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)

Failure of a superoxide generating system, the NADPH oxidase, present in neutrophils and other phagocytes gives rise to chronic granulomatous disease (CGD), a group of single-gene inherited disorders all characterized by an extreme susceptibility to pyogenic infection, with potentially fatal consequences. About 30% of CGD cases are caused by an autosomally inherited deficiency of a 47-Kd cytoplasmic component of the oxidase (p47-phox). Epstein-Barr virus (EBV) immortalized B-lymphocyte lines established from these CGD patients also express this NADPH oxidase defect and consequently are rendered incapable of generating superoxide on stimulation. We have used a p47-phox-deficient EBV-transformed B-cell line as a recipient for retroviral transfer of a functional p47-phox cDNA. The presence and activity of the retrovirally encoded p47-phox in the transduced cells is demonstrated and we show that this restores their capacity to generate superoxide.
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PMID:Restoration of superoxide generation to a chronic granulomatous disease-derived B-cell line by retrovirus mediated gene transfer. 132 10

The effect of cyclosporine on PMA-stimulated superoxide production has been studied on human alveolar macrophages, human neutrophils, cytoplasts and Epstein-Barr-virus-transformed B lymphocytes. Cyclosporine inhibits superoxide production in alveolar macrophages but not in neutrophils and cytoplasts. The respiratory burst of B-lymphocytes was scarcely inhibited by cyclosporine. The activity of NADPH oxidase from macrophages and neutrophils was not directly affected by cyclosporine. These data are considered in relation with the proposed mechanism for cyclosporine action and the stimulation of the respiratory burst.
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PMID:Differential effect of cyclosporine A on respiratory burst by several types of human leukocytic cells. 165 23

Chronic granulomatous disease (CGD) can result from any of four single gene defects involving components of the superoxide (O2-.)-generating phagocyte NADPH oxidase (phox). The phox transmembrane flavocytochrome b558 is composed of two peptides, gp91phox and p22phox. Mutations of gp91phox cause X-linked CGD, whereas mutations of p22phox cause one of the three autosomal recessive forms of CGD. We used the Maloney leukemia virus-based MFG retrovirus vector to produce replication defective retroviruses encoding gp91phox or p22phox. To maximize viral titer MFG retroviruses do not contain internal promoter or resistance elements. Epstein-Barr virus transformed B-lymphocyte cell lines (EBV-B) derived from normal individuals contain phox components and produce O2-., whereas those derived from CGD patients show the CGD defect. Transduction of gp91phox or p22phox-deficient CGD EBV-B lines resulted in correction of O2-. production from a barely detectable baseline to an average 7.2% and 13.8% of normal control, respectively, without any selective regimen to enrich for transduced cells. CD34+ hematopoietic progenitor cells, the therapeutic target for gene therapy of CGD, were isolated from peripheral blood of CGD patients, transduced with MFG-phox retroviruses, and differentiated in culture to mature phagocytes. Transduction of progenitors corrected the gp91phox (seven patients) and p22phox (two patients) CGD phagocyte oxidase defect to 2.5% and 4.9% of normal O2-. production, respectively, representing an 87-fold and 161-fold increase. These studies show correction of flavocytochrome b558-deficient CGD in primary hematopoietic progenitors, providing a basis for development of gene therapy for the X-linked gp91phox and autosomal p22phox-deficient forms of CGD.
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PMID:CD34+ peripheral blood progenitors as a target for genetic correction of the two flavocytochrome b558 defective forms of chronic granulomatous disease. 751 18

We established the system to detect superoxide produced by Epstein Barr virus lymphoblastoid cell line (EB-LCL). Superoxide production of EB-LCL was evaluated by measuring chemiluminescence (CL) enhanced with addition of horseradish peroxidase (HRP). Using this system, we measured CL of EB-LCL established from 13 patients with chronic granulomatous disease (CGD) and 8 normal individuals. Significant elevation of CL was observed in all control EB-LCLs, however, no remarkable CL was seen in any patients' EB-LCLs. We examined the effect of recombinant human interferon gamma (rh-IFN-gamma) and granulocyte colony stimulating factor (G-CSF) on CL of EB-LCL in vitro. With addition of rh-IFN-gamma, CL of normal control EB-LCL was significantly enhanced (p < 0.05), on the other hand, G-CSF was shown to have no effect. No significant CL was observed in any CGD patients' EB-LCLs even with addition of rh-IFN-gamma or G-CSF. It was suggested that superoxide produced by EB-LCL detected in this system was dependent on the same NADPH oxidase system which presents in phagocyte.
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PMID:[Establishment of superoxide production assay system using Epstein-Barr virus transformed cell line with chemiluminescence]. 755 50

Epstein-Barr virus-transformed lymphocytes generate superoxide in response to various agonists in an enzymatic reaction similar to that which occurs in stimulated phagocytes. We generated transformed B lymphoblast cell lines from controls, from four patients with p47-phox-deficient chronic granulomatous disease, and from three parents. The cells from controls and from the parents generated 7.0-35 nmol of O2-/10(7) cells per 30 min in response to phorbol myristate acetate. None of the patient cell lines generated any detectable superoxide. Both p47-phox and p67-phox were detected by immunoblot in the cytosol of control and parent cell lines and, as in neutrophils, these proteins had affinity for GTP-agarose. The patients' cell lines contained no detectable p47-phox by immunoblot. mRNA for both cytosolic proteins was detected in all cell lines. We generated cDNA and obtained multiple clones from two patients by polymerase chain reaction. One patient was a compound heterozygote with each allele resulting in an early stop codon. Clones derived from the other patient demonstrated only a GT deletion at base 75. The cDNA for p47-phox was inserted into an EBV-expression vector and stably transfected cell lines were obtained using hygromycin B selection. Transfected cell lines from a p47-phox-deficient patient generated normal levels of superoxide and had readily detectable cytosolic p47-phox. Thus, B lymphoblasts provide an excellent model system for studies of the NADPH oxidase, for expression of functional recombinant forms of oxidase components, and for initial experimental approaches to genetic reconstitution in CGD.
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PMID:In vitro molecular reconstitution of the respiratory burst in B lymphoblasts from p47-phox-deficient chronic granulomatous disease. 767 2

The NADPH oxidase of phagocytes is known to be expressed in Epstein-Barr-virus-transformed B-lymphocytes, albeit at levels only approx. 5% of those found in neutrophils. We have investigated the basis of this low level of expression and find that all four specific components of the NADPH oxidase are expressed in B-lymphocytes, but only p47-phox protein attains levels equivalent with those found in neutrophils. This component was shown to phosphorylate and translocate to the membrane normally on activation. The other cytosolic component, p67-phox, did show a deficit, and by supplementing a B-cell cytosol extract with recombinant p67-phox, this was shown to account for the somewhat reduced activity of B-cell cytosol in a cell-free oxidase system. The cell-free analysis also clearly located the major deficiency in superoxide-generating capacity of B-lymphocytes to the membrane. Western blotting of membrane proteins revealed major reductions in the amount of cytochrome b558. Analysis of the levels of mRNA for both subunits of cytochrome b558, however, showed levels greater than expected. Significantly more mRNA for gp91-phox was present in B-cells than in undifferentiated HL60 cells, although it was not quite as abundant as in differentiated HL60 cells, which are capable of producing large amounts of superoxide. We conclude that the failure of B-lymphocytes to generate amounts of superoxide equivalent to those generated by neutrophils is primarily due to a post-transcriptionally determined block to the accumulation of cytochrome b558.
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PMID:Low NADPH oxidase activity in Epstein-Barr-virus-immortalized B-lymphocytes is due to a post-transcriptional block in expression of cytochrome b558. 786 1

Rap1A is a GTP-binding protein of the Ras superfamily that is highly abundant in phagocyte membranes. Although Rap1A copurifies with cytochrome b558, a component of the superoxide-generating NADPH oxidase complex of human phagocytes and B lymphocytes, the involvement of Rap1A in the regulation of the oxidative burst in these cells has not been clearly established. Therefore, we have stably transfected human Epstein-Barr virus-transformed B lymphocytes that possess an activable NADPH oxidase complex with cDNAs for mutants of Rap1A "locked" in a GTP-bound (63E) and GDP-bound (17N) state. Both the 17N and 63E mutants of Rap1A inhibited phorbol ester-stimulated O2-. production by 50 and 80%, respectively, while transfection with cDNA for wild-type Rap1A had no effect on the respiratory burst. No effects of the Rap1A mutants on cell viability, proliferation, expression of cell-surface markers, or phorbol 12-myristate 13-acetate-stimulated interleukin-8 generation were detected. These data demonstrate that Rap1A is a regulator of O2-. formation in intact cells. Furthermore, the inhibitory effect of both GTP- as well as GDP-bound mutants indicates that Rap1A functions in a dynamic cycle as opposed to a unidirectional pathway, as is the case for the other NADPH oxidase regulatory GTP-binding protein, Rac.
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PMID:Activated or dominant inhibitory mutants of Rap1A decrease the oxidative burst of Epstein-Barr virus-transformed human B lymphocytes. 803 26

Failure of a superoxide generating system, the NADPH oxidase, present in neutrophils and other phagocytes gives rise to chronic granulomatous disease (CGD), a group of single gene inherited disorders all characterised by an extreme susceptibility to pyogenic infection, with potentially fatal consequences. About 30% of CGD cases are due to an autosomally inherited deficiency of a 47 kDa cytoplasmic component of the oxidase (p47-phox). Epstein-Barr virus (EBV) immortalised B-lymphocyte lines established from these CGD patients also express this NADPH oxidase defect and consequently are rendered incapable of generating superoxide on stimulation. We have utilised a p47-phox-deficient EBV-transformed B cell line as a recipient for retroviral transfer of a functional p47-phox cDNA. The presence and activity of the retrovirally encoded p47-phox in the transduced cells is demonstrated and we show that this restores their capacity to generate superoxide.
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PMID:Chronic granulomatous disease: towards gene therapy. 816 28

Src homology 3 (SH3) domains direct cellular localization and signal transduction through specific protein-protein interactions with proline-rich target sequences. The two SH3 domain in p67-phox, a cytosolic component of the phagocyte NADPH oxidase system, may mediate interactions within the oxidase complex and direct its translocation to membranes. The requirement for SH3 domains in p67-phox was studied both in cell-free and whole cell oxidase assay systems. The amino-terminal domain of p67-phox (amino acids 1-246) that lacks both SH3 domains was active in vitro. Various forms of p67-phox lacking one or both SH3 domains were produced in whole cells using episomal expression vectors to stably transfect p67-phox-deficient Epstein-Barr virus-B cells derived from chronic granulomatous disease patients. Complete restoration of NADPH oxidase activity was achieved with full-length p67-phox cDNA expression. Deletion of either SH3 domain resulted in dramatic reductions of NADPH oxidase activity relative to corrected transfected cells, which correlated with decreases in membrane binding. Deletion of both SH3 domains completely abolished p67-phox membrane binding and oxidase activity. Thus, in contrast to oxidase reconstitution in a cell-free system, we observed a requirement for both SH3 motifs for restoration of oxidase activity and binding of p67-phox to membranes.
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PMID:Role of p67-phox SH3 domains in assembly of the NADPH oxidase system. 820 39

The O2- generating NADPH oxidase of human Epstein-Barr virus immortalized B lymphocytes (EBV-B lymphocytes) and the NADPH oxidase of human neutrophils were compared. The capacity of the oxidase of EBV-B lymphocytes to generate O2- is 100-fold less than that of neutrophils. Like the oxidase of neutrophils, the oxidase of EBV-B lymphocytes is decreased or abolished in chronic granulomatous disease (CGD). Activation of neutrophil oxidase in an heterologous cell-free system, using human neutrophil membranes and EBV-B lymphocyte cytosol from healthy and CGD patients, combined with immunoblotting investigations of the cytosolic activating factors p47 and p67 involved in O2- production, suggests that neutrophils and EBV-B lymphocytes possess similar complements of cytosolic factors p47 and p67. Cytochrome b -245, the major membrane redox component of the O2- generating oxidase, is only slightly expressed in the membrane of EBV-B lymphocytes. A sensitive and specific immunocytochemical method for detection of the two subunits of cytochrome b -245 is described; it shows that both subunits are virtually absent in EBV-B lymphocytes from CGD patients deficient in the large subunit.
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PMID:The O2- generating oxidase of B lymphocytes: Epstein-Barr virus-immortalized B lymphocytes as a tool for the identification of defective components of the oxidase in chronic granulomatous disease. 839 41


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