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.     

Spaceflight results in increase of thick filament but not thin filament proteins in the paramyosin mutant of Caenorhabditis elegans

We have investigated the effect of microgravity during spaceflight on body-wall muscle fiber size and muscle proteins in the paramyosin mutant of Caenorhabditis elegans. Both mutant and wild-type strains were subjected to 10 days of microgravity during spaceflight and compared to ground control groups. No significant change in muscle fiber size or quantity of the protein was observed in wild-type worms; where as atrophy of body-wall muscle and an increase in thick filament proteins were observed in the paramyosin mutant unc-15(e73) animals after spaceflight. We conclude that the mutant with abnormal muscle responded to microgravity by increasing the total amount of muscle protein in order to compensate for the loss of muscle function.     

微重力环境中哺乳动物细胞分子生物学效应研究进展

随着载人航天事业的不断发展,空间失重环境引起的航天员健康问题（心血管疾病、免疫抑制、肌肉萎缩、骨质疏松等）日益突出,这已成为人类探索空间的一大阻碍.越来越多的研究关注到微重力条件下机体及细胞的变化.近期的研究表明,在细胞水平上,微重力会引起细胞降解,改变细胞骨架,并造成细胞在分子水平（如细胞增殖、分化、迁移、粘附、信号转导等过程）的一系列改变.本文对微重力条件下免疫细胞、内皮细胞、骨细胞、癌细胞的相关研究进行了归纳总结,研究结果可为微重力条件下机体及相关细胞的研究提供指导,为治疗或缓解微重力条件造成的疾病提供方法和思路.      

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.     

Evaluation of upper body muscle activity during cardiopulmonary resuscitation performance in simulated microgravity

Performance of efficient single-person cardiopulmonary resuscitation (CPR) is vital to maintain cardiac and cerebral perfusion during the 2–4 min it takes for deployment of advanced life support during a space mission. The aim of the present study was to investigate potential differences in upper body muscle activity during CPR performance at terrestrial gravity (+1Gz) and in simulated microgravity (μG). Muscle activity of the triceps brachii, erector spinae, rectus abdominis and pectoralis major was measured via superficial electromyography in 20 healthy male volunteers. Four sets of 30 external chest compressions (ECCs) were performed on a mannequin. Microgravity was simulated using a body suspension device and harness; the Evetts–Russomano (ER) method was adopted for CPR performance in simulated microgravity. Heart rate and perceived exertion via Borg scores were also measured. While a significantly lower depth of ECCs was observed in simulated microgravity, compared with +1Gz, it was still within the target range of 40–50 mm. There was a 7.7% decrease of the mean (±SEM) ECC depth from 48 ± 0.3 mm at +1Gz, to 44.3 ± 0.5 mm during microgravity simulation (p < 0.001). No significant difference in number or rate of compressions was found between the two conditions. Heart rate displayed a significantly larger increase during CPR in simulated microgravity than at +1Gz, the former presenting a mean (±SEM) of 23.6 ± 2.91 bpm and the latter, 76.6 ± 3.8 bpm (p < 0.001). Borg scores were 70% higher post-microgravity compressions (17 ± 1) than post +1Gz compressions (10 ± 1) (p < 0.001). Intermuscular comparisons showed the triceps brachii to have significantly lower muscle activity than each of the other three tested muscles, in both +1Gz and microgravity. As shown by greater Borg scores and heart rate increases, CPR performance in simulated microgravity is more fatiguing than at +1Gz. Nevertheless, no significant difference in muscle activity between conditions was found, a result that is favourable for astronauts, given the inevitable muscular and cardiovascular deconditioning that occurs during space travel.     

Crustaceans as a model for microgravity-induced muscle atrophy.

Atrophy of skeletal muscles is a serious problem in a microgravity environment. It is hypothesized that the unloading of postural muscles, which no longer must resist gravity force, causes an accelerated breakdown of contractile proteins, resulting in a reduction in muscle mass and strength. A crustacean model using the land crab, Gecarcinus lateralis, to assess the effects of spaceflight on protein metabolism is presented. The model is compared to a developmentally-regulated atrophy in which a premolt reduction in muscle mass allows the withdrawal of the large claws at molt. The biochemical mechanisms underlying protein breakdown involves both Ca(2+)-dependent and multicatalytic proteolytic enzymes. Crustacean claw muscle can be used to determine the interactions between shortening and unloading at the molecular level.     

空间微重力条件下偏晶合金的研究

利用我国首次卫星搭载,进行了偏晶合金Zn—Pb、Al—Pb 的空间微重力下的重熔试验,研究了低于临界温度下Al—Pb 合金重熔过程中的物理规律;在临界温度之上重熔了Zn—Pb 合金,发现了Marangoni 对流对合金凝固过程的影响;在液态烧结条件下制备了均匀的粉末Zn—Pb 合金。在微重力下的研究表明:空间微重力条件对消除由于重力引起的宏观偏折和自然对流十分重要,但在重力消失后,非重力的其它因素的影响又显得格外重要。     

The pleurodele, an animal model for space biology studies.

Pleurodeles waltl, an Urodele amphibian is proposed as a model for space biology studies. Our laboratory is developing three types of experiments in space using this animal: 1) in vivo fertilization and development ("FERTILE" project); 2) influence of microgravity and space radiation on the organization and preservation of specialized structures in the neurons and muscle cells (in vitro; "CELIMENE" PROJECT); 3) influence of microgravity on tissue regeneration (muscle, bone, epidermis and spinal cord).     

Function of the cytoskeleton in gravisensing during spaceflight.

Since astronauts and cosmonauts have significant bone loss in microgravity we hypothesized that there would be physiological changes in cellular bone growth and cytoskeleton in the absence of gravity. Investigators from around the world have studied a multitude of bone cells in microgravity including Ros 17/2.8, Mc3T3-E1, MG-63, hFOB and primary chicken calvaria. Changes in cytoskeleton and extracellular matrix (ECM) have been noted in many of these studies. Investigators have noted changes in shape of cells exposed to as little as 20 seconds of microgravity in parabolic flight. Our laboratory reported that quiescent osteoblasts activated by sera under microgravity conditions had a significant 60% reduction in growth (p<0.001) but a paradoxical 2-fold increase in release of the osteoblast autocrine factor PGE2 when compared to ground controls. In addition, a collapse of the osteoblast actin cytoskeleton and loss of focal adhesions has been noted after 4 days in microgravity. Later studies in Biorack on STS-76, 81 and 84 confirmed the increased release of PGE2 and collapse of the actin cytoskeleton in cells grown in microgravity conditions, however flown cells under 1 g conditions maintained normal actin cytoskeleton and fibronectin matrix. The changes seen in the cytoskeleton are probably not due to alterations in fibronectin message or protein synthesis since no differences have been noted in microgravity. Multiple investigators have observed actin and microtubule cytoskeletal modifications in microgravity, suggesting a common root cause for the change in cell architecture. The inability of the O g grown osteoblast to respond to sera activation suggests that there is a major alteration in anabolic signal transduction under microgravity conditions, most probably through the growth factor receptors and/or the associated kinase pathways that are connected to the cytoskeleton. Cell cycle is dependent on the cytoskeleton. Alterations in cytoskeletal structure can block cell growth either in G1 (F-actin microfilament collapse), or in G2/M (inhibition of microtubule polymerization during G2/M-phase). We therefore hypothesize that microgravity would inhibit growth in either G1, or G2/M.     

In vitro plant cell growth in microgravity and on clinostat.

For the study of gravity's role in the processes of plant cell differentiation in-vitro, a model "seed-seedling-callus" has been used. Experiments were carried out on board the orbital stations Salyut-7 and Mir as well as on clinostat. They lasted from 18 to 72 days. It was determined that the exclusion of a one-sided action of gravity vector by means of clinostat and spaceflight conditions does not impede the formation and growth of callus tissue; however, at cell and subcellular levels structural and functional changes do take place. No significant changes were observed either on clinostat or in space concerning the accumulation of fresh biomass, while the percentage of dry material in space is lower than in control. Both in microgravity (MG) and in control, even after 72 days of growth, cells with a normally developed ultrastructure are present. In space, however, callus tissue more often contains cells in which the cross-section area of a cell, a nuclei and of mitochondria are smaller and the vacuole area--bigger than in controls. In microgravity a considerable decrease in the number of starch-containing cells and a reduction in the mean area of starch grains in amyloplasts is observed. In space the amount of soluble proteins in callus tissue is 1.5 times greater than in control. However, no differences were observed in fractions when separated by the SDS-PAGE method. In microgravity the changes in cell wall material components was noted. In the space-formed callus changes in the concentration of ions K, Na, Mg, Ca and P were observed. However, the direction of these changes depends on the age of callus. Discussed are the possible reasons for modification of morphological and metabolic parameters of callus cells when grown under changed gravity conditions.     

Calcium balance in pea root statocytes under both clinorotation and Ca2+ channel blockers' influence.

We have previously demonstrated that space flight and clinorotation conditions increase cytoplasmic Ca2+ level in pea root statocytes. A rise in [Ca2+]i may be a serious problem for plants in microgravity environment. It is hypothesized that involvement of Ca2+ channel blockers in the growth medium may rescue a plant from abundance of Ca2+ ions. Indeed, combination of clinorotation (2 rpm, 5 days) and any Ca2+ channel blocker (1 micromole D600 or nicardipine, 12 hr) causes decreasing the Ca2+ concentration in pea root statocytes in comparison with clinorotation alone. Redistribution of Ca(2+)-ATPase activities observed under clinorotation comes to normal after D600 application whereas following by nicardipine action the pattern of the cytochemical staining is intermediate between those in stationary control and under clinorotation. Our data support the hypothesis that Ca2+ channel blockers may act as protectors for plants against rise in [Ca2+]i. The role for Ca2+ channels in graviperception and in microgravity effects as well as ways for stabilization of Ca2+ balance in plant cells in space flights are discussed.     

Microgravity effects on "postural" muscle activity patterns.

Changes in neuromuscular activation patterns associated with movements made in microgravity can contribute to muscular atrophy. Using EMG to monitor "postural" muscles, it was found that free floating arm flexions made in microgravity were not always preceded by neuromuscular activation patterns normally observed during movements made in unit gravity. Additionally, manipulation of foot sensory input during microgravity arm flexion impacted upon anticipatory postural muscle activation.     

The mechanics of air-breathing in anuran larvae: implications to the development of amphibians in microgravity.

Because of their rapid development, amphibians have been important model organisms in studies of how microgravity affects vertebrate growth and differentiation. Both urodele (salamanders) and anuran (frogs and toads) embryos have been raised in orbital flight, the latter several times. The most commonly reported and striking effects of microgravity on tadpoles are not in the vestibular system, as one might suppose, but in their lungs and tails. Pathological changes in these organs disrupt behavior and retard larval growth. What causes malformed (typically lordotic) tadpoles in microgravity is not known, nor have axial pathologies been reported in every flight experiment. Lung pathology, however, has been consistently observed and is understood to result from the failure of the animals to inflate their lungs in a timely and adequate fashion. We suggest that malformities in the axial skeleton of tadpoles raised in microgravity are secondary to problems in respiratory function. We have used high speed videography to investigate how tadpoles breathe air in the 1G environment. The video images reveal alternative species-specific mechanisms, that allow tadpoles to separate air from water in less that 150 ms. We observed nothing in the biomechanics of air-breathing in 1G that would preclude these same mechanisms from working in microgravity. Thus our kinematic results suggest that the failure of tadpoles to inflate their lungs properly in microgravity is due to the tadpoles' inability to locate the air-water interface and not a problem with the inhalation mechanism per se.     

Alkylating agent (MNU)-induced mutation in space environment.

In recent years, some contradictory data about the effects of microgravity on radiation-induced biological responses in space experiments have been reported. We prepared a damaged template DNA produced with an alkylating agent (N-methyl-N-nitroso urea; MNU) to measure incorrect base-incorporation during DNA replication in microgravity. We examined whether mutation frequency is affected by microgravity during DNA replication for a DNA template damaged by an alkylating agent. Using an in vitro enzymatic reaction system, DNA synthesis by Taq polymerase or polymerase III was done during a US space shuttle mission (Discovery, STS-91). After the flight, DNA replication and mutation frequencies were measured. We found that there was almost no effect of microgravity on DNA replication and mutation frequency. It is suggested that microgravity might not affect at the stage of substrate incorporation in induced-mutation frequency.     

模拟失重对培养心肌细胞形态和结构的影响

本实验是利用回转器模拟失重对离体培养大鼠乳鼠的心肌细胞形态和结构的影响．在光学显微镜和荧光显微镜下观察发现,细胞的形态由细长梭形变成椭圆形甚至为圆形,并且通过荧光标记后的细胞骨架的排列由纵形变成辐射状．同时在对细胞进行测量发现,细胞体积缩小近40％,细胞长短径比例减少近70％．上述结果提示模拟失重对培养心肌细胞的形态和结构有显著影响．     

Inhibitory effect of simulated microgravity on differentiating preosteoblasts

The bone loss induced by microgravity is partly due to the decrease of mature osteoblasts. In the present study, we employed the random positioning machine (RPM) to simulate microgravity and investigated the acute effects of simulated microgravity on the differentiation of 2T3 preosteoblasts. Following 7 days’ culture under normal (1 g) condition, cells were exposed to simulated microgravity for 24 h. The results showed that 24 h treatment of simulated microgravity significantly decreased alkaline phosphatase (ALP) activity without changing the cell morphology. In addition, the mRNA expressions of osteogenic genes, including runt-related gene 2 (Runx2), osterix, osteocalcin (OC), type I collagen (Col I) and bone morphogenetic protein (BMP), were dramatically downregulated. Moreover, western blot analysis of total extracellular signal-regulated kinase (Erk) and phosphorylated Erk (p-Erk) indicated that p-Erk level, which represents the Erk activation status, was increased. Taken together, our results suggested that acute exposure to simulated microgravity inhibited osteoblast differentiation through modulating the expression of osteogenic genes and the Erk activity. These findings provide new insight for bone loss due to microgravity and unloading.     

Structural and functional organisation of regenerated plant protoplasts exposed to microgravity on Biokosmos 9.

Preparatory experiments for the IML-1 mission using plant protoplasts, were flown on a 14-day flight on Biokosmos 9 in September 1989. Thirty-six hours before launch of the biosatellite, protoplasts were isolated from hypocotyl cells of rapeseed (Brassica napus) and suspension cultures of carrot (Daucus carota). Ultrastructural and fluorescence analysis of cell aggregates from these protoplasts, cultured under microgravity conditions, have been performed. In the flight samples as well as in the ground controls, a portion of the total number of protoplasts regenerated cell walls. The processes of cell differentiation and proliferation under micro-g did not differ significantly from those under normal gravity conditions. However, in micro-g differences were observed in the ultrastructure of some organelles such as plastids and mitochondria. There was also an increase in the frequency of the occurrence of folds formed by the plasmalemma together with an increase in the degree of complexity of these folds. In cell cultures developed under micro-g conditions, the calcium content tends to decrease, compared to the ground control. Different aspects of using isolated protoplasts for clarifying the mechanisms of biological effects of microgravity are discussed.     

Melting and solidification of metallic composites in space

Metallic matrix composites are a relatively new type of strengthened metals. Other properties such as wear resistance or thermal and electrical conductivity can also be modified. One can distinguish solid phase and liquid phase fabrication methods. The latter need only a relatively simple equipment and are particularly suited for complex shapes. The interfacial phenomena, the arrangement of the dispersed particles or fibres and the solidification behaviour of the matrix have to be understood in order to enhance the properties of the composite. The microgravity environment of space drastically influences several phenomena occurring during fabrication such as the fluid motion in the liquid matrix and the transport of the solid particles or fibres. The results of our space experiments in SL1, D1, TEXUS 6,7 and 9 on copper and aluminium based composites are summarized in this context. Two topics are treated more in detail, namely the role of interfacial energies and the expulsion of particles by the solidification front. Further, the relevance of space processing is illustrated for oxide dispersion strengthened metals. Finally the constraints of previous experiments and suggestions for future research are mentioned.     

微重力池沸腾传热研究

对利用中国返回式卫星搭载开展的两次微重力池沸腾空间实验及地基常重力和落塔短时微重力实验的结果进行了评述. 研究发现微重力时丝状加热器沸腾传热会略有强化, 而平板加热器则在高热流条件下明显恶化. 微重力时, 气泡脱落前存在沿加热面的横向运动, 加剧了相邻气泡间的合并, 合并气泡会在其表面振荡作用下从加热面脱落. Marangoni 效应对于微重力气泡行为有重要影响.      

Crystallization of calcium phosphate in microgravity.

Dilute solutions of CaCl2 and KH2PO4 + K2HPO4 were diffusing from either side into a mixing chamber with KCl solution. The microgravity experiment yielded aggregates of large crystals of OCP (Ca8H2(PO4)3,5H2O) and spherolites of smaller, but still visible crystals of HAP (Ca5OH(PO4)3), the stable final phase. Ground-based experiments yielded submicroscopic HAP crystals. Results of calculations of diffusion and crystal growth on the basis of previous knowledge agree well with observations.