Keisuke Yamashita

Publications (9)21.2 Total impact

• Article: Comparative study of aryl hydrocarbon receptor ligand activities of six chemicals in vitro and in vivo.

  Kazumi Sugihara, Takashige Okayama, Shigeyuki Kitamura, Keisuke Yamashita, Mineo Yasuda, Shinichi Miyairi, Yasushi Minobe, Shigeru Ohta
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  ABSTRACT: The aryl hydrocarbon receptor (AhR) ligand activities of six known AhR ligands were compared in vivo and in vitro. The in vivo ligand activity was estimated in terms of induction of cytochrome P450 1A1/2 activities, i.e., ethoxyresorufin-O-dealkylase (EROD) and methoxyresorufin-O-dealkylase (MROD) activities, and in vitro ligand activity was evaluated with a recombinant yeast reporter gene assay. The test chemicals were 3-methylcholanthrene (MC), beta-naphthoflavone (beta-NF), indirubin, indigo, 3,3'-diindolylmethane (DIM) and diphenyl-p-phenylenediamine (DPPD). The first four showed potent AhR ligand activity in vitro, comparable with that of 2,3,7,8-tetrachlorodibenzo-p-dioxin, while DIM and DPPD showed weaker activity. Administration of MC and beta-NF to mice caused significant induction of EROD and MROD activities, while indirubin, indigo and DIM also induced these activities, but less potently. DPPD also induced the activities, but was toxic at higher doses. These enhancing effects were lost or greatly reduced in Ahr-null mice (Ahr (-/-)). Our results suggest that EROD and MROD activity assays are useful for evaluating the AhR ligand activity of chemicals in vivo, where the biodynamics of the chemicals plays an important role.
  Archive für Toxikologie 02/2008; 82(1):5-11. · 4.67 Impact Factor
• Article: Distinct structural requirements for binding of the integrins alphavbeta6, alphavbeta3, alphavbeta5, alpha5beta1 and alpha9beta1 to osteopontin.

  Yasuyuki Yokosaki, Kumi Tanaka, Fumiko Higashikawa, Keisuke Yamashita, Akira Eboshida
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  ABSTRACT: The extracellular matrix protein, osteopontin, is a ligand for several members of the integrin family, including alpha5beta1, alphavbeta3, alphavbeta5 and alpha9beta1. Osteopontin is a substrate for a number of extracellular proteases, including thrombin and the metalloproteases MMP-3 and MMP-7, which cleave osteopontin at sites close to or within the mapped integrin binding sites. Using affinity chromatography and cell adhesion assays, we now identify the integrin alphavbeta6 as an additional osteopontin receptor. Utilizing a series of recombinant forms of osteopontin, we compared the structural requirements for alphavbeta6 binding with those for the 4 other osteopontin-binding integrins. Like alpha5beta1, alphavbeta3 and alphavbeta5 (but not alpha9beta1), alphavbeta6 binds to the RGD site in osteopontin, since RGD peptide or mutation of this site to RAA completely inhibits alphavbeta6-mediated cell adhesion. For both alpha9beta1 and alpha5beta1, the N-terminal fragment generated by thrombin cleavage is a much better ligand than full length osteopontin, whereas thrombin-cleavage does not appear to be required for optimal adhesion to alphavbeta3, alphavbeta5 or alphavbeta6. A recombinant fragment predicted to be generated by MMP cleavage no longer supported alpha5beta1 or alpha9beta1-mediated adhesion, but adhesion mediated by alphavbeta5 or alphavbeta6 was unaffected. Finally, adhesion of alphavbeta5 or alphavbeta6 was inhibited by mutation of two aspartic acid residues upstream of the RGD site, whereas adhesion mediated by alphavbeta3, alpha5beta1 or alpha9beta1 was unaffected by these mutations. These results suggest that the hierarchy of integrin interactions with osteopontin can undergo complex regulation at least in part through the action of extracellular proteases.
  Matrix Biology 10/2005; 24(6):418-27. · 3.30 Impact Factor
• Article: Aryl hydrocarbon receptor-mediated induction of microsomal drug-metabolizing enzyme activity by indirubin and indigo.

  Kazumi Sugihara, Shigeyuki Kitamura, Tsuyoshi Yamada, Takashige Okayama, Shigeru Ohta, Keisuke Yamashita, Mineo Yasuda, Yoshiaki Fujii-Kuriyama, Ken'ich Saeki, Saburo Matsui, Tomonari Matsuda
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  ABSTRACT: Indirubin and indigo, which are thought to be natural ligands for aryl hydrocarbon receptor (AhR), showed marked AhR ligand activities in a reporter gene assay using recombinant yeast. Their activities were comparable with or more potent than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin. When indirubin and indigo were administered to mice, ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in the liver were increased, but subsequently decreased within 2 days. Indirubin was more potent than indigo. Levels of cytochrome P450 1A1/2 proteins and mRNAs in the liver of mice dosed with indirubin were also enhanced. These enhancing effects of indirubin and indigo were not observed in AhR knock-out mice. Ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in rat hepatocytes and HepG2 cells were enhanced by the addition of indirubin or indigo, but less potently than by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Indigocarmine, a sulfate derivative of indigo, which is used as food additive, did not show these inducing effects on drug-metabolizing enzymes. Our results suggest that indirubin and indigo act as inducers for cytochrome P450 1A1/2 mediated by AhR in mammals in vivo.
  Biochemical and Biophysical Research Communications 06/2004; 318(2):571-8. · 2.48 Impact Factor
• Article: Functional analysis of basic transcription element binding protein by gene targeting technology.

  Masanobu Morita, Akira Kobayashi, Toshiharu Yamashita, Tomomasa Shimanuki, Osamu Nakajima, Satoru Takahashi, Shiro Ikegami, Kaoru Inokuchi, Keisuke Yamashita, Masayuki Yamamoto, Yoshiaki Fujii-Kuriyama
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  ABSTRACT: Basic transcription element binding protein (BTEB) is a transcription factor with a characteristic zinc finger motif and is most remarkably enhanced by thyroid hormone T(3) treatment (R. J. Denver et al., J. Biol. Chem. 272:8179-8188, 1997). To investigate the function of BTEB per se and to touch on the effects of T(3) (3,5,3'-triiodothyronine) on mouse development, we generated BTEB-deficient mice by gene knockout technology. Homologous BTEB(-/-) mutant mice were bred according to apparently normal Mendelian genetics, matured normally, and were fertile. Mutant mice could survive for at least 2 years without evident pathological defects. From the expression of lacZ, which was inserted into the reading frame of the BTEB gene, BTEB showed a characteristic tissue-specific expression profile during the developmental process of brain and bone. Dramatically increased expression of BTEB was observed in Purkinje cells of the cerebellum and pyramidal cell layers of the hippocampus at P7 when synapses start to form in the brain. Although general behavioral activities such as locomotion, rearing, and speed of movement were not so much affected in the BTEB(-/-) mutant mice, they showed clearly reduced activity levels in rotorod and contextual fear-conditioning tests; this finding was probably due to defective functions of the cerebellum, hippocampus, and amygdala.
  Molecular and Cellular Biology 05/2003; 23(7):2489-500. · 5.53 Impact Factor
• Article: Fine structural aspects of the urothelium in the mouse ureter with special reference to cell kinetics.

  Keisuke Yamashita
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  ABSTRACT: The present study aimed at clarifying the cell kinetics of the mouse ureteral epithelium by focusing on vesicle maturation in the cells and labeling with bromodeoxyuridine (BrdU). Electron microscopically, superficial cells were characterized by concave plaques in the apical plasma membrane and numerous fusiform vesicles in the cytoplasm. Intermediate cells were laden with ellipsoid vesicles, and basal cells had a few or no round vesicles. From the difference in number and form of vesicles among the three types of cells, it can be inferred that intermediate cells are immature in comparison with superficial cells, and likewise basal cells in comparison with intermediate cells. When BrdU was injected intraperitoneally once a day for seven days, most BrdU-labeled cells were located in the basal layer. Twelve days after the last injection, BrdU was detected in the intermediate or superficial layer in addition to the basal layer. These findings suggest that the basal cell is a progenitor cell giving rise to daughter cells that migrate upward to replace intermediate and superficial cells.
  Hiroshima journal of medical sciences 07/2002; 51(2):41-8.
• Article: In situ localization of nitric oxide synthase and direct evidence of NO production in rat retinal ganglion cells.

  Yuichi Tsumamoto, Keisuke Yamashita, Masaya Takumida, Koji Okada, Satoshi Mukai, Makoto Shinya, Hidetoshi Yamashita, Hiromu K Mishima
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  ABSTRACT: The expression of isoforms of nitric oxide synthase (NOS), enzymes responsible for NO production, and the synthesis of nitric oxide (NO) in rat retinal ganglion cells (RGCs) during synaptogenesis for various phases of the pre- and postnatal developmental periods were investigated. The retinas from prenatal, lactating, young, and adult rats were fixed in paraformaldehyde. The cryosections or paraformaldehyde-fixed ganglion cells purified from rat pups were immunostained for constitutive isoforms of NOS (n and eNOS) and observed with a confocal laser scanning microscope. Synthesis of NO in the RGCs was achieved by in vitro stimulation with glutamate. The intracellular NO levels were measured in real time using diaminofluorescein-2 diacetate, a fluorescence indicator of NO. Immunohistochemical analysis revealed nNOS and eNOS expressed in retinal ganglion cells during the first 2 postnatal weeks. Cultured RGCs also expressed nNOS and eNOS in vitro. Intracellular NO levels in cultured RGCs showed spontaneous fluctuation during a 20-min observation. The presence of both a non-specific NOS inhibitor, L-NAME, and a specific nNOS inhibitor, 7-NI, significantly inhibited (P<0.001) the increase of intracellular NO 6 and 8 min after the introduction of L-arginine and glutamate to the medium. This study revealed that all constitutive NOS isoforms are expressed in RGCs and demonstrated that NO is produced by nNOS mainly through stimulation by glutamate in cultured RGCs.
  Brain Research 04/2002; 933(2):118-29. · 2.73 Impact Factor
• Article: Aryl Hydrocarbon Receptor (AhR)-Mediated Induction of Xanthine Oxidase/Xanthine Dehydrogenase Activity by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

  Kazumi Sugihara, Shigeyuki Kitamura, Tuyoshi Yamada, Shigeru Ohta, Keisuke Yamashita, Mineo Yasuda, Yoshiaki Fujii-Kuriyama
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  ABSTRACT: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant, induced xanthine oxidase and xanthine dehydrogenase (XO/XDH) activities, in addition to ethoxyresorufin–O-dealkylase and methoxyresorufin-O-dealkylase activities in liver of mice. When TCDD was given to mice as a single oral dose of 40 μg/kg, the activities of XO and XDH increased about threefold within 3 days and the increased levels were maintained for 4 weeks. The treatment of mice with 3-methylcholanthrene also induced XO/XDH activities, but phenobarbital and dexamethasone had no effect. The level of aldehyde oxidase, a molybdenum flavoenzyme related to XO/XDH, in mouse liver was also enhanced about 1.5-fold by TCDD treatment. The inducing effect of TCDD and 3-methylcholanthrene was not observed in null mice (AhR−/−), which lack the AhR gene. XO and XDH activities were induced by TCDD in heterozygous mice (AhR+/−). The lipid peroxidation in liver was stimulated by TCDD. The induction of XO and XDH, which produces reactive oxygen species, may contribute to the various toxicities of TCDD.
  Biochemical and Biophysical Research Communications 04/2001; · 2.48 Impact Factor
• Article: Distinct structural requirements for binding of the integrins αvβ6, αvβ3, αvβ5, α5β1 and α9β1 to osteopontin

  Yasuyuki Yokosaki, Kumi Tanaka, Fumiko Higashikawa, Keisuke Yamashita, Akira Eboshida
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  ABSTRACT: The extracellular matrix protein, osteopontin, is a ligand for several members of the integrin family, including α5β1, αvβ3, αvβ5 and α9β1. Osteopontin is a substrate for a number of extracellular proteases, including thrombin and the metalloproteases MMP-3 and MMP-7, which cleave osteopontin at sites close to or within the mapped integrin binding sites. Using affinity chromatography and cell adhesion assays, we now identify the integrin αvβ6 as an additional osteopontin receptor. Utilizing a series of recombinant forms of osteopontin, we compared the structural requirements for αvβ6 binding with those for the 4 other osteopontin-binding integrins. Like α5β1, αvβ3 and αvβ5 (but not α9β1), αvβ6 binds to the RGD site in osteopontin, since RGD peptide or mutation of this site to RAA completely inhibits αvβ6-mediated cell adhesion. For both α9β1 and α5β1, the N-terminal fragment generated by thrombin cleavage is a much better ligand than full length osteopontin, whereas thrombin-cleavage does not appear to be required for optimal adhesion to αvβ3, αvβ5 or αvβ6. A recombinant fragment predicted to be generated by MMP cleavage no longer supported α5β1 or α9β1-mediated adhesion, but adhesion mediated by αvβ5 or αvβ6 was unaffected. Finally, adhesion of αvβ5 or αvβ6 was inhibited by mutation of two aspartic acid residues upstream of the RGD site, whereas adhesion mediated by αvβ3, α5β1 or α9β1 was unaffected by these mutations. These results suggest that the hierarchy of integrin interactions with osteopontin can undergo complex regulation at least in part through the action of extracellular proteases.
  Matrix Biology.
• Article: ダイオキシン胎生期暴露のアサゲザルの発育、生殖への影響に関する研究

  峯生 安田, 寛 隅田, 敬介 山下, 一雄 浅岡, 数美 杉原, Mineo Ysasuda, Hiroshi Sumida, Keisuke Yamashita, Kazuo Asaoka, Kazumi Sugihara, ミネオ ヤスダ, ヒロシ スミダ, ケイスケ ヤマシタ, カズオ アサオカ, カズミ スギハラ
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  ABSTRACT: 厚生労働科学研究費補助金食品・化学物質安全総合研究事業総括・分担研究報告書, 平成14年度, 主任研究者:安田峯生