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)

To understand the expression of NADPH oxidase components during neutrophil maturation, we examined the expression of mRNAs and proteins for NADPH oxidase components, and the superoxide-producing activity using HL-60 cells incubated with dimethyl sulfoxide (DMSO). Northern blot and Western blot analyses revealed that gp91(phox), p67(phox), and p47(phox) were expressed after myelocyte stages, whereas p22(phox), p40(phox), and rac-2 were expressed from the promyelocyte stage. Furthermore, immunocytochemical staining of DMSO-induced HL-60 cells indicated that gp91(phox), p67(phox), and p47(phox) were detected only after myelocyte stages (myelocytes, metamyelocytes, band cells, and segmented cells), whereas p22(phox), p40(phox), and rac-2 were detected from the promyelocyte stage. In addition, nitro blue tetrazolium (NBT) assay showed that superoxide could be produced after myelocyte stages but not produced before promyelocyte stages. Moreover, almost the same results as those with DMSO-induced HL-60 cells were obtained using human bone-marrow cells by immunocytochemical staining and NBT assay, except that p22(phox) was detected by immunocytochemical staining after myelocyte stages in bone-marrow cells. Together, these observations indicate that all the components for NADPH oxidase are expressed, and the superoxide-producing activity is obtained after myelocyte stages during neutrophil maturation.
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PMID:Evaluation of the expression of NADPH oxidase components during maturation of HL-60 cells to neutrophil lineage. 1094 66

Activation of phospholipase D (PLD) occurs in response to various stimuli and results from the activity of two isozymes, hPLD1 and hPLD2. PLD activity appears to be involved in several myeloid cell processes during their development and activation, including proliferation of myeloblasts in the bone marrow and secretion, phagocytosis and NADPH oxidase activation, essential functions of differentiated neutrophils. The present work studies PLD characteristics, activity and both isozyme expression during maturation and differentiation of myeloid cells by using three different systems: leukemic myeloblasts at different stages of maturation, terminally differentiated neutrophils ex vivo and four human myeloid cell lines, NB4, HL-60, PLB 985 and U937, induced to differentiate with alltrans retinoic acid (ATRA), a cyclic adenosine monophosphate (cAMP) analogue or both agents together. HL-60, a bipotential cell line has also been differentiated along the granulocytic pathway with DMSO and the monocytic pathway with 1,25-dihydroxy vitamin D3. In all these systems, PLD activity increases with maturation and differentiation whatever the inducer used and the granulocytic or monocytic pathways. Increase in basal activity which reflects the expression during development of both hPLD1 and hPLD2 appears to be mainly related to the former isozyme expression. Association of PLD characteristic changes with maturation and differentiation was also confirmed using two NB4 clones resistant to these processes. Comparison between PLD characteristics in myeloblasts during maturation and differentiation ex vivo and in vitro in the different cell lines demonstrated that NB4 induced to differentiate with ATRA represents the best model for further studies on the specific roles of each PLD isoform in various functions of differentiated myeloid cells.
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PMID:Modifications in phospholipase D activity and isoform expression occur upon maturation and differentiation in vivo and in vitro in human myeloid cells. 1118 1

The leucocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyses the reduction of oxygen to O(-)(2) at the expense of NADPH. The enzyme is dormant in resting neutrophils but becomes active when the cells are exposed to the appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(phox) becomes phosphorylated on several serines and migrates to the plasma membrane. Protein kinase CK2 is an essential serine/threonine kinase present in all eukaryotic organisms. The leucocyte NADPH oxidase subunit p47(phox) has several putative CK2 phosphorylation sites. In the present study, we report that CK2 is able to catalyse the phosphorylation of p47(phox) in vitro. Phosphoamino acid analysis of phosphorylated p47(phox) by CK2 indicated that the phosphorylation occurs on serine residues. CNBr mapping and phosphorylation of peptides containing the putative site of CK2 indicated that the main phosphorylated residues are Ser-208 and Ser-283 in the Src homology 3 (SH3) domains, and Ser-348 in the C-terminal domain of p47(phox). Dependence of phosphorylation on the conformation of p47(phox) is supported by the finding that p47(phox) undergoes better phosphorylation by CK2 in the presence of arachidonic acid, a known activator of NADPH oxidase which induces conformational changes in p47(phox). In addition, 5,6-dichloro-1-beta-o-ribofuranosyl benzimidazole, a CK2 inhibitor, potentiates formyl-Met-Leu-Phe-induced NADPH oxidase activity in DMSO-differentiated HL-60 cells. Taken together, we propose that CK2 is the p47(phox) kinase, and that phosphorylation of p47(phox) by CK2 regulates the deactivation of NADPH oxidase.
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PMID:Phosphorylation of the leucocyte NADPH oxidase subunit p47(phox) by casein kinase 2: conformation-dependent phosphorylation and modulation of oxidase activity. 1153 39

We studied whether reactive oxygen species (ROS) generated by normal colonic mucosa affect 5-hydroxytryptophan (5-HTP)-evoked 5-HT formation (measured as the sum of 5-HT plus 5-hydroxyindole acetic acid (5-HIAA) accumulation) of guinea pig's isolated colonic mucosa. Catalase (3000-6000 U/ml), a hydrogen peroxide (H2O2) scavenger or diphenylene iodonium (DPI, 10-100 microM), an NADPH oxidase inhibitor, concentration-dependently caused an increase of the sum of 5-HT plus 5-HIAA accumulation in the presence of 5-HTP (10 microM), but these drugs did not significantly affect the 5-HT-metabolite in the colonic mucosa measured as the ratio of 5-HIAA/5-HT. Exogenously applied H2O2 (10-100 microM) concentration-dependently inhibited the sum of 5-HT plus 5-HIAA accumulation. In contrast, neither superoxide dismutase (SOD, 100-300 U/ml), superoxide anion scavenger, nor dimetyl sulfoxide (1-5%, DMSO), a hydroxyl radical scavenger affected the sum of 5-HT plus 5-HIAA accumulation. Moreover, mucosa ROS generation was estimated using the chemiluminescence technique. SOD (100-300 U/ml), catalase (3000-6000 U/ml) or DPI (10-100 microM), concentration-dependently reduced luminol-enhanced chemiluminescence signal from the colonic mucosa, while allopurinol (10-100 microM), a xanthine oxidase inhibitor, did not affect the chemiluminescence signal. These results suggest that ROS is formed through an NADPH oxidase system in the guinea pig colonic mucosa, where it exerts a modulatory effect on mucosal 5-HT formation upon addition of 5-HTP. Thus, ROS formation from normal colonic mucosa could be considered to contribute to the control of 5-HT production in mucosa enterochromaffin cells.
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PMID:Modification of 5-hydroxytryptophan-evoked 5-hydroxytryptamine formation of guinea pig colonic mucosa by reactive oxygen species. 1185 70

The human promyelocytic HL60 cells acquired a neutrophilic phenotype after a 7- to 10-day DMSO treatment. Fc gammaRII was up-regulated. Fc gammaRI was also up-regulated by an additional IFN-gamma treatment. These cells are able to produce O2*- by NADPH oxidase activation in the presence of immune complexes or phorbol-12-myristate-13-acetate (PMA). A change of their PDE4 subtype profile was also observed: PDE4B was the predominant isoenzyme, PDE4D was down-regulated and PDE4A was no longer detectable. Additionally, the more NADPH oxidase was activated by PMA, the less PDE4A was expressed, suggesting that NADPH oxidase activity could be used as a surrogate marker of PDE4A down-regulation. Rolipram and Ariflo (cilomilast), two selective PDE4 inhibitors, dose-dependently inhibited receptor-coupled activation of superoxide. These results suggest that PDE4B is the main subtype involved in regulating superoxide induced by Fc gammaRs activation. Furthermore, these cells, expressing almost exclusively PDE4B subtype, could be useful to identify selective PDE4B inhibitors.
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PMID:DMSO-treated HL60 cells: a model of neutrophil-like cells mainly expressing PDE4B subtype. 1246 39

The epithelial cells that form a barrier lining the lung airway are key regulators of neutrophil trafficking into the airway lumen in a variety of lung inflammatory diseases. Although the lipid mediator leukotriene B(4) (LTB(4)) is known to be a principal chemoattractant for recruiting neutrophils to inflamed sites across the airway epithelium, the precise signaling mechanism involved remains largely unknown. In the present study, therefore, we investigated the signaling pathway through which LTB(4) induces transepithelial migration of neutrophils. We found that LTB(4) induces concentration-dependent transmigration of DMSO-differentiated HL-60 neutrophils and human polymorphonuclear neutrophils across A549 human lung epithelium. This effect was mediated via specific LTB(4) receptors and was inhibited by pretreating the cells with N-acetylcysteine (NAC), an oxygen free radical scavenger, with diphenylene iodonium (DPI), an inhibitor of NADPH oxidase-like flavoproteins, or with PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Consistent with those findings, LTB(4)-induced ERK phosphorylation was completely blocked by pretreating cells with NAC or DPI. Taken together, our observations suggest LTB(4) signaling to transepithelial migration is mediated via generation of reactive oxygen species, which leads to downstream activation of ERK. The physiological relevance of this signaling pathway was demonstrated in BALB/c mice, in which intratracheal instillation of LTB(4) led to acute recruitment of neutrophils into the airway across the lung epithelium. Notably, the response to LTB(4) was blocked by NAC, DPI, PD98059, or CP105696, a specific LTB(4) receptor antagonist.
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PMID:Transepithelial migration of neutrophils in response to leukotriene B4 is mediated by a reactive oxygen species-extracellular signal-regulated kinase-linked cascade. 1279 60

Sanguinarine (SA), a member of the benzo[c]phenanthridine isoquinoline alkaloids, has been shown to possess antimicrobial, anti-inflammatory, and antioxidant properties. We examined the effects of SA on oxidative burst in DMSO-differentiated HL-60 cells, an excellent model for studying oxidative burst. SA inhibited both N-formyl-Met-Leu-Phe (fMLP) and phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst with half-maximal concentration for inhibition (IC(50)) of 1.5 and 1.8 microM, respectively. Despite suggestions of SA antioxidant activity this inhibition cannot be ascribed to radical scavenging property of SA because the IC(50) for superoxide dismutase-like activity in a non-cellular system was 60 microM. TROLOX, a water-soluble vitamin E analog, had IC(50) of 3 microM in the same system. Moreover, cyclic voltammetry measurements show that SA is not an easily oxidizable species, with a peak anodic potential at 700 mV, as compared to TROLOX with peak anodic potential at 200 mV. On the other hand, TROLOX, when used in cell suspension, was much poorer inhibitor of oxidative burst than SA. When testing direct effect of SA on NADPH oxidase in the post-granular fraction of disrupted cells, the IC(50) was found to be 8.3 microM. It is higher than that observed in whole cells, however, the shift may be ascribed to SDS effect on SA activity. We conclude the SA inhibition of oxidative burst is not caused by SA redox activity but most likely is a result of SA affecting the activity of NADPH oxidase directly and in part by preventing the formation of NADPH oxidase protein complex.
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PMID:Sanguinarine is a potent inhibitor of oxidative burst in DMSO-differentiated HL-60 cells by a non-redox mechanism. 1472 51

A novel isoform of the NOX-2 subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has been identified using expressed sequence tag (EST) database mining. The novel isoform, NOX-2S, is a splice variant of NOX-2 and includes a previously unidentified exon, mapped 6.4 kb downstream of exon III, and encodes an in-frame stop codon generating a predicted truncated protein of approximately 12.7 kDa, the smallest reported member of the NOX family. Thus, NOX-2S is predicted to have only two transmembrane domains, however, the new C-terminal sequence includes two new potential protein kinase C (PKC) phosphorylation sites. Expression of NOX-2S mRNA was detected in many mouse tissues, and several human cell lines including the myeloid cell line HL-60, and the B cell line Ramos, indicating that the splice variant is conserved in mouse and man. NOX-2S is found co-expressed together with NOX-2 in all of the tissues and cells under investigation, both nonphagocytic and phagocytic. Induction of the myeloid cell line HL-60 into the neutrophil phagocytic lineage by dimethyl sulphoxide (DMSO), led to a marked increase in NOX-2S and NOX-2 expression in the myelocyte rather than promyelocyte stages of differentiation. Furthermore, in the B-cell line Ramos, differentiated with the cytokine interferon-gamma (IFN-gamma), splicing was altered to increase NOX-2S mRNA generation over NOX-2. Here we have identified NOX-2S, the first reported normally occurring splice variant of NOX-2. The sequence identity between mouse and human NOX-2S strongly implies conservation in function and possibly a role for NOX-2S in the regulation of NADPH oxidase activity.
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PMID:NOX-2S is a new member of the NOX family of NADPH oxidases. 1519 96

Free radical formation has been investigated in diverse experimental models of LPS-induced inflammation. Here, using electron spin resonance (ESR) and the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, we have detected an ESR spectrum of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone radical adducts in the lipid extract of mouse skin treated with LPS for 6 h. The ESR spectrum was consistent with the trapping of lipid-derived radical adducts. In addition, a secondary radical-trapping technique using dimethyl sulfoxide (DMSO) demonstrated methyl radical formation, revealing the production of hydroxyl radical. Radical adduct formation was suppressed by aminoguanidine, N-(3-aminomethyl)benzylacetamidine (1400W), or allopurinol, suggesting a role for both inducible nitric oxide synthase (iNOS) and xanthine oxidase (XO) in free radical formation. The radical formation was also suppressed in iNOS knockout (iNOS(-/-)) mice, demonstrating the involvement of iNOS. NADPH oxidase was not required in the formation of these radical adducts because the ESR signal intensity was increased by LPS treatment in NADPH oxidase knockout (gp91(phox-/-)) mice as much as it was in the wild-type mouse. Nitric oxide (*NO) end products were increased in LPS-treated skin. As expected, the *NO end products were not suppressed by allopurinol but were by aminoguanidine. Interestingly, nitrotyrosine formation in LPS-treated skin was also suppressed by aminoguanidine and allopurinol independently. Pretreatment with the ferric iron chelator Desferal had no effect on free radical formation. Our results imply that both iNOS and XO, but neither NADPH oxidase nor ferric iron, work synergistically to form lipid radical and nitrotyrosine early in the skin inflammation caused by LPS.
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PMID:Free radical production requires both inducible nitric oxide synthase and xanthine oxidase in LPS-treated skin. 1653 16

The present study evaluates electron spin resonance (ESR) and the spin trapper 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) for analysis of superoxide radical production by human neutrophils interacting with viable Staphylococcus aureus and Staphylococcus epidermidis bacteria. To avoid auto-activation due to interaction with glass surfaces, neutrophils were preincubated in plastic tubes until the peak response was reached, and then transferred to a quartz flat cell to record the ESR spectra. The time point for peak response was identified by parallel analysis of the bacteria-neutrophil interaction using luminol amplified chemiluminescence. We found detectable ESR spectra from neutrophils interacting with as few as five bacteria of the weak activating S. epidermidis per neutrophil. Addition of the NADPH oxidase inhibitor diphenylene iodonium totally abolished spectra. Catalase, DMSO or an iron chelator had no impact on the produced spectra and ionomycin, a selective activator of intracellular NADPH oxidase, gave significant ESR spectra. Taken together, our results indicate that DEPMPO is cell permeable and detects NADPH oxidase derived superoxide anions formed in phagosomes or released by human neutrophils phagocytosing viable S. aureus and S. epidermidis. The technique may be used as a sensitive tool to evaluate superoxide anion production in human neutrophils.
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PMID:Evaluation of electron spin resonance for studies of superoxide anion production by human neutrophils interacting with Staphylococcus aureus and Staphylococcus epidermidis. 1760 11


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