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)

Cationic lipid-mediated gene transfer to the mouse lung induces a dose-dependent inflammatory response that is characterized by an influx of leukocytes and elevated levels of the cytokines interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma). We have examined the contribution of plasmid DNA (pDNA) to this observed toxicity, specifically the role of unmethylated CpG dinucleotides, which have been previously shown to be immunostimulatory. We report here that complexes of cationic lipid GL-67 and unmethylated pDNA (pCF1-CAT) instilled into the lungs of BALB/c mice induced highly elevated levels of the cytokines TNF-alpha, IFN-gamma, IL-6, and IL-12 in the bronchoalveolar lavage fluids (BALF). In contrast, BALF of animals administered either GL-67 alone or GL-67 complexed with SssI-methylated pDNA contained low levels of these cytokines. Similar results were observed using a plasmid (pCF1-null) that does not express a transgene, demonstrating that expression of chloramphenicol acetyltransferase (CAT) was not responsible for the observed inflammation. The response observed was dose dependent, with animals receiving increasingly higher amounts of unmethylated pDNA exhibiting progressively higher levels of the cytokines. Concomitant with this increase in cytokine levels were also elevated numbers of neutrophils in the BALF, suggesting a possible cause- and-effect relationship between neutrophil influx and generation of cytokines. Consistent with this proposal is the observation that reduction of neutrophils in the lung by administration of antibodies against Mac-1alpha and LFA-1 also diminished cytokine levels. This reduction in cytokine levels in the BALF was accompanied by an increase in transgene expression. In an attempt to abate the inflammatory response, sequences in the pDNA encoding the motif RRCGYY, shown to be most immunostimulatory, were selectively mutagenized. However, instillation of a plasmid in which 14 of the 17 CpG sites were altered into BALF/c mice did not reduce the levels of cytokines in the BALF compared with the unmodified vector. This suggests that other unmethylated motifs, in addition to RRCGYY, may also contribute to the inflammatory response. Together, these findings indicate that unmethylated CpG residues in pDNA are a major contributor to the induction of specific proinflammatory cytokines associated with instillation of cationic lipid:pDNA complexes into the lung. Strategies to abate this response are warranted to improve the efficacy of this nonviral gene delivery vector system for the treatment of chronic diseases.
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PMID:Contribution of plasmid DNA to inflammation in the lung after administration of cationic lipid:pDNA complexes. 1002 47

Interferon-gamma (IFN-gamma), a multifunctional cytokine produced by activated Th1 lymphocytes, exerts potent effects on the extracellular matrix by regulating fibroblast function. In this study, we examined the modulation of alpha1(I) procollagen gene (COL1A1) expression by recombinant IFN-gamma. The results showed that IFN-gamma stimulated the rapid accumulation of interferon regulated factor (IRF)-1 mRNA, followed by a delayed and dose-dependent inhibition of alpha1(I) procollagen mRNA expression in skin fibroblasts from several different donors. The inhibitory response was abrogated in fibroblasts stably expressing IRF-1 in the antisense orientation. A marked decrease in the amount of heterogeneous nuclear pre-mRNA preceded the inhibition of COL1A1 mRNA expression. In fibroblasts transiently transfected with COL1A1 promoter-chloramphenicol acetyltransferase reporter gene plasmids, IFN-gamma selectively inhibited promoter activity and abrogated its stimulation induced by TGF-beta. The inhibition by IFN-gamma was not due to downregulation of TGF-beta receptor mRNA expression in the fibroblasts or decreased ligand binding to the receptor. IFN-alpha and IFN-beta by themselves had little effect on promoter activity, but IFN-alpha augmented the inhibitory effect of IFN-gamma. Using a series of 5' deletion constructs, a proximal region of the COL1A1 promoter was shown to function as an IFN-gamma response element. This region of the gene harbors overlapping binding sites for transcription factors Sp1, Sp3, and NF-1 but no homologs of previously characterized IFN-gamma response elements. The putative IFN-gamma response region was sufficient to confer inhibition of reporter gene expression by treatment with IFN-gamma. Gel mobility shift analysis showed that two distinct and specific DNA-protein complexes were formed when fibroblast nuclear extracts were incubated with oligonucleotides spanning the IFN-gamma response region. IFN-gamma did not modify the ability of nuclear proteins to bind to this region. The results indicate that IFN-gamma inhibits COL1A1 expression in fibroblasts principally at the level of gene transcription. Inhibition involves IRF-1 and is mediated through a short proximal promoter segment but without an apparent change in promoter occupancy. The findings provide novel insight into the mechanism of IFN-gamma regulation of fibroblast function.
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PMID:Negative modulation of alpha1(I) procollagen gene expression in human skin fibroblasts: transcriptional inhibition by interferon-gamma. 1008 37

Several reports have shown that bicyclic imidazoles, specific inhibitors of the p38 mitogen-activated protein kinase (MAPK), block cytokine synthesis at the translational level. In this study, we examined the role of p38 MAPK in the regulation of the IL-1beta cytokine gene in monocytic cell lines using the bicyclic imidazole SB203580. Addition of SB203580 30 min before stimulation of monocytes with LPS inhibited IL-1beta protein and steady state message in a dose-dependent manner in both RAW264.7 and J774 cell lines. The loss of IL-1beta message was due mainly to inhibition of transcription, since nuclear run-off analysis showed an approximately 80% decrease in specific IL-1 RNA synthesis. In contrast, SB203580 had no effect on the synthesis of TNF-alpha message. LPS-stimulated p38 MAPK activity in the RAW264.7 cells was blocked by SB203580, as measured by the inhibition of MAPKAP2 kinase activity, a downstream target of the p38 MAPK. CCAATT/enhancer binding protein (C/EBP)/NFIL-6-driven chloramphenicol acetyltransferase (CAT) reporter activity was sensitive to SB203580, indicating that C/EBP/NFIL-6 transcription factor(s) are also targets of p38 MAPK. In contrast, transfected CAT constructs containing NF-kappaB elements were only partially inhibited (approximately 35%) at the highest concentration of SB203580 after LPS stimulation. As measured by EMSA, LPS-stimulated NF-kappaB activation was not affected by SB203580. Overall, the results demonstrate, for the first time, a role for p38 MAPK in IL-1beta transcription by acting through C/EBP/NFIL-6 transcription factors.
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PMID:The role of p38 mitogen-activated protein kinase in IL-1 beta transcription. 1022 13

Transcriptional regulators of the Myb family play important roles in cell proliferation, differentiation, and survival. To investigate the role of Myb proteins in the regulation of apoptosis, we studied the apoptotic response of interleukin 2-dependent CTLL-2 cells stably transfected with B-Myb. B-Myb-overexpressing cells showed a diminished cytokine dependence and were resistant to apoptosis induced by doxorubicin, ceramide, and dexamethasone. Overexpression of B-Myb was associated with enhanced expression of bcl-2, which was dependent, at least in part, on increased transcription. In transient transfection assays in T-lymphoblastic cells, B-Myb was able to stimulate the promoter activity of the bcl-2 5' flanking region linked to the chloramphenicol acetyltransferase reporter gene. A segment of the bcl-2 promoter (nucleotides +34 to +58 relative to the transcription initiation site) contained a putative Myb-binding site and was shown to specifically interact with B-Myb and to confer B-Myb responsiveness to a bcl-2/chloramphenicol acetyltransferase reporter construct. These results indicate that B-Myb promotes T cells survival by enhancing the expression of bcl-2 and identify bcl-2 as a B-Myb target gene regulated in a DNA binding-dependent manner.
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PMID:Resistance to apoptosis in CTLL-2 cells overexpressing B-Myb is associated with B-Myb-dependent bcl-2 induction. 1034 57

The rat serine protease inhibitor (Spi) 2 gene family includes both positive (Spi 2.2) and negative (Spi 2.1) acute phase reactants, facilitating modeling of regulation of hepatic acute phase response (APR). To examine the role of signal transducer and activation of transcription (STAT) proteins in the divergent regulation of these model genes after induction of APR, we evaluated the proximal promoters of the genes, focusing on STAT binding sites contained in these promoter elements. Induction of APR by turpentine injection includes activation of a STAT3 complex that can bind to a gamma-activated sequence (GAS) in the Spi 2.2 gene promoter, although the Spi 2.2 GAS site can bind STAT1 or STAT5 as well. To create an in vitro model of APR, primary hepatocytes were treated with combinations of cytokines and hormones to mimic the hormonal milieu of the whole animal after APR induction. Incubation of primary rat hepatocytes with interleukin (IL)-6, a critical APR cytokine, leads to activation of STAT3 and a 28-fold induction of a chloramphenicol acetyltransferase reporter construct containing the -319 to +85 region of the Spi 2.2 promoter. This suggests the turpentine-induced increase of Spi 2.2 is mediated primarily by IL-6. In contrast, although turpentine treatment reduces Spi 2.1 mRNA in vivo and IL-6 does not increase Spi 2.1 mRNA in primary rat hepatocytes, treatment of hepatocytes with IL-6 results in a 5. 4-fold induction of Spi 2.1 promoter activity mediated through the paired GAS elements in this promoter. Differential regulation of Spi 2.1 and 2.2 genes is due in part to differences in the promoters of these genes at the GAS sites. IL-6 alone fails to reproduce the pattern of rat Spi 2 gene expression that results from turpentine-induced inflammation.
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PMID:Regulation of Spi 2.1 and 2.2 gene expression after turpentine inflammation: discordant responses to IL-6. 1036

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

Expression of the gene coding for thymosin beta-4 (Tbeta-4), the major G-actin sequestering peptide in the cell, is regulated mainly at the level of transcription. In this study, we examined the nucleotide sequence of the 5'-flanking region (from -2202 to -881) of the mouse Tbeta-4 gene, and demonstrated that the DNA fragment from -278 to +410 of this gene was capable of directing the expression of a chloramphenicol acetyltransferase reporter gene in NIH3T3 cells. However, expression of the reporter gene in cells cannot be induced by interferon-alpha treatment even though a rapid activation of endogenous Tbeta-4 gene by this cytokine was observed. These results suggest that the projected interferon-stimulated response element (ISRE) might reside in other parts of the mouse Tbeta-4 gene.
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PMID:Characterization of a 5'-flanking region supporting the transcription of mouse thymosin beta-4 in mouse NIH3T3 cells. 1072 45

Alterations in gene expression are a hallmark of cardiac hypertrophy and heart failure. Among these, the decreased expression of the sarcoplasmic reticulum calcium ATPase (SERCA2) has been described. Elevated levels of cytokines in particular, Leukemia Inhibitory Factor (LIF) and Interleukin-6 (IL-6) have been shown to have the capacity to elicit hypertrophic responses in cultured cardiac myocytes. In this study, we investigated the effects of these cytokines (LIF & IL-6) on the regulation of SERCA2 levels in cardiac myocytes. Cultured neonatal rat ventricular myocytes were transfected with a 3.2 kb promoter plasmid construct containing the SERCA2 promoter linked to a chloramphenicol acetyltransferase (CAT) reporter gene, and subsequently treated with 10 ng/ml LIF or 10 ng/ml IL-6. LIF and IL-6 independently caused a significant (p < or = 0.05) 23-36% inhibition in SERCA2 promoter activity. LIF and IL-6 induced inhibition was also evident in SERCA2 mRNA levels as assessed by Northern analysis. Time course of inhibition of SERCA2 mRNA levels showed the most prominent decrease occurring after 48 hours of treatment, with both cytokines having a dose dependent effect on the inhibitory response. Western analysis using a polyclonal antibody to SERCA2 protein indicate a significant, 60% decrease in the amount of total SERCA2 protein in cultured myocytes treated with 10 ng/ml LIF or IL-6. In conclusion, the cytokines LIF and IL-6 downregulate SERCA2 gene expression and protein levels. The molecular mechanism responsible for cytokine induced downregulation of SERCA2 is at least partly transcriptional.
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PMID:Leukemia Inhibitory Factor and Interleukin-6 downregulate sarcoplasmic reticulum Ca2+ ATPase (SERCA2) in cardiac myocytes. 1075 45

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

The expression of several cytokines and adhesion molecules is regulated by the transcription factor NFkappaB, which is activated by tumor necrosis factor alpha (TNF-alpha). In this study, we employed a mouse model of nephritis induced by TNF-alpha to examine whether inhibition of NFkappaB activity using transcription factor decoy oligonucleotides (ODN) blocks cytokine and adhesion molecule expression and attenuates the renal inflammatory response. First, we developed a method for delivering FITC-ODN in vivo into mouse kidney glomeruli by employing HVJ-liposome. Then, in order to study the feasibility of decoy strategy in vivo, the reporter gene chloramphenicol acetyltransferase (CAT) driven by three tandemly repeated NFkappaB binding sequences was transfected into the kidney. Intrapenetorial injection of TNF-alpha stimulated CAT expression in vivo, and the increase in CAT expression was completely abolished by NFkappaB decoy ODN, but not scrambled ODN. Therefore, we examined the effect of NFkappaB decoy ODN transfection on TNF-alpha-induced endogenous interleukin (IL)-1alpha, IL-1beta, IL-6, ICAM-1 and VCAM-1 gene expression as assessed by RT-PCR and Northern blotting. Our present data showed that NFkappaB decoy, but not scrambled, ODN abolished TNF-alpha induced gene expression in vivo, as well as glomerular inflammation as assessed by CD45 staining. Taken together, our results suggest the potential utility of NFkappaB decoy strategy for molecular therapy to glomerular inflammatory diseases.
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PMID:Transcription factor decoy for NFkappaB inhibits TNF-alpha-induced cytokine and adhesion molecule expression in vivo. 1091 4


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