摘要:

AbstractHuman T cells require two discrete signals to initiate their proliferation. In Jurkat T cells the first signal can be provided by the phorbol ester TPA and the second by the calcium ionophore A23187. We have isolated a cDNA from Jurkat T cells representing mRNA induced by TPA but inhibited by simultaneous treatment of the cells with antibody, lectin, or A23187. Sequencing revealed identity of the Jurkat clone to a cDNA, termed ETR101, recently isolated from HL60 promyelocytic leukaemia cells and shown to be an immediate early gene expressed upon TPA stimulation of these cells [Shimizu et al.: J Biol Chem 266:12157, 1991]. The gene is also induced very rapidly upon TPA treatment of Jurkat cells and is superinduced by co‐treatment with cycloheximide. The predicted amino acid sequence encoded by ETR101 has weak homology toJunB andJunD, therefore it is of some interest that these three genes share the chromosomal localization, 19p13.2. The divergent effects of TPA treatment upon cell proliferation and differentiation in different circumstances allow some speculation about a possible role for the ETR101 gene product upon cellular differentiatio

ISSN:0730-2312    

DOI:10.1002/jcb.240540202

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

年代:1994

数据来源: WILEY

摘要:

AbstractA carboxyl‐terminal peptide sequence (“osteostatin”) from parathyroid hormone related protein has been shown to have an inhibitory effect on osteoclastic bone resorption—an action opposite to its amino‐terminal sequence. In this study, we proposed that inhibition of osteoclastic bone resorption by osteostatin was associated with reduction of tartrate resistant acid phosphatase (TRAcP) activity in osteoclasts. Our results have indicated that osteostatin reduced TRAcP activity in a dose dependent manner. This effect of osteostatin was both sensitive (half maximal effect approximately 5 × 10−13M) and potent (maximum inhibition approximately 50% of control). In the first 90 min of treatment, however, reduction of TRAcP activity was erratic but became persistent and progressive when the time course was extended. Moreover, throughout the experimental period the levels of TRAcP activity in the culture medium had fallen significantly. It appears that osteostatin has a biphasic effect on TRAcP activity, inhibiting its secretion and either suppressing its synthesis or increasing its degradation. In addition, osteostatin induced rapid cellular retraction of both human and rat cultured osteoclasts, which was morphologically distinct from that produced b

ISSN:0730-2312    

DOI:10.1002/jcb.240540203

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe higher ordered structure of the chicken α2(I) procollagen gene was analyzed in chromatin isolated from expressing (lung) and nonexpressing (reticulocyte and erythrocyte) tissues. Digestion of DNA with methylation sensitive restriction endonucleases revealed that this gene was methylated in all tissues examined and that no differences existed in the promoter methylation patterns between expressing and nonexpressing tissues. DNAse 1 hypersensitive sites were located between 100–300 bp upstream from the transcription initiation site and within the first intron. These sites were also hypersensitive to the single‐strand specific S1nuclease, implying that this region of the gene in the chromatin is either in an unfolded single‐stranded conformation or under severe conformational stress. These differences in the α2(1) chromatin structure were confirmed by the finding that the promoter was more accessible to restriction endonuclease digestion in the expressing tissues than in the nonexpressing tissues. Digestion of chromatin with Pst I and Sma I revealed that some of these sites in the promoter were differentially protected by DNA‐binding proteins in the two tissue types. These protected sites were located as far upstream as −1,600 and downstream within the first int

ISSN:0730-2312    

DOI:10.1002/jcb.240540204

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

年代:1994

数据来源: WILEY

摘要:

AbstractLectin‐binding patterns of seven human melanoma clones and variants selected from the same parental cell line and differing in their spontaneous metastatic potential in an animal model were compared by flow cytometry and Scatchard analysis. Human melanoma clones and variants with high and low metastatic potential could be distinguished by their peanut agglutinin (PNA)–binding patterns, but not by their wheat germ agglutinin (WGA)–,Ulex europaeusagglutinin I (UEA I)–, and soybean agglutinin (SBA)–binding patterns. Low metastatatic clones and variants proved to be made up of a single poorly peanut agglutinin–binding cell population (2.20–3.52 × 106sites/cell, Ka = 2.48–2.75 × 106M−1). By contrast, highly metastatic variants were found to be constituted by two cellular subpopulations, exhibiting respectively a moderate (2.62–3.72 × 106sites/cell) and a high peanut agglutinin staining (17.68–18.76 × 106sites/cell). One highly metastatic clone was found to be homogeneously constituted by a single population of cells strongly binding this lectin (18.86 × 106sites/cell) with an association constant of 4.06 × 106M−1. Using an EPICS V cytometer, these two subpopulations were sorted from a highly metastatic variant and tested for their metastatic abilities: cells with high PNA binding generated a higher frequency of metastases than did moderately PNA‐binding cells. Following treatment withVibrio choleraeneuraminidase, all cells from all variants and clones were brightly labeled by PNA, collecting in a single peak with similar fluorescence intensities. Electrophoresis of total cellular proteins and subsequent detection with labeled PNA on Western blots show two major PNA‐reactive glycoproteins with apparent molecular weights of 140 and 110 kDa (MAGP1 and MAGP2), expressed only in highly metastatic cells, but which can be strongly labeled by PNA in slightly metastatic cells following a treatment with neuraminidase. These results provide evidence that the expression of terminal galactose (β1–3)N‐acetyl galactosamine structures, positioned on MAGP1 and MAGP2 glycoproteins, is associated with the metast

ISSN:0730-2312    

DOI:10.1002/jcb.240540205

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe heparan sulfate proteoglycans that bind and activate antithrombin III (aHSPGs) are synthesized by endothelial cells as well as other nonvascular cells. We determined the amounts of cell surface–associated and soluble aHSPGs generated by the rat fat pad endothelial (RFP) cell line and the fibroblast (LTA) cell line. The RFP cells exhibit higher levels of cell surface–associated aHSPGs as compared to LTA cells, whereas LTA cells release larger amounts of soluble aHSPGs as compared to RFP cells. After confluence RFP cells show an increase in both cell surface–associated and soluble aHSPGs. In contrast, postconfluent LTA cells maintain a constant level of cell surface–associated and soluble aHSPGs. These observations indicate that different cells types can preferentially accumulate aHSPGs as cell surface–associated or soluble forms which could reflect alternate biological

ISSN:0730-2312    

DOI:10.1002/jcb.240540206

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

年代:1994

数据来源: WILEY

摘要:

AbstractFollowing heat shock the expression of heat shock genes is regulated by the heat shock transcription factor, HSF, known to bind to arrays of the heat shock element, NGAAN, upstream of the heat shock genes. Phosphorylation of HSF is necessary for its activation. We report that the treatment of Chinese hamster HA‐1 cells with 250 nM of okadaic acid (OA), a ser/thr phosphatase inhibitor, leads to an increase in activated HSF after heat shock. This is followed by the activation of the transcription of heat shock genes as assayed by the increase in the synthesis of β‐galactosidase in an HA‐1 cell line containing the heat shock promoter ligated to the β‐galactosidase gene. To investigate the specificity of OA, we used other phosphatase inhibitors. We found that treatment of HA‐1 cells with 500 μM of sodium vanadate, an inhibitor of tyr/phosphatases, resulted in a three to fivefold reduction in HSF activation and binding to the heat shock element following heat shock. Such reduction in HSF activation virtually abolished β‐galactosidase induction. Reduced HSP synthesis was further confirmed by SDS‐PAGE and Western blot analysis using anti–HSP‐70 and 28 antibodies. Sodium vanadate treatment of heat shocked cells greatly reduced levels of thermotolerance. These results show that ser/thr and specifically tyr/phosphatase inhibitors modulate the signal transduction path

ISSN:0730-2312    

DOI:10.1002/jcb.240540207

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe human myeloid cell nuclear differentiation antigen (MNDA) is expressed constitutively in cells of the myeloid lineage, appearing in myeloblast cells in some cases of acute myeloid leukemia and consistently being detected in promyelocyte stage cells as well as in all later stage cells including peripheral blood monocytes and granulocytes. The human myeloid leukemia cell lines, HL‐60, U937, and THP‐1, express similar levels of immunochemically detectable MNDA. Although, the level of MNDA mRNA in primary monocytes is very low it was up‐regulated at 6 h following the addition of interferon α. The effect of interferon α on the MNDA mRNA is also observed in the cell lines HL‐60, U937, and THP‐1. The MNDA mRNA level in primary granulocytes was unaffected by addition of interferon α and other agents including interferon γ, endotoxin, poly (I) · poly (C), and FMLP. The MNDA mRNA level in the myeloid cell lines was also unaffected by the latter four agents. Induction of differentiation in the myeloid cell lines with phorbol ester induces monocyte differentiation which was accompanied by a decrease in MNDA mRNA level. This reduced level of mRNA could then be elevated with subsequent interferon α treatment. The effects of phorbol ester on MNDA mRNA appeared to be associated with induced differentiation since inhibiting cell proliferation did not alter the level of MNDA mRNA and cell cycle variation in MNDA mRNA levels were not observed. The ability of interferon α to up‐regulate MNDA mRNA in phorbol ester treated myeloid cell lines is consistent with the observations made in primary monocytes. Granulocyte differentiation induced by retinoic acid treatment of HL‐60 cells did not alter the MNDA mRNA level which was also unchanged following subsequent treatment with interferon α. The lack of interferon α effects on retinoic acid treated HL‐60 cells is consistent with its inability to influence MNDA mRNA level

ISSN:0730-2312    

DOI:10.1002/jcb.240540208

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

年代:1994

数据来源: WILEY

摘要:

AbstractIt was demonstrated previously that a polyclonal antibody (ES1) raised against EDTA extractable proteins from embryonic chicken heart blocks cardiac endothelial‐mesenchymal transformation in a culture bioassay and stains extracellular matrix at sites of embryonic inductive interactions, e.g., developing heart, limb buds, and neural crest forming region (Krug et al., 1987, Dev Biol 120:348–355; Mjaatvedt et al., 1991, Dev Biol 145:219–230). In the present study, by using an antiserum (ES3) to a similar immunogen, we affinity purified four major EDTA‐soluble proteins. These proteins migrated as 27, 44, 63, and 70 kD molecules under reduced conditions and 27, 41, 52, and 59 kD under nonreduced conditions, respectively, on SDS‐PAGE. Based on several criteria, the protein migrating at 70/59 kD (reduced/nonreduced) was indistinguishable from chicken transferrin (conalbumin): (1) amino acid sequencing showed that eight N‐terminal residues were identical to those of chicken transferrin, (2) acid hydrolysates of both proteins had nearly identical compositions, (3) the protein co‐migrated exactly with chicken transferrin under both reduced and nonreduced conditions, and (4) ES3 IgG recognized both the 70/59 kD protein and chicken transferrin by western blot analysis of nonreduced samples, but not with reduced samples. Immunohistochemistry of chicken embryonic heart with antibodies against transferrin demonstrated that anti‐transferrin immunoreactivity is present in myocardium but absent in cardiac endothelium before the initiation of cardiac endothelial‐mesenchymal formation. However, both cardiac endothelium and migrating mesenchymal cells became immunoreactive with anti‐transferrin at the time transformation occurred. These findings suggest a possible involvement of transferrin in the inductive process of cardiac endothelial‐mesen

ISSN:0730-2312    

DOI:10.1002/jcb.240540209

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe testis‐specific histone H1t gene is known to be transcribed only in pachytene primary spermatocytes during spermatogenesis. Previous studies of the rat histone H1t gene revealed a unique promoter sequence element between the H1/GC box and the H1/CCAAT box. Proteins in crude nuclear extracts of rat testis bind specifically to this sequence element and a temporal correlation exists between the appearance of these DNA binding proteins and the onset of transcription. These discoveries led to a search for histone H1t genes in other mammalian species. The human and monkey histone H1t genes were amplified from genomic DNA using the polymerase chain reaction (PCR). The amplified genes were cloned and the genomic derived inserts were sequenced using linear PCR. Both proximal promoters contained the highly conserved H1/AC box, H1/CCAAT box, and H1/TATA box found in nongerminal H1 genes. Both promoters also contained the H1/GC box and the H1t/CCTAGG sequence element between the H1/GC box and H1/CCAAT box previously seen only in the H1t promoter. Specific amplification of the human H1t gene using template DNA samples from a NIGMS human/rodent somatic cell hybrid mapping panel has shown that the human histone H1t gene is located on chromosome

ISSN:0730-2312    

DOI:10.1002/jcb.240540210

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe influence of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), a potent halogenated aromatic hydrocarbon, on the development of bone tissue‐like organization in primary cultures of normal diploid calvarial‐derived rat osteoblasts was examined. Initially, when placed in culture, these cells actively proliferate while expressing genes associated with biosynthesis of the bone extracellular matrix. Then, post‐proliferatively, genes are expressed that render the osteoblast competent for extracellular matrix mineralization and maintenance of structural as well as functional properties of the mature bone‐cell phenotype. Our results indicate that, in the presence of TCDD, proliferation of osteoblasts was not inhibited but post‐confluent formation of multicellular nodules that develop bone tissue‐like organization was dramatically suppressed. Consistent with TCDD‐mediated abrogation of bone nodule formation, expression of alkaline phosphatase and osteocalcin was not upregulated post‐proliferatively. These findings are discussed within the context of TCDD effects on estrogens and vitamin D‐responsive developmental gene expression during osteoblast differentiation and, from a broader biological perspective, on steroid hormone con

ISSN:0730-2312    

DOI:10.1002/jcb.240540211

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

年代:1994

数据来源: WILEY