The neutrophil carries out its primary function of destroying foreign cells by a complex sequence that begins with binding of a stimulus to membrane receptors initiating membrane and cytoplasmic events that culminate in the activation of contractile elements, oxidative metabolism and degranulation. Microbicidal activity is achieved by the formation of reduced oxygen derivatives and secretion of granule enzymes. We will continue a detailed investigation of the process of activation of the metabolic burst in human neutrophils. The two major areas of study are the role of the Mol glycoproteins in initiation and regulation of the oxidative burst and characterization of the location, activation, and function of the superoxide-forming NADPH oxidase. We will compare O2 metabolism in normal cells, Mol-deficient cells, and normal cells treated with antibodies to Mol. The location, membrane orientation, and cytoskeletal association of Mol will be determined and its role in cytoskeleton- dependent events assessed. In studies of NADPH oxidase we will consider the involvement, subcellular location, and stimulus- indiced translocation of components such as cytochromeb559, flavoproteins, and quinones. Our recently described subcellular arachidonate-activatable oxidase system reconstituted from defined organelles of fractionated unstimulated cells will be characterized. Activation mechanisms will be defined considering roles for Mg2+, Ca2+, adenine and guanine nucleotides, N proteins, phospholipases, and protein kinases. A Mg2+-dependent cytosolic cofactor will be purified, characterized structurally, and used for antibody generation. Biosynthesis, processing and genetic regulation of this cofactor will be studied, cDNA probes developed, and the structural gene cloned. Membrane-associated oxidase components will be purified by detergent solubilization and substrate affinity chromatography. The proposed studies will elucidate cellular and molecular mechanisms of activation of phagocytes and provide valuable probes for further studies of stimulus-response coupling and clinical syndromes of neutrophil dysfunction.
| El Jamali, Amina; Valente, Anthony J; Clark, Robert A (2010) Regulation of phagocyte NADPH oxidase by hydrogen peroxide through a Ca(2+)/c-Abl signaling pathway. Free Radic Biol Med 48:798-810 |
| Lambeth, J David; Krause, Karl-Heinz; Clark, Robert A (2008) NOX enzymes as novel targets for drug development. Semin Immunopathol 30:339-63 |
| El Jamali, Amina; Valente, Anthony J; Lechleiter, James D et al. (2008) Novel redox-dependent regulation of NOX5 by the tyrosine kinase c-Abl. Free Radic Biol Med 44:868-81 |
| Valente, Anthony J; Zhou, Qing; Lu, Zhenhua et al. (2008) Regulation of NOX1 expression by GATA, HNF-1alpha, and Cdx transcription factors. Free Radic Biol Med 44:430-43 |
| Qin, Bin; Cartier, Laetitia; Dubois-Dauphin, Michel et al. (2006) A key role for the microglial NADPH oxidase in APP-dependent killing of neurons. Neurobiol Aging 27:1577-87 |
| He, Weijing; Qiang, Mei; Ma, Wuqiong et al. (2006) Development of a synthetic promoter for macrophage gene therapy. Hum Gene Ther 17:949-59 |
| Banfi, Botond; Malgrange, Brigitte; Knisz, Judit et al. (2004) NOX3, a superoxide-generating NADPH oxidase of the inner ear. J Biol Chem 279:46065-72 |
| Banfi, Botond; Tirone, Fabiana; Durussel, Isabelle et al. (2004) Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5). J Biol Chem 279:18583-91 |
| Banfi, Botond; Clark, Robert A; Steger, Klaus et al. (2003) Two novel proteins activate superoxide generation by the NADPH oxidase NOX1. J Biol Chem 278:3510-3 |
| Li, Sen-Lin; Valente, Anthony J; Qiang, Mei et al. (2002) Multiple PU.1 sites cooperate in the regulation of p40(phox) transcription during granulocytic differentiation of myeloid cells. Blood 99:4578-87 |
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