Browsing by keyword "*NADPH Oxidase"
Now showing items 1-2 of 2
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Mutations in the promoter region of the gene for gp91-phox in X-linked chronic granulomatous disease with decreased expression of cytochrome b558We examined the molecular defect in two kindreds with "variant" X-linked chronic granulomatous disease (CGD). Western blots of neutrophil extracts showed decreased immunoreactive cytochrome b558 components gp91-phox and p22-phox. Analysis of mRNA demonstrated reduced gp91-phox transcripts, with relative preservation of an alternative mRNA species created by transcription initiation in the third exon of the gene. Single strand conformation polymorphism analysis of the 5' flanking region of the patients' gp91-phox genes revealed an electrophoretic abnormality not detected in 40 other gp91-phox genes. Genomic sequencing demonstrated a single base change associated with CGD in each kindred: in one, adenine to cytosine at base pair-57 and in the other, thymidine to cytosine at -55. These mutations are located between the "CCAAT" and "TATA" box consensus sequences involved in eukaryotic gene transcription. Gel shift assays revealed two specific DNA-protein complexes formed between phagocyte nuclear extracts and an oligonucleotide probe representing bases -31 to -68 of the gp91-phox promoter region; the faster-migrating complex could not be formed with oligonucleotides containing either of the promoter mutations. Thus, these promoter region mutations appear to be causally related to the loss of association of a DNA-binding protein and lead to diminished gp91-phox expression, abnormal transcription initiation, and the development of CGD.
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Regulation of the human cellular glutathione peroxidase gene during in vitro myeloid and monocytic differentiationWe have used the HL-60 and PLB-985 myeloid leukemia cell lines to examine the regulation of expression of the important intracellular antioxidant enzyme, glutathione peroxidase (GSH-Px), during phagocytic cell differentiation in vitro. Induction of differentiation along the monocytic pathway by phorbol ester results in an approximately twofold rise in enzyme activity and a parallel increase in the rate of 75Se incorporation into immunoprecipitable GSH-Px protein. Induction along the granulocytic pathway by dimethyl formamide (DMF) results in similar changes in steady-state enzyme levels and rates of GSH-Px protein synthesis. Steady-state levels of GSH-Px gene transcripts also increase more than twofold, approximately in parallel with the enzyme levels. Nuclear run-on transcription assays of GSH-Px mRNA synthesis show ratios of induced to uninduced transcript levels of 2.24 and 1.59 with phorbol myristate acetate (PMA) induction and DMF, respectively, in HL-60 cells, and ratios of 1.34 and 3.46 with PMA and DMF, respectively, in PLB-985 cells. Half lives of GSH-Px mRNA are unchanged or slightly shorter after differentiation of HL-60 cells, and slightly longer after induction of PLB-985. Overall, the present studies show that GSH-Px activity rises during in vitro-induced monocytic or granulocytic differentiation of myeloid cell lines and that the increased expression of the cellular GSH-Px gene occurs through complex mechanisms that include transcriptional up-regulation. This pattern contrasts with the nearly complete cotranslational regulation of GSH-Px expression by exogenous selenium.
