The role of the P450 arachidonic acid (AA) monooxygenase as a major pathway for the metabolism of endogenous AA is now well established, as is the functional relevance of its products and of the enzymes responsible for their biosynthesis. Studies with purified proteins, genetic models of hypertension, or mice carrying disrupted P450 genes have identified members of the CYP2C and CYP4A gene subfamily of P450s as the predominant, and functionally relevant AA epoxygenase and omega hydroxylases in the rat, mouse, and human kidney, respectively. Synthetic chemistry, protein chemistry, and recombinant DNA techniques provide now efficient and routine access to most P450 eicosanoids, specific inhibitors, EET and HETE analogs, antagonists and agonist, purified P450 isoforms, P450 antibodies, cDNAs, as well as plasmid and viral vectors coding for CYP2C AA epoxygenase and CYP 4A omega-hydroxylases. In support of projects 1- 5 and, to optimize productive interactions and resources utilization. Core B will continue to apply established methods of eicosanoid extraction, purification, HPLC analysis, UPLC/MS/MS characterization, protein purification, and recombinant DNA manipulation, for: a) the detection and quantification of eicosanoids in biological samples, b) the biochemical characterization of metabolites generated by cellular, subcellular, or purified protein incubates, c) the storage, purification, and documentation of synthetic standards, specific inhibitors, agonist, and antagonists, d) the storage and documentation of immunospecific probes, e) the partial purification of recombinant enzymes, and f) the amplification, purification and documentation of cDNAs probes. The centralization of these routine tasks in Core B eliminates unnecessary and costly duplications, improves reproducibility, and provide projects 1-5 with efficient and timely access to synthetic standards, biospecific probes and state of the art bioanalytical techniques.
Core B serves important roles in the optimization of resources, as well as a vehicle for the productive exchange of ideas and experiences. As the depository of methods and experimental tools, the Core plays an important function in facilitating interactions and promoting effort coordination among the Program Project scientists.
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