase), sglt (sodium-glucose transporter- ) and glut - and glucose transport. neither ag activity nor sglt activity was increased with either circiliol or circilineol treatment in caco cells compared to positive control. similarly, neither the activity nor the expression level of glut *- was increased in caco cell line with either circiliol or circilineol treatment relative to control. in conclusion, these results strongly suggest that circiliol and circilineol do not possess any anti-diabetic potentials.supported by tubitak z and paubap fbe the purpose of this study was to characterize and assess the impact of a novel magnetite (fe o ) nanosystem functionalized with the natural origin compound eugenol (e) on the pseudomonas aeruginosa virulence, biofilm formation and qs signaling in order to advance research aimed to find alternative and personalized therapeutic approaches for severe infections produced by this opportunistic pathogen. fe o nanoparticles were obtained by a co-precipitation method and functionalized with analytical purity e. functionalized nanoparticles (fe o @e) were characterized by ir, sem, tga and hr tem. one laboratory and p. aeruginosa clinical isolates were utilized in the study. growth and biofilm formation were assessed by an adapted microdilution method followed by absorbance reads and viable count analysis in dynamics ( , , and hours of treatment). soluble virulence factors production was assessed by enzyme activity evaluation of bacteria grown on specific media. the expression of qs core genes was analyzed by qrt-pcr and a luminescence assay. results demonstrated that the average size of the obtained nanosystem ranges - nm, particles are relatively homogenous and have a low tendency to form aggregates. subinhibitory concentrations of fe o @e limited biofilms formation in a time and strain dependent manner, and significantly inhibited the production of toxin pore forming enzymes (haemolysins and lipases) in most strains. the expression of lasi and lasr genes was three fold downregulated, while the expression of pqsr was upregulated in planktonic cultures suggesting that pqs signaling may be involved in virulence modulation after nanoparticle stimulation. the modulation of bacterial virulence and molecular signaling by functional nanoparticles utilized in subinhibitory amounts offer valuable perspectives to develop personalized antimicrobial approaches based on molecular communication control that clearly modulate pathogenicity and progression of the infectious process. p- . . - specimen processing and handling for plasma ammonia measurement b. sarac ßligil , , s. abus ßo glu , f. aky€ urek , b. € ozt€ urk selc ßuk medical school, konya, biochemistry department, selcuk university faculty of medicine, konya, turkey objectives: ammonia requires special processing and handling conditions due to its' unstability. in this study, our aim to investigate a preanalytical factor (delayed analyze) affecting plasma ammonia measurement. design and methods: blood samples were obtained from healthy volunteers. for determining different handling and storage conditions, the following protocols were applied: first protocol (a) transportation on ice and separation (centrifugation at °c) within minutes of collection and analyze immediately. second protocol (b) transportation on ice and separation (centrifugation at °c) within minutes and analyse refrigerated - °c hours (a , b ) and hours (a , b ). all plasma ammonia levels was analyzed enzimatic glutamate dehiydrogenase methods by abbott architect c clinical chemistry analyzer. results: there were statistically alterations in all protocols compared to first protocol. prolonged centrifugation time for plasma ammonia lead to have higher results ( . versus . lg/dl, p < . ). in all protocols including a , a , b , b also cause an elevation in plasma ammonia results ( . , . , . and . ; p = . , p < . , p < . , p < . , respectively). conclusions: ammonia concentration in the blood sample increases over time due to high concentrations in cells as erythrocyte or platelets (three fold). blood samples collected for ammonia determination should be stored on ice, and measured immediately. the wnt/b-catenin signaling pathway has been considered to be a factor in the development and progression of colorectal cancer. many studies have demonstrated that the presence of mutations or polymorphisms in ctnnb (gene encoding b-catenin; mostly mutations in exon ) can lead to aberrant activation of wnt/bcatenin signaling at the onset of various types of malignancies, including colorectal cancer. the aim of our study was to assess ctnnb alteration in the patients with colorectal cancer and compared their tumor and normal tissues. a total of paraffin-embedded colorectal tumor specimens were obtained from department of pathology in cerrahpasa medical faculty. also a total paraffin-embedded normal tissue was used from same cases as a control group. ten-micrometer-thick tissue sections were placed on a glass slide and stained with he. then the tissue sections were dehydrated in graded ethanol solutions and dried without a cover glass. dna was extracted from the tissues with ll of extraction buffer at °c over night. the tubes were boiled for min to inactivate the proteinase k. ctnnb exon was amplified by pcr. sscp is used to observe any difference between the groups. genomic dna was isolated as described above. ll of each pcr products mixed with ll denaturating buffer and were denatured by heating at °c for minutes in, and then were rapidly cooled on ice. the denatured pcr samples are run on % acrylamide/ bis gel in . tbe buffer for . hours at v at room temperature with water cooling system. the gel was stained with silver staining method. migration and adhesion involve continuous modulation of cell motility, beta-catenin play major roles. beta-catenin gene alterations frequency range between and % in colorectal cancer according to the different published studies. in our study no significant differences were found in the ctnnb exon between the tumor group and normal groups. p- . . - theranostic approach in agressive recurrent meningiomafirst experience in turkey m. o. demirkol , b. uc ßar koc ß university, istanbul, american hospital, istanbul, turkey meningiomas arise from the meningothelial cells of the arachnoid membranes of the leptomeninx, which are attached to the inner layer of the dura mater. meningiomas can be classified into three grades (i-iii): grade i meningiomas which are benign, exhibit slow growth; grade ii (atypical) and grade iii (anaplastic) meningiomas, which have a much more aggressive clinical behaviour. meningiomas express non-steroid hormones, including somatostatin. in the brain, somatostatin-a cyclic tetradecapeptide neuropeptide-is believed to act as a neurotransmitter and neuromodulator. somatostatin performs its physiological functions by binding to specific receptors (sstri-sstrv). sstr exhibit high affinity for octreotate (tate). tate is a polar, watersoluble peptide that does not penetrate the intact bbb (brain-blood barrier). pet and scintigraphic imagings can only demonstrate somatostatin receptor positive intracranial lesions if the bbb is disrupted. in this aim, dotatate (dota-dphe , tyr -octreotate, tate) has been labelled with the positron emitter ga and the beta and gama emitter lu. in this case, we conducted a study to evaluate peptide receptor radionuclide therapy (prrt) planning based on pet/ct imaging of meningioma in the department of nuclear medicine and molecular imaging at the amerikan hospital. [ ga] dota -tyr -oc-pet/ct has been established as the imaging modality of choice for the diagnosis and management of patient with skull-base malign meningioma (rapid progress -to mm. from mm. in d.-after fifth operation). due to its high sstr selectivity, [ lu]-tate showed significantly lower uptake/dose delivered to normal tissues, gatate-pet represents the imaging strategy of choice for an accurate assessment of sstr expression levels. although some studies have not shown a clear advantage over pet/ct, there is some evidence that it will have an advantage in selected body sites such as the head and neck, liver, and the pelvis. p- . . - cardiovascular diseases can be treated by using 'tetr-odd-vp ' and 'hre' hypoxia inducible systems a. celik , t. kaya , s. cigdem , m. g€ und€ uz department of medical genetic, turgut ozal university, ankara, faculty of medicine, turgut ozal university, ankara, turkey ischemia is an insufficient supply of blood to a tissue or organ, usually due to a blocked artery by a blood clot. up to now, the number one cause of death worldwide is caused by ischemia and related conditions such as heart attack or stroke. hif- a is a transcription activator that functions as a master regulator of oxygen homeostasis. hif- a protein levels increase under hypoxic conditions as a result of decreased o -dependent prolyl-hydroxylation, ubiqutination and degradation. we aimed to break up clots in blood vessels and to prevent damage caused by ischemia by using hypoxia inducible systems. we added oxygen dependent degredation domain (odd) of hif a between and in front of tetr dna binding domain and vp transactivation domain, so that tetr-odd-vp or odd-tetr-vp could activate transcription of tissue plasminogen activator (tpa) controlled by tetracycline response element (tre), in a hif a independent manner. in addition, we also designed tissue plasminogen activator (tpa) under control of hypoxia response element (hre) of hif a target genes. western blotting and immunofluorescence assay results showed the expression and nuclear localization of tetr-odd-vp and odd-tetr-vp constructs under hypoxic conditions, but not normoxic. in addition, using fluorometric reporter systems and tpa enzymatic assay we proved functionality of these constructs under hypoxic conditions. final apporoach to our project is predicting kinetic enzymatic activity of tpa during break up blood clots by using matlab. the results of the present investigation showed, the developed hypoxia responsible systems that can be engineered into endothelial cells to prevent ischemia related cardiovascular diseases. p- . . - osteogenic potential assessment of some original scaffolds with magnetic properties new advances in bone tissue engineering demand the development of materials that can not only replace bone, but also regenerate the damaged tissue based on external or even internal stimulus. magnetic materials inside bone scaffolds are known to be a promoting factor for bone healing especially when the therapy is accompanied by application of external magnetic stimulation. based on a recent report, the presence of iron oxide in hydroxyapatite can improve the radio opacity and osteoblast proliferation. in this view, this study focuses on the development of silk fibroin-magnetite biocomposites for potential uses in bone tissue bioenegineering. such novel composites possess good mechanical properties, biocompatibility and biomineralization potential by in vitro tests and could become smart arhiectures, able to stimulate bone regeneration. a new culture model was developed by exposing a d cell/ scaffold bioconstruct to a continuous magnetic field during weeks of osteogenic induction. in this view, mc t -e murine osteoblastes progenitor cells were seeded inside the novel silk fibroin-magnetite biocomposites and subjected to osteogenesys in a magnetic field during days. osteogenic specific markers were evaluated every week in the presence and absence of the field. our results showed that the osteogenic marker's expression started earlier when mc t -e cells were exposed to the magnetic field. consequently, in our experimental model, the magnetic field had a benefic effect on the osteogenic differentiation process as mc t -e cells differentiated more efficiently in its presence. these results suggest that the bone healing process could be improved in the presence of a magnetic field. nevertheless, further in vivo studies on animal model should be employed for validation. p- . . - impact of physical activity performed on different times of day on cardiac and skeletal muscle damage in trained and untrained male subjects s. algul, m. kara, o. ozcelik y€ uz€ unc€ u yil university, van, turkey introduction: physical activity elevates creatine kinase (ck) and creatine kinase myocardial band (ck-mb) levels which have been considered to be an indirect marker of skeletal and cardiac muscles damage. purpose: impact of physical activity performed on different times of day on serum levels of ck and ck-mb were investigated in trained and untrained male subjects. materials and methods: trained (n = , . ae . yr, . ae . kg) and untrained (n = , . ae . yr, . ae . kg) subjects performed three soccer matches ( min) in field ( m versus m) in morning (m), afternoon (a) and at night (n) on separate days. the study protocol was approved by the local ethics committee. venous blood samples were taken at onset and at end of match. serum ck and ck-mb levels are measured using autoanalyser. data are expressed as mean ae s.e., compared by wilcoxon-signed rank and mann-whitney u-tests. p < . was accepted as statistically significant. results: ck and ck-mb levels increased in three matches in both groups (p < . ). importantly, there were significant increases in ck-mb levels in a and n exercises compared to m exercise (p < . ) in trained ( . ae . u/l versus . ae . u/l, % (m) . ae . u/l versus ae . u/l, % (a) . ae . u/l versus . ae . u/l, % (n) and also untrained groups ( . ae . u/l versus . ae . u/l, % (m), . ae . u/l versus . ae . u/l, % (a) . ae . u/l versus . ae . u/l, % (n). discussion: increased metabolic stress or muscle damage during physical exercise elevate serum ck and ck-mb levels. however, higher percentage of increase in ck-mb levels in a and n exercise may reflects additional increases in cardiac muscle stress despite the similar skeletal muscle stress as indicated by ck levels. conclusion: considering the observation of higher percentage increase in ck-mb levels in untrained and also trained subjects, the caution should be taken while performing an exercise in a and n time especially in subjects who has cardiac weakness. p- . . - regional assessment of hematological and discrimination indices of complete blood count for beta-thalassemia screening beta-thalassemia is one of the most common genetic abnormality causing health problems worldwide. blood count and film of beta thalassemia trait and iron deficiency anemia have similar features. therefore, several simple screening indices have been developed for differentiating between these diseases. it was to asaimed to assess the hematological parameters and discrimination indices in patients with betathalassemia trait who admitted to our hospital. the parameters of complete blood count (cbc) in subjects ( males and females) diagnosed by mutational analysis (pcr, gene amplification, dna sequencing) between and , were retrospectively screened and the thalassemia status of patients was assessed in terms of discrimination indices (eng-land&fraser (ef), green&king (gk), mentzer (m), ricerca (r), shine&lal (s-l), srivastava (s), ehsani and sirdah). the percentages of being above the cut off value were detected by ef . %, gk . %, m . %, r . %, s-l . %, s . %, ehsani . % and sirdah . %. the percentages of falsely negatives for the indices of ricerca and shine&lal were lower than others. morever, when the first three common mutations of our study were considered, out of , out of and out of patients were up to the cut off values in terms of e&f, g&k, m, s, ehsani and sirdah indices for ivs-i- (g>a), ivsii- (g>a) and heterozygous codon deletion (-aa), respectively. the molecular diagnosis and prenatal detection for families at risk is important because of the difficulties of treatment in this disease. however, the use of discrimination indices may be valuable for distinguishing of thalassemia trait from iron defiency anemia when the equipment of molecular diagnosis are limited. in our study, ricerca and shine&lal had lower falsely negative results than others. nevertheless, further studies to detect diagnostic perfomance of discriminant indices should be conducted. p- . . - novel therapeutic agents in the development of effective drug combinations to treat glioma s. avdieiev , l. gera , r. hodges institute of molecular biology and genetics, kyiv, ukraine, university of colorado, aurora, co, united states glial tumors are driven by multiple molecular aberrations that cannot be controlled by a single targeted agent. so, it is possible to expect that the combined multitarget anti-cancer therapy aimed simultaneously at different elements of tumor formation mechanisms will be more effective and will promote the extension of patients' life. to find out which drug combinations will enable the development of therapeutic regimens with improved effectiveness and decreased toxicity, the cytotoxic effects of several bradykinin antagonists (ba) were analyzed for different glioblastoma (gb) cell lines. among all the ba under investigation, bkm- appeared to be the most effective, with ic values of lm and . lm in rat glioma c and human glioblastoma u cell lines, respectively. bkm- suppressed erk / and akt phosphorylation in u cells. temozolomide (tmz), the firstline anti-gliomic drug used in clinics, has only a temporary positive effect and severe side effects in gb patients. we showed that the combination of bkm- and tmz led to significant potentiation of tmz cytotoxicity at sub-therapeutic concentrations. recombinant proteins with cytotoxic properties are promising agents for complex therapeutic applications. we revealed that the glioma-associated protein chi l inhibited the viability of u cells more effectively than tmz. furthermore, the combination of chi l and bkm- resulted in an additive cytotoxic effect. chi l -mediated decrease of cell viability was associated with a g /s transition arrest. chi l provoked the dramatic reduction of prb phosphorylation and a significant decrease of cyclin d expression, as well as a substantial increase in p level. in addition to the accumulation of p , we observed the upregulation of cdk inhibitor p . therefore, g /s arrest in chi l -treated cells could be realized via activation of prb, downregulation of cyclin d, and activation of p . harmful hereditary mutations in brca and brca are one of the most important risk factors of breast cancer. the aim of this study is to determine the mutations which are associated with breast cancer in people which is diagnosed breast cancer and/or have breast cancer-diagnosed family members by sanger sequencing and thus provide predictive and prognostic utility. our ongoing study is present the genetic variations in brca and brca genes in breast cancer-diagnosed patients, that one of them is male, and person yet healthy whose brca was sequenced by sanger sequencing. the data were analyzed by using seqscape software . and detected variations were compared with literature. in brca , we determined different benign genetic variations and variation with unknown significance and variation which has not in literature. in brca gene of patient and healthy person, benign variations, variations with unknown significance, variations which has not in literature and mutation were determined. this mutation is c. - delgtca and is located in occr. c. - a>c variation in brca gene, was determined in only male patient.c. t>a variation in exon of brca , was observed in only the youngest patient who has no family member with breast cancer and healthy person. while this variation takes place in literature as variation with 'uncertain clinical significance', an in silico program mutation taster speculated as 'disease causing' for this variation. also, almost all of variations with 'unknown significance' literature knowledge were determined in only one and different cases. this situation increases the possibility of being pathogenic of this variations. the our findings until now can contribute to variations with uncertain clinical significance in the literature. also the variations that have not in the literature but we suggest the possible relation with breast cancer as an estimate may be added to literature by expanding the study. p- . . - inhibition of the recombinant human butyrylcholinesterase with paraoxon and coumarin analog of soman organophosphorus compounds (op) represent a class of extremely toxic chemicals that are used as warfare agents. uncontrolled utilization of op is highly dangerous due to their high potential to be efficient poisons in terrorist attacks. current therapy of op poisoning include intravenous administration of atropine and acetylcholine reactivators however, it does not completely eliminate brain damage effects. alternative experimental therapy against op poisoning is utilization of bioscavengers that irreversibly react with op and rapidly inactivate them. recombinant human butyrylcholinesterase (rhbche) is one of the most promising candidates as bioscavenger due to its pharmacokinetic characteristics and broad spectrum of op neutralizing activity. here we investigated in vitro inhibition of rhbche with two model oppesticide paraoxon (pox) and coumarin analog of soman (gd c ). both op lead to rapid and irreversible inactivation of rhbche that was monitored using ellman assay and fluorescence measurements. bimolecular inhibition rate constants dramatically differ between pox and gd c that could be explained by steric hindrance in soman analog. the next steps forward creation of catalytic bioscavengers based on rhbche should be done based on mechanisms of op-rhbche interactions. this work was performed in frame of grant rfme-fi x . non-hodgkin lymphomas (nhls) represent a heterogeneous group of malignancies that arise from the lymphoid system. at the present time exist a lot of drugs for the nhls therapy, but mostly all of them are unsafe and there is no consensus regarding the best treatment protocol. to increase the efficacy and safety of therapeutic b-lymphocyte depletion in lymphomas and leukemia's it would be preferable to induce the death of pathological b cells without affecting normal b cells to prevent side effects. similar to other types of cancer, nhls arise by a multistep accumulation of genetic aberrations that induce a selective growth advantage of the malignant clone. all b-cells of organism have a unique cell surface markerantigen b-cell receptor (bcr). we generate novel approach for personalized non-hodgkin lymphomas therapy based on peptide specific to malignant cells surface receptor. for this purpose we designed new lentiviral peptide library screening technique based on fluorescent reporter cells system. herein aa peptide library was used for screening of nhl's malignant receptor agonist. patients' lymph nodes biopsy samples mrna was used as a source of malignant bcr nucleotide sequence. variable domain of the lymphoma bcr was used for chimeric receptor generation, where bcr vh/vl part responsible for agonist recognition and bottom part of receptor was retranslate signal to the reporter gene. in this embodiment of the method, very large numbers of candidate aa peptides expressing lentivirus and eukaryotic reporter cells are packaged together in a format where each is capable of replication, thereby forging a direct link between genotype and phenotype. after four rounds of screening we discover peptides specifically interacted with malignant bcr's. selected peptide ligands were fused with chimeric antigen receptor for expression on t-cells. modified tcells selectively eliminate nhl's malignant cells ex vivo. this work was supported by grant rfmefi x . introduction: pesticides are used to prevent damage of unwanted insects, rodents, plant, moss and other pests. excessive use of pesticides may cause adverse effects in animals and humans. chlorpyrifosethylene (cpe) is an organophosphate pesticide, used in many agricultural products such as figs, cherries, olives worldwide; caused acute poisoning and chronically oxidative stress. rosadamascena mill (rosaceae) is a rose, used for production of rosewater (rw) and rose oil worldwide. rosaceae products are consumed in food and cosmetic industries. materials and methods: in this study, we investigated that cpe and rw effects on kidney tissues of rats. this study was included adult male rats, divided into groups. each group included rats. i.group: control (regular feed), ii.group: cpe ( . mg/kg/day), iii.group: rw ( mg/kg/day) and iv.group: cpe ( . mg/kg/day) + rw ( mg/kg/day). following days, kidneys were taken after sacrificed. analyzes were performed that malondialdehyde (mda), nitric oxide (no) as oxidant; superoxide dismutase (sod), glutathione reductase (gr) as antioxidant parameters. results: as compared with control, mda and no levels in cpe were a significant increase was determined (p conclusion: cpe is shown that significant increase on oxidant parameters, but significant decrease on antioxidant parameters. rw occurs opposite situation. similarly results of cpe, rw + cpe increased oxidant parameters, but decreased antioxidant parameters. these changes are lower than only cpe. these results showed that positive effects both rw and rw + cpe increasing on antioxidant parameters, also decreasing on oxidant parameters. we provide the comparative analysis of the reduced glutathione (gsh), reactive oxygen species (ros), a-tocopherol levels, an intracellular labile zn + pool and esterase activity of red blood cells of patients with diagnosed components of the metabolic syndrome (ms)arterial hypertension and diabetes mellitus type ii (ah + dm + ). as the comparison groups were selected patients with one diagnosed component of msarterial hypertension (ah + dm À ) or without any diagnosed component of ms (ah -dm -). patients of all investigated groups were at the hospital treatment with a diagnosis coronary heart disease (chd) ii degree. human blood was obtained from normal donors and patients with chd ii stage. cytosolic esterase activity was assessed using calcein-am test. ros level was evaluated using cm-h dcf-da. gsh level was estimated using lowry method. an intracellular labile zn + pool was assessed using fluozin- -am. investigations were performed on the specord m , hplc system lc- prominence (shimadzu) and facscantoii (bd). a significant decrease of the intracellular level of labile zn + in erythrocytes of patients with ah + dm + compare with ah + dm À and ah À dm À was shown. this fact confirms our assumption concerning the important role of zinc homeostasis in the etiopathogenesis of diabetes mellitus type ii. a direct relationship between the intracellular zn + level modification and erythrocytes esterase activity of patients with chd ii degree was observed. moreover, in ah + dm + group of patients this relation was more marked. the unidirectional alteration in the erythrocytes redox state (gsh and a-tocopherol levels reduction, ros formation activation) was revealed at the whole of investigated chd patients groups (ah + dm + , ah + dm À , ah À dm À ). however, the pathological erythrocytes response on in vitro action of the different antioxidants (n-acetylcysteine, ascorbic acid, a-tocopherol, quercetin) had a diverse character that can be a significant test under antioxidant therapy prescription. it is known that long-term social isolation and the disorder of natural circadian rhythm is considered an important stress factors, which cause a variety of metabolic and mental disorders. it should be noted that the impact of the stresses takes up a larger area, according to, review of the action of their mechanism is one of the topical issues of modern science. it is estimated that as a result of stress the metabolic processes change in the organizm. because of this, we've studied the functionality of the antioxidant system in laboratory rat heart muscle cells and blood under psycho-emotional stress. it was found that quantitative changes of nitric oxide (no) was initiated the process of lpo, which caused oxidative stress in the cells and decreased antioxidant enzymes activity, such as catalase,sod, gpx and gr. the results suggested that psycho-emotional stress was accompanied by oxidative stress, causing a reduction in the intensity of energy metabolism in cardiac muscle cells, which was further strengthened by the fact that the activity of the enzymes involved in atp synthesis in mitochondria was reduced. also, we've studied exogenous creatine positive and negative affects on energy metabolism and blood lipid spectrum. based on this, we proposed that psychological stress is one of the factors contributing to the development of various cardiac diseases. the importance of free radical lipid transformations, which differ from the peroxidation processes, was pointed out for the first time in our laboratory studies. we have found that ros can induce free radical destruction processes of sphingolipids with c-c-bond cleavage [lipids, , : - ; lipid insights, , - ] . in case of acylated sphingolipids, they can undergo decomposition with c-c-bond cleavage upon direct uv irradiation [photochem. photobiol., , : - ] . it was of interest to establish the possibility of photosensitized decomposition reactions of not acylated sphingolipids, which do not absorb an ultraviolet. in this work we studied photosensitized reactions of sphingosines containing a free amino group, and low molecular compounds, which simulate their structure, such as aminoalcohols (serinol). as photosensitizers, the salts of transition metals, hydrogen peroxide, and acetone were used. oxygen was removed by bubbling with argon to reduce the probability of side reactions during photolysis of sphingolipids, such as oxidation processes (including oxygen reactions with alkyl radicals). we have shown that the action of uv-radiation on aminoalcohols and sphingosines in aqueous solutions in the presence of photosensitizers induces their destruction with c-c bond rupture. the main carbonyl product of sphingosines free radical destruction was an unsaturated aldehyde - -hexadecanal. it was found, that -hexadecenal possesses a wide spectrum of biological activity: it promotes reorganization of the cell cytoskeleton and modifies the redox state of the cells [febs journal (suppl. ), abstracts: mem. biol. s , lipid signaling & dynamics, p. .] . the results of this study can expand the frontier of research regarding free radical lipid damage, which could contribute to a better understanding of the origins of diseases associated with the activation of free radical processes in living organisms. structural basis for the - - proteindependent inhibition of apoptosis signalregulating kinase protein kinase ask (apoptosis signal-regulating kinase ) is a member of the mitogen-activated protein kinase kinase kinase (map k) family that plays a crucial role in immune and stress responses. the activity of ask is regulated through homo-oligomerization and interaction with other proteins including the - - protein which binds to the phosphorylated motif located at the c-terminus of the kinase domain of ask and suppresses its catalytic activity through unknown mechanism. under stress conditions, ask is dephosphorylated at ser and the - - protein dissociates. this dissociation is then one of the factors that lead to the activation of ask . we performed low-resolution structural analysis of the kinase domain of ask (ask -cd) bound to - - using chemical cross-linking, analytical ultracentrifugation and small angle x-ray scattering. the low-resolution structural analysis shows that ask -cd binds to the - - protein in two to two stoichiometry through a small binding interface involving surface of - - outside its central channel and several regions from the c-lobe of ask -cd. the complex is dynamic and conformationally heterogeneous. phosphorus nmr and time-resolved fluorescence measurements, together with low-resolution structural analysis, indicate that binding of ask -cd to - - modulates conformation of ask 's activation segment. these results suggest that the - - binding suppresses the catalytic activity of ask through direct structural modulation of its activation segment. our study provides new insight into the interaction between the kinase domain of ask and - - and offers a plausible structural explanation for the - - protein-dependent inhibition of ask kinase activity. introduction: thymoquinone ( -methyl- -isopropyl- , -benzoquinone, tq) exerts a great antitumor activity against different types of cancer cells. a growing body of evidence indicates that reactive oxygen species (ros) generation followed by modulation of the akt and mapk pathways is a general mechanisms underlying the tq antitumor action. however, the data of tq effects on the mapk pathway are conflicting. to date, the activation or inhibition of the mapk protein family seems to depend on the cell type and on the tq concentration used. in order to elucidate the antitumor potential of tq against gliomas and the underlying molecular mechanism, tq influence on c rat glioma cells functioning was studied. results: it has been shown that the cultivation of c cells with tq in concentrations of - lm during hours strongly inhibits cell proliferation and induces cell death with id of lm. at the same time, tq induces ros generation and intracellular gsh depletion in a dose-dependent manner, that is followed by the mitochondrial potential decrease. interestingly, ros production has only cytoplasmic, but not mitochondrial origin in cells challenged with tq at the concentrations up to lm. two-electron reduction of tq by dt-diaphorase attenuates tq anticancer efficiency whereby inhibition of dt-diaphorase by dicumarol increases tq-induced c cell death by %. we analyzed mapk pathways involvement in c cells growth inhibition at tq treatment. it has been shown that inhibition of the erk pathway by pd and jnk pathway by sp does not influence on tq-induced effects. on the contrary, the specific phosphoinositide- -kinase (pi k) inhibitor (ly ) abrogates tq-induced growth arrest. conclusion: antitumor effects of thymoquinone on c glioma cells is a result of ros generation and intracellular gsh depletion, that is followed by mitochondria disfunction, and growth arrest via pi k pathway. assessment of oxidative stress and antioxidant defense activity parameters in patients with hiv-infection is of great importance, especially for hiv-positive women of reproductive age planning to have children. data of women of reproductive age with hiv-infection analyzed: patients with hiv-monoinfection (n = ) and patients with co-infection (hiv and hepatitis b and / or c) (n = ). as a control we used the data of healthy women (n = ). serum and hemolysate used as material for the study. lpo-aod products were determined by spectrophotometric and fluorometric methods. average value of initial lpo products -diene conjugates was significantly increased in the group with hiv-co-infected compared to control ( . times; p = . ) and group with hivmonoinfection ( . -fold; p = . ). the level of secondary products -ketodienes and conjugated trienes increased in patients of both groups compared to control ( . times (p = . ) and . -fold (p < . ), respectively). at the same time isolated double bonds and tba-active products content showed no significant changes (p > , ). total antioxidant activity parameter decreased . fold (p = . ) in the group with hiv-monoinfection compared to control. decrease in activity of the primary antioxidant enzyme -superoxide dismutase (p = . , compared to the control and p = . , compared with the group with hiv-monoinfection) and the level of a-tocopherol ( . -fold to control and . fold to hiv-monoinfection) was detected in the group with hiv-coinfection. . -fold higher content of retinol in hiv-coinfection group (compared to the control) revealed. in women with hiv-coinfection the oxidative stress was significantly higher than in women with hiv-monoinfection. suggested to include antioxidant supplements in the complex pathogenetic therapy in women with hiv-coinfection (hiv and hepatitis b and / or c), which will contribute to women's ability to bear children. p- . . - acute different doses of malathion induce cholinesterase inhibition, glucogenic enzymes and histopathological change in rat liver malathion, which is an organophosphorus compound, is a widely used pesticide all over the world. despite its benefits, malathion has many toxic effects on many tissues including liver. we designed to evaluate the acute different doses of malathion on cholinesterase (che) inhibition, gluconeogenic enzymes and histopathological change of rat liver. for this purpose groups were formed. rats in group served as control group animals which were only given corn oil. group , group and group were administered , , mg/kg of malathion, respectively, dissolved in corn oil by oral gavage. the rats were sacrificed after hours following administration and the livers of rats were removed. the liver che, alanine aminotransferase (alt), aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) were studied using autoanalyzer. histopathological investigation was performed using microscope. the liver che activities of group , group and group were inhibition percentage of %, %, and % respectively, comparison of group 's che activity. the liver alt, ast and ldh increased in group and group compare to group and group (p < . ). we also observed that there was vacuolar and hydropic degeneration in liver of group . according to our result, acute administrations of malathion result in hepatotoxic effects with increasing doses. background and aims: an imbalance between free radicals and antioxidants is closely linked to the onset of an acute myocardial infarction (ami). the aim of this study was to investigate the antioxidant status and the lipid peroxidation in patients admitted to hospital immediately after ami. methods: the study population comprised patients with ami and healthy subjects. patients that had an acute myocardial infarction (ami) less than hours since onset were selected for this study. antioxidant status was assessed by lactonase activity of paraoxonase (pon dhc), trolox equivalent antioxidant capacity (teac) and plasma uric acid level. malondialdehyde was used as marker of lipid peroxidation. results: compare with the control group, the levels of mda and pon dhc were significantly higher in group with ami (p < . , respectively p < . ). elevated levels of mda (p < . ) were found in patients with ami compare with the control subjects. ami patients had also statistically significant reduced levels of teac (p < . ) comparative with healthy subjects. no statistically significance was found for plasma uric acid level in subjects from our study. conclusion: a high lipid peroxidation correlated with a decreased teac activity suggest an exacerbated oxidative stress in subjects admitted to hospital immediately after an ami. p- . . - dealing with copper toxicity: new insights into copper detoxification in yeast copper (cu), an essential metal, is a double-edged sword, as its essential nature is counterbalanced by the toxic effect that it can exert on cells when not properly controlled. as such, organisms have evolved defence mechanisms against cu toxicity, and in the yeast saccharomyces cerevisiae, the transcription factor ace orchestrates several of those mechanisms, by activating cu-detoxification genes. in s. cerevisiae iron (fe) uptake is partially dependent on cu, as the membrane multicopper-oxidase fet , which is part of the high-affinity iron uptake system, requires cu as a cofactor. aft , the low iron-sensing transcription factor in yeast, is known to regulate the expression of fet gene. however, we found that a strain lacking fet is more sensitive to cu surplus conditions than a strain carrying the aft gene disrupted. this finding suggests that under such conditions another regulator came into play and controls fet gene expression. we next evaluated whether ace could be the alternative regulator of fet under cu excess. to test this hypothesis we first constructed the double mutant aft ace and assayed its fitness under cu surplus. as expected, the double mutant is much more sensitive to cu than the single aft or ace mutants. we next monitored the expression of fet gene in cells lacking ace , using yeast-one hybrid and qrt-pcr approaches. our data clearly indicates that fet expression is dependent on ace when cu is overly abundant. altogether our data unveil a novel mechanism of cu detoxification relying on the activation of fet by ace in an aft independent way. experiments to understand the consequences of this regulation in terms of cu detoxification are currently being undertaken. in joint degeneracy, reactive oxygen species manifest their toxicity both through intrinsic reactivity and the inflammatory process activation, leading to cartilage dysfunctions, injuries of matrix proteins and cytokines stimulation. the study is focused on the identification of oxidative balance modulation (enzymes and oxygen free radicals) by a bioactive extract obtained from small sea fish. the cellular support was represented by the chondrocyte line chon- derived from human long bones (atcc Ò crl- tm ), that assure the reproducibility of a standardised biological system. the oxidative stress was induced through stimulation with il b, a cytokine-factor that promotes the protein catabolism and also with tnfa, the initiator of pro-inflammatory activation, combined with pma, the activator of protein-kinase c, triggering of oxygen reactive species generating cascades. the antioxidant effect was compared with known antioxidants: vitamin c, x fatty acid, n-acetyl -cysteine. the acellular antioxidant/antiradical screening was done using two complementary techniques for total antioxidant status evaluation and completed by measuring cellular catalase (cat) and superoxidedismutase (sod) activity, correlated with intracellular hydrogen-peroxide and superoxide anion monitoring through flow cytometry. the antioxidant properties of the bioactive extract proved in acellular systems are confirmed at cellular level by the involvement of the product in the enzymatic cascade cat -sod, potentiating the catalytic action of the enzymes, and by the decline of both intracellular reactive oxygen species (the hydrogen peroxide decrease with %, the superoxide anion is reduced with % compared with the stimulated control). the demonstrated antioxidant synergy assures a complete cellular protection induced by the small sea fish extract in human condrocytes. introduction: alzheimer's disease (ad) is a progressive neurodegenerative disorder characterized by memory loss, cognitive impairment. oxidative stress is a contributory factor in ad pathogenesis. glutathione (gsh) is the main antioxidant cellular defence. the ratio of gsh/gssg shows the redox status of gsh, and plays important role in maintaining intracellular redox homeostasis. the current study was carried out to determine oxidative dna damage and ratio of gsh/gssg which plays an important role in protection of target molecules from oxidation in the patients with ad. methods: the study subjects were consisted of patients with ad (n = ) and age matched control group (n = ) who were treated and followed in the cerrahpasa medical faculty hospital, department of neurology and department of internal medicine/ geriatrics. dna strand breaks and h o -induced dna damage were determined in lymphocyte dna with comet assay. the gsh and gssg levels in the erythrocyte lysates were measured by using a commercial spectrophotometric kit. the ratio of gsh/gssg were calculated. statistical analysis was performed with spss software package. results: dna strand breaks and h o -induced dna damage were found to be higher (p = . for all), the ratio of gsh/gssg was found to be lower (p = . ) in the ad group than control group. there was no significant difference between male and female for dna strand breaks and h o -induced dna damage in the ad group, but ratio of gsh/gssg were higher in male when compared with female (p = . ). no significant difference was found between the men of ad group and men of the control group for gsh/gssg ratio whereas women of the ad have a lower gsh/gssg ration than those in the women of the control group (p = . ). conclusion: increased systemic oxidative dna damage and dna susceptibility to oxidation may be resulted from diminished gsh/gssg ratio in ad patients. this finding shows the importance of antioxidant support in ad management. p- . . - validation of a liquid chromatography-tandem mass spectrometry method for the measurement of lipid peroxidation product iso-prostaglandin f a in urine m. kant , m. akıs ß , h. _ is ßlekel , department of medical biochemistry, school of medicine, dokuz eylul university, izmir, department of molecular medicine, school of medicine, dokuz eylul university, izmir turkey -iso-prostaglandin f a ( -iso-pgf a ) is a reliable indicator of lipid peroxidation resulting from oxidative lipid damage and is postulated as a gold standard biomarker for the evaluation of oxidative stress. the aim of this study was to validate non-invasive and highly accurate stable isotope dilution-multiple reaction monitoring liquid chromatography-tandem mass spectrometry (sid-mrm lc-ms/ms) method for identification and quantification of -iso-pgf a . twenty four hour urine samples were collected from healthy volunteers at medical school of dokuz eylul university for analytical performance studies. lc-ms/ms analyses were performed on conventional hplc coupled to a triple quadrupole ion trap mass spectrometer equipped with a turboionspray tm source. analyst software version . were used for data analyses. mrm transitions used were m/z? / for -iso-pgf a and m/z? / for -iso-pgf a-d . analytical performance of the method was evaluated by linearity, selectivity, sensitivity, precision and accuracy studies using pure standards and samples extracted from urine of healthy volunteers. the linear calibration range for -iso-pgf a was determined as ng/ml by using standards ranging from . - ng/ml. analytical sensitivity of the method was determined by lod with s/n of and loq with s/n of . lod and loq for iso-pgf a were . À and À ng/ml, respectively. the assay stability and reproducibility were assessed by the precision and accuracy of intra-and interday measurements (n = ). the intra-and interday cvs for -iso-pgf a were . % and . %, respectively. sid-mrm lc-ms/ms method for absolute quantification of -iso-pgf a was optimized and validated in our laboratory and therefore this highly accurate method can successfully be applied to clinical patient samples. p- . . - synergistic anticancer effects of sulforaphane and cisplatin through the induction of apoptosis and autophagy following oxidative stress in malignant mesothelioma cells malignant mesothelioma is characterized by poor responsiveness to current chemotherapeutic drugs, usually owing to high resistance to apoptosis. here, we investigated chemosensitizing effects of phytochemical resveratrol, in combination with cisplatin, on malignant meothelioma cells. cell viability was evaluated using mtt assay. cell apoptosis was detected with dapi staining, caspase / activity assay and flow cytometry. cell cycle distributions, ros levels and mitochondrial membrane potential were determined using flow cytometry. the expression of cell cycle-, apoptosis-, and autophage-related proteins was measured with western blotting. the combination treatment of cisplatin and resveratrol (cis/res) synergistically induced apoptosis, as evidenced by typical cell morphological changes, the appearance of a sub-g /g peak, an increase in the annexin v(+) cells and the cleavage of caspase- and parp. cis/res increased ros production and depolarization of mitochondrial membrane potential with an increase in the bax/bcl- ratio. these changes were attenuated by pre-treatment with n-acetylcysteine, suggesting that cis/res induced apoptosis through oxidative mitochondrial damage. compared with msto- h cells, cis/res was less efficient in killing h- cells. h- cells exhibited an enhanced autophagy to cis/res, as observed by an increase in viable cells exhibiting intense lysotracker red staining and up-regulation of beclin- and lc a. inhibition of autophagy by bafilomycin a rendered cells more sensitive to cis/res-induced cytotoxicity and this was associated with induction of apoptosis. these data indicate that the increased resistance of h- cells to cis/res is closely related to the activation of self-defensive autophagy, and provide the rationale for targeting the autophagy regulation in the treatment of malignant meothelioma. stress oxidative induced by chemotherapy with cyclophosphamide (cp) causes vulnerability in sperm and decline of fertility. this study was aimed to evaluate the role of ethyl pyruvate (ep) in the amelioration of fertility and growth of primitive embryo in animals that received cp. adult male mice ( - weeks) were randomly divided into groups: control group received normal saline ( . ml/day, ip), cp group received cp ( mg/kg/week,¬ ip), the cp+ep group received ep ( mg/kg/day, ip) along with cp, were treated for days. mice from each group were arranged for evaluation of sperm quality and in vitro fertilization (ivf) too. afterward, the separated oocytes from ovulation stimulated mice were conducted to evaluation of ivf and embryo development. the results revealed that cp caused notable decrease in percentage of fertilization in cp group, but administration of ep along with cp caused an increase in the percentage of fertilization in comparison to cp group. the percentage of the two cell zygotes in cp+ep group, and the percentage of blastocysts in control and cp+ep groups were higher than that in cp group (p < . ). results showed that the total number of arrested embryos in control and cp+ep groups was decreased in comparison to cp group (p < . ). the percentage of arrested embryos type i, ii, and iii in cp+ep group was decreased in comparison to cp group, but that decrease was significant only in types i and ii (p < . ). the average motility, viability, nucleus maturity and sperm morphology were decreased significantly in cp group in comparison with control and ep groups, whereas ep caused significant increase of these parameters (p < . ). also, the percentage of dna damage was increased significantly in cp group in comparison with control and ep groups (p < . ). the results of this study indicated that the ethyl pyruvate was able to suppress free radicals and enhance the ivf and quality of sperm in cp treated animals. malathion, which is a member of organophosphate chemical family, is used to control pests and is a widely used pesticide all over the world. however it is also known to be highly toxic on many tissues including pancreas. to test this we set groups to administer a single dose of malathion dissolved in corn oil via oral gavage at the doses of mg/kg (group ), mg/kg (group ) and mg/kg (group ). only plain corn oil was given to control group (group ). the rats were sacrificed hours after administration of the chemical and the pancreases of rats were removed. in an attempt to evaluate the dose dependent response, we measured amylase and lipase activities, insulin, malondialdehyde (mda), total oxidant status (tos) levels in rat pancreases. all of the parameters were measured spectrophotometrically. we found that pancreas insulin levels significantly increased in group compare to group , besides the insulin levels of group and group were significantly higher than group (p < . ). pancreas amylase and lipase activities significantly decreased in group and group compare to group and group (p < . ). however, there was no significant change in pancreas mda and tos levels (p > . ). according to correlation analysis, when pancreas amylase levels declined, lipase levels were decreased simultaneously and there was a strong positive correlation between them (p < . ). in addition, when the comparison was evaluated as a binary, while pancreas amylase and lipase levels diminished, pancreas insulin levels increased and a strong negative correlation between them was found (p < . ). according to our result, acute administrations of malathion leads to alterations of insulin, amylase, and lipase levels with a dose dependent manner, while it does not to change oxidant status. the aim of this study is to evaluate the potential toxic effects of mancozeb on the stress biomarkers such as catalase (cat) activity, malondialdehyde (mda) level and protein levels in the brain tissue of zebrafish (danio rerio). mancozeb, is a synthetic fungicide contaminating aquatic environments as a potential toxic pollutants, was investigated in the present study for acute toxicity. zebrafish groups (m-low and m-high) were exposed to different doses of mancozeb ( mg l- and . mg l- ) for hours except the control group. catalase (cat) activity, malondialdehyde (mda) level and total protein levels were determined by spectrophotometer. the results showed that cat activity and mda levels were decreased in all experiment groups. protein levels were increased in experiment groups when compared to the control group. in conclusion, the changes in the cat activity and mda levels were time and as well as mancozeb dose-dependent. furthermore, the biochemical parameters of mancozeb exposure on zebrafish, showed that mancozeb has significant effect on the zebrafish and/or aquatic organisims. paracetamol (para), which is antipyretic and analgesic, is widely used around the world. paracetamol can be recommended for moderate or mild pains especially in pregnancy as an analgesic. it is known that, paracetamol can cause hepatotoxicity or nephrotoxicity. we were aimed that in this study to show potential hepatoprotective and nephroprotective effect of betaine against long term paracetamol using at therapeutic doses. it has been prepared groups, control, para and para+-betain groups. paracetamol and betaine were administered by gavage to pregnant rats, from first day to the last day of pregnancy (aprox. day). ml physiological saline (% . nacl solutions), mg/kg/day paracetamol, mg/kg/day paracetamol and mg/kg/day betain was given by orally to control, para and para+betain groups respectively. the day of the birth, newborn rats anesthetized by ether and after decapitated. newborn rat's liver and kidney tissues used for biochemical analysis [malondialdehyde (mda), reduced glutathione (gsh), nitric oxide (no) and paraoxonase-arylesterase (pon-are)] and rat's liver and kidney tissues used for histological studies. we showed that, mda and no levels was significantly increased, while pon activities decreased. on the other hand gsh levels and are activities was decreased but these decline wasn't significant in the liver and kidney para group. these biochemical results showed hepatotoxicity and nephrotoxicity in neonates which can be formed in long term maternal paracetamol using at therapeutic doses. also our histological findings was support these biochemical results. we used betaine against potential hepatotoxic and nephrotoxic effect of long term maternal paracetamol using at therapeutic doses for neonates. betaine has antioxidant properties and also used as a methyl donor for transsulfuration reactions in the cell. biochemical and histological examinations showed that betaine protected the tissue injury relatively. p- . . - lipid rafts are involved in modulation of ca + responses induced by glutoxim and molixan in macrophages pharmacological analogues of oxidized glutathione (gssg) disulfide-containing drugs glutoxim Ò (gssg disodium salt with dmetal nanoaddition, «pharma-vam», st. petersburg) and molixan Ò (complex of glutoxim with inosine nucleoside) have found clinical application as a wide range immunomodulators and hemostimulators. however, the cellular and molecular mechanisms of these drugs action are still unclear. previously we showed for the first time that glutoxim and molixan cause biphasic intracellular ca + concentration ([ca + ] i ) increase due to ca + mobilization from thapsigargin-sensitive ca + stores and subsequent store-dependent ca + entry in rat peritoneal macrophages. it is known that key proteins involved in ca + signaling are localized in discrete plasma membrane lipid rafts domains. lipid rafts are cholesterol and sphingolipids enriched microdomains that function as unique signal transduction platforms. thus, the aim of the present work was to elucidate the possible involvement of lipid rafts in glutoxim and molixan effects on [ca + ] i in macrophages. [ca + ] i measurements were performed with fura- am microfluorimetry. to examine the involvement of lipid rafts in the effect of gssg-based drugs on [ca + ] i we used methyl-bcyclodextrin (mbcd), widely used to remove cholesterol from membranes, thus disrupting the lipid raft domains. we have shown for the first time that macrophage preincubation with mm mbcd for hours before molixan addition causes significant inhibition of both ca + mobilization (on average, by . ae . %) and subsequent ca + entry (on average, by . ae . %), induced by molixan. similar results were obtained in experiments with glutoxim. thus, we have demonstrated for the first time that mbcd significantly decreases both phases of glutoxim-or molixan-induced ca + responses in macrophages. the results suggest that intact rafts are required to initiate complex signaling cascade activated by glutoxim or molixan and leading to [ca + ] i increase in macrophages. plant-derived natural substances (phytochemicals) with potent pro-apoptotic activity towards cancer cells in vitro are considered as promising nutraceuticals in anticancer therapy. nevertheless, due to their relatively low bioavailability, administration of high doses of nutraceuticals that are not achievable in vivo seems to exert potentially negligible physiological effects in clinical trials. thus, there is a need for revealing novel cytophysiological effects of low doses of phytochemicals towards cancer cells. in the present study, we have considered thirty one nutraceuticals and selected four phytochemicals acting at low micromolar range ( to lm) against phenotypically different mcf- , mda-mb- and sk-br- breast cancer cells, namely diosmin, sulforaphane, ursolic acid and betulinic acid. nutraceuticals inhibited cell proliferation and caused changes in the cell cycle that was accompanied by elevated levels of p , p , p and/or p . apoptosis (annexin v staining, multicaspase and mitopotential assays) was observed exclusively when nutraceuticals were used at the concentration of lm, whereas at the concentration of lm, stress-induced premature senescence was noticed (sa-b-gal activity). nutraceuticals diminished the levels of glut- and selected glycolytic enzymes. nutraceuticals promoted oxidative and nitrosative stress as judged by increased production of total reactive oxygen species, total and mitochondrial superoxide, nitric oxide and protein carbonylation. nutraceuticals also stimulated dna single and double strand breaks that was accompanied by atm phosphorylation and to a lesser extent by histone h ax phosphorylation and bp foci formation. taken together, several responses to nutraceutical treatment were observed in breast cancer cells that may reflect the heterogeneous nature of cancer cell populations. this study was supported by grant from the national science center, / /d/nz / . nucleolus is thought to be a stress sensor and oxidative and ribotoxic stimuli may cause the inhibition of rrna synthesis by the inactivation of transcription factor tif-ia/rrn that is accompanied by the relocation of nucleolar proteins and p -based cell cycle arrest and/or apoptosis. as nutraceutical-mediated modulation of nucleolar activity may be considered an attractive anticancer strategy, in the present study, we have investigated the effects of three selected nutraceuticals, namely sulforaphane, ursolic acid and betulinic acid on nucleolus state in three breast cancer cell lines (mcf- , mda-mb- and sk-br- ). we found that low dose nutraceutical treatment resulted in p -mediated inhibition of cell proliferation, a decrease in rrna synthesis, shifts in lamin a/c and b pools, changes in the nucleolar protein levels and their carbonylation, and changes in nucleolus size and number. breast cancer cells differed in erk activity that resulted in different patterns of erk activation/inhibition, phosphorylation status of s and autophagy induction upon nutraceutical stimulation. nutraceuticals also affected dna methylation parameters, namely the levels of dnmt , dnmt a and dnmt b that resulted in both global dna hypo-and hypermethylation. taken together, after nutraceutical treatment, nucleolus-centered cellular response was revealed in breast cancer cells of different phenotypic characteristic that may be considered a potential target of anticancer therapy. this study was supported by grant from the national science center, / /d/nz / . p- . . - rate of apoptosis in human macrophages infected with leishmania tropica promastigotes infection of the cells with parasites or exposing cells to heat stress induces a cellular stress. in the present study human macrophages are infected with leishmania tropica promastigotes and exposed to heat stress. the measurement of cytoplasmic histone-associated dna-fragments was carried out using elisa technique. visualization of apoptotic cells was performed by the terminal deoxynucleotidyl transferase dutp nick end labeling staining method (tunel). degree of oxidative stress on cell is evaluated by measuring nitric oxide (no), malondialdehyde (mda), reducte glutathion (gsh) levels and superoxide dismutase (sod) activities. results of the elisa technique showed that infection of macrophages with promastigotes induced apoptosis rate significantly (p < . ), heat stress however decreased the rate of apoptosis in infected macrophages remarkably (p < . ). high levels of apoptosis rate in infected macrophages and drastic decdrease in apoptosis in heat subjected macrophages infected with promastigotes are confirmed by visualisation of apoptotic cells using tunel method. levels of glutathion (gsh) in infected macrophages decreased significantly (p < . ), while malondialdehyde (mda) levels increased notably (p < . ). however, no statistical significant alterations were detected in the nitric oxide (no) values and superoxide dismutase (sod) activities. results of the present study indicates that infection of human macrophages with leishmania tropica induces a cellular stress response, characterized by decreased values of gsh and increased levels of mda. increased rate of apoptosis in infected macrophages may be due to the increased cellular stress caused by leishmania tropica amastigotes. decreased rate of apoptosis measured in heat exposed macrophages infected with promastigotes indicates an extention in life span of macrophages. measurements of the parameters characterizing the redox and inflammatory processes in blood are essential for diagnosis and prognosis of type diabetes mellitus (t dm), but also for monitoring the effectiveness of medical treatments. along with other biochemical effects, hormonal imbalance leads to modified transport function of erythrocytes due to changes in enzyme systems involved in upholding of cellular homeostasis through a rapid degradation of altered proteins, being the second line of defense against the free radicals, which degrade and eliminate the damaged molecules. some of these enzymes are hemoglobin peroxidase (pa) and esterase (ea). the aim of this research study was to identify new parameters with a potential role of biochemical markers in t dm like hemoglobin peroxidase and esterase activity from erythrocyte. our data showed that pathological processes involved in t dm imply an increased enzymatic activity of pa ( . %), which correlates with increased levels of ea ( . %) and glycated hemoglobin (hba c) ( . %), compared with control group. the variables hba c, pa and ea are correlated: the identified pearson correlation coefficients r = . and r = . respectively, have an associated probability of p < . and p < . a value that indicates a strong positive correlation between the dependent variables pa and ea and independent variable hba c. based on these results we concluded that together with glycosylated hemoglobin assay, all the studied parameters can be successfully used as extra test for diabetes associated with oxidative stress and disorders in erythrocyte functions or blood rheology. the radioprotective effects of propolis and caffeic acid phenethyl ester on radiationinduced oxidative/nitrosative stress in brain tissue s. taysi , e. demir , k. cinar gaziantep university, school of medicine, gaziantep, haran university, sanliurfa, department of neurosurgery, medical school, gaziantep, turkey head and neck cancer patients treated with radiotherapy suffer severe side effects during and following their treatment. efforts to decrease toxicity of irradiation to normal tissue, organs and cells have led to searching for cytoprotective agent. investigations for effective and non-toxic compounds with radioprotective capability led to increasing interest in antioxidant such as propolis and caffeic acid phenethyl ester (cape). the aim of this study was to evaluate the antioxidant and radioprotective effects of propolis and cape on radiation-induced oxidative/nitrosative stress in the brain tissue. fourty sprague-dawley rats were randomly divided into five groups. group (irradiation (ir) + propolis) received total cranium irradiation and propolis was given orally through an orogastric tube daily. group (ir+cape) received total cranium irradiation plus cape, was dissolved in dimethyl sulfoxide (dmso) just before giving to the rats, intraperitoneally (ip) every day. group (ir) received gy of gamma irradiation as a single dose to total cranium plus ml saline daily. group received daily plain dmso. group received daily plain saline. at the end of the day time period, xanthine oxidase (xo), nitric oxide synthase (nos) activities, nitric oxide (no•) and peroxynitrite (onoo -) levels were significantly higher in ir group compared to all other groups. in conclusion, the results suggest the radioprotective ability of propolis and cape involving prevention of radiation-induced oxidative/ nitrosative damage. p- . . - role of leptin and adiponectin in obesityassociated oxidative stress e. becer , a. c ß irakoglu near east university, nicosia, cyprus, istanbul university, istanbul, turkey objective: increased oxidative stress is one of the major characteristics of obesity and obesity-related complications. adipokines also induce the production of reactive oxygen species and generating oxidative stress in physiological and pathological conditions of obesity. the aim of this study was to determine the association of leptin and adiponectin levels with body mass index, lipid parameters and oxidative stress biomarkers in obesity. methods: the study included obese and non-obese subjects. plasma leptin and adiponectin levels (ng/ml) were measured using commercially available enzyme-linked immunosorbent assay kits. serum lipid, superoxide dismutase, malondialdehyde and antropometric parameters were measured. results: obese and non-obese subjects did not differ in age, while plasma glucose, total cholesterol, triglycerides, ldl cholesterol and leptin levels were significantly higher and mean hdl cholesterol and adiponectin levels were significantly lower in obese than non-obese subjects. the plasma leptin (p < . ) and adiponectin (p = . ) levels were significantly correlated with bmi in both obese and non-obese subjects. in obese subjects, leptin levels were significantly correlated with superoxide dismutase (p < . ) and malondialdehyde (p < . ). strikingly, adiponectin was significantly correlated with superoxide dismutase (p = . ) and malondialdehyde (p = . ) levels in obese group. conclusion: our results suggest that leptin and adiponectin levels are associated with defective antioxidant status and increased lipid peroxidation which may have implications in the development of obesity related health problems. clinical trials of biologically active plant substances show a significant preventive effect in cancer, cardiovascular diseases and peptic ulcer disease in the form of both nutritional supplements and therapeutic intervention. anthocyanins contained in dark berries show great antioxidant potential, with the most important including a reduction in oxidative degradation of lipids or tyrosine oxidation by peroxynitrite. our previous studies of the antioxidant properties of extracts from vaccinium corymbosum, aronia melanocarpa and sambucus nigra, however, indicated that their activities largely depend on the method of extraction. while quantitative determination of anthocyanins pointed to a disproportionately larger content of anthocyanins in isolates from lyophylized berries, their scavenging activities against hydroxyl radicals was surprisingly the lowest. inflammatory processes, vascular damage, atherosclerosis and others are caused by oxidativenitrosative stress, so we tested their efficiency to scavenge no degradation products. we found that only purified extracts of lyophilized berries showed the most significant effects against no degradation products, with efficacy of around %. an extract from aronia showed greater than % efficiency, and a net ethanol extract from all the berries showed a % effect. cleaned ethanol extracts showed the lowest effects, while aronia initiated production at a concentration of mg/l. conversely, all acetone extracts consistently initiated no degradation products. these findings are in complete contrast to their determined action against reactive oxygen species. in summary, it follows that a particularly adjusted lyophilized extract of the berries could be responsible for the increased biological activity of no and the observed biological and pharmacological activities of anthocyanins in circulatory disorders. the study was supported by grant vega / / and / / . p- . . - attenuation of dysfunction, oxidative stress and apoptosis by resveratrol in benzo(a)pyrene exposed ins -e / pancreatic beta cell line s. c ß elik, b. baysal faculty of medicine, afyon kocatepe university, afyonkarahisar, turkey diabetes is one of the most important problems in the world. this disease is a very important health problem due to affects many different organs and systems. it has been well established that, environmental pollutants had deleterious effects on glucose metabolism, and caused insulin resistance and type diabetes. with this investigation, it was aimed to investigate the effects of benzo(a)pyrene on pancreatic beta cells and treatment affects of resveratrol. in this study, rat ins- e beta cell line was used. after reaching the appropriate number of cells culture operations by cell culture, benzo(a)pyrene ( lm, hours) application have been made after resveratrol ( lm, hours) application. after incubations oxidative stress, insulin secretion (in cell and in medium), cell proliferation and apoptosis were analysed in cells by biochemical and molecular techniques. b(a)p application resulted in no increase and resveratrol also increased this level of no. resveratrol increased the tas levels decrased by b(a)p, and tos levels were also increased by resveratrol interestingly. osi levels determined with tas and tos levels, has no significant change between groups. gsh levels were decreased by b(a)p while resveratrol increased its' level to control level. mrna expression levels of beta cell functions related genes ins- , ins- and sirt- were increased by resveratrol treatment. insulin levels in cell and in medium were increased after resveratrol treatment. mrna expression level of foxo- gene was increased while pdx- was decreased by resveratrol treatmeant. b(a)p suppressed the mrna expression of p gene, but resveratrol increased. the effects of b(a)p on pancreatic beta cells and the protective effects of resveratrol on this cells were investigated in vitro with this research firstly. the results obtained from this research showed that oxidative changes, functional impairment and carcinogenetic effects of b(a) p in pancreatic beta cells could be blocked by resveratrol. the protective effect of vitamin e (alphatocopherol) on ischemia-reperfusion injury in rat liver ischemia-reperfusion (i/r) process is usually used during transplantation and resection of the liver but liver dysfunction and cellular death due to lack of oxygen in tissues, changes in the balance of oxidant/antioxidant in favor of oxidants, and inflammatory response are inevitable during this process. in the present study, it was aimed to investigate whether vitamin e(alpha-tocopherol), an antioxidant agent, has a protective effect against liver ischemia reperfusion injury in rats by using morphometric methods. for this purpose, adult sprague-dawley male rats were divided into groups as; control, ischemia / reperfusion (i/r), and vitamin e+ischemia/reperfusion (evit +i/r). in experimental groups, the total hepatic ischemia was applied for minutes followed by a hour of reperfusion. in the treatment group, days before ischemia mg / kg dose of vitamin e was administered intraperitoneally once a day. after the termination of the reperfusion, the rats were perfused by cardiac way and liver tissues were dissected. following volume and weight calculations, the livers were subjected to the standard histological preparation methods and embedded in paraffin. serial sections at lm thickness were obtained from these blocks, stained with hematoxylineosin, and analyzed with morphometric methods. in light microscopic examinations of the i/r group, irregularity in lobules, dilatation in central veins and sinusoids, extensive areas of necrosis and pycnotic nuclei were seen in hepatocytes. the volume density of sinusoids to liver parenchyma was estimated as % in the control group, whereas it was % in the i/r group. this value was decreased to % in the evit + i/ r group. however, no significant difference was found among the groups in the lobule area calculated by the point counting method. these results show that intraperitoneal vitamin e administration for days prior to ischemia partially inhibits damage caused by ischemia-reperfusion injury in the liver. the leaves, fruit and bark of annona muricata, a member of the annonaceae family, are commonly used in the traditional medicine of tropical and subtropical regions. in recent years, many studies have shown their anti-cancer, anti-convulsant, antiarthritic, anti-parasitic, anti-malarial, and anti-diabetic activities. it should be noted that these characteristics have been described using different types of extracts from different parts of the plant. our studies have focused on the systematic characterization of activities most easily accessible from an aqueous extract of leaves, with hitherto documented antihypertensive and hepatoprotective effects. we found that the extract shows almost % efficiency against hydroxyl radicals. with increasing concentrations, the effectiveness weakened, reaching a second peak ( %) at a concentration of mg/l. the scavenging activity against no degradation products maintained a continuously increasing trend with a maximum at a concentration of mg/l. surprisingly, the extract initiated peroxynitrite production in a similar trend, except at mg/l, where it scavenged peroxynitrites with relatively high efficiency, up to %. these findings are consistent with the elevated levels of reduced glutathione detected after incubation of liver mitochondria with extract to a maximum concentration of mg/l, with subsequent sharp decline. the activity of glutathione s-transferase was decreased, although not significantly. this indicates a reduction of metabolic processes of compounds, allowing their action over a longer period of time. in a live system, even antihypertensive effects can be observed. however, a significant outflow of gsh to create the gsh adducts of active substances, and particularly s-nitrosoglutathione from increased production of peroxynitrites, can cause liver toxicity. the study was supported by grant vega / / and / / . the role of polyamine metabolism in curcumin induced apoptosis via reactive oxygene species (ros) generation in growth hormone (gh) overexpressed t d breast cancer cells r. genc ß, a. coker gurkan, e. d. arisan, p. obakan yerlikaya, n. palavan unsal, n. palavan unsal t.c istanbul k€ ult€ ur € universitesi, istanbul, turkey autocrine growth hormone (gh) signaling triggers cell proliferation, growth, metastasis and drug resistance in cancer cells. polyamines (pas) play an essential role in cell cycle, proliferation, growth and carcinogenesis of various cancer types such as prostate, colon and breast cancer. odc (ornithine decarboxylase) is the key enzyme in the pa biosynthetic pathway that is under control of antizyme (az) and antizyme inhibitor (azi) activity. pa catabolic enzymes polyamine oxidase (pao) and spermine/spermidine acetyle transferase (ssat) by-products triggers reactive oxygene species (ros) generation and apoptotic cell death. curcumin decreased cell viability in dose and time dependent manner in t d wt and gh+ cells. although forced gh expression induced cell proliferation, lm curcumin inhibited cell invasion. curcumin ( lm) induced apoptosis by acting on intrinsic and extrinsic pathway in both cell lines. moreover, curcumin supressed odc, azi expression in wt and gh+ t d cancer cells. although curcumin decreased az expression in t d wt cancer cell, increased az expression was determined in t d gh cancer cell. pao and ssat expressions were upregulated in t d gh+ cells. concomitantly, putrescine levels were increased in t d gh+ cancer cell compared to wt cells and curcumin depleted spermidine and spermine levels in wt and gh + t d cells. curcumin induced-apoptotic cell death via ros generation and co-treatment of n-acetyl cysteine (nac) overcame curcumin effect. conclusion, forced gh expression triggers cell proliferation and growth via acting on polyamine metabolism and curcumin-triggered ros generation was prevented by nac treatment in t d wt and gh+ breast cancer cells. acknowledgment: this study was supported by tubitak research project (project no: z ). the radio-protective effects of propolis and nigella sativa oil on oxidative/nitrosative stress in liver tissue of rats exposed to total head irradiation s. taysi our purpose is to investigate propolis and nigella sativa oil (nso) for their antioxidant effects on the liver tissue of rats exposed to ionizing radiation. a total of sprague-dawley rats were divided into five groups to test the radioprotective effectiveness of of propolis and nso administered by orogastric tube. appropriate control group was also studied. biochemical parameters in liver tissue of rats were determined by spectrophotometer. xanthine oxidase (xo), nitric oxide synthase (nos), superoxide dismutase (sod) activities, nitric oxide (no•) and malondialdehyde (mda) levels were higher in ir group while glutathione-s-transferase (gst), glutathione peroxidase (gsh-px) level in the ir group were lower in this group when compared to the other groups. gst activity in ir plus propolis group was statistically higher than in all other groups. propolis and nso clearly protect liver tissue from radiationinduced oxidative stress. the effects of royal jelly on the antioxidant parameters in the breast tissues of the rats with breast carcinoma treated with paclitaxel or not effects of royal jelly on the breast tissue antioxidant parameters in rats with breast carcinoma treated with paclitaxel or not. - weeks old female sprague-dawley rats (n = ) included in current study were divided into groups: control group (n = ) with healthy rats; breast cancer group (n = ); breast cancer group (n = ) treated with mg/kg paclitaxel injection (once a week for weeks); breast cancer group (n = ) treated with mg/kg royal jelly (by oral gavage for days); and finally breast cancer group (n = ) treated mg/kg royal jelly in addition to mg/kg paclitaxel injection. rats with breast carcinoma was obtained at th days after a single dose injection of mg/kg n-methyl-n-nitrosourea (mnu). all cancer groups were followed by days with treatment of paclitaxel and/or royal jelly. the antioxidant parameters in rat breast tissues, superoxide dismutase (sod) and catalase (cat) activities were determined by spectrophotometric colorimetric methods and glutathione (gsh) by high performance liquid chromatography (hplc). all the antioxidant parameters decreased in breast cancer group without any treatment (p < . ). but, statistically non significant increases were observed by paclitaxel and royal jelly treatment (p > . ). this study indicated that royal jelly supplementation can not be sufficient to increase the antioxidant parameters in breast cancer. we are going to continue to identify the effects of royal jelly on breast cancer detail. the effects of n-acetylcysteine on microsomal and serum paraoxonase activities at high fat diet induced obese rats obesity is a chronic disease that develops from the interaction between genotype and environmental factors and increase in the accumulation of body fat. it is related with glucose and lipid metabolism disorders, cardiovascular diseases and increased oxidative stress. paraoxanase (pon ) is an enzyme which plays a protective role in oxidative stress, inflammation and liver diseases. it has been suggested that pon has protective effects against high fat diet induced obesity and obesity related disorders. n-asetylcysteine (nac) is a potent antioxidant due to its ability to stimulate glutathione synthesis. the aim of this study was to evaluate the microsomal and serum pon enzyme activities (paraoxonase, arylesterase and lactonase) at high fat diet induced obese rats in the presence of nac. this study consisted of control, obese and nac-supplemented obese ( g/l nac) groups. eighteen sprague-dawley rats were randomized into three groups (n = ). control rats were fed by standart food and obese and nac groups were fed by high fat diet. the microsomal and serum paraoxonase, arylesterase and lactonase activities were determined by colorimetric methods. serum paraoxonase, arylesterase and lactonase activities decreased in obese and nac groups when compared to control groups. on the other hand microsomal paraoxonase, arylesterase and lactonase activities increased in nac group when compared to control and obese groups. however there was no statistically significant difference between the groups. it has been concluded that the microsomal and serum paraoxonase enzyme activities did not change at high fat diet induced obese rats in the presence of n-asetylcysteine. reactive oxygen species, playing an active role in the early and late course of acute pancreatitis, lead to dysfunction of cell membrane and releasing of lysosomal enzymes, and thereby to the injury in pancreatic cells. gallic acid, found in vegetables such as green tea, is an active component which has antioxidant, antiinflammatory, antiviral, anticancer activities. the aim of this study was to investigate the effects of gallic acid in experimental acute necrotizing pancreatitis (anp) model in rats. eighteen male sprague-dawley rats were divided into three groups ( rats in each group). group : sham + saline; group : anp induced by intraductal glycodeoxycholic acid and intravenous cerulein; and group : anp + gallic acid ( mg/kg/day, intraperitoneal). at the end of th hours, pancreas histopathology was examined. the levels of serum amylase as a diagnostic marker of pancreatitis, interleukin- (il- ), total antioxidant status (tas), nitrite + nitrate, total thiols as antioxidant marker and thiobarbituric acid reactive substances (tbars) to measure malondialdehyde (lipid peroxidation product) were determined by spectrophotometric methods. serum amylase, il- , plasma tbars levels were significantly higher but total thiols levels were lower than sham group in anp group without treatment (p < . ). however; tas and nitrite + nitrate levels did not show any significant difference (p > . ). on the other hand, while serum amylase, il- and tbars levels were lower, total thiols levels higher in gallic acid treatment group than in the untreated anp group, but statistically insignificant (p > . ). in conclusion, gallic acid treatment is beneficial but not sufficient to treat the acute necrotizing pancreatitis in rats. p- . . - evaluation of oxidant/antioxidant system parameters, il- and il- levels in amniotic fluid of pregnancies with down sydrome b. _ imge erg€ uder , s. bahsi , t. bahsi , v. topc ßu , a. bakir ankara universty faculty of medicinel, ankara, zekai tahir burak research and training, hospital genetic center, ankara, turkey introduction and aim: down sydrome (ds) can be diagnosed at high-risk of down syndrome pregnancies by invasive prenatal testing. in this study we aimed to demonstrate antioxidant/oxidant system markers, il and il levels in amniotic fluid samples of pregnancies affected by ds. materials and methods: for this purpose we collected amniotic fluid samples from pregnancies affected by down sydrome and normal healthy pregnancies who applied to zekai tahir burak research and training hospital genetic center and were proceeded with amniocenthesis. in the amniotic fluid samples; malondialdehyde (mda), superoxide dismutase (sod), glutathion peroksidase (gsh-px) xhantine oksidase (xo), catalase (cat), adenozine deaminase (ada), nitric oxide (no), nitric oxide senthase (nos) enzymatic activities were evaluated by spectrophotometric methods, il and il levels are evaluated by elisa. for statistical analysis student's t-test and spearman corralation analusis are used. results: it was found that sod levels are significantly elevated in study group compared to control group (p < . ). besides this, in study group, cat and il- levels are found singnificantly lower than control group (p < . ). we couldn't find any significant difference between two groups in terms of mda, gsh-px, xo, no, nos, ada ve il- levels (p > . ). discussion and conclusion: there is an important decrease in inflammation compared to normal pregnancie in the amniotic fluid of pregnancies having ds. based on these results, sod enzyme may be used as a marker for prenatal diagnosis of ds. for this purpose these experiments should be tried on larger sample groups. the aim of our work was to compare prooxidant and antioxidant properties of linalool, which is the oxygenated monoterpene compound reported to be a major volatile component of the essential oil of several aromatic species, in hep g cells. cytotoxicity of linalool was assayed by celltiter-blue Ò cell viability assay. malondialdehyde levels result in membrane damage in hep g cells were assayed by using fluorometric method. hep g cells were incubated with linalool at , and hours. the viability of hep g cells decreased in a manner dependent upon concentration and incubation time. the ic values were calculated . lg/ml ( hours), . lg/ml ( hours) and . lg/ml ( hours). but, cell viability of hep g cells increased when the cells preincubated with linalool at lower concentrations (˂ic ) against h o cytotoxicity. membrane-damaging effects of linalool were increased with accelerating concentrations. on the other hand, membrane damaging effect of h o was decreased when the cells preincubated with linalool before h o incubation. oxygenated monoterpene linalool had both prooxidant and antioxidant properties showing membrane damaging and protective effects on hep g cells depend on concentration. postprandial lipemia is primarily characterized by increasing triglyceride levels after the lipid rich meal. postprandial lipemia may cause oxidative stress by increasing free radical production and increasing oxidative stress could be responsible for the development of many diseases. plasma oxidant-antioxidant status was evaluated in healthy individuals with postprandial hypertriglyceridemia generated by performing oral triglyceride tolerence test (ottt). the study group included subjects ( female and male). ferric reducing ability of plasma (frap), total thiol and thiobarbituric acid reactive substances (tbars) levels were determined by colorimetric methods at fasting and nd, th and th hours following ottt. the levels of frap and thiol were significantly higher in males than females (p = . and . , respectively). thiol levels decreased significantly in both gender at postprandial nd, th and th hours as compared with fasting condition (p = . ). while tbars levels increased at postprandial nd hour, that was only significant for male individuals (p = . ). it has been concluded that postprandial lipemia may change oxidant-antioxidant balance in favor of oxidants and gender is an important criteria while assessing the oxidant-antioxidant status in postprandial lipemia p- . . - ischemia modified albumin and c-reactive protein levels in prediabetes prediabetes is a state of abnormal glucose homeostasis characterized by the presence of impaired fasting glucose, impaired glucose tolerance, or both. the aim of this study was assess serum ischemia modified albumin (ima) in prediabetes and determine its correlation with other risk factors for chronic complications such as inflammation and hyperglycemia. glucose, insulin, total cholesterol, hdl cholesterol, triglycerides, albumin, c-reactive protein (crp) and ima were measured in patients with prediabetes and controls. prediabetes patients had higher levels of ima and crp in comparison with control subjects but there was no significant difference between groups for ima. significant positive correlation was observed between crp and fasting glucose, insulin. there was no significant correlation between ima levels and the parameters tested. we have shown higher level of crp in prediabetes. these results support the hypothesis that chronic inflammation may be involved in development of hyperglycaemia. p- . . - the effects of s _ io nanoparticles of rat uterine smooth muscle specially used in textile field sio nanoparticles on uterus smooth muscle was aimed to be researched. in this study wistar albino female rats were used. rats were separated in groups as control, dose ( lg/ml), dose ( lg/ml) and dose ( lg/ml). nanoparticle's size was chosen as nm. preparations of four groups were evaluated for biochemical and histological examinations. all isolated uterine smooth muscle stripts except the controls were treated with sio for two hours. in biochemical examinations in order to evaluate oxidative stress level of malondialdehit (mda), activity of superoxide dysmutase (sod) and glutathione peroxidase (gsh-px) were measured. in histological examinations via electron microscope ultrastructure of uterus was examined as well as apoptotic cells detected with immunofluorescent labeling method. intergroups differences were defined by statistical analysis. while mda level increased depending on the dosage, sod level was decreased depending on the dosage. gsh-px rate was decreased for each dosage with respect to control. however, no significant difference is detected between groups. in electron microscopic examination no changes were observed in uterus ultrastructure with compare to control. however, in immunofluorescent labeling it was detected that apoptosis increased in dosage groups with compare to control group. as a result, it was thought that application of sio nanoparticles, in nm size and in , and lg/ml dosages caused of oxidative stress and apoptosis. this results suggested that sio has toxic effects on uterine smooth muscle. uterine myomas are the most common benign pelvic tumors arising from myometrium. they are rarely associated with mortality but responsible for significant morbidity and have adverse effects on quality of life especially in reproductive age women. reactive oxygen species and superoxide dismutases, as well as sex steroids play important roles in the reproductive physiology processes. in addition, oxidative stress and impaired antioxidant defense system have been linked to pathophysiology of various diseases including malignant gynecological disorders. clinical investigations indicate that women with myoma may have increased risk of developing malignant tumors particularly sarcomas. the present study aimed to investigate the possible role of oxidant and antioxidant status in myomas. blood and urine samples of myoma patients were collected. activities of erythrocyte antioxidant enzymes [copper-zinc superoxide dismutase (cuzn-sod), catalase (cat), glutathione peroxidase (gpx )] and levels of lipid peroxidation biomarkers [plasma malondialdehyde (mda) and urine -epi-prostaglandin f a ( -epi-pgf a)] were determined. the results were compared with those of controls. the groups were matched in terms of age, body mass index, smoking habit, coexisting chronic diseases, menopausal status and sex steroid hormone levels. all antioxidant enzyme activities were higher ( % for cuzn-sod, p = . ; % for cat, p . ) and the levels of lipid peroxidation biomarkers were lower (% for mda, p = . and % for -epi-pgf a, p > . ) in myoma patients compared to controls. correlation analyses showed a significant negative correlation between erythrocyte cuznsod activity and plasma mda levels (r = - . , p = . ). the decreased lipid peroxidation may be the consequence of elevated antioxidant enzyme activities and the data suggests a protective role of activated antioxidants especially cuznsod and cat in patients with myoma. p- . . - investigation of ischemia-modified albumin levels in patient with acute limb ischemia introduction: acute limb ischemia commonly occurs as a result of embolus caused by cardiac origin and which may end up with limb loss or even death if left untreated. thrombosis are usually seen where the arteries give branches and tendency to atherosclerosis is more serious at these sites. involvement of several arteries in either embolus or in situ thrombosis limits the adequacy of collateral circulation. restriction of blood flow due to arterial stenosis or occlusion often leads patients to complain of muscle pain on walking. any further reduction in blood flow causes ischemic pain at rest, which affects the foot. early recognition may prevent limb loss or death. ischemia can alter the capacity of the amino terminus of the albumin to bind free metal atoms such as cobalt, copper and nickel. this new, chemically changed albumin is called ischemia modified albumin (ima). ima is a sensitive marker of myocardial ischemia, skeletal muscle ischemia, pulmonary embolism, mesenteric ischemia and stroke. therefore, in this study it was aimed to investigate the ima level in acute limb ischemia. materials and methods: in this study, patients with acute limb ischemia (li group; mean age years) and healthy individuals (control group; mean age years) were included. ima levels were detected in control and li group by elisa (organo teknika, avusturya) using ima el _ isa kit. results: ima values were compared with nonparametric methods mann whitney u test, and significantly decreased ima level was statistically significantly different between li group and control group (p < . ). conclusion: there is a significant increase in serum ima in limb ischemia, so that alterations also might be clinically useful in the diagnosis of limb ischemia, but should be supported with further studies. object: polycystic ovary syndrome (pcos) is a multifaceted disorder with a pathogenetic pathway that is not fully understood yet. apart from hormonal derangements, insulin signaling defects and adipose tissue dysfunction, oxidative stress, defined as an imbalance derived from excessive formation of oxidants in the presence of limited antioxidants defenses, has been actively implicated in the etiology of the syndrome. the aim of this study was to determine of serum myeloperoxidase activity (mpo), paraoxonase activity (pon ) and arylesterase activity (are) in patients with pcos. material and methods: the study was carried out on women consisted of patients with pcos and healthy ones as control. serum pon activities were measured spectrophotometrically using diethyl-p-nitrophenylphosphate as substrate. phenylacetate was used as substrate for are measurement, and are activity was determined by measuring absorbance of the resulting phenol at nm. molar absorptivity coefficients were used in the calculation of pon and are activities as nmol phenol/ml serum/min. result: the mpo and are activities were significantly lower in the patient groups when compared with the control group ( . ae . - . ae . u/ml p < . , . ae . - . ae . u/ml p < . , recpectively). the pon activities are higher in the patient group ( . ae . u/ml) compared to the control group ( . ae . u/ml) are found, but are not statistically significant. conclusion: lower serum mpo and are activities might contribute to the increased susceptibility for the development of diseases risk in women with pcos. because free oxygen radicals are thought to contribute to the complication of many chronic diseases, the pcos may be related to oxidative stress. subclinical hypothyroidism, defined as an elevated serum thyroid stimulating hormone level associated with serum thyroid hormone concentrations within the reference range. free radicalmediated oxidative stress has been implicated in the pathogenesis of thyroid disorders. the ischemia-modified albumin (ima) has been proposed as a marker of protein oxidative damage, which has been found to reflect hypoxic stress. this study aimed to investigate the influence of subclinical hypothyroidism on serum ima levels. thirty-one subclinical hypothyroidism patients and control subjects were enrolled in the study. albumin, ima were measured and ima/albumin ratio was calculated. to determine the ima levels the measurement method based on albumin cobalt binding assay was used. serum ima levels of patients with subclinical hypothyroidism were . ae . absu, ima levels of control subjects were . ae . absu. ima levels were significantly higher in patients with subclinical hypothyroidism patients than in control subjects (p < . ). when ima values were normalized for albumin concentrations, the ima/albumin ratio was also significantly elevated in patient group compared to control group (p < . ). ima levels are increased in patients with thyroid dysfunction. elevated levels of ima can be a clinically useful marker of protein oxidative damage in subclinical hypothyroidism. p- . . - the effects on endothelial dysfunction of quercetin in streptozocin-induced diabetic rats excessively produced in pathologic conditions. ultimately, imbalance between oxidants and antioxidants results with oxidative stress (os). in this study, we investigated some os parameters in standard ( % protein, % ( % sucrose) carbohydrate, % lipid) and sucrose ( % protein, % ( % sucrose) carbohydrate, % lipid) diet fed bdnf heterozygous mice liver tissues. the male c bl strain wild type (+/+) and bdnf heterozygous (+/À) mice ( weeks) were obtained. the animals were fed ad libitum by special standard and sucrose diets. twenty four mice were divided into four groups and each group consist six mice. all mice were fed for weeks. first group involved in c bl wild type mice and fed by standard diet. second group contained c bl bdnf heterozygous mice and fed by standard diet. third group consisted c bl wild type mice and fed by sucrose diet. fourth group involved in c bl heterozygous mice and fed by sucrose diet. in first group, mda levels, sod and cat activities were higher than other groups. in second group, cat activities were lower than other groups. but, we could not find any statistically significant differences between all groups about mda, sod, cat levels in bdnf heterozygous mice liver tissues. in conclusion, standard and sucrose diet feeding may not affect mda, sod and cat levels in bdnf heterozygous mice liver tissues. brain-derived neurotrophic factor (bdnf) is member of neurotrophin family which plays critical roles in the development, differention, survival, maintenance of the central and peripheral nervous systems. bdnf also contributes to food intake and body weight control. bdnf heterozygous mice display increased body weight and mild hyperphagia. expression of bdnf is not limited to the brain, it also express some peripheral tissues like adipose tissue, liver, kidney, skeletal muscle, heart. even though roles of bdnf are well known relatively in central nervous systems, effects of this protein is not clear in peripheral tissues. as mentioned before, it is expressed in organs involved in energy, lipid and glucose homeostasis, including the liver, adipose and muscle tissues, but its role there remains unclear. in this study, we aimed to investigate role of bdnf on liver oxidative stress parameters in heterozygous mice model fed fat diet induced obese mice. in this study, we used c bl/ mice inbred strain wild type and bdnf heterozygous (+/À) mice. animals were divided to two groups: wild type (n = ) and bdnf heterozygote mice (n = ). the animals were fed ad libitum by high-fat diet during month. weight gain was recorded every th days. in liver tissues were measured malondialdehyde (mda), superoxide dismutase (sod) and catalase (cat) by spectrophotometric methods. liver mda levels decreased in obese bdnf heterozygous mice compared to obese wild type group and statistically significant difference between groups. bdnf heterozygous mice cat activities were higher than the other group and this difference was statistically significant. there was no statistically significant difference between the groups in terms of sod activities. it has been concluded that the mda levels and sod enzymes activities changed at high-fat diet induced obese bdnf heterozygous mice compared to wild type mice liver tissues. p- . . - disturbances of microelements profile in serum of overweight/obese adult females with acute and persistent pro-inflammatory chlamydia pneumoniae infection p- . . - determination of reactive oxygen species induced dna damage using modified cupric reducing antioxidant capacity (cuprac) colorimetric method s. uzunboy, s. demirci c ß ekic ß, r. apak department of chemistry, istanbul university, faculty of engineering, istanbul, turkey reactive oxygen species (ros) term is a common name of a group of species. hydroxyl radical and singlet oxygen can be taken into account as ros samples. ros may be formed as a result of endogenous or exogenous reasons. although ros have some beneficial functions, they should be balanced by antioxidants (aox). excessive amounts of ros can attack biological macromolecules including dna. dna damage is usually related with mutagenic and carcinogenic changes. that's why determination of dna damage is so important and there are a great many studies in literature comprising different techniques. one of the most common of them is the 'comet assay'. but application of the method and interpretation of the results is not easy. investigation of certain dna damage products is also very common. these methods usually need expensive instrumentation such as using tandem mass spectrometry. on the other hand, depicting total dna damage on a certain product may cause misinterpretations. in the presented study, dna was decomposed by hydroxyl radicals produced by fenton method. in the study while dna is not cuprac reactive the oxidation products can react with the cuprac reagent. the effect of aox was also investigated. for this purpose, selected aox compounds were added to the reaction medium. because of their radical scavenging effect, the cuprac absorbance decreased in the presence of aox. in the presence and absence of aox, absorbance differences were calculated. the calibration graphs between final concentration and absorbance differences were drawn for each aox. gallic acid was determined as the most effective one among the tested aox samples. for statistical comparison with the presented study, tbars was used as reference method. direct use of dna as a probe material to determine oxidative damage may be an advantage to understand dna hazard in biological systems. the proposed method can be applied in all laboratories having a spectrometer as a cost-effective and simple procedure. p- . . - effects of alpha- antagonists on oxidative system of rat heart tissue benign prostate hyperplasia is a progressive process occurring in the stromal and epithelial components of the prostate. alpha- receptor blocking agents are used for relaxation of the smooth muscles in the prostatic stroma. our aim was to investigate the effects of alpha- antagonists on oxidative system of rat heart tissue. male wistar albino rats were divided into groups randomly. ) tamsulosin ( mg/kd/day), ) terazosin ( mg/kg/day), ) doksazosin ( mg/kg/day), ) alfuzosin ( mg/kg/day), ) control. all drugs were administered every other day single doze via oral. rats were sacrificed after days. heart tissue was taken for biochemical analysis. malondialdehyde (mda), nitric oxide (no), protein carbonyl (pc) levels and superoxide dismutase (sod), glutathione peroxidase (gsh-px) enzyme activities were determined in supernatant samples. there was not an significant difference between terazosin, doxazosin, alfuzosin, tamsulosin groups in means of sod, mda and gsh-px levels. no levels were significantly different between tamsulosin group and the control group (p = . ). in addition, tamsulosin group and terazosin group were also significantly different (p = . ). according to these results we can say that tamsulosin group had higher no levels than control and terazosin group. tamsulosin's enhancer effect on no levels leads to relaxation of the heart muscle and vascular relaxation, and so fewer side effects than other alpha antagonists. the effect of rat liver tissue radical metabolism and the protective role of hippophae rhamnoides l. on cold and immobilization stress model cold and immobilization stress is a widely used model for study the changes that occur on oxidant-antioxidant balance. hippophae rhamnoides l. (seabuckthorn; sbt) a unique and valuable plant has recently gained worldwide attention, mainly for its medicinal and nutritional potential. this study was aimed to investigate the protective role of sbt which is a natural herbal product with high antioxidant content on oxidative and nitrosative stress induced by cold and immobilization stress in rats. wistar albino rats were divided into groups randomly. control (i.p. physiological saline), sbt (i.p. mg/kg/ hours sbt), stress (i.p. physiological saline; hours cold + immobilization stress) and sbt + stress (i.p. mg/kg/ hours sbt; hours cold + immobilization stress) groups were formed. nitrotyrosine levels were determined by elisa whereas total antioxidant capacity, total thiol, total glutathione, nitrite-nitrate levels and superoxide dismutase and glutathione peroxidase activities were measured by colorimetric methods. sbt + stress group nitrite-nitrat (p = . ), total glutathione (p = . ) levels and glutathione peroxidase activities (p = . ) were found to be significantly higher whereas superoxide dismutase activity was found to be lower (p = . ) when compared to stress group. there was no significant differences between stress group total thiol and total antioxidant capacity levels compared with stress + sbt group. stress + sbt group oxidative and nitrosative stress marker -nitrotyrosine level was found to be significantly higher when compared with control group (p = . ) whereas there was no significant differences between stress and stress + sbt groups. all this data show that sbt has antioxidant properties on cold and immobilization induced oxidative and nitrosative stress in rat liver tissue. obesity is a major health problem with growing incidence and accompanying complications. its relation with diminished cognitive functions was reported. this study aims to evaluate the effects of obesity induced oxidative stress and metabolic alterations on the cognitive functions of children and adults. children and adolescents with obesity (age: - ); and age and gender matched healthy subjects were enrolled. all subjects completed the battery tests of cnsvs via computer. the scores were compared by using commercial software (ibm spss statistics ). biochemical parameters, malondialdehyde (mda) and protein carbonyl (pc) levels were estimated. mda and pc levels were significantly higher in subjects with obesity ( . ae . lmol/l; . ae . nmol/ml) than the controls ( . ae . lmol/l; . ae . nmol/ml) (< . ). there was statistically significant difference between study and control groups on all cognitive performance domains. significant correlation was detected between mda, pc levels and the cognition indexes. children with obesity should be evaluated for the cognitive functions, together with the metabolic follow-up. obesity induced oxidative stress may be the reason of the diminished cognition in children as well as the changes in the lipid profile and inflammation, but we need larger study groups to lighten these complex process. p- . . - relative contribution of nitric oxide synthase (nos) isoforms to oxidative/nitrosative stress in the cerebral cortex of rat with acute liver failure (alf) acute liver failure (alf) is associated with deregulation of nmda/cgmp/no signaling and oxidative/nitrosative stress in the brain. however, the relative roles of the different nos isoforms and the mechanisms underlying alterations in their activities during alf are not fully clear. here we investigated gene and protein expression of nos isoforms, nos activity, enos uncoupling and total no production in cerebral cortex of rats with thioacetamide (taa)-induced alf. sprague dawley rats ( - g) received three i.p. injections of taa ( mg/kg) at hours intervals. the brain cortex expression nos isoforms (enos/inos/nnos) was measured by real-time pcr and western blot, nos activity was tested by monitoring the conversion of radiolabeled arginine to citrulline. reactive oxygen species (ros) were quantified in the presence of nos substrate l-arginine, using the carboxy-h dcfda probe. no was measured with the griess procedure. the enos expression was decreased, whereas the enos dimmer/monomer ratio and nnos/inos expression were elevated in taa treated rats. while the total nos activity was decreased, the inos activity was elevated and no concentration tended to increase. ros production was elevated by taa. unspecific nos inhibitors l-name and l-nna attenuated ros production in both control and taa rats, but with higher efficiency in the latter case. ca + chelation had almost the same effect as pharmacological nos inhibition suggesting that ca + -independent inos activity is not the main source of ros. incubation with high dose of tetrahydrobiopterin (bh ) with which is critical for enos dimerization and subsequent no production also reduced ros production indicating the enos uncoupling phenomenon in taa cortex. the study points to enos downregulation due to lowered protein expression and uncoupling as a novel mechanism contributing to enhanced superoxide o anion formation, and confirms the role of inos/nnos in enhancing no synthesis in alf-affected brain. introduction: diabetes mellitus (dm) is an endocrine disorder of world which is characterized by altered blood glucose levels and related complications including hepatic injury. myrtus communis l. (mc) is widely used by diabetic patients in the folk medicine of turkey as well as they are used worlwide. it is known that of leaves, oil and fruit of myrtus communis l. (mc) have therapeutic effects on diabetes mellitus (dm). this study was aimed to analyze the possible antidiabetic and hepatoprotective effects of mc berries in streptozotocin (stz) induced diabetic rats. materials and methods: a total of thirty rats composed of six groups as each included five rats were used. mg/kg stz was injected once to animals to induce dm. after stz injection, rats were exposed to three different ethanol extracts of mc berries ( , and mg/kg) by oral gavage during days. alanine aminotransferase (alt) and aspartate aminotransferase (ast) levels were determined in serum and glutathione (gsh), malondialdehyde (mda) levels and superoxide dismutase (sod) activity were determined in liver tissue. results: mc administration provided significant reducement in the altered serum glucose, ast and alt levels in all diabetic groups. mc extract showed significant antioxidant activity by altering sod activity and gsh level and reducing mda levels in diabetic rats compared to controls (p < . ). serum ast and alt levels were reduced by mc administration in all diabetic groups. mc administration provided significance increment in sod activity and gsh level, and significant reduction in mda levels compared to controls (p < . ). the maximum hypoglycemic and antioxidant effects were observed at mg/kg dose of mc. background: human serum paraoxonase (pon ) is a calcium dependent esterase that hydrolyzes organophosphates and also arylesters such as phenyl acetate. pon prevents ldl oxidation by hydrolyzing lipid peroxides. pon is inhibited by various chelating agents, heavy metal ions, and sulfhydryl reagents. in our study we investigated the effect of calcium on ldl oxidation of purified pon q r isoenzymes. methods: pon q r isoenzymes were partially purified from human serum. both allozymic forms were treated by preincubation with mm edta for minutes. ldl oxidation was induced by copper ions. formation of thiobarbituric acid-reacting substances (tbars) was used as a measure of lipid peroxidation. homocysteine thiolactonase (htlase activity) and arylesterase activities were measured spectrophotometrically by using homocysteine thiolactone and phenylacetate as the substrates. results: addition of mm edta to partially purified hdl-pon q r isoenzymes inhibited % of htlase and arylesterase activities. inactivation of pon for arylesterase/htlase activity by the addition of edta did not reduce the abilities of both allozymic forms in protecting ldl from oxidation. conclusion: ca + -dependent inhibition of pon q r arylesterase/htlase by using the metal chelator edta, did not alter pon 's ability to inhibit ldl oxidation. pon 's ability to protect ldl from oxidation may not require calcium. p- . . - evaluation of cholinesterase inhibitory effect, anti-radical and anti-lipid peroxidation activities of mentha pulegium i. hamad , college of applied medical sciences, aljouf university, aljouf, saudi arabia, faculty of medicine, bahri university, khartoum, sudan introduction: many studies indicated that intake of dietary and medicinal plants is effective in preventing or suppressing many diseases, therefore, there is a growing interest in plant'sbioactive compounds. mentha pulegium, is widely used in gulf countries in herbal teas or as additives in commercial spice mixtures for many foods to offer aroma and flavor. the aim of this study is to investigate the in vitro radical activity, the total phenol and flavonoid content, anti-lipid peroxidation and the cholinesterase inhibitory effects of mentha pulegium methanol extract. methods: the acetylcholinesterase and butyrylcholinesterase inhibitory potentials of extracts, were evaluated by colorimetric assay. the in vitro antioxidant activity was measured by dpph assay, the total phenols content was measured by folin-ciocalteau assay, the flavonoids content by the alcl colorimetric method, and the protective effect of menthe mentha pulegium extracts against lipid peroxidation was evaluated using a liposome oxidation system. results: the methanol extract showed a scavenging activity nearly equivalent to vitamin c which is attributed to its high phenolic and flavonoid contents. the extract possessed protective effect against lipid peroxidation in a dose dependent manner. the methanol extract shows very little anticholinesterase activity as compared to the standard compound, physostigmine. conclusion: results presented here indicate that mentha pulegiumpossess strong antioxidant activity and protective effects and they can therefore be used as a natural additive in food, cosmetic and pharmaceutical industries. type diabetes mellitus is a long term metabolic disorder that is characterized by hyperglycemia and insulin resistance. because of the hyperglycemia and free radicals, diabetes can cause cellular instability. micronuclei is a sensitive indicator of genetic damage and a marker of dna damage. micronuclei is also a morphological marker of chromosomal instability. in this study, we aimed to evaluate the frequencies of micronuclei in papanicolaou stained buccal cells of type diabetic patients. a total of type diabetic patients and healthy individuals were involved into our study. buccal smear samples that belong to these individuals were stained by using papanicolaou method for cytologic examination and the stained slides were evaluated by light microscopy (olympus bx- ). cells with micronuclei in each papanicolaou stained buccal smear sample were counted under light microscopy. the frequency of micronucleated epithelial cells were seen as significantly higher in type diabetic patients than the control group (p < . ). one of the boron compounds is sodium perborate tetrahydrate (nabo . h o), which the most widely used solid peroxygen compounds. it is used in safety bleach formulations, detergents and tooth powders. as known these products are commonly used in daily life. however, the actions on blood antioxidant defenses of sodium tetraborate against reactive oxygen species are not identified yet. it is reported that oxidative stress caused by ros damages. in this study, we searched enzyme activities of superoxide dismutase (sod), catalase (cat), glutathione-s-transferase (gst), glutathione reductase (gr), glutathione peroxidase (gsh-px) and glucose- -phosphate dehydrogenase (g pd) also the effect sodium perborate tetra hydrate on activities of these enzymes from human erythrocyte under in vitro conditions. according to our findings sodium perborate tetrahydrate caused significant (p < . ) increasing in the cat activity from red blood cell. the other antioxidant enzyme activities (sod, gst, gr, gsh-px and g pd) did not show any changing by influence of sodium perborate tetrahydrate. metabolism of obese individuals could be exposed to risk of chronic low-grade pro-inflammatory effect and oxidative stress. some inflammatory and oxidative markers have been studied recently. plasma total antioxidant status (tas) and total oxidant status (tos) parameters can be non-invasive markers of diseases such as fatty liver disease, laparoscopic procedures (pneumoperitoneum), accompanying inflammatory condition like urinary tract infection, diabetic neuropathy, chronic hepatitis. the study groups have been comprised of two groups with normal to over-weight children. tas and tos levels were detected and the oxidative stress index (osi) was computed as a marker of the grade of oxidative stress. the over-weight group displayed higher levels of fasting glucose, insulin resistance, the body mass index. also, we know that insulin resistance leads to increased lipolysis and free fatty acid output. higher tos as well as crp is related to the group, also lower tas than other group is shown. crp levels in plasma were positively correlated with insulin and glucose levels. in addition, there was a significant relationship between osi and insulin resistance in the over-weight group. tas and tos are together more accurate sings of oxidative and antioxidative status of people. as well as a raise over weight-related subclinical inflammation and a fall antioxidant capacity is significant even in children. this condition may eventually develop the risk of long-term vascular damage. the effects of hydrogen peroxide pretreatment on antioxidant enzyme activities in calli tissues of two eggplant genotypes under salinity o. yasarkan , e. aky€ uz , g. baysal furtana , s. s. ellialtioglu , r. tipirdamaz nezahat g€ okyigit botanic garden, istanbul, department of biology, faculty of sciences, gazi university, ankara, department of horticulture, faculty of agriculture, ankara university, ankara, department of biology, faculty of sciences, hacettepe university, ankara, turkey the effects of hydrogen peroxide (h o ) pre-treatment on catalase (cat) and superoxide dismutase (sod) were investigated and lipid peroxidation measured as malondialdehyde (mda) content of the calli from salt-sensitive (artvin) and salt-tolerant (mardin) eggplant genotypes under salinity stress. the seeds from each genotypes were germinated on ms medium for weeks and hypocotyl tissues from these plantlets were used as explants for calli induction on ms medium including mg/l , -d and . mg/l kinetin. as for the pre-treatment, the subcultured calli tissues were transplanted on the mediums containing and lm h o for hours and then transplanted on the mediums including mm nacl for hours. antioxidant enzyme analysis and mda measurement was carried out for the control, nacl-only, h o -only and h o pre-treated tissues. pre-treatment with h o decreased the deleterious effects of salt stress on mda contents. in comparison with salt stressed groups, h o pre-treatment with or without nacl reduced mda content especially in artvin. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in lm h o + nacl groups on each genotypes. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in lm h o + nacl groups on each genotypes. the result showed pre-treatment of lm h o induced acclimation of the plants to salinity. in addition, lm h o pre-treatment, as a stress signal, could trigger the activation of antioxidant enzymes in calli and in this way alleviated the oxidative damage in calli growth under salinity. the investigation of effects of ghrelin and cannabinoid cb receptor agonist and antagonist on oxidant and antioxidant mechanisms on brain tissues of penicillininduced epileptic rats the aim of this study is to investigate the individual effects of ghrelin and cannabinoid type (cb ) receptor agonist acea, the antagonist am- and the interaction of these two different systems on oxidant and antioxidant systems in the brain, cerebellum and brain stem tissues of penicillin-induced epileptic rats. in this study male wistar albino rats were used weighing - g. each group was consisted of rats. study groups: : control, :penicilin( iu), :penicillin( iu) + ghrelin( lg), :penicillin( iu) + am- ( . lg), :penicilin ( iu) + acea( . lg), :penicillin( iu) + am- ( . lg) + ghrelin ( lg), :penicillin( iu) + acea( . lg) + ghrelin( lg). than the levels of mda, gpx and sod are measured in plasma and tissue samples of these rats. penicillin was found to be induced lipid peroxidation in the brain, cerebellum and brain stem tissues in our study. ghrelin and acea, which both have anticonvulsant effects, were shown to be effective in reversing the oxidative damage caused by penicillin and proconvulsant am was found to further increase the oxidative stress caused by penicillin in these tissues. ghrelin also was found to suppress the oxidative stress caused by am in the cerebellum tissue but it did not contribute to antioxidant effects produced by acea. since the role of oxidative stress in epilepsy has been established, it may be suggested that ghrelin and acea may have anticonvulsant effects via their antioxidant features. the discovery of inhibitors for enzymes that metabolize endogenous ghrelin and cannabinoids through new studies may contribute to the improvement of seizure resistance in epilepsy. accelerated atherosclerosis in patients with ankylosing spondylitis (as) give rise to increased cardiovascular morbidity and mortality. endothelial dysfunction could be the initial process in the development of atherosclerosis. human endothelial cell-specific molecule- (endocan) is a novel human endothelial cell-specific molecule. therefore, we assessed serum endocan levels and carotid intimamedia thickness (cimt) as a surrogate marker of atherosclerosis in patients with as. a total of patients with a diagnosis of as according to newyork ctriteria and control subjects were included in our study. serum endocan, interleukin- (il- ), tumor necrozis factor-a (tnf-a), c reactive protein (crp) and cimt were measured in all participants. serum endocan, il- , tnf-a levels were measured with elisa. the other parameters were done by routine biochemical methods. as patients exhibited increased serum endocan levels and cimt compared to matched controls (p < . ). whereas, serum il- , tnf-a were similar between grous. in patient with as, there were no significant differences between active and inactive patients by means of il- , tnf-a, endocan and cimt. in as group, cimt correlated with disease duration and age (r = . , p = . ; r = . , p = . ). we could not find any significant correlation between serum endocan levels and parameters studied. our study shows increased cimt in as patients without traditional risk factors such as increased bmi, lipid profile compared to controls. although we found increased circulating endocan levels in patients with as, the other factors could affect increased atherosclerosis in this population because of lack of correlation between endocan and cimt. probable biomarkers could be related to increased cimt in patients with as should be investigated in larger study groups. keywords: ankylosing spondylitis, atherosclerosis, carotid intima media thickness, endocan p- . . - investigation of pentose phosphate pathway and oxidative stress in erthrocyte infected babesia ovis a. bildik, t. karagenc ß, p. a. ulutas, n. aysul, h. aksit adnan menderes university, aydin, turkey introduction: babesia infections occur in cattle, sheep, goat, horse, dog, cat pig and rodents. in this study, the effects of babesia ovis living and present in the erythrocytes to glucose metabolism was researched. at the same time, biochemical parameters were also associated with parasitemia. materials and methods: babesia ovis (israel) cell culture was provided from dr. abel martin gonz alez oliva (portugal). culture passaged or hours according to parasitemia state ( - %). biochemical analyses were performed in erythrocyte culture in which parasitemia between % and %. cell counts and hemoglobin concentration of erythrocytes culture suspension were measured at cell counter instrument and than it was washed times with physiological saline, erythrocyte suspensions were stored at- oc analysis. gssg (oxidize glutathione), gsh (reducte glutathione), nadph, glukoz p dehydrogenaz, gshpx (glutathione peroxidase), gshrx (glutathione reductase) were determined by commercial kits. all experiments were done in duplicate, the results were calculated by the number of erythrocytes. results: parasitemia was positively correlated with gsh, nadph and gshrx (p < . ). a correlation between other biochemical parameters was not observed. discussion: the pentose phosphate pathway in erythrocytes has an important role such as to provide pentose sugar required for the synthesis of nucleic acid, to reduce glutathione, to produce nadph and to protect from methemoglobin accumulation. in studie sthat naturally infected erythrocytes with babesia parasites, it was seen to be caused to oxidative stress, however gsh results in these investigation were obtained differently . conclusion: according to the results of this study that performed in vitro, it can be suggest that their glutathione metabolism and pentose phosphate pathway of parasites may active.key words: babesiosis, gsh, gssg, nadph, g pdh, gshpx, gshrx p- . . - in vitro protective effect of betaine on peroxidative injury caused by ethanol and aspirin exposure on rat brain synaptosomes i. sogut , g. kanbak istanbul bilim university vocational school of health services, istanbul, eskisehir osmangazi university medical school department of biochemistry, eskisehir, turkey aspirin intake of specific daily doses are advised by doctors to postmenapausal women and men above years of age to prevent heart attack and even cancer in recent times. in this study, the aim is to investigate the in vitro cytototoxic effects of different doses of ethanol ( mm, mm ve mm) alone and together with lg/ml aspirin, and possible protective role of mm betaine on rat brain synaptosomes. male sprague dawley rat forebrains were divided into equal pieces and pooled to form study groups. synaptosomal fractions extracted from pooled rat brains were incubated with different doses of ethanol, aspirin and betaine, and malondialdehyde (mda) levels, an important indicator of cellular damage, were measured. a significant increase (p < . ) was observed in mda level of mm ethanol group compared to control group. different doses of ethanol ( mm, mm ve mm) + aspirin exposure significantly increased (p < . ) mda levels compared to controls, whereas betaine administration significantly decreased (p < . ) lipid peroxidation caused by ethanol + aspirin treatment. we conclude that ethanol and ethanol + aspirin administration increases lipid peroxidation in rat brain synaptosomes while betaine helps prevent this peroxidative membrane injury.keywords: aspirin, betaine, ethanol, malondialdehyde (mda) p- . . - analyses of mitochondrial biogenesis in hepatocellular carcinoma treated with berberine f. aygenli, h. c ß imen yeditepe proteomics and mass spectrometry group (yediprot), genetics and bioengineering, yeditepe university, istanbul, turkey objective: berberine (bbr) has been demonstrated to have anticancer activities against various cancer types, particularly hepatoma. in this project, we aimed to reveal the effect of bbr treatment on mitochondrial biogenesis through sirtuins and hif- a in hepatocellular carcinoma cell line, hep b under hypoxia. method: hep b cells were subjected to normoxia ( % o ) and hypoxia ( % o ) in the presence or absence of bbr treatment. the amount of bbr was optimized via cell viability (mts) assay under normoxia. then, immunoblotting experiments were performed to identify the effect of bbr on hif- a, pgc- a, and sirtuins involved in mitochondrial stress. the variation in the oxphos complexes and the level of reactive oxygen species (ros) were also measured to investigate the effect of bbr on mitochondrial energy stress state. results: here, we present that cell viability was significantly decreased at lm. bbr treatment has shown significant reduction in hif- a and sirt which responsible for up-regulation of glycolysis. also, succinate dehydrogenase (cii) and cytochrome c oxidoreductase (ciii) of the oxphos complexes were downregulated without any change in nadh dehydrogenase (ci) or atp synthase (cv). bbr significantly abolished to oxidative stress under hypoxia, which was demonstrated as a reduction in the level of reactive oxygen species by decreasing on sirt expression. bbr induces the overexpression of sirt and its deacetylated-pgc- a, which might be an indicator of being a potent protective agent against hypoxia by normalizing mitochondrial function and inducing mitophagy in impaired mitochondria caused by deficiency of glycolysis and oxphos. conclusion: detailed information about the communication between hif- a and sirtuins and their relation to mitochondrial energy production was provided with the alteration of their activity by bbr treatment. it is highly expected that bbr and its derivatives might become important during the development of supplemental therapies. introduction: reactive oxygen species are involved in a variety of biological phenomena, such as carcinogenesis, inflammation and aging. among the targets of ros, dna appears most important in tumor biology since it is firmly established that cancer is a genetic disease. ros induce several kinds of dna damage, including strand breakage and dna-protein cross-linkage. fruit of zizyphus jujuba, a traditional chinese herb widely consumed in asian countries, has been reported to possess several vital biological activities. this study intends to evaluate their antioxidant activity on glioblastoma cells. materials methods: cell survival was quantified by colorimetric mtt assay. human gliblastoma cells were pretreated with lm h o after minutes lm ziziphus jujuba essential oil was added to the cells for three hours. then, the cell homogenates were taken and glutathione, total oxidant and total antioxidant capacity and nitric oxide levels were estimated using spectrophotometric methods. results: ziziphus jujuba treated cells could prevent intracellular glutathione from being depleted following an exposure of h o . also our data suggest that ziziphus jujuba is effective in preventing h o induced oxidative stress and nitric oxide levels. discussion: some research showed that h o was over produced in the pathological process of acute and chronic neuronal toxicity, the toxicity effect of b-amyloid on the cultured neuron and neuronal cell line was mediated by h o . the traditional medicine recommend several medicinal plants for providing relief from various inflammatory diseases. many research has been reported that the essential oil from seeds of helping to prevent the oxidative stress and neuronal diseases in brain. introduction: toxicity by oxygen radicals has been recommended as a major cause of cancer, heart disease, and aging. oxygen radicals and other oxidants appear to be toxic in large part because they start the chain reaction of lipid peroxidation. most of the analytical techniques for peroxide determination are generally time consuming and not very suitable for routine or on line analysis. we aimed to design a new biosensor for rapid determining of oxidant agent hydrogen peroxide. materials and methods: all reagents were of analytical grade unless stated otherwise, and were purchased from sigma aldrich. firstly, the -hidroxymetacrilate metacriloamidoscystein nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. free nh groups of catalase enzyme make schiff bases between nanopolymer's carbonyl groups, then immobilization was actualysed with cross linking reagent glutaraldehyde. we developed a biosensor system preparing ferrociyanide, selected as a mediator, in the buffer solution. results: polyhemamac nanopolymer and catalase complex were immobilized by glutaraldehite to construct a hydrogen peroxide biosensor. the responses of the biosensor are therefore proportional to the oxidation peaks of the complex at + . v potential. the cyclic voltammograms obtained from the experiments showed that, pottasiumferrociyanide mediator complex positively affected the biosensor responses for hydrogen peroxide determination. discussion and conclusion: as a result of this study, the method developed by the catalase enzyme electrode was found to be more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. since biosensor technology provides economical, practical, specific and sensitive results for the determination of hydrogen peroxide, it was improved very efficiently. p- . . - impact of amoxicillin, gentamicin and cefazolin sodium antibiotics on antioxidant gene expression and enzymatic activities in mouse liver p. g€ uller , h. budak , m. sisecioglu , m. c ß iftci department of chemistry, faculty of science, atat€ urk university, erzurum, department of molecular biology and genetics, faculty of science, atat€ urk university, erzurum, department of chemistry, faculty of arts and sciences, bing€ ol university, bing€ ol, turkey reactive oxygen species (ros) are highly reactive molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. living organisms have the antioxidant defence systems to block harmful effects of ros. the imbalance between oxidants and antioxidants is termed oxidative stress. the antioxidant defence mechanisms are divided into two groups as enzymatic and nonenzymatic defences. enzymatic defence mechanisms consist of enzymes like superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glucose- -phosphate dehydrogenase (g pd) and glutathione s-transferase (gst). the present study was designed to determine the effects of gentamicin, amoxicillin and cefazolin sodium antibiotics on the hepatic antioxidant system and to determine any possible correlation between enzymatic and molecular levels. for this reason, effects of these antibiotics on the transcription of the antioxidant system has been investigated by real time pcr, and then the enzyme activity of these enzymes have been measured in whole liver homogenate obtained from control group and the drug administered groups mice. our results demonstrate that administering antibiotics led to crucial inhibition of all antioxidant enzyme activity. while significant transcriptional activation for sod and cat was seen in the gentamicin treated group, the transcription of gst and g pd was decreased. however transcriptional activation was seen for sod and cat in amoxicillin administered group, the transcription of gst was decreased as compared with the control group. in the cefazolin sodium treated group, while cat and gst transcription were elevated significantly, the expression of sod and g pd were decreased. in conclusion, gentamicin, amoxicillin and cefazolin sodium affect the hepatic antioxidant system at the molecular and protein level. this work was supported by scientific research project of ataturk university of turkey (grant no: / ). p- . . - protective effects of curcumin supplementation on oxidant/antioxidant system changes created by organic phosphorus pesticide poisoning organic phosphorus pesticides (opp), widely used in agriculture or as insecticides in home, cause adverse health effects. chlorpyrifos is one of the most commonly used opp. we aimed to investigate the possible protective effects of curcumin (cur) supplementation, the principal curcuminoid of turmeric, on poisoning symptoms and oxidant/antioxidant system changes caused by chlorpyrifos. adult sprague-dawley rats were used. cur ( , and mg/kg) were administered orally for days. on the sixth day, chlorpyrifos ( mg/kg, s.c.) was administered. twenty four hours after chlorpyrifos administration, body weights, locomotor activities and body temperatures of rats were measured. following the measurements, rats were decapitated and the blood, brain and liver tissue samples were taken and prepared for the biochemical and histopathological measurements. chlorpyrifos administration increased the malondialdehyde (mda) levels but decreased catalase (cat), superoxide dismutase (sod), glutathione reductase (grx) concentrations and reduced/oxidized glutathione (gsh/gssg) ratio in the blood samples, brain and liver tissues compared with the control group (p < . - . ). the concentration of advanced oxidation protein products (aopp) were increased only in the brain tissue after chlorpyrifos administration (p < . ). cur administration reduced all of these changes (p < . - . ). similarly, cur at the doses of mg/kg reduced the decreases in body weight, body temperature and locomotor activity with chlorpyrifos (p < . ). additionally, the histopathological damage scores induced by chlorpyrifos (p < . - . ) were decreased by the administration of cur (p < . - . ). our findings suggest that cur supplementation can ameliorate the poisoning effects of chlorpyrifos via supporting the antioxidant mechanisms and cur could be used for protective purposes against oxidative stress and tissue damage caused by chlorpyrifos. the effect of ogtt applied for screening in pregnancy on adenosine deaminase and xanthine oxidase activity in normal and prediabetic pregnant women z. c. ozmen, k. deveci, i. benli department of biochemistry, gaziosmanpasa university medical faculty, tokat, turkey objective: it was reported that the activities of adenosine deaminase (ada) were different in normal pregnant women and pregnant women with gestational diabetes mellitus (gdm). it was also stated that the activity of xanthine oxidase (xo) was increased in pregnant women with gdm. the objective of this study was to evaluate if glucose have effects on oxidative stress in prediabetic women by affecting ada and ox after g ogtt which was applied in pregnant women for screening. methods: the serum specimens of pregnant women who applied to the outpatient clinic of the obstetrics and gynecology department and had g ogtt, were used in this study. ada and xo activities were analyzed in the serum specimens taken from the normal (n = ) and prediabetic pregnant women (n = ) in the th and th minutes of ogtt. ada and xo activities were measured with the spectrophotometric method and the u/l enzyme activity was calculated. findings: there was no significant difference between the th and th minutes regarding the ada activities in the normal and prediabetic pregnant woman groups. however, we observed a significant difference between th and th minutes regarding the xo enzyme activity in normal pregnant women (p = . ). in normal pregnant women, the median xo enzyme activity in the th minute was . ( . - . ) u/l and it was . ( . - . ) u/l in the th minute. nevertheless, there was no correlation between the xo activity and glucose level. as to the prediabetic pregnant women, there was no significant difference between the xo enzyme activities in th and th minutes. the results of our study showed that the xo activity increased as a response to ogtt in the normal pregnant women compared with the prediabetic pregnant women. this finding made us think that the oxidative stress caused by ogtt did not affect the xo response in prediabetic pregnant women and that there would be some adaptive mechanisms against the chronic exposure to high level glucose. rainbow trout (oncorhynchus mykiss) aquaculture continuously increases in turkey. the objective of the present study is to increase the productivity in fish farming of rainbow trout just via intervention in physical cases without the effects of any chemicals and investigate whether this conditions cause oxidative stress. in this experiment eight tanks were used and rainbow trout larvae were placed in each tank and these tanks were illuminated with light in different wavelengths; natural sunlight, and incandescent long-wave (red light), medium-wave (green light) and shortwave (blue light) led lights. the experiment took days. biochemical changes in rainbow trout exposed to light in different wavelengths (red, green, blue) were analysed via the variations in gr, gst, g pd, gpx, sod and cat enzyme activities, which are significant for enzymatic antioxidant defence system and in ache activity, which plays an important role on central nervous system. maximum activity change in liver tissue was observed for gst and g pd enzymes in fish grown under green light and for sod enzyme in fish grown under blue light. in gill tissue, sod and g pd activities were affected the most, and in brain tissue, these were gst and sod activities. it was observed that the average weight of the fish increased . times under red and blue lights and . times under green light. the highest weight increase was observed under green light, however, antioxidant enzyme activities increased in the liver and gills and decreased in the brain tissue under this light condition. in conclusion, it was observed that productivity was . times under red light when compared with control group and it was determined that the fish grown under red light can tolerate oxidative stress more than other wavelength. p- . . - effect of nigella sativa on biliary obstructioninduced oxidative stress and apoptosis in rats human safety concerns, since that these agents may not only cause acute toxicity via inhibition of acetylcholinesterase but they can also induce delayed toxicity in the nervous system. a key interest to the current work is the potential correlation between gene expression and cytoskeletal protein changes in differentiating neural cells exposed to sub-lethal neurite outgrowth inhibitory concentrations of specific ops, which was addressed by analysing the underlying changes in the levels of cytoskeletal gene expression and protein levels. to assess the molecular effects of op exposure, phenyl saligenin phosphate (psp), chlorpyrifos (cpf) and its metabolite chlorpyrifos oxon (cpfo) were applied at the point of induction of differentiation of rat c glioma and mouse n a neuroblastoma cells and incubated for hours. at sub-lethal concentrations ( , , lm) all three ops used in this study were able to inhibit the development of neurites with no significant effect on cell viability, as determined by neurite outgrowth and mtt reduction assays. to understand the possible effects of ops on cytoskeletal gene expression, primers for genes encoding glial fibrillary acidic protein (gfap), biii tubulin, growth associated protein (gap ) and neurofilament heavy chain (nefh) were optimized for qpcr analysis and the levels of the corresponding proteins were detected by western blot analysis. exposure to ops caused in most cases a reduction in the levels of cytoskeletal proteins, and the results from qrt-pcr analysis also indicated reductions in the gene expression of gfap in c cells, and of nefh and biii tubulin in n a cells, in a dose dependent manner. thus, the observed changes in protein levels are at least partly due to altered gene expression. curcumin is extracted from a perennial herbaceous plant known as curcuma longa. in recent years, considerable interest has been focused on curcumin due to its use to treat a wide variety of disorders without any side effects. earlier studies have shown that curcumin has anti-apoptotic, anti-inflammatory, antiproliferative, antiangiogenic, anticancer and antiplatelet activities. the goal of the present study was to investigate the effects of curcumin on peroxy radical-induced oxidative changes in human platelets. healthy volunteers were enrolled in the study. none of the study participants were on anticoagulation therapy. citrated venous blood samples were centrifuged at g for minute to obtain platelet-rich plasma (prp). the platelet pellet was washed and suspended with tris-nacl buffer. then, platelets were incubated with h o absence and presence of curcumin ( - lg/ ml) for hours at °c. to determine the preventive effects of curcumin on the oxidative stress and apoptosis induced by peroxy radicals in human platelets were determined by measuring levels of of lipid peroxidation, total antioxidant capacity, caspase , and activities, and mitochondrial membrane potential. additionally, we also studied the effects curcumin on platelet aggregation induced by adp. pre-treatment of platelets with curcumin caused a marked reduction in oxidative stress, activation and apoptotic markers in a dose-dependent manner. on the other hand, pre-treatment of platelets with increasing doses of curcumin resulted in inhibition of platelet aggregation induced by adp. in the light of our findings, we suggest that curcumin may have a therapeutic potential to prevent platelet activation related disorders. people have been using mushrooms in the treatment of diseases as well as food, for centuries. most of the edible and inedible mushroom species were used in important medical studies and their effects were begun to be used in the treatment of diseases. this study focuses on the hepatoprotective effects of tricholoma anatolicum, which is endemic specie in turkey, against oxidative stress based on hydrogen peroxide (h o ) on hepg liver cancer cell line. t. anatolicum used in this study was extracted with the help of ultrasonication and fraction methods. then the cytostatic effects of extracts on hepg cells were explored and their hepatoprotective effects were determined. moreover, various concentrations of aqueous extract (ehta) of t. anatolicum were determined by hepg cells's - - hours effect analysis on their cellular morphology, xtt and real-time cell analysis in of xcelligence device. ehta extract's cell pathway (apoptosis and necrosis) effects on hepg cells were determined with flow cytometry method with the help of annexin v-apc and aad fluorescent dye. finally, the phenolic compounds found in ehta extracts were determined with the help of hplc methods. according to xtt cytotoxicity analysis, the ehta extract values were determined as follows: hours ic > lg/ml, hours ic . furthermore, according to the real-time cell analysis made with xcelligence, ehta extracts were found to be; hours ic = . lg/ml, hours ic = . lg/ml, hours ic = . lg/ml. increasing concentrations of ehta extracts were determined to direct hepg cells to apoptosis. moreover, considering the hplc analysis -according to the reference point of mg in g sample-within ehta extracts, catechins and vanillic acid peaked. the final results revealed that t. anatolicum's effect on hepg was cytostatic at low doses, and cytotoxic at high doses. p- . . - relationship between serum ceruloplasmin levels and coronary blood flow background: there is growing evidence that oxidative stres plays an important role in the development of the slow coronary flow (scf) phenomenon. ceruloplasmin (cp) is a copper containing metalloenyzme which has antioxidant functionthrough its ferroxidese activity and is associated with cardiovascular diseases. we aimed to investigate the relationship between scf and serum cp level. methods: patients who underwent elective coronary angiography and had no significant epicardial coronary disease were included in the study. patients who had thrombolysis in myocardial infarction frame counts (tfcs) above the normal cutoffs were considered to have scf and those within normal limits were considered to have normal coronary flow (ncf). a total of patients ( subject as scf and subjects as ncf) were analyzed. ml blood samples were taken from the groups to study ceruloplasmin activity. serum ceruloplasmin levels were determined spectrophotometrically. results: the serum cp levels were statistically lower in scf group than in the ncf group ( ae versus ae ng/ml, p = . ). also there was a significant correlation between serum cp levels and tfcs (r = À . , p = . ). conclusion: the findings of this study suggests that patients with scf had lower serum cp levels correlated with tfcs. we concluded that reduced serum cp levels might represent a biochemical marker of scf. introduction: sleeve gastrectomy (sg) has been used for the surgical treatment of morbid obesity, as a first step or definitive treatment. alterations of thyroid hormones in gastrointestinal surgery were previously studied. the aim of the present study was to determine the effects of triiodothyronine (t ) supplementation on oxidative stress parameters in anastomotic tissue level. materials and methods: twenty-four male wistar albino rats were divided into control (n ), and experimental (n ) groups. rats were underwent a sg, with a hand-sewn suture. experimental group rats received a single dose of t ( mg/ g) in postoperative day. rats were sacrificed on postoperative day . serum thyroid stimulating hormone (tsh), free t (ft ), and free thyroxine (ft ) were analysed using elisa. each tissue was homogenized in ice-cold pbs (ph: . ) and centrifuged at rpm for minutes ( °c) to avoid contamination with cellular debris. the supernatants were used to measure total oxidant status (tos), total antioxidant status (tas), nitric oxide (no) and malondialdehyde (mda) levels. all tissue parameters were analysed by spectrophotometric methods. oxidative stress index (osi) values were calculated. results: rats given t hormone had not decline in ft levels compared with the control groups. a significant decrease in ft levels was found in t given rats on postoperative day . whereas tissue tos levels did not alter by thyroid hormone treatment, tas levels significantly decreased. osi values were not statistically different in tissues. tissue no levels were also similar in both groups. mda levels increased in t given rats compared with the control group. discussion and conclusion: this study showed that anastomosis after sleeve gastrectomy is associated with decreased ft level. although tos levels and osi values were similar in both groups, t supplementation induced lipid peroxidation by increasing tissue mda levels that might deplete tissue antioxidant level. reactive oxygen species (ros) are reactive chemical molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. although intracellular ros level is essential molecules for the signal transduction pathways, elevated intracellular level of ros leads to oxidative stress that causes damage to dna, proteins and lipids. therefore, excessive ros levels have to be eliminated by antioxidant defence systems. tip (tat interacting protein, kda) is a histone acetyltransferases (hats) that catalyses multiple functions in metabolism such as dna repair, apoptosis, etc. we thought that if tip has a role in signal transduction and apoptosis, it might have direct or indirect relationship with the antioxidant system. the present study was designed to determine the impact of tip gene on the hepatic antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), and glutathione reductase (gr) both gene and protein level. for this reason, quantitative gene expression analysis on the antioxidant system has been investigated by real time pcr, quantitative protein expression has been investigated by western blot analysis, and then the activity of these enzymes have been measured in whole liver homogenate collected from control and liver-specific tip conditional knockout mice. additionally, since any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h o ) level in the cell might be an indication for the accumulation of ros, the relative levels of them were also studied. our data showed that the absence of tip affects the antioxidan system both gene and protein level. in conclusion, our initial data suggest that tip may be essential for the (ros) homeostasis and redox regulation. curcumin is a major chemical component produced from the rhizome of the plant curcuma longa. lt has been demonstrated that curcumin has an antioxidant, anti-inflammatory, and antiproliferative effects and, protects tissues against ischemia/reperfusion (i/ r) injury. i/r has detrimental effects on transplanted organs including uteri. the major consequence of l/r injury is oxidative stress leading to the generation of ros. uterine transplantation (ut) has been gaining popularity around the worid in the past few years. the aim of our study was to examine the antiapoptotic effects of curcumin on uterine l/r injury. the rats were randomized into three groups of seven rats each, group i consisted of rats that did not receive any treatment, group ll exposed to . hour of lschemia and hour of reperfusion, group iii of rats that received intraperitonealy curcumin ( mg/kg) . hour before the induction of i/r. then, the rat uterine tissue levels of mda, tac, and activities of caspase , and were measured. furthermore, the apoptotic index was determined immunohistochemically by the tunel method using light microscopy. biochemical analysis results showed that curcumin decreased the mda and caspase- and ieveis, and increased the uterine tissue levels of tac but, caspase activity did not changed by curcumin suggesting that curcumin induces apoptosis via intrinsic apoptotic pathway. on the other hand, an high apoptotic index was observed in i/r group ( . ae . %) and decreased after treatment with curcumin ( . ae . %). in conclusion, we demonstrated the protective effect of curcumin on apoptosis immediately after reperfusion induction in uteri and we can say that curcumin could improve ir injury and decrease apoptotic index. we propose that curcumin may be a novel approach for improvement of uteri i/r injury. glutathione and the related enzymes belong to the defence system of the tissues against chemical and oxidative stress. these enzymes especially glutathione s-transferase are often overexpressed in tumor cells and are regarded as a contributor to their drug resistance and are thought to play an important role in cancer progression. the purpose of this study is to evaluate the protective effects of chlorophylline as an antioxidant molecule which has inhibitory effects on gst p - on chemically-induced breast cancer model. in our previous work, we had observed that this molecule led to proliferation in breast cancer cells. in this study, n-methyl-n-nitrosourea (mnu) used for inducing carcinogenesis in eighteen, -day-old female sprague-dawley rats. chlorophylline and mnu solutions were injected intraperitoneally when the rats were , , and days old. their weight and tumor diameters were measured throughout the months study period. at the end of the study, all animals were sacrificed and determined both glutathione levels and related enzymes activities (gluathione s transferase, glutathione reductase and glutathione peroxidase) in tumor and tissues such as liver, kidney, heart and spleen were studied and analyzed. as a result, in breast cancer model, glutathione and related enzyme activities were protected by chlorophylline treatment whereas mnu made them decreased compared to the control group. in conclusion, chlorophylline with antioxidant features decreased the toxic effect of mnu by regeneration of glutathione and enhancement of its related enzyme activities. the use of antioxidant molecules, because of proliferative effects and defence-oriented behaviours, should be discussed in cancer therapy. p- . . - effect of overexpression of bacillus catalase on lactococcus lactis nisin production z. girgin ersoy, s. tunca gedik gebze technical university, kocaeli, turkey nisin, has been used commercially (e ) in food preservation for approximately years. it's the only bacteriocin which is approved by world health organization as a food additive. the fundamental problem that limits nisin usage in food preservation is low product yield by producer strains. because of high commercial potential of nisin, studies about increasing the production efficiency of nisin is kept in the forefront in recent years. since nisin biosynthesis and bacterial growth are occuring in parallel to each other, conditions that promote growth are also expected to encourage nisin production. it is known that, when supplied with exogenous heme, lactococcus lactis cells can respire under aerobic conditions and produce higher energy which in turn cause higher biomass. however, aerobic conditions also cause oxidative stress since catalase enzyme, which detoxify hydrogen peroxide, is absent in l. lactis. in this study, to complete the missing component of the defence mechanism of l. lactis, catalase (kate) gene of aerobic bacterium bacillus subtilis was overexpressed in facultative anaerobe l. lactis cells. for this, kate gene of b. subtilis was amplified by polymerase chain reaction (pcr). plasmid constructions were established in e. coli by using an e. coli-l. lactis shuttle vector and then the recombinant plasmid was transferred to l. lactis cells by electroporation. the presence of catalase activity in the recombinant strain grown on the solid medium was first detected by dropping hydrogen peroxide directly on the cells, then with enzyme assays. fermentation studies are going on to determine nisin production of the recombinant strain. to the best of our knowledge, this study presents the first preliminary results that shows the effect of overexpression of catalase gene on nisin production. cancer is among the leading causes of morbidity and mortality worldwide. chemotherapy is one of the major cancer treatment strategies. remarkably, natural products have garnered increased attention in the chemotherapy drug discovery field because they are biologically friendly and have high therapeutic effects. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of humic acid. the present study investigated anticancer effects of ha in several human cancer cell lines. ha was purchased from sigma-aldrich. in this study, we used several human cancer cell lines: the human breast cancer cell line, mcf- , colon cancer cell line, ht- , lung adenocarcinoma cell line, a , and servical cancer cell line, hela. the cells were maintained in dmem medium supplemented with % heatinactivated fbs and % penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at •c. five different concentrations ( ug/ml, ug/ml, ug/ ml, ug/ml, ug/ml) were prepared using a stock solution of ha. the cell proliferation and migration was measured. on the other hand, the apoptotic mechanisms induced by ha in cancer cells were investigated using "apoptosis antibody array kit". the effects of ha on cancer cell lines were evaluated over hours. according to our results, ha induced a decrease in ht- , a and hela cell numbers in a dose-dependent and time-dependent manner. contrary to this, ha induced proliferation of mcf- cells in dose dependent manner. ha inhibited cell migration in a dose dependent manner except mcf- cell line. it was also determined apoptotic pathways in cancer cells induced by ha. it was concluded that ha has an inhibitory effect on certain some cancers. since the effect of ha on tumor progression is unknown, further studies are needed to clarify the rol of ha on cancer activity. p- . . - chronic immobilization stress in rats: fluoxetine and amisulpride protects against chronic immobilization stress-induced biochemical alterations in the present study, the effects of amisulpride and fluoxetine on serum total sialic acid (tsa) and lipid bound sialic acid (lsa) levels was investigated in the rats exposed to chronic immobilization stress. the study was administered using male wistar albino rats weighing - g. rats were divided into five groups (n = / group). group i comprised the control group, group ii was exposed with saline + immobilization stress ( minutes daily immobilization stress for days and . ml saline was administered perorally minutes before immobilization), group iii was exposed amisulpride ( mg/kg/day) + immobilization stress, group iv was exposed fluoxetine ( mg/kg/day) + immobilization stress and v. group was exposed amisulpride ( mg/kg/ day) + fluoxetine ( mg/kg/day) + immobilization stress. statistical analysis showed that the saline + stress, amisulpride + stress and amisulpride + fluoxetine + stress groups was significantly higher than the control group with regards to tsa levels (p < . ). whereas, the fluoxetine group was significantly lower than the group regarding tsa levels (p < . ). on the other hand, saline group was significantly higher than the control group with regards to lsa level (p < . ). whereas, no significant differences in lsa levels were observed in the amisulpride, fluoxetine and amisulpride + fluoxetine groups, as compared to the control group (p > . ). the present study demonstrated beneficial effect of fluoxetine and amisulpride on the concentration levels of lsa and tsa in stress. p- . . - protective effect of borax and boric acid on total sialic acid and lipid-bound sialic acid levels against -methylcholanthrene and benzo(a)pyrene induced oxidative stress in rats s. ekin , g. oto, f. g€ ok, y. karakus, d. yildiz y€ uz€ unc€ u yil university, van, turkey the present study was performed to investigate total sialic acid (tsa) and lipid bound sialic acid (lsa) levels as possible in vivo chemoprotective effect of borax (bx) and boric acid (ba) again-st -methylcholanthrene ( -mc) and benzo(a)pyrene (b(a)p) induced oxidative stress in rats. the rats were divided into nine groups of six rats each. group i: control, untreated animals were given % . nacl, group ii: the b(a)p were administered mg/kg via ip. four times. group iii: the -mc-treated animals were administered mg/kg via ip. four times, group iv: ba was given mg/l/day with water. group v: bx was given mg/l/day with water. group vi: b(a)p mg/kg via ip four times + ba mg/l/day dosage with water. group vii: -mc mg/kg via ip four times + ba mg/l/day with water. group viii: b(a)p mg/kg via ip four times + bx mg/l/ day dosage with water. group ix: -mc mg/kg via ip four times + bx mg/l/day with water. the experimental period was continued for days. statistical analysis showed that the -mc + ba group was significantly higher than the control group with regards to tsa and lsa levels p < . , p < . ,p p- . . - effects of aluminum exposure on trace elements in rat tissues b. ozturk kurt, s. ozdemir department of biophysics, cerrahpasa medical faculty, istanbul university, istanbul, turkey aluminum (al) is the most abundant metal and the third most abundant element in the earth's crust. people are constantly exposed to al which is found in most rocks, soils, waters, air and foods, due to a result of an increase in industrialization and improving technology practices. the study was designed to examine the possible effects of aluminum exposure in different durations on trace elements in rat tissues. twenty-four healthy male wistar rats weighed - g were randomly divided into three groups: control group (gc) received only drinking water, short-term group (gs) and long-term group (gl). the study groups were orally exposed to mg/kg body weight alcl in drinking water for and weeks, respectively. at the end of the treatment period, rats were sacrificed and the kidney, liver, brain and cerebellum tissues were removed to analyse the levels of al, ar, b, ni, si, cr, cu, fe, mg, mn, se, cu and zn by icp-oes. the statistically significant increase were determined in cerebellum al, cu, as, b and cr levels in gl according to the gc. while as levels were statistically increased, ni levels were decreased in gl in the kidney and liver. while cu, mg and cr levels were higher, se and b levels were lower in the gs than gc in the brain. there were no significant difference in si and mn levels. as a result of our study, it may be concluded that al accumulation may lead to changes in tissue trace element levels. tacal, o., tacal, Ö., take, g., takic, m.m., taldykbayev, z., talim, b., tamashevski, a., tamer, f., tamer, l., , , taneva, s., taniyan, g., , tanrisev, m., tanriverdi, e.c., tanriverdi, k., tarhan, m., tarhan, t., tartar, s., tas, a., , taskin, a., taskin, t., taskiran, e., taskiran, b., taskoparan, b., taspinar, r., , tastan, Ö., tatli, Ö., tauraite, d., tay, t., tayman, c., taysi, s., , teker, h.t., tekes, s., tekin neijman, s., tekin, m.h., , tekin, g., , tekin, m., tekin, n., tekin, g., telci, d., , telefoncu, a., temel, h.e., , , temelie, m., temizgül, r., temlyakova, e., teplova, v., terashima, r., tercan avci, s., terekhov, s., tereshenko, o., terzi gulel, g., terzi, e., , terzi, e., terzioglu, g., terzioglu, o., testoni, g., tetik vardarli, a., tevdoradze, t., tevzadze, l., tezcan, Ö., tezcanli kaymaz, b., thielens, n., thomaidou, d., thomas, a., thornton, j.d., ticea, a.c., tikhonova, a., tileva, m., , timofeev, v., , timofeev, v., timofeeva, e., tipirdamaz, r., tiryaki, m., , tok, m., tokay, e., toker, a., , tokgun, o., toksoy Öner, e., tokyol, Ç., toman, r., tomasi, a., tombul, n., , tooke, c., topaloglu, h., topbas, m., topcu, b., topcu, c., topcu, g., , , topçu, v., topcu, c., topçuoglu, c., , topel, h., , toprak, b., toprak, m.s., torac, e., tosner, z., tosun, m., , toy, h., toymentseva, a., tozkoparan, b., trabulus, d.c., trantirek, l., trantirkova, s., trchounian, a., , trizna, e., , troshagina, d., tro t, m., truncaite, l., , tsakalidou, e., tsarkov, d., tsarkova, m., , tsigara, e.g., tsigara, m.g., tsverava, l., tsvetkova, e., tsyba, l., tsyganov, d., tsymbal, d., tüfekçi, a.r., , tufekci, a.r., tufenkci, h., tuglu, m.i., , tuglu, i., tuli, a., , , , tuli, a., tulubas, f., tunali, g., tunca gedik, s., , tunca gedik, s., tunçdemir, m., tuncel, h., tuncel, h., tunçer, s., tuncer, e., , tuncer, z.s., tuncer, b., tuncer, e., tuncez akyurek, f., tunçez akyürek, f., tuner, h., tüney kizilkaya, i., tural, b., tural, s., turan, i., turan, m., , turan, v., turan, y., turan, c., turano, p., , türel, s., , turhan, t., , turhan, y., , turk, s., turkan, a., türkcan kayhan, c., turkekul, k., türkel, s., turkeri, o.n., turkeri, n., turkkan, b., turkmen, s., türkmen, n., turkon, h., tutar, y., tüten, a., tutkun, e., , tüylü küçükkilinç, t., tuz, m.u., twardovska, m., tyapkina, o., tzartos, s., photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes evaluation of the sunscreen lichen substances usnic acid and atranorin pleiotropic anticancer activity of selected nutraceuticals against mcf- bucharest, national institute for marine research and development "grigore-antipa uk in some adult and elderly populations the acute and/or persistent infection with the common intracellular respiratory pathogen chlamydia pneumoniae (chl) may be associated with increased risk of developing obesity or cardiovascular disorders. thus, microelements modifying oxidative stress status were determined by icp-ms/ms in the hno diluted serum samples collected from chl-positive adult females (n = ) living in urbanized area in poland. chl infection was confirmed by igg+ antibody elisa and real-time pcr assay. all females were classified under their body-mass index values to the normal-weight (nw), over-weight (ow) and obese group (ob) although there are many drugs currently used in the treatment of peptic ulcer, such a drug providing radical treatment without side effects is not available. since oxidative stress is involved in peptic ulceration, this study was designed to investigate antiulcerogenic and antioxidant effects of hippophae rhamnoides l. ether extract on indomethacine-induced stomach ulcer in rats. materials and methods: thirty-five sprague dawley male rats (weights ranging - g) were randomly divided into groups, as each composed of rats. after hippophae rhamnoides l. leaf ether extracts of mg/kg, mg/kg and mg/kg doses and mg/kg doses of famotidin orally administered, mg/kg doses of indomethacine were orally applied to rats in order to make ulcer. on the sixth hour of indomethacin administration all rats were sacrificed using thiopental ( mg/kg). the stomachs were removed, and ulcer areas were evaluated macroscopically. superoxide dismutase activity (sod), glutathione (gsh) and malondialdehyde (mda) levels in stomach tissues of rats were determined by elisa method with respective kits conclusions: we can conclude that the ether extract of hippophae rhamnoides l. leaves reduces free radical formation and has antiulcerogenic effects on stomach tissue control group; hours torsion/ hours detorsion group (t/d); all other groups were saturated for four days egcg, cape and egcg+cape ( lml/kg). sections were taken from bouine's-fixed and paraffin-embedded testicular tissue blocks and stained with h&e. immunohistochemistry was applied for the detection of pi k, akt and mtorc. intensities were evaluated as mild ( ), moderate ( ) or strong ( ). serum ohdg, plasma mda levels were analyzed using elisa method. results were analyzed by anova statistical test. testis samples in control group exhibited normal histological morphology. disorganization and separation of seminiferous tubule cells and accompanying interstitial edema and vessel dilation were while mda level decreased significantly in cape+egcg group, ohdg level showed significant increase in cape group. in conclusion, cape and egcg exerted protective effects on tt. effects may be achieved through pi k/ akt/mtorc pathway involved in cell proliferation, angiogenesis, apoptosis. prophylactic use of egcg prior to tt surgery improved testicular morphology, therefore could prevent destructive effects of tt lmol/l), c ( . ae . lmol/l)), respectively. conclusions: this study is important for konya region, mitochondrial fatty acid b-oxidation disorders studies subject areas. this study is the first study to assess acylcarnitine levels of patients living in our region. we believe that our results will be useful for future studies. key words: acylcarnitine, mass spectrometry, dried blood spot p- . . - binding of fas and cu(ii) ions to hsa changes its cys thiol group antioxidant capacity and carbonylation pattern with methylglyoxal binding of cu(ii) ion ( . mol/mol hsa) led to increase of k' value if fish oil extract was present, but for other fas k' value decreased. the content of free hsacys -sh decreased for % after cu(ii) ion binding, and during hours incubation at °c, it was further decreased for % (stearic acid, mixfas) and % (myristic, fish oil extract, oleic acid). carbonylation of fa-hsa-cu(ii) complexes with mg ( mol/ mol hsa), lead to decrease in cys -sh content depending on fa present: %- % for myristic and stearic acid, % for oleic acid and mixfas and % for fish oil extract. carbonylation of fa-hsa-cu complexes could contribute to further enhancement of oxidative and carbonyl stress in diabetes as well as other diseases pirto ek p- . . - anti-proliferative and inducing apoptosis of the hydro alcholic achelia. wilhelmsii extract on human breast adenocarcinoma cell lines mcf- and mda-mb- background: vitamin d deficiency is associated with several conditions and/or diseases like inflammation, atherosclerosis, cardiovascular disease and mortality. several studies showed that lower vitamin d levels were associated with high serum levels of inflammatory biomarkers. ykl- is a glycoprotein, secreted by macrophages, neutrophils and different cell types. it is also associated with inflammation and pathological tissue remodeling. in this study, we aimed to evaluate relationship between the vitamin d deficiency and ykl- levels. methods: our study group includes subjects with vitamin d deficiency (group ) and age and sex-matched healthy subjects with normal serum levels of vitamin d (group ). plasma (oh) vitamin d levels were measured with liquid chromatography-tandem mass spectrometry (lc-ms/ms) method. plasma ykl- analysis was performed by elisa. serum hs-crp levels were measured with nephelometric method. results: plasma vitamin d levels below ng/ml were accepted as vitamin d deficiency. although we could not find any significant differences by means of serum hs-crp levels between groups (p > . ), plasma ykl- levels were significantly higher in group than group (p < . ). conclusions: in literature, vitamin d deficiency is associated with inflammation. in our study, we found similar hs-crp levels between groups and higher ykl- levels in group . vitamin d deficiency may be related to increased ykl- levels in terms of causing chronic inflammation.keywords: vitamin d deficiency, ykl- , inflammation. evaluation and comparison of tnf-family ligands and receptors genes in mice and humans by bioinformatics techniques stance called plaque builds up inside the coronary arteries. apelin is a novel endogenous peptide with inotropic and vasodilatory properties and is the ligand for the angiotensin receptor-like (apj) receptor. we aimed to determine genotype and allele frequencies of apj receptor a c gene polymorphism in turkish patients with cad and healthy controls by rflp-pcr. this study was performed on unrelated cad patients and healthy controls. we obtained aa, ac and cc genotype frequencies in cad patients as . %, . % and . %, respectively. in the control group, frequencies of genotypes were found as . % for aa, . % for ac and . % for cc. we did not observe difference in apj receptor a c polymorphism between cad patients and healthy controls (v = . ; df = ; p = . ). the a allele was encountered in % ( ) of the cad and . % ( ) of the controls. the c allele was seen in % ( ) of the cad and . % ( ) of the controls. allele frequencies were not significantly different between groups (v = . ; df = ; p = . ). the frequencies of apj receptor a c genotype were not significantly different between control and patients. individuals with cc genotypes had significantly higher weight, systolic and diastolic blood pressure levels and systolic blood pressure than other genotypes, p ≤ . . in addition, hdl-c level was found decreased, but this reduction was not statistically significant. contrarily, the low levels of weight, sbp, dbp and tc were statistically significant in the subjects with aa genotype in cad. in conclusion, cc genotype carriers may have more risk than other genotypes in the development of hypertension in cad. we suggest that this polymorphism may not be a marker of cad, but it may cause useful in function of the apelin/apj system and may be a genetic predisposing factor for diagnostic processes and can be helpfull in finding new treatment strategies. p- . . - comparative genomics/proteomics analyses of single amino acid repeat containing proteins across different vertebrate taxa a. g. keskus, o. konu department of molecular biology and genetics, bilkent university, ankara, turkeyconsecutive runs of single amino acids lead to overrepresentation of certain physicochemical properties in protein sequences. researchers also demonstrated a link between single amino acid repeat (saar) containing proteins and neurodegenerative diseases as well as biological functions. moreover, saar frequencies were shown to vary across species based on selected orthologous proteomes and/or proteins. hence, analysis of whole proteomes across multiple vertebrate taxa may provide additional species-and sequence-specific trends for saars. in addition, there is a need for testing the observed saar occurrencesthe aim of this study is to evaluate the effect of quercetin (q) on liver injury secondary to cerulein induced-acute pancreatitis (ap). for this reason, rats were randomly divided into four groups ( rats for each group) control group received physiological saline, four time and dimethyl sulfoxide, two time, at hours intervals, intraperitoneally (i.p.). cerulein group received cerulein ( lg/ kg-rat weight, in physiological saline), four times, and dimethyl sulfoxide ( %), two times, at hour intervals, i.p. quercetin pretreatment (q+cer) group received quercetin ( mg/kg-rat weight, in dimethyl sulfoxide) one time, one hour before cerulein treatment and physiological saline, one time, six hour after cerulein treatment. quercetin post-treatment (cer+q) group received dimethyl sulfoxide, one time, one hour before cerulein treatment and quercetin, one time, six hour after cerulein treatment. cerulein treatment increased significantly vascular congestion in hepatic cells. quercetin treatment also decreased significantly vascular congestion. the liver mda and carbonyl levels in cerulein group were significantly higher than the control group (p < . , p < . , respectively). the mda and carbonyl levels in q+cer group decreased significantly compared to the cer group (p < . ). the mda, carbonyl, mpo levels in cer+q group were significantly lower than the cer group (p < . ). the gssg/gsh ratio of q+cer and cer+q groups were significantly lower than the cer group (p < . , p < . , respectively). the sod activity in cer group was significantly lower than the control group, but the sod activity in q+cer and cer+q groups was significantly higher than the cer group (p < . ).this study shows that quercetin treatment was reduced the severity of liver injury secondary to cerulein induced-ap as reflected by changes in the parameters of hepatic oxidant and antioxidant. p- . . - identification of water extract of propolis components by using different columns in gas chromatography-mass spectrometry propolis is a natural material obtained by honey bees from various plants. protective effect of propolis against damages of free radicals is due to different compounds within propolis. the aim of this study is to identify qualitatively and quantitatively the chemical composition of water extract of propolis (wep) provided by erzurum region using rtx- and rtx- ms column of gas chromatography-mass spectrometry (gc-ms) and to compare with two columns.in this study, wep of mg/ml was prepared, cleared by membrane filter of . lm and freezed at À °c. then, it was lyophilized up to dry form and derivatized to apply for gaseous form. mg of dry extract was reacted with ll pyridin + ll bis-trimethylsilyl trifluoroacetamide (bstfa) mixture including % trimethylchlorosilane (tmcs) and incubated for minutes at °c. all analyses were performed with shimadzu gcms-qp ultra by using a flame ionisation detector (fid). rtx- and rtx- ms capillary columns and helium for carrier gas at a flow time of ml/minute were used. injection was applied on split mode at °c. derivatized propolis sample was injected as ll, initial column temperature was adjusted as °c, then increased to °c with increments of °c. total analysis time was determined as minutes. relative percent amount of separated compounds was calculated from total ion chromatogram with computerised integrator. all components were defined by using nist and wiley libraries.peaks obtained from rtx- column were much more than those of rtx- ms. on the other hand, analyses performing with both two columns have similar carbohydrate, aromatic acid and other acid contents.consequently chemical constituents of wep were determined qualitatively and quantitatively with gc-ms. it was concluded that rtx- column among both columns differentiating for polarities may produce more compounds in the propolis analysis. introduction: aquatic environment can be mostly contaminated by mixtures of metals. biochemical parameters have gain importance to characterize the effects of metals on aquatic organisms. glutathione s-transferase (gst) and its substrate glutathione (gsh) are important parameters of antioxidant defense system of fish metabolism due to their vital role in xenobiotic conjugation. objective: the goal of the current study to evaluate the changes in gst and gsh levels in response to cd, zn and cd+zn effects after and exposure days. materials and methods: fish were obtained from cukurova university fish culturing pools (turkey). fish were exposed to . lg/ml of cd (cdcl .h o) and zn (zncl ), and their mixture, for , and days. at the end of the exposure period, liver tissues were dissected and homogenized in a phosphate buffer (ph . ). homogenates were centrifuged at , g ( min, + °c). supernatants were stored at À °c until the analysis. one-way anova was used to compare data (mean ae se) followed by duncan's test (p < . ). results: gst and gsh changes were recorded as decreases after all metal exposures at day . although day exposure was found as less effective, combined effects caused significant decreases in gst and gsh levels. also longer exposure durations were appeared to be more effective in that situation. conclusions: significant decreases in gst and gsh levels could be occurred due to increased reactive oxygen species caused by metals particularly their combined effects. metal type, their single and combined effects and also exposure duration should be also taken into account when considering the antioxidant system response. gst and gsh might be considered as sensitive biomarker in toxicity assessment of metal mixtures.financial support: this study was supported by a grant from c ß ukurova university (turkey).background/aims: the activation of lectin-like oxidized low density lipoprotein receptor- (lox- ) on endothelial cells leads to intracellular oxidative stress and inflammation and a feed-forward cycle of injury in diabetes, since both oxidized low density lipoprotein (oxldls) and glucose increase lox- expression. quercetin (qr) is part of a subclass of flavonoids called flavonols. polyphenolic compounds affect the development of atherosclerosis not only through antioxidant properties but also by modulation of serum lipids, thereby influencing the immune and inflammatory processes associated with the development of atherogenic diseases. we investigated the effects of dietary qr on endothelial dysfunction mediated by oxidized low density lipoprotein (oxldl)/lectin-like oxidized low density lipoprotein receptor- (lox- ) in animal model of type diabetes mellitus. methods: we compared groups of male adult wistar albino rats: a control group, an untreated diabetic group, diabetic groups treated with qr, and qr group. diabetes was induced by a single injection of stz ( mg/kg). animals were kept in standard condition. in day after inducing diabetic, serum was collected for biochemical parameters. glucose, lipid profiles, microalbuminuria, oxldl and lox- levels were determined. results: serum triglyceride, ldl, vldl levels in diabetic control group (without treatment) was significantly higher than control group (normoglycemic untreated group). supplementation with quercetin decreased serum total cholesterol and increased hdl-cholesterol compared with the control group. serum oxldl and lox- levels in diabetic control group (without treatment) were significantly higher than control group (normoglycemic untreated group). conclusions: consumption of quercetin reduced oxldl and lox- levels. thus, quercetin could be effective in improving hyperglycemia, dyslipidemia, and endothelial damage in type diabetes. p- . . - investigation of some oxidative stress parameters in bdnf heterozygous mice liver tissue a. bodur , i. ince , i. abidin , a. alver objectives: the aim of this study was to evaluate the possible protective effects of nigella sativa (ns) against cholestatic oxidative stress and liver damage in the common bile duct ligated rats. methods: a total of male wistar albino rats were divided into three groups:sham control, bile duct ligation (bdl) and bdl+received ns; each group contain animals. the rats in ns treated groups were given ns ( . ml/kg) once a day orally for weeks starting days prior to bdl operation. results: the changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells, and neutrophil infiltration into the widened portal areas were observed in bdl group. treatment of bdl with ns attenuated alterations in liver histology. the alpha smooth muscle actin (a-sma), transforming growth factor beta (tgf-b ) and the activity of tunel in the bdl were observed to be reduced with the ns treatment. bdl significantly increased the tissue hydroxyproline (hp) content, malondialdehyde (mda) levels, and decreased the antioxidant enzyme (superoxide dismutase (sod) and glutathione peroxidase (gpx)) activities. ns treatment significantly decreased the elevated tissue hp content and mda levels and prevented the inhibition of sod and gpx enzymes in the tissues. conclusion: the data indicate that ns attenuates bdl-induced cholestatic liver injury, bile duct proliferation, fibrosis, apoptosis and oxidative stress. the hepatoprotective effect of ns is associated with antioxidative potential. organophosphorous compounds (ops) are used widely as pesticides for agricultural purposes, as oil additives or as flame retardants. however, due to their widespread use, there are major introduction: in this study, the antiulcerogenic effect of a water extract (cawe) obtained from a spices sample, cinnamomum aromaticum, was investigated using indomethacin-induced ulcer models in rats. materials and methods: experimental groups consisted of six rats. antiulcerogenic activities of , , and mg/kg body wt. doses of the cawe were determined by comparing the negative (treated only with indomethacin) and positive (famotidine) control groups. results and discussion: although all doses of the cawe showed significant antiulcerogenic activity as compared to negative control groups, the highest activity was observed with mg/kg body wt. doses ( %). the cawe showed similarly antioxidant activity when compared with trolox and ascorbic acids used as positive antioxidants. in addition, the activities of catalase (cat) and myeloperoxidase (mpo) enzymes were determined in the stomach tissues of rats and compared with those of the negative and positive control groups to expose the effects of these enzymes on antiulcerogenic activity. the enzymatic activities of cat and mpo and lipid peroxidation (lpo) level in indomethacin-administrated tissues were increased significantly by indomethacin in comparison to control groups. these enzymes and lpo level were decreased, however, by the cawe. in contrast to lpo level, cat and mpo activities, glutathione (gsh) level was decreased by indomethacin and increased by all doses of cawe and famotidine. the present results indicate that the cawe has a protective effect in indomethacin-induced ulcers, which can be attributed to its antioxidant potential. introduction: thiol groups (-sh) are important anti-oxidants and essential molecules protecting organism against the harmful effects of oxidative stress. the aim of our study is to evalute thiol-disulphide homeostasis with a novel and automated method in patients with prostate cancer (pc) before and after radical prostatectomy (rp). material and methods: patients with prostate cancer and healty control subjects were enrolled into the study. plasma samples were collected from patients before rp and months after the rp operation. thiol-disulphide homeostasis was determined with a recently developed novel method. prostate specific antigen, albumin, total thiol, native thiol, disulphide and total antioxidant status (tas) were evaulated and compared between the groups. results: native thiol levels were . ae . lmol/l in the control group, . ae . lmol/l in the patients before rp, and . ae . lmol/l in patients after rp. native thiol, total thiol and tas levels were significantly higher in the control group than the patients before rp (p values < . ). native thiol, total thiol and tas levels were higher months after rp compared to before rp in patients, but these changes were not significant statictically (p values . , . and . respectively). discussion and conclusion: our study demonstrated that antioxidant defense mechanism was weakened as indicated by the decreased thiol levels in the patients with pc. increased oxidative stress in prostate cancer patients may cause metabolic disturbance and have a role in the pathogenesis of prostate cancer. p- . . - is there any relation between g oral glucose challenge test and serum total oxidant-antioxidant status in pregnant woman? the purpose of this study was to test the hypothesis that any degree of antepartum screening for gestational diabetes mellitus with oral g glucose challenge test (gct) should be associated with oxidant-antioxidant status.in this prospectif study, oral glucose challenge test was applied to pregnant women aged - years and at - weeks of gestation. plasma glucose concentrations were measured initial, hour and in addition to test hours after ingestion of g glucose. at the same time serum insulin, cortisol, total antioxidant status (tas), total oxidant status (tos) levels were measured and the oxidative stress index (osi) was calculated.ten pregnant women (forty percent) had a positive glucose challenge test (gct). a positive moderate relation with initial and hour serum total antioxidant status (tas) levels (r = . ) and the oxidative stress index (osi) (r = . ) was found. there was a positive weak correlation with initial and hours total oxidant status (tos) levels (r = . ) but statistically significance difference was not found (p > . ).in this study after ingestion of g glucose serum total antioxidant status (tas), serum oxidant status levels (tos) and serum oxidative stress index (osi) levels were higher than the initial levels.the results of this study suggest that antepartum screening for gestational diabetes mellitus with g oral glucose challenge test (gct) weakly associated with oxidant-antioxidant status and to confirm this results the longer follow-up studies with more participants are necessary.wheat (triticum ssp.), cultivated for centuries in the middle-east, central asia, europe, and north-africa, is one leading staple crops around the world, and its marginally grown ancestor einkorn (triticum monococcum ssp. monococcum), possesses rich gene resources for wheat improvement and have bioactive compounds reducing and preventing chronic diseases such as diabetes, cancer, alzheimer, and cardio vascular diseases, beside their nutritional properties. however, as more attention has been given to wheat cultivars with strong gluten, protein content, starch composition, and resistance to biotic and abiotic stresses in bread wheat and yellow-colored pasta product in durum wheat health compounds such as fibers, phytochemicals, and bioactives have been underestimated so far. the aim of this study was, then, to examine the total phenolics and flavonoids, quantify their phenolic acids, a-tocopherol by high performance liquid chromatography (hplc), and their , -diphenyl- -picrylhydrazyl (dpph) scavenging activity of bread (triticum aestivum l.), durum (triticum turgidum ssp. durum desf.) wheat cultivars and einkorn (triticum monococcum ssp. monococcum) wheat populations collected from different provinces (bolu and kastamonu) of turkey. ferulic acid ( . - . -lg/g), p-coumaric ( . - . -lg/g), and total phenolic content (ranged . - . -lmol gae/g) of einkorn populations were significantly higher than bread and durum wheat cultivars. results suggested the possibility of production of einkorn wheat populations, and hopefully cultivars rich in particular health beneficial component(s) may provide benefit to the consumers. in addition, higher phenolic content of einkorn may offer novel wheat genetic resources for the improvement of new wheat cultivars and the development of wheat-based functional foods. oxidative damage due to ischemia and acute kidney injury (aki) after coronary artery bypass graft (cabg) surgery are the leading complication during this process. in the kidney, ischemia/ reperfusion injury contributes to aki that is a clinical syndrome with rapid kidney dysfunction and high mortality rates. some animal and clinical studies have demonstrated an increase in serum and urinary neutrophil gelatinase-associated lipocalin (ngal) expression after renal ischemic injury.in this study, our aim was to investigate the relationship betwwen ngal and oxidative stress parameters due to ischemia caused by total perfusion time (tpt) in patients who have undergone on-pump cabg. materials and methods: the study was conducted in patients who received on-pump cabg at university of istanbul, cerrahpasa medical faculty, department of cardiovascular surgery. blood samples were collected prior to surgery and after hours following the termination of cardiac pulmonary bypass (cpb) . following centrifugation, serum samples were separeted and stored at - c until analysis. serum ngal, ima (ischemia modified alb€ umin), pco (protein carbonyl), nt (nitrotyrosine), lpd (lipid peroxide) levels were determined by elisa procedure. results and discussion: serum ngal, pco, nt levels in after hours following cpb were significantly higher than the before surgery (p < . , p < . , p serum ngal levels in after hours following cpb was found to have positive correlation with ima, pco, nt, and lpo levels (r = . , p < . ; r = . , p < . ; r = . , p < . ; r = . , p < . respectively). ngal levels were positively correlated with total perfusion time after cbp (r = . , p < . ).the results of our study show that, increased ngal levels hours after cpb were positively correlated with oxidative stress parameters and total perfusion time. investigation of the effects of thymoquinone against indomethacine induced gastric damage in rats introduction: incidence and high cost of acute stomach mucosa damages make this issue a very interesting issue for study. for this reason, it is aimed to investigate the effects of different dosage of thymoquinone (tq) against indomethacine induced gastric damage in rats. material and method: in our study, six groups of wistrar male rats were used. groups are named as healthy, ind control, famotidine ( mg/kg) and three different doses of tq ( . , and mg/kg). while any treatment or drug administration will done on healthy group, model was generated to other groups by giving fam or tq with tap water via oral gavage. min later, mg/kg ind administrated to each rat. animals were sacrified about hour later and stomach samples of each groups were collected for macroscopic study and gsh levels measurements. results: lower doses of tq is more effective and all tq groups exhibit reduced ulcer region with respect to the ind control group. gsh level of ind control group is lower than healthy group. the gsh level of tq, especially in lower doses, and fam groups statistically exhibit an increase in gsh level. conclusion: it is observed that ind induced gastric damage cause ulcer and increase in free radical. it is determined that lower doses of tq ( . , and mg/kg) is also exhibit a protective effect on ind induced model. it is tought that quinone in tq structure have a strong redox feature and this feature clean up the free radicals caused by ind, it reduces the oxidative stress and protect the stomach from ulcer. anzer honey is the most famous honey in turkey with many endemic species flowers. the anzer plateau is located rize province of eastern black sea region. in this study, antioxidant and anti-hyaluronidase and anti-urease activities were investigated of the plateau bee pollen. the antioxidant capacity was determined by total phenolic content (tpc), total flavonoids contents (tfc), ferric reducing antioxidant power (frap) and , diphenyl- -picrylhydrazyl (dpph) radical scavenging activity. atherosclerosis is the leading cause of mortality worldwide, and as a chronic inflammatory disease, caused by a complex interplay between inflammatory and oxidative events.quercetin, a plant derived flavonoid and a well-known antioxidant, has shown great promises with regards to its protective effects against oxidative stress.however due to its physicochemical properties, the optimum pharmacokinetic behavior is a challenging issue.herein, we aimed to fabricate quercetin loaded solid lipid nanoparticle (quer-sln) to improve the bioavailability and therapeutics efficiency. furthermore the in-vitro capacity of quer-sln for ameliorating tnf-a induced oxidative stress in human endothelial vein cell (huvec) was evaluated. quer-slns were prepared by simple hot homogenizing method and characterized by means of drug loading (dl), encapsulation efficiency (ee), cytotoxicity, size, zeta potential and morphology. antioxidant activity of plain quercetin and quer-slns were then investigated using intracellular reactive oxygen species (ros) detection method (dcfh-da assay) by facs flowcytometryin conclusion, the results here showed superior control of oxidative stress by quercetin nanosystem as compared to plain quercetin. precirol based slns as a biocompatible/biodegradable lipid, may provide a novel drug delivery system for quercetin with improved beneficiary impact in atherosclerosis. objective: zinc is known as an antioxidant essential trace element. we aimed to evaluate the dose-dependent effects of zinc on the oxidant-antioxidant system in liver, kidney and brain tissues of rats and the histological alterations in the absence of oxidative stress (os). material and methods: thirty-nine female weighing about gr wistar albino rats were divided into four experimental groups as ad libitum (al) diet (control), al diet + mg/kg zn sulfate (low dose; group ), al diet + mg/kg zn sulfate (middle dose; group ) and al diet + mg/kg zn sulfate (high dose; group ). zn sulfate solutions were administered . ml/day orally for days and in day rats were sacrificed and tissues were excised for detecting malondialdehyde (mda), advanced oxidation protein products (aopp), superoxide dismutase (sod), glutathione peroxidase (gsh-px), glutathione reductase (gr), and glutathione-s-transferase (gst) activities. histological evaluation was also performed to confirm the effects of zinc. results: in liver tissues aopp levels decreased in all groups receiving zinc as compared to the control group. liver mda levels were increased in group and ; sod and gsh-px levels were both increased while gst levels were decreased in all groups compared to control. gr levels were increased only in group . in kidney; aopp level was decreased only in group and sod level was only decreased in group as compared to control while gr levels were increased in all doses of zinc. in brain; aopp, gsh-px and gr levels were decreased in all groups receiving zinc as compared to control group. sod activity in brain tissues was increased by the administration of middle dose of zinc (group ). gst level was decreased in only group conclusions: the biochemical and histological findings of this study suggest that zinc has various effects on liver, kidney and brain tissues in the absence of os.key words: zinc, liver, kidney, brain introduction: this study aimed to investigate the effect of diabetes mellitus (dm) on oxidative stress and antioxidant capacity in humour aqueous (ha) and venous serum using total antioxidant capacity (tac) and total oxidative stress (tos) levels in serum and ha in cataract patients. materials and methods: in this study patients were divided into two groups. group was composed of patients with type dm and cataract and group was composed of patients with cataracts who are not accompanied by dm and cataract patients who are not accompanied by systemic diseases. each group consisted of patients, totally patients were included in the study. the ha which was collected from the eyes at the beginning of the cataract surgery and venous blood serum collected from the same patients were analyzed. in both groups, ha and serum tac and tos levels were measured with elisa. results: : serum tac levels in the dm group were significantly lower than in the control group (p < . ). tos serum levels in dm group was statistically higher than the control group (p < . ). differences between tac and tos levels were not statistically significant when compared the two groups' ha results (p > . ). group , divided into two subgroups according to their hba c levels, there was no statistically significant difference between the subgroups when hba c levels were compared with the relationship between serum and ha's tac and tos levels (p > . ). there was not an association between the gender, age and the levels of tac-tos in both groups (p > . ). discussion and conclusion: presences of dm is the only risk factor for increase of oxidative stress and decrease of anti-oxidant capacity in patients without a systemic complication of dm and diabetic retinopathy. in our study, diabetic patients without retinopathy showed similar ha tos and tac levels to healthy individuals, this finding indicates that blood-aqueous barrier is protected in these patients. the effect of ferulic acid against testicular ischemia/reperfusion injury in rats u. sac ßik , g. erbil , z. c ß avdar , c. ural department of histology and embriyology, school of medicine, dokuz eyl€ ul university, izmir, department of molecular medicine, health science of institute, dokuz eyl€ ul university, izmir, turkeytestis torsion is one of the urologic emergencies occurring frequently in neonatal and adolescent period. testis is sensitive to ischemia/reperfusion (i/r) injury and, therefore, ischemia and consecutive reperfusion cause an enhanced formation of reactive oxygen species (ros) that result in testicular cell damage and apoptosis. ferulic acid, known as an antioxsidant, is a phenolic acid found in seeds and leaves of the plants. we aimed to investigate potential protective effect of ferulic acid against testis i/r injury.thirty five wistar rats were randomly divided into groups; control, ethyl alcohol, ischemia, i/r, i/r-ferulic acid groups. animals were exposed to hours of ischemia followed by hours of reperfusion. ferulic acid was administered ( mg/ kg) before reperfusion intravenously. testicular cell damage was examined by h-e staining and pas. tunnel, active caspase- , inos and mpo were evaluated by immunostaining. malondialdehyde (mda), glutathione (gsh) levels, glutathione peroxidase (gpx) and superoxide dismutase (sod) activities were assessed by biochemical methods.histological evaluation showed that ferulic acid pretreatment reduced significantly testicular cell damage and decreased tun-nel, caspase positive cells; inos and also mpo expression. in addition, ferulic acid administration decreased significantly the mda levels increased by i/r. morever, ferulic acid increased significantly the sod activity levels, which was decreased by i/r. there were no statistically significant differences in the levels of gsh and gpx activity in all groups.the present results suggest that ferulic acid is a potentially beneficial agent in protecting testicular i/r. background: in heart failure (hf), angiotensin antagonists (aa), beta-blockers (bb), spironolactone, diuretics and acetylsalicylic acid are often used. top pharmaceutical groups reduce mortality. on the increased oxidative stress (os) in patients with hf, it is known to have beneficial effects of certain groups of drugs. however, the net effect of these drugs in os is unknown. the aim of this study was to investigate the effects of drugs used in hf on os. materials and methods: patients were included in the study. all of the patients had systolic heart failure and all of them were under treatment. drugs used by the patients were recorded. the levels of total antioxidant status (tos), total oxidant status (tas), the enzymatic activity of ceruloplasmin, paraoxonase- and arylestherase were measured according to erel's method. serum total thiol levels were measured with sh modified hu method and the lipid hydroperoxide levels were measured with the ferrous ion oxidation xylenol orange assay. the percentage tos / tas was determined as osi. results: in patients treated with acetylsalicylic acid (asa), spironolactone, beta blocker and furosemide, there were increased tos, decreased tas and osi (p < . ). in patients treated with angiotensin blockers, increased tas and looh, and decreased sh were found (p < . ). in patients treated with nitrates and ccb, tos and osi were found decreased. correlation analysis showed that increased tas correlated with the use of angiotensin blockers, asa, furosemide and beta blocker positively; and with the tos and osi level correlated with the use of spironolactone, asa spironolactone and furosemide (p < . ). conclusion: current medical agents that are being used in hf are effective in reducing os in hf patients. one of the effective mechanisms to reduce the mortality of some of these drugs may decrease os.key words: heart failure, drug use, oxidative stress p- . . - across adjacent ring formed titanium phthalocyanine-mediated photodynamic therapy alters and degrades filamentous actin cytoskeleton and internal membranes photodynamic therapy (pdt) is widely accepted as a promising and minimally invasive treatment strategy due to its applicability on a wide range of cancer diseases. this clinically approved treatment method relies on the dramatic production of singlet oxygen and reactive oxygen species (ros) in target tissue to evoke apoptotic cell death [ ] . we, therefore, focused on the intracellular ros accumulation, internal membrane degradation, filamentous actin cytoskeleton alteration and nucleus morphology changes induced by pdt-mediated across adjacent ring formed titanium phthalocyanine which was previously synthesized bis(ethane- , p-phenol- , -p-phenoxy) phthalocyaninatotitanium (iv).characterization of the synthesized metallophthalocyanine was accomplished by using uv-vis, ir, h-nmr and maldi-tofmass spectroscopies. the dark and pdt-mediated activities of bare and phosphonolipids (max. %) charged titanium phthalocyanine ( . , . , . , and lm) were determined on a human lung carcinoma and hacat human keratinocyte cell lines by using intracellular ros assay, dioc( ), tritc-phalloidin and dapi staining protocols. waltmann pdt l was used as the non-toxic light source at j/cm fluence and mw/ cm fluence rate. the experiments showed that pdt-mediated titanium phthalocyanine leads to significant and concentration dependent reactive oxygen species accumulation. moreover, internal membrane degradation, apoptotic bodies on nucleus and filamentous actin cytoskeleton alteration were observed. consequently, the activity mechanism of pdt-mediated titanium phthalocyanine seems to be in a tight relationship with ros accumulation-mediated internal membrane degradation, filamentous actin cytoskeleton alteration and apoptotic pathways activation. introduction: retinal vein occlusion (rvo) is a common retinal vascular disorder that can affect visual acuity and cause blindness in elder population. sulphur containing aminoacids such as cysteine (cys), cysteinylglycine, glutathione, homocysteine and c-glutamylcysteine are reported to be associated with the pathogenesis of rvo. thiols are organosulfur compounds that are formed of a carbon-bonded sulfhydryl group. sulphur containing aminoacids slightly contribute the composition of plasma thiol pool. thiols can undergo oxidation reaction via oxidants and form disulphide bonds our purpose is to research the relationship between a novel oxidative stress marker serum dynamic thioldisulphide homeostasis and retinal vein occlusion. materials and methods: rvo patients and controls were included in the study. native thiol, total thiol, disulphide levels are measured in the serum samples of rvo and control group by using an automated method described by erel et al. also disulphide/native thiol and disulphide/total thiol ratios were calculated. results: there were no significant difference between the rvo and control group in native thiol, total thiol, disulphide disulphide/native thiol and disulphide/total thiol ratios.(p > . for all) conclusion: our study is the first report evaluating the dynamic thiol-disulphide homeostasis in rvo patients by a newly developed method by erel et al. further large sample sized studies investigating the levels of sulphur containing aminoacids may additionally be planned to verify this study. purpose: the purpose of this study was to evaluate markers of systemic oxidative stress and antioxidant capacity in subjects with severity of osas. methods: a total of osa patients were included in the study ( controls, with mild, with moderate, and with severe osa). patients were grouped according to apnea-hypopnea index (ahi) as mild, moderate and severe osa. patients with ahi< served as control group. known risk factors for oxidative stress, such as age, sex, obesity, smoking, hypelipidemia, and hypertension, were investigated as possible confounding factors. plasma arylesterase, total oxidative stress (tos), total antioxidant capacity (tac), total thiol, catalase (cat) levels were measured for all patients. results: the mean age was . ae . years and . % ( / ) of the study population was female. plasma arylesterase, tos, tac, total thiol, and cat plasma values were not different between mild, moderate, severe osa groups and controls (p > . ). catalase levels were significantly lower in women patients with severe osa compared to healthy women controls (p < . ). there was a negative correlation between ahi and serum total thiol levels (r = À . , p < . ) in severe osa groups. conclusionthe present prospective study provides evidence that osa might be associated with decreased antioxidant burden possibly via catalase way. results: the sera -ohdg, mda and il- were significantly higher in diabetic group than control group (p < . ). although there was a notable positive correlation between mda and -ohdg, there was no a relationship between -ohdg or mda with il- . discussion: in agreement with previous studies our data illustrated that high levels of oxidative stress is associated with increased production of oxidized lipids and nucleobases in diabetic patients compared to control group. also enhanced proinflammatory cytokine, il- , induced inflammmation in these patients.conclusion: oxidative stress and inflammation play pivotal roles in the development of diabetes and can cause major complications in dm. so we suggest that early detection of these measurable indicatores can help to diagnosis the severity or presence of some complication in diabet. the effect of quercetin on erythrocyte glucose- -phosphate dehydrogenase enzyme activity in ethanol treated rats in this study, we aimed to evaluate the effects of ethanol on erythrocyte (g- -p-d) enzyme activity and the effects of quercetin on erythrocyte g- -p-d activity in the recovery of the effects of ethanol.rats were randomly divided into four groups. the control group (n = ) received physiological saline. the quercetin group (n = ) received quercetin ( mg/kg/ day) via i.g. route the alcohol group (n = ) received ethanol ( % v/v, ml/day) via i.g. route. the alcohol + quercetin group (n = ) received ml of ethanol ( % v/v) hours after quercetin treatment ( mg/ kg/day). experimental procedures were peformed for days. erythrocyte g- -p-d activity was found to be higher in the quercetin group than those in the alcohol group (p < . ). in the alcohol group, the erythrocyte g- -p-d activity was found to be significantly decreased than those in the control group (p < . ). statistically significant differences were observed in erythrocyte g- -p-d activity between the alcohol group and the alcohol + quercetin group (p < . ).as a conclusion, our results demonstrate that ethanol decreased erythrocyte g pd activity and quercetin was found to be beneficial in the prevention of toxic effect raised by ethanol.key words: erythrocyte, ethanol, g pd, oxidative stress, quercetin p- . . - effects of the sulphasalazine to the cerebral hypoxia reperfusion injury in rat background: cerebral ischemia/ reperfusion (i/r) injury is still a difficult process to treat and rehabilitate today. this study was designed to investigate beneficial effects of sulfasalazine in cerebral i/r injury in rat. methods: except control group (n = ), wistar albino rats were divided into four groups for acute and chronic stage investigation of i/r injury, and temporary aneurysm clips were attempted to both internal carotid arteries for duration of minutes. four hours later, except control, sham-a, sham-c groups, mg/kg once a day sulfasalazine was administered to animals, orally. animals were sacrified and then necrotic neuronal cells of hippocampal ca , ca , and ca region, and cortical necrotic neurons, perivascular edema, pyknotic neuronal cells, irregularities of intercellular organization (iio) were counted and scaled histopathologically. tissue il- b, il- , malonyldialdehyde (mda), myeloperoxidation (mpo), no, and tnf-a levels were measured by using elisa, too. results: sulfasalazine could reduce perivascular edema, iio, cortical and hippocampal neuronal cell death in both stages. it could decreased mda in acute stage, but not reduce il- b, il- , mpo, no, and tnfa levels. it could increased il- b levels in chronic stage but not affect to il- , mpo, mda, no, tnf-a levels.conclusion: sulfasalazine could improve histopathological architecture of hypoxic tissue in both stages of i/r injury. it could inhibit lipid peroxidation cascades in acute stage but not affect to tissue mpo, no, il- , and tnf-a levels in any stage in rat. these results suggested that therapeutic mechanisms of sulfasalazine should be investigated by using more specific laboratory methods in future studies.key words: antiinflamatory, cerebral hypoxia reperfusion injury, sulfasalazine, stroke. camel and horse milk xanthine oxidase (xo) was found to catalyze the reduction of nitrate and nitrite to nitric oxide (no) under aerobic condition. to date, mammalian nitrate reductase (nar) and nitrite reductase (nir) have not been identified. no, a gas, is found to control a seemingly, limitless range of functions in animals.one assay was used to determine nar and nir activities of milk xo: ( ) nitrite formation from nitrate by nar, and ( ) nitrite utilization by nir. nitrite concentrations were determined by using sulfanylamide and n-( -naphtyl)-ethylenediamine, which form red color measured at nm.these activities of the milk xo require nadh as a physiological electron donor. high xo, nar and nir activities are detected only after heat treatment ( °c, min) of the fresh milk in the presence of molybdate. in both camel and horse milk nar activity of xo was almost two times higher than its nir activity. it is well known that xo can be reversibly converted from the dehydrogenase form to the oxidase through the oxidation of sulfhydryl groups. cysteine and, to a lesser extent, glutathione increased nir activity of milk xo but not its nar activity. the mechanism of this increase of nir activity remains unclear and is currently under study. substitution of tungsten for molybdenum under above conditions gave no detectable nar and nir activity of milk xo. the molybdenum site-directed inhibitor, tungsten inhibited in a dose-dependent manner. therefore, nitrate and nitrite are clear to interact with mo center of xo.camel and horse milk are traditional drinks in central asia and kazakhstan. therefore, it is very important that xo provide a mechanism for generation of no in camel and horse milk where nitric oxide synthase, no producing enzyme, does not exist. p- . . - the influence of phytomedicine on metabolic processes of white rats undergone to ionized radiation the study of peroxide lipids oxidation (plo) process is used as one of stability parameters of organism's changes and as a key mechanism for understanding of adaptation reactions and of pathogenesis of different diseases. it's determined by high biological activity of products which are formed in the plo reactions, in this relation lipids with high contents of fat acids play important role. to investigate the influence of phytomedicine eminium regelii on the metabolic processes (peroxide lipids oxidation) of white rats' organism in conditions of ionized radiation.the animals were exposed to ionizing radiation (gamma-radiation co) on the radiotherapeutic equipment teragam in a dose of gy and received phytomedicine eminium regelii in a dose of . mg/kg orally within days following the ionizing radiation exposure. gamma-rays caused the increase of lipid peroxidation (lpo) primary (dc) and secondary products' (mda) concentrations in spleen, liver, thymus and adrenal glands.treatment by phytomedicine resulted in contents of dc decreased in times in spleen, in times in thymus, in times in adrenal glands, in liver in times, in lymph nodes of small intestine it in times. mda decreased in liver up to and times, in spleen in . times, in thymus in times, in liver in times, in adrenal glands in time, no changes in lymph nodes of small intestine.the effect of phytomedicine treatment of organisms exposed to sublethal dozes of gamma-radiation results in the lpo primary and secondary products concentrations decrease in spleen, liver, thymus and adrenal glands. p- . . - evaluation of serum levels of ischemia modified albumin (ima) in bipolar disorder patients k. € unal , c. topc ßuoglu , m. cingi clinic of biochemistry, ankara polatli duatepe public hospital, ankara, clinic of biochemistry, ankara numune training and research hospital, ankara, clinic of psychiatry, ankara numune training and research hospital, ankara, turkey introduction: bipolar disorder is one of the most debilitating psychiatric disorders characterized by disruptive episodes of mania/hypomania and depression. considering the complex role of biological and environmental factors in the etiology of affective disorders; recent studies have focused on oxidative stress, which may damage nerve cell components and take part in pathophysiology. aim of our study is to contribute these data about oxidative stress in bipolar disorder, by detecting ischemia modified albumin (ima) levels of bipolar disorder patients in remission and also by comparing these results with healthy controls. methods: study population consisted of patients meeting the diagnostic and statistical manual of mental disorders, fifth edition (dsm- ) criteria for bipolar disorder i. healthy subjects were included as control group (hc). serum ischemia modified albumin (ima) levels of all participants were determined. results: statistical analysis on serum ischemia modified albumin (ima) levels did not show any significant difference between bipolar disorder patients in remission and healthy controls.conclusion: studies on oxidative stress in bipolar disorder have reached controversial results up till now. in this study, no statistically significant difference was detected between oxidative parameters of bipolar disorder patients in remission and healthy controls. in order to evaluate oxidative stress in bipolar disorder comprehensively, further studies are needed. keywords: bipolar disorder, ischemia modified albumin (ima), oxidative stress p- . . - xanthine oxidase and adenosine deaminase activity in patients with familial mediterranean fever (fmf) objective: fmf is an autosomal recessive dissease which is characterized by recurrent fever and inflammation of serous membranes. in this study we measured serum adenosine deaminase (ada) and xanthine oxidase (xo) levels in fmf cases. method: serum ada levels were measured with a sensitive colorimetric method described by giusti and xo levels were analysed by the method of worthington in fmf patients and healthy controls. results: there was a significant difference in xo and ada levels between controls and cases. ada and xo levels were higher in patents with fmf. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of ha. the present study was undertaken in order to evaluate the anticancer properties of the ha using a prostate cancer and osteosarcome cell lines pc- , sjsa as an in vitro model system.ha was purchased from sigma-aldrich. the cells were maintained in dmem medium supplemented with % heat-inactivated fbs and % penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at •c. five different concentrations ( ug/ml, ug/ml, ug/ml, ug/ ml, ug/ml) were prepared using a stock solution of ha. we measured cell proliferation and migration to understand of progression effects of ha in pc- and sjsa cell lines in vitro.according to our results, ha treatment caused cytotoxicity and induced cell death in vitro in pc- cells with an ic value of . lg/ml. contrary to this, ha induced proliferation of sjsa cells in dose dependent manner. ha demonstrated the highest proliferatif activity against sjsa cells with an ic value of > lg/ml. on the other hand, cell migration was reduced in pc- cell line and interestingly, migration was accelerated in sjsa cell line.our study may provide new insights into the regulatory effect of ha in cancer, but further studies are needed to clarify the role of ha in cancer pathogenesis. the febs journal (suppl. ) ( )