研究者情報

マスター

アカウント（マスター）

• 氏名

  堀内　基広(ホリウチ　モトヒロ), ホリウチ　モトヒロ

所属（マスター）

• 獣医学研究院　獣医学部門　衛生学分野

所属（マスター）

• 獣医学研究院　獣医学部門　衛生学分野

独自項目

syllabus

• 2021, 大学院共通授業科目（一般科目）：自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, ケミカルハザード、食の安全、毒性学、環境汚染、シェルターメディスン、リスクアセスメント、GIS、生態学、薬理学、病理学、免疫毒性学、分子毒性学、地球規模課題、リスクアナリシス、コンピューターシミュレーション、R、ドッキングシミュレーション、分析化学、環境化学
• 2021, 大学院共通授業科目（一般科目）：複合領域, Inter-Graduate School Classes(General Subject):Inter-Disciplinary Sciences, 修士課程, 大学院共通科目, One Health、感染症、環境汚染、地球環境問題、保全医学、多分野融合、国際機関
• 2021, 大学院共通授業科目（一般科目）：複合領域, Inter-Graduate School Classes(General Subject):Inter-Disciplinary Sciences, 修士課程, 大学院共通科目, One Health、国際的リーダー、国際機関、異分野連携、異分野コミュニケーション、課題解決能力、地球規模課題
• 2021, 研究機器演習（総合成績）, Advanced Seminar on Analytical Machines(Intergraded Grade), 博士後期課程, 国際感染症学院
• 2021, 研究機器演習（総合成績）, Advanced Seminar on Analytical Machines(Intergraded Grade), 博士後期課程, 獣医学院
• 2021, 海外インターンシップA, Internships Abroad A, 博士後期課程, 国際感染症学院, インターンシップ、海外活動、疫学活動、共同研究
• 2021, 海外インターンシップB, Internships Abroad B, 博士後期課程, 国際感染症学院
• 2021, 獣医衛生学特論, Advanced Lecture on Veterinary Hygiene, 博士後期課程, 獣医学院
• 2021, 獣医衛生学特論, Advanced Lecture on Veterinary Hygiene, 博士後期課程, 国際感染症学院
• 2021, 人獣共通感染症対策専門特論, Advanced and Comprehensive Studies on Zoonosis Control, 博士後期課程, 国際感染症学院
• 2021, アカデミックイングリッシュ, Academic English, 博士後期課程, 獣医学院
• 2021, アカデミックイングリッシュ, Academic English, 博士後期課程, 国際感染症学院
• 2021, 応用動物衛生学, Applied Animal Hygiene, 学士課程, 獣医学部, 家畜衛生、環境衛生、疾病制御、動物福祉
• 2021, 微生物学, Microbiology, 学士課程, 医学部, 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
• 2021, 基礎動物衛生学, Basic Animal Hygiene, 学士課程, 獣医学部, 疾病予防、防疫、動物福祉、家畜衛生行政
• 2021, 食品衛生学実習, Practice in Food Hygiene, 学士課程, 獣医学部, 食肉衛生、乳衛生、食中毒、食品の衛生管理、HACCP、衛生指標菌
• 2021, 食品衛生学, Food Hygiene, 学士課程, 獣医学部, 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

PositionHistory

• Ｏｎｅ Ｈｅａｌｔｈリサーチセンター長, 2023年10月1日, 2025年3月31日
• 企画・経営室室員, 2013年4月1日, 2015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
• 教育研究評議会評議員, 2019年4月1日, 2021年3月31日
• 教育研究評議会評議員, 2021年4月1日, 2023年3月31日
• 大学院国際感染症学院長, 2021年4月1日, 2023年3月31日
• 大学院国際感染症学院長, 2023年4月1日, 2025年3月31日
• 獣医学部長, 2017年4月1日, 2019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, 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
• 総長補佐, 2014年4月1日, 2015年3月31日
• 役員補佐, 2013年4月1日, 2014年3月31日

researchmap

プロフィール情報

学位

• 獣医学博士（北海道大学）

プロフィール情報

• 姓

  堀内, ホリウチ

• 名

  基広, モトヒロ

• ID各種

  201301012090071059

対象リソース

http://kaken.nii.ac.jp/d/r/30219216.ja.html

業績リスト

研究キーワード

• カンピロバクター   網羅的遺伝子発現解析   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   ミクログリア   siRNA   PCR   FPLV   Neuro2a   

研究分野

• ライフサイエンス / 獣医学
• ナノテク・材料 / 生物分子化学
• ライフサイエンス / ウイルス学
• ナノテク・材料 / 高分子化学

経歴

• 2021年04月 - 現在 国際感染症学院・学院長
• 2017年04月 - 現在 北海道大学 国際感染症学院 教授
• 2017年04月 - 現在 北海道大学 大学院獣医学研究院 教授
• 2017年04月 - 2021年03月 北海道大学 大学院獣医学研究院・獣医学部 研究院長：学部長
• 2003年08月 - 2017年03月 北海道大学 (連合)獣医学研究科 教授
• 2000年04月 - 2003年07月 帯広畜産大学原虫病研究センター 助教授
• 1996年06月 - 2000年03月 帯広畜産大学原虫病分子免疫研究センター 助教授
• 1997年07月 - 1999年07月 米国国立衛生研究所 訪問研究員
• 1989年01月 - 1996年05月 帯広畜産大学畜産学部 助手
• 1988年04月 - 1988年12月 日本ロシュ（株）

論文

• Extended application of the rapid post-mortem test kit for bovine spongiform encephalopathy to chronic wasting disease.

  Yuichi Matsuura, Kohtaro Miyazawa, Motohiro Horiuchi, Akio Suzuki, Mayumi Yokoyama, Morikazu Imamura, Keigo Ikeda, Yoshifumi Iwamaru

  Microbiology and immunology 66 5 212 - 215 2022年05月 

  Chronic wasting disease (CWD) is a prion disease affecting cervid species primarily in the United States of America and Canada; however, it is now emerging in Scandinavian countries. Although CWD cases have not been reported in Japan, in case of a CWD outbreak occuring, it is critical to prepare for testing a large number of specimens. The present study showed that a rapid post-mortem test kit, which is used for bovine spongiform encephalopathy surveillance in Japan, is valid for the detection of CWD prion.
• Refeeding activates neurons in the dorsomedial hypothalamus to inhibit food intake and promote positive valence.

  Daigo Imoto, Izumi Yamamoto, Hirokazu Matsunaga, Toya Yonekura, Ming-Liang Lee, Kan X Kato, Takeshi Yamasaki, Shucheng Xu, Taiga Ishimoto, Satoshi Yamagata, Ken-Ichi Otsuguro, Motohiro Horiuchi, Norifumi Iijima, Kazuhiro Kimura, Chitoku Toda

  Molecular metabolism 54 101366 - 101366 2021年10月30日 

  OBJECTIVE: The regulation of food intake is a major research area in the study of obesity, which plays a key role in the development of metabolic syndrome. Gene targeting studies have clarified the roles of hypothalamic neurons in feeding behavior, but the deletion of a gene has a long-term effect on neurophysiology. Our understanding of short-term changes such as appetite under physiological conditions is therefore still limited. METHODS: Targeted recombination in active populations (TRAP) is a newly developed method for labeling active neurons by using tamoxifen-inducible Cre recombination controlled by the promoter of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), a member of immediate early genes. Transgenic mice for TRAP were fasted overnight, re-fed with normal diet, and injected with 4-hydroxytamoxifen 1 h after the refeeding to label the active neurons. The role of labeled neurons was examined by expressing excitatory or inhibitory designer receptors exclusively activated by designer drugs (DREADDs). The labeled neurons were extracted and RNA sequencing was performed to identify genes that are specifically expressed in these neurons. RESULTS: Fasting-refeeding activated and labeled neurons in the compact part of the dorsomedial hypothalamus (DMH) that project to the paraventricular hypothalamic nucleus. Chemogenetic activation of the labeled DMH neurons decreased food intake and developed place preference, an indicator of positive valence. Chemogenetic activation or inhibition of these neurons had no influence on the whole-body glucose metabolism. The labeled DMH neurons expressed prodynorphin (pdyn), gastrin-releasing peptide (GRP), cholecystokinin (CCK), and thyrotropin-releasing hormone receptor (Trhr) genes. CONCLUSIONS: We identified a novel cell type of DMH neurons that can inhibit food intake and promote feeding-induced positive valence. Our study provides insight into the role of DMH and its molecular mechanism in the regulation of appetite and emotion.
• Monitoring of chronic wasting disease using real-time quaking-induced conversion assay in Japan.

  Akio Suzuki, Kazuhei Sawada, Temuulen Erdenebat, Takeshi Yamasaki, Minoru Tobiume, Kinuyo Suga, Motohiro Horiuchi

  The Journal of veterinary medical science 2021年09月24日 

  There has been no report on Chronic wasting disease (CWD) cases in Japan to date; however, there is concern about the geographic spread of CWD. To clarify the CWD status in Japan, we conducted CWD monitoring using real-time quaking-induced conversion (RT-QuIC) assay which can detect the low level of CWD prions. A total of 690 obex samples collected from sika deer and Reeves's muntjac in Hokkaido and Honshu was tested for CWD prions. No CWD-positive cases were found, suggesting that CWD is nonexistent in Japan. Our results also indicate that RT-QuIC assay is useful for continuous monitoring of CWD. Furthermore, nucleotide sequence analysis of the PrP gene revealed sika deer in Japan harbor CWD susceptible allele.
• Research Note: Antimicrobial resistance of Campylobacter species isolated from chickens near Ulaanbaatar city, Mongolia.

  Zolzaya Byambajav, Erdenebat Bulgan, Yuji Hirai, Momoko Nakayama, Misaki Tanaka, Yurika Nitta, Akio Suzuki, Takashi Umemura, Bold Altankhuu, Alimaa Tsagaan, Batbaatar Vanaabaatar, Erdenebaatar Janchivdorj, Nyam-Osor Purevdorj, Narantuya Ayushjav, Takeshi Yamasaki, Motohiro Horiuchi

  Poultry science 100 3 100916 - 100916 2021年03月 

  There has been no report on the prevalence of Campylobacter spp. in farm animals in Mongolia. To uncover the prevalence of Campylobacter spp. in chickens in Mongolia and their antimicrobial resistance, in this study, we isolated and characterized Campylobacter spp. from chickens in Mongolia. We collected 71 cloacal swabs of chickens from 5 farms including 4 layer farms and one broiler farm near Ulaanbaatar city and isolated 25 Campylobacter jejuni and 6 Campylobacter coli isolates. All isolates were resistant to tetracycline, and 3 C. coli isolates were resistant to erythromycin. The C. coli isolates possessed either the erm(B) gene or nucleotide substitution at nt 2,075 of 23S rDNA, both of which are known to be associated with erythromycin resistance. Sixteen of the 31 C. jejuni/C. coli isolates (51.6%) were resistant to nalidixic acid and fluoroquinolones. All the fluoroquinolone-resistant isolates possessed amino acid substitution from threonine to isoleucine at codon 86 (nucleotide substitution: ACA to ATA). Multilocus sequence typing and phylogenetic analyses showed a variation in C. jejuni/C. coli in chickens in Mongolia. In addition, some of the C. jejuni isolates seemed to be phylogenetically close to isolates in Asian and Oceanian countries. This is the first report on the characterization of antimicrobial resistance of Campylobacter spp. in farm animals in Mongolia and is valuable for implementation of measures for a prudent use of antimicrobials in farm animals.
• Involvement of N- and C-terminal region of recombinant cervid prion protein in its reactivity to CWD and atypical BSE prions in real-time quaking-induced conversion reaction in the presence of high concentrations of tissue homogenates.

  Akio Suzuki, Kazuhei Sawada, Takeshi Yamasaki, Nathaniel D Denkers, Candace K Mathiason, Edward A Hoover, Motohiro Horiuchi

  Prion 14 1 283 - 295 2020年12月 

  The real-time quaking-induced conversion (RT-QuIC) reaction is a sensitive and specific method for detecting prions. However, inhibitory factors present in tissue homogenates can easily interfere with this reaction. To identify the RT-QuIC condition under which low levels of chronic wasting disease (CWD) and bovine spongiform encephalopathy (BSE) prions can be detected in the presence of high concentrations of brain tissue homogenates, reactivities of various recombinant prion proteins (rPrPs) were tested. Among the tested rPrPs, recombinant cervid PrP (rCerPrP) showed a unique reactivity: the reactivity of rCerPrP to CWD and atypical BSE prions was not highly affected by high concentrations of normal brain homogenates. The unique reactivity of rCerPrP disappeared when the N-terminal region (aa 25-93) was truncated. Replacement of aa 23-149 of mouse (Mo) PrP with the corresponding region of CerPrP partially restored the unique reactivity of rCerPrP in RT-QuIC. Replacement of the extreme C-terminal region of MoPrP aa 219-231 to the corresponding region of CerPrP partially conferred the unique reactivity of rCerPrP to rMoPrP, suggesting the involvement of both N- and C-terminal regions. Additionally, rCerN-Mo-CerCPrP, a chimeric PrP comprising CerPrP aa 25-153, MoPrP aa 150-218, and CerPrP aa 223-233, showed an additive effect of the N- and C-terminal regions. These results provide a mechanistic implication for detecting CWD and atypical BSE prions using rCerPrP and are useful for further improvements of RT-QuIC.
• Selective neuronal vulnerability is involved in cerebellar lesions of Guinea pigs infected with bovine spongiform encephalopathy (BSE) prions: Immunohistochemical and electron microscopic investigations.

  Shoichi Sakaguchi, Sayo Shintani, Kyohei Kamio, Akio Sekiya, Satomi Kato, Yoshikage Muroi, Motohiro Horiuchi, Hidefumi Furuoka

  Neuropathology : official journal of the Japanese Society of Neuropathology 40 2 167 - 179 2020年04月 [査読有り][通常論文]
   

  The cerebellar lesions of bovine spongiform encephalopathy (BSE)-infected guinea pigs were characterized as severe atrophy of the cerebellar cortex associated with the loss of granule cells, decrease in the width of the molecular layer, and intense protease-resistant prion protein (PrPSc ) accumulations that are similar to cerebellar lesions in kuru and the VV2 type of sporadic Creutzfeldt-Jakob disease. The aim of this study is to assess the relationships between the distribution and localization of PrPSc and synapses expressing neurotransmitter transporters in order to reveal the pathogenesis of the disease. We used cell-type-specific immunohistochemical makers recognizing glutamatergic and γ-aminobutylic acid (GABA)ergic terminals to identify terminals impaired with PrPSc accumulations. The distribution of PrPSc accumulations and immunoreactivity of synaptic vesicles were studied throughout the neuroanatomical pathways in cerebellar lesions. Time course study demonstrated that PrPSc accumulation showed a tendency to spread from granular layer to molecular layer. The immunoreactivity of vesicular glutamate transporter 1 (VGluT1) was localized in axon terminals of cerebellar granule cells, and decreased in association with the severity of PrPSc accumulations and loss of granule cells. Immunoreactivities of vesicular glutamate transporter 2 (VGluT2) and vesicular GABA transporter (VGAT) that exist in axon terminals of inferior olivary neurons and GABAergic synapses of Purkinje cells, respectively, were preserved well in these lesions. In brainstem, VGluT1 immunoreactivity decreased selectively in pontine nuclei that are a component of the pontocerebellar pathway, although other neurotransmitter immunoreactivities were preserved well. Our findings suggest that the selective loss of VGluT1-immunoreactive synapses subsequent to PrPSc accumulations can contribute to the pathogenesis of cerebellar lesions of BSE-infected guinea pigs.
• Enhanced phosphorylation of PERK in primary cultured neurons as an autonomous neuronal response to prion infection.

  Misaki Tanaka, Takeshi Yamasaki, Rie Hasebe, Akio Suzuki, Motohiro Horiuchi

  PloS one 15 6 e0234147  2020年 

  Conversion of cellular prion protein (PrPC) into the pathogenic isoform of prion protein (PrPSc) in neurons is one of the key pathophysiological events in prion diseases. However, the molecular mechanism of neurodegeneration in prion diseases has yet to be fully elucidated because of a lack of suitable experimental models for analyzing neuron-autonomous responses to prion infection. In the present study, we used neuron-enriched primary cultures of cortical and thalamic mouse neurons to analyze autonomous neuronal responses to prion infection. PrPSc levels in neurons increased over the time after prion infection; however, no obvious neuronal losses or neurite alterations were observed. Interestingly, a finer analysis of individual neurons co-stained with PrPSc and phosphorylated protein kinase RNA-activated-like endoplasmic reticulum (ER) kinase (p-PERK), the early cellular response of the PERK-eukaryotic initiation factor 2 (eIF2α) pathway, demonstrated a positive correlation between the number of PrPSc granular stains and p-PERK granular stains, in cortical neurons at 21 dpi. Although the phosphorylation of PERK was enhanced in prion-infected cortical neurons, there was no sign of subsequent translational repression of synaptic protein synthesis or activations of downstream unfolded protein response (UPR) in the PERK-eIF2α pathway. These results suggest that PrPSc production in neurons induces ER stress in a neuron-autonomous manner; however, it does not fully activate UPR in prion-infected neurons. Our findings provide insights into the autonomous neuronal responses to prion propagation and the involvement of neuron-non-autonomous factor(s) in the mechanisms of neurodegeneration in prion diseases.
• Bergeyella zoohelcum isolated from oral cavities of therapy dogs.

  Yasukazu Muramatsu, Nami Haraya, Kazuki Horie, Leo Uchida, Takanori Kooriyama, Akio Suzuki, Motohiro Horiuchi

  Zoonoses and public health 66 8 936 - 942 2019年12月 [査読有り][通常論文]
   

  Bergeyella zoohelcum causes rare but severe human clinical diseases, which mostly arise from animal bites. Notably, Bergeyella infections can also occur in older people after prolonged exposure to dogs or cats without biting. We detected B. zoohelcum in oral cavities of therapy dogs in close contact with older people residing in nursing homes. Twenty-two bacterial isolates were identified as B. zoohelcum by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing. Our results showed that MALDI-TOF MS is an effective tool for rapid identification of rarely isolated, difficult-to-identify microorganisms, such as B. zoohelcum, derived from not only human clinical samples but also animal samples. To our knowledge, this is the first report on detection of B. zoohelcum from therapy dogs. We have provided information on dog-assisted therapy to improve the relationship between humans and animals in ageing societies, particularly for preventive healthcare of older people living in nursing care facilities.
• The first isolation and identification of canine parvovirus (CPV) type 2c variants during 2016-2018 genetic surveillance of dogs in Mongolia

  Uyangaa Temuujin, Ariunaa Tserendorj, Jumpei Fujiki, Yoshihiro Sakoda, Erdene-Ochir Tseren-Ochir, Masatoshi Okamatsu, Keita Matsuno, Tumenjargal Sharav, Motohiro Horiuchi, Takashi Umemura, Tungalag Chultemdorj

  INFECTION GENETICS AND EVOLUTION 73 269 - 275 2019年09月 [査読有り][通常論文]
   

  Canine parvovirus type 2 (CPV-2) causes a highly contagious and fatal disease, developing into acute hemorrhagic enteritis and myocarditis, in dogs. CPV-2 has evolved, generating antigenic variants CPV-2a/2b/2c that are globally distributed. However, investigating molecular characterization of CPV-2 among dog populations in Mongolia has been limited. Herein, 42 stool samples were collected from dogs with clinical signs of infection, and conventional PCR assays were employed to detect CPV-2 in 23. Our results indicated that during 2016-2018, the new CPV-2a and 2c subtypes were detected in 34.7% of the samples, and the new CPV-2b subtype was detected in 30.4% of samples. VP2 protein sequence analysis and next-generation sequencing of the complete viral genome confirmed these antigenic types. However, sequence analysis indicated new and unreported mutations, Pro580Thr, and Tyr584His in the CPV-2c subtype. From a PCR-positive sample, CPV-2c was successfully isolated, and we performed an immunofluorescence assay for antigen detection. Additionally, we performed genetic characterization and phylogenetic analysis to investigate genetic diversity among isolates from the region, resulting in high CPV-2 genetic diversity in the Mongolian dog population. Striking similarities were also observed between sequences of the strains isolated from Mongolia and China over a similar time span.
• Estimation of prion infectivity in tissues of cattle infected with atypical BSE by real time-quaking induced conversion assay.

  Kazuhei Sawada, Akio Suzuki, Takeshi Yamasaki, Yoshifumi Iwamaru, Yuichi Matsuura, Kohtaro Miyazawa, Kentaro Masujin, Ryuichiro Atarashi, Motohiro Horiuchi

  The Journal of veterinary medical science 81 6 846 - 850 2019年06月06日 [査読有り][通常論文]
   

  Atypical bovine spongiform encephalopathy (BSE), first identified in 2004, poses a threat due to the potential to spread the disease to cattle and other animals, including humans. Here, we estimated prion titers in various tissues of cattle infected with atypical BSE using a real-time quaking-induced conversion assay that detects amyloid seeding activity of a disease-specific prion protein, PrPSc, a major component of prions. PrPSc was detected both in and outside of nerve tissues, and some of the peripheral nerve tissues contained relatively high prion titers. Low titers of prions were also observed in masseter, jejunum, and adrenal glands. Quantitative data on prion infectivity in tissues of atypical BSE-affected cattle is useful to assess the risk of atypical BSE.
• Enhancement of binding avidity by bivalent binding enables PrPSc-specific detection by anti-PrP monoclonal antibody 132.

  Akio Suzuki, Takeshi Yamasaki, Rie Hasebe, Motohiro Horiuchi

  PloS one 14 6 e0217944  2019年 [査読有り][通常論文]
   

  Anti-prion protein (PrP) monoclonal antibody 132, which recognizes mouse PrP amino acids 119-127, enables us to reliably detect abnormal isoform prion protein (PrPSc) in cells or frozen tissue sections by immunofluorescence assay, although treatment with guanidinium salts is a prerequisite. Despite the benefit of this mAb, the mechanism of PrPSc-specific detection remains unclear. Therefore, to address this mechanism, we analyzed the reactivities of mono- and bivalent mAb 132 to recombinant mouse PrP (rMoPrP) by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). In ELISA, binding of the monovalent form was significantly weaker than that of the bivalent form, indicating that bivalent binding confers a higher binding stability to mAb 132. Compared with other anti-PrP mAbs tested, the reactivity of bivalent mAb 132 was easily affected by a decrease in antigen concentration. The binding kinetics of mAb 132 assessed by SPR were consistent with the results of ELISA. The dissociation constant of the monovalent form was approximately 260 times higher than that of the bivalent form, suggesting that monovalent binding is less stable than bivalent binding. Furthermore, the amount of mAb 132 that bound to rMoPrP decreased if the antigen density was too low to allow bivalent binding. If two cellular PrP (PrPC) are close enough to allow bivalent binding, mAb 132 binds to PrPC. These results indicate that weak monovalent binding to monomeric PrPC diminishes PrPC signals to background level, whereas after exposure of the epitope, mAb 132 binds stably to oligomeric PrPSc in a bivalent manner.
• Tracking and clarifying differential traits of classical- and atypical L-type bovine spongiform encephalopathy prions after transmission from cattle to cynomolgus monkeys.

  Ken'ichi Hagiwara, Yuko Sato, Yoshio Yamakawa, Hideyuki Hara, Minoru Tobiume, Yuko Okemoto-Nakamura, Tetsutaro Sata, Motohiro Horiuchi, Hiroaki Shibata, Fumiko Ono

  PloS one 14 5 e0216807  2019年 [査読有り][通常論文]
   

  Classical- (C-) and atypical L-type bovine spongiform encephalopathy (BSE) prions cause different pathological phenotypes in cattle brains, and the disease-associated forms of each prion protein (PrPSc) has a dissimilar biochemical signature. Bovine C-BSE prions are the causative agent of variant Creutzfeldt-Jakob disease. To date, human infection with L-BSE prions has not been reported, but they can be transmitted experimentally from cows to cynomolgus monkeys (Macaca fascicularis), a non-human primate model. When transmitted to monkeys, C- and L-BSE prions induce different pathological phenotypes in the brain. However, when isolated from infected brains, the two prion proteins (PrPSc) have similar biochemical signatures (i.e., electrophoretic mobility, glycoforms, and resistance to proteinase K). Such similarities suggest the possibility that L-BSE prions alter their virulence to that of C-BSE prions during propagation in monkeys. To clarify this possibility, we conducted bioassays using inbred mice. C-BSE prions with or without propagation in monkeys were pathogenic to mice, and exhibited comparable incubation periods in secondary passage in mice. By contrast, L-BSE prions, either with or without propagation in monkeys, did not cause the disease in mice, indicating that the pathogenicity of L-BSE prions does not converge towards a C-BSE prion type in this primate model. These results suggest that, although C- and L-BSE prions propagated in cynomolgus monkeys exhibit similar biochemical PrPSc signatures and consist of the monkey amino acid sequence, the two prions maintain strain-specific conformations of PrPSc in which they encipher and retain unique pathogenic traits.
• Retrograde Transport by Clathrin-Coated Vesicles is Involved in Intracellular Transport of PrPSc in Persistently Prion-Infected Cells.

  Takeshi Yamasaki, Akio Suzuki, Rie Hasebe, Motohiro Horiuchi

  Scientific reports 8 1 12241 - 12241 2018年08月16日 [査読有り][通常論文]
   

  Intracellular dynamics of an abnormal isoform of prion protein (PrPSc) are tightly associated with prion propagation. However, the machineries involved in the intracellular trafficking of PrPSc are not fully understood. Our previous study suggested that PrPSc in persistently prion-infected cells dynamically circulates between endocytic-recycling compartments (ERCs) and peripheral regions of the cells. To investigate these machineries, we focused on retrograde transport from endosomes to the trans-Golgi network, which is one of the pathways involved in recycling of molecules. PrPSc was co-localized with components of clathrin-coated vesicles (CCVs) as well as those of the retromer complex, which are known as machineries for retrograde transport. Fractionation of intracellular compartments by density gradient centrifugation showed the presence of PrPSc and the components of CCVs in the same fractions. Furthermore, PrPSc was detected in CCVs isolated from intracellular compartments of prion-infected cells. Knockdown of clathrin interactor 1, which is one of the clathrin adaptor proteins involved in retrograde transport, did not change the amount of PrPSc, but it altered the distribution of PrPSc from ERCs to peripheral regions, including late endosomes/lysosomes. These data demonstrated that some PrPSc is transported from endosomes to ERCs by CCVs, which might be involved in the recycling of PrPSc.
• Flow Cytometric Detection of PrPSc in Neurons and Glial Cells from Prion-Infected Mouse Brains.

  Takeshi Yamasaki, Akio Suzuki, Rie Hasebe, Motohiro Horiuchi

  Journal of virology 92 1 2018年01月01日 [査読有り][通常論文]
   

  In prion diseases, an abnormal isoform of prion protein (PrPSc) accumulates in neurons, astrocytes, and microglia in the brains of animals affected by prions. Detailed analyses of PrPSc-positive neurons and glial cells are required to clarify their pathophysiological roles in the disease. Here, we report a novel method for the detection of PrPSc in neurons and glial cells from the brains of prion-infected mice by flow cytometry using PrPSc-specific staining with monoclonal antibody (MAb) 132. The combination of PrPSc staining and immunolabeling of neural cell markers clearly distinguished neurons, astrocytes, and microglia that were positive for PrPSc from those that were PrPSc negative. The flow cytometric analysis of PrPSc revealed the appearance of PrPSc-positive neurons, astrocytes, and microglia at 60 days after intracerebral prion inoculation, suggesting the presence of PrPSc in the glial cells, as well as in neurons, from an early stage of infection. Moreover, the kinetic analysis of PrPSc revealed a continuous increase in the proportion of PrPSc-positive cells for all cell types with disease progression. Finally, we applied this method to isolate neurons, astrocytes, and microglia positive for PrPSc from a prion-infected mouse brain by florescence-activated cell sorting. The method described here enables comprehensive analyses specific to PrPSc-positive neurons, astrocytes, and microglia that will contribute to the understanding of the pathophysiological roles of neurons and glial cells in PrPSc-associated pathogenesis.IMPORTANCE Although formation of PrPSc in neurons is associated closely with neurodegeneration in prion diseases, the mechanism of neurodegeneration is not understood completely. On the other hand, recent studies proposed the important roles of glial cells in PrPSc-associated pathogenesis, such as the intracerebral spread of PrPSc and clearance of PrPSc from the brain. Despite the great need for detailed analyses of PrPSc-positive neurons and glial cells, methods available for cell type-specific analysis of PrPSc have been limited thus far to microscopic observations. Here, we have established a novel high-throughput method for flow cytometric detection of PrPSc in cells with more accurate quantitative performance. By applying this method, we succeeded in isolating PrPSc-positive cells from the prion-infected mouse brains via fluorescence-activated cell sorting. This allows us to perform further detailed analysis specific to PrPSc-positive neurons and glial cells for the clarification of pathological changes in neurons and pathophysiological roles of glial cells.
• Therapeutic effect of autologous compact bone-derived mesenchymal stem cell transplantation on prion disease.

  Zhifu Shan, Yuji Hirai, Momoko Nakayama, Ryo Hayashi, Takeshi Yamasaki, Rie Hasebe, Chang-Hyun Song, Motohiro Horiuchi

  The Journal of general virology 98 10 2615 - 2627 2017年10月 [査読有り][通常論文]
   

  Prion diseases are fatal neurodegenerative disorders of humans and animals and no effective treatments are currently available. Allogenic transplantation of immortalized human mesenchymal stem cells (MSCs) can prolong the survival of mice infected with prions. However, autologous transplantation is an appropriate model for evaluating the effects of MSCs on prion diseases. Therefore, we isolated and purified MSCs from the femur and tibia of mice as compact bone-derived MSCs (CB-MSCs). Flow cytometric analysis showed that CB-MSCs were negative for myeloid stem cell-derived cell markers CD11b and CD45, but positive for molecules such as Sca-1, CD105 and CD90.2, which are reported to be expressed on MSCs. The ability of CB-MSCs to migrate to brain extracts from prion-infected mice was confirmed by an in vitro migration assay. Intra-hippocampus transplantation of CB-MSCs at 120 days post-inoculation marginally but significantly prolonged the survival of mice infected with the Chandler prion strain. The transplantation of CB-MSCs did not influence the accumulation of disease-specific prion protein. However, the CB-MSC transplantation enhanced microglial activation, which appeared to be polarized to the M2-type activation state. These results suggest that autologous MSC transplantation is a possible treatment for prion diseases, while the modification of microglial activation may be a therapeutic target for neurodegenerative diseases.
• Listeria monocytogenes serotype 4b strains replicate in monocytes/macrophages more than the other serotypes

  Rie Hasebe, Ryo Nakao, Aiko Ohnuma, Takeshi Yamasaki, Hirofumi Sawa, Shinji Takai, Motohiro Horiuchi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 79 6 962 - 969 2017年06月 [査読有り][通常論文]
   

  We analyzed the pathogenicity of various serotypes of Listeria monocytogenes using a Balb/c mouse intravenous injection model. The survival rates of mice inoculated with strains NS1/2b (serotype 1/2b), NS3b (serotype 3b) and NS 4b (serotype 4b) were 60, 63.6 and 63.6%, respectively. Although the survival rates were similar, the bacterial growth in the liver of NS3b-infected mice was 144.5-fold higher than that in the liver of NS4b-infected mice. Histopathological analyses suggest that the NS4b strain replicated more in monocytes/macrophages, whereas the NS3b strain replicated more in hepatocytes. These results raise a possibility that the serotype 4b strains replicated more in monocytes/macrophages compared to the other serotype strains. To assess this, we isolated CD11b-positive cells from mouse livers infected with EGDe (serotype 1/2a), NS1/2b, NS3b, NS4b and the serotype 4b strains 51414 and F17 and counted the number of live bacteria in these cells. CD11b-positive cells from the NS4b-, 51414- and F17-infected mice possessed 24.4- to 42.7-fold higher numbers of live bacteria than those from mice infected with EGDe and NS3b strains. These results suggest that serotype 4b strains replicated more in monocytes/macrophages than the other serotypes, and this may be involved in the pathogenicity of serotype 4b strains, particularly in the dissemination of L. monocytogenes through the host body.
• Complement factors alter the amount of PrP(Sc) in primary-cultured mouse cortical neurons associated with increased membrane permeability.

  Rie Hasebe, Misaki Tanaka, Akio Suzuki, Takeshi Yamasaki, Motohiro Horiuchi

  Virology 496 9 - 20 2016年09月 [査読有り][通常論文]
   

  We examined the effects of complement factors on primary-cultured neurons infected with prions. The amount of protease K (PK)-resistant abnormal form of prion protein (PrP(Sc)) reached a maximum level at 12 and 16 days post exposure (dpe) in 22L- and Chandler-infected neurons, respectively. In Chandler-infected neurons, the reaction of complement factors C1q, C3 and C9 significantly increased membrane permeability. This was followed by a decrease of PK-resistant PrP(Sc) at 16 and 20dpe. In contrast, in 22L-infected neurons, the effects of complement factors were observed at 12 and 16dpe, but not at 20dpe. Membrane permeability also increased in 22L-infected neurons by reaction of complement factor C3, but interestingly, the amount of PK-resistant PrP(Sc) initially decreased, and then increased. These results suggest that the reactivity of complement factors in prion-infected neurons depends on the amount of PrP(Sc) and the prion strain.
• Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrPSc-specific immunostaining.

  Misaki Tanaka, Ai Fujiwara, Akio Suzuki, Takeshi Yamasaki, Rie Hasebe, Kentaro Masujin, Motohiro Horiuchi

  The Journal of general virology 97 8 2030 - 2042 2016年08月 [査読有り][通常論文]
   

  We established abnormal isoform of prion protein (PrPSc)-specific double immunostaining using mAb 132, which recognizes aa 119-127 of the PrP molecule, and novel PrPSc-specific mAb 8D5, which recognizes the N-terminal region of the PrP molecule. Using the PrPSc-specific double immunostaining, we analysed PrPSc in immortalized neuronal cell lines and primary cerebral-neuronal cultures infected with prions. The PrPSc-specific double immunostaining showed the existence of PrPSc positive for both mAbs 132 and 8D5, as well as those positive only for either mAb 132 or mAb 8D5. This indicated that double immunostaining detects a greater number of PrPSc species than single immunostaining. Double immunostaining revealed cell-type-dependent differences in PrPSc staining patterns. In the 22 L prion strain-infected Neuro2a (N2a)-3 cells, a subclone of N2a neuroblastoma cell line, or GT1-7, a subclone of the GT1 hypothalamic neuronal cell line, granular PrPSc stains were observed at the perinuclear regions and cytoplasm, whereas unique string-like PrPSc stains were predominantly observed on the surface of the 22 L strain-infected primary cerebral neurons. Only 14 % of PrPSc in the 22 L strain-infected N2a-3 cells were positive for mAb 8D5, indicating that most of the PrPSc in N2a-3 lack the N-terminal portion. In contrast, nearly half PrPSc detected in the 22 L strain-infected primary cerebral neurons were positive for mAb 8D5, suggesting the abundance of full-length PrPSc that possesses the N-terminal portion of PrP. Further analysis of prion-infected primary neurons using PrPSc-specific immunostaining will reveal the neuron-specific mechanism for prion propagation.
• Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment.

  Zhifu Shan, Takeshi Yamasaki, Akio Suzuki, Rie Hasebe, Motohiro Horiuchi

  Prion 10 4 305 - 18 2016年07月03日 [査読有り][通常論文]
   

  Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrP(Sc)). PrP(Sc) is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrP(C)) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrP(Sc) (PrP(Sc)-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrP(Sc) (PrP(Sc)-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrP(Sc) can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119-127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrP(Sc)-sen and PrP(Sc)-res even if all PrP(Sc) molecules were not detected. The analytical dynamic range for PrP(Sc) detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%-11% and 2.5-3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrP(Sc) detection did not affect the following PrP(Sc) detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrP(Sc) detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds.
• PrPSc-Specific Antibody Reveals C-Terminal Conformational Differences between Prion Strains.

  Eri Saijo, Andrew G Hughson, Gregory J Raymond, Akio Suzuki, Motohiro Horiuchi, Byron Caughey

  Journal of virology 90 10 4905 - 4913 2016年05月15日 [査読有り][通常論文]
   

  UNLABELLED: Understanding the structure of PrP(Sc) and its strain variation has been one of the major challenges in prion disease biology. To study the strain-dependent conformations of PrP(Sc), we purified proteinase-resistant PrP(Sc) (PrP(RES)) from mouse brains with three different murine-adapted scrapie strains (Chandler, 22L, and Me7) and systematically tested the accessibility of epitopes of a wide range of anti-PrP and anti-PrP(Sc) specific antibodies by indirect enzyme-linked immunosorbent assay (ELISA). We found that epitopes of most anti-PrP antibodies were hidden in the folded structure of PrP(RES), even though these epitopes are revealed with guanidine denaturation. However, reactivities to a PrP(Sc)-specific conformational C-terminal antibody showed significant differences among the three different prion strains. Our results provide evidence for strain-dependent conformational variation near the C termini of molecules within PrP(Sc) multimers. IMPORTANCE: It has long been apparent that prion strains can have different conformations near the N terminus of the PrP(Sc) protease-resistant core. Here, we show that a C-terminal conformational PrP(Sc)-specific antibody reacts differently to three murine-adapted scrapie strains. These results suggest, in turn, that conformational differences in the C terminus of PrP(Sc) also contribute to the phenotypic distinction between prion strains.
• Temporary upregulation of anti-inflammatory cytokine IL-13 expression in the brains of CD14 deficient mice in the early stage of prion infection.

  Rie Hasebe, Akio Suzuki, Takeshi Yamasaki, Motohiro Horiuchi

  Biochemical and biophysical research communications 454 1 125 - 30 2014年11月07日 [査読有り][通常論文]
   

  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 75dpi, 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 75dpi. These results suggest that CD14 can suppress IL-13 expression in neurons during the early stage of prion infection.
• Genetic diversity in the prion protein gene (PRNP) of domestic cattle and water buffaloes in Vietnam, Indonesia and Thailand.

  Leo Uchida, Agus Heriyanto, Chalermchaikit Thongchai, Tran Thi Hanh, Motohiro Horiuchi, Kanako Ishihara, Yutaka Tamura, Yasukazu Muramatsu

  The Journal of veterinary medical science 76 7 1001 - 8 2014年07月 [査読有り][通常論文]
   

  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 spongiform 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 of livestock in these Southeast Asian countries.
• Characterization of intracellular dynamics of inoculated PrP-res and newly generated PrP(Sc) during early stage prion infection in Neuro2a cells.

  Takeshi Yamasaki, Gerald S Baron, Akio Suzuki, Rie Hasebe, Motohiro Horiuchi

  Virology 450-451 324 - 35 2014年02月 [査読有り][通常論文]
   

  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 (PrP(Sc)) in Neuro2a cells. Within 24h after inoculation, the newly generated PrP(Sc) was evident at the plasma membrane, in early endosomes, and in late endosomes, but this PrP(Sc) was barely evident in lysosomes; in contrast, the majority of the inoculated PrP(Sc) was evident in late endosomes and lysosomes. However, during the subsequent 48 h, the newly generated PrP(Sc) 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 PrP(Sc) generation. These results suggest that the trafficking of exogenously introduced PrP(Sc) from the endo-lysosomal pathway to the endocytic-recycling pathway is important for the establishment of prion infection.
• Comparison of the anti-prion mechanism of four different anti-prion compounds, anti-PrP monoclonal antibody 44B1, pentosan polysulfate, chlorpromazine, and U18666A, in prion-infected mouse neuroblastoma cells.

  Takeshi Yamasaki, Akio Suzuki, Rie Hasebe, Motohiro Horiuchi

  PloS one 9 9 e106516  2014年 [査読有り][通常論文]
   

  Molecules that inhibit the formation of an abnormal isoform of prion protein (PrP(Sc)) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrP(Sc) 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 (PrP(C)) and PrP(Sc) in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrP(Sc) levels without altering intracellular distribution of PrP(Sc). PPS did not change the distribution and levels of PrP(C), whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrP(C) to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrP(Sc) formation. In contrast, CPZ and U18666A initiated the redistribution of PrP(Sc) from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrP(C). The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrP(Sc) redistribution by CPZ or U18666A partly antagonized PrP(Sc) degradation, suggesting that the transfer of PrP(Sc) to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrP(Sc) degradation. This study revealed that precise analysis of the intracellular dynamics of PrP(C) and PrP(Sc) provides important information for understanding the mechanism of anti-prion agents.
• Absence of CD14 delays progression of prion diseases accompanied by increased microglial activation.

  Keiko Sakai, Rie Hasebe, Yusuke Takahashi, Chang-Hyun Song, Akio Suzuki, Takeshi Yamasaki, Motohiro Horiuchi

  Journal of virology 87 24 13433 - 45 2013年12月 [査読有り][通常論文]
   

  Prion diseases are fatal neurodegenerative disorders characterized by accumulation of PrP(Sc), 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 (PrP(Sc)) revealed that deposition of PrP(Sc) 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 β (TGF-β) appeared to be expressed earlier in prion-infected CD14(-/-) mice. In contrast, IL-1β 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.
• Therapeutic effect of peripheral administration of an anti-prion protein antibody on mice infected with prions.

  Natsuo Ohsawa, Chang-Hyun Song, Akio Suzuki, Hidefumi Furuoka, Rie Hasebe, Motohiro Horiuchi

  Microbiology and immunology 57 4 288 - 97 2013年04月 [査読有り][通常論文]
   

  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 PrP(Sc) 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.
• Cutting Edge: Brucella abortus exploits a cellular prion protein on intestinal M cells as an invasive receptor.

  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 (Baltimore, Md. : 1950) 189 4 1540 - 4 2012年08月15日 [査読有り][通常論文]
   

  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 (PrP(C)) 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 PrP(C)-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 PrP(C) on the apical surface of M cells as an uptake receptor.
• Alternative BSE Risk Assessment Methodology for Beef and Beef Offal Imported into Japan

  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 2012年08月 [査読有り][通常論文]
   

  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.
• Characterization of intracellular localization of PrPSc in prion-infected cells using a mAb that recognizes the region consisting of aa 119-127 of mouse PrP

  Takeshi Yamasaki, Akio Suzuki, Takeshi Shimizu, Masahisa Watarai, Rie Hasebe, Motohiro Horiuchi

  JOURNAL OF GENERAL VIROLOGY 93 3 668 - 680 2012年03月 [査読有り][通常論文]
   

  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.
• Reaction of complement factors varies with prion strains in vitro and in vivo

  Rie Hasebe, Gregory J. Raymond, Motohiro Horiuchi, Byron Caughey

  VIROLOGY 423 2 205 - 213 2012年02月 [査読有り][通常論文]
   

  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.
• Identification of Chemoattractive Factors Involved in the Migration of Bone Marrow-Derived Mesenchymal Stem Cells to Brain Lesions Caused by Prions

  Chang-Hyun Song, Osamu Honmou, Hidefumi Furuoka, Motohiro Horiuchi

  JOURNAL OF VIROLOGY 85 21 11069 - 11078 2011年11月 [査読有り][通常論文]
   

  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.
• Predominant Involvement of the Cerebellum in Guinea Pigs Infected with Bovine Spongiform Encephalopathy (BSE)

  H. Furuoka, M. Horiuchi, Y. Yamakawa, T. Sata

  JOURNAL OF COMPARATIVE PATHOLOGY 144 4 269 - 276 2011年05月 [査読有り][通常論文]
   

  This study reports the experimental transmission of bovine spongiform encephalopathy (BSE) to guinea pigs and describes the cerebellar lesions in these animals. Guinea pigs were inoculated intracerebrally with 10% brain homogenates from ME-affected cattle. These animals were designated as the first passage. Second and third passages were subsequently performed. All guinea pigs developed infection at each passage. The mean incubation period of the first passage was 370 days post-infection (dpi) and this decreased to 307 dpi and 309 dpi for the second and third passages, respectively. Mild to severe spongiform degeneration and gliosis were observed in the cerebral cortex, thalamus and brainstem. In addition, the affected animals had marked pathological changes in the cerebellum characterized by severe cortical atrophy associated with Bergmann radial gliosis of the molecular layer and reduction in the width of the granular cell layer. Immunohistochemically, intense PrP(Sc) deposition and scattered plaque-like deposits were observed in the molecular and granular cell layers. Cerebellar lesions associated with severe atrophy of the cortex have not been reported in animal prion diseases, including in the experimental transmission of PrP(Sc) to small rodents. These lesions were similar to the lesions of human kuru or the VV2 variant of sporadic Creutzfeldt-Jakob disease, although typical kuru plaques or florid plaques were not observed in the affected animals. (C) 2010 Elsevier Ltd. All rights reserved.
• Successive Deaths of a Captive Snow Leopard (Uncia uncia) and a Serval (Leptailurus serval) by Infection with Feline Panleukopenia Virus at Sapporo Maruyama Zoo

  Yukiko Sassa, Hideaki Yamamoto, Masami Mochizuki, Takashi Umemura, Motohiro Horiuchi, Naotaka Ishiguro, Takayuki Miyazawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 73 4 491 - 494 2011年04月 [査読有り][通常論文]
   

  Feline parvoviruses were isolated from frozen samples of intestines taken from a snow leopard (Uncia uncia) and a serval (Leptailurus serval) that died successively at Sapporo Maruyama Zoo in Hokkaido, Japan. Isolates possessed an antigenic epitope for both the feline panleukopenia virus (FPLV) and mink enteritis virus, identified with a hemagglutination inhibition test. Sequencing analyses of the VP2 region of the isolates revealed that the two isolates were identical and of the FPLV-type. These results suggested that FPLV was introduced from a feral cat which entered the zoo and transmitted the virus inside the zoo.
• The effects of lysosomal and proteasomal inhibitors on abnormal forms of prion protein degradation in murine macrophages

  Yukiko Sassa, Takeshi Yamasaki, Motohiro Horiuchi, Yasuo Inoshima, Naotaka Ishiguro

  MICROBIOLOGY AND IMMUNOLOGY 54 12 763 - 768 2010年12月 [査読有り][通常論文]
   

  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.
• Transcellular transport of West Nile virus-like particles across human endothelial cells depends on residues 156 and 159 of envelope protein

  Rie Hasebe, Tadaki Suzuki, Yoshinori Makino, Manabu Igarashi, Satoko Yamanouchi, Akihiko Maeda, Motohiro Horiuchi, Hirofumi Sawa, Takashi Kimura

  BMC MICROBIOLOGY 10 165  2010年06月 [査読有り][通常論文]
   

  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.
• A molecular mechanism for Toll-IL-1 receptor domain-containing adaptor molecule-1-mediated IRF-3 activation.

  Tatematsu M, Ishii A, Oshiumi H, Horiuchi M, Inagaki F, Seya T, Matsumoto M

  The Journal of biological chemistry 285 26 20128 - 20136 26 2010年06月 [査読有り][通常論文]
  
• ImmunoAT method: An initial assessment for the detection of abnormal isoforms of prion protein in formalin-fixed and paraffin-embedded tissues

  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 2010年05月 [査読有り][通常論文]
   

  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.
• A novel copper(II) coordination at His186 in full-length murine prion protein

  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 2010年04月 [査読有り][通常論文]
   

  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
• Cell density-dependent increase in the level of protease-resistant prion protein in prion-infected Neuro2a mouse neuroblastoma cells

  Satoshi Nakamitsu, Aya Kurokawa, Takeshi Yamasaki, Masahide Uryu, Rie Hasebe, Motohiro Horiuchi

  JOURNAL OF GENERAL VIROLOGY 91 2 563 - 569 2010年02月 [査読有り][通常論文]
   

  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.
• Confomational Analysis of Soluble Oligomers of GFP Tagged Prion Protein By Fluorescence Fluctuation Spectroscopy

  Hiroshi Sakata, Motohiro Horiuchi, Izumi Takahashi, Masataka Kinjo

  CURRENT PHARMACEUTICAL BIOTECHNOLOGY 11 1 87 - 95 2010年01月 [査読有り][通常論文]
   

  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.
• Generation of monoclonal antibody that distinguishes PrPSc from PrPC and neutralizes prion infectivity

  Motohiro Horiuchi, Ayako Karino, Hidefumi Furuoka, Naotaka Ishiguro, Kumiko Kimura, Morikazu Shinagawa

  VIROLOGY 394 2 200 - 207 2009年11月 [査読有り][通常論文]
   

  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.
• Effect of Transplantation of Bone Marrow-Derived Mesenchymal Stem Cells on Mice Infected with Prions

  Chang-Hyun Song, Osamu Honmou, Natsuo Ohsawa, Kiminori Nakamura, Hirofumi Hamada, Hidefumi Furuoka, Rie Hasebe, Motohiro Horiuchi

  JOURNAL OF VIROLOGY 83 11 5918 - 5927 2009年06月 [査読有り][通常論文]
   

  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.
• The Region Approximately between Amino Acids 81 and 137 of Proteinase K-Resistant PrPSc Is Critical for the Infectivity of the Chandler Prion Strain

  Ryo Shindoh, Chan-Lan Kim, Chang-Hyun Song, Rie Hasebe, Motohiro Horiuchi

  JOURNAL OF VIROLOGY 83 8 3852 - 3860 2009年04月 [査読有り][通常論文]
   

  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.
• 1SA2-02 Intracellular localization of abnormal isoform of prion protein(1SA2 BSJ&ABA Joint Symposium, "Prion and Virus Infections",The 47th Annual Meeting of the Biophysical Society of Japan)

  堀内 基広

  生物物理 49 S3  一般社団法人 日本生物物理学会 2009年
• 3P-065 プリオンタンパク質可溶性オリゴマーの蛍光相関分光法による解析(蛋白質-機能(反応機構,生物活性など),第47回日本生物物理学会年会)

  Sakata Hiroshi, Horiuchi Motohiro, Kinjo Masataka

  生物物理 49 S162  一般社団法人 日本生物物理学会 2009年
• Heat shock cognate protein 70 contributes to Brucella invasion into trophoblast giant cells that cause infectious abortion

  Kenta Watanabe, Masato Tachibana, Satoshi Tanaka, Hidefumi Furuoka, Motohiro Horiuchi, Hiroshi Suzuki, Masahisa Watarai

  BMC MICROBIOLOGY 8 212  2008年12月 [査読有り][通常論文]
   

  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.
• Evaluation of Methods for Removing Central Nervous System Tissue Contamination from the Surface of Beef Carcasses after Splitting

  Naoko Takada, Motohiro Horiuchi, Tetslitaro Sata, Yasushi Sawada

  JOURNAL OF VETERINARY MEDICAL SCIENCE 70 11 1225 - 1230 2008年11月 [査読有り][通常論文]
   

  Since high levels of prions, the causative agent of bovine spongiform encephalopathy (BSE), accumulate in the brain and spinal cord, contamination of beef carcasses with central nervous system tissue (CNST) may Occur at post-slaughter process. In this study, we investigated CNST contamination on the surface of beef carcasses using glial fibrillary acidic protein as a marker after splitting and evaluated the effects of washing procedures on contamination removal. High levels of CNST contamination was detected immediately after splitting, especially in the area close to the spinal Column. This Suggests that spinal cord fragments are attached to carcasses at the time of splitting even though the spinal cords have been removed by vacuum before splitting. Steam cleaning or manually washing with normal pressure water around the spinal column, performed prior to washing with high-pressure water, was found to be effective for reducing the level of CNST contamination. Furthermore, manually washing with high-pressure water could reduce CNST contamination to almost negligible levels. These results are useful for preparation of appropriate sanitation standard operating procedures to reduce the risk of CNST contamination of carcasses for prevention of exposure to BSE prion via the food chain.
• Effect of intraventricular infusion of anti-prion protein monoclonal antibodies on disease progression in prion-infected mice

  Chang-Hyun Song, Hidefumi Furuoka, Chan-Lan Kim, Michiko Ogino, Akio Suzuki, Rie Hasebe, Motohiro Horiuchi

  JOURNAL OF GENERAL VIROLOGY 89 6 1533 - 1544 2008年06月 [査読有り][通常論文]
   

  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.
• Frequencies of PRNP gene polymorphisms in Vietnamese dairy cattle for potential association with BSE

  Y. Muramatsu, Y. Sakemi, M. Horiuchi, T. Ogawa, K. Suzuki, M. Kanameda, T. T. Hanh, Y. Tamura

  Zoonoses and Public Health 55 5 267 - 273 2008年06月 [査読有り][通常論文]
   

  Since 2004, significant associations between bovine spongiform encephalopathy (BSE) susceptibility in cattle and frequencies of insertion/deletion (ins/del indel) polymorphisms within the bovine prion protein gene (PRNP) have been reported. In this study, we investigated the frequencies of indel polymorphisms within two variable sites, a 23-bp indel polymorphism in the promoter region (23indel) and a 12-bp indel polymorphism in intron 1 region (12indel), in the PRNP in 206 Vietnamese dairy cattle and seven Japanese BSE-affected cattle. In Vietnamese dairy cattle, the frequency distributions of del allele and del/del genotypic polymorphisms in the 23indel site, which are thought to be associated with BSE susceptibility, were significantly higher, whereas the frequencies of del allelic and del/del genotypic polymorphisms in the 12indel site, which have been reported to confer BSE susceptibility, were significantly lower. We have provided evidence that Vietnamese dairy cattle have a unique genetic background in the PRNP gene in comparison with cattle or sires previously reported in other countries. © 2008 The Authors.
• Instability of familial spongiform encephalopathy-related prion mutants

  Yasuko Watanabe, Wakako Hiraoka, Yuhei Shimoyama, Motohiro Horiuchi, Mikinori Kuwabara, Osamu Inanami

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 366 1 244 - 249 2008年02月 [査読有り][通常論文]
   

  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.
• Frequencies of PrP genotypes in meat breeds of Japanese sheep and trail of selective breeding in experimental sheep flock

  Jiro Ohara, Tetsuro Togari, Aya Kurokawa, Junko Maeda, Naotaka Ishiguro, Hidefumi Furuoka, Motohiro Horiuchi

  JOURNAL OF VETERINARY MEDICAL SCIENCE 69 12 1325 - 1329 2007年12月 [査読有り][通常論文]
   

  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 genotypes AQ/AR and AR/AR could raise the ratio of scrapie-resistant sheep from less than 50% to 80% within 3 years. However, the use of sires with the AR/AR genotype and the selection of ewes would be required to achieve a higher ratio of scrapie-resistant sheep.
• Detection of prion protein immune complex for bovine spongiform encephalopathy diagnosis using fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

  Fumihiko Fujii, Motohiro Horiuchi, Masayoshi Ueno, Hiroshi Sakata, Issel Nagao, Mamoru Tamura, Masataka Kinjo

  ANALYTICAL BIOCHEMISTRY 370 2 131 - 141 2007年11月 [査読有り][通常論文]
   

  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 65 1513 - 1520 8 2007年08月 [査読有り][通常論文]
  
• Characterization of prion susceptibility in Neuro2a mouse neuroblastoma cell subclones

  Masahide Uryu, Ayako Karino, Yukiko Kamihara, Motohiro Horiuchi

  MICROBIOLOGY AND IMMUNOLOGY 51 7 661 - 669 2007年 [査読有り][通常論文]
   

  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.
• Species-specificity of a panel of prion protein antibodies for the immunohistochemical study of animal and human prion disease

  H. Furuoka, A. Yabuzoe, M. Horiuchi, Y. Tagawa, T. Yokoyama, Y. Yamakawa, M. Shinagawa, T. Sata

  JOURNAL OF COMPARATIVE PATHOLOGY 136 1 9 - 17 2007年01月 [査読有り][通常論文]
   

  Monoclonal antibodies to the prion protein (PrP) have been of critical importance in the neuropathological characterization of PrP-related disease in men and animals. To determine the influence of species-specific amino-acid substitutions recognized by monoclonal antibodies, and to investigate the immunohistochemical reactivity of the latter, analyses were carried out on brain sections of cattle with bovine spongiform encephalopathy, sheep with scrapie, mice infected with scrapie, and human beings with Creutzfeldt-Jakob disease (CJD) or Gerstmann-Straussler-Sheinker disease (GSS). Immunoreactivity varied between the antibodies, probably as the result of differences in the amino-acid sequence of the prion protein in the various species. Some monoclonal antibodies against mouse recombinant PrP gave strong signals with bovine, ovine and human PrPSc, in addition to murine PrPSc even though the amino acid sequences determined by the antibody epitope are not fully identical with the amino-acid sequences proper to the species. On the other hand, in certain regions of the PrP sequence, when the species-specificity of the antibodies is defined by one amino-acid substitution, the antibodies revealed no reactivity with other animal species. In the region corresponding to positions 134-159 of murine PrP, immunohistochemical reactivity or species-specificity recognized by the antibodies may be determined by one amino acid corresponding to position 144 of murine PrP. Not all epitopes recognized by a monoclonal antibody play an important role in antigen-antibody reactions in immunohistochemistry. The presence of the core epitope is therefore vital in understanding antibody binding ability. (c) 2006 Elsevier Ltd. All rights reserved.
• Identification of pH-sensitive regions in the mouse prion by the cysteine-scanning spin-labeling ESR technique

  Yasuko Watanabe, Osamu Inanami, Motohiro Horiuchi, Wakako Hiraoka, Yuhei Shimoyama, Fuyuhiko Inagaki, Mikinori Kuwabara

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 350 3 549 - 556 2006年11月 [査読有り][通常論文]
   

  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.
• Inhibition of PrPSc formation by synthetic O-sulfated glycopyranosides and their polymers

  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 2006年10月 [査読有り][通常論文]
   

  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 PrPSc in cattle with bovine spongifonn encephalopathy slaughtered at abattoirs in Japan

  N Iwata, Y Sato, Y Higuchi, K Nohtomi, N Nagata, H Hasegawa, M Tobiume, Y Nakamura, K Hagiwara, H Furuoka, M Horiuchi, Y Yamakawa, T Sata

  JAPANESE JOURNAL OF INFECTIOUS DISEASES 59 2 100 - 107 2006年04月 [査読有り][通常論文]
   

  Three 80- to 95-month-old Holstein dairy cattle infected naturally with the agent of bovine spongiform encephalopathy (BSE) and slaughtered at abattoirs in Japan were examined for the distribution of disease-specific and protease-resistant prion protein (PrPSc) by immunohistochemistry (IHC) and Western blot (WB) analyses. The cattle showed no clinical signs or symptoms relevant to BSE but were screened as positive by enzyme-linked immunosorbent assay, a rapid test for BSE. This positive result was confirmed by IHC or WB in a specimen of the medulla oblongata. Histopathologically, these cattle showed no vacuolation in tissue sections from the central nervous system except for the medulla oblongata. Both IHC and WB analyses revealed PrPSc accumulation in the brain, spinal cord, satellite and ganglionic cells of the dorsal root ganglia, and the myenteric plexus of the distal ileum. In addition, small amounts of PrPSc were detected in the peripheral nerves of 2 cattle by WB. No PrPSc was demonstrated by either method in the Peyer's patches of the distal ileum; lymphoid tissues including the palatine tonsils, lymph nodes, and spleen; or other tissues. The distribution of PrPSc accumulation in the preclinical stage was different between naturally infected cattle and cattle inoculated experimentally with the BSE agent.
• 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 2006年02月 [査読有り][通常論文]
   

  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.
• Sequence variation of bovine prion protein gene in Japanese cattle (Holstein and Japanese black)

  S Nakamitsu, T Miyazawa, M Horiuchi, S Onoe, Y Ohoba, H Kitagawa, N Ishiguro

  JOURNAL OF VETERINARY MEDICAL SCIENCE 68 1 27 - 33 2006年01月 [査読有り][通常論文]
   

  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 region. we examined 2 sites of insertion/deletion (indel) polymorphisms: a 23-bp indel in the upper region of exon 1, and a 12-bp indel in the putative promoter region of intron 1. A previous report suggests that the 23-bp indel polymorphism is associated with susceptibility to BSE, but we did not find a difference in allele frequency between healthy and BSE-affected Holstein cattle. There were differences in allele frequency between healthy Holstein and JB cattle at the 23- and 12-bp indels and at the SNPs at nucleotide positions 234 and 576, but there was no difference in allele frequency of the octapeptide repeat. We identified a unique PRNP gene lacking a 288-bp segment (96 amino acids) in DNA samples stocked in our laboratory, but this deletion was not found in any of the 1049 cattle examined in the present study. The present results provide data about variations and distribution of the bovine PRNP gene.
• 1P508 Detection of prion protein immune complex using Fluorescence Correlation Spectroscopy and Fluorescence Cross-Correlation Spectroscopy(25. New methods and tools (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

  Fujii Fumihiko, Horiuchi Motohiro, Ueno Masayoshi, Sakata Hiroshi, Nagao Issei, Tamura Mamoru, Kinjo Masataka

  生物物理 46 2 S273  一般社団法人 日本生物物理学会 2006年
• [Prion disease as infectious disease transmissible from animals to human].

  Horiuchi M

  Nihon rinsho. Japanese journal of clinical medicine 63 12 2213 - 2220 12 2005年12月 [査読有り][通常論文]
  
• Conformational change in full-length mouse prion: A site-directed spin-labeling study

  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 2005年09月 [査読有り][通常論文]
   

  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.
• [Prion diseases as zoonosis].

  Horiuchi M

  Uirusu 55 1 45 - 53 1 2005年06月 [査読有り][通常論文]
  
• Effective antigen-retrieval method for immunohistochemical detection of abnormal isoform of prion proteins in animals

  H Furuoka, A Yabuzoe, M Horiuchi, Y Tagawa, T Yokoyama, Y Yamakawa, M Shinagawa, T Sata

  ACTA NEUROPATHOLOGICA 109 3 263 - 271 2005年04月 [査読有り][通常論文]
   

  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.
• Polymorphisms of caprine PrP gene detected in Japan

  Y Kurosaki, N Ishiguro, M Horiuchi, M Shinagawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 67 3 321 - 323 2005年03月 [査読有り][通常論文]
   

  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 information about the caprine PrP gene that may be useful for assessing the risk of goat scrapie.
• Surveillance of chronic wasting disease in sika deer, Cervus nippon, from Tokachi district in Hokkaido

  N Kataoka, M Nishimura, M Horiuchi, N Ishiguro

  JOURNAL OF VETERINARY MEDICAL SCIENCE 67 3 349 - 351 2005年03月 [査読有り][通常論文]
   

  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 (PrPSc) 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 homogeneous, and are not infected with CWD.
• Cell surface retention of PrPc by anti-PrP antibody prevents protease-resistant PrP formation

  M Horiuchi, CL Kim, M Ogino, H Furuoka, M Shinagawa

  Prions: Food and Drug Safety 189 - 190 2005年 [査読有り][通常論文]
  
• Cell-surface retention of PrPC by anti-PrP antibody prevents protease-resistant PrP formation

  CL Kim, A Karino, N Ishiguro, M Shinagawa, M Sato, M Horiuchi

  JOURNAL OF GENERAL VIROLOGY 85 11 3473 - 3482 2004年11月 [査読有り][通常論文]
   

  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.
• Unique amino acid polymorphisms of PrP genes in Mongolian sheep breeds

  A Gombojav, N Ishiguro, M Horiuchi, M Shinagawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 66 10 1293 - 1295 2004年10月 [査読有り][通常論文]
   

  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 breeds, suggesting that these polymorphisms are a common feature among Mongolian sheep breeds.
• Antigenic characterization of an abnormal isoform of prion protein using a new diverse panel of monoclonal antibodies

  CL Kim, A Umetani, T Matsui, N Ishiguro, M Shinagawa, M Horiuchi

  VIROLOGY 320 1 40 - 51 2004年03月 [査読有り][通常論文]
   

  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.
• Experimental transmission of abnormal prion protein (PrPsc) in the small intestinal epithelial cells of neonatal mice

  M. Okamoto, H. Furuoka, M. Horiuchi, T. Noguchi, K. Hagiwara, Y. Muramatsu, K. Tomonaga, M. Tsuji, C. Ishihara, K. Ikuta, H. Taniyama

  Veterinary Pathology 40 6 723 - 727 2003年11月 [査読有り][通常論文]
   

  Using an immunohistochemical method, we attempted to detect the transmission of abnormal prion protein (PrPsc) to the enterocytes of the small intestine of neonatal mice by oral exposure with sheep brain affected by scrapie. Five 1-day-old neonatal mice were exposed by oral inoculation to the homogenized brain of a scrapie-affected sheep. In the small intestine of all mice 1 hour after inoculation, immunoreactivity with antinormal prion protein (PrPc) antibody was seen in the cytoplasm of villus enterocytes. This finding suggests transmission of abnormal PrPsc into the cytoplasm of enterocytes. In control mice treated with normal sheep brain, no PrPc signal was seen in enterocytes of the small intestine. Immunopositivity for neurofilament protein and glial fibrillary acidic protein was seen in the cytoplasm of enterocytes of mice inoculated with scrapie and normal sheep brain. This suggests that the enterocytes of neonatal mice can absorb PrPsc and other macromolecular proteins of the sheep brain affected by scrapie and may be more important than previously thought as a pathway for PrPsc transmission in neonatal animals.
• Cellular prion protein promotes Brucella infection into macrophages

  MH Watarai, S Kim, J Erdenebaatar, S Makino, M Horiuchi, T Shirahata, S Sakaguchi, S Katamine

  JOURNAL OF EXPERIMENTAL MEDICINE 198 1 5 - 17 2003年07月 [査読有り][通常論文]
   

  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.
• Susceptibility of transgenic mice expressing chimeric sheep, bovine and human PrP genes to sheep scrapie

  A Gombojav, Shimauchi, I, M Horiuchi, N Ishiguro, M Shinagawa, T Kitamoto, Miyoshi, I, S Mohri, M Takata

  JOURNAL OF VETERINARY MEDICAL SCIENCE 65 3 341 - 347 2003年03月 [査読有り][通常論文]
   

  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. In contrast, the incubation period was significantly prolonged (p<0.05) in Bo/Mo PrP Tg Prnp(+/+) mice inoculated with KU brain homogenate. The incubation period was significantly longer in all Tg Prnp(+/+) and Prnp(0/0), than in non-Tg mice (p<0.01) inoculated with Y5 brain homogenate. None of the Tg Prnp(0/0). mice inoculated with S2 brain homogenate developed clinical signs and PrPSc was undetectable in their brains. These results suggested that expression of the Sh/Mo PrP or Bo/Mo PrP transgenes does not confer susceptibility to sheep prions upon mice, and thus none of the Tg mouse lines could be a suitable model of sheep scrapie. Hu/Mo PrP Tg Prnp(0/0) mice inoculated with natural and experimental scrapie or mouse prions did not develop clinical signs of scrapie and PrPSc was undetectable. These results suggested that neither sheep nor mouse strains of scrapie are highly transmissible to humans.
• Amino acid Polymorphisms of PrP gene in Mongolian sheep

  A Gombojav, N Ishiguro, M Horiuchi, D Serjmyadag, B Byambaa, M Shinagawa

  JOURNAL OF VETERINARY MEDICAL SCIENCE 65 1 75 - 81 2003年01月 [査読有り][通常論文]
   

  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 to scrapic). Several Yeroo, Orkhon and Khangai sheep raised in Selenge prefecture had histidine at codon 154 (154H). Novel polymorphisms of valine (V) and serine (S) at codon 127, lysine (K) at codon 171, and leucine (L) and arginine (R) at codon 189 were also found in Khalkh, Yeroo and Orkhon sheep. It is not known whether these novel polymorphisms affect scrapie susceptibility.
• Bacteriological and genetic assessment of game meat from Japanese wild boars

  Y Naya, M Horiuchi, N Ishiguro, M Shinagawa

  JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 51 2 345 - 349 2003年01月 [査読有り][通常論文]
   

  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.
• A genetic method to distinguish crossbred inobuta from Japanese wild boars

  N Ishiguro, Y Naya, M Horiuchi, M Shinagawa

  ZOOLOGICAL SCIENCE 19 11 1313 - 1319 2002年11月 [査読有り][通常論文]
   

  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.
• Biological and biochemical characterization of sheep scrapie in Japan

  M Horiuchi, T Nemoto, N Ishiguro, H Furuoka, S Mohri, M Shinagawa

  JOURNAL OF CLINICAL MICROBIOLOGY 40 9 3421 - 3426 2002年09月 [査読有り][通常論文]
   

  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.
• Multiple nuclear pseudogenes of mitochondrial DNA exist in the canine genome

  N Ishiguro, A Nakajima, M Horiuchi, M Shinagawa

  MAMMALIAN GENOME 13 7 365 - 372 2002年07月 [査読有り][通常論文]
   

  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.
• スクレピー感染マウス脳のアミロイド斑について

  古岡 秀文, 堀内 基広, 石原 孝介, 松山 好希, 古林 与志安, 松井 高峯, 品川 森一

  日本獣医学会学術集会講演要旨集 133回 55 - 55 (公社)日本獣医学会 2002年03月 [査読有り][通常論文]
  
• グリシドールによるプリオンの分解不活化(公衆衛生学)

  山本 真理, 堀内 基広, 石黒 直隆, 品川 森一, MATSUO T, KANEKO K

  The journal of veterinary medical science 63 9 983 - 990 社団法人日本獣医学会 2001年09月 [査読無し][通常論文]
   

  伝達性海綿状脳症の病原体プリオンは従来の微生物不活化処理に高い抵抗性を示す.医療機器のプリオン汚染除去を目的として, 3種のエポキシ化合物, β-プロピオラクトン, プロピレンオキサイド及びグリシドール(GLD)のスクレイピープリオンに対する影響を調べた.異常プリオン蛋白(PrP^<Sc>)と抗体の反応性を指標に調べたところ, GLDが有望であった.GLDはプリオン蛋白と結合して, 小分子に断片化することがわかった.3及び5%GLDによりPBS中で室温処理により, プリオンの感染性は, 10^3以上に低下したが十分とは言えなかった.より有効に処理する条件を見いだすために, GLDの効果に及ぼすGLDの濃度, 温度, 塩, pHの影響を調べた.時間の延長と共に調べたGLD5%, 55°C, pH7.8までの範囲では, GLDの濃度, 温度およびpHは高い方が効果的であった.
• Inhibition of Interactions and Interconversions of Prion Protein Isoforms by Peptide Fragments from the C-terminal Folded Domain

  Motohiro Horiuchi, Gerald S. Baron, Liang-Wen Xiong, Byron Caughey

  Journal of Biological Chemistry 276 18 15489 - 15497 2001年05月04日 [査読有り][通常論文]
   

  The formation of protease-resistant prion protein (PrP-res or PrP Sc) involves selective interactions between PrP-res and its normal protease-sensitive counterpart, PrP-sen or PrPC. Previous studies have shown that synthetic peptide fragments of the PrP sequence corresponding to residues 119-136 of hamster PrP (Ha119-136) can selectively block PrP-res formation in cell-free systems and scrapie-infected tissue culture cells. Here we show that two other peptides corresponding to residues 166-179 (Ha166-179) and 200-223 (Ha200-223) also potently inhibit the PrP-res induced cell-free conversion of PrP-sen to the protease-resistant state. In contrast, Ha121-141, Ha180-199, and Ha218-232 were much less effective as inhibitors. Mechanistic analyses indicated that Ha166-179, Ha200-223, and peptides containing residues 119-136 inhibit primarily by binding to PrP-sen and blocking its binding to PrP-res. Circular dichroism analyses indicated that Ha117-141 and Ha200-223, but not non-inhibitory peptides, readily formed high β-sheet structures when placed under the conditions of the conversion reaction. We conclude that these inhibitory peptides may mimic contact surfaces between PrP-res and PrP-sen and thereby serve as models of potential therapeutic agents for transmissible spongiform encephalopathies.
• Specific binding of normal prion protein to the scrapie form via a localized domain initiates its conversion to the protease-resistant state

  Motohiro Horiuchi, Byron Caughey

  EMBO Journal 18 12 3193 - 3203 1999年06月15日 [査読有り][通常論文]
   

  In the transmissible spongiform encephalopathies, normal prion protein (PrP-sen) is converted to a protease-resistant isoform, PrP-res, by an apparent self-propagating activity of the latter. Here we describe new, more physiological cell-free systems for analyzing the initial binding and subsequent conversion reactions between PrP-sen and PrP-res. These systems allowed the use of antibodies to map the sites of interaction between PrP-sen and PrP-res. Binding of antibodies (α219-232) to hamster PrP-sen residues 219-232 inhibited the binding of PrP-sen to PrP-res and the subsequent generation of PK-resistant PrP. However, antibodies to several other parts of PrP-sen did not inhibit. The α219-232 epitope itself was not required for PrP-res binding thus, inhibition by α219-232 was likely due to steric blocking of a binding site that is close to, but does not include the epitope in the folded PrP-sen structure. The selectivity of the binding reaction was tested by incubating PrP-res with cell lysates or culture supernatants. Only PrP-sen was observed to bind PrP-res. This highly selective binding to PrP-res and the localized nature of the binding site on PrP-sen support the idea that PrP-sen serves as a critical ligand and/or receptor for PrP-res in the course of PrP-res propagation and pathogenesis in vivo.
• Immunohistochemical study of some cytoskeletal proteins in hereditary myopathy of the diaphragmatic muscles in Holstein-Friesian cattle

  H Furuoka, A Murakami, M Tsuchihashi, H Yokota, T Doi, Y Kobayashi, T Matsui, M Horiuchi, H Taniyama

  ACTA NEUROPATHOLOGICA 97 2 177 - 184 1999年02月 [査読有り][通常論文]
   

  We have investigated the expression, using immunohistochemical and Western blot methods, of some cytoskeletal proteins including desmin, vimentin, actin, alpha-actinin, and ubiquitin in hereditary myopathy of the diaphragmatic muscles in Holstein-Friesian cattle (the histochemical and electron microscopical aspects have been previously reported). Immunohistochemically, the expression of desmin was observed strongly in the subsarcolemmal regions, but was lacking or faint in the area corresponding to the core-like structures. Vimentin showed al most the same localization as desmin, but no activity could be observed in the core-like structures. In addition, the core-like structures showed strong immunoreactivity for actin and ubiquitin, but no immunoreactivity for alpha-actinin. F-actin stained with phalloidin-tetramethyl-rhodamine was strongly positive in irregular spots that corresponded to the core-like structures, but was negative for desmin positive regions. Western blot analysis of the diseased muscles revealed a significant increase in the amount of desmin and vimentin immunoreactivities and similar amounts of actin and alpha-actinin compared with the control muscles. Two-dimensional electrophoresis revealed no isoforms of desmin, suggesting the absence of abnormal phosphorylated forms of desmin. Since the co-localization of desmin and vimentin and the absence of phosphorylated desmin suggest that the overexpression of desmin may be reflected in the reactive change or regenerating process, the present myopathy should be regarded as an entity separate from desmin-storage myopathy or desmin-related myopathies. We also discuss the possibility that the present myopathy could be considered as myofibrillar myopathy, a recently proposed nosological entity.
• Detection methods of possible prion contaminants in collagen and gelatin

  T. Nemoto, M. Horiuchi, M. Horiuchi, N. Ishiguro, M. Shinagawa

  Archives of Virology 144 177 - 184 1999年01月01日 [査読無し][通常論文]
   

  We describe methods for the preparation of collagen and gelatin samples to detect possible prion contaminants using Western blotting of a major component of prions, PrP(Sc). A commercially available collagen solution containing 2% athero-collagen was spiked with rodent adapted scrapie prion and used as the prion-contaminating collagen. The methods developed center on the enzymatic reduction of the collagen solution viscosity with protease treatments and on the concentration of the prion from the protease-digests with polyethylene glycol-6000 and NaCl. Recovery of the spiked prion as a partially protease-resistant core fragment of PrP(Sc) fluctuated from 30% to 46% of the input amount.
• Differences in the evolutionary pattern of feline panleukopenia virus and canine parvovirus

  Motohiro Horiuchi, Motohiro Horiuchi, Motohiro Horiuchi, Yumi Yamaguchi, Takashi Gojobori, Masami Mochizuki, Hideyuki Nagasawa, Yutaka Toyoda, Naotaka Ishiguro, Morikazu Shinagawa

  Virology 249 440 - 452 1998年09月30日 [査読無し][通常論文]
   

  Canine parvovirus (CPV) suddenly appeared in the late 1970s after which it showed continuous antigenic changes virological and molecular genetic analyses mainly focused on feline panleukopenia virus (FPLV) were conducted in this study because FPLV is the suspected ancestor of CPV; the way in which FPLV evolves may help to explain the emergence of CPV. Analysis of escape mutants against FPLV-specific monoclonal antibody showed that viruses possessing CPV-like properties were not easily detected in FPLV virus stocks Phylogenetic analysis revealed that the nonstructural protein 1 (NS1) and capsid protein 2 (VP2) genes of FPLV changed with time. A similar tendency, however, was not observed in the FPLV VP2 proteins. In contrast, the topology of the phylogenetic tree of VP2 proteins of CPV basically concurred with that of the VP2 genes. Analysis of the ratio of nonsynonymous and synonymous substitutions revealed that synonymous substitutions exceeded nonsynonymous substitutions in both the NS1 and VP2 genes of FPLV, even when the analysis focused on specific regions in the VP2 gene that are known to be located on the capsid surface. Comparison of the CPV VP2 genes revealed that nonsynonymous substitution was found to dominate over synonymous substitution in one specific region in the VP2 gene. These results suggested that FPLV has changed mainly by random genetic drift. In contrast, after the appearance of CPV, changes in the CPV VP2 gene appear to be partly selected by certain positive selection forces. CPV and FPLV are known to be closely related viruses genetically and biologically, but the evolutionary mechanisms of the two viruses appeared to be different.
• ウシプリオンタンパク合成ペプチドに対するニワトリ型モノクローナル抗体(短報)

  松下 かおり, 堀内 浩幸, 古澤 修一, 堀内 基広, 品川 森一, 松田 治男

  The journal of veterinary medical science 60 6 777 - 779 社団法人日本獣医学会 1998年06月 [査読無し][通常論文]
   

  ウシプリオンタンパク(PrP)の合成ペプチドに対するニワトリ型モノクローナル抗体を作成した.ウシPrPペプチドB204(コドン204〜220)をKLHに架橋して免疫したニワトリは, B204に対する抗体を産生した.免疫ニワトリの脾細胞と融合用ニワトリB細胞株MuH1を用いた1回の融合実験で, 19種類のB204特異的ニワトリ型モノクローナル抗体が作成できた.これらの抗体は, B204ペプチドの他4種類の10アミノ酸合成ペプチドを用いた阻害ELISAでアミノ酸配列から予想された動物間(ウシ・マウス・ヒト・ヒツジ・ハムスター)組合せからなる5群に分類できた.これらの結果は, ニワトリ型モノクローナル抗体システムは哺乳動物PrPの免疫学的解析に有用な技術であることを示唆している.
• Characterization of differentially expressed genes in the bovine T lymphoma cell line

  Hideyuki Onodera, Naotaka Ishiguro, Motohiro Horiuchi, Morikazu Shinagawa

  Veterinary Immunology and Immunopathology 62 209 - 219 1998年04月16日 [査読無し][通常論文]
   

  A differential display (D.D.) analysis was made to detect differentially expressed genes in a bovine T lymphoma cell line, BTL-26, derived from the calf type of sporadic bovine lymphosarcoma. A D.D. analysis comparing BTL-26 with the bovine epithelial cell line CKT-1 and healthy bovine thymocytes yielded 24 cDNA clones. The DNA sequencing analysis followed by a homology search showed that 20 of the 24 cDNA clones had no significant homology to any sequences in DNA data base. The remaining four genes were homologous to known sequences. Northern blot hybridization among BTL-26, CKT-1 and healthy bovine thymocytes showed that a cDNA clone, BC8, was differentially expressed in BTL-26. The cloning of full-length cDNA for the BC8 clone and its DNA sequences showed that the BC8 clone is a bovine nuclear domain protein homologous to the human NDP52 gene. Northern blot analysis showed that the BC8 clone bovine NDP52 was predominantly expressed in tumor cell line BTL- 26, compared with the transcripts from several bovine tissues.
• Characterization of the sheep apolipoprotein E (ApoE) gene and allelic variations of the ApoE gene in scrapie Suffolk sheep

  Yusuke Komatsu, Motohiro Horiuchi, Motohiro Horiuchi, Naotaka Ishiguro, Takane Matsui, Morikazu Shinagawa

  Gene 208 2 131 - 138 1998年02月22日 [査読無し][通常論文]
   

  Apolipoprotein E (ApoE) plays a central role in lipid transport and is suggested to be involved in neuronal repair. Human ApoE ε4 allele is known as a risk factor for Alzheimer's disease, and an association of the human ApoE genotype with the human prion disease, Creutzfeldt-Jakob disease, is suggested, albeit controversial. We analyzed the sheep ApoE gene to determine whether any association between the sheep ApoE genotype and the sheep prion disease, scrapie, existed. The sheep ApoE cDNA contained an open reading frame (ORF) consisting of 948 base pairs (bp) that encoded 316 amino acids (aa). The sheep ApoE gene was composed of four exons separated by three introns, and the ORF was encoded by three exons, designated exons 2, 3, and 4. Nucleotide sequence analysis also showed the presence of one G/T nucleotide polymorphism in the ORF that resulted in an Ala/Ser amino-acid substitution at codon 258. PCR-restriction fragment length polymorphism analysis of genomic DNA showed the presence of three sheep ApoE genotypes that were the result of the homologous and heterologous combinations of the two alleles. We analyzed the sheep ApoE genotypic and the allelic frequencies in scrapie and control Suffolk sheep, but they did not significantly differ from those in the control sheep, even though PrP genotype-matched populations were compared. The ApoE genotype appeared not to be associated with the progression of the disease when looking at the age at death. These results indicated that in Suffolk sheep, none of the ApoE genotypes was associated with scrapie.
• Genomic structure of the bovine PrP gene and complete nucleotide sequence of bovine PrP cDNA

  M Horiuchi, N Ishiguro, H Nagasawa, Y Toyoda, M Shinagawa

  ANIMAL GENETICS 29 1 37 - 40 1998年02月 [査読無し][通常論文]
   

  The present authors previously reported the nucleotide sequence of the 5' half of a cDNA encoding bovine prion protein (PrP) and the genomic structure of the bovine PrP gene encoding the 5'-untranslated region. Here they report the extent of intron 2 of the bovine PrP gene and the nucleotide sequence of the 3' half of bovine PrP cDNA that had not been determined before. This newly sequenced 3' half of the bovine PrP cDNA consisted of 2149 bp. The entire 3'-untranslated region (3'-UTR) was found to be encoded by a single exon, exon 3. One nucleotide polymorphism was found in the 3'-UTR. The length of intron 2 was estimated to be about 14 kbp. The structure of bovine PrP gene can be defined by combining the present results and previous reports on the bovine PrP gene.
• DNAループの修復機能に依存しない12塩基反復配列の不安定性 (短報)

  品川 敏恵, 石黒 直隆, 掘内 基広, 品川 森一

  The journal of veterinary medical science 59 11 1071 - 1074 社団法人日本獣医学会 1997年11月 [査読無し][通常論文]
   

  牛のTリンパ腫の約15%では, 12塩基反復配列がc-myb遺伝子に挿入されている. この配列の反復回数が細胞の培養中に増減する現象が観察された. 反復回数の変化がDNAループの修復異常で生じる可能性を探るため, 蛋白のDNAループ結合能と修復能を調べたが, 調査した全ての細胞株で両方の活性が検出された. これらの結果は, 12塩基反復配列の不安定性がDNAループの修復機能と無関係である可能性を示唆している.
• Deletion of c-myb exon 9 induced by insertion of repeats

  Toshie Shinagawa, Naotaka Ishiguro, Motohiro Horiuchi, Takane Matsui, Kosuke Okada, Morikazu Shinagawa

  Oncogene 14 23 2775 - 2783 1997年08月07日 [査読無し][通常論文]
   

  A simple repeat was found to be inserted into exon 9 of the c-myb gene in three out of 20 bovine T lymphomas. The repeat was composed of multiple copies of a 12-nucleotide motif and had no significant homology to the sequences reported so far. Tumor cells containing the repeat expressed two kinds of c-myb mRNA: (1) are that included the repetitive sequence in exon 9, and (2) are that lacked the whole sequence of exon 9. Transfection of an expression vector containing exon and intron sequences and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the mRNA demonstrated that the insertion of the repeat enhanced exon skipping of the transfected minigene. These observations imply that the insertion of the repeat may enhance exon skipping of the c-myb pre-mRNA. Although the transcription-activating activity by the c-Myb with the repeat was low, that by the c-Myb without exon 9 was three- to eightfold higher than the wild-type c-Myb. These data suggest that insertion of the 12-nucleotide repeat in codon 359 may result in c-Myb proteins having high- and low-transcription-activating activity.
• Alternative usage of exon 1 of bovine PrP mRNA

  Motohiro Horiuchi, Motohiro Horiuchi, Naotaka Ishiguro, Hideyuki Nagasawa, Yutaka Toyoda, Morikazu Shinagawa

  Biochemical and Biophysical Research Communications 233 650 - 654 1997年04月28日 [査読無し][通常論文]
   

  Here we report two types of bovine prion protein (PrP) mRNA that possessed different lengths of the 5'-untranslated region and were expressed in various bovine tissues. The two mRNA species were transcribed from identical positions but differed in the usage of the splice site for exon 1/intron. One mRNA possessed exon 1 consisting of 53 nucleotides and the other possessed exon 1 consisting of 168 nucleotides. Usage of exons 2 and 3 was identical for the two mRNA species. The two mRNA species were detected in all but spleen tissue; the mRNA possessing 168-nt exon 1 was not detected in bovine spleen. This is the first report on the tissue-specific alternative splicing of PrP mRNA in any other species. Only a low level of PrP(c) appeared to be present in bovine spleen. These results suggested the possibility that the mRNA possessing 53-nt exon 1 was inefficiently translated into Prp(c); however, in vitro translation analysis showed no marked difference in translational efficiency between the two mRNA species.
• Sensitive enzyme-linked immunosorbent assay for detection of PrP(Sc) in crude tissue extracts from scrapie-affected mice

  Kai Uwe, D Grathwohl, Motohiro Horiuchi, Motohiro Horiuchi, Naotaka Ishiguro, Morikazu Shinagawa

  Journal of Virological Methods 64 205 - 216 1997年03月01日 [査読無し][通常論文]
   

  An enzyme-linked immunosorbent assay (ELISA) was developed that detects PrP(Sc) in crude extracts from brain and spleen tissue of scrapie-affected mice with high sensitivity and specificity. Brain tissue was homogenized in 8% Zwittergent 3-12 and 0.5% Sarkosyl. The homogenate was treated with collagenase and DNase I and then subjected to proteinase K digestion. Precipitates containing PrP(Sc) were obtained by ultracentrifugation. Spleen tissue was homogenized in 4% Triton X-100 and 0.5% Sarkosyl, and the homogenate was treated firstly with collagenase and DNase I, and secondly with proteinase K. PrP(Sc) was then extracted with 6.25% Sarkosyl and precipitated through salting-out with NaCl and by ultracentrifugation. When PrP(Sc) was dissolved in 3-4 M guanidine thiocyanate and adsorbed to microtiter plates, strong and specific reactions to the formation of antigen-antibody complexes could be detected by ELISA. The sensitivity of PrP(Sc)-detection for this ELISA, as measured by serial dilution of scrapie material in tissue homogenates from uninfected animals, was equal or higher than that attained by Western blot. This ELISA is more rapid than Western blot and seems to be more suitable for screening large numbers of animals. It also has potential application for the diagnosis of the transmissible spongiform encephalopathies.
• p53 mutation as a potential cellular factor for tumor development in enzootic bovine leukosis

  Naotaka Ishiguro, Hidefumi Furuoka, Takane Matsui, Motohiro Horiuchi, Morikazu Shinagawa, Masatoshi Asahina, Kousuke Okada

  Veterinary Immunology and Immunopathology 55 351 - 358 1997年03月01日 [査読無し][通常論文]
   

  Mutations of p53 in the lymphocytes from peripheral blood and from tumoral lymph nodes in six naturally occurring bovine leukemia virus (BLV)-infected cows were examined. A point mutation of the p53 gene was found in three of six (50%) BLV-infected cows. These p53 gene mutations resulted in amino acid substitutions of codons 144, 167 and 241. The BLV-infected cow in the tumor stage had abnormally proliferating monoclonal B-lymphocytes having the p53 mutation. However, the mutation was not found in somatic cells, except for tumor cells. These results show that p53 mutation plays an important role in the pathogenesis of BLV-induced neoplasms, and that the B-lymphocyte bearing p53 mutations may be a target cell for tumor formation of enzootic bovine leukosis.
• ウシプリオンタンパクのゲノム構造およびプロモーター領域の解析

  井上 真吾, 田中 成幸, 堀内 基広, 石黒 直隆, 品川 森一

  The journal of veterinary medical science 59 3 175 - 183 社団法人日本獣医学会 1997年03月 [査読無し][通常論文]
   

  ウシ白血球由来のDNAから作製したゲノムライブラリーよりPrP遺伝子のプロモーター領域を含むクローンを分離し, 3,407 bpの塩基配列を決定した. ウシPrP遺伝子の5'側非コード領域のゲノム構造は3つのエクソンと2つのイントロンから成り, その構造はマウス, ラットやヒツジと同様だった. ウシPrP遺伝子の5'側隣接領域は, 転写開始部位から88塩基上流までで78%とG+Cに富み, 転写因子Sp1結合部位と思われる配列が3ヶ所存在したが, CCAAT-boxやTATA-boxは認められなかった. この領域はヒッジPrP遺伝子と高いホモロジー(89%)を示したが, マウス, ラット, ハムスターやヒトのPrP遺伝子とは比較的低い(約46-62%)ものだった. ウシPrP遺伝子の5'側隣接領域のプロモーター活性をCATアッセイにて測定した結果, -88から-30内に主要なプロモーター活性を認めたが, この領域がプロモーター活性を示すためにはイントロン1の存在が不可欠であることが判った. イントロン1を一部欠失させた遺伝子を各種導入したところ, +123から+891内にプロモーター活性に関与する領域が存在することが判った.
• 猫汎白血球減少症ウイルスおよびミンク腸炎ウイルスに特異的なモノクローナル抗体のエピトープの同定(短報)

  堀内 基広, 望月 雅美, 石黒 直隆, 長澤 秀行, 品川 森一

  The journal of veterinary medical science 59 2 133 - 136 社団法人日本獣医学会 1997年02月 [査読無し][通常論文]
   

  猫汎白血球減少症ウイルス中(FPLV)およびミンク腸炎ウイルス(MEV)に特異的なモノクローナル抗体(MAb)を得る目的で, MEV-網走株に対する15株のMAbを作製した. 一つのMAb P2-215はFPLVおよびMEVに特異的であったが, 残りの14のMAbはイヌパルボウイルス(CPV), FPLV, MEVすべてに反応した. CPV/MEVキメラウイルスを用いたエピトープ解析から, MAb P2-215はFPLVおよびMEVに特異的なVP2の93番目のアミノ酸リジンを含むエピトープを認識することが明らかとなった.
• Characterization of differentiation antigens expressed in bovine lymphosarcomas

  Y Sasaki, N Ishiguro, M Horiuchi, M Shinagawa, S Osame, H Furuoka, T Matsui, M Asahina, K Okada

  JOURNAL OF COMPARATIVE PATHOLOGY 116 1 13 - 20 1997年01月 [査読有り][通常論文]
   

  To characterize the cell-surface antigens expressed in tumour cells derived from bovine leukosis and to determine their cell lineages, the immunophenotypes of the tumour cells from 13 bovine lymphosarcomas were examined with 13 monoclonal antibodies (mAbs). Of 13 cattle with lymphosarcomas, four were identified clinically as having the thymic-type sporadic bovine leukosis (SBL) and one as having the skin-type; two had enzootic bovine leukosis (EEL) and six were untypable. Flow cytometric analysis revealed that the tumour cells from nine cases were of T-cell lineage (BoCD(5+) BoCD(2+) or BoCD(2-)) and two were of B-cell lineage (MHC-II+, BoCD5(+), IgM(+)); there were two bovine leukaemia virus-infected cattle (EBL). T-cell rumours appeared to originate from immature (BoCD4(-), BoCD8(-)) T cells, but there was no significant relationship between clinical type (EEL, calf-, skin- and thymic-type) and tumour-cell immunophenotype. (C) 1997 W.B. Saunders Company Limited.
• Differentiation of vaccine virus from field isolates of feline panleukopenia virus by polymerase chain reaction and restriction fragment length polymorphism analysis

  Motohiro Horiuchi, Kazuyo Yuri, Takehisa Soma, Hiromi Katae, Hideyuki Nagasawa, Morikazu Shinagawa

  Veterinary Microbiology 53 3-4 283 - 293 1996年12月 [査読有り][通常論文]
   

  In an attempt to distinguish feline panleukopenia virus (FPLV) live vaccine strains from FPLV field isolates in Japan, we compared restriction fragment length polymorphisms (RFLP) of polymerase chain reaction (PCR)-amplified fragments of live FPLV vaccine strains with those of FPLV Japanese field isolates. On the basis of nucleotide sequence differences between PLI-IV, a live vaccine strain, and FPV-483, a recent field isolate, two restriction enzymes, Dra I and Afa I, were selected for PCR-RFLP analysis of nucleotide (nt) differences at nt 3695 and 4508, respectively, Three live vaccine strains including the PLI-IV strain could be distinguished from the Japanese field isolates by their PCR-RFLP patterns by Afa I, but one live vaccine strain was indistinguishable from the Japanese isolates when Dra I and Afa I were used. The Japanese field isolates were divided into two groups by the profile of PCR-RFLP patterns generated by Dra I and Afa I, suggesting that PCR-RFLP analysis using several enzymes provides a good genetic estimate of strain differentiation, No isolate that shows a Dra I-negative/Afa I-negative pattern has emerged in Japan, indicating the possibility that the live vaccine viruses with a Dra I-negative/Afa I-negative pattern, such as the PLI-IV strain, are candidates for use as live FPLV vaccine strain in Japan where they can be genetically distinguished from field strains.
• Predominant p53 mutations in enzootic bovine leukemic cell lines

  Hiroshi Komori, Naotaka Ishiguro, Motohiro Horiuchi, Morikazu Shinagawa, Yoko Aida

  Veterinary Immunology and Immunopathology 52 53 - 63 1996年06月15日 [査読無し][通常論文]
   

  The role of the p53 tumor suppressor gene in bovine lymphosarcomas, a fragment of about 1100 bp corresponding to approximately 97% of the open reading frame of the p53 gene was first amplified from single-strand cDNA originated from calf thymus by polymerase chain reaction (PCR) and sequenced to obtain the bovine wild-type p53 gene. At the amino acid level, the homologies of the bovine p53 gene with the human, mouse, chicken and cat p53 genes were 80.9%, 72.8%, 52.7% and 82.3%, respectively. Moreover, eight bovine leukemic cell lines were studied for alterations in the p53 gene. These lines showed no significant somatic alterations in Southern blot analysis, and expressed 2.5 kb p53-specific transcripts in Northern blot analysis. In mutation analysis using the reverse transcriptase-PCR technique, we detected three missense point mutations in four of these bovine leukemic cell lines. These mutations occurred in the 'hotspots' of the p53 gene. Thus p53 mutations predominantly occur in BLV-transformed cell lines and seem to be necessary for development of enzootic bovine leukosis (EBL).
• 牛欠失c-myb遺伝子によるトランスフォーム能

  大園 隆美, 石黒 直隆, 堀内 基広, 品川 森一

  The journal of veterinary medical science 58 4 305 - 310 社団法人日本獣医学会 1996年04月 [査読無し][通常論文]
   

  2種類の牛c-myb遺伝子を得た. 1つは, 散発型牛白血病(SBL)胸腺型に由来し75kDa蛋白質(FLMyb)をコードする完全長のc-myb遺伝子であり, 他の1つはSBL子牛型に由来し, 負の制御領域に相当する85アミノ酸を内部欠損した65kDa蛋白質(DELMyb)をコードする遺伝子である. c-myb遺伝子の内部欠損とトランスフォーム能との関係を調べる目的でFLMybとDELMybをコードするレトロベクターを作製し, マウス胎児肝細胞に感染させた. DELMybの高い転写活性能にかかわらず, 半流動寒天およびメチルセルロース中でのコロニー形成能に関してDELMybとFLMybとの間には大きな差はみられなかった. このことは, c-myb遺伝子の内部欠損は転写活性能には有効であるが, 造血系細胞のトランスフォーメーションには直接関与していないことを示している.
• Improvement of PrPSc-detection in mouse spleen early at the preclinical stage of scrapie with collagenase-completed tissue homogenization and Sarkosyl-NaCl extraction of PrPSc

  KUD Grathwohl, M Horiuchi, N Ishiguro, M Shinagawa

  ARCHIVES OF VIROLOGY 141 10 1863 - 1874 1996年 [査読有り][通常論文]
   

  Scrapie in sheep has recently become again a target of control measures and eradication programs. Crucial for the effectiveness of these measures is the detection of infected sheep during the long and potentially hazardous incubation period. However, routine-diagnosis is mostly limited to clinical examinations when disease: becomes apparent, and to postmortem investigations. Through the detection of the scrapie-specific isoform of the pi-ion protein (PrPSc) by Western blot in the spleen and lymph nodes from scrapie-infected mice and sheep, we have shown previously that diagnosis during the preclinical stage is possible. We introduce here an improved method for the diagnosis of mouse scrapie shortly after infection. Through a homogenization procedure that includes a collagenase digestion step, and through extraction and salting-out of PrPSc by Sarkosyl and NaCl, respectively, we were able to detect PrPSc in spleen tissue of intraperitoneally infected mice seven days postinfection. Moreover, the new protocol makes sample-handling easier and reduces the hands-on time. We also successfully enriched PrPSc from spleen tissue through immobilized metal affinity chromatography (IMAC); however, for the diagnosis at the earliest stage of infection, extraction of PrPSc by Sarkosyl and NaCl was more effective.
• BOVINE HERPESVIRUS MENINGOENCEPHALITIS ASSOCIATION WITH INFECTIONS BOVINE-RHINOTRACHEITIS (IBR) VACCINE

  H FURUOKA, N IZUMIDA, M HORIUCHI, S OSAME, T MATSUI

  ACTA NEUROPATHOLOGICA 90 6 565 - 571 1995年12月 [査読有り][通常論文]
   

  During 1992, on a farm in the Tokachi district of Hokkaido, Japan, approximately 20 Holstein-Friesian calves showed neuroparalysis and died within 7-10 days after routine vaccination. Six male calves, aged about 1.5 months, were submitted to our laboratory for pathological examination and diagnosed as acute or subacute necrotizing meningoencephalitis due to bovine herpes virus (BHV) infection. The main necropsy findings included a few hemorrhages or clots, and malacic lesions localized in the cortical to subcortical area of the cerebrum. Histopathological brain lesions were characterized by laminar or focal necrosis of neurons, accompanying macrophages, polymorphonuclear cell infiltration, severe astrogliosis, and perivascular cuffing in all six calves. Nuclear basophilic inclusion bodies, which showed positive reaction with immunocytochemical staining of BHV antigen, were observed in the necrotic neurons, astroglia and oligodendroglia in five affected calves. BHV antigens were also seen in the cell bodies and cell processes of the necrotic neurons, which was indicative of cell-to-cell propagation of infection. There was a general tendency for more severe lesions to be located at the cortex to subcortex of the cerebrum. Milder lesions were observed in the cerebellum and brain stem. These findings suggest that the infectious route to the cerebrum in the present cases was through the olfactory bulbs and/or along the meninges beginning from the ethmoid bone, rather than through the trigeminal ganglia route as had been emphasized in studies dealing with experimental infection.
• A CELLULAR-FORM OF PRION PROTEIN (PRPC) EXISTS IN MANY NONNEURONAL TISSUES OF SHEEP

  M HORIUCHI, N YAMAZAKI, T IKEDA, N ISHIGURO, M SHINAGAWA

  JOURNAL OF GENERAL VIROLOGY 76 2583 - 2587 1995年10月 [査読有り][通常論文]
   

  A cellular form of the prion protein (PrPC) is thought to be a substrate for an abnormal isoform of the prion protein (PrPSc) in scrapie. PrPC is abundant in tissues of the central nervous system, but little is known about the distribution of PrPC in non-neuronal tissues of sheep, the natural host of scrapie. This study investigated the tissue distribution of PrPC in sheep. Although PrPC was abundant in neuronal tissues, it was also detected in non-neuronal tissues such as spleen, lymph node, lung, heart, kidney, skeletal muscle, uterus, adrenal gland, parotid gland, intestine, proventriculus, abomasum and mammary gland. Neither PrPC nor PrP mRNA was detected in the liver. The tissue distribution of PrPC appears to be inconsistent with the tissues which possess scrapie infectivity, suggesting that factor(s) specific to certain cell types may be required to support multiplication of the scrapie agent.
• AMINO-ACID POLYMORPHISMS OF PRP WITH REFERENCE TO ONSET OF SCRAPIE IN SUFFOLK AND CORRIEDALE SHEEP IN JAPAN

  T IKEDA, M HORIUCHI, N ISHIGURO, Y MURAMATSU, GD KAIUWE, M SHINAGAWA

  JOURNAL OF GENERAL VIROLOGY 76 2577 - 2581 1995年10月 [査読有り][通常論文]
   

  We investigated the relationships between amino acid polymorphisms of the prion protein (PrP), restriction fragment length polymorphisms (RFLP) of the PrP gene and the incidence of natural scrapie in Japan. Six variant alleles of the PrP gene were found in healthy sheep. Based on the substitutions at codons 112, 136, 154 and 171, these allelic variants were designated PrPMARQ, PrPTARQ, Prp(MVRQ), PrPMAHQ, PrPMARR and prp(MARH). Each RFLP haplotype (e1h1, e1h2 or e3h1) consisted of multiple alleles including PrPMARQ. Three of these variant alleles were found in scrapie-affected Suffolk sheep. PrPMARQ was associated with high disease incidence, PrPTARQ and PrPMARR were associated with low disease incidence. We found that one scrapie-affected Suffolk was homozygous for PrPMARR and four PrPSc-positive Suffolks carried PrPMVRQ. Both of two scrapie-affected Corriedales and two out of three scrapie-affected cross-breeds between Suffolk and Corriedale carried PrPMARH, suggesting that this allele associates with high incidence of scrapie in Corriedale and its cross-breeds.
• 脳炎症状を呈した牛の脳から分離された牛ヘルペスウイルス1型の制限酵素解析 : ワクチン株との比較

  堀内 基広, 山崎 典子, 古岡 秀文, 松井 高峯, 中川 迪夫, 石黒 直隆, 品川 森一

  The journal of veterinary medical science 57 3 577 - 580 社団法人日本獣医学会 1995年06月 [査読無し][通常論文]
   

  北海道十勝地方の某牧場で肥育仔牛に髄膜脳炎が散発した. 仔牛は牛伝染性鼻気管炎ウイルス生ワクチンをはじめ5種類の生ワクチンを鼻腔内噴霧されていた. 脳炎症状を呈した2頭の牛の脳から11クローンの牛ヘルペスウイルス1型を分離した. 制限酵素Pst I および Hind III によるウイルスゲノムの切断像を758-43ワクチン株のものと比較したが顕著な差は認められなかった. 従って分離株はワクチン株由来であると考えられた.
• A SPONTANEOUS INTERNAL DELETION OF THE C-MYB PROTOONCOGENE ENHANCES TRANSCRIPTIONAL ACTIVATION IN BOVINE T-LYMPHOMA-CELLS

  N ISHIGURO, T OHZONO, T SHINAGAWA, M HORIUCHI, M SHINAGAWA

  JOURNAL OF BIOLOGICAL CHEMISTRY 269 43 26822 - 26829 1994年10月 [査読無し][通常論文]
   

  Three double negative (BoCD4(-), BoCD8(-)) bovine T cell lines, BTL-PC3, BLT-2, and Pr2181, which have been established from bovine lymphosarcomas, were examined for expression and molecular function of bovine c-myb genes. BTL-PC3 expressed 4.0- and 3.6-kilobase c-myb transcripts, and BLT2 and Pr2181 expressed a 3.8-kilobase c-myb message. The c-Myb protein (75 kDa) was detected in Pr2181 but not as a clear band in BLT2, while BTL-PC3 exhibited a 65-kDa c-Myb band in immunoprecipitation tests with anti-Myb antiserum. Nucleotide sequences for c-myb cDNA clones from BTL-PC3 and BLT2 indicated that the predicted bovine wild-type c-Myb from BLT2 consists of 640 amino acids whereas that from BTL-PC3 consists of 555 amino acids lacking 85 internal amino acids. This deleted DNA region (255 base pairs consisting of 85 amino acids) corresponds to the human genomic exon 9 encoding a negative regulatory domain in the c-myb gene. Upon cotransfections with reporter plasmids containing myb binding sites, the internally deleted c-Myb exhibited a 3-fold higher transcriptional activity than the wild-type c-Myb in chloramphenicol acetyltransferase assays. These results indicate that internal DNA deletion in the c-myb gene is directly involved in the enhancement of transcriptional activation in bovine T lymphoma cells.
• 牛散発型白血病由来腫瘍細胞に対して作製したモノクローナル抗体の性状

  品川 敏恵, 石黒 直隆, 堀内 基広, 品川 森一, 松井 高峯

  日本獣医学雑誌 56 5 827 - 833 社団法人日本獣医学会 1994年10月 [査読無し][通常論文]
   

  3種類の散発型牛白血病株, BLT2(胸腺型), BTL-PC3(仔牛型)およびBLS1(皮膚型)をマウスに免疫して作製したモノクローナル抗体(mAb) 38個を用い散発型および地方病型牛白血病に発現している抗原の分布について検討した. 多くのmAbは, 一部の牛および他の動物種由来の単核細胞を除き検索した正常牛リンパ球系細胞に対して, 低い反応性しか示さなかった. mAbのほとんどは, 自然発症例の牛散発型白血病由来末梢単核細胞よりは株化したT細胞株BTL-PC3およびB細胞株BL312とKU-1に強く反応した. 38個のmAbの内27個のmAbで認識する抗原の分子量がウエスタンブロット解析により決定された. mAbC419は, 正常リンパ系細胞には反応せず, 腫瘍化した細胞に特異的に反応することから, 牛散発型白血病細胞の腫瘍関連抗原を認識していることが示唆された.
• モノクローナル抗体S37によるウシTリンパ腫細胞のアポトーシス(短報)

  品川 敏恵, 石黒 直隆, 堀内 基広, 品川 森一

  日本獣医学雑誌 56 5 957 - 959 社団法人日本獣医学会 1994年10月 [査読無し][通常論文]
   

  散発型牛白血病細胞株に対して作製したモノクローナル抗体(mAb)38個の, ウシTリンパ腫細胞(BTL-27)の増殖に対する影響について検討した. その結果, mAB S37が著しい増殖抑制効果を示した. S37の存在下で培養した細胞は, 核の濃縮及びDNAの断片化が観察された. これにより, mAb S37はBTL-27に対しアポトーシスによる細胞死を引き起こすことが明らかとなった.
• スクレイピーに関する調査 : PrP^の検出及びPrP遺伝子型の分析

  小野寺 朝子, 池田 徹也, 堀内 基広, 石黒 直隆, 小沼 操, 平野 紀夫, 見上 彪, 本多 英一, 平井 克哉, 甲斐 一成, 柚木 弘之, 村松 康和, 品川 森一

  日本獣医学雑誌 56 4 627 - 632 社団法人日本獣医学会 1994年08月 [査読無し][通常論文]
   

  現在わが国におけるスクレイピーの汚染状況を把握するために, 1988年から1993年までに北海道, 東北, 関東及び中部地方から集めた主にサフォーク種の羊197頭の脳, 脾臓及びリンパ節からPrP^<sc>の検出を行った. そのうち北海道の16頭及び他地域の2頭からPrP^<sc>が検出されたが, 後者の2頭も北海道から導入された個体であった. 一方, これらの18頭のスクレイピー感染羊におけるPrP遺伝子を含む染色体DNAの制限酵素切断断片の多様性(RFLP)の各型の出現頻度を, 128頭の健康な羊における出現頻度と比較して, RFLPの特異的な型とスクレイピーの自然感染との間に相関があることを確認した. すなわちスクレイピー感染羊におけるRFLP型I型の頻度は健康な羊におけるI型の頻度や, スクレイピー感染羊における他の型の頻度に比べて有意に高くなっていた. 一方II型とVI型の頻度はスクレイピー感染羊に比べて健康な羊で高くなっていた. ゆえに日本のサフォーク種において, I型の羊はスクレイピーに対して感受性であり, II型とVI型の羊は抵抗性であることが示唆された. さらにスクレイピー感受性に関わる遺伝学的な背景を明らかにするために, 我が国における羊PrP遺伝子のRFLP型の分布を161頭の羊で調べた. 各々の地方でRFLP型の分布に若干差が認められた.
• MAPPING OF DETERMINANTS OF THE HOST-RANGE FOR CANINE CELLS IN THE GENOME OF CANINE PARVOVIRUS USING CANINE PARVOVIRUS MINK ENTERITIS VIRUS CHIMERIC VIRUSES

  M HORIUCHI, H GOTO, N ISHIGURO, M SHINAGAWA

  JOURNAL OF GENERAL VIROLOGY 75 1319 - 1328 1994年06月 [査読有り][通常論文]
   

  Feline panleukopenia virus (FPLV), mink enteritis virus (MEV) and canine parvovirus (CPV) are more than 98% similar in DNA and predicted amino acid sequences, but they show different host-cell specificities; CPV is able to replicate in canine cells in culture, whereas FPLV and MEV cannot or replicate only to a low titre. To map the genomic region responsible for the host range of CPV in vitro, CPV/MEV chimeric viruses were generated by transfecting infectious CPV/MEV chimeric plasmids into a cultured feline kidney cell line, and their host cell ranges were analysed. The 60 to 91 map units (m.u.) region of the CPV genome, which contains a part of the capsid protein (VP) gene encoding from amino acid 91 (in the VP2 sequence) to the carboxy terminus of VP protein, was required to impart the ability to replicate in canine cells to MEV, although the chimeric virus containing the 60 to 91 m.u. region of the CPV genome in the MEV background did not replicate in canine cells as efficiently as did CPV derived from the infectious plasmid of CPV. Not only the VP gene, but also a part of the NS gene of CPV were considered to participate in the full expression of the ability to replicate in canine cells. Within the 60 to 91 m.u. region, five of nine amino acid changes between MEV-Abashiri and CPV-Y1 were thought to be phylogenetically CPV-common; however, a recombinant virus containing all five amino acid changes of CPV in the MEV background replicated minimally in canine cells.
• THE SIGNIFICANCE OF PRPSC DETECTION FOR THE DIAGNOSIS OF INSIDIOUS SCRAPIE

  Y MURAMATSU, A ONODERA, M HORIUCHI, N ISHIGURO, M SHINAGAWA

  SLOW INFECTIONS OF THE CENTRAL NERVOUS SYSTEM 724 347 - 349 1994年 [査読無し][通常論文]
  
• DETECTION OF PRP(SC) IN SHEEP AT THE PRECLINICAL STAGE OF SCRAPIE AND ITS SIGNIFICANCE FOR DIAGNOSIS OF INSIDIOUS INFECTION

  Y MURAMATSU, A ONODERA, M HORIUCHI, N ISHIGURO, M SHINAGAWA

  ARCHIVES OF VIROLOGY 134 3-4 427 - 432 1994年 [査読無し][通常論文]
   

  A field sheep insidiously infected with natural scrapie was diagnosed at the preclinical stage through detection of the core fragment of Prp(Sc) (PrPcore) in peripheral lymph nodes by the biopsy method. Three out of 32 euthanized healthy sheep were found positive for PrPcore in the spleen and lymph nodes. Mice that were inoculated with spleen homogenate of one of these sheep showed clinical signs of scrapie and were positive for PrPcore in their brain samples. These results suggest that the detection of PrPcore is significant for diagnosis in the preclinical phase of scrapie.
• 潜伏期の短い家族性スクレイピーの症例(短報)

  品川 森一, 村松 康和, 小野寺 朝子, 松井 高峯, 堀内 基広, 石黒 直隆, 中川 迪夫

  日本獣医学雑誌 55 4 665 - 667 社団法人日本獣医学会 1993年08月 [査読無し][通常論文]
   

  24ヶ月齢で出産した2頭のコリデールヒツジが29及び30ヶ月齢で相次いで運動失調を示し起立不能に陥った. 1例目は原因不明のまま死亡し, 2例目は病理組織学的及び免疫化学的にスクレイピーと診断された. これらの子ヒツジ4頭の内, 3頭が10, 14, 及び19ヶ月齢でスクレイピーを発症した. 親子共に脱毛はなく, そう痒症状を欠いていた. 異常に短い潜伏期及びそう痒症状の欠落について考察する.
• CONSTRUCTION OF AN INFECTIOUS DNA CLONE OF THE Y-1 STRAIN OF CANINE PARVOVIRUS AND CHARACTERIZATION OF THE VIRUS DERIVED FROM THE CLONE

  M HORIUCHI, M SHINAGAWA

  ARCHIVES OF VIROLOGY 130 3-4 227 - 236 1993年 [査読有り][通常論文]
   

  We have cloned genome fragments of canine parvovirus strain Y 1 from replicative-form DNA and double-stranded DNA synthesized from virion DNA in vitro, and constructed a recombinant plasmid containing a full-length Y 1 genome (pCPVY 1). When this recombinant plasmid was transfected into cell cultures, an infectious virus could be recovered. To characterize this pCPVY 1-derived virus, its biological properties were compared with those of the parental strain. No difference was observed between them in antigen expression, viral DNA replication, hemagglutination ability, and virus multiplication, indicating that the virus derived from the infectious plasmid inherited the biological properties of the authentic Y 1 strain. Therefore, this recombinant plasmid appears to be useful for reverse genetics of canine parvovirus.
• Comparative sequence analysis and expression of bovine PrP gene in mouse L-929 cells

  Jun Yoshimoto, Toshiyuki Iinuma, Naotaka Ishiguro, Motohiro Horiuchi, Masakatu Imamura, Morikazu Shinagawa

  Virus Genes 6 4 343 - 356 1992年11月 [査読有り][通常論文]
   

  A cDNA clone encoding bovine scrapie-associated fibril protein, PrP, from a bovine brain cDNA library and six 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 the 165th amino acid from the N-terminus. Bovine PrP cDNA introduced into mouse L-929 cells were stably expressed. The expression level of recombinant bovine PrP in the cells judged by immunofluorescence was higher than that of authentic mouse PrP. The recombinant PrP comigrated with authentic bovine PrP in SDS-polyacrylamide gel electrophoresis, suggesting that the recombinant product was fully glycosylated in L-929 cells. Distinct bundles of the intermediate filaments were frequently seen at the perinuclear region of the cells. © 1992 Kluwer Academic Publishers.
• CHARACTERIZATION OF THE STAGE(S) IN THE VIRUS-REPLICATION CYCLE AT WHICH THE HOST-CELL SPECIFICITY OF THE FELINE PARVOVIRUS SUBGROUP IS REGULATED IN CANINE CELLS

  M HORIUCHI, N ISHIGURO, H GOTO, M SHINAGAWA

  VIROLOGY 189 2 600 - 608 1992年08月 [査読有り][通常論文]
  
• A SPECIFIC RFLP TYPE ASSOCIATED WITH THE OCCURRENCE OF SHEEP SCRAPIE IN JAPAN

  Y MURAMATSU, K TANAKA, M HORIUCHI, N ISHIGURO, M SHINAGAWA, T MATSUI, T ONODERA

  ARCHIVES OF VIROLOGY 127 1-4 1 - 9 1992年 [査読無し][通常論文]
   

  We have investigated restriction fragment length polymorphism (RFLP) on the PrP gene and the frequencies of RFLP patterns in 35 healthy Suffolk sheep randomly collected. According to the combinations of PrP encoding DNA fragments generated by restriction enzymes Eco RI and Hind III, the RFLP patterns were classified into six types and designated as types I to VI. The frequencies of these types were as follows: I, 8.6%; II, 11.4%; III, 17.6%; IV, 11.4%; V, 28.6%; and VI, 22.9%. In 10 sheep diagnosed as having natural scrapie, RFLP types, I, III, IV, and V were determined. To examine the correlation between the RFLP type and the occurrence of scrapie, the frequencies of RFLP types in sheep infected with natural scrapie were compared with those in healthy sheep. It was found that the frequency of type I in the sheep with natural scrapie was 70%, about eight times higher than that in randomly collected healthy sheep. In the 13 experimentally infected sheep that had been used for other purposes, however, no relationship between the RFLP type and onset of scrapie was found.
• マレック病ウイルス血清型2及びCVI-988株感染細胞に存在するウイルス特異抗原の同定

  堀内 基広, 児玉 洋, 見上 彪

  日本獣医学雑誌 52 5 985 - 994 社団法人日本獣医学会 1990年10月 [査読無し][通常論文]
   

  マレック病ウイルスCVI-988株 (血清型1) またはHPRS-24株 (血清型2) 感染細胞に対する単クローン性抗体を産生する24クローンの抗体産生ハイブリドーマを作製し, 単クローン性抗体の性状を間接蛍光抗体法, ウイルス中和試験, 及び免疫沈澱法により解析した. 免疫沈澱法により検出される抗原の分子量に基づいて単クローン性抗体を7群に分類し, 得られた抗体を用いてウイルス特異抗原の解析を行った. 1群及び2群に分類した抗体によって検出される抗原は, これまでに報告がないものであった. すなわち, 1群に分類した抗体により検出される抗原は, CVI-988株及び血清型2ウイルス感染細胞に存在する分子量29/34kdの糖蛋白質であった. 抗原出現の経時的変化の解析から, 本抗原は後期膜抗原に関連していることが示唆された. また, 2群に分類した抗体により検出される抗原は, 血清型2ウイルス感染細胞に存在する分子量37kd, 33kd (SB-1株では34kd) 及び31kdのポリペプチドであった. 本抗原はウイルス感染後早期から感染細胞に出現することから, 初期抗原に関連していることが示唆された. なお, 他の5群に分類した抗体は, 以前に報告があるウイルス特異抗原と類似したものと反応する抗体, 或いは抗体により検出される抗原が十分に解析されなかったものである.
• CHARACTERIZATION OF REPLICATIVE FORM DNA OF THE AUTONOMOUS PARVOVIRUS MINK ENTERITIS VIRUS

  M SHINAGAWA, Y NOMURA, T KARIATUMARI, N ISHIGURO, M HORIUCHI, H GOTO

  MICROBIOLOGY AND IMMUNOLOGY 33 9 721 - 732 1989年 [査読有り][通常論文]
  

MISC

• プリオン病治療実験モデル系確立の試み-免疫療法と細胞治療の可能性-

  堀内基広 臨床評価 44 (4) 2017年
• 北大農場で発生したリステリア症罹患羊の飼育環境からのリステリア属菌の分離とゲノム解析

  長谷部理絵, 青島圭佑, 棟居佳子, 三谷朋弘, 平井佑治, 山崎剛士, 木村享史, 堀内基広 日本獣医学会学術集会講演要旨集 159th 2016年
• MALDI-TOF質量分析システムによる犬口腔内からのBergeyella zoohelcumの検出

  原谷那美, 内田玲麻, 郡山尚紀, 鈴木章夫, 堀内基広, 村松康和 北海道獣医師会雑誌 59 (8) 2015年
• イヌ口腔内からのBergeyella zoohelcumの分離

  村松康和, 原谷那美, 内田玲麻, 郡山尚紀, 鈴木章夫, 堀内基広 日本獣医学会学術集会講演要旨集 158th 2015年
• プリオンの感染と細胞内動態

  山崎剛士, 堀内基広 Dementia Japan 27 (2) 215 -224 2013年 [査読無し][通常論文]
  
• 抗原への二価結合により付与されるmAb132のPrP^Sc特異性

  鈴木章夫, 山崎剛士, 長谷部理絵, 堀内基広 日本ウイルス学会学術集会プログラム・抄録集 60th 2012年
• 異常型プリオンタンパク質の細胞内増殖

  堀内基広, 山崎剛士 Dementia Japan 25 (3) 2011年
• 人獣共通感染症の克服に向けて プリオン病

  堀内基広 最新医学 66 (12) 2011年
• クラスリン依存性エンドサイトーシス阻害剤処理によるPrP^Scの細胞内局在の変化

  山崎剛士, 鈴木章夫, 長谷部理絵, 堀内基広 日本ウイルス学会学術集会プログラム・抄録集 58th 2010年
• 牛海綿状脳症

  中満 智史, 堀内 基広 臨床とウイルス 35 (4) 301 -310 2007年10月10日 [査読無し][通常論文]
  
• ヒト以外のプリオン病-特にウシ海綿状脳症について-

  堀内基広, 中満智史 日本臨床 65 (8) 1513 -1520 2007年 [査読無し][通常論文]
  
• BSE プリオン

  堀内基広 臨床とウイルス 35 (4) 293 -300 2007年 [査読無し][通常論文]
  
• 血漿分画製剤のろ過工程における異常プリオン除去に関する検討

  前野英毅, 川畑嘉之, 室塚剛志, 脇坂明美, 谷藤正明, 堀内基広, 伴野丞計 血液事業 29 (2) 2006年
• BSE診断法の開発と現状 (特集 プリオン病)

  堀内 基広 Virus report 2 (1) 20 -27 2005年06月 [査読無し][通常論文]
  
• ニワトリモノクローナル抗体を用いた異常プリオン蛋白質の検出

  青笹正義, 宮本和慶, 西道教尚, 木村聡太, 品川森一, 佐多徹太郎, 堀内基広, 横山隆, 堀内浩幸, 古沢修一, 松田治男 臨床病理 53 2005年
• 動物由来感染症としてのプリオン病

  堀内基広 日本臨床 63 (12) 2005年
• 感染症病原体と細胞膜との関わり 異常型プリオン蛋白質の生合成と伝達

  堀内基広 膜 30 (2) 78 -83 2005年 [査読無し][通常論文]
  
• 人獣共通感染症としてのプリオン病

  堀内基広 ウイルス 55 (1) 45 -53 2005年 [査読無し][通常論文]
  
• プリオン蛋白質と結合するペプチド性リガンドの探索

  大林浩二, 堀内基広, 石黒直隆, 品川森一 日本獣医学会学術集会講演要旨集 135th 2003年
• 抗PrPモノクローナル抗体パネルによるPrPの構造解析

  金チャンラン, 堀内基広, 石黒直隆, 品川森一 日本獣医学会学術集会講演要旨集 135th 2003年
• 尿崩症を誘発するマウス馴化スクレイピー株の分離と解析

  田村勇耕, 堀内基広, 石黒直隆, 古岡秀文, 品川森一 日本獣医学会学術集会講演要旨集 135th 2003年
• 抗PrP抗体を用いたPrP^Sc検出法の高度化

  堀内基広, 工藤聡子, 品川森一, 梅谷淳 プリオン病及び遅発性ウイルス感染に関する調査研究 平成14年度研究報告書 2003年
• BSEスクリーニング用ELISA(OFR ELISA)の開発とその性能評価

  堀内基広, 梅谷淳, 工藤聡子, 石黒直隆, 横山隆, 品川森一 日本獣医学会学術集会講演要旨集 135th 2003年
• 動物プリオン病の免疫組織化学的染色における抗原賦活化法の検討

  薮添敦史, 古岡秀文, 堀内基広, 田川裕一, 横山隆, 品川森一, 佐多徹太郎 日本獣医学会学術集会講演要旨集 135th 2003年
• 抗体療法-基礎から臨床へ- 抗PrP抗体によるプリオン増殖抑制とプリオン病治療の可能性

  堀内基広 最新医学 58 (12) 2003年
• プリオン病-最近の知見 牛海綿状脳症問題に関する最近の動向

  堀内基広 老年精神医学雑誌 14 (12) 1488 -1494 2003年 [査読無し][通常論文]
  
• プリオン病とその診断 プリオンの検出技術

  堀内基広 臨床検査 46 (12) 2002年
• 感染症 プリオン病 病態の解明を目指して プリオン蛋白質の構造と遺伝子多型

  堀内基広 Mebio 19 (12) 2002年
• プリオン蛋白質とプリオン病

  堀内基広 栄養生理研究会報 46 (1) 2002年
• ガストロピロール原末のスクレイピープリオン汚染の検定

  品川森一, 堀内基広, 石黒直隆 医薬品研究 33 (7) 2002年
• 牛海綿状脳症に関する検査概要と今後の対応

  池田 徹也, 堀内 基広, 古岡 秀文 食品衛生研究 52 (1) 33 -42 2002年01月 [査読無し][通常論文]
  
• プリオンの免疫学的検出法

  品川森一, 堀内基広, 松井高峯 生活衛生 45 (5) 2001年
• プリオン 1. 動物のプリオン病

  堀内基広 ウイルス 51 (2) 145 -150 2001年 [査読無し][通常論文]
  
• Prion protein interconversions and the transmissible spongiform encephalopathies

  Motohiro Horiuchi, Byron Caughey Structure 7 (10) R231 -R240 1999年10月15日 [査読無し][通常論文]
   

  Autocatalytic changes in the conformation and aggregation state of prion protein appear to be fundamental to transmissible spongiform encephalopathies or prion diseases. Here we review the considerable progress that has been made in describing the normal properties of prion protein and the changes that occur during these devastating neurodegenerative diseases.
• スクレイピープリオン蛋白の羊体内分布 ウェスタンブロット法によるPrPSc検出

  高橋未緒, 堀内基広, 石黒直隆, 松井高峯, 古岡秀文, 品川森一 日本獣医学会学術集会講演要旨集 126th 1998年
• スクレイピープリオン蛋白の羊体内分布 抗PrP抗体による免疫組織化学的検索

  鈴木智, 高橋広志, 松井高峯, 堀内基広, 石黒直隆, 品川森一, 古林与志安, 古岡秀文 日本獣医学会学術集会講演要旨集 126th 1998年
• プリオン病について

  堀内基広 食肉の科学 38 (2) 205 -212 1997年 [査読無し][通常論文]
  
• プリオン蛋白質-プリオン病・狂牛病の感染機構の謎に迫る プリオン蛋白の代謝・分布

  品川森一, 堀内基広 実験医学 14 (18) 1996年
• プリオン病の分子遺伝学と生物物理学

  Prusiner Stanley B, 堀内 基広, 品川 森一 蛋白質核酸酵素 40 (16) p2383 -2407 1995年12月 [査読無し][通常論文]
  

講演・口頭発表等

• プリオン感染初代培養神経細胞における神経細胞自律的変化の解析

  田中美咲, 山崎剛士, 長谷部理絵, 鈴木章夫, 堀内基広

  日本獣医学会学術集会講演要旨集 2020年
• Real-time quaking-induced conversion(RT-QuIC)によるCWDおよび非定型BSE検出における組換えシカプリオンタンパク質の有用性

  鈴木章夫, 澤田和平, 山崎剛士, 堀内基広

  日本獣医学会学術集会講演要旨集 2020年
• 異常型プリオンタンパク質生成がニューロンに及ぼす直接的神経変性効果について

  田中美咲, 山崎剛士, 鈴木章夫, 長谷部理絵, 堀内基広

  日本分子生物学会年会プログラム・要旨集(Web) 2019年
• 神経細胞特異的遺伝子発現解析によるプリオン病の神経変性機構の解析

  島倉綾乃, 山崎剛士, 鈴木章夫, 長谷部理絵, 堀内基広

  日本獣医学会学術集会講演要旨集 2018年
• 定型BSEおよび非定型BSEプリオンに感染したマウスの脳のトランスクリプトーム解析

  山崎剛士, 岩丸祥史, 鈴木章夫, 長谷部理絵, 堀内基広

  日本獣医学会学術集会講演要旨集 2018年
• 次世代シーケンサーを用いた,プリオン感染マウス脳由来ミクログリアおよびアストロサイトの活性化状態の解析

  黒田弥乃梨, 山崎剛士, 鈴木章夫, 長谷部理絵, 堀内基広

  日本獣医学会学術集会講演要旨集 2016年
• 異常型プリオンタンパク質(PrP^Sc)特異的染色法によるプリオン持続感染株化細胞とプリオン感染初代培養神経細胞におけるPrP^Scの解析

  田中美咲, 舛甚賢太郎, 舛甚賢太郎, 山崎剛士, 長谷部理絵, 鈴木章夫, 堀内基広

  日本獣医学会学術集会講演要旨集 2016年
• BSEおよびL型BSEの異種間交差伝達実験によるプリオン株の病理学的特徴への種差の影響に関する検討

  榎芙蓉, 木元美樹, 室井喜景, 堀内基広, 古岡秀文

  日本獣医学会学術集会講演要旨集 2015年
• 補体反応はプリオン感染マウス大脳皮質由来初代培養神経細胞の細胞膜透過性を亢進し,異常型プリオンタンパク質の蓄積量を変化させる

  長谷部理絵, 鈴木章夫, 山崎剛士, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2014年
• プリオン病の病態進行に伴うアストロサイト活性化状態の解析

  黒田弥乃梨, 山崎剛士, 鈴木章夫, 長谷部理絵, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2014年
• プリオン感染マウス脳由来アストロサイトの活性化状態の解析

  黒田弥乃梨, 山崎剛士, 鈴木章夫, 長谷部理絵, 堀内基広

  日本獣医学会学術集会講演要旨集 2014年
• 大脳皮質由来初代培養神経細胞におけるプリオン感染の解析

  藤原愛, 山崎剛士, 鈴木章夫, 長谷部理絵, 堀内基広

  日本獣医学会学術集会講演要旨集 2014年
• 北米エルクに由来するCWDプリオンの伝達試験に関する研究

  綿貫亨, 室井喜景, 横山隆, 堀内基広, 古岡秀文

  日本獣医学会学術集会講演要旨集 2014年
• フローサイトメトリーによるプリオン感染細胞の検出

  山崎剛士, 鈴木章夫, 長谷部理絵, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2014年
• プリオンの増殖に関わる細胞内小器官の同定-プリオン感染マウス脳組織の免疫組織学的解析から-

  堀内基広, 斉藤真央, 長谷部理絵, 山崎剛士

  日本ウイルス学会学術集会プログラム・抄録集 2014年
• ウシパイエル板M細胞における解糖系酵素アルドラーゼAを介した異常型プリオン蛋白質取り込み機構

  長澤裕哉, 盛田彰太郎, 日高湧介, 渡邉一史, 渡邊康一, 大和田修一, 北澤春樹, 今村守一, 横山隆, 堀内基広, 坂口末廣, 麻生久

  日本畜産学会大会講演要旨 2014年
• 補体反応はプリオン感染神経細胞の膜透過性を亢進させる

  長谷部理絵, CAUGHEY Byron, 堀内基広

  日本獣医学会学術集会講演要旨集 2013年
• プリオン感染マウスにおけるミクログリア活性化状態の解析

  堀内基広, 蕪木洋之, 長谷部理絵, 山崎剛士

  日本ウイルス学会学術集会プログラム・抄録集 2013年
• 大脳皮質由来初代神経培養系におけるプリオン感染の解析

  藤原愛, 長谷部理絵, 山崎剛士, 鈴木章夫, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2013年
• マウス腸管M細胞における解糖系酵素アルドラーゼAを介した異常型プリオン蛋白質取り込み機構

  長澤裕哉, 盛田彰太郎, 日高湧介, 渡邉一史, 大和田修一, 北澤春樹, 今村守一, 横山隆, 堀内基広, 坂口末廣, 西田教行, 渡邊康一, 野地智法, 麻生久

  日本獣医学会学術集会講演要旨集 2013年
• プリオン感染初期における異常型プリオンタンパク質の新規産生と細胞内動態の解析

  山崎剛士, 鈴木章夫, 長谷部理絵, 堀内基広

  日本獣医学会学術集会講演要旨集 2013年
• プリオンタンパク質凝集体の電子スピン共鳴-スピンラベル法による解析

  稲波修, 山盛徹, 安井博宣, 堀内基広, 平岡和佳子, 古川貢, 中村敏和

  日本獣医学会学術集会講演要旨集 2013年
• BSE感染モルモットにおける接種ルートによるプリオン分布の相違

  中西勇貴, 西村麻紀, 堀内基広, 古岡秀文

  日本獣医学会学術集会講演要旨集 2013年
• Brucella abortusは腸管パイエル板からの侵入にM細胞上のプリオン蛋白質(PrP^c)を利用する

  鈴木道雄, 中藤学, 中藤学, 渡会雅久, 木村昌伸, 堀内基弘, 長谷耕二, 長谷耕二, 飛梅実, 阿戸学, 森川茂, 山田章雄, 山田章雄, 大野博司, 今岡浩一

  日本獣医学会学術集会講演要旨集 2013年
• BSEおよびScrapie由来プリオン経口感染マウスにおける病理学的研究

  藤原理央, 中西勇貴, 堀内基広, 古岡秀文

  日本獣医学会学術集会講演要旨集 2012年
• プリオン感染CD14ノックアウトマウスにおけるミクログリアの活性化とPrP^Sc沈着の解析

  長谷部理絵, 蕪木洋之, 鈴木章夫, 山崎剛士, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2012年
• BSE伝達モルモット小脳におけるプリオン沈着と神経伝達物質に関する病理学的研究

  坂口翔一, 室井喜景, 堀内基広, 古岡秀文

  日本獣医学会学術集会講演要旨集 2011年
• プリオン感染では株により異なる補体因子が反応する

  長谷部理絵, 堀内基広, BYRON Caughey

  日本ウイルス学会学術集会プログラム・抄録集 2010年
• BSE感受性に関わる東南アジアのウシプリオン遺伝子多型

  内田玲麻, HERIYANTO Agus, THONGCHAI Chalermchaikit, HANH Tran Thi, 堀内基広, 石原加奈子, 田村豊

  日本獣医学会学術集会講演要旨集 2010年
• BSE伝達モルモット小脳におけるプリオンの経時的な動態と神経伝達物質への影響

  坂口翔一, 堀内基広, 古岡秀文

  日本獣医学会学術集会講演要旨集 2010年
• プリオン蛋白質の可溶性オリゴマーの蛍光相関分光法による解析

  坂田啓司, 堀内基広, 金城政孝

  日本蛋白質科学会年会プログラム・要旨集 2009年
• 抗PrP抗体の末梢投与によるプリオン病の治療効果

  大澤夏生, 宋昌絃, 鈴木章夫, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2009年
• マウスプリオンタンパク質ヒスチジン186周辺の銅イオン結合部位に関する構造解析

  稲波修, 渡辺康子, 五十嵐学, 伊藤公人, 堀内基広, 桑原幹典, 平岡和佳子

  日本獣医学会学術集会講演要旨集 2009年
• Cd14分子のプリオン病病態への関与

  酒井景子, 長谷部理絵, 宋昌紘, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2009年
• 骨髄由来間葉系幹細胞のプリオン感染脳病変への走化に関与する因子の解析

  宋昌鉉, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2009年
• マウス神経芽腫細胞Neuro2aにおけるプリオンの細胞間伝播にはエクソソーム以外の因子が関与する

  堀内基広, 瓜生匡秀, 山崎剛士, 中満智史, 長谷部理絵

  日本獣医学会学術集会講演要旨集 2008年
• PrP^Scのaa81-aa137の領域はプリオンの感染性に必須である

  堀内基広, 長谷部理絵

  日本ウイルス学会学術集会プログラム・抄録集 2008年
• 間接蛍光抗体によるプリオン持続感染細胞に存在するPrP^Scの検出

  山崎剛士, 瓜生匡秀, 中満智史, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2008年
• プリオン感染マウス脳における骨髄由来間葉系幹細胞の動態

  宋昌鉉, 長谷部理絵, 堀内基宏

  日本ウイルス学会学術集会プログラム・抄録集 2008年
• プリオン持続感染マウスNeuro2aサブクローンにおけるPrP^Scの量的変動について

  中満智史, 黒川彩, 瓜生匡秀, 堀内基広

  日本獣医学会学術集会講演要旨集 2007年
• 抗PrP抗体の脳内投与によるプリオン病の治療効果

  宋昌鉉, 古岡秀文, 鈴木章夫, 長谷部理絵, 前田秋彦, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2007年
• 日本の種雄牛におけるBSE感受性に関するプリオン遺伝子多型の出現頻度

  村松康和, 高橋健一, 早川宏之, 堀内基広, 上野弘志, 田村豊

  日本獣医学会学術集会講演要旨集 2007年
• ポリ6-硫酸化N-アセチルグルコサミン誘導体の合成と抗プリオン活性

  永塚健宏, 永塚健宏, 鵜沢浩隆, 堀内基広, 西田芳弘

  日本糖質学会年会要旨集 2007年
• プリオンタンパク質C末端領域の新たなCu²⁺結合構造の同定

  稲波修, 稲波修, 渡邊康子, 渡邊康子, 堀内基広, 堀内基広, 平岡和佳子, 下山雄平, 桑原幹典, 桑原幹典

  日本獣医学会学術集会講演要旨集 2007年
• プリオンタンパク質のpH感受性における塩橋とヒスチジン残基の役割

  渡邊康子, 渡邊康子, 平岡和佳子, 下山雄平, 堀内基広, 堀内基広, 桑原幹典, 桑原幹典, 稲波修, 稲波修

  日本獣医学会学術集会講演要旨集 2007年
• マウス神経芽腫細胞Neuro2a(N2a)サブクローン間におけるプリオンの細胞間伝播の相違

  瓜生匡秀, 堀内基広

  日本獣医学会学術集会講演要旨集 2007年
• プリオンの増殖とその抑制

  堀内基広, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2007年
• プリオンタンパク質β-シート領域のpH感受性における塩橋とヒスチジン残基の役割

  渡邊康子, 渡邊康子, 平岡和佳子, 下山雄平, 堀内基広, 堀内基広, 桑原幹典, 桑原幹典, 稲波修, 稲波修

  生化学 2007年
• 抗PrP抗体の脳室内投与によるプリオン病進行の遅延効果

  宋昌鉉, 古岡秀文, 金チャンラン, 鈴木章夫, 前田秋彦, 堀内基広

  日本獣医学会学術集会講演要旨集 2007年
• 神経芽腫細胞Neuro2aサブクローンにおけるプリオンの細胞間伝播

  瓜生匡秀, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2007年
• プリオン持続感染細胞における異常型プリオン蛋白質の量的変動

  中満智史, 黒川彩, 瓜生匡秀, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2007年
• 日本で発生したBSE由来PrP^Scの生化学性状の解析

  進藤亮, 山河芳夫, 佐多徹太郎, 横山隆, 古岡秀文, 堀内基広

  日本獣医学会学術集会講演要旨集 2007年
• ベトナムの酪農牛におけるプリオン遺伝子の多形性

  村松康和, 要田正治, 小河孝, 堀内基広, 田村豊

  日本獣医学会学術集会講演要旨集 2006年
• プリオン感受性・非感受性Neuro2aサブクローンを用いたプリオン増殖関連宿主因子の探索

  中満智史, 瓜生匡秀, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2006年
• マウス神経芽腫細胞Neuro2a(N2a)サブクローンで検出される異常型プリオン蛋白質(PrP^Sc)の相違

  瓜生匡秀, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2006年
• Site-Directed Spin Labeling(SDSL)法によるプリオンタンパク質pH感受性領域の解析

  渡邊康子, 稲波修, 稲波修, 堀内基広, 下山雄平, 下山雄平, 中村秀夫, 中村秀夫, 桑原幹典

  日本獣医学会学術集会講演要旨集 2006年
• プリオン感受性細胞と非感受性細胞の遺伝子発現比較解析

  中満智史, 瓜生匡秀, 堀内基広

  日本膜学会年会講演要旨集 2006年
• プリオンタンパク質の抗酸化機能の解明

  渡邊康子, 渡邊康子, 稲波修, 稲波修, 井上哲, 飯塚大輔, 堀内基広, 堀内基広, 桑原幹典

  電子スピンサイエンス学会年会講演要旨集 2006年
• 細胞におけるプリオンの増殖

  堀内基広

  日本膜学会年会講演要旨集 2006年
• 動物プリオン病の病態形成に関与するサイトカインの探索

  古岡秀文, 薮添敦史, 田村勇耕, 堀内基広, 佐多徹太郎

  日本獣医学会学術集会講演要旨集 2006年
• BSE問題とプリオン病研究の現状

  堀内基広

  BMSコンファレンス講演要旨集 2005年
• プリオン感染動物におけるAHSP mRNA量低下の分子機構:造血系細胞に対する直接作用の検証

  勝岡加代子, 大塚弥生, 大塚弥生, 伊東大介, 伊東大介, 大田寛, 大田寛, 堀内基広, 堀内基広, 稲葉睦, 稲葉睦

  日本獣医学会学術集会講演要旨集 2005年
• プリオンタンパク質の銅イオンの新たな結合様式

  稲波修, 稲波修, 堀内基広, 堀内基広, 平岡和佳子, 稲垣冬彦, 下山雄平, 下山雄平, 中村秀夫, 中村秀夫, 桑原幹典

  日本獣医学会学術集会講演要旨集 2005年
• プリオンタンパク質過剰発現細胞における酸化ストレス応答

  井上哲, 稲波修, 飯塚大輔, 堀内基広, 桑原幹典

  日本獣医学会学術集会講演要旨集 2005年
• モンゴル在来ヒツジからのピロプラズマ目原虫DNAの検出

  村松康和, 八幡陽介, 堀内基広, 石黒直隆, 品川森一, 森田千春, 田村豊

  日本獣医学会学術集会講演要旨集 2005年
• マウスプリオンタンパク質α-ヘリックス領域のSite-Directed Spin Labeling(SDSL)法を用いた解析

  渡辺康子, 渡辺康子, 稲波修, 稲波修, 稲波修, 堀内基広, 堀内基広, 下山雄平, 下山雄平, 中村秀夫, 中村秀夫, 桑原幹典

  日本獣医学会学術集会講演要旨集 2005年
• 蛍光相関分光法による未変性条件下での異常型プリオン蛋白質の検出

  堀内基弘, 品川森一

  日本ウイルス学会学術集会プログラム・抄録集 2005年
• マウス神経芽腫細胞Neuro-2aのプリオン感受性はPrP^C以外の因子により規定される

  瓜生匡秀, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2005年
• 現行の牛海綿状脳症検査のシカ慢性消耗病(CWD)診断への応用性

  嶋田希実, 岩丸祥史, 林浩子, 高田益宏, 牛木祐子, 木村久美子, 田川裕一, 堀内基広, 品川森一

  日本獣医学会学術集会講演要旨集 2004年
• 6位硫酸化GlcNAc誘導体が示すシアリダーゼ阻害活性並びに異常プリオン産生阻害活性

  佐々木健二, 西田芳弘, 神原実季恵, 堀内基広, 鵜沢浩隆, 小林一清

  日本化学会講演予稿集 2004年
• プリオンの部位特異的スピンラベル法(SDSL-ESR)による構造変化の解析

  稲波修, 橋田修吉, 飯塚大輔, 井上哲, 堀内基広, 桑原幹典

  日本獣医学会学術集会講演要旨集 2004年
• マウススクレイピーモデルを用いたプリオン蛋白質に対する宿主免疫応答の再考

  片岡那津見, 石黒直隆, 前田秋彦, 堀内基広

  日本獣医学会学術集会講演要旨集 2004年
• ウシプリオン遺伝子の多型解析

  中満智史, 石黒直隆, 前田秋彦, 堀内基広, 宮沢孝幸

  日本獣医学会学術集会講演要旨集 2004年
• Site-directed spin label(SDSL)法によるマウスプリオンタンパク質の銅イオン結合部位周辺の構造解析

  稲波修, 橋田修吉, 飯塚大輔, 堀内基広, 平岡和佳子, 稲垣冬彦, 桑原幹典

  電子スピンサイエンス学会年会講演要旨集 2004年
• 6位硫酸化N-アセチルグルコサミンクラスターの異常プリオン産生阻害活性

  神原実季恵, 西田芳弘, 佐々木健二, 篠田康彦, 小林一清, 堀内基広, 鵜沢浩隆

  高分子学会予稿集(CD-ROM) 2004年
• 「BSEの解明と対策の最前線」プリオン蛋白質とプリオン病

  堀内基広

  日本畜産学会大会講演要旨 2004年
• 人獣共通感染症としてのプリオン病

  堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2004年
• スクレイピー持続感染細胞I3/I5におけるプリオン増殖

  荻野倫子, 菊池宏明, 前田秋彦, 堀内基広

  日本ウイルス学会学術集会プログラム・抄録集 2004年
• 新生子マウスの腸粘膜上皮細胞へのプリオン伝達の試み

  岡本実, 古岡秀文, 堀内基弘, 野口敬紀, 萩原克郎, 村松康和, 辻正義, 朝長啓造, 生田和良

  日本獣医学会学術集会講演要旨集 2003年
• プリオンの環境汚染評価-植物への影響

  三隅智子, 石黒直隆, 堀内基広, 品川森一

  日本獣医学会学術集会講演要旨集 2003年
• 経口ルートによるプリオンの感染成立には消化管リンパ装置の存在が必要である

  堀内基広, 石黒直隆, 品川森一, 古岡秀文, 北村延夫

  日本獣医学会学術集会講演要旨集 2002年
• エポキシ化合物によるプリオン不活化

  山本真理, 品川森一, 石黒直隆, 堀内基広

  日本獣医学会学術集会講演要旨集 2001年
• in vitro試験系による病原性プリオン蛋白質と正常プリオン蛋白質の相互作用の解析

  堀内基広

  日本獣医学会学術集会講演要旨集 2001年
• プリオン蛋白質とプリオン病

  堀内基広

  タンパク質構造討論会講演要旨集 2000年
• スクレイピープリオン蛋白の羊体内分布 抗PrP抗体による免疫組織化学的検索

  鈴木智, 高橋広志, 松井高峯, 堀内基広, 石黒直隆, 品川森一, 古林与志安, 古岡秀文

  日本獣医学会学術集会講演要旨集 1998年
• スクレイピープリオン蛋白の羊体内分布 ウェスタンブロット法によるPrPSc検出

  高橋未緒, 堀内基広, 石黒直隆, 松井高峯, 古岡秀文, 品川森一

  日本獣医学会学術集会講演要旨集 1998年
• 牛プリオン蛋白質PrP遺伝子プロモーター領域の解析

  井上真吾, 田中成幸, 堀内基広, 石黒直隆, 品川森一

  日本獣医学会学術集会講演要旨集 1996年

共同研究・競争的資金等の研究課題

• グリア細胞の極性転換によるプリオン病の病態進行制御に関する研究

  日本学術振興会：科学研究費助成事業

  研究期間 : 2022年04月 -2025年03月 

  代表者 : 堀内 基広
• 非定型ＢＳＥ等動物プリオン病のヒトへの感染リスクの推定と低減に資する研究

  厚生労働省：厚生労働科学研究費補助金

  研究期間 : 2020年04月 -2023年03月 

  代表者 : 堀内基広, 古岡秀文, 新竜一郎, 宮澤光太郎, 飛梅実, 萩原健一, 小野文子
• プリオン病の神経変性機構におけるストレス誘導性転写調節因子ATF3の役割

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2019年04月 -2022年03月 

  代表者 : 堀内 基広

   

  本研究では、ストレス誘導性の転写調節因子ATF3の発現誘導機構、およびATF3が転写調節する分子群の解析により、プリオン病における神経変性機構の核心に迫れると考え、ATF3の神経細胞特異的ノックダウンを計画した。当初、コンディショナルターゲッティングで神経細胞特異的ATF3を欠損する計画であったが、マウスの遺伝背景の差異が大きいことから、神経組織親和性が高いアデノ随伴ウイルスベクター(rAAV-PHP.eB)を用いて経細胞特異的にCRISPR/SaCas9システムでゲノム編集を行う実験系に切り替えた。Sny1 プロモーター下で神経細胞特異的にSaCas9 を発現し、ATF3に対するguide RNAを発現するアデノ随伴ウイルスベクターrAAV-AFT1, rAAV-AFT2を作成した。陰性対照として、オフターゲット効果がないとされるguide RNAを発現するrAAV-NC1, rAAV-NC2, およびSyn1プロモーター下でEGFPを発現するrAAV-EGFPを作成した。rAAV-EGFPをウイルスベクターを脳定位接種法により接種して接種部位の神経細胞でEGFPが発現することを確認した。また、SaCas9を発現するrAAVを初代培養神経細胞に感染させてSaCas9の発現を確認した。現在、予想したゲノム編集が生じるか検討中である。プリオン感染マウスにおけるATF3陽性細胞の定量解析、ATF3神経細胞の割合、および出現部位を解析するために、プリオン感染マウスの脳凍結切片を、NeuN (神経細胞マーカー) とATF3に対する抗体を用いて蛍光多重染色を実施した。ATF3陽性細胞の出現が顕著な視床でが、ATF3陽性細胞の70%以上がNeuN陽性細胞であった。また視床背外側以外では橋灰白質でも病態の進行に伴いATF3陽性細胞が増加していた。
• 食品を介する家畜・家禽疾病のリスク管理に関する研究

  厚生労働省：厚生労働科学研究費補助金

  研究期間 : 2017年04月 -2020年03月 

  代表者 : 新 竜一郎, 柴田 宏昭, 萩原 健一, 飛梅 実, 古岡 秀文, 松浦 裕一, 山﨑 剛士, 鎌田 洋一, 壁谷 英則, 森田 幸雄, 保富 康宏
• ニューロン-グリアのクロストーク解析から迫るプリオン病の神経変性機構

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  研究期間 : 2015年04月 -2019年03月 

  代表者 : 堀内 基広, 小林 篤史, 黒田 弥乃梨, 単 智夫

   

  プリオン病ではミクログリアとアストロサイトが早期から活性化する。グリア細胞の活性化が神経変性に関与することが示唆されているが、その詳細は明らかでない。そこで本研究では、網羅的遺伝子発現解析によりミクログリアとアストロサイトの活性化状態を詳細に解析した。さらに、プリオン感染マウス脳内で神経細胞数の減少が起こる微少領域を同定して、その領域における網羅的遺伝子発現解析を実施した。その結果、小胞体ストレス応答により発現が誘導される転写調節因子ATF3が、神経細胞の脱落が生じる視床背外側で神経細胞に発現することを見いだした。
• 神経変性疾患で治療効果を発揮する間葉系幹細胞亜集団の同定とその解析

  日本学術振興会：科学研究費助成事業

  研究期間 : 2016年04月 -2018年03月 

  代表者 : 堀内 基広

   

  緻密骨由来間葉系幹細胞（CB-MSCs）はプリオン感染マウスの生存期間を延長することから、CB-MSCsは神経変性疾患の治療に利用できる可能性がある。CB-MSCsを移植したプリオン感染マウスでは、予想に反してミクログリアが更に活性化したが、Arg-1などの抗炎症性M2マーカーの発現が上昇していたことから、CB-MSCsはミクログリアの活性化状態を変化させることが示唆された。In vitroにおいてもCB-MSCsの培養上清はプリオン感染マウスから回収したミクログリアの活性化を抗炎症性のM2タイプにシフトさせたことから、CB-MSCsは自然免疫の修飾により治療効果を発揮する可能性が示唆された。
• ミクログリアの機能変化とプリオン病神経病態の関係の解明

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  研究期間 : 2011年04月 -2015年03月 

  代表者 : 堀内 基広, 稲波 修, 長谷部 理絵, 佐々 悠木子, 大島 正伸

   

  我々が確立した異常型プリオンタンパク質の特異染色を用いて、アストロサイトがプリオンの増殖あるいはプリオンの増殖により生じる神経細胞の微細な変化を認識し、その後ミクログリアが活性化するという感染初期における神経病態の一端を明らかにした。 ミクログリアの遺伝子発現解析から、ミクログリアはプリオン感染早期では病気の進行に抑制的に働くが、感染の進行とともに炎症促進性の活性化状態にシフトすることを明らかにした。また、抗炎症性サイトカインは脳におけるプリオンの増殖を抑制することを明らかにした。
• 間葉系幹細胞の神経変性疾患病変部への走化に関わる分子群の同定

  日本学術振興会：科学研究費助成事業 挑戦的萌芽研究

  研究期間 : 2011年 -2012年 

  代表者 : 堀内 基広

   

  プリオン感染動物の神経病変部へMSCsが走化する際に関与するケモカインおよびそれらのレセプターを同定するために、invitro走化試験により走化に関与する因子を解析した。また、プリオン感染マウス脳に移植したMSC上でのレセプターの発現を解析した。その結果、MSCs上に発現するCCR3、CCR5、CXCR3、CXCR4、およびそのリガンドとの相互作用が、MSCsのプリオン病神経病変部への走化に関与することを明らかにした。
• 人獣共通感染症関連する高分子タンパク質重合体の電子スピン二重共鳴法による構造解析

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2009年 -2011年 

  代表者 : 稲波 修, 稲葉 睦, 堀内 基広, 桑原 幹典, 山盛 徹

   

  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を明らかにする目的で研究を行った。実験は家族性プリオン病の変異体D178NのC末端側のヘリックス構造を持つ領域にスピンプローブ(R1)を導入し、酸性下で1M塩酸グアニジン処理により凝集体構造を形成させ、CW-ESRならびに電子スピン二重共鳴法(DEER)によりスピン問距離を計測した。その結果、凝集に関与している構造体はC末端領域の構造が凝集体形成に重要であり、N末端領域は一定の構造を取っておらず、凝集体の形成にも関与していないことが示された。
• 再生医療を応用したプリオン病治療モデルの確立

  日本学術振興会：科学研究費助成事業 挑戦的萌芽研究

  研究期間 : 2008年 -2009年 

  代表者 : 堀内 基広, 宋 昌鉉, 長谷部 理絵

   

  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グ系として用いて、サイトカイン、ケモカインおよび栄養因子およびそれらのレセプターの関与を調べた。その結果、CCR3,CCR4,CCR4およびCXCR4のケモカインレセプターとそのリガンドがhMSCsの走化に関与していることが示唆された。プリオン感染マウスの視床に移植したhMSCsは脳梁を通って、反対側の海馬へと移動するが、移植後、脳梁に存在するhMSCsではCCR3,CCR4,CCR4およびCXCR4のケモカインレセプターが発現していたことから、実際の神経組織においてもCCR3,CCR4,CCR4およびCXCR4およびそのリガンドは、hMSCsのプリオン病病変部の走化に関与することが明らかとなった。
• プリオン病初期病変形成機序の解析

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  研究期間 : 2006年 -2009年 

  代表者 : 堀内 基広, 稲波 修, 木村 和弘, 落合 謙爾, 長谷部 理絵, 瓜生 匡秀, 宋 昌絃, 大島 正伸, 古岡 秀文

   

  DNAマイクロアレイ法を用いて、プリオン感染早期から発現が変動する宿主遺伝子の探索を行い、発現がプリオン感染早期から上昇する遺伝子の病態機序への関与を解析した。その結果、LPSレセプターとして知られるCd14分子が病態進行に関与すること、神経保護作用を有するケモカインとして知られるCxcl10が病気の進行に抑制的に働くことを明らかにした。また、ミクログリアの活性化が病態進行に抑制的に働くことが示唆された。
• プリオン蛋白質構造の原子間力顕微鏡と磁気共鳴法を用いた新たな分子イメージング

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2007年 -2008年 

  代表者 : 稲波 修, 稲葉 睦, 堀内 基広, 桑原 幹典, 浅沼 武敏, 平岡 和佳子, 下山 雄平, 平岡 和佳子, 下山 雄平

   

  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。
• 動物プリオン病の病態と体内伝播を規定する宿主要因の検索と神経免疫機構に関する研究

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2005年 -2007年 

  代表者 : 石黒 直隆, 柳井 徳磨, 桑田 一夫, 堀内 基広, 丸尾 幸嗣

   

  動物プリオン病は感染した宿主により病態が異なる。本研究では、こうした病態の違いが、宿主が有する神経系と免疫系に依存するものと考えPrP遺伝子解析、ウシとヒツジの回腸に投射している外来性神経網の解析、免疫系細胞でのプリオンの蓄積・分解能を解析し以下の成果を得た。 1.ウシ、ヒツジ、ヤギ、シカのPrP遺伝子型解析:プリオンに対する感受性を支配するPrP遺伝子の多型解析を行った。ウシとシカではPrP遺伝子内にアミノ酸置換を示す多型は検出されなかった。日本のヤギではスクレイピーに対して感受性が高い遺伝子型が多く検出された。ヒツジではスクレイピーに抵抗性を示す遺伝子型が多く検出されつつある。 2.トレーサを用いたウシとヒツジでの腸管神経網の解析:ウシとヒツジの回腸遠位部にトレーサを注入し、腸管の神経網を解析した。その結果、腹腔・前腸間膜動脈神経節と脊髄神経節で多くの陽性細胞を得た。幹神経節や迷走神経での陽性細胞は少なかった。回腸にトレーサを注入した揚合は、ウシとヒツジで同様な結果を得た。一方、十二指腸にトレーサを注入した実験では、迷走神経背側核と節状神経節に陽性細胞が検出され、その傾向はヒツジで特に顕著であった。 3.免疫系細胞でのプリオンの取り込みと分解性:BSE感染牛では回腸パイエル板内の濾胞マクロファージ内でプリオンの蓄積が検出されている。そこで、ウシ末梢血由来の単球培養系マクロファージでのプリオンの蓄積性や分解性を検査した。その結果、マクロファージ内では蓄積は観察されなかったが、分解性は効率に起きることが観察された。
• プリオン感染マウスモデルを用いたプリオン病治療候補薬の評価

  日本学術振興会：科学研究費助成事業 特別研究員奨励費

  研究期間 : 2005年 -2006年 

  代表者 : 堀内 基広, KIM C.-I., KIM C.-l

   

  培養細胞レベルでPrP^の産生を抑制する物質は、プリオン病治療薬の候補となりうる。そこで、候補薬物をプリオン感染モデルで治療効果や副作用を評価して、プリオン病治療法の開発に役立てることを目標とした。プリオン病治療薬は中枢神経系組織に到達することが重要であるので、候補物質を浸透圧ポンプを用いて脳室内に直接投与して副作用、延命効果、治療効果の評価を実施した。候補物質の一つである人工合成硫酸化糖(4SGN,p6SGN)を、スクレイピー帯広株を接種して発病初期にあるマウスに浸透圧ポンプを用いて一ヶ月間脳室内に投与したが、陰性対照と比較して有意な延命効果は認められなかった。特定の抗PrP抗体が培養細胞においてプリオンの増殖を阻害することが知られている。そこで、プリオン増殖阻害活性を有する抗体を脳室内に投与した時の副作用を評価した。用いた3種類の抗PrP抗体のうち、1種類の抗体が、海馬錐体細胞を選択的に変性させることが判明したが、他の2種の抗体は顕著な神経変性を惹起しなかった。抗体が認識するエピトープにより神経変性誘発が異なったことから、神経変性作用のある抗体を培養細胞レベルでスクリーニングして排除することを日的に、マウス神経芽腫細胞(N2a)を抗体で刺激してDNAマイクロアレイ法により、抗体刺激により変動する遺伝子群の解析を実施した。しかし、細胞死に関連する遺伝子群が特異的に変化しているという現象は認められなかった。現在、神経毒性を誘発しない抗PrP抗体を脳室内に持続投与し、治療効果の評価を継続中である。
• 動的構造解析法によるプリオンの構造と異常型への変遷機構の解明

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2005年 -2006年 

  代表者 : 稲波 修, 堀内 基広, 苅和 宏明, 稲葉 睦, 桑原 幹典

   

  BSEやCJDなどのプリオン病では、構造異常を呈する異常型プリオンタンパク質(PrPSc)が正常型PrPCと相互作用し、その高次構造を異常型に変換させ、病態を引き起こすと考えられている。このPrPの構造変化機構には、酸性エンドソームが関与していると考えられている。本研究ではsite-directed spin labeling (SDSL)法を用い、αヘリックス1(H1)の裏側、βシート1(S1)とβシート2(S2)の3つのpH感受性領域をESRによる解析で見出した。近年、molecular dynamics (MD) simulationを用いた研究により、酸性pHが引き起こすPrPSc様のβ-sheet-rich構造への転換にアスパラギン酸177(Dl77)とヒスチジン186(H186)が重要な役割を持つと報告されたが、実際には実験的な証明はなされていない。本研究ではさらにD177とR163が形成する塩橋とH176とH186の2つのヒスチジン残基について、β-sheet2(S2)のpH感受性に対する影響をSDSL-ESR法で解析した。その結果、D177が形成する塩橋の形成がS2の中性付近でのβシート構造の維持に重要であることが明らかとなったが、近傍のH176の存在がこの構造安定性にも重要であることが見いだされた。また、H186のCJD変異についてはS2の構造安定化には寄与しておらず、βシート領域以外の別の構造変化がPrps^cの形成の引き金となることが示唆された。以上の結果は、D178Nといったアミノ酸の突然変異で発症する遺伝性CJDやFFIは、S2の構造変化が引き金となりPrPScが形成されると考えられる。また、本研究では、シングルラベルしたプリオンタンパク質にパルスESR法を適応することにより、低pHあるいは塩酸グアニジンにより誘導されるプリオン凝集体で見られる距離情報を得ることが出来ることも見いだした。これは将来、他種類のシングルラベルしたプリオンタンパク質に適応することにより、異常型プリオンの構造情報を得ることが出来る方法として期待できるものである。
• プリオン病における赤芽球系造血細胞の分子病態:生前分子診断のための分子基盤確立

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  研究期間 : 2004年 -2006年 

  代表者 : 稲葉 睦, 堀内 基広, 稲波 修, 梅村 孝司, 山本 雅之, 陰山 聡一

   

  1)AHSP遺伝子の5'上流領域プロモーター領域の解析から、赤芽球系分化誘導時には、翻訳開始点上流-328〜-286bpに位置する3カ所の転写因子GATA結合モチーフがAHSPの転写活性化に必要な最小要素であることが明らかになった。EMSA解析等の結果を加え、赤芽球系前駆細胞におけるAHSPの転写にはGATA-1の作用が必須なことが明らかになった。同時に、PrP^侵襲によるAHSPの発現抑制には、GATA-1による転写制御が関連することが推定された。 2)プリオン感染マウス由来脳乳剤、あるいはPrP^持続感染神経細胞株ScN2a存在下にMELhide8細胞を培養し、AHSP等の発現を解析した。しかし、赤芽球系分化誘導時、AHSPをはじめ、α-グロビン、β-グロビン、GATA-1、EKLF、NF-E2などの転写にはいずれも何ら影響が認められず、また、長期継代後にもMELhide8細胞にはPrP^の蓄積が全くみられなかった。これらの結果から、PrP^はMELhide8細胞に対する直接作用をもたないことが明らかになった。 3)MELhide8細胞におけるAHSP mRNA発現量、ならびに上記GATA依存性のAHSPプロモーターレポーター遺伝子発現は、IL-6の添加で濃度依存性に抑制された。IL-1βにも類似作用が認められたが、その効果はIL-6に比して低かった。これらの作用は、STAT3経路の抑制により明確に減少した。したがって、赤芽球系細胞におけるAHSPの転写抑制は、プリオン病で増加する炎症性サイトカインによるGATA依存性転写のSTAT3系による抑制を介することが示された。 4)牛末梢血赤血球の細胞質にAHSPが存在することが明らかになった。BSE感染個体由来脳を接種した牛群の末梢赤血球AHSP含量は、健康個体のそれと有意差は認められず、早期診断マーカーとすることは困難なことが判明した。
• グリコサミノグリカン類のモジュール合成と生物機能解析

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2004年 -2006年 

  代表者 : 西田 芳弘, 三浦 佳子, 堀内 基広, 鵜沢 浩隆, 鵜沢 浩隆, 小林 一清

   

  ヘパラン硫酸は細胞外マトリックスとして細胞の増殖や分化に重要な役割をしている。また、正常プリオンから異常プリオン蛋白質への転移に係ると考えられているが、複雑な構造を持つことから明確な構造因子(鍵構造)を抽出することができなかった。本研究では、代表者らの糖鎖モジュール化法をグリコサミノグリカン類、特に、ヘパラン硫酸に適用して、構造が明確な硫酸化多糖を合成すること、さらに、合成品をプリオン感染細胞並びにモデルマウスを用いた生物試験に供して、抗プリオン活性を示すグリコサミノグリカンの活性糖の構造因子を明らかにすることを目的とした。その結果、以下のことを明らかにすることができた。 (1)各種硫酸化糖モノマー(モジュラー)を酵素と化学的手法を用いて合成する方法論を確立した。さらに、アクリルアミドのラジカル重合により、ポリアクリルアミド鎖にパラニトアミドフェニル基を介して複数の硫酸化糖が結合した人工ヘパラン硫酸を合成することができた。 (2)持続感染細胞を用いた評価で、ヘパラン硫酸の主要な構成糖である6-sulfo-GlcNAcの人工高分子に抗プリオン活性が確認された(ED_<50> 10Dg/ml)。硫酸基を持たないGlcNAcの高分子には活性が認められなかった(ED_<50>>80Dg/ml)ことから硫酸基が活性に必須であることが明確になった。4-sulfo-GlcNAcの高分子はさらに強い活性(ED_<50> 3-7Dg/ml))を示したこと、その一方で、3-sulfo-GlcNAcは弱い活性しか示さないことから、硫酸基の位置とN-アセチル基の空間配置も重要な因子となることがわかった。 (3)高分子化した6S-GlcNAcと4S-GlcNAcは再現性よく異常プリオンの産生を抑制した。特に非天然型の4S-GlcNAcを含む高分子に高い活性が認められた。同様の結果は長期投与試験においても確認することができた。N-アセチル基をもたない6S-Glcの高分子は長期投与試験でも活性を示さなかったが、4S-GlcNAc、6S-GlcNAcの高分子はそれぞれ6日目と9日目で細胞が異常プリオンを産生することをストップさせた。活性を示した両化合物は細胞膜表面の正常型プリオン蛋白質(Prp^C)を減少させる傾向を示した。しかし、顕著な細胞増殖抑制や毒性は認められなかったことから、人工グリコサミノグリカンを用いたプリオン治療法の開発に新たな可能性を提示することができた。
• 野生のニホンジカは人あるいは他の動物種の感染源になり得るか?

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2004年 -2006年 

  代表者 : 鈴木 正嗣, 梶 光一, 山内 貴義, 有川 二郎, 横山 真弓, 堀内 基広, 木村 享史

   

  1.ニホンジカ(976頭)とニホンイノシシ(87頭)から得た血清を用い,ELISA法とウエスタンブロット法によりE型肝炎に対する血清学的診断を行った.その結果,それぞれの種で2.6%および2.3%が抗体陽性と判断された.イノシシにおいては,RT-PCR法により3頭でHEV-RNAが確認されたが,捕獲状況からこれらは同腹子と考えられた2.野生のニホンイノシシ(88頭)から得た血清を用い,ELISA法と顕微鏡下凝集試験(MAT)により,レプトスピフに対する抗体の検出を行った.その結果,12検体(13.6%)が抗体陽性と判断され,さらにMATでは血清型copenhageni, australis, bratislavaの抗体が検出された.そのため,捕獲ならびに解体にともなう人や猟犬に対する感染リスクが示唆された.3.野生のニホンジカ173頭を材料に,糞便培養法,LAMP法,nested PCR法によりヨーネ菌の検出を試みた.その結果,糞便培養法では菌は確認されなかった.また,LAMP法とnested PCR法でもヨーネ菌DNAは検出されなかった.4.北海道産の野生ニホンジカ(エゾシカ)においては,末梢血よりRickettsia helveticaとEhrlichiamurisの遺伝子が確認された.5.岩手県で捕獲された野生ニホンジカからは,槍形吸虫Dicrocoelium chin...
• 硫酸化糖を分子基盤とする抗BSE試薬の開発と異常プリオン抑制機構の解析

  日本学術振興会：科学研究費助成事業 萌芽研究

  研究期間 : 2004年 -2005年 

  代表者 : 西田 芳弘, 三浦 佳子, 小川 智久, 堀内 基広, 小林 一清

   

  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日間培養した後、PrP^検出用の試料を調整した。陽性対象として、プリオン増殖阻害活性を有する硫酸デキストラン500(DS)を使用した。PrP^の検出用試料の調整は2-2(5)に述べた通りに行った。また、SDS-PAGEおよびウエスタンブロットは共同研究者の堀内らの方法に従って実施した。 (2)結果・成果 硫酸化糖のプリオン増殖阻害活性をPrP^の産生量を指標に評価した。8種の合成硫酸化糖のPrP^産生阻害活性を比べた。陽性対象であるDS500(DS)は1μg/mlの濃度でも、PrP^の産生を70%程度阻害した。調べた硫酸化糖のうち、Hは濃度依存的にPrP^産生を阻害した。検量線グラフからPrP^量を陰性対照の50%にする50%効果濃度(ED_<50>)は約20μg/mlと推定された。また、硫酸化糖Bも100μg/mlでPrP^量が陰性対照の50%以下となり、弱いながらもPrP^産生阻害活性を有する可能性が示唆された。
• 異常型プリオン蛋白質の細胞内増殖の分子機構

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  研究期間 : 2003年 -2005年 

  代表者 : 堀内 基広, 稲葉 睦, 大橋 和彦, 前田 秋彦, 古岡 秀文

   

  本研究では、PrP^産生を阻害する物質の作用機序の解析、および、プリオン増殖に関与する宿主因子や微小環境の同定、からプリオンの細胞内増殖機構を紐解くための研究を行った。 細胞膜上に発現するPrP^Cと反応する4種の抗PrP抗体が、プリオン持続感染細胞におけるPrP^産生を抑制した。細胞膜上のPrP^Cと結合した抗体は細胞膜上に停留する傾向があることから、PrP^Cと抗体が結合して、PrP^Cが通常の分解経路に移行しなくなることが、PrP^産生抑制の原因と考えられた。また、人工硫酸化糖のプリオン増殖抑制能を調べた結果、4-Sulfo-N-acetylglucosamineおよび6-Sulfo-N-acetylglucosamineにPrP^産生抑制活性が認められた。これらを細胞に添加した場合、PrP^Cのエンドサイトーシスが促進され、総PrP^C量が減少した。一方、PrP^産生抑制活性のない硫酸化糖はPrP^Cのエンドサイトーシスを促進しなかった。従って硫酸化糖によるPrP^産生抑制は、PrP^Cの分解促進が原因と考えられた。 マウス神経芽細胞Neuro2aのサブクローンを多数樹立して、プリオン感受性および非感受性に分類した。プリオン非感受性のN2a-1ではPrP^Cの発現は親株やプリオン感受性N2aサブクローンと同程度であった。プリオン感受性サブクローンN2a-3およびN2a-5とN2a-1におけるPrP^の吸着を検討したが、感受性・非感受性サブクローン間でPrP^の吸着には差が認められなかった。N2a-1のようにPrP^Cは発現するがプリオン非感受性の細胞の存在は、PrP^C以外にもプリオン感受性に関与する宿主因子の存在を示す結果である。そこで、樹立したN2aサブクローン間での遺伝子発現をDNAマイクロアレイ法により解析した。プリオン感受性細胞で2倍以上発現が高い遺伝子を36種、プリオン非感受性細胞で2倍以上発現が高い遺伝子を18種、候補遺伝子として選抜した。候補遺伝子に対するsiRNAを用いて標的遺伝子の発現を抑制し、プリオン感受性への影響を調べた。その結果、F2,A1,C5の3遺伝子に対するsiRNAが、N2a-5におけるプリオン増殖を抑制することが明らかとなった。
• 免疫組織化学的手法によるプリオン蛋白の迅速検出法並びに検出感度に関する研究

  日本学術振興会：科学研究費助成事業 萌芽研究

  研究期間 : 2002年 -2003年 

  代表者 : 古岡 秀文, 堀内 基広, 松井 高峯, 古林 与志安, 品川 森一

   

  (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体のエピトープ領域全てが関与するのではなく,限定されたコア領域に依存する可能性があることが推察された.
• 非通常病原体プリオンの分子機構

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2002年 -2002年 

  代表者 : 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) 本年度は、他領域との重複などの問題点があり、新規特定領域への申請は見送ったが、本研究の成果を基礎に来年度以降の申請へむけ、さらなる体制整備を行う予定である。
• プリオンの増殖を規定する正常型-異常型プリオン蛋白質の分子間相互作用に関する研究

  日本学術振興会：科学研究費助成事業 特定領域研究(C)

  研究期間 : 2001年 -2002年 

  代表者 : 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-プVII、VIIIはPrP分子上の構造エピトープを認識する抗体であった。また、グループIXはPrPSc特異的エピトープを認識する抗体である可能性が強く示唆された。aa59-89のリニアエピトープを認識するmAb、aa143-151を認識するmAb、およびaa1555-231領域内の構造エピトープを認識するmAbはプリオン感染細胞におけるPrPScの合成を阻害した。これらの抗体は細胞膜上に発現するPrPCと強く反応することから、抗体が細胞膜上のPrPCと結合することで正常なPrPC代謝経路が影響を受けるために、PrPSc合成の基質となるPrPCの供給が阻害された結果であると考えられる。
• 羊スクレイピー病原体(プリオン)の侵入門戸に関する研究

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2000年 -2002年 

  代表者 : 堀内 基広, 田邊 茂之, 古岡 秀文

   

  本研究では、スクレイピーやBSEの自然状態での主な感染経路である経口感染において、プリオンの侵入門戸を明らかにする目的で、リンパ節やパイエル板など二次リンパ系組織を欠く変異マウス[ALY(alymphoplastic)マウス]と、その野生型であるC57BL/6マウスを用いて、腸管付随リンパ装置が侵入門戸となる可能性について調べた。ALY/ALYマウスおよびその野生型マウスC57BLに、マウススクレイピー病原体を経口、腹腔、および脳内の各ルートで投与して、スクレイピーに対する感受性を調べた。脳内接種ではC57BLは165±5日、ALYでは159±8日でマウスはスクレイピー末期となった。マウス間で有意差は認められなかった。腹腔内接種ではC57BLは251土9日、ALYでは279±6日でマウスはスクレイピー末期となり、ALYマウスで20日程度潜伏期が延長した。経口投与ではC57BLは307±7日でスクレイピー末期となったが、ALYマウスは投与後640日でもスクレイピーを発症しなかった。っまり、ALYマウスは経口ルートではプリオンに感染しなかった。また、経口投与、および腹腔投与後20,40,80日でマウスの消化管、脾臓を採取して、各時点における組織中の感染価をマウスバイオアッセイにて調べた結果、C57BL/6マウスでは調べた全ての時点でプリオン感染価が検出されたが、ALYマウスでは検出されなかった。また、プリオンを経口ルートで投与したC57BL/6の脾臓、消化管(粘膜下リンパ濾胞)ではPrPScが検出された。これらの結果から、パイエル板などの消化管付随リンパ装置が侵入門戸として重要であると考えられた。最近、ALYマウスの原因遺伝子はNF-κB inducing kinaseであることが明らかにされた。これまでALYマウスは主に免疫研究に用いられてきた。プリオンの侵入門戸の一つとして、消化管神経が考えられている。そこで、ALYマウスが消化管神経の構築に異常を伴うか否かを調べたが、消化管神経の走行や密度はC57BLとALYマウスの間で差は認められなかった。本研究の成果から、経口的に取り込まれたプリオンは、消化管上皮に存在するM細胞を経て宿主体内に侵入し、濾胞樹状細胞を経由して、末梢神経へと侵入することが示唆される。プリオンに対する感受性が高い若齢反芻獣のパイエル氏板を覆うドームでは、M細胞が多数存在すること、また若齢反芻獣の回腸遠位部でパイエル氏板が発達していることなどから、回腸遠位部のパイエル氏板がブリオンの侵入門戸となる可能性が高いと考えられる。
• モンゴルにおけるスクレイピーの病原学的,分子遺伝的調査

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2000年 -2002年 

  代表者 : 品川 森一, 堀内 基広, 堀内 基広, 石黒 直隆, 村松 康和

   

  モンゴルは牧羊に関して悠久の歴史を有し、羊の飼育頭数は1200万頭にのぼる。同国は将来、世界の畜産物供給基地にも成りえる可能性を有する。従って、モンゴルにおける家畜プリオン病の疫学調査は、同国の畜産物の安全性を調べる上で重要な知見をもたらすと考えらえる。研究期間中にモンゴル中央部、西部、および東部に赴き、羊スクレイピーの臨床症状などを説明し、該当する羊の存在について聞き取り調査を行なったが、スクレイピーを疑う症状を呈する羊に関する情報は得られなかった。同時に、現地獣医師に神経症状を呈した羊の延髄の採取を依頼した。回収した試料からウエスタンブロットにより異常型プリオン蛋白質(PrPSc)の検出を行なったが、全て陰性であった。回収できた試料の数は十分ではないが、モンゴルにおけるスクレイピーの存在を確認するには至らなかった。モンゴル各地で、9品種(Khalkh、Yeroo、Orkhon、Khangai、Sartuul、Govi-altai、Bayad、Darhad、Sumber-Karakul)、計470頭の静脈血を採取して、有核細胞から染色体DNAを回収した。PCRによりPrP遺伝子の蛋白質コード領域を増幅してPrP遺伝子の塩基配列を解析し、最終的にPrPアミノ酸型を決定した。PrPコドン171のGln/Argのアミノ酸多型はスクレイピー感受性/抵抗性を規定することが知られている...
• 病原性プリオン蛋白質の生成機構に関する基盤研究

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2000年 -2002年 

  代表者 : 品川 森一, 牧野 壮一, 牧野 壮一, 堀内 基広, 古岡 秀文

   

  異常型プリオン蛋白質(PrPSc)は感染因子プリオンの主要構成要素であり、その生成機構はプリオン病病因論の中心となる。プリオン(PrPSc)が外部から侵入すると、正常型プリオン蛋白質(PrPC)と会合して、PrPCをPrPScに転換する。この際、PrPScは鋳型(seed)として働く。本研究では、PrScをseedとするPrPSc生成過程におけるPrPCとPrPScの分子間相互作用を解析した。マウスとハムスターのPrPを用いて、同種のPrPCとPrPScの組み合わせではPrPCはPrPScに転換するが、異種の組み合わせでは転換効率が非常に悪かった。しかしPrPCは異種PrPScと結合し得た。関連する一連の実験結果から、PrPScへの構造転換は、PrPCとPrPScの結合の段階ではなく、主に結合後の構造転換の段階で規定されること、構造転換の宿主特異性はPrP分子の中央部に存在する3アミノ酸の違いにより規定されることを明らかにした。また、PrScの生成に関与する微小環境の条件検討を行ったところ、グリコサミノグリカン(GAG)の構成要素である硫酸多糖がPrPScへの構造転換を促進することを発見した。硫酸多糖は細胞外間マトリックスに多く存在することから、PrPCは細胞膜上に発現することから、プリオン感染動物において細胞外マトリックス中の硫酸多糖がPrPC-PrPSc分子間相互作用に関...
• 羊スクレイピー病原体(プリオン)の多様性に関する研究

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 1997年 -1999年 

  代表者 : 堀内 基広, 松井 高峯, 牧野 壮一, 石黒 直隆

   

  羊のスクレイピー、牛の海綿状脳症(BSE)、およびクロイツフェルト-ヤコブ病(CJD)に代表される伝達性海綿状脳症(プリオン病)の病原体(プリオン)は病原体特異的な核酸を持たないことから、遺伝子型別および抗原型別は病原体型別には応用できない。しかしプリオンは生物学的性状あるいは生化学的性状によりある程度区別可能である。そこで本研究では、日本の羊スクレイピーの病原体に多様性("株")が存在するか否かを明らかにするための一連の実験を行なった。また、プリオンの多様性を規定するPrP以外の宿主の遺伝的要因についても検討を加えた。我が国で過去10年間に日本で発生した羊スクレイピーの11例を選抜し、マウスに接種して伝達性と羊脳内に蓄積したPrP^の蛋白質分解処理抵抗性を調べた。その結果、使用した11例のスクレイピーは生物性状および生化学性状の異なる3群に分類された。つまり日本には複数のスクレイピー病原体が存在することが明らかとなった。本研究のような多数の羊スクレイピーを材料としてその性状を詳細に比較検討した研究はこれまでに英国で報告があるのみで、日本では本研究が初めての例である。本研究で得られた成績は、日本に存在するスクレイピー病原体が動物種を越えて牛や人間に感染する危険性を評価するための重要な知見となる。人ではアポリポプロテインE(ApoE)の特定の遺伝子型が、アルツハイマー病...
• 伝達性海綿状脳症の感染性アミロイド形成に関する研究

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 1996年 -1998年 

  代表者 : 品川 森一, 堀内 基広, 桑山 秀人, 石黒 直隆

   

  感染性プリオンを試験管内で複製することを最終目標として、試験管内で微量のプリオンと多量の正常プリオン蛋白の接触により正常プリオン蛋白の構造を変えることを目論んだ。以前、動物脳から正常プリオン蛋白を多量に精製することに失敗しているため、今回は組換プリオン蛋白を用いることを計画した。さらにプリオン蛋白の精製の困難さから、組換プリオン蛋白のN端にヒスチジンタグを結合し、キレ-トカラムでアフニティ-精製を導入した。今回われわれの用いた系で、プリオンに見られるように,微量のプリオンを添加することにより組換プリオン蛋白のαヘリックス含量が減少し、βシ-ト含量が増加すること、蛋白分解酵素抵抗性に変化すること、プリオン蛋白の一部に相当する合成ペプチドの添加により阻害されること、さらにこのように変化したプリオン蛋白を次の新たなプリオン蛋白に添加することにより、プリオン添加と同様の構造変化を引き起こすことを見出した。唯、この系では,蛋白分解酵素抵抗性に変わったプリオン蛋白の状態がプリオンと同様の構造を反映しているとはいえない可能性が示唆された。この結果、真のプリオン複製に至らなかったが、プリオン複製のために、プリオン蛋白以外の要因が必要か否かを解析するために適した系として使用できる可能性が示唆された。一方、本研究をサポ-トする周辺領域の研究として,プリオン蛋白検出法の改良,牛プリオン遺伝子の完全...
• 伝達性海綿状脳症の高感度早期診断法の開発:PrPScの高感度検出法による早期診断と動物由来薬粧品原料のPrPSc汚染の検出への応用

  文部科学省：科学研究費補助金(試験研究(B), 基盤研究(A))

  研究期間 : 1995年 -1997年 

  代表者 : 品川 森一, 中川 迪夫, 堀内 基広, 久保 正法, 山田 明夫, 古岡 秀文

   

  本研究では、従来から行われているウエスタンブロット(WB)法の試料調整法の改良を行った。その結果、マウスモデルでは、腹腔内接種1週目から脾臓にプリオン蛋白が検出できるようになった。多数検体を検査するためには、より簡便なプリオン検出法が必要であり、マウスモデルを用いてELISA法を開発した。検出感度はウエスタンブロット法のおよそ2倍であった。中枢神経系組織からの試料調整を簡便化し、実際に羊材料に応用可能なことを確かめた上で、この方法の有用性を北海道のと蓄場から分与を受けた羊材料を用いて検証した。また、さらに検出感度を高めるために、ELISAの検出系に光化学反応試薬を導入したところ、およそ20倍ほど検出感度が上昇した。まさらに、コラーゲン中のプリオン検出のための試料調整法も検討し、前処理法としてコラーゲンの粘性を低下させ、大容量から比較的選択的にプリオン蛋白を濃縮することが可能となり、プリオン汚染の検出法に目処がついた。一方、プリオン病は宿主のプリオン蛋白の遺伝子に多型があり、その型によって感受性が異なる。国内の羊プリオン遺伝子型の出現頻度とスクレイピ-との関連を検討した。またプリオンの蓄積は主として中枢神経系であり、少量であるが細網内皮系の組織にも起きる。診断に有用な組織採取部位を決定するためにも、詳細なプリオン蛋白発現の違いを明かにする必要があり、羊体内のプリオン蛋白発現を調...
• 伝達性海綿状脳症関連蛋白質PrPによる神経細胞死の分子機構に関する研究

  文部科学省：科学研究費補助金(奨励研究(A))

  研究期間 : 1995年 -1995年 

  代表者 : 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...
• 遅発性感染症、スクレイピ-における宿主蛋白PrPの機能の解析

  文部科学省：科学研究費補助金(一般研究(B))

  研究期間 : 1992年 -1994年 

  代表者 : 品川 森一, 堀内 基広

   

  伝達性海綿状脳症の感染性はPrPの発現が高い中枢神経系に高く、感染性アミロイドの蓄積も起きる。牛PrP遺伝子を導入したL細胞、L-BPrP3は牛PrPを安定に発現している。このため一般の細胞に比べスクレイピ-病原体の複製増殖が起きるのではないかと期待して、細胞への感染実験をマウス及び羊の病原体を用いて行った。しかし対照として用いたL細胞との間に特に差が認められづ、牛PrP遺伝子の影響はなかった。変異遺伝子については感染実験が完了していない為成績から省いた。羊PrP遺伝子型とスクレイピ-感受性の関連を検討する経過で、発症あるいは感染性アミロイドの形成・蓄積にはPrP以外の遺伝子の関与がある可能性が示唆され、我々の用いた線維芽細胞由来のL-BPrP3はなにか未知の因子が欠けているのではないかと推定するに至り、新たに神経系の細胞に羊PrP遺伝子を高度に発現する細胞を樹立した。PrPの解析を行うには、羊或はうしPrPとだけ反応する免疫抗体、マウスPrPとだけ反応する抗体あるいは両者と反応する抗体が必要となり、これら要求を満たす抗体の作製と、高感度にPrPを検出する方法も開発した。また牛PrPのプロモーターの解析を行い、予想された第一エクソンの上流領域以外に、第一エクソンと第二エクソンの間のイントロン領域にもin vitroでプロモーター活性があることを見いだした。羊PrP遺伝子を扱う...
• 猫パルボウイルス亜群の分子進化と犬パルボウイルス出現機構に関する研究

  文部科学省：科学研究費補助金(奨励研究(A))

  研究期間 : 1993年 -1993年 

  代表者 : 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,...
• 家畜伝達性海綿状脳症に関するサーベイランス:PrPScの検出及びPrP遺伝子型の調査

  文部科学省：科学研究費補助金(総合研究(A))

  研究期間 : 1991年 -1993年 

  代表者 : 品川 森一, 石黒 直隆, 平井 克也, 本多 英一, 見上 彪, 堀内 基広, 小沼 操

   

  1.PrP遺伝子に関連する制限酵素切断断片の多様性:日本で飼育されている羊のスクレイピ-の感受性に関わる遺伝的な背景は知られていない。潜伏期及び感受性に関係するPrPの遺伝子に関連した染色体DNAの制限酵素切断断片に多様性(RFLP)が認められ、このRFLPのパターンと潜伏期あるいは感受性の間に関連があることが明らかにされている。日本の羊におけるRFLP型の調査と特定の型とスクレイピ-の関連の有無を検討した。日本各地から集めた羊の組織から染色体DNAを分離し、EcoRIあるいはHindIIIで消化したDNA断片を羊のPrPコード領域をプローベとしてSouthern Hybridizationを行なった。日本の羊はI〜VIの6型に分けられた。またI〜III型は英国で報告された型と一致していた。スクレイピ-の羊18頭の型はI型に8頭と集中しており、一方II型とVI型は、正常な羊128頭で見られる頻度に比べて低く、抵抗性であることが示された。さらにスクレイピ-感受性にかかわる遺伝学的な背景を明らかにするために、地方別のRFLP型の分布を161頭の羊で調べたところ、ある程度の地域差が認められた。2.PrP^検出によるスクレイピ-汚染状況の調査:1988年から1993年までに北海道、東北、関東および中部地方から集めた主にサフォーク種の羊197頭の脳、脾臓およびリンパ節からPrP...
• 遅発性感染症、スクレイピ-における宿主蛋白PrPの機能の解析:異種及び人工変異PrP遺伝子導入細胞の樹立と各種SAF画分(病原体画分)の調製

  文部科学省：科学研究費補助金(一般研究(B))

  研究期間 : 1990年 -1991年 

  代表者 : 品川 森一, 堀内 基広, 石黒 直隆

   

  ヒツジ及びウシ脳から得たmRNA画分からcDNAライブラリ-を作成しPrPのcDNAのクロ-ニングを行った。ウシでは2096塩基対から成るクロ-ンが得られたが、ヒツジでは成功しなかった。ヒツジでは染色体DNAからこの遺伝子のコ-ド領域を含む制限酵素断片をクロ-ニングした。さらにPCR法によってヒツジ及びウシの複数の個体から遺伝子をクロ-ニングし、比較解析した。これら遺伝子をそれぞれ発現ベクタ-pcDLーSRa296に組み込み、G418耐性マ-カ-を付与して発現プラスミドを作成した。またウシcDNAを基本として、PrPのN端に欠損を持ったもの及び一部アミノ酸置換が起きるように変異させた遺伝子も作成した。これらをリン酸カルシュウム沈澱法によってマウスL細胞に導入し、G418耐性コロニ-を分離した。何れの遺伝子によっても、それらのPrP^cを核周辺に強く発現している細胞が得られた。しかしヒツジPrP遺伝子を導入した細胞は時間と共に発現する細胞が減少した。ウシ遺伝子を入れた細胞のクロ-ニングにより全ての細胞がウシPrP^cを発現し続けるクロ-ンを樹立した。このクロ-ン化細胞に感染脳乳剤あるいはSAF画分を感染させた細胞には細胞質全体に微細顆粒状の蛍光が認められた。対照の感染L細胞でも同様の所見が得られたが、継代によってそのような細胞が速やかに消失した。変異遺伝子を導入した細胞に付いて...
• 牛Tリンパ球抗原レセプタ-遺伝子の分離と構造解析

  文部科学省：科学研究費補助金(一般研究(C))

  研究期間 : 1990年 -1991年 

  代表者 : 石黒 直隆, 堀内 基広

   

  牛T細胞抗原レセプタ-(TCR:α鎖、β鎖、γ鎖、δ鎖)遺伝子を牛末梢血リンパ球および牛胸腺細胞より作成したcDNAライブラリ-より分離し、その構造を解析した。1.牛TCRα鎖は、30個のcDNAクロ-ンの内7クロ-ンが完全長のVーJーC構造を示し、残りは不完全なV領域を有していた。α鎖のC領域は1種類であり、140個のアミノ酸残基からなっていた。2.牛TCRβ鎖は、38個のcDNAクロ-ンの内11クロ-ンが完全長のVーDーJーC構造を示し、DNA塩基配列の相同性から9群のサブファミリ-に分類された。各サブファミリ-は、ヒト由来TCRβ鎖Vファミリ-にそれぞれ高いホモロジ-を示した。牛TCRβ鎖のC領域は2種類存在し、178個のアミノ酸残基からなっていた。3.牛TCRγ鎖は、9クロ-ン分類され、VーJーCの構造を有していたが、完全長の遺伝子は得られなかった。γ鎖C領域は3種類分離されアミノ酸も222個、211個および194個とさまざまであった。4.牛TCRδ鎖は、3クロ-ン分離され、VーDーJーCの構造を有していた。δ鎖のC領域は155個のアミノ酸残基からなっており、マウス、ヒト由来に比べ羊由来C領域に高いホモロジ-を示した。5.牛TCR遺伝子解析中、偶然牛乳酸脱水素酵素遺伝子(LDHーA)を分離した。LDHーA遺伝子は、332個のアミノ酸残基をコ-ドしており、牛TCRα鎖と...
• 非通常病原体(スクレイピ-病原体)感染症の診断法の開発

  文部科学省：科学研究費補助金(試験研究, 試験研究(B))

  研究期間 : 1988年 -1990年 

  代表者 : 品川 森一, 吉野 智男, 太田 千佳子, 石黒 直隆, 百渓 英一, 堀内 基広

   

  1。抗ヒツジPrP抗体の作成:SAF画分からSDSーPAGEにより精製されたヒツジPrPを免疫して作成したウサギ血清は抗体価も特異性も高いものであった。またウシPrPともヒツジのそれと同様に反応した。2。ヒツジ臓器からのPrP検出用試料の調製:マウス臓器の為に用いていた方法をヒツジ組織のために改良を行なった。3。PrPの検出:スクレイピ-と診断あるいは疑われたヒツジ7頭、と畜場で食肉用に屠殺されたヒツジ30頭、外部の農場から帯広畜産大学に導入され、別の研究目的に供されたもの17頭、および実験感染させたもの10頭、合計64頭の臓器からPrPの検出を行なった。臨床的にも病理組織学的にもスクレイピ-と診断されたヒツジ6頭は何れもPrPが検出された。臨床的にスクレイピ-が疑われ病理組織学的に否定された1頭からは検出できなかった。と畜場から分与を受けた臓器からは全例検出できなかった。一見健康で別の研究目的に供されたものの内、3頭のリンパ節および脾臓からPrPが検出された。これらは同じ牧場で生産されたものであった。実験的に静脈内接種した群のヒツジから経時的に臓器採取を行なった。接種後16ヶ月および18ヶ月に実験に供した各2頭の内それぞれ1頭の各種臓器にPrPが検出された。また14ヶ月後にリンパ節をバイオプシ-した3頭中1頭にPrPが検出された。このヒツジは6ヵ月後にスクレイピ-を発症した...