Core--Analytical and Biomolecular

核心--分析与生物分子

• 批准号: 7459645
• 负责人: JORGE CAPDEVILA
• 金额: $ 20.55万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7459645
• 关键词: Agonist; Alkane 1-monooxygenase; Antibodies; Arachidonic Acids; Binding; Biochemical; Biological; Code; Cytochrome P450; Detection; Documentation; Eicosanoids; Enzymes; Funding; High Pressure Liquid Chromatography; Human; Hydroxyeicosatetraenoic Acids; Incubated; Isotopes; Kidney; Libraries; Literature; Maintenance; Mass Spectrum Analysis; Metabolic; Metabolic Pathway; Methodology; Methods; Mixed Function Oxygenases; Molecular Biology; Mus; Numbers; Organ; Phase; Plasmids; Principal Investigator; Productivity; Protein Chemistry; Protein Isoforms; Proteins; Publishing; Quality Control; Radiolabeled; Rattus; Recombinant DNA; Recombinants; Reproducibility; Research Personnel; Resources; Sampling; Standards of Weights and Measures; Subcellular Fractions; Synthesis Chemistry; Techniques; Time; Viral; Viral Vector; analog; cell preparation; expression vector; improved; inhibitor/antagonist; liquid chromatography mass spectrometry; programs; protein purification; radiotracer

Studies of the last 15 years have documented the biochemical and functional relevance of the products and enzymes of the P450 arachidonic acid (AA) monooxygenase branch of the AA cascade. Most of the products of this metabolic pathway have been synthesized and functionally characterized, and 2C and 4A P450 isoforms identified as the predominant AA epoxygenase and omega/omega-1 hydroxylases in the rat, mouse, and human kidney. 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 AA epoxygenase and omega/omega-1 hydroxylases. In support of projects 1-6 and, to optimize productive interactions and resources utilization, Core B will continue to apply established methods of eicosanoid extraction, purification, HPLC analysis, GC/MS, LC/MS, 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 cloned cDNAs. The centralization of these routine tasks in Core B eliminates unnecessary and costly duplications, improves reproducibility, and provides projects 1-6 with efficient and timely access to synthetic standards, biospecific probes and modern bioanalytical techniques.

过去15年的研究记录了AA Cascade的P450蛛网膜酸（AA）单氧酶分支的产物和酶的生化和功能相关性。该代谢途径的大多数产物已合成并在功能上表征，2C和4A P450同工型被鉴定为大鼠，小鼠和人类肾脏中主要的AA Epoxygygany酶和Omega/Omega-1羟化酶。合成化学，蛋白质化学和重组DNA技术现在可为大多数p450类花生类固醇，特定的抑制剂，EET和HETE类似物，拮抗剂和激动剂，纯化的p450同工型，p450抗体，p450抗体，cdnas和cdnas以及质子和元素编码的p450抗体，纯化的p450同种类似物提供有效，常规的访问权限羟化酶。为了支持项目1-6，并为了优化生产性的互动和资源利用，核心B将继续应用既定的类eicosanoid提取，纯化，HPLC分析，GC/MS，LC/MS，蛋白质纯化和重组DNA操纵的既定方法细胞，亚细胞或纯化的蛋白孵化，c）合成标准标准，特定抑制剂，激动剂和拮抗剂的储存，纯化和文献，d）免疫特异性探针的储存和文献，e）部分纯化重组酶的部分纯化，以及cdn cdn cdn cdn cdn cdn cdn cdn cdn cdn cdn。这 这些常规任务在核心B中的集中化消除了不必要和昂贵的重复，改善了可重复性，并为项目提供了有效且及时访问合成标准，生物特异性探针和现代生物分析技术的能力。