H Machida

Publications (2)9.13 Total impact

• Source

  Available from: Ryuji Okamoto

  Article: Molecular cloning and analysis of the 5'-flanking region of the human MYPT1 gene.

  H Machida, M Ito, R Okamoto, K Shiraki, N Isaka, D J Hartshorne, T Nakano
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  ABSTRACT: We have cloned and sequenced a 5 kb genomic fragment in the 5'-flanking region of the human myosin phosphatase target subunit 1. The transcription initiation site (+1) was 268 bp upstream from the translation start site. In this promoter there are no canonical TATA or CAAT box elements but there is a high GC-rich sequence. Basal promoter activity was due to the GC-rich region that contained one Sp1 transcription factor binding site, thus demonstrating that the MYPT1 gene is a housekeeping gene. Luciferase reporter assays showed the presence of two regions for positive elements and one for a negative element.
  Biochimica et Biophysica Acta 03/2001; 1517(3):424-9. · 4.66 Impact Factor
• Article: Arachidonic acid-induced Ca2+ sensitization of smooth muscle contraction through activation of Rho-kinase.

  S Araki, M Ito, Y Kureishi, J Feng, H Machida, N Isaka, M Amano, K Kaibuchi, D J Hartshorne, T Nakano
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  ABSTRACT: Arachidonic acid activates isolated Rho-kinase and contracts permeabilized smooth muscle fibres. Various assays were carried out to examine the mechanism of this activation. Native Rho-kinase was activated 5-6 times by arachidonic acid but an N-terminal, constitutively-active fragment of Rho-kinase, expressed as a glutathione-S-transferase (GST) fusion protein and including the catalytic subunit (GST-Rho-kinase-CAT), was not. GST-Rho-kinase-CAT was inhibited by a C-terminal fragment of Rho-kinase and arachidonic acid removed this inhibition. These results suggest that the C-terminal part of Rho-kinase, containing the RhoA binding site and the pleckstrin homology domain, acts as an autoinhibitor. It is suggested further that activation by arachidonic acid is due to its binding to the autoinhibitory region and subsequent release from the catalytic site. Arachidonic acid, at concentrations greater than 30 microM, increases force in alpha-toxin-permeabilized femoral artery but not in Triton X-100-skinned fibres. The content of Rho-kinase in the latter was lower than in alpha-toxin-treated or intact fibres. The arachidonic acid-induced contraction was not observed at a pCa above 8.0 and was inhibited by Y-27632 and wortmannin, inhibitors of Rho-kinase and myosin light-chain kinase (MLCK), respectively. The activation of Rho-kinase and subsequent phosphorylation of the myosin phosphatase target subunit inhibits myosin phosphatase and increases myosin phosphorylation.
  Pflügers Archiv - European Journal of Physiology 03/2001; 441(5):596-603. · 4.46 Impact Factor