1996 Fiscal Year Annual Research Report

エクオリン遺伝子導入による細胞内カルシウム動態の長期連続測定解析法の開発

Research Project

Project/Area Number	08557006
Section	試験
Research Institution	Hokkaido University
Principal Investigator	本間さと北海道大学, 医学部, 助教授 (20142713)
Co-Investigator(Kenkyū-buntansha)	白川哲夫北海道大学, 歯学部, 講師 (00187527) 近藤孝男名古屋大学, 理学部, 教授 (10124223)
Keywords	生物発光 / エクオリン / 細胞内カルシウム / 遺伝子導入 / サーカディアンリズム
Research Abstract	本年度は以下の実験を行った。実験1:エクオリン遺伝子導入トランスジェニックマウスにおける神経細胞内カルシウム動態の計測。カルシウム結合蛋白であるアポエクオリン遺伝子を、Neuron Specific Enoraseプロモータの下流に組み込んだマウスの脳組織を用い、基質セレンテラジンと反応後、カルシウム添加による発光の有無、発光量の容量依存性、発光の脳内部位特異性について検討した。その結果、トランスジェニックマウスにおいては、大脳組織で小脳や脳幹組織よりもやや強く、かつ遷延する発光が観察された。部位差の成因は不明であり、今後検討したい。ただし、基質と脳組織の反応による化学発光が相当量観察され、エクオリンの発光の容量依存性が判明できなかった。また、NSEプロモータ下流へのエクオリン遺伝子挿入がPCRにて検出できるにも関わらず、発光の観察されない例が多かった。このため、このマウスにおけるアポエクオリン蛋白合成は低いと考えられる。実験2:エクオリン遺伝子導入培養細胞系の樹立トランスジェニックマウス細胞のカルシウム発光が、経時観察可能な量に達さないため、アポエクオリン遺伝子をCMVプロモータの下流に組み込んだベクターを用い培養細胞系の確立を試みた。その結果、NIH3T3細胞への導入で、カルシウム添加で10^2-10^3倍のフラッシュ状発光を示す3細胞系を樹立した。この細胞は、生理的濃度の塩化カリウム刺激にも、容量依存性に発光することが分かった。今回樹立した細胞系での発光は、細胞が独自に合成したカルシウム結合蛋白の発光であるため細胞障害性が全くない。これを利用することにより、生きた細胞での細胞内カルシウム動態観察が、ほぼ無制限に行うこと可能となった。来年度は、さらに、受容体蛋白のco-transfection等により、ホルモンや伝達物質の刺激による細胞内カルシウム動態の研究に応用していきたい。

Research Products

(18 results)

All Other

All Publications (18 results)

[Publications] Hashimoto, S.: "Midday exposure to bright light changes the circadian organization of plasma melatonin rhythm in humans." Neurosci. Lett.(印刷中). (1997)
[Publications] Hahimoto, S.: "Free-running circadian rhythm of melatonin in a sighted young man despite a 24-hr sleep pattern : a non-24-hour circadian syndrome." Psychiat. Clin. Neurosci.(印刷中). (1997)
[Publications] Nakamura, K.: "A sighted man with non-24-hour sleep-wake syndrome showed damped plasma melatonin rhythm." Psychiat. Clin. Neurosci.(印刷中). (1997)
[Publications] Nakamura, K.: "Daily melatonin intake resets circadian rhythms of a sighted man with no-24-hour sleep-wake syndrome who lacks nocturnal melatonin rise." Psychiat. Clin. Neurosci.(印刷中). (1997)
[Publications] Honma, K.: "Sleep and melatonin rhythm in humans." Sleep and Sleep Disorders-from Molecule to Behavior. (印刷中). (1997)
[Publications] Hashimoto, S.: "Vitamin B12 enhances the phase-response of circadian melatonin rhythm to a single bright light exposure in humans." Neruscience Letters. 220. 129-132 (1996)
[Publications] Honma, S.: "Persistence of circadian oscillation while locomotor activity and plasma melatonin levels became aperiodic under prolonged continous light in the rat." Nerusciece Letters. 216. 49-52 (1996)
[Publications] Honma, S.: "Aging affects development and persistence of feeding assoicated circadian rhythm in rat plasma corticosterone." American Journal of Physiology. 271. R1514-R1520 (1996)
[Publications] Honma, S.: "Circadian rhythm and response to light of extracellular glutamate and aspartate in rat suprachiasmative nucleus." American Journal of Physiology. 271. R579-R585 (1996)
[Publications] Yoshihara, T.: "Dissociationm of paraventricular NPY release and plasma corticosterone levels in rats under food deprivation." American Journal of Physiology. 271. E239-E245 (1996)
[Publications] Yoshihara, T.: "Prefeeding release of paraventricular neuropeptide Y is mediated by the ascending noradrenegic neurons in rats." American Journal of Physiology. 270. E596-E600 (1996)
[Publications] Yoshihara, T: "Effects of restricted daily feeding on neuropeptide Y release in the rat paraventricular nucleus." American Journal of Physiology. 270. E589-E595 (1996)
[Publications] Hashimoto, S: "Melatonin rhythm is not shifted by lights which suppress noctural melatonin in humans under entrainment." American Journal of Physiology. 270. R1073-R1077 (1996)
[Publications] Abe. H.: "Substance P receptor regulates the photic induction of Fos-like prontein in the suprachiasmatic nucleus of Syrian hamsters." Brain Research. 708. 135-142 (1996)
[Publications] Yoshihara, T.: "Methamphetamine stimulates the release of neuropeptide Y and noradrenaline from the paraventricular nucleus in rats." Brain Research. 707. 119-121 (1996)
[Publications] T. Kondo: "Circadian rhythms in rapidly dividing cyanobacteria." Science. 275. 224-227 (1997)
[Publications] N. F. Tsinoremas: "A sigma factor that modifies the circadian expression of subset of genes in cyanobacteria." EMBOJ. 15. 2844-2495 (1996)
[Publications] Y. Liu: "Circadian expression of genes involved in the purine biosynthetic pathway of cyuanobacterium Synechococcus sp. strain PPC 7942." Mol. Microbiol.20. 10741-1081 (1996)

1996 Fiscal Year Annual Research Report

エクオリン遺伝子導入による細胞内カルシウム動態の長期連続測定解析法の開発

Principal Investigator

本間 さと 北海道大学, 医学部, 助教授 (20142713)

Research Products

[Publications] Hashimoto, S.: "Midday exposure to bright light changes the circadian organization of plasma melatonin rhythm in humans." Neurosci. Lett.(印刷中). (1997)

[Publications] Hahimoto, S.: "Free-running circadian rhythm of melatonin in a sighted young man despite a 24-hr sleep pattern : a non-24-hour circadian syndrome." Psychiat. Clin. Neurosci.(印刷中). (1997)

[Publications] Nakamura, K.: "A sighted man with non-24-hour sleep-wake syndrome showed damped plasma melatonin rhythm." Psychiat. Clin. Neurosci.(印刷中). (1997)

[Publications] Nakamura, K.: "Daily melatonin intake resets circadian rhythms of a sighted man with no-24-hour sleep-wake syndrome who lacks nocturnal melatonin rise." Psychiat. Clin. Neurosci.(印刷中). (1997)

[Publications] Honma, K.: "Sleep and melatonin rhythm in humans." Sleep and Sleep Disorders-from Molecule to Behavior. (印刷中). (1997)

[Publications] Hashimoto, S.: "Vitamin B12 enhances the phase-response of circadian melatonin rhythm to a single bright light exposure in humans." Neruscience Letters. 220. 129-132 (1996)

[Publications] Honma, S.: "Persistence of circadian oscillation while locomotor activity and plasma melatonin levels became aperiodic under prolonged continous light in the rat." Nerusciece Letters. 216. 49-52 (1996)

[Publications] Honma, S.: "Aging affects development and persistence of feeding assoicated circadian rhythm in rat plasma corticosterone." American Journal of Physiology. 271. R1514-R1520 (1996)

[Publications] Honma, S.: "Circadian rhythm and response to light of extracellular glutamate and aspartate in rat suprachiasmative nucleus." American Journal of Physiology. 271. R579-R585 (1996)

[Publications] Yoshihara, T.: "Dissociationm of paraventricular NPY release and plasma corticosterone levels in rats under food deprivation." American Journal of Physiology. 271. E239-E245 (1996)

[Publications] Yoshihara, T.: "Prefeeding release of paraventricular neuropeptide Y is mediated by the ascending noradrenegic neurons in rats." American Journal of Physiology. 270. E596-E600 (1996)

[Publications] Yoshihara, T: "Effects of restricted daily feeding on neuropeptide Y release in the rat paraventricular nucleus." American Journal of Physiology. 270. E589-E595 (1996)

[Publications] Hashimoto, S: "Melatonin rhythm is not shifted by lights which suppress noctural melatonin in humans under entrainment." American Journal of Physiology. 270. R1073-R1077 (1996)

[Publications] Abe. H.: "Substance P receptor regulates the photic induction of Fos-like prontein in the suprachiasmatic nucleus of Syrian hamsters." Brain Research. 708. 135-142 (1996)

[Publications] Yoshihara, T.: "Methamphetamine stimulates the release of neuropeptide Y and noradrenaline from the paraventricular nucleus in rats." Brain Research. 707. 119-121 (1996)

[Publications] T. Kondo: "Circadian rhythms in rapidly dividing cyanobacteria." Science. 275. 224-227 (1997)

[Publications] N. F. Tsinoremas: "A sigma factor that modifies the circadian expression of subset of genes in cyanobacteria." EMBOJ. 15. 2844-2495 (1996)

[Publications] Y. Liu: "Circadian expression of genes involved in the purine biosynthetic pathway of cyuanobacterium Synechococcus sp. strain PPC 7942." Mol. Microbiol.20. 10741-1081 (1996)

本間さと北海道大学, 医学部, 助教授 (20142713)