Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies have shown that 1-beta-D-arabinofuranosylcytosine (ara-C) induces transcription of the c-jun immediate early response gene in human myeloid leukemia cells. The present work has examined the mechanisms responsible for this effect. Deleted forms of the c-jun promoter were linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into KG-1 cells. The results demonstrate that ara-C-induced c-jun transcription is mediated by an element between positions -74 and -20 upstream to the start site. Electrophoretic mobility shift assays with the fragment f(-74/-20) showed an increase in binding with nuclear proteins from ara-C-treated cells as compared with untreated cells. Competition with an oligonucleotide containing the AP-1 consensus sequence indicated that ara-C stimulates binding of nuclear proteins at the AP-1 site in the c-jun promoter. These findings were confirmed in other gel shift studies with the collagenase enhancer AP-1 consensus sequence and with a DNA fragment containing an altered AP-1 site. The binding of JUN/AP-1 was maximal at 1 hour of ara-C treatment and decreased to baseline levels at 12 hours. The finding that ara-C induces AP-1 binding in the absence of protein synthesis indicated that this agent activates already synthesized JUN/AP-1. To confirm these findings, the AP-1 consensus sequence was introduced 5' to the heterologous SV40 promoter. The results show that AP-1 enhances SV40 promoter activity in ara-C-treated cells. Taken together, these findings indicate that: (1) enhancement of JUN/AP-1 activity in ara-C-treated cells involves a posttranslational modification of JUN/AP-1; and (2) binding of activated JUN/AP-1 to the AP-1 site in the c-jun promoter confers ara-C inducibility of this gene.
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PMID:Activation of the AP-1 transcription factor by arabinofuranosylcytosine in myeloid leukemia cells. 1101 49

Heparin is a potent inhibitor of arterial smooth muscle cell (SMC) migration and proliferation in vivo and in vitro. We propose that heparin affects these SMC functions by interfering with either the expression or the activity of secreted proteases required for cell movement. We have reported that heparin selectively inhibits the expression of tissue-type plasminogen activator in SMCs during mitogenesis. In this study we show that the gene expression of another kind of protease, interstitial collagenase, is induced by fetal bovine serum and is also suppressed by heparin. The inhibitory effect on the induced collagenase mRNA is specific to heparin-like molecules and does not depend on the anticoagulant activity of heparin. The induction of the collagenase gene depends on the protein kinase C pathway, since it can be induced by phorbol esters such as phorbol 12-myristate 13-acetate and blocked by inhibitors such as H-7 and staurosporine. In transient transfection assays with chloramphenicol acetyltransferase constructs containing the phorbol ester-responsive element introduced into baboon SMCs, heparin inhibits transcription induced by serum or phorbol 12-myristate 13-acetate. These results support the conclusion that, in primate SMCs, interstitial collagenase gene transcription mediated by the phorbol ester-responsive element is blocked by heparin.
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PMID:Heparin inhibits collagenase gene expression mediated by phorbol ester-responsive element in primate arterial smooth muscle cells. 131 15

We have examined the effect of interleukin 1 (IL-1) and phorbol esters [12-O-tetradecanoylphorbol-13-acetate (TPA)] on the expression of various components of the AP-1 transcription factor complex during T-cell activation. We previously found that a chloramphenicol acetyltransferase reporter gene driven by the collagenase TPA responsive element was expressed upon stimulation of T-cells by TPA and that this expression was enhanced when IL-1 was added as a costimulant; IL-1 alone had no effect on TPA responsive element-chloramphenicol acetyltransferase expression. In this study, we have found that stimulation of T-cells by IL-1 and TPA is accompanied by activation of a subset of immediate early genes that comprise the AP-1 transcription factor complex. junB and fosB were rapidly induced following stimulation with TPA. Although the levels of other fos-related mRNAs were also elevated, their maximal induction was delayed by approximately 5 h. IL-1 alone had little or no effect, but enhanced TPA induced transcription and steady-state levels of these mRNAs. The expression of fos and jun during T-cell activation was accompanied by increased specific binding of JunB, FosB, and fos-related antigen containing complexes to the TPA responsive element. These findings indicate that the synergistic effect of IL-1 and TPA on AP-1 mediated gene expression is due, in part, to the ability of IL-1 to enhance the expression of genes encoding specific AP-1 transcription factor components.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Induction of AP-1 transcription factor components during T-cell activation by interleukin 1 and phorbol esters. 144 98

Insulin induces a rapid activation of p21ras in NIH 3T3 and Chinese hamster ovary cells that overexpress the insulin receptor. Previously, we suggested that p21ras may mediate insulin-induced gene expression. To test such a function of p21ras more directly, we studied the effect of different dominant inhibitory mutants of p21ras on the induction of gene expression in response to insulin. We transfected a collagenase promoter-chloramphenicol acetyltransferase (CAT) gene or a fos promoter-luciferase gene into NIH 3T3 cells that overexpressed the insulin receptor. The activities of both promoters were strongly induced after treatment with insulin. This induction could be suppressed by cotransfection of two inhibitory mutant ras genes, H-ras(Asn-17) or H-ras(Leu-61,Ser-186). In particular, insulin-induced activation of the fos promoter was inhibited completely by H-ras(Asn-17). These results show that p21ras functions as an intermediate in the insulin signal transduction route leading to the induction of gene expression.
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PMID:Two dominant inhibitory mutants of p21ras interfere with insulin-induced gene expression. 165 21

Using human heart fibroblasts (HHF), we studied the effect of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) on the gene expression of type I collagen, collagenase and tissue inhibitor of metalloproteinases (TIMP). Initially, treatment of HHF with bFGF alone (10 ng/ml) resulted in elevated secretion of collagenase into the culture medium. Subsequent treatment of HHF with TGF-beta in combination with bFGF suppressed collagenase secretion. Northern blot analysis reinforced this observation by revealing an enhancement of the steady-state mRNA level of collagenase in response to bFGF. In order to examine if the collagenase gene was affected by bFGF at the transcriptional level, transfection experiments were carried out with a plasmid containing collagenase promoter linked to chloramphenicol acetyltransferase gene (CAT). Basic FGF stimulated CAT activity by four-fold, indicating increased promoter activity whereas the combination of TGF-beta and bFGF resulted in decreased CAT activity. TGF-beta was shown to increase type I collagen and TIMP mRNA levels by 2.5- and 2.1-fold, respectively. These results suggest that TGF-beta and bFGF may play a pivotal role in regulating collagen metabolism in HHF.
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PMID:Effect of growth factors on collagen metabolism in cultured human heart fibroblasts. 166 Aug 2

Serum amyloid A (SAA) is a major acute-phase plasma protein synthesized by the liver. In addition to the two major plasma isoforms described in humans (SAA1 and SAA2), a third form (SAA3) has been demonstrated in several other species and is distinguished by predominant extrahepatic expression. Two clones, Ch11g5-1-1 and HDg1-1, containing the human SAA3 gene are described in this report. The human SAA3 gene is comparable in organization to the SAA1 and SAA2 genes and shares with them 87% nucleotide identity in the region spanning exon 3 through exon 4. Sequences 5' to exon 3, however, are strikingly different from those in the SAA1 and SAA2 genes. For instance, the sequence deduced for amino acids 1-12 (exon 2) has only 25% identity with human SAA1 and SAA2; it most closely resembles that of rabbit SAA3 isolated from synovial fibroblast cultures (75% identity). Although rabbit SAA3 induces collagenase production in an autocrine fashion the human SAA3 gene is not expressed. This is shown by: (i) a single base insertion in the sequence corresponding to codon 31, (ii) the inability of a 918-bp fragment immediately upstream from SAA3 exon sequences to direct transcription of a chloramphenicol acetyltransferase reporter gene, and (iii) the absence of detectable human SAA3 in mRNA.
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PMID:Nonexpression of the human serum amyloid A three (SAA3) gene. 175 58

Collagenase, the only enzyme active at neutral pH that initiates collagen degradation, is a major gene product of fibroblasts that have been stimulated with a variety of agents, including phorbol esters. To study mechanisms controlling collagenase gene expression, we transiently transfected rabbit synovial fibroblasts with chimeric constructs containing up to 1.2 kb of the rabbit collagenase 5'-flanking DNA linked to the chloramphenicol acetyltransferase gene (CAT). Our data indicate that the magnitude of the phorbol response is directly linked to the size of the promoter fragment and that the smallest piece of promoter DNA conferring phorbol inducibility is 127 bp. Deletional and mutational analysis of this fragment revealed that the AP-1 sequence alone is insufficient for phorbol inducibility and the presence of at least two additional sequences (a PEA3-like element and a sequence that includes 5'-TTCA-3') is required. In addition, a substantial increase in responsiveness is seen when a fragment containing 182 bp of 5'-flanking DNA is transfected, implicating a 36 bp region located between -182 and -149 as an enhancer. We conclude (1) that the AP-1 sequence is necessary but insufficient for expression of collagenase in adult fibroblasts, (2) that phorbol inducibility depends on cooperation among several sequence elements within the collagenase promoter, and (3) that regulation of this promoter is more complex than previously described.
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PMID:The AP-1 sequence is necessary but not sufficient for phorbol induction of collagenase in fibroblasts. 185 Jun 29

Collagenase production by synovial fibroblast-like cells (synoviocytes) plays a major role in cartilage and bone destruction in rheumatoid arthritis. Interleukin-1 (IL-1) increases collagenase secretion by elevating the steady state levels of collagenase mRNA in cultured rheumatoid synoviocytes, while all-trans-retinoic acid (RA) has the opposite effect. We have studied the regulation of collagenase gene transcription by IL-1 and RA in synoviocytes by transient transfection of plasmid constructs containing deletion mutants of the 5'-flanking region of the collagenase gene or the isolated phorbol ester-responsive element ligated to a chloramphenicol acetyltransferase reporter gene. We show that the phorbol ester-responsive element of the collagenase gene mediates both positive and negative regulatory effects, respectively, of IL-1 and RA on transcription. In addition, we show that IL-1 and 12-O-tetradecanoyl-phorbol-13-acetate transiently induce c-jun and c-fos expression and that retinoic acid inhibits IL-1 and 12-O-tetradecanoyl-phorbol-13-acetate induction of c-fos, but not c-jun. These results suggest that RA inhibits collagenase transcription at least in part through inhibition of c-fos.
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PMID:Interleukin-1 stimulates and all-trans-retinoic acid inhibits collagenase gene expression through its 5' activator protein-1-binding site. 217 24

We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested; an optimal level of CAT activity can be obtained using a serum-free, hormonally defined medium. Using these techniques, we have investigated the expression of liver-specific genes transferred into hepatocytes. We show that the L-pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts. Moreover, the use of serum-free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.
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PMID:Transfection of hepatic genes into adult rat hepatocytes in primary culture and their tissue-specific expression. 292 66

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.
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PMID:Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes. 346 23


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