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:1.6.99.6 (NADPH oxidase)
10,295 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Interleukin-15 (IL-15) is a newly described cytokine that shares biological activities with IL-2. We report here results demonstrating the ability of IL-15 to enhance superoxide production and antifungal activity of human monocytes. After 18 and 48 h of treatment with IL-15, human elutriated monocytes manifested enhanced superoxide production in response to either phorbol myristate acetate or opsonized Candida albicans blastoconidia. Similar results were obtained when monocytes were treated with IL-2, but to a lesser extent. Combination studies with IL-15 and IL-2 showed no additive or synergistic effects. Following incubation of monocytes with IL-15 for 18 h, there was no significant increase in mRNA transcripts for components of the NADPH oxidase complex, p40-phox, p47-phox, and gp91-phox, suggesting a posttranscriptional modulation of enhanced superoxide production. Antibodies against the gamma chain of the IL-2 receptor and, to a lesser extent, against the beta chain partially abrogated the IL-15-mediated enhanced superoxide production. Additionally, human monocytes showed enhanced killing activity against C. albicans after 18 h of incubation with IL-15 or IL-2, but this treatment did not enhance the ability of these cells to phagocytose the organism. In addition, the enhanced fungicidal activity seen after 18 h of treatment was no longer detectable after 48 h of cytokine treatment. Culture supernatants from the IL-15-treated monocytes were assayed for the presence of other proinflammatory cytokines. IL-15 treatment did not induce the release of detectable levels of tumor necrosis factor alpha, IL-1beta, or IL-12. Our results indicate that IL-15 upregulates the microbicidal activity of human monocytes against C. albicans.
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PMID:Interleukin-15 augments superoxide production and microbicidal activity of human monocytes against Candida albicans. 942 51

Like neutrophils, Epstein-Barr virus (EBV)-immortalized B lymphocytes express all constituents of the NADPH oxidase complex necessary to generate superoxide anion O2-. The NADPH oxidase activity in EBV-B lymphocytes is only 5% of that measured in neutrophils upon PMA stimulation. Cytochrome b558 is the sole redox membrane component of NADPH oxidase; it is the protein core around which cytosolic factors assemble in order to mediate oxidase activity. In the present study, we have compared the structural and functional properties of cytochrome b558 from EBV-B lymphocytes and neutrophils. Cytochrome b558 from EBV-B lymphocyte plasma membrane, like that from neutrophils, is characterized by a heterodimeric structure with a highly glycosylated beta subunit, known as gp91-phox. While the amount of cytochrome b558 recovered after purification from EBV-B lymphocytes (approximately 0.24 nmol from 1010 cells) was low compared to that recovered from neutrophils (approximately 10 nmol), the biochemical properties of purified cytochrome b558 from both EBV-B lymphocytes and neutrophils were quite similar with respect to their differential spectra, redox potential, and FAD binding site. Once cytochrome b558 was extracted from the EBV-B lymphocyte membrane, it was able to mediate, in a reconstituted system of O2- production the same oxidase turnover as that found for cytochrome b558 extracted from neutrophils. A comparison between membrane bound and soluble cytochrome b558 suggested that the weak oxidase activity measured in intact EBV-B cells might be the result not only of the small amount of expressed cytochrome b558, but also of a defect of the activation process in lymphocyte membrane.
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PMID:Biochemical and immunochemical properties of B lymphocyte cytochrome b558. 957 61

While oxygen free radicals are important mediators of brain injury, questions remain regarding which cell types and enzyme pathways trigger this radical generation. Microglial cells have been hypothesized to be an important source of radical generation; however, the magnitude, kinetics, and mechanism of this process are unknown. Oxygen radical generation by stimulated primary microglia was directly measured and characterized by electron paramagnetic resonance spin trapping. Microglia, when stimulated by phorbol ester or opsonified zymosan, gave rise to EPR spectra characteristic of superoxide. Experiments performed in the presence of superoxide dismutase, catalase, deferoxamine, and dimethyl sulfoxide excluded generation of hydroxyl radicals in significant amounts. Microglial superoxide generation was blocked by the NADPH oxidase inhibitor diphenylene iodonium in a manner similar to that seen in neutrophils, suggesting that a neutrophil like NADPH oxidase was the source of superoxide production. However, microglia produced 20 to 40 times less superoxide compared to a similar number of neutrophils during the first 30 min following stimulation, indicating a marked difference in the regulation of NADPH oxidase activation. Western blots of microglia lysates demonstrated that both large (gp91-phox) and small (p22-phox) NADPH oxidase subunits are expressed in both unstimulated and stimulated microglia. Indirect immunofluorescence demonstrated localization at the membrane surfaces of activated cells. Thus, microglial cells generate superoxide via a neutrophil-like NADPH oxidase but exhibit distinctly different time course and magnitude of activation than that seen in neutrophils.
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PMID:Measurement and characterization of superoxide generation in microglial cells: evidence for an NADPH oxidase-dependent pathway. 960 65

Neutrophils play an essential role in the cellular defense of the bovine mammary gland and compromised leukocyte function has been linked to the development of bovine mastitis. During mastitis, large numbers of leukocytes migrate into the mammary tissues where they become activated, resulting in the assembly of neutrophil membrane and cytosolic proteins to form a superoxide anion-generating complex known as the NADPH oxidase. The key membrane-associated component of the NADPH oxidase is flavocytochrome b, which is a heterodimer of p22-phox and gp91-phox. Currently, only the human, porcine, murine, and rattus p22-phox and the human, porcine, and murine gp91-phox gene sequences are known. Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and analyze expression of bovine flavocytochrome b. Using polymerase chain reaction cloning techniques and a bovine spleen cDNA library we have cloned both of the bovine flavocytochrome b subunits, p22-phox and gp91-phox. Comparison of the bovine sequences with those of other species also revealed important information regarding key structural features of gp91-phox and p22-phox, including location of putative glycosylation sites. This study greatly contributes to our understanding of the potential functional sites of the flavocytochrome b subunits as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases such as mastitis.
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PMID:Cloning and sequencing of the bovine flavocytochrome b subunit proteins, gp91-phox and p22-phox: comparison with other known flavocytochrome b sequences. 966 85

Chronic granulomatous disease (CGD) is a disorder of the lymphohematopoietic system, whereby phagocytes of affected patients are unable to kill microorganisms. CGD is caused by a functional defect in the phagocytic nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase (phox) enzyme complex, leading to a lack of microbicidal metabolites. As a therapeutic approach toward the predominant X-linked form of CGD, we have developed a bicistronic retroviral vector containing the coding sequences of gp91-phox and a cytoplasmically truncated version of the human low-affinity receptor for nerve growth factor (deltaLNGFR). Full reconstitution of superoxide-generating activity was achieved with this vector in a gp91-phox-deficient cell line. Using an optimized gene transfer protocol, up to 85% of the CD34+ cells obtained from the bone marrow of X-CGD patients were transduced. CD15+ cells differentiated in vitro from transduced X-CGD CD34+ cells showed correction of NADPH oxidase activity to 45-52% of normal levels whereas deltaLNGFR expression was found in 40-67% of the CD15+ cells. Moreover, immunoblots prepared from extracts of transduced CD15+ cells revealed gp91-phox protein levels similar to those found in neutrophils derived from normal CD34+ cells. Taking into consideration that superoxide production in only 5 to 10% of wild-type neutrophils is sufficient to protect X-CGD heterozygotes from serious infections, the results achieved in this study shows that for X-CGD patients a curative approach based on the genetic modification of hematopoietic stem/progenitor cells is feasible.
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PMID:Correction of respiratory burst activity in X-linked chronic granulomatous cells to therapeutically relevant levels after gene transfer into bone marrow CD34+ cells. 969 55

We investigated the effects of dexamethasone or indomethacin on the NADPH oxidase activity, cytochrome b558 content, and expression of genes encoding the components gp91-phox and p47-phox of the NADPH oxidase system in the human monocytic THP-1 cell line, differentiated with IFN-gamma and TNF-alpha, alone or in combination, for up to 7 days. IFN-gamma and TNF-alpha, alone or in combination, caused a significant up-regulation of the NADPH oxidase system as reflected by an enhancement of the PMA-stimulated superoxide release, cytochrome b558 content, and expression of gp91-phox and p47-phox genes on both days 2 and 7 of cell culture. Noteworthy was the tremendous synergism between IFN-gamma and TNF-alpha for all studied parameters. Dexamethasone down-regulated the NADPH oxidase system of cytokine-differentiated THP-1 cells as assessed by an inhibition on the PMA-stimulated superoxide release, cytochrome b558 content, and expression of the gp91-phox and p47-phox genes. The nuclear run-on assays indicated that dexamethasone down-regulated the NADPH oxidase system at least in part by inhibiting the transcription of gp91-phox and p47-phox genes. Indomethacin inhibited only the PMA-stimulated superoxide release of THP-1 cells differentiated with IFN-gamma and TNF-alpha during 7 days. None of the other parameters was affected by indomethacin. We conclude that dexamethasone down-regulates the NADPH oxidase system at least in part by inhibiting the expression of genes encoding the gp91-phox and p47-phox components of the NADPH oxidase system.
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PMID:Dexamethasone but not indomethacin inhibits human phagocyte nicotinamide adenine dinucleotide phosphate oxidase activity by down-regulating expression of genes encoding oxidase components. 979 32

We have used a human leukemia cell line that, after homologous recombination knockout of the gp91-phox subunit of the phagocyte respiratory-burst oxidase cytochrome b-558, mimics chronic granulomatous disease (X-CGD) to study the role of oxygen radicals in apoptosis. Camptothecin (CPT), a topoisomerase I inhibitor, induced significantly more apoptosis in PLB-985 cells than in X-CGD cells. Sensitivity to CPT was enhanced after neutrophilic differentiation, but was lost after monocytic differentiation. No difference between the two cell lines was observed after treatment with other apoptosis inducers, including etoposide, ultraviolet radiation, ionizing radiation, hydrogen peroxide, or 7-hydroxystaurosporine. After granulocytic differentiation of both cell lines, CPT still induced apoptosis, suggesting independence from replication in fully differentiated and growth-arrested cells. Pyrrolidine dithiocarbamate (an antioxidant inhibitor of NF-kappaB) and catalase partially inhibited CPT-induced DNA fragmentation in granulocytic-differentiated PLB-985 cells, but had no effect in X-CGD cells. Flow cytometry analysis revealed that reactive oxygen intermediates were generated in CPT-treated PLB-985 cells. These data indicate that oxygen radicals generated by NADPH oxidase may contribute directly or indirectly to CPT-induced apoptosis in human leukemia and in neutrophilic-differentiated cells.
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PMID:Role of oxygen radicals generated by NADPH oxidase in apoptosis induced in human leukemia cells. 983 21

We investigated the NADPH oxidase activity, cytochrome b558 content, and gene expression of gp91-phox and p47-phox in normal Epstein-Barr-virus (EBV)-transformed B lymphocytes, compared to EBV-transformed B lymphocytes from patients with X-linked chronic granulomatous disease (CGD), normal peripheral blood neutrophils or mononuclear cells, and the A301 or C8166 lymphoblastoid cell lines. CGD phenotypes included both "classic" disease with no detectable gp91-phox protein (termed X91(0)) and "variant" phenotype with reduced but detectable gp91-phox protein (X91(-)). Normal EBV-transformed B lymphocytes show a dose-dependent PMA-induced superoxide release. Culturing these cells with IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml), alone or in combination for 7 days, caused a modest increase in their NADPH oxidase activity (P > 0.05 in all situations). Normal EBV-transformed B lymphocytes have lower NADPH oxidase activity and cytochrome b558 content than peripheral blood neutrophils or mononuclear cells (P < 0.05 in all situations). In contrast, they have higher NADPH oxidase activity and cytochrome b558 content than X91(-) CGD EBV-transformed B lymphocytes (P < 0.05 in all situations). A301 or C8166 lymphoblastoid cell lines and X91(0) CGD EBV-transformed B lymphocytes have barely detectable NADPH oxidase activity or cytochrome b558 content (P < 0.05 in all situations). Gene expression studies also show a modest increase in expression and transcription rates of gp91-phox and p47-phox genes in normal EBV-transformed B cells cultured with IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml), alone or in combination for 7 days. We conclude that NADPH oxidase activity and cytochrome b558 content correlate with gp91-phox and p47-phox gene expression in EBV-transformed B lymphocytes.
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PMID:NADPH oxidase activity and cytochrome b558 content of human Epstein-Barr-virus-transformed B lymphocytes correlate with expression of genes encoding components of the oxidase system. 985 26

A membrane-bound cytochrome b558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the superoxide (O2-)-generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. Chronic granulomatous disease (CGD) is characterized by recurrent bacterial infection caused by a defect of the oxidase. Both subunits are absent from phagocytes in typical X-linked recessive CGD patients who are primarily defective in gp91-phox. We report here an atypical case of X-linked CGD in which neutrophils showed a complete absence of O2--forming NADPH oxidase activity, but a small amount (about 10% of control) of both subunits was detected by immunoblot analysis. Spectrophotometric studies of the neutrophils with a recently developed sensitive method gave no evidence for the heme spectrum in the cytochrome b558, of this CGD. Reverse transcription/polymerase chain reaction and sequence analysis revealed a C to T transition replacing histidine at amino acid position 101 (His101) by tyrosine in gp91-phox. These results provide evidence that His101 of gp91-phox is the one of the heme-binding ligands of cytochrome b558.
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PMID:A novel mutation at a probable heme-binding ligand in neutrophil cytochrome b558 in atypical X-linked chronic granulomatous disease. 985 76

Chronic granulomatous disease (CGD) is an uncommon inherited disorder of phagocytic cells in which a defective respiratory burst leads to severe recurrent bacterial and fungal infections. The disease is a consequence of mutations in one of the four molecules that constitute the NADPH oxidase system of electron transport, whose most critical component is an unusual flavocytochrome b localized in the plasma and specific granule membranes. Mutations in the CYBB gene (localized in the short arm of the X chromosome) encoding the beta-subunit of this flavocytochrome (gp91-phox), which is are responsible for 60-65% of all cases of CGD. In this paper, we report the molecular characterization of seven unrelated kindreds native from Colombia and Brazil with CGD caused by gp91-phox deficiency. The exons with the possible mutation were identified by single-strand conformational polymorphism (SSCP) of genomic DNA and then confirmed by DNA sequencing. In one patient we found a substitution of A to G in the penultimate nucleotide of intron 12 (IVS12-2A-->G). In four other cases, four different nonsense mutations were detected: R91X, W106X, R157X, and R290X and the other two patients showed missense substitutions: E225V and C244Y. In six of these kindreds, all mothers were carriers but one that did not present any change in the gp91-phox gene, which indicates a de novo mutation in this kindred. Then, these family-specific mutations in gp91-phox produce different structural defects that alter the expression or function of an essential component of phagocyte oxidase.
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PMID:Molecular analysis of chronic granulomatous disease caused by defects in gp91-phox. 988 86


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