ARACHIDONATE METABOLISM BY ACTIVATED MAST CELLS

活化的肥大细胞进行花生四烯酸代谢

• 批准号: 6109813
• 负责人: Jonathan Peter Arm
• 金额: $ 28.57万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6109813
• 关键词: arachidonate; bone marrow; cell differentiation; enzyme activity; fatty acid metabolism; fatty acid synthase; genetic library; immunofluorescence technique; laboratory mouse; laboratory rabbit; leukotrienes; mast cell; molecular cloning; prostaglandin endoperoxide synthase; prostaglandins; transfection

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.

半胱氨酰白三烯（LTC 4及其衍生物，LTD 4和LTE 4）， 和前列腺素（PG）D2是有效的脂质介质， 生物学特性，这是来自花生四烯酸的代谢 通过5-脂氧合酶（5-LO）和环氧合酶途径， 分别 它们与过敏性疾病的发病机制有关， 疾病，特别是支气管哮喘。 大鼠和小鼠血清肥大 与LTC 4相比，细胞优先产生PGD 2，而大鼠粘膜 表观成熟的肥大细胞优先产生LTC 4， PGD 2. 小鼠IL-3依赖性骨髓源性肥大细胞（mBMMC）， 是一种多能祖细胞， 分化成成熟肥大细胞的能力 受当地微环境的影响。 第一个目标是 项目旨在确定基于组织的元素，无论是可溶性的 细胞因子或细胞相关的，导致分化和/或 BALB/c mBMMC向一种表型成熟， 产生PGD 2，同时下调LTC 4的产生。 分析rna 转录和降解，SDS-PAGE免疫印迹分析，将是 用于研究这些事件的细胞生物学机制。 虽然我们可以评估5-LO的差分调节， 用分子和免疫化学探针研究mBMMC的环氧合酶途径 对于cPLA 2、5-LO、FLAP和环氧合酶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细胞系克隆LTC 4合酶的cDNA，和 通过交叉杂交获得小鼠酶的cDNA。 具体 PGD 2合酶的抗体将用于获得该酶的cDNA 从小鼠肥大细胞的cDNA文库中的ZAP。 的可用性 LTC 4合酶和肥大细胞PGD 2合酶的cDNA将提供一种新的表达载体。 适用于鉴定肽的共有氨基酸序列， 可以产生抗血清并用于SDS-PAGE免疫印迹分析， 也许免疫组织化学和超微结构定位这些 内切酶 这个项目的一个平行目标是描述那些监管机构， 仅针对编码LTC 4合酶和PGD 2的基因的事件 合成酶 为此目的，cDNA将用作探针以克隆 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，顺式作用的 将确定监管要素。