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NIGMS Bioengineering
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ENGINEERING SUPPORT FOR THE MOLECULAR ANATOMY PROGRAM
Norman G. Anderson
Molecular Anatomy Program
Oak Ridge National Laboratory
Oak Ridge, Tennessee
. Sponsored by the U. S. Atomic Energy Commission, the
National Cancer Institute, the National Institute of: General
Medical Sciences, and the National Institute of Allergy and
Infectious Diseases.
Operated for the U. S. Atomic Energy Commission by the
Nuclear Division of Union Carbide Corporation.
DISTRIBUTION OF THIS DOCUMENT IS UNLIMITED
+
Running Head:
Send Proof to:
Engineering for the MAN Program
Dr. Norman G. Anderson
Building K-703
Oak Ridge Gaseous Diffusion Plant
P. O. Box P
Oak Ridge, Tennessee 37830
The fractionation of human blood proteins, largely under the
stimulus of World War II requirements, has passed from a research
effort to the stage of large scale production. While numerous
problems remain' to be solved, the major constituents are well
characterized and clinically useful techniques for identifying
them are available. In adúition, specific antisera may be obtained
which allow euch high resolution nethods as immunoelectrophoresis
to be used routinely with confidence. Approximately 100 different
plasma macromolecules have been described (1). For a large frec-
tion of those which have been extensively examined, disease associ-
ated alterations have been discovered.
By comparison, the proteins which are found in hunan cells are
much less well characterized. If advances comparable to those made
with plasma are to be made, a very concerted effort to develop and
apply new techniques will be required. The object of the Oak Ridge
Molecular Anatomy Program is to begin this effort.
Many enzymes have been studied in human cells and tissues, and
a few of them have been isolated and partially characterized. How-
ever it is instructive to ask what the status of blood plasma frac-
tionation would be if it had been guided solely by enzyme assays.
Viewed through the keyhole of any single assay, or even a large
· number of assays, a very restricted view would have been obtained.
Fortunately broader methods for examining plasma were adopted which
led to the discovery of many proteins whose existerce was not
predicted and in some instances, whose function is not yet fully
understood. In attempting to begin a rational fractionation of
human cells which is to be carried to the molecular level, it .
therefore appears logical to consider certain principles which
appear to have been useful in the past and are applicable now.
If molecular anatomy is to be a logical extension of both
gross and microscopic anatomy, then the classical concept of
a complete description at cach level of dissection should pro-
vail. This implies the development of techniques for "seeing"
the products of dissection at a particular level and also of
dissecting to that level. A clear distinction must be drawn
between the anatomical view considered here and cell dissection
with the aim of isolating a given substance for which one has an
assay. In the latter instance the objective is reached by dis-
carding as much uncharacterized mass as possible. In the former
instance we are interested in describing the mixture to be frac-
tionated as completely as possible, and then in characterizing
each fraction obtained and relating it to the whole.
We require therefore parallel preparative and analytical
techniques, i.e., preparative and analytical techniques which
depend on the same parameters so that the performance of the
first can be evaluated in terms of the second. It is soon dis-
covered that not all of the pairs required have been developed
to a satisfactory level. For example we do not have truly pre-
parative scale centrifuges which parallel the analytical ultra-
centrifuge in the l-128 region. Similarly, no production scale
electrophoretic' separation technique with the resolution of disc
electrophoresis has appeared. Until relatively recently an
analytical counterpart of preparative scale fractionation of
proteins by salting out was not available.
The Molecular Anatomy Program is concerned initially with
the development of techniques for cell production and cell frac-
tionat.on, since the complexity of the problem is beyond present
methodologies. The engineering aspects of the program are con-
cerneå therefore with the development of culture and fractionation
systems and with methods. Underlying the program is the concept
that the basic problem in any field of science is the definition
and description of fundamental units which are describable by the
next subjacent scientific field, This view is widely held in the
non-biological sciences. For example, in the United States the
Atomic Energy Commission has assumed a leading role in developing
the tools and facilities necessary for the production and separa-
tion of subnuclear particles and for the separation (and in the
case of radioactive isotopes, the production) of isotopes. The
scientific and technical back-up required for such efforts are
rather large, and overlap to a surprisingly large degree, the
support requirements for the Molecular Anatomy (or MAN) Program.
It should be pointed out, however, that many of the problems faced
in the MAN program are more complex than those faced by either the
Manhattan Project or by the present AEC. The feasibility studies
for the MAN Program have been carried out at Oak Ridge with support
from all three of the plant areas (the Oak Ridge Gaseous Diffusion
Plant, the Oak Ridge National Laboratory, and the Y-12 Plant). The
problem is to carry on in parallel basic biological studies to de-
fine the underlying problems, and engineering and developmentai work
to develop solutions to these problems, i.e., to see whether the
approach used in the AEC 18 upplicable to parallel problems in the
biome iical sciences. Since we are now concerned with feasibility
studies, only a few problem areas are selected for study and these
are described briefly here.
The major problem areas are the following:
I. Large scale cell culture.
II. Controlled cell breakage.
III. High resolution separation of subcellular fractions
by centrifugal techniques.
IV. Development of techniques for the disassembly of each
subcellular particle species.
V. Development of higher resolution techniques for the
fractionation of mixtures of macromoleculesio.
VI. Development of automated analytical systems for the
analysis of various classes of low molecular weight
substances.
VII. Development of automated systems for deterining
sequences in proteins and nucleic acids.
VIII. Development of automated systems for enzyme assay.
IX. Development of data reduction techniques
X. Integration of results with the aim of developing
clinically useful diagnostic techniques.
Work on the problem of large scale human cell culture at Oak
Ridge has been delayed until many of the separatione systems re-
quired for cell fractionation have been completed. (It should be
noted that intensive work in this area is being successfuly carried
out at Roswell Park.)
The problem of controlled cell breakage is now under investi-
gation. For soft tissues shearing fields of approximately 7000 de
are required (2). However methods for precisely controlling shearing
rate over a relatively wide range have not been developed and systema-
tically applied to human cells. A variety of methods for breaking cells
have been used in the past. A careful intercomparison of these in one
laboratory has not been done.
The major effort to date has been expended on centrifuge develop-
ment.
The result has been a series of centrifugal systems for high
resolution separation of mixtures of particles ranging in size from
· whole cells to macroglobulins. Both the engineering and the biologi-
· cal studies have been published in a recent monograph (3). Over fifty
experimental rotors and centrifuge systems have been built and tested
at Oak Ridge thusfar, and six are commercially available. In
adaition a variety of ancillary problems ranging from the design
of all-plastic non-collapsing centrifuge tubes to integrators for
integrating w't continuously have been developed. Complete con-
tainment systems for use in fractionating infectious materials
have been built and used to develop methods for isolating both
viral and bacterial agents. The viruscs include polio, influenza,
respiratory syncytial, echo 28, hepatiti:s, several adenovirus
strains, and Rauscher lekenia virus. Currently centrifugal tech-
niques for the isolation of Treponema pallidum are being developed.
Three different techniques are used in large scale centrifugal
fractionation. The first is rate zonal centrifugation in large
hollow-bowl rotors divided internally into sector-shaped compart-
ments (4). The largest of these in current use will spin 1.7
liters at 40,000 rpm. Larger rotors with capecities up to 7-8,
liters are now under development. The second is isopycnic zonal
centrifugation which may be done in the large rotors used for rete
studies or in small tubes in the so-called "8-p" method (5). This
method provides a two dimensional separation of a particle suspen-
sion based first, on sedimentation rate, and secondly, on isopycnic
banding density. As the distribution of various known particles in
the resulting sup plot has been examined, it has become clear that
many types of viruses have no counterparts in cells with both the
.
same sedimentation coefficient, and the same buoyant density. As
a result the method may be used to search tissues for many types
* of virus particles (5).
The chief interest in virus isolation however lies in the
field of vaccine purification where the concept that the material
to be injected should be biophysically pure is very slowly gaining
acceptance. Here the initial problem is to concentrate the virus
from relatively large volumes of solution. The physical properties
of the starting material vary rather widely with different vaccines.
In some instances filtration or precipitation can be used to achieve
the required concentration. However the problem of agglutination
and entrapment is accentuated by these methods. Continuous flow
centrifugation in which the material to be isolated is pelleted
directly on the rotor wall also suffers from this problem. We
have therefore developed the technique of continuous-flow-with
banding (6) in which a particle-laden stream flows over the inner
surface of a liquid density gradient in a high centrifugal field.
The virus particles sediment out of the stream and band isopycni.-
cally in the gradient. At the end of the run the gradient is re-
covered by either unloading the rotor by displacement during rota-
tion, or by reorienting the gradient to a vertical orientation by
allowing the rotor to come to rest, and then allowing the gradient :
to drain out the bottom of the rotor. Over 100 liter batches of
•
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culture medium or chorioallantoic fluid may be processed in one
run. Several new vaccines prepared with rotor systems developed
at Oak Ridge are now being evaluated in man.
I review this work to point up an interplay of four elements
which are: basic research, applied research in carefully chosen
areas, engineering, and commerical production of new products
for biomedical purposes. The last is the ultimate test of any
program which is seriously concerned with human disease.
Continuing through the ten interest areas listed, work on
number IV, the disassembly of subcellular particles is being
carried out in part in orienting studies at Oak Ridge, and in
.
part through collaborative contracts with universities. We wish
to determine what additional research and development is required
to solve the problems posed by each major subcellular particle.
As an example, gradient extraction of histones from nuclei requires
that we develop corrosion resistant gradient pumps, seals, and
rotors. All-titanium systems appear to fill this requirement and
have been constructed. For studies on the extraction of very
high-molecular-weight DNA from nuclei rotors in which alkaline
gradients may be used and in which the 'separated fractions are
recovered with minimal shear are required. These are now under
development.
In the fifth area listed, new electrophoretic, salting out,
and other techniques for protein fractionation are under development.
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The use of centrifugal force to stabilize liquid density gradients
during electrophoresis allows the size of a gradient to be greatly
increased, and more efficient cooling to be achieved. The gradient
is held between two discs during centrifugation so that heat trans-
fer is maximized. The technique of gradient resolubilization (7)
when applied to protein fractionation allows increased resolution to..
be obtained when dealing with large volumes of fluid at low tempera-,
tures. The greatest remaining problem in this area, as previously
mentioned, is the development of large scale preparative electro- .
phoresis with resolution comparable to that obtained with disc..
electrophoresis. ..
In the sixth program area certain unfilled requirements .. .
existed at the start of thes program. The automated amino acid
analyzer was commercially available. However comparable systems
for nucleolides and related compounds, and for simple sugars were
not available. Both have therefore been developed in Oak Ridge
and are now in routine use (8, 9). The nucleotide analyzer when
applied to human urine samples yields well over a hundred peaks
which are now being identified (10). Certain difficult problems
remain, including miniaturization of these systems, development
of more rapid methods, and the development of sensors specific
for addition chemical groups. The engineering problems which must
be solved are legion.
**--**. ****** *
Tim
Work on automated systems for protein sequence determination
are underway in several laboratories (11) and will begin in Oak .
Ridge in early 1968. The problem of automated enzyme assey has ..
been considered initially from a basic research viewpoint, and
methods for accurately measuring and transferring small volumes
developed (12). On the basis of this work the development of
miniaturized analyzers will, hopefully, be built.
Data reduction becomes an acute problem as soon as a series
of automated analytical systems are put in routine operation.
With chromatographic methods it is important to know whether the
charts obtained are the sum of a series of Gaussian curves (8)
since if they are, the envelope may be reduced to a series of
such Gaussian curves by computers, and their areas tabulated.
Where two wavelength recording is used, the peaks obtained from
each wavelength curve should be superimposible and the absor-
::
bance ratios should be the same across each peak stripped out.
The trend in data reduction is unmistakably in the direction
::
of the use of small computers which may eventually be part of the
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analytical systems themselves. The hazards of dissociating analy-
tical procedures from computation and evaluation are many, and .
every effort must be made to allow them to proceed either con- .
currently or in close sequence.
This short review does not begin to cover the problems which
must be solved before it will be possible to examine in a systematic
.
manner, molecular alterations at the cell level occurring in human
disease. Certain facts are, however, beginning to emerge which
have a bearing on the planning and execution of the program,
The first of these is that the cooperation of a number of
specialists in very many different fields is required. For this
to be successful, problems must be broken down into pieces which
are intelligible to a given specialist. It is difficult to arrange.
to do this as needed except at a large national laboratory.
The second point is that we require in many instances solutions
to problems which are on the limits of presently available science
and technology. If we transfer a metallurgist, mathematician, or
hydrodynamicist to a biological group indefinitely we soon discover
that he is no longer in touch with his own field, and that he rarely
makes a complete transition to biológy. A multidisciplinary effort
requires that, for the most part, the cooperating members should main-
tain roots in their own discipline.
A third point is that the data required to write a molecular
anatomy of human cells will not accrue in the course of unplanned
research any more than new subnuclear particles will be found by
giving large numbers of small grants. With this program we pass
the point in the biomedical sciences where most laboratories have
equal facilities, and can do similar experiments. The cost and
.
.
complexity preclude multiple duplication. This is possibly the.
most difficult point for individual investigators to accept.
The last point is that the level of effort will have to:
begin to match the complexity of the problems to be solved (13).
Budgeting levels in biomedical research had been partially fixed
before the enormity of the problems involved was fully realized.
.
The space and nuclear energy programs were fortunate in achieving
proper scaling very early.
Basic research in the medical sciences has passed from the
macroscopic, to the microscopic, and now to the molecular level.
The practice of medicine has. of necessity lagged behind since
a considerable period of time elapses before new basic ideas
alter practice. Bioengineering is still largely concerned with
man at the gross anatomical level for the simple reason that many
unsolved engineering problems remain at this level. However in
the space of a few years emphasis and interest will shift to work
at the microscopic and molecular levels and biomedical engineering
will then be a part of, and contribute to current basic researcă
in a very important way.
REFERENCES
•
1. Schultze, H. E., and J. F. Heremans. 1966. Molecular Biology
of Human Proteins. Vol. 1 Nature and Metabolism of Extracellular
Proteins. Elsevier Publishing Co., New York.
2. Anderson, N. G. 1956. A note on "homogenizers" for tissue brel
preparation. J. Biophys. and Biochem. Cytol. 2: 219-220.
3. Anderson, N. G. (ed.). 1966. The Development of Zonal Centri-
fuges. J. Nat. Cancer Inst. Monograph No. 21. 0.8. Gov. Print-
ing Office.
4. Anderson, N. G., 8. P. Barringer, B. F. Babeløy, C. E. Nunley,
M. J. Bàrtkus, W. D. Fisher, and C. T. Rankin, fr. 1966. The
Design and operation of the B-IV Zonal Centrifuge . system. J.
Netl. Cancer Inst. Monograph 21: 137–164.
Anderson, N. G., W. W. Harris, A. A. Barber, . T. Rankin, and
E. L. Candler. 1966. Separation of subcellular components and
viruses by combined rate- and isopycnic-zonal centrifugation.
J. Nat. Cancer Inst. Monograph 21: 253-283.*
6. Anderson, N. G., H. P. Barringer, J. W. Amburgey, G. B. Cline,
C. E. Nunley, and A. 8. Berman. 1966. Continuous-flow centri-
fugation combined with isopycnic banding: Rotors B-VIII and
B-IX. Ibid. 199-216.
Anderson, N. G. 1966. Zonal centrifuges and other separation
systems. Science 154: 103-112.
7.
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8. Anderson, X. G., Green, J. G., Barber, M. L., and Ladd, F. C.
1963. Analytical techniques for cell fractions. III Nucleo-
tides and related compounds. Anal. Biochem. 6: 153-169.
9. Green, J. G. 1966. Automated carbohydrate analyzer: experi-
mental prototype. J. Natl. Cancer Inst. Monograph 21: 447–467.
10. Scott, C. D., J. E. Attrill, and N. G. Anderson. 1967. Auto-
matic, high resolution analysis of urine for its ultravioleta
absorbing constituents. Proc. Soc. Exp. Biol. Med. 125: 181-184.
11. Edman, P. and G. Begg. 1967. A protein Sequenator. European
J. Biochem. 1: 80-91.
12. Anderson, N. Q. 1967. Analytical techniques for cell fractions.
IX Measurement and transfer of small fluid volumes(in prepara-
tion.)
13. Anderson, N. G. · 1967. Molecular Anatomy: Next major science
programme? Science Journal 3: 35-41.
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