Researcher Database

Motohiro Horiuchi
Faculty of Veterinary Medicine Veterinary Medicine Preventive Veterinary Medicine
Professor

Researcher Profile and Settings

Affiliation

  • Faculty of Veterinary Medicine Veterinary Medicine Preventive Veterinary Medicine

Job Title

  • Professor

J-Global ID

Research Interests

  • Campylobacter   Comprehensive gene expression analysis   プリオン   スクレイピー   BSE   伝達性海綿状脳症   PrP   アストロサイト   神経変性   構造転換   合成ペプチド   PrPSc   猫パルボウイルス亜群   モンゴル   再生医療   バイエル氏板   走化性   消化管付髄リンパ装置   濾胞樹状細胞   モノクロナール抗体   神経細胞死   骨髄由来間葉系幹細胞   分子進化   MEV   遺伝子型   ALYマウス   microglia   siRNA   PCR   FPLV   Neuro2a   

Research Areas

  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Nanotechnology/Materials / Molecular biochemistry
  • Life sciences / Virology
  • Nanotechnology/Materials / Polymer chemistry

Academic & Professional Experience

  • 2003/08 - Today Graduate School of Veterinary Medicine, Hokkaido University
  • 2000/04 - 2003/07 Obihiro University of Agriculture and Vetereinary Medicine Associate Prof
  • 1996/06 - 2000/03 Obihiro University of Agriculture and Veterinary Medicine Associate Prof
  • 1997/07 - 1999/07 National Institute of Health Visiting Fellow
  • 1989/01 - 1996/05 Obihiro University of Agriculture and Veterinary Medicine Assistant Prof
  • 1988/04 - 1988/12 Roche Co. Ltd.

Research Activities

Published Papers

  • Rie Hasebe, Akio Suzuki, Takeshi Yamasaki, Motohiro Horiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 454 (1) 125 - 130 0006-291X 2014/11 [Refereed][Not invited]
     
    CD14 deficient (CD14(-/-)) mice survived longer than wild-type (WT) C57BL/6J mice when inoculated with prions intracerebrally, accompanied by increased expression of anti-inflammatory cytokine IL-10 by microglia in the early stage of infection. To assess the immune regulatory effects of CD14 in detail, we compared the gene expression of pro- and anti-inflammatory cytokines in the brains of WT and CD14(-/-) mice infected with the Chandler strain. Gene expression of the anti-inflammatory cytokine IL-13 in prion-infected CD14(-/-) mice was temporarily upregulated at 75 dpi, whereas IL-13 gene expression was not upregulated in prion-infected WT mice. Immunofluorescence staining showed that IL-13 was mainly expressed in neurons of the thalamus at 75 dpi. These results suggest that CD14 can suppress IL-13 expression in neurons during the early stage of prion infection. (C) 2014 Elsevier Inc. All rights reserved.
  • Takeshi Yamasaki, Akio Suzuki, Rie Hasebe, Motohiro Horiuchi
    PLOS ONE 9 (9) e106516  1932-6203 2014/09 [Refereed][Not invited]
     
    Molecules that inhibit the formation of an abnormal isoform of prion protein (PrPSc) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrPSc formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrPC) and PrPSc in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrPSc levels without altering intracellular distribution of PrPSc. PPS did not change the distribution and levels of PrPC, whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrPC to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrPSc formation. In contrast, CPZ and U18666A initiated the redistribution of PrPSc from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrPC. The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrPSc redistribution by CPZ or U18666A partly antagonized PrPSc degradation, suggesting that the transfer of PrPSc to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrPSc degradation. This study revealed that precise analysis of the intracellular dynamics of PrPC and PrPSc provides important information for understanding the mechanism of anti-prion agents.
  • Leo Uchida, Agus Heriyanto, Chalermchaikit Thongchai, Tran Thi Hanh, Motohiro Horiuchi, Kanako Ishihara, Yutaka Tamura, Yasukazu Muramatsu
    JOURNAL OF VETERINARY MEDICAL SCIENCE 76 (7) 1001 - 1008 0916-7250 2014/07 [Refereed][Not invited]
     
    There has been an accumulation of information on frequencies of insertion/deletion (indel) polymorphisms within the bovine prion protein gene (PRNP) and on the number of octapeptide repeats and single nucleotide polymorphisms (SNPs) in the coding region of bovine PRNP related to bovine spongi form encephalopathy (BSE) susceptibility. We investigated the frequencies of 23-bp indel polymorphism in the promoter region (23indel) and 12-bp indel polymorphism in intron 1 region (12indel), octapeptide repeat polymorphisms and SNPs in the bovine PRNP of cattle and water buffaloes in Vietnam, Indonesia and Thailand. The frequency of the deletion allele in the 23indel site was significantly low in cattle of Indonesia and Thailand and water buffaloes. The deletion allele frequency in the 12indel site was significantly low in all of the cattle and buffaloes categorized in each subgroup. In both indel sites, the deletion allele has been reported to be associated with susceptibility to classical BSE. In some Indonesian local cattle breeds, the frequency of the allele with 5 octapeptide repeats was significantly high despite the fact that the allele with 6 octapeptide repeats has been reported to be most frequent in many breeds of cattle. Four SNPs observed in Indonesian local cattle have not been reported for domestic cattle. This study provided information on PRNP or livestock in these Southeast Asian countries.
  • Takeshi Yamasaki, Gerald S. Baron, Akio Suzuki, Rie Hasebe, Motohiro Horiuchi
    VIROLOGY 450 324 - 335 0042-6822 2014/02 [Refereed][Not invited]
     
    To clarify the cellular mechanisms for the establishment of prion infection, we analyzed the intracellular dynamics of inoculated and newly generated abnormal isoform of prion protein (PrPSc) in Neuro2a cells. Within 24 h after inoculation, the newly generated PrPSc was evident at the plasma membrane, in early endosomes, and in late endosomes, but this PrPSc was barely evident in lysosomes; in contrast, the majority of the inoculated PrPSc was evident in late endosomes and lysosomes. However, during the subsequent 48 h, the newly generated PrPSc increased remarkably in early endosomes and recycling endosomes. Overexpression of wild-type and mutant Rab proteins showed that membrane trafficking along not only the endocytic-recycling pathway but also the endo-lysosomal pathway is involved in de novo PrPSc generation. These results suggest that the trafficking of exogenously introduced PrPSc from the endo-lysosomal pathway to the endocytic-recycling pathway is important for the establishment of prion infection. (C) 2013 Elsevier Inc. All rights reserved.
  • Keiko Sakai, Rie Hasebe, Yusuke Takahashi, Chang-Hyun Song, Akio Suzuki, Takeshi Yamasaki, Motohiro Horiuchi
    JOURNAL OF VIROLOGY 87 (24) 13433 - 13445 0022-538X 2013/12 [Refereed][Not invited]
     
    Prion diseases are fatal neurodegenerative disorders characterized by accumulation of PrPSc, vacuolation of neurons and neuropil, astrocytosis, and microglial activation. Upregulation of gene expressions of innate immunity-related factors, including complement factors and CD14, is observed in the brains of mice infected with prions even in the early stage of infections. When CD14 knockout (CD14(-/-)) mice were infected intracerebrally with the Chandler and Obihiro prion strains, the mice survived longer than wild-type (WT) mice, suggesting that CD14 influences the progression of the prion disease. Immunofluorescence staining that can distinguish normal prion protein from the disease-specific form of prion protein (PrPSc) revealed that deposition of PrPSc was delayed in CD14(-/-) mice compared with WT mice by the middle stage of the infection. Immunohistochemical staining with Iba1, a marker for activated microglia, showed an increased microglial activation in prion-infected CD14(-/-) mice compared to WT mice. Interestingly, accompanied by the increased microglial activation, anti-inflammatory cytokines interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta) appeared to be expressed earlier in prion-infected CD14(-/-) mice. In contrast, IL-1 beta expression appeared to be reduced in the CD14(-/-) mice in the early stage of infection. Double immunofluorescence staining demonstrated that CD11b- and Iba1-positive microglia mainly produced the anti-inflammatory cytokines, suggesting anti-inflammatory status of microglia in the CD14(-/-) mice in the early stage of infection. These results imply that CD14 plays a role in the disease progression by suppressing anti-inflammatory responses in the brain in the early stage of infection.
  • Natsuo Ohsawa, Chang-Hyun Song, Akio Suzuki, Hidefumi Furuoka, Rie Hasebe, Motohiro Horiuchi
    MICROBIOLOGY AND IMMUNOLOGY 57 (4) 288 - 297 0385-5600 2013/04 [Refereed][Not invited]
     
    It is generally thought that effective treatments for prion diseases need to inhibit prion propagation, protect neuronal tissues and promote functional recovery of degenerated nerve tissues. In addition, such treatments should be effective even when given after clinical onset of the disease and administered via a peripheral route. In this study, the effect of peripheral administration of an anti-PrP antibody on disease progression in prion-infected mice was examined. mAb 31C6 was administered via the tail veins of prion-infected mice at the time of clinical onset (120 days post-inoculation with the Chandler prion strain) and the distribution of this mAb in the brain and its effect on mouse survival assessed. The antibody was distributed to the cerebellums and thalami of the infected mice and more than half these mice survived longer than mice that had been given a negative control mAb. The level of PrPSc in the mAb 31C6-treated mice was lower than that in mice treated with the negative control mAb and progression of neuropathological lesions in the cerebellum, where the mAb 31C6 was well distributed, appeared to be mitigated. These results suggest that administration of an anti-PrP mAb through a peripheral route is a candidate for the treatment of prion diseases.
  • Yasuhiro Yoshikawa, Motohiro Horiuchi, Naotaka Ishiguro, Mutsuyo Kadohira, Satoshi Kai, Hidehiro Mizusawa, Chisato Nagata, Takashi Onodera, Tetsutaro Sata, Toshiyuki Tsutsui, Masahito Yamada, Shigeki Yamamoto
    JOURNAL OF VETERINARY MEDICAL SCIENCE 74 (8) 959 - 968 0916-7250 2012/08 [Refereed][Not invited]
     
    The Food Safety Commission (FSC) of Japan, established in July 2003, has its own initiative to conduct risk assessments on food stuffs known as "self-tasking assessment". Within this framework, the FSC decided to conduct a risk assessment of beef and beef offal imported into Japan from countries with no previous BSE reports; thus, a methodology was formed to suit to this purpose. This methodology was partly based on the previous assessments of Japanese domestic beef and beef imported from U.S.A./Canada, but some modifications were made. Other organizations' assessment methods, such as those used for BSE status assessment in live cattle by the OIE and EFSA's GBR, were also consulted. In this review, the authors introduce this alternative methodology, which reflects (1) the risk of live cattle in the assessed country including temporal risks of BSE invasion and domestic propagation, with the assessment results verified by surveillance data, and (2) the risk of beef and beef offal consisting of cumulative BSE risk by types of slaughtering and meat production processes implemented and the status of mechanically recovered meat production. Other possible influencing factors such as atypical BSE cases were also reviewed. The key characteristic of the current assessment is a combination of the time-sequential risk level of live cattle and qualitative risk level of meat production at present in an assessed country.
  • Gaku Nakato, Koji Hase, Michio Suzuki, Masanobu Kimura, Manabu Ato, Misaho Hanazato, Minoru Tobiume, Motohiro Horiuchi, Ryuichiro Atarashi, Noriyuki Nishida, Masahisa Watarai, Koichi Imaoka, Hiroshi Ohno
    JOURNAL OF IMMUNOLOGY 189 (4) 1540 - 1544 0022-1767 2012/08 [Refereed][Not invited]
     
    Brucella abortus is a Gram-negative bacterium causing brucellosis. Although B. abortus is known to infect via the oral route, the entry site in the gastrointestinal tract has been unclear. We found that B. abortus was selectively internalized by microfold cells (M cells), a subset of epithelial cells specialized for mucosal Ag uptake. During this process, colocalization of cellular prion protein (PrPC) and B. abortus was evident on the apical surface as well as in subapical vacuolar structures in M cells. Internalization of B. abortus by M cells of PrPC-deficient (Prnp(-/-)) mice was greatly reduced compared with that in wild-type mice. Furthermore, an oral infection study revealed that translocation of B. abortus into the Peyer's patch was significantly lower in Prnp(-/-) than in wild-type mice. These observations suggest that orally infected B. abortus invades the host through M cells by using PrPC on the apical surface of M cells as an uptake receptor. The Journal of Immunology, 2012, 189: 1540-1544.
  • Takeshi Yamasaki, Akio Suzuki, Takeshi Shimizu, Masahisa Watarai, Rie Hasebe, Motohiro Horiuchi
    JOURNAL OF GENERAL VIROLOGY 93 (3) 668 - 680 0022-1317 2012/03 [Refereed][Not invited]
     
    Generation of an abnormal isoform of the priori protein (PrPSc) is a key aspect of the propagation of prions. Elucidation of the intracellular localization of PrPSc in prion-infected cells facilitates the understanding of the cellular mechanism of prion propagation. However, technical improvement in PrPSc-specific detection is required for precise analysis. Here, we show that the mAb 132, which recognizes the region adjacent to the most amyloidogenic region of PrP, is useful for PrPSc-specific detection by immunofluorescence assay in cells pre-treated with guanidine thiocyanate. Extensive analysis of the intracellular localization of PrPSc in priori-infected cells using mAb 132 revealed the presence of PrPSc throughout endocytic compartments. In particular, some of the granular PrPSc signals that were clustered at pen-nuclear regions appeared to be localized in an endocytic recycling compartment through which exogenously loaded transferrin, shiga and cholera toxin B subunits were transported. The granular PrPSc signals at pen-nuclear regions were dispersed to the peripheral regions including the plasma membrane during incubation at 20 degrees C, at which temperature transport from the plasma membrane to pen-nuclear regions was impaired. Conversely, dispersed PrPSc signals appeared to return to pen-nuclear regions within 30 min during subsequent incubation at 37 degrees C, following which PrPSc at pen-nuclear regions appeared to redisperse again to peripheral regions over the next 30 min incubation. These results suggest that PrPSc is dynamically transported along with the membrane trafficking machinery of cells and that at least some PrPSc circulates between pen-nuclear and peripheral regions including the plasma membrane via an endocytic recycling pathway.
  • Rie Hasebe, Gregory J. Raymond, Motohiro Horiuchi, Byron Caughey
    VIROLOGY 423 (2) 205 - 213 0042-6822 2012/02 [Refereed][Not invited]
     
    Roles of complement factors in prion infection of the central nervous system remain unclear. In this study, we assessed the strain-dependent reactivity of complement factors in prion infections of Neuro2a (N2a) cells and mouse brains. N2a cells persistently infected with either Chandler or 22L scrapie strains were cultured in the presence of normal mouse serum (NMS), followed by staining with phosphatidylserine binding protein and early apoptosis marker Annexin V. The proportion of Annexin V positive cells was increased both in Chandler- and 22L-infected cells. Preincubation of NMS with anti-C1q, C3 and/or C9 antibodies reduced Annexin V positive cells in Chandler-infected cells, while only anti-C3 antibodies were effective on 22L-infected cells. The immunohistochemistry showed that deposition of C1q and C3 was different between Chandler- and 22L-infected mouse brains. These results indicate that the reactivity of complement factors differs between prion strains both in vitro and in vivo. (C) 2011 Elsevier Inc. All rights reserved.
  • Chang-Hyun Song, Osamu Honmou, Hidefumi Furuoka, Motohiro Horiuchi
    JOURNAL OF VIROLOGY 85 (21) 11069 - 11078 0022-538X 2011/11 [Refereed][Not invited]
     
    Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to migrate to brain lesions of neurodegenerative diseases; however, the precise mechanisms by which MSCs migrate remain to be elucidated. In this study, we carried out an in vitro migration assay to investigate the chemoattractive factors for MSCs in the brains of prion-infected mice. The migration of immortalized human MSCs (hMSCs) was reduced by their pretreatment with antibodies against the chemokine receptors, CCR3, CCR5, CXCR3, and CXCR4 and by pretreatment of brain extracts of prion-infected mice with antibodies against the corresponding ligands, suggesting the involvement of these receptors, and their ligands in the migration of hMSCs. In agreement with the results of an in vitro migration assay, hMSCs in the corpus callosum, which are considered to be migrating from the transplanted area toward brain lesions of prion-infected mice, expressed CCR3, CCR5, CXCR3, and CXCR4. The combined in vitro and in vivo analyses suggest that CCR3, CCR5, CXCR3, and CXCR4, and their corresponding ligands are involved in the migration of hMSCs to the brain lesions caused by prion propagation. In addition, hMSCs that had migrated to the right hippocampus of prion-infected mice expressed CCR1, CX3CR1, and CXCR4, implying the involvement of these chemokine receptors in hMSC functions after chemotactic migration. Further elucidation of the mechanisms that underlie the migration of MSCs may provide useful information regarding application of MSCs to the treatment of prion diseases.
  • Sassa Y, Yamamoto H, Mochizuki M, Umemura T, Horiuchi M, Ishiguro N, Miyazawa T
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 4 73 (4) 491 - 494 0916-7250 2011/04 [Refereed][Not invited]
  • Yukiko Sassa, Takeshi Yamasaki, Motohiro Horiuchi, Yasuo Inoshima, Naotaka Ishiguro
    MICROBIOLOGY AND IMMUNOLOGY 54 (12) 763 - 768 0385-5600 2010/12 [Refereed][Not invited]
     
    It has been reported that macrophages degrade infectious forms of prion protein (PrPSc). In order to investigate the mechanisms underlying PrPSc degradation in macrophages, the effects of lysosomal and proteasomal inhibitors on macrophage cell lines which were incubated with scrapie-affected brain homogenate were studied. PrPSc degradation was inhibited in the presence of both proteasomal and lysosomal inhibitors. Indirect fluorescence assays to determine the cellular localization of PrPSc were undertaken. PrPSc colocalized with the lysosomal membrane protein Lamp-1 and ubiquitin, a protein that is related to the proteasome. The present data indicate that macrophages might degrade PrPSc via the lysosomal and proteasomal pathways.
  • Rie Hasebe, Tadaki Suzuki, Yoshinori Makino, Manabu Igarashi, Satoko Yamanouchi, Akihiko Maeda, Motohiro Horiuchi, Hirofumi Sawa, Takashi Kimura
    BMC MICROBIOLOGY 10 165  1471-2180 2010/06 [Refereed][Not invited]
     
    Background: West Nile virus (WNV) causes viremia after invasion to the hosts by mosquito bite. Endothelial cells could play an important role in WNV spread from the blood stream into the central nervous system and peripheral tissues. Here, we analyzed the capacity of virus-like particles (VLPs) of the highly virulent NY99 6-LP strain (6-LP VLPs) and the low virulence Eg101 strain (Eg VLPs) to cross cultured human endothelial cells. Results: 6-LP VLPs were transported from the apical to basolateral side of endothelial cells, whereas Eg VLPs were hardly transported. The localization of tight junction marker ZO-1 and the integrity of tight junctions were not impaired during the transport of 6-LP VLPs. The transport of 6-LP VLPs was inhibited by treatment with filipin, which prevents the formation of cholesterol-dependent membrane rafts, suggesting the involvement of raft-associated membrane transport. To determine the amino acid residues responsible for the transport of VLPs, we produced mutant VLPs, in which residues of E protein were exchanged between the 6-LP and Eg strains. Double amino acid substitution of the residues 156 and 159 greatly impaired the transport of VLPs. Conclusion: Our results suggest that a transcellular pathway is associated with 6-LP VLPs transport. We also showed that the combination of the residues 156 and 159 plays an important role in the transport of VLPs across endothelial cells.
  • Yuko Sato, Nozomi Shimonohara, Ken-Ichi Hanaki, Motoki Goto, Yoshio Yamakawa, Motohiro Horiuchi, Hidehiro Takahashi, Tetsutaro Sata, Noriko Nakajima
    JOURNAL OF VIROLOGICAL METHODS 165 (2) 261 - 267 0166-0934 2010/05 [Refereed][Not invited]
     
    The AT-tailing method is a labelling technique that utilises oligo(dA-dT)-dependent signal amplification. In this study, a new immunohistochemical application of the immunoAT method was developed. This method uses an oligo(dA-dT)-conjugated primary antibody (direct immunoAT method) or an oligo(dA-dT)-conjugated secondary antibody (indirect immunoAT method). Fifteen-base oligo(dA-dT)conjugated antibodies (IgG-ATs) were prepared in advance by conjugating maleimide-activated oligo(dA-dT) to IgG via free sulfhydryl residues that had been introduced on the surface of IgG using Traut's reagent. Following the reaction with the target antigen and the IgG-AT, oligo(dA-dT) was elongated by Delta Tth DNA polymerase in the presence of dATP, dTTP and biotinylated dUTP, consequently labelling the antigen-antibody complex with a large amount of biotin. To initially evaluate the immunoAT method, the presence or absence of prion protein (PrP(sc)) was determined in formalin-fixed and paraffin-embedded sections of the medulla oblongata of cattle which had been under active surveillance for bovine spongiform encephalopathy. Sections were examined using direct and indirect immunoAT methods and the EnVision+ system (Dako) under conditions that were identical except for the differing IgG-AT and AT-tailing methods. PrP(sc) detection was consistent using all three methods. The clearest signals were obtained using the indirect immunoAT method, suggesting significant potential for this method. (C) 2010 Elsevier B.V. All rights reserved.
  • Yasuko Watanabe, Wakako Hiraoka, Manabu Igarashi, Kimihito Ito, Yuhei Shimoyama, Motohiro Horiuchi, Tohru Yamamoria, Hironobu Yasui, Mikinori Kuwabara, Fuyuhiko Inagaki, Osamu Inanami
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 394 (3) 522 - 528 0006-291X 2010/04 [Refereed][Not invited]
     
    To explore Cu(II) ion coordination by His(186) in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique) Six moPrP mutants. moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C). and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1). moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1) This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the rutroxide spin probe, suggesting that each interspin distance was within 20 angstrom The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), inoPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12 1 angstrom, 18 1 angstrom, 107 angstrom, and 84 angstrom, respectively In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP(C) (C) 2010 Elsevier Inc All rights reserved
  • Satoshi Nakamitsu, Aya Kurokawa, Takeshi Yamasaki, Masahide Uryu, Rie Hasebe, Motohiro Horiuchi
    JOURNAL OF GENERAL VIROLOGY 91 (2) 563 - 569 0022-1317 2010/02 [Refereed][Not invited]
     
    Cells persistently infected with prions continuously produce protease-resistant prion protein (PrP-res). Here, we show that the FrP-res level in prion-infected Neuro2a (N2a) neuroblastoma cells decreased to 50 % of their initial level over the first 48 h and then recovered by 96 h after seeding. The level of cellular prion protein (PrP(C)) also appeared to fluctuate, but did not influence the fluctuation of the PrP-res level. Prion-infected N2a cells, co-cultured with a higher number of prion-unsusceptible cells, had twice as much PrP-res as those cultured without unsusceptible cells, suggesting that cell density influences the fluctuation of PrP-res as. Direct cell-to-cell contact between cells, rather than soluble factors, was involved in the cell density-dependent increase in the PrP-res level. The cholesterol content, which is known to influence PrP-res formation, also changed depending on cell density. Our data suggest that alterations in cellular microenvironments controlled by cell density influence PrP-res formation.
  • Hiroshi Sakata, Motohiro Horiuchi, Izumi Takahashi, Masataka Kinjo
    CURRENT PHARMACEUTICAL BIOTECHNOLOGY 11 (1) 87 - 95 1389-2010 2010/01 [Refereed][Not invited]
     
    The conversion of prion protein (PrP) from the monomeric cellular isoform to the oligomeric pathological isoform is a crucial event in the pathogenesis of prion diseases. To investigate oligomer formation of PrP, enhanced green fluorescent protein (EGFP)-tagged PrP (EGFP-PrP) without the glycosylphosphatidylinositol (GPI) anchor was prepared and the oligomerization of EGFP-PrP induced by sodium dodecyl sulphate (SDS) was monitored by fluorescence correlation spectroscopy (FCS). The FCS analysis indicated that soluble oligomers were formed at 0.011% SDS. Furthermore, the combination of fluorescence cross-correlation spectroscopy (FCCS) and a panel of anti-PrP monoclonal antibodies (mAbs) revealed the conformational changes in PrP. Our studies provide a method to analyze conformational changes of proteins in solution.
  • Motohiro Horiuchi, Ayako Karino, Hidefumi Furuoka, Naotaka Ishiguro, Kumiko Kimura, Morikazu Shinagawa
    VIROLOGY 394 (2) 200 - 207 0042-6822 2009/11 [Refereed][Not invited]
     
    To establish PrPSc-specific mAbs, we immunized Pmp(-/-) mice with PrPSc purified from prion-infected mice. Using this approach, we obtained mAb 6H10, which reacted with PrPSc treated with proteinase K, but not with PrPSc pretreated with more than 3 M GdnHCl. In contrast, reactivity of pan-PrP mAbs increased with increasing concentrations of GdnHCl used for pretreatment of PrPSc. In histoblot analysis, mAb 6H10 showed a positive reaction on a non-denatured histoblot but reactivity was lower when the histoblot was pretreated by autoclaving. Epitope analysis suggested that the extreme C-terminus of PrP is likely to be part of the epitope for mAb 6H10. MAb 6H10 immunoprecipitated PrPSc from brains of mice, sheep, and cattle infected with prions. Furthermore, pretreatment of purified PrPSc with mAb 6H10 reduced the infectious titer more than 1 log. Taken together, these results suggest that mAb 6H10 recognizes a conformational epitope on PrPSc that is related to prion infectivity. (C) 2009 Elsevier Inc. All rights reserved.
  • Chang-Hyun Song, Osamu Honmou, Natsuo Ohsawa, Kiminori Nakamura, Hirofumi Hamada, Hidefumi Furuoka, Rie Hasebe, Motohiro Horiuchi
    JOURNAL OF VIROLOGY 83 (11) 5918 - 5927 0022-538X 2009/06 [Refereed][Not invited]
     
    Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to migrate to brain lesions in experimental models of ischemia, tumors, and neurodegenerative diseases and to ameliorate functional deficits. In this study, we attempted to evaluate the therapeutic potential of MSCs for treating prion diseases. Immortalized human MSCs (hMSCs) that express the LacZ gene were transplanted into the unilateral hippocampi or thalami of mice, and their distributions were monitored by the expression of beta-galactosidase. In mice infected with prions, hMSCs transplanted at 120 days postinoculation (dpi) were detected on the contralateral side at 2 days after transplantation and existed there even at 3 weeks after transplantation. In contrast, few hMSCs were detected on the contralateral side for mock-infected mice. Interestingly, the migration of hMSCs appeared to correlate with the severity of neuropathological lesions, including disease-specific prion protein deposition. The hMSCs also migrated to a prion-specific lesion in the brain, even when intravenously injected. Although the effects were modest, intrahippocampal and intravenous transplantation of hMSCs prolonged the survival of mice infected with prions. A subpopulation of hMSCs in the brains of prion-infected mice produced various trophic factors and differentiated into cells of neuronal and glial lineages. These results suggest that MSCs have promise as a cellular vehicle for the delivery of therapeutic genes to brain lesions associated with prion diseases and, furthermore, that they may help to regenerate neuronal tissues damaged by prion propagation.
  • Ryo Shindoh, Chan-Lan Kim, Chang-Hyun Song, Rie Hasebe, Motohiro Horiuchi
    JOURNAL OF VIROLOGY 83 (8) 3852 - 3860 0022-538X 2009/04 [Refereed][Not invited]
     
    Although the major component of the prion is believed to be the oligomer of PrPSc, little information is available concerning regions on the PrPSc molecule that affect prion infectivity. During the analysis of PrPSc molecules from various prion strains, we found that PrPSc of the Chandler strain showed a unique property in the conformational-stability assay, and this property appeared to be useful for studying the relationship between regions of the PrPSc molecule and prion infectivity. Thus, we analyzed PrPSc of the Chandler strain in detail and analyzed the infectivities of the N-terminally denatured and truncated forms of proteinase K-resistant PrP. The N-terminal region of PrPSc of the Chandler strain showed region-dependent resistance to guanidine hydrochloride (GdnHCl) treatment. The region approximately between amino acids (aa) 81 and 137 began to be denatured by treatment with 1.5 M GdnHCl. Within this stretch, the region comprising approximately aa 81 to 90 was denatured almost completely by 2 M GdnHCl. Furthermore, the region approximately between aa 90 and 137 was denatured completely by 3 M GdnHCl. However, the C-terminal region thereafter was extremely resistant to the GdnHCl treatment. This property was not observed in PrPSc molecules of other prion strains. Denaturation of the region between aa 81 and 137 by 3 M GdnHCl significantly prolonged the incubation periods in mice compared to that for the untreated control. More strikingly, the denaturation and removal of this region nearly abolished the infectivity. This finding suggests that the conformation of the region between aa 81 and 137 of the Chandler strain PrPSc molecule is directly associated with prion infectivity.
  • Kenta Watanabe, Masato Tachibana, Satoshi Tanaka, Hidefumi Furuoka, Motohiro Horiuchi, Hiroshi Suzuki, Masahisa Watarai
    BMC MICROBIOLOGY 8 212  1471-2180 2008/12 [Refereed][Not invited]
     
    Background: The cell tropism of Brucella abortus, a causative agent of brucellosis and facultative intracellular pathogen, in the placenta is thought to be a key event of infectious abortion, although the molecular mechanism for this is largely unknown. There is a higher degree of bacterial colonization in the placenta than in other organs and many bacteria are detected in trophoblast giant (TG) cells in the placenta. In the present study, we investigated mechanism of B. abortus invasion into TG cells. Results: We observed internalization and intracellular growth of B. abortus in cultured TG cells. A monoclonal antibody that inhibits bacterial internalization was isolated and this reacted with heat shock cognate protein 70 (Hsc70). Depletion and over expression of Hsc70 in TG cells inhibited and promoted bacterial internalization, respectively. IFN-gamma receptor was expressed in TG cells and IFN-gamma treatment enhanced the uptake of bacteria by TG cells. Administering the anti-Hsc70 antibody to pregnant mice served to prevent infectious abortion. Conclusion: B. abortus infection of TG cells in placenta is mediated by Hsc70, and that such infection leads to infectious abortion.
  • Takada N, Horiuchi M, Sata T, Sawada Y
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 11 70 (11) 1225 - 1230 0916-7250 2008/11 [Refereed][Not invited]
  • Chang-Hyun Song, Hidefumi Furuoka, Chan-Lan Kim, Michiko Ogino, Akio Suzuki, Rie Hasebe, Motohiro Horiuchi
    JOURNAL OF GENERAL VIROLOGY 89 (6) 1533 - 1544 0022-1317 2008/06 [Refereed][Not invited]
     
    It is well known that anti-prion protein (PrP) monoclonal antibodies (mAbs) inhibit abnormal isoform PrP (PrPSc) formation in cell culture. Additionally, passive immunization of anti-PrP mAbs protects the animals from prion infection via peripheral challenge when mAbs are administered simultaneously or soon after prion inoculation. Thus, anti-PrP mAbs are candidates for the treatment of prion diseases. However, the effects of mAbs on disease progression in the middle and late stages of the disease remain unclear. This study carried out intraventricular infusion of mAbs into prion-infected mice before and after clinical onset to assess their ability to delay disease progression. A 4-week infusion of anti-PrP mAbs initiated at 120 days post-inoculation (p.i.), which is just after clinical onset, reduced PrPSc levels to 70-80% of those found in mice treated with a negative-control mAb. Spongiform changes, microglial activation and astrogliosis in the hippocampus and thalamus appeared milder in mice treated with anti-PrP mAbs than in those treated with a negative-control mAb. Treatment with anti-PrP mAb prolonged the survival of mice infected with Chandler or Obihiro strain when infusion was initiated at 60 days p.i., at which point PrPSc is detectable in the brain. In contrast, infusion initiated after clinical onset prolonged the survival time by about 8 % only in mice infected with the Chandler strain. Although the effects on survival varied for different prion strains, the anti-PrP mAb could partly prevent disease progression, even after clinical onset, suggesting immunotherapy as a candidate for treatment of prion diseases.
  • Yasuko Watanabe, Wakako Hiraoka, Yuhei Shimoyama, Motohiro Horiuchi, Mikinori Kuwabara, Osamu Inanami
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 366 (1) 244 - 249 0006-291X 2008/02 [Refereed][Not invited]
     
    We examined the influence of D177N (D178N in humans) mutation on the conformational stability of the S2 region of moPrP(C) with varying pHs by using the SDSL-ESR technique. The ESR spectrum of D177N at pH 7.5 was narrower than that of Y161R1, referred to as WT*. The ESR spectrum of D177N did not change when pH in the solution decreased to pH 4.0. Our results suggested that the disappearance of a salt bridge (D177-R163) induced the increase in the instability of S2 region. Moreover, the line shape of the ESR spectrum obtained from H176S neighboring the salt bridge linked to the S2 region was similar to D177N. These results indicate that the protonation of H176 is strongly associated with the stability of S2 region. These findings are important for understanding the mechanism by which the disruption of the salt bridge in the S2 region forms the pathogenic PrPSc structure in hereditary prion disease. (c) 2007 Elsevier Inc. All rights reserved.
  • Ohara J, Togari T, Kurokawa A, Maeda J, Ishiguro N, Furuoka H, Horiuchi M
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 12 69 (12) 1325 - 1329 0916-7250 2007/12 [Refereed][Not invited]
  • Fumihiko Fujii, Motohiro Horiuchi, Masayoshi Ueno, Hiroshi Sakata, Issel Nagao, Mamoru Tamura, Masataka Kinjo
    ANALYTICAL BIOCHEMISTRY 370 (2) 131 - 141 0003-2697 2007/11 [Refereed][Not invited]
     
    Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are powerful techniques tomeasure molecular interactions with high sensitivity in homogeneous solution and living cells. In this study, we developed methods for the detection of prion protein (PrP) using FCS and FCCS. A combination of a fluorescent-labeled Fab' fragment and another anti-PrP monoclonal antibody (mAb) enabled us to detect recombinant bovine PrP (rBoPrP) using FCS because there was a significant difference in the diffusion coefficients between the labeled Fab' fragment and the trimeric immune complex consisting of rBoPrP, labeled Fab' fragment, and another anti-PrP mAb. On the other hand, FCCS detected rBoPrP using two mAbs labeled with different fluorescence dyes. The detection limit for PrP in FCCS was approximately threefold higher than that in FCS. The sensitivity of FCCS in detection of abnormal isoform of PrP (PrPsc) was comparable to that of enzyme-li, hked immunosorbent assay (ELISA). Because FCS and FCCS detect the PrP immune complex in homogeneous solution of only microliter samples with a single mixing step and without any washing steps, these features of measurement may facilitate automating bovine spongiform encephalopathy diagnosis. (c) 2007 Elsevier Inc. All rights reserved.
  • [Prion diseases in animals--bovine spongiform encephalopathy].
    Horiuchi M, Nakamitsu S
    Nihon rinsho. Japanese journal of clinical medicine 8 65 1513 - 1520 0047-1852 2007/08 [Refereed][Not invited]
  • Masahide Uryu, Ayako Karino, Yukiko Kamihara, Motohiro Horiuchi
    MICROBIOLOGY AND IMMUNOLOGY 51 (7) 661 - 669 0385-5600 2007 [Refereed][Not invited]
     
    In this study, we established Neuro2a (N2a) neuroblastoma subclones and characterized their susceptibility to prion infection. The N2a cells were treated with brain homogenates from mice infected with mouse prion strain Chandler. Of 31 N2a subclones, 19 were susceptible to prion as those cells became positive for abnormal isoform of prion protein (PrPSc) for up to 9 serial passages, and the remaining 12 subclones were classified as unsusceptible. The susceptible N2a subclones expressed cellular prion protein (PrPC) at levels similar to the parental N2a cells. In contrast, there was a variation in PrPC expression in unsusceptible N2a subclones. For example, subclone N2a-1 expressed PrPC at the same level as the parental N2a cells and prion-susceptible subclones, whereas subclone N2a-24 expressed much lower levels of PrP mRNA and PrPC than the parental N2a cells. There was no difference in the binding of PrPSc to prion-susceptible and unsusceptible N2a subclones regardless of their PrPC expression level, suggesting that the binding of PrPSc to cells is not a major determinant for prion susceptibility. Stable expression of PrPC did not confer susceptibility to prion in unsusceptible subclones. Furthermore, the existence of prion-unsusceptible N2a subclones that expressed PrPC at levels similar to prion-susceptible subclones, indicated that a host factor(s) other than PrPC and/or specific cellular microenvironments are required for the propagation of prion in N2a cells. The prion-susceptible and -unsusceptible N2a subclones established in this study should be useful for identifying the host factor(s) involved in the prion propagation.
  • Yasuko Watanabe, Osamu Inanami, Motohiro Horiuchi, Wakako Hiraoka, Yuhei Shimoyama, Fuyuhiko Inagaki, Mikinori Kuwabara
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 350 (3) 549 - 556 0006-291X 2006/11 [Refereed][Not invited]
     
    We analyzed the pH-induced mobility changes in moPrP(C) alpha-helix and beta-sheets by cysteine-scanning site-directed spin labeling (SDSL) with ESR. Nine amino acid residues of alpha-helix1 (HI, codon 143-151), four amino acid residues of beta-sheet1 (SI, codon 127-130), and four amino acid residues of beta-sheet2 (S2, codon 160-163) were substituted for by cysteine residues. These recombinant mouse PrPC (moPrP(C)) mutants were reacted with a methane thiosulfonate sulfhydryl-specific spin labeling reagent (MTSSL). The 1/delta H of the central (N-14 hyperfine) component (M-1 = 0) in the ESR spectrum of spin-labeled moPrPC was measured as a mobility parameter of nitroxide residues (R1). The mobilities of E145R1 and Y149R1 at pH 7.4, which was identified as a tertiary contact site by a previous NMR study of moPrP, were lower than those of D143R1, R147R1, and R150R1 reported on the helix surface. Thus, the mobility in the HI region in the neutral solution was observed with the periodicity associated with a helical structure. On the other hand, the values in the S2 region, known to be located in the buried side, were lower than those in the SI region located in the surface side. These results indicated that the mobility parameter of the nitroxide label was well correlated with the 3D structure of moPrP. Furthermore, the present study clearly demonstrated three pH-sensitive sites in moPrP, i.e., (1) the N-terminal tertiary contact site of H1, (2) the C-terminal end of H1, and (3) the S2 region. In particular, among these pH-sensitive sites, the N-terminal tertiary contact region of HI was found to be the most pH-sensitive one and was easily converted to a flexible structure by a slight decrease of pH in the solution. These data provided molecular evidence to explain the cellular mechanism for conversion from PrPC to PrPSc in acidic organelles such as the endosome. (c) 2006 Elsevier Inc. All rights reserved.
  • Satoko Yamaguchi, Yoshihiro Nishida, Kenji Sasaki, Mikie Kambara, Chan-Lan Kim, Naotaka Ishiguro, Takehiro Nagatsuka, Hirotaka Uzawa, Motohiro Horiuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 349 (2) 485 - 491 0006-291X 2006/10 [Refereed][Not invited]
     
    Sulfated glycosaminoglycans (GAGs) and sulfated glycans inhibit formation of the abnormal isoforrn of prion protein (PrPSc) in prion-infected cells and prolong the incubation time of scrapie-infected animals. Sulfation of GAGs is not lightly regulated and possible sites of sulfation are randomly modified, which complicates elucidation of the fundamental structures of GAGs that mediate the inhibition of PrPSc formation. To address the structure-activity relationship of GAGs in the inhibition of PrPSc formation, we screened the ability of various regio selectively O-sulfated glyco pyrano sides to inhibit PrPSc formation in prion-infected cells. Among the glycopyranosides and their polymers examined, monomeric 4-sulfo-N-acetyl-glucosamine (4SGN), and two glycopolymers, poly-4SGN and poly-6-sulfo-N-acetyl-glucosamine (poly-6SGN), inhibited PrPSc formation with 50% effective doses below 20 mu g/ml, and their inhibitory effect became more evident with consecutive treatments. Structural comparisons suggested that a combination of an N-acetyl group at C-2 and an O-sulfate group at either O-4 or O-6 on glucopyranoside might be involved in the inhibition of PrPSc formation. Furthermore, polymeric but not monomeric 6SGN inhibited PrPSc formation, suggesting the importance of a polyvalent configuration in its effect. These results indicate that the synthetic sulfated glycosides are useful not only for the analysis of structure-activty relationship of GAGs but also for the development of therapeutics for prion diseases. (c) 2006 Elsevier Inc. All rights reserved.
  • Distribution of PrP(Sc) in cattle with bovine spongiform encephalopathy slaughtered at abattoirs in Japan.
    Iwata N, Sato Y, Higuchi Y, Nohtomi K, Nagata N, Hasegawa H, Tobiume M, Nakamura Y, Hagiwara K, Furuoka H, Horiuchi M, Yamakawa Y, Sata T
    Japanese journal of infectious diseases 2 59 (2) 100 - 107 1344-6304 2006/04 [Refereed][Not invited]
  • Alymphoplasia mice are resistant to prion infection via oral route.
    M Horiuchi, H Furuoka, N Kitamura, M Shinagawa
    JAPANESE JOURNAL OF VETERINARY RESEARCH 53 (3-4) 149 - 157 0047-1917 2006/02 [Refereed][Not invited]
     
    The major cause of infection in animal prion diseases is thought to be consumption of prion-contaminated stuff. There is evidence that the enteric nerve system (ENS) and gut-associated lymphoid tissues (GATL) are involved in the establishment of prion infection through alimentary tract. To elucidate the initial entry port for prion, we inoculated prion to alymphoplasia (aly) mice showing a deficiency in systemic lymph nodes and Peyer's patches. The aly/aly mice were susceptible to prion infection by intra-cranial inoculation and there were no differences in incubation periods between aly/aly mice and wild-type C57BL/6J mice. Incubation periods in aly/aly mice were about 20 days longer than those in C57BL/6J mice with the intra-peritoneal inoculation. The aly/aly mice were completely resistant to prion infection by per os administration, while C57BL/6J mice were sensitive as they entered the terminal stage of disease around 300 days post inoculation. PrPSc were detected in the intestine and spleen of C57BL/6J mice inoculated with prion intraperitoneally or orally; however PrPSc was not detected in the spleen and intestine of aly/aly mice. Prion infectivity was detected in the intestines and spleens of prion-inoculated C57BL/6J mice, even after the early stages of exposure, while no infectivity was detected in these tissues of prion-inoculated aly/aly mice. No apparent differences were observed in the organization of the enteric nerve system between wild-type and aly/aly mice. These results indicate that GALT rather than ENS acts as the primary entry port for prion after oral exposure.
  • Nakamitsu S, Miyazawa T, Horiuchi M, Onoe S, Ohoba Y, Kitagawa H, Ishiguro N
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 1 68 (1) 27 - 33 0916-7250 2006/01 [Refereed][Not invited]
  • [Prion disease as infectious disease transmissible from animals to human].
    Horiuchi M
    Nihon rinsho. Japanese journal of clinical medicine 12 63 (12) 2213 - 2220 0047-1852 2005/12 [Refereed][Not invited]
  • O Inanami, S Hashida, D Iizuka, M Horiuchi, W Hiraoka, Y Shimoyama, H Nakamura, F Inagaki, M Kuwabara
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 335 (3) 785 - 792 0006-291X 2005/09 [Refereed][Not invited]
     
    The structure of the mouse prion (moPrP) was studied using site-directed spin-labeling electron spin resonance (SDSL-ESR). Since a previous NMR study by Hornemanna et al., [Hornemanna, Korthb, Oeschb, Rieka, Widera, Wuthricha, Glockshubera, Recombinant full-length murine prion protein, mPrP (23-231): purification and spectroscopic characterization, FEBS Lett. 413 (1997) 277-281] has indicated that N96, D143, and T189 in moPrP are localized in a Cu2+ binding region, Helix1 and Helix2, respectively, three recombinant moPrP mutations (N96C, D143C, and T189Q were expressed in an Escherichia coli system, and then refolded by dialysis under low pH and purified by reverse-phase HPLC. By using the preparation, we succeeded in preserving a target cystein residue without alteration of the alpha-helix structure of moPrP and were able to apply SDSL-ESR with a methane thiosulfonate spin label to the full-length prion protein. The rotational correlation times (tau) of 1.1, 3.3, and 4.8 ns were evaluated from the X-band ESR spectra at pH 7.4 and 20 degrees C for N96R1, D143R1, and T189R1, respectively. c reflects the fact that the Cu2+ binding region is more flexible than Helix1 or Helix2. ESR spectra recorded at various temperatures revealed two phases together with a transition point at around 20 degrees C in D143R1 and T189R1, but not in N96R1 With the variation of pH from 4.0 to 7.8, ESR spectra of T189R1 at 20 degrees C showed a gradual increase of tau from 2.9 to 4.8 ns. On the other hand, the pH-dependent conformational changes in N96R1 and D143R1 were negligible. These results indicated that T189 located in Helix2 possessed a structure sensitive to physiological pH changes; simultaneously, N96 in the Cu2+ binding region and D143 in Helix1 were conserved. (C) 2005 Elsevier Inc. All rights reserved.
  • Horiuchi M
    Uirusu 1 55 (1) 45 - 53 0042-6857 2005/06 [Refereed][Not invited]
  • H Furuoka, A Yabuzoe, M Horiuchi, Y Tagawa, T Yokoyama, Y Yamakawa, M Shinagawa, T Sata
    ACTA NEUROPATHOLOGICA 109 (3) 263 - 271 0001-6322 2005/04 [Refereed][Not invited]
     
    For immunohistochemistry of the prion diseases, several pretreatment methods to enhance the immunoreactivity of human and animal abnormal proteinase-resistant prion protein (PrPSc) on the tissue sections have been employed. The method of 121 degrees C hydrated autoclaving pretreatment or the combination method of 121 degrees C hydrated autoclaving with a certain chemical reagent ( formic acid or proteinase K, etc) are now widely used. We found that an improved hydrated autoclaving method at 135 degrees C, more effectively enhanced PrPSc immunoreactivity for the antibodies recognizing the linear epitope. In addition, this method was more effective for the long-term fixation samples as compared with other previous methods. However, this modified method could not retrieve PrPSc antigenic epitopes composed of conformational structures or several discontinuous epitopes. We describe the comparative studies between our improved method and other antigen-retrieval procedures reported previously. Based on the differences of reaction among the antibodies, we also discuss the mechanisms of the hydrated autoclaving methods to retrieve PrPSc immunoreactivity.
  • Kurosaki Y, Ishiguro N, Horiuchi M, Shinagawa M
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 3 67 (3) 321 - 323 0916-7250 2005/03 [Refereed][Not invited]
  • Kataoka N, Nishimura M, Horiuchi M, Ishiguro N
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 3 67 (3) 349 - 351 0916-7250 2005/03 [Refereed][Not invited]
  • CL Kim, A Karino, N Ishiguro, M Shinagawa, M Sato, M Horiuchi
    JOURNAL OF GENERAL VIROLOGY 85 (11) 3473 - 3482 0022-1317 2004/11 [Refereed][Not invited]
     
    The C-terminal portion of the prion protein (PrP), corresponding to a protease-resistant core fragment of the abnormal isoform of the prion protein (PrPSc), is essential for prion propagation. Antibodies to the C-terminal portion of PrP are known to inhibit PrPSc accumulation in cells persistently infected with prions. Here it was shown that, in addition to monoclonal antibodies (mAbs) to the C-terminal portion of PrP, a mAb recognizing the octapeptide repeat region in the N-terminal part of PrP that is dispensable for PrPSc formation reduced PrPSc accumulation in cells persistently infected with prions. The 50% effective dose was as low as similar to 1 nM, and, regardless of their epitope specificity, the inhibitory mAbs shared the ability to bind cellular prion protein (PrPC) expressed on the cell surface. Flow cytometric analysis revealed that mAbs that bound to the cell surface during cell culture were not internalized even after their withdrawal from the growth medium. Retention of the mAb-PrPC complex on the cell surface was also confirmed by the fact that internalization was enhanced by treatment of cells with dextran sulfate. These results suggested that anti-PrP mAb antagonizes PrPSc formation by interfering with the regular PrPC degradation pathway.
  • Gombojav A, Ishiguro N, Horiuchi M, Shinagawa M
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 10 66 (10) 1293 - 1295 0916-7250 2004/10 [Refereed][Not invited]
  • CL Kim, A Umetani, T Matsui, N Ishiguro, M Shinagawa, M Horiuchi
    VIROLOGY 320 (1) 40 - 51 0042-6822 2004/03 [Refereed][Not invited]
     
    We established a panel of monoclonal antibodies (mAbs) against prion protein (PrP) by immunizing PrP gene-ablated mice with the pathogenic isoform of prion protein (PrPSc) or recombinant prion protein (rPrP). The mAbs could be divided into at least 10 groups by fine epitope analyses using mutant rPrPs and pepspot analysis. Seven linear epitopes, lying within residues 56-90, 119-127, 137-143, 143-149, 147-151, 163-169, and 219-229, were defined by seven groups of mAbs, although the remaining three groups of mAbs recognized discontinuous epitopes. We attempted to examine whether any of these epitopes are located on the accessible surface of PrPSc. However, no mAbs reacted with protease-treated PrPSc purified from scrapie-affected mice, even when PrPSc was dispersed into a detergent-lipid protein complex, to reduce the size of PrPSc aggregates. In contrast, denaturation of PrPSc by guanidine hydrochloride efficiently exposed all of the epitopes. This suggests that any epitope recognized by this panel of mAbs is buried within the PrPSc aggregates. Alternatively, if the corresponding region(s) are on the surface of PrPSc, the region(s) may be folded into conformations to which the mAbs cannot bind. The reactivity of a panel of mAb also showed that the state of PrPSc aggregation influenced the denaturation process, and the sensitivity to denaturation appeared to vary between epitopes. Our results demonstrate that this new panel of well-characterized mAbs will be valuable for studying the biochemistry and biophysics of PrP molecules as well as for the immuno-diagnosis of prion diseases. (C) 2004 Elsevier Inc. All rights reserved.
  • MH Watarai, S Kim, J Erdenebaatar, S Makino, M Horiuchi, T Shirahata, S Sakaguchi, S Katamine
    JOURNAL OF EXPERIMENTAL MEDICINE 198 (1) 5 - 17 0022-1007 2003/07 [Refereed][Not invited]
     
    The products of the Brucella abortus virB gene locus, which are highly similar to conjugative DNA transfer system, enable the bacterium to replicate within macrophage vacuoles. The replicative phagosome is thought to be established by the interaction of a substrate of the VirB complex with macrophages, although the substrate and its host cellular target have not yet been identified. We report here that Hsp60, a member of the GroEL family of chaperonins, of B. abortus is capable of interacting directly or indirectly with cellular prion protein (PrPC) on host cells. Aggregation of PrPC tail-like formation was observed during bacterial swimming internalization into macrophages and PrPC was selectively incorporated into macropinosomes containing B. abortus. Hsp60 reacted strongly with serum from human brucellosis patients and was exposed on the bacterial surface via a VirB complex-associated process. Under in vitro and in vivo conditions, Hsp60 of B. abortus bound to PrPC. Hsp60 of B. abortus, expressed on the surface of Lactococcus lactis, promoted the aggregation of PrPC but not PrPC tail formation on macrophages. The PrPC deficiency prevented swimming internalization and intracellular replication of B. abortus, with the result that phagosomes bearing the bacteria were targeted into the endocytic network. These results indicate that signal transduction induced by the interaction between bacterial Hsp60 and PrPC on macrophages contributes to the establishment of B. abortus infection.
  • Gombojav A, Shimauchi I, Horiuchi M, Ishiguro N, Shinagawa M, Kitamoto T, Miyoshi I, Mohri S, Takata M
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 3 65 (3) 341 - 347 0916-7250 2003/03 [Refereed][Not invited]
  • Gombojav A, Ishiguro N, Horiuchi M, Serjmyadag D, Byambaa B, Shinagawa M
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 1 65 (1) 75 - 81 0916-7250 2003/01 [Refereed][Not invited]
  • Y Naya, M Horiuchi, N Ishiguro, M Shinagawa
    JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 51 (2) 345 - 349 0021-8561 2003/01 [Refereed][Not invited]
     
    Bacterial tests were used to assess bacterial contamination of game meat from Japanese wild boars. The bacterial contamination of wild boar meat was less than that of domestic pork, as determined by aerobic plate counts (APC) and coliform counts. None of the meat examined in this study was contaminated by Salmonella or E coli O-157. To detect adulteration by domestic pig meat or European wild boar meat, 46 samples of game meat sold as Japanese wild boar were examined genetically. A total of 17 samples showed genetic haplotypes of European and Asian domestic pigs in the D-loop of mitochondrial DNA (mtDNA), and 16 samples showed nuclear glucosephosphate isomerase-processed pseudogene (GPIP) genotypes of European domestic pigs. The European GP/P genotypes of these samples were confirmed by PCR-RFLP analysis. These results indicate that some game meat sold as Japanese wild boar is adulterated by cross-breeding between pigs and wild boars or by contamination with meat from domestic pigs or European wild boars.
  • N Ishiguro, Y Naya, M Horiuchi, M Shinagawa
    ZOOLOGICAL SCIENCE 19 (11) 1313 - 1319 0289-0003 2002/11 [Refereed][Not invited]
     
    To distinguish pig-wild boar crossbred Inobuta from Japanese wild boar populations, a genetic method by using mitochondrial DNA (mtDNA) haplotypes and the nuclear glucosephosphate isomerase-processed pseudogene (GPIP) was developed. Sixteen mtDNA haplotypes from 152 wild boars from Kyushu, Shikoku and Honshu islands of Japan were distinct from those from Asian and European domestic pigs. Five alleles of GPIP were classified into two groups: 1) alleles GPIP*1, GPIP*3 and GPIP*3a from Japanese wild boars, Asian wild boars and domestic pigs; 2) alleles GPIP*4 and GPIP*4a from European wild boars and domestic pigs. An extensive genetic survey was done to distinguish the crossbred Inobuta from 60 wild boars hunted on Tsushima Island, Goto Islands, and Nagasaki and Ooita Prefectures. The mtDNA haplotypes from the 60 samples showed Japanese wild boars, but four wild boar samples from Nagasaki Prefecture had the European GPIP allele, GPIP*4. These results showed that nuclear DNA polymorphism analysis is useful, in addition to mtDNA haplotype assay, to detect "Inobuta" having the European genotype from Japanese wild boar populations.
  • M Horiuchi, T Nemoto, N Ishiguro, H Furuoka, S Mohri, M Shinagawa
    JOURNAL OF CLINICAL MICROBIOLOGY 40 (9) 3421 - 3426 0095-1137 2002/09 [Refereed][Not invited]
     
    Due to the apparent absence of an agent-specific nucleic acid genome, scrapie strains cannot be classified by genome characterization, which is commonly used for the classification of many viruses. However, scrapie strains can be distinguished to some extent by biological properties such as transmissibility to experimental animals and distribution of neuropathological lesions and by biochemical properties such as the molecular mass and relative protease-resistance of the disease-specific isoform of prion protein (PrPSc). In order to preliminarily characterize the scrapie strains that are prevalent in Japan, we analyzed the transmissibility of sheep scrapie isolates to mice and the relative proteinase K (PK) resistance of the corresponding PrPSc. The results indicate that Japanese scrapie strains can be divided into at least three groups based on biological and biochemical properties. The first group includes isolates which cause disease in mice with an incubation period of similar to400 days and possess PrPSc with relatively high PK resistance. Isolates of the second group contain PrPSc that is highly resistant to PK digestion but transmit poorly to mice. The final group consists of isolates that cause disease in mice with an incubation period of less than 300 days and are associated with PrPSc with reduced PK resistance. Sheep scrapie has occurred sporadically in Japan since 1982, with only similar to60 officially reported cases so far. However, the diversity of scrapie strains in the field suggested by our data raises the concern that a scrapie strain similar to the parental agent of bovine spongiform encephalopathy could exist or emerge in Japan. Thus, continuous surveillance for scrapie will be required to prevent the further spread of scrapie, not only among the sheep population but also to other species, and to eliminate any potential risk of sheep scrapie to public health.
  • N Ishiguro, A Nakajima, M Horiuchi, M Shinagawa
    MAMMALIAN GENOME 13 (7) 365 - 372 0938-8990 2002/07 [Refereed][Not invited]
     
    Many copies of nuclear counterparts of mitochondrial DNA (mtDNA) were found in nuclear DNA from sperm heads of the domestic dog, Canis familiaris, by DNA-DNA hybridization and DNA sequencing. Nuclear counterparts homologous to the mtDNA D-loop region were cloned into lambda phage vectors (EMBL4 and lambdagt11), and nucleotide sequences of seven different mtDNA pseudogenes were then determined. The seven pseudogenes were E3 (474 bp; 82% homology with canine mtDNA), E13 (1867 bp; 67%), 8B (2375 bp; 78%), 12A (2650 bp; 79%), 33 (4131 bp; 86%), 47 (4251 bp; 86%), and E17 (5721 bp; 71%). These seven mtDNA pseudogenes corresponded to portions of cytoplasmic mtDNA containing the genes ile, ND1, leu, 165 rRNA, val, 12S rRNA, phe, D-loop, pro, thr, cytb, and glu. A neighbor-joining phylogenetic tree constructed from 125 rRNA sequences in mtDNA pseudogenes 813, 33, 47, and E17 and in 10 mtDNA fragments from other species showed that these four pseudogenes form a monophyletic clade with canine mtDNA. A neighbor-joining phylogenetic tree based on the 318-bp cytb region showed that the canine pseudogenes existed before the divergence of 17 related canids, and their divergence dates were calculated at around 4.4 to 8.6 million years ago.

MISC

  • OHARA Jiro, TOGARI Tetsuro, KUROKAWA Aya, MAEDA Junko, ISHIGURO Naotaka, FURUOKA Hidefumi, HORIUCHI Motohiro  The journal of veterinary medical science  69-  (12)  1325  -1329  2007/12/25  [Not refereed][Not invited]
     
    The selection of sheep with scrapie-resistant PrP genotypes is one of the control measures for transmissible spongiform encephalopathies in ruminants. In this study, we investigated the frequencies of PrP genotypes in meat breeds in Japan. The nationwide surveillance revealed that nearly half of the Suffolk sheep, a major meat breed in Japan, carried scrapie-susceptible AQ/AQ and AQ/VQ genotypes. In addition, the VQ haplotype, which confers high susceptibility to scrapie within sheep, was also found in Poll Dorset sheep. A trial of selective breeding using sires with scrapie-resistant PrP g...
  • HORIUCHI Motohiro  臨床とウイルス  35-  (4)  293  -300  2007/10/10  [Not refereed][Not invited]
  • NAKAMITSU Satoshi, HORIUCHI Motohiro  臨床とウイルス  35-  (4)  301  -310  2007/10/10  [Not refereed][Not invited]
  • 堀内 基広, 中満 智史  Japanese journal of clinical medicine  65-  (8)  1513  -1520  2007/08  [Not refereed][Not invited]
  • Maeda J, Hongbo Ma, Sun Hong, Chuangwen Ke, Takagi H, Kurane I, Horiuchi M, Takashima I, Maeda A  Medical entomology and zoology  57-  (0)  2006/06/01  [Not refereed][Not invited]
  • NAKAMITSU Satoshi, MIYAZAWA Takayuki, HORIUCHI Motohiro, ONOE Sadao, OHOBA Yasunori, KITAGAWA Hitoshi, ISHIGURO Naotaka  The journal of veterinary medical science  68-  (1)  27  -33  2006/01/25  [Not refereed][Not invited]
     
    To assess relationships between nucleotide polymorphisms of the prion protein (PRNP) gene and susceptibility to bovine spongiform encephalopathy (BSE), we investigated polymorphisms in the open reading frame (ORF) and 2 upper regions of the PRNP gene from 2 Japanese cattle breeds : 863 healthy Holstein cattle, 6 BSE-affected Holstein cattle, and 186 healthy Japanese Black (JB) cattle. In the ORF, we found single-nucleotide polymorphisms (SNPs) at nucleotide positions 234 and 576 and found 5 or 6 copies of the octapeptide repeat, but we did not find any amino acid substitutions. In the upper...
  • HORIUCHI Motohiro  Virus  55-  (1)  45  -53  2005/06/30  [Not refereed][Not invited]
  • 堀内 基広  Virus report  2-  (1)  20  -27  2005/06  [Not refereed][Not invited]
  • KUROSAKI Yasuhisa, ISHIGURO Naotaka, HORIUCHI Motohiro, SHINAGAWA Morikazu  The journal of veterinary medical science  67-  (3)  321  -323  2005/03/25  [Not refereed][Not invited]
     
    Polymorphism of the PrP gene is a primary factor influencing susceptibility and incubation period in natural and experimental scrapie in sheep and goats. Polymorphisms of the caprine PrP gene in Japan were examined in 118 goats. Eight allelic variants and 19 genotypes were obtained. Amino acid polymorphisms were observed at 7 codons : 102, 142, 143, 240, 127, 146 and 211 (the latter 3 are novel polymorphisms). The polymorphisms at codons 142M and 143R, which are associated with the resistance to scrapie, were relatively rare in the present study. Thus, the present results provide informatio...
  • KATAOKA Natsumi, NISHIMURA Masakazu, HORIUCHI Motohiro, ISHIGURO Naotaka  The journal of veterinary medical science  67-  (3)  349  -351  2005/03/25  [Not refereed][Not invited]
     
    Surveillance of chronic wasting disease (CWD) was conducted by performing Western blot analysis of tissue samples from 136 sika deer (Cervus nippon) killed by hunters in the Tokachi district of Hokkaido Island. No prion protein (PrP^<Sc>) associated with CWD was detected in any of the samples. To assess amino acid polymorphisms of the sika deer PrP gene, nucleotide sequencing of the PrP gene was performed. The only amino acid polymorphisms detected were 3 silent mutations at nucleotide positions 63, 225 and 408. These results suggest that sika deer in the Tokachi district are genetically ho...
  • HORIUCHI Motohiro  Membrane  30-  (2)  78  -83  2005/03/01  [Not refereed][Not invited]
  • GOMBOJAV Altangerel, ISHIGURO Naotaka, HORIUCHI Motohiro, SHINAGAWA Morikazu  The journal of veterinary medical science  66-  (10)  1293  -1295  2004/10/25  [Not refereed][Not invited]
     
    To characterize amino acid polymorphisms of sheep prion protein (PrP) gene, DNA from 740 sheep of nine breeds raised in Mongolia was isolated and analyzed. A total of 16 genotypes and seven allelic variants of the PrP gene at codons 112, 136, 154, and 171 were found. The MARQ/MARQ genotype associated with susceptibility to scrapie was found in 82.6% of the sheep while the MARR/MARR genotype associated with resistance to scrapie was found in 1.8% of the sheep. The polymorphisms of valine and serine at codon 127, and leucine and arginine at codon 189 were detected in eight Mongolian sheep bre...
  • 堀内 基広  老年精神医学雑誌  14-  (12)  1488  -1494  2003/12  [Not refereed][Not invited]
  • GOMBOJAV Altangerel, SHIMAUCHI Ikuko, HORIUCHI Motohiro, ISHIGURO Naotaka, SHINAGAWA Morikazu, KITAMOTO Tetsuyuki, MIYOSHI Ichiro, MOHRI Shirou, TAKATA Masuhiro  The journal of veterinary medical science  65-  (3)  s・x, 341  -347  2003/03/25  [Not refereed][Not invited]
     
    The use of Transgenic (Tg) mice expressing chimeric sheep/mouse (Sh/Mo) prion protein (PrP) and chimeric bovine/mouse (Bo/Mo) PrP genes was evaluated as a sheep scrapie model. We also investigated the potential for the transmission of sheep scrapie to a human/mouse (Hu/Mo) PrP Tg mouse line. The Sh/Mo PrP and Bo/Mo PrP Tg Prnp^<+/+> or Prnp^<0/0> mouse lines were inoculated intracerebrally with brain homogenates from three sheep with natural scrapie (KU, Y5 or S2). Incubation periods were slightly shorter in Sh/Mo PrP Tg Prnp^<+/+>, than in non-Tg mice inoculated with KU brain homogenate. I...
  • GOMBOJAV Altangerel, ISHIGURO Naotaka, HORIUCHI Motohiro, SERJMYADAG Dorj, BYAMBAA Badarch, SHINAGAWA Morikazu  The journal of veterinary medical science  65-  (1)  75  -81  2003/01/25  [Not refereed][Not invited]
     
    To characterize amino acid polymorphisms in sheep prion protein (PrP), we analyzed the PrP genes from 271 sheep of 4 breeds (Khalkh, Yeroo, Orkhon and Khangai) raised in central Mongolia (Tuv, Uvurkhangai and Selenge prefectures). A total of 16 genotypes and 8 allelic variants of the PrP gene at codons 112, 136, 154 and 171 were found. At codon 171, 1.8% of the sheep had arginine/arginine (R/R) (resistant to scrapie) and 66.8% had glutamine/glutamine (Q/Q)(susceptible to scrapie). Several Yeroo and Orkhon sheep raised in Selenge prefecture had valine at codon 136 (136V)(highly susceptible t...
  • 池田 徹也, 堀内 基広, 古岡 秀文  食品衛生研究  52-  (1)  33  -42  2002/01  [Not refereed][Not invited]
  • HORIUCHI Motohiro  Virus  51-  (2)  145  -150  2001/12/01  [Not refereed][Not invited]
  • YAMAMOTO M, HORIUCHI M, ISHIGURO N, SHINAGAWA M, MATSUO T, KANEKO K  The journal of veterinary medical science  63-  (9)  983  -990  2001/08/25  [Not refereed][Not invited]
     
    Agents of transmissible spongiform encephalopathy(prion)are known to be extremely resistant to physicochemical inactivation procedures such as heat, radiation, chemical disinfectants such as detergents, alcohols, glutaraldehyde, formalin, and so on. Because of its remakable resistance, it is difficult to inactivate prion. Chemical inactivation seems to be a practical method because it is applicable to large or fixed surfaces and complicated equipment. Here, three epoxides: β-propiolactone, propylene oxide, and glycidol(GLD)were examined of their inactivation ability against scrapie-mouse pr...
  • M Yamada, T Matsui, Y Kobayashi, H Furuoka, M Haritani, M Kobayashi, M Nakagawa  JOURNAL OF VETERINARY MEDICAL SCIENCE  61-  (7)  823  -825  1999/07  [Not refereed][Not invited]
     
    Previously we reported that tissue destruction characterized by the presence of karyopyknotic, karyorrhectic and mitotically arrested cells was seen in alimentary epithelial cells and lymphocytes in the lymphoid and hemopoietic systems of cattle experimentally administered with autumn crocus (Colchicum autumnale L.). This report deals with the mechanism of acute cellular injury following experimental autumn crocus poisoning in cattle as demonstrated by the in situ DNA strand break analysis and electron microscopy. The analyses revealed that cellular injury caused by autumn crocus was closely associated with apoptosis.
  • K Matsushita, H Horiuchi, S Furusawa, M Horiuchi, M Shinagawa, H Matsuda  JOURNAL OF VETERINARY MEDICAL SCIENCE  60-  (6)  777  -779  1998/06  [Not refereed][Not invited]
     
    Chicken monoclonal antibodies (mAbs) were developed against bovine prion protein (PrP) peptide. Chickens immunized with bovine PrP peptide B204 (amino acid residues 204-220) coupled to keyhole limpet hemocyanin produced specific antibodies to the peptide as determined by an enzyme-linked immunosorbent assay (ELISA) using the B204 peptide coupled to ovalbumin as target antigen. From a fusion experiment using the chicken fusion partner cell line MuHI and immune spleen cells, 19 mAbs reactive with B204 were generated. These mAbs were subdivided into five groups based on competitive ELISA using B204 and four 10-amino acid peptides. These five groups included all combinations expected based on comparison of amino acid sequences among the five species, bovine, mouse, human, sheep and hamster, examined. These results indicate that the chicken mAb system is a suitable technique for immunological analysis of PrP in mammals.
  • 堀内 基広  食肉の科学  38-  (2)  205  -212  1997/12/30  [Not refereed][Not invited]
  • T Shinagawa, N Ishiguro, M Horiuchi, M Shinagawa  JOURNAL OF VETERINARY MEDICAL SCIENCE  59-  (11)  1071  -1074  1997/11  [Not refereed][Not invited]
     
    Insertion of a 12-nucleotide repeat in c-myb gene exon 9 was observed in about 15% of sporadic bovine T-lymphomas. The 12-nucleotide repeat in the T-lymphoma cells showed deletion and insertion of the repeat units during cultivation of the cells. To know whether deficiency in DNA loop repair is involved in the instability of the repeat, abilities to bind and correct the loop structure in nuclear extracts were examined. The nuclear extracts of all examined cells had ability to bind and correct the loop structure. These data suggest that instability of the 12-nucleotide repeat in bovine T-lymphoma cells might be independent of deficiency of DNA loop repair function.
  • S Inoue, M Tanaka, M Horiuchi, N Ishiguro, M Shinagawa  JOURNAL OF VETERINARY MEDICAL SCIENCE  59-  (3)  175  -183  1997/03  [Not refereed][Not invited]
     
    We cloned the part of the bovine PrP gene which contains the 5'-flanking region, exon 1, exon 2 and intron 1 to analyze its promoter region. The 5' non-coding region of the bovine PrP gene consisted of three exons and two introns, and its organization was similar to that of the mouse, rat and sheep PrP genes. The 5'-flanking region of the bovine PrP gene from the transcription start site to nucleotide position -88 was (G+C)-rich (78%) and contained three potential binding sites for the transcription factor Spl, but no CCAAT-box or TATA-box. This region showed high homology (89%) with that of the sheep PrP gene, but relatively low homology (approximately 46-62%) with the same region of the mouse, rat, hamster and human PrP genes. The position from -88 to -30 within the 5'-flanking region of the bovine PrP gene showed major promoter activity. However, this region was able to function properly only in collaboration with the region at +123 to +891 of intron 1 of the bovine PrP gene.
  • M Horiuchi, M Mochizuki, N Ishiguro, H Nagasawa, M Shinagawa  JOURNAL OF VETERINARY MEDICAL SCIENCE  59-  (2)  133  -136  1997/02  [Not refereed][Not invited]
     
    To obtain monoclonal antibodies (MAbs) specific to feline panleukopenia virus (FPLV) and mink enteritis virus (MEV), 15 hybridomas secreting MAbs against MEV-Abashiri were established and the properties of the MAbs were analyzed. The cross-reactivity of MAbs revealed that one MAb, P2-215 was specific for FPLV and MEV, whereas the remaining fourteen MAbs reacted with canine parvovirus (CPV), FPLV, and MEV. Epitope analyses using various CPV/MEV chimeric viruses revealed that the MAb P2-215 recognized the epitope comprised of amino acid 93-Lys in VP2, which is known to be FPLV and MEV-specific.
  • T Ohozono, N Ishiguro, M Horiuchi, M Shinagawa  JOURNAL OF VETERINARY MEDICAL SCIENCE  58-  (4)  305  -310  1996/04  [Not refereed][Not invited]
     
    Two kinds of bovine c-Myb, the 75 kDa full-length (FL) c-Myb from the thymic type of sporadic bovine lymphosarcoma (SBL) cells, and 65 kDa deleted (DEL) c-Myb, which is an internally deleted form (85 amino acids corresponding to the negative regulatory domain) from calf-type SBL were found. To investigate the relationship between internal deletion and transforming capacity, we constructed retroviral vectors carrying cDNA that encoded either FL- or DEL-Myb protein, and transfected them to mouse fetal liver cells. In spite of the high trans-activating capacity of DELMyb, DELMyb and FLMyb showed no significant difference in oncogenic capability by measurement of colony formation in soft agar or in methylcellulose medium. Thus, the internal deletion of the c-myb gene is competent for transactivation but not directly relevant to transformation of mouse hemopoietic cells.
  • Prusiner Stanley B, 堀内 基広, 品川 森一  蛋白質核酸酵素  40-  (16)  p2383  -2407  1995/12  [Not refereed][Not invited]
  • M HORIUCHI, N YAMAZAKI, H FURUOKA, T MATSUI, M NAKAGAWA, N ISHIGURO, M SHINAGAWA  JOURNAL OF VETERINARY MEDICAL SCIENCE  57-  (3)  577  -580  1995/06  [Not refereed][Not invited]
     
    Meningo-encephalitis in feedlot cattle sporadically occurred in the Tokachi area in northern Japan. The calves had been vaccinated intranasally with a mixed live-vaccine (infectious bovine rhinotracheitis virus, bovine viral diarrhea-mucosal disease virus, and parainfluenza 3 virus) for which intramuscular inoculation was indicated. Two additional live vaccines, bovine adenovirus type 7 and bovine respiratory syncytical virus, had been inoculated simultaneously. Eleven isolates of bovine herpesvirus type 1 were plaque-purified from two brains with fatal encephalitis; their viral DNAs were examined by restriction endonuclease analysis (REA) using PstI and HindIII. The REA patterns of the virus clones were almost identical to those of the vaccine strain 758-43, suggesting that the isolates from this outbreak of fatal encephalitis originated in the abnormally administrated vaccine.
  • T SHINAGAWA, N ISHIGURO, M HORIUCHI, M SHINAGAWA, T MATSUI  JOURNAL OF VETERINARY MEDICAL SCIENCE  56-  (5)  827  -833  1994/10  [Not refereed][Not invited]
     
    We report here distributions of antigens expressed on sporadic bovine leukosis (SBL) cells by a total of 38 monoclonal antibodies (mAbs) directed against three different types of SBL-cell lines, BLT2 (thymic type), BTL-PC3 (calf type) and BLS1 (skin type). Most mAbs had high reactivities with some bovine lymphoma cell lines and less reactivity with normal lymphocytes except for some peripheral blood mononuclear cells (PBM) in cattle and certain other species. They were more reactive with cultured T-cell line BTL-PC3 and B-cell lines, BL312 and KU-1, than naturally occurring lymphoid tumor cells. Among the 38 mAbs, 27 was determined the molecular weights of their recognized antigens in Western blot analyses. A mAb C419 had high reactivity with lymphoma cell lines but no reactivity with normal lymphocytes, indicating that it recognizes tumor-associated antigens of SBL.
  • T SHINAGAWA, N ISHIGURO, M HORIUCHI, M SHINAGAWA  JOURNAL OF VETERINARY MEDICAL SCIENCE  56-  (5)  957  -959  1994/10  [Not refereed][Not invited]
     
    Influences of 38 monoclonal antibodies (mAbs) on the growth of bovine T lymphoma cells (BTL-27) were examined. One of them, S37, inhibited the cell growth strikingly when it was added to the cultures. S37-treated cells showed DNA fragmentation containing approximately 200-base pair multiple patterns and chromatin condensation. This evidence suggested that mAb S37 induced apoptosis in BTL-27 cells.
  • A ONODERA, T IKEDA, M HORIUCHI, N ISHIGURO, M ONUMA, N HIRANO, T MIKAMI, E HONDA, K HIRAI, K KAI, H YUGI, Y MURAMATSU, M SHINAGAWA  JOURNAL OF VETERINARY MEDICAL SCIENCE  56-  (4)  627  -632  1994/08  [Not refereed][Not invited]
     
    We examined the brains, spleens and/or lymph nodes of 197 mainly Suffolk sheep collected from Hokkaido, and the Tohoku, Kanto and Chubu districts to detect PrP(Sc) and thus to estimate scrapie contamination in Japan. Sixteen sheep in Hokkaido and 2 sheep in other districts that were introduced from Hokkaido were positive for PrP(Sc). By comparison of the frequencies of the restriction fragment length polymorphism (RFLP) types of these 18 scrapie sheep with 128 healthy sheep, we confirmed the association of specific RFLP types of the PrP gene with natural scrapie. The frequency of RFLP type I in scrapie sheep was significantly higher than that in healthy sheep and those of other types in scrapie sheep. In contrast, the frequencies of type II and VI in healthy sheep were higher than those in scrapie sheep. Therefore, type I sheep seemed to be susceptible but type II and VI sheep seemed to be resistant to natural scrapie in Suffolk sheep in Japan. Furthermore, we investigated the distribution of the RFLP types of the PrP gene in 161 sheep in Japan to learn about the genetic background of the susceptibility to scrapie. There were variations in the distribution of the RFLP types in each district.
  • M SHINAGAWA, Y MURAMATSU, A ONODERA, T MATSUI, M HORIUCHI, N ISHIGURO, M NAKAGAWA  JOURNAL OF VETERINARY MEDICAL SCIENCE  55-  (4)  665  -667  1993/08  [Not refereed][Not invited]
     
    Two familial scrapie cases successively occurred in 29-month-old and 30-month-old Corriedale dams and in three of their offspring as early as 10-19 months of age in a small flock. They showed ataxy but no pruritus. Histopathological and immunochemical examinations of those sheep revealed vacuolation of nerve cells in brain tissue and presence of PrP(sc), a component of scrapie-associated fibrils, in brain, spleen and lymph node tissues. We discuss about shortened incubation periods and lacking pruritus in the present scrapie cases.
  • M HORIUCHI, H KODAMA, T MIKAMI  JAPANESE JOURNAL OF VETERINARY SCIENCE  52-  (5)  985  -994  1990/10  [Not refereed][Not invited]
     
    For the identification of serotype-specific antigens of Marek's disease virus (MDV) serotype 1 (MDV1) or serotype 2 (MDV2), a total of 24 hybridoma clones, secreting monoclonal antibodies (MAbs) against CVI-988 (MDV1) or HPRS-24 (MDV2) strain, were established and characterized by immunofluorescence assay, virus neutralization and immunoprecipitation analysis. Based upon the molecular weights (mol. wt.) of the immunoprecipitated polypeptides, the MAbs were subdivided into 7 groups. Among them, two groups of MAbs reacted with antigens that have not been reported, were identified. MAbs belong...

Research Grants & Projects

  • 文部科学省:科学研究費補助金(基盤研究(A))
    Date (from‐to) : 2011 -2012 
    Author : 堀内 基広
  • 文部科学省:科学研究費補助金(挑戦的萌芽研究)
    Date (from‐to) : 2011 -2011 
    Author : 堀内 基広
  • 文部科学省:科学研究費補助金(基盤研究(B))
    Date (from‐to) : 2009 -2011 
    Author : 稲波 修, 稲葉 睦, 堀内 基広, 桑原 幹典, 山盛 徹
     
    平成22年度は初年度で確立した部位特異的スピンラベル法による2点間距離計測技術をプリオン凝集体の構造解析に適用を広げることを目的に研究を推進した。測定系の確立は遺伝的プリオン病の変異体D178Nのαヘリックス2上のT188にスピンプローブ(R1)を導入したD178N/T188R1で行い、連続波ESRにより凝集体では1.8nmであることを明らかにした。pH4.0で1M塩酸グアニジン処理すると凝集体構造を形成することはAFMによるフィラメント構造の観察により確かめられた。本年度は引き続き解析を進め、オクタリピートの最後部分でランダムコイル構造のD178N/S97R1、αヘリックス1のD178N/D144R1、αヘリックス2のD178N/R204R1とD178N/Y225R1について同様に検討した。どの場所でも凝集体のスピン間の距離は1.5nm~2.0nmの間の値が持続波ESR法で検出された。パルスESR法でも計測範囲の短距離限界を示し、周期的で規則正しい構造ではなく、凝集体中の同じアミノ酸残基の分子間の距離は1.5nm~2.0nm程度の間隔を持つランダムに凝集体を形成している可能性を示していると類推された。既に他の研究者によって電子顕微鏡像から得られたシミュレーションでは規則正しい対称構造モデルが提唱されており、オクタリピートを除いたブリオンタンパク質凝集体の構造について、同様方...
  • 文部科学省:科学研究費補助金(萌芽研究, 挑戦的萌芽研究)
    Date (from‐to) : 2008 -2009 
    Author : 堀内 基広, 長谷部 理絵, 宋 昌鉉
     
    プリオン病治療法の確立を目的として、骨髄由来間葉系幹細胞(Bone marrow-derived Mesenchymal stem cells, MSC)のプリオン病の治療への応用について、プリオン感染マウスをモデルとして検討した。ヒト不死化MSCs(hMSCs)を、Chandler株感染マウスが初期の臨床症状を呈する接種後120日に海馬に移植した場合、非移植対照群(150±2日)と比べて潜伏期が有意に延長した(157±6日)。また、hMSCsを尾静脈から移植した場合、非移植対照群(148±6日)と比べて潜伏期が有意に延長した(158±6日)。hMSCsを移植したマウスでは、PrP^の蓄積量は対照群と比較して差は認められなかったが、空胞変性は軽度であった。従って、hMSCsはプリオンの増殖は阻害しないが、病気の進行に抑制的に働くことが明らかとなった。hMSCsはプリオン感染マウス脳内で、神経細胞様、アストロサイト様、およびオリゴデンドロサイト様の細胞に分化したが、その効率は低いことから、hMSCsの移植による潜伏期の延長は、bystander effectによるものと考えられた。hMSCsはいずれのルートで移植した場合でもプリオン病の神経病変へ移行したことから、hMSCsがプリオン病の神経病変部に移行するメカニズムを調べるために、in vitro走化試験をスクリーニン...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(A))
    Date (from‐to) : 2006 -2009 
    Author : Motohiro HORIUCHI, 落合 謙爾, 瓜生 匡秀, 大島 正伸, Osamu INANAMI, Kazuhiro KIMURA, Rie HASEBE, Chang-hyun SON, Kenji OCHIAI, Masahide URYU, Masanobu OOSHIMA, Hidefumi FURUOKA
     
    To identify host genes whose expression is up-regulated in the early stage of prion-infection, DNA microarray analysis was carried out and involvement of the genes in pathobiology of prion diseases was analyzed. The results showed that Cd14 molecule, known as receptor for LPS, influences the progression of the disease, whereas, Cxcl10, known as chemokine with neuroprotective effect, plays a role in antagonizing the disease progression. In addition, microglial activation was suggested to have a protective effect on the disease progression.
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2007 -2008 
    Author : NANAML I, 平岡 和佳子, 下山 雄平, Mutsumi INABA, Motohiro HORIUTCHI, Mikinori KUWABARA, Katetoshi ASANUMA, Wakako HIRAOKA, Yuhei SHINOYAMA
     
    本研究はブリオン病の原因であるブリオンタンパク質の凝集体変化を新しい方法を用いて明らかにし、今後のプリオン病の病態解明や治療薬の評価方法を開発することを目的とした。このプリオン凝集体への変化を起こすには細胞内低pH器官であるエンドソームで起きることから、pHの低下が必須であると考えられていることからpH7.0の生理条件からpH4.0に変化させたときの構造変化を明らかにした。電子スピン共鳴法、ならびに原子間力顕微鏡を用いて家族性プリオン病でよく見られる変異体プリオンD177NはpH4.0、変性下に置くことで変異を持っていない野生型よりも効率よく線維状の凝集体が起きることが示され。凝集の引き金となる領域がタンパク中心部のβシート付近にあることが明らかとなった。今回の結果はプリオン病の原因タンパク質の構造変化を明らかにしただけでなく、今回用いた新たな方法はプリオン関連病の治療薬の評価系に用いることが可能であると考えられる。
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2005 -2007 
    Author : Naotaka ISHIGURO, 丸尾 幸嗣, 柳井 徳磨, 桑田 一夫, 堀内 基広
     
    The pathogenesis of animal prion diseases is influenced by prion strain, genetic factors, natural immunology and several cellular factors in the affected hosts. In this study, genetic factors, nerve-immune system and uptake of PrP^ by macrophages were mainly studied. The following results were obtained.1. Polymorpbisms of PrP gene: Polymorphisms of the 863 cattle, 118 caprine and 136 sika dear (Cervus Nippon) PrP genes in Japan were examined. Several single-nucleotide polymorphisms (SNPs) were found in cattle and sika dear, but not find any amino acid substitutions. Two polymorphisms as...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2005 -2006 
    Author : Osamu INANAMI, 堀内 基広, 苅和 宏明, 稲葉 睦, 桑原 幹典
     
    We examined the influence of D177N (178N in humans) mutation on the conformational stability of the S2 region of moPrPc with varying pHs by using the SDSL-ESR technique. We prepared moPrPc mutants that reacted with methane thiosulfonate spin-probes (Y161R1 and Y161R1/D177N). The ESR spectrum of D177N at pH 7.5 was narrower than that of Y161R1, referred to as WT^*. The ESR spectrum of D177N did not change when pH in the solution decreased from 7.5 to 4.0.These results suggested that the disappearance of a salt bridge (D177-R163) induced the increase in the instability of S2 region. The value...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(A))
    Date (from‐to) : 2004 -2006 
    Author : Mutsumi INABA, 山本 雅之, 陰山 聡一, 堀内 基広, 稲波 修, 梅村 孝司
     
    AHSP is an erythroid-specific molecular chaperone that stabilizes newly synthesised a-globin. Previous studies demonstrated that mRNA levels of AHSP were specifically reduced in hematopoietic tissues of prion-infected animals. The purpose of the present study is to clarify the mechanism for down-regulation of AHSP transcription. A series of truncation and mutation analyses on 2.5-kb 5' upstream region of the AHSP gene in MELhide8 cells and electrophoretic mobility shift assay showed that the minimal 5'-promoter region located at-328-286 to the translation initiation site including a GATA bi...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2004 -2006 
    Author : Yoshihiro NISHIDA, 小林 一清, 三浦 佳子, 堀内 基広, 鵜沢 浩隆
     
    Sulfated glycosaminoglycans (GAGs) and their analogues such as pentosan polysulfate (PPS), dextran sulfate (DS), and heparin inhibit abnormal isoform of prion protein (PrP^) formation in prion-infected cells and prolong the incubation time of scrapie-infected animals. Sulfation of GAGs is not completely regulated and possible sulfation sites are randomly sulfated. This property impedes an elucidation of fundamental structures of GAGs that are involved in diverse biological events on cell surfaces. To address the structure-activity relationship of GAGs in the inhibition of PrP^ forma...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2004 -2006 
    Author : Masatsugu SUZUKI, 梶 光一, 山内 貴義, 有川 二郎, 横山 真弓, 堀内 基広, 木村 享史
     
    1. Nine hundred and seventy six sika deer serum samples were examined. Although 25 (2.6%) of them were positive for anti-HEV IgG, the antibody, titers were very low. HEV RNA was not detected in these samples.2. In 87 wild boar, 2.6% of them were positive for anti-HEV IgG, the antibody. HEV RNA was detected in 2.3% in these samples.3. Twelve serum samples (13.6%) from88wild boar were positive for anti-leptosira IgG.4. By using LAMP and RT-PCR methods, M. paratuberculosis DNA was not detected in 173 samples of wild deer.5. In wild deer blood samples collected in Hokkaido, DNA of Rickettsia He...
  • 文部科学省:科学研究費補助金(萌芽研究)
    Date (from‐to) : 2004 -2005 
    Author : 西田 芳弘, 小林 一清, 三浦 佳子, 小川 智久, 堀内 基広
     
    硫酸化ペントサン、ヘパリンなどのグリコサミノグリカンや硫酸多糖体がプリオンの増殖阻害活性を有することは古くから知られている(Ehlers & Diringer, J Gen Virol.,65:1325-1330,1984. Caughey & Raymond, J Virol.,67:643-650,1993.)。これらは、長年にわたり、プリオン病治療法への応用が期待されてきたが、現在まで、硫酸多糖体を使用したプリオン病治療法は確立されていない。天然の硫酸多糖体は硫酸化やアセチル化の部位や割合などが制御されていないため、硫酸多糖体の構造とプリオン増殖阻害活性の構造活性相関の解析が困難である。プリオン増殖阻害活性を担う硫酸化糖の基本骨格が明らかになれば、これをリード化合物として、新たなプリオン病治療薬が創出できる可能性がある。そこで、本研究では、硫酸化部位を正確に制御した合成硫酸化糖を研究代表者の糖鎖モジュール化法を適用することで合成を行い、硫酸化糖の構造とプリオン増殖阻害活性の関係について解析することを目的とした。(1)合成硫酸化糖によるプリオン増殖阻害試験プリオン持続感染細胞I3/I5-9は、10%FBSを含むOpti-MEM(Invitorgen)で培養した。硫酸化糖はMili-Qに溶解して、使用直前に0.45μmのフィルターで濾過滅菌した。硫酸化糖を加え2日間培養した...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(A))
    Date (from‐to) : 2003 -2005 
    Author : Motohiro HORIUCHI, 古岡 秀文, 大橋 和彦, 稲葉 睦, 前田 秋彦
     
    In this research, we studied on the molecular mechanism of prion propagation in the cells through analyses of the effect of compounds that inhibit PrP^ formation and identification of host factor(s) and microenvironment that are involved in prion propagation.Four different anti-PrP antibodies that react with PrP^C on the cell surface inhibited PrP^ formation in cells persistently infected with prion. The antibody-PrP^C complex on the cell surface was not internalized efficiently and tended to retain on the cell surface. These results suggest that anti-PrP mAb antagonized PrP^ fo...
  • 文部科学省:科学研究費補助金(萌芽研究)
    Date (from‐to) : 2002 -2003 
    Author : 古岡 秀文, 品川 森一, 堀内 基広, 松井 高峯, 古林 与志安
     
    (1)プリオン蛋白の免疫組織化学的検出感度に関する研究昨年度までに開発した免疫組織化学的高感度法とWB法の感度の比較を行った.スクレイピー帯広株脳内接種マウスを用いて経時的なプリオン蛋白の検出により比較を行った.WB法では30日目で脾臓に,60〜90日目に中枢神経系に異常プリオン蛋白が検出できた.一方,免疫組織化学的には脾臓は90日目,中枢神経系では120日目に検出可能であった.WB法ではマウス脾臓全量からの検出であるのに対して,免疫組織化学では約5μmの組織切片を使うことから単純な感度比較はできないが,免疫組織化学的方法は検出感度の点ではWB法に比較して劣ることが明らかになった.定量的な解析は現在実施中である.(2)パネル抗体による免疫組織化学コアエピトープに関する研究市販されている抗体を含め,これまでに開発された17種類の抗体を用いて,抗体の種特異性について,抗体エピトープと動物種間のアミノ酸配列との関連から検討した.動物種のアミノ酸配列と抗体エピトープの不一致が抗体の種特異性を規定する場合と規定しない場合があった.特異性を規定しない場合には,動物種間差でみられるいくつかのアミノ酸の違いは反応性を左右しない.しかしながら,アミノ酸一つの違いが動物種差としての特異性を規定する場合や,同じ領域を認識する抗体にも反応性に差がみられた.このことから,免疫組織化学的抗原抗体反応は抗...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    Date (from‐to) : 2002 -2002 
    Author : 片峰 茂, 堂浦 克美, 金子 清俊, 小野寺 節, 福岡 伸一, 堀内 基広
     
    本邦におけるプリオン研究者の情報交換の促進と将来の共同研究プロジェクト立ち上げのための準備を目的に本研究を遂行した。情報交換に関しては、平成14年10月21日に長崎において班会議を開催し、班員に加えて数名の内外のプリオン研究者による情報交換と討議の場をもった。その結果、個々の班員間の往来及び研究材料の共有などのいくつかの実が挙がっている。例えば、片峰と小野寺は各々が開発したプリオン蛋白遺伝子に関わる遺伝子改変マウスと培養細胞株を共有することにより、プリオン病神経変性の機構解明へ向けた共同研究の進展が図られた。準備研究に関しては、プリオン研究進展に極めて大きな意味をもつ種々のモデル動物、細胞株、抗体、解析システムの開発が行われ、将来の大型共同研究プロジェクトへの準備は整ったと考えられる。以下に特筆される成果を挙げる。(1)プリオン持続感染細胞株の樹立(片峰)(2)プリオン類似蛋白(Dpl)遺伝子トランスジェニックマウスの樹立(片峰)(3)プリオン蛋白(PrP)と相互作用をする分子の同定法の開発(堂浦)(4)異常プリオン蛋白(PrPSc)に特異的立体構造を認識する抗体の確立(堀内)(5)不死化によるPrP欠損神経細胞株の樹立(小野寺)(6)PrPの細胞内挙動の顕微鏡下での追跡法の確立(金子)(7)タンパク質の2次構造変換定理の発見(柳川)(8)微量核酸(RNA)同定法の開発(福岡...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 2000 -2002 
    Author : Motohiro HORIUCHI, 田邊 茂之, 古岡 秀文
     
    Major cause of infection in animal prion diseases is thought to be consumption of prion-contaminated stuff orally. There is evidence that the enteric nerve system (ENS) and lymphoid tissues, especially gut-associated lymphoid tissues (GATL) is involved in the infection of prion via alimentary tract. To elucidate the initial entry port for prion, we inoculated prion to alympholasia (aly) mice that show a deficiency in the systemic lymph nodes and Peyer's patches with various route. The aly/aly mice was susceptible to prion by intra-cranial inoculation, however, they showed reduced susceptibi...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2000 -2002 
    Author : 品川 森一, Motohiro HORIUCHI, 堀内 基広, 石黒 直隆, 村松 康和
     
    Mongolia has a far long history for sheep-farming and is now farming over twelve million sheep. In the future, Mongolia has a potential for the major provider of livestock products among the world. Thus surveillance of prion diseases in animal in Mongolia will provide important information to evaluate the safety of livestock products in the country. During the research term, we visit to central, west, and east part of Mongolia, we did not get a positive indication on the existence of scrapie-suspected sheep in hearing. We also checked PrPSc in the medulla oblongata from sheep showed neurolo...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2000 -2002 
    Author : 品川 森一, Sou-ichi MAKINO, 牧野 壮一, 堀内 基広, 古岡 秀文
     
    An abnormal isoform of prion protein (PrPSc) is a major component of the infectious agent "prion" and the biosynthesis of PrPSc play a central role in pathogenesis of prion diseases. Once prion enters the host, PrPSc binds to normal prion protein (PrPC) and converts PrPC into PrPSc. During the conformational transformation, PrPSc acts as a template or seed. In this study, we analyzed inter-molecular interaction between PrPC and PrPSc that is a key event in PrPSc generation.We found that PrPC bound to heterologous PrPSc but did not convert to PrPSc and that the conversion reaction could be i...
  • 文部科学省:科学研究費補助金(特定領域研究(C))
    Date (from‐to) : 2001 -2001 
    Author : 堀内 基広
     
    病原性プリオン蛋白質(PrPSc)と正常型プリオン蛋白質(PrPC)が直接会合することが、PrPScの増殖の第一段階である。そこで、PrPCとPrPScの結合に関与するPrP分子上のドメインを調べることを目的として、各種PrP合成ペプチドがPrPC-PrPSc間の相互作用を阻害するか否かについて検討した。PrPCがPrPSc存在下でPrPSc様のProteinase K(PK)抵抗性分子(PrP-res)に転換する試験管内転換反応で、PrP合成ペプチドaa117-141、aa166-179、aa200-223はPrPCがPrP-resに転換するのを阻害した。これらのPrPペプチドはPrPCと結合することにより、PrPCとPrPScの結合を阻害することが判明した。結合阻害活性を示すPrPペプチドは反応条件下ではβシート構造をとる傾向があることから、PrPペプチドがPrPScのPrPCへの結合ドメインを模倣することでPrPCと結合し、PrPC上のPrPSc結合ドメインを占拠する結果、PrPCとPrPScの結合を阻害する可能性が示唆された。PrPCとPrPScの分子間相互作用を解析する道具として、PrP分子に対するモノクローナル抗体(mAb)パネルを作製した。計34のmAbは認識するエピトープから9群に分類された。グループI〜VIはPrP分子上のリニアエピトープを認識する抗体、グル...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 1997 -1999 
    Author : 堀内 基広, Takane MATSUI, 牧野 壮一, 石黒 直隆
     
    The agent (prion) of transmissible spongiform encephalopathies (prion diseases) including scrape in sheep, bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease (CJD) in human does not possess the agent-specific nucleic acid as a genome. Therefore genome typing and/or antigenic typing are not applicable for distinction of prion strains. However, prion strains can be distinguishable to some extent by biological and biochemical properties. In this study, we attempted to elucidate whether diversity of sheep prion strains exist in Japan. In addition, we analyzed genetic factor(s) othe...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 1996 -1998 
    Author : Morikazu SHINAGAWA, 堀内 基広, 桑山 秀人, 石黒 直隆
     
    As a final goal of this study is in vitro formation of infectious prion amyloid using mouse PrP^C, in vitro structural conversion of PrP^C using a small amount of mouse prion was carried out. To eliminate the effects of unknown mouse protein contaminating in PrP^C, mouse recombinant PrP^C which possessed a histidine tag at N-terminus of mature PrP^C was used. The recombinant PrP^C converted to a proteinase K (PK) resistant form in the presence of one-thousandth amounts of mouse scrapie prion at pH 5.2 and room temperature for one day and after 14 days the PK-resistance increased more. A dec...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(試験研究(B), 基盤研究(A))
    Date (from‐to) : 1995 -1997 
    Author : Morikazu SHINAGAWA, 中川 迪夫, 堀内 基広, 久保 正法, 山田 明夫, 古岡 秀文
     
    In order to increase the sensitivity for detection of prion protein, the method to prepare samples from tissues for Western blotting was reformed. In mouse scrapie model, prion protein could be detected in the samples prepared from the spleen 1 week after intraperitoneal infection by the new method. It is to be desirable that more convenient method than Western blotting to examine many samples for the presence of prion protein. We developed an ELISA whose sensitivity was about two times higher than that of Western blotting using mouse model. We first confirmed that this ELISA method could b...
  • 文部科学省:科学研究費補助金(奨励研究(A))
    Date (from‐to) : 1995 -1995 
    Author : 堀内 基広
     
    本研究は伝達性海綿状脳症における神経細胞死と本病関連蛋白質(PrP)の関係を分子レベルで解明することを目的として開始した。これまでにマウス神経芽腫細胞(N2a)、およびラット副腎クロム親和性細胞腫(PC12)に羊PrPC(PrPCは正常型PrPを示す)を過発現させることに成功しているので、これら培養細胞を用いて研究を行った。羊PrPC過発現細胞は内在性PrPCに比べ数十倍羊PrPCを発現している。羊PrPCの過発現自体が細胞に及ぼす影響を調べたが、NGF、cAMPの刺激に対する応答などの生物性状にコントロールとなる正常細胞との間に差は認められなかった。また、羊PrPCの過発現自体が細胞の活性に影響するか否かをLDH遊離試験、Ho33258核染色により調べたが、正常細胞との間に差は認められなかった。クロロキン処理により羊PrPCの蓄積を高めた場合でも顕著な差は認められなかった。神経細胞毒性が報告されているマウスPrPコドン106-129の合成ペプチドに相応する羊PrP合成ペプチドを羊PrPC過発現細胞に添加し本ペプチドの細胞致死活性を調べたが、羊PrP合成ペプチドによる羊PrPC過発現細胞の選択的な細胞死は観察されなかった。現在まで、PrPによる神経細胞毒性が観察されたのはマウスおよびラットの初代神経培養細胞に限られており、株化細胞での例はない。本研究においても神経系株化細胞で...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(一般研究(B))
    Date (from‐to) : 1992 -1994 
    Author : Morikazu SHINAGAWA, 堀内 基広
     
    1) Infectious amyloid, which is thought to be an etiological agent of scrapie, consists of PrP^, an isoform of a host membrane protein, PrP^C, which is highly expressed in the central nervous system (CNS). It supposedly is one cause of high accumulation of the infectious amyloid in the CNS.We have established a L-929 derived cell-line expressing exogenous PrP (L-BPrP3) by introducing bovine PrP cDNA.In this study, scrapie replication was examined in this cell-line and in the L-929 cell-line. We expected that L-BPrP3 supports sheep scrapie, however, the replication of sheep scrapie in th...
  • 文部科学省:科学研究費補助金(奨励研究(A))
    Date (from‐to) : 1993 -1993 
    Author : 堀内 基広
     
    本研究は、猫パルボウイルス亜群に属する猫汎白血球減少症ウイルス(FPLV)、ミンク腸炎ウイルス(MEV)、犬パルボウイルス(CPV)の分子遺伝学的な解析を行い、これら3ウイルスの相互関係、ならびにCPV出現機構の解明を目的とした。ウイルスのVPgeneに存在する宿主域決定領域(60-91map units, Horiuchi et al.,J.Gen.Virol.,in press)をPCR法により増幅し、直接塩基配列決定法(Higuchi and Ochman,Nucleic Acide Res.,1989)により塩基配列を決定した。現在までに、FPLV2株(日本;1974,フランス;分離年不明)、CPV4株(日本;1979,1982,1984,1991)、MEV1株(日本,1980)の計7株の塩基配列を決定した。このうち代表的なもの(FPLV1株,CPV3株,MEV1株)についてはDNA Data Base of Japan(DDBJ)に登録した。株数が少なかったため分子系統樹を書くには至らなかったが、塩基配列より予想されるアミノ酸配列の多重アライメントの結果、宿主域決定領域内に系統発生的にCPV特異的と考えられる5個のアミノ酸を同定した[amino acid(aa)93,aa103,aa305,aa564,aa568]。また、Parrishらが米国で報告したaa300,...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(総合研究(A))
    Date (from‐to) : 1991 -1993 
    Author : Morikazu SHINAGAWA, 石黒 直隆, 平井 克也, 本多 英一, 見上 彪, 堀内 基広, 小沼 操
     
    1. Restriction fragment length polymorphism (RFLP) on the sheep-PrP gene : To investigate the RFLP types on the PrP-gene, chromosomal DNAs from sheep tissues collected from various districts in Japan were analyzed using restriction endonucleases EcoRI and HindIII and the coding region of sheep PrP as a probe. Sheep were classified into 6 types. Types I to III corresponded to the types reported in England. Nearly half of the scrapie sheep belonged to type I and the frequency of type II in scrapie sheep was low. Distribution of the types in different parts of Japan showed some variations.2. S...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(一般研究(B))
    Date (from‐to) : 1990 -1991 
    Author : Morikazu SHINAGAWA, 堀内 基広, 石黒 直隆
     
    A cDNA clone encoding bovine scrapie-associated fibril protein, PrP, from a bovine brain cDNA library and 6 amplified genomic DNA clones of bovine PrP were characterized. These clones possessed specific characteristics observed in other animal PrP genes. However, the bovine PrP was divided into two types by the number of repeats. One possessed four octapeptide repetitive sequences like other animal PrP genes and consisted of 256 amino acids, the other had five such repetitive sequences and 264 amino acids. The amino acid sequence of the former bovine PrP agreed with that of sheep PrP up to ...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(一般研究(C))
    Date (from‐to) : 1990 -1991 
    Author : Naotaka ISHIGURO, 堀内 基広
     
    The nucleotide sequences of bovine T-cell receptor (TCR ; alpha, beta, gamma, and delta chains) genes which from cDNA libraries constructed from bovine peripheral blood lymphocytes and thymic lymphocytes were determined.1. Of 30 positive cDNA clones for bovine TCR alpha chain, 20 contained complete and correct rearrangements, whereas 4 contained only one Constant (C) region, and 6 were nonfunctional because of incomplete rearrangement. The bovine Calpha gene is composed of 140 amino acid residues with striking similarity to the Ca region of human and mouse.2. Of 38 CDNA clones for TCR beta ...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(試験研究, 試験研究(B))
    Date (from‐to) : 1988 -1990 
    Author : Morikazu SHINAGAWA, 吉野 智男, 太田 千佳子, 石黒 直隆, 百渓 英一, 堀内 基広
     
    The usefulness of detecting the protein (PrP) consisting of scrapie-associated fibrils in the lymphoreticular organs of sheep as a diagnostic tool was investigated. The PrP was detected by means of a rabbit-anti-sheep PrP polyclonal antibody by Western blot analysis. PrP was detected in samples from the central nervous system of five of six sheep showing clinical signs of natural scrapie infection, in spleen samples from four of the six sheep and in lymph node samples taken from three of the sheep. PrP was detected in the spleen and lymph node samples, but not in the central nervous system ...

Educational Activities

Teaching Experience

  • 感染症学特別研究Ⅰ
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Microbiology
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 医学部
    キーワード : 細菌、ウイルス、グラム陽性菌、グラム陰性菌、抗酸性菌、感染症、ワクチン、プリオン、寄生虫、原虫、マイコプラズマ、クラミジア、スピロヘータ、真菌
  • 感染症学特別演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Practice in Food Hygiene
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 食肉衛生、乳衛生、食中毒、HACCP
  • アカデミックイングリッシュ
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Basic Animal Hygiene
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 食品の衛生管理、食品添加物、食中毒、HACCP、安全性評価、食品衛生行政
  • 人獣共通感染症対策専門特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Food Hygiene
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 疾病予防、防疫、ワクチン、消毒、動物福祉、家畜衛生行政
  • 感染症学特別研究ⅡA
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 感染症学特別研究ⅡB
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 海外インターンシップA
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 海外インターンシップB
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 獣医科学・感染症学基礎科目 研究機器演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 獣医科学・感染症学基礎科目 獣医衛生学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 獣医科学特別研究
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 専門獣医科学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • アカデミックイングリッシュ
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 海外実践疫学演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 海外共同研究演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 人獣共通感染症対策専門特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学特論演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 研究機器演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 獣医衛生学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • アカデミックイングリッシュ
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 獣医科学基礎科目 研究機器演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 獣医科学基礎科目 獣医衛生学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 専門獣医科学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 研究・臨床セミナー
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部
  • アドバンスト演習
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部

Campus Position History

  • 2013年4月1日 
    2014年3月31日 
    役員補佐
  • 2013年4月1日 
    2016年3月31日 
    企画・経営室室員
  • 2014年4月1日 
    2015年3月31日 
    総長補佐
  • 2017年4月1日 
    2019年3月31日 
    教育研究評議会評議員
  • 2017年4月1日 
    2019年3月31日 
    獣医学部長
  • 2017年4月1日 
    2019年3月31日 
    大学院獣医学研究院長
  • 2019年4月1日 
    2021年3月31日 
    教育研究評議会評議員
  • 2019年4月1日 
    2021年3月31日 
    獣医学部長
  • 2019年4月1日 
    2021年3月31日 
    大学院獣医学研究院長

Position History

  • 2013年4月1日 
    2014年3月31日 
    役員補佐
  • 2013年4月1日 
    2016年3月31日 
    企画・経営室室員
  • 2014年4月1日 
    2015年3月31日 
    総長補佐
  • 2017年4月1日 
    2019年3月31日 
    教育研究評議会評議員
  • 2017年4月1日 
    2019年3月31日 
    獣医学部長
  • 2017年4月1日 
    2019年3月31日 
    大学院獣医学研究院長
  • 2019年4月1日 
    2021年3月31日 
    教育研究評議会評議員
  • 2019年4月1日 
    2021年3月31日 
    獣医学部長
  • 2019年4月1日 
    2021年3月31日 
    大学院獣医学研究院長


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