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

昆虫的变态、蜕皮等生命周期受多种神经肽的控制。最近对昆虫神经肽的基础研究使寻找它们的拮抗剂或激动剂成为可能，它们可能是安全和特异的昆虫生长调节剂的候选者。在这项研究中，我们试图寻找一种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，利用BM-N4细胞生物测定系统从微生物代谢产物中进行筛选。由于家蚕毒素诱导的细胞形态发生了特征性的变化，因此可以通过该方法检测到对家蚕毒素的激动性或拮抗性活性。遗憾的是，这次没有在家蚕素受体上发现特定的生物活性物质。然而，我们分离了诺卡丹明作为细胞形态变化的诱导剂，并从Gordona sp.中发现了一个新的酸性多糖。强烈诱导细胞聚集。其次，发展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骨毒素与BM-N4细胞之间的相互作用。由于家蚕毒素明显抑制了固定化生物素化的家蚕毒素与细胞的结合，我们正在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，以鉴定该受体的编码基因，并构建了携带PTTH受体的转基因COS 7细胞。尽管有许多挑战，我们还没有成功克隆。今后，我们将在本研究成果的基础上，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.