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Impaired osteoblast proliferation plays fundamental roles in microgravity‐induced bone loss, and cell cycle imbalance may result in abnormal osteoblast proliferation. However, whether microgravity exerts an influence on the cell cycle in osteoblasts or what mechanisms may underlie such an effect remains to be fully elucidated. Herein, we confirmed that simulated microgravity inhibits osteoblast proliferation. Then, we investigated the effect of mechanical unloading on the osteoblast cell cycle and found that simulated microgravity arrested the osteoblast cell cycle in the G2 phase. In addition, our data showed that cell cycle arrest in osteoblasts from simulated microgravity was mainly because of decreased cyclin B1 expression. Furthermore, miR‐181c‐5p directly inhibited cyclin B1 protein translation by binding to a target site in the 3′UTR. Lastly, we demonstrated that inhibition of miR‐181c‐5p partially counteracted cell cycle arrest and decreased the osteoblast proliferation induced by simulated microgravity. In conclusion, our study demonstrates that simulated microgravity inhibits cell proliferation and induces cell cycle arrest in the G2 phase in primary mouse osteoblasts partially through the miR‐181c‐5p/cyclin B1 pathway. This work may provide a novel mechanism of microgravity‐induced detrimental effects on osteoblasts and offer a new avenue to further investigate bone loss induced by mechanical unloading.  相似文献   

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Effect of microgravity on the cell cycle in the lentil root   总被引:1,自引:0,他引:1  
Characteristics of the cell cycle in cortical regions (0–0.6 mm from the root-cap junction) of the primary root of lentil (Lens culinaris L.) during germination in the vertical position on earth were determined by iododeoxyuridine labelling and image analysis. All cells were in the G1 phase at the beginning of germination and the duration of the first cell cycle was about 25 h. At 29 h, around 14% of the cortical nuclei were still in the G2 or M phases of the first cell cycle, whereas 53 and 33% of the nuclei were respectively in the G1 or S phase of the second cell cycle. In parallel, the cell cycle was analysed in root tips of lentil seedlings grown in space during the IML 2 mission (1994), (1) on the 1-g centrifuge for 29 h, (2) on the 1-g centrifuge for 25 h and placed in microgravity for 4 h, (3) in microgravity for 29 h, (4) in microgravity for 25 h and placed on the 1-g centrifuge for 4 h. The densitometric analysis of nuclear DNA content showed that in microgravity there were less cells in DNA synthesis and more cells in G1 than in the controls on the 1-g centrifuge (flight and ground). The comparison of the sample grown continuously on the 1-g centrifuge in space and of the sample grown first in 1-g and then in microgravity indicated that 4 h of microgravity modified cell cycle, increasing the percentage of cells in the G1 phase. On the contrary, the transfer from microgravity to the 1-g centrifuge (for 4 h) did not provoke any significant change in the distribution of the nuclear DNA content. Thus the effect of microgravity could not be reversed by a 4 h centrifugation. As the duration of the first cell cycle in the lentil root meristem is about 25 h, the results obtained are in agreement with the hypothesis that the first cell cycle and/or the second G1 phase was lengthened in absence of gravity. The difference observed in the distribution of the nuclear DNA content in the two controls could be due to the fact that the 1g control on board was subjected to a period of 15 min of microgravity for photography 25 h after the hydration of the seeds, which indicated an effect of short exposure to weightlessness. The mitotic index of cortical cells was greater on the 1-g centrifuge in space than in any other sample (flight and ground) which could show an effect of the centrifugation on the mitosis.  相似文献   

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Using cytophotometric procedures, we measured the nuclear and nucleolar protein content of successive zones of growth and differentiation in consecutive (1-7 mm) root segments obtained from eight species of the Angiospermae after staining the preparations with Feulgen-Naphthol Yellow S (F-NYS). In meristematic cells the nuclear and nucleolar protein content was found to double during the cell cycle. In species in which differentiation occurs at the same time as nuclear DNA endoreplication, i.e. Vicia faba subsp. minor, V. faba subsp. major, Pisum sativum, Hordeum vulgare and Amaryllis belladonna, the pool of nuclear proteins observed during the G2 phase of the cell cycle was seen in the differentiated zone in nuclei containing 8C DNA. Species in which differentiation is not accompanied by the process of nuclear DNA endoreplication, i.e. Levisticum officinale, Tulipa kaufmanniana and Haemanthus katharinae, exhibited the highest nuclear proteins content during the G2 phase of the cell cycle; comparably high values were not found in the differentiated zone. A decrease in nucleolar protein content was observed during the process of differentiation, this tendency being more evident in the studied species that do not exhibit endoreplication.  相似文献   

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The cell cycle is a temporal program that regulates DNA synthesis and cell division. When we compared the codon usage of cell cycle‐regulated genes with that of other genes, we discovered that there is a significant preference for non‐optimal codons. Moreover, genes encoding proteins that cycle at the protein level exhibit non‐optimal codon preferences. Remarkably, cell cycle‐regulated genes expressed in different phases display different codon preferences. Here, we show empirically that transfer RNA (tRNA) expression is indeed highest in the G2 phase of the cell cycle, consistent with the non‐optimal codon usage of genes expressed at this time, and lowest toward the end of G1, reflecting the optimal codon usage of G1 genes. Accordingly, protein levels of human glycyl‐, threonyl‐, and glutamyl‐prolyl tRNA synthetases were found to oscillate, peaking in G2/M phase. In light of our findings, we propose that non‐optimal (wobbly) matching codons influence protein synthesis during the cell cycle. We describe a new mathematical model that shows how codon usage can give rise to cell‐cycle regulation. In summary, our data indicate that cells exploit wobbling to generate cell cycle‐dependent dynamics of proteins.  相似文献   

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Summary. Fibrillarin and the plant nucleolin homolog NopA64 are two important nucleolar proteins involved in pre-rRNA processing. In order to determine the effects of the altered gravity environment on the nucleolus, we have investigated the location of fibrillarin and NopA64 in nucleolar subcomponents of cress (Lepidium sativum L.) root meristematic cells grown under clinorotation, which reproduces an important feature of microgravity, namely, the absence of the orienting action of a gravity vector, and compared it to the location in control cells grown in normal 1 g conditions. Prior to these experiments, we report here the characterization of cress fibrillarin as a 41 kDa protein which can be isolated from meristematic cells in three nuclear fractions, namely, the soluble ribonucleoprotein fraction, the chromatin fraction, and the nuclear-matrix fraction. Furthermore, as reported for other species, the location of both fibrillarin and NopA64 in the cress cell nucleolus was in zones known to contain complex ribonucleoprotein particles involved in early pre-rRNA processing, i.e., processomes. Under altered gravity, a decrease in the quantity of both fibrillarin and NopA64 compared to controls was observed in the transition zone between fibrillar centers and the dense fibrillar component, as well as in the bulk of the dense fibrillar component. These data suggest that altered (reduced) gravity results in a lowered level of functional activity in the nucleolus. Correspondence and reprints: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, Ramiro de Maeztu 9, 28040 Madrid, Spain.  相似文献   

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Mean diameter of nucleolar bodies (nucleoli without the perinucleolar chromatin) per cell was studied in human leukemic myeloblasts represented by K 562 and Kasumi 1 cell lines which originated from chronic and acute myeloid leukaemia. The measurement of mean diameter of nucleolar bodies in specimens stained for RNA was very simple. Such approach eliminated the variability of the perinucleolar chromatin discontinuous shell which might influence the measured nucleolar size as suggested by earlier studies. Ageing of K 562 myeloblasts produced a significant decrease of cells in S+G2 phase of the cell cycle accompanied by a significant reduction of mean diameter of nucleolar bodies (MDNoBs) per cell. In contrast, treatment of Kasumi 1 myeloblasts with histone deacetylase inhibitor - Trichostatin A - produced a large incidence of resistant cells in S+G2 phase which were characterised by a large increase of MDNoBs. Thus, MDNoBs in leukemic myeloblasts might be a helpful tool to estimate the incidence of cells in the S+G2 phase at the single cell level in smear preparations when the number of cells is very small.  相似文献   

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Dai ZQ  Wang R  Ling SK  Wan YM  Li YH 《Cell proliferation》2007,40(5):671-684
OBJECTIVES: Microgravity is known to affect the differentiation of bone marrow mesenchymal stem cells (BMSCs). However, a few controversial findings have recently been reported with respect to the effects of microgravity on BMSC proliferation. Thus, we investigated the effects of simulated microgravity on rat BMSC (rBMSC) proliferation and their osteogeneic potential. MATERIALS AND METHODS: rBMSCs isolated from marrow using our established effective method, based on erythrocyte lysis, were identified by their surface markers and their proliferation characteristics under normal conditions. Then, they were cultured in a clinostat to simulate microgravity, with or without growth factors, and in osteogenic medium. Subsequently, proliferation and cell cycle parameters were assessed using methylene blue staining and flow cytometry, respectively; gene expression was determined using Western blotting and microarray analysis. RESULTS: Simulated microgravity inhibited population growth of the rBMSCs, cells being arrested in the G(0)/G(1) phase of cell cycle. Growth factors, such as insulin-like growth factor-I, epidermal growth factor and basic fibroblastic growth factor, markedly stimulated rBMSC proliferation in normal gravity, but had only a slight effect in simulated microgravity. Akt and extracellular signal-related kinase 1/2 phosphorylation levels and the expression of core-binding factor alpha1 decreased after 3 days of clinorotation culture. Microarray and gene ontology analyses further confirmed that rBMSC proliferation and osteogenesis decreased under simulated microgravity. CONCLUSIONS: The above data suggest that simulated microgravity inhibits population growth of rBMSCs and their differentiation towards osteoblasts. These changes may be responsible for some of the physiological changes noted during spaceflight.  相似文献   

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Fibrillarin and plant nucleolin homologue NopA64 are two important nucleolar proteins involved in pre-rRNA processing. To understand better the effects of the altered gravity environment on the nucleolus functioning we have investigated the location of fibrillarin and NopA64 in nucleolar subcomponents of cress (Lepidium sativum L.) root meristematic cells grown under simulated microgravity that was compared to the control cells grown in normal conditions at I g. Cress fibrillarin was first shown to have the molecular weight 41 kDa. Both fibrillarin and NopA64 in the cress cell nucleolus are located in the zones known to contain processing pre-rRNA molecules as it has been previously reported in other species. The data confirm participation of these proteins in processomes--RNP complex particles involved in pre-rRNA processing. Under altered gravity a decrease in the quantity of both fibrillarin and NopA64 in the transition zone between fibrillar centres and the dense fibrillar component was observed, compared to control, which could point out to a lowering of the level of early pre-rRNA processing in these experimental conditions. This decrease was also detected in the bulk of the dense fibrillar component. These data support the idea that altered (reduced) gravity results in lowering the level of functional activity of the nucleolus.  相似文献   

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The DNA-binding protein Ku (p70/p80) was originally discovered through the use of human autoimmune sera. In attempts to search out nucleolar proteins in relation to nucleolar dynamic changes, we developed monoclonal antibodies against nuclear proteins. One antibody, termed LL1, received particular attention since asynchronous cells exhibited tremendous differences in their nucleolar fluorescence intensities after immunostaining. The LL1 protein was proven to be the Ku subunit p80 (Ku80) by cDNA cloning and sequencing. Possible correlations between the heterogeneous distribution of Ku80 in nucleoli and the cell cycle were examined. HeLa cells were synchronized at M phase by arrest with nocodazole, or at the G1/S boundary by sequential treatments with thymidine and aphidicolin. These cells were then released by culturing in fresh medium to allow the cell cycle to progress synchronously. Immunofluorescent detection of Ku80 revealed that nucleoli of the cells at the G1/S boundary had very small amounts of Ku80, which was mainly present in the nucleoplasm. Ku80 was gradually accumulated in nucleoli during S phase and reached the maximum at late S or G2 phase. Immunoblotting experiments showed that cell extracts prepared from different phases of the cell cycle had virtually identical amounts of Ku80. These results suggest that Ku80 migrates from nucleoplasm to nucleoli in a cell cycle-dependent manner.  相似文献   

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The patterns of [3H]-leucine and [32P]-phosphate incorporation of proteins extracted with varying molarities of sodium chloride were analyzed from nuclei physically sorted from six fluorescence windows after propidium iodine staining of the G0 + G1 and G2 + M phases of the Chinese hamster ovary (CHO) cell cycle. Eight hundred nanograms of protein were used in each electrophoretic analysis obtained from 200,000 nuclei, a portion of the sample, from each window. Autoradiography was performed in a two-dimensional polyacrylamide gel ultra-microelectrophoresis apparatus (UMEA) designed and fabricated in this laboratory. There was a net reduction and/or loss of [3H]-leucine- and [32P]-phosphate-labeled protein regions from the autoradiographs occurring primarily in the G2 + M phase. Two phosphorylated proteins that were stage specific were observed in partitions of the G2 + M phase. The use of isolated proteins and the coelectrophoresis of these markers demonstrated the similarity in mobility of a number of proteins seen in the autoradiographs of proteins extracted with high and low salt molarities and implied they are synonymous. Coelectrophoresis indicated that a substantial number of high molecular weight proteins that decreased or disappeared at late stages of G2 + M and early mitosis were composed, in part, of nucleolar proteins.  相似文献   

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The cellular response to DNA double‐strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell‐cycle‐dependent manner. Here, we report that the crucial checkpoint signalling proteins—p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2—are phosphorylated rapidly by ATR in an ATM/Mre11/cell‐cycle‐independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM‐deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM‐deficient cells enforce an early G2/M checkpoint that is ATR‐dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM‐deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP‐1), a protein involved in chromatin remodelling, is mediated by DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in a spatio‐temporal manner in addition to ATM. We posit that ATM substrates involved in cell‐cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA‐PKcs in addition to ATM.  相似文献   

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Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.  相似文献   

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