| Co-Investigator(Kenkyū-buntansha) |
IIGO Masayuki School of Medicine, St. Marianna University, Assistant Professor, 医学部, 助手 (10232109)
SHINOHARA Hiroaki Faculty of Engineering, Associate, The University of Okayama, Professor, 工学部, 助教授 (60178887)
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| Research Abstract |
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
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) 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
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