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)

Nitric oxide (NO) is known to have various biologic and pathophysiologic effects on organisms. The molecular mechanisms by which NO exerts harmful effects are unknown, although various O2 radicals and ions that result from reactivity of NO are presumed to be involved. Here we report that adaptive cellular response controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) in hypoxia is suppressed by NO. Induction of erythropoietin and glycolytic aldolase A mRNAs in hypoxically cultured Hep3B cells, a human hepatoma cell line, was completely and partially inhibited, respectively, by the addition of sodium nitroprusside (SNP), which spontaneously releases NO. A reporter plasmid carrying four hypoxia-response element sequences connected to the luciferase structural gene was constructed and transfected into Hep3B cells. Inducibly expressed luciferase activity in hypoxia was inhibited by the addition of SNP and two other structurally different NO donors, S-nitroso-L-glutathione and 3-morpholinosydnonimine, giving IC50 values of 7.8, 211, and 490 microM, respectively. Inhibition by SNP was also observed in Neuro 2A and HeLa cells, indicating that the inhibition was not cell-type-specific. The vascular endothelial growth factor promoter activity that is controlled by HIF-1 was also inhibited by SNP (IC50 = 6.6 microM). Induction generated by the addition of cobalt ion (this treatment mimics hypoxia) was also inhibited by SNP (IC50 = 2.5 microM). Increased luciferase activity expressed by cotransfection of effector plasmids for HIF-1alpha or HIF-1alpha-like factor in hypoxia was also inhibited by the NO donor. We also showed that the inhibition was performed by blocking an activation step of HIF-1alpha to a DNA-binding form.
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PMID:Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia. 963 55

The effects of Chrono-lume (CL) and magnesium sulfate (Mg2+), a component of this luciferin-luciferase reagent, on platelet aggregation were studied in platelet-rich plasma (PRP) obtained from blood anticoagulated with sodium citrate from humans, dogs, cats, horses, and cows. The final added Mg2+ concentration of both solutions ranged from 0.75-3.7 mM. CL and Mg2+ had no effect on maximum aggregation of platelets from humans induced by sub-threshold concentrations of collagen and ADP. In contrast, addition of CL or Mg2+ to canine PRP resulted in a dose-dependent and equal potentiation of platelet aggregation in response to sub-threshold concentrations of collagen, ADP, and thrombin in normal and thrombopathic dogs. The effect of CL on platelet aggregation induced by sub-threshold concentrations of agonists was less pronounced and varied in other species according to the agonist. The reason for the marked difference in sensitivity of human and canine platelets to CL or Mg2+ is not clear, although a difference in releasable cation pools of the platelets from these two species has been recognized. Platelet aggregation studies of animals with suspected thrombopathias should be performed without CL to prevent masking of a platelet function defect.
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PMID:Chrono-lume and magnesium potentiate aggregation of canine but not human platelets in citrated platelet-rich plasma. 968 6

Genetic and biochemical assays were conducted to determine if nitrile induced adult paralysis and germline aneuploidy in female Drosophila melanogaster requires a biochemical activation mechanism which results in the release of free cyanide. Two nitriles predicted to differ substantially in their susceptibility to enzymatic cyanide release were found to be equally effective inducers of aneuploidy. Regardless of differences in chemical structure, nitriles seem to be affecting a common cellular target as judged by the lack of synergistic effects when two nitriles are presented simultaneously. Mitochondrial respiration was not inhibited by acetonitrile under conditions in which sodium cyanide completely blocked respiration. A sensitive luciferase enzyme inhibition assay suggests that some, but not all, nitriles may affect hydrophobic protein interactions. These results suggest that there is no single biochemical mechanism by which all nitriles induce aneuploidy, although the cellular target disrupted is probably the same for each chemical. The implications of these findings for structural alert based pre-screening of mutagens are discussed.
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PMID:Induction by nitriles of sex chromosome aneuploidy: tests of mechanism. 972 15

Gene expression is under the influence of DNA methylation and assembly of chromatin structure. This paper reports the modulation of transgene expression in zebrafish embryos by altering DNA methylation with 5-azacytidine and heterochromatin formation with sodium butyrate, an inhibitor of histone deacetylation. A CMV promoter-luciferase fusion gene construct (pCMVL) microinjected into zebrafish eggs becomes gradually methylated during development, starting at approximately 12 h post-injection. When methylated in vitro by Hpa II methylase prior to injection, the construct is rapidly demethylated in vivo before being de novo methylated. Demethylation is independent of DNA replication, indicating that it is an active DNA repair process. Demethylating activity has been characterized in zebrafish embryo nuclear extracts, in which this activity is heat-labile, sensitive to protease and RNase and requires ATP hydrolysis. Demethylating activity in vitro is dependent on the developmental stage of the embryo from which extracts are prepared. In vivo , luciferase transcripts are detected prior to de novo plasmid methylation. Furthermore, incubation of pCMVL-injected embryos with 5-azacytidine or butyrate immediately after injection inhibits plasmid methylation and extends the period of luciferase expression. When applied after de novo methylation has occurred, both inhibitors prevent methylation of newly replicated DNA and promote transgene expression. These data suggest that methylation of the injected construct during early development induces repression of the transgene, perhaps by converting the construct to a repressive chromatin structure.
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PMID:Modulation of plasmid DNA methylation and expression in zebrafish embryos. 974 49

Reactive oxygen species generated by treatment of smooth muscle cells (SMCs) with either phorbol 12-myristate 13-acetate or with the combination of H2O2 and vanadate strongly induce expression of the class A scavenger receptor (SR-A) gene. In the current studies, cis-acting elements in the proximal 245 bp of the SR-A promoter were shown to direct luciferase reporter expression in response to oxidative stress in both SMCs and macrophages. A composite activating protein-1 (AP-1)/ets binding element located between -67 and -50 bp relative to the transcriptional start site is critical for macrophage SR-A activity. Mutation of either the AP-1 or the ets component of this site also prevented promoter activity in SMCs. Mutation of a second site located between -44 and -21 bp, which we have identified as a CCAAT/enhancer binding protein (C/EBP) element, reduced the inducible activity of the promoter in SMCs by 50%, suggesting that combinatorial interactions between these sites are necessary for optimal gene induction. Interactions between SMC nuclear extracts and the SR-A promoter were analyzed by electrophoretic mobility shift assay. c-Jun/AP-1 binding activity, specific for the -67- to -50-bp site, was induced in SMCs by the same conditions that increased SR-A expression. Moreover, phorbol 12-myristate 13-acetate, H2O2, or the combination of H2O2 and sodium orthovanadate (vanadate) activated c-Jun-activating kinase. The binding activity within SMC extracts specific for the C/EBP site was shown to be C/EBPbeta in SMCs. Taken together, these findings demonstrate that reactive oxygen species regulate the interactions between c-Jun/AP-1 and C/EBPbeta in the SR-A promoter. Furthermore, induction of oxidative stress in THP-1 cells, with a combination of 10 micromol/L vanadate and 100 micromol/L H2O2, induced macrophage differentiation, adhesion, and SR activity. These data suggest that vascular oxidative stress may contribute to the induction of SR-A expression and thereby promote the uptake of oxidatively modified low density lipoprotein by both macrophage and SMCs to produce foam cells in atherosclerotic lesions.
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PMID:Transcriptional activation of scavenger receptor expression in human smooth muscle cells requires AP-1/c-Jun and C/EBPbeta: both AP-1 binding and JNK activation are induced by phorbol esters and oxidative stress. 974 33

Dioctadecylamidoglycylspermine (DOGS, Transfectam) is a cationic lipid able to interact with DNA to form complexes that mediate efficient gene transfer into various eukaryotic cells. The state of condensation of the plasmid changes with the medium composition. We therefore investigated to what extent the DNA condensation buffer influences the transfection efficiency of Transfectam/DNA particles. Our results show that in a variety of cell lines, a greater than 100-fold difference in luciferase gene expression is observed with Transfectam/DNA complexes at a +/- charge ratio of 0.75 depending on the conditions of complex formation. The best transfection conditions consisted of particles formed in RPMI medium, NaHCO3/Na2HPO4 or sodium citrate solutions. Mixing in a 150 mM sodium chloride solution (as recommended) resulted in lower gene expression. When the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was present in the DNA/cationic lipid formulation, the increase in reporter activity was also observed, although to a lower extent. Thus, choosing the optimal conditions for formulating DNA/lipid complexes considerably reduces the amount of lipid and DNA needed to obtain maximum gene transfer.
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PMID:Influence of the DNA complexation medium on the transfection efficiency of lipospermine/DNA particles. 974 67

The lung epithelium resorbs alveolar fluid through combined action of sodium channels and the sodium pump, Na,K-ATPase. The lung often is exposed to hyperoxia in disease states and hyperoxia generates a mixture of reactive oxygen species. In vivo and in vitro exposure of rat lung and alveolar type II cells, respectively, increases gene expression of both alpha-1 and beta-1 subunits of the sodium pump. In contrast to the primary type II cells, several type II cell lines did not increase sodium pump gene expression with hyperoxia, but the renal tubular epithelial MDCK cell line did. Using promoter-receptor constructs transfected into MDCK cells, hyperoxia did not markedly increase transcription of the alpha-1 subunit but doubled transcription of the beta-1 subunit gene. Using 5'-deletion constructs, the region required for the beta-1 increase was localized to a 40-base pair region from -44/-84. The hyperoxic responsiveness of this region was confirmed using constructs with one or two copies of this region placed in minimal promoter-luciferase reporters. This 5' promoter region contains a consensus binding sequence for SP-1, a basal transcription factor but not for binding of other known transcription factors. Thus, hyperoxia induces Na,K-ATPase beta-1 promoter transcription, likely acting through a novel mechanism.
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PMID:Oxidant effects on epithelial Na,K-ATPase gene expression and promoter function. 978

We investigated the possibility that vascular endothelial growth factor (VEGF) treatment could regulate KDR/Flk-1 receptor expression in endothelial cells. Bovine adrenal cortex endothelial cells were incubated with 200 pM rhVEGF165 for 0-7 days. Western blot analysis showed a 3-5-fold increase in total KDR protein following 4-day VEGF treatment. Scatchard analysis revealed that VEGF induced a 2-3-fold increase in high affinity receptor number (5.0 x 10(4)/cell versus 2. 4 x 10(4)/cell) without significantly affecting receptor binding affinity (Kd 76 pM versus 72 pM). Quantitative polymerase chain reaction analysis demonstrated a 3-fold increase in KDR mRNA levels following VEGF exposure. VEGF-induced KDR expression primarily occurred at the transcriptional level as demonstrated by a luciferase reporter assay system. Receptor selective mutants with wild-type KDR binding and decreased Flt-1 binding also induced KDR up-regulation; in contrast, mutants with decreased KDR binding and wild-type Flt-1 binding did not, suggesting that KDR receptor signaling mediated the increase in KDR expression. Inhibition of tyrosine kinase, Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase activities all blocked VEGF-induced KDR up-regulation. Finally, co-incubation of nitric-oxide synthase inhibitors with VEGF had no significant effect on KDR expression, but 100 microM sodium nitroprusside, a NO donor, significantly inhibited VEGF-induced KDR up-regulation, indicating that NO negatively regulates KDR expression. In conclusion, our data demonstrate that VEGF binding to the KDR receptor tyrosine kinase results in an increase in KDR receptor gene transcription and protein expression. Thus, KDR up-regulation induced by VEGF may represent an important positive feedback mechanism for VEGF action in tumor and ischemia-induced angiogenesis.
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PMID:Homologous up-regulation of KDR/Flk-1 receptor expression by vascular endothelial growth factor in vitro. 979 18

Enterocyte differentiation occurs along the crypt-villus axis and is generally thought to involve the transcriptional activation of cell-specific genes, among which is the brush-border structural protein villin. We have examined the molecular mechanisms of villin induction using both in vivo and in vitro systems. Total RNA was purified from rat tissues or cultured cells by the guanidinium thiocyanate method and Northern blot analyses carried out using radiolabeled complementary DNA probes specific for villin or the actin control. Transient transfection (calcium/phosphate method) assays were performed using a luciferase reporter gene containing 2 kb of the human villin gene 5'-flanking region. We have found that the villin mRNA was expressed at high levels in the small intestine, to a lesser degree in the colon, and was not detected in the brain or liver. In HT-29 cells, villin mRNA levels increased 2.5-fold (P<0.001) after 24 hours of sodium butyrate treatment, consistent with the process of enterocyte differentiation. Similarly, villin gene expression was induced in Caco-2 cells during postconfluence differentiation. Transient transfection assays demonstrated marked reporter gene activation (fourfold, P<0.001) in response to sodium butyrate in HT-29 cells, but no activation in the liver cell line HepG2. The effects of sodium butyrate were dose dependent, reaching a maximum at a concentration of 5 mmol/L. We conclude that a 2 kb region of the human villin gene is able to mediate its transcriptional activation during HT-29 cell differentiation. This DNA regulatory region appears to function in a cell type-specific (gut) manner.
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PMID:Transcriptional activation of the human villin gene during enterocyte differentiation. 983 75

Three heterologous promoters (hsp70 and actin 5C from Drosophila melanogaster and IE1 from the immediate early gene of the Bombyx mori baculovirus) were assessed for their ability to drive transient luciferase expression in mosquito cells. Overall, the actin 5C promoter was considerably more effective at driving luciferase expression than either hsp70 or IE1 in cell lines derived from Anopheles, Aedes and Culex species. hsp70 functioned well when induced by heat shock and was also induced to a lesser extent by chemicals such as sodium arsenite. IE1 was also an effective initiator of transcription, particularly in two Anopheles cell lines, but generally it performed less well than the actin 5C promoter and was also outperformed by hsp70 in Anopheles gambiae cells.
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PMID:Comparative analysis of promoters for transient gene expression in cultured mosquito cells. 992 72


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