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年的研究证明了花生四烯酸(AA)级联的P450花生四烯酸(AA)单加氧酶分支的产物和酶的生化和功能相关性。这一代谢途径的大部分产物已经被合成和功能表征，2C和4A P450亚型被鉴定为大鼠、小鼠和人肾脏中主要的AA环氧酶和omega/omega-1羟基酶。现在，合成化学、蛋白质化学和重组DNA技术提供了高效和常规的途径，可以获得大多数P450二十烷类化合物、特定的抑制剂、EET和HETE类似物、拮抗剂和激动剂、纯化的P450亚型、P450抗体、cDNA以及编码AA环氧合酶和omega/omega-1羟基酶的质粒和病毒载体。为了支持项目1-6并优化生产互动和资源利用，核心B将继续应用已建立的二十烷类化合物提取、纯化、高效液相分析、GC/MS、LC/MS、蛋白质纯化和重组DNA操作的方法，用于：a)生物样品中二十烷类化合物的检测和定量，b)细胞、亚细胞或纯化蛋白孵育产生的代谢物的生化特征，c)合成标准、特定抑制剂、激动剂和拮抗剂的储存、纯化和记录，d)免疫特异性探针的储存和记录，e)重组酶的部分纯化，以及f)扩增，克隆的cDNA的提纯和记录。这个 将这些例行任务集中在核心B中，消除了不必要和昂贵的重复，提高了重复性，并为项目1-6提供了高效和及时地获得合成标准、生物特异性探针和现代生物分析技术的机会。