Energetic metabolism response in algae and higher plant species from simulation experiments with the clinostat.

Adenylate state is acknowledged to be among the most convenient approaches in the study of physiological changes in plant cells under simulation of altered gravity condition with the clinostat. Adenylate levels and the ATP/ADP ratio in cytoplasmic and mitochondrial extracts of cultivated cells of Haplopappus gracilis and algae cells of Chlorella vulgaris under initial stages of the fast-rotating and slow-rotating clinorotation, as well as the long-term clinorotation, have been investigated. For analysis of ATP and ADP levels in the plant cells under the clinorotation, we applied a high-sensitive bioluminescence method using the luciferase and piruvate kinase enzyme systems. It has been shown that the adenylate ratio is already increased during at the start of clinorotation with the different speed of rotation in the biological material tested. The considerable changes in mitochondrial ultrastructure of Chlorella cells, as well as the rising ATP level and dropping of the ATP/ADP ratio appear after long-duration clinorotation if compared to control material. It is probably connected with the distinctions in ATP-synthetase functioning in mitochondria of the cells under the clinorotation conditions.     

Comparative characteristic of mitochondria ultrastructural organization in Chlorella cells under altered gravity conditions.

Results from experiments that used cells from the unicellular alga Chlorella vulgaris (strain Larg-1) grown on a clinostat, demonstrated the occurrence of rearrangements in cellular organelles, including changes in the mitochondrial ultrastructure compared to controls. Changes in mitochondrial structure were observed in auto- and heterotrophic regimes of cells grown in altered gravity conditions, especially in long-term experiments. The mitochondrial rearrangements become apparent during cell proliferation, which resulted in an increase in the relative volume of mitochondria per cell: up to 2.7 +/- 0.3% in short-term clino-rotation (2.2 +/- 0.1% in the control) and up to 5.3 +/- 0.4% and 5.1 +/- 0.4% in long-term clinorotation (2.3 +/- 0.2% in the control). The size of the mitochondria and their cristae increased in cells grown under long-time clinorotation. In addition, hypertrophied organelles, not typical for this strain, were observed. These changes in the cells were accompanied by increased electron density of the matrix and a well-ordered topography of the cristae. To examine the separation of oxidative phosphorylation and respiration, an inhibitory agent 2,4-dinitrophenol (2,4-DNP) was applied to cells which resulted in insignificant volume changes of the mitochondria (2.5 +/- 0.4% versus 2.1 +/- 0.2% in the control). The increase of mitochondrial size with regularly arranged cristae, with more condensed matrix and extension of cristae areas of clino-rotated cells, may demonstrate higher functional activity of the mitochondria under altered gravity conditions. Changes observed early in clinorotated cells, in particular the increased level of respiration, adenylate content (especially ATP) and more intensive electron-cytochemical reactions of Mg2(+)-ATPase and succinate [correction of succinat] dehydrogenase (SDH) in mitochondria (including hypertrophic organelles), also suggest increased activity of mitochondria from cells grown under altered gravity conditions compared to controls.     

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.     

Expression of p53-regulated genes in human cultured lymphoblastoid TSCE5 and WTK1 cell lines after spaceflight in a frozen state

The 53 kDa tumor suppressor protein p53 is generally thought to contribute to the genetic stability of cells and to protect cells from DNA damage through the activity of p53-centered signal transduction pathways. To clarify the effect of space radiation on the expression of p53-dependent regulated genes, gene expression profiles were compared between two human cultured lymphoblastoid cell lines: one line (TSCE5) has a wild-type p53 gene status, and the other line (WTK1) has a mutated p53 gene status. Frozen human lymphoblastoid cells were stored in a freezer in the International Space Station (ISS) for 133 days. Gene expression was analyzed using DNA chips after culturing the space samples for 6 h on the ground after their return from space. Ground control samples were also cultured for 6 h after being stored in a frozen state on the ground for the same time period that the frozen cells were in space. p53-Dependent gene expression was calculated from the ratio of the gene expression values in wild-type p53 cells and in mutated p53 cells. The expression of 50 p53-dependent genes was up-regulated, and the expression of 94 p53-dependent genes was down-regulated after spaceflight. These expression data identified genes which could be useful in advancing studies in basic space radiation biology. The biological meaning of these results is discussed from the aspect of gene functions in the up- and down-regulated genes after exposure to low doses of space radiation.     

CNS development under altered gravity: cerebellar glial and neuronal protein expression in rat neonates exposed to hypergravity.

The future of space exploration depends on a solid understanding of the developmental process under microgravity, specifically in relation to the central nervous system (CNS). We have previously employed a hypergravity paradigm to assess the impact of altered gravity on the developing rat cerebellum. The present study addresses the molecular mechanisms involved in the cerebellar response to hypergravity. Specifically, the study focuses on the expression of selected glial and neuronal cerebellar proteins in rat neonates exposed to hypergravity (1.5 G) from embryonic day (E)11 to postnatal day (P)6 or P9 (the time of maximal cerebellar changes) comparing them against their expression in rat neonates developing under normal gravity. Proteins were analyzed by quantitative Western blots of cerebellar homogenates; RNA analysis was performed in the same samples using quantitative PCR. Densitometric analysis of Western blots suggested a reduction in glial (glial acidic protein, GFAP) and neuronal (neuronal cell adhesion molecule, NCAM-L1, synaptophysin) proteins, but the changes in individual cerebellar proteins in hypergravity-exposed neonates appeared both age- and gender-specific. RNA analysis suggested a reduction in GFAP and synaptophysin mRNAs on P6. These data suggest that exposure to hypergravity may interfere with the expression of selected cerebellar proteins. These changes in protein expression may be involved in mediating the effect of hypergravity on the developing rat cerebellum.     

Patterns of cortical microtubules formed in epidermis of Beta vulgaris L. roots under clinorotation.

Changes of cortical microtubules (MTs) from the normal transverse arrangement were observed in epidermal cells of Beta vulgaris roots under clinorotation. We hypothesize that the epidermis is sensitive to clinorotation and that the microtubular cytoskeleton plays a key role in the ensuing growth response.     

Environmental impacts on the developing CNS: CD15, NCAM-L1, and GFAP expression in rat neonates exposed to hypergravity.

We have previously reported that the developing rat cerebellum is affected by hypergravity exposure. The effect is observed during a period of both granule and glial cell proliferation and neuronal migration in the cerebellum and coincides with changes in thyroid hormone levels. The present study begins to address the molecular mechanisms involved in the cerebellar response to hypergravity. Specifically, the study focuses on the expression of cerebellar proteins that are known to be directly involved in cell-cell interactions [protein expressing 3-fucosyl-N-acetyl-lactosamine antigen (CD15), neuronal cell adhesion molecule (NCAM-L1)] and those that affect cell-cell interactions indirectly [glial fibrillary acidic protein (GFAP)] in rat neonates exposed to centrifuge-produced hypergravity. Cerebellar mass and protein expression in rat neonates exposed to hypergravity (1.5 G) from gestational day (G) 11 to postnatal day (P) 30 were compared at one of six time points between P6 and P30 against rat neonates developing under normal gravity. Proteins were analyzed by quantitative western blots of cerebellar homogenates prepared from male or female neonates. Cerebellar size was most clearly reduced in male neonates on P6 and in female neonates on P9, with a significant gender difference; differences in cerebellar mass remained significant even when change in total body mass was factored in. Densitometric analysis of western blots revealed both quantitative and temporal changes in the expression of selected cerebellar proteins that coincided with changes in cerebellar mass and were gender-specific. In fact, our data indicated certain significant differences even between male and female control animals. A maximal decrease in expression of CD15 was observed in HG females on P9, coinciding with maximal change in their cerebellar mass. A shift in the time-course of NCAM-L1 expression resulted in a significant increase in NCAM-L1 in HG males on P18, an isolated time at which cerebellar mass does not significantly differ between HG and SC neonates. A maximal decrease in expression of GFAP was observed in HG males on P6, coinciding with maximal change in their cerebellar mass. Altered expression of cerebellar proteins is likely to affect a number of developmental processes and contribute to the structural and functional alterations seen in the CNS developing under altered gravity. Our data suggest that both cerebellar development and its response to gravitational manipulations differ in males and females.     

Organization of cytoskeleton during differentiation of gravisensitive root sites under clinorotation.

Key role in cell gravisensing is attributed to the actin cytoskeleton which acts as a mediator in signaling reactions, including graviperception. Despite of increased attention to the actin cytoskeleton, major gaps in our understanding of its functioning in plant gravisensing still remain. To fill these gaps, we propose a novel approach focused on the investigation of actin involvement in the development of columella cells and cells in the transition zone of roots submitted to clinorotation. Both statocytes and cells in the transition zone represent the postmitotic cells which take origin in root meristems and are specified into graviperceptive (root cap) and gravireacting (transition zone) root tissues. The aim of the research was to investigate and compare the microfilament arrangements in root cap statocytes and peripheral root tissues (epidermis and cortex cells) in the transition zone and to find out how the actin cytoskeleton is involved in their specification under clinostat conditions. So far, our experiments have shown that under clinorotation the cytoplasmic microfilament network in the cortex cells in the transition zone is significantly enhanced. It is suggested that more abundant cytoplasmic microfilaments could strengthen the cortical actin cytoskeleton arranged parallel with the cortical microtubules, which are found to be partially disorganized in this area. Due to microtubule disorganization, the functioning of cellulose-synthesizing machinery and proper deposition of cell wall might be affected and could cause the alterations in the growth mode. But, in our case growth of the cells in the transition zone under clinorotation was rather stable. Due to our opinion, general stability of cell growth under clinorotation is promoted by mutual functional interrelation between actin and tubulin cytoskeletons. It is suggested that a strengthened cortical actin cytoskeleton restricts the cell growth instead of disorganized microtubules.     

Soybean cotyledon starch metabolism is sensitive to altered gravity conditions.

We have demonstrated that etiolated soybean seedlings grown under the altered gravity conditions of clinorotation (1 rpm) and centrifugation (5xg) exhibit changes in starch metabolism. Cotyledon starch concentration was lower (-28%) in clinorotated plants and higher (+24%) in centrifuged plants than in vertical control plants. The activity of ADP-glucose pyrophosphorylase in the cotyledons was affected in a similar way, i.e. lower (-37%) in the clinorotated plants and higher (+22%) in the centrifuged plants. Other starch metabolic enzyme activities, starch synthase, starch phosphorylase and total hydrolase were not affected by the altered gravity treatments. We conclude that the observed changes in starch concentrations were primarily due to gravity-mediated differences in ADP-glucose pyrophosphorylase activity.     

Hypergravity-induced changes in gene expression in Arabidopsis hypocotyls.

Under hypergravity conditions, the cell wall of stem organs becomes mechanically rigid and elongation growth is suppressed, which can be recognized as the mechanism for plants to resist gravitational force. The changes in gene expression by hypergravity treatment were analyzed in Arabidopsis hypocotyls by the differential display method, for identifying genes involved in hypergravity-induced growth suppression. Sixty-two cDNA clones were expressed differentially between the control and 300 g conditions: the expression levels of 39 clones increased, whereas those of 23 clones decreased under hypergravity conditions. Sequence analysis and database searching revealed that 12 clones, 9 up-regulated and 3 down-regulated, have homology to known proteins. The expression of these genes was further analyzed using RT-PCR. Finally, six genes were confirmed to be up-regulated by hypergravity. One of such genes encoded 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor of terpenoids such as membrane sterols and several types of hormones. The expression of HMGR gene increased within several hours after hypergravity treatment. Also, compactin, an inhibitor of HMGR, prevented hypergravity-induced growth suppression, suggesting that HMGR is involved in suppression of Arabidopsis hypocotyl growth by hypergravity. In addition, hypergravity increased the expression levels of genes encoding CCR1 and ERD15, which were shown to take part in the signaling pathway of environmental stimuli such as temperature and water, and those of the alpha-tubulin gene. These genes may be involved in a series of cellular events leading to growth suppression of stem organs under hypergravity conditions.     

High phosphorylase activity is correlated with increased potato minituber formation and starch content during extended clinorotation.

The major purpose of these experiments were to investigate growth of potato storage organs and starch synthesis in minitubers at slow horizontal clinorotation (2 rpm), which partly mimics microgravity, and a secondary goal was to study the activity and localization of phosphorylase (EC 2.4.1.1) in storage parenchyma under these conditions. Miniplants of Solanum tuberosum L. (cv Adreta) were grown in culture for 30 days for both the vertical control and the horizontal clinorotation. During long-term clinorotation, an acceleration of minituber formation, and an increase of amyloplast number and size in storage parenchyma cells, as well as increased starch content, was observed in the minitubers. The differences among cytochemical reaction intensity, activity of phosphorylase, and carbohydrate content in storage parenchyma cells of minitubers grown in a horizontal clinostat were established by electron-cytochemical and biochemical methods. It is shown that high phosphorylase activity is correlated with increased starch content during extended clinorotation. The results demonstrate the increase in carbohydrate metabolism and possible accelerated growth of storage organs under the influence of microgravity, as mimicked by clinorotation; therefore, clinorotation can be used as a basis for future studies on mechanisms of starch synthesis under microgravity.     

辐射条件下微藻基因表达内参基因的选择

作为空间辐射的一种主要成分,紫外辐射可广泛引起陆地植物与水生生物细胞及其组份的破坏.荧光定量PCR技术广泛应用于各类胁迫环境下,研究目的基因的转录水平.在荧光定量PCR中选用合适的内参基因,能够更加准确地校正和标准化目的基因转录水平.本实验研究了辐射条件下水生生物莱茵衣藻6个传统内参基因18SrRNA,GAPDH,β-actin,β-tubulin,EF1-α和UBC基因表达的稳定性.经GeNorm软件研究分析,在辐射条件下,莱茵衣藻18SrRNA基因表达最不稳定,而选用β-actin和GAPDH作为双内参,可以得到更精确的实验结果.      

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.     

Microgravity effects on neural retina regeneration in the newt.

Data on forelimb and eye lens regeneration in urodeles under spaceflight conditions (SFC) have been obtained in our previous studies. Today, evidence is available that SFC stimulate regeneration in experimental animals rather than inhibit it. The results of control on-ground experiments with simulated microgravity suggest that the stimulatory effect of SFC is due largely to weightlessness. An original experimental model is proposed, which is convenient for comprehensively analyzing neural regeneration under SFC. The initial results described here concern regeneration of neural retina in Pleurodeles waltl newts exposed to microgravity simulated in radial clinostat. After clinorotation for seven days (until postoperation day 16), a positive effect of altered gravity on structural restoration of detached neural retina was confirmed by a number of criteria. Specifically, an increased number of Mullerian glial cells, an increased relative volume of the plexiform layers, reduced cell death, advanced redifferentiation of retinal pigment epithelium, and extended areas of neural retina reattachment were detected in experimental newts. Moreover, cell proliferation in the inner nuclear layer of neural retina increased as compared with control. Thus, low gravity appears to intensify natural cytological and molecular mechanisms of neural retina regeneration in lower vertebrates.     

High magnetic gradient environment causes alterations of cytoskeleton and cytoskeleton-associated genes in human osteoblasts cultured in vitro

The effects of a high magnetic gradient environment (HMGE) on the cytoskeletal architecture and genes associated with the cytoskeleton in osteoblasts (MC3T3-E1 and MG-63 cells) were investigated using confocal microscopy, real-time polymerase chain reaction (PCR) and atomic force microscopy (AFM). The findings showed that, under diamagnetic levitation conditions, the architecture and average height of the cytoskeleton and surface roughness in osteoblasts were dramatically altered. HMGE affects cytoskeleton arrangement and cytoskeleton-associated gene expression.     

Expression of cloned genes of transgenic microorganisms introduced into man-made ecosystems.

Modeling of transgenic microorganism introduction into small man-made ecosystems can help forecast changes in expression of cloned genes under different conditions of existence. Introduction of the E. coli Z905/pPHL7 strain containing a plasmid with luminescent system genes of luminous bacteria led to changes in cell and colony morphology, reduction in metabolic activity of cells, and, as a result, a lower level of expression of cloned gene. A low concentration of nutrients has been shown to favor greatly the phenotypic change of cells of the recombinant strain. Expression of cloned genes changed due to: a lower concentration of plasmid DNA, a change in regulation of cloned genes, and a change in cells of biosynthesis of substrates needed for expression of luminescent genes. The conducted investigations can provide a basis for the use of marker transgenic microorganisms in closed ecosystems of different types. Grant numbers: 99-04-96017, 00-07-9011.     

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.     

Molecular alterations in tumorigenic human bronchial and breast epithelial cells induced by high LET radiation.

Carcinogenesis is a multi-stage process with sequence of genetic events governing the phenotypic expression of a series of transformation steps leading to the development of metastatic cancer. In the present study, immortalized human bronchial (BEP2D) and breast (MCF-10F) cells were irradiated with graded doses of either 150 keV/micrometer alpha particles or 1 GeV/nucleon 56Fe ions. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Cell fusion studies indicated that radiation-induced tumorigenic phenotype in BEP2D cells could be completely suppressed by fusion with non-tumorigenic BEP2D cells. The differential expressions of known genes between tumorigenic bronchial and breast cells induced by alpha particles and their respective control cultures were compared using cDNA expression array. Among the 11 genes identified to be differentially expressed in BEP2D cells, three (DCC, DNA-PK and p21(CIP1)) were shown to be consistently down-regulated by 2 to 4 fold in all the 5 tumor cell lines examined. In contrast, their expressions in the fusion cell lines were comparable to control BEP2D cells. Similarly, expression levels of a series of genes were found to be altered in a step-wise manner among tumorigenic MCF-10F cells. The results are highly suggestive that functional alterations of these genes may be causally related to the carcinogenic process.