Directory of Researchers

SAEKI Keiichi
Graduate School of Agricultural Science / Department of Bioresource Science
Associate Professor
Veterinary Medicine / Zootechnical Science
Last Updated :2024/01/31

Researcher Profile and Settings

Affiliation

  • <Faculty / Graduate School / Others>

    Graduate School of Agricultural Science / Department of Bioresource Science
  • <Related Faculty / Graduate School / Others>

    Faculty of Agriculture / Department of Bioresource Science

Teaching

Research Activities

Research Interests

  • 免疫学
  • ウイルス学
  • prion protein
  • prion

Research Areas

  • Life sciences / Virology
  • Life sciences / Neuroscience - general
  • Life sciences / Veterinary medicine

Published Papers

  • Masahiro Urushidani, Akira Kawayoshi, Tomohiro Kotaki, Keiichi Saeki, Yasuko Mori, Masanori Kameoka

    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is transmitted mainly by droplet or aerosol infection; however, it may also be transmitted by contact infection. SARS-CoV-2 that adheres to environmental surfaces remains infectious for several days. We herein attempted to inactivate SARS-CoV-2 and influenza A virus adhering to an environmental surface by dry fogging hypochlorous acid solution and hydrogen peroxide solution. SARS-CoV-2 and influenza virus were air-dried on plastic plates and placed into a test chamber for inactivation by the dry fogging of these disinfectants. The results obtained showed that the dry fogging of hypochlorous acid solution and hydrogen peroxide solution inactivated SARS-CoV-2 and influenza A virus in CT value (the product of the disinfectant concentration and contact time)-dependent manners. SARS-CoV-2 was more resistant to the virucidal effects of aerosolized hypochlorous acid solution and hydrogen peroxide solution than influenza A virus; therefore, higher concentrations of disinfectants or longer contact times were required to inactivate SARS-CoV-2 than influenza A virus. The present results provide important information for the development of a strategy that inactivates SARS-CoV-2 and influenza A virus on environmental surfaces by spatial fogging.

    Public Library of Science (PLoS), 07 Apr. 2022, PLOS ONE, 17 (4), e0261802 - e0261802

    Scientific journal

  • ネオニコチノイド系農薬クロチアニジンの胎子・授乳期曝露による次世代マウスの免疫系および腸内細菌叢の変化

    村田 碧, 西 美咲, 正田 明日香, 池中 良徳, 佐伯 圭一, 松尾 栄子, 平野 哲史, 万谷 洋平, 横山 俊史, 星 信彦

    日本内分泌撹乱物質学会, Sep. 2021, 環境ホルモン学会研究発表会要旨集, 23回, 38 - 38, Japanese

  • ネオニコチノイド系農薬クロチアニジンの胎子・授乳期曝露が次世代マウスの免疫系および腸内細菌叢に及ぼす影響

    村田 碧, 西 美咲, 正田 明日香, 佐伯 圭一, 松尾 栄子, 万谷 洋平, 横山 俊史, 平野 哲史, 池中 良徳, 星 信彦

    (公社)日本獣医学会, Sep. 2021, 日本獣医学会学術集会講演要旨集, 164回, [JO - 34], Japanese

  • Wuyun Dalai, Eiko Matsuo, Natsumi Takeyama, Junichi Kawano, Keiichi Saeki

    Elucidation of the processes regulating the prion protein gene (Prnp) is an important key to understanding the development of prion disorders. In this study, we explored the involvement of DNA methylation in Prnp transcriptional regulation during neuronal differentiation of embryonic carcinoma P19C6 cells. When P19C6 cells were differentiated into neuronal cells, the expression of Prnp was markedly increased, while CpG methylation was significantly demethylated at the nucleotide region between-599 and-238 from the transcription start site. In addition, when P19C6 cells were applied in a DNA methyltransferase inhibitor, RG108, Prnp transcripts were also significantly increased in relation to the decreased methylation statuses. These findings helped to elucidate the DNA methylation-mediated regulation of Prnp expression during neuronal differentiation.

    JAPAN SOC VET SCI, Mar. 2017, JOURNAL OF VETERINARY MEDICAL SCIENCE, 79 (3), 644 - 648, English

    [Refereed]

    Scientific journal

  • Wuyun Dalai, Eiko Matsuo, Natsumi Takeyama, Junichi Kawano, Keiichi Saeki

    The cellular isoform of the prion protein (PrPc) plays critical roles in the development of prion disorders. Although PrP mRNA is ubiquitously present in a tissue-specific manner, the DNA methylation of PrP gene (Prep) is still unknown. In this study, we demonstrated that the CpG island (CGI, positioned at 218 to +152 bp from the transcriptional start site) including the Prnp core promoter region was completely unmethylated in all tested tissues. On the other hand, CpG methylation in the CGI shore region (positioned at -599 to -238 bp) occurred in various tissue- and site-specific proportions. Interestingly, the correlation analysis between CpG methylation status and PrP mRNA levels showed that one CpG site methylation at 576 was negatively correlated with the PrP mRNA level (Pearson's r= 0.374, P=0.035). Taken together, our results suggest that Prnp is a typical housekeeping gene and various methylation frequencies of the CGI shore region are likely to affect Prnp expression in a tissue-specific manner.

    JAPAN SOC VET SCI, Jan. 2017, JOURNAL OF VETERINARY MEDICAL SCIENCE, 79 (1), 100 - 107, English

    [Refereed]

    Scientific journal

  • Eiko Matsuo, Keiichi Saeki, Polly Roy, Junichi Kawano

    Ibaraki virus (IBAV) is a member of the epizootic hemorrhagic disease virus (EHDV) serogroup, which belongs to the Orbivirus genus of the Reoviridae family. Although EHDV, including IBAV, represents an ongoing threat to livestock in the world, molecular mechanisms of EHDV replication and pathogenesis have been unclear. The reverse genetics (RG) system is one of the strong tools to understand molecular mechanisms of virus replication. Here, we developed a RG system for IBAV to identify the nonessential region of a minor structural protein, VP6, by generating VP6-truncated IBAV. Moreover, several tags were inserted into the truncated region to produce VP6-tagged IBAV. We demonstrated that all VP6-tagged IBAV could replicate in BHK cells in the absence of any helper VP6 protein. Further, tagged-VP6 proteins were first assembled into puncta in cells infected with VP6-tagged IBAV. Our data suggests that, in order to initiate primary replication, IBAV VP6 is likely to accumulate in some parts of infected cells to assemble efficiently into the primary replication complex (subcore). (C) 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license.

    ELSEVIER SCIENCE LONDON, 2015, FEBS OPEN BIO, 5, 445 - 453, English

    [Refereed]

    Scientific journal

  • 腫瘍におけるプリオン蛋白遺伝子発現は間質由来テネイシンCによって調整されている(Stromal tenascin C regulates a prion protein gene expression in tumor tissues)

    佐伯 圭一, オヨン・ダライ, 松尾 栄子, 河野 潤一, 日下部 美帆, 日下部 守昭

    (一社)日本癌学会, Oct. 2013, 日本癌学会総会記事, 72回, 322 - 322, English

  • Development of a New Selective Medium for Isolation of Methicillin-Resistant Staphylococcus aureus

    Etsuko Saito, Yoko Kakita, Hiroyuki Shimoda, Shizuka Shimabukuro, Keiichi Saeki, Akira Shimizu, Junichi Kawano

    A new selective medium containing cephem antibiotics was developed for isolation of methicillin-resistant Staphylococcus aureus (MRSA). MRSA colonies on a medium containing ceftazidime (CAZ) were most easily identifiable and a medium containing cefoperazone (CPZ) was superior in suppressing the growth of other bacteria. With the medium containing a couple of CAZ and CPZ, MRSA and methicillin-resistant coagulase-negative staphylococci (MRCNS) were detected from 2 and 1 of 15 chicken meat samples respectively. The M RSA and MRCNS recovery test showed that the medium was effective for MRSA isolation, suppressing the growth of other bacteria efficiently. These results suggested that the medium containing a couple of CAZ and CPZ was useful for M RSA detection from foods and animals.

    JAPAN SOC VET SCI, Sep. 2011, JOURNAL OF VETERINARY MEDICAL SCIENCE, 73 (9), 1195 - 1197, English

    [Refereed]

    Scientific journal

  • Natsumi Takeyama, Yasuhisa Ano, Guoying Wu, Nobuyoshi Kubota, Keiichi Saeki, Akikazu Sakudo, Eiichi Momotani, Katsuaki Sugiura, Masayoshi Yukawa, Takashi Onodera

    Aims: Insulinoma-associated protein 2 (IA-2) is a member of the protein tyrosine phosphatase family that is localized on the insulin granule membrane. IA-2 is also well known as one of the major autoantigens in Type 1 diabetes mellitus. IA-2 gene deficient mice were recently established and showed abnormalities in insulin secretion. Thus, detailed localization of IA-2 was studied using wild-type and IA-2 gene deficient mice.Main methods: To localize IA-2 expression in mouse neuroendocrine tissues, monoclonal antibodies were generated against IA-2 and western blot and immunohistochemical analyses were carried out in IA-2(+/+) mice. 1A-2(-/-) mice served as a negative control.Key findings: Western blot analysis revealed that the 65 kDa form of IA-2 was observed in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, pituitary gland, muscular layers of the stomach, small intestine, and colon. By immunohistochemical analysis, IA-2 was produced in endocrine cells in pancreatic islets, adrenal medullary cells, thyroid C-cells, Kulchitsky cells, and anterior, intermediate, and posterior pituitary cells. In addition, IA-2 was found in somatostatin-producing D-cells and other small populations of cells were scattered in the gastric corpus. IA-2 expression in neurites was confirmed by the immunostaining of IA-2 using primary cultured neurons from the small intestine and nerve growth factor (NGF)-differentiated PC12 cells.Significance: The IA-2 distribution in peripheral neurons appeared more intensely in neurites rather than in the cell bodies. (c) 2009 Elsevier Inc. All rights reserved.

    PERGAMON-ELSEVIER SCIENCE LTD, May 2009, LIFE SCIENCES, 84 (19-20), 678 - 687, English

    [Refereed]

    Scientific journal

  • Yasuyuki Goto, Chizu Sanjoba, Masahito Asada, Keiichi Saeki, Takashi Onodera, Yoshitsugu Matsumoto

    In murine experimental cutaneous leishmaniasis, parasite infection induces an accumulation of macrophages expressing migration inhibitory factor-related protein 8 (MRP8) and MRP14, two members of the S100 calcium-binding protein family. Although MRP8 and MRP14 are cytoplasmic proteins expressed by myeloid cells, recent studies have demonstrated that NIRP8 and MRP14 have extracellular functions such as chemotactic activities. In this study, we examined whether extracellular NIRP8 and MRP14 interact with Leishmania parasites during infection. By immunohistochemistry, positive staining by MRP8 and MRP14 was detected on amastigotes in skin lesions of Leishmania major-infected mice. Western blot analysis with amastigotes purified from the skin lesions demonstrated that both of these proteins adhered to amastigotes. The adhesion of MRP14 to amastigotes was reproduced in vitro and enhanced in the presence of Ca2+ and Zn2+. MRP14 adhered to not only amastigotes, but also promastigotes, suggesting receptor molecules for NIRI`14 are expressed commonly in both developmental stages. (C) 2008 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, May 2008, EXPERIMENTAL PARASITOLOGY, 119 (1), 80 - 86, English

    [Refereed]

    Scientific journal

  • Accumulation of macrophages expressing MRP8 and MRP14 in skin lesions during Leishmania major infection in BALB/c and RAG-2 knockout mice.

    Goto Y, Sanjoba C, Arakaki N, Okamoto M, SAEKI KEIICHI, Onodera T, Ito M, Matsumoto Y

    Mar. 2007, Parasitol Int., 56 (3), 231 - 234, English

    [Refereed]

    Scientific journal

  • Late-onset olfactory deficits and mitral cell loss in mice lacking prion protein with ectopic expression of Doppel.

    Kim CK, Sakudo A, Taniuchi Y, Shigematsu K, Kang CB, SAEKI KEIICHI, Matsumoto Y, Sakaguchi S, Itohara S, Onodera T

    Feb. 2007, Int. J. Mol. Med., 20 (2), 169 - 176, English

    [Refereed]

    Scientific journal

  • Detection of proteolytic cleavages of diabetes-associated protein IA-2beta in the pancreas and the brain using novel anti-IA-2beta monoclonal antibodies.

    Kawakami T, SAEKI KEIICHI, Takeyama N, Wu G, Sakudo A, Matsumoto Y, Hayashi T, Onodera T

    Feb. 2007, Int. J. Mol. Med., 20 (2), 177 - 185, English

    [Refereed]

    Scientific journal

  • Neurotoxic prion protein (PrP) fragment 106-126 requires the N-terminal half of the hydrophobic region of PrP in the PrP-deficient neuronal cell line.

    Akikazu Sakudo, Izuru Nakamura, Deug-Chan Lee, Keiichi Saeki, Kazuyoshi Ikuta, Takashi Onodera

    The cytotoxicity of aged PrP(106-126) was examined using an immortalized prion protein (PrP) gene-deficient neuronal cell line. The N-terminal half of the hydrophobic region (HR) but not the octapeptide repeat (OR) of PrP was required for aged PrP(106-126) neurotoxicity, suggesting that neurotoxic signals of aged PrP(106-126) are mediated by this region.

    2007, Protein and peptide letters, 14 (1), 1 - 6, English, International magazine

    [Refereed]

    Scientific journal

  • Izuru Nakamura, Guangai Xue, Akikazu Sakudo, Keiichi Saeki, Yoshitsugu Matsumoto, Kazuyoshi Ikuta, Takashi Onodera

    Susceptibility to transmissible spongiform encephalopathy and different alleles of the prion protein gene (PRNP) of humans and sheep are associated. A tentative association between PRNP promoter polymorphisms and bovine spongiform encephalopathy (BSE) susceptibility has been reported in German cattle, whereas none of the known polymorphisms within the bovine PRNP-coding sequence affect BSE susceptibility. In the present study, novel single nucleotide polymorphisms located in the 5'-flanking region of bovine PRNP affecting its expression were demonstrated in Japanese Black cattle. We sequenced exon 1, and the approximately 200-bp 5'-flanking region of the PRNP translation initiation site containing the proximal promoter of PRNP was harvested. We identified 7 single nucleotide polymorphisms: -184A -> G, -141T -> C, -85T -> G, -47C -> A, - 6C -> T, + 17C -> T and + 43C -> T. Six segregated haplotypes in the population were cloned into luciferase-expressing plasmids, transfected into N2a cells, and their reporter activities were measured 48 h after transfection. Six haplotypes showed a decreased expression level including -6C -> T in specific protein 1 binding site (p < 0.05) or -141T -> C (p < 0.01) at 48 h compared with the wild-type haplotype. These results advocate that certain polymorphisms such as specific protein 1 binding site polymorphisms in the bovine PRNP promoter region in Japanese Black cattle could influence promoter activity, suggesting that breeding cattle with such substitutions may be a useful approach in reducing BSE risk.

    KARGER, 2007, INTERVIROLOGY, 50 (3), 190 - 196, English

    [Refereed]

    Scientific journal

  • Serum withdrawal-induced apoptosis in ZrchI prion protein (PrP) gene-deficient neuronal cell line is suppressed by PrP, independent of Doppel.

    Takuya Nishimura, Akikazu Sakudo, Yoriko Hashiyama, Akiko Yachi, Keiichi Saeki, Yoshitsugu Matsumoto, Masaharu Ogawa, Suehiro Sakaguchi, Shigeyoshi Itohara, Takashi Onodera

    Previous studies have shown that cellular prion protein (PrP(C)) plays anti-apoptotic and antioxidative role against cell death induced by serum-deprivation (SDP) in an immortalized prion protein gene-deficient neuronal cell line derived from Rikn prion protein (PrP) gene-deficient (Prnp(-/-)) mice, which ectopically produce excess Doppel (Dpl) (PrP-like glycoprotein). To investigate whether PrP(C) inhibits apoptotic neuronal cell death without Dpl, an immortalized cell line was established from the brain of ZrchI Prnp(-/-) mice, which do not show ectopic expression of Dpl. The results using a ZrchI neuronal Prnp(-/-) cell line (NpL2) showed that PrP(C) potently inhibited SDP-induced apoptotic cell death. Furthermore, PrP(C) expression enhanced the superoxide dismutase (SOD) activity in NpL2 cells. These results indicate that Dpl production did not affect anti-apoptotic and anti-oxidative functions of PrP, suggesting that PrP(C) may be directly correlated with protection against oxidative stress.

    2007, Microbiology and immunology, 51 (4), 457 - 66, English, International magazine

    [Refereed]

    Scientific journal

  • Recent advances in clarifying prion protein functions using knockout mice and derived cell lines

    A Sakudo, T Onodera, Y Suganuma, T Kobayashi, K Saeki, K Ikuta

    Considerable information on the functions of prion protein (PrP) has been accumulated. One experimental approach is the use of PrP gene-knockout mice and derived cell lines. This approach has contributed to elucidating the functions of cellular prion protein (PrPC), such as its anti-oxidative and anti-apoptotic roles. This review will introduce the recent advances in prion biology made possible by, the availability of these tools.

    BENTHAM SCIENCE PUBL LTD, May 2006, MINI-REVIEWS IN MEDICINAL CHEMISTRY, 6 (5), 589 - 601, English

    [Refereed]

    Scientific journal

  • Fusion of Doppel to octapeptide repeat and N-terminal half of hydrophobic region of prion protein confers resistance to serum deprivation

    DC Lee, A Sakudo, CK Kim, T Nishimura, K Saeki, Y Matsumoto, T Yokoyama, SG Chen, S Itohara, T Onodera

    Our previous studies have shown an essential role played by the octapeptide repeat region (OR) and the N-terminal half of hydrophobic region (HR) in the anti-apoptotic activity of prion protein (PrP). As PrP-like protein Doppel (Dpl), which structurally resembles an N-terminally truncated PrP, did not show any anti-apoptotic activity, we examined apoptosis of HpL34 cells expressing Dpl fused to various lengths of the N-terminal region of PrP to investigate whether the PrP/Dpl fusion proteins retain anti-apoptotic function. HpL34 cells expressing DpI fused to PrP(1-124) with the OR and N-terminal half of HR of PrP showed anti-apoptotic function, whereas DpI fused to PrP(1-95) with OR did not rescue cells from apoptotic cell death induced by serum deprivation. These results indicate that the OR and N-terminal half of HR of PrP retains anti-apoptotic activity similar to full-length PrP.

    CENTER ACADEMIC PUBL JAPAN, 2006, MICROBIOLOGY AND IMMUNOLOGY, 50 (3), 203 - 209, English

    [Refereed]

    Scientific journal

  • BSEの分子生物学

    小野寺節、作道章一、佐伯圭一、谷内洋次郎、中村出

    2006, 医薬ジャーナル, 22, 49 - 54, Japanese

    Scientific journal

  • Bovine spongiform encephalopathy in Japan: history and recent studies on oxidative stress in prion diseases.

    Takashi Onodera, Akikazu Sakudo, Guoying Wu, Keiichi Saeki

    With the respect to BSE and vCJD, compliance with the following three rules should strictly be observed: (i) Identification and destruction of all clinically affected cattle; (ii) destruction of all mammalian proteins used in feeding ruminant livestock; and (iii) destruction of all high-risk tissues for use in human consumption. Scrapie in sheep has been documented in the 18th century in the United Kingdom. Through studies of brain-to-brain transmission in the same species in 1935, Cuille et al. successfully isolated the culprit protein from the sheep brain. To transmit said protein from an animal to another, intracerebral inoculation was much more efficient than intraperitoneal or oral route in certain species; i.e. the hamster and mouse. Since discovery of the more efficacious infection route, studies and development of prion research have undergone 4 developmental phases. Phase I depicted discoveries of the pathological features of Creutzfeldt-Jakob disease (CJD) and scrapie with typical lesions of spongiform encephalopathy, while Phase II revealed individual-to-individual (or cross-species) transmissions of CJD, kuru and scrapie in animals. Phases I and II suggested the possible participation of a slow virus in the infection process. In Phase III, Prusiner et al. proposed the 'prion' theory in 1982, followed by the milestone development of the transgenic or gene-targeted mouse in prion research in Phase IV. By strain-typing of prions, CJD has been classified as type 2 or 4 by Parchi et al. and Wadsworth as type-2 or -4 and type-1 or -2, respectively. Wadsworth type 1 is detected in the cerebellum, while Wadsworth type 2 was detected in the prefrontal cortex of 10% of sporadic CJD patients. In 1999, Puoti et al. have reported the co-existence of two types of PrP(res) in a same patient. These reports indicated that PrP(res)-typing is a quantitative rather than a qualitative process, and the relationship between the molecular type and the prion strain is rather complex. In fact, previous findings of Truchot have correlated type-1 distribution with synaptic deposits, and type-2 with arrangement of diffuse deposits in neurons. Although the normal function of PrP(C) has not been fully understood, recent studies have shown that PrP(C) plays a role in copper metabolism, signal transduction, neuroprotection and cell maturation. Further search of PrP(C)-interacting molecules and detailed studies using Prnp(-/-) mice and various type of Prnp(-/-) cell lines under various conditions are the prerequisites in elucidating PrP functions. In the pathogenesis of prion diseases, present results support the hypothesis that 'loss-of-function' of PrP(C) decreases resistance to oxidative stress, and 'gain-of-function' of PrP(Sc) increases oxidative stress. The mechanisms of (i) the 'loss-of-function' of PrP(C) in enhanced susceptibility to oxidative stress and (ii) the 'gain-of-function' of PrP(Sc) in generation of oxidative stress remain to be elucidated, although their mechanisms of action, at least in part, involve the decrease and increase in SOD activity, respectively.

    Last, 2006, Microbiology and immunology, 50 (8), 565 - 78, English, International magazine

    [Refereed]

    Scientific journal

  • Y Baba, K Saeki, T Onodera, K Doi

    We previously reported that the strain difference in the development of porcine-serum (PS)-induced rat hepatic fibrosis was closely related to the difference in the mode of MHC class-II-related genes expression. This study was carried out to clarify the serological and immunohistochemical changes in this hepatic fibrosis model. Six-week-old male Brown Norway (BN) and Wistar rats were injected with 0.5 ml of sterile PS twice a week for up to 8 weeks. The serum levels of PS-specific IgG1, IgG2a, and IgM were elevated more prominently in BN rats than Wistar rats. In the liver, significant increases in the numbers of PS-, OX-6 (RTI.B)-, CD4-, CD8, ED1-, and ED2-positive cells occurred earlier in BN rats than Wistar rats. At 8 weeks, deposition of PS and immunoglobulins was observed in hepatic fibrous septa and renal glomerular mesangium, and IgG1- and IgG2a-positive cells were found in the white pulp of the spleen. The present results suggest that Immoral immunity probably regulated by MHC class 11 molecules and inflammatory cells may be involved in PS-induced hepatic fibrosis in rats. (C) 2005 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, Dec. 2005, EXPERIMENTAL AND MOLECULAR PATHOLOGY, 79 (3), 229 - 235, English

    [Refereed]

    Scientific journal

  • 正常型プリオンタンパクの抗アポト—シス機能における構造的な機能中心の解析

    李 得燦, 作道 章一, 佐伯 圭一, 小野寺 節

    獣医生化学会, Dec. 2005, Veterinary biochemistry, 42, 19 - 23, Japanese

    Scientific journal

  • A Sakudo, DC Lee, Nakamura, I, Y Taniuchi, K Saeki, Y Matsumoto, S Itohara, K Ikuta, T Onodera

    The Prnd-encoded prion protein (PrP)-like protein, Doppel (Dpl), is a homologue of Prnp-encoded PrP, and is N-glycosylated protein with glycosylphosphatidylinositol anchor like PrP. Recently, ectopic expressions of Prnp/Prnd chimeric mRNAs have been identified as the cause of late-onset ataxia observed in several lines of Prnp-knockout mice such as ZrchII, Ngsk, Rcm0, and Rikn mice. However, it remains unclear whether the toxic effect of Dpl expression is a cell-autonomous mechanism but rather reflect a systemic process of heterogeneous cell population in the brain. In this study, the cell-autonomous role of Dpl was estimated by investigating PrP-deficient cells (HpL3-4)-the SV40 large T-antigen immortalized and Rikn Prnp(-/-) mice-derived neuronal cell line expressing Prnp/Prnd chimeric mRNAs. The reverse transcription polymerase chain reaction revealed that serum deprivation did not increase Prnp/Prnd chimeric mRNAs, which in fact was translated into a small amount of Dpl in HpL3-4 cells, whereas serum deprivation induced apoptotic cell death of HpL34 cells. Dpl overexpression enhanced apoptotic cell death, whereas the toxic effect of Dpl on apoptotic cell death was neutralized by PrP expression. These results indicate that Dpl elicited dose-dependently toxic effects on PrP-deficient cells without affecting on PrP-expressing cells, suggesting that the PrP-Dpl interaction can regulate cell death in a cell-autonomous manner. (c) 2005 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, Jul. 2005, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 333 (2), 448 - 454, English

    [Refereed]

    Scientific journal

  • WC Shyu, CP Chen, K Saeki, A Kubosaki, Y Matusmoto, T Onodera, DC Ding, MF Chiang, YJ Lee, SZ Lin, H Li

    Cellular prion protein (PrPC) expression can be regulated by heat-shock stress; and we designed the present stud to determine whether hypoglycemia could affect PrPC expression. RT-PCR and Western blotting were used to measure the expression of PrPC and heat-shock protein (Hsp70) in mouse neuroblastoma (N18) cells cultured 3 hr to 3 days in media deprived of 97.5% (L) or 75% (M) of its glucose. Hypoglycemia caused a concomitant time-dependent and glucose dose-dependent increase in PrPC and Hsp70. In addition, hypoglycemia also increased phosphorylated c-Jun N-terminal kinase (JNK) protein levels in a time-dependent manner. The upregulation of PrPC and Hsp70 under hypoglycemic conditions was disrupted by the specific JNK inhibitor SP600125. It was also found from in vitro studies that hypoglycemic conditions induced higher levels of PrPC promoter activity in PrPC promoters containing a heat-shock element (HSE) than in PrPC promoters lacking HSE. We propose that hypoglycemia-increased PrPC expression might be due to JNK phosphorylation of a heat-shock transcriptional factor, which then interacts with HSE in the promoter of PrPC. (c) 2005 Wiley-Liss, Inc.

    WILEY-LISS, Jun. 2005, JOURNAL OF NEUROSCIENCE RESEARCH, 80 (6), 887 - 894, English

    [Refereed]

    Scientific journal

  • N Vassallo, J Herms, C Behrens, B Krebs, K Saeki, T Onodera, O Windl, HA Kretzschmar

    The cellular prion protein (PrPC) is thought to be involved in protection against cell death, however the exact Cellular mechanisms involved are still controversial. Herein we present data that strongly indicate a functional link between PrPC-expression and phosphatidylinositol 3-kinase (Pl 3-kinase) activation, a protein kinase that plays a pivotal role in cell survival. Both Mouse neuroblastoma N2a cells and immortalized murine hippocampal neuronal cell lines expressing wild-type PrPC had significantly higher Pl 3-kinase activity levels than their respective controls. Moreover, PI 3-kinase activity was found to be elevated in brain lysates from wild-type mice as compared to prion protein-knockout mice. Recruitment of Pl 3-kinase by PrPC was shown to contribute to cellular survival toward oxidative stress by using 3-morpholinosydnonimine (SIN-1) and Serum deprivation. Moreover, both PI 3-kinase activation and cytoprotection by PrPC appeared to rely oil copper binding to the N-terminal octapeptide of PrPC. Thus, we propose a model in which the interaction of copper(II) with the N-terminal domain of PrPC enables transduction of a signal to PI 3-kinase: the latter. in turn, mediates downstream regulation Of Cell Survival. (c) 2005 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, Jun. 2005, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 332 (1), 75 - 82, English

    [Refereed]

    Scientific journal

  • PrP cooperates with STI1 to regulate SOD activity in PrP-deficient neuronal cell line.

    Sakudo A, Lee DC, Li S, Nakamura T, Matsumoto Y, Saeki K, Itohara S, Ikuta K, Onodera T.

    May 2005, Biochem Biophys Res Commun., 328 (1), 14 - 19, English

    [Refereed]

    Scientific journal

  • Infection route-independent accumulation of splenic abnormal prion protein.

    Inoue Y, Yamakawa Y, Sakudo A, Kinumi T, Nakamura Y, Matsumoto Y, Saeki K, Kamiyama T, Onodera T, Nishijima M.

    Mar. 2005, Jpn J Infect Dis, 58 (2), 78 - 82, English

    [Refereed]

    Scientific journal

  • Cellular prion protein suppressess the apoptotic cell death by mediating the intracellular H2O2 in primary culture and immortalized neuronal cells

    Nakamura, I, T Nishimura, K Saeki, Y Matsumoto, T Onodera

    SPRINGER-VERLAG TOKYO, 2005, Prions: Food and Drug Safety, 205 - 205, English

    International conference proceedings

  • Cellular prion protein suppresses the apoptosis in a neuronal cell line established from type-1 prion protein gene-deficient mice

    K Saeki, T Nishimura, A Sakudo, Y Matsumoto, T Onodera

    Lead, SPRINGER-VERLAG TOKYO, 2005, Prions: Food and Drug Safety, 203 - 203, English

    International conference proceedings

  • 動物の検疫および水際対策 : 牛海綿状脳症(BSE)に関する防疫措置

    佐伯 圭一, 小野寺 節

    2005, 日本水産學會誌, 69 (2), 257 - 258, Japanese

    Symposium

  • A Sakudo, DC Lee, T Nishimura, SM Li, S Tsuji, T Nakamura, Y Matsumoto, K Saeki, S Itohara, K Ikuta, T Onodera

    Cellular prion protein PrPC contains two evolutionarily conserved domains among mammals; viz., the octapeptide repeat region (OR; amino acid residue 51-90) and the hydrophobic region (HR; amino acid residue 112-145). Accumulating evidence indicates that PrPC acts as an inhibitor of apoptosis and regulator of superoxide dismutase (SOD) activity. To further understand how PrPC activates SOD and prevents apoptosis, we provide evidence here that OR and N-terminal half of HR mediate PrPC-dependent SOD activation and anti-apoptotic function. Removal of the OR (amino acid residue 53-94) enhances apoptosis and decreases SOD activity. Deletion of the N-terminal half of HR (amino acids residue 95-132) abolishes its ability to activate SOD and to prevent apoptosis, whereas that of the C-terminal half of HR (amino acids residue 124-146) has little if any effect on the anti-apoptotic activity and SOD activation. These data are consistent with a model in which the anti-apoptotic and anti-oxidative function of PrPC is regulated by not only OR but also the N-terminal half of HR. (C) 2004 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, Jan. 2005, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 326 (3), 600 - 606, English

    [Refereed]

    Scientific journal

  • T Nishimura, A Sakudo, Nakamura, I, DC Lee, Y Taniuchi, K Saeki, Y Matsumoto, M Ogawa, S Sakaguchi, S Itohara, T Onodera

    The function of cellular prion protein (PrPC), which is a copper binding protein, remains unclear. To elucidate the mechanisms in which PrPC is involved in neuroprotection, we compared death signals in prion protein gene-deficient (Prnp(-/-)) primary cerebellar granular neurons (CGNs) to those with wild-type (WT) CGNs. When copper was exposed to these CGNs, ZrchI, and Rikn Prnp(-/-) CGNs were more sensitized and underwent apoptotic cell death more readily than WT CGNs. Furthermore, the level of intracellular hydrogen peroxide (H2O2,) in WT CGNs increased by copper toxicity, whereas those in ZrchI and Rikn Prnp(-/-) CGNs did not. These results suggest that PrPC modulates the intracellular H2O2 level as a copper-binding protein to protect CGNs from apoptotic cell death possibly due to inhibiting a Fenton reaction. (C) 2004 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, Oct. 2004, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 323 (1), 218 - 222, English

    [Refereed]

    Scientific journal

  • Targeted disruption of the IA-2 beta gene causes glucose intolerance and impairs insulin secretion but does not prevent the development of diabetes in NOD mice

    A Kubosaki, S Gross, J Miura, K Saeki, M Zhu, S Nakamura, W Hendriks, AL Notkins

    Insulinoma-associated protein (IA)-2beta, also known as phogrin, is an enzymatically inactive member of the transmembrane protein tyrosine phosphatase family and is located in dense-core secretory vesicles. In patients with type I diabetes, autoantibodies to IA-2beta appear years before the development of clinical disease. The genomic structure and function of IA-2beta, however, is not known. In the present study, we determined the genomic structure of IA-2beta and found that both human and mouse IA-2beta consist of 23 exons and span similar to1,000 and 800 kb, respectively. With this information, we prepared a targeting construct and inactivated the mouse IA-2beta gene as demonstrated by lack of IA-2beta mRNA and protein expression. The IA-2beta(-/-) mice, in contrast to wild-type controls, showed mild glucose intolerance and impaired glucose-stimulated insulin secretion. Knockout of the IA-2beta gene in NOD mice, the most widely studied animal model for human type 1 diabetes, failed to prevent the development of cyclophosphamide-induced diabetes. We conclude that IA-2beta is involved in insulin secretion, but despite its importance as a major autoantigen in human type I diabetes, it is not required for the development of diabetes in NOD mice.

    AMER DIABETES ASSOC, Jul. 2004, DIABETES, 53 (7), 1684 - 1691, English

    [Refereed]

    Scientific journal

  • Hyperbaric oxygen enhances the expression of prion protein and heat shock protein 70 in a mouse neuroblastoma cell line

    WC Shyu, SZ Lin, K Saeki, A Kubosaki, Y Matsumoto, T Onodera, MF Chiang, P Thajeb, H Li

    1. Cellular prion protein, PrPC, is a ubiquitous glycoprotein strongly expressed in neurons with an as yet unknown biological function. In previous studies, we demonstrated that PrPC could be regulated by heat shock stress, implying that it might be a stress-responsive protein. Hyperbaric oxygen (HBO) administration is a well-defined model for the study of oxidative stress. 2. This study investigated the effect of HBO on PrPC and Hsp 70 expression in mouse neuroblastoma cell lines (N18), assessing the expression of PrPC and Hsp 70 using RTPCR and Western blotting. HBO administration resulted in a time- and dose-dependent increase in PrPC and Hsp70 expression in N18 cells at both mRNA and protein levels, with a concomitant upregulation of c-Jun N-terminal kinase (JNK). 3. Under HBO treatment, luciferase reporter constructs of the rat PrPC promoter, containing the heat shock element (HSE) also present in Hsp70, expressed higher luciferase activity (3- to 10-fold) than those constructs without HSE. 4. In summary, these data suggest that PrPC and Hsp 70 may be regulated by HBO, through the activation of JNK. Thus, the activated heat shock transcriptional factor 1, phosphorylated by JNK interacted with HSE in the promoter of PrPC resulted in increased gene expression. These findings are vital for future therapeutic approaches in transmissible spongiform encephalopathies and the understanding of the function of the PrPC.

    KLUWER ACADEMIC/PLENUM PUBL, Apr. 2004, CELLULAR AND MOLECULAR NEUROBIOLOGY, 24 (2), 257 - 268, English

    [Refereed]

    Scientific journal

  • Effects of tacrolimus (FK506) on encephalomyocarditic virus-induced diabetes in mice

    S Yama, W Nishioka, Y Hirokami, R Setoguchi, N Takeyama, K Saeki, Y Matsumoto, T Hayashi, K Doi, T Onodera

    The effects of tacrolimus on insulin-dependent diabetes mellitus (IDDM) induced by the D-variant of encephalomyocarditis virus (D-EMCV) have been investigated. Male BALB/c mice were treated with tacrolimus before viral inoculation, and then were inoculated with 10 plaque forming units (PFU) of D-EMCV. The mice continued to be treated with tacrolimus until the animals were sacrificed. D-EMCV-infected mice, which were treated with saline as controls, showed abnormal glucose tolerance test (GTT) values, whereas all infected mice with tacrolimus pretreatment were normal on 7 days-post inoculation (DPI). Histological observations revealed that non-treated tacrolimus D-EMCV-infected mice and which developed diabetes showed severe insulitis in their islets of Langerhans. On the other hand, D-EMCV-infected mice treated with tacrolimus were normal. In D-EMCV-infected mice, viruses in the pancreata were detected at the same level regardless of treatment with tacrolimus or saline. Expressions of TNF-alpha and IFN-gamma mRNA in spleens of tacrolimus-treated D-EMCV-infected mice were lower than that of non-treated tacrolimus D-EMCV-infected mice on 7 DPI. The results suggest that tacrolimus suppresses expressions of TNF-alpha and IFN-gamma mRNAs to prevent the onset of D-EMCV-induced IDDM.

    CENTER ACADEMIC PUBL JAPAN, 2004, MICROBIOLOGY AND IMMUNOLOGY, 48 (1), 7 - 13, English

    [Refereed]

    Scientific journal

  • A Sakudo, DC Lee, E Yoshimura, S Nagasaka, K Nitta, K Saeki, Y Matsumoto, S Lehmann, S Itohara, S Sakaguchi, T Onodera

    Prion protein (PrP) binds copper and exhibits superoxide dismutase-like activity, while the roles of PrP in copper homeostasis remain controversial. Using Zeeman graphite furnace atomic absorption spectroscopy, we quantified copper levels in immortalized PrP gene (Prnp)-deficient neuronal cells transfected with Prnp and/or Prnd, which encodes PrP-like protein (PrPLP/Dpl), in the presence or absence of oxidative stress induced by serum deprivation. In the presence of serum, copper levels were not significantly affected by the expression of PrP and/or PrPLP/Dpl, whereas serum deprivation induced a decrease in copper levels that was inhibited by PrP but not by PrPLP/Dpl. The inhibitory effect of PrP on the decrease of copper levels was prevented by overexpression of PrPLP/Dpl. These findings indicate that PrP specifically stabilizes copper homeostasis, which is perturbed under oxidative conditions, while PrPLP/Dpl overexpression prevents PrP function in copper homeostasis, suggesting an interaction of PrP and PrPLP/Dpl and distinct functions between PrP and PrPLP/Dpl on metal homeostasis. Taken together, these results strongly suggest that PrP, in addition to its antioxidant properties, plays a role in stabilizing cellular copper homeostasis under oxidative conditions. (C) 2003 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, Jan. 2004, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 313 (4), 850 - 855, English

    [Refereed]

    Scientific journal

  • Serum thymic factor prevents LPS-induced pancreatic cell damage in mice via up-regulation of Bcl-2 expression in pancreas

    N Saitoh, A Awaya, A Sakudo, SS Wook, K Saeki, Y Matsumoto, T Onodera

    The protective effect of synthetic serum thymic factor (FTS) nonapeptide on lipopolysaccharide (LPS)-induced pancreatic cell damage in 10-week-old BALB/c male mice was investigated. Mice were divided into three groups. Group I was treated with LPS (10 mug/head; i.p.) (LPS-treated mice). Group II was administered with FTS (50 mug/head; i.p.) 24 hr before treatment with LPS and complemented immediately before LPS injection with FTS (50 mug/head; i.p.) (FTS-administered mice). Group III was only treated with the same volume of saline (control mice). Treatment of LPS in vivo resulted in the destruction of pancreatic acinar cells. In those cells, many apoptotic cells were detected by immunohistochemistry using an anti-single stranded DNA antibody. Immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) revealed that LPS treatment also caused low or a lack of insulin expression in pancreatic islets. In contrast, morphological change was not seen and apoptotic cell death was suppressed in pancreatic cells of FTS-administered mice. Moreover, insulin expression was normal in those mice. FTS administration enhanced expression of Bcl-2 mRNA levels in pancreatic tissues and IL-6 mRNA levels in splenocytes significantly compared with those of LPS treatment at 3 hr after LPS injection. These findings suggest that FTS prevents LPS-induced cell damage via enhancing Bcl-2 expression in the pancreas and systemic IL-6 production.

    CENTER ACADEMIC PUBL JAPAN, 2004, MICROBIOLOGY AND IMMUNOLOGY, 48 (9), 629 - 638, English

    [Refereed]

    Scientific journal

  • Y Nakamura, A Sakudo, K Saeki, T Kaneko, Y Matsumoto, A Toniolo, S Itohara, T Onodera

    The susceptibility of prion protein gene (Prnp)-null cells to coxsackievirus B3 (CVB3) was investigated. Primary cultures of murine Prnp(-/-) brain cells were more sensitive to CVBs than corresponding cells from wild-type mice. The viral susceptibility of a Prnp-null cell line (HpL3-4) derived from the murine hippocampus was compared with that of two established cell lines (HeLa. and HEp-2) that are widely employed for CVB3 studies. After infection with CVB3, HpL3-4 cells showed a very rapid and complete cytopathic effect (CPE). CPE developed earlier and viruses replicated at higher titres in HpL3-4 cells compared with HeLa and HEp-2 cells. Under a semi-solid medium, plaques developed rapidly in CVB3-infected HpL3-4 cells. To confirm the effect of Prnp on virus infection, a Prnp(-/-) cell line and a Prnp-transfected neuronal cell line were analysed. The replication and release of infectious particles of CVB3 in Prnp(-/-) cells were significantly more effective than those of the Prnp-transfected cell line. Levels of type I interferon (IFN) after CVB3 infection were higher in the Prnp-transfected cell line than in Prnp(-/-) cells, whereas apoptotic cells were more obvious in the Prnp(-/-) cells than in those of the Prnp-transfected cell line. These findings suggest that the absence of Prnp retards the induction of CVB3-induced IFNs, resulting in an enhanced CVB3 production and apoptotic cell death. Furthermore, our data indicate that the HpL3-4 cell line may provide a novel and sensitive system for isolation of CVB3 from clinical specimens.

    SOC GENERAL MICROBIOLOGY, Dec. 2003, JOURNAL OF GENERAL VIROLOGY, 84, 3495 - 3502, English

    [Refereed]

    Scientific journal

  • J Yasuda, W Nishioka, A Sakudo, S Yama, R Setoguchi, K Saeki, Y Matsumoto, A Awaya, T Onodera

    Tumor necrosis factor-alpha (TNF-alpha) is a cytokine considered to play a key role in beta-cell destruction in insulin-dependent diabetes mellitus (IDDM). Serum thymic factor (Facteur thymique serique; FTS) is a nonapeptide thymus hormone known to inhibit IDDM in a mouse model. In this study, the effect of TNF-alpha on the murine pancreatic beta-cell line MIN6 was examined. Cell shrinkage and detachment were seen in cells treated with 0-50 ng/ml TNF-alpha for 12 h. Oligonucleosomal DNA fragmentation was determined from non-adherent cells, indicating that the TNF-alpha-induced cell destruction was attributed to apoptosis. Fragmented DNA was quantified by enzyme-linked immunosorbent assay to measure the amount of histone-bound oligonucleosomes. FTS was treated with TNF-alpha and the percentage of fragmented DNA was analyzed. The data indicate a distinct reduction of fragmented DNA at a concentration of 1 ng/ml FTS. Expression of TNF receptor I, inducible form of nitric oxide synthase (iNOS), interleukin-1beta-converting enzyme (ICE), Bcl-2, and nuclear factor KB (NF-kappaB) was analyzed by reverse transcriptase-polymerase chain reaction to investigate the suppressor mechanism of FTS on TNF-alpha-induced apoptosis. FTS treatment suppressed the expression of iNOS and Bcl-2 mRNA in TNF-alpha-treated cells. The expression of NF-kappaB mRNA in TNF-alpha-treated cells was enhanced after FTS treatment, while that of ICE mRNA did not change in TNF-alpha-treated cells with or without FTS treatment. These results suggest that the inhibition of MIN6 cell death by FTS on TNF-alpha-induced apoptosis is caused by a negative feedback mechanism involving the inhibition of iNOS induction. (C) 2003 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, Nov. 2003, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 311 (2), 501 - 505, English

    [Refereed]

    Scientific journal

  • A Sakudo, DC Lee, K Saeki, Y Matsumoto, S Itohara, T Onodera

    Prion protein gene (Prnp)-deficient(Prnp(-/-)) neuronal cells are more susceptible to serum deprivation compared to Prnp(+/+) neuronal cells. However, little is known about the cell death of Prnp(-/-) neuronal cells under serum deprivation. In this study, as a known neuroprotective agent we analyzed the effect of tumor necrosis factor-alpha (TNF-alpha) on the cell death of Prnp(-/-) neuronal cells. Although expression of Bcl-2 and Bcl-x(L) decreased in a time-dependent manner under serum deprivation, treatment with TNF-alpha protected Prnp(-/-) neuronal cells from serum deprivation with an increase in anti-apoptotic proteins Bcl-2 and Bcl-x(L). Nuclear morphological analysis using fluorescence microscopy and flow cytometry analysis showed that gene transfer of bcl-2 or bcl-x(L) significantly inhibited apoptosis induced by serum deprivation. These findings indicate that TNF-alpha attenuated cell death of Prnp(-/-) neuronal cells by induction of Bcl-2 and Bcl-x(L),and that decreases in Bcl-2 and Bcl-x(L) played crucial roles in the apoptosis of Prnp(-/-) neuronal cells. (C) 2003 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, Oct. 2003, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 310 (3), 725 - 729, English

    [Refereed]

    Scientific journal

  • A Kawagishi, A Kubosaki, N Takeyama, A Sakudo, K Saeki, Y Matsumoto, T Hayashi, T Onodera

    Encephalomyocarditis (EMC) virus induces insulin-dependent diabetes and myocarditis in several strains of mice. The T-cell receptor (TCR) Vbeta genes of infiltrating T cells in the pancreas and myocardium of BALB/C mice infected with EMC virus D-variant (EMC-D virus) were analyzed. Using a nested two-step polymerase chain reaction (PCR), TCR Vbeta cDNAs were cloned and sequenced. Two and four kinds of TCR Vbeta clones were obtained from T cells infiltrating into the pancreas and myocardium of BALB/ C mice infected with EMC-D virus, respectively. The infiltrating lymphocytes in the diabetic mice expressed Vbeta 8.1, 8.2, and 8.3 genes predominantly. Previously, the use of Vbeta 8.2 has been reported in autoimmune diseases such as murine experimental allergic encephalomyelitis (EAE) and non-obese diabetic (NOD) mouse. This study suggests that mice infected with EMC virus are a useful animal model for autoimmune diseases such as insulin-dependent diabetes. (C) 2003 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, Oct. 2003, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 310 (3), 791 - 795, English

    [Refereed]

    Scientific journal

  • A Sakudo, M Hamaishi, T Hosokawa-Kanai, K Tuchiya, T Nishimura, K Saeki, Y Matsumoto, S Ueda, T Onodera

    A method for expression and purification of a soluble form of histidine (HIS)-tagged murine prion protein (bacMuPrP), which lacks the entire C-terminal cleavage and glycosyl phosphatidyl inositol (GPI) addition site, has been developed using a recombinant baculovirus expression system and purification with Ni-NTA agarose affinity chromatography. In mammalian sources, PrPC is attached to the cell membrane by a GPI anchor. However, in our system, bacMuPrP was secreted into the media, enabling its easy purification in abundance. Indirect immunofluorescence studies and immunoblot analysis localized not in cell membrane but in the perinuclear endoplasmic reticulum region in cells and is secreted into the media. Tunicamycin treatment revealed non-glycosylated proteins were secreted into the media, suggesting that glycosylation is not necessary for bacMuPrP secretion. Density-gradient sedimentation analysis demonstrated a sedimentation coefficient of secretory bacMuPrP as 2.3 S, indicating a monomeric form. Although affinity-purified PrP from mouse brain or recombinant prion protein (PrP) produced by Escherichia coli and refolded in the presence of copper has been reported to display superoxide dismutase (SOD) activity, bacMuPrP did not show SOD activity. These results suggest that bacMuPrP has a different biochemical and biophysical characterization from mammalian and bacterial-derived PrP. Furthermore, this simple expression system may provide an adequate source for structural, functional, and biochemical analyses of PrP. (C) 2003 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, Aug. 2003, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 307 (3), 678 - 683, English

    [Refereed]

    Scientific journal

  • A Kubosaki, Y Nishimura-Nasu, T Nishimura, S Yusa, A Sakudo, K Saeki, Y Matsumoto, S Itohara, T Onodera

    The purpose of this report was to determine the effect of prion protein (PrP) gene disruption on T lymphocyte function. Previous studies have suggested that normal cellular prion protein (PrPc) binds to copper and Cu2+ is essential for interleukin-2 (IL-2) mRNA synthesis. In this study, IL-2 mRNA levels in a copper-deficient condition were investigated using T lymphocytes from prion protein gene-deficient (PrP0/0) and wild-type mice. Results showed that Cu2+ deficiency had no effect on PrPc expression in Con A-activated splenocytes. However, a delay in IL-2 gene expression was observed in PrP0/0 mouse T lymphocyte cultures using Con A and Cu2+-chelator. These results suggest that PrPc expression may play an important role in rapid Cu2+ transfer in T lymphocytes. The rapid transfer of Cu2+ in murine T lymphocytes could be one of the normal functions of PrPc. (C) 2003 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, Aug. 2003, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 307 (4), 810 - 813, English

    [Refereed]

    Scientific journal

  • A Sakudo, DC Lee, K Saeki, Y Nakamura, K Inoue, Y Matsumoto, S Itohara, T Onodera

    Previous studies have reported a neuroprotective role for cellular prion protein (PrPC) against apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line, but the mechanisms remain unclear. In this study, to investigate the mechanisms by which PrPC prevents apoptosis, the authors compared apoptosis of Prnp(-/-) cells with that of Prnp(-/-) cells expressing the wild-type PrPC or PrPC lacking N-terminal octapeptide repeat region under serum-free conditions. Re-introduction of Prnp rescued cells from apoptosis, upregulated superoxide dismutase (SOD) activity, enhanced superoxide anion elimination, and inhibited caspase-3/9 activation. On the other hand, N-terminally truncated PrPC enhanced apoptosis accompanied by potentiation of superoxide production and caspase-3/9 activation due to inhibition of SOD. These results suggest that PrPC protects Prnp(-/-) cells from apoptosis via superoxide- and caspase-3/9-dependent pathways by upregulating SOD activity. Furthermore, the octapeptide repeat region of PrPC plays an essential role in regulating apoptosis and SOD activity. (C) 2003 Elsevier Inc. All rights reserved.

    ACADEMIC PRESS INC ELSEVIER SCIENCE, Aug. 2003, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 308 (3), 660 - 667, English

    [Refereed]

    Scientific journal

  • 神経内分泌組織におけるIA-2発現解析

    竹山 夏実, 川上 登美子, 佐伯 圭一, 松本 芳嗣, 小野寺 節

    2003, 日仏生物学会誌, 43 (1), English

    Symposium

  • 動物のプリオン病

    佐伯圭一

    2003, 最新医学, 58 (5), 1008 - 1013, Japanese

    [Invited]

    Scientific journal

  • スクレイピー

    佐伯圭一, 小野寺節

    Lead, Oct. 2002, 医学のあゆみ, 203 (10), 951 - 954, Japanese

    Scientific journal

  • Molecular modulation of expression of prion protein by heat shock

    WC Shyu, HJ Harn, K Saeki, A Kubosaki, Y Matsumoto, T Onodera, CJ Chen, YD Hsu, YH Chiang

    Prion diseases (also known as transmissible spongiform encephalopathies) are associated with the conversion of the normal cellular form of the prion protein (PrPC) to an abnormal scrapie-isoform (PrPSc. The conversion of PrPC to PrPSc is post-translational and is owing to protein conformational change. This has led to the hypothesis that molecular chaperones may be involved in the folding of prion proteins, and hence the disease process. By treating human NT-2 cells with heat-shock stress, we found that both the mRNA levels for prion protein (PrP) and heat shock protein 70 (HSP7Q) increased simultaneously after heat treatment. Western-blot analysis of PrP also showed a two-fold increase in PrP protein level 3 after heat treatment. Furthermore, two heat-shock elements (HSEs) were located at the positions of -680 bp (HSE1; GGAACTATTCTTGACATTGCT), and -1653 bp (HSE2; TGAGAACTCAGGAAG) of the rat PrP (RaPrP) gene promoter. Luciferase reporter constructs of the RaPrP promoter with HSE expressed higher luciferase activity (10- to 15-fold) than those constructs without HSE. Electrophoretic gel mobility shift assay (EMSA) and super-shift assay confirmed the interaction of HSE1 and HSE2 with the heat-shock transcription factor-1 (HSTF-1). These results suggest that cellular stress up-regulates both the transcription and translation of PrP through interaction with the HSEs on the PrP gene promoter, resulting in an increase in protein synthesis.

    HUMANA PRESS INC, Aug. 2002, MOLECULAR NEUROBIOLOGY, 26 (1), 1 - 12, English

    [Refereed]

    Scientific journal

  • Targeted disruption of the protein tyrosine phosphatase-like molecule IA-2 results in alterations in glucose tolerance tests and insulin secretion

    K Saeki, M Zhu, A Kubosaki, JP Xie, MS Lan, AL Notkins

    IA-2 is a major autoantigen in type 1 diabetes. Autoantibodies to IA-2 appear years before the development of clinical disease and are being widely used as predictive markers to identify individuals at risk for developing type 1 diabetes. IA-2 is an enzymatically inactive member of the transmembrane protein tyrosine phosphatase family and is an integral component of secretory granules in neuroendocrine cells. To study its function, we generated IA-2-deficient mice. Northern and Western blot analysis showed that neither IA-2 mRNA nor protein was expressed. Physical examination of the IA-2 'animals and histological examination of tissues failed to reveal any abnormalities. Nonfasting blood glucose levels, measured over 6 months, were slightly elevated in male IA-2(-/-) as compared to I-A-2(+/+) littermates, but remained within the nondiabetic range. Glucose tolerance tests, however, revealed statistically significant elevation of glucose in both male and female IA-2(-/-) mice and depressed insulin release. In vitro glucose stimulation of isolated islets showed that male and female mice carrying the disrupted gene released 48% (P < 0.001) and 42% (P < 0.01) less insulin, respectively, than mice carrying the wild-type gene. We concluded that IA-2 is involved in glucose-stimulated insulin secretion.

    AMER DIABETES ASSOC, Jun. 2002, DIABETES, 51 (6), 1842 - 1850, English

    [Refereed]

    Scientific journal

  • Targeted disruption of the protein tyrosine phosphatase-like molecule IA-2 results in alterations in glucose tolerance tests and insulin secretion

    AL Notkins, K Saeki, M Zhu, JP Xie, MS Lan, A Kubosaki

    Lead, AMER DIABETES ASSOC, Jun. 2002, DIABETES, 51 (6), A51 - A51, English

    [Refereed]

    Scientific journal

  • S Shimizu, N Saito, A Kubosaki, S SungWook, N Takeyama, T Sakamoto, Y Matsumoto, K Saeki, Y Matsumoto, T Onodera

    Islet antigen (IA)-2 is a novel autoantigen of insulin-dependent diabetes mellitus (IDDM), and belongs to a new class within the receptor-type protein tyrosine phosphatase (PTP) family characterized by lack of PTP enzymatic activity with conventional substrates. Its expression is restricted primarily to the pancreas, pituitary, and brain with the highest level in the brain. IA-2 mRNA expressions in the brain, pituitary and pancreas of 1-, 4-, and 8-week-old mice were examined. In situ hybridization of the brain revealed that IA-2 mRNA was expressed in the cerebral cortex, hippocampus, thalamus, choroid plexus, hypothalamus, Purkinje cells, and granular layer of the cerebellum. In the pituitary, IA-2 mRNA was located in the anterior and posterior pituitary by in situ hybridization. The pattern of IA-2 mRNA expression in normal male mouse brain at 1, 4, and 8 weeks of age by the Northern blot analysis was similar to that in the pituitary by RT-PCR analysis. The expression level was higher at 4 weeks and lower at I week of age. In the pancreas, IA-2 mRNA expressions detected by RT-PCR were highest at 8 weeks of age. These results indicated that the amount of mRNA expression increased in accordance to development in brain, pituitary, and pancreas. (C) 2001 Academic Press.

    ACADEMIC PRESS INC, Oct. 2001, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 288 (1), 165 - 171, English

    [Refereed]

    Scientific journal

  • Sung Wook Seo, Kazuhiro Hara, Atsutaka Kubosaki, Yukiko Nasu, Takuya Nishimura, Keiichi Saeki, Yoshitsugu Matsumoto, Hideki Endo, Takashi Onodera

    The prion protein (PrP) nucleotide sequences of two ruminants were determined in order to elucidate the differences in susceptibility to spongiform encephalopathy agents in each species. The nucleotide sequences of PrP coding regions of the moufflon and the golden takin encompassed 771 bp in length. The PrP gene sequences of the golden takin were closely related to those of sheep with one amino acid difference. The PrP gene sequence of the moufflon was identical to that of sheep. The similarities between the PrP genes of these two animals and sheep imply that the species barriers between these animals are small or non-existent. These PrP genes could be used to establish transgenic mice with higher susceptibility to prion-related diseases. Copyright© 2002 S. Karger AG, Basel.

    2001, Intervirology, 44 (6), 359 - 363, English

    [Refereed]

    Scientific journal

  • Genomic structure of mouse IA-2: comparison with its human homologue

    K Saeki, J Xie, AL Notkins

    Aims/hypothesis. IA-2 is a transmembrane protein with a tyrosine phosphatase (PTP)-like structure and a major autoantigen in Type I (insulin-dependent) diabetes mellitus. Because the nucleotide sequence of human and mouse IA-2 cDNA are closely related, it seemed likely that the genomic organization of the two molecules would be similar. To test this possibility the current experiments were initiated to characterize and compare the genomic structure of mouse and human IA-2. Methods. IA-2 cDNA was used to screen a 129SVJ mouse genomic library. We selected and mapped 7 overlapping clones. The subcloned inserts were used to determine intron-exon junctions by direct sequencing. Polymerase chain reaction and restriction mapping were used to estimate the size of the introns. Results. The mouse IA-2 gene and the 5' upstream regulatory region were isolated and the intron-exon junctions determined. Mouse IA-2 encompasses approximately 20 kb and encodes 23 exons. Both the 3' and 5' ends were mapped by rapid amplification of cDNA ends (RACE) and a 2 kb 5'-upstream region was shown to have functional promoter activity. Conclusion/interpretation. Comparison of the genomic structure of mouse and human IA-2 shows that they have the same number of exons and nearly identical intron-exon junctions. The region around the major transcription start site of mouse IA-2 is similar to human IA-2 and other transmembrane protein tyrosine phosphatases. It is concluded that human and mouse IA-2 are highly conserved and derived from a common ancestral gene.

    Lead, SPRINGER-VERLAG, Nov. 2000, DIABETOLOGIA, 43 (11), 1429 - 1434, English

    [Refereed]

    Scientific journal

  • Analysis of prion protein mRNA by in situ hybridization in brain and placenta of sheep

    A Kubosaki, A Ueno, Y Matsumoto, K Doi, K Saeki, Y Matsumoto, T Onodera

    In this study, prion protein (PrP) mRNA was focally detected in brain and placenta of pregnant sheep by Northern blot analysis. In addition, host-encoded cellular prion protein (PrPc) was observed in brain and placenta of the ruminant by Western blot analysis as well. Localization of PrP mRNA in pregnant sheep tissues was rendered possible with in situ hybridization. In sheep brain, PrP mRNA was predominantly localized within large neocortical neurons in the cerebrum, Purkinje cells and neurons of the molecular and granule cell layers in the cerebellum, In the placenta, signals were observed in the myometrium, including stratum longitudinale tunicae muscles and circular layers of muscular tunics. In the caruncle and placentome, signals were stronger by in situ hybridization. Since accumulation of the scrapie isoform PrP (PrPSc) is required to PrPc, these results suggest that brain and placenta of sheep may be important organs and sites for the conversion of PrPc to PrPSc. (C) 2000 Academic Press.

    ACADEMIC PRESS INC, Jul. 2000, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 273 (3), 890 - 893, English

    [Refereed]

    Scientific journal

  • Japanese scrapie cases

    T Onodera, K Saeki

    Worldwide attention has been given to scrapie, because bovine spongiform encephalopathy (BSE) could be experimentally transmitted to sheep. This ovine form of BSE was clinically identical to scrapie. In Japanese scrapie cases, a majority of the diseased sheep were from Suffolk, while 8 cases were from Corriedale. It is very likely that sheep-to-sheep transmission of scrapie has taken place in Obihiro, Hokkaido. Normal prion protein may play a role in the morphoregulatory signaling pathway, which orchestrates the specificity of a particular cellular response. Over-expression of normal prion protein in mice cause neurodegenerative disorders. Recently, Prnd was identified downstream of the mouse prion protein gene (Prnp), and encodes 179 amino acids and a prion protein (PrP)-like protein designated doppel (Dpl). Dpl was upregulated in the central nervous system of two PrP-deficient lines of mice, as well as in prionless cell lines. Dpl caused neurodegeneration similar to that caused by PrP. Linked expression of Prnp and Prnd may cause several neurodegenerative disorders.

    Last, NATL INST INFECTIOUS DISEASES, Apr. 2000, JAPANESE JOURNAL OF INFECTIOUS DISEASES, 53 (2), 56 - 61, English

    [Refereed]

    Scientific journal

  • Enhanced expression of cellular prion protein gene by insulin or nerve growth factor in immortalized mouse neuronal precursor cell lines

    C Kuwahara, A Kubosaki, T Nishimura, Y Nasu, Y Nakamura, K Saeki, Y Matsumoto, T Onodera

    In order to understand the fundamental and putative roles of PrPc in the central nervous system, neuronal cell lines were established. Cells were immortalized by recombinant retrovirus vector-mediated transduction of SV40 T-antigen gene,Among these, two cell lines were selected based on their RT-PCR expressions of neuron-specific neurofilament (NF-H, NF-M) and cell morphology. These cell lines showed the properties of neuronal progenitor cells in antigenicity, morphology and responses to differentiating agents. Expression of PrPc was detected by immunocytochemical analysis. These cell lines responded to differentiating agents such as dibutyl cyclic AMP (dcAMP) and phorbol 12-myristate 13-acetate (PMA) before developing into neuronal-like cells. Neurite extensions were observed 20 min after incubation with the differentiating agents. Treatment with nerve growth factor (NGF) and insulin induced cell differentiation and enhanced expression of PrP gene (Prnp) mRNA and protein. The latter phenomenon was not inhibited by wortmannin, which is a specific inhibitor of phosphatidylinositol 3-kinase. These results suggest that PrPc plays an important role in the differentiation-mediated classic signaling pathway of neuronal cell. (C) 2000 Academic Press.

    ACADEMIC PRESS INC, Feb. 2000, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 268 (3), 763 - 766, English

    [Refereed]

    Scientific journal

  • プリオンレスマウス不死化神経細胞におけるEMCV-Bの増殖

    那須 教子, 西村 拓也, 井上 敬一, 糸原 重美, 佐伯 圭一, 松本 芳嗣, 小野寺 節

    2000, 日仏生物学会誌, 40 (1), English

    Symposium

  • Prions prevent neuronal cell-line death

    C Kuwahara, AM Takeuchi, T Nishimura, K Haraguchi, A Kubosaki, Y Matsumoto, K Saeki, Y Matsumoto, T Yokoyama, S Itohara, T Onodera

    MACMILLAN MAGAZINES LTD, Jul. 1999, NATURE, 400 (6741), 225 - 226, English

    [Refereed]

    Scientific journal

  • The effect of Serum Thymic Factor on TNF-α-induced apoptosis in mouse pancreatic beta cell line(MIN6)

    NISHIOKA Wakako, YASUDA Jyunko, AWAYA Akira, SAEKI Keiichi, MATSUMOTO Yoshinori, ONODERA Takashi

    1999, 日仏生物学会誌, 39 (1), 10 - 10, English

    [Refereed]

    Symposium

  • Difference in Bgp-independent fusion activity among mouse hepatitis viruses

    F Taguchi, S Matsuyama, K Saeki

    Mouse hepatitis virus (MHV) utilizes a mouse biliary glycoprotein (Bgp) as a receptor. Co-cultivation of MHV-nonpermissive hamster BHK cells devoid of mouse Bgp with mouse DBT cells infected with MHV-A59 or JHMV induces syncytia formation on BHK cells (Bgp-independent fusion). This study shows the difference in Bgp-independent fusion activity among various MHV strains. Under a phase contrast microscopy, JHMV (cl-2, sp-4) induced the B-independent syncytia on BHK cells similar to those observed on DBT cells, while such syncytia were not seen with the infection of other MHV strains (MHV-I, MHV-3, MHV-A59, MHV-S, srr7, srr11 and srr18). Tiny syncytia detectable only by immunofluorescence were produced with the latter MHV strains except for srr7 which failed to produce syncytia. MHVs except for srr7 grew in BHK cells after Bgp-independent infection. The Bgp-independent fusion by JHMV was inhibited either by anti-S1 or anti-S2 antibodies. These results showed that the JHMV spike protein had a remarkably high Bgp-independent fusion activity.

    Last, SPRINGER-VERLAG WIEN, 1999, ARCHIVES OF VIROLOGY, 144 (10), 2041 - 2049, English

    [Refereed]

    Scientific journal

  • Expression of ovine PrPc mRNA by in situ hybridization.

    Kubosaki A, Kuwahara C, Saeki K, Matsumoto Y, Onodera T

    1998, Anim. Biochem, 35, 19 - 23, Japanese

    Scientific journal

  • Differential receptor-functionality of the two distinct receptor proteins for mouse hepatitis virus

    N Ohtsuka, YK Yamada, K Saeki, F Taguchi

    We compared the virus-binding activity and receptor-functionality of the receptor proteins isolated from mouse hepatitis virus (MHV)-susceptible BALB/c mice (MHVR1) and MI-IV-resistant SJL mice (MHVR2). By using a soluble receptor protein which lacked the transmembrane and intracytoplasmic domains, virus overlay protein blot assay and neutralization tests showed that MHVR1 bound to JHM cl-2 virus with 30-500 times higher efficiency than to MHVR2. MHVR1 was revealed to have 10-30 fold higher receptor-functionality than MHVR2 when examined by measuring virus-binding to the receptor expressed on the cell surface. These findings suggested that the differences in susceptibility between BALB/c and SJL mice may depend upon the genotype of the MI-IV receptor.

    PLENUM PRESS DIV PLENUM PUBLISHING CORP, 1998, CORONAVIRUSES AND ARTERIVIRUSES, 440, 77 - 80, English

    Scientific journal

  • Isolation and characterization of murine coronavirus mutants resistant to neutralization by soluble receptors

    K Saeki, N Ohtsuka, F Taguchi

    Murine coronavirus mutants resistant to neutralization with soluble receptors were isolated to study the receptor-binding site on the S proteins since such mutants were expected to have mutations in an important site for receptor-binding. We have isolated five soluble receptor-resistant (srr) mutants which had mutations of a single amino acid at 3 different positions in S protein. Srr mutant 11 with an amino acid change at position 65 (Leu to His) in the S1 subunit showed an extremely reduced binding by virus overlay protein blot assay. However srr mutants with a mutation at 1114 (Leu to Phe) (srr mutants 3, 4 and 7) or 1163 (Cys to Phe) (srr mutant 18) in the S2 subunit had receptor-binding activity similar to that of wild type cl-2. These results suggest that an amino acid at position 62 located in a conserved region among MHV strains is in particular important for receptor binding. We also discuss why srr mutants with a mutation in S2 showed high resistance to neutralization by soluble receptor, irrespective of their binding to MHV receptors.

    Lead, PLENUM PRESS DIV PLENUM PUBLISHING CORP, 1998, CORONAVIRUSES AND ARTERIVIRUSES, 440, 11 - 16, English

    Scientific journal

  • Identification of spike protein residues of murine coronavirus responsible for receptor-binding activity by use of soluble receptor-resistant mutants

    K Saeki, N Ohtsuka, F Taguchi

    We previously demonstrated by site-directed mutagenesis analysis that the amino acid residues at positions 62 and 214 to 216 in the N-terminal region of mouse hepatitis virus (MHV) spike (S) protein are important for receptor-binding activity (H. Suzuki and F. Taguchi, J. Virol. 70:2632-2636, 1996). To further identify the residues responsible for the activity, we isolated the mutant viruses that were not neutralized with the soluble form of MHV receptor proteins, since such mutants were expected to have mutations in amino acids responsible for receptor-binding activity, Five soluble-receptor-resistant (srr) mutants isolated had mutations in a single amino acid at three different positions: one was at position 65 (Leu to His) (srr11) in the S1 subunit and three were at position 1114 (Leu to Phe) (srr3, srr4, and srr7) and one was at position 1163 (Cys to Phe) (srr18) in the S2 subunit. The receptor-binding activity examined by a virus overlay protein blot assay and by a coimmunoprecipitation assay showed that srr11 S protein had extremely reduced binding activity, while the srr7 and srr18 proteins had binding activity similar to that of wild-type cl-2 protein. However, when cell surface receptors were used for the binding assay, all srr mutants showed activity similar to that of the wild type or only slightly reduced activity, These results, together with our previous observations, suggest that amino acids located at positions 62 to 65 of S1, a region conserved among the MHV strains examined, are important for receptor-binding activity, We also discuss the mechanism by which srr mutants with a mutation in S2 showed high resistance to neutralization by a soluble receptor, despite their sufficient level of binding to soluble receptors.

    Lead, AMER SOC MICROBIOLOGY, Dec. 1997, JOURNAL OF VIROLOGY, 71 (12), 9024 - 9031, English

    [Refereed]

    Scientific journal

  • Epidemiological analysis of ovine scrapie in Japan.

    Onodera T, Saeki, K

    Last, 1997, Bull. Soc. Franco-Japonais Sci. Vet., 8 (1), 3 - 7, English

    International conference proceedings

  • Identification of a promoter region in the rat prion protein gene

    K Saeki, Y Matsumoto, Y Matsumoto, T Onodera

    We have demonstrated the presence of a rat prion protein (RaPrP) gene promoter upstream of multiple initiation sites. A 0.1-kb fragment upstream of the 5'-untranslated region contains specific DNA motifs characteristic of promoter elements including an AP-1 binding site, an inverted CCAAT motif and three inverted Sp-l binding sites. This fragment directs transcription of a luciferase reporter gene in pheochromocytoma cells (PC12) and rat glioma cells (C6), suggesting that it contains the promoter for the RaPrP gene. To more precisely localize the transcription regulatory elements in this region, a series of 5'-deletion mutants were generated. Deletion analysis showed that an inverted CCAAT and adjoining Sp-l binding sequences may play an important role in transcription of the RaPrP gene. (C) 1996 Academic Press, Inc.

    Lead, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, Feb. 1996, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 219 (1), 47 - 52, English

    [Refereed]

    Scientific journal

  • Three-exon structure of the gene encoding the rat prion protein and its expression in tissues

    K Saeki, Y Matsumoto, Y Hirota, Y Matsumoto, T Onodera

    The prion protein (PrP), encoded by a chromosomal gene, is associated with development of the neurodegeneration of prion-induced diseases. Since determination of the complete structure of the gene encoding PrP is important for understanding gene expression in the central nervous system (CNS), the nucleotide (nt) sequence of the isolated whole gene encoding rat PrP (raPrP) was determined. The rat PUP gene (raPrP) spans 16 kilobases (kb) of the rat genome and contains three exons of 19-47 base pairs (bp), 98 bp, and 2 kb separated by two introns of 2.2 kb and 11 kb. The first and second exons are noncoding, while the third exon contains a short 5' untranslated region, the entire 762-bp open reading frame (ORF), and a 3' untranslated region. The putative raPrP promoter in the 5' flanking region contains putative Spl, AP-1, and AP-2 binding sites without a consensus TATA box. This TATA box-deficient feature, coupled with the presence of a high G + C content and Spl-binding sites in the raPrP promoter, characterizes it as a housekeeping gene. Analysis of the raPrP cDNA 5'-end showed that raPrP mRNA transcription was initiated at multiple sites, Northern blot analysis showed that the levels of raPrP mRNA varied among rat tissues, with the highest levels found in the brain and placenta. This determination of raPrP nt sequences, including the introns and the 5' and 3' flanking regions, may make it possible to elucidate cis-acting elements that regulate the expression of this gene in different tissues and cell lines.

    Lead, KLUWER ACADEMIC PUBL, 1996, VIRUS GENES, 12 (1), 15 - 20, English

    [Refereed]

    Scientific journal

  • Analysis of PrPc mRNA by in situ hybridization in brain, placenta, uterus and testis of rats

    K Tanji, K Saeki, Y Matsumoto, M Takeda, K Hirasawa, K Doi, Y Matsumoto, T Onodera

    An amyloid-like isoform of a 33- to 34-kD glycoprotein, termed as the scrapie prion protein (PrPsc), plays a critical role in transmissible spongiform encephalopathies of animals and humans. It has even been suggested to present the responsible infectious agent. This protein is a posttranslationally modified form of the cellular isoform of prion protein (PrPc). Hitherto, little has been known about the functions of PrPc. In order to examine the localization of PrPc mRNA in rat tissues, the in situ hybridization technique was performed. In rat brain, PrPc mRNA was predominantly localized within pyramidal cells of the hippocampus, large neurons of the thalamus and neocortex, and Purkinje cells of the cerebellum. In the placenta, not only PrPc mRNA was localized to a subpopulation of decidual cells at the highest levels, it was also expressed in the amnion and mesodermal layer of the yolk sac. Furthermore, PrPc mRNA was also expressed in the myometrium of the uterus and seminiferous tubule in the testis. However, signals were not obtained in the lung, spleen, liver of prenatals and other fetus tissues. The distribution of rat PrPc mRNA portrayed the levels which were different among the various types of cells, suggesting that its expression may be regulated in a tissue-specific manner.

    KARGER, Nov. 1995, INTERVIROLOGY, 38 (6), 309 - 315, English

    [Refereed]

    Scientific journal

  • ESTABLISHMENT OF HIPPOCAMPAL CELL-LINES OF MICE HOMOZYGOUS FOR TARGETED DELETION OF THE PRN-P GENE

    AM TAKEUCHI, K SAEKI, Y MATSUMOTO, Y MATSUMOTO, K DOI, T YOKOYAMA, S ITOHARA, T ONODERA

    LIPPINCOTT-RAVEN PUBL, 1995, JOURNAL OF NEUROCHEMISTRY, 65, S101 - S101, English

    International conference proceedings

  • Molecular cloning of the prion protein cDNA and its gene expression in rat brain and immortalized neuronal cells.

    Saeki K, Takeuchi AM, Matsumoto Y, Hirota Y, Tabira T, Matsumoto Y, Onodera T

    Lead, 1994, Anim. Biochem, 31, 37 - 42, Japanese

    Scientific journal

  • FUSOGENIC PROPERTIES OF UNCLEAVED SPIKE PROTEIN OF MURINE CORONAVIRUS JHMV

    F TAGUCHI, T IKEDA, K SAEKI, H KUBO, T KIKUCHI

    PLENUM PRESS DIV PLENUM PUBLISHING CORP, 1993, CORONAVIRUSES, 342, 171 - 175, English

    International conference proceedings

MISC

  • 日本国内で発見されたダニ媒介性オルビウイルス,ムコウイルスのリバースジェネティクス系の樹立

    松尾栄子, 若村佳樹, 満田優希, 柳本周, 林昌宏, 佐伯圭一, 江尻寛子, 伊藤(高山)睦代, 伊澤晴彦, 西條政幸, 沢辺京子, 河野潤一

    2019, 日本獣医学会学術集会講演要旨集, 162nd

  • テネイシンC欠損がおよぼすプリオン蛋白遺伝子発現への影響

    烏雲達来, 中山 翔, 松尾 栄子, 河野 潤一, 山崎 智広, 中田 大介, 金 聖大, 紅林 淳一, 礒西 成治, 日下部 守昭, 佐伯 圭一

    (公社)日本獣医学会, Aug. 2012, 日本獣医学会学術集会講演要旨集, 154回, 254 - 254, Japanese

  • 正常プリオン蛋白質におけるアポトーシス抑制機能部位に関する研究

    佐伯圭一, 作道章一, 松本芳嗣, 小野寺節

    20 Aug. 2008, Bull Soc Fr-Jpn Biol, 45/46, 37, Japanese

  • 乳飲みマウスにおける微粒子の腸上皮への取込み

    阿野泰久, 山内啓史, 佐伯圭一, 松本芳嗣, 中山裕之, 小野寺節

    15 Mar. 2007, 日本獣医学会学術集会講演要旨集, 143rd, 217, Japanese

  • Abnormal olfactory function caused by ectopic expression of Doppel in the olfactory bulb of prion protein-deficient mice.

    Kim, C.K, Sakudo, A, Taniuchi, Y, Kang, C.B, Lee, D.C, Saeki, K, Matsumoto, Y, Sakaguchi, S, Itohara, S, Onodera, T

    2007.

    2007, Int. J. Mol. Med., 20, 169-176.

  • Lack of alternations of IA-2β protevtive cleavages in non-obese diabetic (NOD) mice.

    Kawakami, T, Saeki, K, Takeyama, N, Wu, G.Y, Sakudo, A, Matsumoto, Y, Hayashi, T, Onodera, T

    2007.

    2007, Int. J. Mol. Med., 20, 177-185.

  • Expression of hamster PrP and bovine PrP can not prevent murine prion protein gene-deficient cells from apoptosis

    WU Guoying, SAKUDO Akikazu, SAEKI Keiichi, MATSUMOTO Yoshitsugu, ONODERA Takashi

    2006, 日本生体防御学会学術総会講演抄録集, 17th, 63, English

  • 欠損変異プリオン蛋白質の作成によるプリオン蛋白機能解明の試み

    作道章一, 李樹明, 李得燦, 西村拓也, 佐伯圭一, 松本芳嗣, 糸原重美, 辻祥太郎, 小野寺節, 生田和良

    01 Nov. 2004, 日本ウイルス学会学術集会プログラム・抄録集, 52nd, 164, Japanese

  • プリオン遺伝子欠損神経細胞株におけるアポトーシス経路の解析

    谷内安規子, 西村拓也, 作道章一, 李得燦, 中村出, 松本芳嗣, 佐伯圭一, 糸原重美, 小野寺節

    01 Nov. 2004, 日本ウイルス学会学術集会プログラム・抄録集, 52nd, 166, Japanese

  • 正常型プリオン蛋白質による細胞内銅濃度維持

    谷内洋次郎, 作道章一, 李得燦, 吉村悦郎, 長坂征治, 新田佳也子, 佐伯圭一, 糸原重美, 松本芳嗣

    (公社)日本獣医学会, Mar. 2004, 日本獣医学会学術集会講演要旨集, 137回, 98 - 98, Japanese

  • ZchI型(FVB/Prnp-/-)不死化神経細胞株の樹立および,PrPCのアポトーシス抑制能の解析

    中村出, 西村拓也, 李得燦, 作道章一, 佐伯圭一, 松本芳嗣, 小野寺節

    (公社)日本獣医学会, Mar. 2004, 日本獣医学会学術集会講演要旨集, 137回, 98 - 98, Japanese

  • Identification of domains important for anti-apoptotic function of cellular prion protein

    佐伯圭一, 作道章一, 小野寺節

    2004, 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15年度研究報告書, 158 - 162, Japanese

  • 欠損変異プリオン蛋白を用いた正常型プリオン蛋白の機能解析

    李得燦, 作道章一, 西村拓也, 金芝けい, 佐伯圭一, 松本芳嗣, 小野寺節

    31 Jul. 2003, 日本生体防御学会学術総会講演抄録集, 14th, 38, Japanese

  • 欠損変異プリオン蛋白を用いた正常型プリオン蛋白の機能解析

    李得燦, 作道章一, 西村拓也, 徐聖旭, 佐伯圭一, 松本芳嗣, 小野寺節

    (公社)日本獣医学会, Mar. 2003, 日本獣医学会学術集会講演要旨集, 135回, 132 - 132, Japanese

  • スクレイピー (〔2002年〕12月第1土曜特集 プリオン蛋白関連疾患)

    佐伯 圭一, 小野寺 節

    2002.

    Lead, 医歯薬出版, 07 Dec. 2002, 医学のあゆみ, 203 (10), 951 - 954, Japanese

  • アポトーシス抑制因子Bcl‐2 family,Superoxide dismutase(SOD)遺伝子およびプリオン遺伝子の導入による不死化プリオンレス神経細胞死の解析

    作道章一, 李得燦, 佐伯圭一, 糸原重美, 松本芳嗣, 小野寺節

    (公社)日本獣医学会, Mar. 2002, 日本獣医学会学術集会講演要旨集, 133回, 100 - 100, Japanese

  • アポトーシス抑制因子Bcl‐2 family遺伝子の導入による不死化プリオンレス神経細胞死の解析

    作道章一, 李得燦, 佐伯圭一, 糸原重美, 松本芳嗣, 小野寺節

    (公社)日本獣医学会, Sep. 2001, 日本獣医学会学術集会講演要旨集, 132回, 51 - 51, Japanese

  • プリオン蛋白質(PrP)遺伝子欠損マウスおよび神経細胞株におけるPrP様蛋白質(Dpl)遺伝子の発現

    佐伯圭一, 作道章一, 中村優子, 那須教子, 糸原重美, 松本芳嗣, 小野寺節

    05 Mar. 2001, 日本獣医学会学術集会講演要旨集, 131st, 76, Japanese

  • アポトーシス抑制因子Bcl‐2,Bcl‐xL,Superoxide dismutase(SOD)遺伝子の導入による不死化プリオンレス神経細胞死の解析

    作道章一, 中村優子, 佐伯圭一, 糸原重美, 松本芳嗣, 小野寺節

    (公社)日本獣医学会, Mar. 2001, 日本獣医学会学術集会講演要旨集, 131回, 76 - 76, Japanese

  • Atsutaka Kubosaki, Seiichi Yusa, Yukiko Nasu, Takuya Nishimura, Yuko Nakamura, Keiichi Saeki, Yoshitsugu Matsumoto, Shigeyoshi Itohara, Takashi Onodera

    In this study, the authors investigated normal cellular prion protein (PrPC) expression on murine immune systems using prion protein gene-deficient mouse as negative control. Immunocytes expressing PrPC in adult and fetal mice were detected by flow cytometry with the monoclonal antibody against PrPC, 6H4. Cells from thymus and bone marrow reacted positively with 6H4, while spleen cells, peritoneal cells, peripheral blood leukocytes, and intestinal intraepithelial lymphocytes were nonreactive. In thymus, PrPC was observed in CD4(-)CD8(-) double-negative thymocytes. PrPC+ cells of double-negative thymocytes belonged to the CD3(-) subset, but not to the CD3(+) subset. Triple-negative PrPC+ thymocytes expressed CD44 or CD25 antigens. Furthermore, PrPC was observed in c-kit(+) bone marrow cells. In fetuses, PrPC+ cells were observed in the liver and thymus at day 16.0 and 15.0 of gestation, respectively. These results demonstrated that PrPC is expressed on immature immunocytes. (C) 2001 Academic Press.

    Elsevier BV, 2001, Biochemical and Biophysical Research Communications, 282 (1), 103 - 107, English

  • プリオン蛋白質(PrP)遺伝子ノックアウト神経細胞株におけるPrP様蛋白質(Dpl)遺伝子(Prnd)の発現

    佐伯圭一, 作道章一, 中村優子, 那須教子, 糸原重美, 松本芳嗣, 小野寺節

    25 Nov. 2000, 日本分子生物学会年会プログラム・講演要旨集, 23rd, 419, Japanese

  • アポトーシス抑制因子Bcl‐2による不死化プリオンレス神経細胞死の抑制

    原口景子, 作道章一, 窪崎敦隆, 桑原千恵子, 西村拓也, 糸原重美, 佐伯圭一, 松本芳嗣, 小野寺節

    22 Nov. 1999, 日本分子生物学会年会プログラム・講演要旨集, 22nd, 270, Japanese

  • リーシュマニア原虫を用いた哺乳動物遺伝子発現系の検討

    中村 優子, 畑生 俊光, 松本 安喜, 河津 信一郎, Chang K.-P, 佐伯 圭一, 小野寺 節, 松本 芳嗣

    (公社)日本獣医学会, Mar. 1999, 日本獣医学会学術集会講演要旨集, 127回, 93 - 93, Japanese

Books etc

  • 今日から「菌トレ」! オソロしくてオモロい、菌とのくらし 中国語版

    MIYAKO AKIKO, SAEKI KEIICHI

    Supervisor, 聯經出版公司, Apr. 2015, Chinese, 今天開始練菌!:與可怕又有趣的好菌壞菌和平相處

    General book

  • 今日から「菌トレ」!~オソロしくてオモロい、菌とのくらし~

    MIYAKO AKIKO, SAEKI KEIICHI

    Supervisor, 小学館, Jul. 2013, Japanese, ISBN: 9784093883108

    General book

  • 生体防御医学事典

    SUZUKI KAZUO, SAEKI KEIICHI

    Others, Asakura Publishing Co. Ltd., May 2007, Japanese

    Scholarly book

  • 脳とプリオン―狂牛病の分子生物学 (シリーズ・応用動物科学バイオサイエンス)

    ONODERA TAKASHI, SAEKI KEIICHI

    Joint work, 朝倉書店, Sep. 2001, Japanese

    General book

  • 農学21世紀への挑戦~地球を救う50の提案~

    SAEKI KEIICHI, 東京大学大学院農学生命科学研究科編著

    Others, 世界文化社, Mar. 2000, Japanese

    General book

Presentations

  • The influence of the transcriptional regulatory motifs within the 5′ upstream region of mouse prion protein gene on the transcriptional activity.

    Inoue S, Kato R, Matsuo E, Kawano J, Saeki K

    International Union of Microbiological Societies (IUMS), 16 Nov. 2020, English

    Oral presentation

  • マウスプリオン蛋白質遺伝子エクソン1上流域内のHES-1結合配列が遺伝子発現に及ぼす影響

    井上創太, 加藤亮, 松尾栄子, 佐伯圭一

    第73回細菌学会関西支部総会, 14 Nov. 2020, Japanese

    Oral presentation

  • オルビウイルス複製におけるタンパク質翻訳機構の解析

    杉本昌寛, 松田梢, 満田優希, 佐伯圭一, 河野潤一, 松尾栄子

    第73回細菌学会関西支部総会, 14 Nov. 2020, Japanese

    Oral presentation

  • オルビウイルス構造タンパク質VP6の機能解析

    満田優希, 若村佳樹, 松田梢, 佐伯圭一, 河野潤一, 松尾栄子

    第73回細菌学会関西支部総会, 14 Nov. 2020, Japanese

    Oral presentation

  • 流行性出血病(イバラキ病様疾病)発生牧場におけるウイルス中和抗体の追跡調査

    松田梢, 深沢太一, 満田優希, 佐伯圭一, 河野潤一, 松尾栄子

    第73回細菌学会関西支部総会, 14 Nov. 2020, Japanese

    Oral presentation

  • マウスプリオン蛋白質遺伝子エクソン1上流域における転写制御領域の解析

    井上創太, 加藤亮, 七森和, 寺前初音, 木村将徳, 藤山諒太, 松尾栄子, 佐伯圭一

    第163回日本獣医学会学術集会, 14 Sep. 2020, Japanese, DVO-49(井上).pdf, Published, No password

    Oral presentation

  • CCAATエンハンサー結合蛋白質ファミリーによるプリオン蛋白質 遺伝子発現への影響

    木村 将徳, 井上 創太, 加藤 亮, 七森 和, 寺前 初音, 藤山 諒太, 松尾 栄子, 佐伯 圭一

    第163回日本獣医学会学術集会, 14 Sep. 2020, Japanese, DVO-52(藤山).pdf, No password

  • ニホンジカプリオン蛋白質遺伝子における翻訳領域とプロモーター領域に関する研究

    藤山 諒太, 加藤 亮, 七森 和, 井上 創太, 寺前 初音, 木村 将徳, 松尾 栄子, 佐伯 圭一

    第163回日本獣医学会学術集会, 14 Sep. 2020, Japanese, DVO-52(藤山).pdf, No password

    Oral presentation

  • プリオン蛋白質のα開裂に関する研究

    寺前 初音, 七森 和, 井上 創太, 加藤 亮, 木村 将徳, 藤山 諒太, 松尾 栄子, 佐伯 圭一

    第163回日本獣医学会学術集会, 14 Sep. 2020, Japanese, DVO-53(寺前).pdf, No password

    Oral presentation

  • 流行性出血病(イバラキ病様疾病)発生牧場におけるウイルス中和抗体の追跡調査

    松田 梢, 深沢 太一, 佐伯 圭一, 松尾 栄子

    第163回日本獣医学会学術集会, 14 Sep. 2020, Japanese

    Oral presentation

  • 細胞型プリオン蛋白質のα開裂に関する研究

    七森和、加藤亮、井上創太、松尾栄子、佐伯圭一

    神戸大学研究基盤センター若手フロンティア研究会, 19 Dec. 2019, Japanese

    Poster presentation

  • 日本国内で発見されたダニ媒介性オルビウイルス、ムコウイルスのリバースジェネティクス系の樹立

    松尾栄子、柳本周、満田優希、若村佳樹、林昌宏、佐伯圭一、江尻寛子、伊藤(高山)睦代、伊澤晴彦、西條政幸、沢辺京子、河野潤一

    第72回 日本細菌学会関西支部総会, 16 Nov. 2019, Japanese

    Oral presentation

  • Fuzzy self-recognition mechanisms of orbivirus during core assembly in virus inclusion body

    Eiko Matsuo, Adeline Kerviel, Yuki Mitsuda, Chang-Kweng Lim, Keiichi Saeki, Masayuki Saijo, Junichi Kawano, Polly Roy

    第67回日本ウイルス学会学術集会, 29 Oct. 2019, Japanese

  • 日本国内で発見されたダニ媒介性オルビウイルス、ムコウイルスのリバースジェネティクス系の樹立

    松尾栄子、若村佳樹、柳本周、満田優希、林昌宏、佐伯圭一、江尻寛子、伊藤(高山)睦代、伊澤晴彦、西條政幸、沢辺京子、河野潤一

    第162回日本獣医学会学術集会, 12 Sep. 2019, Japanese

    Oral presentation

  • 新規遺伝子組換え技術を用いた流行性出血病ウイルス各血清型に対する迅速な中和抗 体検出法の開発

    MATSUO EIKO, Fukazawa Taichi, SAEKI KEIICHI, KAWANO JUNICHI

    第71回日本細菌学会関西支部総会, Oct. 2018, Japanese, 大阪大学中之島センター10F 佐治敬三メモリアルホール, Domestic conference

    Oral presentation

  • Visualization of Epizootic hemorrhagic Disease Virus Entry and Protein Synthesis

    MATSUO EIKO, Hiroko Omori, SAEKI KEIICHI, KAWANO JUNICHI

    The 66th Annual meeting of the Japanese Society for Virology, Oct. 2018, English, 京都テルサ, Domestic conference

    Oral presentation

  • 新規遺伝子組換え技術を用いた流行性出血病ウイルス各血清型に対する迅速な中和抗 体検出法の開発

    MATSUO EIKO, Fukazawa Taichi, SAEKI KEIICHI, KAWANO JUNICHI

    第161回 日本獣医学会学術集会, Sep. 2018, Japanese, つくば国際会議場, Domestic conference

    Oral presentation

  • Further analysis of Ibaraki virus VP6 to produce fluorescence-labeled orbiviruses

    MATSUO EIKO, Marina Hamaji, Hiroko Omori, Tsuji Hideyuki, Akari Saito, Polly Roy, SAEKI KEIICHI, Takeshi Kobayashi, KAWANO JUNICHI

    13th International dsRNA Virus Symposium, Sep. 2018, English, Houffalize, Belgium, International conference

    Poster presentation

  • マウス肝細胞におけるイバラキウイルス(IBAV)増殖抑制に関する研究

    濱治 麻理奈, SAEKI KEIICHI, KAWANO JUNICHI, MATSUO EIKO

    手フロンティア研究会2017, Dec. 2017, Japanese, 神戸大学百年記念館, Domestic conference

    Poster presentation

  • オルビウイルス構造タンパク質VP6の細胞内局在とgenome packaging機能に関する研究

    MATSUO EIKO, SAEKI KEIICHI, KAWANO JUNICHI, ROY POLLY

    第70回細菌学会関西支部総会, Nov. 2017, Japanese, 大阪府立大学 I-siteなんば 2F, Domestic conference

    Oral presentation

  • マウス肝細胞におけるイバラキウイルス(IBAV)増殖抑制に関する研究

    濱治 麻理奈, SAEKI KEIICHI, KAWANO JUNICHI, MATSUO EIKO

    第65回日本ウイルス学会学術集会, Oct. 2017, Japanese, 大阪国際会議場, Domestic conference

    Poster presentation

  • Biological roles of a loop region of orbivirus VP6 in virus replication

    MATSUO EIKO, SAEKI KEIICHI, KAWANO JUNICHI, Roy Polly

    The 65th Annual meeting of the Japanese Society for Virology, Oct. 2017, English, 大阪国際会議場, Domestic conference

    Oral presentation

  • Enhancement of Ibaraki virus replication by a viral non-structural protein NS2

    saji tomoaki, hirano junki, SAEKI KEIICHI, KAWANO JUNICHI, MATSUO EIKO

    第4回 関西ウイルスクラブ(KVC), Feb. 2017, Japanese, 大阪医科大学, Domestic conference

    [Invited]

    Nominated symposium

  • イバラキウイルスの構造タンパク質VP3の機能解析

    sakura risa, SAEKI KEIICHI, KAWANO JUNICHI, MATSUO EIKO

    若手フロンティア研究会2016, Dec. 2016, Japanese, 神戸大学百年記念館, Domestic conference

    Oral presentation

  • 蛍光標識イバラキウイルス粒子を用いた新規cell binding assay系の確立

    hamaji marina, SAEKI KEIICHI, KAWANO JUNICHI, MATSUO EIKO

    第69回 日本細菌学会関西支部総会, Nov. 2016, Japanese, 大阪市立大学 杉本キャンパス 田中記念館, Domestic conference

    Oral presentation

  • オルビウイルス構造タンパク質VP6の機能解析

    MATSUO EIKO, SAEKI KEIICHI, KAWANO JUNICHI, Roy Polly

    第69回 日本細菌学会関西支部総会, Nov. 2016, Japanese, 大阪市立大学 杉本キャンパス 田中記念館, Domestic conference

    Oral presentation

  • イバラキウイルス非構造タンパク質NS2のウイルス複製促進作用に関する研究

    saji tomoaki, hirano junki, SAEKI KEIICHI, KAWANO JUNICHI, MATSUO EIKO

    第69回 日本細菌学会関西支部総会, Nov. 2016, Japanese, 大阪市立大学 杉本キャンパス 田中記念館, Domestic conference

    Oral presentation

  • Further analysis of Ibaraki virus VP6 to produce fluorescence-labeled orbiviruses

    MATSUO EIKO, HAMAJI MARINA, SAITO AKARI, SAEKI KEIICHI, KAWANO JUNICHI

    The 64th Annual meeting of the Japanese Society for Virology, Oct. 2016, English, 札幌コンベンションセンター, Domestic conference

    Oral presentation

  • Reverse Genetics Systemを用いたイバラキウイルス粒子形成機序の解析

    MATSUO EIKO, SAEKI KEIICHI, KAWANO JUNICHI

    第68回 日本細菌学会関西支部総会, Nov. 2015, Japanese, 京都薬科大学 愛学館, Domestic conference

    Oral presentation

  • Reverse genetics system for Ibaraki virus reveals the essential mechanisms of VP6 in its replication

    MATSUO EIKO, SAEKI KEIICHI, KAWANO JUNICHI

    The 63rd Annual meeting of the Japanese Society for Virology, Nov. 2015, English, 福岡国際会議場, Domestic conference

    Oral presentation

  • Development of Reverse Genetics for Ibaraki Virus to Produce a Viable VP6-Tagged IBAV

    MATSUO EIKO, SAEKI KEIICHI, Roy Polly, KAWANO JUNICHI

    The 12th International Double Stranded RNA Virus Symposium, Oct. 2015, English, Goa Marriott Resort & Spa, Goa, India, International conference

    Oral presentation

  • Mechanisms of Resistance to Ibaraki Virus Replication and Its Persistent Infection in a Murine Hepatocyte Cell Line

    MATSUO EIKO, IWATA YUI, HAMAJI MARINA, SAEKI KEIICHI, KAWANO JUNICHI

    The 14th Awaji International Forum on Infection and Immunity, Sep. 2015, English, Awaji Yumebutai International Conference Center, International conference

    Poster presentation

  • Development of reverse genetics for Ibaraki virus to produce viable VP6-tagged IBAV

    MATSUO EIKO, SAEKI KEIICHI, Roy Polly, KAWANO JUNICHI

    Eighth International Virus Assembly Symposium, May 2015, English, Dubrovnik, Croatia, International conference

    Poster presentation

  • マウス肝細胞におけるイバラキウイルスの持続感染について

    Yui Iwata, Risa Sakura, Shoko Nishimura, Junki Hirano, Yu Murakami, Keiichi Saeki, Junichi, MATSUO EIKO

    第67回日本細菌学会関西支部総会・学術集会, Nov. 2014, Japanese, 兵庫医科大学 3-3講義室(3号館4階), Domestic conference

    Oral presentation

  • イバラキウイルス(IBAV)における新規遺伝子改変法(Reverse Genetics System) を用いた、VP6 変異IBAV の作出

    MATSUO EIKO, Keiichi Saeki, Junichi

    第67回日本細菌学会関西支部総会・学術集会, Nov. 2014, Japanese, 兵庫医科大学 3-3講義室(3号館4階), Domestic conference

    Oral presentation

  • イバラキウイルス(IBAV)におけるReverse Genetics Systemの開発とその応用

    MATSUO EIKO, Keiichi Saeki, Junichi

    第62回日本ウイルス学会学術集会, Nov. 2014, Japanese, パシフィコ横浜 会議センター, Domestic conference

    Oral presentation

  • イバラキウイルス(IBAV)コアタンパク質VP6 とVP3 の相互作用に関する研究

    Yu Murakami, Hideyuki Tsuji, Risa Sakura, Shoko Nishimura, Junki Hirano, Keiichi Saeki, Junichi, MATSUO EIKO

    第67回日本細菌学会関西支部総会・学術集会, Nov. 2014, Japanese, 兵庫医科大学 3-3講義室(3号館4階), Domestic conference

    Oral presentation

  • 広溶菌域ブドウ球菌ファージに関する研究

    Masashi Kishida, Shinichiro Tobi, Yuriko Sugimoto, MATSUO EIKO, Junichi, Keiichi Saeki

    第157回日本獣医学会学術集会, Sep. 2014, Japanese, 北海道大学高等教育推進機構, Domestic conference

    Oral presentation

  • マウス由来株化培養細胞におけるプリオン蛋白遺伝子の発現とDNAメチル化状態

    烏雲達来, Yugo Doi, Natsumi Takeyama, MATSUO EIKO, Junichi, Keiichi Saeki

    第157回日本獣医学会学術集会, Sep. 2014, Japanese, 北海道大学高等教育推進機構, Domestic conference

    Oral presentation

  • マウス肝細胞におけるイバラキウイルスの持続感染について

    Yui Iwata, Risa Sakura, Shoko Nishimura, Junki Hirano, Yu Murakami, Keiichi Saeki, Junichi, MATSUO EIKO

    第157回日本獣医学会学術集会, Sep. 2014, Japanese, 北海道大学高等教育推進機構, Domestic conference

    Oral presentation

  • ブルータングウイルス(BTV)VP6の構造解析を用いた増殖可能なVP6-truncated BTVの作出

    MATSUO EIKO, Esther Leon, Keiichi Saeki, Junichi Kawano, Steve Matthews, Polly Roy

    第157回日本獣医学会学術集会, Sep. 2014, Japanese, 北海道大学高等教育推進機構, Domestic conference

    Oral presentation

  • The essential interaction of VP6 protein with VP3 for recruitment of the replicase complex into orbivirus particle

    MATSUO EIKO, Hideyuki Tsuji, Yu Murakami, Keiichi Saeki, Junichi Kawano, Polly Roy

    The 13th Awaji International Forum on Infection and Immunity in Nara, Sep. 2014, English, Nara Prefectural New Public Hall, International conference

    Oral presentation

  • Stromal tenascin C regulates a prion protein gene expression in tumor tissues.

    SAEKI KEIICHI, Wuyundarai, MATSUO EIKO, KAWANO JUNICHI, Kusakabe Miho, KUSAKABE MORIAKI

    The 72nd Annual Meeting of the Japanese Cancer Association, Oct. 2013, Japanese, PACIFICO YOKOHAMA, Domestic conference

    Poster presentation

  • マウスプリオン蛋白遺伝子発現制御領域のメチル化に関する研究

    Wuyundarai, TAKEYAMA NATSUMI, KUROKAWA TAKURO, DOI YUGO, MATSUO EIKO, KAWANO JUNICHI, SAEKI KEIICHI

    156th Meeting of the Japanese Society of Veterinary Science, Sep. 2013, Japanese, GIFU UNIVERSITY, Domestic conference

    Oral presentation

  • 生体内における感染動態解析のためのEGFP発現大腸菌の作製

    SAKAMOTO NATSUMI, MATSUO EIKO, SAEKI KEIICHI, KAWANO JUNICHI

    第155回日本獣医学会, Mar. 2013, Japanese, 日本獣医学会, 東京, Domestic conference

    Poster presentation

  • 異なる細胞種におけるイバラキウイルスの複製能力の比較

    岩田 友伊, 辻 秀幸, SAEKI KEIICHI, KAWANO JUNICHI, MATSUO EIKO

    第155回日本獣医学会学術集会, Mar. 2013, Japanese, 東京, Domestic conference

    Oral presentation

  • オルビウイルスコアタンパク質VP6とVP3の相互作用に関する研究

    辻 秀幸, 岩田 友伊, SAEKI KEIICHI, KAWANO JUNICHI, Polly Roy, MATSUO EIKO

    第155回日本獣医学会学術集会, Mar. 2013, Japanese, 東京, Domestic conference

    Oral presentation

  • ブルータングウイルス(BTV)コアタンパク質VP3とVP6の結合に関する研究

    MATSUO EIKO, 山崎 清志, Esther Leon, SAEKI KEIICHI, KAWANO JUNICHI, Steve Matthew, TSURUTA HIROKI, Polly Roy

    第65回日本細菌学会関西支部総会, Nov. 2012, Japanese, 神戸, Domestic conference

    Oral presentation

  • ブルータングウイルス(BTV)コアタンパク質VP3とVP6の結合に関する研究

    MATSUO EIKO, Esther Leon, 山崎 清志, SAEKI KEIICHI, KAWANO JUNICHI, Steve Matthew, TSURUTA HIROKI, Polly Roy

    第60回日本ウイルス学会学術集会, Nov. 2012, Japanese, 大阪, Domestic conference

    Oral presentation

  • ブルータングウイルス(BTV)コアタンパク質VP3とVP6の結合に関する研究

    MATSUO EIKO, Esther Leon, SAEKI KEIICHI, KAWANO JUNICHI, Steve Matthew, Polly Roy

    第154回日本獣医学会学術集会, Sep. 2012, Japanese, 盛岡, Domestic conference

    Oral presentation

  • テネイシンC欠損がおよぼすプリオン蛋白遺伝子発現への影響

    Wuyundarai, NAKAYAMA SHO, MATSUO EIKO, KAWANO JUNICHI, YAMAZAKI TOMOHIRO, NAKATA DAISUKE, KIMU SONDE, KUREBAYASHI JUNICHI, ISONISHI NEIHARU, KUSAKABE MORIAKI, SAEKI KEIICHI

    154th Meeting of the Japanese Society of Veterinary Science, Sep. 2012, Japanese, IWATE UNIVERSITY, Domestic conference

    Oral presentation

  • 複製能力欠損ブルータングウイルスを用いた新規ワクチンの開発

    MATSUO EIKO, Cristina CP Celma, Richard Thiery, SAEKI KEIICHI, KAWANO JUNICHI, Stephan Zientara, Polly Roy

    第153回日本獣医学会学術集会, Mar. 2012, Japanese, 大宮ソニックシティ, Domestic conference

    Poster presentation

  • 新規MRSA分離選択培地の開発

    KAKITA YOKO, SHIMODA HIROYUKI, SHIMABUKURO SHIZUKA, SAITO ETSUKO, SAEKI KEIICHI, KAWANO JUNICHI

    150th Meeting of the Japanese Society of Veterinary Science, Sep. 2010, Japanese, OBIHIRO UNIVERSITY OF AGRICULTURE AND VETERINARY MEDICINE, Domestic conference

    Oral presentation

  • 腫瘍組織におけるプリオン蛋白(PrP)遺伝子発現量の比較

    YAMAZAKI TOMOHIRO, MIYAGAWA MOMOKO, FUJITA KOUSUKE, KAWANO JUNICHI, KUREBAYASHI JUNICHI, ISHIKAWA HIROSHI, KUSAKABE MORIAKI, SAEKI KEIICHI

    150th Meeting of the Japanese Society of Veterinary Science, Sep. 2010, Japanese, OBIHIRO UNIVERSITY OF AGRICULTURE AND VETERINARY MEDICINE, Domestic conference

    Oral presentation

Association Memberships

  • 日本獣医学会

Research Projects

  • 佐伯 圭一

    科学研究費補助金/基盤研究(C), Apr. 2012 - Mar. 2015, Principal investigator

    Competitive research funding

  • 佐伯 圭一

    科学研究費補助金/基盤研究(C), 2009, Principal investigator

    Competitive research funding

  • Pathogenesis of BSE and highly sensitive isolation of BSE agent

    ONODERA TAKASBI, MATSUMOTO Yoshitsugu, SAEKI KEIICHI

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), The University of Tokyo, 2005 - 2007

    The chain reaction of BSE epidemics in the UK and Europe and subsequent emergence of vCJD in young adults and teenagers have raised concerns and highlighted the importance of risk assessment in the food chain. Recently, several highly sensitive methods for detecting priors have been developed. Representative of these is PMCA. Originally developed by Claudio Soto and his colleagues, PMCA has been a hot topic of debate in prion meetings all over the world. A broad spectrum of PrP^Sc species have now been successfully amplified using PMCA, including CWD, mouse-adapted scrapie, and BSE. Studies with human sporadic and variant CJD cases show that PMCA amplification efficiency is tightly controlled by the PrP^C substrate genotype at codon 129. PMCA appears to overcome the species barrier encountered during cross-species transmission more rapidly than in vivo. By "forcing" the technique with lower dilutions of the PrP^Sc seed and more amplification rounds, mouse Chandler PrP^Sc can now convert hamster PrP^C or cervid PrP^C, a conversion that might be observable in vivo but only with extremely long incubation periods. Castilla J. showed that using PMCA, PrP^Sc was generated from the healthy brains of 11 different species, including bank voles, mice, cattle, humans, sheep and rabbits, generating a variety of electrophoretic profiles. PMCA was able to detect PrP^Sc in as little as 1 μL of blood from an asymptomatic prion-infected mouse. Given the increasing evidence of human to human transmission via blood products, scientists are waiting for PMCA to be incorporated into a reliable test with the ability to identify blood donors that are asymptomatic carriers. Further advances in amplification technology are to be expected and the replacement of PrP^C by recombinant PrP as a substrate as well as the use of intermittent shaking rather than sonication should circumvent some of the difficulties in the near future.

  • Study for atypical BSE in Italy

    ONODERA Takashi, SAEKI Keiichi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), The University of Tokyo, 2005 - 2006

    A monoclonal antibody (mAb) panel to bovine prion protein (PrP) was studied by immunoblotting and immunohistochemistry for scrapie, and bovine spongiform encephalopathy. The mAb panel is useful because they recognized both normal (PrP^c) and abnormal (PrP^) isoforms of PrP in murine, ovine and bovine brain tissues. Among them, two mAbs recognized a conformational or fragmented PrP epitope. Interestingly, an anti-bovine PrP mAb, 1D12, prepared by immunizing PrP gene-knockout mice with a synthetic polypeptides corresponding to codons 153-166 of the bovine PrP gene showed the novel patterns of reactivity for prion-uninfected neuronal cells. When an enzyme-linked immunosorbent assay-mapping of the mAb epitopes was examined, monoclonal 1D12 reacted with YEDRY and M corresponding to amino acids 156-160 and 165 of bovine PrP. Several patterns of bovine PrP^c distribution in PrP-deficient neuronal cells (HpL3-4) transfected with bovine PrP were observed after different fixation methods. Staining of cell surface was observed after formalin fixation by immunofluorescent assay of 1D12 with confocal microscopy, whereas granules in nucleus were stained after acetone fixation. No reactivity in the nucleus was observed to HpL3-4 or HpL3-4 cells expressing mouse PrP. Reactivity in the nucleus was observed to HpL3-4 cells expressing hamster or bovine PrP. It is the first report to detect the PrPc at both cell surface and nuclei of prion-uninfected cell line. Furthermore, as nuclear PrP^c was specifically recognized by 1D12, amino acids 156-160 and 165 of hamster or bovine PrP may play important role in localization of PrP^c into nucleus.

  • 免疫担当細胞におけるプリオン蛋白質の作用機構に関する研究

    佐伯 圭一

    日本学術振興会, 科学研究費助成事業, 若手研究(A), 東京大学, 2002 - 2004

    [プリオン蛋白(PrP)の機能とアポトーシスの抑制に関する解析] Zn-1型PrP遺伝子欠損マウスおよびRikn型PrP遺伝子欠損マウスおよび野生型マウスより得られた神経細胞初代を用いて銅イオンによって誘導されるアポトーシスについて解析した結果、PrP遺伝子欠損マウス由来神経細胞が有為に高いアポトーシスを起こした。 また、Zn-1型PrP遺伝子欠損マウス、Rikn型PrP遺伝子欠損マウスおよび野生型マウスより得られた脾細胞を用いて無刺激および刺激後の細胞の増殖能について比較検討を行った結果、PMA、イオノマイシン刺激後において野生型マウス由来脾細胞と比較してZn-1型PrP遺伝子欠損マウスおよびRikn型PrP遺伝子欠損マウス由来脾細胞において増殖能が低い結果が得られた。ConA、リポ多糖刺激においては野生型マウス由来脾細胞とPrP遺伝子欠損マウス由来脾細胞間で有為な差は認められなかった。アポトーシス細胞についても解析を行ったが有為な差は認められなかった。 [ウイルス感染時におけるプリオン蛋白の機能解析] Zn-1型、Rikn型PrP遺伝子欠損マウスおよび野生型マウスを用いて、脳心筋炎ウイルス-B株感染について解析した結果、アポトーシス細胞の出現頻度においてPrP遺伝子欠損マウスのほうが、野生型マウスと比較して高頻度であった。

  • research for highly sensitive assay for BSE agents

    ONODERA Takashi, MATSUMOTO Yoshitsugu, SAEKI Keiichi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), The University of Tokyo, 2002 - 2004

    PrP^c is a host protein anchored to the outer surface of neurons and to a lesser extent in lymphocytes. The transmissible agent (PrP^) responsible for scrapie is thought to be a modified form of PrP^c. However, the role of PrP^c in normal uninfected animals is still unknown. Here we describe a novel mouse model for cardiomyopathy, which was observed during the development of prion agent susceptible transgenic (Tg) mouse. These Tg mice were shown to selectively express OrPrP in their tissues, as demonstrated by RT-PCR for mRNA, Western blotting and immunohistochemistry. High levels of OrPrP expression were seen in heart, medium levels in skeletal muscle, low levels in brain, and barely detectable levels in lung, liver, spleen, kidney and thymus. Interestingly, the electrocardiogram of these mice showed that cardiac muscle contraction was significantly prolonged with abnormality of the QRS interval. In addition, the histologic study showed multiple intracytoplasmic vacuolation in the heart muscle. These data suggest that the high expression of OrPrP in heart may cause the cardiac abnormality. Furthermore, an abnormal waveform of ECG was observed in these Tg mice after atropine injection. These mice therefore may be possible to provide a new model to elucidate experimental cardiomyopathy.

  • プリオン蛋白質関連神経細胞死発現機構に関する研究

    佐伯 圭一

    日本学術振興会, 科学研究費助成事業, 特定領域研究, 東京大学, 2002 - 2002

    プリオン蛋白質(PrP)の再発現に伴い細胞のSuperoxide dismutase (SOD)活性を上昇させたとの前年度の結果から、本年度はPrPと酸化ストレスとの関連について調べた。無血清培養条件下におけるPrP欠損神経細胞とPrP再発現細胞株の活性酸素関連分子の量を調べた。SODは2O_2・^-+2H^+→H_2O_2+O_2の反応を触媒する酵素であり、活性酸素(O_2・^-)を消去して過酸化水素(H_2O_2)に変換する。血清除去下での活性酸素の発生量をフリーラジカル特異的蛍光プローブを用いたフローサイトメトリー解析により調べた。Dihydroethidium (DH)により、細胞内活性酸素を蛍光染色しフローサイトメトリーにより測定したところ、PrP欠損神経細胞で見られる活性酸素の発生がPrP再発現細胞株では抑制された。さらに2',7'-dichlorofluorescein diacetate (DCFH-DA)染色により過酸化水素量の測定をおこなったところ、PrP再発現細胞株において過酸化水素の蓄積が認められた。PrP再発現に伴い細胞内の活性酸素が消去され、過酸化水素が蓄積していたことからPrPがSOD活性を調整していかまたはPrP自身がSOD様の酵素活性を持っていることが示唆された。PrPは銅と結合することが報告されているが、銅代謝を制御することによりSODの活性を制御し、活性酸素の消去に関わっている可能性が考えられた。

  • プリオン蛋白質関連神経細胞死発現機構に関する研究

    佐伯 圭一

    日本学術振興会, 科学研究費助成事業, 特定領域研究(C), 東京大学, 2001 - 2001

    プリオン病で見られる神経変性の発現機構には正常型のプリオン蛋白と病原体の本体と見られる異常型のプリオン蛋白の機能が密接に関係していると考えられている。本研究ではプリオン蛋白遺伝子欠損不死化神経細胞株におけるアポトーシス抑制能について検討した。特にアポトーシス抑制関連因子(Bcl-2,Bcl-XL, SOD及びTNF-alpha)との相互作用について検討した。また、プリオン蛋白遺伝子欠損マウス、野生型マウス、プリオン蛋白遺伝子欠損不死化神経細胞株およびプリオン蛋白遺伝子再構築不死化神経細胞株を用いて、EMC-B株、コクサッキーB1,B2,B3,B5,B6株感染時における宿主細胞内プリオン蛋白の機能を推定、検討を行なった。プリオン蛋白遺伝子欠損不死化神経細胞株に上記のBcl-2,Bcl-XL, SOD-1 cDNAを導入したところ、PrP cDNAを導入した細胞株で認められる無血清培地下でのアポトーシス抑制効果が認められた。プリオン蛋白遺伝子を再導入した細胞株におけるBcl-2,Bcl-XL及びSOD蛋白の産生を検討したところ、産生に変化は認められなかった。ところが、細胞内成分および細胞膜成分のSOD活性を測定したところ、SOD活性の上昇を認めた。正常プリオン蛋白は銅結合蛋白であることから、SOD分子に銅を供給する銅シャペロン分子であることが推測され、プリオン蛋白を発現によって細胞内SOD活性が上昇したと考えられた。また、細胞膜成分のSOD活性も上昇したことから、プリオン蛋白自体が膜上でSOD様の活性を持っていると予想された。

  • ウイルス誘発性アポトーシスにおける正常プリオン蛋白の機能に関する研究

    佐伯 圭一

    日本学術振興会, 科学研究費助成事業, 奨励研究(A), 東京大学, 2000 - 2001

    プリオン蛋白欠損不死化神経細胞株を用いてコクサキーウイルスおよび脳心筋炎ウイルスB株(EMC-B)の感受性を検討するとともに、プリオン蛋白遺伝子再導入株を作製し、ウイルスの感受性変化について解析を行なった。プリオン蛋白欠損不死化神経細胞株と他のウイルス分離に一般的に使われている細胞株を比較したところ、プリオン蛋白欠損不死化神経細胞株は脳心筋炎ウイルス及びコクサッキーウイルスそれぞれの感染において感受性を有していた。また、ウイルス増殖が他の細胞株と比較し早く、最終的なウイルス力価も高かった。さらに細胞変性効果出現時期においても他の細胞株と比較して早く、ウイルス分離のための細胞株としてプリオン蛋白欠損不死化神経細胞株の有用性が示された。感染時におけるプリオン蛋白欠損不死化神経細胞の細胞変性効果誘導が、プリオン蛋白の再発現化に伴い影響を受けるのかを解析したところ、プリオン蛋白遺伝子を再導入した細胞は細胞変性効果の出現が遅れ、また、アポトーシス抵抗性を示した。このことより、ウイルス誘発性のアポトーシスにおいてプリオン蛋白が抑制的に働いていることが示唆された。ウイルス感染時における宿主細胞プリオン蛋白の機能を推定するために、EMC-Bウイルス感染後経時的にマウスを屠殺・剖検し、組織標本を作製した後、細胞組織学的、ウイルス病理学的に脳炎症反応の解析を行なった。アポトーシス神経細胞の出現時期は、野生型マウスよりもプリオン蛋白欠損マウスで早期に観察された。また、浸潤細胞のアポトーシスもプリオン蛋白欠損マウスで早期に観察された。これらのことからプリオン蛋白の機能として抗酸化作用が考えられ酸化ストレスによるアポトーシスと密接に関連していることが示唆された。

  • Effect of Prnp gene to prion protein expression

    ONODERA Takashi, SASAKI Keiichi, MATSUMOTO Yoshitsugu

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), THE UNIVERSITY OF TOKYO, 2000 - 2001

    Some of the authors previously demonstrated the relation between cellular prion protein (PrP^c) and apoptosis using immortalized prion protein gene (Prnp)-deficient neuronal cells. However, the mechanism(s) by which PrP^c prevents apoptosis remains unclear. In this study, the authors analyzed apoptosis of Prnp^<-/-> cells using gene transfer of apoptosis-related genes. Transfection of Prnp^<-/-> cells with bcl-2, bcl-x_L or SOD-1, which encodes Cu/Zn superoxide dismutase (SOD), inhibited apoptosis induced by serum deprivation. As serum deprivation decreased Bcl-2 and Bcl-x_L proteins in-Prnp^<-/-> cells, these cells are thought to die via apoptosis pathways regulated by the Bcl-2 family and intracellular superoxides. Re-introduction of Prnp upregulated SOD activity and eliminated superoxide anion generation without inducing changes hi Bcl-2, Bcl-x_L and Cu/Zn SOD expression levels. As PrP^c was bound to copper in the octapeptide repeats, these results suggested that PrP^c inhibited apoptosis by upregulation of SOD activity due to copper metabolism regulation.