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| 1.
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論文
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Hara, Chiho ; Morishita, Koudai ; Takayanagi-Kiya, Seika ; Mikami, Akihisa ; Uchino, Keiro ; Sakurai, Takeshi ; Kanzaki, Ryohei ; Sezutsu, Hideki ; Iwami, Masafumi ; Kiya, Taketoshi ; 木矢, 星歌 ; 櫻井, 武 ; 岩見, 雅史 ; 木矢, 剛智
概要:
金沢大学理工研究域自然システム学系<br />Silkmoth, Bombyx mori, is one of the important model insects in which transgenic techniques and t
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he GAL4/UAS system are applicable. However, due to cytotoxicity and low transactivation activity of GAL4, effectiveness of the GAL4/UAS system and its application in B. mori are still limited. In the present study, we refined the previously reported UAS vector by exploiting transcriptional and translational enhancers, and achieved 200-fold enhancement of reporter GFP fluorescence in the GAL4/UAS system. Enhanced protein expression of membrane-targeted GFP and calcium indicator protein (GCaMP5G) drastically improved visualization of fine neurite structures and neural activity, respectively. Also, with the refined system, we generated a transgenic strain that expresses tetanus toxin light chain (TeTxLC), which blocks synaptic transmission, under the control of GAL4. Ectopic TeTxLC expression in the sex pheromone receptor neurons inhibited male courtship behavior, proving effectiveness of TeTxLC on loss-of-function analyses of neural circuits. In addition, suppression of prothoracicotropic hormone (PTTH) or insulin-like peptide (bombyxin) secretion impaired developmental timing and growth rate, respectively. Furthermore, we revealed that larval growth is sex-differentially regulated by these peptide hormones. The present study provides important technical underpinnings of transgenic approaches in silkmoths and insights into mechanisms of postembryonic development in insects. © 2017 The Author(s).
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| 2.
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Sugimori, Kimikazu ; Shuku, Tomofumi ; Sugiyama, Ayumu ; Nagao, Hidemi ; Sakurai, Takeshi ; Nishikawa, Kiyoshi
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We present a cluster model for the active site of oxidized azurin, and investigate the electronic structure of the activ
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e site of oxidized azurin by using density functional calculations with polarizable continuum model. The singly occupied molecular orbital and spin density in the model widely distribute around the Cu 3dx2-y2 and S(Cys112) 3p orbitals. The dependency of electronic properties such as partial charge density and spin density on the dielectric constant is discussed. We find that partial spin density and charge density on the copper ion become larger, when the dielectric constant increases. © 2005 Elsevier Ltd. All rights reserved.<br />Embargo Period 24 months
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| 3.
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Irukayama-Tomobe, Yoko ; Tanaka, Hirokazu ; Yokomizo, Takehiro ; Hashidate-Yoshida, Tomomi ; Yanagisawa, Masashi ; Sakurai, Takeshi
概要:
金沢大学医薬保健研究域医学系<br />GPR109B (HM74) is a putative G protein-coupled receptor (GPCR) whose cognate ligands have yet to be
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characterized. GPR109B shows a high degree of sequence similarity to GPR109A, another GPCR that was identified as a high-affinity nicotinic acid (niacin) receptor. However, the affinity of nicotinic acid to GPR109B is very low. In this study, we found that certain aromatic D-amino acids, including D-phenylalanine, D-tryptophan, and the metabolite of the latter, D-kynurenine, decreased the activity of adenylate cyclase in cells transfected with GPR109B cDNA through activation of pertussis toxin (PTX)-sensitive G proteins. These D-amino acids also elicited a transient rise of intracellular Ca2+ level in cells expressing GPR109B in a PTX-sensitive manner. In contrast, these D-amino acids did not show any effects on cells expressing GPR109A. We found that the GPR109B mRNA is abundantly expressed in human neutrophils. D-phenylalanine and D-tryptophan induced a transient increase of intracellular Ca2+ level and a reduction of cAMP levels in human neutrophils. Furthermore, knockdown of GPR109B by RNA interference inhibited the D-amino acids-induced decrease of cellular cAMP levels in human neutrophils. These D-amino acids induced chemotactic activity of freshly prepared human neutrophils. We also found that D-phenylalanine and D-tryptophan induced chemotactic responses in Jurkat cells transfected with the GPR109B cDNA but not in mock-transfected Jurkat cells. These results suggest that these aromatic D-amino acids elicit a chemotactic response in human neutrophils via activation of GPR109B. © 2009 by The National Academy of Sciences of the USA.
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| 4.
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Uchio, Naohiro ; Doi, Masao ; Matsuo, Masahiro ; Yamazaki, Fumiyoshi ; Mizoro, Yasutaka ; Hondo, Mari ; Sakurai, Takeshi ; Okamura, Hitoshi
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金沢大学医薬保健研究域医学系<br />GPR7, now known as a receptor of neuropeptide B and neuropeptide W, is expressed in neurons of the s
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uprachiasmatic nucleus (SCN), the mammalian circadian center. By the quantitative in situ hybridization, we demonstrated that GPR7 mRNA showed a significant circadian rhythm in the SCN showing a peak at early subjective night in both light-dark and constant dark. We characterized the circadian feature of GPR7-knockout mice, but the period length and the phase-dependent phase shift to light exposure were not disordered in GPR7-knockout mice. Moreover, the food-anticipatory behavior in restricted feeding schedule was observed in this gene-deleted mouse similar to wild-type. These results indicate that the role of GPR7 may be subtle or limited in relation to the circadian clock despite its robust expression in the SCN.
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| 5.
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Sakurai, Takeshi ; Mieda, Michihiro ; Tsujino, Natsuko
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金沢大学医薬保健研究域医学系<br />The neuropeptides orexin A and orexin B, produced in hypothalamic neurons, are critical regulators o
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f sleep/wake states. Deficiency of orexin signaling results in narcoleptic phenotype in humans, dogs, and rodents. Recently, accumulating evidence has indicated that the orexin system regulates sleep and wakefulness through interactions with neuronal systems that are closely related with emotion, reward, and energy homeostasis. In this review, we will discuss the current understanding of the physiology of the orexin system especially focusing on its roles in the regulation of sleep/wakefulness states. © 2010 New York Academy of Sciences.
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| 6.
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Mieda, Michihiro ; Hasegawa, Emi ; Kisanuki, Yaz Y. ; Sinton, Christopher M. ; Yanagisawa, Masashi ; Sakurai, Takeshi
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金沢大学医薬保健研究域医学系<br />Orexin-A and orexin-B are hypothalamic neuropeptides that play critical roles in the maintenance of
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wakefulness. Intracerebroventricular (ICV) administration of orexin-A has been shown to promote wakefulness and suppress both rapid eye movement (REM) sleep and non-REM (NREM) sleep through the orexin receptor-1 (OX1R) and orexin receptor-2 (OX2R). Here, we elucidated the differential roles of orexin receptors in the regulation of sleep and wakefulness by comparing the effects of ICV orexin-A administration in wild-type, OX1R, and OX2R mice. The effects of orexin-A on wakefulness and NREM sleep were significantly attenuated in both knock-out mice as compared with wild-type mice, with substantially larger attenuation in OX2R mice than in OX1R mice. These results suggest that although the OX2R-mediated pathway has a pivotal role in the promotion of wakefulness, OX1R also plays additional roles in promoting arousal. In contrast, suppression of REM sleep by orexin-A administration was slightly and similarly attenuated in both OX1R and OX2R mice, suggesting a comparable contribution of the two receptors to REM sleep suppression. Histological studies demonstrated differential distributions of each receptor subtype in distinct neuronal populations with specific neurotransmitter identities in brainstem cholinergic/monoaminergic neurons. In the laterodorsal tegmental and pedunculopontine tegmental nuclei especially, cholinergic neurons exclusively expressed OX1R mRNA, but OX2R mRNA was expressed mainly in GABAergic putative interneurons. Thus, each orexin receptor subtype plays differential roles in gating NREM and REM sleep through distinct neuronal pathways. © 2011 by the authors.
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| 7.
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Hondo, Mari ; Nagai, Kanji ; Ohno, Kousaku ; Kisanuki, Yasushi ; Willie, Jon T. ; Watanabe, Takeshi ; Yanagisawa, Masashi ; Sakurai, Takeshi
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金沢大学医薬保健研究域医学系<br />Aim: The effect of orexin on wakefulness has been suggested to be largely mediated by activation of
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histaminergic neurones in the tuberomammillary nucleus (TMN) via orexin receptor-2 (OX2R). However, orexin receptors in other regions of the brain might also play important roles in maintenance of wakefulness. To dissect the role of the histaminergic system as a downstream mediator of the orexin system in the regulation of sleep/wake states without compensation by the orexin receptor-1 (OX1R) mediated pathways, we analysed the phenotype of Histamine-1 receptor (H1R) and OX 1R double-deficient (H1R-/-;OX 1R-/-) mice. These mice lack OX1R-mediated pathways in addition to deficiency of H1R, which is thought to be the most important system in downstream of OX2R. Methods: We used H 1R deficient (H1R-/-) mice, H1R -/-;OX1R-/- mice, OX1R and OX 2R double-deficient (OX1R-/-;OX 2R-/-) mice, and wild type controls. Rapid eye movement (REM) sleep, non-REM (NREM) sleep and awake states were determined by polygraphic electroencephalographic/electromyographic recording. Results: No abnormality in sleep/wake states was observed in H1R-/- mice, consistent with previous studies. H1R-/-;OX 1R-/- mice also showed a sleep/wake phenotype comparable to that of wild type mice, while OX1R-/-; OX 2R-/- mice showed severe fragmentation of sleep/wake states. Conclusion: Our observations showed that regulation of the sleep/wake states is completely achieved by OX2R-expressing neurones without involving H1R-mediated pathways. The maintenance of basal physiological sleep/wake states is fully achieved without both H1 and OX1 receptors. Downstream pathways of OX2R other than the histaminergic system might play an important role in the maintenance of sleep/wake states. © 2009 Scandinavian Physiological Society.
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| 8.
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Xie, Xinmin ; Wisor, Jonathan P. ; Hara, Junko ; Crowder, Tara L. ; LeWinter, Robin ; Khroyan, Taline V. ; Yamanaka, Akihiro ; Diano, Sabrina ; Horvath, Takeshi L. ; Sakurai, Takeshi ; Toll, Lawrence ; Kilduff, Thomas S.
概要:
金沢大学医薬保健研究域医学系<br />Stress-induced analgesia (SIA) is a key component of the defensive behavioral "fight-or-flight" resp
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onse. Although the neural substrates of SIA are incompletely understood, previous studies have implicated the hypocretin/orexin (Hcrt) and nociceptin/orphanin FQ (N/OFQ) peptidergic systems in the regulation of SIA. Using immunohistochemistry in brain tissue from wild-type mice, we identified N/OFQ-containing fibers forming synaptic contacts with Hcrt neurons at both the light and electron microscopic levels. Patch clamp recordings in GFP-tagged mouse Hcrt neurons revealed that N/OFQ hyperpolarized, decreased input resistance, and blocked the firing of action potentials in Hcrt neurons. N/OFQ postsynaptic effects were consistent with opening of a G protein-regulated inwardly rectifying K+ (GIRK) channel. N/OFQ also modulated presynaptic release of GABA and glutamate onto Hcrt neurons in mouse hypothalamic slices. Orexin/ataxin-3 mice, in which the Hcrt neurons degenerate, did not exhibit SIA, although analgesia was induced by i.c.v. administration of Hcrt-1. N/OFQ blocked SIA in wild-type mice, while coadministration of Hcrt-1 overcame N/OFQ inhibition of SIA. These results establish what is, to our knowledge, a novel interaction between the N/OFQ and Hcrt systems in which the corticotropin-releasing factor and N/OFQ systems coordinately modulate the Hcrt neurons to regulate SIA.
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| 9.
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Furutani, Naoki ; Hondo, Mari ; Tsujino, Natsuko ; Sakurai, Takeshi
概要:
金沢大学医薬保健研究域医学系<br />The neuropeptides orexin A and orexin B (also known as hypocretin 1 and hypocretin 2), produced in l
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ateral hypothalamic neurons, are critical regulators of feeding behavior, the reward system, and sleep/wake states. Orexin-producing neurons (orexin neurons) are regulated by various factors involved in regulation of energy homeostasis and sleep/wakefulness states. Bombesin receptor subtype 3 (BRS3) is an orphan receptor that might be implicated in energy homeostasis and is highly expressed in the hypothalamus. However, the neural pathway by which BRS3 regulates energy homeostasis is largely unknown. We examined whether BRS3 is involved in the regulation of orexin neurons. Using a calcium imaging method, we found that a selective BRS3 agonist [Ac-Phe-Trp-Ala-His-(τBzl)-Nip-Gly-Arg-NH2] increased the intracellular calcium concentration of orexin neurons. However, intracellular recordings from slice preparations revealed that the BRS3 agonist hyperpolarized orexin neurons. The BRS3 agonist depolarized orexin neuron in the presence of tetrodotoxin. Moreover, in the presence of GABA receptor blockers, picrotoxin and CGP55845, the BRS3 agonist induced depolarization and increased firing frequency. Additionally, double-label in situ hybridization study revealed that Brs3 mRNA was expressed in almost all orexin neurons and many cells around these neurons. These findings suggest that the BRS3 agonist indirectly inhibited orexin neurons through GABAergic input and directly activated orexin neurons. Inhibition of activity of orexin neurons through BRS3 might be an important pathway for regulation of feeding and sleep/wake states. This pathway might serve as a novel target for the treatment of obesity. © 2010 Springer Science+Business Media, LLC.
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| 10.
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Tanaka, Mari ; Asada, Misako ; Higashi, Atsuko Y. ; Nakamura, Jin ; Oguchi, Akiko ; Tomita, Mayumi ; Yamada, Sachiko ; Asada, Nariaki ; Takase, Masayuki ; Okuda, Tomohiko ; Kawachi, Hiroshi ; Economides, Aris N. ; Robertson, Elizabeth ; Takahashi, Satoru ; Sakurai, Takeshi ; Goldschmeding, Roel ; Muso, Eri ; Fukatsu, Atsushi ; Kita, Toru ; Yanagita, Motoko
概要:
金沢大学医薬保健研究域医学系<br />The glomerular basement membrane (GBM) is a key component of the filtering unit in the kidney. Mutat
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ions involving any of the collagen IV genes (COL4A3, COL4A4, and COL4A5) affect GBM assembly and cause Alport syndrome, a progressive hereditary kidney disease with no definitive therapy. Previously, we have demonstrated that the bone morphogenetic protein (BMP) antagonist uterine sensitization-associated gene-1 (USAG-1) negatively regulates the renoprotective action of BMP-7 in a mouse model of tubular injury during acute renal failure. Here, we investigated the role of USAG-1 in renal function in Col4a3 -/- mice, which model Alport syndrome. Ablation of Usag1 in Col4a3-/- mice led to substantial attenuation of disease progression, normalization of GBM ultrastructure, preservation of renal function, and extension of life span. Immunohistochemical analysis revealed that USAG-1 and BMP-7 colocalized in the macula densa in the distal tubules, lying in direct contact with glomerular mesangial cells. Furthermore, in cultured mesangial cells, BMP-7 attenuated and USAG-1 enhanced the expression of MMP-12, a protease that may contribute to GBM degradation. These data suggest that the pathogenetic role of USAG-1 in Col4a3-/- mice might involve crosstalk between kidney tubules and the glomerulus and that inhibition of USAG-1 may be a promising therapeutic approach for the treatment of Alport syndrome.
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