Possible mechanisms of plant cell wall changes at microgravity.

Space and clinostatic experiments revealed that changes of plant cell wall structure and its function depend on type of tissue and duration of influence. It was shown that clinostat conditions reproduce the part of weightlessness biological effects. It is established that various responses of wall structural-metabolic organization occur at microgravity: changes of cell walls ultrastructure and organelles structure; decrease of synthesis of primary plant cell wall; rearrangements of polysaccharides content. It is shown that mechanisms of plant cell wall changes at microgravity are connected with decrease of cellulose crystallization, activation of pectolytic enzymes and rearrangement of calcium balance of apoplast and cytoplasm.     

Influence of different natural physical fields on biological processes.

In space flight conditions gravity, magnetic, and electrical fields as well as ionizing radiation change both in size, and in direction. This causes disruptions in the conduct of some physical processes, chemical reactions, and metabolism in living organisms. In these conditions organisms of different phylogenetic level change their metabolic reactions undergo changes such as disturbances in ionic exchange both in lower and in higher plants, changes in cell morphology for example, gyrosity in Proteus (Proteus vulgaris), spatial disorientation in coleoptiles of Wheat (Triticum aestivum) and Pea (Pisum sativum) seedlings, mutational changes in Crepis (Crepis capillaris) and Arabidopsis (Arabidopsis thaliana) seedling. It has been found that even in the absence of gravity, gravireceptors determining spatial orientation in higher plants under terrestrial conditions are formed in the course of ontogenesis. Under weightlessness this system does not function and spatial orientation is determined by the light flux gradient or by the action of some other factors. Peculiarities of the formation of the gravireceptor apparatus in higher plants, amphibians, fish, and birds under space flight conditions have been observed. It has been found that the system in which responses were accompanied by phase transition have proven to be gravity-sensitive under microgravity conditions. Such reactions include also the process of photosynthesis which is the main energy production process in plants. In view of the established effects of microgravity and different natural physical fields on biological processes, it has been shown that these processes change due to the absence of initially rigid determination. The established biological effect of physical fields influence on biological processes in organisms is the starting point for elucidating the role of gravity and evolutionary development of various organisms on Earth.     

Effects of altered gravity on plant cell processes: results of recent space and clinostatic experiments.

Space and clinostatic experiments revealed that plant cell structure and metabolism rearrangements depend on taxonomical position and physiological state of objects, growth phase and real or simulated microgravity influence duration. It was shown that clinostat conditions reproduce only a part of microgravity biological effects. It is established that various responses occur in microgravity: 1) rearrangements of cytoplasmic organelles ultrastructure and calcium balance; 2) physical-chemical properties of the plasmalemma are changed; 3) enzymes activity is often enhanced. These events provoke the acceleration of growth and differentiation of cells and their aging as a result; at the same time some responses can be considered as cell adaptation to microgravity.     

Microgravity and bone cell mechanosensitivity.

The capacity of bone tissue to alter its mass and structure in response to mechanical demands has long been recognized but the cellular mechanisms involved remained poorly understood. Bone not only develops as a structure designed specifically for mechanical tasks, but it can adapt during life toward more efficient mechanical performance. Mechanical adaptation of bone is a cellular process and needs a biological system that senses the mechanical loading. The loading information must then be communicated to the effector cells that form new bone or destroy old bone. The in vivo operating cell stress derived from bone loading is likely the flow of interstitial fluid along the surface of osteocytes and lining cells. The response of bone cells in culture to fluid flow includes prostaglandin (PG) synthesis and expression of prostaglandin G/H synthase inducible cyclooxygenase (COX-2). Cultured bone cells also rapidly produce nitric oxide (NO) in response to fluid flow as a result of activation of endothelial nitric oxide synthase (ecNOS), which enzyme also mediates the adaptive response of bone tissue to mechanical loading. Earlier studies have shown that the disruption of the actin-cytoskeleton abolishes the response to stress, suggesting that the cytoskeleton is involved in cellular mechanotransduction. Microgravity, or better near weightlessness, is associated with the loss of bone in astronauts, and has catabolic effects on mineral metabolism in bone organ cultures. This might be explained as resulting from an exceptional form of disuse under near weightlessness conditions. However, under near weightlessness conditions the assembly of cytoskeletal elements may be altered since it has been shown that the direction of the gravity vector determines microtubular pattern formation in vivo. We found earlier that the transduction of mechanical signals in bone cells also involves the cytoskeleton and is related to PGE2 production. Therefore it is possible that the mechanosensitivity of bone cells is altered under near weightlessness conditions, and that this abnormal mechanosensation contributes to disturbed bone metabolism observed in astronauts. In our current project for the International Space Station, we wish to test this hypothesis experimentally using an in vitro model. The specific aim of our research project is to test whether near weightlessness decreases the sensitivity of bone cells for mechanical stress through a decrease in early signaling molecules (NO, PGs) that are involved in the mechanical loading-induced osteogenic response. Bone cells are cultured with or without gravity prior to and during mechanical loading, using our modified in vitro oscillating fluid flow apparatus. In this "FlowSpace" project we are developing a cell culture module that is used to provide further insight in the mechanism of mechanotransduction in bone.     

The first dedicated Life Sciences mission--Spacelab 4.

Spacelab is a large versatile laboratory carried in the bay of the Shuttle Orbiter. The first Spacelab mission dedicated entirely to Life Sciences is known as Spacelab 4. It is scheduled for launch in late 1985 and will remain aloft for seven days. This payload consists of 25 tentatively selected investigations combined into a comprehensive integrated exploration of the effects of acute weightlessness on living systems. An emphasis is placed on studying physiological changes that have been previously observed in manned space flight. This payload has complementary designs in the human and animal investigations in order to validate animal models of human physiology in weightlessness. The experimental subjects include humans, squirrel monkeys, laboratory rats, several species of plants, and frog eggs. The primary scientific objectives include study of the acute cephalic fluid shift, cardiovascular adaptation to weightlessness, including postflight reductions in orthostatic tolerance and exercise capacity, and changes in vestibular function, including space motion sickness, associated with weightlessness. Secondary scientific objectives include the study of red cell mass reduction, negative nitrogen balance, altered calcium metabolism, suppressed in vitro lymphocyte reactivity, gravitropism and photropism in plants, and fertilization and early development in frog eggs. The rationale behind this payload, the selection process, and details of the individual investigations are presented in this paper.     

Protozoa as model systems for the study of cellular responses to altered gravity conditions.

The orientation behavior of Paramecium changed in a similar way after transition to conditions of free-fall in a sounding rocket and after transition to conditions of simulated weightlessness on a fast rotating clinostat. After a period of residual orientation, Paramecium cells distributed themselves randomly 80 s (120 s) after onset of free-fall (simulated weightlessness). Swimming velocity increased significantly; however, the increase was transient and subsided after 3 min in the rocket experiments, while the velocity remained enhanced even during 2 h of rotation on a fast clinostat. Trichocysts were present and without morphological changes in Paramecium cells which had been exposed to a rocket flight, as well as to fast or slow rotation on a clinostat. Regeneration of the oral apparatus of Stentor and morphogenesis of Eufolliculina proceeded normally on the clinostat. The results demonstrate that the clinostat is a useful tool to simulate the conditions of weightlessness on earth and to detect gravisensitive cellular functions.     

The state of gravity sensors and peculiarities of plant growth during different gravitational loads.

In the primary roots of lettuce shoots grown under altered gravitational conditions--180 degrees inversion on the centrifuged clinostat, horizontal clinostat and in dynamic weightlessness--localization of the cellular organelles, cell morphology and peculiarities of growth have been studied. Significant changes took place in the localization of amyloplasts on the horizontal clinostat. The changes of amyloplast position in the cap cells on the horizontal clinostat and under weightlessness are similar. A change of the normal shoot position (180 degrees inversion and horizontal clinostat) causes an inhibition of growth. Weightlessness increases the length of axial organs and cells in the zone of elongation, but decreases the nitotic index in comparison to the centrifuged control. The anlysis of the formation of generative organs has been carried out for Arabidopsis plants grown on board the orbital station Salyut-6. The ability of plants to undergo vegetative growth and to pass through early phases of generative development under weightlessness was confirmed.     

Influence of altered gravity on the cytochemical localization of cytochrome oxidase activity in central and peripheral gravisensory systems in developing cichlid fish.

Cichlid fish larvae were reared from hatching to active free swimming under different gravity conditions: natural environment, increased acceleration in a centrifuge, simulated weightlessness in a clinostat and near weightlessness during space flight. Cytochrome oxidase activity was analyzed semiquantitatively on the ultrastructural level as a marker of regional neuronal activity in a primary, vestibular brainstem nucleus and in gravity receptive epithelia in the inner ear. Our results show, that gravity seems to be positively correlated with cytochrome oxidase activity in the magnocellular nucleus of developing fish brain. In the inner ear the energy metabolism is decreased under microgravity concerning utricle but not saccule. Hypergravity has no effect on cytochrome oxidase activity in sensory inner ear epithelia.     

Functional state of chromatin and proliferative activity of meristematic cells in pea seedlings under various clinostatic conditions.

Data from a complex cytochemical analysis show that the functional state of chromatin and the level of the cell proliferative activity may be reliable cytological criteria for primary structural and functional changes that result in disturbances of plant growth and development. Autoradiographic and cytophotonetric studies made it possible to establish certain differences, induced by fast rotation (50 rev/min) on the clinostat, in the chromatin state and cell reproduction of the pea seedling root meristem for the initial stages of plant development. There were no essential differences for the given parameters under slow (2 rev/min) clinostatic conditions.     

Changes in the central nervous system during long-duration space flight: implications for neuro-imaging.

The purpose of this paper is to review the potential functional and morphological effects of long duration space flight on the human central nervous system (CNS) and how current neuroimaging techniques may be utilized to study these effects. It must be determined if there will be any detrimental changes to the CNS from long term exposure to the space environment if human beings are to plan interplanetary missions or establish permanent space habitats. Research to date has focused primarily on the short term changes in the CNS as the result of space flight. The space environment has many factors such as weightlessness, electromagnetic fields, and radiation, that may impact upon the function and structure of the CNS. CNS changes known to occur during and after long term space flight include neurovestibular disturbances, cephalic fluid shifts, alterations in sensory perception, changes in proprioception, psychological disturbances, and cognitive changes. Animal studies have shown altered plasticity of the neural cytoarchitecture, decreased neuronal metabolism in the hypothalamus, and changes in neurotransmitter concentrations. Recent progress in the ability to study brain morphology, cerebral metabolism, and neurochemistry in vivo in the human brain would provide ample opportunity to investigate many of the changes that occur in the CNS as a result of space flight. These methods include positron emission tomography (PET), single photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI).     

Biological role of gravity: hypotheses and results of experiments on "Cosmos" biosatellites.

In order to reveal the biological significance of gravity, microgravity effects have been studied at the cellular, organism and population levels. The following questions arise. Do any gravity-dependent processes exist in a cell? Is cell adaptation to weightlessness possible; if so, what role may cytoskeleton, the genetic apparatus play in it? What are the consequences of the lack of convection in weightlessness for the performance of morphogenesis? Do the integral characteristics of living beings change in weightlessness? Is there any change in "biological capacity" of space, its resistance to expansion of life? What are the direction and intensity of microgravity action as a factor of natural selection, the driving force of evolution? These problems are discussed from a theoretical point of view, and in the light of results obtained in experiments from aboard biosatellites "Cosmos".     

Changes in the micro-structure of the vestibular apparatus of tadpoles (Rana temporaria) developed in simulated weightlessness

The vestibular apparatus of tadpoles (Rana temporaria) exposed to simulated weightlessness was examined by electron microscopy. Extended exposure to simulated weightlessness is followed by significant alterations in the sensory epithelia and also in the otolith membrane. Large vacuoles, filled with necrobiotic mitochondria and fragments of endoplasmic reticulum, were concentrated in the region where an otolith membrane covers the hair cells but were mostly absent in zones of the epithelia with undifferentiated cells. The number of otoconia in the otolith membrane was diminished. The results were compared with data from space flight experiments and some concordance was noted. The possible connection between some unusual behavior of the tadpoles after weightlessness simulation and the structural alterations in the gravitational sensors was discussed.     

Plant growth, development and embryogenesis during Salyut-7 flight.

The growth and geotropic movements of roots and hypocotyls of lettuce have been studied on board the Salyut 7 station in a stationary position and on the centrifuge at 0.01, 0.1 and 1 g. On the centrifuge at 0.1 and 0.01 g as well as under weightlessness, the final length of hypocotyls was by 8-16% greater than in control plants on the centrifuge at 1 g. The length of roots, however, was reduced by 17% at 0.01 g and under weightlessness; at 0.1 g their growth is much the same as at 1 g. On the Earth, while growing in a vertical position, and in space at 0 < or = g, the roots and hypocotyls deviate from the longitudinal axis of the seed. Average values of deviation eagles on the Earth are always equal to zero, while this is not always the case in space, which indicates the biological effect of microgravity conditions on board a spacecraft. The threshold of geotropic sensitiveness of lettuce hypocotyls, calculated from the linear regression parameters of the dependence of the response geotropic reaction upon the value of the centrifugal force, comprised 2.9 x 10(-3) g. In the Fiton 3 micro-greenhouse under spaceflight conditions, the plants of Arabidopsis thaliana (L) Heynh have, for the first time, undergone a full cycle of individual development. The seeds sown during the flight germinated, performed growth processes, formed vegetative and generative organs and, judging by the final result, they succeeded in fecundation, embryogenesis and ripening. Despite the noted modification of growth and development of plants in space, 42% of formed seeds appeared to be valuable biologically.     

Ultrastructural analysis of organization of roots obtained from cell cultures at clinostating and under microgravity.

Data are presented of a comparative analysis on rhizogenesis in the Arabidopsis thaliana tissue culture growing in a solid nutrient medium under stationary conditions, clinostatic conditions and microgravity. Tissue samples weighing 100 mg. were set in the Petri dishes and placed in a horizontal slow clinostat /2 revs/min/. After 14 days of growth they were analyzed. On clinostating the number of roots formed from the callus cells was approximately one half the control. The formed root cap manifested no essential differences, in comparison with the stationary control, in the number of layers and cell sizes in its layers. In callusogenic roots, formed from clinostated cells, differentiation including root cap cells, proceeds without noticeable deviations from the norm. At the same time, gravireceptor cells do not function under these conditions. This is clearly displayed at a structural level in the location of amyloplasts-statoliths throughout the cytoplasm. The callus cell cultures experienced microgravity for 8 days. The number of formed roots under the influence of this factor was 36% relative to the stationary control. Root cap formation was abnormal. Gravireceptor cells did not formed under microgravity.     

Effects of microgravity on bone and calcium homeostasis.

Mechanical function is known to be of crucial importance for the maintenance of bone tissue. Gravity on one hand and muscular effort on the other hand are required for normal skeletal structure. It has been shown by numerous experimental studies that loss of total-body calcium, and marked skeletal changes occur in people who have flown in space. However, most of the pertinent investigations have been conducted on animal models, including rats and non-human primates, and a reasonably clear picture of bone response to spaceflight has emerged during the past few years. Osteopenia induced by microgravity was found to be associated with reduction in both cortical and trabecular bone formation, alteration in mineralization patterns and disorganization of collagen, and non-collagenous protein metabolism. Recently, cell-culture techniques have offered a direct approach of altered gravity effects at the osteoblastic-cell level. But the fundamental mechanisms by which bone and calcium are lost during spaceflight are not yet fully known. Infrequency and high financial cost of flights have created the necessity to develop on-Earth models designed to mimic weightlessness effects. Antiorthostatic suspension devices are now commonly used to obtain hindlimb unloading in rats, with skeletal effects similar to those observed after spaceflight. Therefore, actual and "simulated" spaceflights, with investigations conducted at whole body and cellular levels, are needed to elucidate pathogeny of bone loss in space, to develop effective countermeasures, and to study recovery processes of bone changes after return to Earth.     

Identification of specific gravity sensitive signal transduction pathways in human A431 carcinoma cells.

Epidermal growth factor (EGF) activates a well characterized signal transduction cascade in human A431 epidermoid carcinoma cells. The influence of gravity on EGF-induced EGF-receptor clustering and early gene expression as well as on actin polymerization and actin organization have been investigated. Different signalling pathways induced by the agents TPA, forskolin and A23187 that activate gene expression were tested for sensitivity to gravity. EGF-induced c-fos and c-jun expression were decreased in microgravity. However, constitutive beta-2 microglobulin expression remained unaltered. Under simulated weightlessness conditions EGF- and TPA-induced c-fos expression was decreased, while forskolin- and A23187-induced c-fos expression was independent of the gravity conditions. These results suggest that gravity affects specific signalling pathways. Preliminary results indicate the EGF-induced EGF-receptor clustering remained unaltered irrespective of the gravity conditions. Furthermore, the relative filamentous actin content of steady state A431 cells was enhanced under microgravity conditions and actin filament organization was altered. Under simulated weightlessness actin filament organization in steady state cells as well as in EGF-treated cells was altered as compared to the 1 G reference experiment. Interestingly the microtubule and keratin organization in untreated cells showed no difference with the normal gravity samples. This indicates that gravity may affect specific components of the signal transduction circuitry.     

Investigations on-board the biosatellite Cosmos-83.

The program of the 5 day flight of the biosatellite Cosmos-1514 (December 1983) envisaged experimental investigations the purpose of which was to ascertain the effect of short-term microgravity on the physiology, growth and development of various animal and plant species. The study of Rhesus-monkeys has shown in that they are an adequate model for exploring the mechanisms of physiological adaptation to weightlessness of the vestibular apparatus and the cardiovascular system. The rat experiment has demonstrated that mammalian embryos, at least during the last term of pregnancy, can develop in microgravity. This finding has been confirmed by fish studies. The experiment on germinating seeds and adult plants has given evidence that microgravity produces no effect on the metabolism of seedlings and on the flowering stage.     

The role of calcium ions in cytological effects of hypogravity

Electron-cytochemical and biochemical methods made it possible to reveal certain differences in ATPase activity stimulation by calcium ions in root apex cells of pea seedlings and moss protonema $\text{Funaria}$$\text{hygrometrica}$ grown under stationary and slow clinostatic (2 rev/min) conditions. It was showed that under clinostatic conditions in comparison with the control variant the ATPase activity decreases in plasmalemma. The protein content in the plasmalemma fraction was also twice as low under these conditions. The root apex cells of the pea seedlings grown under spaceflight conditions were found to contain high concentrations of membrane-bound calcium. The data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance and the system of active calcium ion transport through plasmalemma under hypogravity.     

Light microscopic analysis of the gravireceptor in Xenopus larvae developed in hypogravity.

The paper describes an investigation of the influence of gravity on the early differentiation of gravity receptors in Xenopus embryos and larvae. There is evidence that the expression of crystals in the saccus endolymphaticus was statistically greater when the embryos developed in near weightlessness (hypogravity) than on earth. The function of these crystals is unknown but they may contribute to the functioning of the vestibular apparatus.     

Evaluation of experiments involving the study of plant orientation and growth under different gravitational conditions.

The manifestation of gravitropic reaction in plants has been considered from the phylogenetic point of view. A chart has been suggested according to which it is supposed that the first indications of the ability to identify the direction of the gravitational vector were inherent in the most ancient eukaryotes, which gave rise to green, brown, yellow-green, golden and diatomaceous algae as well as fungi. The experiments on the role of gravity in plant ontogenesis are being continued. The sum total of the data obtained in a number of experiments in space shows that under these conditions a structurally modified but normally functioning gravireceptive apparatus is formed. The data confirming the modification, under changed gravity, of the processes of integral and cellullar growth of the axial organs of seedlings as well as of the anatomo-morphological structure and developmental rates of plants during their prolonged growth in space are presented. It is assumed that this fact testifies to the presence of systems interacting with gravity during plant ontogenesis. At the same time the necessity for further experiments in order to differentiate an immediate biological effect of gravity from the ones conditioned by it indirectly due to the changes in the behavior of liquids and gases is pointed out. The methodological aspects of biological experiments in space as the main source of reliable information on the biological role of gravity are discussed.