Hiroyoshi Matsumura

Kyoto Prefectural University, Kioto, Kyōto, Japan

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Publications (182)548.94 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The tomato mosaic virus (ToMV) resistance gene Tm-1 encodes a protein that shows no sequence homology to functionally characterized proteins. Tm-1 binds ToMV replication proteins and thereby inhibits replication complex formation. ToMV mutants that overcome this resistance have amino acid substitutions in the helicase domain of the replication proteins (ToMV-Hel). A small region of Tm-1 in the genome of the wild tomato Solanum habrochaites has been under positive selection during its antagonistic coevolution with ToMV. Here we report crystal structures for the N-terminal inhibitory domains of Tm-1 and a natural Tm-1 variant with an I91-to-T substitution that has a greater ability to inhibit ToMV RNA replication and their complexes with ToMV-Hel. Each complex contains a Tm-1 dimer and two ToMV-Hel monomers with the interfaces between Tm-1 and ToMV-Hel bridged by ATP. Residues in ToMV-Hel and Tm-1 involved in antagonistic coevolution are found at the interface. The structural differences between ToMV-Hel in its free form and in complex with Tm-1 suggest that Tm-1 affects nucleoside triphosphatase activity of ToMV-Hel, and this effect was confirmed experimentally. Molecular dynamics simulations of complexes formed by Tm-1 with ToMV-Hel variants showed how the amino acid changes in ToMV-Hel impair the interaction with Tm-1 to overcome the resistance. With these findings, together with the biochemical properties of the interactions between ToMV-Hel and Tm-1 variants and effects of the mutations in the polymorphic residues of Tm-1, an atomic view of a step-by-step coevolutionary arms race between a plant resistance protein and a viral protein emerges.
    Proceedings of the National Academy of Sciences of the United States of America. 08/2014;
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    ABSTRACT: A membrane-associated ATPase, PotA, is a component of the spermidine-preferential uptake system in prokaryotes that plays an important role in normal cell growth by regulating the cellular polyamine concentration. No three-dimensional structures of membrane-associated ATPases in polyamine-uptake systems have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of PotA from Thermotoga maritima are reported. Diffraction data were collected and processed to 2.7 Å resolution from both native and selenomethionine-labelled crystals. Preliminary crystallographic analysis revealed that the crystals belonged to the hexagonal space group P3112 (or P3212), with unit-cell parameters a = b = 88.9, c = 221.2 Å, α = 90, β = 90, γ = 120°, indicating that a dimer was present in the asymmetric unit.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 06/2014; 70(Pt 6):738-741. · 0.55 Impact Factor
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    ABSTRACT: We developed a new crystallization technique combining the solution stirring technique and crystallization at the interface between high-concentrated hydrogel and solution (the on-gel stirring technique). The solution stirring effect on the crystal quality of insulin was evaluated by an average difference between temperature factors determined by the slopes of the relative Wilson Plot. Higher-quality insulin crystals could be obtained under the stirred environment. The environmental stability of insulin crystals grown at the interface between hydrogel and solution for temperature change was tested. The dissolution point of a gel-interface crystal was 2 K higher than that of a solution-grown crystal. The quality of an insulin crystal grown by the on-gel stirring technique was finally evaluated and it was better than the solution-grown crystals with/without stirring and the gel-interface grown crystals. These results confirm that the on-gel stirring technique is a practical way to obtain high-quality crystals with improved environmental stability.
    Japanese Journal of Applied Physics 05/2014; 53(6):065502. · 1.07 Impact Factor
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    ABSTRACT: The bacterial cell-division protein FtsA anchors FtsZ to the cytoplasmic membrane. But how FtsA and FtsZ interact during membrane division remains obscure. We have solved 2.2Å resolution crystal structure for FtsA from Staphylococcus aureus. In the crystals, SaFtsA molecules within the dimer units are twisted, in contrast to the straight filament of FtsA from Thermotoga maritima, and the half of S12--S13 hairpin regions are disordered. We confirmed that SaFtsZ and SaFtsA associate in vitro, and found that SaFtsZ GTPase activity is enhanced by interaction with SaFtsA.
    FEBS letters 04/2014; · 3.54 Impact Factor
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    ABSTRACT: SaFtsA and SaFtsZbind by comigration in non denaturing gel electrophoresis (View interaction) SaFtsZ and SaFtsAbind by molecular sieving (View interaction) SaFtsA and SaFtsAbind by x-ray crystallography (View interaction)
    01/2014;
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    ABSTRACT: Copper-containing nitrite reductase (CuNIR) catalyzes the reduction of nitrite (NO2(-)) to nitric oxide (NO) during denitrification. We determined the crystal structures of CuNIR from thermophilic gram-positive bacterium, Geobacillus thermodenitrificans (GtNIR) in chloride- and formate(-)bound forms of wild type at 1.15 Å resolution and the nitrite-bound form of the C135A mutant at 1.90 Å resolution. The structure of C135A with nitrite displays a unique η(1)-O coordination mode of nitrite at the catalytic copper site (T2Cu), which has never been observed at the T2Cu site in known wild-type CuNIRs, because the mobility of two residues essential to catalytic activity, Asp98 and His244, are sterically restricted in GtNIR by Phe109 on a characteristic loop structure that is found above Asp98 and by an unusually short CH-O hydrogen bond observed between His244 and water, respectively. A detailed comparison of the WT structure with the nitrite-bound C135A structure implies the replacement of hydrogen(-)bond networks around His244 and predicts the flow path of protons consumed by nitrite reduction. Based on these observations, the reaction mechanism of GtNIR through the η(1)-O coordination manner is proposed.
    Journal of Biochemistry 11/2013; · 3.07 Impact Factor
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    ABSTRACT: With the recent development in pulsed lasers with ultrashort pulse widths or wavelengths, spatially precise, low-damage processing by femtosecond or deep-UV laser ablation has shown promise for the production of protein single crystals suitable for X-ray crystallography. Femtosecond laser processing of supersaturated solutions can shorten the protein nucleation period or can induce nucleation at low supersaturation, which improves the crystal quality of various proteins including membrane proteins and supra-complexes. In addition to nucleation, processing of protein crystals by femtosecond or deep-UV laser ablation can produce single crystalline micro- or macro-seeds without deterioration of crystal quality. This tutorial review gives an overview of the successful application of laser ablation techniques to nucleation and seeding for the production of protein single crystals, and also describes the advantages from a physico-chemical perspective.
    Chemical Society Reviews 11/2013; · 24.89 Impact Factor
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    ABSTRACT: To investigate the molecular basis of cold adaptation of enzymes, we determined the crystal structure of the tryptophan synthase α subunit (SfTSA) from the psychrophile Shewanella frigidimarina K14-2 by X-ray analysis at 2.6-Å resolution and also examined its physicochemical properties. SfTSA was found to have the following characteristics: (1) The stabilities against heat and denaturant of SfTSA were lower than those of an α subunit (EcTSA) from Escherichia coli. This lower equilibrium stability originated from both a faster unfolding rate and a slower refolding rate. (2) The heat denaturation of SfTSA was completely reversible at pH 7.0, and the solubility of denatured SfTSA was higher than that of denatured EcTSA. The two-state transition of denaturation for SfTSA was highly cooperative, whereas the denaturation process of EcTSA was considerably more complex. (3) The global structure of SfTSA was quite similar to those of α subunits from other species. Relative to those other proteins, SfTSA exhibited an increase in cavity volume and a decrease in the number of ion pairs. SfTSA also lacks a hydrogen bond near loop B, related to catalytic function. These characteristics of SfTSA might provide the conformational flexibility required for catalytic activity at low temperatures.
    Journal of Biochemistry 10/2013; · 3.07 Impact Factor
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    ABSTRACT: FtsA from methicillin-resistant Staphylococcus aureus (MRSA) was cloned, overexpressed and purified. The protein was crystallized using the sitting-drop vapour-diffusion technique. A cocrystal with β-γ-imidoadenosine 5'-phosphate (AMPPNP; a nonhydrolysable ATP analogue) was grown using PEG 3350 as a precipitant at 293 K. X-ray diffraction data were collected to a resolution of 2.3 Å at 100 K. The crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 75.31, b = 102.78, c = 105.90 Å, β = 96.54°. The calculated Matthews coefficient suggested that the asymmetric unit contained three or four monomers.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2013; 69(Pt 8):895-898. · 0.55 Impact Factor
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    ABSTRACT: The spermidine acetyltransferase (SAT) from Escherichia coli catalyses the transfer of acetyl groups from acetyl-CoA to spermidine. SAT has been expressed and purified from E. coli. SAT was crystallized by the sitting-drop vapour-diffusion method to obtain a more detailed insight into the molecular mechanism. Preliminary X-ray diffraction studies revealed that the crystals diffracted to 2.5 Å resolution and belonged to the cubic space group P23, with unit-cell parameters a = b = c = 148.7 Å. They contained four molecules per asymmetric unit.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2013; 69(Pt 8):884-887. · 0.55 Impact Factor
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    ABSTRACT: The crystal structure of peroxiredoxin from the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii (PhPrx) was determined at a resolution of 2.25 Å. The overall structure was a ring-type decamer consisting of five homodimers. Citrate, which was included in the crystallization conditions, was bound to the peroxidatic cysteine of the active site, with two O atoms of the carboxyl group mimicking those of the substrate hydrogen peroxide. PhPrx lacked the C-terminal tail that forms a 32-residue extension of the protein in the homologous peroxiredoxin from Aeropyrum pernix (ApPrx).
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2013; 69(Pt 7):719-22. · 0.55 Impact Factor
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    ABSTRACT: Agarose has been utilized in protein crystallization to control nucleation of protein crystals. It reduces convection, prevents crystal sedimentation, and increases tolerance to environmental perturbations, resulting in high-quality protein crystals. However, crystallographers have seldom used agarose hydrogel because it requires preincubating the crystallization solution at high temperatures where a high-temperature-sensitive protein may be inactivated or aggregated. To overcome this disadvantage, we used a thermoreversible gel polymer (TGP) made from synthetic polymer. TGP turns into hydrogel upon warming and liquefies upon cooling. This novel approach enabled us to prepare the crystallization solutions at low temperature (277–283 K) and to crystallize elastase, glucose isomerase, and lysozyme with TGP. We also found that TGP clearly increased the number of elastase, glucose isomerase, and lysozyme crystals. This approach will provide a wide variety of possibilities for protein crystallization in hydrogels.
    Crystal Growth & Design 04/2013; 13(5):1899–1904. · 4.69 Impact Factor
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    ABSTRACT: The effect of gel–solution interfaces on the femtosecond laser-induced nucleation of proteins was investigated. Using hen egg white lysozyme, we found that the nucleation efficiency could be modulated by the distance between the laser focus and agarose gel surfaces (h) and the gel concentration (c). In particular, laser irradiation near the soft gel surface (h = 50 μm, c = 0.5%) could induce nucleation at very low supersaturation, where no nucleation could be induced in the bulk solutions even with femtosecond laser irradiation. Such enhancement of the nucleation was also confirmed for the membrane protein, acriflavine resistance protein B (AcrB), and an organic compound, N-(4-hydroxyphenyl)acetamide (paracetamol). To gain further insights into the role of gel–solution interfaces, we conducted fast imaging of cavitation bubbles, which are known to locally concentrate supersaturated solutions and thus act as a trigger for laser-induced nucleation. We found that the cavitation bubbles shrank asymmetrically near the soft gel surfaces and then finally collapsed toward the solution side. This is in contrast to the symmetric collapse in bulk solutions, which generates a large amount heat and chemical decomposition at the focus. In addition, cavitation bubbles generated near stiff gel surfaces (c ≥ 2%) caused an axial liquid jet toward the gel surfaces, which would disturb the formation of locally concentrated regions. These results suggest that gel–solution interfaces significantly modulate cavitation bubble dynamics and can be cues to achieve effective nucleation.
    Crystal Growth & Design 03/2013; 13(4):1491–1496. · 4.69 Impact Factor
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    ABSTRACT: Protein crystals are required for X-ray crystallography to determine three-dimensional structures of proteins at atomic resolution. The conventional microscopy is currently used for observation and screening of protein crystals. However, the three-dimensional imaging, which is important for automated treatment of protein crystals, is generally difficult by light microscopy. In addition, the protein crystals in the media are frequently difficult to identify by conventional light microscopy owing to the appearance of salt crystals or amorphous materials. In this work, we successfully demonstrated micro-scale, non-invasive, three-dimensional cross-sectional imaging of protein crystals using ultrahigh resolution optical coherence tomography (UHR-OCT). A low noise, Gaussian like, high power supercontinuum at wavelength of 800 nm was used as the light source. The axial resolution of 2 um in sample and the sensitivity of 95 dB were achieved. Since the protein crystal has homogeneous nano-structure, the optical scattering is negligibly small. Therefore, we used gel-inclusion technique to enhance the intensity of scattered signals, and clear, sharp 3D cross-sectional images of protein crystals were successfully observed. As the gel concentration was increased, the OCT signal intensity was increased. Using this method, the protein crystals surrounded by substantial amount of precipitates could be visualized, which is difficult by conventional light microscopy. The discrimination of protein and salt crystals was also demonstrated by the OCT signal intensity. The wavelength dependence of OCT imaging for protein crystal was examined at wavelength of 800-1700 nm regions. It was confirmed that the finest images were observed using 800 nm wavelength system.
    Proc SPIE 01/2013;
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    ABSTRACT: We developed a spatially precise, soft microseeding method for the production of single protein crystals that are suitable for X-ray crystallographic studies. We used focused femtosecond laser pulses to produce, via multiphoton absorption processes, seed crystals from small regions (1 μm2) of crystals. Hen egg-white lysozyme seed crystals, produced in this manner, grew to be single crystals without any deterioration in their crystallinity. We also validated the technique using polycrystals for the membrane protein, acriflavine resistance protein B, for which single crystals are very difficult to obtain. In addition, we found that the shape of a tetragonal lysozyme crystal prepared from the seed could be controlled by altering the time interval between the initiation of crystallization and laser ablation. We also tried to comprehend the mechanism of femtosecond laser-induced microseeding. We visualized the ablated surfaces of the lysozyme crystals by atomic force microscopy and by laser confocal microscopy combined with differential interference microscopy. The results obtained in this study clearly demonstrate that femtosecond laser ablation of protein crystals is based on a photomechanical process, which ejects crystal fragments with little thermal damage. Femtosecond laser ablation is indeed very promising to produce high quality protein seed crystals from polycrystals or cracked crystals that are not suitable for X-ray diffraction studies.
    Crystal Growth & Design 07/2012; 12(9):4334–4339. · 4.69 Impact Factor
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    ABSTRACT: Orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum (PfOMPDC) catalyses the final step in the de novo synthesis of uridine 5'-monophosphate (UMP) from orotidine 5'-monophosphate (OMP). A defective PfOMPDC enzyme is lethal to the parasite. Novel in silico screening methods were performed to select 14 inhibitors against PfOMPDC, with a high hit rate of 9%. X-ray structure analysis of PfOMPDC in complex with one of the inhibitors, 4-(2-hydroxy-4-methoxyphenyl)-4-oxobutanoic acid, was carried out to at 2.1 Å resolution. The crystal structure revealed that the inhibitor molecule occupied a part of the active site that overlaps with the phosphate-binding region in the OMP- or UMP-bound complexes. Space occupied by the pyrimidine and ribose rings of OMP or UMP was not occupied by this inhibitor. The carboxyl group of the inhibitor caused a dramatic movement of the L1 and L2 loops that play a role in the recognition of the substrate and product molecules. Combining part of the inhibitor molecule with moieties of the pyrimidine and ribose rings of OMP and UMP represents a suitable avenue for further development of anti-malarial drugs.
    Journal of Biochemistry 06/2012; 152(2):133-8. · 3.07 Impact Factor
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    ABSTRACT: The yeast Cyc8p-Tup1p protein complex is a general transcriptional corepressor of genes involved in many different physiological processes. Herein, we present the crystal structure of the Tup1p N-terminal domain (residues 1-92), essential for Tup1p self-assembly and interaction with Cyc8p. This domain tetramerizes to form a novel antiparallel four-helix bundle. Coiled coil interactions near the helical ends hold each dimer together, whereas interdimeric association involves only two sets of two residues located toward the chain centers. A mutagenesis study confirmed that the nonpolar residues responsible for the association of the protomers as dimers are also required for transcriptional repression. An additional structural study demonstrated that the domain containing an Leu(62) → Arg mutation that had been shown not to bind Cyc8p exhibits an altered structure, distinct from the wild type. This altered structure explains why the mutant cannot bind Cyc8p. The data presented herein highlight the importance of the architecture of the Tup1p N-terminal domain for self-association.
    Journal of Biological Chemistry 06/2012; 287(32):26528-38. · 4.65 Impact Factor
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    ABSTRACT: The key enzyme of plant photosynthesis, D-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), must be activated to become catalytically competent via the carbamylation of Lys201 of the large subunit and subsequent stabilization by Mg(2+) coordination. Many biochemical studies have reported that reduced nicotinamide adenine dinucleotide phosphate (NADPH) and 6-phosphogluconate (6PG) function as positive effectors to promote activation. However, the structural mechanism remains unknown. Here, we have determined the crystal structures of activated rice Rubisco in complex with NADPH, 6PG, or 2-carboxy-D-arabinitol 1,5-bisphosphate (2CABP). The structures of the NADPH and 6PG complexes adopt open-state conformations, in which loop 6 at the catalytic site and some other loops are disordered. The structure of the 2CABP complex is in a closed state, similar to the previous 2CABP-bound activated structures from other sources. The catalytic sites of the NADPH and 6PG complexes are fully activated, despite the fact that bicarbonate (NaHCO(3)) was not added into the crystallization solution. In the catalytic site, NADPH does not interact with Mg(2+) directly but interacts with Mg(2+)-coordinated water molecules, while 6PG interacts with Mg(2+) directly. These observations suggest that the two effectors promote Rubisco activation by stabilizing the complex of Mg(2+) and the carbamylated Lys201 with unique interactions and preventing its dissociation. The structure also reveals that the relaxed complex of the effectors (NADPH or 6PG), distinct from the tight-binding mode of 2CABP, would allow rapid exchange of the effectors in the catalytic sites by substrate D-ribulose 1,5-bisphosphate for catalysis in physiological conditions.
    Journal of Molecular Biology 05/2012; 422(1):75-86. · 3.91 Impact Factor
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    ABSTRACT: To grow protein crystals of better quality, there still exists an open question whether a solution flow should be suppressed or intentionally introduced. To obtain a comprehensive understanding of the effects of a solution flow, we directly measured the velocities of individual elementary steps (on {110} faces of tetragonal lysozyme crystals) under a forced solution flow, for the first time, by laser confocal microscopy combined with differential interference contrast microscopy. When we used crystals grown by a two-dimensional (2D) nucleation growth mechanism in a solution of commercial lysozyme (98.5% purity, from Seikagaku Co.), while increasing the solution flow rate, the step velocity decreased monotonically. We confirmed that this decrease in the step velocity with flow rate was due to the enhancement of the mass transfer of impurity (mainly covalently bonded dimer), by the observation using a lysozyme further purified (dimer was removed). In contrast, when we used crystals grown by a spiral growth mechanism in a commercial lysozyme solution, with increasing the flow rate, the step velocity increased and had the maximum at the flow rate of 10 μm/s, and then decreased monotonically. Also, the step velocity was 2–4 times higher than in the case of the 2D nucleation growth. These results demonstrate that the growth of spiral steps is less affected by impurities because the density of spiral steps is much higher than that of 2D island ones.
    Crystal Growth & Design 05/2012; 12(6):2856–2863. · 4.69 Impact Factor
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    ABSTRACT: The genomes of the Tomato mosaic virus and many other plant and animal positive-strand RNA viruses of agronomic and medical importance encode superfamily 1 helicases. Although helicases play important roles in viral replication, the crystal structures of viral superfamily 1 helicases have not been determined. Here, we report the crystal structure of a fragment (S666 to Q1116) of the replication protein from Tomato mosaic virus. The structure reveals a novel N-terminal domain tightly associated with a helicase core. The helicase core contains two RecA-like α/β domains without any of the accessory domain insertions that are found in other superfamily 1 helicases. The N-terminal domain contains a flexible loop, a long α-helix, and an antiparallel six-stranded β-sheet. On the basis of the structure, we constructed deletion mutants of the S666-to-Q1116 fragment and performed split-ubiquitin-based interaction assays in Saccharomyces cerevisiae with TOM1 and ARL8, host proteins that are essential for tomato mosaic virus RNA replication. The results suggested that both TOM1 and ARL8 interact with the long α-helix in the N-terminal domain and that TOM1 also interacts with the helicase core. Prediction of secondary structures in other viral superfamily 1 helicases and comparison of those structures with the S666-to-Q1116 structure suggested that these helicases have a similar fold. Our results provide a structural basis of viral superfamily 1 helicases.
    Journal of Virology 05/2012; 86(14):7565-76. · 5.08 Impact Factor

Publication Stats

1k Citations
548.94 Total Impact Points

Institutions

  • 2014
    • Kyoto Prefectural University
      • Graduate School of Life and Environmental Sciences
      Kioto, Kyōto, Japan
  • 1998–2014
    • Osaka University
      • • Division of Applied Chemistry
      • • Graduate School of Engineering
      • • Department of Beam Materials Science
      Suika, Ōsaka, Japan
  • 2013
    • Saitama University
      • Department of Chemistry
      Saitama, Saitama, Japan
  • 2008–2013
    • National Institute of Advanced Industrial Science and Technology
      • Research Center for Stem Cell Engineering
      Tsukuba, Ibaraki, Japan
  • 2011–2012
    • National Institute of Agrobiological Sciences
      • Division of Plant Sciences
      Tsukuba, Ibaraki-ken, Japan
  • 2010
    • Chiba Institute of Technology
      • Department of Life and Environmental Sciences
      Narashino, Chiba-ken, Japan
  • 2007–2010
    • Nara Institute of Science and Technology
      • Graduate School of Biological Sciences
      Ikoma, Nara, Japan
  • 2004–2008
    • Kyoto University
      • Graduate School of Biostudies
      Kyoto, Kyoto-fu, Japan
  • 2006
    • University of Cambridge
      • Department of Biochemistry
      Cambridge, ENG, United Kingdom
  • 2005
    • Osaka Bioscience Institute
      Ōsaka, Ōsaka, Japan
  • 1997
    • University of Occupational and Environmental Health
      • Department of Environmental Toxicology
      Kitakyūshū, Fukuoka, Japan