REGULATION OF PHAGOCYTE FUNCTION

人类吞噬白细胞是主要的细胞防御 对抗细菌感染。这些细胞经历一系列复杂的过程 反应，包括趋化反应和运动反应， 通过吞噬过程杀死微生物，并通过杀灭细菌 颗粒内容物的分泌和有毒氧气的产生 代谢物。 这一系列组织严密、监管严密的活动 可以在基本层面上理解由传染性病原体引发的 利用许多与正常细胞相同的基本机制 流程。这包括 GTP 结合的广泛参与 白细胞吞噬活性中的蛋白质。 研究表明，NADPH 氧化酶是超氧化物的位点。 人类中性粒细胞的产生，由 ras 相关的 GTP- 调节 结合蛋白 Rac2。该活性以及相关的 GTP 结合 蛋白质由调节 GTP 结合的因素决定 （活跃）和 GDP（非活跃）。因此 GTP 酶激活蛋白 (GAP) 刺激 GTP 水解，同时 GDP 解离刺激剂 (GDS) 和 GDP 解离抑制剂（GDI）调节核苷酸交换。我提议 识别并研究 Rac2 的监管组件，这将 确定 NADPH 氧化酶响应感染的活性 代理。在相关研究中，我将确定Rac蛋白在 调节运动吞噬或细菌摄取的细胞生物学， 和颗粒分泌。 将使用以下方法开发监管 GDS 和 GAP 蛋白的检测方法 重组 Rac2，这些蛋白质将在人类中被鉴定 中性粒细胞。 蛋白质将被纯化至同质性、测序和 克隆的。然后我们将利用这些蛋白质来研究调控 NADPH 氧化酶。该系统将提供一个模型来理解 Rac 然后将扩展到其他蜂窝系统，其中 需要 Rac 和相关蛋白质。 Rac、Rac 突变体和 Rac 的转染和表达方法 将开发进入 HL-60 早幼粒细胞的调节蛋白。 使用 Rac 蛋白的显性失活或组成型活性形式，我们 将决定 Rac 或相关的 Rho 蛋白是否发挥关键作用 在中性粒细胞和/或单核细胞的细胞生物学中的作用。机制 其他对于细菌吸收和杀灭至关重要的系统 Rac 和 Rac 调节蛋白的调节将使用以下方法进行研究 我们在氧化酶系统中的发现作为工作模型。此类研究将 增加对人类吞噬细胞的细胞生物学的了解 分子水平，以及抗感染治疗的新方法。

Human phagocytic white blood cells serve as a major cellular defense against bacterial infection. These cells undergo a complex series of responses, including chemotactic and motile responses, uptake of microorganisms by phagocytotic processes, and bacterial killing by secretion of granule contents and the generation of toxic oxygen metabolites. This highly organized and regulated series of events triggered by infectious agents can be understood at its basic level utilizing many of the same underlying mechanisms as in normal cellular processes. This includes the widespread involvement of GTP-binding proteins in phagocytic white blood cell activities. It has been shown that the NADPH oxidase, the site of superoxide production in human neutrophils, is regulated by the ras-related GTP- binding protein Rac2. The activity of this, and related GTP-binding proteins is determined by factors which regulate the binding of GTP (active) and GDP (inactive). Thus GTPase activating proteins (GAPs) stimulate GTP hydrolysis, while GDP dissociation stimulators (GDS) and GDP dissociation inhibitors (GDI) regulate nucleotide exchange. l propose to identify and investigate regulatory components for Rac2 which would determine the activity of the NADPH oxidase in response to infectious agents. In related studies, l will determine the role of Rac protein in regulating the cell biology of motility phagocytosis or bacterial uptake, and granule secretion. Assays for regulatory GDS and GAP proteins will be developed using recombinant Rac2, and these proteins will be identified in the human neutrophil. The proteins will be purified to homogeneity, sequenced, and cloned. We will then utilize these proteins to investigate the regulation of the NADPH oxidase. This system will provide a model to understand Rac regulation that will then be extended to other cellular systems in which Rac and related proteins are required. Methodologies for the transfection and expression of Rac, Rac mutants and regulatory proteins into HL-60 promyelocytic cells will be developed. Using dominant negative or constitutively active forms of Rac protein, we will determine whether Rac, or the related Rho protein, play critical roles in the cell biology of neutrophils and/or monocytes. The mechanisms by which other systems, crucial for bacterial uptake and killing, are regulated by Rac and Rac regulatory proteins will be investigated using our findings in the oxidase system as a working model. Such studies will lead to increased knowledge of the cell biology of human phagocytes at the molecular level, and to novel approaches in anti-infective therapy.