Masafumi Tsujimoto

Teikyo Heisei University, Edo, Tokyo, Japan

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Publications (227)934.25 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Aminopeptidase B (APB, EC preferentially hydrolyzes the N-terminal basic amino acids of synthetic and peptide substrates, and requires physiological concentration of NaCl for optimal activity. In this study, we used site-directed mutagenesis and molecular modeling to search for an amino acid residue that is critical for the enzymatic properties of human APB. Substitution of Phe297 with Tyr caused a significant decrease in hydrolytic activity toward synthetic and peptide substrates as well as chloride anion sensitivity. Molecular modeling suggests that Phe297 contributes to the construction of the substrate pocket of APB, which is wide enough to hold a chloride anion and allow the interaction of Gln169 with the N-terminal Arg residue of the substrate through bridging with the chloride anion. These results indicate that Phe297 is crucial for the optimal enzymatic activity and chloride anion sensitivity of APB via formation of the optimal structure of the catalytic pocket.
    No preview · Article · Sep 2015 · Biochemistry
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    ABSTRACT: In this study, we examined the role of aminopeptidases with reference to endoplasmic reticulum aminopeptidase 1 (ERAP1) in NO synthesis employing murine macrophage cell line RAW264.7 cells activated by lipopolysaccharide (LPS) and interferon (IFN)-γ and LPS-activated peritoneal macrophages derived from ERAP1 knockout mouse. When NO synthesis was measured in the presence of peptides having N-terminal Arg, comparative NO synthesis was seen with that measured in the presence of Arg. In the presence of an aminopeptidase inhibitor amastatin, NO synthesis in activated RAW264.7 cells was significantly decreased. These results suggest that aminopeptidases are involved in the NO synthesis in activated RAW264.7 cells. Subsequently, significant reduction of NO synthesis was observed in ERAP1 knockdown cells compared with wild-type cells. This reduction was rescued by exogenously added ERAP1. Furthermore, when peritoneal macrophages prepared from ERAP1 knockout mouse were employed, reduction of NO synthesis in knockout mouse macrophages was also attributable to ERAP1. In the presence of amastatin, further reduction was observed in knockout mouse-derived macrophages. Taken together, these results suggest that several aminopeptidases play important roles in the maximum synthesis of NO in activated macrophages in a substrate peptide-dependent manner and ERAP1 is one of the aminopeptidases involved in the NO synthesis. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
    No preview · Article · Jan 2015 · Journal of Biochemistry
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    ABSTRACT: Two novel siderophores, chlorocatechelins A and B, were isolated from a culture broth of Streptomyces sp. Their structures were determined by spectroscopic analysis and degradation study. They contain chloro-substituted catecholate that has not been reported in natural products, whereas this group was conjugated to guanidine to form acylguanidine in chlorocatechelin A. This acylguanidine decomposed in weakly acidic solutions to furnish a less potent siderophore chlorocatechelin B. Chemical and biological insights into acylguanidine are also discussed.
    No preview · Article · Nov 2014 · Organic Letters
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    ABSTRACT: Macrophages play an important role in host defense under several immunological, inflammatory, and/or infectious conditions. In our previous work, we demonstrated that endoplasmic reticulum aminopeptidase 1 (ERAP1) was secreted from macrophages in response to LPS and IFN-γ, and it enhanced their phagocytic activity. In this study, we analyzed the mechanism of LPS/IFN-γ-induced ERAP1 secretion. LPS/IFN-γ-induced secretion of the enzyme from the murine macrophage cell line RAW264.7 was suppressed by polymyxin B. Several agonists of TLRs, such as Pam3CSK4, FSL-1, and ODN1826, induced its secretion. In contrast, neutralizing Abs to IFN-β and TNF-α receptor type 1 suppressed its secretion. Using murine peritoneal macrophages derived from TNF-α and type 1 IFNR knockout mice, we confirmed the involvement of these two cytokines in ERAP1 secretion. In addition, secretion of ERAP1 from both RAW264.7 cells and murine peritoneal macrophages was induced by A23187 and thapsigargin and inhibited by BAPTA-AM and the calmodulin inhibitor W7. These results suggest that LPS/IFN-γ-induced secretion of ERAP1 is mediated by TLRs via induction of intermediate cytokines such as IFN-β and TNF-α, which in turn lead to enhanced cytosolic Ca(2+) levels and calmodulin activation.
    Preview · Article · Mar 2014 · The Journal of Immunology
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    ABSTRACT: Aminopeptidase B (EC, APB) preferentially hydrolyzes N-terminal basic amino acids of synthetic and peptide substrates. APB is involved in the production and maturation of peptide hormones and neurotransmitters such as miniglucagon, cholecystokinin and enkephalin by cleaving N-terminal basic amino acids in extended precursor proteins. Therefore, the specificity for basic amino acids is crucial for the biological function of APB. Site-directed mutagenesis and molecular modeling of the S1 site were used to identify amino acid residues of the human APB responsible for the basic amino acid preference and enzymatic efficiency. Substitution of Gln169 with Asn caused a significant decrease in hydrolytic activity toward the fluorescent substrate Lys-4-methylcoumaryl-7-amide (MCA). Substantial retardation of enzyme activity was observed toward Arg-MCA and substitution with Glu caused complete loss of enzymatic activity of APB. Substitution with Asn led to an increase in IC50 values of inhibitors that interact with the catalytic pocket of APB. The EC50 value of chloride ion binding was also found to increase with the Asn mutant. Gln169 was required for maximal cleavage of the peptide substrates. Molecular modeling suggested that interaction of Gln169 with the N-terminal Arg residue of the substrate could be bridged by a chloride anion. Gln169 is crucial for obtaining optimal enzymatic activity and the unique basic amino acid preference of APB via maintaining the appropriate catalytic pocket structure and thus for its function as a processing enzyme of peptide hormones and neurotransmitters.
    No preview · Article · Jan 2014 · Biochimica et Biophysica Acta
  • Akira Hattori · Masato Maruyama · Hiroshi Fujiwara · Masafumi Tsujimoto

    No preview · Chapter · Dec 2013
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    ABSTRACT: Osteoclast formation is regulated by balancing between receptor activator of nuclear factor-κB ligand (RANKL) expressed in osteoblasts and extracellular negative regulatory cytokines such as interferon-γ (IFN-γ) and interferon-β (IFN-β), which can suppress excessive bone destruction. However, relatively little is known about intrinsic negative regulatory factors in RANKL-mediated osteoclast differentiation. Herein we show the paired-box homeodomain transcription factor Pax6 acts as a negative regulator of RANKL-mediated osteoclast differentiation. Electrophoretic mobility shift and reporter assays found that Pax6 binds endogenously the proximal region of the tartrate-acid phosphatase (TRAP) gene promoter and suppresses nuclear factor of activated T cells c1 (NFATc1)-induced TRAP gene expression. Introduction of Pax6 retrovirally into bone marrow macrophages (BMM) attenuates RANKL-induced osteoclast formation. Moreover, we found that the Groucho family member co-repressor Grg6 contributes to Pax6-mediated suppression of the TRAP gene expression induced by NFATc1. These results suggest that Pax6 interferes with RANKL-mediated osteoclast differentiation together with Grg6. Our results demonstrate that the Pax6 pathway constitutes a new aspect of the negative regulatory circuit of RANKL-RANK signaling in osteoclastogenesis and that the augmentation of Pax6 might therefore represent a novel target to block pathological bone resorption.
    Full-text · Article · Aug 2013 · Journal of Biological Chemistry
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    Akira Hattori · Masafumi Tsujimoto
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    ABSTRACT: The human endoplasmic reticulum aminopeptidase (ERAP) 1 and 2 proteins were initially identified as homologues of human placental leucine aminopeptidase/insulin-regulated aminopeptidase. They are categorized as a unique class of proteases based on their subcellular localization on the luminal side of the endoplasmic reticulum. ERAPs play an important role in the N-terminal processing of the antigenic precursors that are presented on the major histocompatibility complex (MHC) class I molecules. ERAPs are also implicated in the regulation of a wide variety of physiological phenomena and pathogenic conditions. In this review, the current knowledge on ERAPs is summarized.
    Preview · Article · Aug 2013 · Journal of Biochemistry
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    ABSTRACT: Introduction: Evidence elucidating the pathophysiology and pharmacology of conventional drugs, β-2 stimulants and magnesium sulfate, on safety and effectiveness for preeclampsia and preterm labor are rarely found. Both compounds pass through the placental barrier and could exert their adverse effects on the fetus. Exposure to these agents could be problematic long after the birth, and possibly result in diseases such as autism and cardiomyopathy. Since 1970 the possible roles of placental aminopeptidases, which degrade peptide hormones, in preeclampsia and preterm labor have been studied. Areas covered: Many studies reveal that the fetus secretes peptide hormones, such as angiotensin II, vasopressin, and oxytocin, under hypoxia (stress) during the course of its growth, suggesting the critical effects these hormones have during pregnancy. The roles of placental aminopeptidases, the enzymes which degrade fetal hormones without passing through the placental barrier, were clarified. A first-step production system for recombinant aminopeptidases was established, by which engineered recombinant aminopeptidases were used for further experiments testing expected efficacy on controlling the level of hormones. Expert opinion: The authors conclude that both aminopeptidase A and placental leucine aminopeptidase could be potentially safe and effective drugs for patients and their babies in the treatment of preeclampsia and preterm labor.
    Full-text · Article · Aug 2013 · Expert Opinion on Investigational Drugs
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    Shou Takashima · Takumi Matsumoto · Masafumi Tsujimoto · Shuichi Tsuji
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    ABSTRACT: Mouse sialyltransferases are grouped into four families according to the type of carbohydrate linkage they synthesize: -galactoside α2,3- sialyltransferases (ST3Gal-I-VI), -galactoside α2,6-sialyltransferases (ST6Gal-I and ST6Gal-II), N-acetylgalactosamine α2,6-sialyltransferases (ST6GalNAc-I-VI) and α2,8-sialyltransferases (ST8Sia-I-VI). These sialyltransferases feature a type II transmembrane topology and contain highly conserved motifs termed sialylmotifs L, S, III and VS. Sialylmotifs L and S are involved in substrate binding, whereas sialylmotifs III and VS are involved in catalytic activity. In addition to the conventional sialylmotifs, family and subfamily specific sequence motifs have been proposed. In this study, we analyzed the properties and functions of sialylmotifs in characterizing the enzymatic activity of mouse ST8Sia-I and ST8Sia-VI, both of which are α2,8-sialyltransferases involved in the synthesis of either ganglioside GD3 or disialic acid structures on O-glycans, respectively. The ST8Sia-VI-based chimeric enzymes, whose sialylmotif L sequences were replaced with those of ST8Sia-I and ST8Sia-IV (polysialic acid synthetase), were still active toward O-glycans. However, ST8Sia-VI-based chimeric enzymes lost expression or activity when their sialylmotif L sequences were replaced with those of ST3Gal-I and ST6GalNAc-II, suggesting the existence of an ST8Sia family specific motif in the sialylmotif L. The ST8Sia-I- and ST8Sia-VI-based chimeric enzymes lost enzymatic activity when their sialylmotif S sequences were interchanged. Amino acid substitutions in the sialylmotif S of ST8Sia-I and ST8Sia-VI also affected the enzymatic activity in many cases, indicating the crucial and functional importance of the sialylmotif S in substrate binding, which determines the substrate specificity of sialyltransferase. © 2013 The Author 2013. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected] /* */
    Preview · Article · Jan 2013 · Glycobiology
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    ABSTRACT: Small non-coding RNAs, such as microRNAs (miRNAs), are involved in diverse processes, including organ development and tissue differentiation. Exosomes are small membrane vesicles (30-100 nm in diameter) produced by numerous cells. Recently, exosomes have been shown to contain miRNAs. However, the small RNAs contained in exosomes are not fully characterized. In a previous study, we found at least two types of salivary exosome that are different in size and have different proteomes. Studies of salivary exosomal small RNAs are limited to miRNAs. In this study, we examined small RNA transcriptomes using next generation sequencing technology to elucidate a full transcriptome set of small RNAs expressed in the two types of salivary exosomes and in whole saliva (WS). Many types of small RNA, such as miRNA, piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA) and other small RNAs are contained in salivary exosomes and WS. Among these small RNAs we identified novel miRNA candidates.
    No preview · Article · Jan 2013 · Biological & Pharmaceutical Bulletin
  • Seiji Nomura · Masafumi Tsujimoto · Shigehiko Mizutani

    No preview · Article · Jan 2013
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    ABSTRACT: In eukaryotic cells, components of messenger ribonucleoproteins (mRNPs) are often detected in cytoplasmic granules, such as processing bodies (P-bodies) and stress granules (SGs) where translationally repressed mRNAs accumulate. RAP55A, which is an RNA binding component of mRNPs, acts as a translational repressor and localizes to P-bodies and SGs. We found here that a homologous protein RAP55B also localized to P-bodies when expressed in human cultured cells. When RAP55A or RAP55B was highly expressed in the cells, they induced the formation of SG-like large cytoplasmic mRNP granules that contained both P-body and SG components, indicating that RAP55 is important for the assembly of cytoplasmic mRNP granules. In addition, we found that RAP55A associated with protein arginine methyltransferases PRMT1 and PRMT5. Multiple arginine residues of RAP55A were indeed asymmetrically dimethylated in the cell and PRMT1 was shown to be a component of large mRNP granules induced by RAP55A overexpression. Although PRMT1 did not accumulate in P-bodies, siRNA-mediated knockdown of PRMT1 impaired the localization of RAP55A to P-bodies, while other components were still retained in these structures. Thus, our data indicate that RAP55 is important for the assembly of cytoplasmic mRNP granules and that PRMT1 is required for RAP55A to localize to P-bodies.
    No preview · Article · May 2012 · RNA biology
  • Akira Hattori · Yoshikuni Goto · Masafumi Tsujimoto
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    ABSTRACT: Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a final trimming enzyme of N-extended antigenic peptides bound to major histocompatibility complex class I molecules in the endoplasmic reticulum (ER). In our previous work, we found that ERAP1 is secreted from macrophages in response to activation by lipopolysaccharide and interferon-γ. In this paper, we searched for the amino acid sequence of ERAP1 protein important for ER retention by constructing chimeric proteins and found that the sequence between 485 and 615 was significant. Moreover, by comparing the genomic organizations of oxytocinase subfamily members, the exon 10 coding sequence, which might be inserted into the common ancestral gene of the oxytocinase subfamily enzymes during evolution, was found to be important for ER retention of ERAP1. Taken together, our data indicate that ERAP1 contains amino acid sequence important for ER retention.
    No preview · Article · Apr 2012 · Biological & Pharmaceutical Bulletin
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    ABSTRACT: Proteolysis-related genes have diverse functions across taxa and have long been considered as key players for intracellular protein turnover. Growing evidence indicates the biological significance of peptidases in degradation, maturation and modulation of bioactive peptides/proteins. By screening T-DNA tagged lines and functional analysis approaches we unraveled the Arabidopsis leucine aminopeptidase (AtLAP2) function in amino acid turnover. Transcriptomics and metabolomics profiling data suggested involvement of AtLAP2 in specific metabolic pathways. Loss-of-function of AtLAP2 resulted in early-leaf senescent and stress-sensitive phenotypes. Our work indicates an important in planta role for AtLAP2 contributing to a further understanding of the proteases having several implications in higher plants.
    Preview · Article · Oct 2011 · Plant signaling & behavior
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    Yoshikuni Goto · Kenji Ogawa · Akira Hattori · Masafumi Tsujimoto
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    ABSTRACT: Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme with an important role in processing antigenic peptides presented to class I major histocompatibility complex in the endoplasmic reticulum. In this study, we found that endoplasmic reticulum-retained ERAP1 was secreted from macrophages in response to activation by treatment with lipopolysaccharide (LPS) and interferon (IFN)-γ and enhanced their phagocytic activity. Enhancement of the phagocytic activity of murine macrophage RAW264.7 cells induced by LPS/IFN-γ was inhibited by a potent aminopeptidase inhibitor, amastatin. The addition of recombinant wild-type but not inactive mutant ERAP1 to culture medium enhanced phagocytosis. These results suggest that enhancement of phagocytic activity is at least in part mediated by secreted ERAP1 through the generation of active peptides processed by the enzyme. Our data reveal ERAP1-mediated activation of macrophages for the first time and will provide new insights into the role of this enzyme in innate immunity.
    Preview · Article · Jun 2011 · Journal of Biological Chemistry
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    ABSTRACT: Peptidases are known to play key roles in multiple biological processes in all living organisms. In higher plants, the vast majority of putative aminopeptidases remain uncharacterized. In this study, we performed functional and expression analyses of the Arabidopsis LAP2 through cDNA cloning, isolation of T-DNA insertional mutants, characterization of the enzymatic activity, characterization of gene expression and transcriptomics and metabolomics analyses of the mutants. Loss of function of LAP2, one of the 28 aminopeptidases in Arabidopsis, reduced vegetative growth, accelerated leaf senescence and rendered plants more sensitive to various stresses. LAP2 is highly expressed in the leaf vascular tissue and the quiescent center region. Integration of global gene expression and metabolite analyses suggest that LAP2 controlled intracellular amino acid turnover. The mutant maintained free leucine by up-regulating key genes for leucine biosynthesis. However, this influenced the flux of glutamate strikingly. As a result, γ-aminobutyric acid, a metabolite that is derived from glutamate, was diminished in the mutant. Decrements in these nitrogen-rich compounds are associated with morphological alterations and stress sensitivity of the mutant. The results indicate that LAP2 is indeed an enzymatically active aminopeptidase and plays key roles in senescence, stress response and amino acid turnover.
    Preview · Article · May 2011 · New Phytologist
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    ABSTRACT: ER aminopeptidase 1 (ERAP1) customizes antigenic peptide precursors for MHC class I presentation and edits the antigenic peptide repertoire. Coding single nucleotide polymorphisms (SNPs) in ERAP1 were recently linked with predisposition to autoimmune disease, suggesting a link between pathogenesis of autoimmunity and ERAP1-mediated Ag processing. To investigate this possibility, we analyzed the effect that disease-linked SNPs have on Ag processing by ERAP1 in vitro. Michaelis-Menten analysis revealed that the presence of SNPs affects the Michaelis constant and turnover number of the enzyme. Strikingly, specific ERAP1 allele-substrate combinations deviate from standard Michaelis-Menten behavior, demonstrating substrate-inhibition kinetics; to our knowledge, this phenomenon has not been described for this enzyme. Cell-based Ag-presentation analysis was consistent with changes in the substrate inhibition constant K(i), further supporting that ERAP1 allelic composition may affect Ag processing in vivo. We propose that these phenomena should be taken into account when evaluating the possible link between Ag processing and autoimmunity.
    Full-text · Article · Feb 2011 · The Journal of Immunology
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    ABSTRACT: Brassinosteroids (BRs) are growth-promoting steroidal hormones. Despite the importance of BRs in plant biology, the signal that initiates BR biosynthesis remains unknown. Among the enzymes involved in BR biosynthesis in Arabidopsis (Arabidopsis thaliana), DWARF4 catalyzes the rate-determining step. Through both the histochemical analysis of DWF4pro:GUS plants and the direct measurement of endogenous BR content, we discovered that BR biosynthesis is stimulated by auxin. When DWF4pro:GUS was subjected to auxin dose-response tests and a time-course analysis, GUS activity started to increase at an auxin concentration of 10 nm, rising noticeably after 1 h of auxin treatment. In addition, the analysis of the DWF4pro:GUS line in BR- and auxin-mutant backgrounds revealed that the induction by auxin requires auxin-signaling pathways but not BRs, which implies that auxin signaling directly controls BR biosynthesis. Furthermore, chromatin immunoprecipitation assays confirmed that auxin inhibits the binding of the transcriptional repressor, BZR1, to the DWF4 promoter. A microarray analysis that was designed to examine the transcriptomes after treatment with auxin alone or auxin plus brassinazole (a BR biosynthetic inhibitor) revealed that genes previously characterized as being auxin responsive are not properly regulated when BR biosynthesis is disrupted by brassinazole. Therefore, our results support the idea that auxin regulates BR biosynthesis, and that auxin thus relies on synthesized BRs for some of its growth-promoting effects in Arabidopsis.
    Full-text · Article · Feb 2011 · The Plant Journal
  • Kenji Ogawa · Masayuki Funaba · Masafumi Tsujimoto
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    ABSTRACT: Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that plays a critical role in modulating immune response and inflammation. We have investigated the effects of TGF-β1 on the expression of type IV collagenases, matrix metalloproteinase (MMP)-2 and MMP-9, in mouse peritoneal macrophages. TGF-β1 alone enhanced the secretion of MMP-9, while it blocked lipopolysaccharide (LPS)-stimulated MMP-9 production. We have shown that this biphasic effect of TGF-β1 is exerted at the mRNA level of the MMP-9 gene. Although TGF-β1 increased both basal and LPS-induced MMP-2 production at the protein and mRNA levels, the extent of the increase was smaller in LPS-activated macrophages than in control macrophages. The expression of type I and type II receptors for TGF-β was significantly decreased upon activation, suggesting that the lesser effect of TGF-β1 in activated macrophages may result from the decreased expression of TGF-β receptors. In addition, the expression of endogenous TGF-β1 mRNA was decreased significantly in activated macrophages. These findings suggest that activated macrophages not only produce less TGF-β1, but also respond less well to TGF-β to provide for inflammatory response.
    No preview · Article · Feb 2011 · Molecular Biology Reports

Publication Stats

10k Citations
934.25 Total Impact Points


  • 2011-2015
    • Teikyo Heisei University
      Edo, Tokyo, Japan
  • 1999-2011
    • RIKEN
      Вако, Saitama, Japan
  • 2006-2009
    • Saitama University
      • Graduate School of Science and Engineering
      Саитама, Saitama, Japan
  • 2002
    • Nagasaki University
      • Department of Basic Pharmaceutical Sciences
      Nagasaki, Nagasaki, Japan
    • University of Tsukuba
      • Institute of Basic Medical Sciences
      Tsukuba, Ibaraki, Japan
  • 1990-1999
    • Gifu Pharmaceutical University
      • Department of Biology
      Saitama, Saitama, Japan
  • 1983-1996
    • MEDIPOST Biomedical Research Institute
      Sŏul, Seoul, South Korea
  • 1994-1995
    • The University of Tokyo
      • Department of Pharmaceutical Sciences
      Tokyo, Tokyo-to, Japan
  • 1987-1995
    • Japan Research Institute
      Ōsaka, Ōsaka, Japan
  • 1987-1991
    • Setsunan University
      • Faculty of Pharmaceutical Sciences
      Ōsaka-shi, Osaka-fu, Japan
  • 1985-1987
    • CUNY Graduate Center
      New York, New York, United States