Significant Achievements in Space Bioscience 1958-1964 Part 8

_Molecular Biology and Bioinstrumentation_

To support biological investigations in s.p.a.ce and to acc.u.mulate baseline data needed for manned s.p.a.ce flight, NASA has conducted a program in laboratory research and theory. A multidisciplinary approach has included such fields as ecology, physiology, organic and biological chemistry, engineering, electronics, and optics. Emphasis in this program has been placed on qualitative and theoretical rather than purely descriptive research, and the investigation of fundamental biological phenomena at all levels, from the molecular to the total life form.

MOLECULAR BIOLOGY

Research in molecular biology has included chemical, physical, biological, and theoretical investigations of prebiological conditions on Earth and, possibly, on other planets; studies of cellular inclusions; genetic material (DNA and RNA) and coding; as well as energy transfer in biological systems.

The understanding of prebiological conditions on Earth, and possible conditions on other planets, depends upon the nature of the complex chemical species which might be encountered. Scientists have shown that biologically important compounds, such as amino acids, can be generated by applying an electrical discharge, ultraviolet radiation, or heat to a gaseous mixture. Biologically interesting compounds can be removed from such a system by condensation or absorption; however, in the limited time and s.p.a.ce available in such experiments, many compounds are not produced in sufficient quant.i.ty to be measured.

The National Biomedical Research Foundation (NBRF) and the National Bureau of Standards (NBS) are conducting an investigation on equilibria in multielement systems. The distribution of molecular species at equilibrium is independent of the way equilibrium was reached and is dependent only on pressure, temperature, and elemental composition. Many of the conditions which might have arisen naturally can be approximated by thermodynamic equilibrium. Compounds which can be formed at equilibrium need no special mechanism to explain their presence.

However, special mechanisms have to be sought for those compounds which could not be so produced and which would have been required for the structure and nutrition of the first living organisms.

In the absence of precise knowledge of the composition of the primitive planetary atmospheres, equilibrium concentrations with a wide range of temperatures, pressures, and elemental compositions are being investigated by NBRF and NBS. These investigators have postulated that the maximum atmospheric pressure may have approached 100 atm if the primitive Earth was sufficiently hot and if an appreciable portion of the water on Earth's surface today was present on primitive Earth. (If the present oceans were to evaporate, the surface pressure would be approximately 300 atm.) Low pressures of 10?6 atm and temperatures between 500 and 1000 K are being used.

A large range of N, O, C, and H compositions are being investigated for interesting and plausible combinations of factors. In these calculations an IBM 7090 computer is being used to obtain data on a very large number of combinations of chemicals. Other chemical species will be added as the research continues. Some results of this study give an insight into the variety of biologically significant chemicals which might have existed during Earth's primitive prebiological condition or may now exist on the surfaces and in the atmospheres of other planets (refs.

[ref.151]-[ref.153]). The general method described by White et al.

([ref.152]), minimizing the free energy of the system, was used. The solution was approached by an iterative process, starting with an initial guess of concentrations of the compounds. At each step, _M_+1 linear equations are solved where _M_ is the number of elements in the system.

In addition to listing of the concentrations of all compounds included in each problem, the results of three-element problems have been expressed on a triangular composition diagram for convenience. A coa.r.s.e grid of 60 points is used to survey all elemental compositions, with finer grids being used in regions of particular interest. The calculated concentrations of the compounds at each composition are stored, and finally a series of triangular diagrams is printed out, each showing the concentrations of as many as four compounds at the grid points.

Figure 2 shows the results obtained in the C, H, and O systems. Organic compounds in concentrations greater than 10? mole fraction are found everywhere except where free O2, is present. Solid carbon theoretically becomes stable along the lower dashed line at 500 K. However, reactions producing it are very slow. The supersaturated region beyond the line of potential carbon formation was also investigated. A threshold was found where polynuclear aromatic compounds are sufficiently concentrated to form a liquid phase. These conditions may have been involved in the primordial formation of asphaltic petroleum.

[Ill.u.s.tration: Figure 2.-_Equilibrium diagram for the system C-H-O._]

Jukes and a.s.sociates ([ref.154]) at the University of California at Berkeley have been investigating the code for amino acids in protein synthesis, the key for translating the sequence of bases in DNA into the sequence of amino acids in proteins. The amino acid code was solely a matter of theory until Nirenberg and Matthaei ([ref.155]) at the National Inst.i.tutes of Health carried out a crucial experiment. This experiment bridged the last remaining gap separating theoretical genetics and test-tube biochemistry. It now became experimentally possible to search for codes for all 20 amino acids concerned in the synthesis of proteins.

The amino acid bases of DNA are: A, adenine; C, cytosine; G, guanine; T, thymine; and U, uracil, which replaces thymine in RNA. There are only 16 ways of arranging A, C, G, and T in pairs. For this and other reasons it is thought that a triplet of three consecutive bases is needed to code for each amino acid. The sequences of bases in a strand of DNA are known to be unrestricted with respect to the order in which they occur; apparently any one of the four bases can be next to any of the other four, although, of course, each base must be paired with the corresponding complementary base in the adjacent strand. Since the same freedom is true of the amino acid sequences in the polypeptide chains of proteins, any one of the 20 amino acids can occur next to any other.

Moreover, the sequences in DNA are subject to mutational changes in which one base replaces another, or bases are added to or deleted from the DNA. Such rearrangements plus the possibility of lengthening of DNA molecules are numerous enough to account for all the genetics of living forms since the first appearance of life on Earth.

Most of our knowledge is based on experiments with synthetic RNA carried out with extracts of _E. coli_. The majority of the work has been at Nirenberg's laboratory at the National Inst.i.tutes of Health and at Ochoa's laboratory at New York University ([ref.155]). Various combinations of A, C, G, and U were used in preparing the synthetic RNA molecules that are used in experiments to explore the code. These molecules are made by incubating a mixture of ribonucleoside diphosphates with a specific enzyme, polynucleotide phosphorylase. An important property of this enzyme is that it condenses the nucleoside diphosphates into polynucleotide strands containing random sequences depending on the proportion of each base. For example, if the enzyme were furnished with a mixture of 5 parts of A and 1 part of C, it would make strands containing, on the average, 25 sequences of AAA, 5 of AAC, 5 of ACA, 5 of CAA, and 1 each of ACC, CAC, and CCA. The proportion of triplets within the strands of a polynucleotide is reflected in the proportion of amino acids in polypeptides that are obtained in the cell-free system. Most of the present knowledge of the amino acid code is based on this concept. All the proposed codes have been discovered by this experimental approach where synthetic RNA molecules are used as "artificial" messenger RNA.

Representative of another cla.s.s of activities in molecular biology is the examination of pa.s.sive ion flux across axon membranes. This work is being done by Goldman at the National Naval Medical Center. The question of stimulus transmission by nerve tissue is far from simple, and the ion concentrations a.s.sociated with nerve membranes is a significant part of the answer. Because the s.p.a.ce environment may very well produce alterations in these ion potentials, an investigation of their natures and significance becomes extremely important. A working theory is now being developed as a result of this study.

Vital cell processes, chemical transformations, and mechanisms that provide energy for cell maintenance and activity have been studied by Kiesow (refs. [ref.157] and [ref.158]) at the Naval Medical Research Inst.i.tute. The common objective of all phases of this project is the elucidation of reaction steps in which energy and matter are transformed in living systems. Compared with _photo_synthetic organisms, _chemo_synthetic bacteria offer distinct advantages for the study of energy a.s.similation. These studies have led to the following experimental findings.

With the energy from oxidation of nitrite, NO2- to nitrate, NO3- as an _inorganic_ source, and with added _organic_ chemical energy from the hydrolysis of adenosinetriphosphate (ATP) to adenosinediphosphate (ADP) and inorganic phosphate, chemosynthetic bacteria are capable of reducing diphosphopyridinenucleotide (DPN?) to DPNH, in a coupled oxidoreduction-dephosphorylation. Thus, in the crucial step of chemosynthesis, _ATP is consumed, not produced_. However, in simultaneously proceeding cell respiration, the energy donor, DPNH, is oxidized and generates more ATP than is required for DPN? reduction.

This "breeder cycle" for DPNH-with different ratios of cell respiration and biosynthesis-results in a net production of either DPNH, or ATP, or both. Production of DPNH in the cycle leads immediately to the a.s.similation of C4 from HC4O3-. These observations explain the bacteria's energy source without the cla.s.sical hypotheses of either direct phosphorylation or direct CO2 reduction by inorganic chemical or electromagnetic energy. The cycle transforms the free energy of nitrite oxidation into the free energy of the organic compounds. Cell respiration and elementary biosynthesis proceed through structure-bound enzyme systems in the same fraction of subcellular particles. Three components, two cytochromes and one flavoprotein, have been identified.

A thermodynamic a.n.a.lysis of the DPNH "breeder cycle" appears to be attainable by measurements of redox potentials and calorimetric determinations of heats of reaction.

Studies are also being conducted by Pollard and a.s.sociates at Pennsylvania State University in an attempt to formulate a theoretical basis for the description of the processes of synthesis, growth, division, and differentiation of the living cell. Such a theory would be basic to an understanding of very primitive life forms or prebiological material which might be found elsewhere in the universe. For these purposes, studies are being undertaken in macromolecular reproduction which differ from the studies involving cellular genetic material.

Theories concerning the problem of replication of cellular structures and information storage in two-dimensional systems are being developed.

Theories are also being developed about the mechanisms which control and regulate receptor and enzymatic activities within the cell.

One study involved the rate of mutation in cells and disposed of the suggestion that the process of mutation consists of a "tunneling" of proton from one base to another in DNA. Such a suggestion can no longer be advanced as a major explanation of mutations.

Work is also being conducted on the centrifugation of cells of _E.

coli_. It has been shown that cells exposed to as little as 100 g have a modification in their function. This has been looked at from the point of view of thymine uptake, which would be concerned with the formation of DNA, and also from the point of view of the induction of an enzyme, which would correspond to the transcription of the DNA. Preliminary experiments in the latter case indicate considerable centrifugation effect. The thymine uptake is affected, but not nearly as much as formerly thought. Further work is in progress in this area.

Important work has been completed on the cells of _E. coli_ grown on maltose, which can be induced to produce betagalactosidase by the addition of thiomethyl galactoside. If cells are irradiated shortly after induction, the transcription of the DNA ceases and the enzyme produced by the messenger RNA is observed to reach a maximum. This enables the calculation of the half-life of unstable messenger RNA. The half-life for this decay is readily measurable, and values are given over a temperature range of 17 C (5.2 minimum) to 45 C (0.56 minimum).

These agree very well with half-lives measured by others by inducing for short times and measuring the course of enzyme formation. The rate of transcription is involved in the kinetics of cessation of enzyme induction, and the rate of transcription can be measured. Arrhenius plots for this rate and the rate of decay are given, and the activation energies measured are about 16 000 cal/mole. The cessation of transcription is linked to the degradation, possibly of only one strand, of DNA.

Pollard has suggested that one important action of ionizing radiation is concerned with the transcription of the genetic message into RNA.

Clayton and Adler ([ref.159]) showed that induced catalase synthesis in _Rhodopseudomonas spheroides_ is inhibited by low doses of X-rays, giving experimental support to the idea. Pollard and Vogler ([ref.160]), using cells in which the process of induction involved permease, showed that there is some sensitivity to gamma radiation. Novelli et al.

([ref.161]) found a reduced sensitivity as compared with colony formation, but it is still a considerable sensitivity.

The process of induction of an enzyme indicates that the transcription of the genetic message is repressed by something which can be acted on by a small molecule, the inducer, to remove repression and permit the formation of messenger RNA, which then acts to make the enzyme. The messenger RNA undergoes decay through a process which is still not clear. Very elegant measurements by Kepes ([ref.162]) show that for the messenger RNA for betagalactosidase, the half-life is 1.02 min at 37 C and 2.05 min at 25 C. The time of onset of enzyme formation after induction was found to be about 3 minutes.

If the process of transcription is indeed sensitive to ionizing radiation, then the irradiation of cells which have just been induced should show formation of the enzyme to the extent of formation of new messenger RNA within a few minutes, plus the formation of the enzyme while the messenger RNA is decaying. This pattern was found by Clayton and Adler. The experiments conducted by Pollard and a.s.sociates amplify and extend their work and also agree with the work of Kepes ([ref.162]).

BIOINSTRUMENTATION

Fernandez-Moran (refs. [ref.163]-[ref.165]), at the University of Chicago, has devised a new multielectrode electrostatic lens which he has incorporated into an electron microscope. This necessitated the development of a novel high-voltage power source and voltage regulator of extreme stability and accuracy. Some promising work has now been done on superconducting lenses. In a series of experiments with a simple electron microscope without pole pieces, using high-field superconducting niobium-zirconium solenoid lenses in an open air core, liquid helium Dewar, electron microscopic images of test specimens have been recorded while operating at 32 200 gauss in a persistent current mode, with regulated accelerating potentials of 4 to 8 kilovolts. These preliminary experiments have demonstrated the exceptional stability of the images (both short term and long term) over a period of 4 to 8 hours and the relatively high quality of the images.

Progress has been made on the viscosimeter for high intrinsic viscosities. This is now working, and the viscosity of DNA preparations has been measured. It is hoped to use the viscosimeter to study the variation in DNA viscosity as a function of the cell cycle.

An instrument is under development by Wald at the University of Pittsburgh to automatically a.n.a.lyze cytogenetic material and, thus, extend cytogenetic methodology both for research and as a biological monitoring procedure, using automatic electronic scanning and computer a.n.a.lysis of chromosomes. Chromosomal aberrations can thus be monitored under unusual and abnormal conditions such as weightlessness and radiation, since chromosomes are very sensitive to stress situations. In this device a sample will be prepared and automatically inserted under a microscope lens. The device will then scan, identify, and photograph on 35-mm film a predetermined number of mitotic cells and process the film.

The data will be recorded under the direct control of a digital computer. The computer will perform a detailed quant.i.tative a.n.a.lysis of the pictorial data.

Significant effort has been expended in the development of instrumentation for measuring and recording electrophysiological information. One such instrument, developed by the Franklin Inst.i.tute, Philadelphia, Pa., is a temperature-sensing microprobe. This microprobe is an implantable and remote broadcasting instrument. These developments are a.s.sociated, in part, with training programs so that competent individuals may be trained not only in electronics but also in the biological uses of the devices they construct.

A project of interest, conducted at the Stanford Research Inst.i.tute, is the investigation of the uses of an extremely sensitive method for measuring magnetic susceptibility having the possibility of detecting macroscopic quantum effects in macromolecules of biological interest.

Good progress has been made in the first 15 months of a project devoted to the development and initial use of equipment specifically designed for this purpose. A new superconducting circuit, together with superconducting magnetic s.h.i.+elds, has been constructed. This apparatus can measure the magnetic susceptibility of small organic samples at temperatures between 1 and 300 K in fields up to 40 000 gauss. It can detect flux changes of 107 gauss-cm, which is equivalent to detecting a change in specific susceptibility of 1 in 10? in a 100-mg sample under an applied field of 10 000 gauss.

Several hundred preliminary measurements were made on samples of coronene. The most reliable of these were in agreement with published values of the magnetic susceptibility of coronene. Experience during these measurements led to changes which have resulted in an apparatus well suited to the measurements on macromolecules. An improved version of the superconducting circuit now available shows promise of a further improvement in sensitivity by a factor of more than a thousand ([ref.166]).

Living organisms possess many unique processes and systems which are complex and poorly understood. The new theoretical approaches, combined with laboratory studies, are expected to result in advances which will expand both our scientific and technological horizons.

chapter 6

_Flight Programs_

BALLOONS

Biological and medical experiments carried out on balloon flights, both manned and unmanned, antedate the establishment of NASA. Aside from the early use of balloons in flights that could be called simply flight-survival studies, balloons have made important contributions to our present knowledge of the effects of cosmic radiation and to various aspects of s.p.a.ce travel.

The achievements of the Strato-Lab and Man High series by the U.S. Navy and Air Force include a wealth of information on balloon travel and on the survival of man at alt.i.tudes close to and above 100 000 feet.

Generally, balloon launches of animals, which reached a maximum in 1953 when 23 balloons were released, have established the feasibility of a program of extended manned balloon flights to high alt.i.tudes.

Atmospheric life studies outside the area of cosmic radiation effects have been comparatively few. Results from two manned flights, Strato-Lab I and II, indicate that the flights did produce p.r.o.nounced changes in white blood cell count; however, the data suggest that these changes were due to psychological rather than physical stress. Exposure to alt.i.tudes above 90 000 feet for a total of 62 hours did not produce any general behavioral change in two Java monkeys, according to other balloon flights. Many of these flights were effective in testing equipment, telemetering devices, and in pointing the way for other flights.

Stratoscope I and II, originally undertaken by the Office of Naval Research (ONR), are projects involving various astronomical observations with the aid of a balloon-borne telescope and television and camera systems. NASA cooperated with ONR on Stratoscope II (36-inch telescope compared with Stratoscope I's 12-inch telescope) which has already resulted in significant discoveries about the nature of the planets and stars. Water vapor has been identified in the atmosphere of cool red stars and an a.n.a.lysis of the Martian spectra showed a greater abundance of carbon dioxide than had previously been believed. Since the balloon-borne telescope was carried beyond Earth's obscuring atmosphere, the Stratoscope projects have yielded valuable photographs of the Sun, stars, and various planets.