Polarity proteins Bem1 and Cdc24 are components of the filamentous fungal NADPH oxidase complex

Regulated synthesis of reactive oxygen species (ROS) by membrane-bound fungal NADPH oxidases (Nox) plays a key role in fungal morphogenesis, growth, and development. Generation of reactive oxygen species (ROS) by the plant symbiotic fungus, Epichloë festucae, requires functional assembly of a multisubunit complex composed of NoxA, a regulatory component, NoxR, and the small GTPase RacA. However, the mechanism for assembly and activation of this complex at the plasma membrane is unknown. We found by yeast two-hybrid and coimmunoprecipitation assays that E. festucae NoxR interacts with homologs of the yeast polarity proteins, Bem1 and Cdc24, and that the Phox and Bem1 (PB1) protein domains found in these proteins are essential for these interactions. GFP fusions of BemA, Cdc24, and NoxR preferentially localized to actively growing hyphal tips and to septa. These proteins interact with each other in vivo at these same cellular sites as shown by bimolecular fluorescent complementation assays. The PB1 domain of NoxR is essential for localization to the hyphal tip. An E. festucae ΔbemA mutant was defective in hyphal morphogenesis and growth in culture and in planta. The changes in fungal growth in planta resulted in a defective symbiotic interaction phenotype. Our inability to isolate a Δcdc24 mutant suggests this gene is essential. These results demonstrate that BemA and Cdc24 play a critical role in localizing NoxR protein to sites of fungal hyphal morphogenesis and growth. Our findings identify a potential shared ancestral link between the protein machinery required for fungal polarity establishment and the Nox complex controlling cellular differentiation.The NADPH oxidases (Nox) are a widely distributed family of eukaryotic proteins that transfer electrons across biological membranes to catalyze the reduction of molecular oxygen to superoxide (1–3). The multiple Nox isoforms found in eukaryotic cells control various physiological and cellular differentiation processes, including cell proliferation, apoptosis and hormone responses in animals (1, 2), and programmed cell death, hormone signaling and root hair tip growth in plants (4).Fungi have three distinct subfamilies of NADPH oxidase (3, 5). NoxA has the core NADPH oxidase transmembrane and catalytic domains but no additional motifs, whereas NoxB has in addition, an N-terminal extension of ∼40 amino acids that is conserved among fungal species that have this isoform (3, 6). NoxC has a longer N-terminal extension, of 170–250 amino acids, which contains a putative calcium-binding EF-hand motif (3), similar to that found in human Nox5 and the plant Rboh enzymes (3, 5). In Aspergillus nidulans, Podospora anserina and Neurospora crassa, NoxA (Nox1) is required for the development of the sexual fruiting body, indicating that a common function of this isoform is regulation of multicellular development (7–10). NoxB (Nox2) is required for ascospore germination in P. anserina and N. crassa (7, 10). However, in Botrytis cinerea, both NoxA and NoxB are required for formation of the multicellular sclerotial sexual structure, but are dispensable for ascospore germination (11).Fungal NADPH oxidases are also required for cellular growth and differentiation processes associated with plant host infection and colonization. In the symbiotic interaction between the endophytic fungus Epichloë festucae and perennial ryegrass, deletion of noxA, but not noxB, disrupted the highly regulated pattern of growth seen in WT associations (6). In the rice blast pathogen, Magnaporthe oryzae, disruption of either nox1 (noxA) or nox2 (noxB) resulted in loss of plant pathogenicity, due to an inability of the fungus to develop a penetration peg beneath the appressorium (12). Both NoxA and NoxB are important for pathogenicity of B. cinerea, but just NoxB is required for formation of the penetration structure (11).NoxR, a fungal homolog of the phagocytic p67^phox Nox regulator, has been shown to regulate both NoxA and NoxB. In the symbiotic fungus E. festucae, a noxR mutant has a similar disrupted symbiotic interaction phenotype as ΔnoxA (6, 13). In B. cinerea and N. crassa, deletion of noxR (nor-1) resulted in a similar developmental phenotype to the noxA(nox1)/noxB(nox2) double mutant (7, 11). Although NoxA and NoxB have distinct cellular functions, it is not yet known how NoxR regulates these two different Nox isoforms. The N-terminal domain of NoxR is similar to p67^phox, and includes four tetratricopeptide repeat (TPR) motifs required for Rac binding and a putative NADPH oxidase activation domain (3, 13) (Fig. 1A). In contrast, the C terminus of NoxR lacks the protein–protein interaction domains present in p67^phox, including Src Homology 3 (SH3) and a conventional Phox and Bem1 (PB1), for interaction with p47^phox and p40^phox, respectively (1, 14). The absence of these domains in NoxR is consistent with the apparent absence of p47^phox and p40^phox homologs in fungal genome databases (3, 13). However, NoxR does possess a nonconventional PB1 domain in the C terminus of the protein, suggesting that fungi have distinct regulatory components that, upon activation, interact with NoxR to translocate this protein from the cytosol to the plasma membrane to assemble and activate the Nox enzyme complex (3, 13) (Fig. 1A).Open in a separate windowFig. 1.(A) Domain structure of E. festucae NoxR, BemA, Cdc24, and CBS1-containing protein. The tetratricopeptide repeat (TPRs), Nox activation (AD), proline-rich region (PRR), Src homology 3 (SH3), Phox and Bem1 (PB1), phox homology (PX), cystathionine beta-synthase 1 (CBS1), and calponin homology (CH) domains are indicated. (B) Yeast two-hybrid assay of the interactions between PB1 domains of E. festucae NoxR, BemA, Cdc24, and CBS1-containing protein. Yeast strain AH109 was transformed with prey and bait vectors, pGADT7 and pGBKT7, as indicated and plated on to SD medium lacking leucine and tryptophan (Upper, -Leu/-Trp) or lacking leucine, tryptophan, histidine, and adenine (Lower, -Leu/-Trp/-His/-Ade). Growth on the latter indicates an interaction between bait and prey. (C) Summary of interactions between PB1 domains based on results shown in B and Fig. S4A. (D) Yeast two-hybrid assay of interactions between full-length NoxR, BemA, and Cdc24. The plating conditions are the same as in B. (E) COS-7 cells were cotransfected with pEF-BOS-Flag-NoxR and pEF-BOS-Myc, or pEF-BOS-Myc-Cdc24 or pEF-BOS-Myc-BemA, or with pEF-BOS-Flag-BemA and pEF-BOS-Myc-Cdc24. Lysates of the transfected cells (Left) were analyzed by immunoprecipitation (IP) with control IgG (Center) or the anti-Flag monoclonal antibody (Right), followed by immunoblotting (WB) with the anti-Myc (Upper) or anti-Flag (Lower) monoclonal antibodies.The objectives of this study were (i) to identify additional components of the fungal Nox enzyme complex by identifying proteins that interact with the PB1 domain of E. festucae NoxR, (ii) to investigate the subcellular localization of these regulators to identify where the Nox complex is assembled, and (iii) to determine whether these proteins, like NoxA and NoxR, have a role in maintaining the mutualistic symbiotic interaction between E. festucae and its host grass perennial ryegrass.     

Redox regulation of cytokeratin 18 protein by NADPH oxidase 1 in preneoplastic human epithelial cells

Introduction  

A catalytic subunit of NADPH oxidase 1 (Nox1) is implicated to be involved in neoplastic progression in human epithelial cancers. We had previously demonstrated that Nox1 overexpression of immortalized epithelial cells was able to induce the generation of progenitor cells that expressed fetal-type cytokeratins 8 and 18.     

Correlation between NADPH oxidase and protein kinase C in the ROS production by human granulocytes related to age

BACKGROUND: Aging may be defined as gradual and progressive changes in an organism that increase the probability of death. Accumulating evidence now indicates that the sum of deleterious free radical reactions going on continuously throughout cells and tissues constitutes the aging process or is a major contributor to it. OBJECTIVE: The aim of this paper was to study the correlation between NADPH oxidase and protein kinase C (PKC) in the reactive oxygen species (ROS) production related to age. METHODS: The age-induced ROS generation was studied in healthy subjects ranging in age from 20 to 80 years, divided into six age groups: (1) 20-29, (2) 30-39, (3) 40-49, (4) 50-59, (5) 60-69, and (6) 70-80 years. The ROS were quantified using a chemiluminescence assay (luminol dependent) and the results expressed as RLU/s at maximum peak and total chemiluminescence (integral under the curve RLU/s). RESULTS: Our results demonstrate a significant increase of the ROS production from 40 years of age (age groups 3-6). In the age groups 1 and 2, we did not observe a significant difference (p > 0.05). These data suggest an increase of the ROS production from 40 to 49 years of age which may be induced by the PKC activity. The selective PKC inhibitor (calphostin C) abrogated the stimulatory effect of phorbol-12,13-dibutyrate on the ROS production. However, the NADPH oxidase inhibitor diphenylene iodonium did not inhibit the total ROS production by granulocytes in relation to age. CONCLUSIONS: These data suggest a correlation between age-related PKC activity, NADPH oxidase phosphorylation, and ROS production. The above correlations between unspecific and inflammatory responses related to age are discussed.     

Phospholipases and activation of the NADPH oxidase

Abstract: The signal transductional mechanisms regulating the activation of NADPH oxidase, the respiratory burst enzyme in phagocytic cells, are not completely understood. Receptors for most physiologic stimuli trigger the activation of various phospholipases, including phospholipases A₂, C, and D. The lipid mediators formed (arachidonic acid, 1,2-diacylglycerol, and phosphatidic acid) have been implicated as second messengers in the induction of the respiratory burst. In intact cells, we have correlated phospholipase D activation and the production of phosphatidic acid with the activation of NADPH oxidase, using the drug propranolol. Phosphatidic acid activated NADPH oxidase in a cell-free system, but the level of activation was low. 1,2-Diacylglycerol markedly enhanced NADPH oxidase activation by phosphatidic acid. The synergistic effect required the diacyl species, since mono- or tri-acylglycerols were ineffective. Phosphatidic acid could be replaced by either lysophosphatidic acid or phosphatidylserine, but not by phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol, suggesting specificity for an anionic phospholipid. Since other cell-free activators of NADPH oxidase (arachidonic acid, sodium dodecyl sulfate) are also anionic amphiphiles, phosphatidic acid may directly interact with an enzyme component(s). The targets for phosphatidic acid and diacylglycerol in the cell-free system are currently under investigation. These results emphasize the critical importance of phospholipases, particularly phospholipase D, in the regulation of the respiratory burst.     

Fc gamma R-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome

The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b(558) and cytosolic p67(phox), p47(phox), and p40(phox) subunits that undergo membrane translocation upon cellular activation. The function of p40(phox), which binds p67(phox) in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40(phox) and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40(phox) in FcgammaR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcgammaR-induced phagocytosis. YFP-tagged p67(phox) and p40(phox) translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67(phox) and p47(phox) accumulation on nascent and internalized phagosomes did not require p40(phox) or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40(phox) PI3P-binding domain or wortmannin. Translocation of p40(phox) to nascent phagosomes required binding to p67(phox) but not PI3P, although the loss of PI3P binding reduced p40(phox) retention after phagosome internalization. We conclude that p40(phox) functions primarily to regulate FcgammaR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization.     

NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells

The phagocyte NADPH oxidase (NOX2) is critical for the bactericidal activity of phagocytic cells and plays a major role in innate immunity. We showed recently that NOX2 activity in mouse dendritic cells (DCs) prevents acidification of phagosomes, promoting antigen cross-presentation. In order to investigate the role of NOX2 in the regulation of the phagosomal pH in human DCs, we analyzed the production of reactive oxygen species (ROS) and the phagosomal/endosomal pH in monocyte-derived DCs and macrophages (M(diameter)s) from healthy donors or patients with chronic granulomatous disease (CGD). As expected, we found that human M(diameter)s acidify their phagosomes more efficiently than human DCs. Accordingly, the expression of the vacuolar proton ATPase (V-H(+)-ATPase) was higher in M(diameter)s than in DCs. Phagosomal ROS production, however, was also higher in M(diameter)s than in DCs, due to higher levels of gp91phox expression and recruitment to phagosomes. In contrast, in the absence of active NOX2, the phagosomal and endosomal pH decreased. Both in the presence of a NOX2 inhibitor and in DCs derived from patients with CGD, the cross-presentation of 2 model tumor antigens was impaired. We conclude that NOX2 activity participates in the regulation of the phagosomal and endosomal pH in human DCs, and is required for efficient antigen cross-presentation.     

Protective action of NADPH oxidase inhibitors and role of NADPH oxidase in pathogenesis of colon inflammation in mice

AIM: To investigate the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in colon epithelial cells in the pathogenesis of acute and chronic colon inflammation in a mouse model of dextran sulphate sodium (DSS)-induced colitis.METHODS: Balb/c mice were divided into three groups: 8 mice with acute DSS-induced colitis (3.5% DSS solution; 7 d), 8 mice with chronic DSS-induced colitis (3.5% DSS solution for 5 d + water for 6 d; 4 cycles; total: 44 d) and 12 mice without DSS supplementation as a control group. Primary colonic epithelial cells were isolated using chelation method. The cells were cultivated in the presence of mediators (lipopolysaccharide (LPS), apocynin or diphenyleneiodonium). Viability of cells was assessed by fluorescent microscopy. Production of reactive oxygen species (ROS) by the cells was measured fluorometrically using Amplex Red. Production of tumour necrosis factor-alpha (TNF-α) by the colonic epithelial cells was analysed by ELISA. Nox1 gene expression was assessed by real-time PCR.RESULTS: Our study showed that TNF-α level was increased in unstimulated primary colonic cells both in the acute and chronic colitis groups, whereas decreased viability, increased ROS production, and expression of Nox1 was characteristic only for chronic DSS colitis mice when compared to the controls. The stimulation by LPS increased ROS generation via NADPH oxidase and decreased cell viability in mice with acute colitis. Treatment with NADPH oxidase inhibitors increased cell viability and decreased the levels of ROS and TNF-α in the LPS-treated cells isolated from mice of both acute and chronic colitis groups.CONCLUSION: Our study revealed the importance of NADPH oxidase in the pathogenesis of both acute and chronic inflammation of the colon.     

Microvascular display of xanthine oxidase and NADPH oxidase in the spontaneously hypertensive rat

OBJECTIVE: Oxygen free radical production in hypertension may be associated with elevated arteriolar tone and organ injury. Previous results suggest an enhanced level of oxygen free radical formation in microvascular endothelium and in circulating neutrophils associated with xanthine oxidase activity in the spontaneously hypertensive rats (SHR) compared with their normotensive controls, the Wistar Kyoto rats (WKY). The aim of this study was to gain more detailed understanding of where oxidative enzymes are located in the microcirculation. METHODS: An approach was developed to delineate the cellular distribution of two selected oxidative enzymes, xanthine oxidase and nicotinamide adenine dinucleotide phosphate (NADPH) dependent oxidase (protein 67-kDa fraction). Immunolabeling with peroxidase substrate was utilized, which permits full delineation of the primary antibody in all microvascular structures of the mesentery. RESULTS: Xanthine oxidase is present in the endothelium of all segments of the microcirculation, in mast cells, and in parenchymal cells of the mesentery. NADPH oxidase can be detected in the endothelium, leukocytes, and mast cells and with lower levels in parenchymal cells. The mesentery of WKY and SHR has similar enzyme distributions with enhancements on the arteriolar and venular side of the microcirculation that coincide with the sites of enhanced free radical production recently reported. Immune label measurements under standardized conditions indicate that both enzymes are significantly enhanced in the SHR. Adrenalectomy, which serves to reduce the blood pressure and free radical production of the SHR to normotensive levels, leads to a reduction of NADPH and xanthine oxidase to normotensive levels, while supplementation of adrenalectomized SHR with dexamethasone significantly increases the oxidase expression in several parts of the microcirculation to levels above the WKY rats. CONCLUSION: The results indicate that enhanced expression of NADPH and xanthine oxidase in the SHR depends on an adrenal pathway that is detectable in the arteriolar and venular network at high and low pressure regions of the circulation.     

Dual granule localization of the dormant NADPH oxidase and cytochrome b559 in human neutrophils

The subcellular localization of the microbicidal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and associated b-cytochrome was investigated in human neutrophils. In unperturbed neutrophils 85% of b-cytochrome and the major part of membrane-bound components of the NADPH oxidase co-sedimented with markers for specific granules and gelatinase. Using cytochrome b559 as a marker for membrane-bound components of the NADPH oxidase in quantitative studies we observed that, of the remaining 15%, the vast majority co-sedimented with latent alkaline phosphatase, a marker for a newly identified mobilizable intracellular compartment. Only a small fraction co-localized with the plasma membranes. Azurophil granules contained a protease activity which rapidly inactivated the NADPH oxidase components present in other membranes. Stimulation of the neutrophils with formyl-methionyl-leucyl-phenyl-alanine and leukotriene B4 which caused minimal degranulation of specific granules, resulted in translocation of b-cytochrome to the plasma membrane, concomitant with incorporation of alkaline phosphatase into the plasma membrane.     

NADPH氧化酶在糖尿病肾病中的作用

还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶是许多非吞噬细胞中活性氧簇（ROS）产生的主要来源。在高血糖、高血脂、血管紧张素Ⅱ及各种细胞因子、生长因子的作用下,NADPH氧化酶被激活,发生呼吸链级联反应,产生过量的ROS,参与氧化应激,并作为氧化应激的中心环节,通过形成晚期糖基化终末产物（AGE）、激活蛋白激酶C（PKC）、转录因子如核因子（NF）-κB等途径,改变肾脏血液动力学,影响细胞外基质的重构,激活细胞内信号转导等,促进糖尿病肾病的发生发展。调节NADPH氧化酶的活性以抑制肾脏的氧化应激,有望成为延缓肾功能损害的有效治疗措施。     

Functional reconstitution of the phagocyte NADPH oxidase by transfection of its multiple components in a heterologous system

The phagocyte NADPH oxidase system, as previously defined by cell-free reconstitution, is comprised of five essential components, three of which are produced during late phagocytic differentiation--namely, two cytosolic proteins, p47- and p67-phox--and the large subunit of cytochrome b558, gp91-phox. To confirm that these are the only phagocyte-specific components necessary for oxidase activity in whole cells, the recombinant NADPH oxidase was reconstituted in a heterologous cell line. An undifferentiated multipotent leukemic cell line, K562, which expresses endogenous Rac and the small subunit of the flavocytochrome b558 (p22-phox), was cotransfected with episomal expression vectors containing cDNAs for the three other oxidase components. After 4 days of selection, the complete oxidase system was functionally reconstituted in transfected cells stimulated with phorbol myristate acetate or calcium ionophore. These easily transfected cells provide an ideal model system in which several oxidase components can be genetically manipulated and readily expressed. This system can be used to test the effects of mutations associated with any of the genes affected in chronic granulomatous disease and will facilitate studies on structure-function relationships within several oxidase components. This system will also aid in delineation of upstream regulators functioning through various signaling pathways.     

Four novel mutations in the gene encoding gp91-phox of human NADPH oxidase: consequences for oxidase assembly

The superoxide-forming nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase of human phagocytes comprises membrane-bound and cytosolic proteins, which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients with chronic granulomatous disease (CGD) are defective in one of the phagocyte oxidase (phox) components, p47-phox or p67-phox, which reside in the cytosol of resting phagocytes, or gp91-phox or p22-phox, which constitute the membrane-bound cytochrome b(558). In four X-linked CGD patients we have identified novel missense mutations in CYBB, the gene encoding gp91-phox. These mutations were associated with normal amounts of nonfunctional cytochrome b(558) in the patients' neutrophils. In phorbol-myristate-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of p47-phox and p67-phox with the membrane fraction of the cells with Cys369-->Arg, Gly408-->Glu, and Glu568--> Lys substitutions was strongly disturbed. Only a Thr341-->Lys substitution, residing in a region of gp91-phox involved in flavin adenine dinucleotide (FAD) binding, supported a normal translocation. Thus, the introduction or reversal of charge at residues 369, 408, and 568 in gp91-phox destroys the correct binding of p47-phox and p67-phox to cytochrome b(558). Based on mutagenesis studies of structurally related flavin-dependent oxidoreductases, we propose that the Thr341-->Lys substitution results in impaired hydride transfer from NADPH to FAD. Because we found no electron transfer in solubilized neutrophil plasma membranes from any of the four patients, we conclude that all four amino acid replacements are critical for electron transfer. Apparently, an intimate relation exists between domains of gp91-phox involved in electron transfer and in p47/p67-phox binding. (Blood. 2000;95:666-673)     

NADPH氧化酶在酒精性肝病发病机制中的作用

酒精性肝病（ALD）的发病机制有人提出以氧应激和脂质过氧化为中心的“二次打击”假说，NADPH氧化酶（NADPH oxidase，NOX）与自由基形成有着密切关系，参与ALD的多个环节。     

Lipid rafts keep NADPH oxidase in the inactive state in human renal proximal tubule cells

Recent studies have indicated the importance of cholesterol-rich membrane lipid rafts (LRs) in oxidative stress-induced signal transduction. Reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases, the major sources of reactive oxygen species, are implicated in cardiovascular diseases, including hypertension. We tested the hypothesis that NADPH oxidase subunits and activity are regulated by LRs in human renal proximal tubule cells. We report that a high proportion of p22(phox) and the small GTPase Rac1 are expressed in LRs in human renal proximal tubule cells. The D(1)-like receptor agonist, fenoldopam (1 micromol/L per 20 minutes) dispersed Nox subunits within LRs and non-LRs and decreased oxidase activity (30.7+/-3.3%). In contrast, cholesterol depletion (2% methyl-beta-cyclodextrin [beta CD]) translocated NADPH oxidase subunits out of LRs and increased oxidase activity (154.0+/-10.5% versus control, 103.1+/-3.4%), which was reversed by cholesterol repletion (118.9+/-9.9%). Moreover, NADPH oxidase activation by beta CD (145.5+/-9.0%; control: 98.6+/-1.6%) was also abrogated by the NADPH oxidase inhibitors apocynin (100.4+/-3.2%) and diphenylene iodonium (9.5+/-3.3%). Furthermore, beta CD-induced reactive oxygen species production was reversed by knocking down either Nox2 (81.0+/-5.1% versus beta CD: 162.0+/-2.0%) or Nox4 (108.0+/-10.8% versus beta CD: 152.0+/-9.8%). We have demonstrated for the first time that disruption of LRs results in NADPH oxidase activation that is abolished by antioxidants and silencing of Nox2 or Nox4. Therefore, in human renal proximal tubule cells, LRs maintain NADPH oxidase in an inactive state.     

The NADPH oxidase Nox4 and aging in the heart

Oxidative stress in mitochondria is believed to promote aging. Although passive leakage of electron from the mitochondrial electron transport chain has been considered as a major source of oxidative stress in the heart and the cardiomyocytes therein, enzymes actively producing reactive oxygen species may also exist in mitochondria. We have shown recently that Nox4, a member of the NADPH oxidase family, is localized on intracellular membranes, primarily at mitochondria, in cardiomyocytes. Mitochondrial expression of Nox4 is upregulated by cardiac stress and aging in the heart, where Nox4 could become a major source of oxidative stress. This raises an intriguing possibility that Nox4 may play an important role in mediating aging of the heart. Here we discuss the potential involvement of Nox4 in mitochondrial oxidative stress and aging in the heart.     

Up-regulation of NADPH oxidase components and increased production of interferon-gamma by leukocytes from sickle cell disease patients

We have previously demonstrated that mononuclear leukocytes from patients with sickle cell disease (SCD) release higher amounts of superoxide compared with normal controls. The aim of this study was to further study the NADPH oxidase system in these patients by investigating gene expression of NADPH oxidase components, phosphorylation of p47(phox) component, and the release of cytokines related to NADPH oxidase activation in mononuclear leukocytes from patients with SCD. gp91(phox) gene expression was significantly higher in monocytes from SCD patients compared with normal controls (P=0.036). Monocytes from SCD patients showed higher levels of p47(phox) phosphorylation compared with normal controls. INF-gamma release by lymphocytes from SCD patients was significantly higher compared with normal controls, after 48 h culture with phytohemagglutinin (P=0.02). The release of TNF-alpha by monocytes from SCD patients and normal controls was similar after 24 and 48 h culture with lipopolysaccharide (P>0.05). We conclude that monocytes from SCD patients show higher levels of gp91(phox) gene expression and p47(phox) phosphorylation, along with increased IFN-gamma release by SCD lymphocytes. These findings help to explain our previous observation showing the increased respiratory burst activity of mononuclear leukocytes from SCD patients and may contribute to inflammation and tissue damage in these patients.     

辛伐他汀对高糖损伤乳鼠心肌细胞NADPH氧化酶亚基基因表达的影响

取原代培养的SD雄性乳鼠心肌细胞在高糖刺激下加入10-7、10-6和10-5mol/L辛伐他汀作用72 h.结果显示,与对照组比较,高糖组心肌细胞活力明显降低(P＜0.01),乳酸脱氢酶(LDH)活性显著增加(P＜0.01),NADPH氧化酶亚基p22phox、p47phox mRNA表达和活性氧簇(ROS)水平明显增高(均P＜0.05);与高糖组比较,辛伐他汀各组心肌细胞活力明显增加(P＜0.05),LDH活性显著降低(P＜0.05),p22phox、p47phox mRNA表达和ROS水平明显降低,且辛伐他汀浓度对心肌细胞活力的影响呈剂量依赖效应.这些结果提示辛伐他汀能够抑制NADPH氧化酶亚基的基因表达,减轻高糖引起的心肌损伤.     

Regulation of the superoxide-forming NADPH oxidase of human neutrophils is not altered in essential hypertension.

Neutrophils possess a plasma-membrane-bound reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which catalyzes superoxide (O2-) formation and is activated by a variety of stimuli. Recently, neutrophils of patients with essential hypertension (EHT) have been reported to generate O2- at rates up to fourfold higher than those of normotensive (NT) subjects upon exposure to the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe). We studied regulation of O2- formation in neutrophils of 25 EHT subjects and 25 age- and sex-matched NT subjects. The intercellular signal molecules fMet-Leu-Phe, platelet-activating factor and leukotriene B4 activated O2- formation in neutrophils, but the latter two receptor agonists were less effective than the former. fMet-Leu-Phe activated O2- formation with a 50% effective concentration (EC50) of about 30 nmol/l, the effect of the chemotactic peptide being maximal at 0.1-1 mumol/l. fMet-Leu-Phe-induced O2- formation was potentiated by platelet-activating factor, adenosine 5'-[gamma-thio]triphosphate and cytochalasin B and was inhibited by the activators of adenylyl cyclase, isoproterenol, prostaglandin E1 and histamine. 4 beta-Phorbol 12-myristate 13-acetate, 1,2-dioctanoyl-sn-glycerol, gamma-hexachlorocyclohexane and arachidonic acid, which circumvent receptor stimulation, also activated O2- formation. Significant differences between NT and EHT subjects were not evident in respect of any of the parameters studied. Our data suggest that regulation of the neutrophil NADPH oxidase is not disturbed in EHT and that altered O2- formation does not represent a genetic marker for abnormalities in plasma-membrane signal transduction in EHT.