Search for bioactive substances acting on insect neuropeptide receptors and application of them to insect growth requlators.

昆虫的变态和蜕皮等生活史受多种神经肽的调控。昆虫神经肽的基础研究使寻找神经肽的拮抗剂或激动剂成为可能，这些拮抗剂或激动剂有可能成为安全、特异的昆虫生长调节剂。在本研究中，我们试图寻找作用于家蚕毒素或促前胸腺激素（PTTH）受体的生物活性物质。首先，使用BM-N4细胞的生物测定系统在微生物代谢产物中进行筛选。由于细胞的特征性形态学变化是由家蚕毒素诱导的，因此可以通过该测定检测对家蚕毒素的激动或拮抗活性。遗憾的是，这次没有发现针对家蚕毒素受体的特异性生物活性物质。然而，我们分离出诺卡胺素作为诱导细胞形态变化的诱导剂，并发现一种新的酸性多糖，它强烈诱导细胞聚集。接下来，开发了一种新的生物测定方法，利用光学生物传感器系统直接观察bonbyxin和BM-N4细胞之间的相互作用。由于家蚕毒素明显抑制固定化生物素化家蚕毒素与细胞的结合，我们利用该检测系统筛选家蚕毒素拮抗剂，由于PTTH受体的氨基酸序列尚未确定，因此我们进行了该受体的表达克隆，以鉴定编码该受体的cDNA，并构建PTTH受体转基因COS 7细胞。尽管面临许多挑战，我们还没有成功地克隆。今后，我们将在本研究结果的基础上，继续筛选寻找作用于昆虫神经肽受体的生物活性物质。

The life cycle of insects as metamorphosis and ecdysis is controlled by a variety of neuropeptides. Recent basic reserach on insects neuropeptodes has made it possible to search for their antagonists or agonists, which are possible candidates for safe and specific insect growth regulators. In this study, we have attempted to search for a bioactive substance which acts on a receptor of bombyxin or prothoracicotropic hormone (PTTH).First, screening was performed with a bioassay system using BM-N4 cells among metabolites of microorganisms. Since a characteristic morphological change of the cells was induced by bombyxin, an agonistic or antagonistic activity toward bombyxin could be detected by the assay. Unfortunately, no specific bioactive substance on bombyxin receptor was found this time. However, we isolated nocardamine as an inducer of a morphological change of the cells and found a new acidic polysaccharide from Gordona sp. which strongly induced cell aggregation. Next, anew bioassay method with an optical biosensor system was developed to observe an interaction between bonbyxin and BM-N4 cells directly. Since bombyxin clearly inhibited the biding of immobilized biotinylated bombyxin and the cells, we are performing screening bombyxin antagonists using the assay system.As amino acid sequence of PTTH receptor has not been characterized, an expression cloning of the receptor was performed to identify the cDNA encoding the receptor and to construct transgenic COS 7 cells with PTTH receptor. In spite of many challenges, we have not succeeded the cloning yet. From now on, based on the results obtained by this study, we will continue screening search for bioactive substances which act on insect neuropeptide receptors.