2024/12/26 更新

写真a

ナカジマ ケイイチ
中島 恵一
NAKAJIMA Kei-chi
所属
医学部 医学科 基礎医学講座 生化学講座
外部リンク

学位

  • 博士(農学) ( 2000年3月   東京農工大学 )

研究キーワード

  • 生化学

  • 分子生物学

  • 細胞生物学

  • 泌乳生理学

研究分野

  • ライフサイエンス / 細胞生物学

学歴

  • 旭川医科大学   医学部

    - 2021年3月

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    国名: 日本国

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  • 東京農工大学   連合農学研究科

    - 2000年3月

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    国名: 日本国

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経歴

  • 旭川医科大学医学部   生化学講座   講師

    2024年4月 - 現在

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  • 旭川医科大学医学部   生化学講座   助教

    2022年4月 - 2024年3月

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  • 農研機構   主任研究員

    2005年4月 - 2016年3月

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  • 岐阜大学大学院医学研究科   薬理学講座   助教

    2004年4月 - 2005年3月

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  • 岐阜大学医学部   薬理学講座   助教

    2003年6月 - 2004年3月

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論文

  • Inducible deletion of microRNA activity in kidney mesenchymal cells exacerbates renal fibrosis. 国際誌

    Hirofumi Sakuma, Keisuke Maruyama, Tatsuya Aonuma, Yuya Kobayashi, Taiki Hayasaka, Kohei Kano, Satoshi Kawaguchi, Kei-Ichi Nakajima, Jun-Ichi Kawabe, Naoyuki Hasebe, Naoki Nakagawa

    Scientific reports   14 ( 1 )   10963 - 10963   2024年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    MicroRNAs (miRNAs) are sequence-specific inhibitors of post-transcriptional gene expression. However, the physiological functions of these non-coding RNAs in renal interstitial mesenchymal cells remain unclear. To conclusively evaluate the role of miRNAs, we generated conditional knockout (cKO) mice with platelet-derived growth factor receptor-β (PDGFR-β)-specific inactivation of the key miRNA pathway gene Dicer. The cKO mice were subjected to unilateral ureteral ligation, and renal interstitial fibrosis was quantitatively evaluated using real-time polymerase chain reaction and immunofluorescence staining. Compared with control mice, cKO mice had exacerbated interstitial fibrosis exhibited by immunofluorescence staining and mRNA expression of PDGFR-β. A microarray analysis showed decreased expressions of miR-9-5p, miR-344g-3p, and miR-7074-3p in cKO mice compared with those in control mice, suggesting an association with the increased expression of PDGFR-β. An analysis of the signaling pathways showed that the major transcriptional changes in cKO mice were related to smooth muscle cell differentiation, regulation of DNA metabolic processes and the actin cytoskeleton, positive regulation of fibroblast proliferation and Ras protein signal transduction, and focal adhesion-PI3K/Akt/mTOR signaling pathways. Depletion of Dicer in mesenchymal cells may downregulate the signaling pathway related to miR-9-5p, miR-344g-3p, and miR-7074-3p, which can lead to the progression of chronic kidney disease. These findings highlight the possibility for future diagnostic or therapeutic developments for renal fibrosis using miR-9-5p, miR-344g-3p, and miR-7074-3p.

    DOI: 10.1038/s41598-024-61560-y

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  • NG2-positive pericytes regulate homeostatic maintenance of slow-type skeletal muscle with rapid myonuclear turnover. 国際誌

    Takamitsu Tatsukawa, Kohei Kano, Kei-Ichi Nakajima, Takashi Yazawa, Ryoji Eguchi, Maki Kabara, Kiwamu Horiuchi, Taiki Hayasaka, Risa Matsuo, Naoyuki Hasebe, Nobuyoshi Azuma, Jun-Ichi Kawabe

    Stem cell research & therapy   14 ( 1 )   205 - 205   2023年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    BACKGROUND: Skeletal muscle comprises almost 40% of the human body and is essential for movement, structural support and metabolic homeostasis. Size of multinuclear skeletal muscle is stably maintained under steady conditions with the sporadic fusion of newly produced myocytes to compensate for the muscular turnover caused by daily wear and tear. It is becoming clear that microvascular pericytes (PCs) exhibit myogenic activity. However, whether PCs act as myogenic stem cells for the homeostatic maintenance of skeletal muscles during adulthood remains uncertain. METHODS: We utilized PC-fused myofibers using PC-specific lineage tracing mouse (NG2-CreERT/Rosa-tdTomato) to observe whether muscle resident PCs have myogenic potential during daily life. Genetic PC deletion mouse model (NG2-CreERT/DTA) was used to test whether PC differentiates to myofibers for maintenance of muscle structure and function under homeostatic condition. RESULTS: Under steady breeding conditions, tdTomato-expressing PCs were infused into myofibers, and subsequently, PC-derived nuclei were incorporated into myofibers. Especially in type-I slow-type myofibers such as the soleus, tdTomato+ myofibers were already observed 3 days after PC labeling; their ratio reached a peak (approximately 80%) within 1 month and was maintained for more than 1 year. Consistently, the NG2+ PC-specific deletion induced muscular atrophy in a slow-type myofiber-specific manner under steady breeding conditions. The number of myonucleus per volume of each myofiber was constant during observation period. CONCLUSIONS: These findings demonstrate that the turnover of myonuclei in slow-type myofibers is relatively fast, with PCs acting as myogenic stem cells-the suppliers of new myonuclei under steady conditions-and play a vital role in the homeostatic maintenance of slow-type muscles.

    DOI: 10.1186/s13287-023-03433-1

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  • Chemerin Regulates Epithelial Barrier Function of Mammary Glands in Dairy Cows. 国際誌

    Yutaka Suzuki, Sachi Chiba, Koki Nishihara, Keiichi Nakajima, Akihiko Hagino, Won-Seob Kim, Hong-Gu Lee, Tomonori Nochi, Toru Suzuki, Sang-Gun Roh

    Animals   11 ( 11 )   2021年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Epithelial barrier function in the mammary gland acts as a forefront of the defense mechanism against mastitis, which is widespread and a major disorder in dairy production. Chemerin is a chemoattractant protein with potent antimicrobial ability, but its role in the mammary gland remains unelucidated. The aim of this study was to determine the function of chemerin in mammary epithelial tissue of dairy cows in lactation or dry-off periods. Mammary epithelial cells produced chemerin protein, and secreted chemerin was detected in milk samples. Chemerin treatment promoted the proliferation of cultured bovine mammary epithelial cells and protected the integrity of the epithelial cell layer from hydrogen peroxide (H2O2)-induced damage. Meanwhile, chemerin levels were higher in mammary tissue with mastitis. Tumor necrosis factor alpha (TNF-α) strongly upregulated the expression of the chemerin-coding gene (RARRES2) in mammary epithelial cells. Therefore, chemerin was suggested to support mammary epithelial cell growth and epithelial barrier function and to be regulated by inflammatory stimuli. Our results may indicate chemerin as a novel therapeutic target for diseases in the bovine mammary gland.

    DOI: 10.3390/ani11113194

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  • Ninjurin 1 mediates peripheral nerve regeneration through Schwann cell maturation of NG2-positive cells. 国際誌

    Yui Tomita, Kiwamu Horiuchi, Kohei Kano, Takamitsu Tatsukawa, Risa Matsuo, Taiki Hayasaka, Yuri Yoshida, Maki Kabara, Satoshi Yasuda, Keiichi Nakajima, Naoki Nakagawa, Naofumi Takehara, Atsutaka Okizaki, Naoyuki Hasebe, Jun-Ichi Kawabe

    Biochemical and biophysical research communications   519 ( 3 )   462 - 468   2019年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Ninjurin 1 (Ninj1) is identified as a peripheral nerve injury-induced protein. However, the role of Ninj1 in nerve regeneration is unclear. Schwann cells (SCs) and microvasculature are critical for peripheral nerve regeneration. SCs precursors and microvascular pericytes (PCs), which are nerve/glial antigen 2 (NG2)-positive cells are observed in peripheral nervous system. In this study, we investigated the role of Ninj1 in peripheral nerve regeneration using NG2+cell-specific inducible deletion of Ninj1 mouse model. The number of NG2+cells, which were associated with and without microvessels was increased after sciatic nerve crush injury. There was a significant increase in the expression of Ninj1 and EphA7 in the injured nerve tissue. This increase was mostly observed in NG2+cells. Genetic tracing of NG2+cells was performed using tamoxifen (Tam) treatment on NG2CreERT:R26R-tdTomato mice. The sciatic nerve was injured following the Tam-treatment, then tdTomato-expressing SCs were mostly observed in regenerated SCs at 21 days after nerve injury. Ninj1 gene knockout (Ninj1 KO) in NG2+cells was induced using NG2CreERT:Ninj1loxp mice. Tam-treated-NG2CreERT or Tam-nontreated NG2CreERT:Ninj1loxp mice were used as controls. Following Tam-treatment, the sciatic nerve in each group was injured. Ninj1KO significantly attenuated the expression of the myelin binding protein (MBP) as well as the number of myelinated axons. The expression of MBP in cultured SCs was significantly reduced by SiRNA-mediated Ninj1 knockdown (KD). Ninj1KD also attenuated the differentiation of SCs by isolated EphA7+multipotent PCs. The current data indicate that Ninj1 plays a vital role in peripheral nerve regeneration. This is observed particularly in the myelination process of NG2+cells including SCs precursors and multipotent PCs.

    DOI: 10.1016/j.bbrc.2019.09.007

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  • Response of the denitrifier community and its relationship with multiple N2O emission peaks after mature compost addition into dairy manure compost with forced aeration. 国際誌

    Koki Maeda, Fumihito Miyatake, Ryoki Asano, Kei-Ichi Nakajima, Takeki Maeda, Kazunori Iwabuchi

    Chemosphere   206   310 - 319   2018年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    Animal manure is a source of the greenhouse gas nitrous oxide (N2O), therefore understanding the mechanisms underlying its production is essential for developing mitigating strategies and sustainable livestock production system. In this study, microbial communities potentially involved in multiple emission peaks during initial stage of laboratory-scale dairy manure composting with forced aeration system were investigated. Mature compost was used for the bulking agent. Change of overall bacterial community and nitrification-denitrification gene abundance were monitored by using 16S rRNA gene amoA, nirS, nirK or nosZ genes, respectively. Three N2O emission peaks were observed when the temperature reached at 45, 60 and 72 °C, at the same timing of oxygen consumption peaks. The maximum N2O emission peak was 3.86 mg h-1 kg-1 TS when the temperature reached at 60 °C. The shift of bacterial community among these experimental periods was significant, orders Flavobacteriales, Burkholderiales and Xanthomonadales increased, while orders belong to Bacillales, Lactobacillales, Clostridiales and Bacteroidales decreased. In addition, abundance of two denitrification genes (nirS and nosZ) significantly increased during this period. Clone library analysis of these genes showed that significantly increased sequences belonged to Pseudomonas-like clusters for both genes, indicates that denitrifiers possesses these genes are involved for these N2O emission peaks caused by mature compost addition.

    DOI: 10.1016/j.chemosphere.2018.04.169

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  • Downregulated angiopoietin-like protein 8 production at calving related to changes in lipid metabolism in dairy cows. 国際誌

    Misato Nakano, Yutaka Suzuki, Satoshi Haga, Eri Yamauchi, Dahye Kim, Koki Nishihara, Keiichi Nakajima, Takafumi Gotoh, Seungju Park, Myunggi Baik, Kazuo Katoh, Sanggun Roh

    Journal of animal science   96 ( 7 )   2646 - 2658   2018年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Acute physiological adaptation of lipid metabolism during the postpartum transition period of cows facilitates peripheral metabolic regulation. Hepatokines, which are hormones secreted from hepatocytes, are presumed to play a critical role in systemic metabolic regulation. Angiopoietin-like protein 8 (ANGPTL8) has been identified as a novel hepatokine associated with circulating triglyceride concentrations in mice and humans. However, regulation of ANGPTL8 and its physiological effects is still unknown in cattle. The present study aimed to reveal changes in ANGPTL8 expression and secretion during the periparturient period, and to investigate its regulatory effect on adipocytes and mammary epithelial cells. In the peripartum period, liver ANGPTL8 mRNA expression was lesser on the day of parturition and 1 wk postpartum than it was 1 wk before parturition (P < 0.05). Moreover, plasma ANGPTL8 concentrations decreased on the day of parturition as compared with that 1 wk before parturition (P < 0.05). In addition, ANGPTL8 expression in cultured bovine hepatocytes was downregulated after oleate and palmitate treatment but upregulated after insulin treatment (P < 0.05). ANGPTL8 decreased hormone-sensitive lipase (HSL) expression in differentiated adipocytes and cluster of differentiation 36 (CD36), fatty acid synthase (FAS), acetyl-coa carboxylase (ACC), and stearoyl-coa desaturase (SCD) in cultured bovine mammary epithelial cells (P < 0.05). These data suggest that hepatic ANGPTL8 production was downregulated postpartum when the cows experienced a negative energy balance. This downregulation was associated with increased concentrations of NEFA and decreased concentrations of insulin in lactating cows, and it facilitated lipid mobilization from adipose tissue to the mammary glands. We speculate that ANGPTL8 might have beneficial effects in reverting or improving the physiological adaptation and pathological processes of lipid metabolism during the peripartum period.

    DOI: 10.1093/jas/sky162

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  • Innate immune response of bovine mammary epithelial cells to Mycoplasma bovis

    Satoshi Gondaira, Hidetoshi Higuchi, Hidetomo Iwano, Koji Nishi, Takanori Nebu, Keiichi Nakajima, Hajime Nagahata

    JOURNAL OF VETERINARY SCIENCE   19 ( 1 )   79 - 87   2018年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:KOREAN SOC VETERINARY SCIENCE  

    Mycoplasma spp. are contagious bacteria, and mycoplasmal mastitis is a serious productivity problem on dairy farms. Bovine mammary epithelial cells (bMECs) have an important role in the elimination of pathogens, but the effect of Mycoplasma bovis on bMECs has not been fully described. To elucidate the immune response against intramammary infection by M. bovis, we undertook microarray analysis to examine and profile mRNA expression in bMECs after stimulation with M. bovis. We also compared the effects of M. bovis, Staphylococcus aureus, and Escherichia coli on immune-related mRNA expression in bMECs. Transcriptome analysis indicated a significant decrease in the level of mRNA-encoding lysine-specific demethylase 4D, suggesting that the immune response is suppressed by a decrease in histone demethylase activity. Interleukin (IL)-1 beta, IL-6, tumor necrosis factor alpha, toll-like receptor (TLR) 2, and TLR4 mRNA expression levels were significantly increased in bMECs stimulated with heat-killed M. bovis, but the expression levels were lower than those following stimulation by heat-killed S. aureus or E. coli. Our results suggest that M. bovis weakly affects mRNA expression in bMECs compared to the effects of E. coli or S. aureus. Moreover, live M. bovis may induce suppression of the immune response in bMECs.

    DOI: 10.4142/jvs.2018.19.1.79

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  • Relative Contribution of nirK- and nirS- Bacterial Denitrifiers as Well as Fungal Denitrifiers to Nitrous Oxide Production from Dairy Manure Compost. 国際誌

    Koki Maeda, Sakae Toyoda, Laurent Philippot, Shohei Hattori, Keiichi Nakajima, Yumi Ito, Naohiro Yoshida

    Environmental science & technology   51 ( 24 )   14083 - 14091   2017年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The relative contribution of fungi, bacteria, and nirS and nirK denirifiers to nitrous oxide (N2O) emission with unknown isotopic signature from dairy manure compost was examined by selective inhibition techniques. Chloramphenicol (CHP), cycloheximide (CYH), and diethyl dithiocarbamate (DDTC) were used to suppress the activity of bacteria, fungi, and nirK-possessing denitrifiers, respectively. Produced N2O were surveyed to isotopocule analysis, and its 15N site preference (SP) and δ18O values were compared. Bacteria, fungi, nirS, and nirK gene abundances were compared by qPCR. The results showed that N2O production was strongly inhibited by CHP addition in surface pile samples (82.2%) as well as in nitrite-amended core samples (98.4%), while CYH addition did not inhibit the N2O production. N2O with unknown isotopic signature (SP = 15.3-16.2‰), accompanied by δ18O (19.0-26.8‰) values which were close to bacterial denitrification, was also suppressed by CHP and DDTC addition (95.3%) indicating that nirK denitrifiers were responsible for this N2O production despite being less abundant than nirS denitrifiers. Altogether, our results suggest that bacteria are important for N2O production with different SP values both from compost surface and pile core. However, further work is required to decipher whether N2O with unknown isotopic signature is mostly due to nirK denitrifiers that are taxonomically different from the SP-characterized strains and therefore have different SP values rather than also being interwoven with the contribution of the NO-detoxifying pathway and/or of co-denitrification.

    DOI: 10.1021/acs.est.7b04017

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  • Retinoic acid modulates lipid accumulation glucose concentration dependently through inverse regulation of SREBP-1 expression in 3T3L1 adipocytes. 国際誌

    Mabrouk Attia Abd Eldaim, Shinya Matsuoka, Yuko Okamatsu-Ogura, Akihiro Kamikawa, Mohamed Mohamed Ahmed, Akira Terao, Kei-Ichi Nakajima, Kazuhiro Kimura

    Genes to cells : devoted to molecular & cellular mechanisms   22 ( 6 )   568 - 582   2017年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    It is well known that retinoic acid (RA) suppresses adipogenesis, although there are some contradicting reports. In this study, we examined the effect of extracellular glucose on RA-induced suppression of adipogenesis in 3T3L1 cell culture. When the cells were cultured in normal glucose medium (NG), the addition of RA suppressed lipid accumulation. However, when cultured in high glucose medium (HG), addition of RA to the cells enhanced lipid accumulation. These changes were accompanied by parallel alterations in fatty acid synthase (FAS) and sterol regulatory element-binding protein (SREBP)-1 gene expression. Transfection of SREBP-1 siRNA suppressed RA-induced enhancement of lipid accumulation and FAS expression in the cells cultured with HG. Transfection of the nuclear form of SREBP-1a cDNA into the cells cultured with NG inhibited RA-induced suppression of lipid accumulation and FAS expression. Moreover, RA- and HG-induced SREBP-1a expression occurred at the early phase of adipogenesis and was dependent on glucocorticoid to induce liver X receptor (LXR) β, peroxisomal proliferator-activated receptor (PPAR) γ and retinoid X receptor (RXR), the key nuclear factors influencing the SREBP-1a gene expression. These results suggest that RA suppresses and enhances lipid accumulation through extracellular glucose concentration-dependent modulation of SREBP-1 expression.

    DOI: 10.1111/gtc.12498

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  • The cell wall component lipoteichoic acid of Staphylococcus aureus induces chemokine gene expression in bovine mammary epithelial cells.

    Yoshio Kiku, Yuya Nagasawa, Fuyuko Tanabe, Kazue Sugawara, Atsushi Watanabe, Eiji Hata, Tomomi Ozawa, Kei-Ichi Nakajima, Toshiro Arai, Tomohito Hayashi

    The Journal of veterinary medical science   78 ( 9 )   1505 - 1510   2016年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JAPAN SOC VET SCI  

    Staphylococcus aureus (SA) is a major cause of bovine mastitis, but its pathogenic mechanism remains poorly understood. To evaluate the role of lipoteichoic acid (LTA) in the immune or inflammatory response of SA mastitis, we investigated the gene expression profile in bovine mammary epithelial cells stimulated with LTA alone or with formalin-killed SA (FKSA) using cap analysis of gene expression. Seven common differentially expressed genes related to immune or inflammatory mediators were up-regulated under both LTA and FKSA stimulations. Three of these genes encode chemokines (IL-8, CXCL6 and CCL2) functioning as chemoattractant molecules for neutrophils and macrophages. These results suggest that the initial inflammatory response of SA infection in mammary gland may be related with LTA induced chemokine genes.

    DOI: 10.1292/jvms.15-0706

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  • Isotopically enriched ammonium shows high nitrogen transformation in the pile top zone of dairy manure compost

    Koki Maeda, Sakae Toyoda, Midori Yano, Shohei Hattori, Makoto Fukasawa, Keiichi Nakajima, Naohiro Yoshida

    BIOGEOSCIENCES   13 ( 4 )   1341 - 1349   2016年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:COPERNICUS GESELLSCHAFT MBH  

    Nitrogen isotope ratios (delta N-15) of NH4+ in dairy manure compost piles with and without bulking agent (10% w/w) were compared to understand the effects of the use of bulking agent on nitrogen conversion during manure composting. The delta N-15-NH4+ values in each of three pile zones (top, side and core) were also compared. At the end of the process, piles with bulking agent showed significantly higher delta N-15 values (17.7 +/- 1.3 parts per thousand) than piles without bulking agent (11.8 +/- 0.9 parts per thousand), reflecting the significantly higher nitrogen conversion and NH3 loss in the former. The samples from the top zone, especially in the piles with bulking agent, showed very high NH4+ concentrations with significantly high 15N (delta N-15: 12.7-29.8 parts per thousand) values, indicating that extremely high nitrogen conversion, nitrification-denitrification activity of the microbes and NH3 volatilization occurred in this zone.

    DOI: 10.5194/bg-13-1341-2016

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  • 反芻動物におけるヘパトカインとしてのChemerinとANGPTL8の発現調節 査読

    鈴木, 裕, 中野, 美智, 芳賀, 聡, 中島, 恵一, 加藤, 和雄, 盧, 尚建

    栄養生理研究会報   59 ( 2 )   59 - 68   2015年10月

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)   出版者・発行元:家畜栄養生理研究会  

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    その他リンク: http://id.ndl.go.jp/bib/026859396

  • Cytokine mRNA profiling and the proliferative response of bovine peripheral blood mononuclear cells to Mycoplasma bovis

    Satoshi Gondaira, Hidetoshi Higuchi, Hidetomo Iwano, Keiichi Nakajima, Kazuhiro Kawai, Shuhei Hashiguchi, Satoru Konnai, Hajime Nagahata

    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY   165 ( 1-2 )   45 - 53   2015年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER  

    Mycoplasma bovis is known as a significant pathogen and cause of large economic losses in beef and dairy calves worldwide. Numerous factors appear to play an important role in the development of disease during infection with M. bovis, e.g., inhibition of immune cell proliferation and induction of lymphocyte apoptosis. However, the mechanisms involved in M. bovis infections have not been explored and remain incompletely understood. We investigated the major cytokine mRNA expression in bovine PBMC stimulated with M. bovis, for comparison, Staphylococcus aureus and Escherichia coli, which are the representative mastitis-causing pathogens. Here we demonstrated that live M. bovis significantly induced tumor necrosis factor alpha (TNF-alpha), interleukin 12p40 (IL-12), and interferon gamma (IFN-gamma) mRNA expression in bovine peripheral blood mononuclear cells (PBMC) at a multiplicity of infection (MOI) of 1000 but not at an MOI of 10 and 100. Live M. bovis at MOIs of 1, 10, and 100 induced significant bovine PBMC proliferative responses compared with unstimulated bovine PBMC. Furthermore, we showed that the cultural supernatant of M. bovis induced a significant increase in TNF-alpha, IL-6, and IL-10 mRNA expression in bovine PBMC. Our results suggest that M. bovis weakly affects the cellular integrity of bovine PBMC and induces clear proliferative responses and associated cytokine production in them. However, large numbers of live M. bovis are required to induce an immune response in bovine PBMC. (C) 2015 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.vetimm.2015.03.002

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  • Short communication: opposing effects of lactoferrin on the proliferation of fibroblasts and epithelial cells from bovine mammary gland. 国際誌

    K Nakajima, F Itoh, M Nakamura, A Kawamura, T Yamazaki, T Kozakai, N Takusari, A Ishisaki

    Journal of dairy science   98 ( 2 )   1069 - 77   2015年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Lactoferrin is present in several physiologic fluids, including milk and colostrum. Recently, evidence has accumulated that lactoferrin acts as a regulator of cell proliferation. Lactoferrin mRNA and protein levels in bovine mammary glands are known to markedly increase after cessation of milking. To clarify the role of bovine lactoferrin (bLF) in mammary involution and remodeling during dry periods, we investigated whether bLF affects the proliferation of cultured cells derived from bovine mammary gland and examined the mechanism underlying the proliferative response to bLF. Addition of bLF to the culture medium increased the proliferation of bovine mammary stromal fibroblasts (bMSF), but decreased that of bovine mammary epithelial cells (bMEC). Proliferation was significantly increased in the bMSF treated with bLF (100μg/mL or greater) as compared with unstimulated cells. The maximal proliferative effect of bLF on bMSF occurred at 1,000μg/mL, such that the proliferation of the bLF-stimulated bMSF was approximately 2.5 times that of unstimulated cells. The bLF increased the production of proliferating cell nuclear antigen and rapid phosphorylation of the p44/p42 mitogen-activated protein kinase in bMSF, but not in bMEC. The bLF-induced proliferation and production of proliferating cell nuclear antigen in bMSF was suppressed by U0126, a specific inhibitor of mitogen-activated protein kinase. Furthermore, treatment with bLF for 24h decreased the mRNA levels of the 3 isoforms of transforming growth factor β in bMSF (16-66%) but upregulated those in bMEC (122-157%). These opposite effects of bLF on the proliferation of epithelial and fibroblast cells and their expression of transforming growth factor β may play a crucial role in bovine mammary involution and remodeling.

    DOI: 10.3168/jds.2014-8430

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  • 初産牛の一乳期および乳期別TMR給与法が産乳性,栄養,体重および収益に及ぼす影響

    中村 正斗, 中島 恵一, 早坂 貴代史

    日本畜産学会報   86 ( 4 )   465 - 472   2015年

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    記述言語:日本語   出版者・発行元:公益社団法人 日本畜産学会  

    ホルスタイン種初産牛における,TMR(完全混合飼料)の乳期別2種飼養(2群区)に対する一乳期1種飼養(1群区)の有効性を検討するために,それらの産乳性,栄養,体重および収益を比較した.TMRはグラスサイレージ,配合飼料,大豆粕からなり,1群区は一乳期をTDN72%,CP18%で,2群区は泌乳前期を1群区と同一とし,泌乳後期をTDN69%,CP15%で,各区9頭に不断給飼した.2群区は1群区に比べ,泌乳後期の産乳量,乾物摂取量,TDNとCPの充足率が減少し,泌乳持続性および乾乳時の体重とボディコンディションスコアが有意に低かった.2群区は,305日間の平均増体重がRichards成長曲線に基づく標準増体重を下回り,栄養不足が観察された.1群区は2群区に比べ乳飼比は上がるが,乳代から飼料費を控除した305日間の収益差額は1頭当たり14千円増益と試算された.

    DOI: 10.2508/chikusan.86.465

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  • Mycoplasma species isolated from intramammary infection of Japanese dairy cows. 国際誌

    H Higuchi, S Gondaira, H Iwano, K Hirose, K Nakajima, K Kawai, K Hagiwara, Y Tamura, H Nagahata

    The Veterinary record   172 ( 21 )   557 - 557   2013年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1136/vr.101228

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  • Fatty acid-binding protein expression in the gastrointestinal tract of calves and cows

    Hideaki Hayashi, Sachiko Maruyama, Madoka Fukuoka, Takaharu Kozakai, Keiichi Nakajima, Takenori Onaga, Seiyu Kato

    ANIMAL SCIENCE JOURNAL   84 ( 1 )   35 - 41   2013年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY  

    Fatty acid-binding protein (FABP) has high affinity for long-chain fatty acids and appears to participate in the metabolism and intracellular transport of lipids. Liver- and intestinal-type FABP (L-FABP and I-FABP, respectively) are expressed in the small intestine. However, in the gastrointestinal tract of ruminants, expression and localization of FABPs are unknown. In this study, we investigated the expression of I-FABP and L-FABP in the gastrointestinal tract of cattle. I- and L-FABP had higher messenger RNA (mRNA) and protein expression levels in the duodenum and jejunum relatively to other gastrointestinal regions in both calves and cows. Furthermore, L-FABP mRNA and protein expression were high in the colon. Both these protein types were confirmed to be in the cytosol of jejunal epithelial cells, where they were found in the villi rather than in the crypts. We concluded that duodenal and jejunal FABPs might be involved in the metabolism of fatty acids mainly in epithelial cells in cattle.

    DOI: 10.1111/j.1740-0929.2012.01038.x

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  • 乾乳期間短縮が次乳期の乳量・乳成分に及ぼす影響

    中村 正斗, 中島 恵一, 高橋 雄治, 塩野 浩紀

    日本畜産学会報   84 ( 3 )   349 - 359   2013年

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    記述言語:日本語   出版者・発行元:公益社団法人 日本畜産学会  

    ホルスタイン種乳牛の乾乳期間短縮が,次乳期の乳量・乳成分に及ぼす影響を明らかにするため,分娩前2ヵ月から分娩後10ヵ月まで比較検討した.短縮区は泌乳後期牛15頭を用い,搾乳日数を延長し,分娩予定の30日前に乾乳し,対照区は平均乾乳期間101日の乳牛16頭を用いた.分娩後3ヵ月間は両区ともグラスサイレージ(GS)と配合飼料を混合して不断給飼した.その後は日本飼養標準にしたがって,GS, コーンサイレージ,配合飼料,大豆粕を給与し,乾草を不断給飼した.305日乳量は,乾乳期間の効果および産次の効果がともに有意で,2産のウシでは短縮区の乳量が有意に減少した.305日乳量に短縮区の搾乳延長による乳量を加えた総乳量は,3産以上のウシが有意に多かったが,区間に差はなく,乾乳期間短縮による乳量減少は相殺された.305日の乳成分率と乳中体細胞数は区間に差がなかった.短縮区の分娩後の体重回復は対照区に比べ有意に早くなり,乳量減少の影響が示唆された.

    DOI: 10.2508/chikusan.84.349

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    その他リンク: http://id.ndl.go.jp/bib/024855148

  • Alg14 organizes the formation of a multiglycosyltransferase complex involved in initiation of lipid-linked oligosaccharide biosynthesis. 国際誌

    Jishun Lu, Tetsuo Takahashi, Atsuko Ohoka, Kei-ichi Nakajima, Ryo Hashimoto, Nobuaki Miura, Hiroyuki Tachikawa, Xiao-Dong Gao

    Glycobiology   22 ( 4 )   504 - 16   2012年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS INC  

    Protein N-glycosylation begins with the assembly of a lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER) membrane. The first two steps of LLO biosynthesis are catalyzed by a functional multienzyme complex comprised of the Alg7 GlcNAc phosphotransferase and the heterodimeric Alg13/Alg14 UDP-GlcNAc transferase on the cytosolic face of the ER. In the Alg13/14 glycosyltransferase, Alg14 recruits cytosolic Alg13 to the ER membrane through interaction between their C-termini. Bioinformatic analysis revealed that eukaryotic Alg14 contains an evolved N-terminal region that is missing in bacterial orthologs. Here, we show that this N-terminal region of Saccharomyces cerevisiae Alg14 localize its green fluorescent protein fusion to the ER membrane. Deletion of this region causes defective growth at 38.5°C that can be partially complemented by overexpression of Alg7. Coimmunoprecipitation demonstrated that the N-terminal region of Alg14 is required for direct interaction with Alg7. Our data also show that Alg14 lacking the N-terminal region remains on the ER membrane through a nonperipheral association, suggesting the existence of another membrane-binding site. Mutational studies guided by the 3D structure of Alg14 identified a conserved α-helix involved in the second membrane association site that contributes to an integral interaction and protein stability. We propose a model in which the N- and C-termini of Alg14 coordinate recruitment of catalytic Alg7 and Alg13 to the ER membrane for initiating LLO biosynthesis.

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  • Bovine milk lactoferrin induces synthesis of the angiogenic factors VEGF and FGF2 in osteoblasts via the p44/p42 MAP kinase pathway. 国際誌

    Kei-Ichi Nakajima, Yosuke Kanno, Masato Nakamura, Xiao-Dong Gao, Asami Kawamura, Fumiaki Itoh, Akira Ishisaki

    Biometals   24 ( 5 )   847 - 56   2011年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    Lactoferrin (LF) belongs to the transferrin family and is present in several physiological fluids, including milk and colostrum. LF has recently been identified as an anabolic factor for bone. Here we investigated whether bovine LF (bLF) induces synthesis of angiogenic factors by osteoblasts. If so, we examined the underlying mechanism. We found that bLF purified from milk increased the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) in murine osteoblast-like MC3T3-E1 cells and primary murine osteoblasts in a time- and dose-dependent manner. Furthermore, bLF increased VEGF and FGF2 protein levels in MC3T3-E1 cells. In addition, treatment of MC3T3-E1 cells with bLF rapidly induced phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase. The bLF-mediated increases in VEGF and FGF2 mRNA and protein were inhibited by U0126, a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Taken together, our results strongly suggest that bLF induces VEGF and FGF2 synthesis in a p44/p42 MAP kinase-dependent manner in MC3T3-E1 cells.

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  • 泌乳持続性向上に関するウシ乳腺の細胞生物学的解析 : ラクトフェリンが乳腺を構成する細胞の増殖に及ぼす影響

    中島, 恵一, 中村, 正斗, 伊藤, 文彰

    栄養生理研究会報   55 ( 2 )   121 - 122   2011年10月

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)   出版者・発行元:家畜栄養生理研究会  

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    その他リンク: https://agriknowledge.affrc.go.jp/RN/2010814867

  • Plasminogen/plasmin modulates bone metabolism by regulating the osteoblast and osteoclast function. 国際誌

    Yosuke Kanno, Akira Ishisaki, Eri Kawashita, Naoyuki Chosa, Keiichi Nakajima, Tatsuji Nishihara, Kuniaki Toyoshima, Kiyotaka Okada, Shigeru Ueshima, Kenji Matsushita, Osamu Matsuo, Hiroyuki Matsuno

    The Journal of biological chemistry   286 ( 11 )   8952 - 60   2011年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg(-/-) mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg(-/-) OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg(-/-) mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg(-/-) OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg(-/-) mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.

    DOI: 10.1074/jbc.M110.152181

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  • 乾乳期短縮が泌乳前期の乳量・乳成分,血液成分,疾病発生および繁殖性に及ぼす影響

    中村 正斗, 中島 恵一, 高橋 雄治

    日本畜産学会報   82 ( 1 )   25 - 34   2011年

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    記述言語:日本語   出版者・発行元:公益社団法人 日本畜産学会  

    ホルスタイン種乳牛の乾乳期30日への短縮が,次の泌乳前期の乳量・乳成分,TDN充足率,血液成分,疾病発生および繁殖性に及ぼす影響を明らかにするため,分娩前2ヵ月から分娩後3ヵ月まで飼養試験を行った.短縮区は泌乳後期牛14頭を用い,グラスサイレージ(GS)主体給与で搾乳日数を延長,分娩予定の30日前に乾乳し,乾乳後は分娩まで乾草,GS,配合飼料を給与した.対照区は平均乾乳期間110日の乾乳牛14頭を用い,分娩前1ヵ月までは乾草のみ,それ以後は短縮区と同じ飼料構成で給与した.分娩後は両区ともGSと配合飼料を混合して不断給餌した.その結果,乾乳期30日への短縮は,泌乳前期の乳量が抑えられ(<I>P</I> < 0.05),乳タンパク質率が増加し(<I>P</I> < 0.05),体重減少やボディコンディションスコアの低下が小さく(<I>P</I> < 0.05),血液成分では血糖値が上昇傾向(<I>P</I> < 0.10),遊離脂肪酸濃度が低下(<I>P</I> < 0.05)を示し,泌乳前期の栄養状態の改善が推察された.また短縮区は対照区に比べ泌乳前期のTDN充足率が上昇する傾向(<I>P</I> < 0.10)を示した.繁殖性と疾病発生に及ぼす乾乳期短縮の悪い影響は認められなかったが,短縮区の在胎日数が短かった(<I>P</I> < 0.05).

    DOI: 10.2508/chikusan.82.25

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    その他リンク: https://search.jamas.or.jp/link/ui/2011157438

  • Cloning, expression analysis, and regulatory mechanisms of bovine chemerin and chemerin receptor. 国際誌

    S-H Song, K Fukui, K Nakajima, T Kozakai, S Sasaki, S-G Roh, K Katoh

    Domestic animal endocrinology   39 ( 2 )   97 - 105   2010年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Recently, we reported that chemerin, a new adipokine, is highly expressed in the adipose tissue, up-regulated during adipocyte differentiation, and regulates adipogenesis via its own receptor in mice. The objectives of this study were to clone chemerin and its receptor from the adipose tissues of Japanese Black cattle and to investigate the expression of these genes in 16 different tissues. We compared the gene expression of chemerin and its receptor between adipocytes and stromal-vascular (S-V) cells (non-adipocytes) prepared from subcutaneous adipose tissue. In addition, we investigated the mRNA expression levels of chemerin and its receptor in bovine differentiated adipocytes. The DNA sequences of bovine chemerin and its receptor were determined, and they were found to be highly homologous to those of humans, mice, and pigs. The amino acid sequences predicted for the full-length cDNA of bovine chemerin and its receptor were also similar to those of humans, mice, and pigs, suggesting that these genes have similar functions. Bovine chemerin mRNA was highly expressed in the adipose and liver tissues, and the transcripts of chemerin receptor were widely expressed in several tissues including adipose, muscle, liver, and brain tissues. The expression of bovine chemerin mRNA was higher in adipocytes than in S-V cells prepared from adipose tissue. The transcripts of chemerin and its receptor were up-regulated during adipocyte differentiation. Treatment with tumor necrosis factor (TNF)-alpha (10 ng/mL) in bovine differentiated adipocytes increased the mRNA expression of chemerin and its receptor. These results indicate that chemerin, a new adipokine highly expressed in the adipocytes of bovine adipose tissue, is the TNF-alpha-up-regulated gene with a role in adipogenesis.

    DOI: 10.1016/j.domaniend.2010.02.007

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  • Synergistic Effect of Dexamethasone and Prolactin on VEGF Expression in Bovine Mammary Epithelial Cells via p44/p42 MAP Kinase

    Kei-Ichi Nakajima, Masato Nakamura, Akira Ishisaki, Takaharu Kozakai

    ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES   22 ( 6 )   788 - 793   2009年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ASIAN-AUSTRALASIAN ASSOC ANIMAL PRODUCTION SOC  

    Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis under various physiological and pathological conditions. We found that the VEGF isoforms VEGF120. VEGF164, and VEGF188 were expressed in the bovine mammary gland and bovine mammary epithelial cells (bMECs). Expression of VEGF in the mammary gland was significantly higher during the lactation period than during the dry period. Although dexamethasone or prolactin alone had little effect on the expression of VEGF, that in dexamethasone-treated cells was significantly induced after additional treatment with prolactin. Furthermore, the VEGF expression induced by the combination of dexamethasone and prolactin was reduced by PD98059 in a dose-dependent manner. This combination also stimulated the phosphorylation of p44/p42 MAP kinase in these cells. These results strongly suggest that the combination of dexamethasone and prolactin stimulates VEGF expression in bMECs via p44/p42 MAP kinase.

    DOI: 10.5713/ajas.2009.80519

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  • The Effects of Fasting and Grazing on Na-glucose Cotransporter-1 (SGLT-1) Gene Expression of Rectal Epithelia in Beef Cattle

    T. Kozakai, K. Imura, K. Nakajima, S. Sakanoue, N. Watanabe

    ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES   22 ( 2 )   232 - 237   2009年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ASIAN-AUSTRALASIAN ASSOC ANIMAL PRODUCTION SOC  

    The expression of SGLT-1 mRNA has been reported in the small intestine of mammals and the rectum of chickens. However, the expression and functional significance of SGLT-1 in bovine rectum is not known. In this study, we studied the effects of fasting and grazing on SGLT-1 gene expression in biopsy epithelial tissue of bovine rectum. In Japanese Black beef cattle, i) SGLT-1 gene expression was measured by quantitative real-time PCR in the biopsy rectal epithelia samples obtained through an endoscope, ii) SGLT-1 gene expression in the rectal epithelial tissues increased at 48 and 72 h after fasting correlating with a decrease in body weight. iii) SGLT-1 gene expression decreased after one month from the start of grazing (May to June) and then stabilized until the end of the grazing period (June to October) in the rectal epithelial tissues of grazing cattle. In conclusion, it is clear that SGLT-1 gene expression in the rectal epithelial tissue is increased by a restricted dietary condition.

    DOI: 10.5713/ajas.2009.80114

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  • Possible involvement of prolactin in the synthesis of lactoferrin in bovine mammary epithelial cells. 国際誌

    Kei-ichi Nakajima, Masato Nakamura, Xiao-Dong Gao, Takaharu Kozakai

    Bioscience, biotechnology, and biochemistry   72 ( 4 )   1103 - 6   2008年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS LTD  

    Bovine mammary epithelial cells (bMECs) synthesize lactoferrin, which is secreted into milk. Our results suggest that prolactin stimulated secretion of lactoferrin in primary bMECs and their clonal cell line under serum-free conditions. Prolactin also stimulated mRNA expression of lactoferrin in the clonal cell line. This effect was reduced by AG-490, suggesting that the prolactin-stimulated mRNA expression of lactoferrin was mediated by Janus kinase (JAK)2.

    DOI: 10.1271/bbb.70713

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  • Molecular cloning and expression analysis of cDNA encoding bovine adipogenin

    Yeon-Hee Hong, Yumi Ogihara, Daisuke Hishikawa, Chizu Gotoh, Tomoyo Iga, Yasuki Suzuki, Sang-Houn Song, Keiichi Nakajima, Takaharu Kozakai, Shin-ichi Sasaki, Sang-Gun Roh

    ANIMAL SCIENCE JOURNAL   77 ( 6 )   613 - 619   2006年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY  

    Adipose tissue plays a central role in energy homeostasis. In a previous report, we reported adipogenin, an adipocyte-specific membrane protein whose expression is higher in fat and increases during the adipocyte differentiation of 3T3-L1 cells, as a candidate gene for involvement in adipogenesis. Here, we isolated the bovine adipogenin gene using in silico cloning and the target region for polymerase chain reaction amplification in the database. cDNA, including a 246 base pair (bp) open reading frame, was identified in the adipose tissues of cattle. In silico gene analysis showed that the bovine adipo-genin-encoded protein has a predicted MW of 9882.45, encoding 81 amino acids and located on bovine chromosome 13. The deduced amino acid sequence of bovine adipogenin conserved 86%, 66% and 78% identity with that in pigs, mice and humans, respectively. A semi-quantitative reverse transcriptase polymerase chain reaction showed that bovine adipogenin mRNA was more highly expressed in subcutaneous, perirenal and mescentric adipose tissue than in non-adipose tissue. The level of bovine adipogenin mRNA is dramatically elevated during the adipocyte differentiation of four different kinds of preadipocytes prepared from subcutaneous, perirenal, mesenteric and parametrial adipose tissues. Our results suggest that adipogenin plays an important role in the regulation of adipocyte development in cattle and may be a strong candidate gene for obesity in mammals.

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  • Differential roles of MAP kinases in atorvastatin-induced VEGF release in cardiac myocytes. 国際誌

    Keiichi Nakajima, Hidetaka Suga, Hiroyuki Matsuno, Akira Ishisaki, Kouseki Hirade, Osamu Kozawa

    Life sciences   79 ( 12 )   1214 - 20   2006年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Statins, specific inhibitors of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, are now widely used for treatment of patients with hypercholesterolemia. In addition to the reduction of cholesterol biosynthesis, accumulating evidence indicates that statins have several pleiotropic effects especially on cardiovascular system. However, the exact role of statin in cardiac myocytes remains unclear. In the present study, we investigated whether atorvastatin induces vascular endothelial growth factor (VEGF) release in cardiac myocytes, and the underlying mechanism. We observed that atorvastatin significantly stimulated VEGF release in a dose-dependent manner. It induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase but not SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). The atorvastatin-induced VEGF release was enhanced by PD98059, which is a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Further, it was significantly reduced by SB203580, a specific inhibitor of p38 MAP kinase. Furthermore, the atorvastatin-induced phosphorylation of p38 MAP kinase was attenuated by SB203580, whereas it was enhanced by PD98059. Taken together, these results suggest that the atorvastatin-induced VEGF release in cardiac myocytes is positively regulated by p38 MAP kinase and negatively regulated byp44/p42 MAP kinase and that the atorvastatin-induced phosphorylation of p38 MAP kinase is regulated by p44/p42 MAP kinase in these cells.

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  • Lack of alpha2-antiplasmin improves cutaneous wound healing via over-released vascular endothelial growth factor-induced angiogenesis in wound lesions. 国際誌

    Y Kanno, K Hirade, A Ishisaki, K Nakajima, H Suga, T Into, K Matsushita, K Okada, O Matsuo, H Matsuno

    Journal of thrombosis and haemostasis : JTH   4 ( 7 )   1602 - 10   2006年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    BACKGROUND: The fibrinolytic system is supposed to play an important role in the degradation of extracellular matrices for physiological and pathological tissue remodeling; however, the detailed mechanism regarding how this system affects cutaneous wound healing remains to be clarified. METHODS AND RESULTS: We performed experimental cutaneous wounding in mice with a deficiency of alpha(2)-antiplasmin (alpha(2)AP), which is a potent and specific plasmin inhibitor. We found that an accelerated wound closure was observed in alpha(2)AP-deficient (alpha(2)AP-/-) mice in comparison with wild type (WT) mice. Moreover, we observed that a greater increase of angiogenesis occurred in the process of wound healing in alpha(2)AP-/- mice than in the WT mice. Intriguingly, mRNA expression of vascular endothelial growth factor (VEGF), which is the best characterized positive regulator of angiogenesis, in wound lesions was found to show a greater increase in the early phase of the healing process in alpha(2)AP-/- mice than in WT mice. In addition, the amount of released-VEGF from the explanted fibroblasts of alpha(2)AP-/- mice increased dramatically more than in the WT mice. Finally, the intra-jugular administration of anti-VEGF antibody clearly suppressed the increased angiogenesis and accelerated wound closure in the wound lesion of alpha(2)AP-/- mice. CONCLUSION: The lack of alpha(2)AP markedly causes an over-release of VEGF from the fibroblasts in cutaneous wound lesions, thereby inducing angiogenesis around the area, and thus resulting in an accelerated-wound closure. CONCLUSIONS: This is the first report to describe the crucial role that alpha(2)AP plays following angiogenesis in the process of wound healing. Our results provide new insight into the role of alpha(2)AP on cutaneous wound healing.

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  • Possible involvement of phosphatidylinositol 3-kinase/Akt signal pathway in vasopressin-induced HSP27 phosphorylation in aortic smooth muscle A10 cells. 国際誌

    Hidetaka Suga, Keiichi Nakajima, En Shu, Yosuke Kanno, Kouseki Hirade, Akira Ishisaki, Hiroyuki Matsuno, Kumiko Tanabe, Shinji Takai, Shigeru Akamatsu, Kanefusa Kato, Yutaka Oiso, Osamu Kozawa

    Archives of biochemistry and biophysics   438 ( 2 )   137 - 45   2005年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We previously reported that p38 mitogen-activated protein (MAP) kinase takes a part in arginine vasopressin (AVP)-induced heat shock protein 27 (HSP27) phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) is involved in the phosphorylation of HSP27 in these cells. AVP time-dependently induced the phosphorylation of PI3K and Akt. Akt inhibitor, 1l-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, partially suppressed the phosphorylation of HSP27. The AVP-induced HSP27 phosphorylation was attenuated by LY294002, a PI3K inhibitor. The combination of Akt inhibitor and SB203580, a p38 MAP kinase inhibitor, completely suppressed the AVP-induced phosphorylation of HSP27. Furthermore, LY294002 or Akt inhibitor did not affect the AVP-induced phosphorylation of p38 MAP kinase and SB203580 did not affect the phosphorylation of PI3K or Akt. These results suggest that PI3K/Akt plays a part in the AVP-induced phosphorylation of HSP27, maybe independently of p38 MAP kinase, in aortic smooth muscle A10 cells.

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  • alphaB-crystallin is phosphorylated during myocardial infarction: involvement of platelet-derived growth factor-BB. 国際誌

    En Shu, Hiroyuki Matsuno, Shigeru Akamastu, Yosuke Kanno, Hidetaka Suga, Keiichi Nakajima, Akira Ishisaki, Shinji Takai, Kanefusa Kato, Yasuo Kitajima, Osamu Kozawa

    Archives of biochemistry and biophysics   438 ( 2 )   111 - 8   2005年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    alphaB-crystallin is the most abundant low-molecular-weight heat shock protein in heart and recent studies have demonstrated that it plays a cardioprotective role during myocardial infarction both in vivo and in vitro. On the other hand, platelet-derived growth factor (PDGF), a potent serum mitogen, has been reported to improve cardiac function after myocardial infarction. In the present study, using a mouse myocardial infarction model, we investigated whether alphaB-crystallin is phosphorylated during myocardial infarction and the implication of PDGF-BB. Phosphorylation of alphaB-crystallin at Ser-59 was time dependently induced and plasma PDGF-BB levels were concomitantly increased. Moreover, PDGF-BB-stimulated phosphorylation of alphaB-crystallin was suppressed by SB203580, a specific inhibitor of p38 mitogen-activated protein (MAP) kinase, in primary cultured cardiac myocytes. Our results indicate that PDGF-BB induces phosphorylation of alphaB-crystallin via p38 MAP kinase during myocardial infarction.

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  • Akt regulates thrombin-induced HSP27 phosphorylation in aortic smooth muscle cells: function at a point downstream from p38 MAP kinase. 国際誌

    Keiichi Nakajima, Kouseki Hirade, Akira Ishisaki, Hiroyuki Matsuno, Hidetaka Suga, Yosuke Kanno, En Shu, Yasuo Kitajima, Yoshihiro Katagiri, Osamu Kozawa

    Life sciences   77 ( 1 )   96 - 107   2005年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We previously reported that p38 MAP kinase takes part in thrombin-induced HSP27 phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether Akt is involved in the phosphorylation of HSP27 and the role of adenylyl cyclase-cAMP system. Thrombin time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) and Akt in aortic smooth muscle A10 cells. SB203580, a p38 MAP kinase inhibitor, significantly suppressed the thrombin-induced phosphorylation of Akt and the Akt inhibitor suppressed the phosphorylation of HSP27. Furthermore, the thrombin-induced phosphorylation of HSP27, p38 MAP kinase and Akt were decreased by dibutyryl-cAMP (DBcAMP). These results strongly suggest that Akt functions the thrombin-induced phosphorylation of HSP27 at a point downstream from p38 MAP kinase in aortic smooth muscle cells and the adenylyl cyclase-cAMP system is upstream regulator of the HSP27 phosphorylation in these cells.

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  • Methotrexate suppresses inflammatory agonist induced interleukin 6 synthesis in osteoblasts. 国際誌

    Minoru Yoshida, Yosuke Kanno, Akira Ishisaki, Haruhiko Tokuda, Kouseki Hirade, Keiichi Nakajima, Yoshihiro Katagiri, Katsuji Shimizu, Osamu Kozawa

    The Journal of rheumatology   32 ( 5 )   787 - 95   2005年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    OBJECTIVE: Interleukin 6 (IL-6) is a pleiotropic cytokine that plays a crucial role in the pathogenesis of rheumatoid arthritis (RA). In bone metabolism, it is known that IL-6 is produced and secreted by osteoblasts, and that IL-6 induces osteoclast formation and stimulates bone resorption. Various bone inflammatory agonists such as tumor necrosis factor-alpha (TNF-alpha), IL-1alpha, prostaglandin D2 (PGD2), PGE2, and PGF2alpha, which play important roles in the pathogenesis of RA, induce IL-6 synthesis in osteoblast-like MC3T3-E1 cells. Low dose methotrexate (MTX) is currently used for treatment of patients with RA. We investigated the effect of MTX on IL-6 synthesis induced by these agents in MC3T3-E1 cells. METHODS: Cultured cells were pretreated with various doses of MTX, and then stimulated by these inflammatory agonists. The IL-6 in the conditioned medium was measured by IL-6 enzyme immunoassay. RESULTS: MTX significantly suppressed IL-6 synthesis stimulated by these agonists in a dose-dependent manner, although MTX alone had no effect on the levels of IL-6. In addition, MTX significantly inhibited the enhancement by IL-17 of TNF-alpha-stimulated IL-6 synthesis. MTX reduced the levels of IL-6 induced by 12-O-tetradecanoylphorbol 13-acetate, a direct activator of protein kinase C (PKC), suggesting that MTX inhibits PKC signals for IL-6 synthesis. CONCLUSION: MTX suppresses IL-6 synthesis stimulated by various inflammatory agonists in osteoblasts.

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  • Adenylyl cyclase-cAMP system inhibits thrombin-induced HSP27 in vascular smooth muscle cells. 国際誌

    Kouseki Hirade, Kumiko Tanabe, Masayuki Niwa, Akira Ishisaki, Keiichi Nakajima, Mitsuhiro Nakamura, Tadashi Sugiyama, Yoshihiro Katagiri, Kanefusa Kato, Osamu Kozawa

    Journal of cellular biochemistry   94 ( 3 )   573 - 84   2005年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We previously reported that thrombin stimulates the induction of heat shock protein (HSP) 27 via p38 mitogen-activated protein (MAP) kinase activation in aortic smooth muscle A10 cells. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on the thrombin-stimulated induction of HSP27 in A10 cells. Forskolin, a direct activator of adenylyl cyclase, reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the HSP27 accumulation. Furthermore, dibutyryl-cAMP (DBcAMP), a permeable analog of cAMP, significantly suppressed the accumulation of HSP27. On the other hand, calphostin C, an inhibitor of protein kinase C (PKC), reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. Moreover, forskolin reduced the p38 MAP kinase phosphorylation induced by the 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, and significantly suppressed the TPA-stimulated accumulation of HSP27. These results indicate that adenylyl cyclase-cAMP system has an inhibitory role in thrombin-stimulated HSP27 induction in aortic smooth muscle cells, and the effect seems to be exerted on the thrombin-induced PKC- p38 MAP kinase signaling pathway.

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  • Adenylyl cyclase-cAMP system inhibits thyroid hormone-stimulated osteocalcin synthesis in osteoblasts. 国際誌

    Yosuke Kanno, Akira Ishisaki, Minoru Yoshida, Keiichi Nakajima, Haruhiko Tokuda, Osamu Numata, Osamu Kozawa

    Molecular and cellular endocrinology   229 ( 1-2 )   75 - 82   2005年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    It is generally recognized that thyroid hormone modulates osteoblast cell function. We have previously shown that triiodothyronine (T(3)) activates p38 mitogen-activated protein (MAP) kinase, resulting in the synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on thyroid hormone-stimulated osteocalcin synthesis in these cells. Dibutyryl-cAMP (DBcAMP) reduced the osteocalcin synthesis stimulated by T(3). Forskolin and cholera toxin suppressed the osteocalcin synthesis while dideoxyforskolin, a forskolin derivative that does not activate adenylyl cyclase, had little effect on the synthesis. KT5720, a selective inhibitor of protein kinase A, reversed the inhibitory effect of forskolin or DBcAMP. DBcAMP and forskolin markedly reduced the phosphorylation of p38 MAP stimulated by T(3). Pituitary adenylate cyclase-activating polypeptide (PACAP) significantly inhibited the T(3)-stimulated osteocalcin synthesis. These results strongly suggest that the adenylyl cyclase-cAMP system has an inhibitory role in thyroid hormone-stimulated osteocalcin synthesis via suppression of p38 MAP kinase activation in osteoblasts.

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  • Possible involvement of protein kinase C activation in differentiation of human umbilical vein endothelium-derived cell into smooth muscle-like cell. 国際誌

    Akira Ishisaki, Hironaka Tsunobuchi, Keiichi Nakajima, Toru Imamura

    Biology of the cell   96 ( 7 )   499 - 508   2004年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We previously reported that when deprived of fibroblast growth factor, human umbilical vein endothelium-derived cells (HUVE-DCs) are capable of differentiating into smooth muscle-like cells through activin A-induced, Smad-dependent signaling, and that maintenance of the endothelial-cell phenotype and differentiation into smooth muscle-like cells are reciprocally controlled by fibroblast growth factor-1 and activin A (Ishisaki et al., 2003). Here, we examined how protein kinase C (PKC), which plays pivotal roles in the regulation of cellular proliferation and differentiation in numerous cell types, might affect the above differentiation. We found that phorbol-12-myristate-13-acetate-induced down-regulations of some PKCs accompany suppressions of the expressions of smooth muscle cell markers in HUVE-DCs deprived of fibroblast growth factor. Moreover, the PKC-inhibitors Gö6850 and Gö6983 suppressed the differentiation of HUVE-DCs into smooth muscle-like cells. These results strongly suggest that activation of PKC is involved in the above differentiation.

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  • Vasopressin phosphorylates HSP27 in aortic smooth muscle cells. 国際誌

    Shigeru Akamatsu, Keiichi Nakajima, Akira Ishisaki, Hiroyuki Matsuno, Kumiko Tanabe, Mariko Takei, Motoki Takenaka, Kouseki Hirade, Naoki Yoshimi, Hidetaka Suga, Yutaka Oiso, Kanefusa Kato, Osamu Kozawa

    Journal of cellular biochemistry   92 ( 6 )   1203 - 11   2004年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Administration of arginine vasopressin (AVP) time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) at Ser-15 and Ser-85 in smooth muscle of aorta in vivo. The AVP-induced phosphorylation of HSP27 at Ser-15 and Ser-85 was inhibited by a V1a receptor antagonist but not by a V2 receptor antagonist. In cultured aortic smooth muscle A10 cells, AVP markedly stimulated the phosphorylation of HSP27 at Ser-15 and Ser-85. The AVP-induced phosphorylation of HSP27 was attenuated by SB203580 and PD169316, inhibitors of p38 mitogen-activated protein (MAP) kinase, but not by PD98059, a MEK inhibitor. These results strongly suggest that AVP phosphorylates HSP27 via p38 MAP kinase in aortic smooth muscle cells.

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  • Effect of a synthetic matrix metalloproteinase inhibitor (ONO-4817) on neointima formation in hypercholesterolemic hamsters. 国際誌

    Hiroyuki Matsuno, Akira Ishisaki, Keiichi Nakajima, Osamu Kozawa

    Journal of cardiovascular pharmacology   44 ( 1 )   57 - 65   2004年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Degradation of extracellular matrix (ECM) proteins plays an important role in the development of vascular remodeling. We investigated the alteration of matrix metalloproteinases (MMPs) on the development of neointima formation and the effect of a newly synthesized MMP inhibitor using hypercholesterolemic hamsters. Endothelial injury was achieved by a catheter in the hamster carotid artery. Two weeks after the injury, neointima was detected in all hamsters. Oral administration (twice a day) of ONO-4817 was started 2 hours before injury and continued for the next 2 weeks. The neointimal area, with appearance of maze-like structures, was markedly reduced by 52.4 +/- 8.4% by treatment with ONO-4817 at a dose of 20 mg/kg per day. The treatment by ONO-4817 (20 mg/kg per day) significantly reduced the indexes of histone H1 on day 1, 5, and 10 and the BrdU index of intimal smooth muscle cells on day 5 and 10, but not on day 1. Whereas DNA synthesis was not reduced by ONO-4817, in vitro SMC migration on the other hand was reduced dose dependently. According to the results of western-blotting analysis, the expressions of MMPs were increased 1 week after injury. Especially, MMP-12 was not detected in hamsters without cholesterol diet, but it was much increased after injury in hypercholesterolemic hamsters. Additionally, active form of MMP-12 increased in the injured artery of hypercholesterolemic hamsters. In conclusion, inhibition of MMPs results in the suppression of neointima following vascular injury via both prevention of SMC migration and SMC proliferation of late phase in hypercholesterolemic hamsters. MMP-12 plays an important role on vascular stenosis in hypercholesterolemia and ONO-4817 could be a useful compound for the therapy for this field.

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  • Involvement of SAPK/JNK in prostaglandin E(1)-induced VEGF synthesis in osteoblast-like cells. 国際誌

    Y Kanno, H Tokuda, K Nakajima, A Ishisaki, T Shibata, O Numata, O Kozawa

    Molecular and cellular endocrinology   220 ( 1-2 )   89 - 95   2004年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We previously reported that prostaglandin E(1) (PGE(1)) activates both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase via cAMP-dependent protein kinase in osteoblast-like MC3T3-E1 cells, and that p38 MAP kinase but not p42/p44 MAP kinase is involved in PGE(1)-induced synthesis of vascular endothelial growth factor (VEGF). In the present study, we investigated the involvement of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in the PGE(1)-induced VEGF synthesis in MC3T3-E1 cells. PGE(1) induced the phosphorylation of SAPK/JNK. SP600125, a specific inhibitor of SAPK/JNK, markedly reduced the PGE(1)-induced VEGF synthesis. Forskolin, a direct activator of adenylyl cyclase, elicited the phosphorylation of SAPK/JNK, and 8bromo-cAMP, a plasma membrane-permeable cAMP analogue-stimulated VEGF synthesis was significantly reduced by SP600125. SP600125 suppressed the PGE(1)-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p38 MAP kinase induced by PGE(1). The phosphorylation of c-Jun induced by PGE(1) was also inhibited by SP600125. SB203580, a p38 MAP kinase inhibitor, failed to reduce the PGE(1) induced phosphorylation of SAPK/JNK. A combination of SP600125 and SB203580 suppressed the PGE(1)-stimulated VEGF synthesis in an additive manner. These results strongly suggest that PGE(1) activates SAPK/JNK in osteoblasts, and that SAPK/JNK plays a part in PGE(1)-induced VEGF synthesis.

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  • Involvement of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) in prostaglandin F2alpha-induced heat shock protein 27 in osteoblasts. 国際誌

    H Tokuda, M Niwa, A Ishisaki, K Nakajima, H Ito, K Kato, O Kozawa

    Prostaglandins, leukotrienes, and essential fatty acids   70 ( 5 )   441 - 7   2004年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We have reported that prostaglandin F2(alpha) (PGF2(alpha)) activates p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells, and that p44/p42 MAP kinase plays a role in the PGF2(alpha)-induced heat shock protein 27 (HSP27). In the present study, we investigated the involvement of stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), a member of the MAP kinase superfamily, in PGF2(alpha)-induced HSP27 in MC3T3-E1 cells. PGF2(alpha) time dependently induced the phosphorylation of SAPK/JNK. SP600125, a specific inhibitor of SAPK/JNK, markedly reduced the PGF2(alpha)-stimulated HSP27 accumulation. The inhibitory effect of SP600125 was dose dependent in the range between 0.1 and 30 microM. SP600125 reduced the PGF2(alpha)-increased level of HSP27 mRNA. SP600125 suppressed the phosphorylation of SAPK/JNK induced by PGF2(alpha), but did not affect the PGF2(alpha)-induced phosphorylation of p44/p42 MAP kinase. On the other hand, PD98059, a specific inhibitor of the upstream kinase of p44/p42 MAP kinase, which reduced the phosphorylation of p44/p42 MAP kinase stimulated by PGF2(alpha), had little effect on the PGF2(alpha)-induced phosphorylation of SAPK/JNK. These results strongly suggest that SAPK/JNK plays a part in PGF2(alpha)-induced HSP27 in addition to p44/p42 MAP kinase in osteoblasts.

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  • Simvastatin enhances the regeneration of endothelial cells via VEGF secretion in injured arteries. 国際誌

    Hiroyuki Matsuno, Mariko Takei, Hideharu Hayashi, Keiichi Nakajima, Akira Ishisaki, Osamu Kozawa

    Journal of cardiovascular pharmacology   43 ( 3 )   333 - 40   2004年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The search for a novel therapy for endothelial regenerating is an area of intensive investigation. Recent experimental and clinical evidence strongly suggests that 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) have several physiological effects independent of low-density lipoprotein cholesterol reduction. We here report that the carotid arterial blood flow after endothelial injury in hamsters treated with simvastatin was restored, in contrast to the situation in nontreated hamsters. Histologic observations showed a prompt recovery of endothelial cells with a much higher DNA synthesis index in repaired endothelium of hamsters treated with simvastatin. The amount of secreted vascular endothelial cell growth factor (VEGF) by cultured vascular smooth muscle cells from hamsters treated with simvastatin was significantly increased. Mevalonate reduced the amount of VEGF secretion by simvastatin in vitro. Finally, an injection of either an anti-VEGF antibody or an anti-VEGF receptor-1 (Flt-1) antibody, but not anti-VEGF receptor-2 (Flk-1), reduced the prompt endothelial healing. Simvastatin regulates endothelial regenerating by an over-release of VEGF and by this may result in prompt endothelial healing after vascular injury. Our results provide new insights into the role of statin and VEGF in the pathogenesis of vascular diseases.

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  • Platelet-derived growth factor-BB phosphorylates heat shock protein 27 in cardiac myocytes. 国際誌

    Motoki Takenaka, Hiroyuki Matsuno, Akira Ishisaki, Keiichi Nakajima, Kouseki Hirade, Mariko Takei, Eisuke Yasuda, Shigeru Akamatsu, Naoki Yoshimi, Kanefusa Kato, Osamu Kozawa

    Journal of cellular biochemistry   91 ( 2 )   316 - 24   2004年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    It is recognized that heat shock protein 27 (HSP27) is highly expressed in heart. In the present study, we investigated whether platelet-derived growth factor (PDGF) phosphorylates HSP27 in mouse myocytes, and the mechanism underlying the HSP27 phosphorylation. Administration of PDGF-BB induced the phosphorylation of HSP27 at Ser-15 and -85 in mouse cardiac muscle in vivo. In primary cultured myocytes, PDGF-BB time dependently phosphorylated HSP27 at Ser-15 and -85. PDGF-BB stimulated the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily. SB203580, a specific inhibitor of p38 MAP kinase, reduced the PDGF-BB-stimulated phosphorylation of HSP27 at both Ser-15 and -85, and phosphorylation of p38 MAP kinase. However, PD98059, a specific inhibitor of MEK, or SP600125, a specific inhibitor of SAPK/JNK, failed to affect the HSP27 phosphorylation. These results strongly suggest that PDGF-BB phosphorylates HSP27 at Ser-15 and -85 via p38 MAP kinase in cardiac myocytes.

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  • A peptide isolated from alpha B-crystallin is a novel and potent inhibitor of platelet aggregation via dual prevention of PAR-1 and GPIb/V/IX. 国際誌

    H Matsuno, A Ishisaki, K Nakajima, K Kato, O Kozawa

    Journal of thrombosis and haemostasis : JTH   1 ( 12 )   2636 - 42   2003年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    BACKGROUND: The ability of low-molecular-weight heat shock protein (HSP) to modulate thrombin-induced platelet aggregation has been investigated. OBJECTIVES: We examined the inhibitory effects on platelet aggregation of nine amino acid sequences isolated from HSP20 or alpha B-crystallin and their various derivatives. METHODS AND RESULTS: Platelet aggregation induced by various agonists was performed. These findings indicated that a peptide (Trp-Ile-Arg-Arg-Pro-Phe-Phe-Pro-Phe) from alpha B-crystallin significantly inhibits platelet aggregation induced by thrombin, TRAP (a protease activated receptor-1 agonist) and botrocetin, ristocetin (a stimulator of the platelet glycoprotein Ib/V/IX-von Willebrand factor axis), but not a protease-activated receptor-4 agonist, collagen and ADP. The inhibitory activity against thrombin or botrocetin is mainly linked to Arg-Arg-Pro-Phe or Trp-Ile-Arg-Arg-Pro, respectively, among nine amino acids. Additionally, during in vivo experiments, Trp-Ile-Arg-Arg-Pro-Phe-Phe-Pro-Phe shows a significant antithrombotic effect without marked bleeding. CONCLUSION: Our results provide the basis for a potential new aspect of antiplatelet compound for the therapy of thrombosis and cardiovascular disease.

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  • Lack of alpha 2-antiplasmin promotes re-endothelialization via over-release of VEGF after vascular injury in mice. 国際誌

    Hiroyuki Matsuno, Akira Ishisaki, Keiichi Nakajima, Kiyotaka Okada, Shigeru Ueshima, Osamu Matsuo, Osamu Kozawa

    Blood   102 ( 10 )   3621 - 8   2003年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We here report that the arterial blood flow after endothelial injury in mice deficient in alpha 2-antiplasmin (alpha 2-AP-/- mice) was well maintained compared with that of wild-type mice. Moreover, the development of neointima 4 weeks after injury in alpha 2-AP-/- mice was significantly decreased. Histologic observations showed a prompt recovery of endothelial cells with a much higher proliferating index in repaired endothelium in alpha 2-AP-/- mice. The amount of secreted vascular endothelial growth factor (VEGF) by explanted vascular smooth muscle cells (SMCs) from alpha 2-AP-/- mice was significantly increased. In separate experiments using a human endothelial cell (EC) line, we could demonstrate that plasminogen binds to ECs and that this binding can be prevented by alpha 2-AP. Finally, an injection of either an anti-VEGF receptor-1 antibody or alpha 2-AP reduced the prompt endothelial healing. alpha 2-AP is the main inactivator of plasmin, which cleaves extracellular matrix-bound VEGF to release a diffusible proteolytic fragment. Lack of alpha 2-AP, therefore, could lead to a local over-release of VEGF by the continuously active plasmin in the injured area, which could result in a prompt re-endothelialization after vascular injury. Our results provide new insight into the role of alpha 2-AP and VEGF in the pathogenesis of re-endothelialization following vascular injury.

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  • Affinity selection of DNA-binding proteins from yeast genomic DNA libraries by improved lambda phage display vector. 国際誌

    Hiroko Hagiwara, Sumiko Kunihiro, Keiichi Nakajima, Motoaki Sano, Haruhiko Masaki, Midori Yamamoto, Jeong Won Pak, Yan Zhang, Kumiko Takase, Ichiro Kuwabara, Ichiro N Maruyama, Masayuki Machida

    Journal of biochemistry   132 ( 6 )   975 - 82   2002年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Phage display is a useful means of identifying and selecting proteins of interest that bind specific targets. In order to examine the potential of phage display for the genome-wide screening of DNA-binding proteins, we constructed yeast genomic libraries using lambda foo-based vectors devised in this work. After affinity selection using GAL4 UAS(G) as a probe, phages expressing GAL4 were enriched approximately 5 x 10(5)-fold from the library. Approximately 90% of polypeptides encoded in correct translation reading frames by the selected phages were known or putative polynucleotide-binding proteins. This result clearly indicates that the modified lambda phage display vector in combination with our enrichment technique has great potential for the enrichment of DNA-binding proteins in a sequence-specific manner.

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  • Molecular cloning of genomic DNA for fructose-1,6-bisphosphatase. From Aspergillus oryzae. 国際誌

    M Sano, K Nakajima, K Takase, M Yamamoto, M Machida

    Bioscience, biotechnology, and biochemistry   64 ( 8 )   1747 - 50   2000年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We have cloned and sequenced an Aspergillus oryzae genomic DNA fragment that encodes a fructose-1,6-bisphosphatase gene (fbpA) with the aim of studying transcriptional regulation mechanisms involved in basic metabolism. Expression of fbpA was repressed in the presence of glucose, but not in the presence of pyruvate or sodium acetate in the medium. The CreA and FacB element found in the fbpA 5'-flanking region may be important in fbpA regulation.

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  • Comprehensive cloning and expression analysis of glycolytic genes from the filamentous fungus, Aspergillus oryzae. 国際誌

    K Nakajima, S Kunihiro, M Sano, Y Zhang, S Eto, Y C Chang, T Suzuki, Y Jigami, M Machida

    Current genetics   37 ( 5 )   322 - 7   2000年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We cloned all the glycolytic genes from Aspergillus oryzae and analyzed their transcriptional regulation by the carbon source in the medium. The deduced amino-acid sequences of the glycolytic genes showed high identity (approximately 41-93%) to those from other lower eukaryotes. Genomic Southern hybridization indicated that all the genes existed as a single copy in the genome. Comparison of mRNA levels between mycelia grown on glucose and on pyruvate showed that most of the A. oryzae glycolytic genes were induced by glucose in the medium. The overall expression profiles of the A. oryzae glycolytic genes resembled those of Saccharomyces cerevisiae. The expression of one of the phosphofructokinase genes (pfkB), however, was repressed by glucose while both PFK1 and PFK2 were induced in S. cerevisiae. These findings indicate that further analysis of the transcriptional regulation of the A. oryzae glycolytic genes will be useful for investigating the evolutionary change of transcription regulation in lower eukaryotes and to construct promoters for industrial applications.

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  • Molecular cloning and characterization of rpbA encoding RNA polymerase II largest subunit from a filamentous fungus, Aspergillus oryzae. 国際誌

    K Nakajima, Y C Chang, T Suzuki, Y Jigami, M Machida

    Bioscience, biotechnology, and biochemistry   64 ( 3 )   641 - 6   2000年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    We have cloned rpbA encoding the RNA polymerase II largest subunit (polIIL) from a filamentous fungus, Aspergillus oryzae. The rpbA product included eight highly conserved regions and the carboxyl-terminal domain (CTD). A. oryzae polIIL CTD with 184 amino acids was composed of 25 CTD consensus repeats, which was a similar number to those of lower eukaryotes. The amino acids in each repeat of A. oryzae polIIL, however, conformed less to the CTD consensus than those of polIILs from other lower eukaryotes.

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  • Current progress in the analysis of transcriptional regulation in the industrially valuable microorganism Aspergillus oryzae 査読

    Nakajima K, Sano M, Machida M

    Biotechnology and Bioprocess Engineering   5   253 - 262   2000年

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    担当区分:筆頭著者  

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  • 筋小胞体Caポンプのリン酸化中間体の反応とA-ドメインのはたらきについて

    大保貴嗣, 中島恵一, ADAMO Hugo P., 山崎和生, 川辺淳一

    日本生体エネルギー研究会討論会講演要旨集   49th   2023年

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  • NG2陽性周細胞は筋幹細胞として遅筋線維の維持に寄与している

    竜川貴光, 竜川貴光, 鹿野耕平, 鹿野耕平, 鹿野耕平, 堀内至, 堀内至, 松尾梨沙, 中島恵一, 矢澤隆志, 鹿原真樹, 鹿原真樹, 江口良二, 東信良, 川辺淳一, 川辺淳一, 川辺淳一

    日本血管生物医学会学術集会プログラム・抄録集   30th (CD-ROM)   2022年

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  • 日常生活状態における骨格筋組織の再生・維持に毛細血管周細胞が重要である

    鹿野耕平, 内田紗瑛子, 鈴木勇太, 田中洋子, 田丸祐也, 中島恵一, 堀内至, 澤田潤, 長谷部直幸, 川辺淳一

    日本老年医学会雑誌   56   2019年

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  • Ninjurin1は,NG2陽性シュワン前駆細胞の成熟化を調整することにより末梢神経再生に関わる

    富田唯, 富田唯, 堀内至, 堀内至, 鹿野耕平, 鹿野耕平, 吉田有里, 吉田有里, 竜川貴光, 竜川貴光, 中島恵一, 早坂太希, 早坂太希, 鹿原真樹, 中川直樹, 竹原有史, 長谷部直幸, 長谷部直幸, 川辺淳一, 川辺淳一

    日本分子生物学会年会プログラム・要旨集(Web)   42nd   2019年

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  • ラクトフェリンがウシ乳腺線維芽細胞の増殖及び細胞増殖因子の発現に及ぼす影響

    中島 恵一

    日本乳房炎研究会proceedings   20   93 - 96   2016年

  • 給与飼料の養分含量が初産泌乳牛の血液成分と2産次の子牛体重に及ぼす影響

    中村正斗, 中島恵一, 早坂貴代史

    日本畜産学会大会講演要旨   121st   2016年

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  • 培養系における乳腺上皮細胞機能に及ぼすIRAPの効果

    大塚浩通, 中島恵一, 富岡美千子, 鈴木豊, 田島誉士

    日本獣医学会学術集会講演要旨集   159th   2016年

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  • 乾乳期間短縮が乳牛の分娩前後の血液性状,白血球構成及び疾病発生に及ぼす影響

    青山嘉朗, 菊佳男, 高橋雄治, 中村正斗, 中島恵一, 塩野浩紀

    日本獣医師会獣医学術学会年次大会講演要旨集   2015 (CD-ROM)   2016年

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  • 黄色ブドウ球菌のリポタイコ酸は免疫調整因子の発現を増強する

    菊佳男, 長澤裕哉, 田邉扶由子, 菅原和恵, 渡部淳, 秦英司, 尾澤知美, 中島恵一, 新井敏郎, 林智人

    農研機構動物衛生研究部門成果情報(Web)   2016   2016年

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  • 胚移植により受胎した経産牛の初回移植での成否と血漿代謝性ホルモン濃度の関係

    伊藤文彰, 橋本知子, 山崎武志, 中島恵一, 萩谷功一, 梅田世奈, 杉山あかね, 矢代直樹, 青野晃, 菅原真子, 芹田友香, 笹井洋二, 舛田正博, 田鎖直澄

    日本畜産学会大会講演要旨   119th   2015年

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  • 初産牛の栄養管理にはTMRの乳期別2種飼養より一乳期1種飼養が適している

    中村正斗, 中島恵一, 早坂貴代史

    農研機構北海道農業研究センター成果情報(Web)   2015   2015年

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  • 乾乳期間短縮した乳牛の分娩前後の血液性状及び白血球構成について

    澤井紀子, 菊佳男, 高橋雄治, 中村正斗, 中島恵一, 塩野浩紀

    日本獣医師会獣医学術学会年次大会講演要旨集   2014   2015年

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  • 経産牛の初回胚移植受胎の成否と泌乳形質の関係に及ぼす分娩季節の影響

    伊藤文彰, 山崎武志, 中島恵一, 萩谷功一, 梅田世奈, 舛田正博

    日本畜産学会大会講演要旨   120th   2015年

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  • 泌乳期と乾乳期の乳牛における内分泌因子ケメリンのシグナル調節とその役割

    鈴木裕, 中島恵一, 芳賀聡, 中野美和, 宮地慎, 石崎宏, 盧尚建

    日本畜産学会大会講演要旨   120th   2015年

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  • 給与飼料の栄養濃度が初産牛の乳量および体重変化に及ぼす影響

    中村正斗, 中島恵一, 早坂貴代史

    日本畜産学会大会講演要旨   119th   2015年

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  • ヒツジにおける唾液中グレリンの分泌動態

    林英明, 佐藤利洋, 江口麻夏, 伊藤文彰, 中島恵一, 杉野利久, 児島将康, 寒川賢治

    日本畜産学会大会講演要旨   119th   2015年

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  • インプリンティング遺伝子が乳腺組織発達に及ぼす影響

    米倉真一, 米倉真一, 大畑真輝, 大林佳人, 土屋萌, 徳武優佳子, 中島恵一

    日本畜産学会大会講演要旨   119th   2015年

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  • ラクトフェリンによるウシ乳腺を構成する細胞の増殖制御

    中島恵一, 伊藤文彰, 中村正斗, 山崎武志, 田鎖直澄

    農研機構北海道農業研究センター成果情報(Web)   2014   2014年

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  • M.bovis刺激に対するウシ乳腺上皮細胞の免疫応答性について

    権平智, 中島恵一, 岩野英知, 樋口豪紀, 永幡肇

    家畜衛生学雑誌   40 ( 3 )   2014年

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  • ラクトフェリンによる乳腺上皮細胞と乳腺線維芽細胞の増殖調節作用

    中島恵一, 中村正斗, 伊藤文彰

    日本畜産学会大会講演要旨   118th   2014年

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  • 2産以上の乳牛の乾乳期間を30日に最短縮しても次乳期の産乳性は低下しない

    中村正斗, 中島恵一, 高橋雄治, 塩野浩紀, 菊佳男

    農研機構北海道農業研究センター成果情報(Web)   2013   2013年

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  • ウシの内分泌・泌乳調節系の解明と制御の試み

    伊藤文彰, 中島恵一

    日本畜産学会大会講演要旨   116th   2013年

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  • 胚移植により受胎した経産牛の初回移植での受胎と泌乳形質の関係

    伊藤文彰, 橋本知子, 山崎武志, 中島恵一, 萩谷功一, 梅田世奈, 杉山あかね, 矢代直樹, 青野晃, 菅原真子, 芹田友香, 笹井洋二, 舛田正博

    日本畜産学会大会講演要旨   117th   2013年

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  • 乾乳期短縮が次期泌乳期の乳量・乳成分に及ぼす影響

    中村正斗, 中島恵一, 高橋雄治, 塩野浩紀

    日本畜産学会大会講演要旨   115th   2012年

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  • 乾乳期間30日への短縮が初乳量と初乳成分に及ぼす影響

    中村正斗, 中島恵一, 高橋雄治, 塩野浩紀

    日本畜産学会大会講演要旨   114th   2011年

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  • ラクトフェリンが乳腺上皮細胞と乳腺線維芽細胞の増殖に及ぼす影響

    中島恵一, 中村正斗, 河村あさみ, 伊藤文彰

    日本畜産学会大会講演要旨   114th   2011年

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  • 酪農の経営改善に貢献する泌乳持続性の高い乳用牛への改良

    武田尚人, 久保田哲史, 萩谷功一, 山崎武志, 中島恵一, 佐分淳一, 宮本明夫, 川島千帆, 笹井洋二, 富樫研治

    農研機構北海道農業研究センター成果情報(Web)   2011   2011年

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  • 乾乳期間30日への短縮は泌乳前期の乳量を抑制し乳牛の栄養状態を改善する

    中村正斗, 中島恵一, 高橋雄治, 塩野浩紀

    農研機構北海道農業研究センター成果情報(Web)   2010   2010年

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  • 乾乳期短縮が乳量・乳成分と繁殖性に及ぼす影響

    中村正斗, 中島恵一, 高橋雄治

    日本畜産学会大会講演要旨   112th   2010年

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  • ウシ乳腺上皮細胞の血管内皮増殖因子発現に及ぼす泌乳関連ホルモンの作用

    中島恵一, 中村正斗, 伊藤文彰, 小酒井貴晴

    研究成果情報 北海道農業   2009   2010年

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  • Plasminogen/Plasminの骨芽細胞及び破骨細胞機能調節による骨代謝制御

    菅野陽介, 石崎明, 河下映里, 中島恵一, 岡田清孝, 上嶋繁, 松尾理, 松野浩之

    生化学   2010年

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  • ラクトフェリンによる血管新生促進因子の発現誘導

    中島恵一, 中村正斗, 河村あさみ, 石崎明

    ミルクサイエンス   59 ( 2 )   2010年

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  • ウシ乳腺上皮細胞の血管内皮増殖因子(VEGF)発現誘導におけるMAPキナーゼの関与

    中島恵一, 中村正斗, 伊藤文彰, 小酒井貴晴

    日本畜産学会大会講演要旨   112th   2010年

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  • カシューナッツ殻液が消化管上皮細胞のNa/モノカルボン酸共輸送体1に及ぼす影響

    小酒井貴晴, 小池聡, 小林泰男, 中島恵一

    生化学   2010年

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  • 乾乳期短縮が乳生産とエネルギー充足に及ぼす影響の産次による違い

    中村正斗, 中島恵一, 高橋雄治

    日本畜産学会大会講演要旨   110th   2009年

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  • ウシ第一胃上皮細胞における酪酸によるNa/モノカルボン酸共輸送体1の発現制御

    小酒井貴晴, 木村真芙友, 林英明, 加藤清雄, 中島恵一

    日本畜産学会大会講演要旨   110th   2009年

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  • 安全・安心な畜産物生産技術の開発-抗生物質に依存しない減投薬飼養管理システムの構築-第3編 乳牛の低ピーク・高持続型泌乳管理システムの開発 第1章 育種改良モデルの構築と実証 1 低ピーク高持続性乳牛作出のための選抜モデルの構築(1)低ピーク・高持続性泌乳能力に関与するDNA一塩基多型

    小酒井貴晴, 中島恵一, 山崎武志, 武田尚人

    農林水産省農林水産技術会議事務局研究成果   ( 470 )   2009年

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  • 離乳前後のウシ消化管各部位におけるFatty Acid Binding Protein(FABP)発現

    林英明, 福岡まどか, 丸山祥子, 小酒井貴晴, 中島恵一, 翁長武紀, 加藤清雄

    日本獣医学会学術集会講演要旨集   146th   2008年

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  • 高泌乳牛における乾乳期短縮が泌乳前期の乳生産とエネルギー充足に及ぼす影響

    中村正斗, 中島恵一, 高橋雄治

    日本畜産学会大会講演要旨   109th   2008年

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  • ウシ消化管におけるNa/モノカルボン酸共輸送体1(SMCT1)の遺伝子発現様式

    小酒井貴晴, 木村真芙友, 林英明, 加藤清雄, 中島恵一

    日本畜産学会大会講演要旨   109th   2008年

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  • ウシ胃腸管各部位におけるFABP(Fatty Acid-Binding Protein)の発現

    林英明, 丸山祥子, 小酒井貴晴, 中島恵一, 翁長武紀, 加藤清雄

    日本畜産学会大会講演要旨   109th   2008年

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  • ウシおよびラット乳腺組織におけるモノカルボン酸輸送体(MCTs)の発現に関する研究

    川口正人, 林英明, 小酒井貴晴, 中島恵一, 加藤清雄

    日本獣医学会学術集会講演要旨集   146th   2008年

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  • ウシナトリウム依存性短鎖脂肪酸輸送体のクローニングと消化管各部位における発現差異

    小酒井貴晴, 木村真芙友, 林英明, 加藤清雄, 中島恵一

    生化学   2007年

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  • 黒毛和種牛の消化管上皮生検組織におけるアクアポリン水輸送体およびNa-グルコース共輸送体の遺伝子発現

    小酒井貴晴, 伊村啓, 中島恵一, 坂上清一, 渡辺也恭

    日本畜産学会大会講演要旨   107th   2007年

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  • ホルスタイン種における成長ホルモン受容体SNPと泌乳持続性の関係

    小酒井貴晴, 中島恵一, 山崎武志, 武田尚人, 別府哲朗, 笹井洋二, 須田芳人, 菅原真子, 富樫研治

    日本畜産学会大会講演要旨   107th   2007年

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  • ウシ乳腺上皮細胞のラクトフェリン合成に及ぼすプロラクチンの影響

    中島恵一, 中村正斗, 小酒井貴晴

    日本畜産学会大会講演要旨   108th   2007年

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  • ホルスタイン種泌乳牛の消化管各部位におけるナトリウム/グルコース共輸送体1(SGLT1)の遺伝子発現

    伊村啓, 日置彩子, 林英明, 加藤清雄, 中島恵一, 小酒井貴晴

    北海道畜産学会大会講演要旨   61st   2006年

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  • 表面流式人工湿地による排水中無機態窒素の処理

    森岡理紀, 中村正斗, 中島恵一

    研究成果情報 北海道農業   2005   2006年

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  • 体細胞数が多い分房は乳房汚染により炎症反応が亢進し易い

    中村正斗, 矢用健一, 伊藤秀一, 森岡理紀, 中島恵一

    畜産草地研究成果情報   ( 5 )   2006年

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  • 体細胞数が多い分房は乳房汚染により炎症反応が亢進し易い

    中村正斗, 矢用健一, 伊藤秀一, 森岡理紀, 中島恵一

    研究成果情報 北海道農業   2005   2006年

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  • ウシ乳腺上皮細胞における血管内皮増殖因子(VEGF)の発現解析

    中島恵一, 中村正斗, 小酒井貴晴

    日本畜産学会大会講演要旨   106th   2006年

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  • 表面流式人工湿地による排水中無機態窒素の処理

    森岡理紀, 中村正斗, 中島恵一

    畜産草地研究成果情報   ( 5 )   2006年

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  • 反すい動物の小腸フィステルを介した内視鏡による小腸上皮組織バイオプシー技術の開発

    小酒井貴晴, 山崎武志, 中島恵一, 小林泰男, 花田正明, 青木康浩, 大下友子, 秋山典昭

    日本畜産学会大会講演要旨   106th   2006年

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  • Adenylyl cyclase-cAMP system inhibits thrombin-induced HSP27 in vascular smooth muscle cells

    K Hirade, K Tanabe, M Niwa, A Ishisaki, K Nakajima, M Nakamura, T Sugiyama, Y Katagiri, K Kato, O Kozawa

    JOURNAL OF PHARMACOLOGICAL SCIENCES   97   287P - 287P   2005年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

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  • Upregulation by methotrexate of prostaglandin D2-stimulated heat shock protein 27 induction in osteoblasts

    M Yoshida, M Niwa, H Matsuno, A Ishisaki, K Nakajima, K Hirade, K Shimizu, K Kato, O Kozawa

    JOURNAL OF PHARMACOLOGICAL SCIENCES   94   186P - 186P   2004年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:JAPANESE PHARMACOLOGICAL SOC  

    Web of Science

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  • DNAマイクロアレイを応用したphage displayによる出芽酵母転写因子の網羅的解析

    萩原央子, 国広澄子, 山本みどり, 中島恵一, 高瀬久美子, 正木春彦, 桑原一郎, 丸山一郎, 町田雅之

    日本分子生物学会年会プログラム・講演要旨集   26th   2003年

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  • ファージディスプレイライブラリーからのDNA結合タンパク質の探索

    萩原央子, 中島恵一, 佐野元昭, 町田雅之, ZHANG Y

    バイオテクノロジーシンポジウム予稿集   18th   2000年

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  • phage displayによる酵母ゲノムライブラリーからのDNA結合性因子の探索

    中島恵一, 萩原央子, ZHANG Y, 桑原一郎, 丸山一郎, 町田雅之

    日本分子生物学会年会プログラム・講演要旨集   23rd   2000年

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  • 麺カビAsperegillus oryzaeのRNA polymerase II largest subunit遺伝子のクローニングと解析

    中島恵一, ZHANG Y, 鈴木智雄, 地神芳文, 町田雅之

    日本分子生物学会年会プログラム・講演要旨集   20th   1997年

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講演・口頭発表等

  • 毛細血管を構成する周皮細胞は骨格筋の維持に寄与する

    中島恵一, 竜川貴光, 鹿野耕平, 川辺淳一

    第59回 日本生化学会北海道支部例会 

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    開催年月日: 2022年7月

    記述言語:日本語   会議種別:ポスター発表  

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共同研究・競争的資金等の研究課題

  • ウシの黄色ブドウ球菌性乳房炎の慢性化におけるCXCL8の持続的放出の役割

    研究課題/領域番号:15K07731  2015年4月 - 2018年3月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    渡部 淳, 木村 和弘, 木村 享史, 秦 英司, 中島 恵一, 林 智人

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    配分額:4,940,000円 ( 直接経費:3,800,000円 、 間接経費:1,140,000円 )

    黄色ブドウ球菌性乳房炎が慢性化する過程で観察されるケモカインCXCL8の持続的放出について、ウシ乳腺上皮細胞における CXCL8の発現は主に黄色ブドウ球菌リポテイコ酸等によって誘発され、ラクトフェリンのエラスターゼ分解産物である催炎性ラクトフェリン由来ペプチドもCXCL8発現に部分的に関与することが示唆された。また同乳房炎においてCXCL8同様に持続的に放出される好中球エラスターゼは好中球の細胞死に伴なって放出され、その細胞死には壊死や好中球細胞外トラップ形成が関わることが示唆された。

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  • 乾乳期のウシ乳腺組織における再構築メカニズムの解明

    研究課題/領域番号:15K07713  2015年4月 - 2016年3月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    中島 恵一, 樋口 豪紀, 石崎 明

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    配分額:4,810,000円 ( 直接経費:3,700,000円 、 間接経費:1,110,000円 )

    ウシ乳腺は乾乳期に退行し、再構築されると考えられているが、そのメカニズムについては未だに不明な部分が多い。本研究では、ウシ乳腺組織で乾乳期特異的に発現する遺伝子を探索・同定するとともに、ウシ乳腺組織を構成する細胞の相互作用を明らかにすることを目的とした。本年度は以下の成果が得られた。

    1)申請者らはこれまでに、乾乳期のウシ乳腺においてBMPタンパク質、BMP受容体の発現が増加することを明らかにした。本研究において乳腺の退行に伴ってラクトフェリンの分泌量が増加することを見出した。そこで、ラクトフェリンがBMPのmRNA発現に及ぼす影響を調べた結果、ラクトフェリン処理により乳腺上皮細胞におけるBMP2およびBMP4のmRNA発現が増加した。さらに、BMP4が乳腺上皮細胞におけるbeta-caseinの発現を抑制したことから、BMPが乾乳期におけるウシ乳腺の退行・再構築に関与することが示唆された。
    2)マイクロアレイ法により泌乳中期と乾乳期の乳腺組織で発現する遺伝子を比較し、発現が変動する因子を抽出した。その中から細胞外に分泌される因子に着目し、リアルタイムPCR法によってそれらの発現変動を確認した結果、乾乳期におけるCXCL13およびCXCL9の発現は泌乳中期の約4倍であった。一方、乳タンパク質であるalpha-lactalbuminの発現は、乳合成活性に伴って増加し、泌乳中期に乾乳期の約30倍に達した。
    3)乳腺上皮細胞、乳腺線維芽細胞をコラーゲンゲル中で共培養し、細胞塊の形成を観察したところ、乳腺上皮細胞単独での培養と比較して両細胞の共培養では、細胞塊が大きくなり、長時間持続することが観察された。
    4)血液中に漏出するalpha-lactalbuminが、ウシ乳腺の退行・再構築を推定するマーカーの候補となることを示した。

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  • ウシ乳腺組織におけるマイコプラズマの高度免疫回避機能に関する細胞生物学的研究

    研究課題/領域番号:15K07749  2015年4月 - 2016年3月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    樋口 豪紀, 鈴木 一由, 岩野 英知, 中島 恵一

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    配分額:4,810,000円 ( 直接経費:3,700,000円 、 間接経費:1,110,000円 )

    M. bovisの高度免疫回避機構の解明を試みた。①ウシ乳腺感染培養モデルの構築を行った。 ②好中球のCL反応はMOI依存性に有意な上昇が確認された。③PMAによって誘導される好中球のNET形成能はM. bovisの添加によって著しく阻害されることが明らかになった。④ M. bovisを好中球に添加することにより、対照群ではMOIの増加に伴ってアポトーシスが誘導された。この時、好中球のcaspase-3および9のmRNA発現量の増加が認められた。⑤ウシ乳腺感染培養モデルにより、M. bovisに暴露された好中球およびリンパ球との培養により乳腺上皮細胞にアポトーシスが誘導された。

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  • ケメリンと他のアディポカインの相互作用の解析によるウシ体脂肪蓄積と脂質代謝の解明

    研究課題/領域番号:25292158  2013年4月 - 2016年3月

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    盧 尚建, 鈴木 啓一, 中島 恵一, 萩野 顕彦, 芳賀 聡

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    配分額:19,240,000円 ( 直接経費:14,800,000円 、 間接経費:4,440,000円 )

    本研究では、Chemerinについて離乳期と乳牛の泌乳期の代謝移行期における生理的機能を解明することを目的とした。肝臓におけるChemerinのタンパク質発現は離乳後に低下していたが、Chemerinは離乳期のインスリン分泌調節には関与せず、肝臓におけるプロピオン酸からの糖新生を調節した。乳牛ではChemerinの肝臓における発現量および血中量は分娩直後に低下していたが、この変化は肝臓の糖新生調節に直接的には関与しないことが明らかになった。以上のことから、ウシにおいてChemerinの発現・分泌は代謝移行期にダイナミックに変化し、脂質代謝または糖代謝の調節に関与することが明らかになった。

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  • 内分泌型FGFがウシの乳生産に及ぼす影響の解明

    2012年4月 - 2013年3月

    北海道科学技術総合振興センター 

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  • ウシ乳腺組織の免疫学的多様性とその発現調節機構に関する細胞生物学的研究

    研究課題/領域番号:21580380  2009年4月 - 2012年3月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    樋口 豪紀, 岩野 英知, 中島 恵一, 小酒井 貴晴

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    配分額:4,810,000円 ( 直接経費:3,700,000円 、 間接経費:1,110,000円 )

    本研究の展開により、乳腺感染の新たな研究モデルとして「三次元ウシ乳腺感染モデル」が構築された。本モデルにより、これまで乳汁合成細胞として認識されてきた乳腺上皮細胞の免疫学的多様性、特に乳腺感染にともなう免疫担当細胞との相互作用が明らかとなった。乳腺免疫全体における乳腺上皮細胞および免疫担当細胞の役割を包括的・網羅的に解析し、さらに細胞内情報伝達系の解析によりその制御機構を明らかにした。本申請課題は予防獣医医療における発展的研究課題であり、その成果は国内における1次産業の恒常的維持・発展に帰する大きな社会的意義を持つものと考えられる。

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  • 泌乳持続性向上のためのウシ乳腺組織の細胞間シグナル伝達因子の解析

    2009年4月 - 2010年3月

    秋山記念生命科学振興財団 

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  • 泌乳持続性向上のためのウシ乳腺上皮細胞におけるアポトーシス抑制因子の解析

    研究課題/領域番号:18780220  2006年 - 2008年

    日本学術振興会  科学研究費助成事業  若手研究(B)

    中島 恵一

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    配分額:3,800,000円 ( 直接経費:3,500,000円 、 間接経費:300,000円 )

    本研究では泌乳牛ホルスタイン種の泌乳持続性向上のために、乳汁を合成する乳腺上皮細胞の活性化およびアポトーシスに関わる因子の解析を行った。乳腺上皮細胞において低分子量heat shock proteinであるalpha-B-クリスタリンが乳タンパク質の発現に関与していることを明らかにした。また、乳腺の血管新生を促進する血管内皮増殖因子VEGFの発現が、乳タンパク質と同様に泌乳関連ホルモンにより制御されることを明らかにした。

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  • 遺伝子ノックアウトマウスを用いた骨代謝における線溶系因子の分子基盤の解析

    研究課題/領域番号:16590873  2004年4月 - 2006年3月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    小澤 修, 石崎 明, 松野 浩之, 中島 恵一

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    配分額:3,600,000円 ( 直接経費:3,600,000円 )

    骨粗鬆症は高齢化社会を迎えた我が国において医学的にまた社会的に大きな問題となっている。しかし、骨粗鬆症の成因および病態の詳細は未だ明らかではなく、その解明は急務となっている。骨代謝は骨形成を担う骨芽細胞と骨吸収を担う破骨細胞によって、そのバランスが保たれている。しかし、これまでに線溶系因子の活性の変化により、骨芽細胞や破骨細胞の機能がどのように影響されるかを明かとした報告はなく、線溶系がどのようなメカニズムで骨代謝を調節するかについて判然としていない。
    私共は線溶系各因子の遺伝子欠損マウスを用いて線維素溶解系の活性化および不活性化がどのように骨代謝に影響を及ぼすかについて検討した。この検討により骨髄細胞の破骨細胞への分化能力および分化後の骨吸収能力に関してはコントロールである野生型マウスとurokinase-typeプラスミノーゲンアクチベータ受容体の遺伝子欠損マウスとの間に有意差は認められなかった。一方、これらの細胞による石灰化能力に関しては、urokinase-typeプラスミノーゲンアクチベータ受容体の遺伝子欠損マウス由来の骨髄細胞の方が、野生型マウス由来の骨髄細胞と比較して有意に高い石灰化能力を有することが明らかとなった。加えて、線溶系因子の活性化の程度が高い場合には、大腿骨海綿骨密度を高くし、また、低い場合には大腿骨海綿骨密度を低くする機構が作用することが明らかとなった。一方、プラスミノーゲンノックアウトマウスの脛骨から採取した骨髄細胞の破骨細胞分化能力と骨破壊能力は野生型マウスのそれらよりも高く、一方、α2-アンチプラスミンノックアウトマウスの脛骨から採取した骨髄細胞の破骨細胞分化能力と骨破壊能力は野生型マウスのそれらよりも低いことが明らかとなった。これらの結果から、線溶系の活性の変化による大腿骨海綿骨密度の変化は、破骨細胞の分化や骨破壊能力の違いによるものであることが示唆された。

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  • 心筋細胞・血管平滑筋細胞からの血圧調節遺伝子の同定と創薬への応用

    研究課題/領域番号:16790416  2004年4月 - 2006年3月

    日本学術振興会  科学研究費助成事業  若手研究(B)

    中島 恵一

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    配分額:3,400,000円 ( 直接経費:3,400,000円 )

    1 BMP(Bone morphogenetic protein)処理したマウス血管平滑筋細胞と未処理の細胞からTotal RNAを抽出し、遺伝子サブトラクション法を行った。その結果、HSP27(heat shock protein 27)遺伝子及びαBクリスタリン遺伝子が濃縮された。HSP27遺伝子はTGF-beta処理の有無によるサブトラクション法においても濃縮された。現在、培養細胞内でのHSP27とSmadとの相互作用について解析中である。その他の遺伝子については、サブトラクション法により得られたクローンの数が膨大であったため、Smad-binding活性を指標にTwo-hybridシステムによる2次スクリーニングを行った。その結果、得られた遺伝子について現在解析中である

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  • 遺伝子ノックアウトマウスを用いた骨リモデリングにおける線溶系因子の分子基盤の解析

    研究課題/領域番号:16591482  2004年4月 - 2006年3月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    石崎 明, 小澤 修, 松野 浩之, 中島 恵一

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    配分額:3,500,000円 ( 直接経費:3,500,000円 )

    骨代謝は骨を形成する骨芽細胞の働きと骨を吸収する破骨細胞の働きによって、そのバランスが保たれている。しかし、これまでに線溶系因子の活性の変化により、骨芽細胞や破骨細胞の機能がどのように影響されるかを明確にした例はなく、線溶系がどのようなメカニズムで骨代謝を調節するかについては不明である。
    そこで、今回我々は、線溶系各因子の遺伝子ノックアウトマウスを利用して、線溶系の活性化の程度による骨密度の変化や、骨芽細胞や破骨細胞の増殖・分化能力の変化を調べることにより、線溶系による骨代謝調節メカニズムについて明らかにしたいと考えた。具体的には、骨粗鬆症モデルである卵巣除去手術(OVX)による大腿骨海綿骨密度低下について、各線溶系遺伝子ノックアウトマウスを用いて観察し、それぞれの結果を比較検討した。その結果、プラスミノーゲンノックアウトマウスと野生型マウスではOVXによる明らかな大腿骨海綿骨密度の低下が認められたが、α2-アンチプラスミンノックアウトマウスでは同様な結果は認められなかった。また、プラスミノーゲンノックアウトマウスの大腿骨海綿骨密度はOVX処理と未処理に関わらず、野生型マウスよりも明らかに低い値を示していた。これらの結果から、線溶系因子の活性化の程度が高い場合には、大腿骨海綿骨密度を高くし、また、低い場合には大腿骨海綿骨密度を低くする機構か働いていると考えられた。一方、プラスミノーゲンノックアウトマウスの脛骨から採取した骨髄細胞の破骨細胞分化能力と骨破壊能力は野生型マウスのそれらよりも高く、逆に、α2-アンチプラスミンノックアウトマウスの脛骨から採取した骨髄細胞の破骨細胞分化能力と骨破壊能力は野生型マウスのそれらよりも低いことが判明した。これらの結果から、線溶系の活性の変化による大腿骨海綿骨密度の変化は、破骨細胞の分化や骨破壊能力の違いによるものであると予測された。

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  • 新たな遅筋特異的幹細胞の発見と骨格筋維持における役割の解明

    基盤研究(C)

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    骨格筋再生、特に恒常状態での骨格筋維持には未だ不明な点が多い。申請者が所属する研究チームでは、毛細血管周細胞群の中から骨格筋分化能を有する周細胞(毛細血管幹細胞 CapSCs)を発見し、周細胞欠損を誘導させた成体マウスの解析により、周細胞は骨格筋の中でも、特に、遅筋の維持に重要であることを見出した。本研究では、CapSCsの遅筋分化の機序を明らかにするとともに、全身の遅筋維持における周細胞の関与ならびに運動能・代謝能に及ぼす影響を解明する。

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  • 毛細血管幹細胞による骨格筋分化の証明と分子メカニズムの解明

    研究活動スタート支援

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    毛細血管周囲に存在する周細胞の一部は、細胞表面マーカーとしてNG2(neuron-glial antigen 2)を発現していることが知られている。申請者が所属する研究チームでは、毛細血管のNG2陽性周細胞から多分化能を有した毛細血管幹細胞(Capillary Stem Cell:CapSCと命名)を発見している。CapSCは血管細胞へ分化するだけでなく、重症筋ジストロフィーマウスの細胞移植実験において、骨格筋線維への分化を介して高い病態改善効果を示した。つまり、CapSCは、生体内で筋幹細胞として働く可能性があるとともに、骨格筋再生不全を呈する疾患の細胞治療に臨床応用することが期待できる。そこで本研究では、CapSCを含むNG2陽性周細胞が、骨格筋の維持・再生に寄与することをNG2陽性周細胞欠損誘導マウスで証明するとともに、CapSCによる骨格筋分化の分子メカニズムを明らかにする。

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