ARACHIDONATE METABOLISM BY ACTIVATED MAST CELLS

半胱氨基白三烯(LTC4及其衍生物LTD4和LTE4)， 和前列腺素(PG)D2是强效的脂质介质，具有明显的 来自花生四烯酸代谢的生物学特性 通过5-脂氧合酶(5-LO)和环氧合酶途径， 分别进行了分析。它们被认为与过敏的发病机制有关。 疾病，特别是支气管哮喘。大鼠和小鼠浆膜肥大 与LTC4相比，细胞优先产生PGD2，而大鼠粘膜 明显成熟的肥大细胞优先产生LTC4 PGD2。小鼠依赖IL-3的骨髓源性肥大细胞(MBMMC)， 优先产生LTC4的是一种多能祖细胞 分化为成熟的肥大细胞取决于两种表型 受当地微环境的影响。这样做的第一个目的是 该项目寻求确定基于组织的元素，是否可溶 细胞因子或细胞相关，导致分化和/或 BALB/c-MBMMC向优先表型分化的研究 生成PGD2，同时下调LTC4的生成。核糖核酸的分析 转录和降解，以及SDS-PAGE免疫印迹分析，将 用于研究这些事件的细胞生物学机制。 尽管我们可以评估5-LO和5-LO的不同监管 用分子和免疫化学探针研究MBMMC的环氧合酶途径 对于cPLA2，5-LO，Flat，以及环氧合酶I和II，我们缺乏探针 每个序列中的末端酶。因此，第二个目标是 从人KG-1细胞系中克隆LTC4合成酶基因，并 目的：通过杂交技术获得小鼠酶的全长基因。特定的 将使用抗PGD2合成酶的抗体来获得该酶的cdna 从lambda-ZAP中的小鼠肥大细胞cDNA文库中获得。是否可以使用 LTC4合成酶和肥大细胞PGD2合成酶的cDNA将提供 适用于识别与其结合的多肽的共同氨基酸序列 制备的抗血清可用于SDS-PAGE免疫印迹分析和 可能这些细胞的免疫组织化学和超微结构定位 酵素。该项目的一个平行目标是划定这些监管机构 仅针对编码LTC4合成酶和PGD2的基因的事件 合成酶。为此，将使用cDNA作为探针来克隆 这些酶的基因。基因组组织将被表征， 外显子和5‘侧翼区将被测序，顺式作用 将定义监管要素。

The cysteinyl leukotrienes (LTC4, and its derivatives, LTD4 and LTE4), and prostaglandin (PG) D2 are potent lipid mediators with distinct biologic properties, which are derived from the metabolism of arachidonic acid by the 5-lipoxygenase (5-LO) and cyclooxygenase pathways, respectively. They have been implicated in the pathogenesis of allergic disease, in particular bronchial asthma. Rat and mouse serosal mast cells preferentially generate PGD2 compared to LTC4, whereas rat mucosal mast cells of apparent maturity preferentially generate LTC4 relative to PGD2. The mouse, IL-3 dependent bone marrow-derived mast cell (mBMMC), which preferentially generates LTC4 is a pluripotent progenitor capable of differentiation into mature mast cells of either phenotype depending upon the influence of the local microenvironment. The first aim of this project seeks to identify tissue-based elements, whether soluble cytokines or cell-associated, that lead to the differentiation and/or maturation of BALB/c mBMMC toward a phenotype that preferentially generates PGD2, whilst downregulating LTC4 generation. Analysis of RNA transcription and degradation, and SDS-PAGE immunoblot analysis, will be used to investigate the cellular biologic mechanisms of these events. Although we can assess the differential regulation of the 5-LO and cyclooxygenase pathways of mBMMC with molecular and immunochemical probes for cPLA2, 5-LO, FLAP, and cyclooxygenase I and II, we lack the probes for the terminal enzymes in each sequence. The second aim is therefore to clone the cDNA for LTC4 synthase from the human KG-1 cell line, and to obtain the cDNA for the mouse enzyme by cross-hybridization. Specific antibody to PGD2 synthase will be used to obtain the cDNA for this enzyme from a mouse mast cell cDNA library in lambda-ZAP. The availability of cDNAs for LTC4 synthase and mast cell PGD2 synthase will provide a consensus amino acid sequence suitable for identifying peptides to which antiserum can be raised and used for SDS-PAGE immunoblot analysis and perhaps immunohistochemistry and ultrastructural localization of these enzymes. A parallel aim of this project is to delineate those regulatory events directed only at the genes which encode LTC4 synthase and PGD2 synthase. To this end the cDNAs will be used as probes to clone the genes for these enzymes. The genomic organization will be characterized, the exons and 5' flanking region will be sequenced, and the cis-acting regulatory elements will be defined.