SIGNAL TRANSDUCTION IN NEUTROPHIL MEDIATED HEART INJURY

描述：（改编自申请人摘要）总体目标 这一提议是为了表征特定的信号转导机制， 在中性粒细胞刺激后被激活， 损伤 中性粒细胞释放致命的氧自由基（如超氧化物 阴离子），其能够加剧组织损伤，如在 这些细胞在缺血后心肌坏死期间的浸润， 再灌注损伤 磷脂酶D（PLD）是磷脂酶D的核心。 产生PA，一种假定的第二信使，参与释放 超氧阴离子的中性粒细胞。 作者发现PLD活性可以 以其活性形式特异性免疫沉淀 来自细胞裂解物的抗磷酸酪氨酸抗体。 的分子量 嗜中性粒细胞PLD以及细胞内定位与 其他哺乳动物细胞的研究结果。 此外，PLD活动是 发现在超氧化物存在的条件下， 释放被激活。 因此，现在假设一部小说 人中性粒细胞中PLD亚型通过酪氨酸调节 磷酸化，这使得酶变得活跃并合成PA， 进而引发氧自由基的释放 的具体目标 本研究的主要内容是：（1）一种新型粒细胞PLD的纯化和鉴定 通过柱色谱法和分子生物学技术，使用 测序的纯化蛋白（PY-PLD）以及现有的哺乳动物 PLD cDNA序列（PLD 1）。 (2)探讨粒细胞PLD的发生机制 调节，特别是磷酸化激酶， 磷酸化和小GTP结合蛋白的作用，使用 用抗表位标记蛋白的抗体进行免疫沉淀， 体外酶测定。 (3)阐明PY-PLD在NADPH中的作用 氧化酶系统，通过在体外用PLD抗体阻断超氧化物释放 和体内。 这些研究将使人们更清楚地了解 参与心脏损伤的氧化过程，并提出新的治疗方法 干预措施。

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.