Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 1311-base pair human tumor necrosis factor (TNF) alpha promoter region was fused to the luciferase (Luc) reporter gene and studied in a transient transfection system in three TNF producing cell lines, the U937 macrophage cell line, the MLA 144 T cell line, and the 729-6 B cell line. This full length promoter construct can be induced by phorbol 13-myristate acetate (PMA) in each of these cell types. Analysis of a series of 5'-truncations showed several peaks of basal and PMA induced activity suggesting the presence of several positive and negative regulatory elements. A PMA responsive element was localized to a region between -95 and -36 bp relative to the transcription start site. Within this region, single AP-2- and AP-1-like consensus sequences were noted. These AP-2 and AP-1 sites were each modified with a double point mutation. A modest (20-50%) reduction in TNF promoter activity was observed with the AP-2 site mutation. However, mutation of the AP-1 site markedly diminished both the basal and PMA-activated promoter activity. Also co-transfections of the wild-type promoter construct with an AP-1/c-jun expression vector resulted in augmented basal and PMA-induced promoter activity.
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PMID:The regulation of the human tumor necrosis factor alpha promoter region in macrophage, T cell, and B cell lines. 142 62

To investigate a possible role of cytokines in parvovirus-mediated suppression of tumorigenesis, we tested in cell culture whether parvoviruses are able to induce interferon (IFN)-beta, tumor necrosis factor (TNF)-alpha or interleukin-6 (IL-6). Infection of rodent or human cells with the parvoviruses minute virus of mice (MVM), H-1 or adeno-associated virus (AAV) types 2 or 5 failed to induce expression of the luciferase or beta-galactosidase reporter genes transfected into these cells as constructs containing an IFN-beta promoter. Parvoviruses did weakly induce synthesis of TNF-alpha and of IL-6 in cell culture and could slightly enhance synthesis of these cytokines when induced by other agents. These in vitro data suggest that the rather unspecific tumor-suppressive properties of parvoviruses are unlikely to be attributable to stimulation of the synthesis of IFN, TNF or IL-6.
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PMID:Parvoviruses are inefficient in inducing interferon-beta, tumor necrosis factor-alpha, or interleukin-6 in mammalian cells. 152 25

Recent findings have indicated that megakaryocytes may be susceptible to human immunodeficiency virus (HIV) infection, suggesting a potential role for megakaryocytes as viral reservoirs in HIV-infected patients. We report that the megakaryocytic cell line Dami could be productively infected with the HTLV III-B strain of HIV-1, in 26 different experiments (results of 16 experiments are reported); productive infection lasted up to 30 weeks. Despite a lack of detectable surface expression of the CD4 molecule and very low levels of CD4 mRNA, between 40% and 60% of megakaryocytic cells produced viral proteins after contact with HIV-1. Neither cytopathogenic effects nor syncytial formation was observed. Production of high levels of functional viral particles was indicated by analysis of p24 protein levels, reverse transcriptase activity, ultrastructural studies, and the capacity of supernatants from infected Dami cells to infect the Molt-4 T-lymphocytic cell line. HIV-1 RNA and protein levels in infected Dami cells were enhanced by treatment with tumor necrosis factor-alpha (TNF-alpha), and decreased by treatment with interferon-alpha (IFN-alpha) and IFN-gamma. Transient transfection of the megakaryocytic cells with various constructs of the HIV-1 promoter (LTR) linked to the luciferase reporter gene suggested that the effect of TNF-alpha was related, as in monocytic and T-cell lines, to transactivation of the enhancer region of the HIV-1 LTR. These findings indicate that signals provided by the immune system may modulate HIV-1 expression in cells of the megakaryocytic lineage.
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PMID:Productive human immunodeficiency virus-1 infection of megakaryocytic cells is enhanced by tumor necrosis factor-alpha. 158 16

By using human CD4+ lymphoblastoid T cells transiently cotransfected with human immunodeficiency virus (HIV) and cytomegalovirus (CMV), we tested whether modulation of T-cell activation through the protein kinase C (PKC) or the protein kinase A (PKA) pathway synergized with CMV immediate-early (IE) proteins in HIV long terminal repeat (LTR) transactivation. Stimulation with phorbol myristate acetate, tumor necrosis factor, or cross-linked antibodies to CD3 and CD28 resulted in modest enhancement (two- to fourfold) of the activity of a luciferase expression vector under control of the HIV LTR. Cotransfection of a vector expressing the CMV IE1 and IE2 proteins under the control of their own promoter enhanced HIV LTR activity 16- to 49-fold. Combination of any one of the above stimuli and CMV IE expression amplified HIV LTR activity 99- to 624-fold. Stimulation of PKA-dependent pathways with forskolin, 8-bromo cyclic AMP, or prostaglandin E2 had a minimal effect on HIV LTR activity, whereas such stimuli resulted in synergistic amplification in cells cotransfected with CMV IE (three- to fivefold increases over the effects of CMV IE alone). This synergism was independent of the NF-kappa B binding motifs within the HIV LTR. CMV IE2, but not IE1, protein induced HIV transactivation and synergized with signals modulating T-cell activation. The intense synergism observed was superior to the increase in IE protein expression following PKC activation by phorbol myristate acetate. Treatment of cells with PKC inhibitor GF109203X blocked most of the observed synergism.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Modulation of T-cell activation through protein kinase C- or A-dependent signalling pathways synergistically increases human immunodeficiency virus long terminal repeat induction by cytomegalovirus immediate-early proteins. 165 49

The capacity of cells of the human bronchial epithelium to express the gene for interleukin-8 (IL-8) was evaluated in bronchial epithelium derived cell lines, HS-24 and BET-1A, using tumor necrosis factor-alpha (TNF) as a model inflammatory stimulus. As in other epithelium, TNF markedly increased the level of the 1.8-kilobase IL-8 mRNA transcripts in both bronchial epithelial cell lines. In HS-24 cells, nuclear run-on analyses showed the IL-8 gene transcription rate was dramatically increased, more than 30-fold, after TNF stimulation. The half-life of IL-8 mRNA transcripts in these cells was approximately 40 min and did not change after TNF stimulation, suggesting that TNF up-regulated IL-8 gene expression mainly at the transcriptional level. DNase I hypersensitivity site mapping of chromatin DNA in resting HS-24 cells demonstrated two hypersensitivity sites within 400 base pairs (bp) 5' to exon I and one site within exon I. However, after TNF stimulation, the exon I hypersensitivity site disappeared and a new site approximately 120 bp 5' to exon I emerged. Consistent with these observations, transfection studies with HS-24 cells using fusion genes composed of the 5'-flanking sequences of the IL-8 gene and a luciferase reporter gene demonstrated potent promoter activity in a 174-bp segment (-130 to +44 relative to the transcription start site), which also exhibited a response to TNF, while a segment from -112 to +44 showed very low promoter activity and no response to TNF. Thus, human bronchial epithelial cells can express the IL-8 gene, with expression in response to the inflammatory mediator TNF regulated mainly at the transcriptional level, and with elements within the 5'-flanking region of the gene that are directly or indirectly modulated by the TNF signal.
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PMID:Interleukin-8 gene expression in human bronchial epithelial cells. 191 68

The rat angiotensinogen gene is induced in the course of the hepatic acute-phase response. We demonstrate that monocyte conditioned medium can stimulate transcription of a stably introduced reporter construct driven by 615 base pairs of the angiotensinogen 5'-flanking sequence, as well as the endogenous gene, in Reuber H35 cells. Point mutations of a cis-acting element, located 545 base pairs from the transcription start site and sharing sequence identity with known nuclear factor kappa B (NF kappa B)-binding sites, led to loss of cytokine inducibility. When cloned upstream of a minimal promoter, this cis-acting element imparted transcriptional inducibility by monocyte conditioned medium, interleukin-1, and tumor necrosis factor on a luciferase reporter gene in HepG2 cells. Two distinct proteins bound this element in vitro: a heat-stable, constitutively present, hepatic nuclear protein that gave rise to a DNase I-protected footprint covering the functionally defined element; and a binding protein of different mobility, induced by monocyte conditioned medium, which also recognized the NF kappa B-binding site of the murine kappa light-chain enhancer. UV cross-linking showed this inducible protein to have an apparent molecular mass of 50 kilodaltons, similar to that described for NF kappa B and distinct from the constitutively present protein that was shown by Southwestern (DNA-protein) blot to have a molecular mass of 32 kilodaltons. Methylation interference analysis showed that the induced species made contact points with guanine residues in the NF kappa B consensus sequence typical of NF kappa B. Induction of this binding activity did not require new protein synthesis, and 12-O-tetradecanoylphorbol-13-acetate could mimic the induction by cytokines. We thus provide direct evidence for involvement of NF kappa B or a similar factor in the hepatic acute-phase response and discuss the potential role of the presence of a constitutive nuclear factor binding the same cis element.
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PMID:An inducible 50-kilodalton NF kappa B-like protein and a constitutive protein both bind the acute-phase response element of the angiotensinogen gene. 210 65

Since human immunodeficiency virus (HIV) nef has been suggested to exert regulatory effects on HIV long terminal repeat (LTR) activity, we transiently transfected HIV LTR chloramphenicol acetyltransferase or luciferase expression vectors into a human astrocytoma clone (U-373nef) that constitutively expresses the HIV nef gene. In these cells, basal HIV LTR activity, as well as tumor necrosis factor-induced or tat-driven activity, was similar to that in control cells. Lack of any detectable effect of HIV nef on LTR activity was not the result of mutations in integrated nef DNA, as was shown by polymerase chain reaction. These data suggest that the role of nef in HIV genome transcription does not necessarily involve a direct influence on HIV LTR activity.
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PMID:Constitutive expression of human immunodeficiency virus (HIV) nef protein in human astrocytes does not influence basal or induced HIV long terminal repeat activity. 218 77

Stimulation of the human monocytic cell line Mono Mac 6 with lipopolysaccharide (LPS) leads to rapid and transient expression of cytokines like tumor necrosis factor (TNF). When such cells are precultured for 2 days with a low dose of LPS (20 ng/ml) followed by stimulation with a high dose of LPS (1 microgram/ml), expression of the TNF gene is minimal, i.e. the cells are tolerant. In nuclear run-on analysis, such tolerant cells show only a low degree of transcription, indicating that tolerance operates at or upstream of the transcription level. The CD14 LPS receptor is, however, up-regulated (not down-regulated) in tolerant cells, and LPS can, in fact, still lead to activation of tolerant cells as evidenced by mobilization of the transcription factor nuclear factor kappa B (NF-kappa B). Resolution of the NF-kappa B complex in gel shift analysis shows that the binding protein, mobilized in naive Mono Mac 6 cells, consists mainly of p50-p65 heterodimers, while in tolerant cells, the p50 homodimer is predominant. This increase in p50 homodimers coincides with an increase in p105 mRNA, suggestive of a transcriptional up-regulation of p50. Reporter gene analysis reveals that the NF-kappa B complex mobilized in tolerant cells is functionally inactive in that NF-kappa B-dependent luciferase constructs containing the human immunodeficiency virus long terminal repeat or the TNF 5'-region show only minimal transactivation after LPS stimulation. Similar to Mono Mac 6 cells, primary blood monocytes, when precultured with a low dose of LPS, also become tolerant and produce little TNF after LPS stimulation. The tolerant blood monocytes also up-regulate CD14, and they mobilize NF-kappa B with a predominance of p50 homodimers. Taken together, these results demonstrate that tolerance to LPS is determined by post-receptor mechanisms that involve an altered composition of the NF-kappa B complex.
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PMID:Tolerance to lipopolysaccharide involves mobilization of nuclear factor kappa B with predominance of p50 homodimers. 751 28

The bovine endothelial nitric oxide synthase gene plus 2.9 kilobases of 5'-flanking sequence has been isolated and characterized. The gene spans 20 kilobases and contains 26 exons and 25 introns. Two transcription start sites have been determined by primer extension analysis which are located 170 and 240 base pairs upstream, respectively, from the methionine translational initiation codon. Evidence supporting the upstream boundary region for transcriptional initiation was also obtained by reverse transcription-polymerase chain reaction. The 5'-flanking region lacks a typical TATA box but contains numerous putative transcription factor binding sites. These include consensus sequences for an AP-1 site, an NF-1 site, a tumor necrosis factor responsive element, two sterol regulatory elements, 3 acute-phase response element, two sterol regulatory elements, 3 acute-phase response elements, 6 GATA motifs, 16 CACCC boxes, 5 Sp1 sites, 15 estrogen half-palindromic motifs, and 9 fluid shear stress-responsive elements. The isolated gene promoter directs basal transcription of a luciferase reporter gene when transiently transfected into bovine aortic endothelial cells. High sequence homology of the promoter region to the human endothelial nitric oxide synthase gene promoter (75% nucleotide identity in 1.6 kilobases of 5'-flanking sequence) suggests evolutionary conservation of transcriptional regulation. Isolation and characterization of the bovine endothelial nitric oxide synthase gene should facilitate further investigation of mechanisms by which gene expression is regulated.
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PMID:Organization of the bovine gene encoding the endothelial nitric oxide synthase. 751 47

To understand the roles of intracellular calcium levels on gelatinase/type IV collagenase expression, we analyzed the effects of calcium ionophores on the expression of 92- and 72-kDa gelatinases (MMP-9 and MMP-2) in human fibrosarcoma cells (HT-1080). Calcium ionophores ionomycin and A23187 reduced the levels of pericellular gelatinolytic activity in both untreated and phorbol 12-myristate 13-acetate (PMA) or tumor necrosis factor-alpha (TNF alpha)-stimulated cells as determined by degradation of radiolabeled gelatin. Gelatin zymography and immunoblotting revealed a dose-dependent decrease in the levels of secreted 92-kDa gelatinase, which was paralleled by a decrease of its mRNA. Treatment of cells with thapsigargin caused similar decreases of 92-kDa gelatinase mRNA and protein. The decrease of 92-kDa gelatinase expression was due to lower transcription rate as determined by transfection assays with 92-kDa gelatinase/luciferase construct. The expression of 72-kDa gelatinase was only slightly decreased by ionophores. Treatment of HT-1080 cells with PMA, TNF alpha, or concanavalin A resulted in the conversion of 72-kDa gelatinase proenzyme to its presumed 64- and 62-kDa active forms as determined by gelatin zymography and immunoblotting. Simultaneous treatment with the ionophores or thapsigargin resulted in inhibition of PMA-induced gelatinase activation. The expression of membrane-type matrix metalloproteinase, a potential activator of 72-kDa gelatinase, was not affected by ionophores. The results indicate that calcium ionophores decrease gelatinolysis by repressing both the expression of 92-kDa gelatinase and the activation of the 72-kDa gelatinase.
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PMID:Calcium ionophores decrease pericellular gelatinolytic activity via inhibition of 92-kDa gelatinase expression and decrease of 72-kDa gelatinase activation. 761 67


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