BIOLOGY OF THE HUMAN NEUTROPHIL PLASMA MEMBRANE

人类中性粒细胞质膜的生物学

基本信息

批准号：
3126799
负责人：
Mark S Klempner
金额：
$ 25.59万
依托单位：
TUFTS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
1981
资助国家：
美国
起止时间：
1981-01-01 至 1992-03-31
项目状态：
已结题

项目摘要

Neutrophils (PMN) and macrophages provide host defense against infection by ingesting and killing microorganisms. These essential functions depend on the creation of a toxic intracellular environment created by the fusion of lysosomes with the organism containing phagosome. One of the goals of this project is to better define the events which regulate this fusion process. Changes in Ca++ concentration and pH have been intimately linked to PMN and macrophages activation and are a major focus of the proposed studies. We have identified a new Ca++ pumping enzyme in human PMN lysosomes which functions at physiologic Ca++, ATP, and H+ concentrations and have proposed that this Ca++ pump plays a major role in activating phagocytes. Using isotopic and fluorescent methods, we will characterize the regulation of calcium uptake by PMN lysosomes and the release of calcium from these organelles. These studies will provide information on normal control mechanisms for triggering phagocytes and will form the basis of a strategy for isolating this Ca++ -ATPase. A combination of affinity and standard chromatographic methods are proposed. Failure to create a phagolysosomal environment sufficiently toxic to kill pathogenic microorganisms results in morbid infectious diseases. Intrinsic cell defects are one class of this failure and the Chediak-Higashi syndrome is a prominent example. The hallmark of this human disorder is giant, abnormal lysosomes which fuse defectively with the phagosome. This results in recurrent, often lethal, infections. While the fundamental defect is unknown, we have recently described an abnormal Ca++ pump in CHS PMN lysosomes. Using human and beige mouse PMN and macrophages lysosomes, we will further examine the nature of this pump and determine whether this represents the first example of an abnormal ion translocating pump which is the proximate cause of human disease. Organisms, including Yersinia, Legionella, and Mycobacteria which survive and multiply within phagolysosomes, cause morbid infectious diseases. Using a model of Yersinia pestis organisms which have been genetically engineered to report the Ca++ and pH conditions in their environment, we have begun to define the ionic constitution of the phagolysosome. Our goal is to identify characteristics of the organism-containing phagolysosome which can be mainipulated as part of a new antimicrobial strategy. We will genetically engineer additional mutants of Y. pestis and use these probes to examine whether modifying the ionic environment alters the virulence of the pathogen.

中性粒细胞 (PMN) 和巨噬细胞提供宿主防御通过摄入和杀死微生物而感染。这些必不可少的功能依赖于细胞内有毒物质的产生溶酶体与生物体融合所创造的环境含有吞噬体。该项目的目标之一是更好地定义调节这种融合过程的事件。 Ca++ 浓度和 pH 值的变化密切相关与 PMN 和巨噬细胞激活相关，是主要关注点拟议的研究。我们已经确定了一种新的 Ca++ 泵送人中性粒细胞溶酶体中的酶，其生理功能 Ca++、ATP 和 H+ 浓度，并提出这 Ca++泵在激活吞噬细胞中起主要作用。使用同位素和荧光方法，我们将表征 PMN 溶酶体对钙的吸收和释放的调节来自这些细胞器的钙。这些研究将提供有关触发的正常控制机制的信息吞噬细胞并将形成分离该细胞的策略的基础 Ca++-ATP 酶。亲和力与标准的结合提出了色谱方法。未能创建吞噬溶酶体环境的毒性足以杀死病原体微生物导致病态传染病。固有的细胞缺陷是此类故障的一类，Chediak-Higashi 综合症就是一个突出的例子。这个人类的标志疾病是巨大的、异常的溶酶体，它们与吞噬体。这会导致反复的、通常是致命的感染。虽然根本缺陷尚不清楚，但我们最近发现描述了 CHS PMN 溶酶体中异常的 Ca++ 泵。使用人类和米色小鼠 PMN 和巨噬细胞溶酶体，我们将进一步检查该泵的性质并确定是否这是异常离子易位的第一个例子泵是人类疾病的直接原因。生物体，包括存活下来的耶尔森氏菌、军团菌和分枝杆菌并在吞噬溶酶体内繁殖，引起病态感染疾病。使用鼠疫耶尔森氏菌模型，该模型具有经过基因改造以报告 Ca++ 和 pH 条件在他们的环境中，我们已经开始定义离子吞噬溶酶体的构成。我们的目标是确定含有生物体的吞噬溶酶体的特征可以作为新的抗菌策略的一部分进行操作。我们将基因改造鼠疫耶尔森氏菌的其他突变体并使用这些探针检查是否改变了离子环境改变病原体的毒力。