NEUROENDOCRINE PACERS, PEPTIDES, RECEPTORS, ANTIBODIES

提出了一个广泛的跨学科研究计划，该计划将 专注于了解一个模型神经内分泌系统 这是一种生殖行为，用来产卵 关联的行为。这项研究将结合 六个机构的个人，每个机构都专门从事不同的 生物和化学分支(神经生理学、免疫学、 受体生物化学、多肽结构和化学合成)。 使用了各种技术，包括核磁共振、X射线衍射、 作为功能的单抗的产生- 干扰(FI)高特异性试剂、微测序和 电气技术。我们选择了一种海洋软体动物--海兔 与哺乳动物相比，这项工作具有一定的优势 神经内分泌系统，以及过去的教训， 可概括的神经生物学原理通常得到更多 从对“简单”系统的研究中获得高效。 海兔产卵的指挥是由 神经肽激素，产卵激素(ELH)，由 袋细胞(BC)神经元，在30分钟的放电中释放 自发产卵前的同步动作电位。 这种“后放电”在体外可以通过几秒钟的 电刺激。这种持久的放电是由 CAMP依赖的蛋白质磷酸化调节至少两个 独立的K通道。 本申请中描述的研究涉及：1) 鉴定、提纯和微测序这些蛋白质， 细胞质和膜，它们介导了特殊的 两种神经内分泌起搏器的电生理特性 系统。我们将开发单抗作为荧光探针，用于细胞内 注射，以确定介导起搏器的蛋白质 机械装置。2)确定构效关系 在BCS制造的神经肽产卵激素(ELH)中， 通过使用合成ELH的片段和修饰，在 结合行为产卵测试， 电生理测试和涉及分离的立方体的测试 性腺。此外，ELH和趣味的核磁共振波谱 溶液中的类似物和晶体的X射线衍射将是 执行以获取有关第二和第三级的信息 这些多肽的结构。3)鉴定、提纯和 对来自非神经组织的ELH受体进行微测序 性腺，产生对它的抗体并使用该抗体 确定神经系统中的结合部位。

A broad interdisciplinary research program is proposed that would concentrate on understanding one model neuroendocrine system that commands a reproductive act, the laying of eggs with associated behaviors. This research would combine the talents of individuals at six institutions, each specializing in a different branch of biology and chemistry (neurophysiology, immunology, receptor biochemistry, peptide structure and chemical synthesis). Diverse techniques are used including NMR, X-ray diffraction, the generation of monoclonal antibodies (MAbs) as function- interfering (FI) highly specific reagents, microsequencing and electrical techniques. We choose a marine mollusk, Aplysia, for this work because of certain advantages, over mammalian neuroendocrine systems, and the lessons of the past, that generalizable neurobiological principles are often obtained more efficiently from research on "simple" systems. The command of egg-laying in Aplysia is mediated by the neuropeptide hormone, egg-laying hormone (ELH), synthesized by the bag cell (BC) neurons, and released in a 30 minute discharge of synchronous action potentials before spontaneous egg-laying. This "afterdischarge" can be triggered in vitro by a few seconds of electrical stimulation. This long-lasting discharge is mediated by cAMP-dependent protein phosphorylation modulating at least two independent K+-channels. The research described in this application is concerned with: 1) Identifying, purifying and microsequencing those proteins, cytoplasmic and membrane, that mediate the special electrophysiological properties in two neuroendocrine pacemaker systems. We will develop MAbs as FI-probes, for intracellular injection, in order to identify the proteins mediating pacemaker mechanisms. 2) Determining the structure-activity relationships of the neuropeptide egg-laying hormone (ELH), made by the BCs, by using fragments and modifications of synthetic ELH, in conjunction with behavioral egg-laying assays, electrophysiological assays, and assays involving cubes of isolated gonad. In addition NMR spectroscopy of ELH and interesting analogs in solution and X-ray diffraction of crystals will be performed to gain information on the secondary and tertiary structure of these peptides. 3) Identifying, purifying and microsequencing the ELH receptor from non-neural tissue, the gonad, generating antibodies to it and using the antibody to determine binding sites in the nervous system.