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)

f-Met-Leu-Phe-stimulated luminol-enhanced chemiluminescence was found to be repeatedly defective in some MDS patients. This defect was not attributed to myeloperoxidase deficiency, nor to a defect in NADPH oxidase function, because PMA chemiluminescence was found to be normal in these individuals. An arbitrary value of 7 mV (half the mean control value) was chosen to subdivide the group: MDS patients with values < 7 mV had a mean f-Met-Leu-Phe chemiluminescence response of 2.5 +/- 0.5 compared to MDS patients with values > 7 mV who had a mean response of 15.6 +/- 1.6 mV, P < 0.01 (healthy controls 14 +/- 2 mV). The characteristics of the f-Met-Leu-Phe receptor and initial calcium flux results suggested that the receptor itself was normal in number and function in low f-Met-Leu-Phe responders. The rate of superoxide generation, which is calcium-dependent, was also found to be in the normal range in low f-Met-Leu-Phe responders, although total superoxide production was reduced in some of these patients. When MDS neutrophils with a low f-Met-Leu-Phe response were stimulated with PMA, chemiluminescence was normal, suggesting normal activity of the NADPH-oxidase complex. Furthermore, myeloperoxidase activity was reduced in only three out of the 11 low f-Met-Leu-Phe responders. Following priming with GM-CSF, f-Met-Leu-Phe chemiluminescence was 27 +/- 1.6 mV in low f-Met-Leu-Phe responders compared to controls (87.7 +/- 11 mV, P < 0.005). Thus, although responses were improved, they were not as marked as in control neutrophils. These data suggest that a subgroup of MDS patients have a low f-Met-Leu-Phe chemiluminescence response which is not due to a defect in the f-Met-Leu-Phe receptor or oxidase activity, and in the majority of cases MPO activity is normal. Initial patient survival data suggest that these patients may have an increased risk of infective mortality. It is proposed that defective f-Met-Leu-Phe chemiluminescence results from a putative defect in cell-signalling mechanism upstream of PKC, and GM-CSF priming only partially improves responsiveness.
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PMID:Identification of a subgroup of myelodysplastic patients with a neutrophil stimulation-signalling defect. 791 69

Previously we have shown that reactive oxygen species (ROS) formation induced by phorbol ester in association with vanadate is essential for protein tyrosine phosphorylation and phospholipase A2 (PLA2) activation. Here we show that the interaction of beta-glucan particles (glucanp) or zymosan with complement receptor type 3 (CR3) leads, when associated with vanadate, to a cascade of reactions culminating in PLA2 activation. Vanadate + zymosan (or glucanp) markedly enhance protein tyrosine phosphorylation in bone marrow derived macrophages (BMMs), whereas neither of the agents alone has any effect. The enhancement was due to both sustained activation of protein tyrosine kinase (PTK) and inactivation of protein tyrosine phosphatase (PTP) as assessed in lysates of treated cells. Zymosan elevates membranal PKC, an effect that is potentiated by vanadate. Activation of both PTK and PKC leads to the activation of NADPH oxidase and to ROS formation. The formed ROS together with vanadate are potent inactivators of PTP leading to amplification of tyrosine phosphorylation and myelin basic protein kinase (MBP-K) activation. The activation of the cascade of protein kinases eventually leads to activation of PLA2. All the activation steps, i.e., activation of PTK, NADPH oxidase, MBP-K,PLA2 and the inactivation of PTP are sensitive to the NADPH oxidase inhibitor diphenyleneiodonium (DPI), to antioxidants and to PKC inhibitors. Thus, ROS formation (in the presence of vanadate) is critical for protein phosphorylation processes constituting the regulatory pathway of PLA2 activation by ligand-CR3 interaction.
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PMID:A role for reactive oxygen species in zymosan and beta-glucan induced protein tyrosine phosphorylation and phospholipase A2 activation in murine macrophages. 803 63

Legionella pneumophila may subvert monocyte defenses by several mechanisms including the inhibition of phagosome-lysosome fusion or the impairment of oxidative metabolism. We have investigated the effect of L. pneumophila Knoxville 1, a virulent strain that does not inhibit phagosome-lysosome fusion, on the oxidative responsiveness of human monocytes. Infection of monocytes with L. pneumophila for 48 h resulted in marked inhibition of superoxide generation stimulated by phorbol myristate acetate (PMA) but not by zymosan, a particulate agonist. Evidence is provided that L. pneumophila interfered with the transductional pathway (i.e., protein kinase C, PKC) leading to activation of the NADPH oxidase in monocytes. The phosphorylation of 34-, 48-, 62-, 68-, and 80-kDa proteins stimulated by PMA was markedly inhibited in infected monocytes. In addition, the expression of both alpha and beta PKC isotypes was partially inhibited in infected monocytes. Taken together, our data suggest that the down-modulation of PKC isotypes plays a role in the inhibition of PMA-stimulated superoxide generation.
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PMID:Legionella pneumophila inhibits superoxide generation in human monocytes via the down-modulation of alpha and beta protein kinase C isotypes. 812 Apr 47

Previous studies have shown that intracellular killing of bacteria by monocytes is stimulated by interaction between IgG and Fc gamma receptors (Fc gamma R) in the membrane of these cells. In the present study anti-Fc gamma R monoclonal antibodies (mAb) were used to investigate the relative contributions of the various classes of Fc gamma R to the intracellular killing of Staphylococcus aureus by human monocytes and the biochemical pathways involved. Anti-Fc gamma RI or anti-Fc gamma RII mAb, but not anti-Fc gamma RIII mAb, efficiently stimulated the intracellular killing of bacteria by monocytes. Cross-linking Fc gamma RI or Fc gamma RII, but not Fc gamma RIII, on monocytes with mouse anti-Fc gamma R mAb followed by bridging with F(ab')2 fragments of goat anti-mouse IgG enhanced this process. Since the NADPH oxidase inhibitor diphenyleneiodonium blocked the Fc gamma R-mediated intracellular killing of S. aureus, oxygen-dependent bactericidal mechanisms are most probably involved. Cross-linking Fc gamma RI or Fc gamma RII but not binding of the mAb to the Fc gamma R on monocytes activated phospholipase C, as demonstrated by the increase in the intracellular concentration of inositol-(1,4,5)-triphosphate. The enhanced intracellular killing stimulated by cross-linking Fc gamma R on monocytes was completely blocked by U-73122, an inhibitor of phospholipase C-dependent processes. Protein kinase C activity, but not the rise in the cytosolic free Ca++ concentration or pertussis toxin-sensitive G proteins, is essential for the Fc gamma R-mediated intracellular killing of bacteria by monocytes. Together, these results demonstrate that cross-linking Fc gamma RI or Fc gamma RII is equally effective in stimulating the intracellular killing of bacteria by monocytes and that this stimulation is a phospholipase C-dependent process.
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PMID:Stimulation of the intracellular killing of Staphylococcus aureus by human monocytes mediated by Fc gamma receptors I and II. 822 59

Protein kinase C (PKC) inhibitors, staurosporine or 1,5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited NADPH oxidase activity and phosphorylation of 47 kDa protein (p47) in PMA-stimulated neutrophils in a dose-dependent manner. These PKC inhibitors, at the same doses, did not affect oxidase activity and caused only partial inhibition of p47 phosphorylation in OZ-stimulated neutrophils. There was residual (20%) phosphorylated p47 in the membranes of OZ-stimulated cells in the presence of PKC inhibitors, at concentrations which caused total inhibition of oxidase activity and p47 phosphorylation in PMA-stimulated neutrophils. In the presence of ionomycin, which increased intracellular calcium ion concentrations, staurosporine was less effective in inhibiting both superoxide generation and p47 phosphorylation stimulated by PMA, similar to its effect in OZ-stimulated cells. The results indicate that some phosphorylation of p47 always accompanied oxidase activation induced by PMA or OZ, though the degree of phosphorylation of membrane-bound p47 does not directly correlate with rates of superoxide production.
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PMID:The requirement of p47 phosphorylation for activation of NADPH oxidase by opsonized zymosan in human neutrophils. 830 97

Neutrophils possess at least two phospholipid-dependent forms of protein kinase C, a classical Ca/PS/DG-dependent beta-isotype of protein kinase C and a Ca-independent but PS/DG-dependent novel protein kinase C (nPKC) which we now demonstrate to have different substrate specificities. Activation of human neutrophils triggers assembly of an NADPH oxidase in the membrane and generation of O2-. A role for the major Ca-dependent isotype beta-PKC in neutrophils is proposed in stimulus-induced phosphorylation and association of a cytosolic 47 kDa protein (p47-phox) with the membrane NADPH oxidase. In this study we demonstrate that purified beta-PKC and nPKC have very different substrate specificities; beta-PKC but not nPKC phosphorylated both endogenous and recombinant p47-phox. In addition, beta-PKC but not nPKC phosphorylated [ser25]PKC(19-31), the substrate peptide based on a sequence in the Ca-dependent alpha, beta and gamma-isotypes. Pseudosubstrate(19-36), derived from the C-terminus of Ca-dependent PKC isotypes, inhibited beta-PKC but not nPKC activity using either Histone IIIS or peptide(19-31) as substrate. Pseudosubstrate(19-36) also inhibited beta-PKC catalyzed phosphorylation of endogenous and recombinant p47-phox. Pseudosubstrate(19-36) also inhibited the O2- generation triggered by GTP gamma S in electroporated neutrophils by 50%. 32P-Labelled neutrophils electroporated in the presence of GTP gamma S showed phosphorylation of multiple cytosolic proteins including a 47 kDa band, and phosphorylation of membrane-associated 34 kDa, 47 kDa and 54 kDa proteins. Pseudosubstrate(19-36) inhibited phosphorylation of p47-phox in the membrane but not in the cytosol. These findings suggest translocatable, Ca-dependent isotypes of PKC such as beta-PKC may play a role in the phosphorylation of membrane associated p47-phox and the assembly or maintenance of an active NADPH oxidase.
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PMID:Protein kinase C isotypes and signal-transduction in human neutrophils: selective substrate specificity of calcium-dependent beta-PKC and novel calcium-independent nPKC. 847 29

We examined the effects of okadaic acid, a protein phosphatase 1 and 2A inhibitor, on superoxide generation in human neutrophils. Superoxide generation induced by fMLP was inhibited by low-dose okadaic acid (10-100 nM), but it had no effect on superoxide synthesis by PMA, and the fMLP-induced rise of the intracellular Ca2+ concentration was not affected by low-dose okadaic acid. These findings suggested that the inhibitory mechanism of okadaic acid might involve PKC-independent and Ca(2+)-independent pathways in fMLP induced NADPH oxidase activation. Both fMLP-stimulated phosphorylation of serine residues in p47phox and its translocation to the plasma membrane were suppressed by low-dose okadaic acid. On the other hand, PMA-induced phosphorylation and translocation of p47phox were not affected by such a low dose of okadaic acid. These findings suggested that fMLP induced phosphorylation of serine residues in p47phox was regulated by protein phosphatase 2A, and its phosphorylation was necessary for translocation and superoxide generation in fMLP-activated human neutrophils.
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PMID:Involvement of protein phosphatase 2A in PKC-independent pathway of neutrophil superoxide generation by fMLP. 865 38

Effects of the farnesylcysteine mimetic, farnesylthiosalicylate on the activation of myeloid cells were studied. In dimethyl-sulfoxide-differentiated HL60 cells and in human neutrophils farnesylthiosalicylate (< or = 20 microM) dose-dependently elevated cytosolic Ca2+ concentrations, suggesting phospholipase-C-mediated release of the ion from intracellular stores. In human neutrophils, in addition to the production of inositol trisphosphate, farnesylthiosalicylate induced activation of the NADPH oxidase and translocation of the cytosolic oxidase components p47-phox and p67-phox to the membrane. The calcium signal, inositol-trisphosphate production and superoxide generation elicited by farnesylthiosalicylate were partially blocked by treatment of the cells with pertussis toxin, consistent with participation of pertussis-toxin-sensitive and pertussis-toxin-resistant elements. In HL60 cells, farnesylthiosalicylate (< or = 20 microM) did not activate NADPH oxidase but dose-dependently augmented PMA-elicited activity of the enzyme. This effect was resistant to pertussis-toxin treatment. In vitro augmentation of PKC-mediated phosphorylation of histone and cytosolic p47-phox by farnesylthiosalicylate and the finding that downregulation of PKC abrogated potentiation of NADPH oxidase activity by farnesylthiosalicylate were compatible with the involvement of PKC in the response of HL60 cells to farnesylthiosalicylate. It is suggested that the effects of farnesylthiosalicylate on myeloid cells reflect interaction of the analog with prenylcysteine-docking sites on cellular signaling elements.
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PMID:Activation of signaling pathways in HL60 cells and human neutrophils by farnesylthiosalicylate. 902 78

The effect of race-specific elicitors on NADPH oxidase was examined in vivo by treating tomato cells with elicitor-containing intercellular fluids prepared from infected tomato leaves inoculated with specific Cladosporium fulvum races. Treatment of Cf-4 or Cf-5 cells with intercellular fluids from incompatible but not from compatible races of C. fulvum increased oxidase activity and the amount of p67-phox, p47-phox, and rac2 in the plasma membrane. Comparison of these three components in the cytosol and plasma membrane indicated that elicitors promoted the translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells carrying the appropriate resistance gene. Protein kinase C activators and inhibitors did not affect enzyme activity or the binding of these three components to the plasma membrane. In contrast, staurosporine, calmodulin antagonists, and EGTA inhibited elicitor-induced oxidase activity and the translocation of the cytosolic components. The assembly process involves a Ca(2+)-dependent protein kinase that catalyzes the phosphorylation of p67-phox and p47-phox, facilitating their translocation to the plasma membrane. Our data suggest that although both plants and animals share common elements in eukaryotic signal transduction, the involvement of different protein kinases mediating the activation of phosphorylation of p67-phox and p47-phox may reflect the unique spatial and temporal distribution of signal transduction pathways in plants.
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PMID:Race-specific elicitors of Cladosporium fulvum promote translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells. 906 55

Activation of the neutrophil NADPH oxidase requires translocation of cytosolic proteins p47(phox), p67(phox), and Rac to the plasma membrane or phagosomal membrane, where they assemble with membrane-bound flavocytochrome b. During this process, it appears that p47(phox) undergoes conformational changes, resulting in the exposure of binding sites involved in assembly and activation of the oxidase. In the present study, we have directly evaluated activation-induced conformational changes in p47(phox) using tryptophan fluorescence and circular dichroism spectroscopy. Treatment of p47(phox) with amphiphilic agents known to activate the NADPH oxidase (SDS and arachidonic acid) caused a dose-dependent quenching in the intrinsic tryptophan fluorescence of p47(phox), whereas treatment with a number of other amphiphilic agents that failed to activate the oxidase had no effect on p47(phox) fluorescence. In addition, the concentration range of activating agents required to induce changes in fluorescence correlated with the concentration range of these agents that induced maximal NADPH oxidase activity in a cell-free assay system. We next determined if activation by phosphorylation caused the same type of conformational changes in p47(phox). Protein kinase C phosphorylation of p47(phox) in vitro resulted in comparable quenching of fluorescence, which also correlated directly with NADPH oxidase activity. Finally, the circular dichroism (CD) spectrum of p47(phox) was significantly changed by the addition of SDS, whereas treatment with a non-activating detergent had no effect on the CD spectrum. These results support the conclusion that activation by amphiphilic agents results in changes in the secondary structure of p47(phox). Thus, our studies provide direct evidence linking conformational changes in p47(phox) to the NADPH oxidase activation/assembly process and also further support the hypothesis that amphiphile-mediated activation of the NADPH oxidase induces changes in p47(phox) that are similar to those mediated by phosphorylation in vivo.
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PMID:Analysis of activation-induced conformational changes in p47phox using tryptophan fluorescence spectroscopy. 936 11


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