[Cell biology researches aboard the robotic space vehicles: preparation and performance].

The article reviews the unique aspects of preparation and performance of cell biology experiments flown on robotic space vehicles Bion and Foton, and gives an overview of key findings in researches made under the author's leadership over the past decades. Described are the criteria of selecting test objects, and the conditions required for preparation and implementation of space and control (synchronous) experiments. The present-day status and issues of researches into cell responsivity to space microgravity and other factors are discussed. Also, potentialities of equipment designed to conduct experiments with cell cultures in vitro and populations of single-celled organisms are presented, as well as some ideas for new devices and systems. Unveiled are some circumstances inherent to the development and performance of space experiments, setting up laboratory facilities at the launch and landing site, and methods of safe transportation and storage of biosamples. In conclusion, the author puts forward his view on biospecies, equipment and areas of research aboard future space vehicles.

[Problems of exobiology: the origin of life on Earth]. / Problemy ekzobiologii: vozniknovenie zhizni na Zemle.

The basic problem of exobiology is origin and evolution of life as a space phenomenon. Consideration is given to the facts for the space origin, spreading in the interstellar space of and invasion of the surface of planets by organic compounds, constituents of archetypes of living systems. The authors bring up to discussion the issues of life development under the conditions of Earth, and formation of the main properties of the living organisms differing in the level of organization. Outlined are some international projects on exobiological research in experiments with bio-objects on space platforms.

[Evolution of the interaction fo living systems with the environment]. / Evoliutsiia vzaimodeistviia zhivykh sistem s okruzhaiushchei sredoi.

Basic principles of interaction of living systems with environmental factors and different phases of the organic evolution are presented. The general strategy of adaptation of living systems on various levels of organization to changes in the environment is considered. It is shown that in the course of evolutionary adaptation the living systems not only altered their morphophysiological status but also actively adjusted the environment modifying its physical/chemical parameters. Special attention is given to the hereditary and external factors in of structural and functional evolution living organisms. Environmental factors are shown to gain significance in structural/functional and behavioral formation of living organisms as the organic world has evolved.

Cytogenetic characteristic of osteogenic cells in vitro as perspective predictors of osteopenia under microgravity.

Mechanical stimulation of bone tissue determined by earth gravity is one of the main factors mediating the nature, rate and direction of functional adaptation of the bone system in the process of onto- and phylogenesis. Theoretically expected losses of bone mass under condition of mechanical load deficit under microgravity (osteopenia, osteoporosis) may become a factor that limits the duration of space flights. As a result of long-term studies some properties and regularities of change in human tissue after prolonged space flights (for 5-7 months) were established.

[The cell as a gravity-dependent biomechanic system]. / Kletka kak gravitatsionno-zavisimaia biomekhanicheskaia sistema.

In the period of 1995-1997 experimental and theoretical studies with various biomechanic objects, i.e. individual cells and cell associations, were performed under changed gravity (0.00001-5 g). Experimental investigations were conducted using clinostats and centrifuges to model effects of hypo- and hypergravity, and aboard space vehicles in real microgravity. Cell cultures in vitro including fibroblasts and osteoblasts on a solid glass or plastic substrate served as objects of the studies. Changes in value and direction of the gravity vector were found to modify the morphophysiological characteristics of cells: structural organization (spatial rearrangement of the intracell component, changes in forms, sizes and quantity of cells) and functional activity (alterations in energy expenditure and intensity of intracellular metabolism). The data suggest that there should be mechanisms of gravitational sensitivity in living systems on the cellular level. As was stated, sensitivity of unicellular free-living organisms to gravity is mostly defined by the motor activity determined by the level of general metabolism. Morphological characteristics (form, size and mass) are of secondary importance. Theoretical analysis resulted in correction of one of the principle postulates of gravitational biology stating a direct link between size (mass) and gravitational sensitivity of organism. Described were consistent patterns of growth, development, and behavior of unicellular cultures in gravitational fields. Strengthening of the force of gravity (hypergravity) leads to eventual deceleration of cell growth and diminution of biomass gain. On the other hand, the spaceflight environment (microgravity) stimulates growth mechanisms. In our opinion, behind these gravitational effects are altered levels of energy spent by cells to overcome the force of gravity. Opposite trends were observed in experiments with cell cultures in vitro. During space microgravity, fibroblast cultures on the solid substrate decreased the growth rate, and inhibited cell division and migration within the substrate. Compared to the Earth's gravity, under elevated gravity these parameters were noticeably higher. It was demonstrated that the main cause of the unfavorable effects of space microgravity on the cellular level is decay in the adherence of cells to the substrate. Explored were also the most probable mechanisms of the effects of changed gravity on the cell as a biomechanic structure. Specialized and non-specialized graviceptors of various types of cells were crypt-analyzed and classified. In future, investigations should be angled for elucidation of the role of intracellular components in perception and implementation of the gravitational stimulus, and description of quantitative characteristics of energy exchange and metabolism in cells as a function of gravity force and direction.

[The growth movements of moss protonemata under clinostatic and microgravity conditions]. / Rostovye dvizheniia protonemy mkhov v usloviiakh klinostatirovaniia i mikrogravitatsii.

Populations of dark-grown protonemata of moss Ceratodon purpureus wt-4 (Germany) and wt-U (Ukraine) were rotated on clinostat or flown in space (experiment "Protonema" aboard Bion-11, December 24, 1996-January 7, 1997) to determine the effects of altered gravity on orientation of protonemata growing filaments. Protonemata had been cultivated 8 days in vertical stationary position at dark to be transported to microgravity or placed in clinostat for the period of 14 days. In the ground control, protonemata demonstrated the negatively gravitropic growth (straight upwards in a bundle of compact filaments). The horizontal or circular rotation in clinostat and exposure to microgravity made filaments grow every each way within the substrate plane but with an apparent trend to rightward curling resulting in "spiral galaxies".

Gravisensitivity of cells of several types.

NASA: Researchers examined time-related changes in growth, motility, and some morphological-functional characteristics of cells and cell association in unicellular organisms, plants, and human and mice cell cultures. Post-flight analysis included studies of cell formation, division rate, chemical composition, and ultrastructural organization of cells and their major organelles in weightlessness and hypergravity. Unicellular organisms used in the studies were the ciliates Tetrahymena pyriformis, Dileptus anser, Bursaria truncatella, and Loxodes striatus; flagellate Euglena gracilis and Chlamydomonas reinhardtii; and the sarcodina Amoeba proteus.^ieng

[The role of gravitational force in the evolution of living systems (the biomechanical and energy aspects)]. / Rol' sily tiazhesti v évoliutsii zhivykh sistem (biomekhanicheskie i énergeticheskie aspekty).

Possible pathways of the origin and development of adaptive mechanisms in the living systems of various levels of organization are considered from the positions of biomechanics and bioenergetics. Main attention is paid to the specific feature of functional rearrangements in the living organisms during change of habitat, at the stage of their exit from water to land. The following problems of interaction between the living systems and gravity during evolution are discussed: cellular level of organization and transition to multicellularity, formation and improvement of the skeletal and skeletal-motor systems in plants and animals, and thermal homeostasis of the living organisms in the gravity field. We showed a leading role of gravity in organization of the morphofunctional status of the living organisms during their evolution.

[Patterns in the growth and functioning of single-cell organisms under conditions of altered gravitational force]. / Zakonomernosti rosta i funktsionirovaniia odnokletochnykh organizmov v usloviiakh izmenennoi sily tiazhesti.

The results of studies over many years on various types of unicellular free-swimming organisms in conditions of altered gravity (hyper-, hypo-, and microgravity ranging from 10(-5) to 5 g) were reviewed. Laws governing their growth and functioning under these conditions were established. In general, gravisensing of unicellular free-swimming organisms depends on their metabolic activity and mobility, on the one hand, and on the environmental conditions of the population, on the other. A working hypothesis on the priority of ecophysiological properties (environment, metabolic activity, and mobility) over morphological properties (mass, dimensions, and shape) in receiving and processing of gravity stimuli at the cellular level has been formulated.

[The general principles of cellular gravisensing]. / Obshchie printsipy gravichuvstvitel'nosti kletok.

A review of the results of studies over many years into cellular mechanisms underlying gravireception in different cell types, which were carried out by the author in the laboratory during simulation of the effects of altered gravity using a clinostat and centrifuge and under the conditions of space flight. Comparative analyses of the obtained results with the published data has been performed. It was shown that besides specific features of adaptation of different cell types to altered gravity, including microgravity, there are common pathways of adaptation of the structural-functional organization of the cell to this factor. An integral molecular mechanism underlying cell gravisensing was revealed. A working hypothesis on general principles of cell gravisensing has been formulated.

The role of signal systems in cell gravisensitivity.

Reception of physical environmental signals caused by alteration of the gravitation field leads to the shift of morpho-physiological cell characteristics. The gravity influence on a cell may be direct or non direct, its extent varying in dependence of the cell model applied. Direct influences are more pronounced in vitro, while non direct influences are usually expressed in the community of unicellular organisms (in vivo). Gravity affects morphogenesis processes, such as locomotion, adhesion, intercellular contacts, etc. At the same time, all the processes named are under control of cell integral systems of the signal transduction. Minor disturbances in this system coming from the environment, due to amplification, may provide significant modulations of the signals. So, studies of this system at the level of molecular cell reception is of great interest. Results of flight and model experiments are discussed in the present manuscript.

Physico-chemical characteristics of biomembranes and cell gravisensitivity.

The resistance of living systems to the action of environmental factors is known to be largely determined by molecular organization of biomembranes constituting the basis of the cell per se and of all intracellular organelles. Gravity as one of the environmental factors, plays a definite role in the vital activity of organisms. Therefore, the problem of altered gravity impact on biological objects should be considered in close relation to the functional state of membranes and contractible elements of cytoskeleton. Moreover, the involvement of membrane structures and cytoskeleton in the processes of reception and realization of gravitational stimulus allows us to evaluate the extent of the direct or indirect influence of gravity on cell functioning in the gravitational field. The results of experimental studies having been conducted up to this time on a variety of cells and cell organelles under altered gravity conditions demonstrated noticeable alterations in the molecular organization of the membranes.

[Effects of altered gravity on the culture of unicellular eucaryotic organisms, Bursaria truncatella (Ciliophora)]. / Vliianie izmenennoi sily tiazhesti na kul'turu odnokletochnykh éukarioticheskikh organizmov Bursaria truncatella (Ciliophora).

The paper reports the results of experiments with centrifugation and clinostating. Growth rate, cellular division and several morphofunctional characteristics of unicellular organisms of infusoria Bursaria truncatella in culture were studied under normal (1 g), elevated (hypergravity at 2 and 5 g), and compensated gravity. The data point to certain changes in the functional activity and morphology of cells consequent to long-time cultivation under these conditions. The observed regularities in the dynamics of B.truncatella growth and shifts in its physiology and morphology due to hypergravity or compensated gravity support our earlier proposed working hypothesis about the dominance of functional activity over morphological properties in sensitivity of unicellular organisms to perception and realization of the gravitational stimulus.

[Biomechanical aspects of the process of formation of food vacuoles in the infusoria Bursaria truncatella under changed gravity]. / Biomekhanicheskie aspekty protsessa obrazovaniia pishchevykh vakuolei u infuzorii Bursaria truncatella v usloviiakh izmenennoi sily tiazhesti.

Results from clinostatic and centrifugal laboratory experiments in which there has been evaluated an activity of digestive process in infusoria Bursaria truncatella by the content of vacuole numbers and its change in the cell under changed gravity are presented. It is indicated that the extended clinostatic exposure of infusoria stimulates their digestive activity and an increased gravity (centrifugation 2 g, 5 g) inhibits this process. In these examinations, an effort was made to explain the obtained results started from the propositions of cell biomechanics and bioenergetics. The mechanisms of gravity influence on this process have been proposed.

[Cell growth and motility in culture (in vitro) under microgravity conditions. The Fibroblast Experiment]. / Rost i podvizhnost' kletok v kul'ture (in vitro) v usloviiakh mikrogravitatsii. Eksperiment "Fibroblast".

The experiment "Fibroblast" was performed in 1992 on biosatellite "Cosmos-2229" in onboard device "Biobox" designed by the order of European Space Agency. The main objective was elucidation of the mechanisms responsible for the effect of space flight factors, mostly microgravity, on cell culture. We studied time-related changes in growth, motility and some morphological characteristics of the cells in monolayer cultures on a solid substrate and in three-dimensional cultures supported by sponge gels. Studies were carried out on connective tissue cells isolated from the mouse embryos. Comparative after-flight analysis of the cell cultures exposed to space flight and of those under the normal gravity conditions (1 g) on the Earth has revealed some differences. The space flight conditions, mainly microgravity, induced marked changes in morphological characteristics and functional activity of the cultured fibroblasts: changes in the nucleus size and shape, retardation of cell growth and division rate. We believe that these changes may be due to weakening of intercellular contacts and cell adhesion to the substrate. These findings are important both for general biology and space medicine, specifically for the problems of tissue regeneration and wound healing under the conditions of long-term space flight.

[The ecological-morphological characteristics of the growth and distribution of cultures of single-cell organisms in a gravitational field]. / Ekologo-morfologicheskie osobennosti rosta i raspredeleniia kul'tur odnokletochnykh organizmov v gravitatsionnom pole.

The paper analyses and summarizes the experimental results obtained by the authors and data accumulated in the literature during recent years in order to reveal the general regularities of the growth, distribution and behavioral parameters of the cells under conditions of reduced gravity ranged from 10(-5) to 5 g. It is indicated that an intensity of gravity effect on these parameters of unicellular organisms depends greatly on the environments, types of cells and their metabolic levels. The effects of changed gravity at the cellular level are the result of shifting the primary physiological characteristics and physicochemical parameters of the culture and cell environments as an integrated biological system.

The effect of microgravity on the development of plant protoplasts flown on Biokosmos 9.

An experiment using plant protoplasts has been accepted for the IML-1 Space Shuttle mission scheduled for 1991. Preparatory experiments have been performed using both fast and slow rotating clinostats and in orbit to study the effect of simulated and real weightlessness on protoplast regeneration. Late access to the space vehicles before launch has required special attention since it is important to delay cell wall regeneration until the samples are in orbit. On a flight on Biokosmos 9 ("Kosmos-2044") in September 1989 some preliminary results were obtained. Compared to the ground control, the growth of both carrot and rapeseed protoplasts was decreased by 18% and 44% respectively, after 14 days in orbit. The results also indicated that there is less cell wall regeneration under micro-g conditions. Compared to the ground controls the production of cellulose in rapeseed and carrot flight samples was only 46% and 29% respectively. The production of hemicellulose in the flight samples was 63% and 67% respectively of that of the ground controls. In both cases all samples reached the stage of callus development. The peroxidase activity was also found to be lower in the flight samples than in the ground controls, and the number of different isoenzymes was decreased in the flight samples. In general, the regeneration processes were retarded in the flight samples with respect to the ground controls. From a simulation experiment for IML-1 performed in January 1990 at ESTEC, Holland, regenerated plants have been obtained. These results are discussed and compared to the results obtained on Biokosmos 9. Protoplast regeneration did not develop beyond the callus stage in either the flight or the ground control samples from the Biokosmos 9 experiment.