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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human immunodeficiency virus (HIV-1) utilizes the NF-kappaB/Rel proteins to regulate transcription through NF-kappaB binding sites in the HIV-1 long terminal repeat (LTR). Normally, NF-kappaB is retained in the cytoplasm by inhibitory IkappaB proteins; after stimulation by multiple activators including viruses, IkappaBalpha is phosphorylated and degraded, resulting in NF-kappaB release. In the present study, we examined the effect of tetracycline-inducible expression of transdominant repressors of IkappaBalpha (TD-IkappaBalpha) on HIV-1 multiplication using stably selected Jurkat T cells. TD-IkappaBalpha was inducibly expressed as early as 3 h after doxycycline addition and dramatically reduced both NF-kappaB DNA binding activity and LTR-directed gene activity. Interestingly, induced TD-IkappaBalpha expression also decreased endogenous IkappaBalpha expression to undetectable levels by 24 h after induction, demonstrating that TD-IkappaBalpha repressed endogenous NF-kappaB-dependent gene transcription. TD-IkappaBalpha expression also sensitized Jurkat cells to tumor necrosis factor-induced apoptosis. De novo HIV-1 infection of Jurkat cells was dramatically altered by TD-IkappaBalpha induction, resulting in inhibition of HIV-1 multiplication, as measured by p24 antigen, reverse transcriptase, and viral RNA. Given the multiple functions of the NF-kappaB/IkappaB pathway, TD-IkappaBalpha expression may interfere with HIV-1 multiplication at several levels: LTR-mediated transcription, Rev-mediated export of viral RNA, inhibition of HIV-1-induced pro-inflammatory cytokines, and increased sensitivity of HIV-1-infected cells to apoptosis.
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PMID:Inducible expression of IkappaBalpha repressor mutants interferes with NF-kappaB activity and HIV-1 replication in Jurkat T cells. 951 41

The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. Cytokines induce NFkappaB activity in activated but not in quiescent HSCs with subsequent expression of NFkappaB-responsive genes, such as intercellular adhesion molecule (ICAM)-1 and interleukin (IL)-6. We investigated the effect of proteasome inhibitors and an IkappaB super-repressor on the cytokine mediated activation of NFkappaB, ICAM-1, and IL-6 in activated HSCs. Culture-activated HSCs were stimulated with IL-1beta or tumor necrosis factor alpha (TNFalpha) in the presence or absence of proteasome inhibitors, ALLN or MG-132, or after infection with an adenovirus expressing the IkappaB super-repressor (Ad5IkappaB) or beta-galactosidase (Ad5LacZ) as a control. NFkappaB activity was evaluated by immunofluorescence and by electrophoretic mobility shift assay. The steady state level of cytoplasmic IkappaB protein was measured by Western Blot. ICAM-1 and IL-6 expression was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbant assay. Proteasome inhibitors, which block the degradation of IkappaB, and the Ad5IkappaB, which provides an exogenous nondegradable IkappaB, block the stimulation of NFkappaB activity by TNFalpha and IL-1beta in activated HSCs. These reagents block the subsequent nuclear translocation of p65 NFkappaB and induction of ICAM-1 and IL-6 by cytokines. The specificities of the proteasome inhibitors and the IkappaB super-repressor are demonstrated by their failure to block c-Jun N-terminal kinase induction by cytokines. Cytokine-induced stimulation of NFkappaB, ICAM-1, and IL-6 is blocked by proteasome inhibitors and Ad5IkappaB in activated HSCs. Inhibition of IkappaBalpha degradation is a potential target for anti-inflammatory therapy in the liver and might influence the activation process of HSCs following fibrotic stimuli.
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PMID:Inhibition of NFkappaB in activated rat hepatic stellate cells by proteasome inhibitors and an IkappaB super-repressor. 958 6

Evidence is rapidly accumulating that low-activity-reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases homologous to that in phagocytic cells generate reactive oxygen species as signaling intermediates in both endothelium and vascular smooth muscle. We therefore explored the possibility of such an oxidase regulating growth of airway smooth muscle (AWSM). Proliferation of human AWSM cells in culture was inhibited by the antioxidants catalase and N-acetylcysteine, and by the flavoprotein inhibitor diphenylene iodonium (DPI). Membranes prepared from human AWSM cells generated superoxide anion (O) measured by superoxide dismutase-inhibitable lucigenin chemiluminescence, with a distinct preference for NADPH instead of reduced nicotinamide adenine dinucleotide as substrate. Chemiluminescence was also inhibited by DPI, suggesting the presence of a flavoprotein containing oxidase generating O as a signaling molecule for cell growth. Examination of human AWSM cells by reverse transcriptase-polymerase chain reaction consistently demonstrated transcripts with sequences identical to those reported for p22(phox). Transfection with p22(phox) antisense oligonucleotides reduced human AWSM proliferation. Inhibition of NADPH oxidase activity with DPI prevented serum-induced activation of nuclear factor-kappaB (NF-kappaB), and overexpression of a superrepressor form of the NF-kappaB inhibitor IkappaBalpha significantly reduced human AWSM growth. These findings suggest that an NADPH oxidase containing p22(phox) regulates growth-factor responsive human AWSM proliferation, and that the oxidase signals in part through activation of the prototypical redox-regulated transcription factor NF-kappaB.
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PMID:NADPH oxidase promotes NF-kappaB activation and proliferation in human airway smooth muscle. 1188 Mar 5

We used a molecular genetics approach to investigate the role of nuclear factor-kappaB (NF-kappaB) in neointimal hyperplasia induced by flow interruption of carotid artery in mice. Wild type mice (WT mice) and mice rendered deficient in p105, the precursor of p50, one of the components of the multimeric transcription factor NF-kappaB (NF-kappaB knockout mice; KO mice), were subjected to a complete ligation of the left common carotid artery. Morphometric analysis of the structural alteration caused by the disruption of the arterial blood flow was performed 14 days after surgery. Furthermore the expression of intercellular adhesion molecule-1 (ICAM-1) in injured arteries was evaluated 4 days after artery ligation by the means of reverse transcriptase polymerase chain reaction (RT-PCR) and quantification of the ICAM-1 protein levels. In a separate experiment normal mice were randomly assigned to receive a recombinant adeno-associated virus (rAAV) encoding the gene for the NF-kappaB inhibitory protein IkappaBalpha (rAAV-IkappaBalpha), or the beta-galactosidase gene (rAAV-LacZ), both at a dose of 10(11) copies and 2 weeks later were subjected to the complete ligation of the left carotid artery. NF-kappaB activity (studied by means of electrophoretic mobility shift assay-EMSA), IkappaBalpha expression (evaluated by Western blot analysis) ICAM-1 evaluation (RT-PCR and quantification of the protein levels) and a morphometric analysis were evaluated in the injured arteries. Disruption of the arterial blood flow caused a marked neointimal hyperplasia. The mean intimal area was 0.023+/-0.002 mm(2) in wild type mice compared with 0.002+/-0.001 mm(2) in NF-kappaB knockout mice. ICAM-1 expression was 1.7+/-0.8 relative amount of ICAM-1 mRNA in wild type mice compared with 0.4+/-0.06 relative amount of ICAM-1 mRNA in NF-kappaB knockout mice. ICAM-1 protein levels were also significantly reduced in NF-kappaB knockout mice. Injured arteries treated with rAAV-IkappaBalpha had a greater expression of IkappaBalpha and lower NF-kappaB activity, when compared with vessels treated with rAAV-LacZ. Furthermore, ICAM-1 expression was markedly attenuated by the treatment with rAAV-IkappaBalpha (rAAV-LacZ=1.6+/-0.8 relative amount of ICAM-1 mRNA; rAAV-IkappaBalpha=0.55+/-0.04 relative amount of ICAM-1 mRNA). ICAM-1 protein levels were also significantly decreased in rAAV-IkappaBalpha treated mice. Finally the mean intimal area was 0.028+/-0.003 mm(2) in left carotid arteries treated with rAAV-LacZ whereas it was 0.003+/-0.004 mm(2) in vessels treated with rAAV-IkappaBalpha. Our data indicate that NF-kappaB plays a crucial role in neointimal hyperplasia induced by flow cessation in the mouse carotid artery, and in addition suggest that rAAV-mediated gene transfer of IkappaBalpha might represent a novel therapeutic approach to the treatment of restenosis.
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PMID:Crucial role of nuclear factor-kappaB in neointimal hyperplasia of the mouse carotid artery after interruption of blood flow. 1253 35

Neutrophil adhesion to extracellular matrix is necessary for an effective inflammatory response. Adhesion may accelerate neutrophil activation by affecting intracellular signaling pathways. The nuclear transcription factor kappaB (NF-kappaB) controls several cellular functions, including inflammation, proliferation, and cell survival. We explored the role of adhesion in NF-kappaB activation in human neutrophils. Cells were stimulated with tumor necrosis factor-alpha (TNF-alpha), granulocyte macrophage-colony-stimulating factor (GM-CSF), interleukin-8 (IL-8), and formyl-methionyl-leucyl-phenylalanine (fMLP). All four initiated neutrophil adherence to and spreading on fibronectin. GM-CSF and IL-8 did not activate NF-kappaB in suspended neutrophils but rapidly activated NF-kappaB under adherent conditions on matrix, as shown by IkappaB kinase activity assay, IkappaBalpha degradation, electromobility shift assay, and quantitative reverse transcriptase-PCR. In contrast, TNF-alpha activated NF-kappaB both in suspended cells and adherent cells. fMLP did not activate NF-kappaB in either suspended or adherent cells. Specific beta(2) integrin blockade prevented NF-kappaB activation by GM-CSF and IL-8 on fibronectin. Co-stimulating CD18 and CD11b with activating antibodies resulted in NF-kappaB activation by GM-CSF and IL-8 in suspended cells. We inhibited actin polymerization with cytochalasin and blocked the non-receptor kinase Syk with piceatannol. Both maneuvers prevented the co-stimulatory NF-kappaB-activating signal by beta(2) integrins. Thus, in addition to beta(2) integrin ligand binding, NF-kappaB activation depended on the formation of the receptor-associated intracellular focal adhesion complex. We conclude that beta(2) integrins may provide co-stimulatory signals allowing some soluble mediators to activate the NF-kappaB pathway even when they are not capable of doing so in suspension. This effect may become important when human neutrophils leave the circulating blood and migrate through extracellular matrix during inflammation.
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PMID:Integrins and cytokines activate nuclear transcription factor-kappaB in human neutrophils. 1461 35

Pigment epithelium-derived factor (PEDF) protects immature cerebellar granule cell neurons (CGCs) against apoptosis induced by K+ and serum deprivation. However, the precise mechanism of this protection remains unknown. We recently reported that the transcription factor nuclear factor kappa B (NF-kappaB) is activated in PEDF-treated CGCs. Although it is well known that NF-kappaB blocks apoptotic cell death through the induction of pro-survival factors, the effects of PEDF on the expression of these factors are not fully understood. In this study, we employed the use of reverse transcriptase-polymerase chain reaction to analyze the gene expression of certain pro-survival genes and found that genes such as c-IAP1, c-IAP2, FLIPs, A1/Bfl-1 and Mn-SOD were induced in PEDF-treated neurons. On the other hand, no induction was observed of the pro-apoptotic Bcl-2 family members Bax and Bid at any time from 3 to 24 h following PEDF addition. Furthermore, phosphorylation of cyclic AMP-responsive element binding protein (CREB) and increment of nuclear cyclic AMP-response element (CRE)-like DNA binding were observed in PEDF-treated CGCs. The anti-apoptotic effect of PEDF was blocked by overexpression of dominant negative CREB or a mutated form of IkappaBalpha. These results suggested that induction of both CRE- and NF-kappaB-dependent genes is required for the observed neuroprotective effects of PEDF on CGCs.
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PMID:Pigment epithelium-derived factor induces pro-survival genes through cyclic AMP-responsive element binding protein and nuclear factor kappa B activation in rat cultured cerebellar granule cells: Implication for its neuroprotective effect. 1589 82

Fibroblast-like synoviocytes (FLS) play a key role in the genesis of rheumatoid arthritis (RA). FLS are among the most versatile cells with the potential to activate an array of genes that are able to initiate and propagate inflammation in RA-affected joints. Controlling activation of FLS might hold the key to restraining inflammation in RA-affected joints. In this study, we investigate the effect and mechanisms of short-term hyperthermia on a series of proinflammatory genes in FLS. In vitro experiments demonstrate that exposure of FLS to elevated temperatures for the duration of 30 min prevents activation of a series of genes with proinflammatory properties. Exposure to hyperthermia reduces IL-1beta-induced prostaglandin E2 release, suppresses activation of the adhesion molecules VCAM-1, ICAM-1, the cytokines TNFalpha, IL-1alpha, IL-1beta, IL-8 as well as COX-2 protein synthesis. Real time reverse transcriptase-polymerase chain reaction showed that hyperthermia altered gene expression at the transcriptional level. The amount and the duration of inhibition is gene-specific and lasts for up to 25 h. As to the mechanism of inhibition, electrophoretic mobility shift assay experiments demonstrated that exposure of FLS to hyperthermia prevents IL-1beta-induced NF-kappaB translocation and subsequent DNA binding. Many mechanisms have been shown to be involved in hyperthermia-mediated effects on NF-kappaB-DNA interactions. We demonstrate by Western blot experiments that in FLS, hyperthermia prevents the phosphorylation and subsequent degradation of IkappaBalpha, therefore retaining the NF-kappaB complex in the cytoplasm. Carefully controlled in vivo tests are certainly needed before one can take full advantage of those phenomena; however, the ease by which the temperature in joints can be modulated might offer an opportunity for manipulating inflammatory processes in joints by simple balneological means.
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PMID:Short-term hyperthermia prevents activation of proinflammatory genes in fibroblast-like synoviocytes by blocking the activation of the transcription factor NF-kappaB. 1698 13

Diabetic mellitus confers a major risk of congenital malformations, and is associated with diabetic embryopathy, affecting multiple organs including the kidney. The DNA paired box-2 (Pax-2) gene is essential in nephrogenesis. We investigated whether high glucose alters Pax-2 gene expression and aimed to delineate its underlying mechanism(s) of action using both in vitro (mouse embryonic mesenchymal epithelial cells (MK4) and ex vivo (kidney explant from Hoxb7-green florescent protein (GFP) mice) approaches. Pax-2 gene expression was determined by reverse transcriptase-polymerase chain reaction, Western blotting, and immunofluorescent staining. A fusion gene containing the full-length 5'-flanking region of the human Pax-2 promoter linked to a luciferase reporter gene, pGL-2/hPax-2, was transfected into MK4 cells with or without dominant negative IkappaBalpha (DN IkappaBalpha) cotransfection. Fusion gene expression level was quantified by cellular luciferase activity. Reactive oxygen species (ROS) generation was measured by lucigenin assay. Embryonic kidneys from Hoxb7-GFP mice were cultured ex vivo. High D(+) glucose (25 mM), compared to normal glucose (5 mM), specifically induced Pax-2 gene expression in MK4 cells and kidney explants. High glucose-induced Pax-2 gene expression is mediated, at least in part, via ROS generation and activation of the nuclear factor kappa B signaling pathway, but not via protein kinase C, p38 mitogen-activated protein kinase (MAPK), and p44/42 MAPK signaling.
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PMID:Reactive oxygen species and nuclear factor-kappa B pathway mediate high glucose-induced Pax-2 gene expression in mouse embryonic mesenchymal epithelial cells and kidney explants. 1698 13

NF-kappaB is a crucial transcription factor tightly regulated by protein interactions and post-translational modifications, like phosphorylation and acetylation. A previous study has shown that trichostatin A (TSA), a histone deacetylase inhibitor, potentiates tumor necrosis factor (TNF) alpha-elicited NF-kappaB activation and delays IkappaBalpha cytoplasmic reappearance. Here, we demonstrated that TSA also prolongs NF-kappaB activation when induced by the insulino-mimetic pervanadate (PV), a tyrosine phosphatase inhibitor that initiates an atypical NF-kappaB signaling. This extension is similarly correlated with delayed IkappaBalpha cytoplasmic reappearance. However, whereas TSA causes a prolonged IKK activity when added to TNFalpha, it does not when added to PV. Instead, quantitative reverse transcriptase-PCR revealed a decrease of ikappabalpha mRNA level after TSA addition to PV stimulation. This synthesis deficit of the inhibitor could explain the sustained NF-kappaB residence in the nucleus. In vivo analysis by chromatin immunoprecipitation assays uncovered that, for PV induction but not for TNFalpha, the presence of TSA provokes several impairments on the ikappabalpha promoter: (i) diminution of RNA Pol II recruitment; (ii) reduced acetylation and phosphorylation of histone H3-Lys(14) and -Ser(10), respectively; (iii) decreased presence of phosphorylated p65-Ser(536); and (iv) reduction of IKKalpha binding. The recruitment of these proteins on the icam-1 promoter, another NF-kappaB-regulated gene, is not equally affected, suggesting a promoter specificity of PV with TSA stimulation. Taken together, these data suggest that TSA acts differently depending on the NF-kappaB pathway and the targeted promoter in question. This indicates that one overall histone deacetylase role is to inhibit NF-kappaB activation by molecular mechanisms specific of the stimulus and the promoter.
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PMID:Histone deacetylase inhibitor trichostatin A sustains sodium pervanadate-induced NF-kappaB activation by delaying IkappaBalpha mRNA resynthesis: comparison with tumor necrosis factor alpha. 1740 87

Helicobacter pylori infection leads to gastroduodenal inflammation, peptic ulceration, gastric lymphoma and gastric cancer. Certain herbal remedies have been used to treat gastric disease. In this study, we examined the anti-inflammatory effect of San-Huang-Xie-Xin-Tang (SHXT) and its main component baicalin on Helicobacter pylori-infected human gastric epithelial AGS cell. AGS cells were treated with Helicobacter pylori at a bacterium/cell ratio of 300:1. mRNA expression and protein levels were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and western blot analysis, respectively. Interleukin-8 (IL-8) level and the translocation of nuclear factor kappa B (NF-kappaB) were measured by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked DNA-protein interaction assay (ELDIA), respectively. Nitric oxide production was measured by Griess reagent. We found that SHXT and baicalin inhibited Helicobacter pylori-induced cyclooxygenase-2 (COX-2) enhancement and IkappaBalpha degradation in both mRNA and protein levels. SHXT and baicalin also inhibited Helicobacter pylori-induced inducible nitric oxide synthase (iNOS) and IL-8 mRNA expression, and decreased NO and IL-8 production. Furthermore, SHXT and baicalin inhibited nuclear translocation of p50 subunit of NF-kappaB in Helicobacter pylori-infected AGS cells. Based on the above findings, SHXT and baicalin might exert anti-inflammatory and gastroprotective effects in Helicobacter pylori-induced gastric inflammation.
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PMID:San-Huang-Xie-Xin-Tang inhibits Helicobacter pylori-induced inflammation in human gastric epithelial AGS cells. 1753 3


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