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論文
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Tanaka, Shigeo M.
概要:
In bone cells, intracellular Ca2+ (iCa2+) functions as a second messenger in the mechanotransduction pathway. Its responses to mechanical stimulation can be observed microscopically on a rigid flat surface with a Ca2+ fluorescent
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indicator, generally, under fluid flow. However, bone cells are physiologically exposed to dynamic loading accompanied by bone matrix deformation. In this situation, microscopic methods of observing iCa2+ responses cannot be used because of the loss of focus or movement of cells out of the observation area. The purpose of this study was to develop a compact optical device for the observation of iCa2+ responses of osteoblasts to dynamic loadings accompanied by substrate deformation. This system comprised four light emitting diodes (LEDs) and a photodetector (PD) placed underneath a culture chamber, specifically designed for tissue-level iCa2+ observations. This device was used to study the frequency dependence of iCa2+ responses of osteoblasts to dynamic loading. MC3T3-E1 osteoblasts were cultured three-dimensionally in a collagen sponge scaffold with the fluorescent Ca2+ indicator Fluo-4 AM and mechanically stimulated by a 0.2% deformation of the sponge at 0.2, 2, or 20 Hz for 150 s. Our device succeeded in detecting temporal changes in the intensity of emitted fluorescent light, showing a frequency-dependent increase in fluorescence intensity. This device may contribute to further understanding of the mechanosensing and mechanotransduction mechanisms in bone under near-physiological conditions.
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| 2.
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論文
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Sugiyama, Chie ; Nakamichi, Noritaka ; Ogura, Masato ; Honda, Eriko ; Maeda, Sayaka ; Taniura, Hideo ; Yoneda, Yukio
概要:
金沢大学大学院自然科学研究科分子作用学<br />Activation of ionotropic glutamate (Glu) receptors, such as N-methyl-d-aspartate receptors, is
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shown to modulate the gene transcription mediated by the transcription factor activator protein-1 (AP1) composed of Fos and Jun family proteins in the brain, while little attention has been paid to the modulation of AP1 expression by metabotropic Glu receptors (mGluRs). In cultured rat cortical neurons, where constitutive expression was seen with all groups I, II and III mGluR subtypes, a significant and selective increase was seen in the DNA binding activity of AP1 120 min after the brief exposure to the group II mGluR agonist (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) for 5 min. In cultured rat cortical astrocytes, by contrast, a significant increase was induced by a group I mGluR agonist, but not by either a group II or III mGluR agonist. The increase by DCG-IV was significantly prevented by a group II mGluR antagonist as well as by either an intracellular Ca2+ chelator or a voltage-sensitive Ca2+ channel blocker, but not by an intracellular Ca2+ store inhibitor. Moreover, DCG-IV significantly prevented the increase of cAMP formation by forskolin in cultured neurons. Western blot analysis revealed differential expression profiles of Fos family members in neurons briefly exposed to DCG-IV and NMDA. Prior or simultaneous exposure to DCG-IV led to significant protection against neuronal cell death by NMDA. These results suggest that activation of the group II mGluR subtype would modulate the gene expression mediated by AP1 through increased intracellular Ca2+ levels in cultured rat cortical neurons. © 2007.
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論文
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Nakamichi, Noritaka ; Yoneda, Yukio
概要:
金沢大学大学院自然科学研究科分子作用学<br />In contrast to other ionotropic glutamate receptors, N-methyl-d-aspartate (NMDA) receptor chann
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els are rather stable after the simulation. Brief exposure to NMDA at 50 μM rapidly increased the fluorescence intensity for increased intracellular free Ca2+ levels in a reversible- and concentration-dependent manner in rat cortical neurons cultured for 3-15 days in vitro (DIV), while EC50 values were significantly decreased in proportion to cellular maturation from 3 to 15 DIV. Although a constant increase was persistently seen in the fluorescence throughout the sustained exposure to NMDA for 60 min irrespective of the cell maturation from 3 to 15 DIV, the second brief exposure for 5 min resulted in a less efficient increase in the fluorescence than that found after the first brief exposure for 5 min in a manner dependent on intervals between the two repetitive brief exposures. In vitro maturation significantly shortened the interval required for the reduced responsiveness to the second brief exposure, while in immature neurons prolonged intervals were required for the reduced responsiveness to the second brief exposure to NMDA. Moreover, brief exposure to NMDA led to a marked decrease in immunoreactivity to extracellular loop of NR1 subunit in cultured neurons not permeabilized in proportion to the time after washing. These results suggest that cellular maturation would facilitate the desensitization process to repeated stimulation by NMDA, without markedly affecting that to sustained stimulation, through a mechanism related to the decreased number of NMDA receptors expressed at cell surfaces in cultured rat cortical neurons. © 2006 Elsevier Ltd. All rights reserved.
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