Publications (31)93.06 Total impact
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Article: Comprehensive Synthesis of Photoreactive (3-Trifluoromethyl)diazirinyl Indole Derivatives from 5- and 6- Trifluoroacetylindoles for Photoaffinity Labeling.
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ABSTRACT: 5- and 6-trifluoromethyldiazirinyl indoles were synthesized from corresponding bromoindole derivatives for the first time. They acted as mother skeletons for the comprehensive synthesis of various bioactive indole metabolites. These can be used in biological functional analysis as diazirine-based photoaffinity labels.The Journal of Organic Chemistry 09/2012; 77(19):8581-7. · 4.45 Impact Factor -
Article: Characterization of the modes of binding between human sweet taste receptor and low-molecular-weight sweet compounds.
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ABSTRACT: One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2-hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein-coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2-hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling-based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands.PLoS ONE 01/2012; 7(4):e35380. · 4.09 Impact Factor -
Article: Isolation and Biochemical Characterization of Rubelase, a Non-Hemorrhagic Elastase from Crotalus ruber ruber (Red Rattlesnake) Venom.
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ABSTRACT: A novel non-hemorrhagic basic metalloprotease, rubelase, was isolated from the venom of Crotalus ruber ruber. Rubelase hydrolyzes succinyl-L-alanyl-L-alanyl-L-alanyl p-nitroanilide (STANA), a specific substrate for elastase, and the hydrolytic activity was inhibited by chelating agents. It also hydrolyzes collagen and fibrinogen. However, hemorrhagic activity was not observed. By ESI/Q-TOF and MALDI/TOF mass spectrometry combined with Edman sequencing procedure, the molecular mass of rubelase was determined to be 23,266 Da. Although its primary structure was similar to rubelysin (HT-2), a hemorrhagic metalloprotease isolated from the same snake venom, the circumstances surrounding putative zinc binding domain HEXXHXXGXXH were found to be different when the three-dimensional computer models of both metalloproteases were compared. The cytotoxic effects of rubelase and rubelysin on cultured endothelial and smooth muscle cells were also different, indicating that the substitution of several amino acid residues causes the changes of active-site conformation and cell preference.Toxins. 07/2011; 3(7):900-10. -
Article: Development of a stigmatic mass microscope using laser desorption∕ionization and a multi-turn time-of-flight mass spectrometer.
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ABSTRACT: A novel stigmatic mass microscope using laser desorption∕ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG, has been developed. Stigmatic ion images of crystal violet masked by a fine square mesh grid with a 12.7 μm pitch as well as microdot patterns with a 5 μm dot diameter and a 10 μm pitch made with rhodamine B were clearly observed. The estimated spatial resolution was about 3 μm in the linear mode with a 20-fold ion optical magnification. Separating stigmatic ion images according to the time-of-flight, i.e., the mass-to-charge ratio of the ions was successfully demonstrated by a microdot pattern made with two different dyes, crystal violet and methylene blue. Stigmatic ion images of a microdot pattern made with crystal violet were observed after circulation in MULTUM-IMG, and the pattern of the ion image was maintained after ten cycles in MULTUM-IMG. A section of a mouse brain stained with crystal violet and methylene blue was observed in the linear mode, and the stigmatic total ion image of crystal violet and methylene blue agreed well with the optical microphotograph of the hippocampus for the same section.Journal of Biomedical Optics 04/2011; 16(4):046007. · 3.16 Impact Factor -
Article: Identification and modulation of the key amino acid residue responsible for the pH sensitivity of neoculin, a taste-modifying protein.
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ABSTRACT: Neoculin occurring in the tropical fruit of Curculigo latifolia is currently the only protein that possesses both a sweet taste and a taste-modifying activity of converting sourness into sweetness. Structurally, this protein is a heterodimer consisting of a neoculin acidic subunit (NAS) and a neoculin basic subunit (NBS). Recently, we found that a neoculin variant in which all five histidine residues are replaced with alanine elicits intense sweetness at both neutral and acidic pH but has no taste-modifying activity. To identify the critical histidine residue(s) responsible for this activity, we produced a series of His-to-Ala neoculin variants and evaluated their sweetness levels using cell-based calcium imaging and a human sensory test. Our results suggest that NBS His11 functions as a primary pH sensor for neoculin to elicit taste modification. Neoculin variants with substitutions other than His-to-Ala were further analyzed to clarify the role of the NBS position 11 in the taste-modifying activity. We found that the aromatic character of the amino acid side chain is necessary to elicit the pH-dependent sweetness. Interestingly, since the His-to-Tyr variant is a novel taste-modifying protein with alternative pH sensitivity, the position 11 in NBS can be critical to modulate the pH-dependent activity of neoculin. These findings are important for understanding the pH-sensitive functional changes in proteinaceous ligands in general and the interaction of taste receptor-taste substance in particular.PLoS ONE 01/2011; 6(4):e19448. · 4.09 Impact Factor -
Article: Characterization of the beta-D-glucopyranoside binding site of the human bitter taste receptor hTAS2R16.
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ABSTRACT: G-protein-coupled receptors mediate the senses of taste, smell, and vision in mammals. Humans recognize thousands of compounds as bitter, and this response is mediated by the hTAS2R family, which is one of the G-protein-coupled receptors composed of only 25 receptors. However, structural information on these receptors is limited. To address the molecular basis of bitter tastant discrimination by the hTAS2Rs, we performed ligand docking simulation and functional analysis using a series of point mutants of hTAS2R16 to identify its binding sites. The docking simulation predicted two candidate binding structures for a salicin-hTAS2R16 complex, and at least seven amino acid residues in transmembrane 3 (TM3), TM5, and TM6 were shown to be involved in ligand recognition. We also identified the probable salicin-hTAS2R16 binding mode using a mutated receptor experiment. This study characterizes the molecular interaction between hTAS2R16 and beta-D-glucopyranoside and will also facilitate rational design of bitter blockers.Journal of Biological Chemistry 09/2010; 285(36):28373-8. · 4.77 Impact Factor -
Article: Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin.
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ABSTRACT: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. As a result, a 2 mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.Biochimica et Biophysica Acta 09/2010; 1800(9):986-92. · 4.66 Impact Factor -
Article: Mutational deglycosylation of the Fc portion of immunoglobulin G causes O-sulfation of tyrosine adjacently preceding the originally glycosylated site.
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ABSTRACT: Mutagenesis directed to a specific glycosylation site has been widely used to examine biological roles of individual glycans. However, occurrence of any post-translational modification on such deglycosylated mutants has not yet been well characterized. Here we performed mass spectrometric analyses of the Fc fragment of an unglycosylated mutant of mouse immunoglobulin G2b, whose conserved N-glycosylation site, i.e. Asn297, was substituted with alanine. We found that a major part of this mutant is sulfated at Tyr296, which adjacently precedes the originally glycosylated site. Our findings demonstrate that mutational deglycosylation can induce an unexpected post-translational modification in the protein.FEBS letters 08/2010; 584(15):3474-9. · 3.54 Impact Factor -
Article: Photoactive ligands probing the sweet taste receptor. Design and synthesis of highly potent diazirinyl D-phenylalanine derivatives.
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ABSTRACT: Some D-amino acids such as d-tryptophan and D-phenylalanine are well known as naturally-occurring sweeteners. Photoreactive D-phenylalanine derivatives containing trifluoromethyldiazirinyl moiety at 3- or 4-position of phenylalanine, were designed as sweeteners for functional analysis with photoaffinity labeling. The trifluoromethyldiazirinyl D-phenylalanine derivatives were prepared effectively with chemo-enzymatic methods using L-amino acid oxidase and were found to have potent activity toward the human sweet taste receptor.Bioorganic & medicinal chemistry letters 02/2010; 20(3):1081-3. · 2.65 Impact Factor -
Article: Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region.
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ABSTRACT: G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.Journal of Biological Chemistry 07/2008; 283(26):17753-6. · 4.77 Impact Factor -
Article: Comparison of mass spectra of peptides in different matrices using matrix-assisted laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG.
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ABSTRACT: The mass spectra of peptides obtained with different matrices were compared using a matrix-assisted laser desorption/ionization (MALDI) ion source and a multi-turn time-of-flight (TOF) mass spectrometer, MULTUM-IMG, which has been developed at Osaka University. Two types of solid matrices, alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), and a liquid matrix made from a mixture of 3-aminoquinoline and CHCA were used. When measuring the peak signal intensity of human angiotensin II [M+H]+ from a fixed sample position, the liquid matrix produced a stable signal over 1000 laser shots, while the signal obtained with CHCA and DHB decayed after about 300 and 100 shots, respectively. Significant differences in the mass resolving power were not observed between the spectra obtained with the three matrices. Signal peak areas were measured as a function of the cycle number in a multi-turn ion trajectory, i.e., the total flight time over a millisecond time scale. For both [M+H]+ of human angiotensin II and bovine insulin, the decay of the signal peak area was the most significant with CHCA, while that measured with DHB was the smallest. The results of the mean initial ion velocity measurements suggested that the extent of metastable decomposition of the analyte ions increased in order of DHB, the liquid matrix, and CHCA, which is consistent with the difference in the decay of the signal peak area as the total flight time increased.Rapid Communications in Mass Spectrometry 06/2008; 22(10):1461-6. · 2.79 Impact Factor -
Article: Structural investigation of the antibiotic sporaviridin: 11—Molecular secondary ion mass spectral studies on the constituent pentasaccharides viridopentaoses
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ABSTRACT: Molecular secondary ion mass spectra of three pentasaccharides, viridopentaoses A, B and C, using various matrices, are discussed. The appearance of the molecular ion species is dependent upon the relative proton affinities between the sample and the organic matrix material. However, the presence of sodium ion, rather than proton, also greatly influences the appearance of the molecular ion species. The glycosidic linkages are mainly cleaved between the glycosidic oxygen atom and the anomeric carbon atom to give informative sugar sequence ions. These fragmentations have been confirmed by the linked scanning technique (B/E).Biological Mass Spectrometry 03/2008; 20(9):582 - 588. · 3.41 Impact Factor -
Article: Microbial production of sensory-active miraculin.
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ABSTRACT: Miraculin (MCL), a tropical fruit protein, is unique in that it has taste-modifying activity to convert sourness to sweetness, though flat in taste at neutral pH. To obtain a sufficient amount of MCL to examine the mechanism involved in this sensory event at the molecular level, we transformed Aspergillus oryzae by introducing the MCL gene. Transformants were expressed and secreted a sensory-active form of MCL yielding 2 mg/L. Recombinant MCL resembled native MCL in the secondary structure and the taste-modifying activity to generate sweetness at acidic pH. Since the observed pH-sweetness relation seemed to reflect the imidazole titration curve, suggesting that histidine residues might be involved in the taste-modifying activity. H30A and H30,60A mutants were generated using the A. oryzae-mediated expression system. Both mutants found to have lost the taste-modifying activity. The result suggests that the histidine-30 residue is important for the taste-modifying activity of MCL.Biochemical and Biophysical Research Communications 09/2007; 360(2):407-11. · 2.48 Impact Factor -
Article: Analogue Chromophore Study of the Influence of Electronic Perturbation on Color Regulation of Photoactive Yellow Proteint†
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ABSTRACT: We report a unique ΛAmax shift of the absorption maximum of a photoactive yellow protein (PYP) analogue reconstituted with a fluorinated chromophore (F-PYP). The difference in ΛAmax between the free chromophore and the protein was significantly larger than that with the native chromophore. We concluded that the unusual ΛAmax shift is caused by the electronegative character of the fluorine atom and not by steric hindrance. This result suggests that formation of a hydrogen bond between the fluorine atom and one or more amino acid residues could neutralize its electron-withdrawing character. The properties of analogues of PYP with brominated and methylated chromophore could be explained as an effect of steric hindrance.Photochemistry and Photobiology 10/2006; 82(6):1422 - 1425. · 2.41 Impact Factor -
Article: Synthesis of diazirinyl photoprobe carrying a novel cleavable biotin.
ChemBioChem 06/2005; 6(5):814-8. · 3.94 Impact Factor -
Article: Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis.
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ABSTRACT: The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction. The major low molecular weight components of the venom were identified as gamma-aminobutyric acid and glutamic acid by micro-liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectrometry analysis. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass analysis revealed that the A. samariensis venom contained the various proteins with weights of 4-100 kDa. A biological assay using Joro spiders (Nephila clavata) clearly showed that the high molecular weight fraction of the venom prepared by ultrafiltration exerted as potent non-lethal long-term paralysis as the whole venom, whereas the low molecular weight fraction was devoid of any paralytic activity. These results indicated that several venomous proteins in the high molecular weight fraction are responsible for the paralytic activity. Furthermore, we determined the primary structure of one component designated As-fr-19, which was a novel multiple-cysteine peptide with high sequence similarity to several sea anemone and snake toxins including dendrotoxins, rather than any insect toxic peptides identified so far. Taken together, our data showed the unprecedented molecular and toxicological profiles of wasp venoms.Biochemical and Biophysical Research Communications 06/2005; 330(4):1048-54. · 2.48 Impact Factor -
Article: Separation and identification of food dyes by thin‐layer chromatography/liquid secondary ion mass spectrometry
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ABSTRACT: In order to separate and identify rapidly the 11 food dyes that are permitted in Japan, a thin-layer chromatographic/liquid secondary ion (LSI) mass spectrometric method was established. The characteristic features of these food dyes under fast atom bombardment and LSI mass spectrometric conditions were carefully investigated using various matrices. While glycerol enhanced reduction of them, magic bullet depressed the reduction to give the molecular ion species with the theoretical isotopic abundances. The two solvent systems, methanol-methyl ethyl ketone-10% sodium sulphate (1:1:3.5) (solvent A) and methanol-acetonitrile-10% sodium sulphate (3:3:10) (solvent B) were selected for separation of the xanthene dyes and the others, respectively by use of a reversed-phase thin-layer chromatographic plate prepared by ourselves. A method was also devised to concentrate a diffused spot on the plate using thioglycerol. The established thin-layer chromatographic/LSI mass spectrometric method was finally applied to identification of an unknown food dye from a candy.Biological Mass Spectrometry 04/2005; 20(9):522 - 528. · 3.41 Impact Factor -
Article: Tachykinin and tachykinin receptor of an ascidian, Ciona intestinalis: evolutionary origin of the vertebrate tachykinin family.
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ABSTRACT: Tachykinins (TKs) are the most prevalent vertebrate brain/gut peptides. In this study, we originally identified authentic TKs and their receptor from a protochordate, Ciona intestinalis. The Ciona TK (Ci-TK) precursor, like mammalian gamma-preprotachykinin A (gamma-PPTA), encodes two TKs, Ci-TK-I and -II, including the -FXGLM-NH(2) vertebrate TK consensus. Mass spectrometry of the neural extract revealed the production of both Ci-TKs. Ci-TK-I contains several Substance P (SP)-typical amino acids, whereas a Thr is exceptionally located at position 4 from the C terminus of Ci-TK-II. The Ci-TK gene encodes both Ci-TKs in the same exon, indicating no alternative generation of Ci-TKs, unlike the PPTA gene. These results suggested that the alternative splicing of the PPTA gene was established during evolution of vertebrates. The only Ci-TK receptor, Ci-TK-R, was equivalently activated by Ci-TK-I, SP, and neurokinin A at physiological concentrations, whereas Ci-TK-II showed 100-fold less potent activity, indicating that the ligand selectivity of Ci-TK-R is distinct from those of vertebrate TK receptors. Ci-TK-I, like SP, also elicited the typical contraction on the guinea pig ileum. The Ci-TK gene was expressed in neurons of the brain ganglion, small cells in the intestine, and the zone 7 in the endostyle, which corresponds to the vertebrate thyroid gland. Furthermore, the Ci-TK-R mRNA was distributed in these three tissues plus the gonad. These results showed that Ci-TKs play major roles in sexual behavior and feeding in protochordates as brain/gut peptides and endocrine/paracrine molecules. Taken together, our data revealed the biochemical and structural origins of vertebrate TKs and their receptors.Journal of Biological Chemistry 01/2005; 279(51):53798-805. · 4.77 Impact Factor -
Article: Tachykinin and Tachykinin Receptor of an Ascidian, Ciona intestinalis
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ABSTRACT: Tachykinins (TKs) are the most prevalent vertebrate brain/gut peptides. In this study, we originally identified authentic TKs and their receptor from a protochordate, Ciona intestinalis. The Ciona TK (Ci-TK) precursor, like mammalian γ-preprotachykinin A (γ-PPTA), encodes two TKs, Ci-TK-I and -II, including the -FXGLM-NH2 vertebrate TK consensus. Mass spectrometry of the neural extract revealed the production of both Ci-TKs. Ci-TK-I contains several Substance P (SP)-typical amino acids, whereas a Thr is exceptionally located at position 4 from the C terminus of Ci-TK-II. The Ci-TK gene encodes both Ci-TKs in the same exon, indicating no alternative generation of Ci-TKs, unlike the PPTA gene. These results suggested that the alternative splicing of the PPTA gene was established during evolution of vertebrates. The only Ci-TK receptor, Ci-TK-R, was equivalently activated by Ci-TK-I, SP, and neurokinin A at physiological concentrations, whereas Ci-TK-II showed 100-fold less potent activity, indicating that the ligand selectivity of Ci-TK-R is distinct from those of vertebrate TK receptors. Ci-TK-I, like SP, also elicited the typical contraction on the guinea pig ileum. The Ci-TK gene was expressed in neurons of the brain ganglion, small cells in the intestine, and the zone 7 in the endostyle, which corresponds to the vertebrate thyroid gland. Furthermore, the Ci-TK-R mRNA was distributed in these three tissues plus the gonad. These results showed that Ci-TKs play major roles in sexual behavior and feeding in protochordates as brain/gut peptides and endocrine/paracrine molecules. Taken together, our data revealed the biochemical and structural origins of vertebrate TKs and their receptors.Journal of Biological Chemistry 12/2004; 279(51):53798-53805. · 4.77 Impact Factor -
Article: Isolation and structural characterization of siderophores, madurastatins, produced by a pathogenic Actinomadura madurae.
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ABSTRACT: Madurastatins Al (1), A2 (2) and A3 (3), novel pentapeptides that were acylated with salicylic acid at the N-terminus, were isolated from the culture broth of a pathogenic Actinomadura madurae IFM 0745 strain. These structures were mainly determined by 2D NMR and MS/MS spectral techniques. The strain produced simultaneously madurastatins B1 (4) and B2 (5) consisting of Ser and salicylic acid moieties. Compounds 1 and 4 had an antibacterial activity against Micrococcus luteus, indicating that the presence of the aziridine ring is essential for such activity. Because 1 has a strong affinity with ferric ion due to the presence of two hydroxamic acids and a salicylic acid, it is considered to be a siderophore that is a low molecular weight iron chelater. The production of siderophores may be one of the characteristics of pathogenic microorganisms.The Journal of Antibiotics 03/2004; 57(2):125-35. · 1.65 Impact Factor
Top co-authors
Top Journals
Institutions
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2004–2012
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Suntory Institute for Bioorganic Research
Ōsaka-shi, Osaka-fu, Japan
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2007–2011
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The University of Tokyo
- Department of Applied Biological Chemistry
Tokyo, Tokyo-to, Japan
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2005
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Meijo University
Nagoya-shi, Aichi-ken, Japan
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