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:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Proinflammatory cytokines prime the membrane-bound NADPH oxidase of neutrophils and monocytes of mice suffering from experimental arthritis so as to attain an activated state, which, upon a second stimulus, releases 6-fold increased levels of reactive oxygen species (ROS) than do unprimed phagocytes. Enhanced NADPH oxidase activity deregulates ROS-dependent signal transduction pathways of inflammation, which play a crucial role in the pathogenesis of arthritis. The antiarthritic reactivity of two inhibitors of NADPH oxidase, diphenylene iodoniumchloride (DPI) and staurosporine, was tested in male DBA/1 x B10A(4R) hybrid mice suffering from potassium peroxochromate arthritis. Daily doses of 2.8 mumol/kg of DPI or 30 nmol/kg of staurosporine sufficed to inhibit the arthritis by 50%. A complete inhibition was obtained with 10 mumol/kg of DPI, and 100 nmol/kg of staurosporine suppressed the arthritis by 85%. The onset, progression, and remission of arthritis correlated to both the activity of phagocytic NADPH oxidase (r = 0.750) and to overt disease symptoms as judged by the arthritis index. Our data support the hypothesis that oxidative stress plays a pivotal role in the pathology of arthritis, which can be therapeutically targeted by NADPH oxidase inhibitors.
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PMID:Antiinflammatory effects of NADPH oxidase inhibitors. 762 63

Neutrophil NADPH oxidase is a membrane-bound enzyme complex responsible for the reduction of oxygen to superoxide anion in the respiratory burst. Impaired neutrophil function has often been reported in myeloproliferative diseases and myelodysplastic syndromes (MDSs), but its laboratory features have not been well characterized. Traditionally, NADPH oxidase activity has been evaluated with a microscopic method by nitroblue tetrazolium salt reduction to blue-black insoluble formazan granules identified in positive neutrophils by microscopy. We investigated neutrophil NADPH oxidase in 22 patients with chronic myeloproliferative disorders (cMPDs) and 15 patients with MDSs, using the microscopic method as well as a photometric microplate assay, which monitored the cytochemical reaction for 30 min. The relationship between cMPD and patient susceptibility to infections was also investigated. In the photometric assay, the mean enzyme activity in MDSs and cMPDs was lower than in normal subjects. NADPH oxidase activity was greater in cMPDs (except myelofibrosis) than in MDSs (except chronic myelomonocytic leukemia), and these differences were less evident with microscopy. Moreover, for cMPDs, patients with susceptibility to infections showed a lower NADPH oxidase activity than patients without infections.
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PMID:Neutrophil NADPH oxidase activity in chronic myeloproliferative and myelodysplastic diseases by microscopic and photometric assays. 892 99

Detergent-mediated activation of the phagocyte superoxide-generating NADPH oxidase requires the participation of at least four proteins: the membrane-bound heterodimeric cytochrome b558 and three cytosolic components, p47-phox, p67-phox and a Rac1/Rac2 protein. Peptides corresponding to sequences of different subunits of NADPH oxidase have been used as probes of the mechanism and sequence of assembly of the active complex. In the present study effects of mastoparans on activation of NADPH oxidase were investigated. Mastoparans are wasp venom cationic amphiphilic tetradecapeptides capable of modulation of various cellular activities. Natural mastoparans, as well as several synthetic mastoparan analogues, unrelated to oxidase components, blocked activation of the oxidase in the cell-free system (EC50 = 1.5 microM) and in guanosine 5'-[gamma-thio]triphosphate (GTP[S])/ATP-stimulated neutrophils permeabilized with streptolysin O. In the cell-free system the effect was not relieved by raising the detergent concentration and could not be ascribed to changes in critical micellar concentration values of the activating SDS or arachidonate. Chromatography of neutrophil cytosol on an immobilized mastoparan column suggested interaction of cytosolic p47-phox and p67-phox with the peptide. In spite of this interaction mastoparan did not interfere with translocation of p47-phox and p67-phox to the cell membranes.
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PMID:The assembly of neutrophil NADPH oxidase: effects of mastoparan and its synthetic analogues. 765 16

Activation of the human NADPH oxidase requires the interaction of at least four cytosolic proteins and one membrane-bound heterodimeric protein. Src homology 3 (SH3) domains and their proline-rich counterstructures have been shown to play an important role in protein-protein interactions. Because it was found that the cytosolic oxidase components p67phox, p47phox, and p40phox reside in a complex in resting neutrophils, we studied the role of SH3 domains in their interaction by use of an overlay technique. Wild-type and mutated 35S-labeled p67phox and p47phox were used to detect immobilized cytosolic proteins on a protein blot. A specific association of native p67phox to blotted p47phox and blotted p40phox was found. These interactions were not disturbed by deleting the only proline-rich region (amino acids 227-231) in p67phox. We also found a specific association of native p47phox with blotted p67phox. Deletions in a putative SH3-binding region of p47phox completely abrogated the interaction with p67phox. Other results suggest that the C terminus of p47phox exposes this SH3-binding domain for interaction with p67phox. Similar results were obtained when the binding of cytosolic p67phox to wild-type or mutated p47phox were studied in solution. Interestingly, mutants of p47phox unable to bind to p67phox were fully capable of supporting superoxide production under cell-free activation conditions. We conclude that an interaction between the C-terminal proline-rich region of p47phox and the second SH3 domain of p67phox is not required for oxidase activity in the cell-free assay.
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PMID:Interactions between the cytosolic components p47phox and p67phox of the human neutrophil NADPH oxidase that are not required for activation in the cell-free system. 774 54

Chronic granulomatous disease (CGD) is due to a functional defect of the O2(-)-generating NADPH oxidase of neutrophils. Mutations resulting in CGD have been shown to occur in only four genes, thus identifying the main components of the oxidase complex, namely the two subunits of a membrane-bound cytochrome b and two cytosolic factors of activation of 67 kD (p67phox) and 47 kD (p47phox). The present study deals with the biochemical and genetic analysis of the defect in a patient suffering from a p67phox-deficient form of CGD. The p67phox deficiency was ascertained by immunochemistry and the ability of recombinant p67phox to restore NADPH oxidase activity using a cell-free system of oxidase activation. The cellular extracts from the proband contained no p67phox protein and no p67phox mRNA when assayed by Western and Northern blot analysis. However, reverse transcription of mRNA and subsequent cDNA amplification by polymerase chain reaction using specific p67phox primers showed that trace amounts of a p67phox mRNA deleted for exon 3 were synthesized in the patient immortalized B lymphocytes. Sequence analysis of the genomic DNA showed a T-to-C transition at position +2 of intron 3. This point mutation in the consensus 5' splice site of the intron 3 was probably responsible for lack of accumulation of mRNA and also for the skipping of exon 3 detected in the few mRNA molecules that escaped cellular degradation.
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PMID:A mutation located at the 5' splice junction sequence of intron 3 in the p67phox gene causes the lack of p67phox mRNA in a patient with chronic granulomatous disease. 780 98

Blepharismin is an endogenous photosensitizing pigment found in the protozoan Blepharisma. This pigment inhibited the generation of superoxide anion (O2-.) in neutrophils not only via a diacylglycerol-induced protein kinase C (PKC)-dependent reaction but also by an arachidonate-induced PKC-independent reaction. The inhibition was light and concentration dependent for both reactions. Light-activated inhibition was strong at wavelengths between 520 and 570 nm but not above 610 nm. PKC activity in neutrophils and from rat brain was inhibited by blepharismin in a light- and concentration-dependent manner. Moreover, arachidonate-activated NADPH oxidase activity in a cell-free system was also inhibited by the pigment in a light- and concentration-dependent manner. These results suggest that blepharismin inhibits NADPH oxidase activation through the non-specific inhibition of various membrane-bound enzymes and that this inhibition may also be correlated with that of PKC.
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PMID:Photoactivated inhibition of superoxide generation and protein kinase C activity in neutrophils by blepharismin, a protozoan photodynamically active pigment. 787 58

Chronic granulomatous disease (CGD) results from defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, central to which is the membrane-bound cytochrome b-245. The cytochrome is composed of two protein subunits, the larger (gp91-phox) being deficient in X-linked CGD. In this study, we have analyzed expression of the cytochrome subunits in B-cell lines from two autosomal CGD patients for whom the disease is caused by deficiency of p22-phox, the smaller subunit. We report the presence of a 65-kD precursor of gp91-phox in the membrane fraction of both p22-phox-deficient cell lines, corresponding to the core protein with N-linked carbohydrate side chains in the high mannose form. Expression of p22-phox in these cells resulted in functional correction of NADPH oxidase. In addition, gp91-phox in the reconstituted cells was processed to its terminally glycosylated form. These data suggest that the association of the 65-kD gp91-phox precursor with p22-phox is a prerequisite for processing of the carbohydrate side chains to the complex form in the Golgi. The detection of this precursor will enable characterization of mutations disrupting the subunit interaction (either naturally occurring or derived by in vitro mutagenesis) and so aid in structure-function analysis of cytochrome b-245. Reconstitution of p22-phox-deficient cells shows the potential of gene therapy for this autosomal form of CGD.
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PMID:p22-phox-deficient chronic granulomatous disease: reconstitution by retrovirus-mediated expression and identification of a biosynthetic intermediate of gp91-phox. 791 88

Professional phagocytes, neutrophils, possess a unique membrane-associated NADPH oxidase system, dormant in resting cells, which becomes activated upon exposure to the appropriate stimuli and catalyzes the one-electron reduction of molecular oxygen to superoxide, O2-. Oxidase activation involves the assembly, in the plasma membrane, of membrane-bound and cytosolic constituents of the oxidase system, which are disassembled in the resting state. The oxidase system consists of two plasma membrane-bound components; low-potential cytochrome b558, which is composed of two subunits of 22 kDa and 91 kDa, and a flavoprotein related to the electron transport between NADPH and heme-binding domains of the oxidase. Recent reports have indicated that FAD-binding sites of the oxidase are contained in cytochrome b558 (flavocytochrome b558). At least two cytosolic components, 67 kDa protein and a phosphorylated 47 kDa protein, are known to translocate to the plasma membrane, ensuring assembly of an active O2(-)-generating NADPH oxidase system. More recently, the membrane (Raps) and cytosolic (Racs) GTP-binding proteins have been established as essential to oxidase assembly. It is the purpose of this review to focus on recent data concerning the regulatory mechanisms which lead to organization and activation of the neutrophil NADPH oxidase system.
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PMID:Activation factors of neutrophil NADPH oxidase complex. 801 44

Rho-related proteins are members of the ras superfamily of small GTP-binding proteins. Their function in fibroblasts has been analysed using microinjection of living cells. Rho appears to link plasma membrane receptors to the assembly of focal adhesions and actin stress fibres. The closely related protein rac, on the other hand, links receptors to the polymerization of actin at the plasma membrane to form membrane ruffles and pinocytotic vesicles. In phagocytic cells, rac has been shown to be required for activation of a membrane-bound NADPH oxidase in response to receptor activation. These systems provide the basis for a working model for the mechanism of action of the rho family of small GTPases.
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PMID:Cellular responses regulated by rho-related small GTP-binding proteins. 810 28

The phagocyte superoxide-generating NADPH oxidase, a multicomponent, membrane-bound electron transport chain, consists of cytochrome b558, p47-phox, p67-phox, and p21rac1 or p21rac2. The mechanisms of oxidase assembly are poorly understood. In previous studies using a cell-free NADPH oxidase system, we showed that preincubation of neutrophil membrane with neutrophil cytosol containing p47-phox, but not p67-phox, led to formation of a long-lived NADPH oxidase intermediate. This suggested that p47-phox interacted with cytochrome b558 in the early stages of oxidase assembly while p67-phox participated in a later stage. Peptides containing the sequence RGVHFIF (corresponding to amino acids 559-565 of the 91-kDa subunit of cytochrome b558) inhibit NADPH oxidase activity by blocking the early interaction between p47-phox and cytochrome b558. In the present study, we examined whether p21rac facilitated the interaction between p47-phox and cytochrome b558. We preincubated pure recombinant p47-phox with neutrophil membrane containing cytochrome b558 in the cell-free system. Superoxide-generating activity was subsequently reconstituted by adding pure rp67-phox and partially purified p21rac. RGVHFIF inhibited superoxide production if added to the cell-free system during preincubation of rp47-phox with membrane. RGVHFIF was markedly less inhibitory if added to the cell-free system after membrane was preincubated with pure rp47-phox. In contrast to p47-phox, preincubation of membrane with either p21rac or rp67-phox conferred no protection from inhibition of superoxide-generating activity by RGVHFIF added after preincubation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:p21rac does not participate in the early interaction between p47-phox and cytochrome b558 that leads to phagocyte NADPH oxidase activation in vitro. 811 10


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