Formation and vesiculation of biomembranes during spaceflight.

Shuttle flight, sounding rocket flight, and parabolic flight experiments demonstrate the formation of bilayer membrane vesicles (liposomes) in reduced gravity, following the dilution of detergent from detergent-phospholipid mixed micelles. The reduction in detergent concentration initiates assembly of bilayer membrane sheets, which are sensitive to solution disturbances. An increase in disturbances by forced dilution results in small diameter liposomes (< 150 nm), in both ground and flight samples. In the absence of forced dilution, liposomes remain small at 1-g, but exhibit much larger diameters at 0-g (1000-2000 nm). Our spaceflight data reveal that membrane assembly and vesiculation are strongly influenced by gravity-induced solution disturbances (e.g., convection currents), which limit vesicle diameter.     

Studies of the interaction of gravity with biological membranes using alamethicin doped planar lipid bilayers as a model system.

Gravity interacts with biological systems on different levels of complexity. For the understanding of the action of gravity on such systems at higher degrees of organisation, the investigation of interactions on the membrane and even on the molecular level is crucial. To do such studies, planar lipid bilayers with incorporated transport mediating molecules, i.e. membranes of defined biochemical composition, are close to perfect model systems. In our experiments we have used painted planar lipid bilayers doped with alamethicin. Alamethicin is especially suitable for such studies because of its high sensitivity to applied external forces, which is a result of its special pore forming mechanism. Additional, different to most other transport mediating molecules, a big amount of data from the literature is available about the dependency of alamethicin pores on physical and chemical membrane parameters. We found that the conductance of alamethicin doped bilayers is dependent on the angle of the bilayer with the gravitational vector and that it furthermore can be reduced significantly under hyper gravity conditions in a centrifuge. The effect of gravity here is not an effect on the pore conductance or the membrane-aqueous solution interface, but it is due to an interaction of gravity with the pore forming mechanism, as can be shown by investigating the dependency of the alamethicin pore kinetics on the applied forces.     

Gravity effects on biological systems.

Theoretical investigations of the membrane-solution interface predict different effects of gravity on vertically and horizontally oriented planar membranes. Single channel events of gramicidin incorporated into phosphatidylserine planar bilayer membranes were measured in 0.1 M KCl solution, pH 7, at room temperature. The potential difference across the membrane was set to +/- 70 mV. The mean channel current was observed to be about 20% higher in horizontally oriented membranes compared to vertical membranes. This is in good agreement with the theoretical considerations and demonstrates that gravity does affect membrane processes by interaction with the membrane-solution interface which is a ubiquitous structure in biological systems.     

Transient effects of microgravity on early embryos of Xenopus laevis.

In order to study the role of gravity on the early development of the clawed toad Xenopus laevis, we performed an experiment on the Maser-6 sounding rocket launched from Kiruna (Sweden) on 4 Nov 1993. The aim was to find out whether a short period of microgravity during fertilization and the first few minutes of development does indeed result in abnormal axis formation as was suggested by a pilot experiment on the Maser 3 in 1989. On the Maser 6 we used two new technical additions in the Fokker CIS unit, viz. a 1-g control centrifuge and a video recording unit which both worked successfully. The 1-g control centrifuge was used to discriminate between the influences of flight perturbations and microgravity. After fertilization shortly before launch, one of the first indications of successful egg activation, the cortical contraction, was registered in microgravity and on earth. Analysis of the video tapes revealed that the cortical contraction in microgravity starts earlier than at 1 g on earth. After recovery of the eggs fertilized in microgravity and culture of the embryos on earth, the morphology of the blastocoel has some consistent differences from blastulae from eggs fertilized in the 1-g centrifuge of the rocket. However from the gastrula stage onward, the microgravity embryos apparently recover and resume normal development: the XBra gene is normally expressed, and histological examination shows normal axis formation.     

Effects of spaceflight (STS-87) on tropisms and plastid positioning in protonemata of the moss Ceratodon purpureus.

Apical cells of moss protonemata represent a single-celled system that perceives and reacts to light (positive and negative phototropism) and to gravity (negative gravitropism). Phototropism completely overrides gravitropism when apical cells are laterally irradiated with relatively high red light intensities, but below a defined light intensity threshold gravitropism competes with the phototropic reaction. A 16 day-long exposure to microgravity conditions demonstrated that gravitropism is allowed when protonemata are laterally illuminated with light intensities below 140 nmol m-2s-1. Protonemata that were grown in darkness in microgravity expressed an endogenous tendency to grow in arcs so that the overall culture morphology resembled a clockwise spiral. However this phenomenon only was observed in cultures that had reached a critical age and/or size. Organelle positioning in dark-grown apical cells was significantly altered in microgravity. Gravisensing most likely involves the sedimentation of starch-filled amyloplasts in a well-defined area of the tip cell. Amyloplasts that at 1-g are sedimented were clustered at the apical part of the sedimentation zone in microgravity. Clustering observed in microgravity or during clino-rotation significantly differs from sedimentation-induced plastid aggregations after inversion of tip cells at 1-g.     

Leaf senescence under various gravity conditions: relevance to the dynamics of plant hormones.

Effects of simulated microgravity and hypergravity on the senescence of oat leaf segments excised from the primary leaves of 8-d-old green seedlings were studied using a 3-dimensional (D) clinostat as a simulator of weightlessness and a centrifuge, respectively. During the incubation with water under 1-g conditions at 25 degrees C in the dark, the loss of chlorophyll of the segments was found dramatically immediately after leaf excision, and leaf color completely turned to yellow after 3-d to 4-d incubation. In this case kinetin (10 micromolar) was effective in retarding senescence. The application of simulated microgravity conditions on a 3-D clinostat enhanced chlorophyll loss in the presence or absence of kinetin. The loss of chlorophyll was also enhanced by hypergravity conditions (ca. 8 to 16 g), but the effect was smaller than that of simulated microgravity conditions on the clinostat. Jasmonates (JAs) and abscisic acid (ABA) promoted senescence under simulated microgravity conditions on the clinostat as well as under 1-g conditions. After 2-d incubation with water or 5-d incubation with kinetin, the endogenous levels of JAs and ABA of the segments kept under simulated microgravity conditions on the clinostat remained higher than those kept under 1-g conditions. These findings suggest that physiological processes of leaf senescence and the dynamics of endogenous plant hormone levels are substantially affected by gravity.     

Some recent data on chemical protection against ionizing radiation.

Once introduced in the organism, the radioprotectors are fastly degraded and that increases their toxicity, shortens their duration of action and renders them inactive after oral delivery. So, it was tried to protect them by their incorporation in vectors. When a cysteamine-liposomal suspension was orally delivered, it showed a radioprotective activity for about 4 hours. By using 35S cysteamine, it was noted that its plasmatic concentration was increased. Freeze-drying of these preparations was a good mean of conservation if the samples were stored at 4 degrees C. A good and sustained activity was also obtained after oral delivery of WR-2721 entrapped in microspheres. Otherwise, it was shown that after interacting with the polar heads of phospholipids, under determined conditions of pH and in fluid phase, aminothiols can penetrate inside the membrane and be entrapped in the internal medium of liposomes and as they penetrate, they can lessen the diffusion of oxygen in the lipidic bilayers.     

Studies on precellular evolution: the encapsulation of polyribonucleotides by liposomes.

Liposomes are 5 to 50 micron vesicles with an internal aqueous environment, whose amphiphilic lipidic components self-assemble into systems with at least one double-layered membrane. Liposomes have been suggested as possible models of precellular systems formed in the early Archean Earth from lipids of non-enzymatic origin. Since it is generally accepted that RNA molecules preceded double-stranded DNA molecules as genetic material, we have studied the encapsulation of polyribonucleotides within liposomes made from dipalmitoyl phosphatidylcholine, and from egg yolk phosphatidylcholine to which cholesterol was added in some cases. The liposomes were prepared under anoxic conditions following the reverse phase evaporation method described by Szoka and Papahadjopoulos. Quantitative determinations show that approximately 50% of the available lipids form liposomes, and that up to 5% of the polyribonucleotides can be entrapped by them. We have also studied the encapsulation of polyribonucleotides in the presence of 1) urea and cyanamide, two non-electrolytes that have been used as prebiotic condensing agents, and 2) of Zn++ and Pb++, two cations employed in the non-enzymatic template-directed synthesis of polyribonucleotides from activated nucleotides.     

Calcium signaling in plant cells in altered gravity.

Changes in the intracellular Ca2+ concentration in altered gravity (microgravity and clinostating) evidence that Ca2+ signaling can play a fundamental role in biological effects of microgravity. Calcium as a second messenger is known to play a crucial role in stimulus-response coupling for many plant cellular signaling pathways. Its messenger functions are realized by transient changes in the cytosolic ion concentration induced by a variety of internal and external stimuli such as light, hormones, temperature, anoxia, salinity, and gravity. Although the first data on the changes in the calcium balance in plant cells under the influence of altered gravity have appeared in 80th, a review highlighting the performed research and the possible significance of such Ca2+ changes in the structural and metabolic rearrangements of plant cells in altered gravity is still lacking. In this paper, an attempt was made to summarize the available experimental results and to consider some hypotheses in this field of research. It is proposed to distinguish between cell gravisensing and cell graviperception; the former is related to cell structure and metabolism stability in the gravitational field and their changes in microgravity (cells not specialized to gravity perception), the latter is related to active use of a gravitational stimulus by cells presumebly specialized to gravity perception for realization of normal space orientation, growth, and vital activity (gravitropism, gravitaxis) in plants. The main experimental data concerning both redistribution of free Ca2+ ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravisensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane surface tension --> alterations in the physicochemical properties of the membrane --> changes in membrane permeability, --> ion transport, membrane-bound enzyme activity, etc. --> metabolism rearrangements --> physiological responses. An analysis of data available on biological effects of altered gravity at the cellular level allows one to conclude that microgravity environment appears to affect cytoskeleton, carbohydrate and lipid metabolism, cell wall biogenesis via changes in enzyme activity and protein expression, with involvement of regulatory Ca2+ messenger system. Changes in Ca2+ influx/efflux and possible pathways of Ca2+ signaling in plant cell biochemical regulation in altered gravity are discussed.     

Free and membrane-bound calcium in microgravity and microgravity effects at the membrane level.

The changes of [Ca2+]i controlled is known to play a key regulatory role in numerous cellular processes especially associated with membranes. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown aboard orbital station "Salyut 6". These results: 1) indicate that observed Ca(2+)-binding sites of membranes also consist in proteins and phospholipids; 2) suggest that such effects of space flight in membrane Ca-binding might be due to the enhancement of Ca2+ influx through membranes. In model presented, I propose that Ca(2+)-activated channels in plasma membrane in response to microgravity allow the movement of Ca2+ into the root cells, causing a rise in cytoplasmic free Ca2+ levels. The latter, in its turn, may induce the inhibition of a Ca2+ efflux by Ca(2+)-activated ATPases and through a Ca2+/H+ antiport. It is possible that increased cytosolic levels of Ca2+ ions have stimulated hydrolysis and turnover of phosphatidylinositols, with a consequent elevation of cytosolic [Ca2+]i. Plant cell can response to such a Ca2+ rise by an enhancement of membranous Ca(2+)-binding activities to rescue thus a cell from an abundance of a cytotoxin. A Ca(2+)-induced phase separation of membranous lipids assists to appear the structure nonstable zones with high energy level at the boundary of microdomains which are rich by some phospholipid components; there is mixing of molecules of the membranes contacted in these zones, the first stage of membranous fusion, which was found in plants exposed to microgravity. These results support the hypothesis that a target for microgravity effect is the flux mechanism of Ca2+ to plant cell.     

Evaluation of using two-phase frictional pressure drop correlations for normal gravity to microgravity and reduced gravity

The calculation of two-phase frictional pressure drop (TPFPD) is required by two-phase systems operating under microgravity and reduced gravity. There are a large number of correlations for the TPFPD in tubes under normal gravity. However, it is hard to find out a TPFPD correlation obtained from microgravity and/or reduced gravity conditions, and thus people have to use TPFPD correlations for normal gravity to calculate TPFPD under microgravity and reduced gravity. It is necessary to evaluate the feasibility of such practice. This paper offers a comprehensive review of the TPFPD correlations for normal gravity and an up-to-data survey of the TPFPD experimental study under microgravity and reduced gravity. There are 23 TPFPD correlations for normal gravity reviewed and 135 experimental data under microgravity obtained from the literature. These experimental data are used to evaluate the reviewed TPFPD correlations. It is found that the smallest mean absolute relative deviation (MARD) of the correlations is greater than 34%. Using TPFPD correlations for normal gravity to reduced gravity and microgravity may be acceptable for the first approximation, but correlations intended for microgravity and reduced gravity are needed and more experiments are desired to obtain more data with high accuracy.     

Changes in membrane lipid composition in azuki bean epicotyls under hypergravity conditions: Possible role of membrane sterols in gravity resistance

Seedlings of azuki bean (Vigna angularis Ohwi et Ohashi) were cultivated under hypergravity conditions, and changes in membrane lipid composition in their epicotyls were analyzed. Under hypergravity conditions at 300g, the levels of total sterols, phospholipids, and fatty acids per fresh weight were kept higher, as compared with 1g controls. In particular, sterol levels were prominently increased by hypergravity. On the other hand, hypergravity did not clearly influence the levels of each phospholipid and glycolipid class, or their fatty acid compositions. Thus, the effect of hypergravity on membrane lipid metabolism was specific for sterol biosynthesis. In various regions of azuki epicotyls, high growth rate was associated with high sterol levels. Hypergravity suppressed elongation growth and stimulated lateral expansion of azuki epicotyls. In the presence of lovastatin, an inhibitor of sterol biosynthesis, at 30 μM, such changes in growth parameters occurred even under 1g conditions, suggesting that lovastatin made epicotyls hypersensitive to the gravitational force. These results support the hypothesis that membrane sterols are involved in maintenance of normal growth capacity of plant organs against gravity.     

Simulated microgravity does not alter epithelial cell adhesion to matrix and other molecules.

Microgravity has advantages for the cultivation of tissues with high fidelity; however, tissue formation requires cellular recognition and adhesion. We tested the hypothesis that simulated microgravity does not affect cell adhesion. Human colorectal carcinoma cells were cultured in the NASA Rotating Wall Vessel (RWV) under low shear stress with randomization of the gravity vector that simulates microgravity. After 6-7 days, cells were assayed for binding to various substrates and compared to cells grown in standard tissue culture flasks and static suspension cultures. The RWV cultures bound as well to basement membrane proteins and to CEA, an intercellular adhesion molecule, as control cultures did. Thus, microgravity does not alter epithelial cell adhesion and may be useful for tissue engineering.     

Enzyme activities and membrane lipids in Artemia cysts after a long duration space flight

In the Free Flyer Biostack Experiment (L.D.E.F. mission) investigations have shown that biological objects in a resting state can survive more than 5.5 years of exposure to the space factors in particular microgravity and cosmic rays. We have measured enzyme activities involved in metabolic pathways of sugar and lipid degradation and determined phospholipid composition. Pyruvate kinase and glucose-6-phosphate dehydrogenase activities in space-exposed cysts were higher than in earth controls after 1 hour incubation. In controls, total phospholipids remained unchanged, on the contrary they increased significantly in space-exposed cysts. The rate of metabolism of various phospholipid components was unchanged in controls allowing the development while the level of most of them decreased in space-exposed cysts except for phosphatidylcholine. Enzyme activities (acetylhydrolase, phospholipase A₂ and lyso phospholipase) involved in phospholipid degradation increased; however, activities were much higher in space-exposed cysts. In conclusion, the long duration space flight resulted in an increase of the metabolic activity correlated with a faster development within the first 20 hours of post flight incubation.     

Kinetics of amyloplast movement in cress root statocytes under different gravitational loads.

In order to investigate the movement of a statolith complex along the longitudinal axis of root cap statocytes under different mass accelerations, a series of experiments with Lepidium sativum L. in an automatically operating centrifuge during the Bion-11 satellite flight and on a centrifuge-clinostat have been performed. During spaceflight, roots were grown for 24 h under root-tip-directed centrifugal 1-g acceleration, then exposed to microgravity for 6, 12 and 24 min and chemically fixed. During the first 6 min of microgravity, the statoliths moved towards the cell center with a mean velocity of 0.31 +/- 0.04 micrometers/min, which decreased to 0.12 +/- 0.01 micrometers/min within subsequent 12-24 min period. The mean relative position of the statolith complex in respect to the distal cell wall (% of total cell length) increased from 24.0 +/- 0.5% in 1 g-grown roots to 38.8 +/- 0.8% in roots exposed for 24 min to microgravity, but remained smaller than in roots grown continuously in microgravity (48.0 +/- 0.7%). The properties of the statolith movement away from the distal pole of the statocyte were studied in roots grown for 24 h vertically under 1 g and then placed for 6 min on a fast rotating clinostat (50 rpm) or 180 degrees inverted. After 2 min of both treatments, the mean relative position of the statoliths increased by about 10% versus its initial position. Later on, the proximal displacement of amyloplasts slowed down under simulated weightlessness, while it proceeded at a constant velocity under 1 g inversion. In roots grown on the clinostat and then exposed to 1 g in the longitudinal direction, amyloplast sedimentation away from the central region of statocyte was similar at the beginning of distal and proximal 6-min 1-g stimulation. However, at the end of this period statolith displacement was more pronounced in proximal direction as compared to distal. It is proposed that statolith position in the statocyte of a vertical root is controlled by the force of gravity, however, the intracellular forces, first of all those generated by the network of the cytoskeleton, are manifested when an usual orientation of the organ is changed or the statocytes are exposed to microgravity and clinorotation.     

Urodelean amphibians in studies on microgravity: effects upon organ and tissue regeneration.

Results obtained from nine experiments performed onboard Russian biosatellites have shown that microgravity promotes tissue regeneration in the newt, Pleurodeles waltl. The effect has been reproduced in all flights and on a clinostat as well for eye tissues (lens and retina), limbs and tail. The effect was demonstrated in 1.5- to 2-fold increase in cell proliferation in the early stages of regeneration in space flight. Animals "flown" intact and operated after flight regenerated faster than control ones and showed long-lasting micro-"g" effect. The most recent experiment flew aboard the Bion-11 biosatellite. This test was performed for study on microgravity effect on neural retina regeneration after optic nerve lesioning in the newt. Obtained results confirmed our previous information about intensification of regenerative processes in detached neural retina in urodela exposed to simulated weightlessness (Grigoryan et al., 1998). In particular, we found the increase and activation of cell populations participating in neural retina restoration and maintenance of retinal structure. Our findings suggest that promoting effect of microgravity upon regeneration could be influenced by several factors, largely influenced by a response of the whole organism to changed gravity vector. We hypothesized the synthesis of the specific range of stress proteins induced by micro-"g" and their regulative role in cell proliferation. Such a hypothesis for the existence of "altered gravity stress proteins" is discussed.     

Long-term effects of microgravity and possible countermeasures.

It is well known that long-term exposure to microgravity causes a number of physiological and biochemical changes in humans; among the most significant are: 1) negative calcium balance resulting in the loss of bone; 2) atrophy of antigravity muscles; 3) fluid shifts and decreased plasma volume; and 4) cardiovascular deconditioning that leads to orthostatic intolerance. It is estimated that a mission to Mars may require up to 300 days in a microgravity environment; in the case of an aborted mission, the astronauts may have to remain in reduced gravity for up to three years. Although the Soviet Union has shown that exercise countermeasures appear to be adequate for exposures of up to one year in space, it is questionable whether astronauts could or should have to maintain such regimes for extremely prolonged missions. Therefore, the NASA Life Sciences Division has initiated a program designed to evaluate a number of methods for providing an artificial gravity environment.     

The ASTROCULTURE(TM) flight experiment series, validating technologies for growing plants in space.

A flight experiment, ASTROCULTURE(TM)-1 (ASC-1), to evaluate the operational characteristics and hardware performance of a porous tube nutrient delivery system (PTNDS) was flown on STS-50 as part of the U.S. Microgravity Laboratory-1 mission, 25 June to 9 July, 1992. This experiment is the first in a series of planned ASTROCULTURE(TM) flights to validate the performance of subsystems required to grow plants in microgravity environments. Results indicated that the PTNDS was capable of supplying water and nutrients to plants in microgravity and that its performance was similar in microgravity to that in 1g on Earth. The data demonstrated that water transfer rates through a rooting matrix are a function of pore size of the tubes, the degree of negative pressure on the 'supply' fluid, and the pressure differential between the 'supply' and 'recovery' fluid loops. A slightly greater transfer rate was seen in microgravity than in 1g, but differences were likely related to the presence of hydrostatic pressure effects at 1g. Thus, this system can be used to support plant growth in microgravity or in partial gravity as on a lunar or Mars base. Additional subsystems to be evaluated in the ASTROCULTURE(TM) flight series of experiments include lighting, humidity control and condensate recovery, temperature control, nutrient composition control, CO2 and O2 control, and gaseous contaminant control.     

微重力下胶原蛋白纤维化及羟基磷灰石结晶的初步研究

在长期空间飞行过程中, 骨质丢失是一个严重问题. 羟基磷灰石(HAP)晶体是骨骼的主要成分, 骨骼中的胶原蛋白纤维在HAP生长结晶过程中起到关键作用. 研究了胶原蛋白纤维化过程在模拟微重力和常重力条件下的变化, 对以胶原 蛋白纤维作为模板生长出的HAP晶体形貌进行了观察. 结果表明, 不同浓度胶原蛋白溶液中形成的胶原蛋白纤维, 其内部孔隙数量和尺寸在模拟微重力条件下要明显大于常重力条件下, 胶原蛋白纤维内部孔隙的分布也不同于常重力条 件下的结果. 以模拟微重力条件下形成的胶原蛋白纤维为模板生长出的HAP 晶体主要为立方体状, 而以常重力条件下形成的胶原蛋白纤维为模板生长出的 HAP晶体形貌主要为板状. 该结果有助于未来进一步阐明空间骨质丢失的机理.      

Physical parameters affecting living cells in space

The question is posed: Why does a living cell react to the absence of gravity? What sensors may it have? Does it note pressure, sedimentation, convection, or other parameters?

If somewhere in a liquid volume sodium ions are replaced by potassium ions, the density of the liquid changes locally: the heavier regions sink, the lighter regions rise. This may contribute to species transport, to the metabolism. Under microgravity this mechanism is strongly reduced. On the other hand, other reasons for convection like thermal and solutal interface convection are left. Do they affect species transport?

Another important effect of gravity is the hydrostatic pressure. On the macroscopic side, the pressure between our head and feet changes by 0.35 atmospheres. On the microscopic level the hydrostatic pressure on the upper half of a cell membrane is lower than on the lower half. This, by affecting the ion transport through the membrane, may change the surrounding electric potential. It has been suggested to be one of the reasons for graviperception.

Following the discussion of these and other effects possibly important in life sciences in space, an order of magnitude analysis of the residual accelerations tolerable during experiments in materials sciences is outlined. In the field of life sciences only rough estimates are available at present.