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.      

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.     

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.     

Protein crystal growth--microgravity aspects.

Protein crystals, grown under reduced gravity conditions, are either superior or inferior in their structural perfection than their Earth-grown counterparts. A reduction of the crystals' quality due to low-gravity effects on the growth processes cannot be understood from existing models. In this paper we put forth a rationale which predicts either advantages or disadvantages of microgravity growth. This rationale is based on the changes in the effective solute and impurity supply rates in microgravity and their effects on the intrinsic growth rate fluctuations that arise from the coupling of bulk transport to nonlinear interfacial kinetics and cause severe inhomogeneities. Depending on the specific diffusivity and kinetic coefficient of a protein and the impurities in the solution, either transport enhancement through forced flow or transport suppression under reduced gravity can result in a reduction of the kinetic fluctuations and, thus, growth with higher structural perfection. Investigating this mechanism of microgravity effects, we first demonstrate a one-to-one correspondence between these fluctuations, that are due to the bunching of growth steps, and the formation of defects in the crystals. We have confirmed the forced flow aspects of this rationale in ground-based experiments with lysozyme utilizing flowing solutions with varying, well characterized impurity contents.     

Spaceflight experiments with Arabidopsis starch-deficient mutants support a statolith-based model for graviperception.

In order to help resolve some of the controversy associated with ground-based research that has supported the starch-statolith theory of gravity perception in plants, we performed spaceflight experiments with Arabidopsis in Biorack during the January 1997 and May 1997 missions of the Space Shuttle. Seedlings of wild-type (WT) Arabidopsis, two reduced-starch strains, and a starchless mutant were grown in microgravity and then were given either a 30, 60, or 90 minute gravity stimulus on a centrifuge. By the 90 min 1-g stimulus, the WT exhibited the greatest magnitude of curvature and the starchless mutant exhibited the smallest curvature while the two reduced starch mutants had an intermediate magnitude of curvature. In addition, space-grown plants had two structural features that distinguished them from the controls: a greater number of root hairs and an anomalous hypocotyl hook structure. However, the morphological changes observed in the flight seedlings are likely to be due to the effects of ethylene present in the spacecraft. (Additional ground-based studies demonstrated that this level of ethylene did not significantly affect gravitropism nor did it affect the relative gravitropic sensitivity among the four strains.) Nevertheless, this experiment on gravitropism was performed the "right way" in that brief gravitational stimuli were provided, and the seedlings were allowed to express the response without further gravity stimuli. Our spaceflight results support previous ground-based studies of these and other mutants since increasing amounts of starch correlated positively with increasing sensitivity to gravity.     

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.     

The effect of gravity on plant germination.

An axis clinostat was constructed to create micro and negative gravity also a rotated flat disk was constructed with different rotation rates to give increased gravity, by centrifugal force up to 48 g. Rice seeds were grown on agar in tubes at the constant air temperature of 20 degrees C under an average light condition of 110 micromol/m2/sec(PPF). Humidity was not controlled but was maintained above 90%. Since the tube containers were not large enough for long cultivation, shoot and root growth were observed every 12 hours until the sixth day from seeding. The lengths of shoots and roots for each individual plant were measured on the last day. The stem lengths were increased by microgravity but the root lengths were not. Under the negative gravity, negative orthogeotropism and under microgravity, diageotropism was observed. No significant effect of increased gravity was observed on shoot and root growth.     

Skeletogenesis in sea urchin larvae under modified gravity conditions.

From many points of view, skeletogenesis in sea urchins has been well described. Based on this scientific background and considering practical aspects of sea urchin development (i.e. availability of material, size of larvae, etc.), we wanted to know whether orderly skeletogenesis requires the presence of gravity. The objective has been approached by three experiments successfully performed under genuine microgravity conditions (in the STS-65 IML-2 mission of 1994; in the Photon-10 IBIS mission of 1995 and in the STS-76 S/MM-03 mission of 1996). Larvae of the sea urchin Sphaerechinus granularis were allowed to develop in microgravity conditions for several days from blastula stage onwards (onset of skeletogenesis). At the end of the missions, the recovered skeletal structures were studied with respect to their mineral composition, architecture and size. Live larvae were also recovered for post-flight culture. The results obtained clearly show that the process of mineralisation is independent of gravity: that is, the skeletogenic cells differentiate correctly in microgravity. However, abnormal skeleton architectures were encountered, particularly in the IML-2 mission, indicating that the process of positioning of the skeletogenic cells may be affected, directly or indirectly, by environmental factors, including gravity. Larvae exposed to microgravity from blastula to prism/early pluteus stage for about 2 weeks (IBIS mission), developed on the ground over the next 2 months into normal metamorphosing individuals.     

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.     

Transport phenomena and crystal growth of the GeSe-Xenon system for different gravitational conditions

Previous chemical vapor transport experiments of the GeSe-GeI₄ system performed under reduced gravity conditions /1/ yielded crystals of considerably improved surface and bulk morphology. In addition, the mass transport rates observed in microgravity environment were significantly greater than predicted. A quantitative thermodynamic analysis of the solid-gas phase reactions of the GeSe-GeI₄ system revealed the multi-component, multi-reaction nature of the vapor phase /2/. Continued transport studies on ground of the GeSe-GeI₄ system in the presence of inert gases provided experimental evidence for the existence of a boundary layer /2/ and its thickness dependence on GeI₄ pressure in closed tube systems. Systematic transport rate measurements for different orientations of the density gradient relative to the gravity vector demonstrated the effects of ampoule inclination on mass flux /3/. Based on a computational model for simultaneous chemical vapor transport, sublimation, and Stefan flow /3/, the excellent agreement of predicted with ground-based experimental mass transport rates over wide pressure ranges /3/ confirmed the validity of the model and the discrepancy between observed and expected transport rates of the GeSe-GeI₄ system in microgravity.     

Signs of Müller cell gliotic response found in the retina of newts exposed to real and simulated microgravity

The effects of real and simulated microgravity on the eye tissue regeneration of newts were investigated. For the first time changes in Müller glial cells in the retina of eyes regenerating after retinal detachment were detected in newts exposed to clinorotation. The cells divided, were hypertrophied, and their processes were thickened. Such changes suggested reactive gliosis and were more significant in animals exposed to rotation when compared with desk-top controls. Later experiments onboard the Russian biosatellite Bion-11 showed similar changes in the retinas that were regenerating in a two-week spaceflight. In the Bion-11 animals, GFAP, the major structural protein of retinal macroglial cells, was found to be upregulated. In a more recent experiment onboard Foton-M3 (2007), GFAP expression in retinas of space-flown, ground control (kept at 1 g), and basal control (sacrificed on launch day) newts was quantified, using microscopy, immunohistochemistry, and digital image analysis. A low level of immunoreactivity was observed in basal controls. In contrast, retinas of space-flown animals showed greater GFAP immunoreactivity associated with both an increased cell number and a higher thickness of intermediate filaments. This, in turn, was accompanied by up-regulation of stress protein (HSP90) and growth factor (FGF2) expressions. It can be postulated that such a response of Müller cells was to mitigate the retinal stress in newts exposed to microgravity. Taken together, the data suggest that the retinal population of macroglial cells could be sensitive to gravity changes and that in space it can react by enhancing its neuroprotective function.     

An active role of the amyloplasts and nuclei of root statocytes in graviperception.

Three main phases are discerned in the gravitropic reaction: perception of a gravitational stimulus, its transduction, and fixation of the reaction resulting in bending of an organ. According to the starch-statolith hypothesis of Nemec and Haberlandt, amyloplasts in the structurally and functionally specialized graviperceptive cells (statocytes) sediment in the direction of a gravitational vector in the distal part of a cell while a nucleus is in the proximal one. If amyloplasts appear to act as gravity sensors, the receptors, which interact with sedimented amyloplasts, and next signaling are still unclear. An analysis of the structural-functional organization of cells in different root cap layers of such higher plants as pea, Arabidopsis thaliana, and Brassica rapa grown under 1 g, on the clinostats, and in microgravity, allows us to support the hypothesis that amyloplasts function as statoliths in statocytes, but they may not be only the passive statolithic mass. We propose that amyloplasts fulfill a more complex function by interacting with a receptor, which is a nucleus, in transduction of some signal to it. Gravity-induced statolith movement in certain order leads to a new functional connection between gravity susceptors--amyloplasts and a receptor--a nucleus receiving some signal presumedly of a mechanical or biochemical nature from the amyloplasts. During gravitropism, sugar signaling could induce expression of genes encoding auxin transport proteins in a nucleus giving the nucleus an intermediate role in signal trunsduction following perception.     

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.     

Advances of Microgravity Sciences

Advances of microgravity sciences in China are introduced. The research works include ground-based study and space experiments. In the recent years, the main means still are theoretical analysis, numerical simulation, ground-based experiment, and short-time microgravity experiments of drop tower. Besides, many space experiment projects are arranged. SJ-10 recoverable satellite will carry out 19 scientific experiment projects. Nine of them are for microgravity Sciences. The other ways for space microgravity experiment are with the help of Chinese Shenzhou spacecraft, Chinese Tiangong space laboratory, and Chinese space station in the near future. The Chinese space station will become main platform of Chinese microgravity sciences experiment in space.      

Size and cell number of the utricle in kinetotically swimming fish: a parabolic aircraft flight study.

Humans taking part in parabolic aircraft flights (PAFs) may suffer from space motion sickness (SMS, a kinetosis). Since it has been repeatedly shown earlier that some fish of a given batch also reveal a kinetotic behavior during PAFs (especially so-called spinning movements and looping responses) and due to the homology of the vestibular apparatus among all vertebrates, fish can be used as model systems to investigate the origin of susceptibility to motion sickness. Therefore, we examined the utricular maculae (they are responsible for the internalization of gravity in teleosteans) of fish swimming kinetotically at microgravity in comparison with animals from the same batch who swam normally. On the histological level, it was found that the total number of both sensory and supporting cells of the utricular maculae did not differ between kinetotic animals as compared to normally swimming fish. Cell density (sensory and supporting cells/100 micrometers2), however, was reduced in kinetotic animals (p<0.0001), which seemed to be due to malformed epithelial cells (increase in cell size) of the kinetotic specimens. Susceptibility to kinetoses may therefore originate in malformed sensory epithelia.     

Gravity effects on connective tissue biosynthesis by cultured mesenchymal cells.

Quantitative and qualitative aspects of collagen synthesis under microgravity, normal gravity and hypergravity conditions were investigated during the spacelab D-2 mission by incubating human fibroblast cultures with [3H]-proline for 0, 4, 7, 10 and 20 hours. Quantitative analysis revealed an increase of collagen synthesis under microgravity conditions, being 40% higher than 1g controls. Hypergravity samples at 1.44g, 6.6g and 10g showed a decrease in collagen synthesis with increasing g, being down to about 15% at 10g. The relative proportion of collagen from total protein synthesized, the secretion of collagen by the cells, proline hydroxylation of individual collagen alpha-chains and the relative proportions of collagens I, III and V synthesized were not affected at any of the applied conditions.     

A ground-based study for a shuttle BRIC experiment on gravity effects on gene expression.

A BRIC (Biological Research In a Canister) experiment to investigate the effects of reduced gravity at the molecular level using Arabidopsis has been initiated. In preparation for a space flight experiment, a series of ground-based studies were conducted. Results from these studies indicate that: 1) up to 20,000 seeds can be germinated on a 100 mm diameter Petri plate, 2) nylon membrane is the best surface for recovery of plant material after freezing, 3) depending on the age of the seedlings at the time of freezing, 20 to 40 g of tissue can be obtained from Petri plates that fit in a single canister; 4) tissue from one canister yields adequate amounts of RNA to perform differential display to isolate gravity-regulated genes. Our results indicate that the proposed BRIC experiment is feasible and can provide valuable information on the possible effects of microgravity on gene regulation.     

A drop-tower experiment to determine the threshold of gravity for inducing motion sickness in fish.

It has been repeatedly shown earlier that some fish of a given batch reveal motion sickness (a kinetosis) at the transition from 1 g to microgravity. In the course of parabolic aircraft flight experiments, it has been demonstrated that kinetosis susceptibility is correlated with asymmetric inner ear otoliths (i.e., differently weighed statoliths on the right and the left side of the head) or with genetically predispositioned malformed cells within the sensory epithelia of the inner ear. Hitherto, the threshold of gravity perception for inducing kinetotic behavior as well as the relative importance of asymmetric otoliths versus malformed epithelia for kinetosis susceptibility has yet not been determined. The following experiment using the ZARM drop-tower facility in Bremen, Germany, is proposed to be carried out in order to answer the aforementioned questions. Larval cichlid fish (Oreochromis mossambicus) will be kept in a camcorder-equipped centrifuge during the microgravity phases of the drops and thus receive various gravity environments ranging from 0.1 to 0.9 g. Videographed controls will be housed outside of the centrifuge receiving 0 g. Based on the video-recordings, animals will be grouped into kinetotically and normally swimming samples. Subsequently, otoliths will be dissected and their size and asymmetry will be measured. Further investigations will focus on the numerical quantification of inner ear supporting and sensory cells as well as on the quantification of inner ear carbonic anhydrase reactivity. A correlation between: (1) the results to be obtained concerning the g-loads inducing kinetosis and (2) the corresponding otolith asymmetry/morphology of sensory epithelia/carbonic anhydrase reactivity will further contribute to the understanding of the origin of kinetosis susceptibility. Besides an outline of the proposed principal experiments, the present study reports on a first series of drop-tower tests, which were undertaken to elucidate the feasibility of the proposal (especially concerning the question, if some 4.7 s of microgravity are sufficient to induce kinetotic behavior in larval fish).     

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.