ISSN:0730-2312    

DOI:10.1002/jcb.240490302

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe molecular basis for adipose‐specific gene expression is not known. To approach the problem of adipocyte gene expression, we have analyzed in detail the capacity of the 5′‐flanking region of the adipocyte P2 (aP2) gene to direct cell‐type specific gene expression. Although the proximal promoter containing AP‐1 and C/EBP binding sites is capable of directing differentiation‐dependent gene expression in cultured adipocytes, these constructs are essentially inactive in the tissues of transgenic mice. We found that −5.4 kb of the 5‐flanking region were required to direct heterologous gene (chloramphenicol acetyl transferase; CAT) expression to the adipose tissue of transgenic mice. By deletion analysis, we identified a 520 bp enhancer at −5.4 kb of the aP2 gene. We show that this enhancer can direct high levels of gene expression specifically to the adipose tissue of transgenic mice. This enhancer also functions in a differentiation‐dependent manner in cultured adipocytes and cannot be transactivated in preadipocytes by C/EBP. Molecular analysis indicates that several cis‐ and transacting acting elements, though not C/EBP, contribute to the specificity and pot

ISSN:0730-2312    

DOI:10.1002/jcb.240490303

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

年代:1992

数据来源: WILEY

摘要:

AbstractTo study hematopoietic differentiation a variety of in vitro systems have been established using hematopoietic precursors derived from various explanted adult and fetal tissues. In this prospective we describe and discuss the potential of a novel system for studying the earliest stages of hematopoietic development. In addition, some of the applications of this system as a unique in vitro model for studying other developmental systems are discussed. Murine embryonic stem cells (ESC), which are totipotent and can be maintained undifferentiated indefinitely in vitro, have the capacity to differentiate in vitro into hematopoietic precursors of most, if not all, of the colony forming cells found in normal bone marrow. This potential can be exploited to study the control of the early stages of hematopoietic induction and differentiation. Recent results have indicated that there is a strong transcriptional activation, in a well defined temporal order, of many of the hematopoietically relevant genes. Examples of the genes expressed early during the induction of hematopoiesis include erythropoietin (Epo) and its receptor as well as the Steel (SI) factor (SLF) and its receptor (c‐kit). Several other genes, including CSF‐1, IL‐1, and G‐CSF were expressed during the later stages of hematopoietic differentiation. Contrasting with these observations, IL‐3 and GM‐CSF were not expressed during the first 24 days of ES cell differentiation suggesting that neither factor is necessary for the induction of hematopoietic precursors. Although these studies are just beginning, this system is easily manipulated and gives us an approach to understanding the control of the induction and differentiation of the hematopoietic system in ways not previous

ISSN:0730-2312    

DOI:10.1002/jcb.240490304

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe well‐known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible on human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum‐free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]‐thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 × 104and 8 × 104cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl‐cAMP and 8‐bromo‐cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin.These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density. Moreover, they confirm the existence of cAMP independent pathways for thyroid cell growth. They also provide an explanation for the equivocal effects observed in vitro on human thyroid cell growth after stimula

ISSN:0730-2312    

DOI:10.1002/jcb.240490305

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

年代:1992

数据来源: WILEY

摘要:

AbstractExpression of rat protein kinase C‐δ (PKC‐δ ) and PKC‐ξ in insect cells using recombinant baculovirus resulted in the production of proteins with a molecular size of approximately 76 kD and 78 kD, respectively, as determined by immunoblotting with subtype‐specific antisera. Although the PKC‐ξ cDNA encoded for 592 amino acids, a 76 kD protein was also generated by in vitro transcription/translation. Extracts of cells expressing PKC‐δ were able to bind phorbol ester to levels comparable to extracts of cells expressing PKC‐α. No phorbol ester binding was, however, detected in insect cell extracts expressing PKC‐ξ. However, similar levels of protein kinase activity were detected in lysates of cells expressing PKC‐δ or PKC‐ξ when protamine sulfate was used as exogenous substrate. Compared to protamine sulfate, both, myelin basic protein (MBP) or histone, were poor substrates for PKC‐δ and PKC‐ξ. In contrast to PKC‐ξ, the PKC‐δ enzyme activity phosphorylated MBP or histone in a phosphatidylserine‐(PS)/diacylglycerol(DG)‐dependent manner, albeit not to the same extent as PKC‐α. Lack of stimulation of the enzyme activity of PKC‐ξ by PS/DG, was confirmed by endogenous phosphorylation of insect cell proteins by PKC‐ξ, whereas several insect cell proteins were phosphorylated by PKC‐δ in a PS/DG‐dependent manner, including a protein of 78 kD.Our data demonstrate that the 76 kD PKC‐ξ, in contrast to PKC‐δ, is unable to bind phorbol esters and displays a protein kinase activity that is independent of PS of PS/DG. In addition, staurosporine was about 2–4 order of magnitudes less effective in inhibiting the p

ISSN:0730-2312    

DOI:10.1002/jcb.240490306

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

年代:1992

数据来源: WILEY

摘要:

AbstractWe previously reported that in cultured adipose cell lines insulin increased selectively the expression of Glut 1, in contrast to in vivo regulation where variations in insulinemia have been shown to affect only GLUT 4. We have addressed here the question of the long‐term regulation of GLUT 1 and GLUT 4 in fat cells by using primary cultures of rat adipocytes. Epididymal fat cells were isolated by collagenase and cultured 4 days in DMEM supplemented with BSA 1%, FCS 1%, and glucose 10 mM. GLUT 1 and GLUT 4 proteins were assessed in total cellular membranes by Western blotting, using specific antibodies against their respective C‐terminal peptides. GLUT 1 steadily increased over culture time to reach at day 3, a level 3‐fold higher than the initial value. In contrast, GLUT 4 decreased sharply and stabilized at day 3, at 30% of the initial value. The changes in GLUT 1 and GLUT 4 mRNAs with culture time were parallel to changes in the corresponding proteins, suggesting a pre‐translational level of regulation. The expression of the lipogenic enzyme, fatty acid synthetase (FAS), highly expressed in fat cell, decreased over time following a pattern closely parallel to that of GLUT 4. Chronic exposure to insulin added at day 2 had no effect on GLUT 4 expression but increased the expression of GLUT 1 and FAS by 70% and 36%, respectively. Glucose consumption was stable over 4 days of culture, while lactate production increased from 24 to 36% of glucose utilization, in agreement with the loss in FAS. Glucose consumption increased only slightly with insulin (160%), in good keeping with the low levels of expression of both GLUT 4 and FAS in these cultured cells. These data indicate that culture alters oppositely the expression of GLUT 1 and GLUT 4 in rat adipocytes and suggest that factor(s) other than insulin predominate in their regulation

ISSN:0730-2312    

DOI:10.1002/jcb.240490307

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

年代:1992

数据来源: WILEY

摘要:

AbstractIn situ hybridization and Northern analysis of heme oxygenase (HO) mRNA was used to determine the induction and expression of HO by various environmental agents. Exposure of Hep3B cells to hemin (10 μM) for as little as 5 min resulted in significant production of HO transcripts and mRNA expression as seen by in situ hybridization. We followed the pattern of HO transcript accumulation by heme and results indicate that the peak of induction of HO by heme was reached between 10 and 20 minutes. Other metalloporphyrins were all effective in inducing HO mRNA after 1 h exposure. On the other hand, CoCl2caused accumulation of HO mRNA at a later time than seen with the metalloporphyrins. However, lipopolysaccharide (LPS) gave a more immediate effect on HO induction which was somewhat similar to heme in its time course. Direct measurements of HO activity revealed that enzyme activity could be detected after about 20 min exposure to hemin, and this activity was inhibited by tin protoporphyrin (SnPP). The different pattern of HO mRNA induction by LPS as contrasted with CoCl2suggests that LPS may act through a different translational factor, or stimulate free radical formation and the subsequent release of heme and induction of HO. These results indicate that heme causes accumulation of HO mRNA by a different mechanism than that of CoCl2. Finally, LPS shares a concomitant effect on induction of HO as an acute phase reactant type protein

ISSN:0730-2312    

DOI:10.1002/jcb.240490308

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

年代:1992

数据来源: WILEY

摘要:

AbstractTrypanosoma cruzipresents six histones electrophoretically resolved in three gel systems. Indirect evidence shows that one of these histones, name, corresponds to H4in other species. We present evidence that histones is H4by sequencing its amino terminal end. The amino terminal ofT. cruzihistone H4, unlike that of other H4s examined thus far is not blocked. Moreover, this protein presents two variants. This partial amino acid sequence ofT. cruzihistone H4differs greatly from homologous sequences of human, yeast, orTetrahymena.Since the conservatism of the core histones (H2A, H2B, H3, and H4) is clearly illustrated by comparative sequence analyses, the data shown here demonstrates thatT. cruzihistone H4is the most divergent reported. Quantitative analysis of the data suggests that the rate of substitutions in the histone H4amino terminal sequence varies among different lineages. We postulate a slow‐down in the evolutionary rate of histone H4amino terminal domain in the metazoa branch related perhaps to the appearance of a novel function for this domai

ISSN:0730-2312    

DOI:10.1002/jcb.240490309

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

年代:1992

数据来源: WILEY

摘要:

AbstractMigration of endothelial cells is requisite to wound repair and angiogenesis. Since the glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is associated with remodeling, cellular migration, and angiogenesis in vitro, we questioned whether SPARC might influence the motility of endothelial cells. In this study we show that, in the absence of serum, exogenous SPARC inhibits the migration of bovine aortic endothelial cells induced by bFGF. Similar results were obtained from two different assays, in which cell migration was measured in a Boyden chamber and in monolayer culture after an experimental wound. Without bFGF, the migration of endothelial cells was unaffected by SPARC. The inhibitory effect of SPARC on cell motility was dose‐dependent, required the presence of Ca2+, was mimicked by synthetic peptides from the N‐ and C‐terminal Ca2+‐binding domains of the protein, and was not seen in the presence of serum. Modulation of the activities of secreted and cell‐associated proteases, including plasminogen activators and metalloproteinases, appeared not to be responsible for the effects that we observed on the motility of endothelial cells. Moreover, a molecular interaction between SPARC and bFGF was not detected, and SPARC did not interfere with the binding of bFGF to high‐affinity receptors on endothelial cells. Finally, in culture medium that contained serum, SPARC inhibited the incorporation of [3H]‐thymidine into newly synthesized DNA, both in the absence and presence of bFGF. However, DNA synthesis was not affected by SPARC when the cells were plated on gelatin or fibronectin in serum‐free medium. We propose that the combined action of a serum factor and SPARC regulates both endothelial cell proliferation and migration and coordinates these events during morphogenetic processes such as wound repair a

ISSN:0730-2312    

DOI:10.1002/jcb.240490310

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

年代:1992

数据来源: WILEY

摘要:

AbstractSodium butyrate (NaB) can induce teratocarcinoma cell differentiation as retinoic acid (RA). However, the function of these two agents seems to be a little different [Kosaka et al., Exp Cell Res, 192 : 46–51, 1991]. F9 cells treated with NaB synthesize both tissue‐type (tPA) and urokinase‐type (uPA) plasminogen activator, though RA induces only tPA production. Urokinase‐type PA is demonstrated to exist in association with membrane and to localize its activity to the close environment of the cell surface. This may cause the specific cell morphology and characteristics of differentiated F9 cells induced w

ISSN:0730-2312    

DOI:10.1002/jcb.240490311

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

年代:1992

数据来源: WILEY