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Nuclear Fraction (nuclear + fraction)
Selected AbstractsSequential activation of Notch family receptors during mouse spermatogenesisDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2003Shintaro Mori The expression pattern of Notch family receptors during mouse spermatogenesis was examined by immunohistochemistry. The entire cytoplasm of spermatogonia, spermatocytes and spermatids showed staining with antibodies against extracellular domains of Notch1, 2 and 4. In contrast, the nuclei of spermatogonia showed staining with an antibody against the intracellular domain of Notch3, and the nuclei of spermatocytes and spermatids showed staining with antibodies against the intracellular domains of Notch1 and 4. During regeneration of spermatogonia in busulfan-treated mice, the nuclei of all proliferating cells showed staining for the intracellular domain of Notch3. Western blot analysis showed that the molecular weights of the intracellular domains of Notch1 and 3 localizing in the nuclear fraction were smaller than those in the cytoplasmic fraction. This was consistent with the theory that the intracellular domain of Notch was cleaved in the cytoplasm and translocated to the nucleus. These results suggest that different Notch signals are sequentially activated during mouse spermatogenesis and control the proliferation and differentiation of spermatogenic stem cells. [source] Identification of different isoforms of eEF1A in the nuclear fraction of human T-lymphoblastic cancer cell line specifically binding to aptameric cytotoxic GT oligomersFEBS JOURNAL, Issue 15 2003Barbara Dapas GT oligomers, showing a dose-dependent cytotoxic effect on a variety of human cancer cell lines, but not on normal human lymphocytes, recognize and form complexes with nuclear proteins. By working with human T-lymphoblastic CCRF-CEM cells and by using MS and SouthWestern blotting, we identified eukaryotic elongation factor 1 alpha (eEF1A) as the main nuclear protein that specifically recognizes these oligonucleotides. Western blotting and supershift assays confirmed the nature of this protein and its involvement in forming a cytotoxicity-related complex (CRC). On the contrary, normal human lymphocytes did not show nuclear proteins able to produce CRC in a SouthWestern blot. Comparative bidimensional PAGE and Western-blotting analysis for eEF1A revealed the presence of a specific cluster of spots, focusing at more basic pH, in nuclear extracts of cancer cells but absent in those of normal lymphocytes. Moreover, a bidimensional PAGE SouthWestern blot demonstrated that cytotoxic GT oligomers selectively recognized the more basic eEF1A isoform expressed only in cancer cells. These results suggest the involvement of eEF1A, associated with the nuclear-enriched fraction, in the growth and maintenance of tumour cells, possibly modulated by post-translational processing of the polypeptide chain. [source] Nuclear and nuclear envelope localization of dystrophin Dp71 and dystrophin-associated proteins (DAPs) in the C2C12 muscle cells: DAPs nuclear localization is modulated during myogenesisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2008R. González-Ramírez Abstract Dystrophin and dystrophin-associated proteins (DAPs) form a complex around the sarcolemma, which gives stability to the sarcolemma and leads signal transduction. Recently, the nuclear presence of dystrophin Dp71 and DAPs has been revealed in different non-muscle cell types, opening the possibility that these proteins could also be present in the nucleus of muscle cells. In this study, we analyzed by Immunofluorescence assays and Immunoblotting analysis of cell fractions the subcellular localization of Dp71 and DAPs in the C2C12 muscle cell line. We demonstrated the presence of Dp71, ,-sarcoglycan, ,-dystrobrevin, ,-dystroglycan and ,-syntrophin not only in plasma membrane but also in the nucleus of muscle cells. In addition, we found by Immunoprecipitation assays that these proteins form a nuclear complex. Interestingly, myogenesis modulates the presence and/or relative abundance of DAPs in the plasma membrane and nucleus as well as the composition of the nuclear complex. Finally, we demonstrated the presence of Dp71, ,-sarcoglycan, ,-dystroglycan, ,-dystrobrevin and ,-syntrophin in the C2C12 nuclear envelope fraction. Interestingly, ,-sarcoglycan and ,-dystroglycan proteins showed enrichment in the nuclear envelope, compared with the nuclear fraction, suggesting that they could function as inner nuclear membrane proteins underlying the secondary association of Dp71 and the remaining DAPs to the nuclear envelope. Nuclear envelope localization of Dp71 and DAPs might be involved in the nuclear envelope-associated functions, such as nuclear structure and modulation of nuclear processes. J. Cell. Biochem. 105: 735,745, 2008. © 2008 Wiley-Liss, Inc. [source] Hypoxia-inducible factor and nuclear factor kappa-B activation in blood,brain barrier endothelium under hypoxic/reoxygenation stressJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Ken A. Witt Abstract This investigation focuses on transcription factor involvement in blood,brain barrier (BBB) endothelial cell-induced alterations under conditions of hypoxia and post-hypoxia/reoxygenation (H/R), using established in vivo/ex vivo and in vitro BBB models. Protein/DNA array analyses revealed a correlation in key transcription factor activation during hypoxia and H/R, including NF,B and hypoxia-inducible factor (HIF)1. Electrophoretic mobility shift assays confirmed NF,B and HIF1 binding activity ex vivo and in vitro, under conditions of hypoxia and H/R. Hypoxia- and H/R-treated BBB endothelium showed increased HIF1, protein expression in both cytoplasmic and nuclear fractions, in ex vivo and in vitro models. Co-immunoprecipitation of HIF1, and HIF1, was shown in the nuclear fraction under conditions of hypoxia and H/R in both models. Hypoxia- and H/R-treated BBB endothelium showed increased expression of NF,B-p65 protein in both cytoplasmic and nuclear fractions. Co-immunoprecipitation of NF,B-p65 with NF,B-p50 was shown in the nuclear fraction under conditions of hypoxia and H/R in the ex vivo model, and after H/R in the in vitro model. These data offer novel avenues in which to alter and/or investigate BBB activity across model systems and to further our understanding of upstream regulators during hypoxia and H/R. [source] Alteration of argyrophilic nucleolar organizer region associated (Ag-NOR) proteins in apoptosis-induced human salivary gland cells and human oral squamous carcinoma cellsJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 4 2001Yasuhiro Morimoto Abstract: The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-associated proteins (Ag-NOR proteins) varies with cell activity, including ribosomal biogenesis occurring in proliferating cells. Proteins associated with some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carcinoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG and SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cells treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to transfer membranes and staining for Ag-NOR proteins by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39kDa, and 37 kDa) were detected in the proteins obtained from the control cells. The level of the 110-kDa protein decreased in the proteins prepared from OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fractionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction prepared from the apoptotic cells, while the 110-kDa protein decreased in the nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleolin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa protein. In the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytosol fraction of apoptotic cells, while the level of the 110-kDa protein decreased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells incubated with cytosol fractions prepared from HSG and SCC-25 cells were identical to those of the HSG cells. The alternation of AgNORs in apoptosis-induced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, which exhibited highly fluorescent condensed chromatin in OA-treated HSG cells. Silver grains representing AgNORs were not detected in the cells undergoing apoptosis. The dual-imposition view confirmed that AgNORs, which are visible as dots in nucleoli in the control cells, disappeared from the apoptotic nuclei of HSG cells. Our results indicate that the 110-kDa nucleolar Ag-NOR protein is associated with apoptosis and is cleaved during apoptosis. [source] A novel mechanism for mitogenic signaling via pro,transforming growth factor , within hepatocyte nucleiHEPATOLOGY, Issue 6 2002Bettina Grasl-Kraupp Transforming growth factor (TGF) ,, an important mediator of growth stimulation, is known to act via epidermal growth factor receptor (EGF-R) binding in the cell membrane. Here we show by immunohistology, 2-dimensional immunoblotting, and mass spectrometry of nuclear fractions that the pro-protein of wild-type TGF-, occurs in hepatocyte nuclei of human, rat, and mouse liver. Several findings show a close association between nuclear pro-TGF-, and DNA synthesis. (1) The number of pro-TGF-,+ nuclei was low in resting liver and increased dramatically after partial hepatectomy and after application of hepatotoxic chemicals or the primary mitogen cyproterone acetate (CPA); in any case, S phase occurred almost exclusively in pro-TGF-,+ nuclei. The same was found in human cirrhotic liver. (2) In primary culture, 7% of hepatocytes synthesized pro-TGF-,, which then translocated to the nucleus; 70% of these nuclei subsequently entered DNA replication, whereas only 2% of pro-TGF-,, hepatocytes were in S phase. (3) The frequency of hepatocytes coexpressing pro-TGF-, and DNA synthesis was increased by the hepatomitogens CPA or prostaglandin E2 and was decreased by the growth inhibitor TGF-,1. (4) Treatment with mature TGF-, increased DNA synthesis exclusively in pro-TGF-,, hepatocytes, which was abrogated by the EGF-R tyrosine kinase inhibitor tyrphostin A25. In conclusion, TGF-, gene products may exert mitogenic effects in hepatocytes via 2 different signaling mechanisms: (1) the "classic" pathway of mature TGF-, via EGF-R in the membrane and (2) a novel pathway involving the presence of pro-TGF-, in the nucleus. [source] Hypoxia-inducible factor and nuclear factor kappa-B activation in blood,brain barrier endothelium under hypoxic/reoxygenation stressJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Ken A. Witt Abstract This investigation focuses on transcription factor involvement in blood,brain barrier (BBB) endothelial cell-induced alterations under conditions of hypoxia and post-hypoxia/reoxygenation (H/R), using established in vivo/ex vivo and in vitro BBB models. Protein/DNA array analyses revealed a correlation in key transcription factor activation during hypoxia and H/R, including NF,B and hypoxia-inducible factor (HIF)1. Electrophoretic mobility shift assays confirmed NF,B and HIF1 binding activity ex vivo and in vitro, under conditions of hypoxia and H/R. Hypoxia- and H/R-treated BBB endothelium showed increased HIF1, protein expression in both cytoplasmic and nuclear fractions, in ex vivo and in vitro models. Co-immunoprecipitation of HIF1, and HIF1, was shown in the nuclear fraction under conditions of hypoxia and H/R in both models. Hypoxia- and H/R-treated BBB endothelium showed increased expression of NF,B-p65 protein in both cytoplasmic and nuclear fractions. Co-immunoprecipitation of NF,B-p65 with NF,B-p50 was shown in the nuclear fraction under conditions of hypoxia and H/R in the ex vivo model, and after H/R in the in vitro model. These data offer novel avenues in which to alter and/or investigate BBB activity across model systems and to further our understanding of upstream regulators during hypoxia and H/R. [source] Effects of polyamines on DNA synthesis using various subcellular DNA polymerases extracted from normal rat liver, tumour-bearing rat liver, and tumour cellsCELL BIOCHEMISTRY AND FUNCTION, Issue 1 2001Takahiko Taguchi Abstract The effects of polyamines on DNA synthesis in vitro using various subcellular DNA polymerase fractions from normal and tumour-bearing rat livers, and tumour cells were investigated. When nuclear and mitochondrial DNA polymerase fractions were used, DNA synthesis on activated DNA was increased 3.5,8-fold by the addition of 20,mM putrescine or cadaverine. However, DNA synthesis was not stimulated by the addition of spermidine or spermine at any concentration tested. In contrast, DNA synthesis using the cytoplasmic DNA polymerase fraction was not stimulated at various concentrations of any of the four polyamines tested. The stimulatory effects of putrescine and cadaverine were absent when nuclear fractions from tumour-bearing rat liver or from tumour cells were used. In addition, in vitro DNA synthesis was not stimulated by 20,mM putrescine or cadaverine when nuclear extracts from the livers of rats administered putrescine subcutaneously were used. The specific activities of DNA polymerases extracted from tumour cells and tumour-bearing rat liver were already fully stimulated. These results suggest that DNA polymerases in tumour cells and tumour-bearing liver cells are stimulated by trapped putrescine produced in tumour cells and are thus no longer activated by exogenous putrescine. Copyright © 2001 John Wiley & Sons, Ltd. [source] The role of various Bcl-2 domains in the anti-proliferative effect and modulation of cellular glutathione levels: a prominent role for the BH4 domainCELL PROLIFERATION, Issue 1 2003R. W. M. Hoetelmans Reduced cell proliferation and increased levels of cellular glutathione (GSH) are characteristic for cells that overexpress the anti-apoptotic Bcl-2 protein. We investigated the influence of various Bcl-2 domains on both these characteristics. Rat CC531 colorectal cancer cells were stably transfected with the human bcl- 2 gene (CCbcl2 cells) or with bcl- 2 gene constructs missing a coding sequence for a func-tional domain, BH1 (CC,BH1 cells), BH3 (CC,BH3 cells), BH4 (CC,BH4 cells) or the transmembrane region (CC,TM cells). We measured GSH levels in exponentially and confluent growing bcl- 2-transfected cell populations. The fraction of S-phase cells during exponential growth was significantly reduced in CCbcl2, CC,BH1, CC,BH3, and CC,TM cells compared with parental CC531, neo-transfected CC531 and CC,BH4 cells. GSH levels in these bcl -2 transfectants were significantly higher than in the parental line measured at 50% confluence; at 100% confluence they reached a similar level as found in parental cells. Independently from the presence of BH1, BH3 or TM domains, overexpression of Bcl-2 reduces cellular proliferation under conditions of increased GSH levels. This apparent link is lost in CC,BH4 cells; these cells are not reduced in cellular proliferation and harbour significantly higher GSH levels than found in the other transfectants. Studies on the subcellular localization revealed an extremely low expression of the Bcl-2 protein lacking the N-terminal BH4 domain in nuclear fractions. Nuclear translocation of Bcl-2 requires the presence of the BH4 domain and seems prominent in reducing cellular proliferation. [source] G2 -phase radiation response in lymphoblastoid cell lines from Nijmegen breakage syndromeCELL PROLIFERATION, Issue 2 2002A. Antoccia The relationship between G2 -phase checkpoint activation, cytoplasmic cyclin-B1 accumulation and nuclear phosphorylation of p34CDC2 was studied in Nijmegen breakage syndrome cells treated with DNA damaging agents. Experiments were performed on lymphoblastoid cell lines from four Nijmegen breakage syndrome patients with different mutations, as well as on cells from an ataxia telangiectasia patient. Lymphoblastoid cell lines were irradiated with 0.50,2 Gy X-rays and the percentage of G2 -phase accumulated cells was evaluated by means of flow cytometry in samples that were harvested 24 h later. The G2 -checkpoint activation was analysed by scoring the mitotic index at 2 and 4 h after treatment with 0.5 and 1 Gy X-rays and treatment with the DNA double-strand break inducer calicheamicin-,1. Cytoplasmic accumulation of cyclin-B1 was evaluated by means of fluorescence immunostaining or Western blotting, in cells harvested shortly after irradiation with 1 and 2 Gy. The extent of tyrosine 15-phosphorylated p34CDC2 was assessed in the nuclear fractions. Nijmegen breakage syndrome cells showed suboptimal G2 -phase checkpoint activation respect to normal cells and were greatly different from ataxia telangiectasia cells. Increased cytoplasmic cyclin-B1 accumulation was detected by both immunofluorescence and immunoblot in normal as well as in Nijmegen breakage syndrome cells. Furthermore, nuclear p34CDC2. phosphorylation was detected at a higher level in Nijmegen breakage syndrome than in ataxia telangiectasia cells. In conclusion, our data do not suggest that failure to activate checkpoints plays a major role in the radiosensitivity of Nijmegen breakage syndrome cells. [source] | |||||||||||||