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:2.7.11.13 (protein kinase C)
49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Seleno-glutathione peroxidase (GSHPx) is considered to be the major enzymatic activity in charge of removing excess cytosolic and mitochondrial H2O2 in most tissues including brain. Intracellular GSHPx activity is therefore hypothesized to be one important factor that contributes to minimize hydroxyl radical formation via Fenton-type reactions. An animal model was developed to challenge this hypothesis in vivo and evaluate the role of GSHPx in hydroperoxide metabolism and oxidative stress homeostasis. Three lines of transgenic mice, homozygous for the integration of 1 to 3 GSHPx transgene copies, have been generated. The transgene was placed under transcriptional control of a metallothionein promoter (hMT-IIA). This promoter was chosen because metallothionein expression, normally low in most tissues, can be induced by several inflammatory cytokines, protein kinase C activators, and stress agents including heavy metals. The data reported here provide information on the constitutive expression of GSHPx mRNA and enzyme in various brain regions of healthy untreated adult tg-MT-GPx mice. Northern and/or Western analysis indicated that transgenic GSHPx was expressed constitutively in all brain regions investigated in tg-MT-GPx-6 mice, including the cerebral cortex, brainstem, hippothalamus, cerebellum, substantia nigra, and striatum. Similar results were obtained with the two other transgenic lines, tg-MT-GPx-11 and -13. Depending on the brain region, the GSHPx immunoreactivity detected in tissue extracts with an immunoaffinity-purified polyclonal antibody was about 2- to 5-fold stronger in transgenic extracts than in their non-tg counterparts (western blots). In contrast, the corresponding increase in GSHPx activity measured in these extracts was smaller, for example, about 1.5-fold in transgenic mesencephalon. Immunocytochemical data indicated that GSHPx-like staining was distinctly more intense in transgenic midbrain brain sections than in corresponding non-tg sections. Interestingly, only a subset of the cells displayed higher density staining that most likely reflects increased amounts of GSHPx protein. This observation suggests that the stained cells, not yet identified, may have larger GSHPx activity increments than the cell-average increments measured in tissue extracts. Current work is in progress to determine whether transgenic GSHPx expression may be induced by inflammatory processes or perturbations of heavy metal metabolism.
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PMID:Transgenic glutathione peroxidase mouse models for neuroprotection studies. 783 20

We have reported previously that hydrogen peroxide induces arachidonic acid release from prelabeled vascular smooth muscle cells. Here, we studied the effect of hydrogen peroxide on the phosphorylation of cytosolic phospholipase A2 in these cells. Hydrogen peroxide induced a rapid, time-dependent increase in the phosphorylation of cytosolic phospholipase A2. Hydrogen peroxide also increased arachidonic acid release from prelabeled cells in a time-dependent manner similar to that of phosphorylation of cytosolic phospholipase A2. Protein kinase C depletion significantly inhibited the hydrogen peroxide-stimulated cytosolic phospholipase A2 phosphorylation and arachidonic acid release. Hydrogen peroxide caused a time-dependent increase in mitogen activated protein kinase activity. Taken together, these findings suggest that cytosolic phospholipase A2 may, at least in part, contribute to arachidonic acid release induced by hydrogen peroxide and this effect appears to be mediated by protein kinase C and mitogen activated protein kinase.
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PMID:Hydrogen peroxide activation of cytosolic phospholipase A2 in vascular smooth muscle cells. 785 86

To examine the effects of oxidants on the airway epithelial barrier functions, human tracheal epithelial cells were cultured on porous filter membrane. Glucose oxidase (GO; 10 U/ml), hydrogen peroxide (H2O2; 4 x 10(-3) M), and xanthine (5 x 10(-4) M) plus xanthine oxidase (20 mU/ml) (X-XO) significantly increased electrical conductance across epithelial membrane (G), short-circuit current (Isc) measured with Ussing's chamber methods, and [3H]mannitol flux through the cultured epithelium. Increases in G and Isc induced by oxidants were significantly inhibited by catalase (1,000 U/ml) and the protein kinase C inhibitor staurosporine (10(-7) M), but superoxide dismutase (SOD; 100 U/ml) was without effect. GO, H2O2, and X-XO inhibited the epithelial cell growth, [3H]thymidine incorporation by the cells, and epithelial repair of artificially produced focal epithelial defects (1-2 mm diam) on plastic vessels. Catalase also inhibited effects induced by oxidants on cell growth and proliferation. These results suggest that oxidants reduce tracheal epithelial barrier functions by damaging tight junctions and inhibiting cell proliferation, and these effects of oxidants on epithelial cells may be mediated by H2O2 rather than superoxide anion and by activation of protein kinase C.
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PMID:Oxidants affect permeability and repair of the cultured human tracheal epithelium. 786 48

Free radical formation and subsequent lipid peroxidation may participate in the pathogenesis of tissue injury, including the brain injury induced by hypoxia or trauma and cardiac injury arising from ischemia and reperfusion. However, the exact cellular mechanisms by which the initial oxidative insult leads to the ultimate tissue damage are not known. A number of reports have indicated that protein kinase C (PKC) may be activated following oxidative stress and that this enzyme may play an important role in the steps leading to cellular damage. In this work, we have examined in a cell model whether PKC is activated following oxidative exposure. UC11MG cells, a human astrocytoma cell line, were treated with H2O2. Incubation with 0.5 mM H2O2 increased malondialdehyde levels by as early as 15 minutes. To assess the effects of H2O2 treatment on PKC activation, we measured phosphorylation of an endogenous PKC substrate, the MARCKS (myristoylated alanine-rich C kinase substrate) protein. Treatment of cells with 0.2-1.0 mM H2O2 resulted in a rapid increase in MARCKS phosphorylation. Phosphorylation was stimulated approximately 2.5-fold following treatment with 0.5 mM H2O2 for ten minutes. Treatment with phorbol 12-myristate 13-acetate, a PKC activator, increased MARCKS phosphorylation approximately 4-fold. The H2O2-induced MARCKS phosphorylation was inhibited by the addition of the kinase inhibitors H-7 and staurosporine. Furthermore, specific down-regulation of PKC by phorbol ester also inhibited H2O2-induced MARCKS phosphorylation. These results indicate that PKC is rapidly activated in cells following an oxidative exposure and that this cell system may be a good model to further investigate the role of PKC in regulating oxidative damage in the cell.
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PMID:Oxidant-induced activation of protein kinase C in UC11MG cells. 788 45

Reactive oxygen intermediates (ROIs) have been found to be the messengers in the activation of the kappa B transcription regulator in mitogen- or cytokine-stimulated cells, operating in conjunction with or independently of various other mechanisms; these include Ca(++)-dependent and PKC-dependent cytoplasmic signaling pathways. We have recently reported that low-dose ionizing radiation induces NF-kappa B in human lymphoblastoid 244B cells. Since ionizing radiation generates free radicals in cells, we have investigated whether the ROIs generated by ionizing radiation induce NF-kappa B activity, and also whether they do so by a similar mechanism as in cells treated with PMA or H2O2. The results not only confirm a previous observation from our laboratory that low-dose ionizing radiation (0.1-2.0 Gy) activates kappa B transcription factor transiently with a maximal induction at 0.5 Gy exposure, but also demonstrate mechanistically that the activation of NF-kappa B by low-dose ionizing radiation can be inhibited considerably by the antioxidant N-acetyl-L-cysteine, indicating that at least the major part of the activation process is mediated by ROIs. These findings support the idea that ROIs can regulate the kappa B elements which in turn can serve as response elements for oxidant stress.
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PMID:Induction of nuclear factor kappa B after low-dose ionizing radiation involves a reactive oxygen intermediate signaling pathway. 793 61

The phospholipase D (PLD)-mediated synthesis of phosphatidylethanol (PtdEtOH) and the hydrolysis of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) were examined in drug-sensitive and multidrug-resistant lines of MCF-7 human breast carcinoma cells. In drug-sensitive (MCF-7/WT) cells, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) failed to enhance either the synthesis of PtdEtOH or the hydrolysis of either phospholipid. In the drug-resistant (MCF-7/MDR) cells, 100 nM PMA greatly enhanced both the synthesis of PtdEtOH (approximately 21-fold) and the hydrolysis of PtdEtn (approximately 29-fold), but had no effect on the hydrolysis of PtdCho. The PLD activators sphingosine and H2O2 were found to elicit only a slight (1.28-1.4-fold) stimulatory effect on PtdCho hydrolysis in both the MCF-7/WT and MCF-7/MDR cell types, and had only a small effect on PtdEtn hydrolysis in the MCF-7/WT cells as well. However, these agents significantly (approximately 2.6-3.5-fold) stimulated PtdEtn hydrolysis in the MCF-7/MDR cells. These data indicate that MCF-7/MDR cells contain a PtdEtn-specific PLD activity which can be selectively stimulated by PMA, sphingosine and H2O2.
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PMID:Phorbol ester selectively stimulates the phospholipase D-mediated hydrolysis of phosphatidylethanolamine in multidrug-resistant MCF-7 human breast carcinoma cells. 794 88

We have shown that hyperoxic exposure of immature rats induces airway smooth muscle layer thickening and cell turnover parallel to that found in the airways of patients with bronchopulmonary dysplasia and chronic, severe asthma. We hypothesized that reactive oxygen species could promote the observed airway remodeling by directly stimulating signal transduction pathways that regulate cell growth. To test this hypothesis in cultured cells, we assessed the effects of hydrogen peroxide (H2O2) on mitogen-activated protein (MAP) kinase activation in bovine tracheal myocytes. The MAP kinases are a family of 40 to 46 kD cytosolic serine/threonine kinases that participate in the transduction of mitogenic signals to the cell nucleus. Quiescent cells were exposed to H2O2 (25 to 200 microns; 2 to 60 min), after which SDS-PAGE of cell extracts was performed. Western analysis using an anti-MAP kinase antiserum revealed a decrease in the mobility of the 42 and 44 kD MAP kinase bands after H2O2 exposures of 5 to 30 min, reflecting the phosphorylation at threonine and tyrosine residues required for enzymatic activity. MAP kinase activation was demonstrated by kinase renaturation assays, which showed an almost 4-fold increase in 42 and 44 kD MAP kinase activity. Down-regulation of protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu) partially reduced H2O2-stimulated MAP kinase activity, suggesting that H2O2 induces MAP kinase activation via both PKC-dependent and PKC-independent pathways. Western analysis using a phosphotyrosine monoclonal antibody revealed increased tyrosine phosphorylation of proteins with approximate molecular weights of 72 and 125 kD after H2O2 exposure, demonstrating that H2O2 can stimulate the tyrosine phosphorylation of multiple cytosolic proteins, including MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hydrogen peroxide stimulates mitogen-activated protein kinase in bovine tracheal myocytes: implications for human airway disease. 794 86

Human antioxidant-response element (hARE) containing two copies of the AP1/AP1-like elements arranged as inverse repeat is known to mediate basal and beta-naphthoflavone-induced transcription of the type 1 NAD(P)H:quinone oxidoreductase (NQO1) gene. Band-shift assays revealed that beta-naphthoflavone increased binding of nuclear proteins at the hARE. Super shift assays identified Jun-D and c-Fos proteins in the band-shift complexes observed with control and beta-naphthoflavone-treated Hepa-1 nuclear extracts. Hepa-1 cells stably transformed with hARE-tk-chloramphenicol acetyl transferase (CAT) recombinant plasmid were used to demonstrate that, in addition to beta-naphthoflavone, a variety of antioxidants, tumor promoters and hydrogen peroxide (H2O2) also increased expression of hARE-mediated CAT gene. beta-naphthoflavone induction of the CAT gene expression in Hepa-1 cells was found insensitive to inhibitors of protein kinase C and tyrosine kinases. However, binding of regulatory proteins at the hARE and the CAT gene expression in Hepa-1 cells were increased by dithiothreitol, 2-mercaptoethanol and diamide. Treatment of the Hepa-1 cells with N-ethylmaleimide reduced binding of proteins at the hARE and interfered with expression and beta-naphthoflavone induction of the CAT gene. These results suggested a role of sulfhydryl modification of hARE binding (Jun and Fos) proteins which mediate basal and induced expression of the NQO1 gene. We also report that in-vitro-translated products of the proto-oncogenes, Jun and Fos, bind to the hARE in band-shift assays. The incubation of Jun and Fos proteins with small amounts of nuclear extract from dimethylsulfoxide-treated (control) or beta-naphthoflavone treated Hepa-1 cells prior to band-shift assays increased the binding of Jun and Fos proteins to the hARE. Interestingly, the increase in binding of Jun and Fos proteins to the hARE was more prominent with beta-naphthoflavone-treated nuclear extract as compared to the control nuclear extract. In addition, incubation of control nuclear extract with beta-naphthoflavone, microsomes and NADPH increased the binding of Jun and Fos proteins to the hARE. Evidence from in vitro studies indicate the presence of unknown nuclear factor(s) that receive signals from metabolites of beta-naphthoflavone and modulate Jun and Fos binding to the AP1 site contained within the hARE.
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PMID:Human antioxidant-response-element-mediated regulation of type 1 NAD(P)H:quinone oxidoreductase gene expression. Effect of sulfhydryl modifying agents. 795 57

Using a sensitive flow cytometric assay, which measures the intracellular oxidation of 2'7' dichlorofluorescein (DCFH) by H2O2, we have assessed, at a single-cell level, the effects of a variety of physiological priming agonists and cytochalasin B (CB) on purified populations of neutrophils stimulated at different points along the signal response transduction pathway. Pretreatment of purified neutrophils with the physiological priming agonists monocyte interleukin-8 (IL-8), granulocyte-monocyte colony-stimulating factor (GM-CSF), platelet-activating factor (PAF), IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6, and non-stimulatory doses of formyl-methionyl-leucyl-phenylalanine (FMLP), resulted in an increased percentage of cells generating an oxidative burst in response to subsequent receptor stimulation with FMLP. CB had a similar but much more pronounced effect on cellular recruitment to a receptor-mediated responsive state. Activation of protein kinase C (PKC) using the phorbol ester phorbol myristate acetate (PMA) resulted in a heterogeneous response, with all cells generating H2O2, but with two populations differing in their magnitude of response. Physiological priming agonists had no effect on the heterogeneity of the PMA response. However, pretreatment with CB dramatically altered the PMA response, producing a homogeneous population highly responsive to stimulation with PKC. In contrast, direct stimulation of G proteins with fluoride (A1F-4) was primed both by physiological priming agonists and by CB. These results demonstrate that priming of neutrophils by physiological agonists involves changes at the level of signal transduction which enable a previously non-responsive cell to respond to a secondary stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Priming of the oxidative burst in human neutrophils by physiological agonists or cytochalasin B results from the recruitment of previously non-responsive cells. 795 84

The effects of hydrogen peroxide D-alpha-tocopherol and of D-beta-tocopherol on proliferation, protein kinase C and activator protein-1 (AP-1) activation have been studied in vascular smooth muscle cells. Cell proliferation, when activated by foetal calf serum, was inhibited by D-alpha-tocopherol. Protein kinase C activity was stimulated by hydrogen peroxide in a manner similar to phorbol myristate acetate; in the latter case, but not in the former, D-alpha-tocopherol inhibited the reaction. Hydrogen peroxide prevented phorbol-myristate-acetate-stimulated AP-1 binding to DNA but stimulated it if protein kinase C was down-regulated or inhibited. D-alpha-Tocopherol promoted AP-1 activation in quiescent cells but prevented its activation by phorbol myristate acetate. None of the described effects of D-alpha-tocopherol were shared by D-beta-tocopherol, suggesting a non-antioxidant mechanism as the basis of its action. The data show that hydrogen peroxide and D-alpha-tocopherol affect more than one element in the cell signal-transduction cascade.
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PMID:Modulation of activator protein-1 (AP-1) transcription factor and protein kinase C by hydrogen peroxide and D-alpha-tocopherol in vascular smooth muscle cells. 800 57


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