SIGNAL TRANSDUCTION IN NEUTROPHIL MEDIATED HEART INJURY

中性粒细胞介导的心脏损伤中的信号转导

• 批准号: 2618335
• 负责人: JULIAN G. CAMBRONERO
• 金额: $ 9.7万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2618335
• 关键词: SIGNAL; TRANSDUCTION; NEUTROPHIL; MEDIATED; HEART

DESCRIPTION: (Adapted from the applicant's abstract) The overall goal of this proposal is to characterize specific signal transduction mechanisms that are activated after neutrophil stimulation and are involved in heart injury. Neutrophils release lethal oxygen radicals (such as superoxide anions) that are capable of exacerbating tissue damage, as encountered after the infiltration of these cells during post-ischemic myocardial necrosis and reperfusion injury. The enzyme phospholipase D (PLD) is central to the production of PA, a putative second messenger involved in the release of superoxide anions by neutrophils. The authors found that a PLD activity can be specifically immunoprecipitated in its active form with anti-phospho-tyrosine antibodies from cell lysates. The molecular weight of neutrophil PLD as well as the intracellular localization are different from those reported for other mammalian cells. Additionally, the PLD activity is found increased in adherent neutrophils in conditions in which superoxide release is activated. As a result, it is now hypothesized that a novel isoform of PLD in human neutrophils is regulated through tyrosine phosphorylation, which allows the enzyme to become active and synthesize PA, that in turn triggers the release of oxygen radicals. The Specific Aims of this proposal are: (1) Purify and characterize a novel granulocyte PLD isoform by column chromatography and by molecular biology techniques, using the sequenced purified protein (PY-PLD) as well as the existing mammalian PLD cDNA sequence (PLD1). (2) Determine the mechanism of granulocyte PLD regulation, particularly the phosphorylating kinase, the site(s) of phosphorylation and the role of small GTP-binding proteins, using immunoprecipitation with antibodies against epitope-tagged proteins and in vitro enzymatic assays. (3) Elucidate the role of PY-PLD in the NADPH oxidase system, by blocking superoxide release with PLD antibodies in vitro and in vivo. These studies will result in a clearer understanding of the oxidative processes involved in heart injury and suggest novel therapeutic interventions.

描述：(改编自申请人的摘要)总体目标 这项提议是为了描述特定的信号转导机制 在中性粒细胞刺激后被激活，并参与心脏 受伤。中性粒细胞释放致命的氧自由基(如超氧化物 阴离子)，能够加剧组织损伤，如在 缺血后心肌坏死过程中这些细胞的浸润 再灌注损伤。磷脂酶D(PLD)是 PA的产生，可能是参与释放的第二信使 中性粒细胞产生的超氧阴离子。作者发现，PLD活动可以 以其活性形式特异性地免疫沉淀与 细胞裂解产物中的抗磷酸化酪氨酸抗体。乳清蛋白的分子量 中性粒细胞PLD以及细胞内定位不同于 其他哺乳动物细胞的报告也是如此。此外，PLD活动是 发现在超氧化物条件下贴壁的中性粒细胞增加 释放已激活。因此，现在人们假设一部小说 人中性粒细胞中PLD亚型受酪氨酸调节 磷酸化，使酶变得活跃并合成PA， 这反过来会触发氧自由基的释放。的具体目标 本研究的主要内容是：(1)分离纯化一种新型粒细胞PLD 通过柱层析和分子生物学技术，使用 测序纯化蛋白(PY-PLD)及现存哺乳动物 PLD cDNAs序列(PLD1)。(2)粒细胞性PLD发病机制的探讨 调控，特别是磷酸化激酶，位点(S) 磷酸化和小GTP结合蛋白的作用，使用 抗表位标记蛋白抗体的免疫沉淀法 体外酶分析。(3)阐明PY-PLD在NADPH中的作用 PLD抗体体外阻断超氧化物释放的氧化酶系统 在活体内。这些研究将使我们更清楚地了解 参与心脏损伤的氧化过程并提出新的治疗方法 干预措施。