RELATION OF GAMMA GLOBULIN STRUCTURE TO HYPERSENSITIVITY

丙种球蛋白结构与超敏反应的关系

• 批准号: 3124625
• 负责人: KIMISHIGE ISHIZAKA
• 金额: $ 26.22万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1974
• 资助国家: 美国
• 起止时间: 1974-09-01 至 1989-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3124625
• 关键词: allergens; antigen antibody reaction; complement fixation tests; density gradient ultracentrifugation; disulfide bond; electron microscopy; enzyme structure; histamine release; human tissue; hypersensitivity; immunochemistry; immunoglobulin E; mast cell; tissue /cell culture

The purpose of this research project is to elucidate the biochemical mechanisms of IgE-dependent mediator release from mast cells, which cause allergic diseases. Our efforts will be focused to analyze biochemical events which are caused by the bridging of IgE-receptors on mast cells. Recently, we established a culture system to obtain human basophilis in the culture of the mononuclear cells in umbilical cord blood. Cultured basophils could be sensitized with human IgE for anti-IgE-induced histamine release. Thus, i) we shall identify growth factor(s) for human basophils and characterize the cultured human basophils. ii) Experiments will be carried out to determine that membrane-associated enzymes, such as serine protease and methyltransferase, are involved in the activation of cultured basophils, and that this process will lead to Ca2+ uptake and mediator release. iii) The affinity of human IgE and rodent IgE for IgE-receptors on cultured human basophilis will be determined, and iv) IgE-receptors on the cells will be identified by SDS gel electrophoresis. v) We shall determine the substrate specificity of membrane-associated serine protease, which is activated upon bridging of IgE receptors. vi) Possible relationship between the proteolytic enzyme and Alpha or Beta subunits of IgE-receptors will be determined. vii) The possibility is considered that the activation of the protease results in the formation of a kinin-like substance, which in turn activates the other membrane-associated enzymes for mediator release. We shall determine whether kallikrein-like enzymes and/or bradykinin may induce the activation of methyltransferases and adenylate cyclase in rodent mast cells and human basophils. viii) The possible role of phospholipid methylation in the enhancement of turnover of phosphatidylinositol will be investigated by using rodent mast cells and human cultured basophils. ix) We shall analyze intracellular Ca2+ movement and Ca++ influx into mast cells (basophils) induced by bridging of IgE-receptors. A fluorescent quinoline Ca++ indicator, Quin 2, will be employed for the anlysis. x) Finally, biochemical mechanisms of desensitization will be investigated in mouse mast cells. We shall test the hypothesis that desensitization is due to activation of membrane-associated enzymes, and that different enzymes are involved in the activation of mast cells for mediator release and desensitization. Similar experiments on desensitization will be carried out with cultured human basophils in future years.

本研究项目的目的是阐明生物化学 肥大细胞释放IgE依赖性介质的机制， 过敏性疾病 我们的工作将集中在分析生化 由肥大细胞上IgE受体的桥接引起的事件。 最近，我们建立了一个培养系统，以获得人嗜碱性粒细胞， 脐带血单个核细胞的培养。 培养 嗜碱性粒细胞可被人IgE对抗IgE诱导的组胺致敏 release. 因此，i）我们将鉴定人嗜碱性粒细胞的生长因子 并鉴定培养的人嗜碱性粒细胞。 （二）实验将 以确定膜相关酶，如丝氨酸 蛋白酶和甲基转移酶，参与激活培养的 嗜碱性粒细胞，这一过程将导致Ca 2+摄取和介导 release. iii）人IgE和啮齿动物IgE对IgE受体的亲和力 iv）IgE-受体， 通过SDS凝胶电泳鉴定细胞。 （五）我们将 确定膜相关丝氨酸蛋白酶的底物特异性， 其在IgE受体桥接时被激活。 （六）可能 蛋白水解酶和α或β亚基之间的关系 将测定IgE受体。 （七）有可能认为， 蛋白酶的活化导致形成激肽样蛋白酶， 一种物质，它反过来激活其他膜相关酶 释放调解人 我们将确定类激肽释放酶 和/或缓激肽可以诱导甲基转移酶的活化， 啮齿类动物肥大细胞和人类嗜碱性粒细胞中的腺苷酸环化酶。 （八） 磷脂甲基化可能在增强细胞代谢中的作用 磷脂酰肌醇将通过使用啮齿动物肥大细胞进行研究， 人类培养的嗜碱性粒细胞。 ix）我们将分析细胞内Ca 2+运动 和Ca++内流到肥大细胞（嗜碱性粒细胞）诱导的桥接 IgE受体。 荧光喹啉Ca++指示剂Quin 2将在 用于分析。 x）最后， 将在小鼠肥大细胞中研究脱敏。 我们将测试 脱敏是由于激活 膜相关的酶，不同的酶参与， 激活肥大细胞以释放介质和脱敏。 类似 将用培养的人进行脱敏实验 在未来的几年里，