Search

Abstract: 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 7days’ culture under normal (1 g) condition, cells were exposed to simulated microgravity for 24h. The results showed that 24h 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. [Copyright &y& Elsevier]

Abstract: Arabidopsis gene expression microarray (44K) was used to detect genes highly induced under simulated microgravity stress (SMS). Ten SMS-inducible genes were selected from the microarray data and these 10 genes were found to be abundantly expressed in 3-week-old plants. Nine out of the 10 SMS-inducible genes were also expressed in response to the three abiotic stresses of drought, touch, and wounding in 3-week-old Arabidopsis plants respectively. However, WRKY46 was elevated only in response to SMS. Six other WRKY genes did not respond to SMS. To clarify the characteristics of the genes expressed at high levels in response to SMS, 20 cis-elements in the promoters of the 40 selected genes including the 10 SMS-inducible genes, the 6 WRKY genes, and abiotic stress-inducible genes were analyzed and their spatial positions on each promoter were determined. Four cis-elements (M/T-G-T-P from MYB1AT or TATABOX5, GT1CONSENSUS, TATABOX5, and POLASIG1) showed a unique spatial arrangement in most SMS-inducible genes including WRKY46. Therefore the M/T-G-T-P cis-element patterns identified in the promoter of WRKY46 may play important roles in regulating gene expression in response to SMS. The presences of the cis-element patterns suggest that the order or spatial positioning of certain groups of cis-elements is more important than the existence or numbers of specific cis-elements. Taken together, our data indicate that WRKY46 is a novel SMS inducible transcription factor and the unique spatial arrangement of cis-elements shown in WRKY46 promoter may play an important role for its response to SMS. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Hematopoietic progenitor cell proliferation can be altered in either spaceflight or under simulated microgravity experiments on the ground, however, the underlying mechanism remains unknown. Our previous study showed that exposure of the human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO to conditions of simulated microgravity significantly inhibited the cellular proliferation rate and induced cell apoptosis. We postulated that the downregulation of the erythropoietin receptor (EPOR) expression in UT-7/EPO cells under simulated microgravity may be a possible reason for microgravity triggered apoptosis. In this paper, a human EPOR gene was transferred into UT-7/EPO cells and the resulting expression of EPOR on the surface of UT-7/EPO cells increased approximately 61% (p <0.05) as selected by the antibiotic G418. It was also shown through cytometry assays and morphological observations that microgravity-induced apoptosis markedly decreased in these UT-7/EPO–EPOR cells. Thus, we concluded that upregulation of EPOR in UT-7/EPO cells could inhibit the simulated microgravity-induced cell apoptosis in this EPO dependent cell line. [Copyright &y& Elsevier]

Abstract: Gene expression analysis using microarrays has proved to be an important method in life science. The opportunity to grow higher plants on the International Space Station (ISS) opens up the possibility for gene expression profiling of plants grown in microgravity. The work presented focuses on how to meet the scientific requirements of plant growth and the sample preservation, given the technical and operational constraints associated with space research. The growth chamber (Multigen-2 Science Testing Unit) and a protocol suggested to be used in the European Modular Cultivation System (EMCS) Multigen-2 experiment on the ISS to grow and later preserve Arabidopsis seedlings, were tested on ground. The results showed that most of the plants developed normally. In order to avoid high population stress the number of seedlings per growth area should be reduced. The RNAlater preservation method to be used in the space experiment was compared with a quick freeze in Liquid Nitrogen (LN2). The RNA from samples preserved in RNAlater at room temperature for 24h was slightly more degraded than the RNA from the LN2 preserved samples (RNA integrity number, RIN: 7.7 and 8.6, respectively). However, the RNA quality and quantity was satisfactory for microarray analysis. Of the genes analysed, 74 genes (0.28%) were significantly differentially expressed, most of them showing moderate to low regulation. Among the genes induced in the RNAlater preserved samples, three salt inducible transcription factors (ZAT10, SZF1 and SZF2) were identified, suggesting that the high salt concentration in RNAlater causes salt stress before the transcription stopped. In conclusion, the Multigen-2 preservation protocol tested here will allow for the genes regulated by microgravity in the space experiment to be revealed. The results do indicate that not all the biological processes are stopped instantly by the RNAlater. The limited diffusion indirectly caused by the microgravity may potentially result in a different degree of salt stress in the test compared to the 1×g control during the space experiment. This has to be accounted for during the evaluation of the results. Since slightly degraded RNA was observed, further optimalisation of the preservation protocol will be performed. [Copyright &y& Elsevier]

Author: Purpose: This study was aimed to explore the effect of flow shear stress on the expression of Cbfa1 in human osteosarcoma cells and to survey its functional alteration in simulated microgravity. After culture for 48h in two different gravitational environments, i.e. 1G terrestrial gravitational condition and simulated microgravity condition, human osteosarcoma cells (MG-63) were treated with 0.5 or 1.5Pa fluid shear stress (FSS) in a flow chamber for 15, 30, and 60min, respectively. The total RNA in cells was isolated. RT-PCR analysis was made to examine the gene expression of Cbfa1. The total protein of cells was extracted and the expression of Cbfa1 protein was detected by means of Western blotting. Results: MG-63 cells cultured in 1G condition reacted to FSS treatment with an enhanced expression of Cbfa1. Compared with no-FSS control group, Cbfa1 mRNA expression increased significantly at 30 and 60min with the treatment of FSS (P <0.01). And there was remarkable difference on the Cbfa1 mRNA expression between the treatments of 0.5 and 1.5Pa FSS at 30 or 60min (P <0.01). Cbfa1 protein expressions had a trend to increase at 30min with the treatment of FSS and they increased significantly at 60min with the treatment of 0.5 or 1.5Pa FSS (P <0.05). As to the cells cultured in simulated microgravity by using clinostat, the expression of Cbfa1 was significantly different between 1G and simulated microgravity conditions at each test time (P <0.05). Compared with no-FSS control group cultured in simulated microgravity, Cbfa1 mRNA expression increased significantly at 30 and 60min with the treatment of FSS (P <0.05). And Cbfa1 protein expression increased significant at 60min with the treatment of 1.5Pa FSS under simulated microgravity conditions (P <0.05). Conclusions: FSS can significantly increase the gene and protein expression of Cbfa1 in human osteosarcoma cells. And this inducible function of FSS was adversely affected by simulated microgravity. [Copyright &y& Elsevier]

Abstract: Within the next decades, an increasing number of human beings will be brought into space to carry out technical and scientific tasks. There, they will be exposed simultaneously to combined stimuli, especially microgravity and radiation. In the endeavour to assess the risks for humans during long-duration space missions, it is necessary to understand already at the cellular level the complex interplay of these parameters. Cellular stress protection responses lead to an increased transcription of several genes via the modulation of transcription factors. The activation of the nuclear factor κB (NF-κB) pathway as a possible anti-apoptotic route represents such an important cellular stress response. It is hypothesized that the activation of NF-κB and the subsequent expression of NF-κB-dependent genes is involved in the cellular response to components of the cosmic radiation. Irradiation of human embryonic kidney cells (HEK/293) with α-particles (2.1MeV, LET ∼160keV/μm) was performed at the PTB, Braunschweig, Germany. Using the microbeam facility, cells were exposed to nuclear hits or, for the purpose of comparison, to a diffuse irradiation of the whole cell. After irradiation the following biological endpoints were determined: (i) cell survival (by means of the colony forming ability test), and (ii) quantitative RT-PCR analysis of selected NF-κB target genes (IκBα GADD45β, bcl-2, and bcl-XL). One nuclear α-particle traversal reduces the probability to survive to ∼75%. Exposure to two α-particles per nucleus resulted in an upregulation of the expression of the GADD45β gene. After exposure of HEK cells to five nuclear hits, about 43% of the irradiated cells survived, and the transcriptional response was not significant. Ten nuclear hits activated the IκBα expression, this increased IκBα production might be involved in the termination of the radiation-induced NF-κB activation. Diffuse irradiation increased the transcription of IκBα and GADD45β. Expression of the antiapoptotic genes bcl-2 and bcl-XL remained almost unchanged after α-particle irradiation. These findings suggest a role for GADD45β in the cellular radiation response to low fluences, while IκBα is enrolled at higher fluences. [Copyright &y& Elsevier]

Abstract: Immediate-early genes serve as useful neurobiological tools for mapping brain activity induced by a sensory stimulation. In this study, we have examined brain activity related to gravity perception of medaka (Oryzias latipes) by use of c-fos. The gene, which is homologous to the c-fos genes of other vertebrates, was identified in medaka. Functionally important domains are highly conserved among all the vertebrate species analyzed. Intraperitoneal administration of kainic acid transiently induced the c-fos mRNAs in medaka brains. The results indicate that the expression of c-fos can be utilized as a suitable anatomical marker for the increased neural activities in the central nervous system of medaka. Fish were continuously exposed to 3 g hypergravity by centrifugation. Investigation of c-fos mRNA expression indicated that c-fos mRNA significantly increased 30 min after a start of 3 g exposure. The distribution of its transcripts within the brains was analyzed by an in situ hybridization method. The 3-g treated medakas displayed c-fos positive cells in their brainstem regions, which are related to vestibular function, such as torus semicircularis, nucleus tangentialis, posterior octavu nucleus, and inferior olive. Our results established a method to follow the effect of gravity stimulation, which can be used to investigate gravity perception. [Copyright &y& Elsevier]