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)

The tumor necrosis factor (TNF) promoter and 3'-untranslated region (3'-UTR) each contain sequence elements that mediate a response to bacterial endotoxin. Although the promoter contains sequences that permit augmented TNF gene transcription in response to LPS, the 3'-UTR contains sequences that normally confer translational repression, but which allow "derepression" to occur after cell contact with endotoxin. We now show that both genetic elements act in concert during activation of TNF gene expression in macrophages. In order to do so, we have made use of chloramphenicol acetyltransferase reporter constructs in which the TNF promoter and 3'-UTR are represented either independently or in combination with one another. Suppression of chloramphenicol acetyltransferase and TNF mRNA synthesis, observed after treatment of the macrophages with dexamethasone, 2-aminopurine, pentoxifylline, or dibutyryl cAMP, has also been studied in detail. Each class of inhibitor suppresses TNF biosynthesis through a separate mechanism. Interestingly, suppression by pentoxifylline is manifested partly (but not entirely) at the level of transcription, and depends upon the presence of both the TNF promoter and 3'-UTR. The data suggest that other sequences within the TNF gene could also be required for the full effect of pentoxifylline, which may act to prevent processing of the primary transcript. The suppressive effect of dexamethasone is manifested both at the level of transcription and at the level of translation, and is mediated both by sequences present in the TNF promoter and by sequences present in the 3'-UTR. Suppression by 2-aminopurine is solely dependent upon promoter sequences.
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PMID:Interactive effects of the tumor necrosis factor promoter and 3'-untranslated regions. 184 73

Lipoproteins from two pathogenic spirochetes (Borrelia burgdorferi and Treponema pallidum) induced the biosynthesis of TNF in murine macrophages and in permanently transformed macrophages of the cell line RAW 264.7. Induction was studied by measuring the secretion of biologically active TNF and by measuring the activity of the reporter enzyme chloramphenicol acetyltransferase (CAT) produced within macrophages transfected with an endotoxin-responsive CAT construct. Several lines of evidence indicated that the induction of TNF and CAT was attributable to the spirochete lipoproteins rather than to contaminating or endogenous LPS: 1) the dose response curves observed for the lipoproteins were markedly different from those obtained with LPS; 2) lipoprotein-mediated activation was unaffected by amounts of polymyxin B that completely neutralized the induction of TNF and CAT by LPS, 3) low concentrations of the lipoproteins induced TNF in macrophages from endotoxin-unresponsive C3H/HeJ mice as effectively as in macrophages from normal C3H/HeN mice, and 4) isolated spirochete lipoproteins, but not a non-lipoprotein immunogen, were potent inducers of CAT in the transformed macrophages. Moreover, LPS was not detected in the B. burgdorferi lipoprotein mixtures by Limulus amebocyte lysate assay. Proteolytic digestion of the intact bacterial protein preparations only modestly diminished their ability to activate the cells, suggesting that small lipopeptides comprise the biologically active portions of the molecules, as is the case with the murein lipoprotein of Escherichia coli. Through their ability to induce TNF production by macrophages, spirochete lipoproteins may play important roles in the development of the local inflammatory changes and the systemic manifestations that characterize syphilis and Lyme disease.
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PMID:Lipoproteins of Borrelia burgdorferi and Treponema pallidum activate cachectin/tumor necrosis factor synthesis. Analysis using a CAT reporter construct. 189 Mar 8

We have prepared a construct (designated CATTNF) in which the mouse TNF (cachectin) coding sequence is replaced by a sequence encoding chloramphenicol acetyltransferase (CAT), with preservation of the TNF promoter and 3'-untranslated sequences known to be important in the regulation of gene expression. When activated by LPS, permanently transfected RAW 264.7 (mouse macrophage) cells synthesize large quantities of CAT. Unlike TNF itself, CAT is nonsecreted and quite stable in the macrophage cytoplasm. Fewer than 1,000 LPS-induced macrophages can easily be detected by CAT assay. Cells maintain the ability to respond to LPS in vivo; as such, when injected intravenously, they accurately report conditions required for the production of TNF in diverse tissues. These cells may thus be used for the detection of cachectin/TNF synthesis in mice under conditions in which endogenously produced cachectin/TNF would be undetectable. Studies of the expression of CATTNF in nonmacrophage cell lines have revealed that the modified TNF gene is constitutively expressed in L-929 cells, but that its expression is tightly suppressed in HeLa cells and in NIH 3T3 cells. This finding would suggest that certain non-macrophage cells are potentially capable of utilizing the TNF promoter and translating the TNF mRNA; however, the endogenous gene has been developmentally silenced.
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PMID:A CAT reporter construct allows ultrasensitive estimation of TNF synthesis, and suggests that the TNF gene has been silenced in non-macrophage cell lines. 201 May 47

An intact cAMP response element (CRE) in the upstream regulatory sequence of IL-1 beta (-2755/-2762) has been shown to be essential for maintaining full IL-1 beta inducibility following treatment with LPS, PMA, or TNF-alpha. In the present study, using the recombinant plasmid pIL-1(4.0 kb)-chloramphenicol acetyltransferase, containing 4.0 kb of the IL-1 beta upstream regulatory sequence, we have demonstrated that dibutyryl cAMP treatment alone is capable of induction. Due to the critical nature of the CRE for the induction of IL-1 beta transcription, an effort was made to determine the importance of the cAMP signaling pathway(s) by determining whether CRE binding protein (CREB) and other CREB/activating transcription factor (ATF) family members that responded to cAMP were associated with the DNA-protein complex that forms at this site. Nuclear extracts prepared from LPS-treated THP-1 5A cells were fractionated by ammonium sulfate precipitation and heparin-Sepharose chromatography, and the resulting fractions were characterized in electrophoretic gel mobility shift assays. These purification steps resulted in an approximately 100-fold enrichment of the proteins binding to the CRE site. Western blot analysis of isolated fractions, using CREB- and ATF-1-specific Ab showed an increased level of these proteins in the enriched fractions. Tryptic digest and DNase I protection studies showed the presence of CREB protein in the complex at the CRE site. Supershift electrophoretic gel mobility shift assays and immunoprecipitation analysis provided further evidence that both CREB and ATF-1 are present in the complex. In addition, an increase in CREB phosphorylation was observed when THP-1 5A cells were treated with dibutyryl cAMP, LPS, or both. These studies confirm the importance of a cAMP signaling pathway(s) in the regulation of IL-1 beta at the transcriptional level.
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PMID:Cyclic AMP signaling pathways are important in IL-1 beta transcriptional regulation. 759 50

The observation that the major membrane immunogens of the spirochetal pathogens. Treponema pallidum and Borrelia burgdorferi are lipoproteins prompted studies to investigate macrophage activation by the 47-kDa lipoprotein of T. pallidum and the acylated outer surface protein A (OspA) of B. burgdorferi. Both lipoproteins induced the synthesis of biologically active TNF-alpha and chloramphenicol acetyltransferase in a murine macrophage cell line transfected with a chloramphenicol acetyltransferase reporter gene controlled by a TNF promoter (TB2 cells). Nonacylated forms of these polypeptides did not induce cell activation. Comparison between purified OspA and B. burgdorferi cellular lipids revealed that the former was the more potent inducer of TNF-alpha. Synthetic lipohexapeptides corresponding to the N-termini of the 47-kDa lipoprotein of T. pallidum and OspA also activated TB2 cells in a dose-dependent fashion, whereas the nonlipidated hexapeptides were without effect, further underscoring the importance of protein acylation to cell activation. Among several lines of evidence supporting that macrophage stimulation by LPS and lipopeptides proceeds via different mechanisms, the most notable was that lipopeptides activated peritoneal macrophages from LPS-nonresponsive C3H/HeJ mice. The potential for spirochetal lipoproteins to function as general macrophage activators was demonstrated by the ability of the synthetic analogues to induce IL-1 beta, IL-6, and IL-12, in addition to TNF, in murine and/or human macrophages. Our findings indicate that spirochetal lipoproteins may be important immunomodulators in syphilis and Lyme disease and that the synthetic lipopeptides will be useful surrogates for studying immune mechanisms operative in the two spirochetal diseases.
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PMID:Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytes/macrophages. 787 55

Latent cytomegalovirus (CMV) infection is often reactivated in the lung. We postulated that this reactivation could occur by stimulation of the CMV major immediate early (IE) promoter by other viruses that infect the lung. The specific aim of this study was to investigate whether adenovirus early proteins could stimulate the CMV IE promoter in inflammatory cells. We transfected the monocyte/macrophage THP-1 cell line and the T-lymphocyte Jurkat cell line with plasmids coding for adenovirus E1A 12S or 13S proteins, along with a plasmid containing the CMV IE promoter region linked to the chloramphenicol acetyltransferase (CAT) reporter gene. In unstimulated THP-1 cells, the E1A 13S gene product increased CMV IE CAT activity by 18-fold compared with cells containing the control E1A plasmid. This effect was not seen in cells transfected with the E1A 12S plasmid. There was a similar effect of the E1A 13S gene product in LPS-stimulated THP-1 cells. In unstimulated Jurkat cells, the E1A 13S gene product stimulated CMV IE CAT activity by 19-fold compared with cells containing the E1A control plasmid; the E1A 12S gene product had no effect. There was a similar effect of the 13S E1A gene product in phorbol myristate acetate-stimulated Jurkat cells. These findings demonstrate that the CMV IE promoter can be stimulated by early viral proteins of adenovirus in inflammatory cells. These observations could be important for understanding the reactivation of latent CMV infection.
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PMID:Adenovirus E1A 13S gene product up-regulates the cytomegalovirus major immediate early promoter. 813 60

We have examined the hypothesis that TNF may play a pathogenetically important role in the hemolytic uremic syndrome. Specifically, we considered the possibility that shigatoxin, which eventuates this syndrome, might induce TNF biosynthesis, and/or that TNF and shigatoxin might sensitize animals, each to the toxic effects of the other agent. Shigatoxin was found to sensitize mice to the lethal effect of LPS and to the lethal effect of TNF. On the other hand, pretreatment of animals with either TNF or LPS did not noticeably sensitize mice to the lethal effect of shigatoxin. Intraperitoneal injections of shigatoxin did not induce the production of detectable quantities of TNF in the plasma of mice. When shigatoxin was injected into transgenic mice bearing a chloramphenicol acetyltransferase (CAT) reporter gene that indicates TNF synthesis, CAT activity was induced within the kidney, but not in other tissues. We therefore conclude that shigatoxin acts to induce TNF synthesis within the kidney, and at the same time increases renal sensitivity to the toxic effects of TNF. While this mouse model does not reproduce the hemolytic uremic syndrome as it occurs in humans, it does suggest that local synthesis of TNF within the kidney may contribute to renal injury induced by shigatoxin.
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PMID:A reporter transgene indicates renal-specific induction of tumor necrosis factor (TNF) by shiga-like toxin. Possible involvement of TNF in hemolytic uremic syndrome. 822 26

Ultraviolet (UV) light induces the biosynthesis of chloramphenicol acetyltransferase (CAT) in the skin of mice bearing the CATTNF reporter transgene. Moreover, nuclear run-on assays indicate that UV light induces transcription of the TNF gene in RAW 264.7 macrophages. These observations suggest that the TNF gene (and/or its mRNA product) responds to signals elicited by UV light. To identify transcriptional UV response elements within the TNF promoter, and to determine whether a posttranscriptional response might also exist, a series of reporter constructs using a CAT coding sequence attached to various portions of the TNF promoter and 3' untranslated region were devised and transfected into several cultured cell lines. All cells tested were found to be UV responsive, and in NIH 3T3 cells, induction was found to depend upon two general regions of the promoter. The more distal region encompassed nucleotides (nt) -1059 through -451 with respect to the cap site, while the more proximal region spanned nt -403 through -261. A negative element, blocking the UV response, was interposed (nt -451 through -403). As with the response to LPS, the response to UV irradiation appears to involve translational activation in macrophages. However, the UV and LPS signaling pathways have little in common with one another, as indicated by three observations. First, no difference in responsiveness was observed on comparison of TNF gene induction in macrophages derived from C3H/HeN as opposed to C3H/HeJ mice. Second, cell fusion studies showed that while the LPS signaling pathway is extinguished by fusion of RAW 264.7 cells with NIH 3T3 cells, the UV signaling pathway remained intact. Finally, induction did not depend upon the NF-kappa B binding sites that are known to be required for LPS response in macrophages, since mutation of these sites did not impair the UV response.
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PMID:Analysis of tumor necrosis factor promoter responses to ultraviolet light. 828 22

IL-4 and IL-10 inhibit the cytokine production and mRNA expression by monocytes/macrophages. To investigate the molecular mechanism of the inhibitory effect on transcriptional or post-transcriptional regulation of IL-6 gene expression by IL-4 and IL-10, we studied IL-6 production, expression level of IL-6 mRNA, IL-6 promoter activity, transcriptional activity of NF-kappaB and NF-IL-6, and IL-6 mRNA stability in human monocytic cell lines, THP-1 and U937, stimulated by PMA and LPS in the absence or the presence of IL-4 or IL-10. Both IL-4 and IL-10 were seen to inhibit IL-6 production and the expression of IL-6 mRNA in both monocytic cell lines studied. In chloramphenicol acetyltransferase assays, utilizing the transient transfection of a chloramphenicol acetyltransferase reporter plasmid containing the IL-6 gene promoter, IL-4, but not IL-10, suppressed the transcriptional activity of the IL-6 gene promoter stimulated by PMA and LPS. Electrophoretic mobility shift assays showed that IL-4, but not IL-10, inhibited nuclear NF-kappaB activity, and that IL-4 and IL-10 did not affect NF-IL-6 activity. On the other hand, IL-10 enhanced the degradation of IL-6 mRNA in a mRNA stability assay. These results suggest that IL-4 may inhibit the transcription of the IL-6 gene by affecting NF-kappaB binding activity, while IL-10 may inhibit the IL-6 mRNA levels post-transcriptionally, without suppressing promoter activity. Therefore, we conclude that IL-4 and IL-10 inhibit IL-6 production by different mechanisms in human monocytic cell lines.
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PMID:Differential regulation of IL-6 gene transcription and expression by IL-4 and IL-10 in human monocytic cell lines. 878 24

Leukocytes adhere to target cells through their integrins and play a crucial role in self-defense, inflammation, and differentiation. Intercellular adhesion molecule-1 (ICAM-1; CD54) is a representative ligand for integrins and is expressed on many cell types, some of which are targets for leukocyte adhesion. Recent studies suggest that adhesion molecules function not only as a cellular glue, but also as a signal transducer. However, it remains to be clearly defined whether engagement of ICAM-1 is able to induce activation signals in target cells. In rheumatoid synovium, synovial cells are known to express abundant ICAM-1 and produce multiple inflammatory cytokines, such as IL-1beta. In this study, we provide the first evidence that ICAM-1 engagement induces activation of the transcription factor AP-1 and transcription of the IL-1beta gene using a specific Ab to cross-link ICAM-1 on a rheumatoid synovial cell line (E11 cells). This evidence includes ICAM-1 cross-linking-dependent induction of 1) in situ IL-1beta transcription and protein synthesis, 2) transiently transfected chloramphenicol acetyltransferase (CAT) reporter plasmids containing both the IL-1beta LPS-responsive enhancer (between -3134 and -2729) as well as multiple copies of an AP-1 site from this enhancer (between -3117 and -3111), and 3) the binding of a Jun/Fos family complex to this AP-1 site. Thus, ICAM-1 not only functions as a glue for integrin binding, but also as a transducer for AP-1 activation signals important for IL-1beta gene transcription.
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PMID:Cross-linking of intercellular adhesion molecule 1 (CD54) induces AP-1 activation and IL-1beta transcription. 894 19


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