摘要:

AbstractThe early response geneornithine decarboxylase(odc) is indispensable for normal and malignant cell growth. Although DNA methylation is generally associated with chromatin condensation and gene inactivation, theodcgene is heavily methylated at CCGG‐sequences in animal cell lines. In this work we analyzed the chromatin structure and the DNA methylation status at the CpG‐rich promoter sequences at theodclocus in mouse 3T3 fibroblasts. We show that the proximal promoter region of theodclocus is not hypermethylated, while the distal promoter sequences appear to have a few methylated CCGG‐sites and display methylation polymorphism. Furthermore, it was found that the 5′ promoter region ofodcis constitutively more sensitive to micrococcal nuclease than the coding and 3′ regions of theodcgene. Stimulation of the cells with serum resulted in an appearance of a DNase I sensitive site at the promoter region. The chromatin structure of the mid‐coding and 3′ regions of theodcgene also underwent structural changes that were accompanied by the rapid accumulation ofodcmRNA. Such changes were not detected in the chromatin structure ofglyceraldehyde‐3‐phosphate dehydrogenase(gadph) gene, whose expression remains invariant upon serum stimulation. These data suggest that the chromatin structure may play an important role in the rapid transcriptional activation ofodcand other immediate early genes during serum stimulation. © 19

ISSN:0730-2312    

DOI:10.1002/jcb.240550202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe effect of phorbol 12‐myristate 13‐acetate (PMA) on Ca2+‐ATPase activity in rat liver nuclei was investigated. Ca2+‐ATPase activity was calculated by subtracting Mg2+‐ATPase activity from (Ca2+‐Mg2+)‐ATPase activity. The nuclear Ca2+‐ATPase activity was significantly increased by the presence of PMA (2–20 μM) in the enzyme reaction mixture; the maximum effect was seen at 10 μM. The PMA (10 μM)‐increased Ca2+‐ATPase activity was not blocked by the presence of staurosporine (2 μM) or dibucaine (2 and 10 μM), an inhibitor of protein kinase. Meanwhile, vanadate (20 and 100 μM) caused a significant reduction in the nuclear Ca2+‐ATPase activity increased by PMA (10 μM). The present finding suggests that PMA has an activating effect on liver nuclear Ca2+‐ATPase independent of protei

ISSN:0730-2312    

DOI:10.1002/jcb.240550203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have recently found the calcium dependent glycogenolytic effect of pancreastatin on rat hepatocytes and the mobilization of intracellular calcium. To further investigate the mechanism of action of pancreastatin on liver we have studied its effect on guanylate cyclase, adenylate cyclase, and phospholipase C, and we have explored the possible involvement of GTP binding proteins by measuring GTPase activity as well as the effect of pertussis toxin treatment of plasma liver membranes on the pancreastatin stimulated GTPase activity and the production of cyclic GMP and myo‐inositol 1,4,5‐triphosphate.Pancreastatin stimulated GTPase activity of rat liver membranes about 25% over basal. The concentration dependency curve showed that maximal stimulation was achieved at 10−7M pancreastatin (EC50= 3 nM). This stimulation was partially inhibited by treatment of the membranes with pertussis toxin. The effect of pancreastatin on guanylate cyclase and phospholipase C were examined by measuring the production of cyclic GMP and myo‐inositol 1,4,5‐triphosphate respectively. Pancreastatin increased the basal activity of guanylate cyclase to a maximum of 2.5‐fold the unstimulated activity at 30°C, in a time‐ and dose‐dependent manner, reaching the maximal stimulation above control with 10−7M pancreastatin at 10 min (EC50= 0.6 nM). This effect was completely abolished when rat liver membranes had been ADP‐ribosylated with pertussis toxin. On the other hand, adenylate cyclase activity was not affected by pancreastatin. Phospholipase C activity of rat liver membranes was rapidly stimulated (within 2–5 min) at 30°C by 10−7M pancreastatin, reaching a maximum at 15 min. The dose response curve showed that with 10−7M pancreastatin, maximal stimulation was obtained (EC50= 3 nM). GTP (10−5M) stimulated the membrane‐bound phospholipase C as expected. However, the incubation of rat liver membranes with GTP partially inhibited the stimulation of phospholipase C activity produced by pancreastatin, whereas GTP enhanced the activation of phospholipase C by vasopressin. This inhibition by GTP was dose dependent and 10−5M GTP obtained the maximal inhibition (about 40%). the inhibitory effect of GTP on the stimulatory effect of pancreastatin on phospholipase C activity was completely abolished when rat liver membranes had previously been ADP‐ribosylated with pertussis toxin. The presence of 8‐Br‐cGMP mimics the effect of GTP, whereas GMP‐PNP increased both basal and pancreastatin‐stimulated phospholipase C, suggesting a role of the cyclic GMP as a feed‐back regulator of the synthesis of myo‐inositol 1,4,5‐triphosphate. However, the pretreatment of membranes with pertussis toxin did not modify the production of myo‐Inositol 1,4,5‐triphosphate stimulated by pancreastatin.In conclusion, pancreastatin activates guanylate cyclase activity and phospholipase C involving different pathways, pertussis toxin‐sensitive,

ISSN:0730-2312    

DOI:10.1002/jcb.240550204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractTranscriptional regulation of gene expression in vivo in bone, associated with normal development or skeletal disorders, to date, has not been studied. We report the successful isolation of nuclei that are transcriptionally active from normal and osteopetrotic rat bone. Transcription rates of cell growth and bone‐related genes (including histone H4,c‐fos,c‐jun, TGFβ1, β2macroglobulin, collagen, fibronectin, osteocalcin, osteopontin, and tartrate resistent acid phosphatase) change as a function of calvarial development from birth to 6 weeks and are selectively modified in osteopetrotic animals. Additionally, nuclei isolated from intact bone yield promoter binding factors. Bone nuclei, which transcribe faithfully and contain the normal complement of nuclear protein factors, offer a powerful approach for investigating in vivo gene regulation in skeletal development and pathology. © 1994 Wiley

ISSN:0730-2312    

DOI:10.1002/jcb.240550205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractMurine long‐term bone marrow cultures (LTBMCs) were used to generate hematopoietic cells free from marrow stromal cells. These progenitor cells were treated with GM‐CSF (5 U/ml) with or without rat bone osteocalcin or rat serum albumin in either α‐MEM with 2% heat‐inactivated horse serum alone (α) or supplemented with 10% L‐cell‐conditioned medium (as a source of M‐CSF) (L10). Few substrate‐attached cells survived in basal α medium, but when treated with L10 medium or GM‐CSF, they survived and proliferated. Osteocalcin did not significantly affect survival or proliferation. Subcultures of cells treated with GM‐CSF had large numbers of multinucleated cells, more than half of which were tartrate‐resistant acid phosphatase–positive (TRAP). Osteocalcin further promoted the development of TRAP‐positive multinucleated cells; a dose of 0.7 μg/ml osteocalcin promoted osteoclastic differentiation by 60%. Using a novel microphotometric assay, we detected significantly more tartrate‐resistant acid phosphatase activity in the osteocalcin plus GM‐CSF group (75.6 ± 14.2) than in GM‐CSF alone (53.3 ± 7.3). In the absence of M‐CSF, GM‐CSF stimulated tartrate‐resistant acid phosphatase activity, but osteocalcin did not have an additional effect. These studies indicate that osteocalcin promotes osteoclastic differentiation of a stromal‐free subpopulation of hematopoietic progenitors in the presence of GM‐CSF and L‐cell‐conditioned medium. These results are consistent with the hypothesis that this bone‐matrix constituent pl

ISSN:0730-2312    

DOI:10.1002/jcb.240550206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractA novel cell regulatory sialoglycopeptide (CeReS‐18), purified from the cell surface of bovine cerebral cortex cells has been shown to be a potent and reversible inhibitor of proliferation of a wide array of fibroblasts as well as epithelial‐like cells and nontransformed and transformed cells. To investigate the possible mechanisms by which CeReS‐18 exerts its inhibitory action, the effect of the inhibitor on the posttranslational regulation of the retinoblastoma susceptibility gene product (RB), a tumor suppressor gene, has been examined. It is shown that CeReS‐18 mediated cell cycle arrest of both human diploid fibroblasts (HSBP) and mouse fibroblasts (Swiss 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late G1 arrest site. Although their normal nontransformed counterparts are sensitive to cell cycle arrest mediated by CeReS‐18, cell lines lacking a functional RB protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are less sensitive. The refractory nature of these cells is shown to be independent of specific surface receptors for the inhibitor, and another tumor suppressor gene (p53) does not appear to be involved in the CeReS‐18 inhibition of cell proliferation. The requirement for a functional RB protein product, in order for CeReS‐18 to mediate cell cycle arrest, is discussed in light of regulatory events associated with density‐dependent growth inhibition. © 1994

ISSN:0730-2312    

DOI:10.1002/jcb.240550207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractIn order to titrate the dependence of individual steps in protein transport on signal peptide hydrophobicity, we have examined a series of mutants which involve replacement of the hydrophobic core segment of theEscherichia colialkaline phosphatase signal peptide. The core regions vary in composition from 10:0 to 0:10 in the ratio of alanine to leucine residues. Thus, a nonfunctional polyalanine‐containing signal peptide is titrated with the more hydrophobic residue, leucine. Analysis of this series identified a midpoint for rapid precursor processing between alanine to leucine ratios of 6:4 and 5:5 [Doud et al. (1993): Biochemistry 32:1251–1256]. Examination of precursors that are processed more slowly indicates a lower limit of signal peptide hydrophobicity that permits membrane association and translocation. Analysis of precursors that are processed rapidly defines an intermediate range of hydrophobicity that is optimum; above this level precursors become insensitive to transport inhibitors such as sodium azide and carbonyl cyanide 3‐chlorophenylhydrazone (CCCP) in parallel with substantial inhibition of β‐lactamase processing. Our data indicate that there is a surprisingly narrow range of signal peptide hydrophobicity which both supports transport of the protein to which it is attached and which does not have such a high affinity for the transport pathway that it disrupts the appropriate balance of other secreted proteins. © 1994 Wiley

ISSN:0730-2312    

DOI:10.1002/jcb.240550208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractVitamin D responsive transcription of the bone‐specific osteocalcin gene differs markedly in osteosarcoma cells and normal diploid osteoblasts. In osteoblasts the osteocalcin gene is transcribed, and upregulated by Vitamin D, only in post‐proliferative cells, but in osteosarcoma cells expression is constitutive. This distinction in transcriptional regulation of the osteocalcin gene correlates with striking differences in the relative representation of two principal Vitamin D‐dependent protein/DNA complexes designated V1 and V2 at the Vitamin D responsive element in the osteocalcin promoter. Formation of both complexes is Vitamin D dependent and they contain the Vitamin D receptor as well as an RXR related protein. Pore size exclusion and sedimentation velocity analyses suggest that the V1 and V2 complexes represent oligomeric protein assemblies (respectively, tetramers and trimers), and reflect primarily DNA‐directed association of the monomeric protein components at the osteocalcin Vitamin D responsive element. UV crosslinking and methylation interference analyses of the V1 and V2 complexes at the osteocalcin Vitamin D responsive element indicate differences in protein/DNA recognition. For example, the V1 complex interacts with both steroid half‐elements, whereas the V2 complex appears to recognize the proximal half‐element. Our findings suggest variations in protein/protein and protein/DNA interactions of the VDR and RXR related complexes V1 and V2 at the osteocalcin Vitamin D responsive element that reflect unique properties of the osteosarcoma and normal diploid osteoblast phenotype. © 1994 Wil

ISSN:0730-2312    

DOI:10.1002/jcb.240550209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractEffects of protein kinase C (PKC) inhibitor and activator on 1,25(OH)2D3‐induced gene expression were examined in rat intestinal epithelial cells, IEC‐6 cells. A potent PKC inhibitor, H‐7 (20 μM), completely abated 1,25(OH)2D3‐induced 24‐hydroxylase gene expression at 3 and 6 h. The effect of H‐7 was dose dependent with IC50around 5 μM. Other protein kinase inhibitors, HA‐1004 and H‐89 (20 μM), had no effects. Furthermore, the activation of PKC by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) potentiated the effect of 1,25(OH)2D3by 1 h. TPA appeared to exert its effect at a transcriptional step, since mRNA stability was not affected by TPA treatment. At 3 h after the treatment of the cells with H‐7 and TPA, vitamin D receptor (VDR) contents estimated by3H‐1,25(OH)2D3binding capacity were 72.4 and 63.2% of vehicle‐treated cells without significant changes of binding affinities, suggesting that the effect of H‐7 and TPA was not the result of changes in VDR content or its binding affinity. In conclusion, PKC is involved in 1,25(OH)2D3‐induced 24‐hydroxylase gene expression in IEC‐6 cells between 1,25(OH)2D3‐VDR binding and VDR‐induced

ISSN:0730-2312    

DOI:10.1002/jcb.240550210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe avian oviduct receptor binding factor‐1 (RBF‐1) is a 10 kDa nuclear matrix protein that was originally identified through its ability to effect high affinity interaction of activated progesterone receptor (PR) with chromatin. In the present study, the RBF‐1 is shown to not be restricted to reproductive tissues (e.g., oviduct) but present in all avian tissues examined by Western blot analysis with a monoclonal antibody prepared against purified RBF‐1. The heart and pancreas had the highest and lowest RBF‐1 levels, respectively; the concentration ranging by ∼ 50‐fold in these tissues. The 10 kDa size of the RBF‐1 detected in all tissues suggests no significant tissue‐specific differences in the protein. This was consistent with the finding that purified hepatic and oviductal RBF‐1 have identical amino‐terminal sequence. Using a recently isolated cDNA to RBF‐1, the levels of RBF‐1 mRNA were found to correlate well with the ubiquitous presence of the protein as well as tissue‐specific differences in concentration. The presence of RBF‐1 in non‐progesterone responsive tissues suggests the possibility that RBF‐1 may not be specifically involved in PR‐DNA interactions but may play a more diverse role, possibly involving other steroid receptors such as the glucocortico

ISSN:0730-2312    

DOI:10.1002/jcb.240550211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY