Significant Achievements in Space Bioscience

NASA SP-92

Significant Achievements in

Space Bioscience

1958-1964

Scientific and Technical Information Division 1966

NATIONAL AERONAUTICS AND SPACE ADMINISTRATION

Washington, D.C.

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C., 20402—Price 55 cents

Foreword

This volume is one of a series which summarize the progress made during the period 1958 through 1964 in discipline areas covered by the Space Science and Applications Program of the United States. In this way, the contribution made by the National Aeronautics and Space Administration is highlighted against the background of overall progress in each discipline. Succeeding issues will document the results from later years.

The initial issue of this series appears in 10 volumes (NASA Special Publications 91 to 100) which describe the achievements in the following areas: Astronomy, Bioscience, Communications and Navigation, Geodesy, Ionospheres and Radio Physics, Meteorology, Particles and Fields, Planetary Atmospheres, Planetology, and Solar Physics.

Although we do not here attempt to name those who have contributed to our program during these first 6 years, both in the experimental and theoretical research and in the analysis, compilation, and reporting of results, nevertheless we wish to acknowledge all the contributions to a very fruitful program in which this country may take justifiable pride.

Homer E. Newell

Associate Administrator for

Space Science and Applications, NASA

Preface

This summary of certain aspects of the space biology program of the National Aeronautics and Space Administration brings together some results of NASA research and NASA-sponsored research under grants and contracts from 1958 through 1964. Closely related research even though not sponsored by NASA is also included.

The space biology program has had a late start in comparison with the space physics program, and only a token program existed before 1962. Much of the present research involves preparation of space-flight experiments and obtainment of adequate baseline information. Perhaps half the research results reported are derived from the NASA program. Additional information is included from many other sources, especially the U.S. Air Force with its long history of work in aviation and aerospace medicine.

Relatively few biological space-flight experiments have been undertaken. These have been to test life-support systems and to demonstrate, before manned space flight, an animal's capability to survive. Few critical biological experiments have been placed in orbit by NASA, but a biosatellite program will soon make a detailed study of the fundamental biological effects of weightlessness, biorhythms, and radiation.

The search for extraterrestrial life has been limited to ground-based research and planning for planetary and lunar landings. Life-detection experiments have been developed and tested, and an important and exciting program is being planned to detect and study extraterrestrial life, if it exists.

Interest in space biology has been slow in developing, and there has been some caution and controversy in the scientific community. However, increased interest is starting to push forward the frontier of this new and important scientific field, and future outlook appears to be optimistic.

This summary was written and compiled by the members of the Bioscience Programs Division of the Office of Space Science and Applications. The report was edited and chapters [1], [3], 6, and [7] were written by Dale W. Jenkins, Chief, Environmental Biology; [chapter 2], by Gregg Mamikunian, Staff Scientist, Exobiology; chapters [4] and [8], by Richard E. Belleville, Chief, Behavioral Biology; and [chapter 5], by George J. Jacobs, Chief, Physical Biology.

Contents

• [1 BACKGROUND] 1
• [2 EXOBIOLOGY] 5
• [3 ENVIRONMENTAL BIOLOGY] 23
• [4 BEHAVIORAL BIOLOGY] 43
• [5 MOLECULAR BIOLOGY AND BIOINSTRUMENTATION] 57
• [6 FLIGHT PROGRAMS] 65
• [7 MANNED SPACE FLIGHT] 77
• [8 SIGNIFICANCE OF THE ACHIEVEMENTS] 111
• [REFERENCES] 119

[chapter 1]

Background

The biological program of the National Aeronautics and Space Administration had a late start. A small life sciences group, organized in 1958, was concerned with life support and use of primates for system and vehicle testing for the Mercury program. Three small suborbital flights of biological materials were flown in space.

The Bioscience Program Office of the Office of Space Science and Applications was organized in 1962. The goals of the Bioscience Program are: (1) to determine if extraterrestrial life exists anywhere in the solar system and to study its origin, nature, and level of development, if it is present; (2) to determine the effects of space and planetary environments on Earth organisms, including man; (3) to conduct biological research to develop life support and protective measures for extended manned space flight; and (4) to develop fundamental theories in biology relative to origin, development, and relationship to environment. Research and development has been carried out to design life-detection experiments and instruments for future flights to Mars and to develop experiments to study the effects of the space environment on living organisms. A biosatellite program, started in 1963, has the first of six flights scheduled for 1966.

Space exploration has demanded a rigorous development, especially in the biosciences area. Investigation of the solar system for exotic life forms, the environmental extremes to which Earth organisms (including man) are being exposed, the possibilities for modification of planetary environments by biological techniques yet to be developed, and the problems of communication in biosystems are areas which have required refinement of the theoretical framework of biology before progress could be made rapidly enough to keep pace with technological advances in transportation.

Of all the sciences, biology alone has not yet benefited from comparisons with the universe beyond Earth. It is reasonable to suppose that breakthroughs might be made in biology on the basis of comparisons with life from other worlds. Organisms elsewhere may have found alternatives to processes we think of as basic characteristics of life.

In contrast, physical science has advanced sufficiently to provide a great body of laws which may be expressed in mathematical terms, and by which phenomena may be predicted with complete accuracy. A well-known characteristic of biological phenomena is variability. The Darwinian concept of evolution is perhaps the only pervading generalization in biology. This concept has been supported by evidence of a hereditary mechanism in the discovery of genes and gene mutations.

Space bioscience represents the convergence of main disciplines with a single orientation, whose direction is determined by the problems of manned space travel which have, in turn, created a host of bioengineering problems concerned with supporting man in space.

Foremost among these questions is the possibility of the existence of extraterrestrial life. The field which is concerned with the search for extraterrestrial life has come to be called "exobiology." In addition to the challenge of great technological problems which must be solved, exobiology is so closely related to the central scientific questions in biological science that it is considered by some to be the most significant pursuit in all of science.

One of the major opportunities already presented by the advances in propulsion systems is the ability to escape from the influence of the Earth, which has made possible the study of organism-environment relationships, particularly the role that environmental stimuli play in the establishment and maintenance of normal organization in living systems.

Transcending even these formidable objectives of space bioscience is an objective shared by all life sciences, the discovery of nature's scheme for coding the messages contained in biological molecules. Extraterrestrial biology seeks to find not only evidence of life now present, but the vestigial chemicals of its previous existence. The ways and means have already been made available to study molecules on whose long, recorded messages is written the autobiography of evolution—the history of living organisms extending back to the beginnings of life. On this same basis, it is now within the realm of science to foresee the means of predicting the development of life from primordial, nonliving chemical systems. Closely allied to the search for extraterrestrial life is research which seeks to identify the materials and the conditions which are the prerequisites of life.

Space bioscience research is now extending human knowledge of fundamental biological phenomena, both in space and on Earth, just as the physical sciences explore other aspects of the universe. The accomplishment of bioscience objectives is totally dependent upon advances in the technology of space flight. A highly developed launch-vehicle capability is essential to accomplish the long-duration missions required in the search for extraterrestrial life.

Life on other planets in the solar system (with emphasis on Mars) will be investigated by full exploitation of space technology which will allow both remote (orbiter) and direct (lander) observations of the planetary atmosphere, surface, and subsurface. Certain characteristics of terrestrial life, such as growth and reproduction, provide a basis for relatively simple experiments which may be used on early missions to detect the existence of life on Mars. Later missions will provide extensive automatic laboratory capabilities for analyzing many samples taken from various depths and locations. Because of the hypothetical nature of current experiment designs, it is likely that visual observations of the planet will be required. Many technical problems are involved in storing and transmitting the large amounts of data over planetary distances. Such visual observations might very well be crucial in interpreting results from other experiments. Critical to all exploration of the Moon and planets are the requirements to: (1) prevent contamination of the environment with Earth organisms and preserve the existing conditions of the planet for biological exploration; (2) provide strict quarantine for anything returned to Earth from the Moon and planets.

The biological exploration of Mars is a scientific undertaking of the greatest significance. Its realization will be a major milestone in the history of human achievement. The characterization of life, if present, and study of the evolutionary processes involved and their relationship to the evolution of terrestrial life would have a great scientific and philosophical impact. What is at stake is nothing less than knowledge of our place in nature.

Extended Earth orbital flights with subhuman specimens will be used to determine the effects on Earth organisms of prolonged weightlessness, radiation, and removal from the influence of the Earth's rotation. Such flights of biosatellites and other suitable spacecraft are expected to: (1) establish biological specifications for extending the duration of manned space flight; (2) provide a flexible means of testing unforeseen contingencies, thus providing an effective biological backup for manned missions; (3) yield experimental data more rapidly by virtue of the greater number and expendability of subjects; (4) anticipate possible delayed effects appearing in later life or in subsequent generations, through use of animal subjects with more rapid development and aging; (5) develop and test new physiological instrumentation techniques, surgical preparations, prophylactic techniques, and therapeutic procedures which are not possible on human subjects; and (6) provide a broad background of experience and data which will permit more accurate interpretations of observed effects of space flight on living organisms, including man.

[chapter 2]

Exobiology

The possibility of discovering an independent life form on a planet other than Earth presents an unequaled challenge in the history of scientific search. Therefore, the detection of life within the solar system is a major objective of space research in the foreseeable future.

The scientific data presently available concerning the possible existence of a Martian life form and the chemical constitution of the surface of Mars are disappointingly few. In fact, it is impossible to make a statement about any of the many surface features, other than the polar caps, with any degree of certainty. The observational results have been accounted for by many conflicting hypotheses which can only be resolved by the accumulation of new evidence.

The arguments supporting the existence of Martian life ([ref.1]) are based on the following observations:

1. The various colors, including green, exhibited by the dark areas
2. The seasonal changes in the visual albedo and polarization of the dark areas
3. The ability of the dark areas to regenerate after an extensive "duststorm"
4. The presence of absorption bands at 3.3µ-3.7µ, attributed to organic molecules

Conflicting interpretations of the above observations have been advanced. The argument based on the colors is inconclusive, and several workers have suggested that the color is a contrast effect with the bright-reddish continents. The meager quantitative data have been discussed by Öpik ([ref.2]) who has reduced Kozyrev's photometric observations of the very dark area of Syrtis Major to intrinsic reflectivities by allowing for the estimated atmospheric attenuation and reflectivity. Kuiper ([ref.3]) similarly demonstrated the absence of the near-infrared reflection maximum, which is characteristic of most green plants, indicating that chlorophyll was not responsible for the color.

The second and third arguments remain the most cogent. However, serious limitations are imposed on the second if the severity of the Martian climate is considered. Föcas ([ref.4]) has photometrically measured the seasonal changes in the fine structure of the dark areas of Mars and concludes that—

1. The dark areas of Mars show periodic variation of intensity following the cycle of the darkening element
2. The average intensity of the dark area, not including the action of the darkening waves, increases from the poles toward the equator
3. The action of each of the darkening waves decreases from the poles toward the equator. This decrease is balanced in the equatorial zone by the combined action of the two darkening waves alternately originating at the two poles. The mechanism of the darkness-generating element seems to be constant for all latitudes during the Martian year.

The variation in intensity has been explained recently by nonlife mechanisms for Depressio Hellespontica (an area showing one of the greatest seasonal changes) ([ref.2]). Similar nonlife mechanisms may be applicable to the other dark regions, and, thus, the "darkening" can be used only as circumstantial evidence in support of a Martian life form.

If inorganic interpretations of the seasonal albedo variation are accepted, then an inorganic interpretation must also be advanced for the polarization variation. Two possibilities can be suggested:

1. A change in surface texture, caused by varying absorption of atmospheric constituents, causing both the albedo and polarization to change in the manner observed
2. A change in surface texture, in which the surface material becomes rougher, which also explains the observed polarization data ([ref.5])

The third argument against the regenerative feature of the dark areas being a life process has been advanced by Kuiper ([ref.6]). It is based on atmospheric circulation causing dust, presumably lava, to be blown on the dark areas of Mars during the late summer, autumn, and winter, and then removed during the spring. Mamikunian and Moore have recently advanced the similar explanation that carbonaceous chondrites or asteroidal matter may induce the observed phenomenon if they are abundant on the planet's surface. The pulverized chondritic material will exhibit a high degree of opacity due to localization and, hence, a change in polarization characteristics and a decrease in polarization following mixing of the chondritic material with indigenous surface minerals.

The fourth observational argument, the Sinton bands ([ref.7]), has been shown to be at least doubtful. Rea, Belsky, and Calvin ([ref.8]) recorded infrared reflection spectra for a large number of inorganic and organic samples, including minerals and biological specimens, for the purpose of interpreting the 3µ-to-1µ spectrum of Mars. These authors state that a previous suggestion that the Martian "bands" be attributed solely to carbohydrates is not a required conclusion. At the same time they fail to present a satisfactory alternate explanation, and the problem remains unsolved. More recently, Rea et al. ([ref.9]) noted the similarity between the 3.58µ and 3.69µ minima in the Martian infrared spectra and those of D₂O-HDO-H₂O mixtures and, particularly, of HDO.

With all this marked disagreement in interpreting the observational data concerning Mars, it becomes clearly evident that an experimental approach to the detection of life on Mars should provide the maximum positive information possible. Some life-detection experiments developed with NASA support have been summarized by Quimby ([ref.10]).

The schema of the biological exploration of a planet is to conduct a series of complementary experiments proceeding from general to specific. The general experiments will examine gross characteristics of the planet's environment and surface for determining the probability of an active biota (life). Data from the general experiments will be significant in—

1. Defining the nature of specific experiments in which life detection is the major objective; and
2. Providing a high degree of confidence in undertaking specific experiments, since indications from the gross characterization of the planet in question will influence the choice and design of the specific experiments.

The biological exploration of planets is then to be defined as the search for those parameters relevant to the origin, development, sustenance, and degradation of life in a planetary environment. This definition will give rise to a critical question for each progressively specific and complex experiment to determine—

1. The existence of life on the planet
2. The degree of similarity or dissimilarity (structure and function) with respect to terrestrial life
3. The origin of this planetary life

The immediate objective of the biological explorations of the planet is to define the state of the planetary surface, which may exhibit the following properties:

1. A prebiota (defined as the absence of life)
2. An active biota (defined as the presence of life)
3. An extinct biota (defined as evidence of former life)

The identification and the detailed characterization of each of the above stages of planetary development constitute the subject matter of the biological exploration of the planets and, specifically, Mars.

THE EXPERIMENTAL INVESTIGATION OF CHEMICAL EVOLUTION

Attempts have been made to simulate and approximate models of primitive Earth conditions for abiogenic synthesis, and successful synthesis of essential biochemical constituents necessary for maintaining life has been partly accomplished.

Urey ([ref.11]) has clearly pointed out the possible role of a reducing atmosphere in the synthesis of prebiological organic molecules. Miller ([ref.12]) synthesized a variety of amino acids in a reducing atmosphere by means of an electrical discharge. A variety of organic compounds have been synthesized by the action of various energy sources upon reducing atmospheres, and several investigators have extended the Urey-Miller-type reactions to synthesize nucleic acid components ([ref.13]), adenosine triphosphate ([ref.14]), and a host of biologically essential organic compounds.

It is likely that in the synthesis of organic moieties, simple and specific molecules were first produced when the planets had a reducing atmosphere. Further complexity or degradation of the organic compounds produced varied, depending on the geochemical changes of the planet's surface, the atmospheric constituents, the degree of interaction between surface and atmosphere, and the rate of the organic synthesis. Oparin ([ref.15]) presented the most detailed mechanisms for the spontaneous generation of the first living organism arising in a sea of organic compounds synthesized in a reducing atmosphere on Earth.

It is generally accepted that, under favorable conditions, life can arise by spontaneous generation. A primary requirement for this initiation is that there be abundant organic compounds concentrated in one or more specific zones. These simple organic molecules would undergo modification to develop a greater structural complexity and specificity, finally giving rise to a "living" organism. Therefore, because of the ease with which organic compounds can be synthesized under reducing conditions, planetary surfaces may contain an abundant source of similar organic matter. However, difficulties arise in postulating steps for further organization or modification of the above synthesized organic matter into a living state. Most of the original organic matter produced in the primary reducing atmospheres of the various planets may have been quite similar. However, major variations between planets, in chemical evolution beyond the prebiotic stage, must have been the rule rather than the exception.

The primary interest in this area of research has been the realization of the possible existence of organic molecules on planetary surfaces and, particularly, Mars. Pertinent synthesis may be either biological or abiological. Research conducted in the simulation of cosmochemical synthesis has used most of the available solar spectrum. Simulation experiments devised to study the effects of these energies on the assumed early atmosphere of the Earth have yielded products that play a dominant role in molecular and biochemical organization of the cell.

Calvin ([ref.16]) irradiated water and carbon dioxide in a cyclotron, obtaining formaldehyde and formic acid. Miller ([ref.17]) found that when methane, ammonia, water, and hydrogen were subjected to a high-frequency electrical discharge, several amino acids were produced along with a variety of other organic compounds.

Corroborating experiments established that the synthesis of amino acids occurred readily. The apparent mechanism for the production of amino acids is as follows: aldehydes and hydrogen cyanide are synthesized in the gas phase by the electrical discharge. These substances react together and also together with ammonia in the water phase of the system to give hydroxy and amino nitriles, which are then hydrolyzed to hydroxy and amino acids. Among the major constituents were aspartic acid, glutamic acid, glycine, α-alanine, and β-alanine.

The "Miller-Urey" reaction mixture has been extended and several modifications introduced. Oró ([ref.18]) introduced hydrogen cyanide into the system as the primary gas component. Adenine was obtained when Oró heated a concentrated solution of hydrogen cyanide in aqueous ammonia for several days at temperatures up to 100° C. Adenine is an essential component of nucleic acids and of several important coenzymes. Guanine and urea were the two other products identified in the hydrogen cyanide reaction. Oró further obtained guanine and uracil as products of nonenzymatic reactions by using certain purine intermediates as starting materials.

Ponnamperuma ([ref.19]) also obtained adenine upon irradiation of methane, ammonia, hydrogen, and water, using a high-energy electron beam as the source of energy of irradiation. These results indicate that adenine is very readily synthesized under abiotic conditions. Adenine, among the biologically important purines and pyrimidines, has the greatest resonance energy, thus making its synthesis more likely and imparting greater radiation stability to the molecule.

The formation of adenine and guanine, the purines in RNA and DNA, by a relatively simple abiological process lends further support to the hypothesis that essential biochemical constituents of life may have originated on Earth by a gradual chemical evolution and selection. In this respect, the examination of planetary surfaces—specifically Mars—presents practical implications for current research on the problem of chemical evolution.

When Ponnamperuma et al. ([ref.14]) exposed adenine and ribose to ultraviolet light in the presence of phosphate, adenosine was produced. When the adenine and ribose were similarly exposed in the presence of the ethyl ester of polyphosphoric acid, adenosine diphosphate (ADP) and adenosine triphosphate (ATP) were produced. The abiological formation of ATP was a major stride along the path of chemical evolution, since ATP is the principal free energy source of living organisms.

Oparin ([ref.15]) postulated that α-amino acids could have been formed nonbiologically from hydrocarbons, ammonia, and hydrogen cyanide at a time when the Earth's atmosphere contained these substances in high concentrations. Oparin's hypothesis has received strong experimental support, as evidenced by the work of Miller ([ref.12]). Bernal ([ref.20]) has emphasized the role played by ultraviolet light in the formation of organic compounds at a certain stage of the Earth's evolution.

Generally it has been believed that the first proteins or foreprotein were nonbiologically formed by the polycondensation of preformed free amino acids ([ref.21]). Akabori ([ref.22]) proposed a hypothesis for the origin of the foreprotein and speculated that it must have been produced through reactions consisting of the following three steps.

The first step is the formation of aminoacetonitrile from formaldehyde, ammonia, and hydrogen cyanide.

CH₂O + NH₃ + HCN ————> H₂N—CH₂—CN + H₂O

The second is the polymerization of aminoacetonitrile on a solid surface, probably absorbed on clay, followed by the hydrolysis of the polymer to polyglycine and ammonia.

x H₂N—CH₂—CN ————> (—NH—CH₂—C—)_x

||

NH

|

| + x H₂O

V

(—NH—CH₂—CO—)_x + x NH₃

The third step is the introduction of side chains into polyglycine by the reaction with aldehydes or with unsaturated hydrocarbons. Akabori has demonstrated experimentally the formation of cystinyl and cysteinyl residue in his above-postulated mechanism.

Fox's theory of thermal copolymerization ([ref.23]) suggests that proteins or like molecular units could have been formed in the Earth's crust, under geothermal conditions. The accumulated amino acids were heat polymerized and transported into the primary oceans for further modifications. Fox has obtained polymers consisting of all 18 amino acids usually present in proteins. The polymerization is generally done at 160° C to 200° C, although in the presence of polyphosphoric acid it can be accomplished at temperatures below 100° C. Molecular weights increased from 3600 in a proteinoid made at 160° C to 8600 in one made at 190° C.

Fox showed that when hot saturated solutions of thermal copolymers containing the 18 common amino acids were allowed to cool, large numbers of uniform, relatively firm, and elastic spherules separate. These range from 0.2µ to 60µ in diameter and are quite uniform within each preparation. Various chemical observations suggest the presence of peptide bonds in the structural organization of these proteinoids. Continuing observations of these microspheres have established further characteristics that point to the possibility of their interpretation as a kind of primitive protein macromolecule with self-organizing properties, such that a primitive form of cell, with boundary and other properties, might form.

In laboratory experiments the behavior of gram-negative and gram-positive microspheres in dilute alkali parallels that of gram-negative and gram-positive bacteria ([ref.23]). Furthermore, time-lapse studies indicate that the proteinoid microspheres undergo a septate kind of fission, mimicking cell division as shown in figure 1. Cytochemical studies show that the microsphere's boundary is membranelike in having a primitive selectivity. Electron micrographs of sections of stained microspheres also indicate the presence of a boundary.

Oparin ([ref.15]) states that the type of organization peculiar to life could only result from the evolution of a multimolecular organic system separated from its environment by a distinct boundary but constantly interacting with this environment. In his concept of coacervates as precell models, Oparin ([ref.24]) indicates that present-day protoplasm possesses a number of features similar to coacervate structure. These coacervates could represent the starting point for evolution leading to the origin of life. Moreover, in the course of their evolution the initial systems may gradually become more complex. Oparin also showed ([ref.15]) that mixing solutions of different proteins and other substances of high molecular weight produced these coacervate droplets. These droplets are characterized by the formation of a surface layer with altered structure and mechanical properties, thus providing a somewhat selective barrier in which to house a molecular system capable of replication. However, these coacervates are unstable structurally.

Figure 1.—Protenoid microspheres undergoing septate fission. Small microspheres and filamentous associations thereof are also shown ([ref.25]).

The NASA program has further provided considerable impetus for continuing research with respect to the chemical evolution of life, since its life-detection experiments may encounter prebiological molecules in their search for extraterrestrial life on other planetary surfaces.

In the area of exobiological research, the significant accomplishments to date have been—

1. The reconstruction of some of the pathways which may have led to the origin of life, by means of laboratory simulation of processes yielding prebiological organic molecules
2. The developments in experimental and theoretical biology; specifically, the role of nucleic acid-protein interactions in storage and transmission of information both within living cells and from generation to generation of cells
3. The suspected role of DNA in information storage and the development of new concepts of the coding mechanism in DNA that may lead to a universal biological theory embracing evolutionary, as well as homeostatic, adaptation to environment and learned behavioral systems

With the essential biochemical constituents of life and the mechanism of replication beginning to be understood, the challenge for the synthesis of living matter by abiogenic experimental techniques has become to many scientists the ultimate goal of the scientific era.

NASA has established an exobiology laboratory at Ames Research Center in addition to the sizable support of research at various academic centers of excellence for the continuation of abiogenic synthesis.

Although research on organochemical evolution is in its infancy, the data from relatively few experiments have already created an immense enthusiasm for knowledge of the biochemical pathways of evolution. This kind of research will ultimately elucidate the terrestrial evolution of life and, perhaps, the nature of life on other planetary bodies and the distribution of life in our galaxy.

This program, with its vast demands on the scientific community at large, is coordinated with related endeavors of a number of Federal agencies. It is allied with certain biochemical studies at the National Institutes of Health for the eventual elucidation of the dynamic pathways in cosmochemical synthesis of life's essential biochemical constituents.

METEORITES AND ORGANIC GEOCHEMISTRY

Meteorites

A significant area of exobiological research is the investigation of a special class of stony meteorites known as "carbonaceous chondrites." It is increasingly apparent that almost all life-detection concepts rely on the eventual analysis of the solid materials that may be available on Mars and other planetary surfaces. Cosmic dust and meteorites are two classes of material bodies that reach the Earth from outer space. The carbonaceous chondrites are the only extraterrestrial materials known to contain organic carbon.

The study of meteorites has generated an astonishing diversity of hypotheses. There is agreement at only one point: that meteorites are preserved chunks of very ancient, perhaps primordial, planetary matter and that when we are able to understand the curious structures and chemical and isotopic variations in the meteorites, we will also know a great deal about early planetary (and perhaps preplanetary) history.

Meteorites provide a more representative sample of average planetary matter than the highly differentiated crust of the Earth. Although it is known that the meteorite parent bodies ceased to be geochemically active shortly after their formation, some 4½ billion years ago, there is no consensus on the nature of the meteorite parent bodies, not even on such basic properties as size, location, and multiplicity. This is not surprising because the meteorite samples commonly available for study represent only about 10^-23 to 10^-26 of the parent body.

Carbonaceous Meteorites

Analysis and characterization of the chemical constituents (organic) of carbonaceous chondrites, including the possible mechanism of their formation, may be expected to improve methods of analyzing samples from the Moon and planets and of interpreting remote automated biological analyses on the planets' surfaces.

Carbon has been detected in all meteorites analyzed; however, both the amount and forms present vary considerably. Among the forms of meteorite carbon are diamond, graphite, cohenite (Fe,Ni,Co)₃C, moissanite SiC, calcite CaCO₃, dolomite (Ca,Mg)CO₃, bruennerite (Mg,Fe)CO₃. A summary of the results of carbon analyses on large numbers of meteorites is given in [table I] ([ref.26]).

Most meteorites possess only traces of carbon, and studies of this carbon indicate that it is composed largely of graphite, cohenite, and moissanite, with some diamond. However, studies of the carbon in the carbonaceous chondrites have failed to detect any of these forms. Some carbonates are present in a minority of the carbonaceous group, but account for only a small percentage of the total carbon (perhaps about 10 percent of the total C in type I only).

The carbonaceous chondrites contain organic carbon. The word "organic" is not used in a biological sense, merely as a chemical term to describe compounds of carbon other than carbonates, bicarbonates, and carbides. No evidence has been found of any form of carbon other than organic, except for traces of carbonates.

Various studies have demonstrated possible methods of estimating the total amount of organic matter present in meteorites. Wiik ([ref.27]) has suggested that organics can be estimated by measuring the loss of weight on ignition. Unfortunately, this method has several disadvantages and gives very low values. Corrections must be made for weight gains due to oxidation of reduced constituents, such as FeO, Fe, Ni, and Co, and for weight losses due to H₂O, S, etc. The water loss is exceedingly difficult to estimate, as part comes from the combustion of organic hydrogen and part comes from the loss of mineral-bound water. The carbon also proves difficult to combust completely, and high temperatures (over 1000° C) are required for efficient conversion to CO₂.

In one study the major fraction of organic matter removed proved to have a carbon content of about 47 percent ([ref.28]). Thus, if all the meteorite carbon is present as organic matter of approximately this composition, total organics must be approximately double the carbon content; that is, 2 percent by weight carbon indicates 4 percent by weight organic matter. This estimate may be too low, for Mueller ([ref.29]) has extracted a major organic fraction containing only 24 percent carbon; however, this work has not been confirmed for other meteorites.

Briggs and Mamikunian ([ref.26]) have pointed out that only 25 percent of the organic matter has been extracted, and only about 5 percent of this has been chemically characterized. Most of this 5 percent is a complex mixture of hydroxylated aromatic acids together with hydrocarbons of the aliphatic, napalicyclic, and aromatic series. Small amounts of amino acids, sugars, and fatty acids are also present.

Thus far, these chemical analyses point to an abiogenic origin for the organic matter, and no conclusive evidence exists of biological activity on the meteorite parent body. Microbiological investigations of samples of the carbonaceous chondrites have yielded only inconclusive evidence on the problem of "organized elements."

Several of these microstructures from different carbonaceous chondrites are illustrated in a paper by Mamikunian and Briggs ([ref.30]). It has been difficult to identify the organized structures, and most do not have morphologies identical to known terrestrial micro-organisms. However, they may prove to be a variety of mineral grains, droplets of organic matter and sulfur, as well as a small amount of contaminating terrestrial debris.

A comparison between the photographs of the organized elements observed in the Orgueil and Ivuna meteorites and the synthetic proteinoid microspheres observed by Fox ([ref.25]) point to similarities between the two. One inference from this finding is that the organized elements in carbonaceous chondrites were never alive but, rather, should be considered as natural experiments in molecular evolution. Also, these similarities strengthen the belief that the laboratory experiments are similar to the natural experiments in space.

In cooperation with the Smithsonian Astrophysical Observatory, NASA has a network to track meteors in the Midwest (South Dakota, Nebraska, Kansas, Oklahoma, Iowa, Missouri, and Illinois). Photographs of meteor trails are used for scientific study, and attempts are made to track and recover meteorites for examination for traces of organic material of extraterrestrial origin.

Fundamental research in terrestrial organic geochemistry has shown that ancient sediments and drill core samples subjected to organic analysis contain certain stable biochemical components of past life. This preserved record is significant not only in studies of early-life chemical pathways but also in studies of the interaction of organic matter with the geological factors. Since life on any planetary body will interact with the soil, or surface material, it is of interest to understand the relationship.

CONCEPTS FOR DETECTION OF EXTRATERRESTRIAL LIFE

It is not possible to present completely convincing evidence for the existence of extraterrestrial life. The problem often reduces to probabilities and to estimates of observational reliability. In almost all cases the evidence is optimistically considered strongly suggestive of—or, at the worst, not inconsistent with—the existence of extraterrestrial life. Alternatively, there is a pessimistic view that the evidence advanced for extraterrestrial life is unconvincing, irrelevant, or has another, nonbiological explanation.

In studies of the laboratory synthesis of life-related compounds and its significance concerning the origin of life, several results seem to suggest that organochemical synthesis is a general process, occurring perhaps on all planets which retain a reducing atmosphere. The temperature ranges must be such that precursors and reaction products are not thermally dissociated. The reaction rates for the synthesis of more complex organic molecules diminish to a negligible value when the temperature range is below 100° C.

Besides the planetary parameter of temperature, an even more fundamental necessity for a living state exists—a liquid solvent system. For terrestrial life forms, water serves this purpose. Water has this and other properties of biological significance because of hydrogen bonding between adjacent molecules in the liquid state.

Ultraviolet radiation could serve as an extraterrestrial energy source for organic synthesis. Research shows that, while an atmosphere is important, living systems can survive a wide range of ambient pressures and are little affected by a wide range of magnetic field strengths.

Oxygen is not a prerequisite for all living systems. While it is sometimes concluded that free oxygen is needed for all but the simplest organisms, less efficient metabolic processes coupled with higher food collection efficiency—or a more sluggish metabolism—would seem to do just as well. Earth is the only planet in the solar system on which molecular oxygen is known to be present in large amounts. Since plant photosynthesis is the primary source of atmospheric oxygen, it seems safe to infer that no other planet has large-scale plant photosynthesis accompanied by the production of oxygen.

The possibility of the existence of extraterrestrial life raises the important question of man's being able to detect it. Research on extraterrestrial life detection is predicated on the ability to develop ways to detect it even when the living systems are based on principles entirely different from those on Earth.

The substitution of various molecules for those of known biological significance to living organisms as we know them has been investigated; the substitution of NH₂ for OH in ammonia-rich environments leads to a diverse, and biologically very promising, chemistry. The hypothesis that silicon may replace carbon does not support the construction of extraterrestrial genetics based on silicon compounds. (Silicon compounds participate in redistribution reactions which tend to maximize the randomness of silicon bonding, and the stable retention of genetic information over long time periods is thus very improbable.)

Evidence relevant to life on Mars has been summarized by Sagan (ch. 1 of [ref.10]):

The Origin of Life

In the past decade, considerable advances have been made in our knowledge of the probable processes leading to the origin of life on Earth. A succession of laboratory experiments has shown that essentially all the organic building blocks of contemporary terrestrial organisms can be synthesized by supplying energy to a mixture of the hydrogen-rich gases of the primitive terrestrial atmosphere. It now seems likely that the laboratory synthesis of a self-replicating molecular system is only a short time away from realization. The syntheses of similar systems in the primitive terrestrial oceans must have occurred—collections of molecules which were so constructed that, by the laws of physics and chemistry, they forced the production of identical copies of themselves out of the building blocks in the surrounding medium. Such a system satisfies many of the criteria for Darwinian natural selection, and the long evolutionary path from molecule to advanced organism can then be understood. Since nothing except very general primitive atmospheric conditions and energy sources are required for such syntheses, it is possible that similar events occurred in the early history of Mars and that life may have come into being on that planet several billions of years ago. Its subsequent evolution, in response to the changing Martian environment, would have produced organisms quite different from those which now inhabit Earth.

Simulation Experiments

Experiments have been performed in which terrestrial micro-organisms have been introduced into simulated Martian environments, with atmospheres composed of nitrogen and carbon dioxide, no oxygen, very little water, a daily temperature variation from +20° to -60° C, and high ultraviolet fluxes. It was found that in every sample of terrestrial soil used there were a few varieties of micro-organisms which easily survived on "Mars." When the local abundance of water was increased, terrestrial micro-organisms were able to grow. Indigenous Martian organisms may be even more efficient in coping with the apparent rigors of their environment. These findings underscore the necessity for sterilizing Mars entry vehicles so as not to perform accidental biological contamination of that planet and obscure the subsequent search for extraterrestrial life.

Direct Searches for Life on Mars

The early evidence for life on Mars—namely, reports of vivid green coloration and the so-called "canals"—are now known to be largely illusory. There are three major areas of contemporary investigation: visual, polarimetric, and spectrographic.

As the Martian polar ice cap recedes each spring, a wave of darkening propagates through the Martian dark areas, sharpening their outlines and increasing their contrast with the surrounding deserts. These changes occur during periods of relatively high humidity and relatively high daytime temperatures. A related dark collar, not due to simple dampening of the soil, follows the edge of the polar cap in its regression. Occasional nonseasonal changes in the form of the Martian dark regions have been observed and sometimes cover vast areas of surface.

Observations of the polarization of sunlight reflected from the Martian dark areas indicate that the small particles covering the dark areas change their size distribution in the spring, while the particles covering the bright areas do not show any analogous changes.

Finally, infrared spectroscopic observations of the Martian dark areas show three spectral features which, to date, seem to be interpretable only in terms of organic matter, the particular molecules giving rise to the absorptions being hydrocarbons and aldehydes. [However, see p. 7 and Rea et al. ([ref.9]).]

Taken together, these observations suggest, but do not conclusively prove, that the Martian dark areas are covered with small organisms composed of familiar types of organic matter, which change their size and darkness in response to the moisture and heat of the Martian spring. We have no evidence either for or against the existence of more advanced life forms. There is much more information which can be garnered from the ground, balloons, Earth satellites, Mars flybys, and Mars orbiters, but the critical tests for life on Mars can only be made from landing vehicles equipped with experimental packages....

Results of Kaplan et al. ([ref.31]) indicate that Mars has no detectable oxygen, but does contain small amounts of water vapor, more abundant carbon dioxide, possibly a large surface flux of solar ultraviolet radiation, and estimated daily temperature variations of 100° C at many latitudes. Studies have shown that terrestrial micro-organisms can survive these extremely harsh environments. Furthermore, a variety of physiological and ecological adaptations might enable the biota to survive the low nighttime temperatures and intracellular ice crystallization.

Less evidence is available to support the possibility of extraterrestrial life on other planets. The Moon has no atmosphere, and extremes of temperature characterize its surface. However, the Moon could have a layer of subsurface permafrost beneath which liquid water might be trapped. The temperatures of these strata might be biologically moderate.

Studies by Davis and Libby ([ref.32]) on the atmosphere of Jupiter support the possibility of the production of organic matter in its atmosphere in a manner analogous to the processes which may have led to the synthesis of organic molecules in the Earth's early history. It is difficult to assess the possibility that life has evolved on Jupiter during the 4- or 5-billion-year period in which the planet has retained a reducing atmosphere.

The question of extraterrestrial life and of the origin of life is interwoven. Discovery of the first and analysis of its nature may very well elucidate the second.

The oldest form of fossil known today is that of a microscopic plant similar in form to common algae found in ponds and lakes. Scientists know that similar organisms flourished in the ancient seas over 2 billion years ago. However, since algae are a relatively complex form of life, life in some simpler form could have originated much earlier. Organic material similar to that found in modern organisms can be detected in these ancient deposits as well as in much older Precambrian rocks.

Although the planets now have differing atmospheres, in their early stages the atmospheres of all the planets may have been essentially the same. The most widely held theory of the origin of the solar system states that the planets were formed from vast clouds of material containing the elements in their cosmic distribution.

It is believed that the synthesis of organic compounds preceding the origin of life on Earth occurred before its atmosphere was transformed from hydrogen and hydrides to oxygen and nitrogen. This theory is supported by laboratory experiments of Calvin ([ref.16]), Miller ([ref.33]), and Oró ([ref.34]).

The Earth's present atmosphere consists of nitrogen and oxygen in addition to relatively small amounts of other gases; most of the oxygen is of biological origin. Some of the atmospheric gases, in spite of their low amounts, are crucial for life. The ultraviolet-absorbing ozone in the upper atmosphere and carbon dioxide are examples of such gases.

Significant in the search for extraterrestrial life are the data (e.g., planet's temperature) transmitted by Mariner II, which was launched from Cape Canaveral on August 27, 1962, and flew past Venus on December 14, 1962. Mariner II's measurements showed temperatures on the surface of Venus of the order of 800° F, too hot for life as known on Earth.

The question "Is life limited to this planet?" can be considered on a statistical basis. Although the size of the sample (one planet) is small, the statistical argument for life elsewhere is believed by many to be very strong. While Mars is generally considered the only other likely habitat of life in our solar system, Shapley ([ref.35]) has calculated that more than 100 million stars have planets sufficiently similar in composition and environment to Earth to support life. Of course, yet unknown factors may significantly reduce or even eliminate this probability.

SPACECRAFT STERILIZATION

The search for extraterrestrial life with unmanned space probes requires the total sterilization of the landing capsule and its contents. Scientists agree that terrestrial organisms released on other planets would interfere with exobiological explorations (refs. [ref.36]-[ref.43]). Any flight that infects a planet with terrestrial life will compromise a scientific opportunity of almost unequaled proportions. Studies on microbiological survival in simulated deep-space conditions (low temperature, high ultraviolet flux, and low dose levels of ionizing radiation) indicate that these conditions will not sterilize contaminated spacecraft (refs. [ref.44]-[ref.48]). Furthermore, many terrestrial sporeformers and some vegetative bacteria, especially those with anaerobic growth capabilities, readily survive in simulated Martian environments (refs. [ref.49]-[ref.54]). It has been estimated that a single micro-organism with a replication time of 30 days could, in 8 years of such replication, equal in number the bacterial population of the Earth. This potential could result not only in competition with any Martian life, but in drastic changes in the geochemical and atmospheric characteristics of the planet. To avoid such a disaster, certainly the first, and probably many succeeding landers on Mars, must be sterile—devoid of terrestrial life ([ref.55]). Since the space environment will not in itself kill all life aboard, the lander must leave the Earth in a sterile condition.

The sterility of an object implies the complete absence of life. The presence of life or the lack of sterility may be proven; but the absence of life or sterility cannot be proven, for the one viable organism that negates sterility may remain undetected. Many industrial products which must be guaranteed as sterile cannot be tested for sterility in a nondestructive manner. A similar situation exists in determining the sterility of a spacecraft. Certification of sterility—based on experience with the sterilizing process used, knowledge of the kinetics of the death of micro-organisms, and computation of the probability of a survivor from assays for sterility—is the only accurate approach to defining the sterility of such treated items.

Macroscopic life can be readily detected and kept from or removed from the spacecraft, but the detection and removal of microscopic and submicroscopic life is an extremely difficult task. The destruction of micro-organisms can be achieved by various chemical and physical procedures. Sterilizing agents have been evaluated not only for their ability to kill microbial life on surfaces and sealed inside components, but also for the agents' effects on spacecraft reliability as well (refs. [ref.56]-[ref.59]). Of the available agents, only heat and radiation will penetrate solid materials. Radiation is expensive, hazardous, difficult to control, and apparently damages more materials than does heat. Heat, therefore, has been selected as the primary method of spacecraft sterilization and will be used, except in specific instances where radiation may prove to be less detrimental to the reliability of critical parts ([ref.60]).

The sterilization of spacecraft is a difficult problem if flight reliability is not to be impaired. The development of heat-resistant parts will enable the design and manufacture of a heat-sterilizable spacecraft. Without careful microbiological monitoring of manufacture and assembly procedures, many bacteria could be trapped in parts and subassemblies. To permit sterilization at the lowest temperature-time regimen that will insure kill of all organisms, the microbiological load inside all parts and subassemblies must be held to a minimum.

The role of industrial clean rooms in reducing the biological load on spacecraft is currently being defined. NASA-supported studies indicate that biological contamination in industrial clean rooms for extended time periods is about 1 logarithm less (tenfold reduction), compared with conditions in a well-operated microbiological laboratory ([ref.61]). With the use of clean-room techniques and periodic decontamination by low heat cycles or ethylene oxide treatment, it should be possible to bring a spacecraft to the point of sterilization with about 10⁶ organisms on board ([ref.60]).

The sterilization goal established for Mars landers is a probability of less than 1 in 10 000 (10^-4) that a single viable organism will be present on the spacecraft. Laboratory studies of the kinetics of dry-heat kill of resistant organisms show that at 135° C the number of bacterial spores can be reduced 1 logarithm (90 percent) for every 2 hours of exposure (refs. [ref.58] and [ref.62]). The reduction in microbial count needed is the logarithm of the maximum number on the spacecraft (10⁶) plus the logarithm of the reciprocal of the probability of a survivor (10⁴), or a total of 10 logarithms of reduction in microbial count. Thus, with an additional 2 logarithms added as a safety factor, a total of 12 logarithms of reduction in count has been accepted as a safe value which can be achieved by a dry-heat treatment of 135° C for 24 hours. This is the heat cycle that is currently under study and being developed for use in spacecraft sterilization ([ref.60]). However, other heat treatments at temperatures as low as 105° C for periods of 300 hours or longer are under study ([ref.63]).

Based on results to date, it is reasonable to believe that a full complement of heat-sterilizable hardware will be available when needed for planetary exploration. Every effort is being made to improve the state of the art to a point where spacecraft can not only withstand sterilization temperatures, but will be even more reliable than the present state-of-the-art hardware that is not heated.

[chapter 3]

Environmental Biology

BIOLOGICAL EFFECTS OF WEIGHTLESSNESS AND ZERO GRAVITY

High priority has been given to studies of weightlessness. Gravity is one of the most fundamental forces that acts on living organisms, and all life on Earth except the smallest appears to be oriented with respect to gravity, although certain organisms are more responsive to it than others. The gravity force on Earth is 1 g, but this force may be experimentally varied from zero g, or weightlessness, to many thousands of g's.

Zero gravity or decreased gravity occurs during freefall, in parabolic trajectory, or during orbit around the Earth. Gravitational force decreases by the square of the distance away from the Earth's center. It is reduced about 5 percent at about 200 nautical miles' altitude. Gravitational force greater than 1 g can be obtained by acceleration, deceleration, or impact. It also can be increased by using a centrifuge which adds a radial acceleration vector to the 1 g of Earth.

On the ground, the biological effects of gravity have been studied at 1 g, and experimentally, forces of many g have been produced. In addition, modifications of the effects of the 1-g force have been induced by suspension of the organism in water or by horizontal immobilization of an erect animal such as man. The biological effects of such modification have been of significant value in understanding some of the possible consequences of human exposure to the zero-g environment of space.

Weightlessness in an Earth-orbiting satellite occurs when the continuous acceleration of Earth's gravity is exactly counterbalanced by the continuous radial acceleration of the satellite. In such a weightless state, organisms are liberated from their natural and continuous exertion against 1 g, but this liberation may carry with it certain serious physical penalties.

Some of the physical processes which probably have the greatest biological effects are (1) convective flow of fluid, e.g., protoplasmic streaming, transport of nutrient materials, oxygen, waste products, and CO₂ from the immediate environment of the cell, and (2) sedimentation occurring within cells; substances of higher density sediment in a gravitational field, and those of lighter density rise. A separation of particles of different densities probably occurs. The removal of gravity would change a distribution of particles like mitochondria by 10 percent ([ref.64]).

Gravity has effects on the physical processes involved in mitosis and meiosis. Study under weightlessness might contribute to our understanding of the general cellular information-relay process.

A gravitational effect is known in the embryonic development of the frog Rana sylvatica. After fertilization, the eggs rotate in the gravitational field so that the black animal hemisphere is uppermost. Development becomes abnormal if this position is disturbed. If the egg is inverted following the first cleavage and held in this position, two abnormal animals result, united like Siamese twins. This phenomenon appears to be related to the gravitational separation of low- and high-density components of the egg. The size of the egg is about 1 to 2 mm and is suspended in water of about the same density. This system is very sensitive to gravity; and, under weightlessness, the separation of different density components might be irregular, leading to aberrant development. When certain aquatic insect eggs are inverted, subsequent development results in shortened abnormal larvae.

The directional growth of plant shoots and plant roots is probably due to this sedimentation phenomenon, particularly the effect on movement of auxins ([ref.65]).

Free convection flow is a major transport process, and under its influence the mixing of substances is much more effective than when diffusion operates alone. Free convection flow is a macroscopic phenomenon which increases not only with g, but varies also approximately with the five-fourths power of the bulk concentration involved. Whether or not convection is important at the microscopic level remains an experimentally unsolved question. The Grashoff number limits free convection to the macroscopic domain. It would appear in weightlessness that the contribution of free convective flow would be small and that only diffusion should occur. This phenomenon would cause equilibration to occur much more slowly than that occurring with free convection and diffusion. The absence of convective transfer raises a problem as to how nutrients may be obtained and waste products removed in living cells during weightlessness. In a liquid substrate, nutrients and oxygen would be depleted, and waste products would accumulate around the cell.

Absence of gravity may have far-reaching consequences in the homeostatic aspects of cell physiology. The outstanding characteristics of living cells which are most likely to be influenced by the absence of gravity are the ability of the cell to maintain its cytoplasmic membrane in a functional state, the capacity of the cell to perform its normal functions during the mitotic cycle, and the capacity of the cytoplasm to maintain the constant reversibility of its sol-gel system ([ref.66]).

Two-phase systems, e.g., air-in-water and air-in-oil, possess entirely different characteristics at zero g than at 1 g. These physical differences in phase interaction could well be suspected of interfering with the orientation and flow pattern of cell constituents, thus hindering the cellular processes involved in the movement, metabolism, and storage of nutrients and waste.

On the basis of theoretical calculations, weightlessness can be expected to have some effect even on one individual cell if its size exceeds 10 microns in diameter ([ref.64]). Cell colonies might be affected. In larger cells there may be a redistribution of enzyme-forming systems which give rise to polarization. The low surface tension of the cell membrane lends itself to hydrostatic stress distortion, implying an alteration in permeability and thus an almost certain alteration of cell properties under low gravity conditions.

Another aspect of gravity that affects the growth and development of living organisms is the directionality of the gravitational field. In fact, some plants are so sensitive that they are able to direct their growth with as little stimulus as a 1×10^-6 gravitational field. Investigations of plant growth in altered gravitational fields are underway at Argonne National Laboratory and Dartmouth College.

The Argonne Laboratory has designed and developed a 4-pi, or omnidirectional, clinostat. By rotating a plant so that the force of gravity is distributed evenly over all possible directions, the directional effects of gravity are eliminated, simulating some aspects of the zero-g state. It was shown that certain plants grew more slowly and had fewer and smaller leaves, while others had about 25 percent greater replication of fronds and had greater elongation of certain plant parts. It will be extremely interesting to compare these effects under zero-g conditions in orbiting spacecraft.

The effect of gravity in transporting growth hormones in plants has been demonstrated at Dartmouth College using radiocarbon-labeled growth hormones. Plant geotropisms and growth movements have been studied and biosatellite experiments developed.

Anatomy is considered a derivative adaptation to gravity ([ref.67]). A large background of plant research exists on the effect of orientation on plant responses. Information from clinostat experiments is considered susceptible of extrapolation to low gravity conditions because the threshold period for gravitational triggering is relatively long.

Once over critical minimum dimensions, the major effects of low gravity would be assumed to occur in those heterocellular organisms that develop in more or less fixed orientation with respect to terrestrial gravity and which respond to changes in orientation with relatively long induction periods; these are the higher plant orders. On the other extreme are the complex primates which respond rapidly, but whose multiplicity of organs and correlative mechanisms are susceptible to malfunction and disorganization. It may be suggested that the heterocellular lower plants and invertebrates will be less affected. Perturbations of the environment to which the experimental organism is exposed must be limited or controlled to reduce uncertainties in interpretation of the results. At the same time, the introduction of known perturbations may assist in isolating the effects due solely to gravity. Study of de novo differentiation and other phenomena immediately after syngamy may be of particular importance. Study of anatomical changes after exposure of the organism to low gravity is important.

BIOLOGICAL EFFECTS OF SPACE RADIATION[¹]

Radiation sources in space are of three types: galactic cosmic radiation, Van Allen belts, and solar flares with an intense proton flux. Cosmic radiation has higher energy levels than radiation produced by manmade accelerators.

The Panel on Radiation Biology, while recognizing the need for radiobiological studies of an applied nature with reference to manned flight programs, stated that it would be shortsighted for the United States to confine its efforts to the solution of immediate problems since, in the long run, successful exploration of space will be aided by the contributions of basic research. Both the immediate biological research program and the continuing program for basic studies should be built upon the large body of existing knowledge of radiation effects. The attitude that all radiobiological experiments need be repeated in the space environment should be resolutely rejected. Since fundamental radiobiology cannot be performed easily in space, it has been recommended that, wherever possible, these investigations be carried out in ground laboratories in preference to flying laboratories.

Space environment does vary from the terrestrial environment, but the variations are not so great as to lead to the expectation of strikingly different biological effects of radiation in space. However, it is conceivable that radiations whose effects are well known under terrestrial conditions may have some unsuspected biological effects when combined with unusual features of the space environment: e.g., zero g. Previous space radiobiological studies have depended solely on very low and inaccurately measured doses of ambient space radiation. It has been difficult to distinguish between the observed response levels and the random noise; thus, experiments have been inconclusive.

Biological Effects of Heavy Ions and Mesons

The biological effects of heavy ions (especially Z>2) and mesons are of specific interest to space radiobiology.

Controlled Radiobiological Experiments in Space

There is the remote possibility that the radiobiological response may be modified by factors as yet unknown and perhaps not susceptible to terrestrial study. Experiments have been designed to settle this matter including the exposure of biological materials during space flight which meet the following criteria of reliability: (1) the use of well-known biological systems, e.g., mutation induction or chromosome breakage; (2) the use of a sufficient number of individuals in the experiment to guarantee statistical precision on the results; (3) the exposure of the system to known quantities and qualities of radiation; (4) the use of adequate controls.

High-altitude balloon ascents of the 1930's initiated study of the biological effects of cosmic rays. They were limited to the exploration of secondary cosmic radiation effects. After World War II, the research extended to the use of V-2 rockets fired from the White Sands Proving Ground. Interest returned to balloons and a significant program was underway by 1950, first using mice and then hamsters, fruit flies, cats, and dogs. These flights gave no evidence of radiation damage. However, it was realized that the flights were too far south to obtain a significant exposure, and more northerly flights began in 1953. Mice and guinea pigs were flown on these later flights. Chase ([ref.68]) showed the most unequivocal results to that time, a statistically significant increase in light hairs on black animals and the streaks of white hair up to 10 times wider than expected. Brain lesions were detected in the guinea pigs flown on Man High in 1957. Many other types of biological material were sent aloft in an effort to further corroborate existing information and to investigate genetic and developmental effects of cosmic radiation.

From the earlier V-2 rocket flights to the Jupiter missile launchings of the monkeys Able and Baker, cosmic-ray research was continued, but the short flight durations of these vehicles did not provide substantial information. The USAF Discoverer satellite program has given impetus to cosmic-ray research and provided for longer "staytimes."

It has been difficult to separate radiation effects from other space-flight factors: therefore, some of the alterations observed are still subject to debate. Vibration, acceleration, and weightlessness appear to be the three most important additional parameters. Measurements of radiation dosage have been made by chemical and photographic dosimetry, ion chambers, and biological dosimetry. All evidence to date indicates that radiation exposure levels are not hazardous to man at present orbital altitudes up to 200 nautical miles. Most biological materials flown so far have been for the express purpose of investigating space-radiation levels and effects. The biological materials have ranged from tissue cultures to entire organisms and from phage and bacterial cells to man. The studies have required much of the space and weight resources allotted biology by the U.S.S.R. and the United States. They have been accompanied by ground-based controls.

The Vostok series provided the following data:

1. A small, but statistically significant, increase was observed in the percentage of chromosome aberrations in the rootlet cells of air-dried wheat and pea seeds after germination. In this case only, the increase did not depend on flight duration.
2. Lysogenic bacteria exhibited an increase of genetic alterations and increased phage production. Length of flight was associated with increased bacteriophage production by the lysogenic bacteria. There was an increase of recessive lethals coupled with nonconvergence of chromosomes (sex linked) in the fruit fly. A stimulation of cell division in wheat and pea seeds was observed. Cultures of human cells exposed to space-flight factors did not differ significantly from terrestrial controls with respect to such indicators as proliferation rate, percentage of mortality and morphological, antigenic, and cultural properties. Repeated flights of the identical HeLa cells revealed that there was a longer latent period for restoration of growth capacity than in cells carried into space once or not flown at all.
3. The most definite radiation effects observed were only revealed in genetic tests. No harmful influence on those characteristics affecting the viability of the organism has been discovered.

The Air Force Discoverer series launched from the west coast had a few successful flights incorporating organisms. With severe environmental stress and long recovery times, data on radiation exposure were equivocal up to Discoverer XVII and XVIII when cultures of human tissue were flown, recovered, and assessed for radiation exposure effects. Comparison with ground-based controls revealed no measurable differences.

Radiation dosimetry from the Mercury series established that minimal exposures were encountered at those orbital altitudes. A typical example is the MA-8 flight of W. M. Schirra, Jr., during which the body surface dosage was less than 30 millirads.

NASA has supported fundamental radiation studies at the Oak Ridge National Laboratory and the Lawrence Radiation Laboratory. Emphasis has been placed on the biological effects of high-energy proton radiation and particulate radiation from accelerators.

At the NASA Ames Research Center extensive fundamental studies are being carried out on the effects of radiation, especially in the nervous system. It has been demonstrated that deposits accumulate in the brain following exposure to large doses of ionizing particle radiation as well as after X-irradiation. These deposits, referred to as a "chemical lesion," result from an accumulation of glycogen. The formation of these deposits during exposure to large doses of X-irradiation was not increased in environments of 99.5 percent oxygen and increased atmospheric pressure.

SIMULATION OF PLANETARY (MARTIAN) ENVIRONMENTS

Attempts have been made to simulate to some degree the various parameters of the Martian environment, such as atmospheric composition, pressure, radiation flux, temperatures, and the day-night as well as seasonal cycles. Certain factors for Mars cannot yet be simulated, such as soil composition, gravitational field, magnetic field, and electrical field.

Caution is required in interpreting all simulation experiments. How Earth organisms respond to simulated Martian environments probably has nothing to do with life on Mars, but these experiments may show whether or not anything in the environment of Mars makes life as we know it impossible. We must expect that on Mars, life will have evolved and have adapted over long periods of time under conditions which are quite different from conditions on Earth. The simulation experiments also provide some information about the possibility of contaminating the planet Mars, or any planet, with organisms from Earth. In addition, they give us some clues about the possibilities of adaptation and evolution of life under these conditions.

From an evolutionary point of view, if life has developed on Mars, we expect it to have evolved at least to a microbial stage. On Earth, micro-organisms are the most ubiquitous and numerous forms of life. This fact should be considered in studying extraterrestrial bodies.

Micro-organisms have been selected as the best test organisms, and bacteria and fungi have been used because they are durable and easy to grow. Also, because of their rapid growth, many generations can be studied in a relatively short period of time. The organisms include chemoautotrophic bacteria, which are able to synthesize their cell constituents from carbon dioxide by energy derived from inorganic reactions; anaerobic bacteria, which grow only in the absence of molecular oxygen; photoautotrophic plants such as algae, lichens, and more complex seed plants; and small terrestrial animals.

Organisms have been collected from tundra, desert, hot springs, alpine, and saline habitats to obtain species with specialized capabilities to conserve water, balance osmotic discrepancies, store gases, accommodate to temperature extremes, and otherwise meet stresses. An attempt is made in these simulation experiments to extend these processes across the possible overlapping microenvironments which Earth and Mars may share.

Scientists have developed various special environmental simulators, including "Mars jars" and "Marsariums." These have made possible controlled temperatures, atmospheres, pressures, water activities, and soil conditions for duplicating assumed Martian surface. A complex simulator, developed by Young et al. ([ref.52]), reproduces the formation of a permafrost layer with some water tied up in the form of ice beneath the soil surface. This simulator serves as a model to study the wave of darkening, thus supporting the hypothesis that the pole-to-equator wave of darkening is correlated with the availability of subsurface water. The simulator is a heavily insulated 2-cu-ft capacity chamber with an internal pressure of 0.1 atm. The chamber contains a soil mixture of limonite and sand and an atmosphere of carbon dioxide and nitrogen. With the use of a liquid nitrogen heat exchanger at one end and an external battery of infrared lamps at the other end, the temperature simulates that of Mars from pole to equator. Thermocouples throughout the soil monitor the temperatures in the chamber.

Zhukova and Kondratyev ([ref.69]) designed a structure measuring 100×150×180 cm. Micro-organisms were placed at the surface of a copper bar made in a special groove separated by glass cloth. Copper was selected as one of the best heat-conduction materials permitting a rapid change of temperature. The lower end of the bar was immersed into a mixture of dry ice and ethyl alcohol, which made it possible to create a temperature of -60° C. Heating was performed by an incandescent spiral.

As the knowledge concerning the Martian environment becomes more refined, scientists can more accurately simulate this environment under controlled conditions in the laboratory. Determination of the effects of the Martian environment on Earth organisms will permit better theorization on the forms of life we might find on Mars and will permit us to estimate the potential survival of Earth contaminants on Mars.

However, until the environmental conditions of Mars are defined more accurately, the experiments must be changed continually to fit newly determined conditions. Therefore, existing simulation data are made less valid for comparison. The data resulting from the simulation experiments for Mars have been compiled in [table II], and the experiments are summarized below.

The earliest simulation studies were carried out by the Air Force, and the studies during the past 6 years have been supported by NASA. Recently, these studies have received less support or have been terminated in favor of critical studies on the effects of biologically important environmental extreme factors on Earth organisms. These critical studies permit establishing the extreme environmental factor parameters in which Earth life can grow or survive. These data will have valuable application to the consideration of life on any planet, to the design of life-detection instruments, to the sterilization of space vehicles, and to the problem of contamination of planets.

Some exploratory experimental studies are in progress to study the capabilities of organisms to grow under the assumed conditions on Jupiter. These include studies at high pressure with liquid ammonia, methane, and other reducing compounds.

Early experiments simulating Martian conditions using soil bacteria were carried out by Davis and Fulton ([ref.70]) at the Air Force School of Aviation Medicine, San Antonio, Tex. Mixed populations of soil bacteria were put in "Mars jars" with the following conditions: 65-mm Hg pressure, 1 percent water or less, nitrogen atmosphere, sandstone-lava soil, and a temperature day-night cycle of +25° to -25° C. The moisture was controlled by desiccating the soil and adding a given amount of water. Experiments, conducted up to 10 months, demonstrated that obligate aerobes died quickly. The anaerobes and sporeformers survived. Although a small increase in the total number of organisms indicated growth, the increases in the number of bacteria may have been due to breaking up clumps of dirt.

Roberts and Irvine ([ref.71]) reported that, in a simulated Martian environment, colony counts of a sporeforming bacterium, Bacillus cereus, increased when 8 percent moisture was added. Moisture was considered more important than temperature or atmospheric gases inasmuch as a simulated Martian microenvironment containing 8 percent moisture permitted germination and growth of endospores of Clostridium sporogenes. Increases in colony counts of Bacillus cereus appeared to be influenced by temperature cycling ([ref.72]).

Studies of the effects of simulated Martian environments on sporeforming anaerobic bacteria were carried out by Hawrylewicz et al. ([ref.49]). They showed that the encapsulated facultative anaerobe, Klebsiella pneumoniae, survived under simulated Martian atmosphere for 6 to 8 months, but were less virulent than the freshly isolated organisms. Spores of the anaerobe Clostridium botulinum survived 10 months in the simulator. Hagen et al. ([ref.53]) found that the addition of moisture to dry-simulated Martian soil did not improve the survival of Bacillus subtilis or Pseudomonas aeruginosa. Bacillus cereus spores survived, with added organic medium plus moisture, but no germination of the spores resulted.

Hawrylewicz et al. ([ref.49]) put rocks from Antarctica bearing various lichens in simulated Martian conditions in a large desiccator. They found that the algal portion of a lichen, Trebouxia erici, showed only slight resistance to the Martian environment. They also pointed out the effect moisture had on the physical condition of lichens. The undersurface of a lichen has great water-absorbing capability, and the slightest amount of moisture on a rock surface is absorbed by the lichen which can turn green in 15 minutes.

Scher et al. ([ref.51]) exposed desert soils to simulated environmental conditions and diurnal cycles of Mars. The atmosphere consisted of 95 percent nitrogen and 5 percent carbon dioxide (no oxygen) and was dried, using calcium sulfate as a desiccant. The total atmospheric pressure was 0.1 atm. The temperature ranged from -60° to +20° C in 24-hour cycles. One hour was spent at the maximum and at the minimum temperatures. The chambers were irradiated with ultraviolet, 2537 Å, with a dose of 10⁹ ergs/cm², which is comparable to a daily dose found on Mars, and easily exceeds the mean lethal dose for unprotected bacteria. Soil aliquots were removed weekly and incubated at 30° C. The scoring was done both aerobically and anaerobically. Sporeforming obligate and facultative anaerobes, including Clostridium, Bacillus, and Planosarcina, and nonsporeforming facultative anaerobes, including Pseudomonas and Rhodopseudomonas, were found. The experimental chambers were frozen and thawed cyclically up to 6 months. Organisms that were able to survive the first freeze-thaw cycle were able to survive the entire experiment. The ultraviolet irradiation did not kill subsurface organisms, and a thin layer of soil served as an ultraviolet shield. All of the samples showed survivors.

Young et al. ([ref.52]) assumed that water is present on Mars, at least in microenvironments, and that nutrients would be available. The primary objective of their experiments was to determine the likelihood of contaminating Mars with Earth organisms should a space probe from Earth encounter an optimum microenvironment in terms of water and nutrients. The experiments used bacteria in liquid nutrient media. The environment consisted of a carbon dioxide-nitrogen atmosphere, and the temperature cycling was -70° to +25° C, with a maximum time above freezing of 4½ hours. Aerobacter aerogenes and Pseudomonas sp. grew in nutrient medium under Martian freezing and thawing cycles. Atmospheric pressure was not a significant factor in the growth of bacteria under these conditions.

Silverman et al. ([ref.47]) studied bacteria and a fungus under extreme—but not "Martian"—conditions. Spores of five test organisms (B. subtilis var. niger, B. megaterium, B. stearothermophilus, Clostridium sporogenes, and Aspergillus niger) and soils were exposed while under ultrahigh vacuum to temperatures of from -190° to +170° C for 4 to 5 days. Up to 25° C there was no loss in viability; at higher temperatures, differences in resistivity were observed. At 88° C, only B. subtilis and A. niger survived in appreciable numbers; at 107° C, only A. niger spores survived; none were recoverable after exposure to 120° C. B. subtilis survived at atmospheric pressure and 90° C for 5 days, but none of the other spores were viable alter 2 days. Four groups of soil organisms (mesophilic, aerobic, and anaerobic bacteria, molds, and actinomycetes) were similarly tested in the vacuum chamber. From one sample only actinomycetes survived 120° C, while one other soil sample yielded viable bacteria after exposure to 170° C. Several organisms resisted 120° C in ultrahigh vacuum for 4 to 5 days. When irradiated with gamma rays from a cobalt 60 source, differences were observed between vacuum-dried spores irradiated while under vacuum and those exposed to air immediately before irradiation. A reduction of from one-third to one-ninth of the viability of spores irradiated in vacuum occurred with vacuum-treated spores irradiated in air.

Siegel et al. ([ref.73]), in approximate simulations of Martian environments, studied tolerances of certain seed plants, such as cucumbers, corn, and winter rye, to low temperatures and lowered oxygen tensions. Lowered oxygen tensions enhanced the resistance of seedlings, particularly cucumber and rye to freezing, and lowered the minimum temperature required for germination. Germination of seeds in the absence of liquid water has also been studied. In this case, seeds of xerophytes have been suspended in air at 75-mm Hg pressure above water. The air was thus saturated. Germination was slow but did occur.

Siegel et al. (refs. [ref.73] and [ref.74]) found that the growth rate of several higher plants was enhanced by certain gases usually thought to be toxic, such as N₂O. This finding is significant inasmuch as the presence of nitrogen oxides in the Martian atmosphere has been cited as evidence for the nonexistence of plants on that planet by Kiess et al. ([ref.75]). Exploratory survival tests showed that various mature plants, as well as the larvae, pupae, and adult specimens of a coleopteran insect, were undamaged when exposed to at least 40 hours of an atmosphere containing 96.5 percent N₂O, 0.7 percent O₂, and 2.8 percent N₂.

Lichens are of interest because of their ability to survive and thrive under extreme environmental conditions on Earth. Biological activity of slow-growing lichens was detected by metabolic gas exchange, CO₂ detection being especially convenient. Siegel points out that this method is sensitive and nondestructive, to be preferred to staining techniques, which at present are limited because they are only semiquantitative, subjective, and destructive of the lichen.

A Russian study of simulated planetary environments has been performed with good simulation but for periods of only 2 to 6 hours. Comments on simulation experiments made by Zhukova and Kondratyev ([ref.69]) are presented as follows:

On the basis of modern conceptions on Martian conditions it is difficult to imagine that higher forms of animals or plants exist on the planet. A Martian change of seasons similar to that of our planet empowers us to think that there is a circulation of an organic substance on Mars, which cannot exist without participation of microbic forms of life. Microorganisms are the most probable inhabitants of Mars although the possibility is not excluded that their physiological features will be very specific. That is why the solution of the problem concerning the character of life on Mars is of exceptional interest. But still the answer to this question can be verified only by simulating Martian conditions, taking into account the information obtained from astrophysicists.

Experiments aimed at creating artificial Martian climatic conditions have been started quite recently; their number is not large since they cannot be combined with the results of numerous experiments investigating the effect of extreme factors on microorganisms. The result of the effect of such physicochemical parameters of the medium as pressure, sharp temperature changes, the absence of oxygen and insolation, depends on their combination and simultaneity. These examples convincingly show that while simulating Martian conditions one should strive to the most comprehensive complex of simultaneously acting factors. The creation of individual climatic parameters acting successively leads to absolutely different, often opposite results. It should be mentioned also that refusal to imitate insolation and the performance of experiments with specimens of soil which itself has protective effect on cells of microorganisms, but not with pure culture of bacteria, are usual shortcomings in the bulk of studies on this problem.

It appears that organisms from Earth might survive in large numbers when introduced to Martian environment. Whether these organisms will be capable of growth and explosive contamination of the planet in a biological sense or not is highly questionable. The likelihood of an organism from Earth finding ideal conditions for growth on Mars seems extremely low. However, the likelihood of an organism from Earth serving as a contaminant for any life-detection device flown to Mars for the purpose of searching out carbon-based life is considerably higher. The chance that life has originated and evolved on Mars is a completely separate question and much more difficult to answer.

It would be interesting to attempt to determine possible evolutionary trends which might occur on a planet by means of selection of organisms in a simulated planetary environment. Rapid genetic selection combined with radiation and chemicals to speed up mutation rate under these conditions should reveal possible evolutionary trends under the planetary environmental conditions. This could be attempted after the planetary environments are more accurately defined.

EXTREME AND LIMITING ENVIRONMENTAL PARAMETERS OF LIFE

The question of the existence of extraterrestrial life is one of the most important and interesting biological questions facing mankind and has been the subject of much controversial discussion and conjecture. Many of the quantitative, and even qualitative, environmental constituents of the planets also are still subjects of controversy and speculation. Best guesses about a relatively unknown planetary environment, combined with lack of information about the capabilities of Earth life to grow in extreme environments, do not provide the basis for making informed scientific estimates.

Life on Earth is usually considered to be relatively limited in its ability to grow, reproduce, or survive in extreme environmental conditions. While many common plants and animals (including man) are quite sensitive to, or incapable of, surviving severe chemical and physical changes or extremes of environment, a large number of micro-organisms are highly adapted and flourish in environments usually considered lethal. Certain chemoautotrophic bacteria require high concentrations of ammonia, methane, or other chemicals to grow. Anaerobic bacteria grow only in the absence of oxygen.

Besides adapting to the extremes of environments on Earth, life is also capable of growing and reproducing under extreme environmental conditions not normally encountered: e.g., from a few rad of radiation in normal habitats to 10⁶ or more rad from artificial sources, from 0.5 gauss of Earth magnetism to 167 000 gauss in manmade magnetic fields, and from 1-g force of gravity to 110 000 g. The extreme ranges of physical and chemical environmental factors for growth, reproduction, and survival for Earth micro-organisms are phenomenally large.

Life is ubiquitous on Earth and is found in almost every possible environment, including the most severe habitats, from the bottom of the ocean to the highest mountain tops and from cold Arctic habitats to hot springs, as well as in volcanic craters, deep wells, salt flats, and mountain snowfields. Earth life has become adapted to, and has invaded, nearly every habitat, no matter how severe. The physiological and morphological adaptations of life are exceedingly diverse and complex.

Surprisingly, the extreme parameters or ranges of the physical and chemical environmental factors permitting growth, reproduction, and other physiological processes of Earth organisms have not been critically compiled. A partial compilation of certain selected environmental factors has been made by Vallentyne ([ref.76]). A compilation of available published data on certain environmental extremes, particularly from recent NASA-supported research (compiled by Dale W. Jenkins, in press), is presented in tables [III] to [VI]. These data can serve as a starting point for a more intensive literature review by specialists, critical evaluation, standardization of end points, and especially to point out areas where critical experimentation is urgently needed.

This critical compilation involves a review of a very broad and complex range of subjects involved in many different disciplines with widely scattered literature. Since the effects of many of the specific environmental factors are harmful, it is difficult to select a point on a scale from no effect to death and use some criteria to say that normal or even minimal growth and reproduction are occurring. The effects of environmental factors are dependent on (1) the specific factor, times, (2) the concentration or energy, times, (3) the time of exposure or application of the factor. Many reports, especially older ones, do not give all of the necessary data to permit proper evaluation. A complicating factor is that the effect of each factor depends on the other factors before, during, and after its application. The condition of the organism itself is a great variable. Proper evaluation requires the critical review by a variety of biological specialists, physicists, and chemists.

To determine the potential of Earth organisms to survive or grow under other planetary environmental conditions, a number of experiments have been carried out attempting to simulate planetary environments, especially of Mars, as reviewed previously. While the results are of real interest, they do not provide much basic information. Further, as the Martian environment is more accurately defined, the experimental conditions are changed. In addition, some experimenters have altered certain factors, such as water content, to allow for potential microhabitats or for areas which might contain more water at certain times.

[chapter 4]

Behavioral Biology

EFFECTS OF THE SPACE ENVIRONMENT ON BEHAVIOR

NASA was established in 1958, shortly after the Russian launching of the second Earth satellite Sputnik II, the first vehicle to carry life into orbit around the Earth. This accomplishment was preceded by the pioneering work of Henry et al. ([ref.77]), in which animals were exposed briefly to low-gravity states in Aerobee rockets. A motion-picture camera photographed the behavior of two white mice in rotating drums during this series of flights, which marked the first time that simple psychological tests were made on animals in the weightless condition. While this behavioral experiment was relatively simple, it provided the basic concepts for recent studies which involved rotation of animals during the weightless state. Subsequent flights such as Project MIA (Mouse-in-Able) reflected a preoccupation with physiologic measures (refs. [ref.78] and [ref.79]), although the flights of Baker and Able included preflight and postflight performance studies ([ref.80]). Able's behavior was recorded in detail on in-flight film, but none of the behavior was programed or under experimental control.

The first flights in which behavior or performance was explicitly programed were those of Sam and Miss Sam in flights of the Little Joe rocket with the Mercury capsule, launched from Wallops Island in 1959 and 1960 ([ref.81]). The first major space achievement in the behavioral sciences was the successful in-flight measurement of the behavior of the chimpanzee Ham in early 1961, in which the pretrained animal performed throughout the flight. The second achievement along these lines was in 1962 when the chimpanzee Enos made several orbits around Earth and performed continuously on a complex behavioral task. The tasks which the animals performed during these flights have been described in detail by Belleville et al. ([ref.82]), and the results of the in-flight performance have been presented by Henry and Mosely ([ref.83]). These early flights provided much of the technological framework on which current biological experiments on organisms during flights of extended duration are based. Due largely to the efforts of Grunzke (refs. [ref.84] and [ref.85]), the apparatus needed to sustain animals during space flight, such as zero-g watering and feeding devices, are now commonplace ([ref.86]). Advanced systems of programing stimulus presentations and recording responses, developed for Project Mercury, may now be seen in many basic research laboratories throughout the country.

Several other noteworthy advances have been made as an outgrowth of the Mercury animal flights. Immediately before the orbital flight MA-5, in which the chimpanzee Enos was employed, it was unexpectedly found that this 5-year-old animal was hypertensive. Subsequent centrifuge studies showed that its vascular responses exceeded those of a control group. Consideration of the animal's preflight experience led to speculation concerning the origin of this hypertension. An explanation of the high-blood-pressure responses detected in Enos has been pursued by Meehan et al. ([ref.87]). Persistent hypertension has been produced in other laboratory chimpanzees restrained in the same manner as those participating in space flight and exposed to demanding performance tasks, a demonstration which has important implications for prolonged manned space flight and for cardiovascular medicine in general.

Studies more directly concerned with behavior and performance have been extended from those of Project Mercury. These extensions have been in the following directions: (1) the establishment and maintenance of complex behavioral repertoires under conditions of full environmental control, (2) the refinement of behavioral techniques for assessing sensory and motor processes, and (3) the maintenance of sustained performance under conditions of long-term isolation and confinement and preliminary extension of such experimental analysis to man.

Numerous studies with primate subjects, including several at Ames Research Center, have been devoted to developing methods for maintaining optimum performance in environments with limited sources of stimulation. Monkeys, baboons, and chimpanzees, for example, have been isolated for periods of longer than 2 years with no decrement in performance on complicated behavioral tasks ([ref.88]). The behavioral techniques used in these studies are closely related to those employed on human subjects under NASA sponsorship at the University of Maryland ([ref.89]). The essence of these techniques is in the proper programing of environmental stimuli ([ref.90]). It is not sufficient to provide the subject with his physiological requirements for survival, but he must be given the psychological motivation for using these provisions. This statement, of course, is an oversimplification of the problem, but it serves to illustrate the essence of these experimental programs.

Gravity has long been known as one of the major factors influencing various life processes and the orientation of both plants and animals. One of the most challenging problems of space research has been to define this influence more precisely. Related to the effect of gravity on living processes is the problem of the effects of weightlessness. Of particular interest to psychologists are the possible modifications an altered gravitational environment might produce in behavioral patterns basic to the animal's maintenance and survival, such as eating, sensory and discriminative processes, development and maturation, and learning capacity ([ref.91]).

One prominent method of studying gravitational effects is to simulate an increase in gravity by centrifugation. Smith et al. ([ref.92]) and Winget et al. ([ref.93]) have investigated the effects of long-term acceleration on birds, primarily chickens, while Wunder (refs. [ref.94] and [ref.95]) and his coworkers (refs. [ref.96]-[ref.99]) have used fruit flies, mice, rats, hamsters, and turtles. The general findings are that, when animals are subjected to a prolonged period of acceleration of moderate intensity, they exhibit decreased growth, delayed maturation, and an increase in the size of certain muscles and organs, dependent on the species. With regard to the decreased growth effect, the data of these investigators show some exceptions. When the gravitational increase is kept below a certain limit, growth was greater than that of controls in the fruit fly, turtle, mouse, and chicken. The limit below which enhancement of growth was observed varied with the species studied.

The data on food intake do not present a consistent picture. Wunder ([ref.94]) found that food intake in accelerated mice was markedly reduced from that of nonaccelerated control animals. Smith, however, found that in chickens, food intake increased up to 36 percent over controls and has derived an exponential relation between food intake and acceleration. After six generations of selective breeding, Smith has produced a strain of chickens better adapted to prolonged exposure to high g.

A very relevant finding of their research with birds was that exposure to chronic acceleration in some way appears to interfere with habituation to rotatory stimulation. Chickens who were being subjected to chronic acceleration were given repeated rotatory stimulation tests to estimate their labyrinthine sensitivity. This study revealed that centrifuged animals showed a marked reduction in labyrinthine sensitivity. This result appeared to persist after the acceleration was terminated. In animals who developed gait or postural difficulties as a result of acceleration, there was no evidence of a postnystagmus in response to the rotatory stimulation test, which the investigators point out may be evidence of a lesion in the labyrinth or its neural pathways.

Smith has implicated social factors as interfering with acceleration effects. His subjects were typically accelerated four or six to a cage. When groups were mixed midway through the experiment, they exhibited a higher mortality rate and incidence of acceleration symptoms than did groups whose constituency remained unchanged.

At the U.S. Naval School of Aerospace Medicine, numerous studies have been conducted on the effects of slow rotation on the behavior and physiology of humans and animals ([ref.100]). Rotation initially produces decrements in performance, but adaptation to a rotating environment ensues quite rapidly (refs. [ref.101]-[ref.103]). Perceptual distortion, nystagmus, nausea, and other signs of discomfort are common responses to slow rotation. These symptoms are generally reduced with continued exposure (adaptation). Interestingly, however, adaptation is delayed when the subjects are exposed to a fixed reference outside their rotating environment.

At NASA-Ames, rodents have been used in experiments by Weissman and Seldeen to delimit the stimulus effects of rotation. In these experiments the subjects must discriminate between different speeds of rotation in order to obtain food reinforcement. The results thus far provide evidence that these animals are capable of discriminating between the different speeds at which they are being rotated. The range of speeds studied was 0-25 rpm, with tests of discrimination being made at intervals of less than 5 rpm. Experiments such as these will lead to the development of techniques for measuring rotational sensitivity in many species, including man.

The optimum configuration of manned spacecraft will depend, in part, upon biomedical considerations. A voluminous literature now exists on the possible hazards to man of prolonged exposure to zero-g conditions. Should prolonged weightlessness prove to be a serious detriment to health, consideration must be given to design concepts which provide artificial gravity.

No data exist on the minimum gravity requirements necessary to sustain basic biological functions for extended periods. A limit of 0.2 g has been given as the lower level at which man can walk unaided ([ref.104]). It has also been recommended that angular velocity be maintained at the lowest possible level in order to minimize the occurrence of vestibular disturbances. These recommendations are based on human-factor requirements, rather than upon biological considerations, which may significantly modify these values. In recent studies, a technique has been devised which promises to provide reliable criteria for biological acceptability, since it is based on fundamental biological and behavioral principles.

As animals progress up the evolutionary stale, their survival depends less and less upon stereotyped physiological reactions which occur in reflex fashion, in response to environmental stimulation. In higher organisms, survival depends more upon the capacity of organisms to modify their behavior. At the highest levels of functional efficiency, the ultimate form of adaptation is seen—the manipulation of the environment by the organism. Developments in behavioral science now permit us to utilize the adaptive behavior of animals to investigate many problems of biological interest. Recent studies on the self-selection of gravity levels represent a further attempt to exploit the adaptive capacities of animals, in order to provide information relevant to problems of space exploration.

One such project allows animals to select their own gravity environment in an apparatus designed to create g-forces through centrifugal action by rotation at 60 rpm ([ref.105]). The surface of this centrifuge is parabolic, so that the resultant of the centrifugal g and the Earth's gravity is always normal to the surface. When the animal moves away from the center, increasing the radius of rotation, it is exposed to increasing gravity. Motion toward the center reduces the gravity level. By this means, an animal is free to select its own gravity environment.

When the animal moves toward or away from the center, he is moving from one tangential velocity to another. He is therefore acted upon by a third force—due to Coriolis acceleration. The effects of Coriolis forces are a major problem difficult to eliminate in studies such as these, but they must be taken into account in the design of spacecraft which produce artificial gravity by rotation. Motion of the head in any direction not parallel to the centrifugal force vector would result in bizarre stimulation of the semicircular canals and consequent motion sickness. This effect is likely to become even more pronounced if the sensitivity of these organs is increased by prolonged exposure to reduced gravity. Methods such as these are currently being developed for conducting a refined psychophysical analysis of gravity, including studies by Lange and Broderson on the perception of angular, linear, and Coriolis acceleration.

The results of animal studies such as these will be of great value in arriving at a decisive judgment concerning the need for artificial gravity in a manned orbiting space station, or other vehicles designed for long-term occupancy.

To aid in the interpretation of in-flight data, other studies are underway to determine the functions of the vestibular system, as a principal brain center related to orientation in space and to the physiology of posture and movement, as well as with the influences of acceleration, rotation, and weightlessness. Experiments are presently being conducted on monkeys and cats in order to trace these complex neurological connections and to determine their functional organization.

BIOLOGICAL INFORMATION SYSTEMS

The nature of memory has been the subject of considerable speculation in the past. It has long been felt intuitively that retention of information in the central nervous system involves either an alteration of preexisting material or structure, or, alternatively, synthesis of materials not present previously. The cellular site of operational alteration was unknown but, again intuitively, was felt to be closely associated with the synapses. The problems faced by early investigators were great; but nevertheless much information relevant to the question of biological information storage was obtained. With the relatively recent advent of more refined tools and methodologies, there has been rapid progress.

A significant amount of the work which has been conducted in the area of biological information and communication systems is easily classified as "basic research" (refs. [ref.106]-[ref.109]). This discussion will be limited to those aspects closely related to the fields of molecular biology and experimental psychology, which seem to have universal application to all known animal life forms. Studies involving the basic principles of acquisition, processing, storage, and retrieval of information in living systems are emphasized.

Early Work

Early speculations on the operational nature of memory have been based upon relatively little experimental evidence. Charles Darwin observed that domestic rabbits had smaller brains than their wild counterparts, and attributed this to lack of exercise of their intellect, senses, and voluntary movements. Unfortunately, subsequent studies of the brains of men with greatly differing intellectual capability did not substantiate the hypothesis. Idiots sometimes had larger brains than geniuses. Later, an idea proposed by Ramon y Cajal came into favor. Since brain cells did not increase in number after birth, he proposed that memory involved the establishment of new and more extended intercortical connections. Unfortunately, methods were not available to test this hypothesis adequately and it has remained until quite recently in the realm of conjecture.

Another major hypothesis was that there were two or more stages in the information storage process. The final form the information took in the brain was called a brain engram, or memory trace. However, prior to the formation of the engram, a transitory process denoted as "reverberational memory" was postulated to exist for a relatively short time (minutes to hours) (refs. [ref.106] and [ref.107]). This hypothesis was used by Pauling to explain why an elderly chairman of a board could brilliantly summarize a complex 8-hour meeting and yet, after its conclusion and his return to his office, not even remember having attended the meeting. Thus, this individual's reverberational memory functioned well, but advanced years had seriously impaired his brain's ability to form a permanent engram. Similar, although less dramatic, observations in other situations are not uncommon. A wide variety of experiments have been conducted to study this aspect of memory and to relate it to the process whereby the information is transformed to a more stable form (refs. [ref.110]-[ref.112]).

More recently, the concept of a specific biochemical activity during the process of long-term storage of information has gained considerable favor. Initially, neither the site nor the nature of the change was well defined. Quite recent studies by Krech et al. (refs. [ref.113] and [ref.114]), Bennett et al. ([ref.115]), Rosenzweig et al. (refs. [ref.116] and [ref.117]) support the view that alteration of the levels of acetylcholinesterase at cortical synapses play an important role in information storage. These studies will be discussed in a later section. However, these authors do not claim that the changes observed are unambiguously related to the storage of memory. It may well be that the alterations observed are in some way related to this process but are still secondary to some other, more basic, process.

An alternative hypothesis is that the information resides in its ultimate form in some more central structure of the neurone than the synapse. (It has even been postulated that the basic information is stored in nonneuronocortical material.) Perhaps Halstead was the first to postulate the involvement of nucleoprotein in this process ([ref.107]). From the biochemist's point of view, this is an extremely attractive hypothesis. Both proteins and nucleic acids possess sufficient possible permutations of structure to permit storage of a lifetime's accumulation of information in an organ the size of the brain. From the previously known ability of the nucleic acids to code genetic information, they are the prime suspects. However, from the known regulatory ability of nucleic acids in specific protein synthesis, it is possible that the final repository is protein.

Recent Biochemical Studies

Among the foremost investigators of the chemistry and biochemistry of the central nervous system is Holger Hyden at the University of Göteborg, Sweden. He and others (refs. [ref.118]-[ref.120]) have for many years performed elegant microanalytical studies of single nerve cells. The evidence which Hyden has obtained is consistent with the hypothesis that the initial electrical reverberations in the brain induce a change in the molecular structure of the ribonucleic acid (RNA) of the neurones which, in turn, leads to a subsequent deposition of specific proteins. It is well known from other investigations that a major role of RNA in any type of cell is to specify and mediate synthesis of the protein enzymes of the cells. Thus, in this hypothesis, it is only necessary to postulate the modification of brain RNA by the activities associated with reverberational memory. Particularly pertinent to this hypothesis are observations that—

1. Large nerve cells have a very high rate of metabolism of RNA and proteins, and, of the somatic cells, are the largest producers of RNA.
2. Vestibular stimulation by passive means leads to an increase in the RNA content of the Deiters nerve cells of rabbits ([ref.121]). The protein content of these cells is also increased.
3. Changes in the RNA composition of neurones and glia of the brainstem occur during a learning situation. Animals were trained over a period of 4 to 5 days to climb a steeply inclined wire to obtain food. The big nerve cells and the glia of their lateral vestibular apparatus were analyzed, since the Deiters neurones present in this structure are directly connected to the middle ear. The amount of RNA was found to be increased in the nerve cells; and, more significantly, the adenine-to-uracil ratio of both the nuclear RNA of nerve cells and glia cells became significantly increased ([ref.119]). A variety of control experiments were conducted. Although there was an increase in RNA content of these cells in animals exposed to passive stimulation, there was no change in the ratio of adenine to uracil. Nerve cells from the reticular formation, another portion of the brain, had only an increased content of RNA with no base-ratio change. Animals subjected to a stress experiment involving the vestibular nucleus showed only an increase in content of RNA. Littermates living in cages on the same diet as learning animals showed no change in content of RNA. Thus, it would appear that the change in the base ratio of the RNA synthesized is not due to increased neurone function per se, but is more directly related to the learning process. The fact that this was nuclear RNA implies that it was immediately related to chromosomal DNA.
4. Neuronal RNA with changed cytosine-guanine ratios synthesized during a short period of induced protein synthesis could be blocked by actinomycin D. It was concluded, therefore, that the RNA was immediately DNA dependent and directly related to the genetic apparatus.

Rats which were normally right handed were forced to modify their handedness in order to obtain food. The RNA of nerve cells in that part of the cortex, whose destruction destroys the ability to transfer handedness, was analyzed. A significant increase in RNA of nerve cells of the fifth to sixth cortical layers on the right side of the brain was observed. The corresponding nerve cells on the opposite side of the same brain served as controls. There was an increase in RNA and a significant increase in the purine bases relative to the pyrimidine bases in the learning side of the cortex. When the animals were not forced to learn a new procedure, only an increase of RNA was observed, with no change in base ratio.

Frank Morrell, head of the Neurology Department at Stanford Medical School, has also been active in this field during the past 6 years. He has found that if a primary epileptic lesion is induced on one side of the cortex, a secondary mirror lesion eventually develops in the contralateral homologous cortex. This secondary lesion, which showed self-sustaining epileptiform discharge, could be isolated, whereupon the epileptiform discharge disappeared. This was interpreted as learned behavior of the secondary lesion. From changes in the staining properties of the secondary lesion, Morrell concluded that changes in RNA had occurred in the cell. Changes in the composition of the RNA could not be shown by these techniques.

At the University of California at Berkeley, Drs. Rosenzweig, Bennett, and Krech have conducted extensive studies related to this topic. These investigators have directed their efforts toward demonstrating alterations in the cerebral cortex of animals exposed to continuing learning situations or continuously deprived of sensory stimulation. In a recent publication ([ref.116]), which also summarizes a considerable amount of previous work, they report studies which demonstrate the following:

1. Rats given enriched experience develop, in comparison with their restricted littermates, greater weight and thickness of cortical tissue and an associated proportional increase in total acetylcholinesterase activity of the cortex.
2. The gain in weight of cortical tissue is relatively larger than the increase in enzymatic activity. Acetylcholinesterase activity increases in other portions of the brain even though tissue weight decreases.
3. The changes appear in a variety of lines of rats, although differing in amount between strains.
4. The changes are observed in both the young and adult animals.

The previous studies were comparisons between experience-enriched animals and animals maintained in isolation. Animals which were housed in colonies, but given no special treatment, showed intermediate effects in those situations studied.

The Berkeley group emphasized that the finding of changes in the brain subsequent to experience does not prove that the changes have anything to do with memory storage, but do establish the fact that the brain can respond to environmental pressure. However, the results are compatible with the hypothesis that long-term memory storage involves the formation of new somatic connections among neurones. Calculations of the amount of additional material required to permit this to exist are compatible with the increases observed.

A number of investigators have studied the effects of antimetabolites and drugs on the learning process. Since their specific metabolic effects are known in other tissues, the rationale is that if these materials do interfere with memory, then specific types of metabolic activities may be implicated in the deposition of the engram.

One of the initial studies of this type was conducted by Dingman and Sporn ([ref.122]), presently at the National Institute of Mental Health. They showed that 8-azaguanine, a purine antagonist, injected intra-cisternally was incorporated into the RNA of the brains of rats. Associated with this incorporation was an impairment of the maze-learning ability of the animals. These findings have been confirmed.

Flexner and his associates injected puromycin, an inhibitor of protein synthesis, into the brains of mice, which were then trained to perform in a maze. Losses of short-term or long-term memory were obtained, depending upon the site of the injection. The results indicate that the hippocampal region is the site of recent memory.

The hippocampal region is of interest in connection with memory processes for a number of other reasons. Adey et al. ([ref.123]) and his group observed a transient fall in electrical impedance in this region when cats learned to perform in a T-maze in response to a visual cue. It was supposed that the electrodes were situated within glial cells of the dendritic zone of the hippocampal pyramidal cell layer. Extinction of the learned habit abolished the briefly evoked impedance changes, which subsequently reappeared with retraining.

A number of other studies more or less indirectly implicate RNA in the learning processes. For instance, in retinal cells of rabbits raised in darkness, there was virtually no ribonucleoprotein as compared with normal amounts in the cells of animals raised in light ([ref.124]). Further, maintenance of normal electrical activity of isolated perfused cat brains is highly dependent upon the presence of the ribonucleic acid precursors, uridine and cytidine, in the perfusate ([ref.125]), and severe derangements occur if any of a variety of pyrimidine antagonists are added ([ref.126]). Brief electrical stimulation of cat cortical tissue causes an increase in nucleic acid cytidine and adenine, thus indicating a synthesis of altered polynucleotides. Finally, injections of RNA in animals have shown interesting effects. When given at a dose of 116 mg/kg daily for 1 month, rats showed an enhanced response and greater resistance to extinction in a shock-motivated behavioral response. It has been shown by another group that injections of RNA enhance the ability of young animals to learn various tasks.

Planaria have been used in a variety of studies which seem to bear on the problem of memory. Quite recent evidence by Bennett, Calvin, and their associates has cast somewhat of a pall over the studies; nevertheless, the work may have some validity. Interest in the use of flatworms, particularly planaria, for study of memory began with a demonstration by McConnell that these simple animals could undergo conditioning ([ref.127]). Subsequently, it was found that some conditioning was retained when the animal was transected and allowed to regenerate. The retention of training was found in both new animals, although the very simple brain, really only two ganglia, was in the head section ([ref.128]).

Apparently, some diffusely distributed component of the animal was responsible for retention of learning. Evidence has accumulated to indicate that this material is RNA. Among this evidence is the following:

1. The two halves of a trained planaria were allowed to regenerate in a solution containing RNA-destroying enzymes. Whereas the head ends retained some training, no retention was observed in the animals derived from the tail end ([ref.129]).
2. When pieces of trained planaria were fed to untrained animals, the untrained cannibal required a shorter time to become trained to a criterion. It would appear that the digestive system of planaria is so simple that the material responsible for the transfer of the information was not broken down.
3. When RNA, obtained from trained planaria, is injected into the digestive tract of untrained animals, there is a transfer of information.

NEUROPHYSIOLOGY[²]

Neurophysiological studies concern the functions of the nervous system—in particular the central nervous system (CNS)—under normal, simulated, and actual flight conditions. Of paramount importance is the maintenance of equilibrium and orientation in three-dimensional space. The ability of man and his close relatives among the vertebrates to maintain these functions depends on an integrated sensory input from the vestibular organ; the eyes; the interoceptors of the muscles, tendons, joints, and viscera; and the exteroceptors of the skin.

Certain parameters of the environmental and space-flight conditions drastically affect man's ability to maintain equilibrium and spatial orientation. Centrifugal forces modify or reverse the directional vector of gravity. Linear acceleration may increase enormously, as may angular stimulation. The sensory organs listed above are unreliable under such conditions. The very organ which is designed specifically to furnish information on spatial orientation may malfunction in man while he is in flight. Thus, with respect to sensory orientation, these labyrinthine organs are by no means precision instruments.

The use of classical histological methods and the observation of equilibrium disturbances resulting from operative interference with the internal ear have in the past been the two principal sources of knowledge concerning the structure and function of the labyrinth, but the answers given to various questions vary considerably in their value. The development of electrophysiological techniques and the refinement in recent years of the ultrastructural analysis by means of the electron microscope may allow more precise experimental studies of the correlation of function and structure.

Before considering vestibular impulses in their bulbar and descending spinal pathways, a recent study concerning the generation of impulses in the labyrinth must be mentioned. Von Bekesy's finding ([ref.131]) of the direct current potentials in the cochlea aroused speculation about the existence of similar labyrinthine potentials. Such dc potentials were also detected in the semicircular canal of the guinea pig by Trincker ([ref.132]), who measured the potential changes in the endolymph, surface of the cupula, or side of the crista during cupular deflection. It seems likely, however, that the effects do not represent the physicochemical changes in the cupula but the electrical potentials in the nerve and nerve endings of the crista. Attempts at differentiating these effects have failed so far. Great expectations are brought by the advances of microchemistry, microphysiology, and physical chemistry with regard to the excitatory processes, the generation of the nerve impulse. Quite apart from a need to understand vestibular nerve discharges and patterns more adequately in such terms, the analysis of the vestibular system has in the past revealed general biological principles which were not readily discernible through the examination of other tissues ([ref.133]).

The neural connections of the vestibular organ consist of numerous chains of neurons, reciprocally linked in many ways and having their synapses in various anatomical nuclei. All the chains work in intimate collaboration, and the final pattern of reflex responses is attributable largely to the highly complex integrating activity of the center. The labyrinthine function is automatic, carried out in a reflex fashion: in other words, mostly below the level of consciousness. The brain centers through which the labyrinth elicits the various appropriate muscular reactions of the head, body, limbs, and eyes—the righting, the postural, and the ocular reflexes—represent an intricate mechanism. Before we can hope for a satisfactory understanding of their functional organization, we will have to know their anatomy in more detail. Thus, we are confronted with a fruitful field for the exploration of basic mechanisms of neuronal activity. Major advances dining the last years have provided us with new information about the neuroanatomy of the vestibular system (refs. [ref.134]-[ref.137]).

Vestibular impulses entering the brainstem ascend and descend the neuroaxis and cross the midline. It was previously believed that the vestibular apparatus had only subcortical projections. Recently, however, it has been established by means of electrophysiological methods that the organ is represented by a projection area in the cerebral cortex of some animals (refs. [ref.138]-[ref.141]). The use of brief electrical stimulation of the vestibular nerve in order to elicit a cortical response has been of great value for the mapping of these areas.

Among a great variety of sensory receptors, the vestibular ones are capable of evoking the most widespread somatovisceral effects throughout the body. Moreover, vestibular effects seem to be imperious and less dependent upon the state of readiness of the nervous system. As a consequence of the extensive distribution of vestibular effects, there are many opportunities for central integration. Proprioceptive and vestibular systems are both known to be active in posture and locomotion; streams of impulses arising from the receptors in each of these systems must converge to influence the activity of the final common path. The state of the motor centers of the spinal cord, as affected by vestibular stimulation, has been tested by dorsal root and other sensory input interventions. These experiments have provided us with insight into the mechanisms concerned with the vestibular control of spinal reflexes (refs. [ref.142]-[ref.146]).

It has long been known that the vestibular apparatus is essential for the development of motion sickness. Commonplace subjective experience of nausea relates to visceral changes mediated through autonomic efferent pathways and may ultimately involve rhythmic somatic nerve discharges to skeletal muscles responsible for retching and vomiting. However, very little is known about the central nervous mechanisms responsible for elaboration of the whole syndrome. Since the maintenance of vestibular bombardment for some length of time seems essential for the development of motion sickness, one would presume this to be an instance of slow temporal summation. Experimental findings demonstrate a powerful effect of temporal summation upon somatic motor outflow during vestibular stimulation ([ref.147]), and not upon parasympathetic outflow.

The practical implication of these studies is closely related to physiological effects of weightlessness. Based on experimental evidence from short weightless periods obtained in aircraft, it was concluded that "when the exposure becomes longer, there may develop minor physiologic disturbances which, if cumulative or irritating, may cause or enhance psychiatric symptoms" ([ref.148]). Although the zero-g condition, per se, does not cause spatial disorientation if visual cues are provided, the astronauts reported a temporary loss of orientation during the orbital flight while they were engaged in activities which diverted their attention. However, no disturbing sensory inputs were observed during the weightless period. Violent head maneuvers within the limited mobility of the helmet were performed in every direction without illusions or vertigo. The subjective sensations of "tumbling forward" after sustainer engine cutoff reported by the Mercury astronauts, and Titov's motion sickness attacks, which were particularly dismaying during head movements, were well within the entire range of psychosomatic experiences already obtained during aerodynamic trajectories ([ref.149]). Interestingly enough it now appears that the otolithic output in mammals including man is the differential of linear acceleration, and therefore unaffected by zero g.

Of interest in this connection are the problems which may be encountered during and following long-term exposure to weightlessness. Although there is no evidence of adverse effects on operative behavior, the possibility of biological disturbances on a cellular or subcellular level, which may cause a deterioration of the somatic basis, has been repeatedly stressed. Whether effects of this sort will occur or whether the organism will be able to adapt is still an open question. Since motion sensitivity based on vestibular stimulation differs widely among individuals, the selection of astronauts may solve the problem of zero-g vestibular disturbance. Reports from the MA-8 (Sigma 7) and Vostok III and IV flights seem to support this assumption. Moreover, experiments are being made in the slow rotation room at the Naval School of Aviation Medicine to study the Coriolis effects which arise when "artificial gravity" is produced by angular acceleration. Since man can adapt to wave motion on shipboard within a few days, a similar process may be expected to occur in the case of long-term weightlessness ([ref.150]).

[chapter 5]

Molecular Biology and Bioinstrumentation

To support biological investigations in space and to accumulate baseline data needed for manned space 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 space available in such experiments, many compounds are not produced in sufficient quantity 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 coarse 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^-20 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.

Figure 2.—Equilibrium diagram for the system C-H-O.

Jukes and associates ([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 Institutes 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 Institutes 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 class of activities in molecular biology is the examination of passive 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 associated with nerve membranes is a significant part of the answer. Because the space 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 Institute. 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 photosynthetic organisms, chemosynthetic bacteria offer distinct advantages for the study of energy assimilation. 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 assimilation of C¹⁴ from HC¹⁴O₃—. These observations explain the bacteria's energy source without the classical 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 analysis of the DPNH "breeder cycle" appears to be attainable by measurements of redox potentials and calorimetric determinations of heats of reaction.