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1.
The NADPH oxidase complex of phagocytic leukocytes: a biochemical and cytochemical view 总被引:10,自引:4,他引:6
The NADPH oxidase complex catalyzes the formation of superoxide (O2
–) in phagocytic leukocytes. This paper reviews recent advances in our understanding of this enzyme system. Recent studies have defined conditions for reconstitution of this enzymatic activity with purified proteins in a cell-free system. The role of the individual proteins that make up the active complex, their regulation and the effects of mutations in these proteins are discussed. While these studies represent major achievements, it is clear from cytochemical investigations that additional levels of complexity exist in the modulation of the NADPH oxidase complex in vivo. A major role for cytochemical analysis in understanding the cell biological aspects of the generation of reactive oxygen species is discussed.Portions of this review were presented at the 36th Symposium of the Society for Histochemistry, 21 September 1994, Heidelberg, Germany 相似文献
2.
A. W. Segal 《Protoplasma》1995,184(1-4):86-103
Summary Phagocytic cells of the immune system contain an oxidase that is important for the killing and digestion of engulfed microbes. This is an electron transport chain that transfers electrons from NADPH in the cytosol to oxygen to form superoxide and hydrogen peroxide in the phagocytic vacuole. Absence or abnormality of this oxidase results in the syndrome of CGD, characterised by a profound predisposition to infection. The electron transport chain consists of a flavocytochrome b located in the plasma membrane and membrane of the specific granules. It is composed of a and b-subunits, with apparent molecular masses of 23 kDa and 76–92 kDa, respectively. The b-subunit is a member of the FNR family of reductases with FAD and NADPH binding sites. Based upon the crystal structure of FNR we have constructed a model of the more hydrophilic C terminal half of this b-subunit, which acts as a guide to the organisation of the molecule, and provides a template on which to map mutations in CGD. The location of the heme is uncertain. Electron transport is dependent upon an activation complex of cytosolic proteins including p40
phox
, p47
phox
, and p67
phox
, and the small GTP binding protein, p21
rac
. This oxidase system is important for the killing and digestion of bacteria and fungi. This might be accomplished in a number of ways. The oxidase produces superoxide and hydrogen which might be toxic themselves. The hydrogen peroxide can act as substrate for myeloperoxidase which can oxidise chloride and iodide to chlorine and iodine and their hypohalous acids. The proteins contained within the cytoplasmic granules are also very important in the killing process. These are neutral proteinases that require a neutral or slightly alkaline pH for optimal activity. The oxidase transports electrons, unaccompanied by protons, across the wall of the phagocytic vacuole, resulting in an elevation of the vacuolar pH, thereby optimising conditions for killing and digestion of engulfed organisms by these neutral proteinases. 相似文献
3.
4.
The present paper concerns 4 groups of haemolymph cells of fleas (proleukocytes, leukocytes, trophic cells and oenocytoids), results of observations on their phagocytic activity during parenteral infection of insects with bacteria, bacilli, and cells' response to the infection with Microsporidia. 相似文献
5.
Rada BK Geiszt M Hably C Ligeti E 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2005,360(1464):2293-2300
NADPH oxidase of phagocytic cells transfers a single electron from intracellular NADPH to extracellular O2, producing superoxide (O.-2), the precursor to several other reactive oxygen species. The finding that a genetic defect of the enzyme causes chronic granulomatous disease (CGD), characterized by recurrent severe bacterial infections, linked O.-2 generation to destruction of potentially pathogenic micro-organisms. In this review, we focus on the consequences of the electrogenic functioning of NADPH oxidase. We show that enzyme activity depends on the possibilities for compensating charge movements. In resting neutrophils K+ conductance dominates, but upon activation the plasma membrane rapidly depolarizes beyond the opening threshold of voltage-gated H+ channels and H+ efflux becomes the major charge compensating factor. K+ release is likely to contribute to the killing of certain bacteria but complete elimination only occurs if O.-2 production can proceed at full capacity. Finally, the reversed membrane potential of activated neutrophils inhibits Ca2+ entry, thereby preventing overloading the cells with Ca2+. Absence of this limiting mechanism in CGD cells may contribute to the pathogenesis of the disease. 相似文献
6.
Interleukin-10 inhibits neutrophil phagocytic and bactericidal activity 总被引:10,自引:0,他引:10
Lauri L. Laichalk Jean M. Danforth Theodore J. Standiford 《FEMS immunology and medical microbiology》1996,15(4):181-187
Abstract Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells, which is dependent upon the coordinated expression of both pro- and anti-inflammatory cytokines. Interleukin-10 (IL-10) is a recently described cytokine with potent anti-inflammatory properties in vivo and in vitro. In this study we investigated whether IL-10 could directly regulate the ability of neutrophils (PMN) to phagocytose and kill bacteria. Initial studies demonstrated that human recombinant IL-10 (hrIL-10) inhibited the ability of PMN to phagocytose Escherichia coli in vitro. Inhibition of phagocytosis occurred in the absence of changes in CR1 (C3b) or Fc receptor expression, as treatment of PMN with IL-10 failed to induce significant changes in FcγIIR, FcγIIIR or CR1 cell surface expression. However, incubation of PMN with IL-10 resulted in a dose-dependent decrease in CD11b (Mac-1) expression. In addition to effects on PMN phagocytosis, hrIL-10 significantly attenuated PMN microbicidal activity, as bactericidal assays revealed that co-incubation of PMN with hrIL-10 resulted in a marked decrease in killing of phagocytosed bacteria. Furthermore, IL-10 inhibited the production of superoxide from PMA-stimulated PMN, suggesting that the detrimental effects of IL-10 on PMN microbicidal activity were due, in part, to suppression of respiratory burst. In summary, our studies indicate that IL-10 inhibits PMN-dependent phagocytosis and killing of E. coli in vitro, and suggest that this cytokine may impair effective antibacterial host defense in vivo. 相似文献
7.
Localization of the 47 kDa phosphoprotein involved in the respiratory-burst NADPH oxidase of phagocytic cells. 总被引:11,自引:1,他引:10
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A 47 kDa phosphoprotein is involved in the respiratory-burst oxidase of phagocytic cells. After stimulation of neutrophils with phorbol myristate acetate, this phosphoprotein was identified in both the cytosol and membranes. Peptide mapping of the two forms resulted in identical patterns of phosphopeptides. Dose-response curves for accumulation of phosphoprotein in the two sites were very similar, whereas the detection of the phosphoprotein in the cytosol preceded that in the membranes. The membrane-associated 47 kDa phosphoprotein was absent from the neutrophils of patients with X-chromosome-linked chronic granulomatous disease, which lack cytochrome b-245, and intermediate levels were detected in the membranes of their heterozygote carrier mothers. Activation of the neutrophil oxidase system appears to be dependent upon phosphorylation of the cytosolic 47 kDa protein and its association with cytochrome b-245 in the membranes. It is probably the cytosolic factor required for reconstitution of the active oxidase in cell-free systems. 相似文献
8.
Intracellular localization and preassembly of the NADPH oxidase complex in cultured endothelial cells 总被引:17,自引:0,他引:17
The phagocyte-type NADPH oxidase expressed in endothelial cells differs from the neutrophil enzyme in that it exhibits low level activity even in the absence of agonist stimulation, and it generates intracellular reactive oxygen species. The mechanisms underlying these differences are unknown. We studied the subcellular location of (a) oxidase subunits and (b) functionally active enzyme in unstimulated endothelial cells. Confocal microscopy revealed co-localization of the major oxidase subunits, i.e. gp91(phox), p22(phox), p47(phox), and p67(phox), in a mainly perinuclear distribution. Plasma membrane biotinylation experiments confirmed the predominantly (>90%) intracellular distribution of gp91(phox) and p22(phox). After subcellular protein fractionation, approximately 50% of the gp91(phox) (91-kDa band), p22(phox), p67(phox), and p40(phox) pools and approximately 30% of the p47(phox) were present in the 1475 x g ("nucleus-rich") fraction. Likewise, approximately 50% of total NADPH-dependent O(2)() production (assessed by lucigenin (5 microm) chemiluminescence) was found in the 1475 x g fraction. Co-immunoprecipitation studies and measurement of NADPH-dependent reactive oxygen species production (cytochrome c reduction assay) demonstrated that p22(phox), gp91(phox), p47(phox), p67(phox), and p40(phox) existed as a functional complex in the cytoskeletal fraction. These results indicate that, in contrast to the neutrophil enzyme, a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as a preassembled intracellular complex associated with the cytoskeleton. 相似文献
9.
K A Sanders K M Sundar L He B Dinger S Fidone J R Hoidal 《Journal of applied physiology》2002,93(4):1357-1364
It has been hypothesized that O(2) sensing in type I cells of the carotid body and erythropoietin (EPO)-producing cells of the kidney involves protein components identical to the NADPH oxidase system responsible for the respiratory burst of phagocytes. In the present study, we evaluated O(2) sensing in mice with null mutant genotypes for two components of the phagocytic oxidase. Whole body plethysmography was used to study unanesthetized, unrestrained mice. When exposed to an acute hypoxic stimulus, gp91(phox)-null mutant and wild-type mice increased their minute ventilation by similar amounts. In contrast, p47(phox)-null mutant mice demonstrated increases in minute ventilation in response to hypoxia that exceeded that of their wild-type counterparts: 98.0 +/- 18.0 vs. 20.0 +/- 13.0% (n = 11, P = 0.003). In vitro recordings of carotid sinus nerve (CSN) activity demonstrated that resting (basal) neural activity was marginally elevated in p47(phox)-null mutant mice. With hypoxic challenge, mean CSN discharge was 1.5-fold greater in p47(phox)-null mutant than in wild-type mice: 109.61 +/- 13.29 vs. 72.54 +/- 7.65 impulses/s (n = 8 and 7, respectively, P = 0.026). Consequently, the hypoxia-evoked CSN discharge (stimulus-basal) was approximately 58% larger in p47(phox)-null mutant mice. Quantities of EPO mRNA in kidney were similar in gp91(phox)- and p47(phox)-null mutant mice and their respective wild-type controls exposed to hypobaric hypoxia for 72 h. These findings confirm the previous observation that absence of the gp91(phox) component of the phagocytic NADPH oxidase does not alter the O(2)-sensing mechanism of the carotid body. However, absence of the p47(phox) component significantly potentiates ventilatory and chemoreceptor responses to hypoxia. O(2) sensing in EPO-producing cells of the kidney appears to be independent of the gp91(phox) and p47(phox) components of the phagocytic NADPH oxidase. 相似文献
10.
Dikalov SI Li W Doughan AK Blanco RR Zafari AM 《American journal of physiology. Regulatory, integrative and comparative physiology》2012,302(10):R1134-R1142
Production of superoxide (O(2)(·-)) by NADPH oxidases contributes to the development of hypertension and atherosclerosis. Factors responsible for activation of NADPH oxidases are not well understood; interestingly, cardiovascular disease is associated with both altered NADPH oxidase activity and age-associated mitochondrial dysfunction. We hypothesized that mitochondrial dysfunction may contribute to activation of NADPH oxidase. The effect of mitochondrial inhibitors on phagocytic NADPH oxidase in human lymphoblasts and whole blood was measured at the basal state and upon PKC-dependent stimulation with PMA using extracellular 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium or mitochondria-targeted 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine spin probes and electron spin resonance (ESR). Intracellular cytosolic calcium [Ca(2+)](i) was measured spectrofluorometrically using fura-2 AM. Incubation of lymphoblasts with the mitochondrial inhibitors rotenone, antimycin A, CCCP, or ruthenium red (an inhibitor of mitochondrial Ca(2+) uniporter) did not significantly change basal activity of NADPH oxidase. In contrast, preincubation with the mitochondrial inhibitors prior to PMA stimulation of lymphoblasts resulted in two- to three-fold increase of NADPH oxidase activity compared with stimulation with PMA alone. Most notably, the intracellular Ca(2+)-chelating agent BAPTA-AM abolished the effect of mitochondrial inhibitors on NADPH oxidase activity. Cytosolic Ca(2+) measurements with fura-2 AM showed that the mitochondrial inhibitors increased [Ca(2+)](i), while BAPTA-AM abolished the increase in [Ca(2+)](i). Furthermore, depletion of cellular Ca(2+) with thapsigargin attenuated CCCP- and antimycin A-mediated activation of NADPH oxidase in the presence of PMA by 42% and 31%, correspondingly. Our data suggest that mitochondria regulate PKC-dependent activation of phagocytic NADPH oxidase. In summary, increased mitochondrial O(2)(·-) and impaired buffering of cytosolic Ca(2+) by dysfunctional mitochondria result in enhanced NADPH oxidase activity, which may contribute to the development of cardiovascular diseases. 相似文献
11.
Mott R 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2006,361(1467):393-401
I survey the state of the art in complex trait analysis, including the use of new experimental and computational technologies and resources becoming available, and the challenges facing us. I also discuss how the prospects of rodent model systems compare with association mapping in humans. 相似文献
12.
The multi-subunit NADPH oxidase complex plays a crucial role in host defense against microbial infection through the production of reactive oxygen species. Activation of the NADPH oxidase requires the targeting of a cytoplasmic p40-p47-p67(phox) complex to the membrane bound heterodimeric p22-gp91(phox) flavocytochrome. This interaction is prevented in the resting state due to an auto-inhibited conformation of p47(phox). The X-ray structure of the auto-inhibited form of p47(phox) reveals that tandem SH3 domains function together to maintain the cytoplasmic complex in an inactive form. Further structural and biochemical data show that phosphorylation of p47(phox) activates a molecular switch that relieves the inhibitory intramolecular interaction. This permits p47(phox) to interact with the cytoplasmic tail of p22(phox) and initiate formation of the active, membrane bound enzyme complex. 相似文献
13.
The superoxide-generating oxidase of phagocytic cells. Physiological, molecular and pathological aspects. 总被引:32,自引:0,他引:32
Professional phagocytes (neutrophils, eosinophils, monocytes and macrophages) possess an enzymatic complex, the NADPH oxidase, which is able to catalyze the one-electron reduction of molecular oxygen to superoxide, O2-. The NADPH oxidase is dormant in non-activated phagocytes. It is suddenly activated upon exposure of phagocytes to the appropriate stimuli and thereby contributes to the microbicidal activity of these cells. Oxidase activation in phagocytes involves the assembly, in the plasma membrane, of membrane-bound and cytosolic components of the oxidase complex, which were diassembled in the resting state. One of the membrane-bound components in resting phagocytes has been identified as a low-potential b-type cytochrome, a heterodimer composed of two subunits of 22-kDa and 91-kDa. The link between NADPH and cytochrome b is probably a flavoprotein whose subcellular localization in resting phagocytes remains to be determined. Genetic defects in the cytochrome b subunits and in the cytosolic factors have been shown to be the molecular basis of chronic granulomatous disease, a group of inherited disorders in the host defense, characterized by severe, recurrent bacterial and fungal infections in which phagocytic cells fail to generate O2- upon stimulation. The present review is focused on recent data concerning the signaling pathway which leads to oxidase activation, including specific receptors, the production of second messengers, the organization of the oxidase complex and the molecular defects responsible for granulomatous disease. 相似文献
14.
Robert A. Linder Fabian Seidl Kimberly Ha Ian M. Ehrenreich 《Molecular biology of the cell》2016,27(1):209-218
Quantitative traits are often influenced by many loci with small effects. Identifying most of these loci and resolving them to specific genes or genetic variants is challenging. Yet, achieving such a detailed understanding of quantitative traits is important, as it can improve our knowledge of the genetic and molecular basis of heritable phenotypic variation. In this study, we use a genetic mapping strategy that involves recurrent backcrossing with phenotypic selection to obtain new insights into an ecologically, industrially, and medically relevant quantitative trait—tolerance of oxidative stress, as measured based on resistance to hydrogen peroxide. We examine the genetic basis of hydrogen peroxide resistance in three related yeast crosses and detect 64 distinct genomic loci that likely influence the trait. By precisely resolving or cloning a number of these loci, we demonstrate that a broad spectrum of cellular processes contribute to hydrogen peroxide resistance, including DNA repair, scavenging of reactive oxygen species, stress-induced MAPK signaling, translation, and water transport. Consistent with the complex genetic and molecular basis of hydrogen peroxide resistance, we show two examples where multiple distinct causal genetic variants underlie what appears to be a single locus. Our results improve understanding of the genetic and molecular basis of a highly complex, model quantitative trait. 相似文献
15.
Babior BM 《IUBMB life》2000,50(4-5):267-269
The best known NADPH oxidase is that of phagocytes-neutrophils and monocytes. In these cells, the enzyme manufactures large quantities of O2- and other reactive oxidants that are used for the purpose of killing invading microorganisms. Recent studies, however, have suggested that a number of other tissues contain NADPH oxidases. In contrast to the very vigorous production of oxidants by phagocytes, rates of oxidant production by these other cell types are quite low. Oxidant production by these cells is generally thought to serve a signaling function. 相似文献
16.
17.
The aim of the present study was to investigate possible mechanisms that could be involved in the deactivation of the assembled, catalytically active NADPH oxidase of phagocytic cells and thereby lead to termination of O(2)(.-) production. Our major findings are the following: (1) Addition of GDP to the active oxidase is able to reduce O(2)(.-) production both in the fully purified and in a semi-recombinant cell-free activation system. (2) p67(phox) inhibits GTP hydrolysis on Rac whereas p47(phox) has no effect on Rac GTPase activity. (3) Soluble regulatory proteins (GTPase activating protein, guanine nucleotide dissociation inhibitor, and the Rac-binding domain of the target protein p21-activated kinase) inhibit activation of the NADPH oxidase but have no effect on electron transfer via the assembled enzyme complex. (4) Membrane-associated GTPase activating proteins (GAPs) have access also to the assembled, catalytically active oxidase. Taken together, we propose that the GTP-bound active form of Rac is required for sustained enzyme activity and that membrane-localized GAPs have a role in the deactivation of NADPH oxidase. 相似文献
18.
19.
K. N. Markvicheva A. Yu. Gorokhovatskii N. M. Mishina N. N. Mudrik L. M. Vinokurov S. A. Luk’yanov V. V. Belousov 《Russian Journal of Bioorganic Chemistry》2010,36(1):124-129
Until recently, the production of reactive oxygen species by NADPH oxidase has been considered only in the context of the
oxidative damage to pathogens inside the phagosome. However, homologues of phagocytic NADPH oxidase have been found in almost
all cell types, where they produce hydrogen peroxide and thereby regulate the initial intracellular stages of MAP kinase cascades.
In the present work, the activation of two MAP kinase cascades, p38 and Erk1/2, during phagocytosis has been studied. It was
found that phagocytosis activates both cascades. The activation of Erk1/2 is dependent, and the activation of p38 is not dependent,
on the activity of NADPH oxidase. Therefore, the activation of MAP kinases in phagocytes during phagocytosis occurs by a mechanism
similar to that operating in nonphagocytic cells, indicating the universality of the function of NADPH oxidases in different
cell types. 相似文献
20.
2,6-Dichlorophenolindophenol-reducing activity of phagocytosis-associated NADPH oxidase 总被引:1,自引:0,他引:1
The 2,6-dichlorophenolindophenol (DCIP)-reducing activity of the phagocytosis-associated NADPH oxidase was investigated using homogenates and a membrane fraction (F2) of elicited guinea pig peritoneal macrophages stimulated by phorbol myristate acetate. Essentially all of the stimulation-specific DCIP reduction under aerobic conditions could be inhibited when high concentrations of superoxide dismutase (SOD), about 10 times those usually used to inhibit the superoxide (O-2)-mediated cytochrome c reduction, were used. SOD inhibited the DCIP reduction by chemically generated O2- in the same manner as the stimulation-specific DCIP reduction by the macrophage F2, and the concentration of SOD necessary for 50% inhibition was about 10 times that for the reduction of cytochrome c. Under anaerobic conditions, however, the NADPH oxidase could reduce DCIP, though the rate was slow because we could not use a sufficiently high DCIP concentration. The observations indicate that the NADPH oxidase preferentially reduces oxygen under aerobic conditions, though the oxidase can reduce DCIP in the anaerobic state. 相似文献