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Restoration of gravitropic sensitivity in starch-deficient mutants of Arabidopsis by hypergravity.

Despite the extensive study of plant gravitropism, there have been few experiments which have utilized hypergravity as a tool to investigate gravisensitivity in flowering plants. Previous studies have shown that starch-deficient mutants of Arabidopsis are less sensitive to gravity compared to the wild-type (WT). In this report, the question addressed was whether hypergravity could restore the sensitivity of starch-deficient mutants of Arabidopsis. The strains examined include a WT, a starchless mutant and a reduced-starch mutant. Vertical orientation studies with dark-grown seedlings indicate that increased centrifugal acceleration improves orientation relative to the acceleration vector for all strains, even the WT. For starchless roots, growth of seedlings under constant 5 g acceleration was required to restore orientation to the level of the WT at 1 g. In contrast, approximately 10 g was required to restore the orientation of the starchless mutant hypocotyls to a WT level at 1 g. Examination of plastid position in root cap columella cells of the starchless mutant revealed that the restoration of gravitropic sensitivity was correlated with the sedimentation of plastids toward the distal cell wall. Even in WT plants, hypergravity caused greater sedimentation of plastids and improved gravitropic capability. Collectively, these experiments support the hypothesis of a statolith-based system of gravity perception in plants. As far as is known, this is the first report to use hypergravity to study the mechanisms of gravitropism in Arabidopsis.

Arabidopsis↗

Effects of hypergravity on the cell shape and on the organization of cytoskeleton and extracelluar matrix molecules of in vitro human dermal fibroblasts.

In vitro human dermal fibroblasts were submitted to normal gravity (1 g) or to chronic hypergravity ranging from 2 to 20 g for 8 days. Changes only appeared above 15 g. The majority of 20 g-subjected cells showed fine filipods in the shape of a star whereas most control cells had rounded shapes and spread by forming lamellipodia. Indirect immunofluorescence staining of vinculin, alpha-actinin and actin stress fibers showed changes of the arrangement anchoring points of stress fibers under hypergravity. Tubulin staining showed that the centrosomal material generally located above the nucleus in control cells had migrated to the nucleus side in 20 g-exposed cells. After 8 d of culture under 20 g hypergravity the thickness of fibronectin network seemed to be increased and bundles of fibrils appeared linking ordered arrays of fibers. The fibrils of collagen I formed better delimited and thicker bundles of fibers. We may assume that 20 g hypergravity can induce changes in fibroblast cell shape, migration way, and anchorage leading to a reorganization of extracellular matrix without concomitant change of cell proliferation.

Actin Cytoskeleton↗

Effects of hypergravity on adherent human cells.

In recent years, accumulating evidence has shown that microgravity or hypergravity may affect cell growth and differentiation. Since it is not easy to carry out researches in space or to simulate weightlessness on earth, we conducted experiments on simulated hypergravity (2 to 15 g) by using a centrifuge (radius: 80 cm; speed motor: 180 rpm). We looked for the effects of chronic hypergravity (7 to 10 days) on cultures of three human cell lines: lung or dermic fibroblasts and lung adenocarcinoma A 549 cells. The results showed a significant decrease (10-20%, P<0.05) in cell proliferation connected to a significant decrease (20-50%, P<0.01) in culture DNA content under hypergravity, but only for lung fibroblasts. The protein content was never disturbed. Dermic fibroblast elastase activity was enhanced (8-13%, P<0.02) under 15 g. Total phospholipid content as well as relative amounts of phospholipid components, analysed by thin layer chromatography, were unchanged in A 549 cells.

Adenosarcoma↗

Hypergravity-induced increase in the apoplastic pH and its possible involvement in suppression of beta-glucan breakdown in maize seedlings.

Elongation growth of both coleoptiles and mesocotyls of maize (Zea mays L. cv. Cross Bantam T51) seedlings was inhibited under basipetal hypergravity (300 g) conditions. Hypergravity increased the pH of the apoplastic fluid of coleoptiles from 5.0 to 5.5 and mesocotyls from 5.2 to 5.7. When beta-1,3:1,4-D-glucanases (beta-glucanases) extracted from cell walls of the 1-g control coleoptiles and mesocotyls were assayed at pH 5.0 and 5.5 for coleoptiles, and at 5.2 and 5.7 for mesocotyls, respectively, the activity in the increased pH conditions was significantly lower than that in the control pH conditions. During the autolysis of the enzymically active cell wall preparations obtained from 1-g control organs, a molecular mass downshift of hemicellulosic polysaccharides occurred in cell walls. This downshift was suppressed in the increased pH conditions as compared with the control pH conditions. It was reported that hypergravity increased the molecular mass of hemicellulosic polysaccharides by decreasing the beta-glucanase activity, and thereby decreased the mechanical extensibility of cell walls in maize coleoptiles and mesocotyls. These results suggest that, in maize coleoptiles and mesocotyls, hypergravity-induced increase in the pH in the apoplastic fluid is involved in the reduction of the activity of beta-glucanases which, in turn, causes an increase in the molecular mass of hemicellulosic polysaccharides and inhibits elongation growth.

Cell Wall↗

Induction of early response genes by hypergravity in cultured mouse osteoblastic cells (MC3T3-E1).

Hypergravity as low as 50g transiently stimulated cultured mouse osteoblastic cells (MC3T3-E1) to induce early response genes such as c-fos and egr-1, whereas expression of c-jun was marginally affected. The maximum induction of c-fos required more than 90g, but egr-1 induction became maximum below 50g. Staurosporin inhibited the induction of c-fos by hypergravity almost completely at a concentration of 0.1 microM, but it inhibited the induction of egr-1 only partially. In cells pretreated with 12-O-tetradecanoylphorbol 13-acetate, induction of c-fos by hypergravity was almost completely abolished, whereas that of egr-1 was not affected. Activity of protein kinase C seemed to be activated in cells centrifuged at 900g. These results indicate that hypergravity stimulates multiple signal transduction cascades that are connected with the expression of early response genes.

Animals↗

Influence of hypergravity on the development of monoaminergic systems in the rat spinal cord.

We have investigated in this study the influence of a moderate hypergravity (1.8 G) on the development of monoaminergic projections to the spinal cord in the rat. Pregnant dams and their offspring were submitted to hypergravity from day 11 of gestation to postnatal day 15. Some animals were sacrificed at birth, other at postnatal day 15 and other after 8 months of normal gravity. In newborn animals, a substantial delay of the development of monoaminergic projections to the spinal cord was evidenced. In 15 days and 8 months animals, the pattern of innervation appeared anarchic, with numerous dystrophic profiles, mainly of serotonergic system. Ultrastructural examination of serotonergic projections revealed a paucity of synapses, and the frequent enveloping of serotonergic boutons by thin astrocytic profiles. We conclude that rats submitted to hypergravity during the critical period of onset of monoaminergic projections to the spinal cord are affected durably in the organization and the ultrastructure of these projections. Future studies are directed to the functional analysis of hypergravity animals, and to the influence of microgravity on the same system.

Animals↗

A mild stress due to hypergravity exposure at young age increases longevity in Drosophila melanogaster males.

Drosophila melanogaster flies were exposed to hypergravity starting at two days of age, the range of gravity levels used being 2.58-7.38 g. No longevity change was observed for exposures of less than 14 days. The longevity of males increased if they were submitted to hypergravity for durations ranging from 14 to 24 days. This increase in longevity was never observed in females. The positive effect of exposure to hypergravity has been replicated in two laboratories using two wild-type strains and different rearing conditions. A short hypergravity exposure seems to be a mild stress, yielding positive effects on longevity. This is in accordance with two previous studies showing a slight longevity increase after heat shock in the nematode Caenorhabditis elegans and in Drosophila melanogaster.

Animals↗

Effects of hypergravity exposure on the developing central nervous system: possible involvement of thyroid hormone.

The present study examined the effects of hypergravity exposure on the developing brain and specifically explored the possibility that these effects are mediated by altered thyroid status. Thirty-four timed-pregnant Sprague-Dawley rats were exposed to continuous centrifugation at 1.5 G (HG) from gestational Day 11 until one of three key developmental points: postnatal Day (P) 6, P15, or P21 (10 pups/dam: 5 males/5 females). During the 32-day centrifugation, stationary controls (SC, n = 25 dams) were housed in the same room as HG animals. Neonatal body, forebrain, and cerebellum mass and neonatal and maternal thyroid status were assessed at each time point. The body mass of centrifuged neonates was comparatively lower at each time point. The mass of the forebrain and the mass of the cerebellum were maximally reduced in hypergravity-exposed neonates at P6 by 15.9% and 25.6%, respectively. Analysis of neonatal plasma suggested a transient hypothyroid status, as indicated by increased thyroid stimulating hormone (TSH) level (38.6%) at P6, while maternal plasma TSH levels were maximally elevated at P15 (38.9%). Neither neonatal nor maternal plasma TH levels were altered, suggesting a moderate hypothyroid condition. Thus, continuous exposure of the developing rats to hypergravity during the embryonic and neonatal periods has a highly significant effect on the developing forebrain and cerebellum and neonatal thyroid status (P < 0.05, Bonferroni corrected). These data are consistent with the hypothesized role of the thyroid hormone in mediating the effect of hypergravity in the developing central nervous system and begin to define the role of TH in the overall response of the developing organism to altered gravity.

Animals↗

Reduction of G1 phase duration and enhancement of c-myc gene expression in HeLa cells at hypergravity.

We have found that hypergravity stimulates the proliferation of HeLa cells through reduction of the G1 phase duration, concomitant with enhancement of c-myc gene expression. HeLa cells were grown in monolayer in culture flasks that were centrifuged to generate a constant 18, 35 or 70 g at 37 degrees C for up to 4 days. The cell proliferation was enhanced at 18, 35 and 70 g, most notably at 35 g. Cell cycle analyses with [3H]thymidine (TdR)-colcemid treatment showed that the cell generation time in the 35 g culture was reduced by 17% as compared to the control, which was attributed to a 26% reduction of the G1 phase duration. No differences were observed in the duration of the S, G2 and M phases or in the [3H]TdR incorporation per S phase cell between the 35 g culture and the control. The induction of c-myc gene expression was investigated by RNA blot hybridization during a 15-360 min exposure of cells to 18, 35 and 70 g. Elevated levels of c-myc mRNA were observed after a 15-min exposure, and maintained after a 360-min exposure at all hypergravities examined. The highest induction rate of c-myc mRNA was 3.8-fold higher than the control after a 120-min exposure to 35 g. The 35 g condition was the most effective hypergravity for stimulating both cell proliferation and c-myc gene expression. Our study suggests that the appropriate level of hypergravity stimulates HeLa cell proliferation by reducing the G1 phase duration without affecting DNA synthesis rate, mediated through induction of c-myc gene expression.

Cell Cycle↗

Ultrastructure of the parathyroid gland of magnesium-treated golden hamster exposed to a hypergravity environment: a stereological study.

The ultrastructure of the parathyroid glands of magnesium-treated golden hamsters exposed to a 5 gravity environment was studied. In the parathyroid glands of the magnesium-treated animals exposed to a hypergravity environment, the Golgi complexes and cisternae of the granular endoplasmic reticulum were increased as compared to those of the magnesium-treated animals and decreased as compared to those of animals exposed to a hypergravity environment, but were almost the same as those of the control animals. In the control and experimental animals, the chief cells were rich in free ribosomes and mitochondria. In addition, numerous secretory granules were situated close to the plasma membrane in the magnesium-treated animals exposed to a hypergravity environment. These observations suggest that the synthesis of parathyroid hormone may be stimulated in the parathyroid glands of magnesium-treated hamsters exposed to hypergravity environment.

Animals↗

[Effects of hypergravity on migration, proliferation and function of mouse osteoblastic cell line MC3T3-E1].

The purpose of this study was to investigate the hypergravity-induced responses and their mediators of osteoblastic cell line, MC3T3-E1. The synchronized G1 or the S Phase cells were exposed to 5 and 18 x g hypergravity at 37 degrees C. The migration velocity was measured and the morphology was observed. MC3T3-E1 cells were cultured for 1, 2 or 3 days at 37 degrees C, exposing to 5, 10, 20 and 40 x g hypergravity. The proliferation, prostaglandin E2 (PGE2) production rate and alkaline phosphatase (ALPase) activity were measured. The results were as follows: 1) In the G1 phase of the cell cycle, the migration of MC3T3-E1 cells was increased by 18 x g. In the S phase, the morphology altered depending on the g-stress. 2) The proliferation of MC3T3-E1 cells was enhanced at 20 and 40 x g but reduced at 10 x g. The proliferation of HeLa cells and JTC-12 cells was also enhanced at 40 x g. 3) Indomethacin (10(-5)M) reduced the proliferation of MC3T3-E1 cells induced by 40 x g. But indomethacin (10(-5)M) did not reduce the proliferation of HeLa cells. 4) The increase of the released PGE2 from the cells depended on the time (1-8h) and the gravity (1-40 x g). 5) The increase of the ALPase activity of MC3T3-E1 cells also depended on the gravity. These results suggest that the hypergravity enhanced the proliferation of MC3T3-E1 cells via PGE2-mediated mechanism.

Alkaline Phosphatase↗

Electron microscopic study of the parathyroid gland of the calcium-treated hamster subjected to hypergravity environment.

The ultrastructure of the parathyroid glands of calcium-treated golden hamsters subjected to 5 gravity environment was studied. In the calcium-treated animals exposed to hypergravity environment, the Golgi complexes and cisternae of the granular endoplasmic reticulum were significantly decreased compared with those of the animals exposed to hypergravity environment only and appeared to increase compared with those of the calcium-treated animals, but were almost similar to those of the control animals. In addition, many chief cells contained some prosecretory granules in the Golgi areas, some secretory granules situated close to the plasma membrane and many lysosomes. The morphology of the parathyroid glands in the calcium-treated animals exposed to hypergravity environment resembled that of the control animals. These results suggest that the parathyroid glands suppressed by treatment of calcium and stimulated in response to hypergravity environment may indicate the secretory activity of the parathyroid glands of the control animals.

Animals↗

Effects of hypergravity on "whole-blood" cultures of human lymphocytes.

The purpose of this paper is to present a detailed description of the effects of hypergravity on the mitogenic response of human lymphocytes to concanavalin A. The effect on cultures of lymphocytes isolated from peripheral blood are compared with those on whole-blood cultures obtained by diluting fresh blood with culture medium 1:10. Whole-blood cultures of lymphocytes from crew members will be investigated inflight on the Spacelab missions D-1 in 1985 and SLS-1 in 1987. In hypergravity there is an increase in lymphocyte activation of up to 500%. A similar increase can be induced by pre-incubating the cultures in hypergravity prior to exposure to concanavalin A at 1 G. The effect is less evident in cultures of isolated lymphocytes. The influence of autologous plasma and erythrocytes has also been investigated. Plasma and hypergravity have a synergistic and positive effect on lymphocyte activation, i.e. cultures of separated lymphocytes show the highest activation when incubated at 10 G and supplemented with autologous plasma. Conversely, erythrocytes depress lymphocyte activation.

Adaptation, Physiological↗

Hypergravity and estrogen effects on avian anterior pituitary growth hormone and prolactin levels.

Developing female chicks with fractured right radii were maintained for 14 d at either earth gravity (1 G) or a hypergravity state (2 G). The birds at 1 G were divided into groups which received daily injections of 1) saline, 2) 200 micrograms estrone, and 3) 400 micrograms estrone for 14 d. The 2-G birds were divided into three similarly treated groups. All 2-G birds showed significantly lower body weights than did 1-G birds. Anterior pituitary (AP) glands were excise; and analyzed for growth hormone and prolactin content by analytical electrophoresis. The 1-G chicks receiving either dose of daily estrogen showed increased AP growth hormone levels, whereas hypergravity alone did not affect growth hormone content. Chicks exposed to daily estrogen and hypergravity displayed reduced growth hormone levels. AP prolactin levels were slightly increased by the lower daily estrogen dose in 1-G birds, but markedly reduced in birds exposed only to hypergravity. Doubly-treated chicks displayed normal prolactin levels. Reduced growth in 2-G birds might be due, in part, to reduced AP levels of prolactin and/or growth hormone.

Animals↗

Regeneration of guinea pig facial nerve: the effect of hypergravity.

Exposure to moderate hypergravity improves the regenerative capacity of sectioned guinea-pig facial nerve. The improvement in regeneration is tri-directional as follows: a) an average 1.7 fold increase in rate of regeneration in guinea pigs subjected to hypergravity; b) a 25% enhancement of facial muscle activity following the exposure to hypergravity; and c) improvement in the quality of regeneration from an esthetic standpoint. A good correlation was recorded between the histological structure of the severed nerve at the end of the regeneration and the clinical results.

Animals↗

Otoconial alterations after embryonic development in hypergravity.

The relation between prolonged hypergravity and structural adaptation of otoconia was studied in hamsters (n = 56). Three groups of hamsters (n = 27), were conceived and born in a centrifuge: group 1 (n = 10) 1 month under 2.5 G, group 2 (n = 9) 5 months under 2.5 G and 4 months under 1 G, group 3 (n = 8) 1 month under 2.5 G and 8 months under 1 G. Control hamsters (n = 29) were conceived and born under 1 G (1 month old, n = 7; 9 months old, n = 22). Histological study of the otoconial layers (energy dispersive x-ray element analysis and scanning electron microscopy) showed similar calcium content, size, and shape in utricular and saccular otoconia in all groups. Different were the utricular otoconial size classes, large, medium-sized, and small. The area with small otoconia increased in group 1 (p = 0.002). In group 2, the large otoconial area decreased (p = 0.001) and the medium-sized one increased (p < 0.001). In group 3, the large otoconial area decreased (p = 0.003) and the medium-sized one increased (p = 0.007). For age-related effects we found group 1 with an increased area of large otoconia (p = 0.001) and a decreased medium-sized one compared to groups 2 (p < 0.001) and 3 (p = 0.02). Hypergravity during formation of otoconia does not affect calcium content, size, or shape, but changes relative size of the areas with large, medium-sized, or small otoconia and the development of these areas. This resulted in a structural adaptation to hypergravity.

Animals↗

Exploration and motor activity in juvenile and adult rats exposed to hypergravity at 1.8 G during development: a preliminary report.

Pups from gestating rats exposed to hypergravity (1.8 G) or to normal gravity at the perinatal period were evaluated for motor activity, exploration and social interactions during juvenile and adult stages. By comparison to controls, the hypergravity group had shorter latencies before choosing a maze arm in a T-maze and a lower number of exploratory pokes in a hole board. During dyadic encounters, the hypergravity group had a lower number of self-grooming episodes and shorter latencies before crossing under the opposing rat. In contrast, no intergroup differences were observed during exploration of an elevated plus-maze and a light-dark box. These results indicate that exposure to 1.8 G during development appears to decrease exploratory tendencies in the hole board and fear-related responses in T-maze and social interaction tests.

Aging↗

Ultrastructural aspects of otoliths and sensory epithelia of fish inner ear exposed to hypergravity.

The present electron microscopical investigations were directed to the question, whether alterations in the gravitational force might induce structural changes in the morphology of otoliths or/and inner ear sensory epithelia of developing and adult swordtail fish (Xiphophorus helleri) that had been kept either under long-term moderate hypergravity (8 days; 3g) or under short-time extreme hypergravity (10 minutes up to 9g). The otoliths of adult and neonate swordtail fish were investigated by means of scanning electron microscopy (SEM). Macular epithelia of adult fish were examined both by SEM and transmission electron microscopy (TEM). The saccular otoliths (sagittae) of normally hatched adult fish revealed an enormous inter- (and even intra-; i.e. left vs. right) individual diversity in shape and size, whereas the otoliths of utricles (lapilli) and lagenae (asterisci) seemed to be more constant regarding morphological parameters. The structural diversity of juvenile otoliths was found to be less prominent as compared to the adults, differing from the latter regarding their peculiar crystalline morphology. Qualitative differences in the fine structure (SEM) of otoliths taken from adult and larval animals kept under 3g in comparison to 1g controls could not be observed. The SEM and TEM investigations of sensory epithelia also did not reveal any effects due to 3g stimulation. Even extreme hypergravity (more than 7g) for 10 minutes did not result in distinct pathological changes.

Aging↗