Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A proinflammatory cytokine cascade, including IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and IL-8, is activated in response to infection or immunologic insult. Besides their immunologic effects, several of these mediators stimulate bone resorption and inhibit bone formation. Osteocalcin, the most abundant noncollagenous protein present in bone, is an osteoblast-specific product whose production closely correlates with bone formation, and which has also been implicated in control of bone resorption. IL-1 and TNF have previously been shown to down-regulate osteocalcin production in vitro and in vivo, although the mechanism of this inhibition is unknown. In the present studies, IL-1 beta and TNF-alpha both inhibited 1,25-dihydroxyvitamin D3-stimulated production of osteocalcin protein and mRNA by ROS 17/2.8 osteosarcoma cells, whereas IL-6 had no effect on protein and only weakly inhibited mRNA. To determine if down-regulation was exerted at the transcriptional level, an osteocalcin promoter-chloramphenicol acetyltransferase (CAT) fusion gene was constructed (PHOC-CAT). After transient transfection of PHOC-CAT into ROS 17/2.8 osteosarcoma cells, reporter CAT activity was up-regulated by vitamin D at concentrations above 10(-12) M. In screening studies, TNF-alpha (-57%) and IL-6 (-37%) inhibited vitamin D-stimulated osteocalcin transcription, whereas IL-1 alpha, IL-1 beta, and IL-8 had no effect. Other immune cytokines and growth factors, including IL-2, IL-3, IL-7, and M-CSF, also failed to regulate osteocalcin transcription. Despite their lack of promoter regulation, IL-1 alpha and IL-1 beta also stimulated PGE2 production by ROS 17/2.8, further confirming the ability of the host cell to respond to these mediators. In dose-response experiments, down-regulation by TNF-alpha was significant at concentrations as low as 0.14 pM (0.1 U/ml), whereas approximately 10(4)-fold higher concentration of IL-6 was required to exert a similar effect. TNF-alpha-mediated down-regulation was unaffected by indomethacin. These data demonstrate that of these cytokines, TNF-alpha alone potently down-regulates osteocalcin promoter function, whereas IL-1 acts post-transcriptionally, possibly by reducing mRNA stability. Heterogeneity therefore exists among the proinflammatory cytokines with respect to the level at which control of osteocalcin expression is exerted.
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PMID:Proinflammatory cytokines tumor necrosis factor-alpha and IL-6, but not IL-1, down-regulate the osteocalcin gene promoter. 130 41

The interaction of IFN-alpha with IL-1 beta or TNF-alpha on hepatitis B surface antigen (HBsAg) expression was analysed in hepatitis B virus (HBV)-DNA integrated PLC/PRF/5 and non-integrated HuH-7 human hepatoma cells. Secretion of HBsAg in PLC/PRF/5 cells was reduced by IFN-alpha, IL-1 beta or TNF-alpha, and synergistically depressed when IFN-alpha was used in combination with IL-1 beta or TNF-alpha. By Northern blot analysis, the levels of HBsAg mRNA were suppressed by IFN-alpha in combination with IL-1 beta or TNF-alpha. In the chloramphenicol acetyltransferase plasmid transfection assay, IFN-alpha in combination with IL-1 beta or TNF-alpha caused a much greater suppression of HBV enhancer activity than IFN-alpha, IL-1 beta or TNF-alpha alone in both hepatoma cells. These findings suggest that the interaction of IFN-alpha with IL-1 beta or TNF-alpha synergistically represses HBV enhancer activity, resulting in depressed expression of HBsAg.
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PMID:Interaction of interferon-alpha with interleukin-1 beta or tumor necrosis factor-alpha on hepatitis B virus enhancer activity. 131 44

Cultured glomerular mesangial cells (GMCs) can be activated at the transcriptional level by a variety of physiologically relevant factors including cytokines, endotoxin and glycosylated end products. The mechanism with which the signal is transduced from the membrane to the nucleus of these cells is largely unclear. In vascular endothelial cells, the signal transduction pathway involves activation of the pleuripotent transcription factor, NF-kappa B, and leads to increased expression of a variety of genes including vascular cell adhesion molecule-1 (VCAM-1). Here, we demonstrate that TNF-alpha and IL-1 beta transiently induced VCAM-1 mRNA expression in a time dependent manner. TNF-alpha also induced the specific interaction of proteins from GMC nuclei with an oligonucleotide bearing the NF-kappa B binding sites in the VCAM-1 promoter. Electrophoretic mobility shift and supershift analysis indicated that the p65 subunit of NF-kappa B is a component of this induced complex. Finally, reporter activity driven by a VCAM-1 promoter-chloramphenicol acetyltransferase reporter construct increased 8-10 fold following TNF-alpha incubation, or p65 cotransfection. Thus, the p65 subunit of NF-kappa B is activated in GMCs exposed to cytokine and can mediate induction of gene expression.
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PMID:Nuclear factor-kappa B mediates induction of vascular cell adhesion molecule-1 in glomerular mesangial cells. 752 98

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

Pretreatment of L929 cells with IFN enhances Sendai virus-mediated induction of IL-6, TNF-alpha, and IFNA and IFNB genes. The priming effect could be demonstrated at both the RNA and protein levels and the former did not require cellular protein synthesis. Priming increased the Sendai virus-mediated induction of a murine IFNA4 promoter-chloramphenicol acetyltransferase gene hybrid plasmid (A4CAT) in transiently transfected cells, and deletion analysis showed that the identical DNA sequence was required for the inducibility in primed and unprimed cells. Cotransfection of A4CAT plasmid with interferon regulatory factor-1 (IRF-1) expression plasmid increased CAT expression, however, the IRF-1-mediated expression was further enhanced by priming. These results show that the identical inducible element present in the promoter region of IFNA4 gene is required for both the inducibility and the priming effect; however, no direct correlation between the enhancement of expression of the IRF-1 gene and enhancement of expression of IFN, IL-6, and TNF-alpha genes in the primed cells was observed. We suggest that priming facilitates inducer-mediated posttranscriptional modulation of various transcriptional factors that play a role in stimulation of transcription of these genes.
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PMID:Priming does not change promoter sequence requirements for IFN induction or correlate with the expression of IFN regulatory factor-1. 768 28

Tuberculosis has emerged as an epidemic fueled by the large number of individuals infected with the human immunodeficiency virus, especially those who are injecting drug users. We found a striking increase from 4- to 208-fold in p24 levels in bronchoalveolar lavage fluid from involved sites of Mycobacterium tuberculosis infection vs uninvolved sites in three HIV+ patients. We used an in vitro cell culture model to determine if tuberculosis could activate replication of HIV-1. Mononuclear phagocyte cell lines U937 and THP-1 infected with HIV-1JR-CSF, in vitro and stimulated with live M. tuberculosis H37Ra, had a threefold increase in p24 in culture supernatants. Using the HIV-1 long terminal repeat with a chloramphenicol acetyltransferase (CAT) reporter construct, live M. tuberculosis increased transcription 20-fold in THP-1 cells, and cell wall components stimulated CAT expression to a lesser extent. The nuclear factor-kappa B enhancer element was responsible for the majority of the increased CAT activity although two upstream nuclear factor-IL6 sites may also contribute to enhanced transcription. Antibodies to TNF-alpha and IL-1 inhibited the increase in CAT activity of the HIV-1 long terminal repeat by M. tuberculosis from 21-fold to 8-fold. Stimulation of HIV-1 replication by M. tuberculosis may exacerbate dysfunction of the host immune response in dually infected individuals.
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PMID:Mycobacterium tuberculosis enhances human immunodeficiency virus-1 replication by transcriptional activation at the long terminal repeat. 773 95

A critical role of tumor necrosis factor (TNF)-alpha in irritant contact dermatitis and in the challenge phase of allergic contact dermatitis has recently been demonstrated in vivo. As in situ hybridization studies have indicated that keratinocytes were the cellular source of TNF-alpha in these reactions, we studied the mechanisms of TNF-alpha mRNA induction in keratinocytes by agents that induce contact dermatitis. Murine la-/CD3- epidermal cells were stimulated with phorbol myristate acetate (PMA), dimethylsulfoxide (DMSO), sodium dodecyl sulfate (SDS) and NiSO4, all of which up-regulated epidermal cell TNF-alpha mRNA production. In contrast, trinitrobenzenesulfonic acid and trinitrochlorobenzene did not significantly up-regulate TNF-alpha mRNA. These results were confirmed with murine keratinocyte cell lines. In keratinocytes transfected with a chloramphenicol acetyltransferase construct containing the -1059 to +138 base pair TNF-alpha promoter, increased promoter activity was observed upon stimulation with PMA and DMSO. In addition, PMA stimulation did not affect the stability of TNF-alpha mRNA. The PMA- but also the DMSO- and SDS- induced up-regulation of TNF-alpha mRNA was abolished by an inhibitor of protein kinase C (PKC). In contrast, NiSO4 up-regulated TNF-alpha mRNA by a PKC-independent mechanism, did not increase TNF-alpha promoter activity, but markedly increased the stability of the TNF-alpha mRNA. Co-stimulation with PMA and NiSO4 induced a marked increase in TNF-alpha mRNA over that obtained with each agent alone. Thus, whereas PKC-dependent irritants act by up-regulating TNF-alpha promoter activity, nickel acts via post-transcriptional regulation. Our results also establish that some irritants and irritant sensitizers directly induce TNF-alpha in keratinocytes without intermediate Langerhans cell-derived signals.
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PMID:Nickel and skin irritants up-regulate tumor necrosis factor-alpha mRNA in keratinocytes by different but potentially synergistic mechanisms. 779 16

The molecular mechanism(s) by which three cytokines (IFN-gamma, TNF-alpha, TGF-beta) affect class II MHC gene expression in primary rat microglia was examined. IFN-gamma is a potent inducer of the class II gene, and this induction is unaffected by treatment with either TNF-alpha or TGF-beta. Transient transfection of primary rat microglia with an HLA-DRA promoter linked to the chloramphenicol acetyltransferase reporter gene (DRA-CAT) demonstrated that IFN-gamma acts at the transcriptional level to induce class II MHC gene expression, and that TNF-alpha and TGF-beta have no influence on IFN-gamma-induced promoter activity. Experiments using a series of DRA substitution mutants that individually affect the W, X1, X2, or Y elements, as well as a double mutation in both X1 and X2, indicate that all four of these elements are required for responsiveness of the DRA promoter to IFN-gamma. The effect of IFN-gamma and TNF-alpha on DNA binding proteins by microglia was examined. A constitutive complex with specificity for the X2 box was detected in extracts from unstimulated microglia. IFN-gamma treatment changed this complex to migrate with slower mobility, and TNF-alpha had no effect on either the constitutive or IFN-gamma-induced complexes. These studies provide information on the molecular regulation of the class II MHC gene in microglia, a cell type critically involved in immune regulation within the central nervous system.
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PMID:Class II MHC gene expression in microglia. Regulation by the cytokines IFN-gamma, TNF-alpha, and TGF-beta. 787 54

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

The participation of cell ceramide in tumor necrosis factor (TNF)-alpha-stimulated NF-kappa B activation in Jurkat T cells and HL-60 cells was studied. TNF-alpha readily stimulated NF-kappa B activity in both cell lines as assayed by electrophoretic mobility shift assay and the use of a human immunodeficiency virus-chloramphenicol acetyltransferase reporter construct. However, TNF-alpha stimulation did not increase cell ceramide levels in either cell line. The exogenous addition of a short chain ceramide, N-acetylsphingosine, to Jurkat cells had no effect on NF-kappa B activity. When Jurkat T cells were exposed to the glucosylceramide synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, endogenous ceramide levels increased 4-fold. The increase in ceramide, however, did not result in NF-kappa B activation nor did it potentiate TNF-alpha or phorbol ester-stimulated activity. We conclude that TNF-alpha-induced NF-kappa B activation occurs in Jurkat and HL-60 cell lines that do not demonstrate an increase in TNF-alpha-induced ceramide. Increasing ceramide levels by the addition of short chain ceramides or the use of a glucosylceramide synthase inhibitor can be dissociated from activation of NF-kappa B by TNF-alpha.
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PMID:Dissociation of endogenous cellular ceramide from NF-kappa B activation. 813 72


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