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Microgravity Research Opportunities for the 1990s: Chapter 5 Microgravity Research Opportunities for the 1990s PART II—SCIENTIFIC ISSUES 5 Biological Sciences and Biotechnology INTRODUCTION Progress in biology research is often a consequence of the investigator's skill in maintaining experimental control through the use of diverse chemical, physical, genetic, and immunological techniques. Major advances in molecular biology and biotechnology are critically dependent on refinements of existing techniques and the development of new and better ones. Microgravity research in the biological sciences can help to further these advances. It can also provide fundamental knowledge important to NASA's overall goals. To do so, REPORT MENU however, a systematic program is needed to identify and explore those cellular and NOTICE biomolecular processes, mechanisms, structures, and assemblies that are affected by MEMBERSHIP transfer to the microgravity environment. For the several areas of biology and PREFACE EXECUTIVE SUMMARY biotechnology, such as cell culture, cell fusion, electrophoretic separation, and protein PART I crystal growth, in which the effects of microgravity have been demonstrated, further CHAPTER 1 research is needed to identify and explore mechanisms by which gravitational effects are CHAPTER 2 realized. New methods and techniques to take advantage of the potential of microgravity PART II applications need to be developed. CHAPTER 3 CHAPTER 4 CHAPTER 5 CHAPTER 6 CHAPTER 7 JUSTIFICATION FOR MICROGRAVITY RESEARCH PART III CHAPTER 8 Gravity affects biological systems through its influence on the transfer of mass and APPENDIX A heat, particularly in the area of fluid dynamics and transport, as well as its less well APPENDIX B understood impact on cell structure and function. The basis of modern research in the biological sciences is the study of molecules, molecular assemblies, organelles, cells, and cell assemblies in controlled fluid and chemical environments. The impetus for microgravity research is that it may lead to new knowledge about biological systems, to improvements in current experimental techniques, and to the development of new experimental approaches to biological problems. file:///C|/SSB_old_web/mgoppch5.htm (1 of 14) [6/18/2004 11:17:11 AM]

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Microgravity Research Opportunities for the 1990s: Chapter 5 The NASA research program in the biological sciences and biotechnology has focused on three primary areas of biological interest: (1) separation physics aimed at providing improved resolution and sensitivity in preparative and bioanalytical techniques; (2) cell biology, cell function, and cell-cell interactions; and (3) physical chemistry of biological macromolecules and their interactions, including studies of protein crystal growth directed at supporting crystallographic structure determinations. Brief discussions of microgravity research in each of these areas provide an introduction to the work that has been done and to some of the realized or anticipated scientific and technological benefits. Separation physics.1 Progress in the biological sciences and biotechnology has been limited to some extent by the failure of fluid-based techniques of current separation and analytical methods to achieve the necessary resolution, a failure exacerbated by the effects of gravity. Two such effects are density-dependent thermal convection and sedimentation, both of which perturb processes used to separate and purify proteins, fractionate cell components and organelles, or separate mixtures of naturally or genetically engineered cells. Cell biology.2 Modern research in the biological sciences and biotechnology relies on the manipulation of cells of living organisms. In the case of biotechnology, the purpose of these manipulations may be to produce useful molecules-both naturally occurring and of artificial origin-in useful quantities; to develop new organisms or new biological molecules for specific uses; or to improve yields of plant and animal products through genetic alteration. Recombinant techniques, for example, make it possible to produce natural or artificially mutated versions of proteins exhibiting a wide range of activities and uses, scientific and medical, in heretofore extraordinary quantities. The techniques essential to these manipulations are applied in aqueous environments and are subject to fluid dynamics and transport processes. Gravitational effects may have important consequences-experimental in the case of scientific research and financial in the case of biotechnological production. Examples include fermentation processes; compartmental targeting of expressed products within the cell; and the ultimate purity, structural integrity, and activity of a protein product. Particle sedimentation under the influence of gravity, for example, can interfere with aggregation processes such as those mediating cell-cell interactions, cell fusion, cell agglutination, and cellular interactions with substrates. Molecular structure.3 A full understanding of the functions of biological macromolecules, and of the chemical and physical effects that they organize and manage to achieve these functions, is not possible without detailed knowledge of their three- dimensional architectures. Nor is it possible to engineer new proteins, whether for medical uses or as complex biomaterials, without an ability to relate molecular structure and function. Protein crystallography, currently the principal method for determining the structure of complex biological molecules, requires relatively large, well-ordered single crystals of useful morphology. Crystals with these qualities may be difficult to produce for a variety of reasons, some of which may be influenced by gravity, through density-driven convection and sedimentation. Protein crystal growth experiments conducted aboard the shuttle have provided persuasive evidence that improvements can, in fact, be realized for a variety of protein samples. Another justification for microgravity research concerns the importance of biological research to technological developments supporting crewed space missions. A number of ambitious goals for human exploration of the solar system have been advocated. These include a permanently inhabited base on the Moon and a program of crewed missions to Mars. file:///C|/SSB_old_web/mgoppch5.htm (2 of 14) [6/18/2004 11:17:11 AM]

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Microgravity Research Opportunities for the 1990s: Chapter 5 Experience from a variety of extended missions in Earth orbit has shown that prolonged exposure to microgravity can have profound effects on human physiology and that return to terrestrial gravity may require long periods of readjustment and reconditioning. Research in the life sciences has begun to pinpoint some of the physiological systems most affected by habituation to reduced-gravity conditions, but little is known about the effects of microgravity on the underlying cellular processes. Additional biological science research is required in support of life sciences and human physiology goals if we are to meet the full range of life-support challenges presented by human space exploration. These challenges range from the development of methods for ameliorating the physiological effects of microgravity and the return to terrestrial gravity to the development of technologies for meeting nutritional, respiratory, and waste disposal needs. This research should explore microgravity effects on cells and cell processes, particularly those involving human physiological systems at risk, as well as survey microgravity effects across a wide range of model systems and organisms. A final justification for microgravity research in the biological sciences is the anticipation that new knowledge will be accumulated by the optimized production of small amounts of microgravity-derived biomaterials such as purified proteins or protein crystals. Such precious specimens may induce the research community to conduct experiments in novel and previously inaccessible areas of inquiry. Microgravity research may also facilitate otherwise labor-intensive, and therefore seldom-attempted, efforts on Earth and make precious reagents and biomaterials accessible to a wider community. It is possible that knowledge gained in microgravity experiments will permit improvements in terrestrial strategies in such areas as bioseparations and materials processing. It is also possible that methods and processes may be discovered that are possible only in space. FUNDAMENTAL RESEARCH AREAS NASA has for some time nurtured a program of flight experiments to delineate specific advantages and limitations of microgravity research with regard to biological sciences. Experiments were designed to produce results that were anticipated from model experiments or analyses but could not be tested in terrestrial laboratories. Because of broad interest in these studies, scientists in a wide range of disciplines, in both academia and industry, were encouraged to participate and judge the potential benefits of biology and biotechnology research in microgravity. Typical of innovative science, preliminary experiments in electrophoresis, phase partitioning, protein crystal growth, and cell culture yielded unexpected results. In some cases, additional or unanticipated advantages of a microgravity environment appeared; in other cases, secondary effects masked in terrestrial experiments became dominant. Research areas in the biological sciences that have demonstrated potential benefit from microgravity experimentation are presented below. Investigations of Mechanisms of Macromolecular Interactions at the Subcellular Level Biotechnology is for the most part applied molecular biology in the sense that molecular events occurring within the cell, or among groups of cells, are altered or manipulated in some way to achieve a desired end. The methods involved may be based file:///C|/SSB_old_web/mgoppch5.htm (3 of 14) [6/18/2004 11:17:11 AM]

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Microgravity Research Opportunities for the 1990s: Chapter 5 on recombinant DNA technology, cell fusion, or a number of other cellular or subcellular approaches. The objectives may be to produce altered cell types in culture, specific immunoglobulins, drugs, commercial enzymes, proteins, other pharmacological agents, and even whole organisms having improved properties. In order to apply molecular biology at the cellular and subcellular level, where microgravity is intended as a probe or a tool, one must know with some assurance exactly which processes and mechanisms are, in fact, affected by gravity. At present, no adequate knowledge base exists. The underlying premise for performing biology and biotechnology experiments in space, and for the development of molecular biology tools based on the absence of gravity, is that the microgravity environment must have direct effects on cells and subcellular events. A similar premise underlies research to understand the short- and long-term physiological effects of microgravity on humans and to develop methods and procedures to minimize or eliminate any negative impacts of the effect of microgravity. The following questions must be answered: What are the effects of microgravity on cells and subcellular events, what are their magnitudes, and what are the consequences? Even if the consequences are insignificant, the answers are needed in order to understand these systems fully. A concerted effort should be made to determine convincingly whether microgravity has an observable effect on the growth, development, and structure of living cells. Observations and biochemical assays should be conducted on organisms exposed for various periods of time to a microgravity environment to determine if there are any gross changes in character and, if so, to what general categories of events these effects should be ascribed. Following a careful cataloging of microgravity effects on cells, studies should be extended to more complex cell-cell systems and the study of longer-term trends. These might include, depending on the earlier results, investigations of cell-cell and cell-matrix interactions and how they are affected by microgravity, as well as the transport of materials between cells. Mixed cell systems should be examined to determine if intercellular communication is affected. Other areas for investigation include the secretory and transport properties of cultured endocrine cells, the development of nerve cells, plant cell-wall structural changes, chemotaxis, virus propagation, adhesion processes, and the assembly of organized macromolecular aggregations such as viruses, membranes, and cell organelles. In the long term, processes such as multigenerational changes, embryogenesis, evolution, and complex cell interactions approaching the organism level must be addressed. This level of sophistication would also include viral, fungal, and bacterial infection of cells and the role that microgravity plays in alterations of these interactions. Growth of Biological Macromolecular Crystals Macromolecular crystallography has proved to be a powerful tool for basic research in biochemistry and molecular biology. As a result, it has attracted the interest and support of the pharmaceutical, chemical, and biotechnology industries, particularly for use in structure-based drug design and protein engineering. Crystals of an appropriate size and quality are a prerequisite for determination of the three-dimensional structures of proteins file:///C|/SSB_old_web/mgoppch5.htm (4 of 14) [6/18/2004 11:17:11 AM]

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Microgravity Research Opportunities for the 1990s: Chapter 5 and nucleic acids. The major obstacle in the application of crystallographic methods to proteins and in the application of protein structure in structure-based drug design is growing suitable crystals. Initial research in crystal growth under microgravity conditions concentrated on the preparation of metals, alloys, and electronic materials and the identification of model systems. Results of these experiments suggested that growth in microgravity might also have beneficial effects on the preparation of protein crystals. Rationales included the elimination of sedimentation, which might create more nearly isotropic or uniform growth conditions and thereby produce improvements in size and shape, and the elimination of density-driven convection, which might create conditions closer to diffusion-limited growth and thereby improve crystalline order and extend x-ray scattering limits to higher resolution. An extensive program of experiments in protein crystal growth in microgravity has been conducted in recent years to test these possibilities. This program has been international in scope and has employed a variety of orbital vehicles, but the majority of these experiments have been sponsored by NASA and conducted aboard the space shuttle. In recent years, successful macromolecular crystallization experiments have been performed by U.S. investigators using a number of instruments of U.S. and European design. In early Vapor Diffusion Apparatus (VDA) experiments, crystals of g-interferon, porcine elastase, and isocitrate lyase grew larger, displayed more uniform morphologies, and yielded diffraction data of higher resolution than equivalent crystals grown on Earth.4 Similar results were obtained for canavalin5 and positive results continue to accumulate from this apparatus.6 On USML-1, experiments in the glovebox, using a modified vapor diffusion technique, yielded crystals of malic enzyme of substantially enhanced properties.7,8 In the Cryostat device provided by the German Space Agency DARA, crystals were obtained by liquid-liquid diffusion of satellite tobacco mosaic virus (STMV) that were larger and diffracted to a higher resolution than the best obtained in the laboratory. Data from these crystals allowed structure determination of STMV at 1.8-Å resolution, the highest ever achieved for any virus crystal.9-11 On IML-1 in the Cryostat12 and on IML-2 in the European Space Agency (ESA) Advanced Protein Crystallization Facility (APCF), which also supports liquid-liquid diffusion experiments, a number of morphological alterations to crystals of canavalin and another larger virus, turnip yellow mosaic virus (TYMV), were clearly demonstrated. A third technique, temperature-induced batch crystallization, has recently produced larger, higher-resolution crystals of insulin.13 A group of U.S. investigators also carried out protein crystal growth experiments on the Russian Mir, using a number of different crystallization devices. In agreement with the results cited above, they reported that experiments on 5 of 21 proteins produced results superior to those obtained on Earth.14,15 An interesting aspect of the results seen so far is that when the same protein is crystallized by a variety of different techniques in microgravity, a range of crystalline samples may be expected,16 pointing up the need for multiple flight experiments and optimization. These experiments have provided persuasive evidence that growth in microgravity can produce protein crystals of larger size, better shape, and higher quality than have been obtained on Earth. They also show that benefits from microgravity crystal growth can be crucial to success in protein structure determination.17,18 (See Figure 5.1 and Plates 5.1 and 5.2.) file:///C|/SSB_old_web/mgoppch5.htm (5 of 14) [6/18/2004 11:17:11 AM]

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Microgravity Research Opportunities for the 1990s: Chapter 5 FIGURE 5.1 Crystal of malic enzyme form parasitic nematode Ascaris suum grown in interactive glovebox experiments aboard the United States Microgravity Laboratory-1 flight in 1992. While this crystal (0.20 to 0.25 mm on an edge) is about 20 to 25% of the volume of good crystals grown on Earth, resolution limits from this crystal exceed by about 0.5 Å the best data ever collected from crystals grown on Earth. On the other hand, although protein crystal growth experiments in microgravity have yielded a variety of encouraging and successful results, they have not shown that protein crystals uniformly display improved properties when grown in microgravity. A reason for this result might be the mismatch between protein and technique. Experiments testing a variety of proteins in a particular apparatus have been illuminating. These experiments could be expanded to include a broader range of proteins, such as membrane proteins, receptor- ligand complexes, glycoproteins, and other problematic macromolecules, to understand better the limits of a given technique and what might be done to overcome them. It is also important, however, that a broader range of techniques be explored in repetitive microgravity experiments. More experiments that use liquid-liquid diffusion or batch methods, for example, will provide a better understanding of the differences among techniques and the results expected to be produced in microgravity. Another reason that not all protein crystals grown in microgravity have shown improved properties might be that crystallization conditions have not been optimized for growth in a microgravity environment. Past results have shown that, more often than not, optimum conditions for growth on Earth are not optimum for growth in microgravity and that the periods of time required to allow crystal growth to go to completion are too short for optimal growth in microgravity. The glovebox experiments in protein crystal growth on USML-1 were the first to offer opportunities for iterative experiments in microgravity with an experienced specialist to interpret results and design subsequent experiments. The results of these experiments indicated that iteration can increase the production of crystals of high quality in microgravity.19,20 A goal of future experiments should be to provide a better understanding of the file:///C|/SSB_old_web/mgoppch5.htm (6 of 14) [6/18/2004 11:17:11 AM]

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Microgravity Research Opportunities for the 1990s: Chapter 5 fundamental forces and mechanisms governing protein crystal growth on Earth and in microgravity. While it may continue to be important to survey the crystal growth performance of a wide variety of proteins and techniques in space, it is becoming increasingly necessary as well to observe, measure, and manipulate crystal growth processes so that we understand what they entail. A systematic evaluation of which macromolecules derive the greatest benefits, and from which techniques, requires repetitive experiments with a single method to optimize growth parameters for specific proteins, as well as an expansion of experiments to a wider variety of proteins and methods. Analyses of chemical and physical characteristics of macromolecules that impact their organization into crystals merit support. Although a diverse mix of macromolecules might be desirable, even essential, in future crystal growth experiments, a systematic approach to these experiments is the only means of delineating the dominant factors involved in protein crystal growth in microgravity. Automation and remote control of protein crystal growth will likely be needed for future microgravity experiments. Automated systems that permit dynamic monitoring and control of key variables such as temperature, protein concentration, ionic strength, pH, and precipitant concentration will be useful for both ground-based and space experiments that are directed at better understanding of protein crystal growth processes. Nevertheless, at least for the near term, NASA may want to consider support for additional missions such as USML-1 in which an experienced observer is in control of iterative protein crystal growth experiments. In summary, the success of initial experiments suggests that further research is needed and an expanded program of protein crystal growth experiments deserves support. It would be helpful if this research were to focus somewhat more sharply on gaining a clearer understanding of the physical and chemical phenomena involved in the nucleation and growth of protein crystals, in addition to developing an expanded range of techniques and methods to secure the benefits of microgravity growth for a broader range of macromolecular crystals. This implies increased basic research in protein crystal growth on Earth not only to expand understanding of the fundamental scientific principles, but also to support and develop the cadre of scientists needed to design and conduct the fundamental crystal growth experiments in microgravity. Separation and Purification of Biological Macromolecules and Assemblies Biological systems typically contain a large variety of proteins and nucleic acid molecules that have widely differing properties and that are often combined in macromolecular complexes, oligomers, and unique molecular assemblies such as viruses and ribosomes. Purification of macromolecules, complexes, or even cells is often a necessary first step to more detailed biological characterization and biotechnology applications. This is true whether isolation is directly from natural sources, such as blood sera, or from manufactured mixtures, such as culture media, fermentation products, hybridoma cultures, or synthetic peptide solutions. Furthermore, to exploit the potential of genetic engineering and engineered cells, it is necessary to purify products from the heterogeneous mixtures in which they are synthesized. Associated separation and purification challenges are often major obstacles to advanced biochemical studies and biotechnological applications. file:///C|/SSB_old_web/mgoppch5.htm (7 of 14) [6/18/2004 11:17:11 AM]

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Microgravity Research Opportunities for the 1990s: Chapter 5 Analytical separations commonly use electrophoresis in water-based gels to good advantage; gels of this type are generally recognized as providing the highest resolution for analytical separations of proteins and analyses of nucleic acid sequences. Density-driven thermal convection and sedimentation are well-known phenomena limiting the resolution of electrokinetic separations on Earth, and suppression of these phenomena in microgravity environments might be expected to eliminate the need for gels and make it possible to achieve the ultimate resolving power of electrophoretic experiments in free fluids. The results of electrophoresis experiments carried out in microgravity with well-characterized molecules and particles have shown that buoyancy-driven phenomena are diminished, but have revealed new phenomena at work that may affect purification processes.21 One of the significant findings has been the role of electrohydrodynamic effects in electrophoresis, where in certain experiments in microgravity they appear to significantly disrupt resolution.22 More fundamental research, much of it in the fields of fluid dynamics and transport phenomena, is needed if investigators are to learn how to moderate or eliminate disadvantages due to electrohydrodynamic effects in microgravity. Further, while it is important to use microgravity to minimize negative effects caused by gravitational acceleration, it would also be useful to harness the unique features of microgravity fluid processes, such as virtually unlimited capillary rise or interfacial tension effects, for the enhancement of separation technology. Separation processes also depend, in some cases, on subtle differences in the interactions between a molecule of interest and different solvents. Investigations of partitioning in aqueous polymer two-phase systems on recent shuttle missions have demonstrated the dependence of separation efficiency on several variables, including volume fractions of the component phases; container geometry; physical properties of the component phases, such as interfacial tension and relative phase viscosities; and surface tension interactions between aqueous phases and container walls.23 Some of these variables can be minimized; interactions with container walls, for example, may be reduced by special coatings. The importance of improvements in resolution in chromatographic and electrophoretic separations to progress in the biochemical and biological sciences should not be underestimated. For example, biological sciences and biotechnology will be influenced significantly over the next decade by the mapping of the human genome-an effort that will require the isolation and purification of DNA fragments on a scale not encountered previously. For other applications, resolution requirements for protein purification have become increasingly stringent as the role of impurities in recombinant products has become more evident. A continued program of microgravity research in the separation and purification of biological macromolecules should be supported where it can contribute to improved techniques and improved results in terrestrial and microgravity environments. Cell Culture, Growth, and Differentiation The culture and manipulation of living cells, particularly the more fragile mammalian and plant cells, can be scientifically and technologically rewarding, but it can also be technically challenging. Some of these cell types, which naturally occur as part of specialized tissues and organs, are dependent for their survival and proper function on the environment provided by other cells and tissues. While the unique functions of differentiated cells make them especially valuable for research or for the generation of specific products, file:///C|/SSB_old_web/mgoppch5.htm (8 of 14) [6/18/2004 11:17:11 AM]

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Microgravity Research Opportunities for the 1990s: Chapter 5 such cells, as a direct consequence of their interdependence, frequently exhibit sensitivity to shear, have complex metabolic requirements, and lose cell functions when cultured in artificial environments. Such sensitivities can complicate the utilization of these cells to generate desired products or responses. The culture of cells in microgravity offers a number of potential benefits: sedimentation and density-driven convection are virtually absent, and changes in physical and chemical properties of the fluids constituting and nourishing the culture media can be exploited to benefit the culture system. The resulting environment may, therefore, be relatively quiescent and free of the steep velocity gradients in fluids and the sedimentation effects characteristic of ground-based cell culture systems. Improvement of the culture of shear-sensitive mammalian and plant cells would permit the manipulation of cells in ways that are difficult or impossible to accomplish on Earth. Such effects could be particularly important in processes that are dependent on cellular interactions, such as cell fusion or the assembly of three-dimensional multicellular systems. Prototype culture systems have been developed that are compatible with microgravity operations and that simulate some of the fluid effects found in microgravity. Mammalian cell cultures have been used to test the feasibility and potential for extended microgravity research. Preliminary results indicate that culture devices developed with NASA support provide a low fluid shear environment with minimal sedimentation.24 Delicate mammalian cells have been cultured to very high densities in these instruments, and unique associations of cells into tissue-like aggregates have been observed. Limitations on the development of multicellular structures due to gravitationally induced disturbances have also been observed. Microgravity will enable maintenance of a culture environment that supplies minimum shear and maximum freedom for three-dimensional cellular association. Cell Fusion and Membrane Assembly The fusion of cells to produce viable hybrids-a low-frequency event with current terrestrial technologies-is another area that could benefit from the microgravity environment. Fused cells are used for genetic studies where natural crosses are impossible or impractical. Fusion is the key step to monoclonal antibody production. Cell fusion requires the interaction of two different cell types and the annealing of the cell membranes to yield one hybrid cell. The fusion of cell membranes is usually accomplished by treatment with electric current or chemicals, either or both of which may be harmful to the cell. The cells to be fused are usually of different densities, so sedimentation tends to separate the cells during the fusion process and leads to aborted fusion or cell death. Microgravity could reduce the tendency of the cells to separate due to sedimentation and density differences. This might permit a reduction in the intensity and duration of the high-frequency aligning field in the case of electrofusion and, possibly, in the concentrations of fusing agents used for chemically induced cell fusion. Similar considerations may be operative in the synthesis of liposomes and the deposition of biomembranes. Further studies concerning the influence of a microgravity environment on cell fusion are needed. Results from sounding rocket and spacelab experiments25,26 suggested that studies should be extended to typical mammalian fusion partners. The effects of such factors as cell viability, growth phase, composition of growth medium, and shear environment on the fusion process should be evaluated. Other technologies should be investigated in both ground-based studies and microgravity to determine whether improvements are observed in microgravity similar to those observed with electrofusion techniques. Basic research utilizing model artificial membranes and related biopolymer file:///C|/SSB_old_web/mgoppch5.htm (9 of 14) [6/18/2004 11:17:11 AM]

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Microgravity Research Opportunities for the 1990s: Chapter 5 analogues may provide a better understanding of the processes and mechanisms at work in the fusion of natural cell membranes and could possibly lead to the development of more durable artificial membranes. A thorough characterization of the factors influencing fusion efficiency will improve Earth-based fusion technologies and will benefit the development of microgravity-based cell fusion activities. EXPERIMENTAL REQUIREMENTS Research areas in the biological sciences with expected sensitivity to the gravitational environment involve processes that typically take hours, days, or even weeks. The minimum duration of microgravity required for these experiments is, thus, greater than that available from drop towers, parabolic airplane flights, or suborbital rockets. If experiments in the biological sciences are to be conducted in microgravity, they must be carried out aboard the shuttle, the space station, or other orbiters such as free-flyers. Little is known about the limits below which accelerations, or frequencies and durations of accelerations, must be maintained so that the biological effects of microgravity are not obscured. It must be true, of course, that if the overall acceleration environment of the experimental system is kept to a fraction of the stochastic acceleration environment of its molecular components, then interference with studies of biological effects of microgravity is avoided. In the case of the shuttle, it is known that the background acceleration environment in orbit, the so-called g-jitter, can reach levels that interfere with biological experiments. Acceleration environments will vary from vehicle to vehicle and acceleration limits will vary from experiment to experiment, so that the best match of experiment to vehicle will have to be judged on a case-by-case basis. It may also be that acceleration environments that are a constant fraction of unit gravity may be helpful to studies in some areas of the biological sciences. It is important, however, that acceleration environments be monitored, not only so that experiments can be interpreted properly but also so that realistic acceleration scenarios can be included in the experimental design. RECOMMENDATIONS AND CONCLUSIONS In support of the biological and biotechnical research areas described above, priority should be given in the order listed to the following research activities: 1. More research should be devoted to quantifying the role of gravity in protein crystal growth processes and to identifying those mechanisms affected by gravity. 2. Further experimentation is needed in both terrestrial and microgravity environments to develop new methods and materials that take advantage of microgravity for biochemical separations. 3. The potential advantages of the microgravity environment for the study of cellular interactions, cell fusion, and multicellular assembly processes should be explored to identify candidate cell systems that show maximum benefit from culture in a microgravity environment. file:///C|/SSB_old_web/mgoppch5.htm (10 of 14) [6/18/2004 11:17:11 AM]

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Microgravity Research Opportunities for the 1990s: Chapter 5 4. Cellular and biomolecular processes, structures, assemblies, and mechanisms that might be affected by gravity should be systematically assayed to explore the effects of microgravity on these systems. The objective should be a comprehensive database of cell types and their experiences in microgravity environments. The first two topics have some demonstrated successes and the greater likelihood of future successes. The other two topics are exploratory. REFERENCES 1. Vanderhoff, J.W., and C.J. van Oss. 1979. Electrophoretic separation of biological cells in microgravity. Pp. 257-273 in Electrokinetic Separation Methods, P.G. Righetti, C.J. van Oss, and J.W. Vanderhoff, eds. Elsevier/North-Holland Biomedical Press, Amsterdam. 2. Cogoli, A. 1987. Cell cultures in space: From basic research to biotechnology II. Pp. 285-290 in Proceedings of the Third European Symposium on Life Sciences Research in Space, Graz, Austria, September 14-18. ESA SP-271. 3. Bugg, C.E. 1986. The future of protein crystal growth. J. Cryst. Growth, 76:535- 544. 4. DeLucas, L.J., et al. 1989. Protein crystal growth in microgravity. Science, 246:651-654. 5. McPherson, A., A. Greenwood, and J. Day. 1991. The effect of microgravity on protein crystal growth. Adv. Space Res., 11(7):343-356. 6. DeLucas, L.J., et al. 1994. Recent results and new hardware developments for protein crystal growth in microgravity. J. Cryst. Growth, 135:183-195. 7. DeLucas, L.J., et al. 1993. Protein crystal growth results from the United States Microgravity Laboratory-1 mission. J. Phys. D: Appl. Phys., 26:B100-B103. 8. DeLucas, L.J., et al. 1993. Protein crystal growth results from the United States Micro-gravity Laboratory-1 Mission. NASA Reference Publication (conference proceedings) from the USML-1 and USMP-1 Joint L+1 Science Review. Huntsville, Ala., September 22- 24. 9. Day, J., and A. McPherson. 1992. Macromolecular crystal growth experiments on International Microgravity Laboratory-1. Protein Science, 11:1254-1268. 10. McPherson, A. 1992. Effects of a microgravity environment on the crystallization of biological macromolecules. Pp. 619-626 in Proceedings of the VIIIth European Symposium on Materials and Fluid Sciences in Microgravity, Vol. II, J.C. Legros, ed. Free University of Brussels, Brussels, Belgium. file:///C|/SSB_old_web/mgoppch5.htm (11 of 14) [6/18/2004 11:17:11 AM]

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Microgravity Research Opportunities for the 1990s: Chapter 5 11. McPherson, A. 1993. Virus and protein crystal growth on Earth and in microgravity. J. Phys. D: Appl. Phys., 26:104-112. 12. Day, J., and A. McPherson. 1992. Macromolecular crystal growth experiments on International Microgravity Laboratory-1. Protein Science, 11:1254-1268. 13. Long, M.M., et al. 1994. Protein crystal growth in microgravity-temperature- induced large-scale crystallization of insulin. Proceedings of the International Symposium of Microgravity Sciences and Applications, Beijing, China, May 10-13, 1993. Microgravity Sci. Technol., 7:196-202. 14. Stoddard, B.L., R.K. Strong, G.K. Farber, A. Arrott, and G. Petsko. 1991. Design of apparatus and experiments to determine the effect of microgravity on the crystallization of biological macromolecules using the Mir space station. J. Cryst. Growth, 110:312-316. 15. Strong, R.K., B.L. Stoddard, A. Arrott, and G.K. Farber. 1992. Long duration growth of protein crystal in microgravity aboard the Mir space station. J. Cryst. Growth, 119:200-214. 16. DeLucas, L.J., et al. 1993. Protein crystal growth results from the United States Microgravity Laboratory-1 mission. J. Phys. D: Appl. Phys., 26:B100-B103. 17. Ealick, S.E., et al. 1991. Three-dimensional structure of recombinant human interferon-g. Science, 252:698-702. 18. He, X.M., and D.C. Carter. 1992. Atomic structure and chemistry of human serum albumin. Nature, 358:209-215. 19. DeLucas, L.J., et al. 1993. Protein crystal growth results from the United States Microgravity Laboratory-1 mission. NASA Reference Publication (conference proceedings) from the USML-1 and USMP-1 Joint L+1 Science Review. Huntsville, Ala., September 22- 24. 20. Day, J., and A. McPherson. 1992. Macromolecular crystal growth experiments on International Microgravity Laboratory-1. Protein Science, 11:1254-1268. 21. Hymer, W.C., et al. 1987. Continuous flow electrophoretic separation of proteins and cells from mammalian tissues. Cell Biophysics, 10:61-85. 22. Rhodes, P.H., R.S. Snyder, and G.O. Roberts. 1989. Electrodynamic distortion of sample streams in continuous flow electrophoresis. J. Colloid Interface Sci., 129:78-90. 23. Van Alstine, J.M., S. Bamberger, J.M. Harris, R.S. Snyder, J.F. Boyce, and D.E. Brooks. 1990. Phase partitioning experiments on Shuttle flight STS-26. Pp. 399-407 in Proceedings of the VIIth European Symposium on Materials and Fluid Sciences in Microgravity, Oxford, United Kingdom, September 10-15. ESA SP-295. 24. Gmünder, F.K., and A. Cogoli. 1988. Cultivation of single cells in space. Applied Microgravity Technology, 1:115-122. file:///C|/SSB_old_web/mgoppch5.htm (12 of 14) [6/18/2004 11:17:11 AM]

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Microgravity Research Opportunities for the 1990s: Chapter 5 25. Baumann, T., W. Kreis, W. Mehrle, R. Hampp, and E. Reinhard. 1990. Regeneration and characterization of protoplast-derived cell lines from Digitalis lanata EHRH and Digitalis purpurea L. suspension cultures after electrofusion under microgravity conditions. Pp. 405-410 in Proceedings of the Fourth European Symposium on Life Sciences Research in Space, Trieste, Italy, May 28-June 1. ESA SP-307. 26. German Ministry of Research and Technology (BMFT). 1987. Proceedings of the Norderney Symposium on Scientific Results of the German Spacelab Mission D1, P.R. Sahm, R. Jansen, and M.H. Keller, eds. Wissenschaftliche Projektfuerung D1, Cologne, Germany. PLATE 5.1 Crystals of satellite tobacco mosaic virus (STMV) grown in microgravity during the flight of International Microgravity Laboratory-1 in January 1992. X-ray diffraction measurements demonstrated the diffraction of the crystals to a resolution greater than 1.8- Å Bragg spacings, significantly improved over those grown in Earth laboratories. PLATE 5.2 Droplets of protein solution in the Vapor Diffusion Apparatus (VDA) on shuttle mission STS-26. Droplets, about 40 l in size, are poised on double-barreled syringes and are formed by the mixing of protein and precipitant solutions isolated in separate barrels prior to launch. Droplets equilibrate by vapor diffusion against a reservoir of precipitant stabilized in microgravity in a porous high-molecular-weight polyethylene plug (not shown). file:///C|/SSB_old_web/mgoppch5.htm (13 of 14) [6/18/2004 11:17:11 AM]