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 promoter of the mouse inducible nitric oxide synthase (iNOS) has a putative octamer motif (ATGCAAAA) which exists 24 bp upstream from the TATA box and is mismatched at a single residue from the consensus octamer motif. To examine whether this site is involved in iNOS expression, we constructed various deletions and site-directed mutants of the iNOS promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene, transfected the constructs into RAW 264.7 macrophages, and stimulated the cells with interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS). CAT activity was not induced by LPS in constructs containing only the octamer motif (-71 to +82), but was induced with constructs containing the octamer motif and the upstream sequences of the NF-kappaB site (-91 to +82). However, a site-directed mutation of the octamer motif in the context of the -91 to +82 promoter construct or an extended promoter construct (-1542 to +82) abolished IFN-gamma and/or LPS-induced CAT activity. Similar results were obtained from site-directed mutants at either the NF-kappaB site or both the NF-kappaB site and octamer motif in these two constructs. In addition, we demonstrated that the conversion of the iNOS octamer motif into a consensus sequence increased CAT activity. Electrophoretic mobility shift assay (EMSA) performed with the NF-kappaB site or the octamer motif-containing oligonucleotide probe revealed that NF-kappaB binding was induced by LPS treatment, while the Oct-1 binding was constitutive. Competition assays performed with octamer-related oligonucleotide competitors derived from the immunoglobulin-kappaB or SV40 promoter confirmed the identity of the iNOS promoter sequence as being a Oct-1 binding site. EMSA carried out using a probe containing both the NF-kappaB site and the octamer motif identified two LPS-induced complexes. Competition assays with each NF-kappaB site or octamer motif competitor revealed that NF-kappaB and Oct-1 were present in these two complexes. These data suggest that, besides the NF-kappaB site, the octamer motif is essential for the maximal expression of the iNOS gene in murine macrophages, and the direct interaction of Oct-1 and NF-kappaB is important for the regulation of this gene.
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PMID:Octamer motif is required for the NF-kappaB-mediated induction of the inducible nitric oxide synthase gene expression in RAW 264.7 macrophages. 1010 79

We have studied the effects of lipopolysaccharide (LPS) on the Newcastle disease virus (NDV)-mediated induction of cytokine genes expression. Raw cells treated with LPS before or after virus infection showed down-regulation in the expression of interferon A and, to a lesser extent, interferon B genes. In contrast, induction of the interleukin (IL)-6 gene was enhanced. The effects of LPS were not a result of the suppression of virus replication, because the transcription of viral nucleocapsid gene was not affected. Consistent with these findings, LPS also suppressed the NDV-mediated induction of chloramphenicol acetyltransferase reporter gene driven by murine interferon A4 promoter in a transient transfection assay. Furthermore, LPS inhibited virus-mediated phosphorylation of interferon regulatory factor (IRF)-3 and the consequent translocation of IRF-3 from cytoplasm to nucleus. The LPS-mediated inhibition of IFNA gene expression was much weaker in infected Raw cells that constitutively overexpressed IRF-3. The nuclear translocation of IRF-7 in infected cells was also inhibited by LPS. These data suggest that LPS down-regulates the virus-mediated induction of IFNA genes by post-translationally targeting the IRF-3 and IRF-7 proteins.
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PMID:Lipopolysaccharide inhibits virus-mediated induction of interferon genes by disruption of nuclear transport of interferon regulatory factors 3 and 7. 1036 58

The activation status of a recently identified STAT (signal transducers and activators of transcription) factor, LIL-Stat (lipopolysaccharide [LPS]/IL-1-inducible Stat) in adult T-cell leukemia (ATL) cells was investigated by electrophoretic mobility shift assays using nuclear extracts of leukemic cells from 7 patients with ATL and a GAS (gamma interferon activation site)-like element termed LILRE (LPS/IL-1-responsive element), which is found in the human prointerleukin 1beta (IL1B) gene. Spontaneous DNA binding of LIL-Stat was observed in all ATL cells examined. However, in normal human peripheral lymphocytes, DNA binding of LIL-Stat was detected only after stimulation with IL-1. These results demonstrated that LIL-Stat is constitutively activated in ATL cells. Furthermore, our transient transfection studies using LILRE chloramphenicol acetyltransferase (CAT) reporters argue that LIL-Stat in ATL cells functions as a transcriptional activator through binding to the LILRE in the IL1B gene. (Blood. 2000;95:2715-2718)
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PMID:Constitutive activation of LIL-Stat in adult T-cell leukemia cells. 1075 55

Inflammatory cytokines cause the down-regulation of multiple cytochrome P450 mRNAs, but the transcriptional mechanisms involved are not known. We investigated the role of a putative negative NF-kappaB-responsive element, nkappaB-RE1, in the down-regulation of the CYP2C11 gene in rat hepatocytes. This sequence spans the transcription start site of CYP2C11, from positions -2 to +8. Electrophoretic mobility shift assays showed that nuclear extracts from livers of rats treated with bacterial lipopolysaccharide, or from hepatocytes treated with interleukin-1beta, formed a protein complex with an oligonucleotide probe containing the nkappaB-RE1, and that this complex contained predominantly the p50 subunit of NF-kappaB. Binding of NF-kappaB to the nkappaB-RE1 probe was of lower affinity than to a probe containing the prototypic NF-kappaB enhancer of the immunoglobulin kappa chain gene. Mutations in the 5'-end of the nkappaB-RE1, and to a lesser extent the 3'-end, reduced the affinity of NF-kappaB for this element. Introduction of the 5'-mutation into nkappaB-RE1 abolished the response of the -200-CYP2C11-chloramphenicol acetyltransferase reporter construct to interleukin-1 or lipopolysaccharide. We conclude that nkappaB-RE1 is a functional negative regulatory element that participates in the inflammatory suppression of CYP2C11.
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PMID:Suppression of CYP2C11 gene transcription by interleukin-1 mediated by NF-kappaB binding at the transcription start site. 1077 59

The present study underlines the importance of p21(ras) in regulating the inducible nitric oxide synthase (iNOS) in primary astrocytes. Bacterial lipopolysaccharides induced the GTP loading of p21(ras), and the expression of a dominant-negative mutant of p21(ras) (Deltap21(ras)) inhibited lipopolysaccharide-induced GTP loading in rat primary astrocytes. To delineate the role of p21(ras) in the induction of iNOS, we examined the effect of Deltap21(ras) on the expression of iNOS and the production of nitric oxide. It is interesting that expression of Deltap21(ras) markedly inhibited the production of nitric oxide and the expression of iNOS in lipopolysaccharide- and proinflammatory cytokine (tumor necrosis factor-alpha, interleukin-1beta; interferon-gamma)-stimulated rat and human primary astrocytes. Inhibition of iNOS promoter-derived chloramphenicol acetyltransferase activity by Deltap21(ras) suggests that p21(ras) is involved in the transcription of iNOS. As activation of nuclear factor-kappaB (NF-kappaB) is necessary for the transcription of iNOS, we examined the effect of Deltap21(ras) on the activation of NF-kappaB. Expression of Deltap21(ras) inhibited the DNA binding as well as the transcriptional activity of NF-kappaB in activated astrocytes, suggesting that Deltap21(ras) inhibits the expression of iNOS by inhibiting the activation of NF-kappaB. These studies also suggest that inhibitors of p21(ras) may be used as therapeutics in nitric oxide- and cytokine-mediated neuroinflammatory diseases.
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PMID:Expression of a dominant-negative mutant of p21(ras) inhibits induction of nitric oxide synthase and activation of nuclear factor-kappaB in primary astrocytes. 1082 Jan 88

In the present study, the mechanism by which dexamethasone (DEX) inhibited IL-1beta gene expression in bacterial lipopolysaccharide (LPS)-activated RAW 264.7 cells was investigated. The decrease in LPS-induced IL-1beta mRNA expression was demonstrated by quantitative reverse transcription polymerase chain reaction (RT-PCR). Since the promoter in IL-1beta gene contains binding motifs for NF-kappaB/Rel, AP-1, NF-IL6, and CREB/ATF, which appear to be important in LPS-mediated IL-1beta induction, the effects of DEX on the activation of these transcription factors were examined. Treatment of DEX to RAW 264.7 cells induced a dose-related inhibition of NF-kappaB/Rel and AP-1 in chloramphenicol acetyltransferase activity, while neither NF-IL6 nor CREB/ATF activation was affected by DEX. Treatment of RAW 264.7 cells with DEX inhibited DNA binding of NF-kappaB/Rel and AP-1 proteins to their cognate DNA sites as measured by electrophoretic mobility shift assay (EMSA). DEX treatment caused a significant reduction in nuclear c-rel, p65, and p50 protein contents, and these decreases were paralleled by the accumulation of cytoplasmic c-rel, p65, and p50. DEX treatment of RAW 264.7 cells did not inhibit the nuclear translocation of c-jun and c-fos. We found that the inhibition of IL-1beta production by DEX is not related to p38, which is important in the IL-1beta induction. These results suggest that DEX may inhibit IL-1beta gene expression by a mechanism involving the blocking of LPS-induced NF-kappaB/Rel and AP-1 activation.
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PMID:Dexamethasone inhibits IL-1 beta gene expression in LPS-stimulated RAW 264.7 cells by blocking NF-kappa B/Rel and AP-1 activation. 1093 15

Thiopental is one of the intravenous anesthetics used widely. Several reports have demonstrated that thiopental inhibits the immune responses. We investigated whether or not thiopental inhibits the production of tumor necrosis factor-alpha (TNF-alpha) induced by lipopolysaccharide (LPS) in human glioma cells (A-172). Moreover, we determined whether or not thiopental modulates activation of the transcription factor NF-kappaB, a factor that regulates expression of the genes that code for proinflammatory cytokines in A-172 cells and in experimental murine brain inflammation. Thiopental inhibited TNF-alpha production induced by LPS in A-172 cells. Electrophoretic mobility shift assays demonstrated that thiopental inhibited NF-kappaB activation induced by LPS in A-172 cells. In experimental murine brain inflammation induced by intracerebroventricular injection of LPS, intraperitoneal injection of thiopental inhibited NF-kappaB activation. Western blot analysis indicated that this inhibition was linked to preservation of IkappaBalpha protein expression in A-172 cells. The chloramphenicol acetyltransferase assay revealed that NF-kappaB-dependent reporter gene expression was suppressed in A-172 cells exposed to thiopental. These findings are consistent with the idea that thiopental exerts antiinflammatory effects in cultured cells and experimental murine brain inflammation, through suppression of TNF-alpha production via inhibition of NF-kappaB activation.
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PMID:Thiopental inhibits NF-kappaB activation in human glioma cells and experimental brain inflammation. 1148 44

Since nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been known to be involved in inflammatory and autoimmune-mediated tissue destruction, modulation of NO synthesis or action represents a new approach to the treatment of inflammatory and autoimmune diseases. Caffeic acid phenethyl ester (CAPE), an active component of honeybee propolis, has been identified to show anti-inflammatory, anti-viral and anti-cancer activities. The present study, therefore, examined effects of CAPE on iNOS expression and activity of iNOS enzyme itself. Treatment of RAW 264.7 cells with CAPE significantly inhibited NO production and iNOS protein expression induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). CAPE also inhibited iNOS mRNA expression and nuclear factor-kappa B (NF-kappaB) binding activity in a concentration-dependent manner. Furthermore, transfection of RAW 264.7 cells with iNOS promoter linked to a chloramphenicol acetyltransferase reporter gene, revealed that CAPE inhibited the iNOS promoter activity induced by LPS plus IFN-gamma through the NF-kappaB sites of the iNOS promoter. In addition, CAPE directly interfered with the catalytic activity of murine recombinant iNOS enzyme. These results suggest that CAPE may exert its anti-inflammatory effect by inhibiting the iNOS gene expression at the transcriptional level through the suppression of NF-kappaB activation, and by directly inhibiting the catalytic activity of iNOS.
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PMID:Caffeic acid phenethyl ester inhibits nitric oxide synthase gene expression and enzyme activity. 1173 36

Propolis obtained from honeybee hives has been used in Oriental folk medicine as an anti-inflammatory, anti-carcinogenic, or immunomodulatory agent. However, the molecular basis for anti-inflammatory properties of propolis has not yet been established. Since nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been known to be involved in inflammatory and autoimmune-mediated tissue destruction, modulation of NO synthesis or action represents a new approach to the treatment of inflammatory and autoimmune diseases. The present study, therefore, examined effects of ethanol extract of propolis (EEP) on iNOS expression and activity of iNOS enzyme itself. Treatment of RAW 264.7 cells with EEP significantly inhibited NO production and iNOS protein expression induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). EEP also inhibited iNOS mRNA expression and nuclear factor-kappa B (NF-kappaB) binding activity in a concentration-dependent manner. Furthermore, transfection of RAW 264.7 cells with iNOS promoter linked to a chloramphenicol acetyltransferase (CAT) reporter gene, revealed that EEP inhibited the iNOS promoter activity induced by LPS plus IFN-gamma through the NF-kappaB sites of the iNOS promoter. In addition, EEP directly interfered with the catalytic activity of murine recombinant iNOS enzyme. These results suggest that EEP may exert its anti-inflammatory effect by inhibiting the iNOS gene expression via action on the NF-kappaB sites in the iNOS promoter and by directly inhibiting the catalytic activity of iNOS.
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PMID:Ethanol extract of propolis inhibits nitric oxide synthase gene expression and enzyme activity. 1200 5

Bacterial lipopolysaccharide (LPS) induces a rapid and transient increase in transcription of the tumor necrosis factor-alpha (TNF-alpha) gene in cells of monocyte/macrophage lineage. This study examines the role of potential regulatory elements within the proximal promoter region of the mouse TNF-alpha gene in LPS induction and cyclic AMP (cAMP)-mediated inhibition of TNF-alpha in RAW 264.7 murine macrophage-like cells. Transfection of proximal promoter chloramphenicol acetyltransferase (CAT) reporter constructs demonstrated that this region is LPS inducible in murine RAW 264.7 cells, with a 5.9-fold increase over nonstimulated transfectants. Site-specific mutations of the ETS, activated protein-1 (AP-1)/cAMP-responsive element (CRE)-like, or NF-kappaB-like motifs within this region caused a reduction in the LPS response by 52%, 46%, and 51%, respectively. LPS induction of the proximal promoter-CAT reporter construct was reduced by >40% by the addition of 8-bromo-cAMP (8-Br-cAMP). To determine the role of the proximal promoter region in the context of the entire TNF-alpha gene, we produced a hemagglutinin (HA)-tagged genomic TNF-alpha construct that contains a deletion of the proximal promoter region. Transfection of this construct into RAW 264.7 cells demonstrated a decrease in LPS-induced transcripts as well as a lack of response to cAMP. This suggested an essential role for this regulatory region in LPS-induced activation and cAMP inhibition of mouse TNF-alpha gene transcription in murine macrophages.
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PMID:The proximal promoter region is essential for lipopolysaccharide induction and cyclic AMP inhibition of mouse tumor necrosis factor-alpha. 1206 Apr 92


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