Researcher Profile and Settings

Affiliation

• Faculty of Veterinary Medicine　Veterinary Medicine　Applied Veterinary Sciences

Job Title

• Professor

Degree

• PhD（Hokkaido University）

Research funding number

• 70241370

J-Global ID

• 200901085983212151

Research Interests

• 獣医生化学   分子遺伝学   実験動物学   Veterinary Biochemistry   Molecular Genetics   Laboratory Animal Science   

Research Areas

• Life sciences / Experimental pathology
• Life sciences / Veterinary medicine
• Life sciences / Laboratory animal science

Educational Organization

•  Bachelor's degree program　School of Veterinary Medicine
•  Doctoral (PhD) degree program　Graduate School of Veterinary Medicine

Academic & Professional Experience

• 2022/04 - Today Faculty of Veterinary Medicine Hokkaido University Laboratory of Laboratory Animal Science and Medicine Department of Applied Veterinary Sciences Professor
• 2013/11 - 2022/12 Faculty of Veterinary Medicine, Hokkaido University Attending Veterinarian of Animal Facility
• 2013/11 - 2022/03 Faculty of Veterinary Medicine, Hokkaido University Laboratory of Laboratory Animal Science and Medicine Department of Applied Veterinary Sciences Associate Professor
• 2012/04 - 2013/10 Institute for Genetic Medicine, Hokkaido University Laboratory of Animal Experiments Director
• 2008/07 - 2013/10 Institute for Genetic Medicine, Hokkaido University Disease Model Innovation Associate Professor
• 2007/04 - 2013/10 Institute for Genetic Medicine, Hokkaido University Laboratory of Animal Experiments Associate Professor
• 2005/10 - 2007/03 Institute for Genetic Medicine, Hokkaido University Laboratory of Animal Experiments Associate Professor
• 2004 - 2005 Graduate School of Medicine, Hokkaido University Laboratory of Histology and Cytology Assistant Professor
• 1997 - 2004 Faculty of Agriculture, Iwate University Laboratory of Physiology, Department of Veterinary Medicine Associate Professor
• 1996 - 1997 University of Texas MD Anderson Cancer Center Visiting Fellow
• 1992 - 1997 Graduate School of Veterinary Medicine, Hokkaido University Laboratory of Biochemistry Assistant Professor
• 1995 - 1995 University of São Paulo School of Medicine Visiting Fellow

Education

•        - 1992/03  Hokkaido University  Graduate School of Veterinary Medicine
•        - 1992  Hokkaido University  Graduate School, Division of Veterinary Medicine
•        - 1989  Hokkaido University  Faculty of Veterinary Medicine
•        - 1987/03  Hokkaido University  School of Veterinary Medicine  Veterinary Medicine

Association Memberships

• JAPANESE ASSOCIATION FOR LABORATORY ANIMAL SCIENCE   THE JAPANESE BIOCHEMICAL SOCIETY   THE MOLECULAR BIOLOGY SOCIETY OF JAPAN   THE JAPANESE SOCIETY OF VETERINARY SCIENCE   The Japanese College of Laboratory Animal Medicine (JCLAM)   The Japanese Association for Laboratory Animal Medicine (JALAM)   JAPAN VETERINARY MEDICAL ASSOCIATION   日本実験動物医学専門医協会   日本実験動物医学会   日本実験動物学会   日本分子生物学会   日本生化学会   日本獣医学会   

Research Activities

Published Papers

• A Highly Conserved Region in BRCA2 Suppresses the RAD51-Interaction Activity of BRC Repeats.

  Zida Zhu, Taisuke Kitano, Masami Morimatsu, Kazuhiko Ochiai, Toshina Ishiguro-Oonuma, Kosuke Oosumi, Xianghui Lin, Koichi Orino, Yasunaga Yoshikawa

  Veterinary sciences 10 (2) 2023/02/10 

  Mammary tumors are the most prevalent type of tumors in female dogs. Breast cancer 2, early onset (BRCA2) malignant mutations are associated with tumorigenesis in humans and dogs. BRCA2 plays a pivotal role in homologous recombination repair by recruiting RAD51 recombinase to DNA damage sites to maintain genome stability. To recruit RAD51, BRCA2 must interact with RAD51 via BRC repeats, but the regulation of this interaction has been unclear. In this study, we focused on a highly conserved region (HCR) near BRC repeats. Using co-immunoprecipitation and mammalian two-hybrid assay, we found that HCR suppressed the RAD51-interaction activity of BRC repeats and that substitutions of HCR phosphorylation sites affected it. In canine tumor samples, we found ten mutations, including a novel HCR mutation (I1110M) from canine tumor samples. The effect of four HCR mutations, including I1110M, on the RAD51-interaction activity of BRC repeats was tested. One of the HCR mutations found in canine mammary tumors increased the interaction, but the two mutations found in human breast cancers decreased it. This study suggested that the HCR regulated the RAD51-interacting activity of BRC repeats through HCR phosphorylation and that mutations in HCR may be related to tumorigenesis in both dogs and humans.
• Canine Mammary Tumor Cell Lines Derived from Metastatic Foci Show Increased RAD51 Expression but Diminished Radioresistance via p21 Inhibition.

  Kei Shimakawa, Kazuhiko Ochiai, Sachi Hirose, Eri Tanabe, Masaki Michishita, Motoharu Sakaue, Yasunaga Yoshikawa, Masami Morimatsu, Tsuyoshi Tajima, Masami Watanabe, Yoshikazu Tanaka

  Veterinary sciences 9 (12) 2022/12/17 

  Due to the high incidence of mammary tumors in dogs, it is important to elucidate the pathogenesis of these tumors in veterinary medicine. Radiation therapy is often used to treat mammary tumors that target DNA lesions. RAD51 is a key molecule that repairs DNA damage via homologous recombination. We examined the relationship between RAD51 expression and radiosensitivity in mammary tumor cell lines. CHMp and CHMm from the same individual were selected based on the differences in RAD51 expression. The radiosensitivity of both cell lines was examined using MTT and scratch assays; CHMm, which has high RAD51 expression, showed higher sensitivity to radiation than CHMp. However, the nuclear focus of RAD51 during DNA repair was formed normally in CHMp, but not in most of CHMm. Since irradiation resulted in the suppression of cell cycle progression in CHMp, the expression of p21, a cell cycle regulatory factor, was detected in CHMp after 15 Gy irradiation but not in CHMm. These results indicate that functional expression is more important than the quantitative expression of RAD51 in canine mammary tumor cells in response to DNA damage.
• In vitro anticancer effects of alpelisib against PIK3CA‑mutated canine hemangiosarcoma cell lines.

  Marika Maeda, Kazuhiko Ochiai, Masaki Michishita, Masami Morimatsu, Hiroki Sakai, Nayuta Kinoshita, Motoharu Sakaue, Eri Onozawa, Daigo Azakami, Masami Yamamoto, Katsumi Ishioka, Takuya Sadahira, Masami Watanabe, Yoshikazu Tanaka

  Oncology reports 47 (4) 2022/04 

  Hemangiosarcoma (HSA) is a malignant neoplasm that occurs in humans and canines with a poor prognosis owing to metastatic spread, despite effective treatment. The frequency of spontaneous HSA development is higher in canines than in humans. Therefore, canine HSA is a useful model of intractable human disease, which requires early detection and an effective therapeutic strategy. A high frequency of the p110α phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit alpha (PIK3CA) mutations is detected in a comprehensive genome‑wide analysis of canine cases of HSA. The present cloned the full‑length cDNA of canine PIK3CA and identified a mutation in codon 1047 from canine cases of HSA and cell lines that were established from these. The enforced expression of the 1047th histidine residue (H1047)R or L mutants of canine PIK3CA in HeLa cells enhanced epidermal growth factor receptor (EGFR) signaling via Akt phosphorylation. PIK3CA mutant canine HSA cell lines exhibited the hyperphosphorylation of Akt upon EGF stimulation as well. Alpelisib, a molecular targeted drug against PIK3CA activating mutations, exerted a significant antitumor effect in canine PIK3CA‑mutated HSA cell lines. By contrast, it had no significant effect on canine mammary gland tumor cell lines harboring PIK3CA mutations. On the whole, the findings of the present study suggest that alpelisib may be highly effective against PIK3CA mutations that occur frequently in canine HSA.
• BRCA2 C-Terminal RAD51-Binding Domain Confers Resistance to DNA-Damaging Agents

  Zida Zhu, Taisuke Kitano, Masami Morimatsu, Arisa Tanaka, Ryo Morioka, Xianghui Lin, Koichi Orino, Yasunaga Yoshikawa

  International Journal of Molecular Sciences 23 (7) 2022/04 [Refereed][Not invited]
   

  Breast cancer type 2 susceptibility (BRCA2) protein is crucial for initiating DNA damage repair after chemotherapy with DNA interstrand crosslinking agents or X-ray irradiation, which induces DNA double-strand breaks. BRCA2 contains a C-terminal RAD51-binding domain (CTRBD) that interacts with RAD51 oligomer-containing nucleofilaments. In this study, we investigated CTRBD expression in cells exposed to X-ray irradiation and mitomycin C treatment. Surprisingly, BRCA2 CTRBD expression in HeLa cells increased their resistance to X-ray irradiation and mitomycin C. Under endogenous BRCA2 depletion using shRNA, the sensitivities of the BRCA2-depleted cells with and without the CTRBD did not significantly differ. Thus, the resistance to X-ray irradiation conferred by an exogenous CTRBD required endogenous BRCA2 expression. BRCA2 CTRBD-expressing cells demonstrated effective RAD51 foci formation and increased homologous recombination efficiency, but not nonhomologous end-joining efficiency. To the best of our knowledge, our study is the first to report the ability of the BRCA2 functional domain to confer resistance to X-ray irradiation and mitomycin C treatment by increased homologous recombination efficiency. Thus, this peptide may be useful for protecting cells against X-ray irradiation or chemotherapeutic agents.
• イヌとヒトで異なるRAD51分子構造がDNA相同組換え修復効率におよぼす影響の検討

  植村 光希, 落合 和彦, 道下 正貴, 吉川 泰永, 前田 まりか, 森松 正美, 近江 俊徳, 田中 良和

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [IO - 11] 1347-8621 2021/09
• 癌抑制遺伝子Brca2のイントロン1における発現調節機構の解析

  上妻 創, 吉川 泰永, 森松 正美, 落合 和彦, 折野 宏一

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [IO - 17] 1347-8621 2021/09
• イヌとヒトで異なるRAD51分子構造がDNA相同組換え修復効率におよぼす影響の検討

  植村 光希, 落合 和彦, 道下 正貴, 吉川 泰永, 前田 まりか, 森松 正美, 近江 俊徳, 田中 良和

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [IO - 11] 1347-8621 2021/09
• 癌抑制遺伝子Brca2のイントロン1における発現調節機構の解析

  上妻 創, 吉川 泰永, 森松 正美, 落合 和彦, 折野 宏一

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 164回 [IO - 17] 1347-8621 2021/09
• The number of glutamines in the N-terminal of the canine androgen receptor affects signalling intensities.

  Kazuhiko Ochiai, Samak Sutijarit, Mitsuki Uemura, Masami Morimatsu, Masaki Michishita, Eri Onozawa, Marika Maeda, Takanori Sasaki, Masami Watanabe, Yoshikazu Tanaka, Toshinori Omi

  Veterinary and comparative oncology 19 (2) 399 - 403 2021/06 [Refereed]
   

  Most male dogs are castrated at young ages, making them easy to rear following androgen deprivation. Although the incidence of canine prostate cancer is low, several patients have resistance to androgen therapy and poor clinical prognosis. These outcomes are similar to those of end-stage human androgen-independent prostate cancer. The androgen receptor (AR) of canines has two polyglutamine (polyQ) sequences (Q × 10 and Q × 23) at its N-terminal. The length of polyQ may be a risk factor for the development of prostate cancer in dogs; however, there is no evidence to support this. Hence, we artificially created polyQ deletion mutants of canine AR and evaluated their effects on AR signalling. The deletions of Q × 10 and Q × 23 were associated with significant reductions in AR signalling intensities. The Q × 10 mutants, which increase or decrease Q sequentially, also altered AR signalling. Furthermore, the Q × 10 deletion mutant, compared with the Q × 10 control, altered the intensities of the binding of polyQ to the C-terminal of AR, which contains a ligand-binding domain; this was not observed with the Q × 9, 11, and 12 variants. The number of glutamines in the N-terminals of canine ARs may influence AR signalling intensities and contribute to the risk of prostate cancer in dogs.
• Identification of the core motif of the BRCA2 C-terminal RAD51-binding domain by comparing canine and human BRCA2.

  Yasunaga Yoshikawa, Masami Morimatsu, Kazuhiko Ochiai, Toshina Ishiguro-Oonuma, Ryo Morioka, Kento Okuda, Koichi Orino

  The Journal of veterinary medical science 83 (5) 759 - 766 2021/05/09 [Refereed]
   

  Mammary tumors are the most common tumors in women and non-spayed female dogs. One of the reasons for mammary tumors is mutations of the tumor suppressor gene, BRCA2. BRCA2 participates in homologous recombination repair by interacting with the RAD51 recombinase. BRCA2 has two RAD51-binding domains, consisting of BRC repeats and the C-terminal RAD51-binding domain, respectively. Although several studies have addressed the function of the C-terminal RAD51-binding domain of human BRCA2, the amino acid sequences required for the RAD51-interaction activity remain unclear. In this study, the C-terminal RAD51-binding domains of canine and human BRCA2 were compared; the canine domain displayed a weaker interaction with RAD51. This difference was attributed to the C-terminal portion of the domain via a comparison between canine and human domains. Furthermore, peptides shorter than those previously reported displayed RAD51-interacting activity, and a core motif of this domain consisting of 25 amino acids was identified. Since a mutation (S3323N) was reported in the core motif of this domain, the effect of this mutation was evaluated. The mutant exhibited similar RAD51-binding activity as that of the wild-type protein, suggesting that the mutation was functionally neutral. These data suggested that the C-terminal portion of the BRCA2 C-terminal RAD51-binding domain influenced its RAD51-interaction activity, and a minimum core motif of 25 amino acids was identified in this domain. These data may help clarify BRCA2 function, as well as the tumorigenic effects of BRCA2 mutation.
• The response of adipose tissues to Mycoplasma pulmonis and Sendai virus infection in C57BL/6 and DBA/2 mice.

  Tussapon Boonyarattanasoonthorn, Keisuke Sato, Yuko Okamatsu-Ogura, Masami Morimatsu, Takashi Agui

  The Journal of veterinary medical science 83 (3) 403 - 411 2021/03/11 [Refereed]
   

  Adipose tissues in mammals are categorized into white and brown adipose tissues in which cellular morphology, cell functions, and tissue distribution are different. White adipose tissue (WAT) plays a major role in energy reservation, while brown adipose tissue (BAT) mainly relates to the thermoregulation of the body. One interesting function of adipose tissue is the response to the infection, especially the pathogens that cause pneumonia. We have previously reported that DBA/2 (D2) mice are susceptible to pathogens causing pneumonia, Mycoplasma (M.) pulmonis and Sendai virus (SeV), whereas C57BL/6 (B6) mice are resistant to them. Furthermore, morphological alteration of mediastinal fat tissue (MFT) was seen after infection of M. pulmonis in D2 mice but not in B6 mice. In this study, we aimed to exhibit the difference in adipose tissue response in other areas, including interscapular brown adipose tissue (iBAT), inguinal white adipose tissue (ingWAT), and perigonadal WAT (perigoWAT) between resistant strain, B6 and susceptible strain, D2 after challenging them with M. pulmonis and SeV. Compared with B6 mice, D2 mice showed an increase in fat-associated lymphoid cluster in MFT, an increase in BAT in both iBAT and ingWAT after M. pulmonis and SeV infection. The results of this study indicate that pneumonia caused by M. pulmonis and SeV infection induces browning of adipocyte, suggesting that BAT plays a role in pathogen infection and inflammation.
• Reduced translation efficiency due to novel splicing variants in 5' untranslated region and identification of novel cis-regulatory elements in canine and human BRCA2.

  Yasunaga Yoshikawa, Hajime Kozuma, Masami Morimatsu, Kaori Sugawara, Koichi Orino

  BMC molecular and cell biology 22 (1) 2 - 2 2021/01/06 [Refereed]
   

  BACKGROUND: Breast cancer 2, early onset (BRCA2) is a tumor suppressor gene. The protein encoded by this gene plays an important role in homologous recombination (HR)-mediated DNA repair. Deleterious mutations in BRCA2 and downregulation of its expression have been associated with tumorigenesis in dogs and humans. Thus, regulation of BRCA2 expression level is important for maintaining homeostasis in homologous recombination. RESULTS: In this study, the mechanisms that regulate the expression of BRCA2 were proposed. Novel splicing variants were identified in the 5' untranslated region (UTR) of canine and human BRCA2 in canine testis, canine ovary, and canine and human cultured cell lines. In cultured cells, the ratio of BRCA2 splicing variants at the 5' UTR was altered by serum starvation. These novel splicing variants, excluding one of the canine splicing variants, were found to reduce the translational efficiency. Additionally, the DNA sequence in human BRCA2 intron 1 harbored novel cis-regulatory elements. Three silencer and two enhancer cis-regulatory elements were identified in human BRCA2 intron 1. CONCLUSIONS: This study demonstrates that BRCA2 expression level is regulated via 5' UTR splicing variants and that the BRCA2 intron 1 region harbors cis-regulatory elements.
• Novel canine isocitrate dehydrogenase 1 mutation Y208C attenuates dimerization ability.

  Shota Kawakami, Masaki Michishita, Motoharu Sakaue, Masami Morimatsu, Mitsuki Uemura, Nobuaki Kashiwagi, Marika Maeda, Yukino Machida, Daigo Azakami, Ai S Egusa, Eri Onozawa, Katsumi Ishioka, Masami Watanabe, Yoshikazu Tanaka, Toshinori Omi, Kazuhiko Ochiai

  Oncology letters 20 (6) 351 - 351 2020/12 [Refereed]
   

  Isocitrate dehydrogenase 1 (IDH1) mutations are common in gliomas, acute myeloid leukemia, and chondrosarcoma. The mutation 'hotspot' is a single arginine residue, R132. The R132H mutant of IDH1 produces the 2-hydroxyglutarate (2-HG) carcinogen from α-ketoglutarate (α-KG). The reduction of α-KG induces the accumulation of hypoxia-inducible factor-1α subunit (HIF-1α) in the cytosol, which is a predisposing factor for carcinogenesis. R132H is the most common IDH1 mutation in humans, but mutations at the R132 residue can also occur in tumor tissues of dogs. The current study reported the discovery of a novel Tyr208Cys (Y208C) mutation in canine IDH1 (cIDH1), which was isolated from 2 of 45 canine chondrosarcoma cases. As the genomic DNA isolated from chondrosarcoma tissue was mutated, but that isolated from blood was not, Y208C mutations were considered to be spontaneous somatic mutations. The isocitrate dehydrogenase activity of the Y208C mutant was attenuated compared with that of wild-type (WT) cIDH1, but the attenuation of Y208C was less intense than that of the R132H mutation. The induction of HIF-1α response element activity and cell retention of HIF-1α were not increased by Y208C overexpression. In silico and cell biological analysis of IDH1 dimerization revealed that the Y208C mutation, but not the R132H mutation, attenuated binding activity with WT cIDH1. These data suggested that the attenuation of dimerization by the Y208C mutation may cause tumorigenesis through different mechanisms other than via 2-HG production by the IDH1 R132 mutation.
• The canine RAD51 mutation leads to the attenuation of interaction with PALB2.

  Mitsuki Uemura, Kazuhiko Ochiai, Masami Morimatsu, Masaki Michishita, Eri Onozawa, Daigo Azakami, Yumiko Uno, Yasunaga Yoshikawa, Takanori Sasaki, Masami Watanabe, Toshinori Omi

  Veterinary and comparative oncology 18 (2) 247 - 255 1476-5810 2020/06 [Refereed][Not invited]
   

  RAD51 forms a complex with BRCA2 and plays a central role in the DNA damage response pathway that is associated with homologous recombination. The structures of RAD51 and its homologues are highly conserved from prokaryotes to higher eukaryotes. Although a large number of BRCA2 mutations have been reported, there are only a few reports on the mutations of RAD51, which have been shown in humans and dogs. However, several mutations of canine RAD51 were identified from mammary gland tumour tissues in a recent study. Some of these mutations seem to have an influence on the homo-oligomerization or interaction with "Partner and localizer of BRCA2" (PALB2). In this study, we cloned the canine PALB2 homologue and investigated the effect on its interaction with the RAD51 mutants to evaluate the alteration in the function of RAD51 mutants. The A209S and T225S mutants of RAD51 show an attenuation of the interaction between RAD51 and PALB2. These results indicate that the canine RAD51 mutations can potentially alter the homologous recombination pathways in response to DNA damage in dogs.
• Profiling of cellular immune responses to Mycoplasma pulmonis infection in C57BL/6 and DBA/2 mice.

  Tussapon Boonyarattanasoonthorn, Yaser Hosny Ali Elewa, Hassan T Tag-El-Din-Hassan, Masami Morimatsu, Takashi Agui

  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 73 55 - 65 1567-1348 2019/09 [Refereed][Not invited]
   

  Mycoplasma infections cause respiratory tract damages and atypical pneumonia, resulting in serious problems in humans and animals worldwide. It is well known that laboratory inbred mouse strains show various susceptibility to Mycoplasma pulmonis (M. pulmonis) infection, which causes murine respiratory mycoplasmosis. In this study, we aimed to demonstrate the difference in cellular immune responses between resistant strain, C57BL/6NCrSlc (B6) and susceptible strain, DBA/2CrSlc (D2) after challenging M. pulmonis infection. D2 mice showed higher amount of bacterial proliferation in lung, higher pulmonary infiltration of immune cells such as neutrophils, macrophages, and lymphocytes, and higher levels of interleukin (IL)-1β, IL-6, IL-17A, and tumor necrosis factor-α in bronchoalveolar lavage fluid than did B6 mice. The results of this study suggest that D2 mice are more susceptible than B6 mice to M. pulmonis infection due to a hyper-immune inflammatory response.
• Null mutation of the endothelin receptor type B gene causes embryonic death in the GK rat.

  Jinxi Wang, Ruihua Dang, Yoshiki Miyasaka, Kousuke Hattori, Daisuke Torigoe, Tadashi Okamura, Hassan T Tag-Ei-Din-Hassan, Masami Morimatsu, Tomoji Mashimo, Takashi Agui

  PloS one 14 (6) e0217132  1932-6203 2019 [Refereed][Not invited]
   

  The Hirschsprung disease (HSCR) is an inherited disease that is controlled by multiple genes and has a complicated genetic mechanism. HSCR patients suffer from various extents of constipation due to dysplasia of the enteric nervous system (ENS), which can be so severe as to cause complete intestinal obstruction. Many genes have been identified as playing causative roles in ENS dysplasia and HSCR, among them the endothelin receptor type B gene (Ednrb) has been identified to play an important role. Mutation of Ednrb causes a series of symptoms that include deafness, pigmentary abnormalities, and aganglionosis. In our previous studies of three rat models carrying the same spotting lethal (sl) mutation on Ednrb, the haplotype of a region on chromosome (Chr) 2 was found to be responsible for the differing severities of the HSCR-like symptoms. To confirm that the haplotype of the responsible region on Chr 2 modifies the severity of aganglionosis caused by Ednrb mutation and to recreate a rat model with severe symptoms, we selected the GK inbred strain, whose haplotype in the responsible region on Chr 2 resembles that of the rat strain in which severe symptoms accompany the Ednrbsl mutation. An Ednrb mutation was introduced into the GK rat by crossing with F344-Ednrbsl and by genome editing. The null mutation of Ednrb was found to cause embryonic death in F2 progeny possessing the GK haplotype in the responsible region on Chr 2. The results of this study are unexpected, and they provide new clues and animal models that promise to contribute to studies on the genetic regulatory network in the development of ENS and on embryogenesis.
• Analysis for genetic loci controlling protoscolex development in the Echinococcus multilocularis infection using congenic mice.

  Md Atiqul Islam, Daisuke Torigoe, Yayoi Kameda, Takao Irie, Hirokazu Kouguchi, Ryo Nakao, Md Abdul Masum, Osamu Ichii, Yasuhiro Kon, Hassan T Tag-El-Din-Hassan, Masami Morimatsu, Kinpei Yagi, Takashi Agui

  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 65 65 - 71 1567-1348 2018/11 [Refereed][Not invited]
   

  The resistance/susceptibility to Echinococcus multilocularis infection in mice is genetically controlled. However, genetic factors responsible for these differences remain unknown. Our previous study in genetic linkage analysis has revealed that there is a significant quantitative trait locus (QTL) for the establishment of cyst (Emcys1), and a highly significant QTL for the development of protoscolex of E. multilocularis larvae (Empsc1), on mouse chromosomes 6 and 1, respectively. The current study aimed to confirm these QTLs and narrow down the critical genetic region that controls resistance/susceptibility to E. multilocularis infection by establishing congenic and subcongenic lines from C57BL/6 (B6) and DBA/2 (D2) mice. For protoscolex development phenotype, two congenic lines, B6.D2-Empsc1 and D2.B6-Empsc1 were developed, where responsible QTL, Empsc1 was introgressed from D2 into B6 background and vice versa. For cyst establishment phenotype, two congenic lines, B6.D2-Emcys1 and D2.B6-Emcys1 were developed, where responsible QTL, Emcys1 was introgressed from D2 into B6 background and vice versa. Because there was no significant difference in cyst establishment between B6.D2-Emcys1 and D2.B6-Emcys1 mice after challenge with E. multilocularis, it is suggested that the Emcys1 does not solely control the cyst establishment in mouse liver. However, infection experiments with B6.D2-Empsc1 and D2.B6-Empsc1 mice showed a significant difference in protoscolex development in the cyst. It confirms that the Empsc1 controls phenotype of the protoscolex development in the cyst. Subsequently, two subcongenic lines, B6.D2-Empsc1.1 and B6.D2-Empsc1.2 from B6.D2-Emcys1 and one subcongenic line, D2.B6-Empsc1.1 from D2.B6-Empsc1 were developed to narrow down the critical region responsible for protoscolex development. From the results of infection experiments with E. multilocularis in these subcongenic mice, it is concluded that a gene responsible for protoscolex development is located between D1Mit290 (68.1 cM) and D1Mit511 (97.3 cM).
• Functional analysis of duck, goose, and ostrich 2'-5'-oligoadenylate synthetase.

  Hassan T Tag-El-Din-Hassan, Masami Morimatsu, Takashi Agui

  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 62 220 - 232 1567-7257 2018/08 [Refereed][Not invited]
   

  Up-to-date the flavivirus infection in avian taxa is not clearly defined. Several reports have demonstrated that many viruses belonging to Flaviviridae may cause diseases in poultry species; however, the susceptibility of other avian species is variable and still unclear. In human and mice, the 2'-5'-oligoadenylate synthetase (OAS) proteins are associated with resistance to the flavivirus infection as well as other virus infections. However, the avian OAS proteins are rarely studied. In our previous studies, we confirmed that the chicken OAS-like protein (chOASL) expressed OAS-enzymatic activity (the classical OAS/RNase L-dependent pathway) as well as the anti-flavivirus activity (the putative OAS/RNase L-independent pathway). Therefore, the current study aimed at functional analysis of avian OAS proteins from duck, goose, and ostrich. The duOASL, goOASL, and osOAS1 proteins expressed enzymatic activity as well as chOASL, whereas osOASL expressed little enzymatic activity. On the other hand, duOASL, goOASL, and osOASL possessed significant antiviral activity against West Nile virus (WNV)-replicon replication as well as chOASL, whereas osOAS1 did not. In addition, similar to chOASL, their antiviral activity was independent of RNase L activation. These results suggest that OASL is the only OAS protein in the duck and goose as well as chicken and possesses both OAS-enzymatic and anti-flavivirus activities, whereas the ostrich possesses both OAS1 and OASL proteins with sharing the functional activities, OAS-enzymatic and anti-flavivirus activities, respectively. It is of interest that the ostrich undergoes differential process in OAS gene evolution from other poultries and thus possesses different molecular mechanism in antiviral activity.
• Endogenous Leu332Gln mutation in p53 disrupts the tetramerization ability in a canine mammary gland tumor cell line.

  Kazuhiko Ochiai, Daigo Azakami, Masami Morimatsu, Hinako Hirama, Shota Kawakami, Takayuki Nakagawa, Masaki Michishita, Ai S Egusa, Takanori Sasaki, Masami Watanabe, Toshinori Omi

  Oncology reports 40 (1) 488 - 494 1021-335X 2018/07 [Refereed][Not invited]
   

  Mutations in the p53 gene are associated with more than half of all human cancers. These mutations often cause a disruption of the tumor-suppressor function of p53 and induce genomic instabilities. Wild‑type p53 requires tetramerization to function as an initiator of cell cycle arrest and apoptosis. Although alterations in p53 tetramerization caused by mutation have been well studied, there are few cell lines containing an endogenous mutation in the tetramerization domain of p53. Here, we report the discovery of a canine mammary gland tumor cell line CTB‑m2, which contains the Leu332Gln (L332Q) mutation corresponding to Leu344 in the tetramerization domain of human p53. Although CTB‑m2 cells are genetically heterozygous for the Leu332Gln mutation, the mutant mRNA was almost exclusively expressed. CTB‑m2 cells showed enhanced cell proliferation compared to wild‑type p53-expressing CTB‑m cells of the same lineage. A p53 tetramerization reporter assay showed that the ability of the p53 mutant to form tetramers was significantly lower than that of wild‑type p53. An immunoblot analysis of cross-linked p53 oligomerized forms demonstrated that the L332Q mutant lacked the ability to form tetramers but retained the ability to form dimers. These data suggest that the p53 mutant cell line CTB‑m2 could be a useful tool for analyzing the precise tetramerization mechanisms of p53 and verifying the effects of therapeutic agents against tumors expressing p53 mutants that lack the ability to tetramerize.
• Immune cellular responses to sendai virus infection in D2.B6-Sen1Sen2Sen3 congenic mice, of which three quantitative trait loci responsible for the resistance to infection were introgressed from C57BL/6 mouse into DBA/2 mouse

  Raghda M. F. Abbas, Hassan T. Tag-El-Din-Hassan, Tussapon Boonyarattanasoonthorn, Keisuke Aoshima, Masami Morimatsu, Takashi Agui

  Japanese Journal of Veterinary Research 66 (2) 93 - 103 0047-1917 2018/05/01 [Refereed][Not invited]
   

  It has been reported that C57BL/6 (B6) mice are resistant to the Sendai virus (SeV) infection, whereas DBA/2 (D2) mice are susceptible, the cause of susceptibility in D2 mice is hyper-inflammatory cytokine production, and three quantitative trait loci (QTLs), Sen1, Sen2, and Sen3 are identified to be responsible for this difference. We previously have verified that the introgression of B6-derived these three QTLs into D2-genetic background increases survival rate and suppresses cytokine production by generating D2.B6-Sen1Sen2Sen3 congenic mice. In this study, we investigated immune cellular responses of D2.B6-Sen1Sen2Sen3 mice after SeV infection. Body weight loss, viral load, immune cells in broncho-alveolar lavage fluid (BALF), and histopathological index of SeV-infected male D2. B6-Sen1Sen2Sen3 mice were comparable to those of B6 mice except for the number of neutrophils in BALF. In contrast, female D2.B6-Sen1Sen2Sen3 mice were divided into survived and non-survived mice after SeV infection. Viral load and macrophage number in BALF in SeV-infected female D2. B6-Sen1Sen2Sen3 mice were comparable to those of B6 mice, whereas the number of total cells, neutrophils, and lymphocytes in BALF were remained in the level of D2 mouse. There was a correlation between body weight loss and these immune cellular responses in SeV-infected female D2.B6-Sen1Sen2Sen3 mice. These results indicate that the introgression of B6 alleles of these three QTLs into D2-genetic background resulted in resistance to SeV infection by optimizing the aggressive immune cellular responses that seen in D2 mice, although there may be other loci responsible for difference between B6 and D2 mice.
• R132 mutations in canine isocitrate dehydrogenase 1 (IDH1) lead to functional changes.

  Shota Kawakami, Kazuhiko Ochiai, Daigo Azakami, Yuiko Kato, Masaki Michishita, Masami Morimatsu, Toshina Ishiguro-Oonuma, Eri Onozawa, Masami Watanabe, Toshinori Omi

  Veterinary research communications 42 (1) 49 - 56 0165-7380 2018/03 [Refereed][Not invited]
   

  Glioma is the second most common intracranial neoplasia in dogs, but the pathogenic mechanisms remain unclear. In humans, isocitrate dehydrogenase 1 (IDH1) is frequently mutated in gliomas. Although almost all human IDH1 mutations have been identified as involving the Arg132 codon, few studies have reported structural, functional, and mutational information for canine IDH1. Therefore, in this study, we cloned the canine IDH1 homologue and used PCR mutagenesis to substitute the wildtype (WT) Arg132 with His (R132H) or Ser (R132S). WT and mutated IDH1 were overexpressed in HeLa cells, and their presence was confirmed by immunoblotting and immunocytochemistry using mutation-specific antibodies. The IDH1 activity between WT, R132H, and R132S transfectants was compared by measuring the production of NADH and NADPH. NADPH production in R132H and R132S transfectants was lower than that in WT, but NADH levels were not significantly different. Finally, we detected increased expression of hypoxia inducible factor 1 alpha (HIF-1α) in the R132H and R132S transfectants. These results indicated that the canine IDH1 Arg132 mutation has the potential to induce carcinogenesis in canine somatic cells.
• Verification of genetic loci responsible for the resistance/susceptibility to the Sendai virus infection using congenic mice.

  Raghda Mohamed Fathi Abbas, Daisuke Torigoe, Yayoi Kameda, Hassan T Tag-El-Din-Hassan, Nobuya Sasaki, Masami Morimatsu, Takashi Agui

  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 57 75 - 81 1567-1348 2018/01 [Refereed][Not invited]
   

  Sendai virus (SeV) is one of the most important pathogens in the specific-pathogen free rodents. It is known that there are some inbred mouse strains susceptible or resistant to SeV infection. The C57BL/6 (B6) and DBA/2 (D2) mice are representative of the resistant and susceptible strains, respectively. Previous study with the quantitative trait locus (QTL) analysis identified three QTLs responsible for resistance or susceptibility to SeV infection on different chromosomes and indicated that resistance or susceptibility to SeV infection was almost predicted by genotypes of these three QTLs. In this paper, to verify the above hypothesis, congenic lines were generated as follows; B6-congenic lines carrying one of the D2 alleles of three QTLs and combination of these three QTLs, and D2-congenic lines carrying single or combination of B6 alleles of three QTLs. All these congenic lines were then challenged with SeV infection. D2 congenic lines introgressed single or combination of B6 alleles of QTLs changed to resistance to SeV infection. Especially, a D2 triple-congenic line became resistant as similar level to B6-parental strain. However, B6-congenic lines introgressed single or combination of D2 alleles of QTLs all remained to be resistant to SeV infection. Both IL-6 and TNF-α in broncho-alveolar lavage fluid of D2 triple-congenic line were decreased to the similar level of B6 mice, suggesting that this is a part of factors that D2 triple-congenic line became resistant to the similar level of B6 mice. Data obtained from these congenic mice verified that three QTLs identified previously were indeed responsible for the resistance/susceptibility to SeV infection in B6 and D2 mice.
• Comparison of the antiviral potential among soluble forms of herpes simplex virus type-2 glycoprotein D receptors, herpes virus entry mediator A, nectin-1 and nectin-2, in transgenic mice

  Yoshikazu Fujimoto, Yukiko Tomioka, Kinuyo Ozaki, Keiko Takeda, Haruka Suyama, Sayo Yamamoto, Hiroki Takakuwa, Masami Morimatsu, Toshimitsu Uede, Etsuro Ono

  JOURNAL OF GENERAL VIROLOGY 98 (7) 1815 - 1822 0022-1317 2017/07 [Refereed][Not invited]
   

  Herpesvirus entry mediator A (HVEM), nectin-1 and nectin-2 are cellular receptors of glycoprotein D (gD) of herpes simplex virus type-2 (HSV-2). It has been shown that soluble forms of HSV gD receptors have the antiviral potential in cultured cells and transgenic mice. Here, to compare antiviral potential of soluble forms of HVEM, nectin-1 and nectin-2 against HSV-2 infections in vivo, transgenic mice expressing fusion proteins consisting of the entire ectodomain of HVEM, nectin-1 or nectin-2 and the Fc portion of human IgG (HVEMIg, nectin-1Ig and nectin-2Ig, respectively) were intraperitoneally infected with HSV-2. In the infection with 3 MLD50 (50 % mouse lethal dose), effective resistance was not observed in transgenic mice expressing nectin-21g. In a transgenic mouse line with high expression of nectin-11g, significant protection from the infection with 30 and 300 MLD50 was observed (survival rate of 100 and 71 %, respectively). On the other hand, transgenic mice expressing HVEMIg showed a complete resistance to the lethal infection even with 300 MLD50 (survival rate of 100 %). These results demonstrated that HVEMIg could exert effective antiviral activities against HSV-2 infections in vivo as compared with other soluble forms of HSV gD receptors.
• Cell-cycle arrest in mature adipocytes impairs BAT development but not WAT browning, and reduces adaptive thermogenesis in mice

  Yuko Okamatsu-Ogura, Keigo Fukano, Ayumi Tsubota, Junko Nio-Kobayashi, Kyoko Nakamura, Masami Morimatsu, Hiroshi Sakaue, Masayuki Saito, Kazuhiro Kimura

  SCIENTIFIC REPORTS 7 (1) 6648  2045-2322 2017/07 [Refereed][Not invited]
   

  We previously reported brown adipocytes can proliferate even after differentiation. To test the involvement of mature adipocyte proliferation in cell number control in fat tissue, we generated transgenic (Tg) mice over-expressing cell-cycle inhibitory protein p27 specifically in adipocytes, using the aP2 promoter. While there was no apparent difference in white adipose tissue (WAT) between wild-type (WT) and Tg mice, the amount of brown adipose tissue (BAT) was much smaller in Tg mice. Although BAT showed a normal cellular morphology, Tg mice had lower content of uncoupling protein 1 (UCP1) as a whole, and attenuated cold exposure-or beta 3-adrenergic receptor (AR) agonist-induced thermogenesis, with a decrease in the number of mature brown adipocytes expressing proliferation markers. An agonist for the beta 3-AR failed to increase the number of proliferating brown adipocytes, UCP1 content in BAT, and oxygen consumption in Tg mice, although the induction and the function of beige adipocytes in inguinal WAT from Tg mice were similar to WT mice. These results show that brown adipocyte proliferation significantly contributes to BAT development and adaptive thermogenesis in mice, but not to induction of beige adipocytes.
• Canine REIC/Dkk-3 interacts with SGTA and restores androgen receptor signalling in androgen-independent prostate cancer cell lines

  Yuiko Kato, Kazuhiko Ochiai, Shota Kawakami, Nobuhiro Nakao, Daigo Azakami, Makoto Bonkobara, Masaki Michishita, Masami Morimatsu, Masami Watanabe, Toshinori Omi

  BMC VETERINARY RESEARCH 13 (1) 170  1746-6148 2017/06 [Refereed][Not invited]
   

  Background: The pathological condition of canine prostate cancer resembles that of human androgen-independent prostate cancer. Both canine and human androgen receptor (AR) signalling are inhibited by overexpression of the dimerized co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein a (SGTA), which is considered to cause the development of androgen-independency. Reduced expression in immortalised cells (REIC/Dkk-3) interferes with SGTA dimerization and rescues AR signalling. This study aimed to assess the effects of REIC/Dkk-3 and SGTA interactions on AR signalling in the canine androgen-independent prostate cancer cell line CHP-1. Results: Mammalian two-hybrid and Halo-tagged pull-down assays showed that canine REIC/Dkk-3 interacted with SGTA and interfered with SGTA dimerization. Additionally, reporter assays revealed that canine REIC/Dkk-3 restored AR signalling in both human and canine androgen-independent prostate cancer cells. Therefore, we confirmed the interaction between canine SGTA and REIC/Dkk-3, as well as their role in AR signalling. Conclusions: Our results suggest that this interaction might contribute to the development of a novel strategy for androgen-independent prostate cancer treatment. Moreover, we established the canine androgen-independent prostate cancer model as a suitable animal model for the study of this type of treatment-refractory human cancer.
• Analysis of the Relationship Between Enzymatic and Antiviral Activities of the Chicken Oligoadenylate Synthetase-Like

  Hassan T. Tag-El-Din-Hassan, Nobuya Sasaki, Daisuke Torigoe, Masami Morimatsu, Takashi Agui

  JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 37 (2) 71 - 80 1079-9907 2017/02 [Refereed][Not invited]
   

  The oligoadenylate synthetase (OAS) is well known as an antiviral factor against the flavivirus infection in mammals. It is known that the oligoadenylate synthetase-like (ChOAS-L) gene is only present in the chicken genome. It has been shown in the previous report that the ChOAS-L possesses enzymatic activity to convert ATP into 2'-5'-linked oligoadenylates and antiviral activity against West Nile virus (WNV) replicon. Therefore, this study aimed to investigate the relationship between enzymatic and antiviral activities of ChOAS-L. Eight mutated ChOAS-L proteins were generated using either the site-directed mutagenesis or standard polymerase chain reaction protocol. The wild-type and mutated proteins were ectopically expressed in 293FT cells to analyze the enzymatic activity and in BHK-21 and BALB/3T3 cells to analyze the antiviral activity using WNV replicon. The results revealed that all mutated proteins showed no enzymatic activity except for ChOAS-L-A Delta UbL2. However, all mutated proteins showed antiviral activity to inhibit the replication of the WNV replicon except for ChOAS-L-A Delta UbL1/UbL2, which showed a partial inhibition compared to the wild-type ChOAS-L-A or other mutated proteins. These results suggest that the ChOAS-L expresses the antiflavivirus activity in a manner independent of enzymatic activity. Our results propose reconsideration of the mechanism of antiviral activity against the flavivirus replication of ChOAS-L.
• The role of mouse 2 ',5 '-oligoadenylate synthetase 1 paralogs

  Enas Elkhateeb, Hassan T. Tag-El-Din-Hassan, Nobuya Sasaki, Daisuke Torigoe, Masami Morimatsu, Takashi Agui

  INFECTION GENETICS AND EVOLUTION 45 393 - 401 1567-1348 2016/11 [Refereed][Not invited]
   

  The interferon-induced oligoadenylate synthetase (OAS) family is one of the most important immune response proteins to the viral infection. The OAS protein binds dsRNA and is activated to produce 2',5'-oligoadenylates, which lead to the activation of latent form of RNase L, resulting in degradation of cellular and viral RNA and inhibition of viral replication. In mice, the Oas gene family locates on chromosome 5. The mouse Oas gene locus undergoes a recent series of duplication event, leading to the presence of eight paralogs of Oas1 genes (Oas1a through Oas1h) that forms Oas gene cluster with the Oas2, Oas3 and two OasL (OasL1 and OasL2) genes. Previous studies demonstrated that the mouse Oas1b gene conferred resistance to the flavivirus infection in mice; however, the antiviral activity of other mouse Oas1 gene family is still unknown. Therefore, in the present study, we have evaluated the mouse Oas1 paralogs regarding the enzymatic activity and antiviral activity against the two neurotropic flaviviruses, West Nile virus and tick-borne encephalitis virus. The mouse Oas1 genes were cloned from C57BL/6J (B6) as well as the Oas1b derived from feral mouse strain, MSM. The obtained results demonstrated that only Oas1a and Oas1g showed enzymatic activity. Although MSM-derived Oas1b showed antiviral activity to both viruses, all B6-derived OAS paralogs did not show antiviral activity. These results suggest that Oas1a and Oas1g play a role in potentiating viral RNA-induced interferon response in the cell, whereas the Oas1b works as a specific anti-flavivirus factor unless it is mutated. However, the role of other paralogs is unknown and should wait for further investigation. (C) 2016 Elsevier B.V. All rights reserved.
• A soluble form of Siglec-9 provides a resistance against Group B Streptococcus (GBS) infection in transgenic mice

  Mitsumasa Saito, Sayo Yamamoto, Kinuyo Ozaki, Yukiko Tomioka, Haruka Suyama, Masami Morimatsu, Ken-ichi Nishijima, Shin-ichi Yoshida, Etsuro Ono

  MICROBIAL PATHOGENESIS 99 106 - 110 0882-4010 2016/10 [Refereed][Not invited]
   

  Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns. A key GBS virulence factor is its capsular polysaccharide (CPS), possessing terminal sialic acid residues that suppress host immune response and provide a survival advantage to the pathogen. CPS binds to Siglec-9 expressed on neutrophils, which is expected to down-regulate the immune responsiveness of neutrophils. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of CPS to Siglec-9 on immune cells, leading to provide antibacterial benefit against GBS infection in the transgenic mouse line expressing sSiglec-9 (sSiglec-9 Tg). The sSiglec-9 in the sera of sSiglec-9 Tg bound to the sialylated-GBS strains belonging to serotypes Ia, Ib, II, III, IV and V in whole GBS cell ELISA. When GBS cells of serotype III that is a common serotype in late-onset GBS disease (LOD) were intraperitoneally inoculated into sSiglec-9 Tg, sSiglec-9 Tg showed a significant resistance as compared with non-transgenic littermates. Furthermore, GBS serotype III organisms were not detected in cultures of the blood from surviving mice (<1 x 10(3) CFU/ml). These results indicated that sSiglec-9 Tg mice were more efficient in eliminating GBS and survived better after the intraperitoneal challenge with GBS serotype III bacteria. (C) 2016 Elsevier Ltd. All rights reserved.
• Cross-protective potential of anti-nucleoprotein human monoclonal antibodies against lethal influenza A virus infection

  Yoshikazu Fujimoto, Yukiko Tomioka, Hiroki Takakuwa, Gen-Ichiro Uechi, Toshiyo Yabuta, Kinuyo Ozaki, Haruka Suyama, Sayo Yamamoto, Masami Morimatsu, Le Quynh Mai, Tetsu Yamashiro, Toshihiro Ito, Koichi Otsuki, Etsuro Ono

  JOURNAL OF GENERAL VIROLOGY 97 (9) 2104 - 2116 0022-1317 2016/09 [Refereed][Not invited]
   

  The nucleoprotein (NP) possesses regions that are highly conserved among influenza A viruses, and has therefore been one of the target viral proteins for development of a universal influenza vaccine. It has been expected that human or humanized antibodies will be made available for the prophylaxis, pre-emptive and acute treatment of viral infection. However, it is still unclear whether anti-NP human antibody can confer protection against influenza virus infection. In this study, we generated transgenic mice expressing anti-NP human mAbs derived from lymphocytes of a patient infected with H5N1 highly pathogenic avian influenza (HPAI) virus, and experimental infections were conducted to examine antiviral effects of the anti-NP antibodies against H5N1 HPAI viral infections with a high fatality rate in mammals. Transgenic mouse lines expressing the anti-NP human mAbs at more than 1 mg ml(-1) showed marked resistance to H5N1 virus infections. In addition, resistance to infection with an H1N1 subtype that shows strong pathogenicity to mice was also confirmed. Although the anti-NP mAbs expressed in the transgenic mice did not neutralize the virus, the mAbs could bind to NP located on the surface of infected cells. These results suggested a possibility that the non-neutralizing anti-NP human mAbs could induce indirect antiviral effects, such as antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. Taken together, these results demonstrated that anti-NP human mAbs play an important role in heterosubtypic protection against lethal influenza virus infections in vivo.
• A soluble form of human nectin-2 impairs exocrine secretion of pancreas and formation of zymogen granules in transgenic mice

  Yukiko Tomioka, Yoshikazu Fujimoto, Kanji Nakai, Kinuyo Ozaki, Sayo Yamamoto, Haruka Suyama, Masami Morimatsu, Toshihiro Ito, Etsuro Ono

  Biochemistry and Biophysics Reports 5 196 - 202 2405-5808 2016/03/01 [Refereed][Not invited]
   

  Transgenic mouse lines expressing a soluble form of human nectin-2 (hNectin-2Ig Tg) exhibited distinctive elevation of amylase and lipase levels in the sera. In this study, we aimed to clarify the histopathology and to propose the transgenic mouse lines as new animal model for characteristic pancreatic exocrine defects. The significant increase of amylase and lipase levels in sera of the transgenic lines approximately peaked at 8 weeks old and thereafter, plateaued or gradually decreased. The histopathology in transgenic acinar cells was characterized by intracytoplasmic accumulation of abnormal proteins with decrease of normal zymogen granules. The hNectin-2Ig expression was observed in the cytoplasm of pancreatic acinar cells, which was consistent with zymogen granules. However, signals of hNectin-2Ig were very weak in the transgenic acinar cells with the abnormal cytoplasmic accumulaion. The PCNA-positive cells increased in the transgenic pancreas, which suggested the affected acinar cells were regenerated. Acinar cells of hNectin-2Ig Tg had markedly small number of zymogen granules with remarkable dilation of the endoplasmic reticulum (ER) lumen containing abundant abnormal proteins. In conclusion, hNectin-2Ig Tg is proposed as a new animal model for characteristic pancreatic exocrine defects, which are due to the ER stress induced by expression of mutated cell adhesion molecule that is a soluble form of human nectin-2.
• Tumor suppressor REIC/DKK-3 and co-chaperone SGTA: Their interaction and roles in the androgen sensitivity

  Kazuhiko Ochiai, Masami Morimatsu, Yuiko Kato, Toshina Ishiguro-Oonuma, Chihiro Udagawa, Oumaporn Rungsuriyawiboon, Daigo Azakami, Masaki Michishita, Yuichi Ariyoshi, Hideo Ueki, Yasutomo Nasu, Hiromi Kumon, Masami Watanabe, Toshinori Omi

  ONCOTARGET 7 (3) 3273 - 3286 1949-2553 2016/01 [Refereed][Not invited]
   

  REIC/DKK-3 is a tumor suppressor, however, its intracellular physiological functions and interacting molecules have not been fully clarified. Using yeast two-hybrid screening, we found that small glutamine-rich tetratricopeptide repeat-containing protein a (SGTA), known as a negative modulator of cytoplasmic androgen receptor (AR) signaling, is a novel interacting partner of REIC/DKK-3. Mammalian two-hybrid and pull-down assay results indicated that the SGTA-REIC/DKK-3 interaction involved the N-terminal regions of both REIC/DKK-3 and SGTA and that REIC/DKK-3 interfered with the dimerization of SGTA, which is a component of the AR complex and a suppressor of dynein motor-dependent AR transport and signaling. A reporter assay in human prostate cancer cells that displayed suppressed AR signaling by SGTA showed recovery of AR signaling by REIC/DKK-3 expression. Considering these results and our previous data that REIC/DKK-3 interacts with the dynein light chain TCTEX-1, we propose that the REIC/DKK-3 protein interferes with SGTA dimerization, promotes dynein-dependent AR transport and then upregulates AR signaling.
• Molecular cloning of canine co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) and investigation of its ability to suppress androgen receptor signalling in androgen-independent prostate cancer

  Yuiko Kato, Kazuhiko Ochiai, Masaki Michishita, Daigo Azakami, Rei Nakahira, Masami Morimatsu, Toshina Ishiguro-Oonuma, Yasunaga Yoshikawa, Masato Kobayashi, Makoto Bonkobara, Masanori Kobayashi, Kimimasa Takahashi, Masami Watanabe, Toshinori Omi

  VETERINARY JOURNAL 206 (2) 143 - 148 1090-0233 2015/11 [Refereed][Not invited]
   

  Although the morbidity of canine prostate cancer is low, the majority of cases present with resistance to androgen therapy and poor clinical outcomes. These pathological conditions are similar to the signs of the terminal stage of human androgen-independent prostate cancer. The co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) is known to be overexpressed in human androgenindependent prostate cancer. However, there is little information about the structure and function of canine SGTA. In this study, canine SGTA was cloned and analysed for its ability to suppress androgen receptor signalling. The full-length open reading frame (ORF) of the canine SGTA gene was amplified by RT-PCR using primers designed from canine-expressed sequence tags that were homologous to human SGTA. The canine SGTA ORF has high homology with the corresponding human (89%) and mouse (81%) sequences. SGTA dimerisation region and tetratricopeptide repeat (TPR) domains are conserved across the three species. The ability of canine SGTA to undergo homodimerisation was demonstrated by a mammalian two-hybrid system and a pull-down assay. The negative impact of canine SGTA on androgen receptor (AR) signalling was demonstrated using a reporter assay in androgen-independent human prostate cancer cell lines. Pathological analysis showed overexpression of SGTA in canine prostate cancer, but not in hyperplasia. A reporter assay in prostate cells demonstrated suppression of AR signalling by canine SGTA. Altogether, these results suggest that canine SGTA may play an important role in the acquisition of androgen independence by canine prostate cancer cells. (C) 2015 Elsevier Ltd. All rights reserved.
• Nfkbiz regulates the proliferation and differentiation of keratinocytes

  Toshina Ishiguro-Oonuma, Kazuhiko Ochiai, Kazuyoshi Hashizume, Toshihiko Iwanage, Masami Morimatsu

  JAPANESE JOURNAL OF VETERINARY RESEARCH 63 (3) 107 - 114 0047-1917 2015/08 [Refereed][Not invited]
   

  Nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-kappa B) inhibitor zeta (Nfkbiz) is a nuclear inhibitor of NF-kappa B (I kappa B) protein that is also termed as molecule possessing ankyrin repeats induced by lipopolysaccharide, interleukin-1-inducible nuclear ankyrin repeat protein, or I kappa B zeta; We found previously that disrupting the Nfkbiz gene resulted in atopic dermatitis-like lesions in mice, suggesting an important role for Nfkbiz in the skin. In this study, we examined the cellular function of Nfkbiz in keratinocytes. Immunohistochemical analyses for Ki-67 revealed that Nfkbiz(-/-) keratinocytes were hypoproliferative. In skin from Nfkbiz(-/-) mice, the expression of the keratinocyte differentiation markers K10 and filaggrin were reduced, although that of K14 was unchanged. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed that the frequency of apoptosis was comparable between control and Nfkbiz(-/-) keratinocytes. Interestingly, the subcellular localization of the NF-kappa B subunits and the transcriptional activity of NF-kappa B were not changed in Nfkbiz(-/-) keratinocytes. These findings indicate a novel possible role of Nfkbiz in controlling the proliferation and differentiation of epidermal keratinocytes through NF-kappa B-independent mechanisms.
• Reduced canine BRCA2 expression levels in mammary gland tumors

  Yasunaga Yoshikawa, Masami Morimatsu, Kazuhiko Ochiai, Toshina Ishiguro-Oonuma, Seiichi Wada, Koichi Orino, Kiyotaka Watanabe

  BMC VETERINARY RESEARCH 11 159  1746-6148 2015/07 [Refereed][Not invited]
   

  Background: Mammary tumors are the most common tumor type in intact female dogs. Recently, the breast cancer 2 early onset (BRCA2) gene was proposed to be associated with tumorigenesis in dogs. The expression level of BRCA2 is important for its DNA repair function in mammalian cells, and its expression level is linked to tumorigenesis in mammary tissue. However, the expression of canine BRCA2 in mammary tumors is unclear. Results: BRCA2 mRNA levels were compared between seven mammary gland samples and seventeen mammary tumor samples isolated from dogs. The expression level of canine BRCA2 in mammary tumor samples was lower than levels in mammary gland samples. We attempted to identify why the BRCA2 expression level was decreased in mammary tumor samples by promoter sequencing analysis; however, we did not find any mutations in the canine BRCA2 promoter that altered BRCA2 transcription levels. We did detect two types of BRCA2 splice variants in 8 mammary tumor samples. One of the variants induced a frame-shift mutation that could lead to nonsense-mediated mRNA decay, a ubiquitous cellular mechanism that eliminates mRNA containing a premature termination codon. Conclusions: Reduced expression of canine BRCA2 mRNA in mammary tumor samples is a possible mechanism to explain mammary tumor development in dogs. One possible reason for reduced BRCA2 mRNA levels in these tumor samples was nonsense-mediated mRNA decay, not mutations in the BRCA2 promoter region. While it remains unclear why canine BRCA2 expression levels are reduced in mammary tumor samples, this study found that the expression level of BRCA2 was associated with canine mammary tumorigenesis.
• Identification of multiple genetic loci in the mouse controlling immobility time in the tail suspension and forced swimming tests

  Ahmed F. Abou-Elnaga, Daisuke Torigoe, Mohamed M. Fouda, Ragab A. Darwish, Usama A. Abou-Ismail, Masami Morimatsu, Takashi Agui

  JAPANESE JOURNAL OF VETERINARY RESEARCH 63 (2) 53 - 62 0047-1917 2015/05 [Refereed][Not invited]
   

  Depression is one of the most famous psychiatric disorders in humans in all over the countries and considered a complex neurobehavioral trait and difficult to identify causal genes. Tail suspension test (TST) and forced swimming test (FST) are widely used for assessing depression-like behavior and antidepressant activity in mice. A variety of antidepressant agents are known to reduce immobility time in both TST and FST. To identify genetic determinants of immobility duration in both tests, we analyzed 101 F-2 mice from an intercross between C57BL/6 and DBA/2 strains. Quantitative trait locus (QTL) mapping using 106 microsatellite markers revealed three loci (two significant and one suggestive) and five suggestive loci controlling immobility time in the TST and FST, respectively. Results of QTL analysis suggest a broad description of the genetic architecture underlying depression, providing underpinnings for identifying novel molecular targets for antidepressants to clear the complex genetic mechanisms of depressive disorders.
• The role of IFN-gamma in regulating Nfkbiz expression in epidermal keratinocytes

  Toshina Ishiguro-Oonuma, Kazuhiko Ochiai, Kazuyoshi Hashizume, Masami Morimatsu

  BIOMEDICAL RESEARCH-TOKYO 36 (2) 103 - 107 0388-6107 2015/04 [Refereed][Not invited]
   

  Nfkbiz is an inhibitor of nuclear factor KB (IkB) protein localized to the nucleus. We previously found that Nfkbiz gene-disrupted mice showed atopic dermatitis-like lesion, implying the important role of Nfkbiz in skin homeostasis. The purpose of this study was to examine the effect of interferon (IFN)-gamma on Nfkbiz expression in keratinocytes. IFN-gamma induced Nfkbiz expression at a comparable level to IL-1. Promoter analysis revealed that interferon-stimulated response element (ISRE) located in the Nfkbiz promoter region is important for responding to the stimulation. Interestingly, IFN-gamma and IL-1 displayed synergism in terms of inducing Nfkbiz expression. By using selective inhibitors, we found that Janus activated kinase (JAK) 1 and nuclear factor (NF)-kB are important for Nfkbiz expression after IFN-gamma stimulation and for synergism between IFN-gamma and ILL These findings indicate a possible important role of Nfkbiz in modulating the progression of inflammatory diseases in which IFN-gamma and IL-1 are abundant.
• Polymorphisms of canine BRCA2 BRC repeats affecting interaction with RAD51

  Kazuhiko Ochiai, Toshina Ishiguro-Oonuma, Yasunaga Yoshikawa, Chihiro Udagawa, Yuiko Kato, Masami Watanabe, Makoto Bonkobara, Masami Morimatsu, Toshinori Omi

  BIOMEDICAL RESEARCH-TOKYO 36 (2) 155 - 158 0388-6107 2015/04 [Refereed][Not invited]
   

  Mutations in the breast cancer susceptibility gene BRCA2 leading to the failure of interactions with the recombinase RAD51 are associated with an increased risk of cancer in humans. This interaction depends on the eight BRC repeat (BRC1-8) sequences in BRCA2. We previously reported that canine BRC3 has two polymorphisms (T1425P and K1435R) influencing the interaction with RAD51, and 1435R was identified in mammary tumor dog samples. In this study, we investigated the sequence variations of BRC3 and 4 in 236 dogs of five breeds. Allele frequencies of 1425P and 1435R were 0.063 and 0.314, respectively, and there was no other polymorphism in the sequenced region. A mammalian two-hybrid assay using BRC3-4 sequences demonstrated that 1425P allele reduced the binding strength with RAD51 but 1435R had no effect. These results may provide an insight into the functions of not only individual but also multiple BRC repeats of BRCA2 in dogs.
• Hallmarks of Hepatitis C Virus in Equine Hepacivirus

  Tomohisa Tanaka, Hirotake Kasai, Atsuya Yamashita, Kaori Okuyama-Dobashi, Jun Yasumoto, Shinya Maekawa, Nobuyuki Enomoto, Toru Okamoto, Yoshiharu Matsuura, Masami Morimatsu, Noboru Manabe, Kazuhiko Ochiai, Kazuto Yamashita, Kohji Moriishi

  JOURNAL OF VIROLOGY 88 (22) 13352 - 13366 0022-538X 2014/11 [Refereed][Not invited]
   

  Equine hepacivirus (EHcV) has been identified as a closely related homologue of hepatitis C virus (HCV) in the United States, the United Kingdom, and Germany, but not in Asian countries. In this study, we genetically and serologically screened 31 serum samples obtained from Japanese-born domestic horses for EHcV infection and subsequently identified 11 PCR-positive and 7 seropositive serum samples. We determined the full sequence of the EHcV genome, including the 3' untranslated region (UTR), which had previously not been completely revealed. The polyprotein of a Japanese EHcV strain showed approximately 95% homology to those of the reported strains. HCV-like cis-acting RNA elements, including the stem-loop structures of the 3' UTR and kissing-loop interaction were deduced from regions around both UTRs of the EHcV genome. A comparison of the EHcV and HCV core proteins revealed that Ile(190) and Phe(191) of the EHcV core protein could be important for cleavage of the core protein by signal peptide peptidase (SPP) and were replaced with Ala and Leu, respectively, which inhibited intramembrane cleavage of the EHcV core protein. The loss-of-function mutant of SPP abrogated intramembrane cleavage of the EHcV core protein and bound EHcV core protein, suggesting that the EHcV core protein may be cleaved by SPP to become a mature form. The wild-type EHcV core protein, but not the SPP-resistant mutant, was localized on lipid droplets and partially on the lipid raft-like membrane in a manner similar to that of the HCV core protein. These results suggest that EHcV may conserve the genetic and biological properties of HCV. IMPORTANCE EHcV, which shows the highest amino acid or nucleotide homology to HCV among hepaciviruses, was previously reported to infect horses from Western, but not Asian, countries. We herein report EHcV infection in Japanese-born horses. In this study, HCV-like RNA secondary structures around both UTRs were predicted by determining the whole-genome sequence of EHcV. Our results also suggest that the EHcV core protein is cleaved by SPP to become a mature form and then is localized on lipid droplets and partially on lipid raft-like membranes in a manner similar to that of the HCV core protein. Hence, EHcV was identified as a closely related homologue of HCV based on its genetic structure as well as its biological properties. A clearer understanding of the epidemiology, genetic structure, and infection mechanism of EHcV will assist in elucidating the evolution of hepaciviruses as well as the development of surrogate models for the study of HCV.
• DNA相同組換えタンパク質RAD51の動物種特異的アミノ酸配列が機能に及ぼす影響の解析

  落合 和彦, 閑野 大輝, 宇田川 智野, 呰上 大吾, 大沼 俊名, 吉川 泰永, 森松 正美, 近江 俊徳

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 157回 517 - 517 1347-8621 2014/08
• A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice

  Yukiko Tomioka, Masami Morimatsu, Ken-ichi Nishijima, Tatsufumi Usui, Sayo Yamamoto, Haruka Suyama, Kinuyo Ozaki, Toshihiro Ito, Etsuro Ono

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 450 (1) 532 - 537 0006-291X 2014/07 [Refereed][Not invited]
   

  Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation. (C) 2014 Elsevier Inc. All rights reserved.
• Sensitization to laboratory animal allergens among students and researchers exposed to laboratory rodents in Hokkaido University

  Aya Yoshimura, Manabu Musashi, Takeaki Kaneko, Shunsuke Ohnishi, Chieko Orito, Yukako Kawahara, Satoshi Hashino, Masami Morimatsu, Satoshi Konno, Jiro Arikawa, Tetsuya Ishii, Masaya Sawamura, Ichiro Ueda

  Japanese Journal of Allergology 63 (8) 1132 - 1139 1347-7935 2014 [Refereed][Not invited]
   

  Background: Based on a case who developed anaphylaxis after mouse bite which occurred at Hokkaido University, we studied on allergic sensitization prevalence for laboratory animals among students and researchers who are exposed to laboratory rodents and rabbit, for the purpose of allergy prevention, particularly anaphylaxis. Methods: We carried out the health check-up on laboratory animal allergy (LAA) by questionnaires and specific-IgE antibody test for 555 rodents and/or rabbit handlers from whom informed consent was obtained. Result: Prevalence of positive IgE antibody higher than class 1 to mice, rats, hamsters, guinea pigs, and/or rabbits in the examinees was 14.1% (62/441), 17.9% (50/279), 18.8% (6/32), 17.4% (4/23), and 11.3% (12/106), respectively. Moreover, among users of mouse, those who had allergic symptoms during contact with animals resulted in significantly higher positive rate for anti-mouse IgE antibody test than the other (38.1% vs 8.8%, p< 0.01). Conclusion: Health check-up including measurement of specific-IgE antibody against laboratory animals is useful for understanding allergic sensitization.
• Abnormal spermatogenesis and reduced fertility in transgenic mice expressing the immediate-early protein IE180 of pseudorabies virus

  Yukiko Tomioka, Masami Morimatsu, Satoshi Taharaguchi, Sayo Yamamoto, Haruka Suyama, Kinuyo Ozaki, Naoki Iwamori, Etsuro Ono

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 440 (4) 683 - 688 0006-291X 2013/11 [Refereed][Not invited]
   

  Transcription factors of alphaherpesviruses not only control the expression of their own viral genes, but also influence the gene expression of mammalian cells. In the course of breeding of the transgenic mouse line (TgIE96) expressing the immediate-early protein IE180 of pseudorabies virus belonging to the subfamily Alphaherpesvirinae, we found that TgIE96 male mice suffered from severe breeding difficulties. Testes of TgIE96 were smaller than that of non-transgenic littermates and abnormal spermatogenesis such as morphological, numerical and functional anomalies of spermatozoa were found in the transgenic mouse line. Expression of IE180 was detected in the germ cells at all stages, especially spermatocytes, and fewer Sertoli cells. In addition, expression of IE180 was also detected in the germinal cells of C57BL/6 mice inoculated with PRV into their testes. These results suggest that IE180 of PRV induces male infertility by abnormal spermatogenesis, which effect morphological, numerical, and functional anomalies of spermatozoa, in transgenic mice. (C) 2013 Elsevier Inc. All rights reserved.
• Molecular cloning and tumour suppressor function analysis of canine REIC/Dkk-3 in mammary gland tumours

  Kazuhiko Ochiai, Masami Watanabe, Daigo Azakami, Masaki Michishita, Yasunaga Yoshikawa, Chihiro Udagawa, Pornphimon Metheenukul, Thippayarat Chahomchuen, Hiroshi Aoki, Hiromi Kumon, Masami Morimatsu, Toshinori Omi

  VETERINARY JOURNAL 197 (3) 769 - 775 1090-0233 2013/09 [Refereed][Not invited]
   

  REIC/Dkk-3, a member of the human Dickkopf (Dkk) family, plays a role as a suppressor of growth in several human cancers. In this study, the tumour suppression function of canine REIC/Dkk-3 was investigated. The full-length open reading frame of the canine REIC/Dkk-3 homologue was cloned and the tissue distribution of REIC/Dkk-3 mRNA was determined, along with the subcellular localisation of the REIC/Dkk-3 protein in canine cancer cell lines. Expression of REIC/Dkk-3 was lower in mammary gland tumours and in canine mammary carcinoma cell lines than in normal mammary gland tissue. Overexpression of REIC/Dkk-3 induced apoptosis in canine mammary carcinoma cell lines. These results show that expression of REIC/Dkk-3 is downregulated in canine mammary tumours and that one of the functions of this gene is induction of apoptosis. (C) 2013 Elsevier Ltd. All rights reserved.
• I kappa B zeta Is a Transcriptional Key Regulator of CCL2/MCP-1

  Dominic G. Hildebrand, Eva Alexander, Sebastian Hoerber, Simon Lehle, Kerstin Obermayer, Niels-Arne Muenck, Oliver Rothfuss, Julia-Stefanie Frick, Masami Morimatsu, Ingo Schmitz, Johannes Roth, Jan M. Ehrchen, Frank Essmann, Klaus Schulze-Osthoff

  JOURNAL OF IMMUNOLOGY 190 (9) 4812 - 4820 0022-1767 2013/05 [Refereed][Not invited]
   

  CCL2, also referred to as MCP-1, is critically involved in directing the migration of blood monocytes to sites of inflammation. Consequently, excessive CCL2 secretion has been linked to many inflammatory diseases, whereas a lack of expression severely impairs immune responsiveness. We demonstrate that I kappa B zeta, an atypical I kappa B family member and transcriptional coactivator required for the selective expression of a subset of NF-kappa B target genes, is a key activator of the Ccl2 gene. I kappa B zeta-deficient macrophages exhibited impaired secretion of CCL2 when challenged with diverse inflammatory stimuli, such as LPS or peptidoglycan. These findings were reflected at the level of Ccl2 gene expression, which was tightly coupled to the presence of I kappa B zeta. Moreover, mechanistic insights acquired by chromatin immunoprecipitation demonstrate that I kappa B zeta is directly recruited to the proximal promoter region of the Ccl2 gene and is required for transcription-enhancing histone H3 at lysine-4 trimethylation. Finally, I kappa B zeta-deficient mice showed significantly impaired CCL2 secretion and monocyte infiltration in an experimental model of peritonitis. Together, these findings suggest a distinguished role of I kappa B zeta in mediating the targeted recruitment of monocytes in response to local inflammatory events. The Journal of Immunology, 2013, 190: 4812-4820.
• イヌ乳腺腫瘍関連遺伝子BRCA2のBRC repeat 3に存在するSNPsの頻度および機能に及ぼす影響の検討

  落合 和彦, 塩入 弓絵, 染田 沙織, 大澤 有加, 宇田川 智野, 森松 正美, 吉川 泰永, 盆子原 誠, 土田 修一, 近江 俊徳

  日本獣医学会学術集会講演要旨集 (公社)日本獣医学会 155回 251 - 251 1347-8621 2013/03
• Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

  Yasunaga Yoshikawa, Kazuhiko Ochiai, Masami Morimatsu, Yu Suzuki, Seiichi Wada, Takahiro Taoda, Satomi Iwai, Seishiro Chikazawa, Koichi Orino, Kiyotaka Watanabe

  PLOS ONE 7 (10) e45833  1932-6203 2012/10 [Refereed][Not invited]
   

  Mammary tumors are the most common tumor type in both human and canine females. Mutations in the breast cancer susceptibility gene, BRCA2, have been found in most cases of inherited human breast cancer. Similarly, the canine BRCA2 gene locus has been associated with mammary tumors in female dogs. However, deleterious mutations in canine BRCA2 have not been reported, thus far. The BRCA2 protein is involved in homologous recombination repair via its interaction with RAD51 recombinase, an interaction mediated by 8 BRC repeats. These repeats are 26-amino acid, conserved motifs in mammalian BRCA2. Previous structural analyses of cancer-associated mutations affecting the BRC repeats have shown that the weakening of RAD51's affinity for even 1 repeat is sufficient to increase breast cancer susceptibility. In this study, we focused on 2 previously reported canine BRCA2 mutations (T1425P and K1435R) in BRC repeat 3 (BRC3), derived from mammary tumor samples. These mutations affected the interaction of canine BRC3 with RAD51, and were considered deleterious. Two BRC3 mutations (K1440R and K1440E), reported in human breast cancer patients, occur at amino acids corresponding to those of the K1435R mutation in dogs. These mutations affected the interaction of canine BRC3 with RAD51, and may also be considered deleterious. The two BRC3 mutations and a substitution (T1430P), corresponding to T1425P in canine BRCA2, were examined for their effects on human BRC3 function and the results were compared between species. The corresponding mutations and the substitution showed similar results in both human and canine BRC3. Therefore, canine BRCA2 may be a good model for studying human breast cancer caused by BRCA2 mutations.
• Molecular epidemiology of avian influenza viruses circulating among healthy poultry flocks in farms in northern Vietnam

  Hiroki Takakuwa, Tetsu Yamashiro, Mai Q. Le, Lien S. Phuong, Hiroichi Ozaki, Ryota Tsunekuni, Tatsufumi Usui, Hiroshi Ito, Masami Morimatsu, Yukiko Tomioka, Tsuyoshi Yamaguchi, Toshihiro Ito, Toshiyuki Murase, Etsuro Ono, Koichi Otsuki

  PREVENTIVE VETERINARY MEDICINE 103 (2-3) 192 - 200 0167-5877 2012/02 [Refereed][Not invited]
   

  Repeated epizootics of highly pathogenic avian influenza (HPAI) virus subtype H5N1 were reported from 2003 to 2005 among poultry in Vietnam. More than 200 million birds were killed to control the spread of the disease. Human cases of H5N1 infection have been sporadically reported in an area where repeated H5N1 outbreaks among birds had occurred. Subtype H5N1 strains are established as endemic among poultry in Vietnam, however, insights into how avian influenza viruses including the H5N1 subtype are maintained in endemic areas is not clear. In order to determine the prevalence of different avian influenza viruses (AIVs), including H5N1 circulating among poultry in northern Vietnam, surveillance was conducted during the years 2006-2009. A subtype H5N1 strain was isolated from an apparently healthy duck reared on a farm in northern Vietnam in 2008 and was identified as an HPAI. Although only one H5N1 virus was isolated, it supports the view that healthy domestic ducks play a pivotal role in maintaining and transmitting H5N1 viruses which cause disease outbreaks in northern Vietnam. In addition, a total of 26 AlVs with low pathogenicity were isolated from poultry and phylogenetic analysis of all the eight gene segments revealed their diverse genetical backgrounds, implying that reassortments have occurred frequently among strains in northern Vietnam. It is, therefore, important to monitor the prevalence of influenza viruses among healthy poultry between epidemics in an area where AlVs are endemic. (C) 2011 Elsevier B.V. All rights reserved.
• Establishment of a PCR analysis method for canine BRCA2

  Yasunaga Yoshikawa, Masami Morimatsu, Kazuhiko Ochiai, Kento Okuda, Takahiro Taoda, Seishiro Chikazawa, Asako Shimamura, Toshinori Omi, Makoto Bonkobara, Koichi Orino, Kiyotaka Watanabe

  BMC Research Notes 5 173 - 182 1756-0500 2012 [Refereed][Not invited]
   

  Background: Mammary tumors are the most common tumor type in both human and canine females. In women, carriers of mutations in BRCA2, a tumor suppressor gene product, have a higher risk of breast cancer. Canine BRCA2 has also been suggested to have a relationship with mammary tumors. However, clearly deleterious BRCA2 mutations have not been identified in any canine mammary tumors, as appropriate methods to detect mutations or a consensus BRCA2 sequence have not been reported. Findings. For amplification and sequencing of BRCA2, we designed 14 and 20 PCR primer sets corresponding to the BRCA2 open reading frame (ORF) and all 27 exons, respectively, including exon-intron boundaries of the canine BRCA2 regions, respectively. To define the consensus canine BRCA2 ORF sequence, we used established methods to sequence the full-length canine BRCA2 ORF sequence from two ovaries and a testis obtained from individual healthy mongrel dogs and partially sequence BRCA2 genomic sequences in 20-56 tumor-free dogs, each aged over 6 years. Subsequently, we compared these sequences and seven previously reported sequences, and defined the most common base sequences as the consensus canine BRCA2 ORF sequence. Moreover, we established a detection method for identifying splicing variants. Unexpectedly, we also identified novel splicing variants in normal testes during establishment of these methods. Conclusions: The present analysis methods for determining the BRCA2 base sequence and for detecting BRCA2 splicing variants and the BRCA2 ORF consensus sequence are useful for better understanding the relationship between canine BRCA2 mutation status and cancer risk. © 2010 Yoshikawa et al licensee BioMed Central Ltd.
• Interactions between canine RAD51 and full length or truncated BRCA2 BRC repeats

  K. Ochiai, Y. Yoshikawa, T. Oonuma, Y. Tomioka, K. Hashizume, M. Morimatsu

  VETERINARY JOURNAL 190 (2) 293 - 295 1090-0233 2011/11 [Refereed][Not invited]
   

  In humans, mutations in the gene for the breast cancer susceptibility protein BRCA2 affect its interactions with the recombinase RAD51 and are associated with an increased risk of cancer. This interaction occurs through a series of eight BRC repeat sequences in BRCA2. A mammalian two-hybrid assay using individual BRC repeats demonstrated that BRC6 did not bind to RAD51, whereas there was strong (BRC1, 2 and 4), intermediate (BRC8), or weak (BRC3, 5 and 7) binding of other BRC repeats to RAD51. In serial deletion mutation experiments, binding strengths were increased when the C-terminal BRC repeat was removed from BRC1-8, BRC1-5 and BRC1-3. These results may provide an insight into the effects of missense or truncation mutations in BRCA2 in canine tumours. (C) 2010 Elsevier Ltd. All rights reserved.
• A nucleoside anticancer drug, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (TAS106), sensitizes cells to radiation by suppressing BRCA2 expression

  Shunsuke Meike, Tohru Yamamori, Hironobu Yasui, Masato Eitaki, Akira Matsuda, Masami Morimatsu, Masakazu Fukushima, Yasundo Yamasaki, Osamu Inanami

  MOLECULAR CANCER 10 92 - 100 1476-4598 2011/07 [Refereed][Not invited]
   

  Background: A novel anticancer drug 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, TAS106) has been shown to radiosensitize tumor cells and to improve the therapeutic efficiency of X-irradiation. However, the effect of TAS106 on cellular DNA repair capacity has not been elucidated. Our aim in this study was to examine whether TAS106 modified the repair capacity of DNA double-strand breaks (DSBs) in tumor cells. Methods: Various cultured cell lines treated with TAS106 were irradiated and then survival fraction was examined by the clonogenic survival assays. Repair of sublethal damage (SLD), which indicates DSBs repair capacity, was measured as an increase of surviving cells after split dose irradiation with an interval of incubation. To assess the effect of TAS106 on the DSBs repair activity, the time courses of gamma-H2AX and 53BP1 foci formation were examined by using immunocytochemistry. The expression of DNA-repair-related proteins was also examined by Western blot analysis and semi-quantitative RT-PCR analysis. Results: In clonogenic survival assays, pretreatment of TAS106 showed radiosensitizing effects in various cell lines. TAS106 inhibited SLD repair and delayed the disappearance of gamma-H2AX and 53BP1 foci, suggesting that DSB repair occurred in A549 cells. Western blot analysis demonstrated that TAS106 down-regulated the expression of BRCA2 and Rad51, which are known as keys among DNA repair proteins in the homologous recombination (HR) pathway. Although a significant radiosensitizing effect of TAS106 was observed in the parental V79 cells, pretreatment with TAS106 did not induce any radiosensitizing effects in BRCA2-deficient V-C8 cells. Conclusions: Our results indicate that TAS106 induces the down-regulation of BRCA2 and the subsequent abrogation of the HR pathway, leading to a radiosensitizing effect. Therefore, this study suggests that inhibition of the HR pathway may be useful to improve the therapeutic efficiency of radiotherapy for solid tumors.
• Valine 1532 of human BRC repeat 4 plays an important role in the interaction between BRCA2 and RAD51

  Kazuhiko Ochiai, Yasunaga Yoshikawa, Kumiko Yoshimatsu, Toshina Oonuma, Yukiko Tomioka, Eichi Takeda, Jiro Arikawa, Katsumi Mominoki, Toshinori Omi, Kazuyoshi Hashizume, Masami Morimatsu

  FEBS LETTERS 585 (12) 1771 - 1777 0014-5793 2011/06 [Refereed][Not invited]
   

  The breast cancer susceptibility protein BRCA2 is essential for recombinational DNA repair. BRCA2 specifically binds to RAD51 via eight BRC repeat motifs and delivers RAD51 to double-stranded DNA breaks. In this study, a mammalian two-hybrid assay and competitive ELISA showed that the interaction between BRC repeat 4 (BRC4) and RAD51 was strengthened by the substitution of a single BRC4 amino acid from valine to isoleucine (V1532I). However, the cancer-associated V1532F mutant exhibited very weak interaction with RAD51. This study used a comparative analysis of BRC4 between animal species to identify V1532 as an important residue that interacts with RAD51. Structured summary of protein interactions: cRAD51 physically interacts with cRAD51 by two hybrid (View interaction) fBRC4 physically interacts with cRAD51 by two hybrid (View interaction) cBRC4 physically interacts with cRAD51 by two hybrid (View interaction) hBRC4 physically interacts with hBRC4 and hRAD51 by competition binding (View Interaction 1, 2) hBRC4 physically interacts with cRAD51 by two hybrid (View interaction) hBRC4 binds to hRAD51 by enzyme linked immunosorbent assay (View interaction) (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
• Motor-Coordination-Dependent Learning, More than Others, Is Impaired in Transgenic Mice Expressing Pseudorabies Virus Immediate-Early Protein IE180

  Juan C. Lopez-Ramos, Yukiko Tomioka, Masami Morimatsu, Sayo Yamamoto, Kinuyo Ozaki, Etsuro Ono, Jose M. Delgado-Garcia

  PLOS ONE 5 (8) e12123  1932-6203 2010/08 [Refereed][Not invited]
   

  The cerebellum in transgenic mice expressing pseudorabies virus immediate-early protein IE180 (TgIE96) was substantially diminished in size, and its histoarchitecture was severely disorganized, resulting in severe ataxia. TgIE96 mice can therefore be used as an experimental model to study the involvement of cerebellar circuits in different learning tasks. The performance of three-month-old TgIE96 mice was studied in various behavioral tests, including associative learning (classical eyeblink conditioning), object recognition, spatial orientation (water maze), startle response and prepulse inhibition, and passive avoidance, and compared with that of wild-type mice. Wild-type and TgIE96 mice presented similar reflexively evoked eyeblinks, and acquired classical conditioned eyelid responses with similar learning curves for both trace and delay conditioning paradigms. The two groups of mice also had similar performances during the object recognition test. However, they showed significant differences for the other three tests included in this study. Although both groups of animals were capable of swimming, TgIE96 mice failed to learn the water maze task during the allowed time. The startle response to a severe tone was similar in both control and TgIE96 mice, but the latter were unable to produce a significant prepulse inhibition. TgIE96 mice also presented evident deficits for the proper accomplishment of a passive avoidance test. These results suggest that the cerebellum is not indispensable for the performance of classical eyeblink conditioning and for object recognition tasks, but seems to be necessary for the proper performance of water maze, prepulse inhibition, and passive avoidance tests.
• Discoidin Domain Receptor 2 (DDR2) Is Required for Maintenance of Spermatogenesis in Male Mice

  Kiyoshi Kano, Ayami Kitamura, Takashi Matsuwaki, Masami Morimatsu, Kunihiko Naito

  MOLECULAR REPRODUCTION AND DEVELOPMENT 77 (1) 29 - 37 1040-452X 2010/01 [Refereed][Not invited]
   

  Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase (RTK). We recently identified homozygous smallie mutant mice (BKS.HRS. Ddr2(slie/slie)/J, Ddr2(slie/slie) mutants), which lack a functional DDR2. Ddr2(slie/slie) mutant mice are dwarfed and infertile due to peripheral dysregulation of the endocrine system. To understand the role of DDR2 signaling in spermatogenesis, we studied the expression of several receptors, enzymes, and proteins related to spermatogenesis in wild-type and Ddr2(slie/slie) mutant mice at 10 weeks and 5 months of age. DDR2 were expressed in adult wild-type male mice in Leydig cells. The number of differentiated spermatozoa in the seminal fluid was significantly lower in the Ddr2(slie/slie) mutant mice than in the wild-type mice. The number of TUNEL-positive cells was significantly greater in 5-month-old Ddr2(slie/slie) mutants. Testosterone was significantly reduced at 5 months age, but LH was similar in both types of mice at both 10 weeks and 5 months of age. The expression levels of LH receptors (Lhcgr), StAR, P450scc, and Hsd3 beta 6 were significantly different between the two types of mice at 10 weeks of age, but they were significantly reduced in 5-month-old Ddr2(slie/slie) mutants compared to wild-type mice of the same age. DDR2 was expressed in the Leydig cells of adult wild-type male mice. In conclusion, our results indicated that DDR2 signaling plays a critical role in the maintenance of male spermatogenesis.
• Fusion protein consisting of the first immunoglobulin-like domain of porcine nectin-1 and Fc portion of human IgG1 provides a marked resistance against pseudorabies virus infection to transgenic mice

  Yukiko Tomioka, Masami Morimatsu, Keiko Amagai, Minako Kuramochi, Yuki Watanabe, Shigeto Kouda, Toshio Wada, Noritaka Kuboki, Etsuro Ono

  MICROBIOLOGY AND IMMUNOLOGY 53 (1) 8 - 15 0385-5600 2009/01 [Refereed][Not invited]
   

  Nectin-1 is a Ca(2+)-independent Ig-like cell-cell adhesion molecule and an alphaherpesvirus receptor that binds to virion glycoprotein D by the first Ig-like domain. We have investigated the antiviral potentials of soluble forms of porcine nectin-1 to PRV infection by generating transgenic mice expressing different types of fusion protein. Previously, we reported that mice transgenic for a chimera that carried the entire ectodomain of porcine nectin-1 fused to the Fc portion of porcine IgG1 were more resistant than those transgenic for a chimera that carried the first Ig-like domain fused to the Fc portion. Recently, we generated transgenic mice expressing a fusion protein made of the first Ig-like domain fused to the Fc portion of human IgG1, and reported that they showed a microphthalmia. Here, two transgenic mouse lines expressing the fusion protein were challenged with PRV for comparing their resistances with those of transgenic mice expressing different types of fusion protein. Surprisingly, both transgenic mouse lines showed a high resistance to the viral infection, especially via the i.n. route. Significant resistance of the embryonic fibroblasts was also observed. Altogether, these findings indicated that the fusion protein consisting of the first Ig-like domain fused to the human Fc portion provided a marked resistance against PRV infection to the transgenic mice.
• Novel variations and loss of heterozygosity of BRCA2 identified in a dog with mammary tumors

  Yasunaga Yoshikawa, Masami Morimatsu, Kazuhiko Ochiai, Masashi Nagano, Yukiko Tomioka, Nobuo Sasaki, Kazuyoshi Hashizume, Toshihiko Iwanaga

  AMERICAN JOURNAL OF VETERINARY RESEARCH 69 (10) 1323 - 1328 0002-9645 2008/10 [Refereed][Not invited]
   

  Objective-To establish novel polymorphic markers for analysis of loss of heterozygosity (LOH), so as to study the possible involvement of BRCA2 in mammary tumors obtained from dogs. Sample Population-Blood samples, mammary gland specimens, or mammary tumors from 3 tumor-bearing dogs and 10 tumor-free dogs. Procedures-Nucleotide sequence analysis was performed with a DNA autosequencer. Loss of heterozygosity analysis was performed for markers established in the present study. The expression level of canine BRCA2 was quantified by real-time PCR analysis. Results-3 novel microsatellite markers with high heterozygosity rates (> 50%) were established, and the previously reported marker for canine BRCA2 gene locus was improved. These markers were used for the analysis of DNA from formalin-fixed and paraffin-embedded samples. By use of these markers, LOH in canine BRCA2 was identified as a result of recombination. In mammary tumor DNA that corresponded to the LOH-positive dog, the level of canine BRCA2 expression was decreased compared with that of nonneoplastic mammary gland tissue; the open reading frame contained 4 missense variations, 1 insertion variation, and 1 silent variation, some of which were localized to functional domains. Conclusions and Clinical Relevance-3 novel polymorphic markers were developed for LOH analysis of canine BRCA2 and identified a dog with LOH with some variations in the functional domains. These markers could be useful for assessing the relevance of BRCA2 variation in mammary tumors of dogs.
• Microphthalmia and lack of vitreous body in transgenic mice expressing the first immunoglobulin-like domain of nectin-1

  Kazuhiko Yoshida, Yukiko Tomioka, Satoru Kase, Masami Morimatsu, Kyoko Shinya, Shigeaki Ohno, Etsuro Ono

  GRAEFES ARCHIVE FOR CLINICAL AND EXPERIMENTAL OPHTHALMOLOGY 246 (4) 543 - 549 0721-832X 2008/04 [Refereed][Not invited]
   

  Background Nectins are Ca2+-independent immunoglobulin (Ig)-like cell-cell-adhesion molecules. We have generated transgenic mice expressing a series of soluble forms of nectin-1, and investigated special effects of each soluble form of nectin-1 in vivo. In the course of generating transgenic mice expressing a soluble form of nectin-1 consisting of the first Ig-like domain of nectin-1 and the Fc portion of human IgG1 (PHveC-VhIg), we found that all of the transgenic founder mice showed a microphthalmia. The purpose of this study is to examine functions of the extracellular domains of nectin-1 in eye development using transgenic technology. Methods Eyes of four different transgenic mouse lines expressing each soluble form of nectin-1 were analyzed histologically. Tissue sections were processed with hematoxylin-eosin staining and indirect immunoperoxidase technique. Results All of five transgenic mouse founders expressing PHveC-VhIg, and of three lines expressing PHveC-VpIg made of the first Ig-like domain fused to porcine Fc portions at 5 weeks showed a microphthalmia, but not all of the transgenic mouse lines expressing PHveCIg or PHveCpIg made of the entire ectodomain fused to human or porcine Fc portions. In the abnormal eyes, the vitreous body was almost absent. In PHveC-VhIg-expressing mice at postnatal day 6, each vitreous space was very small. In the neonatal transgenic mice, the vitreous body was almost the same as that of control mice, and PHveC-VhIg was expressed in the optic nerve, conjunctival epithelium, ciliary body, corneal and lens epithelium. At this stage, nectin-1, -3 and -4 were stained in the optic nerve of control mice as well as in that of the transgenic mice. Nectin-1 is faintly stained in the epithelium of the cornea and lens epithelium, but not in the ciliary body. Conclusion Soluble forms of the first Ig-like domain of nectin-1 (PHveC-VhIg and PHveC-VpIg), but not those of the entire ectodomain (PHveCIg and PHveCpIg), lead to microphthalmia and lack of vitreous body in the transgenic mice.
• Cerebellar pathology in transgenic mice expressing the pseudorabies virus immediate-early protein IE180

  Yukiko Tomioka, Taisuke Miyazaki, Satoshi Taharaguchi, Saori Yoshino, Masami Morimatsu, Toshimitsu Uede, Etsuro Ono, Masahiko Watanabe

  EUROPEAN JOURNAL OF NEUROSCIENCE 27 (8) 2115 - 2132 0953-816X 2008/04 [Refereed][Not invited]
   

  Pseudorabies virus is an alphaherpesvirus causing fatal neurological diseases in animals. Pseudorabies virus carries a gene encoding immediate-early (IE) protein IE180, which controls the transcription of other viral and host cell genes. Previously, we reported that transgenic expression of IE180 in mice causes severe ataxia and cerebellar deformity. Here we identified profound abnormalities in adult IE180 transgenic mice, including malpositioning of Purkinje cells (PCs), granule cells (GCs) and Bergmann glia (BG), impaired dendritogenesis and synaptogenesis in PCs, disoriented BG fibers, absence of molecular layer interneurons, and increased apoptosis of neurons and glia. In accordance with the cellular defects, we found the expression of IE180 in PCs, GCs and astrocytes during cerebellar development. We next examined transgenic mice expressing truncated IE180 mutants: dlN132 lacking the acidic transcriptional active domain, dlC629 lacking the nuclear localization signal and dlC1081 having all known domains but lacking the carboxyl-terminal sequence. Despite similar expression levels of the transgenes, ataxia and cerebellar defects were only manifested in the dlC1081 transgenic mice but their phenotypes were milder compared with the IE180 transgenic mice. In the dlC1081 transgenic mice, cerebellar neurons and glia were normally positioned but cerebellar size was severely reduced due to GC deficits. Interestingly, dlC1081 was mainly expressed in the GCs with low expression in a few BG. Taken together, the present findings clarified a causal relationship between cerebellar pathology and cellular expression of IE180, and further afforded an experimental insight into different symptomatic severity as a consequence of different cellular defects caused by such cytotoxic viral agents.
• Comparison of the antiviral potentials among the pseudorabies-resistant transgenes encoding different soluble forms of porcine nectin-1 in transgenic mice

  Etsuro Ono, Yukiko Tomioka, Yuki Watanabe, Keiko Amagai, Masami Morimatsu, Kyoko Shinya, Pierre Cherel

  JOURNAL OF GENERAL VIROLOGY 88 (Pt 10) 2636 - 2641 0022-1317 2007/10 [Refereed][Not invited]
   

  Nectin-1 is an alphaherpesvirus receptor that binds to virion glycoprotein D by the first immunoglobulin (lg)-like domain. The possibility of making animals resistant to pseudorabies virus (PRV) infection has been investigated by generating transgenic mice expressing soluble forms of porcine nectin-1. Previously, transgenic mice were generated that expressed a fusion protein made of the entire ectodomain of nectin-1 fused to the Fc portion of human IgG, or the first lg-like domain fused to the Fc portion of porcine IgG. Here, the contribution of the second and third lg-like domains of nectin-1 was analysed by generating transgenic mice expressing the entire ectodomain of nectin-1 fused to the porcine Fc portion. Transgenic mice expressing each of three different fusion proteins were challenged with PRV for comparison of their resistance. Altogether, mice transgenic for a chimera that carried the entire ectodomain were more resistant than those transgenic for a chimera that carried the first lg-like domain.
• Role of NF-kappa B in constitutive expression of MAIL in epidermal keratinocytes

  Toshina Oonuma, Masami Morimatsu, Kazuhiko Ochiai, Toshihiko Iwanaga, Kazuyoshi Hashizume

  JOURNAL OF VETERINARY MEDICAL SCIENCE 69 (3) 279 - 284 0916-7250 2007/03 [Refereed][Not invited]
   

  Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear I kappa B protein that is also known as interleukin-1-inducible nuclear ankyrin repeat protein and inhibitor of nuclear factor kappa B zeta (I kappa B zeta). We previously observed that MAIL-deficient mice were affected by atopic dermatitis-like skin lesions and demonstrated the importance of MAIL in the skin. In this study, we investigated MAIL expression in mouse keratinocytes. MAIL mRNA was constitutively expressed in the skin epidermis. MAIL expression was also confirmed in primary keratinocytes and the PAM212 keratinocyte cell line. The inhibitors of nuclear factor kappa B (NF kappa B)-Bay 11-7082 and the I kappa B alpha M supersuppressor-considerably downregulated MAIL expression in the keratinocytes. Immunoreactivity for NF-kappa B components was localized in the cytoplasm and nucleus of normal unstimulated keratinocytes. The expression level of MAIL in the skin did not change following lipopolysaccharide (LPS) administration to mice. Interestingly, in accordance with the in vivo findings, the MAIL expression level did not change following LPS stimulation even in primary keratinocytes; however, MAIL expression was strongly increased by interleukin-1 stimulation. These results collectively suggest that the constitutive expression of MAIL in keratinocytes is controlled, at least in part, by NF-kappa B and that there may be LPS-specific repressive mechanisms that inhibit MAIL induction.
• Immunohistochemical and in situ hybridization analysis of galectin-3, a beta-galactoside binding lectin, in the urinary system of adult mice

  Junko Nio, Hiromi Takahashi-Iwanaga, Masami Morimatsu, Yasuhiro Kon, Toshihiko Iwanaga

  HISTOCHEMISTRY AND CELL BIOLOGY 126 (1) 45 - 56 0948-6143 2006/07 [Refereed][Not invited]
   

  Galectin is an animal lectin that has high affinity to beta-galactoside of glycoconjugates. In the present study, cellular expression of galectin subtypes in the urinary system of adult mice was examined by in situ hybridization and immunohistochemistry. The major subtype expressed in the murine urinary system was galectin-3, which was expressed continuously from the kidney to the distal end of the urethra. The renal cortex expressed galectin-3 more intensely than the medulla. Renal galectin-3 immunoreactivity was strongest in the cortical collecting ducts, where principal cells were the sole cellular source. All cell layers of the transitional epithelium from the renal pelvis to the urethra strongly expressed galectin-3 at the mRNA and protein levels. An electron microscopic study demonstrated diffuse cytoplasmic localization of galectin-3 in principal cells of the collecting ducts and in the bladder epithelial cells. Urethral galectin-3 expression at the pars spongiosa decreased in intensity near the external urethral orifice, where the predominant subtype of galectin was substituted by galectin-7. The muscular layer of the ureter and urinary bladder contained significant signals for galectin-1. Taken together, the observations indicate that the adult urinary system shows intense and selective expression of galectin-3 in epithelia of the uretic bud- and cloaca-derivatives.
• Spermatogonial cell-mediated activation of an I kappa B zeta-independent nuclear factor-kappa B pathway in sertoli cells induces transcription of the Lipocalin-2 gene

  RS Fujino, K Tanaka, M Morimatsu, K Tamura, H Kogo, T Hara

  MOLECULAR ENDOCRINOLOGY 20 (4) 904 - 915 0888-8809 2006/04 [Refereed][Not invited]
   

  In spermatogenesis, Sertoli cells serve as supporting cells for the proliferation and differentiation of germ cells. However, it appears that Sertoli cell function is regulated by adjacent spermatogonial cells in the testis because expression of lipocalin-2 mRNA, which encodes an iron-siderophore-binding protein, is barely detectable in Sertoli cells of germ cell-deficient W/W-v mice, and more abundantly expressed in jsd/jsd mice. By employing a coculture system comprising immortalized Sertoli cells ( designated as Sertoli-B) and c-Kit(+) spermatogonial cells from 7-d-old mouse testis, we found that lipocalin-2 gene transcription in Sertoli cells is induced by a factor secreted from spermatogonial cells. Transfection of Sertoli-B cells with a series of reporter constructs encompassing an upstream region of the mouse lipocalin-2 gene revealed that a nuclear factor (NF)-kappa B binding consensus sequence in the proximal region of lipocalin-2 gene is responsible for transcriptional activation. A major NF-kappa B component, p65, bound to this region and translocated from the cytoplasm to the nucleus upon stimulation with spermatogonial cell-conditioned medium. Moreover, short interference RNA directed to p65 or a dominant-negative form of I kappa B alpha suppressed the spermatogonial cell factor-mediated transcription of lipocalin-2. However, NF-kappa B-activating inflammatory molecules, such as IL-1 beta and lipopolysaccharide, did not induce lipocalin-2 mRNA in Sertoli-B cells and the expression of lipocalin-2 was unaffected in the testis of I kappa B zeta-deficient mice. These results demonstrate that spermatogonial cells regulate lipocalin-2 gene expression in Sertoli cells in a manner distinct from that employed by immune cells.
• Elevated plasma concentrations of haptoglobin in European brown bears during hibernation

  K Mominoki, M Morimatsu, M Kaijalainen, E Hohtola, R Hissa, M Saito

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY 142 (4) 472 - 477 1095-6433 2005/12 [Refereed][Not invited]
   

  Haptoglobin (Hp), a hemoglobin-binding protein, is known as an acute phase protein and increases during the acute phase of inflammation in most mammals. We reported previously in brown bears that the mean Hp concentrations were higher in blood samples obtained in winter than those in spring. To examine a possible relation of the seasonal variations of Hp to hibernation, in the present study, we measured the plasma concentrations of Hp as well as some other acute phase proteins (alpha(2)-macroglobulin, alpha(1)-antitrypsin, C-reactive protein) in 6 European brown bears (Ursus arctos), from which blood samples were obtained at 5-6 different months of year including February, the time of hibernation. The Hp concentrations showed clear seasonal variations, being highest in February. The alpha(2)-macroglobulin concentrations also showed a similar but much smaller rise in February, but those of alpha(1)-antitrypsin and C-reactive protein did not show any seasonal variations. Our results suggest that the seasonal variation of plasma Hp concentration in brown bears is associated with a hibernation-specific mechanism more than that of acute phase response. (C) 2005 Elsevier Inc. All rights reserved.
• Analysis of genetic variations in the exon 27 region of the canine BRCA2 locus

  Y Yoshikawa, M Morimatsu, K Ochiai, M Nagano, Y Yamane, N Tomizawa, N Sasaki, K Hashizume

  JOURNAL OF VETERINARY MEDICAL SCIENCE 67 (10) 1013 - 1017 0916-7250 2005/10 [Refereed][Not invited]
   

  Mammary tumors are the most common tumor type in women as well as in female dogs. The BRCA2 gene encodes a large nuclear protein that is involved in DNA repair, and mutations in the human BRCA2 confer an increased risk of female mammary tumors. The BRCA2 protein acts as a tumor suppressor, and inactivation of BRCA2 by loss of heterozygosity is implicated in mammary carcinogenesis. In this study, to establish an appropriate polymorphic marker for loss of heterozygosity analysis of the canine BRCA2, we analyzed the genomic sequences of the exon 27 regions of 30 mammary-tumor-bearing and 21 tumor-free dogs. In addition to 10204ins/delAAA, which is the only polymorphism previously identified for the canine BRCA2 locus, we discovered four novel single nucleotide polymorphisms. The analysis of these five polymorphisms revealed the presence of four allele types. Since 10204ins/delAAA was the most common of the five polymorphisms identified, we developed a PCR-based assay method to assay for this polymorphism. We believe that this method is valuable for loss of heterozygosity analysis of the canine BRCA2 gene in tumor pathogenesis.
• Histochemical demonstration of a Na+-coupled transporter for short-chain fatty acids (Slc5a8) in the intestine and kidney of the mouse

  Kumiko Takebe, Junko Nio, Masami Morimatsu, Shin-Ichiro Karaki, Atsukazu Kuwahara, Ikuo Kato, Toshihiko Iwanaga

  BIOMEDICAL RESEARCH-TOKYO 26 (5) 213 - 221 0388-6107 2005/10 [Refereed][Not invited]
   

  Short-chain fatty acids in the intestinal lumen affect colonic cell proliferation as well as function as an energy source for intestinal epithelial cells. A novel transporter of monocarboxylates, Slc5a8, is expressed abundantly in the colon, where it may participate in the Na+-coupled absorption of short-chain fatty acids produced by bacterial fermentation of dietary fiber. The present study examined the cellular localization of Slc5a8 in the murine gastrointestinal tract and kidney by in situ hybridization and immunohistochemistry. The hybridization signals were recognized in the terminal ileum and whole length of the large intestine, and were especially intense in the distal colon and rectum. The immunoreactivity of Slc5a8 was restricted to the striated border (the brush border) of enterocytes, and was not present in goblet cells, Paneth cells, or lamina propria cells. In the kidney, proximal tubules of both the cortex and the outer stripe of the outer medulla intensely expressed Slc5a8 mRNA, while the distal portions, including the loop of Henle, lacked the signals. The renal Slc5a8 immunoreactivity was localized only in the brush border of proximal tubules, not along the basolateral membrane. Thyroid follicular cells were immunoreactive for Slc5a8, with predominant labeling on the apical membrane. No other organs, including the esophagus, stomach, liver, pancreas, and salivary glands contained any notable signals of Slc5a8. These findings on the cellular and subcellular localization of Slc5a8 under normal conditions are helpful for understanding the physiological and pathological roles of Slc5a8.
• Insertion/deletion polymorphism in the BRCA2 nuclear localization signal

  Y Yoshikawa, M Morimatsu, K Ochiai, M Nagano, Y Yamane, N Tomizawa, N Sasaki, K Hashizume

  BIOMEDICAL RESEARCH-TOKYO 26 (3) 109 - 116 0388-6107 2005/06 [Refereed][Not invited]
   

  Mutations in human BRCA2 confer an increased risk of female breast cancer. In this study, we found a novel insertion/deletion polymorphism (10204insAAA causing amino acid change M33321K) in canine BRCA2, which is located in the putative second nuclear localization signal (NLS2) and C-ten-ninal Rad51-binding region. The nuclear localization of the insAAA C-terminus was more efficient than localization of the delAAA sequence when NLS1 was mutated. Strong, comparable Rad51 binding was observed for both the insAAA and delAAA C-termini. Dogs with the insertion/deletion polymorphism will provide a new model for studying the function of BRCA2.
• Targeted disruption of MAIL, a nuclear I kappa B protein, leads to severe atopic dermatitis-like disease

  T Shiina, A Konno, T Oonuma, H Kitamura, K Imaoka, N Takeda, K Todokoro, M Morimatsu

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (53) 55493 - 55498 0021-9258 2004/12 [Refereed][Not invited]
   

  MAIL (molecule-possessing ankyrin repeats induced by lipopolysaccharide) is a nuclear IkappaB protein that is also termed interleukin-1-inducible nuclear ankyrin repeat protein or inhibitor of nuclear factor kappaB (IkappaB) zeta. In this study, we generated Mail(-/-) mice to investigate the roles of MAIL in whole organisms. Mail(-/-) mice grew normally until 4-8 weeks after birth, when they began to develop lesions in the skin of the periocular region, face, and neck. MAIL mRNA and protein were constitutively expressed in the skin of wild type controls, especially in the keratinocytes. Serum IgE was higher in Mail(-/-) mice than in normal. Histopathological analysis indicated that the Mail(-/-) skin lesions appeared to be atopic dermatitis ( AD) eczema with inflammatory cell infiltration. In addition, markedly elevated expression of some chemokines such as thymus and activation-regulated chemokine was detected in the Mail(-/-) skin lesions, similar to that observed in the skin of patients with AD. In Mail(-/-) mice, MAIL-deficient keratinocytes might be activated to produce chemokines and induce intraepidermal filtration of inflammatory cells, resulting in the onset of the AD-like disease. These findings suggest that MAIL is an essential molecule for homeostatic regulation of skin immunity. The Mail(-/-) mouse is a valuable new animal model for research on AD.
• Brca2 C-terminus interacts with Rad51 and contributes to nuclear focus formation in double-strand break repair of DNA

  K Ochiai, M Morimatsu, Y Yoshikawa, B Syuto, K Hashizume

  BIOMEDICAL RESEARCH-TOKYO 25 (6) 269 - 275 0388-6107 2004/12 [Refereed][Not invited]
   

  In humans and mice, the interaction between the breast cancer susceptibility protein, BRCA2, and RAD51 recombinase is essential for DNA repair by homologous recombination, the failure of this process can predispose to cancer. Cells with mutated BRCA2 are hypersensitive to ionizing radiation (IR) and exhibit defective DNA repair. Using yeast and mammalian two-hybrid assays, we demonstrate that canine Rad51 protein interacts specifically with the C-terminus of canine Brca2. In support of the biological significance of this interaction, we found that radiation-induced focus formation of Rad51 in COS-7 cells was compromised by forced expression of the C-terminus of canine Brca2. A similar result was obtained for the murine C-terminus. These data suggest that the C-terminal domain of canine Brca2 functions to bind Rad51 and that this domain contributes to the IR-induced assembly of the Rad51 complex in vivo.
• Transcriptional regulation of the MAIL gene in LPS-stimulated RAW264 mouse macrophages

  T Ito, M Morimatsu, T Oonuma, T Shiina, H Kitamura, B Syuto

  GENE 342 (1) 137 - 143 0378-1119 2004/11 [Refereed][Not invited]
   

  IkappaB inhibits nuclear factor kappa B (NF-kappaB), which is known to regulate the expression of various genes, including genes involved in inflammation. Recently, a novel IkappaB family protein, 'molecule possessing ankyrin repeats induced by lipopolysaccharide' (MAIL), was identified. MAIL is a nuclear-acting, inducible protein, unlike typical IkappaB proteins. However, the mechanism of its induction by lipopolysaccharide (LPS) is unclear. Using the LPS-reactive region located upstream from the MAIL gene, we investigated the mechanism of MAIL induction. MAIL expression was strongly regulated by NF-kappaB and partly regulated by CREB. Furthermore, deletion, point mutation and binding analyses revealed that the NF-kappaB binding site located at -229 to -220 bp is a n essential target of MAIL expression. Overexpression of MAIL protein suppressed the LPS-induced promoter activity of the MAIL gene. These data indicate that MAIL expression is strongly upregulated by NF-kappaB, and it is controlled, at least in part, by an autoregulation mechanism. (C) 2004 Elsevier B.V. All rights reserved.
• Cloning of bovine MAIL and its mRNA expression in white blood cells of Holstein cows

  D Yamaji, H Kitamura, K Kimura, Y Matsushita, H Okada, T Shiina, M Morimatsu, M Saito

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 98 (3-4) 175 - 184 0165-2427 2004/04 [Refereed][Not invited]
   

  Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is known as an IkappaB protein induced after administration of bacterial lipopolysaccharide, (LPS) to mice. In the present study, we cloned bovine MAIL cDNA and examined its mRNA expression in white blood cells isolated from Holstein cows. Bovine MAIL had more than 80% amino acid identities with murine and human MAILs, highly conserved ankyrin-repeat motifs and PEST-like sequences. Bovine MAIL mRNA was undetectable in isolated peripheral white blood cells, but rapidly induced (< 1h) after stimulation by LPS and lipid A in vitro in a dose-dependent manner. The lipid A-induced MAIL mRNA expression was found in polymorphonuclear cells, monocytes/macrophages and total lymphocytes, but not in T-lymphocytes. MAIL mRNA was also induced in vivo in peripheral blood leukocytes of cows after intramammary injection of Escherichia coli derived from coliform, mastitis. Thus, bovine MAIL, as rodent MAILs, is induced by inflammatory stimuli in specific immune cells in vitro and in vivo, suggesting a role in inflammatory responses to bacterial infection in cattle. (C) 2003 Elsevier B.V. All rights reserved.
• Association of the nucleocapsid protein of the Seoul and Hantaan hantaviruses with small ubiquitin-like modifier-1-related molecules

  BH Lee, K Yoshimatsu, A Maeda, K Ochiai, M Morimatsu, K Araki, M Ogino, S Morikawa, J Arikawa

  VIRUS RESEARCH 98 (1) 83 - 91 0168-1702 2003/12 [Refereed][Not invited]
   

  We performed yeast two-hybrid screening of a human kidney cell cDNA library to study the biological role of the hantavirus nucleocapsid protein (NP). We found that Seoul virus (SEOV) and Hantaan virus (HTNV) NPs were associated with small ubiquitin-like modifier (SUMO)-1-interacting proteins PIAS 1, PIASxbeta, HIPK2, CHD3, and TTRAP, which interacted with the SUMO-1 conjugating enzyme (Ubc-9) and SUMO-1 in the yeast two-hybrid assay. Interactions between the HIPK2, CHD3, and TTRAP proteins and SEOV NP were also shown in a mammalian two-hybrid assay. However, there was no interaction between PIAS proteins and NP, which was probably due to the inhibitory effect of PIAS on transcription in the mammalian two-hybrid assay. Nevertheless, a co-expression experiment suggested the existence of a PIAS-NP interaction in the cytoplasm. The region spanning amino acids 100-125 of SEOV NP, which represents a critical region for NP-NP polymerization. was found to be responsible for the interaction with SUMO-1-related molecules in both the yeast and mammalian two-hybrid assays. These results add to the information on interactions of hantavirus NP and host cellular proteins. (C) 2003 Elsevier B.V. All rights reserved.
• Properties of the tumor suppressor gene Brca2 in the cat

  T Oonuma, M Morimatsu, K Ochiai, B Syuto

  JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (10) 1123 - 1126 0916-7250 2003/10 [Refereed][Not invited]
   

  Mammary tumors are common in cats. As mutations in human Brca2 confer an increased risk of breast cancer, the full-length cDNA of the feline homologue of Brca2 was sequenced to obtain a basis for studying the relationship between its function and susceptibility to mammary tumors. The feline Brca2 cDNA is 10 kb long, and encodes 3,371 amino acids. The amino acid sequence of feline Brca2 shares low homology with the Brca2 of other mammals, e.g., 53% homology with the murine protein. Analysis of the expression pattern of the feline Brca2 gene revealed that, as previously reported for other mammals, it is transcribed in various tissues, including the mammary gland.
• Role of CXCR4 and SDF-1 in mammary tumor metastasis in the cat

  T Oonuma, M Morimatsu, T Nakagawa, R Uyama, N Sasaki, M Nakaichi, H Tamamura, N Fuji, S Hashimoto, H Yamamura, B Syuto

  JOURNAL OF VETERINARY MEDICAL SCIENCE 65 (10) 1069 - 1073 0916-7250 2003/10 [Refereed][Not invited]
   

  It has recently been suggested that the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12) promote metastasis of various cancers in humans. Since feline mammary tumors also metastasize to distant organs frequently, we used real-time quantitative PCR to examine the expression of feline CXCR4 (fCXCR4) in ten feline mammary tumor cell lines and seven feline mammary tumor tissues, and also the expression of feline SDF-1 (fSDF-1) in various organs. Cell lines derived from metastatic regions expressed more fCXCR4 than those derived from primary tumors. Mammary tumor tissues overexpressed more fCXCR4 than normal mammary tissues. Organs with high levels of fSDF-1 expression represent common sites of metastasis. Migration assays using the feline mammary tumor cell line NAC were also performed to test the activity of TN14003 and TC14012, antagonists of human CXCR4, to antagonize fCXCR4 expressed on NAC cells. TN14003 and TC14012 inhibited migration of NAC cells. We conclude that fCXCR4 may be a therapeutic target for feline mammary tumors.
• Bacterial lipopolysaccharide-induced expression of the I kappa B protein MAIL in B-lymphocytes and macrophages

  H Kitamura, Y Matsushita, T Iwanaga, K Mori, K Kanehira, D Fujikura, M Morimatsu, M Saito

  ARCHIVES OF HISTOLOGY AND CYTOLOGY 66 (1) 53 - 62 0914-9465 2003/03 [Refereed][Not invited]
   

  Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL), a recently cloned nuclear IkappaB protein induced by lipopolysaccharide (LPS) stimulation in lymphoid organs, is involved in the regulation of inflammatory responses. The present in situ hybridization and immunohistochemical analyses revealed the distinct expression of the MAIL mRNA and protein in B-lymphocytes of the white pulp of the spleen and cortical lymphoid follicles of lymph nodes in LPS-injected mice. MAIL signals were also localized in F4/80-positive macrophages in these organs. LPS clearly induced MAIL expression in cultured B-lymphocytes and monocytes/macrophages, but only faintly so in T-lymphocytes, fibroblasts, and endothelial cells. MAIL was also induced by inflammatory cytokines such as interleukin-1 and -6, and tumor necrosis factor in cultured cells. Northern blot, Western blot, and in situ hybridization analyses showed that the major expression product of the Mail gene was a long splicing variant (MAIL-L) rather than a short one, both in lymphoid organs and cultured cells. These results collectively indicate that LPS induces MAIL-L predominantly in B-lymphocytes and macrophages.
• The multimerization of hantavirus nucleocapsid protein depends on type-specific epitopes

  K Yoshimatsu, BH Lee, K Araki, M Morimatsu, M Ogino, H Ebihara, J Arikawa

  JOURNAL OF VIROLOGY 77 (2) 943 - 952 0022-538X 2003/01 [Refereed][Not invited]
   

  Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.
• Cellular expression of gut chitinase mRNA in the gastrointestinal tract of mice and chickens

  M Suzuki, W Fujimoto, M Goto, M Morimatsu, B Syuto, T Iwanaga

  JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 50 (8) 1081 - 1089 0022-1554 2002/08 [Refereed][Not invited]
   

  Recently, the second mammalian chitinase, designated acidic mammalian chitinase (AMCase), has been identified in human, mouse, and cow. In contrast to the earlier identified macrophage-derived chitinase (chitotriosidase), this chitinase is richly expressed in the gastrointestinal (GI) tract, suggesting its role in digestion of chitin-containing foods as well as defense against chitin-coated microorganisms and parasites. This in situ hybridization study first revealed cellular localization of the gut-type chitinase in the mouse and chicken. In adult mice, the parotid gland, von Ebner's gland, and gastric chief cells, all of which are exocrine cells of the serous type, expressed the gut chitinase mRNA. In the chicken, oxyntico-peptic cells in glandular stomach (proventriculus) and hepatocytes expressed the chitinase mRNA. Because cattle produce the gut chitinase (chitin-binding protein b04) only in the liver, the gut chitinases in mammals and birds have three major sources of production, i.e., the salivary gland, stomach, and liver. During ontogenetic development, the expression level in the parotid gland and stomach of mice increased to the adult level before weaning, whereas in the stomach of chickens intense signals were detectable in embryos from incubation day 7.
• Characterization of binding sites for diisopropyl phosphorofluoridate in spinal cord cytosol

  R Kamata, M Morimatsu, T Suzuki, T Takewaki, H Kobayashi

  ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 12 (1) 55 - 58 1382-6689 2002/08 [Refereed][Not invited]
   

  Gel filtration chromatography was performed on cytosol preparation of hen spinal cord to find molecular target(s) for organophosphorus-induced delayed neurotoxicity (OPIDN). Three binding peaks of [H-3]diisopropyl phosphorofluoridate (DFP), an organophosphate that induces OPIDN, were separated from the cytosol preparation. The activities of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) that has been proposed as a screening method for OPIDN eluted in the fractions within these two DFP binding peaks. However, the other peak had none of the activities of AChE and NTE. Therefore, this DFP binding proteins in cytosol may be peculiar to the pathogenesis of OPIDN. (C) 2002 Elsevier Science B.V. All rights reserved.
• The identification and properties of chitin-binding protein b01 (CBPb01)

  M Suzuki, M Morimatsu, B Syuto

  JOURNAL OF VETERINARY MEDICAL SCIENCE 64 (6) 477 - 481 0916-7250 2002/06 [Refereed][Not invited]
   

  Bovine serum contains N-acetyl-D-glucosamine (GlcNAc)-sensitive opsonin inhibitory factors. In the present study, a major component of chitin-binding protein (chitin-binding protein b01, CBPb01) was purified from bovine serum, and identified CBPb01 as bovine IgM by its subunit structure, antigenic properties, and partial sequences. The results of a lectin-binding assay showed that the heavy chain of CBPb01 had a GlcNAc structure, but the commercial IgM did not. It is possible that CBPb01 interconnects through its GlcNAc structure, subsequently forming complexes. We also demonstrated that CBPb01 had opsonin-inhibitory activity, and that this activity was dependent on the binding of CBPb01 to GlcNAc on the zymosan surface. These findings indicate the presence of a kind of IgM that recognizes GlcNAc structure in the regulation of opsonization.
• Bacterial lipopolysaccharide induces mRNA expression of an I kappa B MAIL through toll-like receptor 4

  H Kitamura, K Kanehira, T Shiina, M Morimatsu, BD Jung, S Akashi, M Saito

  JOURNAL OF VETERINARY MEDICAL SCIENCE 64 (5) 419 - 422 0916-7250 2002/05 [Refereed][Not invited]
   

  Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear IkappaB protein recently identified as a molecule appearing in immunocompetent organs after administration of bacterial lipopolysaccharide (LPS). Participation of Toll-like receptor (TLR) 4, which is a major form of LPS receptors, in the LPS-induced MAIL expression was investigated. When a human myelomonocytic cell line U937 was treated with phorbol 12-myristate 13-acetate for 3 days, the LPS-induced MAIL expression was much potentiated in parallel with an increase in TLR4 expression. The MAIL induction was attenuated when the cells were treated with a neutralizing antibody against TLR4. The in vivo induction of MAIL in the spleen was smaller in mice having a missense mutation of the Tlr4 gene than in normal control mice. These results collectively indicate that TLR4 contributes, at least in part, MAIL induction after LPS stimulation.
• A novel serum chitinase that is expressed in bovine liver

  M Suzuki, M Morimatsu, T Yamashita, T Iwanaga, B Syuto

  FEBS LETTERS 506 (2) 127 - 130 0014-5793 2001/10 [Refereed][Not invited]
   

  Chitinases are ubiquitous chitin-fragmenting hydrolases. They are synthesized by a vast array of organisms, including those not composed of chitin. Here, we describe a novel serum chitinase (chitin-binding protein b04, CBPb04), which is expressed in bovine liver. Although CBPb04 is secreted as an endocrine chitinase, it shows higher homology with human gastrointestinal tract exocrine chitinase (AMCase) than with macrophage endocrine chitinase (human chitotriosidase). This suggests that cows have a specific defense against chitin-containing microorganisms. CBPb04 mRNA is expressed in hepatocytes. This is the first report of a hepatogenic mammalian chitinase. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
• Genomic organization, chromosomal localization, and promoter analysis of the mouse Mail gene

  T Shiina, M Morimatsu, H Kitamura, T Ito, S Kidou, K Matsubara, Y Matsuda, M Saito, B Syuto

  IMMUNOGENETICS 53 (8) 649 - 655 0093-7711 2001/10 [Refereed][Not invited]
   

  The Mail (molecule possessing ankyrin repeats induced by lipopolysaccharide) protein is a member of the I kappaB family. It has six ankyrin repeats that are conserved in other I kappaB proteins, such as I kappaB-alpha and Bcl-3. Mail mRNA expression is induced rapidly following lipopolysaccharide (LPS) injection, most notably in the spleen, lung, and lymph nodes of mice, where immune cells, such as lymphocytes and macrophages, are abundant. In this study, we cloned and characterized the Mail gene. The isolated genomic clones span approximately 30 kb and encompass the entire gene. Comparisons with Mail cDNA revealed that the Mail gene consists of 14 exons. Several splice junctions encoding ankyrin repeats are conserved among Mail and other I kappaB family genes. Southern hybridization showed that Mail is a single-copy gene. Using fluorescence in situ hybridization analysis, mouse and rat Mail genes were mapped to Chromosome (Chr) 16C 1.2-C1.3 and Chr 11q21.1, respectively. Primer extension determined the transcription start site of Mail. Sequence analysis of the proximal promoter region revealed the presence of a TATA box and putative transcription factor-binding sites, such as those for NF-kappaB and NF-IL6. This region is sufficient to drive high-level reporter gene expression in LPS-stimulated transfected cells.
• Cloning and sequencing full length of canine Brca2 and Rad51 cDNA

  K Ochiai, M Morimatsu, N Tomizawa, B Syuto

  JOURNAL OF VETERINARY MEDICAL SCIENCE 63 (10) 1103 - 1108 0916-7250 2001/10 [Refereed][Not invited]
   

  Mammary tumors are the most common neoplasm in female dogs. Canis canis, and in women. Mutations in human Brca2 confer an increased risk of female breast cancer. Previous studies have shown that the Brca2 tumor suppressor protein interacts with the recombinational repair protein Rad51. We cloned the full-length cDNA of the canine homologues of Brca2 and Rad51 to obtain a basis for studying their relationship with susceptibility to mammary tumors. The canine Brca2 and Rad51 cDNAs are 11 and 1.5 kb long, encoding 3,471 and 339 amino acids, respectively. The amino acid sequence of canine Brca2 showed 68% homology with the human protein., and 58% homology with a murine protein. There were highly conserved regions in the C-terminus of all three proteins, where the Rad51 interacting domain and putative nuclear localization signals are located. Comparing with the partial genomic sequence previously reported, we found possible nuclear polymorphisms in exon 11, some of which result in amino acid substitutions. On the other hand, canine Rad51 protein had extremely high homology (99%) to the human and murine proteins. Expression of both Brca2 and Rad51 was detected in the mammary gland, suggesting that these two genes interact in the canine mammary gland.
• Changes in sodium or glucose filtration rate modulate expression of glucose transporters in renal proximal tubular cells of rat

  S Vestri, MM Okamoto, HS de Freitas, RA dos Santos, MT Nunes, M Morimatsu, JC Heimann, UF Machado

  JOURNAL OF MEMBRANE BIOLOGY 182 (2) 105 - 112 0022-2631 2001/07 [Refereed][Not invited]
   

  Renal glucose reabsorption is mediated by luminal sodium-glucose cotransporters (SGLTs) and basolateral facilitative glucose transporters (GLUTs). The modulators of these transporters are not known, and their substrates glucose and Na+ are potential candidates. In this study we examined the role of glucose and Na+ filtration rate on gene expression of glucose transporters in renal proximal tubule. SGLT1, SGLT2, GLUT1 and GLUT2 mRNAs were assessed by Northern blotting; and GLUT1 and GLUT2 proteins were assessed by Western blotting. Renal cortex and medulla samples from control rats (C), diabetic rats (D) with glycosuria, and insulin-resistant 15-month old rats (I) without glycosuria; and from normal (NS), low (LS), and high (HS) Na+-diet fed rats were studied. Compared to C and I rats, D rats increased (P < 0.05) gene expression of SGLT2 by similar to 36%, SGLT1 by similar to 20%, and GLUT2 by similar to 100%, and reduced (P < 0.05) gene expression of GLUT1 by more than 50%. Compared to NS rats, HS rats increased (P < 0.05) SCLT2, GLUT2, and GLUT1 expression by <similar to>100%. with no change in SGLT1 mRNA expression, and LS rats increased (P < 0.05) GLUT1 gene expression by <similar to>150% with no changes in other transporters. In summary, the results showed that changes in glucose or Na+ filtrated rate modulate the glucose transporters gene expression in epithelial cells of the renal proximal tubule.
• Isolation, characterization, and cDNA cloning of chicken turpentine-induced protein, a new member of the scavenger receptor cysteine-rich (SRCR) family of proteins

  K Iwasaki, M Morimatsu, O Inanami, E Uchida, B Syuto, M Kuwabara, M Niiyama

  JOURNAL OF BIOLOGICAL CHEMISTRY 276 (12) 9400 - 9405 0021-9258 2001/03 [Refereed][Not invited]
   

  Acute-phase serum proteins were induced by administrating a chicken with turpentine oil. One of these proteins was a new protein that appeared in front of albumin in polyacrylamide disc gel electrophoresis using a 4.5-16% gel. To purify this protein, turpentine-administrated chicken serum was fractionated by ammonium sulfate precipitation at 50% saturation, and the supernatant fraction was chromatographed on a DEAE-Toyopearl 650S column. The purified protein is a mannose-glycoprotein, and its N-terminal sequence, determined by the Edoman method, is not homologous from that of other reported acute-phase proteins. An analysis of physiological function with two different test systems, chemiluminescence measurement and electron spin resonance spectroscopy, showed that the purified protein has antioxidant, activity and inhibits superoxide (O-2(radical anion)) mediated by activation of the receptor. In support of these results, the complete amino acid sequence of 18-B is homologous to the scavenger receptor cysteine-rich (SRCR) family of proteins that participate in the regulation of leukocyte function. 18-B is composed of four SRCR domains, which is different from the previously characterized SRCR family of proteins such as Sp alpha, CD6, and CD163. These findings indicate that turpentine-induced 18-B, a new member of scavenger receptor cysteine-rich family, may be implicated in regulation of cell function in a manner of inhibition of the overproduction of the reactive oxygen species.
• MAIL, a novel nuclear I kappa B protein that potentiates LPS-induced IL-6 production

  H Kitamura, K Kanehira, K Okita, M Morimatsu, M Saito

  FEBS LETTERS 485 (1) 53 - 56 0014-5793 2000/11 [Refereed][Not invited]
   

  We have identified and characterized a novel member of the ankyrin-repeat family named 'molecule possessing ankyrin-repeats induced by lipopolysaccharide' (MAIL). The C-terminal portion of MAIL shared high sequence homology,vith the I kappaB family. Intraperitoneal injection of lipopolysaccharide (LPS) into mice rapidly (< 0.5 h) induced MAIL mRNA in various tissues, particularly in the spleen, lymph node, and lung. Ectopically expressed MAIL was localized in the nucleus, and remarkably potentiated the LPS-induced mRNA expression and secretion of interleukin (IL)-6 in Swiss 3T3 cells, These findings indicate that MAIL is one of the nuclear I<kappa>B proteins and an activator of IL-6 production. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
• The evaluation of the potential of botulinum C3 enzyme as an exogenous differentiation inducing factor to neurons

  Y Watanabe, M Morimatsu, B Syuto

  JOURNAL OF VETERINARY MEDICAL SCIENCE 62 (5) 473 - 478 0916-7250 2000/05 [Refereed][Not invited]
   

  Botulinum C3 enzyme produced by Clostridium botulinum type C and D strains modifies Rho proteins. In a previous study, we observed that the LDH isozyme pattern of neurons treated with C3 enzyme was different from that induced with endogenous growth factor of neurons such as NGF [21]. This type of change is considered to have an advantage in the medical use of C3 enzyme for neural disorder. To determine the functional similarity of C3-treated neurons to control and NGF-treated neurons, we examined the responses of C3-treated neurons to various drugs, including some neurotransmitters, by measuring the rise of intracellular Ca ions into the neurons. The time course of the rise of intracellular Ca ions induced by high concentration of potassium in the C3-treated neurons was similar to that in the NGF-treated neurons. The C3-treated neurons responded to glutamic acid, aspartic acid, kainic acid, gamma-aminobutylic acid, muscarine and ACh with similar time courses and magnitudes as the control neurons. These results suggest that the C3 enzyme induces the functional differentiation of neurons, and that C3 enzyme has the potential for the medical use as an exogenous differentiation-inducing factor of neurons.
• Canine leptin: cDNA cloning, expression and activity of recombinant protein

  M Iwase, K Kimura, N Sasaki, R Komagome, K Ishioka, M Morimatsu, T Murakami, M Saito

  RESEARCH IN VETERINARY SCIENCE 68 (2) 109 - 114 0034-5288 2000/04 [Refereed][Not invited]
   

  Leptin, the product of the ob gene, is one of the key molecules for the regulation of appetite and whole-body energy balance, and thereby for the pathogenesis of obesity. In an attempt to clarify the roles of leptin in obesity and/or related diseases in companion animals, canine leptin CDNA was cloned by amplifying reverse-transcriptase products of RNA extracted from the adipose tissue of the beagle. A CDNA clone of about 3 kbp contained a 501 bp open reading frame coding a 167-amino acid protein with a 21-amino acid signal peptide. The sequence of a 146-amino acid mature leptin was more than 79 per cent identical to those of other mammals. Northern blot analysis revealed abundant expression of leptin mRNA in adipose tissue, but not in other tissues, in adult beagles. When Chinese hamster ovary cells expressing the rat leptin receptor were stimulated with recombinant canine leptin produced by E. coli, some intracellular signal transduction proteins were phosphorylated, indicating that the recombinant leptin was biologically active. The data reported herein will be helpful for further studies of leptin of the dog in health and disease. (C) Harcourt Publishers Ltd.
• Botulinum C3 enzyme changes the lactate dehydrogenase isozyme pattern of primary culture of neurons

  Y Watanabe, M Morimatsu, B Syuto

  JOURNAL OF VETERINARY MEDICAL SCIENCE 62 (3) 249 - 254 0916-7250 2000/03 [Refereed][Not invited]
   

  Changes in the lactate dehydrogenase (LDH) isozyme pattern of primary culture of neurons treated with botulinum C3 enzyme were examined in order to elucidate the functional changes accompanying the morphological change that follows ADP-ribosylation of Rho protein. Primary neurons were prepared from the cerebrum of ICR mouse embryos on day 15. Neurons were cultured in MEM with 10% fetal calf serum at 37 degrees C. In the neurons treated with C3 enzyme, a typical morphological change was observed after 24 hr, and the LDH isozyme pattern was changed after 72 hr. The ratio of H-subunit to M-subunit in LDH was decreased by C3 treatment, suggesting the induction of a state of lower intracellular oxygen consumption in neurons in the primary cultures.
• Mutated human beta 3-adrenergic receptor (Trp64Arg) lowers the response to beta 3-adrenergic agonists in transfected 3T3-L1 preadipocytes

  K Kimura, N Sasaki, A Asano, J Mizukami, S Kayahashi, T Kawada, T Fushiki, M Morimatsu, T Yoshida, M Saito

  HORMONE AND METABOLIC RESEARCH 32 (3) 91 - 96 0018-5043 2000/03 [Refereed][Not invited]
   

  Wild-type or mutated human beta 3-adrenergic receptor (Trp64Arg) cDNAs were stably expressed in mouse 3T3-L1 cells. Saturation binding study using a beta-adrenergic ligand revealed that there was no significant difference in the receptor density and the equilibrium dissociation constant between the two cell lines. However, the ability of the mutant beta 3-adrenergic receptor to accumulate cyclic AMP (cAMP) in response to isoproterenol was much reduced and Kact for cAMP accumulation was lowered as compared to the wild type receptor. The amount of alpha subunit of stimulatory GTP-binding protein (GS alpha) and adenylyl cyclase activity in response to forskolin were not different in the two cell lines. The responses of the mutant receptor to epinephrine, norepinephrine and L-755,507, a highly specific agonist for human beta 3-adrenergic receptor, were also reduced, but the reduction of Kact for L-755,507 was more evident than other agonists tested. The cAMP accumulation in response to some conventional beta 3 agonists was less than 10% of that to isoproterenol even in the cells expressing the wild type receptor. These results suggest that the Trp64Arg mutant beta 3-adrenergic receptor has less ability to stimulate adenylyl cyclase, and that lipolytic activity through the beta 3-adrenergic receptor by catecholamines in subjects carrying this mutation might be suppressed.
• Cells deleted for Brca2 COOH terminus exhibit hypersensitivity to gamma-radiation and premature senescence

  M Morimatsu, G Donoho, P Hasty

  CANCER RESEARCH 58 (15) 3441 - 3447 0008-5472 1998/08 [Refereed][Not invited]
   

  The putative Brca2-MmRad51 interaction is analyzed in mouse cells deleted for the COOH terminus of Brca2 (amino acids 3140-3328), which contains a region that associates with MmRad51 by yeast two-hybrid. These cells are hypersensitive to gamma-radiation (suggesting defective recombinational repair) but not UV light (suggesting intact nucleotide excision repair) and maintain the G(1)-S and G(2)-M checkpoints after exposure to gamma-irradiation. Cells deleted for the COOH terminus of Brca2 progress through the cell cycle at a similar rate as wild-type cells but undergo senescence more rapidly. These data support the hypothesis that deletion of Brca2 stimulates cancer by defective MmRad51-mediated DNA repair and not by defective cell cycle regulation.
• Comparison of amino acid sequence of the C-terminal domain of insulin-responsive glucose transporter (GLUT4) in livestock mammals

  H Abe, Y Kawakita, T Miyashige, M Morimatsu, M Saito

  JOURNAL OF VETERINARY MEDICAL SCIENCE 60 (6) 769 - 771 0916-7250 1998/06 [Refereed][Not invited]
   

  The nucleotide sequence of cDNA coding the 38-amino acid of the C-terminal domain of insulin-responsive glucose transporter (GLUT4) was determined by the method of reverse transcription-polymerase chain reaction in the sheep, goat and pig, and compared with that of bovine which have been shown to have a unique amino acid conversion of Asn508 to His. The deduced amino acid sequence was completely identical in the three species, and did not have the amino acid conversion at position 508. Western blot analysis confirmed that an antiserum raised against a rat C-terminal peptide cross-reacted efficiently with GLUT4 of these livestock mammals.
• Central IL-1 differentially regulates peripheral IL-6 and TNF synthesis

  H Kitamura, S Okamoto, Y Shimamoto, M Morimatsu, A Terao, M Saito

  CELLULAR AND MOLECULAR LIFE SCIENCES 54 (3) 282 - 287 1420-682X 1998/03 [Refereed][Not invited]
   

  Centrally given interleukin (IL)-1 is known to induce a rapid rises in blood IL-6. To extend this and to examine the mechanism by which this occurs, the effects of intracerebroventricular (icv) injection of human recombinant IL-1 beta on mRNA expression of IL-6 and tumour necrosis factor (TNF) in the spleen and liver were examined in rats. Icy injection of IL-1 produced a rapid rise of the tissue mRNA levels of IL-6 and TNF in both organs, prior to and/or in parallel with an increase in their serum levels. Pretreatment with chlorisondamine, a ganglionic blocking agent, inhibited the IL-6 responses, while it had little influence on the TNF responses. The results suggest that brain IL-1 induces peripheral production of IL-6, but not of TNF, through autonomic nervous system activation.
• Adrenergic activation of vascular endothelial growth factor mRNA expression in rat brown adipose tissue: implication in cold-induced angiogenesis

  A Asano, M Morimatsu, H Nikami, T Yoshida, M Saito

  BIOCHEMICAL JOURNAL 328 179 - 183 0264-6021 1997/11 [Refereed][Not invited]
   

  Cold exposure produces adaptive hyperplasia and growth of brown adipose tissue (BAT), the major site of non-shivering thermogenesis in rodents, associated with increased angiogenesis in this tissue. Vascular endothelial growth factor (VEGF), one of the most potent angiogenic factors, was found to be expressed abundantly in BAT of the rat. When rats were exposed to cold at 4 degrees C, the VEGF mRNA level in BAT was increased by 2-3-fold in 1-4 h, but returned to the basal level within 24 h. VEGF expression in other tissues such as heart, kidney and lung did not change after cold exposure. The cold-induced increase in VEGF mRNA was abolished by surgical sympathetic denervation, but mimicked by administration of noradrenaline or a beta(3)-adrenoceptor agonist CL316,243, indicating the critical role of the beta-adrenergic pathway in VEGF expression in BAT. Among three isoforms of VEGF, the mRNA of a short form (VEGF120) lacking heparin-binding activity was preferentially increased after cold exposure and treatment with the adrenergic agonists. These results suggest that cold exposure activates the sympathetic nerves and leads to a rapid increase in synthesis of VEGF in BAT, which in turn stimulates the proliferation of surrounding vascular endothelial cells.
• Immobilization stress increases hepatic IL-6 expression in mice

  H Kitamura, A Konno, M Morimatsu, BD Jung, K Kimura, M Saito

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 238 (3) 707 - 711 0006-291X 1997/09 [Refereed][Not invited]
   

  When mice were subjected to restriction of movement in a small cylinder (immobilization stress), the serum interleukin (IL)-6 level rose in 1 h, following increased expression of IL-6 mRNA in both the liver and the spleen. The IL-6 mRNA induction was much greater in the liver than in the spleen when compared on a whole-organ basis. Intraperitoneal injection of bacterial lipopolysaccharide (LPS) also increased IL-6 mRNA expression in these organs, but more preferentially in the spleen. Immunohistochemical examinations of liver tissue using an antibody against murine IL-6 revealed that immobilization stress induced IL-6 mainly in hepatic parenchymal cells, whereas LPS injection did so only in sinusoidal mononuclear cells. These results indicate that immobilization stress induces IL-6 production in the liver, especially in hepatic parenchymal cells, probably by a different mechanism from that for IL-6 induction by LPS. (C) 1997 Academic Press.
• Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2

  SK Sharan, M Morimatsu, U Albrecht, DS Lim, E Regel, C Dinh, A Sands, G Eichele, P Hasty, A Bradley

  NATURE 386 (6627) 804 - 810 0028-0836 1997/04 [Refereed][Not invited]
   

  Inherited mutations in the human BRCA2 gene cause about half of the cases of early-onset breast cancer. The embryonic expression pattern of the mouse Brca2 gene is now defined and an interaction identified of the Brca2 protein with the DNA-repair protein Rad51. Developmental arrest in Brca2-deficient embryos, their radiation sensitivity, and the association of Brca2 with Rad51 indicate that Brca2 may be an essential cofactor in the Rad51-dependent DNA repair of double-strand breaks, thereby explaining the tumour-suppressor function of Brca2.
• Molecular cloning and mRNA expression of the bovine insulin-responsive glucose transporter (GLUT4)

  H Abe, M Morimatsu, H Nikami, T Miyashige, M Saito

  JOURNAL OF ANIMAL SCIENCE 75 (1) 182 - 188 0021-8812 1997/01 [Refereed][Not invited]
   

  Insulin-responsive glucose transporter GLUT4, is a member of the glucose transporter family (GLUT) and is present exclusively in muscle and adipose tissue. It is a target of insulin action in humans and rodents. To clarify the molecular structure of bovine GLUT4, its GLUT4 cDNA was cloned by the RT-PCR method. Several cDNA clones corresponding to the different regions of GLUT4 were obtained by amplifying reverse-transcriptase products of RNA extracted from Holstein cattle skeletal muscle. Nucleotide sequence analysis of the cDNA clones revealed that bovine GLUT4 cDNA was composed of 2,656 base pairs with a coding region for a 509 amino acid protein. The deduced amino acid sequence was 64% and 92% identical with bovine GLUT1 (GLUT ubiquitously expressed in all tissues) and rat GLUT4, respectively. Although the amino acid sequence of the GLUT4 COOH-terminal region is highly conserved among the species so far reported, one amino acid (Asp) of this region was replaced by His in bovine GLUT4. The tissue distribution of GLUT4, was also examined by Northern blot analysis using a probe prepared from the bovine cDNA. GLUT4 mRNA was detected in skeletal muscle, heart, and adipose tissue, but not in liver, kidney, lung, brain, or spleen. Such a distribution is essentially the same as in humans and rodents, suggesting that GLUT4 is an insulin-responsive glucose transporter in cattle.
• Seasonal variations of blood haptoglobin level of brown bears in Japan

  K Mominoki, H Tsuruga, M Morimatsu, M Saito

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-PHYSIOLOGY 114 (4) 349 - 353 0300-9629 1996/08 [Refereed][Not invited]
   

  Haptoglobin (Hp), a hemoglobin-binding protein, is known as an acute phase protein increasing in blood during inflammation in most mammals. On the basis of our previous studies on purification and characterization of bear Hp (Comp. Biochem. Physiol. 110B, 785-789, 1995), in this study, we developed an immunoassay method to measure serum Hp level in bear, and measured the concentration of Hp in blood samples collected from 84 reared and 25 wild brown bears in Hokkaido, Japan. The mean serum Hp concentration was 0.94 +/- 0.25 mg/ml in wild bears, which is nearly equal to those reported in other species. In reared bears, the Hp concentration was apparently higher (3.82 +/- 0.29 mg/ml), although total protein and albumin concentrations were nearly equal in the two groups. A significant seasonal variation of serum Hp, low in spring and high in autumn and winter, was found in reared bears. Possible factors participating in the seasonal variation were discussed with special references to hibernation.
• Expression and localization of insulin-regulatable glucose transporter (GLUT4) in rat brain

  M Kobayashi, H Nikami, M Morimatsu, M Saito

  NEUROSCIENCE LETTERS 213 (2) 103 - 106 0304-3940 1996/08 [Refereed][Not invited]
   

  The localization of glucose transporters (GLUTs) was examined in various regions of the rat brain. The mRNA of GLUT1 and GLUT3 were found ubiquitously in every brain region (cortex, hippocampus, midbrain, striatum, hypothalamus, medulla oblongata and cerebellum). The mRNA and protein of GLUT4, an insulin-regulatable glucose transporter in peripheral tissues, were also identified, particularly abundantly in the cerebellum. In situ hybridization analysis revealed that GLUT4 mRNA was present in some discrete cells, such as Purkinje cells in the cerebellum, the vestibular nucleus in the medulla oblongata and also in ependymal cells along the cerebral ventricles. The GLUT4 mRNA level in the cerebellum changed little in fasted or experimentally induced diabetic rats while those in adipose tissues decreased much. The results suggest that insulin-sensitive glucose uptake may occur in some specific cells of the brain but is regulated in a different manner from those in peripheral cells.
• Seasonal variations of blood haptoglobin level of brown bears in Japan.

  K Mominoki, H Tsuruga, M Morimatsu, M Saito

  Comparative biochemistry and physiology. Part A, Physiology 114 (4) 349 - 53 1096-4940 1996/08 

  Haptoglobin (Hp), a hemoglobin-binding protein, is known as an acute phase protein increasing in blood during inflammation in most mammals. On the basis of our previous studies on purification and characterization of bear Hp (Comp. Biochem. Physiol. 110B, 785-789, 1995), in this study, we developed an immunoassay method to measure serum Hp level in bear, and measured the concentration of Hp in blood samples collected from 84 reared and 25 wild brown bears in Hokkaido, Japan. The mean serum Hp concentration was 0.94 +/- 0.25 mg/ml in wild bears, which is nearly equal to those reported in other species. In reared bears, the Hp concentration was apparently higher (3.82 +/- 0.29 mg/ml), although total protein and albumin concentrations were nearly equal in the two groups. A significant seasonal variation of serum Hp, low in spring and high in autumn and winter, was found in reared bears. Possible factors participating in the seasonal variation were discussed with special references to hibernation.
• PLASMA HAPTOGLOBIN RESPONSE TO INTRACEREBROVENTRICULAR ADMINISTRATION OF CYTOKINES IN RATS

  H KITAMURA, Y SHIMAMOTO, M MORIMATSU, A TERAO, M SAITO

  BIOMEDICAL RESEARCH-TOKYO 16 (5) 353 - 356 0388-6107 1995/10 [Refereed][Not invited]
   

  To examine effects of brain cytokines on hepatic acute phase protein synthesis, the plasma level of haptoglobin (Hp) was measured after intracerebroventricular (icv) administration of human recombinant interleukin (Il)-1 beta, IL-6 and tumor necrosis factor (TNF)-alpha in rats. Icv injection of IL-1 or TNF (100 ng/rat, 3 times at every 3 h) produced a significant increase in plasma Hp level 12-24 h after the first injection. Neither intraperitoneal injection of these cytokines at the same doses nor icv injection of IL-6 affected the plasma Hp level. These results indicate that brain IL-1 and TNF, but not IL-6, can induce the acute phase response of some plasma proteins.
• STIMULATION OF HAPTOGLOBIN SYNTHESIS BY INTERLEUKIN-6 AND TUMOR-NECROSIS-FACTOR, BUT NOT BY INTERLEUKIN-1, IN BOVINE PRIMARY CULTURED-HEPATOCYTES

  N NAKAGAWATOSA, M MORIMATSU, M KAWASAKI, H NAKATSUJI, B SYUTO, M SAITO

  JOURNAL OF VETERINARY MEDICAL SCIENCE 57 (2) 219 - 223 0916-7250 1995/04 [Refereed][Not invited]
   

  The hepatic synthesis of acute phase proteins in ruminants has been suggested to be regulated by some mechanisms different from those in other species such as rodents and human. To explore possible regulatory factors unique to ruminants, we examined effects of interleukin (IL)-6, IL-1 and tumor necrosis factor (TNF), on haptoglobin (Hp) synthesis using a primary culture system of bovine hepatocytes. After bovine primary cultured hepatocytes were incubated in the presence of various concentrations of the cytokines, the synthesis and mRNA level of haptoglobin and albumin were measured by labeling with [S-35]-methionine and immunoprecipitation, and by Northern blot analysis, respectively. Hp synthesis was dose-dependently increased by recombinant human (rh) IL-6, and also by rhTNF-alpha, but to a less extent, while it was not affected by rhlL-1 beta. The stimulatory effect is mainly pretranslational, because mRNA level of Hp changed in parallel with protein synthesis. In contrast, albumin synthesis was suppressed by these three cytokines similarly. These results are inconsistent with the previously proposed view that TNF and IL-l overlap in their pathways leading to the transcriptional activation of many acute phase protein genes. In conclusion, there is a species-specific unique signaling system, especially for TNF, in transcriptional activation of bovine Hp gene.
• HAPTOGLOBIN IN CARNIVORA - A UNIQUE MOLECULAR-STRUCTURE IN BEAR, CAT AND DOG HAPTOGLOBINS

  K MOMINOKI, N NAKAGAWATOSA, M MORIMATSU, B SYUTO, M SAITO

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 110 (4) 785 - 789 0305-0491 1995/04 [Refereed][Not invited]
   

  Haptoglobin (Hp), a hemoglobin-binding protein in plasma, consists of alpha and beta subunits and has a tetra-chain arrangement (beta-alpha-alpha-beta) connected by disulfide bridges in most mammals so far examined, Dog Hp has been reported to be unique compared with other Hps in respect that (1) the two alpha beta units are joined by a non-covalent interaction rather than a disulfide bridge and (2) the alpha chain has an oligosaccharide-binding sequence (Asn-X-Ser/Thr) and is glycosylated, To determine whether the unique structures of dog Hp are common in the Carnivora, we purified Hps from sera of bear and cat, and analyzed their subunit structure and partial amino acid sequences, The analyses by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and non-reducing conditions, revealed that bear and cat Hps have similar subunit arrangements to dog Hp, suggesting the absence of a disulfide bridge between two a chains, This was confirmed by amino acid sequence analysis of the alpha chains: that is, Cys(15) participating in the inter-a chain disulfide bridge was replaced by Val in bear or Leu in cat and dog, Thus, the unique subunit arrangement of Hp reported in dog may be common in the Carnivora, In contrast to dog Hp, however, alpha chains of bear and cat Hps were found not to have the typical oligosaccharide binding sequence on their alpha chains and were not glycosylated.
• Haptoglobin in Carnivora: a unique molecular structure in bear, cat and dog haptoglobins.

  K Mominoki, N Nakagawa-Tosa, M Morimatsu, B Syuto, M Saito

  Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 110 (4) 785 - 9 1096-4959 1995/04 

  Haptoglobin (Hp), a hemoglobin-binding protein in plasma, consists of alpha and beta subunits and has a tetra-chain arrangement (beta-alpha-alpha-beta) connected by disulfide bridges in most mammals so far examined. Dog Hp has been reported to be unique compared with other Hps in respect that (1) the two alpha beta units are joined by a non-covalent interaction rather than a disulfide bridge and (2) the alpha chain has an oligosaccharide-binding sequence (Asn-X-Ser/Thr) and is glycosylated. To determine whether the unique structures of dog Hp are common in the Carnivora, we purified Hps from sera of bear and cat, and analyzed their subunit structure and partial amino acid sequences. The analyses by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and non-reducing conditions, revealed that bear and cat Hps have similar subunit arrangements to dog Hp, suggesting the absence of a disulfide bridge between two alpha chains. This was confirmed by amino acid sequence analysis of the alpha chains: that is, Cys15 participating in the inter-alpha chain disulfide bridge was replaced by Val in bear or Leu in cat and dog. Thus, the unique subunit arrangement of Hp reported in dog may be common in the Carnivora. In contrast to dog Hp, however, alpha chains of bear and cat Hps were found not to have the typical oligosaccharide binding sequence on their alpha chains and were not glycosylated.
• HYPERPLASIA OF BROWN ADIPOSE-TISSUE AFTER CHRONIC STIMULATION OF BETA-3-ADRENERGIC RECEPTOR IN RATS

  NAGASE, I, N SASAKI, K TSUKAZAKI, T YOSHIDA, M MORIMATSU, M SAITO

  JAPANESE JOURNAL OF VETERINARY RESEARCH 42 (3-4) 137 - 146 0047-1917 1994/12 [Refereed][Not invited]
   

  When mammals are exposed to a cold environment for a long time, the capacity of nonshivering thermogenesis by brown adipose tissue (BAT) increases in association with the increased expression of some specific proteins and tissue hyperplasia, which are totally dependent on sympathetic innervation to this tissue. To clarify roles of the beta-adrenergic mechanism in BAT hyperplasia, the effects of chronic administration of various beta-adrenergic agonists on BAT were examined in rats, especially focusing on some agonists to the beta 3-adrenoceptor which is present specifically in adipocytes. Chronic administration of noradrenaline or isoproterenol for 7-10 days produced a marked increase in the tissue contents of DNA, total protein, mitochondrial uncoupling protein, and insulin-regulatable glucose transporter protein. The trophic effects of noradrenaline and isoproterenol were mimicked by chronic administration of beta 3-adrenergic agonists, such as CL316,243, BRL 26830A, and ICI D7114. These results suggest that the beta 3-adrenoceptor plays important roles for hyperplasia of BAT, and thereby increasing in the capacity of thermogenesis.
• ISOLATION AND PRIMARY CULTURE OF BOVINE HEPATOCYTES - ALBUMIN SYNTHESIS AND ADRENERGIC ACTIVATION OF GLYCOGENOLYSIS

  N NAKAGAWATOSA, M MORIMATSU, K MOMINOKI, H NAKATSUJI, B SYUTO, M SAITO

  JOURNAL OF VETERINARY MEDICAL SCIENCE 56 (1) 125 - 129 0916-7250 1994/02 [Refereed][Not invited]
   

  We describe a technique for isolation and primary culture of bovine hepatocytes, and their metabolic characterization. Hepatocytes were isolated from the caudate lobe of bovine liver by perfusion with 0.25 mM ethyleneglycol tetraacetic acid and 0.05% collagenase. The viability and yield of the cells were 70-92% and 0.1-3.6 x 10(7) cells/g liver, respectively. When the isolated hepatocytes were cultured in Williams' medium E, they began to spread in 3 hr and formed monolayers in 24 hr. These monolayers were retained for at least 6 days. To monitor the metabolic activities specific to liver, synthesis and secretion of albumin were measured by labeling with [S-35]-methionine and immunoprecipitation. This activity was low in isolated hepatocytes, but increased after culturing 1-3 days, and decreased again after 6 days. Glycogenolytic activity was also assessed by measuring glucose release to the medium by stimulation with epinephrine. The glycogenolytic response to epinephrine was also enhanced by culturing the hepatocytes 1-3 days, but was decreased after 6 days. Since the isolated bovine hepatocytes retained the liver-specific activities of albumin synthesis and glycogenolysis for several days in culture, these cells are useful for cellular and molecular studies on the functions of bovine liver.
• AVIDITY OF ANTIBODY AND AGGLUTINABILITY OF ANTIBODY-SENSITIZED LATEX IN LATEX AGGLUTINATION-TEST

  S YAMAMOTO, K TAGATA, Y ISHIKAWA, H SANTSUKA, M YAMADA, M MORIMATSU, M NAIKI

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 36 (3) 257 - 264 0165-2427 1993/04 [Refereed][Not invited]
   

  This paper describes the avidity of IgG antibody used for preparation of latex sensitized with IgG antibody (IgG-sensitized latex) and the agglutinability of IgG-sensitized latex in slide reversed passive latex agglutination (RPLA). Using immunodiffusion techniques, it was found that anti-canine C-reactive protein (CRP) sera from four rabbits immunized with canine CRP had the same antibody titers. However, the antibodies had different levels of avidity. When lattices were sensitized under the same condition with the IgG antibodies of different avidity levels separated from the above-mentioned antisera using Protein A and canine CRP-Sepharose 4B immunosorbent, these demonstrated different patterns of agglutinability in slide RPLA. The latex sensitized with IgG antibody of higher avidity demonstrated a stronger agglutinability.
• EFFICIENT PREPARATION OF MONOSPECIFIC ANTI-CANINE C-REACTIVE PROTEIN SERUM AND PURIFICATION OF CANINE C-REACTIVE PROTEIN BY AFFINITY-CHROMATOGRAPHY

  S YAMAMOTO, N ABE, H SANTSUKA, T SHIDA, K KISHIDA, S KUWAJIMA, M YAMADA, M MORIMATSU, M NAIKI

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 36 (3) 293 - 301 0165-2427 1993/04 [Refereed][Not invited]
   

  The canine C-reactive protein (CRP) fraction isolated from canine acute-phase serum on a phosphorylcholine-Sepharose 4B column was further subjected to Sephacryl S-300 gel filtration. A canine CRP fraction not containing IgM was then obtained. The antisera, obtained after several immunizations with this canine CRP fraction, contained nonspecific antibodies that reacted with albumin, transferrin and IgG in addition to CRP. This antiserum could be easily changed to monospecific canine CRP serum, when it was subjected to absorption for only 15 min using glutaraldehyde-insolubilized normal canine serum protein containing 3.5 mug ml-1 of CRP. Pure canine CRP was isolated with a recovery rate of 95% from canine acute-phase serum by affinity chromatography using specific anti-canine CRP antibody.
• PURIFICATION AND IDENTIFICATION OF A SERUM-PROTEIN INCREASED BY ANTHELMINTIC DRUGS FOR DIROFILARIA-IMMITIS IN DOGS

  N TOSA, M MORIMATSU, M NAKAGAWA, F MIYOSHI, E UCHIDA, M NIIYAMA, B SYUTO, M SAITO

  JOURNAL OF VETERINARY MEDICAL SCIENCE 55 (1) 27 - 31 0916-7250 1993/02 [Refereed][Not invited]
   

  Polyacrylamide gel electrophoretic analysis of canine serum protein has revealed that the administration of anthelmintics elicits an increase in a certain serum protein. This protein, named PT60, was partially purified by ammonium sulfate fractionation and preparative electrophoresis. The purified PT60 gave a single band with the molecular size of 53 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions. After reduction with 2-mercaptoethanol, two bands appeared at 35 kDa and 17 kDa, indicating that PT60 consists of two subunits which are linked with each other by disulfide bonds. PT60 had the capacity to bind to hemoglobin. In an immunodiffusion test, an antiserum against PT60 cross-reacted with canine haptoglobin (Hp). N-terminal amino acid sequences of two PT60 subunits were identical to those of alpha and beta subunits of canine Hp, respectively. Thus, PT60 was identified as Hp.
• INDUCTION OF ACUTE PHASE PROTEIN BY RECOMBINANT HUMAN INTERLEUKIN-6 (IL-6) IN CALVES

  Y NAKAJIMA, E MOMOTANI, T MURAKAMI, Y ISHIKAWA, M MORIMATSU, M SAITO, H SUZUKI, K YASUKAWA

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 35 (3-4) 385 - 391 0165-2427 1993/01 [Refereed][Not invited]
   

  Interleukin-6 (IL-6) is a major inducer of acute phase proteins in human and murine species. However, the effects of IL-6 have not yet been investigated in cattle. Following continuous infusion of recombinant human IL,6, serum concentrations of bovine haptoglobin and fibrinogen increased in a manner similar to those in cattle with acute phase reaction. In contrast, C-reactive protein and alpha1-acid glycoprotein, as well as the other hematologic parameters, did not change significantly. Intravenous administration of recombinant human IL-6 resulted in only a mild and transient increase of bovine haptoglobin. These results suggest that the regulation of acute phase protein production in cattle is similar, but not identical, to that observed in human and murine species.
• BOVINE HAPTOGLOBIN - SINGLE RADIAL IMMUNODIFFUSION ASSAY OF ITS POLYMERIC FORMS AND DRAMATIC RISE IN ACUTE-PHASE SERA

  M MORIMATSU, M SARIKAPUTI, B SYUTO, M SAITO, S YAMAMOTO, M NAIKI

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 33 (4) 365 - 372 0165-2427 1992/09 [Refereed][Not invited]
   

  Using purified bovine haptoglobin (Hp) and specific antisera, a single radial immunodiffusion (SRID) assay method has been developed to measure the serum Hp level in cattle. Bovine Hp is a highly polymerized protein showing heterogeneous molecular forms in serum. After treatment with cysteine or glutathione, Hp was partially reduced to a homogeneous form, suitable for SRID assay. This method gives values comparable to those obtained by hemoglobin-binding capacity assay, and has the advantage of being simple and convenient. Although serum Hp was not detectable in healthy cattle, it was found more than 50-fold after invasive surgery, indicating that Hp is a characteristic acute-phase protein in cattle.
• PREPARATION OF LATEX SENSITIZED WITH RABBIT IGG ANTIBODY FOR SLIDE REVERSED PASSIVE AGGLUTINATION

  S YAMAMOTO, K TAGATA, Y ISHIKAWA, H FUJISE, H NAGAHATA, M YAMADA, T SAKANO, M MORIMATSU, M NAIKI

  VETERINARY RESEARCH COMMUNICATIONS 16 (4) 265 - 272 0165-7380 1992/08 [Refereed][Not invited]
   

  A method is described for preparing latex particles sensitized with IgG antibody (IgG-sensitized latex) applicable to the slide reversed passive agglutination (RPLA) test. Soap-free latex (latex) was sensitized with IgG which had been isolated from rabbit anti-bovine lactoferrin serum using protein A Sepharose CL 4B. Unadsorbed protein-binding sites on the surface of latex were blocked with bovine serum albumin (BSA). IgG-sensitized latex that gave better agglutination in RPLA could be selectively obtained by centrifugation at 19 900g for 15 min in 0.01 mol/L glycine buffer (pH 7.3; specific gravity 1.042) containing 3% NaCl, 5% saccharose and 2% choline chloride. By dispersing this IgG-sensitized latex in 0.01 mol/L glycine buffer (pH 7.3) containing 1-2% BSA, a uniformly suspended, highly reactive, readily agglutinable preparation was obtained.
• LATEX AGGLUTINATION-TEST - A SIMPLE, RAPID AND PRACTICAL METHOD FOR BOVINE SERUM CRP DETERMINATION

  M SARIKAPUTI, M MORIMATSU, S YAMAMOTO, B SYUTO, M SAITO, M NAIKI

  JAPANESE JOURNAL OF VETERINARY RESEARCH 40 (1) 1 - 12 0047-1917 1992/05 [Refereed][Not invited]
   

  A semi-quantitative latex agglutination test for bovine serum CRP levels has been established by mixing diluted serum (or diluted standard serum) with a 1% latex suspension containing 0.489-mu-m latex particles coated with affinity-purified antibody at a ratio of 20-mu-g/mg latex. The agglutination was performed on a glass slide in a moist chamber at room temperature with 45 min. incubation. This test is reliable, reproducible and the results correlate with those of the single radial immunodiffusion (SRID) test. The effect of low temperature storage on CRP concentration revealed a 30% degradation of CRP during 2 years storage at 4-degrees-C. The possible role of EDTA addition to prevent a decrease in serum CRP concentration by freezing and thawing is also discussed.
• ISOLATION OF C-REACTIVE PROTEIN FROM CAT SERUM

  A WATANABE, M MORIMATSU, K YOSHIMATSU, S YAMAMOTO, A TERAO, K TSUKAZAKI, M SAITO, M NAIKI

  JOURNAL OF SMALL ANIMAL PRACTICE 33 (2) 71 - 77 0022-4510 1992/02 [Refereed][Not invited]
   

  C-reactive protein (CRP) was isolated from sera from healthy cats by calcium-dependent affinity chromatography on phosphorylcholine derivatives of bovine albumin-coupled Toyopearl, followed by an anion-exchange chromatography using DEAE cellulose. It was identified as CRP by its immunochemical cross reactivity with human CRP. The molecular weight of cat CRP was approximately 100 kilodaltons (kDa) and composed of two glycosylated subunits (23 kDa) and three non-glycosylated subunits (20 kDa) with non-covalent association. Under electron microscopic examination, cat CRP had a pentameric disc-like configuration which is characteristic of CRP. Immunoelectrophoresis and isoelectric focusing showed that cat CRP is an acidic alpha-1-globulin (pI 4.1 to 4.3). Serum concentrations of CRP in cats and kittens were measured by single radial immunodiffusion. In 12 healthy cats from various sources, values ranged from 38 to 168-mu-g/ml. In kittens, serum CRP levels also showed a wide distribution, 81 per cent of them were less than 40-mu-g/ml.
• ISOLATION OF CANINE C-REACTIVE PROTEIN AND CHARACTERIZATION OF ITS PROPERTIES

  S YAMAMOTO, K TAGATA, H NAGAHATA, Y ISHIKAWA, M MORIMATSU, M NAIKI

  VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 30 (4) 329 - 339 0165-2427 1992/01 [Refereed][Not invited]
   

  C-reactive protein (CRP) was isolated from the acute phase serum of dogs subjected to surgical stimulation. Its properties were characterized. Canine CRP was isolated by ion-exchange chromatography using DEAE-Sephaeel and DEAE-Sephadex A-50 and affinity chromatography using protein A-Sepharose CL 4B in combination with agar-block electrophoresis. In immunoelectrophoresis, canine CRP had the same gamma-mobility as human gamma-type CRP. The molecular weight of purifined canine CRP was estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis to be approximately 157 000 and 155 000 respectively. This CRP was a thermolabile protein which completely lost its antigenicity by heating at 70-degrees-C for 15 min. The serum concentration of CRP in normal beagle dogs ranged from 0.198 to 0.826-mu-g ml-1 (0.486 +/- 0.170-mu-g ml-1). The concentration was acutely increased by surgery as it was in man and was rapidly decreased with convalescence. Dogs can be a useful animal model for investigation of the mechanism of CRP production and the function of CRP.
• ELEVATION OF BOVINE SERUM C-REACTIVE PROTEIN AND SERUM AMYLOID-P COMPONENT LEVELS BY LACTATION

  M MORIMATSU, A WATANABE, K YOSHIMATSU, T FUJINAGA, M OKUBO, M NAIKI

  JOURNAL OF DAIRY RESEARCH 58 (3) 257 - 261 0022-0299 1991/08 [Refereed][Not invited]
   

  C-reactive protein (CRP) and serum amyloid P component (SAP), which are known to increase in sera from humans and many other animals with acute inflammation caused by infection, toxic drug administration or injury, were previously purified from bovine serum. These serum levels were determined by enzyme-linked immunosorbent assay (ELISA) using specific antiserum to bovine CRP or SAP which was prepared by immunizing rabbits and goats with each purified protein. Among 68 healthy Holstein cows, 45 non-lactating cows had levels of CRP and SAP of 20.6 +/- 1.4 and 27.6 +/- 1.3-mu-g/ml respectively; 23 lactating cows had higher levels of CRP and SAP (76.0 +/- 13.6 and 38.3 +/- 5.5-mu-g/ml respectively). In the latter group, there was a high correlation between milk yield and serum CRP levels (P < 0.001). From these observations, it was assumed that lactation might stimulate CRP synthesis rather than SAP synthesis in bovine liver as an acute phase reaction, and that CRP might be called a lactation-associated protein.
• ISOLATION AND CHARACTERIZATION OF BOVINE HAPTOGLOBIN FROM ACUTE PHASE SERA

  M MORIMATSU, B SYUTO, N SHIMADA, T FUJINAGA, S YAMAMOTO, M SAITO, M NAIKI

  JOURNAL OF BIOLOGICAL CHEMISTRY 266 (18) 11833 - 11837 0021-9258 1991/06 [Refereed][Not invited]
   

  A macromolecular hemoglobin-binding protein, which was not detectable in normal bovine sera but appeared during acute phase inflammation, was purified, characterized, and designated as bovine haptoglobin (Hp). The purified protein had a molecular mass of 1,000-2,000 kDa, and was composed of two kinds of peptides, a 20-kDa peptide (alpha-chain) and a 35-kDa glycopeptide (beta-chain) linked by disulfide bonds. Amino acid composition and N-terminal sequence analyses revealed that both peptides were homologous to each counterpart of human Hp. Studies using some reducing reagents proved that highly polymerized Hp in serum was composed of 2-20 polymerized forms of alpha-2-beta-2 tetramer. Hp could bind one molecule of hemoglobin/alpha-2-beta-2 unit. Hp with smaller sizes obtained from native Hp by partial reduction with cysteine showed almost the same Hb-binding capacity.
• A NEW PURIFICATION PROCEDURE FOR BOVINE C-REACTIVE PROTEIN AND SERUM AMYLOID-P COMPONENT

  M SARIKAPUTI, M MORIMATSU, B SYUTO, M SAITO, M NAIKI

  INTERNATIONAL JOURNAL OF BIOCHEMISTRY 23 (10) 1137 - 1142 0020-711X 1991 [Refereed][Not invited]
   

  1. A new purification procedure was started with salting-out fractionation of serum proteins at 45-75% saturated ammonium sulfate concentration, followed by HE agarose affinity chromatography by which calcium-dependently bound CRP and SAP were purely eluted with EDTA-containing buffer. 2. Pure CRP and SAP were finally separated by DEAE-5PW HPLC. 3. This procedure gave recovery of 15 and 26%, and fold purification of 2650 and 2400 for CRP and SAP. respectively. 4. Each subunit of CRP and SAP had one intrasubunit disulfide bond, determined by reduction and carboxymethylation.
• ISOLATION AND CHARACTERIZATION OF C-REACTIVE PROTEIN AND SERUM AMYLOID-P COMPONENT FROM BOVINE SERUM

  M MORIMATSU, H SAKAI, K YOSHIMATSU, O MINOWA, S YAMAMOTO, K YATOMI, T FUJINAGA, M NAIKI

  JAPANESE JOURNAL OF VETERINARY SCIENCE 51 (4) 723 - 732 0021-5295 1989/08 [Refereed][Not invited]
  

Conference Activities & Talks

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  上妻 創, 吉川 泰永, 森松 正美, 落合 和彦, 折野 宏一

  第164回日本獣医学会学術集会  2021/09
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  第164回日本獣医学会学術集会  2021/09
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  第163回日本獣医学会学術集会  2020/09
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  第163回日本獣医学会学術集会  2020/09
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  第163回日本獣医学会学術集会  2020/09
• MUC16発現腫瘍に対する可溶型Siglec-9の抗腫瘍効果の検討  [Not invited]

  植田有希, 富岡幸子, 竹内崇師, 八木田晴香, 尾崎絹代, 山本沙代, 森松正美, 笛吹達史, 小野悦郎

  第162回日本獣医学会学術集会  2019/09
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  第162回日本獣医学会学術集会  2019/09
• BRCA2のC末端RAD51結合領域におけるRAD51との結合に影響を与えるアミノ酸残基の同定  [Not invited]

  吉川泰永, 森松正美, 落合和彦, 田中杏里咲, 折野宏一

  第162回日本獣医学会学術集会  2019/09
• イヌRAD51変異が複合体形成におよぼす影響の検討  [Not invited]

  植村光希, 落合和彦, 森松正美, 柏木伸旭, 宇埜友美子, 道下正貴, 呰上大吾, 小野沢栄里, 近江俊徳

  第162回日本獣医学会学術集会  2019/09
• がん抑制遺伝子p53に変異を有するイヌ乳腺腫瘍由来細胞の性状解析  [Not invited]

  中沢綾香, 石黒(大沼)俊名, 落合和彦, 森松正美, 木崎景一郎

  第161回日本獣医学会学術集会  2018/09
• 腫瘍抑制遺伝子BRCA2のエキソン11 における新規腫瘍関連変異の検索  [Not invited]

  吉川泰永, 大隅恒佑, 森松正美, 折野宏一

  第161回日本獣医学会学術集会  2018/09
• イヌIDH1の新規変異が機能に及ぼす影響の検討  [Not invited]

  川上翔太, 落合和彦, 道下正貴, 森松正美, 近江俊徳

  第161回日本獣医学会学術集会  2018/09
• イヌ乳腺腫瘍細胞株で生じたp53遺伝子変異の検出と機能解析  [Not invited]

  落合和彦, 平間日奈子, 森松正美, 川上翔太, 道下正貴, 中川貴之, 呰上大吾, 稲垣健志, 坂本敦司, 近江俊徳

  第161回日本獣医学会学術集会  2018/09
• C57BL/ 6 マウスよりセンダイウイルス感染抵抗性遺伝子座を導入されたDBA/ 2 コンジェ ニックマウスのセンダイウイルス感染に対する免疫応答  [Not invited]

  Raghda Abbas, Hassan Tag-EL-Din-Hassan, Tussapon Boonyarattanasoonthorn, 青島 圭佑, 森松 正美, 安居院 高志

  第161回日本獣医学会学術集会  2018/09
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  The 20th Seoul National University-Hokkaido University Joint Symposium  2017/11
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  6th World Waterfowl Conference  2017/10
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  第160回日本獣医学会学術集会  2017/09
• コンジェニックマウスを用いたセンダイウイルス感染感受性/抵抗性遺伝子座の証明  [Not invited]

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  第160回日本獣医学会学術集会  2017/09
• 多包条虫（エキノコックス）中間宿主の感受性および抵抗性遺伝子座を導入したコンジェニックマウス系統の作製とその解析  [Not invited]

  Md Islam, 孝口裕一, 入江隆夫, 亀田弥生, 鳥越大輔, 中尾 亮, Tag-El-Din-Hassan HT, 森松正美, 八木欣平, 安居院高志

  第160回日本獣医学会学術集会  2017/09
• RNA-Seq解析による多包条虫の原頭節発育に関与する宿主側遺伝子の検索  [Not invited]

  伊達 衆, 中尾 亮, Md Islam, 孝口 裕一, 入江 隆夫, 森松 正美, 安居院 高志, 八木 欣平, 片倉 賢

  第160回日本獣医学会学術集会  2017/09
• Analysis of The Molecular Mechanism for The Enzymatic and Antiviral Activities of The Chicken Oligoadenylate Synthetase L (ChOAS-L)  [Not invited]

  Hassan T. Tag-El-Din-Hassan, Nobuya Sasaki, Daisuke Torigoe, Masami Morimatsu, Takashi Agui

  Avian Genetics and Immunity  2017/06
• 可溶型ヒトネクチン-2発現トランスジェニックマウスに見られた膵外分泌異常  [Not invited]

  富岡幸子, 藤本佳万, 中井寛治, 尾崎絹代, 山本沙代, 八木田晴香, 森松正美, 竹内崇師, 伊藤壽啓, 小野悦郎

  第159回日本獣医学会学術集会  2016/09
• Antiviral activity analysis of the Ostrich 2’-5’ Oligoadenylate Synthetase-Like (OsOAS-L)  [Not invited]

  Hassan T. Tag-El-Din-Hassan, Nobuya Sasaki, Daisuke Torigoe, Masami Morimatsu, Takashi Agui

  World Poultry Congress 2016  2016/09
• 癌抑制遺伝子BRCA2のイントロンにおける新規サイレンサー配列の同定と転写因子の特定  [Not invited]

  角本俊介, 吉川泰永, 森松正美, 落合和彦, 石黒-大沼俊名, 折野宏一

  第158回日本獣医学会学術集  2015/09
• イヌ乳腺腫瘍サンプルにおける癌抑制遺伝子 BRCA2 発現量の減少  [Not invited]

  吉川泰永, 森松正美, 落合和彦, 石黒-大沼俊名, 和田成一, 折野宏一

  第158回日本獣医学会学術集会  2015/09
• マウスのアセチルコリンに対する感受性を決定する遺伝因子の解析  [Not invited]

  田中聖一, 一之瀬岳夫, 松山 充, 高屋敷優子, 永島 博, 鳥越大輔, 森松正美, 安居院高志

  日本実験動物学会総会  2015/05
• DNA相同組換えタンパク質RAD51の動物種特異的アミノ酸配列が機能に及ぼす影響の解析  [Not invited]

  落合和彦, 閑野大輝, 宇田川智野, 呰上大吾, 大沼俊名, 吉川泰永, 森松正美, 近江俊徳

  第157回日本獣医学会学術集会  2014/09
• シアル酸結合レクチンSiglec-9可溶型分子はMUC1発現腫瘍の増殖を抑制する  [Not invited]

  富岡幸子, 森松正美, 西島謙一, 笛吹達史, 山本沙代, 八木田晴香, 尾崎絹代, 伊藤壽啓, 小野悦郎

  第157回日本獣医学会学術集会  2014/09
• がん抑制遺伝子REIC／Dkk‐３イヌホモログの構造と乳腺腫瘍に対するアポトーシス誘導能の解析  [Not invited]

  落合和彦, 渡部昌実, 宇田川智野, 呰上大吾, 森松正美, 大沼俊名, 近江俊徳

  第156回日本獣医学会学術集  2013/09
• オーエスキー病ウイルス前初期蛋白質IE180発現トランスジェニックマウスにおける精子発生異常の解析  [Not invited]

  富岡幸子, 森松正美, 田原口智士, 小野悦郎

  日本獣医学会学術集会講演要旨集  2013/03
• イヌ乳腺腫瘍関連遺伝子BRCA2のBRC repeat 3に存在するSNPsの頻度および機能に及ぼす影響の検討  [Not invited]

  落合和彦, 塩入弓絵, 染田沙織, 大澤有加, 宇田川智野, 森松正美, 吉川泰永, 盆子原誠, 土田修一, 近江俊徳

  日本獣医学会学術集会講演要旨集  2013/03
• 動物実験従事者の動物アレルギー  [Not invited]

  吉村彩, 武藏学, 金子壮朗, 大西俊介, 折戸智恵子, 川原由佳子, 森松正美, 今野哲, 有川二郎

  Campus Health  2013/03
• イヌ乳腺腫瘍で発見した癌関連遺伝子産物BRCA2における変異BRC repeat 3の機能解析  [Not invited]

  吉川泰永, 森松正美, 落合和彦, 和田成一, 垰田高広, 近澤征史朗, 岩井聡美, 折野宏一, 渡辺清隆

  日本獣医学会学術集会講演要旨集  2012/08
• 放射線治療抵抗性がんに対するRAD51の制御を基軸とした改変型BRCによる新規治療戦略の開発に向けた基礎研究  [Not invited]

  落合和彦, 森松正美, 吉川泰永, 宇田川智野, 多田尚美, 近江俊徳

  日本獣医学会学術集会講演要旨集  2012/08
• 癌抑制遺伝子BRCA2のPCR法による変異解析法の確立  [Not invited]

  吉川泰永, 森松正美, 鈴木優, 西村百合香, 落合和彦, 折野宏一, 垰田高広, 近澤征史郎, 岩井聡美, 島村麻子, 新井敏郎, 渡辺清隆

  日本獣医学会学術集会講演要旨集  2011/08
• イヌ乳腺腫瘍関連遺伝子産物BRCA2のBRC repeat4のアミノ酸がDNA相同組換え修復に必須なRAD51ホモ会合体形成に与える影響  [Not invited]

  落合和彦, 吉川泰永, 近江俊徳, 森松正美

  日本獣医学会学術集会講演要旨集  2011/08
• 核酸代謝拮抗剤TAS106による相同組換え修復阻害作用を介した放射線増感効果  [Not invited]

  女池俊介, 山盛徹, 安井博宣, 松田彰, 森松正美, 福島正和, 山崎靖人, 稲波修

  日本獣医学会学術集会講演要旨集  2011/08
• イヌおよびヒトBRCA2のBRC repeat3領域におけるミスセンス変異の比較  [Not invited]

  吉川泰永, 森松正美, 落合和彦, 折野宏一, 和田成一, 垰田高広, 近澤征史郎, 岩井聡美, 渡辺清隆

  日本分子生物学会年会プログラム・要旨集(Web)  2011
• 核酸代謝拮抗剤TAS106による相同組換え修復阻害作用を介した放射線増感効果  [Not invited]

  女池俊介, 山盛徹, 安井博宣, 松田彰, 森松正美, 福島正和, 山崎靖人, 稲波修

  日本放射線影響学会大会講演要旨集  2010/10
• イヌ乳腺腫瘍における癌抑制遺伝子BRCA2の新規変異の発見と機能解析  [Not invited]

  鈴木優, 吉川泰永, 森松正美, 落合和彦, 折野宏一, 峠田高広, 近澤征史朗, 渡辺清隆

  日本獣医学会学術集会講演要旨集  2010/09
• イヌ乳腺腫瘍における癌抑制遺伝子BRCA2におけるスプライシングバリアントの発見  [Not invited]

  吉川泰永, 森松正美, 落合和彦, 折野宏一, 峠田高広, 近澤征史朗, 渡辺清隆

  日本獣医学会学術集会講演要旨集  2010/09
• イヌ乳腺腫瘍関連遺伝子Brca2のBRC repeat4のアミノ酸配列がRad51との相互作用に及ぼす影響  [Not invited]

  落合和彦, 吉川泰永, 大沼俊名, 橋爪一善, 森松正美

  日本獣医学会学術集会講演要旨集  2010/09
• イヌ乳腺腫瘍で発見したBRCA2のBRC repeat3におけるミスセンス変異の解析  [Not invited]

  吉川泰永, 鈴木優, 森松正美, 落合和彦, 折野宏一, 垰田高広, 近澤征史朗, 渡辺清隆

  生化学  2010
• 可溶型ネクチン‐1発現トランスジェニックマウスに見られた行動学的・神経生理学的変化  [Not invited]

  小野悦郎, HORVATH Szatmar, FARKAS Tamas, JANKA Zoltan, 富岡幸子, 森松正美, 尾崎絹代, 山本沙代, TOLDI Jozsef

  日本実験動物学会総会講演要旨集  2009/04
• Behavioral and Neurophysiological Changes in Transgenic Mice Expressing Different Levels of Soluble Nectin-1 Protein  [Not invited]

  ONO ETSURO, HORVATH SZATMAR, FARKAS TAMAS, JANKA ZOLTAN, TOMIOKA YUKIKO, MORIMATSU MASAMI, OZAKI KINUYO, YAMAMOTO SAYO, TOLDI JOZSEF

  Exp Anim  2009/04
• イヌ乳腺腫瘍における癌抑制遺伝子BRCA2の新規変異の検索  [Not invited]

  吉川泰永, 鈴木優, 森松正美, 落合和彦, 折野宏一, 垰田高広, 渡辺清隆

  日本獣医学会学術集会講演要旨集  2009
• 仮性狂犬病ウイルス前初期蛋白IE180発現マウスにおける小脳形成不全の病理組織学的解析  [Not invited]

  富岡幸子, 宮崎太輔, 田原口智士, 森松正美, 渡辺雅彦, 小野悦郎

  日本獣医学会学術集会講演要旨集  2008/03
• 家族性乳癌原因遺伝子産物BRCA2のC末端RAD51結合領域におけるunclassified variantsの解析  [Not invited]

  吉川泰永, 森松正美, 岩永敏彦

  生化学  2008
• 家族性乳癌原因遺伝子BRCA2のC末端RAD51結合領域におけるunclassified variantの解析  [Not invited]

  吉川泰永, 森松正美, 岩永敏彦

  生化学  2007
• 仮性狂犬病ウイルスの転写調節因子IE180の発現による神経病理発生メカニズムの解析  [Not invited]

  富岡幸子, 宮崎太輔, 田原口智士, 森松正美, 渡辺雅彦, 小野悦郎

  生化学  2007
• オーエスキー病ウイルスC末端欠失変異型前初期蛋白IE180発現マウスの解析  [Not invited]

  富岡幸子, 宮崎太輔, 田原口智士, 吉野さおり, 森松正美, 渡辺雅彦, 上出利光, 小野悦郎

  日本獣医学会学術集会講演要旨集  2006/08
• 表皮ケラチノサイトにおけるMAIL(INAP/IκBζ)の発現調節機構の解析  [Not invited]

  大沼俊名, 森松正美, 岩永敏彦, 橋爪一善

  日本獣医学会学術集会講演要旨集  2006/08
• イヌ乳腺腫瘍関連遺伝子BRCA2の多型マーカーの確立  [Not invited]

  吉川泰永, 森松正美, 落合和彦, 富岡幸子, 永野昌志, 冨沢伸行, 山根義久, 佐々木伸雄, 橋爪一善, 岩永敏彦

  日本獣医学会学術集会講演要旨集  2006/08
• 乳腺腫瘍発症機構の基礎研究~遺伝性乳癌原因遺伝子とケモカイン受容体を標的として  [Not invited]

  森松正美

  日本獣医学会学術集会講演要旨集  2006/03
• A spermatogonial stem cell-derived factor induces the transcription of lipocalin-2/24p3 gene in Sertoli cells by an I.KAPPA.B.ZETA.-independent NF-.KAPPA.B pathway  [Not invited]

  HARA TAKAHIKO, FUJINO RYU-SUKE, TANAKA KIYOKO, MORIMATSU MASAMI, TAMURA KAZUHIRO, KOGO HIROSHI

  生化学  2006
• Expression and function of MAIL in the skin  [Not invited]

  OONUMA TOSHINA, MORIMATSU MASAMI, SHIINA TAKAHIKO, KONNO AKIHIRO, KITAMURA HIROSHI, TIWANAGA OSHIHIKO, HASHIZUME KAZUYOSHI

  生化学  2006
• Identification and characterization of polymorphic markers within canine BRCA2 gene  [Not invited]

  YASUNAGA YOSHIKAWA, MORIMATSU MASAMI, OCHIAI KAZUHIKO, NAGANO MASASHI, TOMIOKA YUKIKO, SASAKI NOBUO, HASHIZUME KAZUYOSHI, IWANAGA TOSHIHIKO

  生化学  2006
• 家族性乳がん原因遺伝子産物BRCA2の核移行シグナルに認められた腫よう発症頻度を高める多型の機能解析  [Not invited]

  吉川泰永, 森松正美, 落合和彦, 永野昌志, 冨沢伸行, 山根義久, 佐々木伸雄, 橋爪一善, 岩永敏彦

  日本分子生物学会年会講演要旨集  2005/11
• Targeted Disruption of MAIL, a Nuclear IkB Protein, Leads to Severe Atopic Dermatitis-like Disease  [Not invited]

  SHIINA TAKAHIKO, KONNO AKIHIRO, OONUMA TOSHINA, KITAMURA HIROSHI, IMAOKA KOICHI, TAKEDA NAOKI, TODOKORO KAZUO, MORIMATSU MASAMI

  Exp Anim  2005/04
• がん抑制遺伝子産物Brca2のイヌ型BRC repeat4の構造と機能  [Not invited]

  落合和彦, 森松正美, 橋爪一善

  日本獣医学会学術集会講演要旨集  2004/08
• イヌ乳腺腫よう関連遺伝子産物イヌBrca2の核移行シグナルに存在する多型配列  [Not invited]

  吉川泰永, 森松正美, 落合和彦, 永野昌志, 冨沢伸行, 山根義久, 橋爪一善

  日本獣医学会学術集会講演要旨集  2004/08
• ウシPlacental Lactogen(PL)遺伝子上流領域のクローニングおよびヒツジPL遺伝子のシス因子との比較  [Not invited]

  門野祐紀, 森松正美, 橋爪一善

  日本獣医学会学術集会講演要旨集  2004/08
• ネコ乳腺腫よう細胞株における腫ようマーカーの測定とGFP発現細胞株の作成  [Not invited]

  横井佐知, 森松正美, 大沼俊名, 中川貴之, 佐々木伸雄, 中市統三, 橋爪一善

  日本獣医学会学術集会講演要旨集  2004/08
• ウマけん組織に発現する遺伝子scleraxisの検索  [Not invited]

  根本学, 森松正美, 山本欣郎, 橋爪一善

  日本獣医学会学術集会講演要旨集  2004/08
• 赤外線サーモグラフィによる自然発情牛および鈍性発情牛の観察  [Not invited]

  田中成佳, 大沢健司, 平田統一, 高橋正弘, 首藤文栄, 森松正美, 橋爪一善

  日本獣医学会学術集会講演要旨集  2004/03
• ウシ乳房炎でのサイトカインならびにToll‐like receptor 4の遺伝子発現  [Not invited]

  河木英愛, 福田茂夫, 菊佳男, 伊藤寿浩, 森松正美, 吉野知男, 岡田洋之

  日本獣医学会学術集会講演要旨集  2002/08
• ニワトリおよびカエルの消化器におけるキチン分解酵素の組織発現  [Not invited]

  藤本和歌子, 鈴木雅子, 森松正美, 岩永敏彦

  日本獣医学会学術集会講演要旨集  2002/08
• ネコ乳腺腫ようの転移におけるケモカインレセプターの役割について ネコ乳腺腫よう細胞株におけるfCxcr4の発現  [Not invited]

  大沼俊名, 森松正美, 中川貴之, 宇山理奈, 佐々木伸雄, 首藤文栄

  日本獣医学会学術集会講演要旨集  2002/08
• 医用材料に結合する血清タンパク質の好中球に対する作用  [Not invited]

  佐々木秀樹, 森松正美, 首藤文栄

  日本獣医学会学術集会講演要旨集  2002/08
• イヌ乳腺腫よう関連遺伝子Brca2とRad51のほ乳動物細胞内での相互作用の解析  [Not invited]

  落合和彦, 森松正美, 冨沢伸行, 首藤文栄

  日本獣医学会学術集会講演要旨集  2002/08
• 子牛の実験的エンドトキシン血症における標的細胞でのTLR4と炎症性サイトカイン遺伝子発現の動態  [Not invited]

  三井隆行, 菊佳男, 福田茂夫, 田中隆志, 伊藤寿浩, 森松正美, 首藤文栄, 高橋淳吉, 吉野知男

  日本獣医学会学術集会講演要旨集  2002/08
• バーク抽出物のマウスに対する効果  [Not invited]

  富山美奈子, 小藤田久義, 森松正美, 首藤文栄

  日本獣医学会学術集会講演要旨集  2002/08
• ウシのToll‐like receptor4(TLR4)のクローニングと機能解析  [Not invited]

  伊藤寿浩, 森松正美, 首藤文栄

  日本獣医学会学術集会講演要旨集  2001/09
• ネコ乳腺腫よう関連遺伝子Brca2のクローニング  [Not invited]

  大沼俊名, 森松正美, 落合和彦, 橋本志津, 山村穂積, 首藤文栄

  日本獣医学会学術集会講演要旨集  2001/09
• 新規核内IkBタンパク質MAILの同定および発現・機能解析  [Not invited]

  北村浩, 金平克史, 岩永敏彦, 藤倉大輔, 山地大介, 松下由紀子, 森松正美, 斉藤昌之

  日本獣医学会学術集会講演要旨集  2001/09
• ウシ肝臓に発現する新規血清キチナーゼの同定とクローニング  [Not invited]

  鈴木雅子, 森松正美, 山下哲郎, 岩永敏彦, 首藤文栄

  日本獣医学会学術集会講演要旨集  2001/09
• イヌにおける乳腺腫よう関連遺伝子Brca2の変異検出法  [Not invited]

  宇賀聡, 落合和彦, 橋本志津, 冨沢伸行, 森松正美, 首藤文栄

  日本獣医学会学術集会講演要旨集  2001/09
• 新規核内IκBタンパク質MAILのゲノム構造,染色体マッピングおよびプロモーター解析  [Not invited]

  椎名貴彦, 森松正美, 伊藤寿浩, 北村浩, 斉藤昌之, 松原和純, 松田洋一, 首藤文栄

  日本獣医学会学術集会講演要旨集  2001/09
• イヌの乳腺腫よう関連遺伝子Brca2とRad51の相互作用の解析  [Not invited]

  落合和彦, 森松正美, 冨沢伸行, 首藤文栄

  日本獣医学会学術集会講演要旨集  2001/09
• 医用材料に結合する血清タンパク質の分析  [Not invited]

  佐々木秀樹, 鈴木雅子, 森松正美, 首藤文栄

  日本獣医学会学術集会講演要旨集  2001/09
• ウシMAILのクローニングと発現解析  [Not invited]

  山地大介, 北村浩, 松下由紀子, 椎名貴彦, 森松正美, 斉藤昌之

  日本獣医学会学術集会講演要旨集  2001/09
• LPSによるB細胞およびマクロファージでの新規IkBタンパク質MAILの誘導  [Not invited]

  北村浩, 金平克史, 岩永敏彦, 椎名貴彦, 松下由紀子, 藤倉大輔, 赤司祥子, 森松正美, 斉藤昌之

  生化学  2001/08
• ウシ血清中のキチン結合性タンパク質 CBPb04(ウシ血清キチナーゼ)の分離・精製と固定  [Not invited]

  鈴木雅子, 森松正美, 山下哲郎, 首藤文栄

  キチン・キトサン研究  2001/07
• ニワトリのテレピン油誘導タンパク質がヘテロフィル細胞の活性酸素産生能に及ぼす影響  [Not invited]

  岩崎健, 稲波修, 内田英二, 三好健二郎, 森松正美, 首藤文栄, 桑原幹典, 新山雅美

  日本獣医学会学術集会講演要旨集  2000/09
• 新規キチン結合性タンパク質の分離,精製と同定  [Not invited]

  鈴木雅子, 山下哲郎, 落合和彦, 森松正美, 首藤文栄

  日本獣医学会学術集会講演要旨集  2000/09
• 炎症により誘導されるマウス新規遺伝子142.5のゲノム解析  [Not invited]

  椎名貴彦, 伊藤寿浩, 北村浩, 金平克史, 斉藤昌之, 森松正美, 首藤文栄

  日本獣医学会学術集会講演要旨集  2000/09
• イヌ乳腺腫よう関連遺伝子の機能解析Brca2およびRad51のクローニング  [Not invited]

  落合和彦, 森松正美, 冨沢伸行, 首藤文栄

  日本獣医学会学術集会講演要旨集  2000/09
• 低濃度C3の初代培養神経細胞に対する効果  [Not invited]

  山本祐紀子, 渡辺康子, 田端義巌, 森松正美, 首藤文栄

  日本獣医学会学術集会講演要旨集  2000/09
• ストレスメディエーターとしてのサイトカイン 末梢および脳内インターロイキン1と交感神経系の役割  [Not invited]

  北村浩, 鄭培東, 木村和弘, 寺尾晶, 森松正美, 斉藤昌之

  日本獣医学会学術集会講演要旨集  2000/09
• ストレス応答するマウス脳内遺伝子のクローニングと発現解析  [Not invited]

  北村浩, 金平克史, 沖田圭介, 岩永敏彦, 椎名貴彦, 藤倉大輔, 森松正美, 斉藤昌之

  生化学  2000/08
• 炎症刺激に応答するマウス脳内遺伝子のクローニングと発現解析  [Not invited]

  金平克史, 北村浩, 沖田圭介, 岩永敏彦, 藤倉大輔, 森松正美, 斉藤昌之

  日本獣医学会学術集会講演要旨集  2000/03
• ストレス刺激に応答するマウス脳内遺伝子の検索と解析  [Not invited]

  北村浩, 沖田圭介, 金平克史, 藤倉大輔, 岩永敏彦, 森松正美, 斉藤昌之

  日本分子生物学会年会プログラム・講演要旨集  1999/11
• ウシ血清中のキチン結合性巨大分子タンパク質の同定  [Not invited]

  鈴木雅子, 山下哲郎, 夏目彩子, 森松正美, 首藤文栄

  日本獣医学会学術集会講演要旨集  1999/09
• C.botulinum菌体外酵素C3による初代培養神経細胞の機能分化  [Not invited]

  渡辺康子, 坂田和実, 森松正美, 新貝りゅう蔵, 首藤文栄

  日本獣医学会学術集会講演要旨集  1999/09
• ウシ血清中のセルロース結合性IgM(CebIgM)の糖およびLPSに対する親和性  [Not invited]

  夏目彩子, 鈴木雅子, 森松正美, 首藤文栄

  日本獣医学会学術集会講演要旨集  1999/09
• 炎症刺激に応答するマウス新規遺伝子142.5のゲノム構造解析  [Not invited]

  椎名貴彦, 北村浩, 森松正美, 落合和彦, 金平克史, 斉藤昌之, 首藤文栄

  日本獣医学会学術集会講演要旨集  1999/09
• テレピン油投与により誘導されるニワトリの血中タンパク質の比較  [Not invited]

  岩崎健, 内田英二, 三好健二郎, 新山雅美, 市川舜, 森松正美, 首藤文栄

  日本獣医学会学術集会講演要旨集  1999/09
• ウシ血清中のC反応性タンパク質と血清アミロイドP成分の分子特性  [Not invited]

  沢向共子, 森松正美, 首藤文栄

  日本獣医学会学術集会講演要旨集  1999/09
• マウスにおいてLPS刺激によって誘導される新規遺伝子142.5のヒトホモログのクローニング  [Not invited]

  落合和彦, 北村浩, 森松正美, 椎名貴彦, 沖田圭介, 金平克史, 斉藤昌之, 首藤文栄

  日本獣医学会学術集会講演要旨集  1999/09
• アフリカツメガエルの空気中匂い分子受容器特異的蛋白質の分子生物学的解析  [Not invited]

  鈴木恵子, 山下哲郎, 森松正美, 重茂克彦, 岡達三, 首藤文栄

  日本獣医学会学術集会講演要旨集  1999/09
• Effect of Bovine Serum Chitin Binding Proteins on Bovine PMN.  [Not invited]

  SUZUKI MASAKO, NATSUME AYAKO, MORIMATSU MASAMI, SHUTO BUN'EI

  キチン・キトサン研究  1999/07
• 子ウシの成長にともなう骨格筋・脂肪組織の糖輸送タンパク質の消長について  [Not invited]

  阿部啓之, 河北由美, 甫立京子, 森松正美, 斉藤昌之

  日本畜産学会大会講演要旨  1999/03
• Acute phase proteins and hibernation of brown bears Comparison of haptoglobin and other acute phase proteins.  [Not invited]

  MOMINOKI KATSUMI, MORIMATSU MASAMI, SAITO MASAYUKI

  日本獣医学会学術集会講演要旨集  1998/08
• A new acute phase protein of chickens induced by administration of turpentine oil.  [Not invited]

  IWASAKI TAKESHI, UCHIDA EIJI, NIIYAMA MASAMI, ICHIKAWA SHUN, MORIMATSU MASAMI, SHUTO FUMIE

  日本獣医学会学術集会講演要旨集  1998/08
• Combination of nerve cell directional antibodies to synaptosome.  [Not invited]

  AOYAGI KITAKO, TABATA YOSHITAKA, WATANABE YASUKO, MORIMATSU MASAMI, SHUTO FUMIE

  日本獣医学会学術集会講演要旨集  1998/08
• Bovine serum CRP concentration increase by PG administration.  [Not invited]

  SAWAMUKAI KYOKO, EBIHARA MEGUMI, MORIMATSU MASAMI, MIYAKE YOICHI, SHUTO FUMIE

  日本獣医学会学術集会講演要旨集  1998/08
• Relationship between morphological changes and metabolic system changes in the primary culture nervous system cells.  [Not invited]

  WATANABE YASUKO, MORIMATSU MASAMI, SHUTO FUMIE

  日本獣医学会学術集会講演要旨集  1998/08
• Zymosan opsonization control by CBP01.  [Not invited]

  SUZUKI MASAKO, SUZUKI KEIKO, EBIHARA MEGUMI, MORIMATSU MASAMI, SHUTO FUMIE

  日本獣医学会学術集会講演要旨集  1998/08
• Noninvasive measurement of brain activity of dogs using near infrared rays.  [Not invited]

  MORIMATSU MASAMI, INOUE HIROKI, TOMISAWA NOBUYUKI, MOMINOKI KATSUMI, TAMURA MAMORU, SHUTO FUMIE

  日本獣医学会学術集会講演要旨集  1998/08
• Analysis of specific protein molecules in the aerial smell molecule receptor of Xenopus.  [Not invited]

  SUZUKI KEIKO, MORIMATSU MASAMI, SHUTO FUMIE

  日本獣医学会学術集会講演要旨集  1998/08
• Retrieval and identification of gene in brain which responds to inflammation stimulus.  [Not invited]

  KITAMURA HIROSHI, OKITA KEISUKE, IWANAGA TOSHIHIKO, MORIMATSU MASAMI, SAITO MASAYUKI

  日本獣医学会学術集会講演要旨集  1998/08
• Mechanism of opsonization of zymosan by serum protein.  [Not invited]

  EBIHARA MEGUMI, SUZUKI MASAKO, SAWAMUKAI KYOKO, SUZUKI KEIKO, MORIMATSU MASAMI, SHUTO FUMIE

  日本獣医学会学術集会講演要旨集  1998/08
• Analysis of separation and refinement and function of IgG combining with chitin in cattle serum.  [Not invited]

  ONIYANAGI REIKO, MUGISHIMA AKIKA, SUZUKI KEIKO, MORIMATSU MASAMI, SHUTO FUMIHIDE

  日本獣医学会学術集会講演要旨集  1997/09
• Molecular properties and opsonized mechanism of zymosan binding protein.  [Not invited]

  EBIHARA MEGUMI, TAKEUCHI YUKA, ONIYANAGI REIKO, SUZUKI KEIKO, MORIMATSU MASAMI, SHUTO FUMIHIDE

  日本獣医学会学術集会講演要旨集  1997/09
• Connection between gene recombined molecule Rad51 and mammary cancer gene product Brca2.  [Not invited]

  MORIMATSU MASAMI, SHARAN S K, LIM D S, SANDS A, BRADLEY A, HASTY P, SHUTO FUMIHARU

  日本獣医学会学術集会講演要旨集  1997/09
• Participation of Rho in the metabolic system in primary cultured nervous system cells.  [Not invited]

  WATANABE YASUKO, SUZUKI KEIKO, TABATA YOSHIKANE, AOYAGI KITAKO, MORIMATSU MASAMI, SHUTO FUMIHARU

  日本獣医学会学術集会講演要旨集  1997/09
• Comparison of the serum chitin connective macromolecule protein.  [Not invited]

  SUZUKI MASAKO, MUGISHIMA AKIKA, MORIMATSU MASAMI, SHUTO FUMIHIDE

  日本獣医学会学術集会講演要旨集  1997/09
• Separation and pharmacokinetics of combination fragment of the Clostridium botulinum C1 toxin.  [Not invited]

  TABATA YOSHIIWA, AOYAGI KITAKO, MORIMATSU MASAMI, SHUTO FUMIHIDE

  日本獣医学会学術集会講演要旨集  1997/09
• Comparison of the C-terminal region of sugar transport protein type 4 (GLUT4) of domestic animals.  [Not invited]

  ABE HIROYUKI, KAWAKITA YUMI, MIYASHIGE TOSHIKAZU, MORIMATSU MASAMI, SAITO MASAYUKI

  日本畜産学会大会講演要旨  1997/08
• Identification of adenylate cyclase combined with D1 dopamine receptor.  [Not invited]

  KIMURA KAZUHIRO, OKADA NAOKO, MORIMATSU MASAMI, SAITO MASAYUKI

  神経化学  1996/09
• cDNA cloning of dog .BETA.3 adrenergic receptor.  [Not invited]

  SASAKI NORIYASU, MORIMATSU MASAMI, SAITO MASAYUKI

  日本獣医学会学術集会講演要旨集  1996/08
• Expression regulation of vascular endothelial cell growth factor in rat brown fat tissue.  [Not invited]

  ASANO JUN, MORIMATSU MASAMI, KIMURA KAZUHIRO, SAITO MASAYUKI

  日本獣医学会学術集会講演要旨集  1996/08
• Relationship between seasonal variation of haptoglobin concentration in brown bear blood and hibernation.  [Not invited]

  MOMIKI KATSUMI, MORIMATSU MASAMI, TSURIGA HIFUMI, SAITO MASAYUKI

  日本獣医学会学術集会講演要旨集  1996/08
• Identification of adenylate cyclase which combines with D1 dopamine receptor.  [Not invited]

  OKADA NAOKO, MORIMATSU MASAMI, KIMURA KAZUHIRO, SAITO MASAYUKI

  日本獣医学会学術集会講演要旨集  1996/08
• Action of immunity cytokine on hypothalamus - sympathetic nervous system.  [Not invited]

  TERAO AKIRA, KITAMURA HIROSHI, MORIMATSU MASAMI, SAITO MASAYUKI

  日本獣医学会学術集会講演要旨集  1996/08
• Identification of adenylate cyclase conjugating with D1 dopamine receptor.  [Not invited]

  OKADA HISAKO, MORIMATSU MASAMI, KIMURA KAZUHIRO, SAITO MASAYUKI

  日本分子生物学会年会プログラム・講演要旨集  1996/07

MISC

• がん抑制遺伝子BRCA2の新規転写調節機構の解明

  望月千尋, 上妻創, 朱子達, 北野泰佑, 折野宏一, 森松正美, 吉川泰永  日本獣医学会学術集会講演要旨集  166th-  2023
• 癌抑制遺伝子BRCA2の翻訳効率に影響を与えるスプライシングバリアント

  吉川泰永, 森松正美, 上妻創, 折野宏一  日本獣医学会学術集会講演要旨集  163rd-  2020
• イヌRAD51変異が複合体形成におよぼす影響の検討

  植村 光希, 落合 和彦, 森松 正美, 柏木 伸旭, 宇埜 友美子, 道下 正貴, 呰上 大吾, 小野沢 栄里, 近江 俊徳  日本獣医学会学術集会講演要旨集  162回-  479  -479  2019/08  [Not refereed][Not invited]
  
• がん抑制遺伝子p53に変異を有するイヌ乳腺腫瘍細胞に対する血管新生阻害薬の抗腫瘍効果の解析

  中沢綾香, 石黒(大沼)俊名, 落合和彦, 森松正美, 木崎景一郎  日本獣医学会学術集会講演要旨集  162nd-  2019
• BRCA2のC末端RAD51結合領域におけるRAD51との結合に影響を与えるアミノ酸残基の同定

  吉川泰永, 森松正美, 落合和彦, 田中杏里咲, 折野宏一  日本獣医学会学術集会講演要旨集  162nd-  2019
• イヌ乳腺腫瘍細胞株で生じたp53遺伝子変異の検出と機能解析

  落合 和彦, 平間 日奈子, 森松 正美, 川上 翔太, 道下 正貴, 中川 貴之, 呰上 大吾, 稲垣 健志, 坂本 敦司, 近江 俊徳  日本獣医学会学術集会講演要旨集  161回-  431  -431  2018/08  [Not refereed][Not invited]
  
• C57BL/6マウスよりセンダイウイルス感染抵抗性遺伝子座を導入されたDBA/2コンジェニックマウスのセンダイウイルス感染に対する免疫応答

  Abbas Raghda, Tag-EL-Din-Hassan Hassan, Boonyarattanasoonthorn Tussapon, 青島 圭佑, 森松 正美, 安居院 高志  日本獣医学会学術集会講演要旨集  161回-  476  -476  2018/08  [Not refereed][Not invited]
  
• AAALACによる認証 (日本実験動物環境研究会平成29年度総会 第61回研究会 シンポジウム)

  森松 正美  実験動物と環境  26-  (1)  47  -51  2018/04  [Not refereed][Not invited]
  
• がん抑制遺伝子p53に変異を有するイヌ乳腺腫瘍由来細胞の性状解析

  中沢綾香, 石黒(大沼)俊名, 落合和彦, 森松正美, 木崎景一郎  日本獣医学会学術集会講演要旨集  161st-  2018
• 腫瘍抑制遺伝子BRCA2のエキソン11における新規腫瘍関連変異の検索

  吉川泰永, 大隅恒佑, 森松正美, 折野宏一  日本獣医学会学術集会講演要旨集  161st-  2018
• RNA‐Seq解析による多包条虫の原頭節発育に関与する宿主側遺伝子の検索

  伊達衆, 中尾亮, ISLAM Md, 孝口裕一, 入江隆夫, 森松正美, 安居院高志, 八木欣平, 片倉賢  日本獣医学会学術集会講演要旨集  160th-  344  2017/08/30  [Not refereed][Not invited]
  
• 多包条虫(エキノコックス)中間宿主の感受性および低抗性遺伝子座を導入したコンジェニックマウス系統の作製とその解析

  ISLAM Md, 孝口裕一, 入江隆夫, 亀田弥生, 鳥越大輔, 中尾亮, TAG‐EL‐DIN‐HASSAN Hassan, 森松正美, 八木欣平, 安居院高志  日本獣医学会学術集会講演要旨集  160th-  517  2017/08/30  [Not refereed][Not invited]
  
• イヌ乳腺腫瘍サンプルにおける癌抑制遺伝子BRCA2発現量の減少

  吉川泰永, 森松正美, 落合和彦, 石黒(大沼)俊名, 和田成一, 折野宏一  日本獣医学会学術集会講演要旨集  158th-  2015
• 癌抑制遺伝子BRCA2のイントロンにおける新規サイレンサー配列の同定と転写因子の特定

  角本俊介, 吉川泰永, 森松正美, 落合和彦, 石黒(大沼)俊名, 折野宏一  日本獣医学会学術集会講演要旨集  158th-  2015
• 北海道大学における動物実験実施者等の実験動物への感作状況

  吉村 彩, 武藏 学, 金子 壮朗, 大西 俊介, 折戸 智恵子, 川原 由佳子, 橋野 聡, 森松 正美, 今野 哲, 有川 二郎, 石井 哲也, 澤村 正也, 上田 一郎  アレルギー  63-  (8)  1132  -1139  2014/09  [Not refereed][Not invited]
   

  【目的】北海道大学で発生したマウス咬傷によるアナフィラキシー事例を踏まえ,アレルギー予防対策の構築を目的として,動物実験を実施する学生及び職員の動物アレルギーの感作状況を調査した.【方法】齧歯類等の取扱者で同意を得た555名を対象に問診票と実験動物5種に対する特異的IgE抗体と好酸球数測定によるアレルギー健診を実施した.【結果】特異的IgE抗体陽性率(陽性者数/取扱者数)は,マウス14.1%(62/441名),ラット17.9%(50/279名),ハムスター18.8%(6/32名),モルモット17.4%(4/23名),ウサギ11.3%(12/106名)であった.マウス取扱者においては,動物に接触した時に何らかのアレルギー症状が現れる場合は,抗マウスIgE抗体陽性率が有意に高いことも判明した(38.1% vs 8.8%,p<0.01).【結論】動物取扱者の感作状況を把握するために,特異的IgE抗体検査を含む健診を実施することが有用であることが示された.(著者抄録)
• 動物実験施設におけるアナフィラキシーの発生と防止対策

  小原徹, 高尾尊身, 佐加良英治, 朱宮正剛, 米川博通, 室田宏之, 森松正美  実験動物と環境  21-  (41)  47  -53  2013/04/01  [Not refereed][Not invited]
  
• 動物実験従事者の動物アレルギー

  吉村 彩, 武藏 学, 金子 壮朗, 大西 俊介, 折戸 智恵子, 川原 由佳子, 森松 正美, 今野 哲, 有川 二郎  CAMPUS HEALTH  50-  (1)  316  -316  2013/03  [Not refereed][Not invited]
  
• 動物実験従事者の動物アレルギー

  吉村 彩, 武藏 学, 金子 壮朗, 大西 俊介, 折戸 智恵子, 川原 由佳子, 森松 正美, 今野 哲, 有川 二郎  CAMPUS HEALTH  49-  (4)  89  -89  2012/09  [Not refereed][Not invited]
  
• イヌ乳腺腫瘍で発見した癌関連遺伝子産物BRCA2における変異BRC repeat 3の機能解析

  吉川泰永, 森松正美, 落合和彦, 和田成一, 垰田高広, 近澤征史朗, 岩井聡美, 折野宏一, 渡辺清隆  日本獣医学会学術集会講演要旨集  154th-  339  -339  2012/08/31  [Not refereed][Not invited]
  
• 放射線治療抵抗性がんに対するRAD51の制御を基軸とした改変型BRCによる新規治療戦略の開発に向けた基礎研究

  落合 和彦, 森松 正美, 吉川 泰永, 宇田川 智野, 多田 尚美, 近江 俊徳  日本獣医学会学術集会講演要旨集  154回-  338  -338  2012/08
• 遺伝性乳がん原因遺伝子BRCA2の動物種間における機能比較

  落合和彦, 吉川泰永, 近江俊徳, 森松正美  日本病態生理学会雑誌  21-  (1)  33  -40  2012/05/20  [Not refereed][Not invited]
  
• 癌抑制遺伝子BRCA2のPCR法による変異解析法の確立

  吉川泰永, 森松正美, 鈴木優, 西村百合香, 落合和彦, 折野宏一, 垰田高広, 近澤征史郎, 岩井聡美, 島村麻子, 新井敏郎, 渡辺清隆  日本獣医学会学術集会講演要旨集  152nd-  299  -299  2011/08/31  [Not refereed][Not invited]
  
• イヌ乳腺腫瘍関連遺伝子産物BRCA2のBRC repeat 4のアミノ酸がDNA相同組換え修復に必須なRAD51ホモ会合体形成に与える影響

  落合 和彦, 吉川 泰永, 近江 俊徳, 森松 正美  日本獣医学会学術集会講演要旨集  152回-  318  -318  2011/08
• イヌおよびヒトBRCA2のBRC repeat3領域におけるミスセンス変異の比較

  吉川泰永, 森松正美, 落合和彦, 折野宏一, 和田成一, 垰田高広, 近澤征史郎, 岩井聡美, 渡辺清隆  日本分子生物学会年会プログラム・要旨集(Web)  34th-  3P-0142 (WEB ONLY)  2011  [Not refereed][Not invited]
  
• イヌ乳腺腫瘍で発見したBRCA2のBRC repeat 3におけるミスセンス変異の解析

  吉川 泰永, 鈴木 優, 森松 正美, 落合 和彦, 折野 宏一, 垰田 高広, 近澤 征史朗, 渡辺 清隆  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  83回・33回-  3P  -0612  2010/12
• イヌ乳腺腫瘍における癌抑制遺伝子BRCA2の新規変異の発見と機能解析

  鈴木 優, 吉川 泰永, 森松 正美, 落合 和彦, 折野 宏一, 垰田 高広, 近澤 征史朗, 渡辺 清隆  日本獣医学会学術集会講演要旨集  150回-  312  -312  2010/09
• イヌ乳腺腫瘍における癌抑制遺伝子BRCA2におけるスプライシングバリアントの発見

  吉川 泰永, 森松 正美, 落合 和彦, 折野 宏一, 垰田 高広, 近澤 征史朗, 渡辺 清隆  日本獣医学会学術集会講演要旨集  150回-  313  -313  2010/09
• イヌ乳腺腫瘍関連遺伝子Brca2のBRC repeat4のアミノ酸配列がRad51との相互作用に及ぼす影響

  落合 和彦, 吉川 泰永, 大沼 俊名, 橋爪 一善, 森松 正美  日本獣医学会学術集会講演要旨集  150回-  313  -313  2010/09
• イヌ乳腺腫瘍における癌抑制遺伝子BRCA2の新規変異の検索

  吉川 泰永, 鈴木 優, 森松 正美, 落合 和彦, 折野 宏一, 垰田 高広, 渡辺 清隆  日本獣医学会学術集会講演要旨集  148回-  283  -283  2009/09
• 家族性乳癌原因遺伝子産物BRCA2のC末端RAD51結合領域におけるunclassified variantsの解析

  吉川泰永, 森松正美, 岩永敏彦  生化学  2008
• 家族性乳癌原因遺伝子BRCA2のC末端RAD51結合領域におけるunclassified variantの解析

  吉川 泰永, 森松 正美, 岩永 敏彦  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  80回・30回-  2T3  -11  2007/11
• イヌの乳腺腫瘍で発見された癌抑制遺伝子BRCA2におけるヘテロ接合性の消失(LOH)

  吉川泰永, 森松正美, 落合和彦, 富岡幸子, 永野昌志, 佐々木伸雄, 橋爪一善, 岩永敏彦  日本獣医学会学術集会講演要旨集  144th-  174  2007/08/27  [Not refereed][Not invited]
  
• イヌ乳腺腫瘍関連遺伝子BRCA2の多型マーカーの確立

  吉川泰永, 森松正美, 落合和彦, 富岡幸子, 永野昌志, 冨沢伸行, 山根義久, 佐々木伸雄, 橋爪一善, 岩永敏彦  日本獣医学会学術集会講演要旨集  142nd-  155  2006/08/31  [Not refereed][Not invited]
  
• 短鎖脂肪酸トランスポーターslc5a8の消化管と腎臓における発現

  武部久美子, 仁尾純子, 森松正美, 桑原厚和, 岩永敏彦  機能性食品と薬理栄養  3-  (5)  303  -308  2006/07/30  [Not refereed][Not invited]
  
• 家族性乳がん原因遺伝子産物BRCA2の核移行シグナルに認められた腫よう発症頻度を高める多型の機能解析

  吉川泰永, 森松正美, 落合和彦, 永野昌志, 冨沢伸行, 山根義久, 佐々木伸雄, 橋爪一善, 岩永敏彦  日本分子生物学会年会講演要旨集  28th-  179  2005/11/25  [Not refereed][Not invited]
  
• Structure and function of breast cancer susceptibility gene Brca2 in the dog

  OCHIAI KAZUHIKO, MORIMATSU MASAMI, YOSHIKAWA YASUNAGA, UGA SATOSHI, SHUTO BUN'EI, HASHIZUME KAZUYOSHI  獣医生化学  41-  (2)  51  -59  2004/10/30  [Not refereed][Not invited]
  
• イヌ乳腺腫よう関連遺伝子産物イヌBrca2の核移行シグナルに存在する多型配列

  吉川泰永, 森松正美, 落合和彦, 永野昌志, 冨沢伸行, 山根義久, 橋爪一善  日本獣医学会学術集会講演要旨集  138th-  182  2004/08/01  [Not refereed][Not invited]
  
• Effects of the Bark Extract on the Mouse

  TOMIYAMA MINAKO, KOFUJITA HISAYOSHI, MORIMATSU MASAMI, SHUTO BUN'EI  獣医生化学  40-  (2)  47  -53  2003/10/30  [Not refereed][Not invited]
  
• 医用材料に結合するタンパク質の好中球に対する作用

  佐々木 秀樹, 森松 正美, 首藤 文榮  獣医生化学 = Veterinary biochemistry  39-  (2)  37  -37  2002/10/31
• Cloning of bovine MAIL and its mRNA expression in peripheral white blood cells.

  YAMAJI DAISUKE, KITAMURA HIROSHI, MATSUSHITA YUKIKO, SHIINA TAKAHIKO, MORIMATSU MASAMI, SAITO MASAYUKI  獣医生化学  39-  (2)  13  -18  2002/10/31  [Not refereed][Not invited]
  
• 子牛の実験的エンドトキシン血症における標的細胞でのTLR4と炎症性サイトカイン遺伝子発現の動態

  三井隆行, 菊佳男, 福田茂夫, 田中隆志, 伊藤寿浩, 森松正美, 首藤文栄, 高橋淳吉, 吉野知男  日本獣医学会学術集会講演要旨集  134th-  149  -149  2002/08/20  [Not refereed][Not invited]
  
• ウシ乳房炎でのサイトカインならびにToll-like receptor 4の遺伝子発現

  河木 英愛, 福田 茂夫, 菊 佳男, 伊藤 寿浩, 森松 正美, 吉野 知男, 岡田 洋之  日本獣医学会学術集会講演要旨集  134回-  149  -149  2002/08
• 創傷治癒過程におけるキチン結合性タンパク質の作用機構

  鈴木 雅子, 森松 正美, 山下 哲郎, 岩永 敏彦, 首藤 文榮  獣医生化学 = Veterinary biochemistry  38-  (2)  2001/10/31  [Not refereed][Not invited]
  
• LPS応答分子MAILのウシホモログとmRNA発現解析

  山地 大介, 北村 浩, 森 浩志, 岩永 敏彦, 松下 由紀子, 椎名 貴彦, 森松 正美, 斉藤 昌之  獣医生化学 = Veterinary biochemistry  38-  (2)  2001/10/31  [Not refereed][Not invited]
  
• Identification and expressional and functional analysis of MAIL, a novel IkB family member.

  KANEHIRA KATSUSHI, KITAMURA HIROSHI, IWANAGA TOSHIHIKO, FUJIKURA DAISUKE, MORIMATSU MASAMI, SAITO MASAYUKI  獣医生化学  38-  (2)  19  -25  2001/10/31  [Not refereed][Not invited]
  
• 新規核内IkBタンパク質MAILの同定および発現・機能解析

  北村浩, 金平克史, 岩永敏彦, 藤倉大輔, 山地大介, 松下由紀子, 森松正美, 斉藤昌之  日本獣医学会学術集会講演要旨集  132nd-  140  2001/09/07  [Not refereed][Not invited]
  
• LPSによるB細胞およびマクロファージでの新規IkBタンパク質MAILの誘導

  北村浩, 金平克史, 岩永敏彦, 椎名貴彦, 松下由紀子, 藤倉大輔, 赤司祥子, 森松正美, 斉藤昌之  生化学  73-  (8)  1042  2001/08/25  [Not refereed][Not invited]
  
• Purification and identification of chitin binding protein CBPb04 (bovine serum chitinase)

  SUZUKI Masako, MORIMATSU Masami, YAMASHITA Tetsuro, SYUTO Bunei  キチン・キトサン研究 = Chitin and chitosan research  7-  (2)  182  -183  2001/07/01
• ストレス応答するマウス脳内遺伝子のクローニングと発現解析

  北村浩, 金平克史, 沖田圭介, 岩永敏彦, 椎名貴彦, 藤倉大輔, 森松正美, 斉藤昌之  生化学  72-  (8)  1017  2000/08/25  [Not refereed][Not invited]
  
• Detection of the opsonization-inhibiting factors in bovine serum.

  SUZUKI MASAKO, TAKEUCHI YUKA, EBIHARA MEGUMI, MORIMATSU MASAMI, SHUTO BUN'EI  獣医生化学  37-  (1)  11  -15  2000/04/30  [Not refereed][Not invited]
  
• 炎症刺激に応答するマウス脳内遺伝子のクローニングと発現解析

  金平克史, 北村浩, 沖田圭介, 岩永敏彦, 藤倉大輔, 森松正美, 斉藤昌之  日本獣医学会学術集会講演要旨集  129th-  165  -165  2000/03/01  [Not refereed][Not invited]
  
• Preparation of a chimera molecule composed of botulinum neurotoxin heavy chain and IgG against the botulinum neurotoxin light chain

  Y Tabata, K Yotsuya, Y Torii, K Aoyagi, M Morimatsu, B Syuto  JAPANESE JOURNAL OF INFECTIOUS DISEASES  53-  (1)  32  -45  2000/02  [Not refereed][Not invited]
  
• ストレス刺激に応答するマウス脳内遺伝子の検索と解析

  北村浩, 沖田圭介, 金平克史, 藤倉大輔, 岩永敏彦, 森松正美, 斉藤昌之  日本分子生物学会年会プログラム・講演要旨集  22nd-  482  1999/11/22  [Not refereed][Not invited]
  
• Effect of Bovine Serum Chitin Binding Proteins on Bovine PMN

  SUZUKI M., NATSUME A, MORIMATSU M, SYUTO B  キチン・キトサン研究 = Chitin and chitosan research  5-  (2)  138  -139  1999/07/15
• Molecular characterization of airborne odorant receptor organ specific protein in Xenopus laevis daudin.

  SUZUKI KEIKO, YAMASHITA TETSURO, MORIMATSU MASAMI, OKA TATSUZO, SHUTO BUN'EI  家畜生化学  36-  (1)  37  -42  1999/04/30  [Not refereed][Not invited]
  
• The Molecular Characteristics and Biological Activities of the Chitin Binding Proteins in Animal Sera

  SUZUKI M., NAKAO A., MUGISHIMA A., ONIYANAGI R., MORIMATSU M, SYUTO B  キチン・キトサン研究 = Chitin and chitosan research  4-  (2)  232  -233  1998/05/15
• Haptoglobin in the brown bear (Ursus arctos): molecular structure and hibernation related seasonal variations.

  MOMINOKI KATSUMI, MORIMATSU MASAMI, SAITO MASAYUKI  家畜生化学  34-  (2)  45  -53  1997/12  [Not refereed][Not invited]
  
• Glucose metabolism and glucose transporters in cattle.

  ABE HIROYUKI, MORIMATSU MASAMI, SAITO MASAYUKI  家畜生化学  33-  (2)  1  -10  1996/12  [Not refereed][Not invited]
  
• Regulation of Adipose Tissue-specific Gene Expression in Cattle: Structure and tissue distribution of insulin-regulatable glucose transporter.

  SAITO MASAYUKI, MORIMATSU MASAMI, ABE HIROYUKI  食肉に関する助成研究調査成果報告書  14(1995)-  185  -189  1996/12  [Not refereed][Not invited]
  
• D1ドーパミン受容体と結合するアデニル酸シクラーゼの同定

  木村 和弘, 岡田 尚子, 森松 正美, 斉藤 昌之  神経化学  35-  (3)  540  -541  1996/09/05  [Not refereed][Not invited]
  
• D1ドーパミンレセプターと共役するアデニル酸シクラーゼの同定

  岡田 尚子, 森松 正美, 木村 和弘, 斉藤 昌之  日本分子生物学会年会プログラム・講演要旨集  19-  (0)  507  -507  1996/08/01  [Not refereed][Not invited]
  
• Spermatogonial cell-mediated activation of an IkappaBzeta-independent nuclear factor-kappaB pathway in Sertoli cells induces transcription of the lipocalin-2 gene.

  Fujino R-S, Tanaka K, Morimatsu M, Tamura K, Kogo H, Hara T  Mol Endocrinol.  20-  (4)  904  -915  1996  [Not refereed][Not invited]
  
• Regulation of Adipose Tissue-specific Gene Expression in Cattle: Implication in the control of intramuscular fat deposition.

  SAITO MASAYUKI, MORIMATSU MASAMI, ABE HIROYUKI  食肉に関する助成研究調査成果報告書  13(1994)-  160  -166  1995/12  [Not refereed][Not invited]
  
• Control mechanism of acute phase protein synthesis and pathobiochemistry.

  MORIMATSU MASAMI, NAKAGAWA NORIKO, KAWASAKI MASAMI, SAITO MASAYUKI  北海道獣医師会雑誌  39-  (9)  173  -177  1995/09  [Not refereed][Not invited]
  
• Isolation and Charatrerization of Chitin-Binding Protein in Bovine Serum.

  NAKAO ATSUKO, YOTSUYA KAORU, MORIMATSU MASAMI, OKADA KEIJI, INANAMI OSAMU, SHUTO BUN'EI  家畜生化学  32-  (1)  31  -36  1995/03  [Not refereed][Not invited]
  
• The function of C-reactive protein on milk production and fertilization in the cow.

  SHIOKAWA TSUGUAKI, MORIMATSU MASAMI, INANAMI OSAMU, OKADA KEIJI, SHIGA AKIO, SHUTO BUN'EI  家畜生化学  32-  (1)  37  -41  1995/03  [Not refereed][Not invited]
  
• A tequnique for Isolation and Primary Culture of Bovine Hepatocytes.

  NAKAGAWA NORIKO, MORIMATSU MASAMI, MOMINOKI KATSUMI, NAKATSUJI HIROKO, SHUTO BUN'EI, SAITO MASAYUKI  家畜生化学研究会報  30-  (2)  51  -56  1993/10  [Not refereed][Not invited]
  
• ウシ初代培養肝細胞を用いたハプトグロビン合成調節機構の解析

  中川紀子, 森松正美, 川崎昌美, 中辻浩喜, 首藤文栄, 斉藤昌之  日本獣医学会学術集会講演要旨集  116th-  77  1993/08  [Not refereed][Not invited]
  
• ウシ肝細胞の分離と初代培養

  中川紀子, 森松正美, 中辻浩喜, 首藤文栄, 斉藤昌之  日本獣医学会学術集会講演要旨集  115th-  110  1993/04  [Not refereed][Not invited]
  
• Comparative Study on the Molecular Properties of Mammalian Haptoglobins.

  MORIMATSU MASAMI, NAKAGAWA NORIKO, MOMINOKI KATSUMI, SHUTO BUN'EI, NAIKI MASAHARU, SAITO MASAYUKI  家畜生化学研究会報  30-  (1)  23  -30  1993/03  [Not refereed][Not invited]
  
• ウシにおける外科手術侵襲後の急性相反応

  山下和人, 泉沢康晴, 森松正美, 伊藤隆司, 斉藤昌之, 児玉道, 小谷忠生  日本獣医学会学術集会講演要旨集  116th-  1993
• Acute Phase Response of Haptoglobin in Cattle Assessed by a Single Radial Immunodiffusion Method.

  MORIMATSU MASAMI, TOSA NORIKO, NAIKI MASAHARU, SAITO MASAYUKI  家畜生化学研究会報  (29)  61  -68  1992/09  [Not refereed][Not invited]
  
• ウシの急性炎症における血清ハプトグロビン濃度の変動

  森松正美, SARIKAPUTI M, 島田尚美, 首藤文栄, 斎藤昌之, 藤永徹, 中辻浩喜, 大久保正彦, 内貴正治  日本獣医学会学術集会講演要旨集  111th-  302  1991/03  [Not refereed][Not invited]
  
• Isolation, characterization, and changes in the serum levels of bovine C-reactive protein and serum amyloid P component.

  MORIMATSU MASAMI, SARIKAPUTI M, NAIKI MASAHARU  家畜生化学研究会報  (25)  29  -36  1990/11  [Not refereed][Not invited]
  

Industrial Property Rights

• 特許第4355582号:哺乳類(雌)の発情時期のスクリーニング方法  2009/08/07

  大澤 健司, 田中 成佳, 森松 正美, 首藤 文榮  独立行政法人科学技術振興機構  201103031545253565
• 特許第4163549号:MAIL遺伝子ノックアウトマウス  2008/08/01

  森松 正美, 椎名 貴彦  独立行政法人科学技術振興機構  201103096251158275
• 特開2005-204750:哺乳類(雌)の発情時期のスクリーニング方法  2005/08/04

  大澤 健司, 田中 成佳, 森松 正美, 首藤 文榮  独立行政法人科学技術振興機構  200903084374874319
• 特開2004-357607:エンドトキシン認識に関わる新規な分子及びそれをコードするポリヌクレオチド  2004/12/24

  森松 正美, 伊藤 寿浩  独立行政法人 科学技術振興機構  200903045202987310
• 特開2004-321112:MAIL遺伝子ノックアウトマウス  2004/11/18

  森松 正美, 椎名 貴彦  独立行政法人 科学技術振興機構  200903061291557036

Research Grants & Projects

• NFkB情報伝達系が癌発症に及ぼす影響におけるNFKBIZの機能解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2022/04 -2025/03 

  Author : 森松 正美
• 感受性責任遺伝子探索による多包虫症の寄生体・宿主相互作用の分子機序の解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2020/04 -2023/03 

  Author : 安居院 高志, 野中 成晃, 森松 正美, 八木 欣平

   

  本研究は多包虫（エキノコックス）感染に置いて嚢胞内に原頭節を有する感受性の系統（DBA/2 (D2) ）と原頭節を有さない抵抗性の系統（C57BL/6 (B6)）が存在することから、原頭節の有無を制御している原因を明らかにすることである。昨年度までにB6を遺伝的背景にD2由来の染色体断片に入れ替えた（サブ）コンジェニックマウスを用いてエキノコックス感染実験を行った結果、責任遺伝子の存在領域を3.3 cMまで狭めることができた。この範囲内には55個の遺伝子が存在していた。候補領域内の全遺伝子55個について別の研究で作成されたRNAseqのデータを参照しSNPの有無を確認すると、34個の遺伝子においてB6とD2との間でSNPが存在していた。この34個についてEnsemblデータベースを利用しアミノ酸置換を伴っているかを確認したところ21個の遺伝子でアミノ酸置換が確認できた。D2-B6間で翻訳領域にアミノ酸置換が見られる遺伝子は10個であった。従ってこの10個の遺伝子の中に責任遺伝子が存在する可能性が示唆された。これら10個についてB6、D2、BALB/c、AKRの4つのマウス系統でSNPを比較した。その結果ある遺伝子にのみD2特異的なSNPが確認された。この遺伝子はある酵素をコードするものであった。そこでこの遺伝子をB6及びD2マウスより単離し、これを発現ベクターに組み込み、それを線維芽細胞株にトランスフェクトしその酵素活性の違いを検討した。しかし、酵素活性の測定方法の確立に時間を要し未だ結論を得ない状況である。
• 腫瘍におけるBRCA２の変異と結合分子群の機能解明を目的とした新規モデルの作出

  日本学術振興会：科学研究費補助金（基盤研究(B)）

  Date (from‐to) : 2018 -2021 

  Author : 森松 正美
• Anti-tumor mechanism of soluble form of sialic acid-recognizing protein, Siglec-9

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2017/03 

  Author : Tomioka Yukiko, TAKEUCHI Takashi

   

  It was revealed that proliferation and malignant transformation of MUC1-positive tumor were prevented in the transgenic mice expressing a soluble form of sialic acid-binding immunoglobulin-like lectin 9 (sSiglec-9). In addition, it was suggested that this anti-tumor mechanism indirectly acted through the immune system of the host. On the other hand, sSiglec-9 acivated a proliferation of MUC16-positive tumor rather than inhibited, surprisingly. According to the above results, it was shown Siglec-9 interacted with the mucin expressed in tumor and to participate in a tumor proliferation and malignant transformation. These knowledge make the beginning to found the molecular targeted therapy for cancers through Siglec-9.
• 組換え酵素Rad51と癌抑制タンパク質BRCA2の結合の種間比較に基づく分子創薬

  日本学術振興会：科学研究費補助金（基盤研究(C)）

  Date (from‐to) : 2014 -2016 

  Author : 森松 正美
• Mechanisms of development of AIDS encephalopathy using a new transgenic mouse model expressing HIV-1 Nef

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/04 -2014/03 

  Author : TOMIOKA Yukiko, ONO Etsuro, MORIMATSU Masami

   

  Transgenic mice expressing Human Immunodeficiency Virus 1 (HIV-1) accessory protein Nef (Nef Tg) showed the severe neurological symptom including motor ataxia, hindquarter paralysis, and epileptic seizure. Histopathologically, prominent astrocytosis, microgliosis, vacuolation of the white matter, lymphocytic infiltration were observed in the brain and spinal cord of Nef Tg. In addition, expression of some cytokines such as IP-10 was increased in the brain of Nef Tg. These pathological changes were similar to those of AIDS encephalopathy in human, which indicated that Nef protein plays an important role in neuropathogenesis of HIV-1 infection.
• Tumorigenic effects of BRCA2 mutation and genomic instability in dogs

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2011 -2013 

  Author : MORIMATSU Masami, OCHIAI Kazuhiko

   

  Mammary tumors are the most common tumor type in both human and canine females. In women, carriers of mutations in BRCA2, a tumor suppressor gene product, have a higher risk of breast cancer. Canine BRCA2 has also been suggested to have a relationship with mammary tumors. However, clearly deleterious BRCA2 mutations have not been identified in any canine mammary tumors, and effects of BRCA2 mutations on tumorigenicity and genomic instability in dogs are unknown. In this study, 1. a PCR analysis method for canine BRCA2 was established, 2. effects of the missense mutations in canine BRCA2 on BRC repeats were elucidated, 3. tumour suppressor function of canine REIC/Dkk-3 in mammary gland tumors was analyzed.
• Biological function and gene regulation of epididymal-specific carboxylesterase (CES4)

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2010 -2012 

  Author : YAMASHITA Tetsuro, MORIMATSU Masami

   

  The biological function of an epididymal-specific carboxylesterase, CES4, was studied using Ces4 knock out mouse. The metabolome analysis of epididymal cell extracts revealed that the most of the glutathione was identified as oxidative form in the epididymis of KO mouse. The expression of oxidative stress-responsive heme oxygenase-1 (HO-1) was also increased in the KO mouse tissue. These data indicated that the epididymis of KO mouse was exposed by oxidative stress.
• Regulation Mechanisms of the body size in mice

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2009 -2011 

  Author : KANO Kiyoshi, MORIMATSU Masami

   

  We analyzed phenotypes of DDR2 transgenic mice and ATDC5 cell lines with a focus on growth and size control. In previous study, cartilage cell proliferation decreased in DDR2 KO mice, and we analyzed also the function of DDR2 in cartilage tissue. We produced Ddr2 cDNA transgenic mice, which were specifically overexpressed only in the cartilage cells, but there were no prominent phenotypes in cartilage tissue of the transgenic mice. To understand the role of Ddr2 in chondrocyte proliferation and differentiation, we next performed knockdown experiments using Ddr2 miRNA in ATDC5 cells. We found that DDR2 might repress cell proliferation and differentiation in cartilage cells. DDR2 is suggested to have important function especially in the reproductive organs by the expression in various tissues.
• Analysis of a novel NFkB inhibitor deficient mouse as a model for atopic dermatisis

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2007 -2009 

  Author : Masami MORIMATSU, Yukiko TOMIOKA, Katsuhiko OMOE, Kiyoshi KANO, Sadatoshi MAEDA

   

  MAIL (molecule possessing ankyrin-repeats induced by LPS) is a nuclear IkappaB protein that is also termed inhibitor of nuclear factor kappaB (IkappaB) zeta. We generated Mail-deficient mice that appeared to develop atopic dermatitis (AD). In this study, we tried to rescue embryonic lethality of the mice. We investigated expression and function of MAIL in the skin to elucidate its role in AD. Possible interactions of Mail with some genes were also investigated.
• Expression and role of sugar-recognition molecule galectin in the digestive tract

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2005 -2006 

  Author : IWANAGA Toshihiko, MORIMATSU Masami

   

  Galectin is an animal lectin that recognizes β-galatosides of glycoconjugates and is abundant in the gut and urogenital tract. This study revealed the cellular expression of galectin subtypes in the digestive tract, kidney and ovary of mice by in situ hybridization and immunohistochemistry. Signals for five subtypes (galectin-2,-3,-4/6, and-7) were detected exclusively in the epithelia of digestive tract. In the glandular stomach, galectin-2 and-4/6 were predominantly expressed from the gastric pits to neck of gastric glands, where mucous cells were the main cellular sources. The small intestine exhibited intense, maturation-associated expressions of galectin-2,-3, and-4/6. In the large intestine, galectin-4/6 were predominated, and the upper half of crypts simultaneously contained transcripts of galectin-3. Stratified epithelium from the lip to forestomach and anus intensely expressed galectin-7 with weak expressions of galectin-3. In the urinary system, the major subtype was galectin-3, which was expressed in the collecting ducts and transitional epithelium continuously from the kidney to the distal end of the urethra, suggesting selective expression of galectin-3 in epithelia of uretic bud and cloaca-derivatives. Galectin-1 and-3 were predominant in the ovary. The corpus luteum at particular stages of regression intensely expressed both types of galectins. Galectin-3 was restricted to regressing corpus luteum and always coincident to the expression of a progesterone degradation enzyme. The signal intensity of galectin-1 first increased at the starting point of regression followed by increasing expression of galectin-3. This finding suggest that galectin-1 and-3 may mediate the progesterone production and metabolism in luteal cells via different mechanisms. Because multi-functions of galectins, information on their cell/stage-specific expression contributes to a better understanding of the functions and pathological involvements of galectins.
• 新規アトピー性皮膚炎原因遺伝子の機能解析

  文部科学省：科学研究費補助金(萌芽研究)

  Date (from‐to) : 2005 -2006 

  Author : 森松 正美

   

  研究代表者らは炎症刺激で誘導される遺伝子をスクリーニングする過程で機能未知の遺伝子を同定し、MAILと命名した。ジーンターゲティングによりこの遺伝子を破壊したマウスを作製したところ、ホモ型欠損マウスの顔面を中心にアトピー性皮膚炎様の病変が認められた。本研究の第1の目的は、申請者が開発したMAIL破壊マウスを用いてMAILの機能を解析するとともに、これを疾患モデル動物として確立することである。第2の目的は、このモデルマウスの原因遺伝子が、ヒトでもアトピー性皮膚炎と関係があるか否かを探ることである。1.マウスの交配と維持:MAILのホモ欠損型が胎生期致死となることが原因でマウスの数が不足している点が研究を進める上で支障となっていた。交配規模を拡大し,産子の遺伝型をPCRにより判定して欠損マウスを得た。2.マウスの病態解析:MAILがケラチノサイトの初代培養や株化細胞で無刺激の状態でも発現していることを明らかにした。この発現には転写因子NF-kappaBが重要な役割を果たすことがわかった。この発現は,炎症性サイトカインによって上昇することが判明し,MAILが皮膚炎病態に関わっていることが示唆された。さらに皮膚炎を発症しているMAIL破壊マウスの皮膚組織を検索したところ,アトピー性皮膚炎に関与すると考えられているTh2系サイトカインの産生が認められた。3.ヒトの遺伝子解析:ヒトの皮膚生検試料におけるMAILの発現をin situ hybridizationで検索したが,はっきりした発現は認められなかった。今後,検出感度を上昇させるなどの方法の改善が望まれる。さらに,ヒトMAIL遺伝子の多型と皮膚炎との関係を調べるために,MAILのcDNAをRT-PCRで増幅するためのプライマーを設計し,疫学的解析を実施するための基盤を築いた。
• Molecular and clinical analysis for metastasis and invasion of cancers in small animals

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2003 -2006 

  Author : SASAKI Nobuo, TSUJIMOTO Hajime, NISHIMURA Ryohei, NAKAYAMA Hiroyuki, MORIMATSU Masami, MOCHIZUKI Manabu

   

  In this study, we tried to analyze the mechanism of tumor metastasis and local invasion in small animals using 5 cell lines established in our laboratory. In canine mammary cancer, expression and function of E-cadherin and catenin were compared between 4 pairs of cell lines established from the primary or metastatic lesions of the same patients. As a result, there were no differences in their expression but their function was higher in cell lines established from the metastatic lesions than those from primary lesions. This may indicate that the cells in the metastasis with low E-cadherin function are easily released from the primary lesions. In addition, sLe(x), an adhesion molecule to the endothelial cells, was alos highly expressed in one of the cell lines established from the thoracic effusion due to metastasis. Our previous data showed sLe(x) was 60% positive in the mammary tumor tissues, but not in normal mammary gland. From these data, sLe(x) may play a role in the metastasis of canine mammary tumors. Currently one of this mammary tumor cells was cloned and microarray analysis was conducted to clarify the genetic change in the cells from primary and metastatic lesions in nude mice model. The result may suggest some important molecules including NfκB and hipoxia related one, which will be analyzed in the future. In feline mammary tumors, we established 8 cell lines including 2 pairs from primary and metastatic lesions of the same patient. Using these cell lines, expression of a chemokine receptor CXCR4 and its ligand SDF-1 were compared between cell lines. Their expression was higher in those originated from metastasis than primary lesions, suggesting they may also play a role in tumor metastasis in feline mammary tumors. In addition, tumor-suppressing molecule, Braca2, was also analyzed in relation to the malignancy of this tumor. Retinoids, vitamin A derivatives, may control the growth of canine mast cell tumors. This tumor growth-inhibitory effect of retinoids against canine mast cell tumors was mediated by a receptor RAR alpha and via inducing apoptosis in both in vitro model and in vivo nude mice model, as in human acute promyelocytic leukemia. These results suggest the retinoids may be a potential chemotherapeutic agent for canine mast cell tumors. For canine osteosarcoma, gene therapy using wild type p53 transfection using adenovirus as a vector virus. This trial showed some efficacy in nude mice model and even in the canine patients with ostesarcoma. For canine melanoma, spindle check point function is currently investigated to analyze its relationship to chemotherapeutic response of the tumor.
• ウシ肝cDNAマイクロアレイの評価と妊娠ウシ肝の特異発現遺伝子の検索

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2004 -2005 

  Author : 橋爪 一善, 木崎 景一郎, 高坂 哲也, 森松 正美

   

  ウシ肝臓由来cDNAクローンを基に作製したウシ肝cDNAマイクロアレイの有効性を未知クローンの配列、機能、発現動態の解析、検証することから、ウシ肝の生理状態の把握および疾病診断に適用できる汎用的なアレイの開発を目的として、より広範な遺伝子情報を集積した約10,000オリゴ情報からなるアレイを作製した。 発現遺伝子動態の解析には体細胞クローンウシと通常の方法で作出したウシ肝臓を用いた。体細胞クローンウシでは病理および組織学的に変異を示す肝臓が認められるが、著しい病理変異を示さない肝臓であっても数%の遺伝子の発現変動を認め、cDNAアレイに含む数多くの遺伝子情報は、現在のところ機能が十分明確でなくとも肝臓の機能診断に有効な情報であることが示唆された。また、機能および遺伝子の全配列が不明であったcDNAクローンからその発現動態およびin silico解析により、2種の新規遺伝子であるプロラクチン関連タンパク質(PRP)遺伝子を同定し、PRP-VIIIおよび-IXと命名するとともに、in situ hybridizationにより主たる発現細胞が胎盤の二核細胞であることを証明した。加えて、これまで開発してきたウシ肝cDNAマイクロアレイおよび子宮・胎盤cDNAマイクロアレイ上の共通する遺伝子の検証およびin silico解析することから、約10,000オリゴを配置したウシオリゴアレイを作製した。このオリゴアレイは約2,620個の肝由来クローン情報および約1,730個の子宮・胎盤由来クローンを軸として、Gene Bank Database情報を基に抽出した約5,600遺伝子の60塩基配列からなる個別遺伝子情報を集積したものである。尚、本アレイの作製は、これまでアレイ開発で連携してきた農業生物資源研究所のグループとの共同成果である。
• Regulation of host defense and development by innate-immunity signal transduction system

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2003 -2005 

  Author : Masami MORIMATSU, Kumiko YOSHIMATSU, Yoshio YAMAMOTO

   

  In innate immune system, specific pattern molecules such as LPS bind to Toll-like receptors and activate signal transduction pathway involving NF-kappaB. Recently we identified a novel nuclear IkB molecule, MAIL. In this study we focused on MAIL and other NF-kappaB related molecules.1. Analysis of MAIL deficient miceEmbryonic lethality was observed for about 90% of MAIL deficient mice. Hypoplasia of the liver was suggested to be a possible cause for this embryonic lethality. Remaining 10% of MAIL deficient mice were born alive. These mice developed severe atopic dermatitis-like disease.2. Analysis of transcriptional regulation of the MAIL geneWe cloned LPS-reactive upstream region of the mouse MAIL gene, and analyzed its promoter function by using a series of mutated sequences. An NF-kappaB binding site located at -229 to -220 bp was revealed to be an essential target of MAIL expression. Overexpression of MAIL protein suppressed the LPS-induced promoter activity of the MAIL gene. These data indicate that MAIL expression is upregulated by NF-kappaB, and it is controlled, at least in part, by an autoregulation mechanism.3. Analysis of an NF-kappaB target gene product, BRCA2BRCA2, an NF-kappaB target molecule, plays essential roles for cell growth and embryogenesis through its DNA repair and recombination function. We demonstrated that BRCA2 proteins from the mouse and dog interact with Rad51 recombinase. We found a novel insertion/deletion polymorphism in canine BRCA2, which is located in a nuclear localization signal. This polymorphism was associated with nuclear localization efficiency and mammary tumor morbidity.
• ジーンターゲティングによる新規アトピー性皮膚炎モデル動物の開発

  文部科学省：科学研究費補助金(萌芽研究)

  Date (from‐to) : 2003 -2004 

  Author : 森松 正美

   

  最近、ある機能未知の遺伝子を同定して機能解析のためにジーンターゲティングによりこの遺伝子を破壊したマウスを作製したところ、ホモ型欠損マウス(仮にAllergic Dermatitis、ALDマウスと呼ぶ)の顔面を中心にアトピー性皮膚炎様の病変が認められた。本研究の目的は、外見的に皮膚炎を認めるALDマウスについてその病態を解析し、これを疾患モデル動物として確立することである。1.マウスの交配と維持:研究の途中で岩手大学から北海道大学に拠点を移したが、いずれの場所でも問題なく微生物学的に清浄な環境でマウスを飼育した。繁殖規模を拡大すると同時に近交系への戻し交配を進めた。また、万が一の事故に備えて受精卵を凍結保存させた。2.病態の解析:皮膚病変部位の組織学的検索を行い、白血球系細胞の細胞浸潤による炎症反応を認めた。皮膚病変部では、ケモカインであるTARCとeotaxin、ケモカイン受容体であるCCR3の増加が認められた。これらはヒトのアトピー性皮膚炎とよく似た所見であり、MAIL欠損マウスが良いモデルとなる可能性を示すものである。正常マウスでのMAILの発現を免疫組織化学およびリアルタイムPCRで検索したところ、表皮ケラチノサイトでの発現を認めた。この結果は、ケラチノサイトのMAILが皮膚の恒常性に重要であり、これが欠損すると皮膚炎を発症することを示唆している。マウスの系統によって免疫応答に差があることが報告されているので、Th1優勢のC57BL/6、およびTh2優勢のBALB/cへの戻し交配を進めたが、いずれの系統でもMAIL欠損マウスで皮膚炎が認められた。以上より、本遺伝子操作マウスを疾患モデル動物として応用するための基盤の整備を進めることができた。
• Development of hantavirus vaccine and rapid diagnosis kit by using soluble recombinant envelope glycoproteins

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2001 -2003 

  Author : MORIMATSU Kumiko, ONO Eriko, MORIMATSU Masami, KARIWA Hiroaki, ARIKAWA Jiro

   

  1. It was clarified that recombinant Hantavirus envelope glycoproteins G1 and G2 (GPs) had the cell-fusion activity by using CAG promotor-expression system. Fusion-responsible epitope on GPs was found to locate in the neutralization (FRNT)-relating epitope. Therefore, the recombinant GPs was considered to possess neutralization-and fusion-relating epitopes. In addition, hantavirus nucleocapsid (N) protein was also expressed in mammalian cells by using same vector. As the result the N protein may suppress that expression and transportation of the envelope protein. It was also shown that the virus like particle was not formed by GPs and N protein alone. 2. By using recombinant GPs, pseudotype vesicular stomatitis virus (VSV) enveloped with hantavirus GPs altered to VSV G protein was produced (VSVΔG*HTN). Similar pseudotype VSV was produced with recombinant GPs of Seoul virus (VSVΔG*SEO). These pseudotypes were applied to rapid neutralization assay as safety alternatives of authentic viruses. These pseudotype virus particles were also applied to vaccination as alternatives to authentic virion. Mice were immunized with soluble recombinant GPs or pseudotype virion. In mice immunized with soluble recombinant GPs, FRNT antibody was not detected. Contrary, in mice immunized with VSVΔG*HTN virion, FRNT antibody was detected. Challenge administration of hantavirus was carried out by s.c. inoculation of 4 FFU of hantavirus. These mice did not show the elevation of anti-N antibody and hantavirus specific CD8 T cell response, indicating that the FRNT antibody could protect mice from hantavirus infection. On the other hand, all control mice immunized with VSVΔG*G or PBS could not escape from hantavirus infection. These results indicated that packaged recombinant GPs on VSV particle were possible to induce protective FRNT antibody. This study shows that novel approach for the development of vaccination of viruses that have difficulties in preparation of authentic virus particles.
• DNA repair defect, tumorigensis, and hereditary breast cancer gene Brca2 -Studies in vitro and in vivo, and association with Rad51.

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 1999 -2001 

  Author : Masami MORIMATSU, 冨澤 伸行, Shuji TSUDA, Bunei SYUTO, Katsuhiko NISHIMORI, Nobuyuki TOMIZAWA

   

  To establish the bases of developing new methods for prevention, diagnosis, and therapy of breast cancer , we investigated in vitro and invivo functions of the Brca2 and other tumor associated genes. Especially, researches for the significance of the interaction of Brca2 and Rad51, application of Brca2 analysis in veterinary medicine, and roles IkB MAIL were carried out.1. Cloning and mutation analysis of canine Brca2Full length (about 10 kbp) of the cDNA for canine Brca2 was cloned and sequenced. For mutation analysis, DNA fragments were obtained by PCR and used for protein truncation tests.2. Cloning and expression of feline Brca2Full length (about 10 kbp) of the cDNA for feline Brca2 was cloned and sequenced. Expression of feline Brca2 was confirmed in mamnary gland.3. Interaction of Brca2 and Rad51Interaction of C-terminus of canine Brca2 with Rad51 was demonstrated by yeast two-hybrid assay. Transactivation activity of canine Brca2 was also demonstrated.4. Characterization of a novel IkB protein, MAILWe cloned, sequenced, and analyzed the expression of the MAIL gene encoding a novel IkB protein.
• Study for the development of new gene targeting method utilizing epitope tags

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 1999 -2001 

  Author : Masami MORIMATSU, Kumiko YOSHIMATSU, Kazushige OGAWA, Bunei SYUTO, 吉村 佳典, Ichiro MIYOSHI, Yoshimichi YOSHIMURA

   

  To establish the bases for developing new methods of gene targeting, we investigated the efficiency of gene knock-in and the use of specific markers called epitope-tags. By using this approach, gene of interest is to be analyzed by antibodies against epitope-tags. We selected some genes for targeting experiments. Detailed characterization of candidates for epitope-tags was also carried out.1. Investigation of a novel endotoxin-inducible gene, MAIL, as a target.MAIL is suitable for targeting experiment because it is known that transcription of the gene is markedly up-regulated by endotoxin stimulation and that the gene product is specifically localized in the nucleus. We cloned and sequenced the genomic DNA of MAIL. Targeted disruption of MAIL gene was also performed.2. Investigation of genes encoding chitinase as a target.Because of tissue-specificity of chitinase gene, we choose this gene as a target. We cloned, sequenced, and analyzed expression pattern of chitinase gene, and established the basis for gene targeting.3. Investigation of other target genes.Inducible genes, such as B-13, and tissue-specific genes, such as glucose transporters, were investigated as targets.4. Analysis of epitope-tags.Antigenic specificity and cellular function of candidate epitope-tags from hantavirus proteins were examined.5. Developing knock-in method.We tried two-step targeting method for gene knock-in. However, expected recombinants were not obtained.Further research is needed to clarify the cause of the failure.
• Fundamental studies on the protective mechanisms of a novel serum lectin (CBPb01)

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1999 -2000 

  Author : SYUTO Bunei, MORIMATSU Masami

   

  The activation of the neutorophil function, such as phagocytosis and superoxide production induced by opsonins, is a very important system in the nonspecific defense, and its mechanism has been studied extensively. On the other hand, we have hypothesized that some kinds of regulatory proteins to the neutrophil activation system are present in serum and play the important roles as a regulatory systems. This hypothesis motivates us to isolate and to characterize a regulatory protein in the bovine serum. In this study, a bovine serum protein which binds to chitin (chitin binding protein, BPb01) and suppresses the superoxide generation in neutorphils has been purified and identified. The outline of the studies is as follows. 1) A protein with high affinity to N-acetyl-D-glucosamine (GlcNAc) was separated and was named CBPb01. 2) Purification and identification : the amino acid sequence of CBPb01 peptides were identical to the reported amino acid sequence of bovine IgM. 3) Constituent carbohydrates : lectin binding analysis revealed that CBPb01 contains mannose, GlcNAc and fucose. 4) Inhibition of superoxide generation by CBPb01 : about 40% of superoxide generation was decreased, when a neutrophils were stimulated with the opsonized zymosan that was treated with CBPb01. This decrease was completely inhibited by adding GlcNAc. We have concluded that CBPb01 is not a lectin but a GlcNAc recognizing IgM that regulate the superoxide generation in neutrophils. 5) Inhibition mechanism for the superoxide generation by CBPb01 : although CBPb01 binds to neutrophils, the superoxide generation was not decreased. These results suggest that CBPbO1 inhibits the active structure of protein complex formed with opsonins, while it does not involve the process of the down stream from phospholipase C to NADPH oxidase activation in the superoxide generation system. 6) This is the first report of the chitin-binding bovine IgM that controls a superoxide generation in neutrophils.
• 近赤外光を用いたイヌの脳活動の無侵襲計測 - イヌのこころの動きを測る

  文部科学省：科学研究費補助金(萌芽的研究)

  Date (from‐to) : 1999 -1999 

  Author : 森松 正美, 冨澤 伸行

   

  脳の多彩な精神活動は,神経細胞によるエネルギー代謝とそれにともなう酸素の消費によって支えられている.脳活動が変動すると脳の血流量や血液内ヘモグロビン(Hb)の酸素化度が変化するため,これらを測定することは,動物の精神活動を探る糸口となることが期待される.本研究ではイヌの精神活動の分析に,近赤外分光法による脳Hb酸素化度の測定が利用可能か否かを検討した.1.麻酔下のイヌによる脳血流測定の基礎的解析脳血流量を測定する際に筋血流量の影響が障害となる可能性があるため,麻酔下のイヌを用いて検討した.プローブを外科的処置により露出させた頭蓋骨表面に装着した場合と,皮膚表面からあてた場合とで測定値を比較し,測定における筋血流量の影響を調べた.呼気中酸素分圧や頚動脈の開閉等を行って検討した.プローブを頭部正中に装着すれば,筋血流量の変化の影響を抑えて脳血流量を測定できる可能性が高いことが示された.さらに,脳特異的血流変化が検出できるかどうかを検討した.脳は二酸化炭素に感受性が高いため,これを吸入させてその影響を調べたところ,脳に特徴的な変化が観察された.2.無侵襲・無麻酔・無拘束のイヌにおける脳血流測定イヌの情動変化を近赤外分光法で捉えることができるか否かを検討するため,イヌにとって望まれる状況と望まれない状況を設定し脳血流の変化を観察した.餌を与えて喜ばせた場合,Hb酸素化度の低下が認められた.別離不安の見られるイヌについて,飼い主がイヌから離れて隠れたところ,Hb酸素化度の一過性の上昇とそれに続く比較的長時間の低下が観察された
• 急性相タンパク質の新規動物資源生産管理および野生動物生態調査への応用

  文部科学省：科学研究費補助金(奨励研究(A))

  Date (from‐to) : 1997 -1998 

  Author : 森松 正美

   

  生体が、感染、炎症、ストレス等による侵襲を受けると、肝臓から血液中に特定の一群の生体防御関連タンパク質、すなわち急性相タンパク質が分泌される。この研究の目的は、急性相タンパク質の測定による動物の健康管理を新規家畜や野生動物に応用することにより、基礎研究及び関連産業の発展への貢献をはかることである。本研究では、クマおよびシカの急性相タンパク質に焦点をあてて、免疫測定法を確立するとともに飼育個体や野生個体における変動を解析して,これらの新規家畜や野生動物の健康管浬に利用することを計画した。平成10年度は,急性相タンパク質測定法の確立,試料の収集と測定の実施を中心に以下のごとく検討した.クマの急性相タンパク質:クマの急性相タンパク質のうち,ハプトグロビン(Hp),α2-マクログロブリン(α2M),α1-アンチトリプシン(α1-AT)およびC-反応性タンパク質(CRP)について単純放射免疫拡散法(SRID法)あるいはウエスタンプロット法による測定を行った。野生個体の捕獲例では,外見的に正常な個体に比べ外傷を負った個体で顕著なハブトグロビンの増加が認められた。健康な飼育個体で冬眠との関係に着目して調べたところ,冬眠に伴ってHpとα2Mが増加したが,α1-ATとCRPは変化しなかった。クマの健康管理に急性相タンパク質の測定が有用であると思われるが,冬眠など生理的な変化の影響について留意する必要がある。シカの急性相タンパク質:シカの血清をポリアクリルアミドゲル電気泳動により分析したところ,健康状態によって顕著に変化するタンパク質を見いだした。これを分離して分析し,シカHpと同定した。クマの例と同様に特異的な免疫測定法を確立して調べた結果,シカでHpが炎症性の刺激により急激に増加する良いマーカーとなりうろことが示された。
• Neuroendocrine mechanism of stress-induced immunosuppression and roles of cytokines.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1995 -1996 

  Author : SAITO Masayuki

   

  Various types of stressor suppress immune activities. Previously, we have confirmed a significant role of the sympathetic nervous system (SNS) in the stress-induced immunosuppression. In this study, we examined the effects of brain stimulation on the sympathetically mediated immunosuppression in rats. When the ventromedial hypothalamus (VMH), a putative hypothlamic center linked to the SNS,was stimulated electrically, the proliferative activity of splenic lymphocytes was reduced. The suppressive effect of VMH was not affected by adrenalectomy, but abolished by ganglionic blockade and surgical sympathetic denervation. These results suggest a critical role of the VMH in the stress-induced immunosuppression. Similar suppressive effects were also observed when interleukin-1 (IL-1), a representative cytokine produced in immune cells and brain cells, was given intracerebroventricularly. Brain IL-1 was also found to accelerate noradrenaline turnover in various peripheral organs, implying increased activity of the SNS.There are reports that the stress-induced immunosuppression was attenuated by intracranial injection of anti-IL-1 antibody. Collectively, it was concluded that brain IL-1 and the VMH play important roles in the stress-induced immunosuppression.
• ウシ肝細胞における急性相タンパク質の遺伝子発現調節機構の解析

  文部科学省：科学研究費補助金(奨励研究(A))

  Date (from‐to) : 1995 -1995 

  Author : 森松 正美

   

  急性相タンパク質は、炎症性サイトカインが肝細胞を刺激することにより合成・分泌される血清タンパク質の総称であり、生体の防御・免疫機能に関与している。申請者はこれまでウシの急性相タンパク質の血中レベルの変動等について研究を進め、ラット等の実験動物やヒトでこれまで報告されてきたものとは異なるいくつかの特徴的な現象を見いだしてきた。本研究ではこれを更に発展させ,この現象の根幹となる遺伝子発現調節機構をin vitroの肝細胞培養実験系を用いて解明することを試み,いくつかの新知見を得ることができた。[1]ウシ急性相タンパク質の型別(I型とII型)の検討近年、ラットやヒト由来の材料を用いた研究結果に基づき、急性相タンパク質は作用するサイトカインの種類によりI型(インターロイキン(IL)-1と腫瘍壊死因子(TNF)が例外無くともに作用し,IL-6も効果があることがある)とII型(IL-6でのみ誘導される)に分類されるのが一般的となってきた。申請者は,これがウシの急性相タンパク質にもあてはまるか否かを明らかにするため,ウシのハプトグロビン(Hp),C-反応性タンパク質(CRP),alpha 1-酸性糖タンパク質(al-AG),alpha 2-マクログロブリン(a2-MG)のcDNAをそれぞれクローニングしてこれをプローブとし,サイトカインで刺激したウシの初代培養肝細胞におけるこれら急性相タンパク質のmRNA発現量をNorthernハイブリダイゼーション法で解析した。その結果,ラットやヒトでI型急性相タンパク質と考えられているHp,CRP,al-AGがいずれもTNFとIL-6で誘導されるがIL-1では誘導されないことを明らかにした.すなわち,これまでラットやヒトで提唱されているI型とII型の分類はウシにあてはまらないことを発見し,ウシに特有の急性相タンパク質合成調節機構が存在することが示唆された。[2]ウシ特有の上記機構解明の試み上記(1)についてHp,CRP,al-AGがラットやヒトでI型であることを考えると、ウシでもこれらが基本的にはI型類似だが、何らかの因子の欠陥によりIL-1に不応答であると予想できる。このウシ肝細胞に欠けている因子を遺伝子相補により単離することを最終目標としてそれに不可欠なウシ肝細胞株の樹立を試みた。ウシ初代培養肝細胞にSV40ウイルス由来の大型T抗原(LT)を導入して不死化することを試みた。哺乳動物細胞発現ベクターあるいはアデノウイルスに組み換えたLTを導入したところ,長期培養に耐える細胞がいくつか得られたが,残念ながらこれらでは急性相タンパク質遺伝子の発現を認めなかった。現在,不死化の方法を変更してさらにウシ肝細胞株樹立を試みている。
• ウシハプトグロビン遺伝子の構造および転写調節機構の解析

  文部科学省：科学研究費補助金(奨励研究(A))

  Date (from‐to) : 1994 -1994 

  Author : 森松 正美

   

  ハプトグロビン(Hp)は,肝臓で合成・分泌される血清タンパク質であり,急性炎症時に血中レベルが上昇して生体の防御・免疫機能に関与する急性相タンパク質のひとつである.本研究では,ウシHpに着目し,その遺伝子進化の背景をさぐるとともに,特徴的な遺伝子転写調節機構について,いくつかの新知見を得ることに成功した.1.ウシHp遺伝子の進化申請者はこれまでの研究成果から,ウシのHpは特徴的な遺伝子重複による分子進化を経て形成されたと予想した.本研究では,ウシゲノムDNAをサザンブロット法で解析して制限酵素地図を作製したところ,ゲノム上に単一コピーで存在することが明らかとなり,重複前の遺伝子は著しく変異したか,あるいはすでに欠失したと予想した.さらに,他の哺乳動物のHpをタンパク質レベルで調べた結果から,ウシで発見した遺伝子重複による分子進化は,広く反芻動物に認められることを明らかにした.なお,当初の実験計画では,ウシHpのゲノムDNAをクローニングして全塩基配列を決定する計画であったが,残念ながらまだクローニングには成功しておらずこの実験は継続中である.2.ウシHp遺伝子の転写調節ウシ肝臓におけるmRNA量を調べたところ,Hpの発現は,正常では極めて低いが炎症性疾患で急激に上昇することを明らかにした.さらに,ウシ初代培養肝細胞実験系で調べたところ,炎症性サイトカインのうち,インターロイキン(IL)-6と腫瘍壊死因子(TNF)が,Hp遺伝子の転写を誘導したが,IL-1は効果がなかった.このことは,これまでに報告された急性相タンパク質では,TNFで誘導されるものは例外なくIL-1でも誘導され,両者が共通の細胞内情報伝達系を活性化するとされてきたことと極めて対照的な新知見である.また,泌乳ホルモンであるプロラクチンがウシHpの遺伝子発現を誘導することを,あらゆる急性相タンパク質の中で初めて発見した.
• Molecular mechanisms of the central actions of interleukin-1

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1993 -1994 

  Author : SAITO Masayuki, MORIMATSU Masami

   

  It has been established that the central nervous system and the peripheral immune system interact in a biderectional manner. Interleukin-1 (IL-1) is one of the cytokines for the communication between these two systems. In this study, to clarify the cellular and molecular mechanisms for the central action of IL-1, we examined the effects of IL-1 on norepinephrine (NE) turnover (an index of noradrenergic neuronal activity) in the brain and peripheral organs in rats, and obtained the following results : 1. When IL-1 was given intraperitoneally, it increased NE turnover in the hypothalamus and also in some peripheral organs such as spleen and lung. These responses were mimicked by an intracerebroventricular (icv) injection of minute amounts of IL-1, suggesting a direct action of IL-1 to the brain. 2. The stimulative effects of IL-1 were completely blocked by the pretreatment with either an antibody against corticotropin-releasing hormone (CRH) or indomethacin, an inhibitor of prostaglandin (PG) biosynthesis, suggesting important roles of brain CRH and PGs for the IL-1 action. 3. An icv injection of CRH could activate NE turnover in every organs in the same way as the IL-1 injection. In contrast, PGs given intracerebroventricularly were effective for NE turnover in some limited organs : that is , PGE2 for spleen and lung, and PGD2 only for the hypothalamus. Thus, the signal of IL-1 may be converted to the production of PGD2 and PGE2 to activate the noradrenergic neurons projecting to the hypothalamus and peripheral organs, respectively. The molecular mechanisms for such differential regulation of PG production in the brain remain to be further investigated.
• ウシの初代培養肝細胞を用いた急性相タンパク質の遺伝子発現・合成調節機構の解析

  文部科学省：科学研究費補助金(奨励研究(A))

  Date (from‐to) : 1993 -1993 

  Author : 森松 正美

   

  急性相タンパク質は、主に肝臓により合成・分泌され、急性炎症時に特徴的な血中動態を示しながら生体の防御・免疫機能に関与している。本研究では、ウシ肝細胞の初代培養系を確立して急性相タンパク質の合成調節機構を細胞・分子レベルで解明するための基礎を築くとともにいくつかの新知見を得ることに成功した。1.ウシ肝細胞の初代培養の確立(1)ウシから肝尾状葉を摘出後、コラゲナーゼを灌流して結合組織を消化し、肝細胞を分離する方法を確立した。肝摘出前にヘパリンをウシに注射して血液凝固を抑制することにより実験の再現性が向上した。また、灌流するコラゲナーゼの濃度は0.05%が、灌流時間は10〜15分間が適当であった。(2)分離した肝細胞を培養することにより、タンパク質合成とグリコゲン分解の活性が上昇した。培養条件はラットのものを応用したが、ラットの場合と同様に、肝特異的代謝活性は培養開始後一週間で再び低下した。2.初代培養肝細胞を用いた急性相タンパク質の合成調節機構の解析(1)まず、ウシの急性炎症で血中に最も急激に上昇するハプトグロビン(Hp)について調べた。肝細胞の培養上清に放出されたHpを特異抗体で免疫沈降して測定した。調べた炎症性サイトカインのうち、インターロイキン6(IL-6)と腫瘍壊死因子(TNF)はHpの合成を誘導したが、IL-1では変化がなかった。特に、TNFが誘導する点は、ラット等とは異なる点であり、in vivoにおける急激な上昇と関係があるものと推察できる。(2)急性炎症のみならず泌乳に関連して変動するC-反応性タンパク質(CRP)について、上記Hpと同様に免疫沈降実験を行った。しかし、現在までのところCRPの検出に成功していない。これについては、検出感度を上昇させるべく特異抗体の再調製を行うとともに、遺伝子発現レベルでの変化の解析を行っているところである。
• 炎症反応の制御に関わる細胞情報伝達機構
• 相同組換え反応の分子機構
• 家畜の急性相タンパク質の合成・分泌調節機構
• Signal transduction mechanisms in the inflammatory response
• Molecular mechanisms of homologous recombination.
• Mechanisms of the synthesis and secretion of acute-phase proteins in domestic animals.

Educational Activities

Teaching Experience

Position History

• 2012年4月1日 

  - 

  2013年10月31日 

  遺伝子病制御研究所附属動物実験施設長

Committee Membership

• 2023/02 - Today   Editorial Board Member "Animals"
• 2022/06 - Today   Japanese College of Laboratory Animal Medicine (JCLAM)   Committee for Information Technology
• 2022/05 - Today   Japanese Association for Laboratory Animal Science (JALAS)   Assessment and Verification Committee
• 2022/05 - Today   Japanese Association for Laboratory Animal Science (JALAS)   Board of Directors
• 2022/04 - Today   Japanese College of Laboratory Animal Medicine (JCLAM)   Board of Directors
• 2020 - Today   Japanese Association for Laboratory Animal Science (JALAS)   Committee for the Act on Welfare and Management of Animals and its related matters
• 2020 - Today   Japanese Association for Laboratory Animal Science (JALAS)   Committee for Training System of Laboratory Animal Manager
• 2019/03 - Today   Editorial Board Member of "Biomedical Research"
• 2018 - Today   Japanese Association for Laboratory Animal Science (JALAS)   Committee for laboratory animal welfare and ethics
• 2016 - Today   Japanese Association for Laboratory Animal Science (JALAS)   Committee for the Training of Site Visitors in Verification and Validation Programs for Proper Conduct of Animal Experimentations
• 2014 - Today   Japanese Association for Laboratory Animal Medicine (JALAM)   Board of Directors
• 2020 -2022   Japanese Association of Laboratory Animal Facilities of National University Corporation   Committee for Academic Information and Public Relations
• 2019 -2022   Japanese Association of Laboratory Animal Facilities of National University Corporation   Committee for Medium Size Animals
• 2016 -2022   Japanese Association for Laboratory Animal Science (JALAS)   Committee for the Review of Association Articles, Bylaws and Regulations
• 2019 -2021   Japanese College of Laboratory Animal Medicine （JCLAM)   Committee for Examination Review
• 2018 -2019   Japanese Association for Laboratory Animal Science (JALAS)   Board of Directors
• 2012 -2015   Japanese College of Laboratory Animal Medicine （JCLAM)   Examination Committee/Examination Test-writing Committee
• 2015   Japanese Association for Laboratory Animal Medicine and Japanese College of Laboratory Animal Medicine   Joint Ad hoc Committee of for the Development of the Position Statement for Analgesia, Anesthesia, and Euthanasia of Rodent Fetuses and Neonates

Social Contribution

Social Contribution

Social Contribution

• AAALAC International Ad Hoc Specialist

  Date (from-to) : 2017/09/01

  Role : Advisor

Academic Contribution

• 日本実験動物学会外部検証専門員

  Date (from-to) :2018/07/10

  Role: Academic research planning

  Type: Academic research