Hiroshi Kobayashi

Chiba University, Chiba-shi, Chiba-ken, Japan

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Publications (36)86.63 Total impact

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    ABSTRACT: Many previous studies in animal models and clinical investigations have suggested that statins are useful chemotherapeutics against rheumatoid arthritis, whereas in vitro experiments using synovial cell lines showed no significant effect of statins on cell proliferation until now. Since synovial fluid in rheumatoid joint knee was found to be acidic, we examined the effect of statins on human synovial sarcoma cell line SW982 cells in acidic medium. Statins suppressed the proliferation of SW982 cells at pH6.7, while the suppression was very weak in pH7.5 medium. It was shown that the suppression was caused by the decrease in geranylgeranyl diphosphate, suggesting that a geranylgeranylated protein(s) has an essential role in cell proliferation of SW982 cells under acidic conditions. Our present data clearly implied that statins had high efficacy against SW982 cells in acidic medium whose pH is close to that of rheumatoid arthritis loci in patients. These results lead us to anticipate that screening of chemicals having high therapeutic efficacy in acidic medium promotes the development of new microenvironment-dependent medicines for chemotherapies against rheumatoid arthritis.
    International immunopharmacology 06/2013; · 2.21 Impact Factor
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    ABSTRACT: We examined anti-tumor effects of zoledronic acid (ZOL), one of the bisphosphonates agents clinically used for preventing loss of bone mass, on human mesothelioma cells bearing the wild-type p53 gene. ZOL-treated cells showed activation of caspase-3/7, -8 and -9, and increased sub-G1 phase fractions. A combinatory use of ZOL and cisplatin (CDDP), one of the first-line anti-cancer agents for mesothelioma, synergistically or additively produced the cytotoxicity on mesothelioma cells. Moreover, the combination achieved greater anti-tumor effects on mesothelioma developed in the pleural cavity than administration of either ZOL or CDDP alone. ZOL-treated cells as well as CDDP-treated cells induced p53 phosphorylation at Ser 15, a marker of p53 activation, and up-regulated p53 protein expression levels. Down-regulation of p53 levels with siRNA however did not influence the ZOL-mediated cytotoxicity but negated the combinatory effects by ZOL and CDDP. In addition, ZOL treatments augmented cytotoxicity of adenoviruses expressing the p53 gene on mesothelioma. These data demonstrated that ZOL-mediated augmentation of p53, which was not linked with ZOL-induced cytotoxicity, played a role in the combinatory effects with a p53 up-regulating agent, and suggests a possible clinical use of ZOL to mesothelioma with anti-cancer agents.
    PLoS ONE 01/2013; 8(3):e60297. · 3.53 Impact Factor
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    ABSTRACT: Although it is now well known that some diseased areas, such as cancer nests, inflammation loci, and infarction areas, are acidified, little is known about cellular signal transduction, gene expression, and cellular functions under acidic conditions. Our group showed that different signal proteins were activated under acidic conditions compared with those observed in a typical medium of around pH 7.4 that has been used until now. Investigations of gene expression under acidic conditions may be crucial to our understanding of signal transduction in acidic diseased areas. In this study, we investigated gene expression in mesothelioma cells cultured at an acidic pH using a DNA microarray technique. After 24 h culture at pH 6.7, expressions of 379 genes were increased more than twofold compared with those in cells cultured at pH 7.5. Genes encoding receptors, signal proteins including transcription factors, and cytokines including growth factors numbered 35, 32, and 17 among the 379 genes, respectively. Since the functions of 78 genes are unknown, it can be argued that cells may have other genes for signaling under acidic conditions. The expressions of 37 of the 379 genes were observed to increase after as little as 2 h. After 24 h culture at pH 6.7, expressions of 412 genes were repressed more than twofold compared with those in cells cultured at pH 7.5, and the 412 genes contained 35, 76, and 7 genes encoding receptors, signal proteins including transcription factors, and cytokines including growth factors, respectively. These results suggest that the signal pathways in acidic diseased areas are different, at least in part, from those examined with cells cultured at a pH of around 7.4.
    Genes. 01/2013; 4(1):65-85.
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    ABSTRACT: Interferons (IFNs) have been tested for the therapeutic effects in various types of malignancy, but mechanisms of the anti-tumors effects and the differential biological activities among IFN members are dependent on respective cell types. In this study, we examined growth inhibitory activities of type I and III IFNs on 5 kinds of human mesothelioma cells bearing wild-type p53 gene, and showed that type I IFNs but not type III IFNs decreased the cell viabilities. Moreover, growth inhibitory activities and up-regulated expression levels of the major histocompatibility complexes class I antigens were greater with IFN-β than with IFN-α treatments. Cell cycle analyses demonstrated that type I IFNs increased S- and G2/M-phase populations, and subsequently sub-G1-phase fractions. The cell cycle changes were also greater with IFN-β than IFN-α treatments, and these data collectively showed that IFN-β had stronger biological activities than IFN-α in mesothelioma. Type I IFNs-treated cells increased p53 expression and the phosphorylation levels, and activated apoptotic pathways. A combinatory use of IFN-β and cisplatin or pemetrexed, both of which are the current first-line chemotherapeutic agents for mesothelioma, produced synergistic anti-tumor effects, which were also evidenced by increased sub-G1-phase fractions. These data demonstrated firstly to our knowledge that IFN-β produced synergistic anti-tumor effects with cisplatin or pemetrexed on mesothelioma through up-regulated p53 expression.
    PLoS ONE 01/2013; 8(8):e72709. · 3.53 Impact Factor
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    ABSTRACT: INTRODUCTION:: Genetic characterization of malignant mesothelioma shows a homozygous deletion of the INK4A/ARF locus, which results in inactivation of the p53 pathways. METHODS:: We examined possible antitumor effects of adenoviruses with a deletion of the E1B-55kD gene (Ad-delE1B55) on mesothelioma and investigated combinatory actions with the first-line chemotherapeutic agents. RESULTS:: Ad-delE1B55 produced cytotoxicity on mesothelioma cells, which was associated with p53 phosphorylation, pRb dephosphorylation, and cleavage of caspases. Ad-delE1B55-infected cells displayed hyperploidy at the cell-cycle analysis and showed enlarged nuclear configurations. Combination of Ad-delE1B55 plus cisplatin or pemetrexed produced antitumor effects in vitro. Furthermore, Ad-delE1B55 and cisplatin showed combinatory effects in an orthotopic animal model. CONCLUSIONS:: Cell death caused by Ad-delE1B55 is attributable to cell-cycle arrest at M-phase checkpoint followed by activated apoptotic pathways, and combination of the first-line chemotherapeutic agents and the oncolytic adenovirus is a potential therapeutic for mesothelioma.
    Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 12/2012; 7(12):1850-1857. · 4.55 Impact Factor
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    ABSTRACT: We examined whether zoledronic acid (ZOL), the third generation of bisphosphonates, produced cytotoxic effects on human mesothelioma cells in vitro and in vivo, and investigated a possible involvement of p53, Ras, and extracellular signal-regulated kinase1/2 (ERK1/2) pathways. Cytotoxicity and cell cycles were assessed with a colorimetric assay and flow cytometry, respectively. Expression levels of apoptosis-linked proteins and prenylation of small guanine-nucleotide-binding regulatory proteins were tested with p53-small interfering RNA, an ERK kinase1/2-inhibitor, and prenyl alcohols. The antitumor activity was examined in an orthotopic animal model. ZOL treatments suppressed growth of mesothelioma cells bearing the wild-type p53 gene through apoptosis induction accompanied by activation of caspases, or S-phase arrest by up-regulated cyclin A and B1. ZOL induced p53 phosphorylation and subsequent activation of the downstream pathways. Down-regulated p53 expression with the small interfering RNA, however, showed that both apoptosis and S-phase arrest were irrelevant to the p53 activation. Geranylgeranyl but not farnesyl pyrophosphate inhibited ZOL-induced apoptosis and S-phase arrest, and the geranylgeraniol supplement decreased ZOL-mediated Rap1A but not Ras unprenylation. Inhibition of ERK1/2 pathways suppressed ZOL-induced apoptosis but not S-phase arrest. We further demonstrated that ZOL, administrated intrapleurally, inhibited the tumor growth in the pleural cavity. These data indicate that ZOL induces apoptosis or S-phase arrest, both of which are independent of p53 activation and Ras unprenylation, and suggest that ZOL is a possible therapeutic agent to mesothelioma partly through non-Ras- and ERK1/2-mediated pathways.
    Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 04/2012; 7(5):873-82. · 4.55 Impact Factor
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    ABSTRACT: Besides amino acid decarboxylation, the ADP biosynthetic pathway was reported to enhance survival under extremely acidic conditions in Escherichia coli (Sun et al., J. Bacteriol. 193∶ 3072-3077, 2011). E. coli has two pathways for ATP synthesis from ADP: glycolysis and oxidative phosphorylation. We found in this study that the deletion of the F(1)Fo-ATPase, which catalyzes the synthesis of ATP from ADP and inorganic phosphate using the electro-chemical gradient of protons generated by respiration in E. coli, decreased the survival at pH 2.5. A mutant deficient in hemA encoding the glutamyl tRNA reductase, which synthesizes glutamate 1-semialdehyde also showed the decreased survival of E. coli at pH 2.5. Glutamate 1-semialdehyde is a precursor of heme synthesis that is an essential component of the respiratory chain. The ATP content decreased rapidly at pH 2.5 in these mutants as compared with that of their parent strain. The internal pH was lowered by the deletion of these genes at pH 2.5. These results suggest that respiration and the F(1)Fo-ATPase are still working at pH 2.5 to enhance the survival under such extremely acidic conditions.
    PLoS ONE 01/2012; 7(12):e52577. · 3.53 Impact Factor
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    ABSTRACT: ATP participates in many cellular metabolic processes as a major substrate to supply energy. Many systems for acidic resistance (AR) under extremely acidic conditions have been reported, but the role of ATP has not been examined. To clarify whether or not ATP is necessary for the AR in Escherichia coli, the AR of mutants deficient in genes for ATP biosynthesis was investigated in this study. The deletion of purA or purB, each of which encodes enzymes to produce AMP from inosinate (IMP), markedly decreased the AR. The content of ATP in these mutants decreased rapidly at pH 2.5 compared to that of the wild type. The AR was again decreased significantly by the mutation of adk, which encoded an enzyme to produce ADP from AMP. The DNA damage in the purA and purB mutants was higher than that in the wild type. These results demonstrated that metabolic processes that require ATP participate in survival under extremely acidic conditions, and that one such system is the ATP-dependent DNA repair system.
    Journal of bacteriology 06/2011; 193(12):3072-7. · 3.94 Impact Factor
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    ABSTRACT: The roles of OmpC and OmpF in acidic resistance (AR) were examined. When ompC and ompF were deleted, AR was decreased. The decreased level of AR seen in the mutant that was deficient in ompC and ompF was elevated by the addition of glutamate, but not by the addition of arginine or lysine. The expression levels of adiA and cadB were diminished by the deletion of ompC and ompF, and the conversion of arginine to agmatine and lysine to cadaverine by intact cells were reduced in the mutant. The expression of gadA/gadB was not affected by the deletion of ompC and ompF. These results suggest that the transport of arginine, lysine, and their decarboxylated products through OmpC and/or OmpF is essential for the survival of Escherichia coli cells under extremely acidic conditions.
    Biological & Pharmaceutical Bulletin 01/2011; 34(3):330-4. · 1.85 Impact Factor
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    ABSTRACT: Escherichia coli has three major K(+) uptake systems, Trk, Kup, and Kdp, which have been studied extensively at near neutral pH. However, the function of these transporters under acidic conditions is not well understood, although growth and survival under acidic conditions are important for bacterial pathogenesis. In this study, we examined the expression and activity of Kdp under acidic conditions and found that the transport activity of Kdp is decreased at low pH and that the expression of kdp is regulated by the internal K(+) concentration in a pH-independent manner. Consequently, the low activity of Kdp was compensated for by the induction of its elevated expression by low K(+) accumulation via Kdp at acidic pH.
    Biological & Pharmaceutical Bulletin 01/2011; 34(3):426-9. · 1.85 Impact Factor
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    ABSTRACT: In tumor cell masses, the extracellular pH decreases below 6.5. The effect of external acidic pH on the efficacy of 24 chemical compounds including molecular-targeted inhibitors and anti-tumor reagents was investigated in human cancer cells. Lovastatin showed no cytotoxicity in mesothelioma or pancreatic carcinoma cells at concentrations up to 10 μM and pH around 7.4, but 10 μM lovastatin decreased the survival of these cells below 40% at acidic pH. Lovastatin inhibits HMG-CoA reductase, resulting in a decrease in the levels of cholesterol and prenylated proteins. An inhibitor of the former pathway showed pH-independent cytotoxic activity, whereas an inhibitor of the latter pathway had stronger activity at acidic pH. The inhibitory efficacy of cantharidin also increased at acidic pH. On the other hands, no pH dependency or slightly impaired efficacy at low pH conditions was observed in other 20 reagents, and especially, the activity of aphidicolin was suppressed under acidic conditions. These results suggested that screening under acidic conditions would be useful for developing new chemotherapeutic reagents.
    Cancer letters 11/2010; 297(2):182-9. · 4.86 Impact Factor
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    ABSTRACT: The first enantioselective total synthesis of tangutorine has been achieved, wherein a Pd-catalyzed asymmetric allylic amination using a chiral diaminophosphine oxide (DIAPHOX) preligand was the key step.
    Organic Letters 02/2010; 12(4):872-5. · 6.14 Impact Factor
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    Hiroshi Kobayashi, Ryuzo Azuma, Takuo Yasunaga
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    ABSTRACT: Cellular signal transduction is initiated by the binding of extracellular ligands to membrane receptors. Receptors are often expressed in excess, and cells are activated when a small number of receptors bind ligands. Intracellular signal proteins are activated at a high level soon after ligand binding, and the activation level decreases in a negative feedback manner without ligand clearance. Why are excess receptors required? What is the physiological significance of the negative feedback regulation? To answer these questions, we developed a Monte Carlo simulation program to kinetically analyze signal pathways using the model in which ligands are bound to receptors and then membrane complexes with other membrane proteins are formed. Our simulation results showed that excess receptors are not required for cell activation when the dissociation constant (Kd) of the ligand-receptor complex is 10-10 M or less. However, such low Kd values cause delayed signal shutdown after ligand clearance from the extracellular space. In contrast, when the Kd was 10-8 M and the ligand level was less than 1 muM, excess receptors were required for prompt signal propagation and rapid signal cessation after ligand clearance. An initial increase in active cytosolic signal proteins to a high level is required for rapid activation of cellular signal pathways, and a low level of active signal proteins is essential for the rapid shutdown of signal pathways after ligand clearance. The present kinetic analysis revealed that excess receptors and negative feedback regulation promote activation and cessation of signal transduction with a low amount of extracellular ligand.
    Cell Communication and Signaling 02/2009; 7:3. · 5.09 Impact Factor
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    ABSTRACT: The major histone-like Escherichia coli protein, HU, is composed of alpha and beta subunits respectively encoded by hupA and hupB in Escherichia coli. A mutant deficient in both hupA and hupB grew at a slightly slower rate than the wild type at pH 7.5. Growth of the mutant diminished with a decrease in pH, and no growth was observed at pH 4.6. Mutants of either hupA or hupB grew at all pH levels tested. The arginine-dependent survival at pH 2.5 was diminished approximately 60-fold by the deletion of both hupA and hupB, whereas the survival was slightly affected by the deletion of either hupA or hupB. The mRNA levels of adiA and adiC, which respectively encode arginine decarboxylase and arginine/agmatine antiporter, were low in the mutant deficient in both hupA and hupB. The deletion of both hupA and hupB had little effect on survival at pH 2.5 in the presence of glutamate or lysine, and expression of the genes for glutamate and lysine decarboxylases was not impaired by the deletion of the HU genes. These results suggest that HU regulates expression of the specific set of genes required for growth and survival in acidic environments.
    Current Microbiology 02/2009; 58(5):443-8. · 1.52 Impact Factor
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    ABSTRACT: In solid tumor and inflammation loci, low pH conditions have been observed as a consequence of either a lack of sufficient vascularization or excess activity of tumor cells, and T cells have been reported to infiltrate tumors and inflammation sites. However, it remains unclear how extracellular acidic environments affect immune cell function. A previous report proposed that a different signal transduction cascade might occur under low pH conditions in Jurkat T cells (Fukamachi T, Saito H, Kakegawa T, Kobayashi H. Different proteins are phosphorylated under acidic environments in Jurkat cells. Immunol Lett 2002;82:155-8). In this study, we investigated the protein phosphotyrosine level in Jurkat and Jurkat mutant cells under different pH conditions. The ZAP-70 phosphorylation level increased under acidic environments. P38 MAPK was more activated at acidic pH. The level of active p38 was low in mutant P116 deficient in ZAP-70, and interestingly the level remained consistently low at all pH values tested. The activation of ERK was not stimulated at low pH. These results suggest that extracellular low pH stimulates or enhances TCR signaling via ZAP-70 and p38.
    Immunology Letters 02/2008; 115(2):105-9. · 2.34 Impact Factor
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    ABSTRACT: In vertebrates, mRNAs containing a 5'-terminal oligopyrimidine (TOP) motif are coordinately post-transcriptionally regulated. Binding of specific proteins to this element has been proposed to downregulate expression of TOP mRNAs at the level of translational initiation. We previously reported that rapamycin induces binding activity to the TOP element of ribosomal protein (r-protein) L32 mRNA. In this study, we adapt DEAE-cellulose/oligo dT-cellulose tandem column chromatography to purify TOP element-binding proteins from bovine submaxillary lymph nodes (SLN). We also show by northwestern blot analysis that two proteins of molecular weight 47kDa (47BP) and 43kDa (43BP) specifically bind to a (32)P-labeled riboprobe containing TOP regulatory element of the r-protein L32. Microsequencing of the purified 47BP revealed an internal sequence of 15 amino acids identical to the consensus sequence of the 2x RBD-Gly family. Western blot analysis of the cytoplasm fractions using an AUF1 antibody revealed that these two proteins are p45 AUF1 and p42 AUF1. Increases of the four isoforms of AUF1 protein were observed in 100,000g supernatant fractions of rapamycin-administered rat SLN. Furthermore, decreases of p45 AUF1 and p42 and/or p40 AUF1 were observed in the polysomal fractions of BJAB cells in which translation of TOP mRNAs was selectively suppressed by rapamycin treatment. Taken together, these results suggest that AUF1 is a TOP mRNA-binding protein that may participate in the translational suppression of TOP mRNAs resulting from rapamycin treatment.
    Archives of Biochemistry and Biophysics 10/2007; 465(1):274-81. · 3.37 Impact Factor
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    Hiroshi Kobayashi, Ryuzo Azuma, Akihiko Konagaya
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    ABSTRACT: The physiological significance of membrane protein clustering for signal transduction was examined theoretically using a Monte Carlo computer simulation. Simulation results revealed that pre-stimulation clustering of membrane proteins enhanced signal transduction. Membrane protein clustering induced by the binding of external stimuli provided no kinetic advantage in terms of formation rate or maximum quantity of active membrane receptor complexes. These data suggested that membrane proteins associate weakly in the clustering areas of non-stimulated cells, and that their association is strengthened upon binding of extracellular stimuli to the membrane receptor. Additionally, the number of cytosolic proteins recruited to membrane receptor complexes was not increased by the membrane complex clustering, except when cytosolic signal proteins were localized to a narrow area such as a tunnel that ran from the membrane cluster to the nucleus. Simulations were carried out on a conventional personal computer under Windows XP or 2000 operating systems. Since neither special computing hardware nor special training is required, our simulation procedure could be easily adapted for kinetic analysis of any signal transduction pathway.
    Signal Transduction 07/2007; 7(4):329 - 339.
  • Hiroshi Kobayashi, Ryuzo Azuma, Akihiko Konagaya
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    ABSTRACT: Cellular signal transduction is initiated by the binding of extracellular ligands to membrane receptors. However, the concentration of ligand required for cellular activation is often lower than that required for receptor binding; receptors are often expressed in excess. To elucidate the physiological significance of this phenomenon, we have developed a Monte Carlo simulation program for kinetic analysis of ligand-receptor formation. Our present simulation showed that an excess amount of the receptors was not required for signal activation when the dissociation constant (Kd) of the ligand-receptor complex (LR) was low (10~9 or less). However, a low Kd value caused delayed LR dissociation after clearance of ligand from the extracellular space; no signal shutdown took place. These data indicate that an excess amount of receptors with high Kd (10~8 or more) was required for prompt signal propagation at physiological ligand concentrations and rapid signal cessation following ligand clearance. Our simulations were conducted using a conventional personal computer with a CPU running at 2.6 GHz under Windows XP or 2000 operating systems, and single simulation runs typically took less than two hours. Our simulation program could be readily implemented for kinetic analysis of any signal transduction system with various parameters, and could be used by any investigator because special computing hardware and training are not required.
    Advanced Information Networking and Applications Workshops, 2007, AINAW '07. 21st International Conference on; 06/2007
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    ABSTRACT: We previously reported that an IkappaB-beta COOH terminal region protein (designated CTIB) was essential for the proliferation of CHO cells under acidic stress (Lao et al., 2005. J Cell Physiol 203(1):186-192). In order to investigate the mechanisms underlying the requirement of CTIB for acidic adaptation, CTIB was silenced with an RNAi technique in CHO cells. CTIB silencing resulted in those cells completely failing to proliferate and maintain intracellular pH (pHi) homeostasis at an extracellular pH (pHe) of 6.3. An increased activation of p38 MAP kinase was induced by CTIB silencing at the low pH value. CTIB was only present in the cytoplasm and co-immunoprecipitation of the cytoplasmic fraction revealed that the loss of CTIB led to a loss of p65 in the immunoprecipitate complex. CTIB silencing reduced both the decrease in p65 and the increase in p50 in the nucleus when the cells were incubated at pHe 6.3. In cells with CTIB silenced, the transcriptions of p65, p105, and IL1-beta were suppressed, and decreases in both the transcription and activity of MnSOD were observed at pHe 6.3. Suppression of these genes suggested a suppressed NF-kappaB activity since p105, IL1-beta, and MnSOD were target genes of NF-kappaB. Our data demonstrated that CTIB functioned to prevent the over-accumulation of p65 in the nucleus, ensuring the appropriate composition of the NF-kappaB complex in the nucleus to respond to stimuli under acidic conditions.
    Journal of Cellular Physiology 05/2006; 207(1):238-43. · 4.22 Impact Factor
  • Advances in experimental medicine and biology 02/2006; 584:219-28. · 1.83 Impact Factor

Publication Stats

166 Citations
86.63 Total Impact Points

Institutions

  • 1999–2013
    • Chiba University
      • • Graduate School of Pharmaceutical Sciences
      • • Graduate School of Science
      • • Faculty of Pharmaceutical Sciences
      Chiba-shi, Chiba-ken, Japan
  • 2012
    • Northeast Institute of Geography and Agroecology
      • Guangzhou Institutes of Biomedicine and Health
      Beijing, Beijing Shi, China
  • 2007
    • Tokyo Institute of Technology
      • Department of Mathematical and Computing Sciences
      Tokyo, Tokyo-to, Japan
  • 2006
    • RIKEN
      Вако, Saitama, Japan