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PubMed2006

Inositol 1,4,5-trisphosphate and Ran expression during simulated and real microgravity.

In order to gain further insight into the signal transduction pathway concerning gravitropism, we studied the expression profiles of mRNA in etiolated sunflower (Helianthus annuus L.) seedlings. Differential-display reverse transcriptase PCR product assayed by capillary electrophoresis revealed the small GTPase Ran, regulating nuclear import and export of proteins. Parallel analysis of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) release by a highly advanced system of metal-dye detection combined with high-performance liquid chromatography provided evidence that the second messenger Ins(1,4,5)P3 is modulated by changes of the gravity vector. Investigations by fast clinorotation and sounding rockets established a positive correlation between the Ins(1,4,5)P3 level and the expression rate of Ran mRNA during simulated and real microgravity. Since an asymmetric distribution of auxin during graviresponse is suggested to induce differential cell elongation, additional information on the perception and transduction pathways was achieved by auxin stimulation experiments. While we were able to demonstrate an auxin-dependent production of Ins(1,4,5)P3, the expression of Ran mRNA was not affected by auxin. Finally, besides the phosphoinositide system as one element of the signal transduction chain linking graviperception to graviresponse, a Ran-mediated interaction model of extracellular microgravity signal perception and intercellular transduction pathway is proposed.

Chromatography, High Pressure Liquid↗
PubMed2003

[Osteoporosis and genes in space environment].

Based on observations of extreme and accelerated loss of bone density in astronauts flying on longer space missions, scientists coordinated by the European Space Agency, ESA, have since the mid 1990'ies been focusing on potential causes for this alarming finding. The experimental conditions on board space craft are however very restrictive, for which reason research into human physiology in the space environment has gone its own ways regarding how to create solid and statistically significant data. Space observations can profit markedly from ground-based simulations studies, and some interesting data on countermeasures against loss of bone matter, that have been tested in the recently completed 90-days bed rest study, are reported here. Paired with information on different phenotypes observed in such studies, the data reported looks very promising. This bed rest study has generated a set of extremely interesting data, which in terms of resolution and quality allows the researchers to consider this study as a significant step ahead, and as a crucial steppingstone in defining the next round of space based studies of bone metabolism. In addition the findings in the bed rest study are directly applicable to long-term bed ridden patients in terms of the need to maintain activity as a means to improve the recovery prognosis.

Adaptation, Physiological↗
PubMed2004

Microgravity effect on testicular functions.

In mammals spaceflight influences spermatogenesis since spermatogonial germ cell proliferation, compared to synchronous controls, is lightly decreased in irradiated or flown rats. Moreover, changes of the plasmatic testosterone production was described either in flight rats, or in rats maintained in simulated microgravity conditions. The hormonal levels of the astronauts change as it has been previously described, including hormones involved in the regulation of spermatogenesis such as testosterone and luteinizing hormone (LH). In microgravity conditions, human testosterone levels decreased whereas circulating LH levels increased. To study the effect of simulated microgravity on mammalian spermatogenesis we have utilized the Rotary Cell Culture System (RCCS) and we have cultured testicular fragments isolated from prepuberal rats in a chemically defined medium for three days under microgravity conditions. As control we have cultured the same amount of fragments at unit gravity. The morphology of the samples has been studied and the number of proliferating cells has been counted in control samples and in samples maintained in RCCS. The results indicate that the number of duplicating cells in the tubules was significantly increased in the microgravity-cultured fragments. The amount of testosterone secreted in the culture medium has been also evaluated and in RCCS samples the amount of the hormone was higher respect to the control samples.

Animals↗
PubMedDate not supplied

The role of cytoskeleton in cell changes under condition of simulated microgravity.

Single cells and cell culture are very good model for estimation of primary effects of gravitational changes. It is suggested that cell cytoskeleton plays a key role in mechanisms of adaptation to mechanical influences including gravitational ones. Our results demonstrated that cultured cells of human vascular endothelium (correction of endotheliun) are highly sensitive to hypogravity (clinorotation) and respond by significant decrease of cell proliferative activity. Simultaneously it was noted that the formation of confluent monolayer appeared early in cultures exposed to simulated microgravity due to accelerated cells spreading. Long-term hypogravity (several hours or days) leads to significant changes of cell cytoskeleton revealed as microfilament thinning and their redistribution within cell. Such changes were observed only in monolayer cells and not in cell suspensions. Gravitational forces as known to be modificators of cell adhesive ability and determine their mobility. Hypogravity environment stimulated endothelial cell migration in culture: 24-48 hrs pre-exposition to hypogravity significantly increased endothelial cell migration resulting in 2-3-fold acceleration of mechanically injured monolayer repair. Obtained results suggest that the effects of hypogravity on cultured human endothelial cells are, possibly, associated with protein kinase C and/or adenylate cyclase activity and are accompanied by noticeable functional cell changes.

Actin Cytoskeleton↗
PubMed1998

[Effects of clinorotation on cytosolic free calcium level in embryonic chick brain cells].

Cytosolic free Ca2+ ([Ca2+]i) in suspended brain cells of chicken embryos hatched for 6-18 d (E6-E18) was measured by using the fura-2/AM fluorescence spectroscopy. The biological effects of microgravity on the embryonic chicks were simulated by revolving on a clinostat during their hatch. The experimental results showed that level of [Ca2+]i in E8-E17 embryonic brain cells decreased after revolving for different period. [Ca2+]i in brain cells of E10 and E13 chick embryos reduced significantly (P < 0.01) after revolving 4-7 h and 24 h respectively. When E10 and E13 chick embryos were incubated again as long as being revolved, [Ca2+]i in brain cells rose but were still lower than the controls. The levels of cytosolic free calcium in brain cells of revolved E17 embryonic chicks increased after being colorpunctured by red light. These results demonstrate that the decrease of [Ca2+]i in embryonic chick brain cells caused by clinorotation is reversible, and colorpuncture can accelerate this recovery.

Animals↗
PubMed2004

Effect of simulated microgravity on PGE2-induced edema and hyperalgesia in rat paws: pharmacological data and biochemical correlates.

The aim of the present study has been to investigate the effect of conditions of modeled microgravity using a three-dimensional clinostat (Random Positioning Machine, RPM) on edema and thermal hyperalgesia induced by prostaglandin E2 (PGE2) in the hind paw of rat. Our results showed that RPM reduced PGE2-evoked edema associated to a significant decrease in hyperalgesia compared to ground control animals. To further characterize the mechanisms by which RPM induces anti-inflammatory and anti-hyperalgesic action, we performed biochemical assays of PGE2 and Western immunoblot experiments to assess whether the intraplantar administration of exogenous PGE2 modifies the expression of the iNOS. These results showed that RPM diminished the levels of PGE2 in exudates of paws previously treated with PGE2, but did not influence the iNOS expression.

Animals↗
PubMed1989

Calcium gradient in plant cells with polarized growth in simulated microgravity.

Plant cells characterized by apical growth, for example, root hairs and apical cells of moss protonema, are a convenient model to address the problem of gravity response mechanisms including initiation of cell polarity. The fluorescent calcium probe, chlorotetracycline, allowed us to display the calcium distribution gradient in these cells. Irradiation by red light led to a sharp decrease in the Ca2+ ion activity in cells. During clinostatting in darkness the pattern of calcium influx and distribution changes inconsiderably as compared with control; in root hairs calcium is detected mainly in their apices and bases as in control. Addition of chlorpromazine to the medium probably increases the influx and accumulation of Ca2+ ions. Under data obtained confirm speculations on the Ca2+ ion functional role for the apical growth of plant cells and may suggest the participation of gravity in redistribution or activation of ion channels, calcium channels included, in the plasmalemma.

Bryopsida↗
PubMed2006

Gene expression alterations in activated human T-cells induced by modeled microgravity.

Studies conducted in real Space and in ground-based microgravity analog systems (MAS) have demonstrated changes in numerous lymphocyte functions. In this investigation we explored whether the observed functional changes in lymphocytes in MAS are associated with changes in gene expression. NASA-developed Rotating Wall Vessel (RWV) bioreactor was utilized as a MAS. Activated T lymphocytes were obtained by adding 100 ng/ml of anti-CD3 and 100 U/ml of IL-2 in RPMI medium to blood donor mononuclear cells for 4 days. After that the cells were washed and additionally cultured for up to 2 weeks with media (RPMI, 10% FBS and 100 U/ml IL-2) replacement every 3-4 days. Flow cytometry analysis had proven that activated T lymphocytes were the only cells remaining in culture by that time. They were split into two portions, cultured for additional 24 h in either static or simulated microgravity conditions, and used for RNA extraction. The gene expression was assessed by Affymetrix GeneChip Human U133A array allowing screening for expression of 18,400 genes. About 4-8% of tested genes responded to MG by more than a 1.5-fold change in expression; however, reproducible changes were observed only in 89 genes. Ten of these genes were upregulated and 79 were downregulated. These genes were categorized by associated pathways and viewed graphically through histogram analysis. Separate histograms of each pathway were then constructed representing individual gene expression fold changes. Possible functional consequences of the identified reproducible gene expression changes are discussed.

Apoptosis↗
PubMedDate not supplied

Early cardiovascular adaptation to zero gravity simulated by head-down tilt.

The early cardiovascular adaptation to zero gravity, simulated by head-down tilt at 5 degrees, was studied in a series of 10 normal young men. The validity of the model was confirmed by comparing the results with data from Apollo and Skylab flights. Tilt produced a significant central fluid shift with a transient increase in central venous pressure, later followed by an increase in left ventricular size without changes in cardiac output, arterial pressure, or contractile state. The hemodynamic changes were transient with a nearly complete return to the control state within 6 hr. The adaptation included a diuresis and a decrease in blood volume, associated with ADH, renin and aldosterone inhibition.

Adaptation, Physiological↗
PubMed2004

Low oxygen [correction of oxygene] tension may defence the bone tissue from unloading simulated osteopenia.

The main task of the present study is to investigate the influences of normobaric gas mixture with lowered PO2 (90-110 mm Hg) on the bones metabolism in rats after hind limb unloaded (HLU). We observed increased the glycosaminoglicans (GAG) concentration in blood serum rats with hind limb unloaded in atmospheric air (AA). An increased activity of acid phosphatase (AcP) and tartratresistant acid phosphatase (TRAcP) in blood serum of rats after HLU was observed. The activities of the alkaline phosphatase (AIP) in blood serum did not change. An increased osteocalcin (OST) and C-terminal Propeptide of type I Collagen (CICP) concentration in blood serum, but parathyroid hormone (PTH) decrease comparatively with the control. The above mentioned biochemistry markers in animals after 28 days unloading in normobaric gas mixture (NGM) were more stable and in spite of the same procedure in AA, all dates were near to the controls.

Acid Phosphatase↗
PubMed2004

Clinostating effects on Apogee wheat resistance to wheat streak mosaic virus.

The effects of vertical and horizontal clinostating with container velocity 2 rpm and platform velocity 1 rpm on Apogee wheat resistance to wheat streak mosaic virus (WSMV) were studied. For the first time the yield of grain was obtained from both, healthy and the WSMV-infected wheat plants under conditions of simulated microgravity (clinorotation) which is accounted for by the inclusion of various mechanisms in the process of plant adaptation to the factors of stresses. For the first time the stages of viral infection development were elucidated under the conditions of prolonged clinostating and the presence of the WSMV was detected in wheat plants on the 18th day after inoculation employing the method of polymerase chain reaction (PCR). In the test variant with vertical clinostating (R=1,0) the least favorable conditions for viral reproduction were identified. In the same variant the increases of the total nitrogen content in the ears were observed which may be an evidence of interferon-like protein synthesis in plant similar to the interferon system functioning in animals.

Biomass↗
PubMed2000

Cardiovascular responses to simulated microgravity in Sprague-Dawley rats.

Microgravity is known to induce orthostatic intolerance and baroreflex impairment in astronauts. Cardiovascular responses observed in 30 degrees head-down tilt rat models, whether 24 hr whole body suspension (WBS) or 7 day tail-suspension (TS), mimic observations made during exposure to microgravity. We evaluated the cardiovascular effects of simulated microgravity and the subsequent post-suspension in rats using the above models. Mean arterial pressure (MAP) of both WBS and TS rats did not change during suspension. In both models, MAP decreased post-suspension and this response lasted for 6 hrs. Salt-loaded animals did not show a post-suspension reduction in MAP. Plasma ionized calcium was decreased at 2 hr of WBS, with no change in sodium, potassium, magnesium, glucose, or hematocrit. Body weight changes were similar for all animals whether under suspension or control conditions. Both rat models demonstrate post-suspension hypotension and these results support the notion that salt-loading may have some beneficial effects in ameliorating this hypotension.

Animals↗
PubMed2001

Shear stress enhances microcin B17 production in a rotating wall bioreactor, but ethanol stress does not.

Stress, including that caused by ethanol, has been shown to induce or promote secondary metabolism in a number of microbial systems. Rotating-wall bioreactors provide a low stress and simulated microgravity environment which, however, supports only poor production of microcin B17 by Escherichia coli ZK650, as compared to production in agitated flasks. We wondered whether the poor production is due to the low level of stress and whether increasing stress in the bioreactors would raise the amount of microcin B17 formed. We found that applying shear stress by addition of a single Teflon bead to a rotating wall bioreactor improved microcin B17 production. By contrast, addition of various concentrations of ethanol to such bioreactors (or to shaken flasks) failed to increase microcin B17 production. Ethanol stress merely decreased production and, at higher concentrations, inhibited growth. Interestingly, cells growing in the bioreactor were much more resistant to the growth-inhibitory and production-inhibitory effects of ethanol than cells growing in shaken flasks.

Bacteriocins↗
PubMed1998

Simulated microgravity conditions enhance differentiation of cultured PC12 cells towards the neuroendocrine phenotype.

We are studying microenvironmental cues which contribute to neuroendocrine organ assembly and tissue-specific differentiation. As our in vitro model, we cultured rat adrenal medullary PC12 pheochromocytoma cells in a novel cell culture system, the NASA rotating wall vessel (RWV) bioreactors. This "simulated microgravity" environment in RWV bioreactors, characterized by randomizing gravitational vectors and minimizing shear stress, has been shown to favor macroscopic tissue assembly and to induce tissue-specific differentiation. We hypothesized that the unique culture conditions in the RWV bioreactors might enhance the in vitro formation of neuroendocrine organoids. To test our hypothesis, we evaluated the expression of several markers of neuroendocrine differentiation in cultures of PC12 cells maintained for up to 20 d in the slow turning lateral vessel (STLV) type RWV. PC12 cell differentiation was assessed by morphological, immunological, biochemical and molecular techniques. PC12 cells, cultured under "simulated microgravity" conditions, formed macroscopic, tissue-like organoids several millimeters in diameter. Concomitantly, the expression of phenylethanolamine-N-methyl transferase (PNMT), but not of other catecholamine synthesizing enzymes, was enhanced. Increased PNMT expression, as verified on both the gene and protein level, was accompanied by an increase in the specific activity of the enzyme. Furthermore, after 20 d in culture in the STLV, we observed altered patterns of protein tyrosine phosphorylation and prolonged activation of c-fos, a member of the AP-1 nuclear transcription factor complex. We conclude that culture conditions in the RWV appear to selectively activate signal transduction pathways leading to enhanced neuroendocrine differentiation of PC12 cells.

Animals↗
PubMed2001

Renal function of rats in response to 37 days of head-down tilt.

Spaceflight induces changes in human renal function, suggesting similar changes may occur in rats. Since rats continue to be the prime mammalian model for study in space, the effects of chronic microgravity on rat renal function should be clarified. Acute studies in rats using the ground-based microgravity simulation model, head-down tilt (HDT), have shown increases in glomerular filtration rate (GFR), electrolyte excretion, and a diuresis. However, long term effects of HDT have not been studied extensively. This study was performed to elucidate rat renal function following long-term simulated microgravity. Chronic exposure to HDT will cause an increase in GFR and electrolyte excretion in rats, similar to acute exposures, and lead to a decrease in the fractional excretion of filtered electrolytes. Experimental animals (HDT, n=10) were tail-suspended for 37 days and renal function compared to ambulatory controls (AMB, n=10). On day 37 of HDT, GFR, osmolal clearance, and electrolyte excretion were decreased, while plasma osmolality and free water clearance were increased. Urine output remained similar between groups. The fractional excretion of the filtered electrolytes was unchanged except for a decrease in the percentage of filtered calcium excreted. Chronic exposure to HDT results in decreased GFR and electrolyte excretion, but the fractional excretion of filtered electrolytes remained primarily unaffected.

Animals↗
PubMed2005

Simulated microgravity using the Random Positioning Machine inhibits differentiation and alters gene expression profiles of 2T3 preosteoblasts.

Exposure to microgravity causes bone loss in humans, and the underlying mechanism is thought to be at least partially due to a decrease in bone formation by osteoblasts. In the present study, we examined the hypothesis that microgravity changes osteoblast gene expression profiles, resulting in bone loss. For this study, we developed an in vitro system that simulates microgravity using the Random Positioning Machine (RPM) to study the effects of microgravity on 2T3 preosteoblast cells grown in gas-permeable culture disks. Exposure of 2T3 cells to simulated microgravity using the RPM for up to 9 days significantly inhibited alkaline phosphatase activity, recapitulating a bone loss response that occurs in real microgravity conditions without altering cell proliferation and shape. Next, we performed DNA microarray analysis to determine the gene expression profile of 2T3 cells exposed to 3 days of simulated microgravity. Among 10,000 genes examined using the microarray, 88 were downregulated and 52 were upregulated significantly more than twofold using simulated microgravity compared with the static 1-g condition. We then verified the microarray data for some of the genes relevant in bone biology using real-time PCR assays and immunoblotting. We confirmed that microgravity downregulated levels of alkaline phosphatase, runt-related transcription factor 2, osteomodulin, and parathyroid hormone receptor 1 mRNA; upregulated cathepsin K mRNA; and did not significantly affect bone morphogenic protein 4 and cystatin C protein levels. The identification of gravisensitive genes provides useful insight that may lead to further hypotheses regarding their roles in not only microgravity-induced bone loss but also the general patient population with similar pathological conditions, such as osteoporosis.

Animals↗
PubMed2000

Effect of different gravity environments on DNA fragmentation and cell death in Kalanchoe leaves.

Different gravity environments have been shown to significantly affect leaf-plantlet formation and asexual reproduction in Kalanchoë daigremontiana Ham. and Perr. In the present work, we investigated the effect of gravity at tissue and cell levels. Leaves and leaf-plantlets were cultured for different periods of time (min to 15 d) in different levels of gravity stimulation: simulated hypogravity (1 rpm clinostats; 2 x 10(-4) g), 1 g (control) and hypergravity (centrifugation; 20 and 150 g). Both simulated hypogravity and hypergravity affected cell death (apoptosis) in this species, and variations in the number of cells showing DNA fragmentation directly correlated with nitric oxide (NO) formation. Apoptosis in leaves was more common as gravity increased. Apoptotic cells were localized in the epidermis, mainly guard cells, in leaf parenchyma, and in tracheary elements undergoing terminal differentiation. Exposures to acute hypergravity (up to 60 min) showed that chloroplast DNA fragmentation occurred prior to nuclear DNA fragmentation, marginalization of chromatin, nuclear condensation, and nuclear blebbing. Addition of sodium nitroprusside (NO donor) mimicked centrifugation. NO and DNA fragmentation decreased with N(G)-monomethyl-L-arginine (NO-synthase inhibitor). The variations in NO levels, nucleoid DNA fragmentation, and cell death show how chloroplasts, cells and leaves may respond (and adapt) to gravity changes.

Apoptosis↗
PubMed2004

Escherichia coli growth under modeled reduced gravity.

Bacteria exhibit varying responses to modeled reduced gravity that can be simulated by clino-rotation. When Escherichia coli was subjected to different rotation speeds during clino-rotation, significant differences between modeled reduced gravity and normal gravity controls were observed only at higher speeds (30-50 rpm). There was no apparent affect of removing samples on the results obtained. When E. coli was grown in minimal medium (at 40 rpm), cell size was not affected by modeled reduced gravity and there were few differences in cell numbers. However, in higher nutrient conditions (i.e., dilute nutrient broth), total cell numbers were higher and cells were smaller under reduced gravity compared to normal gravity controls. Overall, the responses to modeled reduced gravity varied with nutrient conditions; larger surface to volume ratios may help compensate for the zone of nutrient depletion around the cells under modeled reduced gravity.

Colony Count, Microbial↗