Synthesis and large production of glucocorticoid action enhancers and development of new steroid therapy.

本研究的重点是从肠道细菌中大量生产糖皮质激素作用增强剂及其化学合成方法的建立。此外，我们还试图通过使用这些调节剂来改善类固醇治疗。我们发现了两种糖皮质激素作用的增强剂。一种是来自肠道细菌的糖皮质激素敏感性增强剂(GSA)，它能增强靶细胞对糖皮质激素作用的敏感性，其化学结构被确定为假尿苷1-N-油酰基磷酸盐。第二类是糖皮质激素活性增强剂，如佛波酯、表皮生长因子和甘油二酯，它们在体内直接和/或间接激活蛋白激酶C。另一方面，蛋白激酶C的抑制剂抑制糖皮质激素的作用。在研究期间，我们对产生GSA的肠道细菌奇异葡萄球菌的培养条件进行了改进。最低限度的Essena…中含有高浓度组氨酸在细胞生长的指数后期，较多的L培养基能诱导GSA。我们也建立了大规模酶法合成假尿苷5‘-磷酸的方法，但到目前为止还没有办法将油酰胺的NH_2末端结合到假尿苷5’-磷酸的磷酸位置上。在糖皮质激素作用机制的研究中，我们发现蛋白激酶C的抑制剂，如H-7，能抑制糖皮质激素受体和热休克蛋白在胞浆中的解离，从而抑制糖皮质激素受体向细胞核的转位，并抑制肝细胞的糖皮质激素作用。根据这些结果，我们推测蛋白激酶C在糖皮质激素的作用中是必不可少的，蛋白激酶C的激活剂增强了糖皮质激素的作用。在这些结果的基础上，我们现在正在使用GPAs和GSA来抑制小鼠L5178Y和L1210淋巴性白血病细胞的生长，作为类固醇治疗的临床应用之一。较少

Establishments of large production of glucocorticoid action enhancers from enteric bacteria and of the methods of chemical synthesis of them are focused in this research. In addition we tried to improve steroid therapy by using of these modulators. We found two kinds of the enhancers of glucocorticoid actions. One is glucocorticoid sensitivity amplifier (GSA) from enteric bacteria, which enhances sensitivity for glucocorticoid actions in target cells and the chemical structure of it was determied to be pseudouridiny1-N-oleoylphosphamate. The second is glucocorticoid potency amplifiers (GPAs), such as phorbol ester, epidermal growth factor and diacylglycerols, all of which activate protein kinase C directly and/or indirectly in vivo. On the other hand, inhibitors of protein kinase C supperssed glucocorticoid actions.In the research period, we improved culture conditions of p. mirabilis, one of the enteric bacteria, which produces GSA. High concentrations of histidine in mimimal essentia … More l medium induced GSA at late exponential phase of cell Growth. We also established enzymatical synthesis of pseudouridine 5'-phosphate in large scale but there are no way to bind NH_2-terminal position of oleamide to phosphate position of pseudouridine 5'-phosphate until now. We are continuing to condensation of them.In the studies of mechanism of glucocorticoid action, we found that inhibitor of protein kinase C, such as H-7, inhibited dissociation of glucocorticoid receptor and heat shock protein in cytosol, resuting in the inhibition of translocation of glucocorticoid receptor into nuclei and in the inhibition of glucocorticoid action in hepatocytes. From these results, we speculate that protein kinase C is essential in the glucocorticoid actions and activators of proteinkinase C enhance glucocorticoid actions. On the basis of these results, we are now using GPAs and GSA in the inhibition of growth of L5178Y and L1210 lymphatic leukemia cells in mice as one of the clinical use of steroid therapy. Less