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Print ISSN : 0919-2719
Volume 12 , Issue 2+3+4
December
Showing 1-4 articles out of 4 articles from the selected issue
Review
  • Kazuya Kikuchi
    2004 Volume 12 Issue 2+3+4 Pages 55-60
    Published: 2004
    Released: October 28, 2005
    JOURNALS FREE ACCESS
    Fluorescence resonance energy transfer (FRET) has been used extensively as the designing principle for fluorescent sensor molecules, in both GFP based sensors and synthetic organic sensors. One of the most significant advantages of designing a sensor molecule with FRET modulation is that it can enable ratiometric measurement in living cells. The design strategy for the development of small molecular FRET sensor is described; it is based on changing the two factors in the Förster equation: donor and acceptor distance and overlap integral of donor emission and acceptor absorbance spectra. A careful design strategy is necessary in designing small molecule-based sensor molecules in order to avoid close contact of donor fluorophore and acceptor fluorophore in aqueous solution. Furthermore, a strategy to design FRET sensor molecules to monitor cellular activity of protein tyrosine phosphatases with modulating overlap integral is introduced.
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Regular Article
  • Iyuki Namekata, Reiko Yamagishi, Yoshimitsu Kato, Ryu Nakamura, Hikaru ...
    2004 Volume 12 Issue 2+3+4 Pages 61-69
    Published: 2004
    Released: October 28, 2005
    JOURNALS FREE ACCESS
    Two-dimensional and line-scan images of cytoplasmic and nuclear free Ca2+ movements in cardiac myocytes were obtained during normal Ca2+ transients and abnormal Ca2+ oscillations with confocal microscopy and Ca2+ sensitive fluoroprobes. When the myocardial cells were field-stimulated at 0.5 Hz, nuclear Ca2+ was observed to rise and fall following cytoplasmic Ca2+ with an obvious delay. Isoproterenol significantly decreased, while cyclopiazonic acid significantly increased, the time required for Ca2+ decay; the changes were larger in the cytoplasm than in the nucleus. When the cells were voltage-clamped at 0 mV for 3 sec, no difference in the steady state Ca2+ concentration was observed between the cytoplasm and the nucleus. Nuclear Ca2+ was also observed to increase following a Ca2+ wave—a local increase in free Ca2+ propagating within the cytoplasm—with a delay. When arrhythmic Ca2+ oscillations in the cytoplasm were induced by aconitine, they were propagated into the nucleus; treatment with KB-R7943 abolished the abnormal Ca2+ oscillations both in the cytoplasm and in the nucleus. Thus, we demonstrated in isolated myocardial cells that normal and abnormal Ca2+ oscillations in the cytoplasm, although insulated by the nuclear envelope, are propagated into the nucleus, a mechanism through which factors affecting cytoplasmic Ca2+ may influence intranuclear events.
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  • Yosuke Okada, Atsushi Suzuki, Sachiko Takagi, Hiroaki Hirai, Ryoichi S ...
    Type: scientific monograph
    Subject area: Infomation Science
    2004 Volume 12 Issue 2+3+4 Pages 71-83
    Published: 2004
    Released: October 28, 2005
    JOURNALS FREE ACCESS
    Polyglutamylation is a major post-translational modification of tubulin in neurons, and a change in glutamylated tubulin level has been reported during differentiation of neurons. In this study, we produced monoclonal antibodies (mAbs) with different specificities for α- and β-subunits of tubulin and investigated polyglutamylation of tubulin during differentiation of neural precursor cells (neurospheres) using these antibodies.
    We raised a mAb to carp brain tubulin (K9) and two mAbs to the synthetic peptide A (corresponding to the polyglutamylation site of α-tubulin) (E10 and PG1). K9 reacted with tubulin carrying more than one unit of polyglutamylated chains while PG1 and E10 reacted with tubulin carrying more than two and three chains, respectively. Using ELISA and immunoblot analysis, we revealed that K9 recognizes a conformational change of the conservative region between the two subunits of tubulin. We also found that PG1 specifically recognizes α-tubulin under high NaCl concentrations, and reacts with both subunits under low NaCl concentrations. Using this property of PG1, we observed a change in the localization of α-tubulin during differentiation of neural precursor cells prepared from rat embryonic brain. Polyglutamylated tubulin was not detected in neurospheres, but was detected in cells cultured for one day in medium containing retinoic acid. Poly-glutamylated β-tubulin was detected in cells cultured for 5 days; polyglutamylated α-tubulin was localized in the cell body while β-tubulin was seen in neurites. After 7 days, localization of polyglutamylated species changed. Polyglutamylated α-tubulin was localized in both the cell body and neurites. These results indicate that polyglutamylation is important in neuronal differentiation and that each polyglutamylated tubulin subunit plays a different role in differentiation of neurons.
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  • Rintaro Suzuki, Kazuo Tomizawa, Kazuo Suzuki, Masaru Tanokura
    Type: scientific monograph
    Subject area: Infomation Science
    2004 Volume 12 Issue 2+3+4 Pages 85-90
    Published: 2004
    Released: October 28, 2005
    JOURNALS FREE ACCESS
    Myeloperoxidase (MPO) has been identified as one of the major target of anti-neutrophil cytoplasmic antibody (ANCA), and ANCA with specificity for MPO is called MPO-ANCA. Binding of MPO-ANCA to MPO is a trigger for many inflammatory diseases, but the details of the interaction between two molecules are unknown. We used the result of an epitope mapping study and molecular modeling techniques to identify the MPO-ANCA binding site of MPO. The structural features of MPO suggest that the most likely region for the interaction on the molecule lies adjacent to the peroxidase active site.
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