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)

Alveolar macrophages (AM) from pathogen-free rabbits were unable to release reactive oxygen intermediates (ROI) unless they were conditioned in serum for 24-48 h before triggering with membrane-active agents. The degree of serum conditioning of AM depended upon the concentration of serum used; optimal ROI release was obtained at or above 7.5% fetal bovine serum (FBS). FBS, autologous rabbit serum, pooled rabbit serum, and pooled human serum were each capable of conditioning AM for release of ROI. Serum conditioning of AM requires synthesis of new protein(s); and the enzyme required for ROI production, NADPH oxidase, was only detectable in serum-conditioned cells. Moreover, serum-conditioned cells lost their ability to release ROI after transfer to serum-free medium, while cells maintained in serum-free medium acquired the capacity to release ROI after their transfer to serum-containing medium, demonstrating the reversibility of the phenomenon. Initial purification data indicate that conditioning is mediated by a discrete serum constituent, which precipitates 40-80% saturated ammonium sulfate, does not bind to Cibacron Blue columns, and has a molecular weight of 30,000 to 50,000, as determined by molecular exclusion chromatography. Unlike gamma interferon, which also enhances ROI release by macrophages, our serum-conditioning factor is not acid labile, retaining 67% of its activity after 120 min incubation at pH 2.0. Moreover, it does not appear to be a contaminating endotoxin, since LPS neither conditioned AM for ROI production, nor triggered ROI production by serum-conditioned AM. We propose that such a conditioning requirement may normally protect the lung against ROI-mediated tissue injury. However, during a pulmonary inflammatory reaction initiated by other mediator systems, the resulting transudation of plasma proteins into the alveolar spaces may condition AM in situ for ROI production.
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PMID:Serum factor requirement for reactive oxygen intermediate release by rabbit alveolar macrophages. 298 90

The effect of LPS on the respiratory burst in resident rat peritoneal macrophages has been examined. Rat macrophages secreted high levels of both O2- and H2O2 in response to triggering with phorbol esters, opsonized zymosan, and immune complexes. After culture in vitro with LPS these macrophages exhibited a marked diminution in their capacity to secrete high levels of respiratory burst products. The LPS-mediated loss of secretory activity was apparent after 2 hr of exposure to LPS and was inhibitable by polymyxin B in a dose-dependent fashion. The effect was not selective for any triggering agent type as inhibition of secretory activity occurred after triggering with PMA, zymosan and immune complexes. PGE2 added at levels secreted by the macrophages in response to LPS also inhibited respiratory burst product secretion. In addition, indomethacin prevented the LPS-mediated inhibition of secretion. Because the inhibition of secretion was common to all triggering agents tested, this suggested that the basis for the impaired secretion was at a level other than the receptor for the triggering agent. Both LPS and PGE2 treatment of the macrophages increased the Km of the oxidase for NADPH (1.7- to 2.3-fold) without affecting significantly the Vmax of the enzyme. These data suggest that stimulation of rat peritoneal macrophages by LPS results in an impaired ability to secrete respiratory burst products as a result of a PGE2-mediated decrease in NADPH oxidase affinity and that this alteration is independent of alterations in tumoricidal activity.
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PMID:Rat macrophage treatment with lipopolysaccharide leads to a reduction in respiratory burst product secretion and a decrease in NADPH oxidase affinity. 303 38

Mouse peritoneal macrophages respond to environmental stimuli in different ways depending on their state of differentiation. Macrophages from mice with bacillus Calmette--Guerin (BCG) infection produced large amounts of H2O2 in response to phorbol diesters (PDEs), while those from noninfected mice produced little or no H2O2. The effects of PDEs on cells are mediated by specific cellular receptors for these ligands. The purpose of this study was to determine if the varying responses of macrophages from different groups of mice were caused by differences in their receptors for the PDE ligands. By all parameters studied, the binding of [20-3H]phorbol 12,13-dibutyrate ( [3H]PDBu) was similar in all macrophages irrespective of their ability to produce H2O2 in response to PDEs. Binding of [3H]PDBu was rapid at 23 degrees C reaching a maximum at 10-20 min with a subsequent decline to 50-60% of maximum by 30-60 min. Binding was slower at 0 degrees C reaching a maximum at 90-120 min. The binding was reversible, with dissociation kinetics paralleling association kinetics. The binding was saturable; the Kd's (45 to 91 nM) and number of binding sites (about 7-14 X 10(5)/cell or 11-12 pmol/mg protein) were essentially the same for the different classes of macrophages. The binding was specific, and analogs of PDBu inhibited [3H]PDBu binding to macrophages with potencies comparable to their potencies in causing in vivo tumor promotion and elicitation of other cellular responses in vitro. The ligands [3H]PDBu and [3H]PMA were degraded to comparable degrees by macrophages from normal or BCG-infected mice. Macrophages from C3H/HeJ and C3H/HeN mice, although known to differ in their abilities to respond to stimuli such as lymphokines and LPS, did not differ in their ability to produce H2O2 in response to PDEs or in their receptors for PDEs. Results of this study suggest that in vivo "activation" of macrophages in mice infected with BCG is not associated with a change in the cells' receptors for PDEs, but may be associated with "postreceptor" changes such as linkage of the PDE receptor with NAD(P)H oxidase, a change in NAD(P)H oxidase, or induction of synthesis of NAD(P)H oxidase.
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PMID:Phorbol diester-induced H2O2 production by peritoneal macrophages. Different H2O2 production by macrophages from normal and BCG-infected mice despite comparable phorbol diester receptors. 630 16

Reactive oxygen intermediates (ROIs) play an important role in inflammatory processes as mediators of injury and potentially in signal transduction leading to gene expression. Cyclooxygenase (COX) is a rate-limiting enzyme in prostanoid biosynthesis, and its recently cloned inducible form, COX-2, is induced by proinflammatory cytokines. This study linked ROIs to the signaling pathways that induce COX-2 expression. The hydroxyl radical scavengers DMSO (1%), as well as di- and tetramethylthiourea, inhibited IL-1-, TNF alpha-, and LPS-induced COX-2 expression in rat mesangial cells. The suppression of COX-2 mRNA expression correlated with the COX-2 protein level. In comparison with the prolonged induction of the inducible gene encoding protein-tyrosine phosphatase by hydrogen peroxide, the COX-2 gene was only transiently induced. Protein-tyrosine phosphatase is also induced by heat shock and chemical stress, whereas COX-2 is not. Superoxide was a more potent inducer for COX-2 than hydrogen peroxide. In addition, NADPH stimulated COX-2 expression, and an inhibitor of NADPH oxidase blocked COX-2 expression induced by TNF alpha. COX-2 and KC gene expression costimulated by IL-1 were inhibited differentially by the scavengers. These studies demonstrate that oxidant stress is a specific and important inducer of COX-2 gene expression. This induction may contribute to the deleterious amplification of prostanoids in inflammation and compound the direct effects of ROI production.
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PMID:Involvement of reactive oxygen intermediates in cyclooxygenase-2 expression induced by interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide. 770 75

Reactive oxygen species (ROS) produced by NADPH oxidase activation in neutrophils play a major role in mediating sepsis-induced acute lung injury. To provide insight into whether the NADPH oxidase inhibitor apocynin might attenuate oxidant-induced lung injury, we examined the effect of apocynin on (1) sepsis-induced lung injury in guinea pigs, (2) ROS generation by LPS-stimulated neutrophils measured by chemiluminescence (CL), and (3) LPS-stimulated neutrophil-mediated human umbilical vein endothelial cell (HUVEC) injury assessed by 51Cr release. Sepsis-induced lung injury in guinea pigs was assessed by comparing 125I-labeled albumin concentrations in lung tissue and bronchoalveolar lavage (BAL) fluid relative to plasma (L/P and BAL/P), lung wet-to-dry weight ratios, and the number of neutrophils in BAL fluid. The lung wet-to-dry weight ratio, L/P, and the number of neutrophils in BAL fluid decreased after pretreatment and post-treatment with apocynin. BAL/P decreased upon pretreatment but not upon post-treatment with apocynin. Apocynin at concentrations from 10 to 100 micrograms/ml significantly reduced LPS-stimulated neutrophil CL and neutrophil-mediated HUVEC 51Cr release. We conclude that the NADPH oxidase inhibitor apocynin attenuates (1) sepsis-induced lung injury in guinea pigs, (2) neutrophil ROS generation measured by CL, and (3) neutrophil-mediated HUVEC injury assessed by 51Cr release.
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PMID:Effect of the NADPH oxidase inhibitor apocynin on septic lung injury in guinea pigs. 795 74

Small numbers of CD34+ primitive hematopoietic progenitors are found in normal human peripheral blood. These cells differentiate to myeloid or lymphoid lineage under the influence of growth factors. We investigated the effects of IL5 and other growth factors on the production of eosinophils from peripheral blood CD34+ cells. CD34+ cells were plated in agarose with different combinations of cytokines. At 14 days of growth a triple stain technique was used to identify eosinophil, monocyte and neutrophil colonies. IL5 alone did not support colony growth. In contrast GM-CSF and IL3 alone or together supported the generation of more than 50% eosinophil colonies. Addition of IL5 increased the fraction of eosinophil colonies to over 70%. Under the best conditions (IL3 + GM-CSF + IL5), the addition of interferon-a or LPS inhibited colony growth by 51% and 58%, respectively. Since IL5 alone did not support colony growth from CD34+ cells, we determined when IL5 responsive cells appeared in culture. Cells were grown initially with IL3 + GM-CSF, washed, and plated with IL5 alone. Only when progenitors were grown at least 3 days, could IL5 serve as the single growth factor supporting pure eosinophil colony growth (47 colonies/104 cells plated at day 3 and 134 colonies/104 cells at day 7). Growth of CD34+ in liquid culture for 28 days in the presence of IL3, GM-CSF and IL5 resulted in almost 250 fold increase in cell number, yielding a population of 83% maturing eosinophils. We used our culture system and the sensitive technique of RT-PCR to analyze the kinetics of production of mRNA transcripts encoding several eosinophil proteins. Freshly isolated CD34+ cells contained no eosinophil granule protein transcripts and barely detectable amounts of some oxidase protein transcripts. At day 3 of culture no cells recognizable by histochemical staining as eosinophils could be detected, but transcripts for all five eosinophil granule proteins were present. These transcripts increased several fold during the entire culture period. Similar kinetics were seen for the NADPH oxidase protein transcripts. These studies demonstrate that within 3 days of culture, peripheral blood CD34+ cells can become committed to the eosinophil lineage as demonstrated by responsiveness to IL5 and production of specific protein transcripts.
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PMID:Growth and differentiation of eosinophils from human peripheral blood CD 34+ cells. 902 88

Bacterial LPS is a pluripotent agonist for PMNs. Although it does not activate the NADPH-dependent oxidase directly, LPS renders PMNs more responsive to other stimuli, a phenomenon known as "priming." Since the mechanism of LPS-dependent priming is incompletely understood, we investigated its effects on assembly and activation of the NADPH oxidase. LPS pretreatment increased superoxide (O2-) generation nearly 10-fold in response to N-formyl methionyl leucyl phenylalanine (fMLP). In a broken-cell O2--generating system, activity was increased in plasma membrane-rich fractions and concomitantly decreased in specific granule-rich fractions from LPS-treated cells. Oxidation-reduction spectroscopy and flow cytometry indicated LPS increased plasma membrane association of flavocytochrome b558. Immunoblots of plasma membrane vesicles from LPS-treated PMNs demonstrated translocation of p47-phox but not of p67-phox or Rac2. However, PMNs treated sequentially with LPS and fMLP showed a three- to sixfold increase (compared with either agent alone) in plasma membrane-associated p47-phox, p67-phox, and Rac2, and translocation paralleled augmented O2- generation by intact PMNs. LPS treatment caused limited phosphorylation of p47-phox, and plasma membrane-enriched fractions from LPS- and/or fMLP-treated cells contained fewer acidic species of p47-phox than did those from cells treated with PMA. Taken together, these studies suggest that redistribution of NADPH oxidase components may underlie LPS priming of the respiratory burst.
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PMID:Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly. 943 18

In response to bacterial endotoxin (lipopolysaccharide, LPS) monocytes synthesize and express on their surface tissue factor (TF) which triggers the blood coagulation cascade. Since LPS stimulates active oxygen species production by these cells, we investigated the roles of superoxide anion and nitric oxide in the induction of TF in human blood monocytes. Scavengers of reactive oxygen intermediates such as N-acetyl cysteine or pyrrolidine dithiocarbamate were able to block TF induction. In addition, inhibition of NADPH oxidase and/or NO synthase which are major sources of active oxygen species in phagocytes also blocked TF induction. The restoration of TF expression, in monocytes treated with inhibitors of reactive oxygen production, by N,N'-dimethyl-gamma, gamma'-dipyridylium dichloride and/or sodium nitrosylpentacyanoferrate (III), which generate respectively O2- and NO, suggests that these two radicals participate in the induction of TF at the surface of blood monocytes stimulated by LPS.
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PMID:Role of oxygen radicals in tissue factor induction by endotoxin in blood monocytes. 948 74

Ethanol increases human and animal susceptibility to opportunistic lung infections in part by suppression of endotoxin (LPS) and bacteria-mediated upregulation of inducible nitric oxide synthase (iNOS) in alveolar macrophages (AM). LPS and cytokine-induced NOS mRNA are dependent on NF-kappaB/Rel (NFkappaB) and Activator Protein-1 (AP-1), which are regulated in turn by protein kinase C and tyrosine kinase-dependent phosphorylation. ETOH does not directly inhibit NFkappaB or AP-1, in vivo, but rather inhibits LPS-induced activation of the MEKK/MAP kinase system and inhibition of inhibitory protein IkappaBalpha required for formation of AP-1 and NFkappaB, respectively. in AM. Both transcription factors are involved iNOS mRNA transcription. LPS-induced upregulation of MEKK/MAP tyrosine kinase upregulates NADPH oxidase activity and oxygen free radical formation required for activation of NFkappaB and AP-1 and phosphorylation of IkappaBalpha. LPS downregulates endogenous calcium-sensitive PKC isozymes (PKCdelta), which repress iNOS mRNA expression. ETOH inhibits LPS-induced upregulation of iNOS mRNA by preventing its ability to decrease PKCdelta and upregulate tyrosine kinase-mediated phosphorylation. This effect of ETOH is prevented by inhibitors of PKC and tyrosine kinase. The data support the hypothesis that ETOH inhibits LPS-induced upregulation of iNOS mRNA by interfering with the phosphorylation processes involved in activation of the nuclear transcription factors NFkappaB and AP-1.
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PMID:Role of PKC and tyrosine kinase in ethanol-mediated inhibition of LPS-inducible nitric oxide synthase. 966 19

Trehalose dimycolate (TDM), a glycolipid present in the cell wall of Mycobacterium spp., is a powerful immunostimulant. We have developed an original model of macrophage activation where TDM is injected in vivo to prime peritoneal macrophages. These primed macrophages do not express inducible NO synthase (NOS II), however, they can be fully activated, i.e. induced to express NOS II and to develop a NOS II-dependent antiproliferative activity, following in vitro exposure to low concentrations of LPS. In a previous paper, we have shown that TDM-priming of mouse peritoneal macrophages is mediated by the sequential production of IL-12 and IFN-gamma. In the present paper, we investigated the role of TNF in the priming of macrophages by TDM. By semi-quantitative RT-PCR, we have shown that TDM injection induced transcription of TNF-alpha in peritoneal cells. TNF-mRNA levels peaked 5 hours after TDM injection and remained elevated for at least 32 hours. TNF expression was absolutely necessary for macrophage priming, as injection of an anti-TNF monoclonal antibody, 4 h before and 20 hours after TDM injection, prevented LPS-dependent activation of macrophages in vitro. This result was confirmed by the inability of TDM to prime macrophages from LT-alpha/TNF-alpha knockout (LT/TNFKO) mice. In addition, analysis of LT/TNFKO mice treated with TDM revealed that induction of the IL-12 transcript in their peritoneal cells and expression of a functional NADPH oxidase in macrophages are TNF-independent events.
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PMID:Tumor necrosis factor is required for the priming of peritoneal macrophages by trehalose dimycolate. 1058 20


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