RELATION OF GAMMA GLOBULIN STRUCTURE TO HYPERSENSITIVITY

本研究项目的目的是阐明生物化学。 肥大细胞释放IgE依赖的介质的机制 过敏性疾病。我们的努力将集中在分析生化 肥大细胞上的IgE受体桥接所引起的事件。 最近，我们建立了一套培养系统，以获得人的嗜碱性 脐带血单个核细胞的培养。文化修养 人IgE可致敏嗜碱性粒细胞产生抗IgE诱导的组胺 放手。因此，i)我们将鉴定人嗜碱性粒细胞的生长因子(S 并对培养的人类嗜碱性粒细胞进行鉴定。Ii)实验将是 以确定膜相关酶，如丝氨酸 蛋白水解酶和甲基转移酶参与了培养细胞的激活 嗜碱性细胞，这一过程将导致钙摄取和介体 放手。III)人IgE和啮齿动物IgE对IgE受体的亲和力 将测定培养的人嗜碱性粒细胞，以及iv)IgE受体 用十二烷基硫酸钠凝胶电泳法对细胞进行鉴定。V)我们将 确定膜相关丝氨酸蛋白酶的底物专一性， 它在IgE受体桥接时被激活。六)可能性 蛋白水解酶与α、β亚基的关系 将测定免疫球蛋白受体。Vii)考虑的可能性是 蛋白水解酶的激活导致激动素样蛋白的形成 物质，进而激活其他与膜相关的酶 释放调解人。我们将确定激肽释放酶样酶 和/或缓激肽可诱导甲基转移酶的激活和 啮齿动物肥大细胞和嗜碱性粒细胞中的腺苷环化酶。Viii) 磷脂甲基化在促进细胞周转中的可能作用 磷脂酰肌醇将通过使用啮齿动物肥大细胞和 人类培养的嗜碱性粒细胞。九)我们将分析细胞内钙离子的运动 和钙离子进入肥大细胞(嗜碱性粒细胞)的桥联 免疫球蛋白受体。一种荧光喹啉钙离子指示剂Quin 2将被 受雇于分析。X)最后，生物化学机制 脱敏作用将在小鼠肥大细胞中进行研究。我们将测试 假设脱敏是由于激活 膜相关酶，不同的酶参与了 肥大细胞的激活以促进介质的释放和脱敏。类似 脱敏实验将在培养的人身上进行 未来几年的嗜碱性粒细胞。

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.