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:1.14.14.3 (luciferase)
38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Numerous environmental stimuli alter cell functions by the induction of intracellular reactive oxygen species, such as superoxide and hydrogen peroxide (H2O2). These redox alterations can change the activity of kinases and phosphatases responsible for controlling intracellular signal transduction cascades important in determining how cells react to their environment. One such well known pathway includes nuclear factor-kappaB (NFkappaB); however, the exact redox-sensitive factors important in controlling H2O2-mediated activation of NFkappaB remain unclear. In the present study, we have investigated how intracellular clearance of H2O2, using a recombinant adenovirus expressing glutathione peroxidase-1 (GPx-1), modulates NFkappaB activation following UV irradiation, tumor necrosis factor-alpha, or H2O2 treatment of MCF-7 cells. Findings from these studies demonstrate that GPx-1 overexpression can down-regulate NFkappaB DNA binding, and transcriptional activation of an NFkappaB-dependent luciferase reporter, to varying extents following these environmental stimuli. Studies using dominant negative adenoviral vectors expressing IKKalpha(KM) and IKKbeta(KA) suggest that GPx-1-mediated H2O2 clearance appears to preferentially inhibit the activity of IKKalpha, but not IKKbeta. These studies demonstrate for the first time that redox regulation of NFkappaB activation by intracellular H2O2 may be specific for a unique subunit in the IKK complex. Such findings suggest that IKK kinases or IKK phosphatases may have unique redox-regulated components. These studies have shed mechanistic insight into the potential application of redox-modulating gene therapies aimed at altering NFkappaB activation following environmental injury.
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PMID:GPx-1 gene delivery modulates NFkappaB activation following diverse environmental injuries through a specific subunit of the IKK complex. 1149 54

To investigate mechanisms of rat glutathione S-transferase P1 gene (rGSTP1) expression regulation during chemical carcinogenesis. we studied enhancer elements located in the region between -2.5 kb to -2.2 kb. The region was upstream from the start site of transcription and was divided into two major fragments, GPEI and GPEII. The GPEII fragment was further divided into two smaller fragments, GPEII- I and GPEII-2. Using a luciferase reporter system, we identified a strong enhancer of GPEI and a weak enhancer of GPEII in HeLa and a rat hepatoma cell line CBRH79 19 cell. The enhancer of GPEII was located within the GPEII-I region. Chemical stimulation by glycidyl methatylate (GMA) and phorbol 12-o-tetradecanoate 13-acetate (TPA) analysis revealed that induction of rGSTP1 expression was mainly through GPEI. Although H2O2 could enhance GPEII enhancer activity, the enhancement is not mediated by the NF-kappaB factor that bound the NF-kappaB site in GPEII. Using electrophoretic mobility shift assays (EMSA) and the UV cross-linking assays, we found that HeLa and CBRH7919 cells had proteins that specifically bound GPEI core sequence and a 64 kDa protein that interacted with GPEII-1. The cells from normal rat liver did not express the binding proteins. Therefore, the trans-acting factors seem to be closely related to GPEI, GPEII enhancer activities and may play an important role in high expression of rGSTPI gene.
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PMID:The effect of chemical carcinogenesis on rat glutathione S-transferase P1 gene transcriptional regulation. 1171 May 60

The promoter and enhancer elements of the mouse erythropoietin (mEpo) gene, which have high homology with those of the human erythropoietin (hEpo) gene, were fused with luciferase. The construct was transfected into erythropoietin-producing hepatoma cell line (Hep3B) cells by lipofectin with lacZ as an internal standard. The wild type (TGATA) showed a 39.5-fold increase in induction by hypoxia. Mouse GATA-2 inhibited the hypoxic induction of the wild-type (m3), promoterluciferase construct but not the hypoxic induction of the mutant (m4, 5) promoter-luciferase constructs. N(G)-monomethyl L-arginine (L-NMMA) inhibited the hypoxic induction of the m3 promoter-luciferase construct, but this inhibition was recovered by L-arginine. H2O2 also inhibited the hypoxic induction of the m3 promoter-luciferase construct, but this inhibition was recovered by catalase. Gel shift assays performed on nuclear extracts of 293 cells overexpressing mGATA-1, -2, and -3 revealed that mGATA-1, -2, and -3 bind to the TGATA element of the mEpo promoter. These results indicate that mGATA binds to the TGATA site of the mEpo promoter and negatively regulates mEpo gene expression. Negative regulation of mEpo gene by GATA transcriptional factors is discussed.
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PMID:GATA suppresses erythropoietin gene expression through GATA site in mouse erythropoietin gene promoter. 1204 67

The gp91phox homologue Nox1 produces H2O2, which induces cell growth, transformation, and tumorigenicity. However, it has not been clear whether H2O2 effects are mediated indirectly via a generally oxidizing cellular environment or whether H2O2 more directly targets specific signaling pathways. Here, we investigated signaling by H2O2 induced by Nox1 overexpression using a luciferase reporter regulated by the antioxidant response element ARE4. Surprisingly, Nox1-derived H2O2 activated the reporter gene 15-fold with no effect on the redox state of the major thiol antioxidant substances, glutathione and thioredoxin. H2O2 signaling to ARE4 was mediated by activation of both the c-Jun N-terminal kinase and ERK1/2 pathways modulated by Ras. Thus, "redox signaling" resulting in kinase signaling pathways is distinct from "oxidative stress," and is mediated by discrete, localized redox circuitry.
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PMID:H2O2-dependent activation of GCLC-ARE4 reporter occurs by mitogen-activated protein kinase pathways without oxidation of cellular glutathione or thioredoxin-1. 1463 94

Exposure to certain particulate hexavalent chromium [Cr(VI)] compounds, such as lead chromate (PbCrO4), has been associated with lung cancer and respiratory tract toxicity. Previous studies indicate that the solubility of Cr(VI)-compounds is an important factor in Cr(VI)-induced carcinogenesis. The present study investigates reactive oxygen species (ROS) generation by PbCrO4 particles and cellular responses using RAW 264.7 cells. A mixture containing PbCrO4 and RAW 264.7 cells generated hydroxyl radical ((.)OH), using cellularly generated H2O2 as a precursor, as measured by electron spin resonance (ESR) spin trapping in combination with H2O2 and (.)OH scavengers, catalase and sodium formate. The effect of ascorbic acid on (.)OH radicals was also measured using ESR. Confocal microscopy showed that particles could become either bound to the cell surface or engulfed over a 120 min time period. H2O2 generation and O2 consumption were also increased after treatment of the cells with PbCrO4. Both NF-kappaB and AP-1 were activated after exposure to PbCrO4 particles as measured by the NF-kappaB or AP-1 luciferase reporter plasmid assay. Our investigation thus demonstrated that the RAW 264.7 cells phagocytized the PbCrO4 particles leading to accumulation of the particles within vacuoles in the cytoplasm. These particles could induce chronic production of ROS and activation of NF-kappaB and AP-1. Such induction of transcription pathways may be involved in the inflammatory and carcinogenic responses induced by Cr(VI)-containing particles.
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PMID:PbCrO4 mediates cellular responses via reactive oxygen species. 1497 58

Light-emitting bacteria are the most abundant and widespread luminescent organisms. Most species of such bacteria live in marine environments. However, until recently, biological role of bacterial luminescence remained unknown. Recent studies indicated that light produced in bacterial cells may stimulate DNA repair. Therefore, it is not surprising that agents that cause DNA damage induce expression of lux genes. Moreover, it was proposed previously that bacterial luciferases may be involved in detoxification of reactive oxygen species. Recently, this hypothesis was confirmed experimentally. Here we investigated effects of hydrogen peroxide on light emission by various strains of luminescent bacteria. We found that luminescence of strains with luciferase of fast kinetics of reaction decreased at considerably lower concentrations of H2O2 than that of strains with luciferase of the slow kinetics. The action (either direct or indirect) of luciferases as anti-oxidants seemed to be independent of activity of catalase, which was found to be different in various strains. Therefore, it seems that luciferases of the slow kinetics are more efficient in detoxification of reactive oxygen species than those of the fast kinetics.
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PMID:Effects of hydrogen peroxide on light emission by various strains of marine luminescent bacteria. 1516 91

Plants have a range of mechanisms to protect against oxidative damage induced by excess light and environmental stress. One of these processes consists of the detoxification of reactive oxygen species by the ascorbate peroxidase (APX) family of enzymes, which convert H2O2 into H2O. Two of the genes encoding APX in Arabidopsis are induced by high light, namely APX1 and APX2. We have applied a genetic approach to understanding the mechanisms of photoprotection, using APX2 as an indicator of oxidative stress. Transgenic plants containing the reporter gene luciferase linked to the APX2 promoter were EMS mutagenized and kindly provided to us by Mullineaux and colleagues. We have screened this mutagenized seed to identify mutants with aberrant photoprotection, based on altered luminescence resulting from altered luciferase activity. Here we describe the screen and steps involved in the identification of the mutations in an effort to identify novel photoprotective genes and products.
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PMID:Identifying photoprotection mutants in Arabidopsis thaliana. 1518 87

The coding region of cDNA and genomic DNA, with its promoter region, of zebrafish metallothionein (zMT) gene homologous to the piscine MT-II was obtained. The A/T-rich promoter region contains four metal regulatory elements (MREs), three activator protein 1 (AP1) and one specific protein 1 (Sp1) binding sites. The four MREs are organized into two clusters, a distal cluster with one MRE lying around 740 bp upstream of the transcription start point and a proximal cluster with three MREs located close to the TATA box. The metal induction ability of the promoter was assessed by transient luciferase gene expression assays in HepG2 cells. The zMT promoter was inducible by Zn2+, Cd2+, Cu2+ and Hg2+ ions in decreasing inducibility, while inert to Ni2+, Pb2+ and Co2+ ions, and H2O2 treatment in vitro. Deletion of the putative cis-acting elements in the promoter region revealed that the distal MRE (MREd) was important in mediating metal inducibility. Despite the binding of HepG2 cell nuclear protein factors to all MREs as confirmed by electrophoretic mobility shift assay (EMSA), the proximal MREs did not provide significant contribution to metal induction of zMT gene in HepG2 cells. The metal inducibility of zMT promoter required the cooperative effect of at least three MRE sites.
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PMID:Cloning of zebrafish metallothionein gene and characterization of its gene promoter region in HepG2 cell line. 1524 16

The aim of this research was to analyze the effects and the modes of action of elemental sulfur (S(0)) in bioluminescence and respiration of Vibrio fischeri cells and the enzymes crude luciferase, pure catalase, and alcohol dehydrogenase (ADH). Metallic copper removed sulfur and reduced the toxicity of acetone extracts of sediment samples analyzed in the bioluminescence test. The sulfur inhibition of cell bioluminescence was noncompetitive with decanal, the luciferase substrate; reversible, with maximum toxicity after 15 min (EC(50) = 11.8 microg/L); and almost totally recovered after 2 h. In vitro preincubation of crude luciferase extract with sulfur (0.28 ppm) weakly inhibited bioluminescence at 5 min, but at 30 min the inhibition reached 60%. Increasing the concentration of sulfur in the parts per million concentration range in vitro decreased bioluminescence, which was not constant, but depended on exposure time, and no dead-end/total inhibition was observed. The redox state of enzymes in the in vitro system significantly affected inhibition. Hydrogen peroxide restored fully and the reducing agent dithiothreitol, itself toxic, restored only partially luciferase activity in the presence of sulfur. Sulfur (5.5 ppm) slightly inhibited ADH and catalase, and dithiothreitol enhanced sulfur inhibition. High sulfur concentrations (2.2 ppm) inhibited the bioluminescence and enhanced the respiration rate of V. fischeri cells. Elemental sulfur data were interpreted to show that sulfur acted on at least a few V. fischeri cell sites: reversibly modifying luciferase at sites sensitive to/protected by oxidative and reducing agents and by affecting electron transport processes, resulting in enhanced oxygen consumption. Sulfur together with an enzyme reducing agent inhibited the oxidoreductive enzymes ADH and catalase, which have --SH groups, metal ion cofactors, or heme, respectively, in their active centers.
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PMID:Elemental sulfur: toxicity in vivo and in vitro to bacterial luciferase, in vitro yeast alcohol dehydrogenase, and bovine liver catalase. 1526 10

Site-directed mutagenesis was used to generate three cysteine mutants of GSTp, C(47/101), C(14/47/101) and C(14/47/101/169). GSTp, C(47/101), C(14/47/101) and C(14/47/101/169) were transfected into 293 cells separately and GST activity was determined by using CDNB as substrate. Data showed that each cysteine mutant inhibited endogenous GST catalyzatic activity and had remarkable dominant negative effect. The expression vectors of wide type GSTp and its cysteine mutants were co-transfected with c-Jun, NF-kappaB, or p21 luciferase reporting vector, into 293 cells separately, luciferase activity showed that C(14/47/101) and C(14/47/101/169) can dramatically activate c-Jun and p21 transcriptional activity. Each cysteine mutant can increase endogenous p21 level, and also increased mortality rate of 293 cells when exposed to H2O2. These results suggest that cysteine residues of GSTp play an important role in protecting cells against oxitative stress.
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PMID:[The effects of cysteines on the function of human glutathion S-transferase pi(GSTp) under cell oxidative stress]. 1532 18


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