The search for novel biologically active peptides that control bone metabolism.

为了发现调节骨代谢的新型生物活性肽，我使用体外系统生物测定法筛选成骨细胞中的钙离子动员和cAMP升高。当我开始这个项目时，关于调节大鼠胃骨代谢的肽的论文已经发表。在那篇论文中，他们发现了胃中存在一种新型肽的证据。我用胃的提取物作为筛选的起始材料。取50个胃标本，用1M醋酸溶液提取。提取液采用柱层析分离；C-18 Sep-Pak柱，离子交换柱和凝胶过滤。我发现了一些凝胶过滤色谱的活性成分。首先，我在实验中使用钙离子动员作为生物活性标记物。我成功地对这些活性物质进行了纯化和分离，并测定了氨基酸序列。分析表明，其活性物质为神经介质C…More （NMC）、缓激肽和t -激肽。我在这个项目中首先证明了NMC可能作为骨代谢调节剂，而t -激肽，被认为是血浆中的特定分子形式，也存在于胃中。接下来，分离出一些具有cAMP升高活性的组分并进行序列分析。分析结果表明，活性物质的氨基酸序列与VIP（血管活性肠多肽）相同。但其中一种含有一些氨基酸，与VIP不同。这些结果提示VIP存在一些未识别的氨基酸翻译后修饰或剪接变异体，但我需要做进一步的精确分析。我正在使用我开发的体内和体外试验系统对这四种生物活性肽的生理和生物功能进行检查。此外，我计划做这个项目，以发现新的生物活性肽，调节或控制骨代谢。少

To discover novel biologically active peptides that regulate the bone metabolism, I performed screening using in vitro systematic bioassay for a Ca ion mobilization and cAMP elevating in osteoblastic cells. When I started this project, the paper regarding the peptides that regulate the bone metabolism from rat stomach had been published. In that paper, they discovered the evidence that there is a novel peptide in the stomach. I used the extract of stomach as starting material for screening. Fifty stomachs were collected and extracted in 1M acetic acid solution. The extract was separated by follwing column chromatography ; C-18 Sep-Pak column, ion-exchange column and gel filtration. I found several active fractions of gel filtration chromatography. First I used Ca ion mobilization as a bioactive marker in the assay. I succeeded in purification and isolation of these active substances and determined the amino acid sequences. The analyses showed that the active substances are neuromedin C … More (NMC), bradykinin and T-kinin. I first demonstrated in this project that NMC might act as bone metabolism modulator and T-kinin, which was thought to be specific molecular form in plasma, is also present in stomach. Next, some fractions that have cAMP elevating activity were isolated and carried out sequence analyses. The analyses showed that all amino acid sequences of active substances are identical to VIP (vasoactive intestinal polypeptide). But one of them contained some amino acids, which were not identical to VIP.These Results suggested there are some post-translational modification of amino acid or splicing variants of VIP that have unidentified, although I need to perform further precise analysis. I am doing examination of physiological and biological functions of these four biologically active peptides using in vitiro and in vivo assay system I developed. Furthermore, I am planing to do this project to discover novel biologically active peptides that regulate or control bone metabolism. Less