Development and Application of Selective Elcectochemical Analysys System for Peptides and Amines.

Oka开发了一种新的电化学记录技术来检测GnRH肽能神经元释放的GnRH肽。我们设计了一种新型的碳纤维微电极，并在体外测量了小鱼脑末梢神经GnRH神经元的GnRH肽释放。我们记录了GnRH神经元的自发安培尖峰电流。由于安培峰值显示出与真正的GnRH溶液相似的电压依赖性，因此它们被认为是由胞外化和氧化的GnRH肽产生的电流。我们现在正在与Iigo合作，确认安培峰值与GnRH释放到孵育介质中并将其与放射免疫测定法测量相关联。Shinohara通过设计复杂的表面修饰碳电极和自组装单层电极开发了新的电化学记录技术，这将使生物活性胺和肽的测量变得方便和快速。他把我们的注意力集中在Trp上，它是人类必需的氨基酸，是哺乳动物神经递质和激素的前体分子。利用色氨酸是疏水的这一事实，他成功地用一种表面被疏水分子修饰的电极对色氨酸的氧化进行了选择性测量。他进一步研究了含trp肽（如GnRH）与电极表面修饰之间的关系。(2)他通过在电极上组装辅酶衍生物和重组载脂蛋白酶来固定酶的取向。他通过介质测量了酶的催化电流，实现了酶底物的安培生物传感。Iigo采用灌注培养法培养ayu松果体，通过测量褪黑激素分泌的节律性来研究生物钟特征。当光-暗循环被逆转时，褪黑素的日分泌节律（暗时高，明时低）被重置为一个新的光周期。光脉冲照射6小时后，在恒定的黑暗条件下，褪黑素分泌节律出现相依赖的相移；在主观黑暗期的前半段，他们对光脉冲的反应表现出相位延迟，而在后半段，他们对光脉冲的反应表现出相位提前。相比之下，主观光周期的光脉冲不会引起相移。因此，他揭示了ayu松果体中生物钟的某些方面。他现在正在尝试使用筱原在这个研究项目中开发的电化学测量装置来实时测量褪黑激素的分泌节奏。少

Oka developed a new electrochemical recording technique to detect GnRH peptide release from the GnRH peptidergic neurons. We have devised a new type of carbon fiber microelectrode and measured the release of GnRH peptide from the terminal nerve GnRH neurons of a small fish brain in vitro. We recorded spontaneous amperometric spike currents from the GnRH neurons. Since the amperometric spikes showed voltage-dependence similar to that of an authentic GnRH solution, they were suggested to be the currents that result from the exocytosed and oxidated GnRH peptides. We are now collaborating with Iigo to confirm and correlate the amperometric spikes with the release of GnRH into the incubation medium that are to be measured with the radioimmunoassay.Shinohara has developed new electrochemical recording techniques by devising sophisticated surface-modified carbon electrodes and self-assembled monolayer electrodes that will enable handy and rapid measurement of bioactive amines and peptides. (1 … More ) He has focused our attention on Trp, which is an essential amino acid for human beings and is a precursor molecule of mammalian neurotransmitters and hormones. By taking advantage of the fact that Trp is hydrophobic, he has succeeded in a selective measurement of oxidation of Trp by using an electrode that has been surface-modified by hydrophobic molecules. He has furthermore examined the relationship between the Trp-containing peptides, such as GnRH, and the surface-modification of the electrode. (2) He has orientation-fixed the enzymes by assembling co-enzyme derivatives on the electrode and by reconstituting apoenzymes. He measured the catalytic currents of the enzymes through the mediators and realized the amperometric biosensing of enzyme substrates.Iigo cultured the pineal gland of ayu by Perfusion-culture method and examined the characteristics of the biological clock by measuring the rhythmicity of melatonin secretion. The daily rhythm of melatonin secretion, which shows higher level during the dark and lower level during the light, was reset to a new photo-periodicity when the light-dark cycle was reversed. After exposure to 6-hour light pulses, under constant dark conditions, the melatonin secretion rhythm showed phase-dependent phase shifts; they showed phase delay in response to the light pulses during the 1st half of the subjective dark period, while they showed phase advance in response to those during the 2nd half. In contrast, the light pulses during the subjective light period did not evoke phase shifts. Thus, he has revealed some aspects of the biological clock that resides in the pineal gland of ayu. He is now trying to measure the melatonin secretion rhythms in real-time by using the electrochemical measurement device which Shinohara developed during this research project. Less