MF研究者総覧

研究者データベース

詳細
浦　和寛(ウラ　カズヒロ)
水産科学研究院　海洋応用生命科学部門　増殖生物学分野
准教授
researchmap個人ページ
Last Updated :2024/05/09

基本情報

所属

  • 水産科学研究院　海洋応用生命科学部門　増殖生物学分野

職名

  • 准教授

学位

  • 博士（水産学）（北海道大学）

ホームページURL

J-Global ID

研究キーワード

  • プロテアアーゼ   ウニ   セルラーゼ   棘皮動物   人工餌料開発   消化酵素   エゾバフンウニ   消化吸収   卵巣   雌特異タンパク質   精巣   栄養細胞   局在   精製   主要卵黄タンパク質   特異抗体   コラーゲン   イトヨ   骨再生   内分泌   生理学   骨代謝   バイオマーカー   タンパク質   主要卵黄タンパク質(MYP)   組織再生医療   ウロコ   角膜再生   細胞・組織   BMP   

研究分野

  • ライフサイエンス / 水圏生産科学
  • ライフサイエンス / 水圏生命科学
  • 環境・農学 / 化学物質影響
  • 環境・農学 / 放射線影響

担当教育組織

職歴

  • 2010年 - 現在 北海道大学 水産科学研究科(研究院) 准教授

研究活動情報

論文

  • Linyan Shi, Kazuhiro Ura, Yasuaki Takagi
    Osteoarthritis and Cartilage Open 6 2 100450 - 100450 2024年06月
  • Naoya Terauchi, Dawei Meng, Wen Li, Haruto Inada, Kazuhiro Ura, Yasuaki Takagi
    Fisheries Science 89 4 527 - 535 2023年05月12日
  • Md Rashidul Islam, Wen Li, Yumi Ogata, Takeya Yoshioka, Kazuhiro Ura, Takagi Yasuaki
    Sustainable Chemistry and Pharmacy 31 100944 - 100944 2023年04月
  • W. Li, K. Ura, Y. Takagi
    Current Research in Food Science 5 698 - 709 2022年 [査読有り]
  • Cloning of cDNA encoding a newly recognized apolipoprotein-like protein and its expression in the northern sea urchin Mesocentrotus nudus.
    T. Yuhi, O. Nishimiya, K. Ohno, A. Takita, T. Inoguchi, K. Ura, Y. Takagi
    Fisheries Science 88 259 - 273 2022年01月 [査読有り]
  • Wen Li, Naoya Terauchi, Dawei Meng, Nobuyuki Miyamoto, Naonobu Tsutsumi, Kazuhiro Ura, Yasuaki Takagi
    Sustainable Chemistry and Pharmacy 23 100499 - 100499 2021年10月
  • Wen Li, Taishi Kobayashi, DaWei Meng, Nobuyuki Miyamoto, Naonobu Tsutsumi, Kazuhiro Ura, Yasuaki Takagi
    Food Bioscience 41 100991 - 100991 2021年 [査読有り]
  • Xi Zhang, Huiyan Zhang, Shigeru Toriumi, Kazuhiro Ura, Yasuaki Takagi
    Aquaculture 529 735641 - 735641 2020年12月
  • Dawei Meng, Wen Li, Kazuhiro Ura, Yasuaki Takagi
    International Journal of Biological Macromolecules 148 182 - 191 2020年04月 [査読有り][通常論文]
  • Purity and properties of gelatins extracted from the head tissue of the hybrid kalamtra sturgeon
    Md. Rashidul Islam, Tomoharu Yuhi, Dawei Meng, Takeya Yoshioka, Yumi Ogata, Kazuhiro Ura, Yasuaki Takagi
    Food Science and technology 142 110944  2020年 [査読有り][通常論文]
  • Major yolk protein binds folic acid in a sea urchin
    Kazuhiro Ura, Takahiro Gotoh, Yudai Kitano, Osamu Nishimiya, Yasuaki Takagi
    Aquacult. Sci. 68 4 309 - 315 2020年 [査読有り][通常論文]
  • M. R. Islam, T. Yuhi, K. Ura, Y. Takagi
    Appl. Sci. 10 19 6660 - 6660 2020年 [査読有り]
     
    To develop a new method for extracting gelatin from the sturgeon head, the conditions for pretreatment and extraction were optimized. Treatment at 65 °C (3–3.5 h) was enough to separate the head into mixed tissues (skin, scales, pectoral fins, muscle, bones, gills, and small cartilage pieces), skull cartilage, and liquid. From the intensities of α-, β- and γ-bands and yields, the optimized pretreatment conditions for type A and type B gelatin were 0.05 M HCl (1 h) and 0.1 M NaOH (1 h), respectively. The best extraction conditions were: (1) for type A gelatin: with distilled water (DW) (w/v 1:5) at 35 °C, pH 7 when stirring at 200 rpm for 3 h, and (2) for type B gelatin: with DW (w/v 1:5) at 50 °C, pH 8 when stirring at 200 rpm for 1 h. After the decalcification of extracted residues with 0.05 M HCl for 3 h, re-extraction of gelatin was possible. Under the best extraction conditions, yields of type A and B gelatins were 5.01 and 7.25% (dry gelatin weight/wet sample weight), respectively. Thus, it is possible to extract an industrial amount of gelatin from sturgeon heads, making them valuable by-products.
  • 配合飼料によるキタムラサキウニ養殖 北海道・岩手県における実証試験
    浦 和寛, 萩野裕貴, 中川紅実, 濱 遥香
    養殖ビジネス 41 - 44 2019年12月 [査読無し][招待有り]
  • N.Sato, T.Furuta, T.Takeda, Y.Miyabe, K.Ura, Y.Takagi, H.Yasui, Y.Kumagai, H.Kishimura
    Journal of Food Biochemistry 43 2 e12709 - e12709 2019年02月 [査読有り][通常論文]
  • Moroi S, MiuraT, Tamura T, Zhang X, Ura K, Takagi Y
    Mater. Sci. Engineer. C 104 109925  2019年 [査読有り][通常論文]
  • Nishimiya O, Teraoka Y, Gotoh T, Yuhi T, Higuchi I, Ura K, Takagi Y
    Fish. Sci. 85 1 127 - 135 2019年 [査読有り][通常論文]
     
    The major yolk protein in sea urchins is a transferrin-like protein. In this report, a new component was detected in gonad extracts of Strongylocentrotus intermedius and Mesocentrotus nudus, which cross-reacts with antiserum against egg yolk proteins. We tentatively named them egg yolk-related proteins siYRP and mnYRP. The siYRP was purified from testis of S. intermedius by ammonium sulfate fractionation, anion exchange chromatography, affinity chromatography and gel filtration. Purified siYRP was >900kDa in size. The siYRP purified on SDS-PAGE under reducing conditions gave seven bands, corresponding to 93, 213 and >250kDa. Purified mnYRP displayed similar structural characteristics as siYRP. Purified siYRP and mnYRP were identified by tandem mass spectrometry and renamed as siVitellogenin (Vtg)-like and mnVtg-like proteins, respectively. Both Vtg-like proteins were confirmed to be lipoglycoproteins by staining with Sudan black and periodic acid-Schiff. A specific antiserum against the siVtg-like protein was raised in rabbit. Antiserum against the siVtg-like protein immunostained siVtg-like and mnVtg-like proteins. Immunochemical methods using the antiserum revealed that siVtg-like and mnVtg-like proteins were present in the ovary, testis and unfertilized eggs of both species. These results indicated that Vtg-like proteins have important physiological functions for gonadal growth and gametogenesis in sea urchins.
  • Meng D, Tanaka H, Kobayashi T, Hatayama H, Zhang X, Ura K, Yunoki S, Takagi Y
    Int. J. Biol. Macromol. 131 572 - 580 2019年 [査読有り][通常論文]
     
    Non-mammalian collagens have attracted increasing attention for industrial and biomedical use. We have therefore evaluated extraction conditions and the biochemical properties of collagens from aquacultured sturgeon. Pepsin-soluble type I and type II collagen were respectively extracted from the skin and notochord of bester sturgeon by-products, with yields of 63.9 ± 0.19% and 35.5 ± 0.68%. Collagen extraction efficiency was improved by an alkaline pretreatment of the skin and notochord (fewer extraction cycles were required), but the final yields decreased to 56.2 ± 0.84% for type I and 31.8 ± 1.13% for type II. Alkaline pretreatment did not affect the thermal stability or triple-helical structure of both types of collagen. Types I and II collagen formed re-assembled fibril structures in vitro, under different conditions. Alkaline pretreatment slowed down the formation of type I collagen fibrils and specifically inhibited the formation of thick fibril-bundle structures. In contrast, alkaline pretreatment did not change type II collagen fibril formation. In conclusion, alkaline pretreatment of sturgeon skin and notochord is an effective method to accelerate collagen extraction process of types I and II collagen without changing their biochemical properties. However, it decreases the yield of both collagens and specifically changes the fibril-forming ability of type I collagen.
  • Quantitative changes of major yolk protein in the coelomic fluid and gonads of the sea urchin, Mesocentrotus nudus, during the reproductive cycle.
    Ura K, Suzuki N, Numata H, Tsue S, Satoh M, Takei N, Higuchi I, Yuhi T, Nishimiya O, Takagi Y
    北海道大学水産科学研究彙報 69 29 - 36 2019年 [査読無し][通常論文]
  • Zhang X, Adachi S, Ura K, Takagi Y
    Int. J. Biol. Macromol. 137 809 - 820 2019年 [査読有り][通常論文]
  • Li W, Kobayashi T, Moroi S, Kotake H, Ikoma T, Saeki H, Ura K, Takagi Y
    Carbohydr. Polym. 214 303 - 310 2019年 [査読有り][通常論文]
  • Yamamoto, Ryoko, Minami, Hisanori, Matsusaki, Hiromi, Sakashita, Mami, Morita, Naoki, Nishimiya, Osamu, Tsutsumi, Naonobu, Hosokawa, Masashi, Itabashi, Yutaka, Matsui, Toshiro, Ura, Kazuhiro
    JOURNAL OF FUNCTIONAL FOODS 47 40 - 47 2018年08月 [査読有り][通常論文]
     
    The beneficial health effects of edible sea urchin consumption in mice fed a normal (ND) or high-fat diet (HFD) were investigated in this study. Notably, sea urchin-administered (250, 500, 1000 mg/kg) mice exhibited lower body, liver, and visceral fat weights, lower plasma levels of aspartate aminotransferase and alanine aminotransferase, and lower hepatic triacylglycerol levels than those fed carboxymethyl cellulose (CMC). Despite the high levels of cholesterol found in sea urchins, intake of these organisms had no effect on plasma cholesterol levels among the mice tested. Additionally, sea urchin consumption resulted in enhanced levels of arachidonic acid and docosahexaenoic acid within mouse livers. Lastly, mice fed HFD with sea urchin (500 mg/kg) exhibited increased mRNA expression of uncoupling protein-1 within brown adipose tissue, compared with those fed HFD with CMC. In conclusion, consumption of sea urchin might provide a protective effect against the development of obesity and/or nonalcoholic fatty liver disease.
  • Hajime Uchida, Yutaka Itabashi, Ryuichi Watanabe, Ryoji Matsushima, Hiroshi Oikawa, Toshiyuki Suzuki, Masashi Hosokawa, Naonobu Tsutsumi, Kazuhiro Ura, Donato Romanazzi, Matthew R. Miller
    Food Chemistry 252 84 - 91 2018年06月30日 [査読有り][通常論文]
     
    Using high-performance liquid chromatography coupled to high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC/ESI-Q-TOF-MS), we have developed a new method for detection and identification of furan fatty acids (F-acids), which are widely distributed in living organisms and foods as minor lipid components and are known to have antioxidant and anti-inflammatory effects. For this purpose, total fatty acids prepared from the testis lipids of Japanese chum salmon (Oncorhynchus keta) were examined without any concentration or isolation of F-acids. In negative ESI mode, F-acids gave a prominent [M−H]− ion, by which individual F-acids could be detected and identified. High-resolution extracted ion chromatograms clearly showed the occurrence of five major F-acid homologs as already reported by GC/MS. The method was successfully applied to several fish samples and revealed the occurrence of F-acids for the first time in the two New Zealand fish, hoki (Macruronus novaezelandiae) and school shark (Galeorhinus galeus).
  • Yuki Okashita, Heng Wang, Shiori Tsue, Osamu Nishimiya, Kazuhiro Ura, Yasuaki Takagi
    FISHERIES SCIENCE 83 5 803 - 810 2017年09月 [査読有り][通常論文]
     
    In the present study, we examined the localization of the major yolk protein (MYP) in the intestine of the sea urchin Strongylocentrotus intermedius. First, partial MYP complementary DNA was isolated from the sea urchin intestine. The expression level of MYP messenger RNA (mRNA) along the sea urchin digestive tract is highest in the intestine, so we performed in situ hybridization and immunohistochemical analysis using this tissue. No MYP mRNA was detected in the luminal epithelium, connective tissue, muscle tissue, or coelomic epithelium by in situ hybridization analysis. Positive immunohistochemical staining was observed in the luminal epithelium, inner epithelium and connective tissue, the signal being strongest in the latter. We conclude that MYP synthesized in the inner epithelial cells is moved to and stored in connective tissue and the luminal epithelium, before being secreted into the body cavity and the inner digestive cavity of the sea urchin.
  • The reproductive cycle and transcription-level changes in the major yolk protein of wild northern sea urchin, Mesocentrotus nudus, in southern Hokkaido.
    K. Ura, H. Wang, T. Hori, S. Aizawa, S. Tsue, M. Satoh, N. Takei, K. Hoshino, I. Higuchi, S. Sanuki, T. Yuhi, O. Nishimiya, Y. Takagi
    Aquacult. Sci. 65 232 - 237 2017年 [査読有り][通常論文]
  • Noriko Azuma, Seishi Hagihara, Masaki Ichimura, Yasuaki Takagi, Kazuhiro Ura, Shinji Adachi
    ICHTHYOLOGICAL RESEARCH 64 1 139 - 144 2017年01月 [査読有り][通常論文]
     
    Genetic characterization was performed in five individuals of wild Amur sturgeon Acipenser schrenckii, and/or its presumed hybrid caught around Hokkaido, using a mitochondrial DNA (mtDNA) marker and two markers of nuclear DNA (nDNA). Genetic analyses indicated that two of the five fish had the mtDNA haplotype of Kaluga, Huso dauricus, whereas the nDNA markers indicated signs for both A. schrenckii and H. dauricus genotypes, referring to a hybrid origin. The other three fish were plausibly pure A. schrenckii. The results indicated the importance of combined usage of mtDNA and nDNA markers for correct species identification in sturgeon.
  • 東典子, Seishi Hagihara, Masaki Ichimura, Yasuaki Takagi, Kazuhiro Ura, Shinji Adachi
    Ichthyological Research 64 1 139 - 144 2017年01月 [査読有り][通常論文]
  • X. Zhang, N. Azuma, S. Hagihara, S. Adachi, K. Ura, Y. Takagi
    Gene 579 1 8 - 16 2016年03月 [査読有り][通常論文]
     
    To characterize type I and II collagen in the Amur sturgeon at the molecular level, mRNAs encoding the pro alpha chain of both types of collagen were cloned and sequenced. Full sequences of both were obtained, and the molecular phylogeny based on the deduced amino acid sequence indicated that the correct sequences of the target genes were obtained. Analyses of primary structure of the pro alpha chains revealed that type I and II collagen share the basic structure of the prom chain of fibril collagen, but have different characteristics, especially in residues related to thermal stability. In the triple helical domain, Gly-Pro-Pro sequence stabilizing the tripeptide unit was more frequent in type II than in type I, and Gly-Gly, which likely decline in thermal stability, was more frequent in type I than in type II. These results suggested that the denaturation temperature of type II would be remarkably higher than type I. The spatial pattern of gene expression was analyzed by quantitative real-time PCR, which showed that relatively ubiquitous type I gene and strongly skewed distribution of type II gene, which highly expressed only in vertebra, snout cartilage, and notochord. This pattern was similar to the distribution pattern of each collagen protein detected by previous biochemical analyses using Amur and Bester sturgeons. The present study is the first report of the cloning of the full-length cDNAs for both of type I and type II collagen in the Amur sturgeon, and is the first comparative analysis of type I and II collagens in a sturgeon species at the molecular level. The results provide basic and general information on collagens in sturgeons. (C) 2015 Elsevier B.V. All rights reserved.
  • Heng Wang, Kazuhiro Ura, Yasuaki Takagi
    FISHERIES SCIENCE 81 6 1127 - 1134 2015年11月 [査読有り][通常論文]
     
    Major yolk protein (MYP) is the most abundant protein found in the yolk granules of sea urchin eggs. The structure of MYPs stored in the eggs of two sea urchin species from Strongylocentrotus intermedius and Mesocentrotus nudus were investigated. Egg-localized major yolk protein (EGMYP) extracted from the eggs of two species of sea urchins was assessed using disc-polyacrylamide gel electrophoresis and immunoelectrophoresis. The molecular weight of native EGMYP was 595 in S. intermedius eggs and 625 kDa in M. nudus eggs. The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis under reducing conditions revealed that the EGMYP of S. intermedius separated into four bands (approximately 172, 116, 74 and 68 kDa), while the EGMYP of M. nudus resolved as a set of bands ranging from 175 to 58 kDa (approximately 175, 165, 153, 115, 102, 90, 78, 65 and 58 kDa). Yolk granules from eggs were isolated using sucrose density ultracentrifugation and examined using electron microscopy. The structures of purified EGMYP extracted from eggs and from yolk granules were similar in both species. The EGMYP stored in the eggs of the sea urchins was a glycoprotein complex, the protein structure of which, however, varied between the two species.
  • Norihiko Komatsu, Nobuhiro Ogawa, Kurin Iimura, Kazuhiro Ura, Yasuaki Takagi
    FISHERIES SCIENCE 80 6 1249 - 1256 2014年11月 [査読有り][通常論文]
     
    Fish scales are a potential source of collagen for fabricating scaffolds for cells during tissue engineering because fish collagen has a low risk of zoonosis. Since the assembly of collagen fibrils has a significant impact on the functionality of the scaffold, the ability to replicate the fibril assembly of human tissues is critical. To determine the mechanism of fish collagen fibril assembly, we first identified non-collagenous proteins (NCPs), the potential regulators of fibril assembly in vivo, and then used tandem mass spectrometry to analyze the NCPs contained in the basal plates of goldfish Carassius auratus scales, a collagenous plate which is characterized by a plywood-like assembly of collagen fibrils similar to that found in the cornea. We identified a 19-kDa acidic protein as dermatopontin, the NCP which is a possible regulator of fibril assembly in the mammalian cornea. We cloned a goldfish dermatopontin cDNA of 1,074 bp containing an open reading frame encoding 196 amino acids. Reverse transcription-PCR revealed that dermatopontin mRNA was expressed in a wide range of tissues, including scale, skin, fin, eye, and skeletal muscle. In situ hybridization revealed that dermatopontin mRNA was expressed primarily in the basal plate-producing hyposquamal scleroblasts of the scales, suggesting that the dermatopontin is linked to the collagen fibril assembly of the basal plate.
  • X. Zhang, M. Ookawa, Y. Tan, K. Ura, S. Adachi, Y. Takagi
    Food Chem. 160 305 - 312 2014年10月 [査読有り][通常論文]
     
    Collagens purified from Bester sturgeon organs were characterised biochemically, and their fibril-forming abilities and fibril morphologies formed in vitro clarified. Yields of collagens were 2.1%, 11.9%, 0.4%, 18.1%, 0.4%, 0.8% and 0.03% (collagen dry weight/tissue wet weight) from scales, skin, muscle, swim bladder, digestive tract, notochord and snout cartilage, respectively. Using SDS-PAGE and amino acid composition analyses, collagens from scales, skin, muscle, the swim bladder and digestive tract were characterised as type I, and collagens from the notochord and snout cartilage as type II. Denaturation temperatures of the collagens, measured using circular dichroism, were 29.6, 26.8, 29.0, 32.9, 31.6 and 36.3 degrees C in scales, skin, muscle, swim bladder, digestive tract, and notochord, respectively. For fibril formation, swim bladder and skin collagen showed a more rapid rate of increase in turbidity, a shorter time to attain the maximum turbidity, and formed thicker fibrils compared with porcine tendon type I collagen. (C) 2014 Elsevier Ltd. All rights reserved.
  • Takuro Nakajima, Haruka Shimura, Miyuki Yamazaki, Yasuhiro Fujioka, Kazuhiro Ura, Akihiko Hara, Munetaka Shimizu
    AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY 307 4 R414 - R425 2014年08月 [査読有り][通常論文]
     
    Landlocking of salmon relaxes selective pressures on hypoosmoregulatory ability (seawater adaptability) and may lead to the abandonment of its physiological system. However, little is known about the mechanism and consequence of the process. Biwa salmon is a strain/subspecies of Oncorhynchus masou that has been landlocked in Lake Biwa for an exceptionally long period (about 500,000 years) and has low ability to adapt to seawater. We compared activity of gill Na+, K+-ATPase (NKA) of Biwa salmon with those of anadromous strains of the same species (masu and amago salmon) during downstream migration periods and after exogenous hormone treatment. Gill NKA activity in anadromous strains increased during their migration periods, while that in Biwa salmon remained low. However, treatments of Biwa salmon with growth hormone (GH) and cortisol increased gill NKA activity. Cortisol treatment also improved the whole body seawater adaptability of Biwa salmon. Receptors for GH and cortisol responded to hormonal treatments, whereas their mRNA levels during downstream migration period were essentially unchanged in Biwa salmon. Circulating levels of cortisol in masu salmon showed a peak during downstream migration period, while no such increase was seen in Biwa salmon. The present results indicate that Biwa salmon can improve its seawater adaptability by exogenous hormonal treatment, and hormone receptors are capable of responding to the signals. However, secretion of the endogenous hormone (cortisol) was not activated during the downstream migration period, which explains, at least in part, their low ability to adapt to seawater.
  • Kawamata Hiroshi, Kuwaki Shunsuke, Mizuno Masanori, Urasawa Kazunari, Mishina Tomobumi, Ikoma Toshiyuki, Tanaka Junzo, Zhang Xi, Adachi Shinji, Ura Kazuhiro, Azuma Noriko, Takagi Yasuaki, Nozaki Ryusuke
    生物物理 54 1 S162  一般社団法人 日本生物物理学会 2014年
  • Yongkai Tan, Kurin Iimura, Tetsuro Sato, Kazuhiro Ura, Yasuaki Takagi
    Gene 516 2 277 - 284 2013年03月10日 [査読有り][通常論文]
     
    There has been significant interest in the expression and function of dermatopontin (DPT) in mammals owing to recent evidence pointing to its critical role in collagen fibrillogenesis. Despite this interest, limited information is available about the site/s of DPT mRNA expression or changes in expression in vivo. We used reverse-transcription polymerase chain reaction and in situ hybridization to evaluate the spatial and temporal pattern of DPT mRNA expression in zebrafish, Danio rerio, a widely used vertebrate model. We observed that DPT transcripts were expressed in zebrafish embryos at all developmental stages in a range of tissues, including the brain and optic neuron cells. Based on our results, we hypothesize that DPT may also play a role in neural functions in vivo. © 2012 Elsevier B.V.
  • X. Zhang, K. Shimoda, K. Ura, S. Adachi, Y. Takagi
    JOURNAL OF FISH BIOLOGY 81 6 1985 - 2004 2012年11月 [査読有り][通常論文]
     
    In the larval bester, a hybrid sturgeon of beluga Huso huso and sterlet Acipenser ruthenus, development of cartilage around the notochord began 7 days post hatch (dph) (14.0 mm, total length, L-T). The vertebral cartilage develops in the following sequence: basidorsals and basiventrals, neural canals, neural spines and ribs. The development of ribs remained incomplete in the largest specimen (181 dph, 179 mm L-T) that was examined. Endoskeletal development of the fins began 4 dph for the dorsal and anal fins, 6 dph for the pectoral fin and 10 dph for the caudal and pelvic fins. Complete elements of all fins were observed by 91 dph and complete ossification of fin rays was observed by 122 dph in the double-stained specimens. Observation of the histological sections, however, suggested that ossification occurred soon after the formation of the organic matrix in the fin rays. Dorsal scutes were first visible by 25 dph, followed by the lateral and ventral scutes, which were visible by 37 and 44 dph, respectively. The number of scutes was fixed at 44, 59 and 91 dph and ossification was complete by 59 (dorsal) and 91 dph (lateral and ventral scutes) in the double-stained specimens. Ossification occurred soon after the formation of the scute organic matrix in the histological sections. Four types of scales were observed in the H. huso x A. ruthenus hybrid. Median predorsal, preanal and small scales on the anterior section of the head were visible by 59 dph. Scales on the caudal fin were visible by 91 dph and a variable assemblage of scales anterior to the anal fin was visible by 122 dph. Both the scutes and scales developed in a process that is similar to that of intramembranous ossification. (C) 2012 The Authors Journal of Fish Biology (C) 2012 The Fisheries Society of the British Isles
  • Syuto Hasegawa, Kazuhiro Ura, Hiroyuki Tanaka, Takao Ojima, Yasuaki Takagi
    FISHERIES SCIENCE 78 5 1107 - 1115 2012年09月 [査読有り][通常論文]
     
    We isolated a cellulase from the digestive organs of the short-spined sea urchin Strogylocentrotus intermedius using a combination of ion-exchange chromatography and gel filtration together with an assay for carboxymethylcellulase activity. The isolated cellulase was stained as a single band by Congo red. The molecular weight of the isolated cellulase, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, was 59 kDa. The isolated cellulase exhibited hydrolytic activity toward carboxymethyl cellulose, with an optimum temperature and pH of 30 A degrees C and pH 8.0, respectively. The thermal stability of the enzyme was characterized by determining the temperature at which activity decreased by 50 % with treatment for 30 min at pH 7.0 and found to be 32 A degrees C. Cellulase activity remained at a high level at 5-20 A degrees C, which is the growth temperature of the short-spined sea urchin. These results confirm that the short-spined sea urchin should preferably be reared at a water temperature of < 20 A degrees C.
  • Kurin Iimura, Hidekazu Tohse, Kazuhiro Ura, Yasuaki Takagi
    JOURNAL OF EXPERIMENTAL ZOOLOGY PART B-MOLECULAR AND DEVELOPMENTAL EVOLUTION 318B 3 190 - 198 2012年05月 [査読有り][通常論文]
     
    Teleost fish scale is a dermal skeleton equipped with a strong regenerative ability. Owing to this regenerative ability, teleost fish scale can be used as a model for the regeneration of the dermal skeleton. However, there is insufficient fundamental knowledge of the regeneration, and this limits the usage of fish scale. In this study, as a first step toward understanding the molecular mechanism of the cellular differentiation during scale regeneration, we cloned the cDNAs for osteoblast-related proteins (Runx2, Sparc, and Bgp) in goldfish, and analyzed their expressions during scale regeneration. The expression profiles of these genes during scale regeneration were similar to those during mammalian osteoblastic differentiation. Specifically, runx2 expression was increased at the earliest time point, followed by sparc expression and then bgp expression. In the earlier stages, these genes were expressed in cells that formed cellular condensations and the flat cells surrounding them in the scale pocket. As the regeneration proceeded, the expressions became restricted to the episquamal, hyposquamal, and marginal scleroblasts and the cells around the marginal area of the regenerating scale. These results strongly suggest that (1) the differentiation mechanism of scleroblasts is similar to that of mammalian osteoblasts and odontoblasts, (2) scleroblast differentiation occurs around the cellular condensations at the early regeneration stage and is restricted to the marginal area of the scale at the later stage, and (3) the differentiation mechanisms are similar between the episquamal scleroblasts that produce the external layer and the hyposquamal scleroblasts that produce the basal plate. J. Exp. Zool. (Mol. Dev. Evol.) 318:190198, 2012. (c) 2012 Wiley Periodicals, Inc.
  • Kazuhiro Ura, Syuto Hasegawa, Eri Tanaka, Takahiro Gotoh, Takao Ojima, Yasuaki Takagi
    ANATOMICAL RECORD-ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY 295 1 73 - 77 2012年01月 [査読有り][通常論文]
     
    Subtilase, a major protease in the short-spined sea urchin (Strongylocentrotus intermedius), was isolated and used as antigen for the subsequent production of a specific polyclonal antibody. Immunoreactive cells were observed by immunohistochemical analysis in granules in the anterior and posterior stomach and the anterior intestine. These granules, which were most numerous in the anterior stomach, also stained intensely with methylene blue-Azure II. However, granules in cells of the esophagus, posterior intestine, and rectum were not stained by this antibody. We conclude that subtilase mainly localizes in the stomach and anterior intestine of the sea urchin. Anat Rec, 2012. (C) 2011 Wiley Periodicals, Inc.
  • Nobuhiro Ogawa, Kazuhiro Ura, Yasuaki Takagi
    FISHERIES SCIENCE 76 2 189 - 198 2010年03月 [査読無し][通常論文]
     
    The external layer of a teleost fish scale is composed of type I collagen, an amorphous matrix substance and hydroxyapatite crystals. Calcification of this layer can be inhibited in the scale-regenerating process under calcium-and phosphate-deficient (CaDPD) conditions, and can be facilitated by incubation in physiological saline. The aim of this study was to evaluate this model of calcification using histological and quantitative analysis in order to promote further understanding of the mechanism of calcification in fish scales. We found that the external layer of the scales produced under CaDPD conditions contained more densely aligned collagen fibrils with a small amount of the amorphous matrix substance. The CaDPD scale contained only one-third of the amount of calcium and phosphate present in the control fish. After 4 hours of incubation, a two-to threefold increase in calcium content and a 1.5-fold increase in phosphate content were observed. Calcification proceeded in the external layer, and mineral deposits grew only in the electron-dense matrix substance. We conclude that this model would be suitable for studying the early process of fish scale calcification that occurs in the noncollagenous substance. The electron-dense substance may contain key molecules that promote calcification.
  • 小林 豊, 高村 巧, 下野 功, 浦 和寛, 都木靖彰
    水産増殖 57 2 271 - 278 水産増殖談話会 2009年 [査読有り][通常論文]
     
    岩手県大槌産，岩手県山田産，北海道鹿部産，北海道知内産の2年貝養殖ホタテガイの貝殻中のLi，Mn，SrおよびBa濃度を誘導結合プラズマ質量分析装置を用いて測定したところ，産地により微量元素組成に有意な差が認められた。MANOVAの判別分析を用いて産地識別を試みたところ，48個体中9個体で誤判別が発生し，判別的中率は81.3%であった。大槌産と他の産地との間で誤判別される例が多かったが，そのほかの産地間の誤判別は少なかった。同様の方法を用いて岩手県八幡平産および北海道七飯産の養殖ニジマスの耳石中の微量元素濃度を定量した結果，その組成にも産地により有意な差があった。判別的中率は95.5%であった。生産年による微量元素組成の変動が小さければ，貝殻および耳石微量元素組成による産地識別が可能であると考えられる。
  • 山野 恵祐, 東藤 孝, 浦 和寛
    水産総合研究センター研究報告 26 26 129 - 134 水産総合研究センター 2008年12月 [査読無し][通常論文]
     
    近年のウニ類の漁獲量は一万数千トンを推移しており、北海道を中心とした北日本ではエゾバフンウニ、キタムラサキウニ、九州を中心とした西日本ではアカウニが主要な漁獲対象となっている。これらのウニの資源増大を図るため、種苗生産・放流も長年に渡って実施されている。ウニ類の漁業生産はもっぱら漁獲に依っており、一部、養殖が行われているケースもあるが、養殖による生産量は極めて少なく漁業統計には現れない程度に過ぎない。食品としてのウニを見た場合、ウニは水産物の中でも特異な性質を有している。すなわちウニ類では生殖巣（卵巣及び精巣）だけが食される。また食用に適した生殖巣は、栄養物を生殖巣内に十分に蓄積しているが成熟期に入る前の未成熟な状態のものである。このため食品としての品質や歩留まりは成熟状態と密接に関連しており、良質の生殖巣を得られる時期、いわゆる旬は、種毎に限られた季節となる。本課題では、ウニの生殖巣の分化及び生殖巣の発達・成熟に関する研究を実施した。
  • Yuichi Ozaki, Koichi Ishida, Koji Saito, Kazuhiro Ura, Shinji Adachi, Kohei Yamauchi
    FISHERIES SCIENCE 73 3 574 - 584 2007年06月 [査読有り][通常論文]
     
    Specific antibodies against follicle-stimulating hormone beta subunit (FSH beta), prolactin (PRL), and somatolactin (SL) of the Japanese eel Anguilla japonica were produced. These antibodies, as well as antibodies against luteinizing hormone beta subunit (LH beta) and growth hormone (GH) produced previously, were used to examine changes in the production of pituitary hormones in female eels during maturation induced by salmon pituitary homogenate (SPH) injection. Immunohistochemical observations showed a decrease in FSH production after SPH injection, suggesting that SPH inhibits FSH production. In contrast, LH production increased markedly with maturation. The number of GH producing cells decreased gradually during maturation, possibly because of inhibition by exogenous GH present in the SPH and/or endogenous insulin-like growth factor-I produced by the stimulation of salmon GH. Although changes in the number of PRL producing cells with maturation were not evident, the number of SL producing cells showed a peak at the late vitellogenic stages, and thereafter decreased to the migratory nucleus stage. These results suggest that GH and SL are involved in sexual maturation in SPH injected eels, as in other fishes.
  • Yasuaki Takagi, Kazuhiro Ura
    JOURNAL OF NANOSCIENCE AND NANOTECHNOLOGY 7 3 757 - 762 2007年03月 [査読有り][通常論文]
     
    The corneal stroma is composed of multiple lamellae, each containing closely packed collagen fibrils. The orientation of fibrils in a lamella is parallel, but those in different lamellae are orthogonal. As a result, the corneal stroma has a characteristic orthogonal plywood-like structure. Such a highly-regulated three-dimensional arrangement of collagen fibrils gives strength and transparency to the corneal stroma, but it also presents a challenge in the fabrication of materials to replace it. A bioinspired technology is required to process such materials, but the regulatory mechanism of collagen-fibril orientation is still unknown. The low regenerating activity of the corneal stroma seems to be a major factor preventing progress in this field of research. A similarly highly-ordered arrangement of collagen fibrils can be seen in the basal plates of teleost fish scales. Moreover, the scales have high regenerating ability. When a scale is mechanically lost, a new scale is rapidly regenerated. The cells that produce the basal plates are extremely activated; thus, production of the highly-ordered collagen fibrils is very rapid. Therefore, the regenerating scales should be a uniquely helpful biological model for studying the regulatory mechanism of collagen-fibril orientation. Fish-scale collagen has another advantage for use as a biomaterial: the low probability of zoonotic infection. Therefore, scale collagen is a most promising biomaterial for fabricating three-dimensionally arranged collagen fibers to substitute for the corneal stroma. Three tasks that must be clarified for the bioinspired production of a corneal substitute from fish scale collagen are proposed.
  • OHIRA YASUHARU, SHIMIZU MOTOHIRO, URA KAZUHIRO, TAKAGI YASUAKI
    Fisheries science : FS 73 1 46 - 54 2007年02月01日 [査読無し][通常論文]
  • Scale regeneration and calcification in the gold fish Carassius auratus: quantitative and morphological process.
    Y. Ohira, M. Shimizu, K. Ura, Y. Takagi
    Fish. Sci. 73 574 - 584 2007年 [査読有り][通常論文]
  • 浦 和寛, 原 彰彦, 山内 晧平
    水産増殖 54 4 429 - 435 水産増殖談話会 2006年12月20日 [査読無し][通常論文]
     
    サクラマスの血清タンパク質において、銀化変態時に出現する分子量約80,000の銀化特異タンパク質を免疫生化学的手法により検出するとともに、このタンパク質に対する特異抗体を得た。抗体を用いてサクラマスの銀化変態期における銀化特異タンパク質の血中量の変化を調べた。その結果、銀化変態期に血中甲状腺ホルモン量の変化に伴い銀化特異タンパク質の血中量は増加し銀化最盛期に最大値を示した。また、銀化前の稚魚に甲状腺ホルモンを投与すると、この銀化特異タンパク質の血中量は増加した。これらの結果から、この銀化特異タンパク質はサクラマスの銀化度合いを調べるための生物学的指標の一つになる可能性が示された。また、このタンパク質は甲状腺ホルモンにより合成されることが明らかとなった。
  • Y Sawada, M Hattori, N Sudo, K Kato, Y Takagi, K Ura, M Kurata, T Okada, H Kumai
    AQUACULTURE RESEARCH 37 8 805 - 812 2006年06月 [査読有り][通常論文]
     
    A previous study elucidated that an extreme hypoxia during somitogenesis induced the most frequent skeletal malformation centrum defects in red sea bream (RSB), Pagrus major. In this study, details of the hypoxic conditions to induce them in RSB, dissolved oxygen (DO) concentration and exposure time to hypoxia, were investigated. Fertilized eggs were exposed to seawater of six DO concentrations (0%, 10%, 25%, 50%, 75% and 100% of saturation) for seven different periods (5, 10, 30, 60, 120, 240 and 360 min) during somitogenesis. Somitic disturbances in newly hatched larvae were induced by exposure to 0% and 10% DO concentration for 10 and 120 min and longer respectively. Rearing eggs exposed to hypoxic condition of 10% DO for 240 min for 40 days post-hatch showed that the location and the frequency of somitic disturbances in larvae and centrum defects in juveniles were significantly correlated (P < 0.01). Dissolved oxygen concentration of the interstitial water in the egg high density layer formed at the water surface in a stationary state abruptly decreased to 3.7% within 7 min. Centrum defect induction by exposure of eggs to extreme low DO concentrations for a short period, which is the probable situation in the practical juvenile production, suggests that careful maintenance of DO concentration is important in the incubating water of fertilized eggs during egg sorting and transportation, where eggs are made into a pile and undergo hypoxia, for the prevention of centrum defects.
  • K. Ura, A. Hara, K. Yamauchi
    Aquacult. Sci. 54 429 - 436 2006年 [査読有り][通常論文]
  • H. Shimada, N. Tominaga, S. Kohra, H. Ishibashi, Y. Mitsui, K. Ura, K. Arizono
    Trace Elements and Electrolytes 20 4 240 - 243 2003年12月 [査読有り][通常論文]
     
    Previously, we reported that the induction of vitellogenin mRNA in the larvae of Caenorhabditis elegans (C. elegans) can be used as a biomarker for short-term screening of environmental endocrine disrupters. Therefore, the present study was designed to determine if the induction of metallothionein (MT) mRNA in the larvae of C. elegans would be a biomarker for short-term screening of heavy metals. The larvae were exposed to various concentrations of cadmium (Cd), mercury (Hg), zinc chloride (Zn), cupper chloride (Cu) or lead acetate (Pb) for 3 h. Cd (1, 10 and 100 μM) and Hg (0.01, 0.1, 1 and 10 μM) exposures resulted in the marked induction of MT-I and MT-II mRNAs in the larvae of C. elegans as measured by reverse transcription polymerase chain reaction. The Cd-and Hg induction of MT-II mRNA was higher than that of MT-I mRNA, and concentration-dependent increase was observed in MT-II but not in MT-I. Time course analyses for MT-I and MT-II mRNA expressions with Cd and Hg were also determined. Cd induction of MT-I and MT-II mRNAs reached a peak at 2 h after the exposure (10 μM), and the levels of MT-II were higher than that of MT-I. For Hg, an initial peak of induction of MT-II mRNA occurred 15 min after the exposure (0.1 μM), and the levels reached maximum by 2 h. The initial peak of induction of MT-I mRNA occurred much later (∼ 2 h after Hg exposure) than MT-II mRNA. These results indicated that the induction of MT-II mRNA in the larvae of C. elegans can be used as a potential biomarker for short-term exposure to Cd and Hg.
  • N Tominaga, K Ura, M Kawakami, T Kawaguchi, S Kohra, Y Mitsui, T Iguchi, K Arizono
    JOURNAL OF HEALTH SCIENCE 49 1 28 - 33 2003年02月 [査読有り][通常論文]
     
    In this paper, Caenorhabditis elegans (C. elegans) is proposed as a model organism for studying chemical effects over multiple generations. We investigated whether C. elegans responds to vertebrate steroid hormones. We found that estrogenic steroids, especially estradiol (E2), have a cholesterol-like potency in supporting the reproduction of C. elegans. In contrast, testosterone (TS) and diethylstilbestrol (DES) did not display this potency. On the other hand, E2, TS and DES supressed the fecundity rate of C. elegans, when culture carried out with cholesterol. Moreover effect of TS accumulated over generation, in contrast to the other chemicals tested. These data suggested that with convenient biomarkers such as fecundity, C. elegans might be an effective model organism for studying chemical actions, including the disruption of reproduction.
  • H Shimada, N Tominaga, S Kohra, H Ishibashi, Y Mitsui, K Ura, K Arizono
    TRACE ELEMENTS AND ELECTROLYTES 20 4 240 - 243 2003年 [査読有り][通常論文]
     
    Previously, we reported that the induction of vitellogenin mRNA in the larvae of Caenorhabditis elegans (C. elegans) can be used as a biomarker for short-term screening of environmental endocrine disrupters. Therefore, the present study was designed to determine if the induction of metallothionein (MT) mRNA in the larvae of C elegans would be a biomarker for short-term screening of heavy metals. The larvae were exposed to various concentrations of cadmium (Cd), mercury (Hg), zinc chloride (Zn), cupper chloride (Cu) or lead acetate (Pb) for 3 h. Cd (1, 10 and 100 muM) and Hg (0.0 1, 0.1, 1 and 10 muM) exposures resulted in the marked induction of MT-1 and MT-II mRNAs in the larvae of C. elegans as measured by reverse transcription polymerase chain reaction. The Cd-and Hg induction of MT-II mRNA was higher than that of MT-I mRNA, and concentration-dependent increase was observed in MT-II but not in MT-I. Time course analyses for MT-I and MT-II mRNA expressions with Cd and Hg were also determined. Cd induction of MT-I and MT-II mRNAs reached a peak at 2 h after the exposure (10 muM), and the levels of MT-II were higher than that of MT-1. For Hg, an initial peak of induction of MT-II mRNA occurred 15 min after the exposure (0.1 muM), and the levels reached maximum by 2 h. The initial peak of induction of MT-1 mRNA occurred much later (similar to 2 h after Hg exposure) than MT-II mRNA. These results indicated that the induction of MT-II mRNA in the larvae of C. elegans can be used as a potential biomarker for short-term exposure to Cd and Hg.
  • K Ura, T Kai, S Sakata, T Iguchi, K Arizono
    JOURNAL OF HEALTH SCIENCE 48 6 583 - 586 2002年12月 [査読有り][通常論文]
     
    To evaluate the toxicity of environmental chemicals to invertebrates, a static bioassay was developed in the laboratory using the Caenorhabditis elegans (C. elegans). First, reproducibility of this aquatic acute toxicity test system was confirmed. In order to estimate chemical toxicities in C. elegans, worms were subsequently exposed to eleven different xenobiotics. Mortality after 24 hr was adopted as the endpoint of toxicity. We found that benzo[a]pyrene, nonylphenol, benzophenone, bisphenol A and cadmium chloride affected viability of C. elegans. These data suggest that C. elegans is a suitable toxicity test organism for environmental xenobiotic chemicals, and that lethality can be used as a testing endpoint.
  • K Mochida, T Matsubara, T Andoh, K Ura, S Adachi, K Yamauchi
    MOLECULAR REPRODUCTION AND DEVELOPMENT 62 1 57 - 68 2002年05月 [査読有り][通常論文]
     
    Our previous study shows that seminal plasma of a teleost, the Nile tilapia, contains a glycoprotein Mr = 120,000 named as SPP (Seminal plasma glycoprotein)120 which forms a homopolymer that has sperm immobilizing activity. In order to elucidate the mechanisms of the formation of the homopolymer and the immobilization of sperm, molecular cloning of SPP120 was conducted. The cDNA for SPP120 contains a complete open reading frame encoding 797 amino acid residues with 14 potential N-glycosylation sites. The predicted amino acid sequence of SPP120 contains a partial von Willebrand factor type D domain and a zona pellucida domain, that are involved in protein-protein adhesion that form filamentous structures in various kinds of cells. This result suggests that SPP120 forms a homopolymer via these domains in seminal plasma and probably interacts with spermatozoa. Northern blotting reveals that the gene is also expressed in ovary, even in ovulated eggs. The results of in situ hybridization indicate that in testis the gene is expressed in Sertoli cells and epithelial cells of sperm ducts, and the localization corresponds to that of the protein analyzed by immunohistochemistry. In the ovary, the gene is expressed at the perinucleolus stage of oocytes; however, the protein is not detected in any cells other than oocytes. (C) 2002 Wiley-Liss, Inc.
  • T Matsuno, K Ura, R Sonoda, Y Kohara, H Uesugi, K Arizono, T Iguchi, N Tominaga
    SENSORS AND MATERIALS 14 7 395 - 406 2002年 [査読有り][通常論文]
     
    We attempted to establish a system for sensing chemical substances using gene expression patterns in Caenorhabditis elegans (C elegans). The target chemical substances used in this study were steroid hormones: estradiol and testosterone. These hormones are known to act as endocrine disruptors when the substances which come from outside are received by biological systems as an external (xenobiotic) factor. The effects of these substances on expression patterns were measured using the cDNA microarray. The hierarchical clustering method was applied to analyse the response patterns. They were classified according to the similarity of their responses. We found that each cluster, which was a group of similar response patterns, corresponds to one kind of chemical substance. This means that the C elegans cDNA microarray can be utilized as a chemical sensing system.
  • Shinya Mizuno, Kazuhiro Ura, Yoshifumi Onodera, Haruhisa Fukada, Naoyuki Misaka, Akihiko Hara, Shinji Adachi, Kohei Yamauchi
    Zoological Science 18 6 853 - 860 2001年08月 [査読有り][通常論文]
     
    We developed a quantitative PCR assay to investigate transcript levels of gill cortisol receptor (CR) in masu salmon (Oncorhynchus masou). Using this system, we examined changes in transcript levels of gill CR during smoltification of wild masu salmon while tracking serum cortisol and growth hormone (GH) concentrations patterns. The masu salmon were parr in January and February, and thereafter smoltified, migrating from the river to the sea in May. Gill CR transcript levels were very low in January and February, but thereafter increased and reached a maximum in April (5 fold increase over levels observed in January). In May, when smolt enter the sea, the gill CR transcript levels decreased. Serum cortisol concentrations were also low from January to March and increased to the peak in April. These changes correlated well with changes in CR transcript levels from March to April. In contrast, serum GH concentration began to increase in January, peaked in March and decreased from March to May. These results elucidated patterns in tran-script levels of gill CR during smoltification of wild masu salmon and suggested that gill CR transcription was positively regulated by cortisol until serum cortisol level reached a peak, and negatively done after the peak in masu salmon.
  • 浦 和寛
    MRIレポート : 長崎大学水産学部附属海洋資源教育研究センター年報 : annual report of Marine Research Institute, Nagasaki University 3 長崎大学 2001年04月 [査読無し][通常論文]
  • S Mizuno, K Ura, T Okubo, Y Chida, N Misaka, S Adachi, K Yamauchi
    FISHERIES SCIENCE 66 4 670 - 677 2000年08月 [査読有り][通常論文]
     
    Ultrastructural changes in gill chloride cells during smoltification were examined in wild and hatchery-reared masu salmon. On the filament, two types of chloride cells (alpha and beta) and accessory cells were observed in wild fish from January (parr) to May (full-smolt), while only alpha and accessory cells (alpha-a cells) were observed in hatchery-reared fish from March (pre-smolt) to May (full-smolt). Among alpha-a cells of both types of fish, development of rough endoplasmic reticulum (ER) and Golgi apparatus was detected in pre-smelt before full-smelt. However, ultrastructural changes were not revealed in beta cells of wild fish during smoltification. During smoltification, the alpha-a cell number increased and beta cell number decreased. On the lamella, only one type of chloride cell was observed in both wild and hatchery-reared fish during smoltification. Their ultrastructural changes were almost the same as those in filament alpha-a cells, and their number declined during smoltification. Although there was almost no difference between changes in ultrastructure or in the number of chloride cells in both wild and hatchery-reared fish during smoltification, the chloride cell number of hatchery-reared fish varied widely in comparison to that in wild fish. The present study is the first report for wild salmonids of ultrastructural changes in gill chloride cells during smoltification, and indicated that there were some ultrastructural differences between gill chloride cells of wild masu salmon and hatchery-reared fish.
  • K. Ura, S. Mizuno, T. Okubo, Y. Chida, S. Adachi, K. Yamauchi
    Fish Physiol. Biochem. 17 1-6 397 - 403 1997年12月 [査読有り][通常論文]
     
    Changes in immunoreactivity of Na+/K+-ATPase alpha-subunit in gill sections of wild masu salmon (Oncorhynchus masou) during the parr-smelt transformation (smoltification) were compared with changes in gill Na+/K+-ATPase specific activity. Gill Na+/K+-ATPase specific activity increased from April and peaked in May. Immunohistochemical analysis, using an antiserum against a synthetic oligopeptide based on the conserved region of the Na+/K+-ATPase alpha-subunit, revealed that immunoreactivity was confined to chloride cells in the surface layer of primary lamellae and the proximal end of secondary lamellae. The size and number of these cells increased gradually from February to May; however, the number of chloride cells of the secondary lamellae decreased in May. These data suggest that the synthesis of Na+/K+-ATPase and the proliferation of chloride cells occur prior to the elevation of enzyme activity. Moreover, it is likely the proliferation and hypertrophy of chloride cells on primary lamellae prepare smelts for entry into seawater and migration in the ocean.
  • N Hiramatsu, M Shimizu, H Fukada, M Kitamura, K Ura, H Fuda, A Hara
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-PHARMACOLOGY TOXICOLOGY & ENDOCRINOLOGY 118 2 149 - 157 1997年10月 [査読有り][通常論文]
     
    A specific and sensitive enzyme linked immunosorbent assay (ELISA) and a single radial immunodiffusion (SRID) were developed for measurement of serum vitellogenin (Vg) levels in Sakhalin taimen (Hucho perryi). Regarding specificity for serum Vg, an antiserum raised against lipovitellin of taimen (a-Lv) was adequate for both assays. ELISA and SRID could detect Vg in serum at concentrations as low as 10 ng/ml and 25 mu g/ml, respectively. In estrogen administration experiments, the level of serum Vg began clearly increasing within 12 to 24 hr after injection of immature females with estradiol-17 beta (E-2) The appearance and levels of Vg in males treated with E-2 were delayed and smaller, respectively, than for females. Vg levels varied throughout natural vitellogenesis from 0-4 mu g/ml (3 years old) to approximately 30 mg/ml (5-6 years old). We observed an early transitory peak of serum Vg levels (primary reaction) at the time of early vitellogenesis and chronic high Vg levels (for 6-7 months) in winter period before ovulation. Changes of serum E-2 levels were correlated with Vg levels. However, E-2 levels decreased a month earlier than Vg levels near ovulation. It appears that the duration of vitellogenesis in taimen is considerably longer than that in other salmonids, lasting more than 2 years. (C) 1997 Elsevier Science Inc.
  • 深田 陽久, 玄 浩一郎, 平松 尚志, 浦 和寛, 原 彰彦
    北海道大學水産學部研究彙報 47 2 31 - 39 北海道大学 1996年12月 [査読無し][通常論文]
  • K Ura, K Soyano, N Omoto, S Adachi, K Yamauchi
    ZOOLOGICAL SCIENCE 13 2 219 - 227 1996年04月 [査読有り][通常論文]
     
    A specific polyclonal antibody against Na+, K+-ATPase alpha-subunit was developed using a synthetic oligopeptide as antigen. By Western blot analysis under non-reducing conditions, this antibody recognized a protein band of approximately 150 kDa corresponding to the intact form (alpha beta-complex) of Na+, K+-ATPase in rabbit kidney. Furthermore, this antibody recognized a 150 kDa protein band corresponding to the intact form of Na+, K+-ATPase and some bands of about 60-65 kDa corresponding to fragments of the alpha-subunit in gill and kidney of masu salmon. This antibody did not recognize the a-subunit under reducing conditions. By immunohistochemical analysis, cells immunoreactive with this antibody were observed in renal tubular epithelial cells in kidney sections of rabbit, masu salmon, eel and rockfish. In addition, large spherical eosinophilic cells in gills of masu salmon, eel and rockfish were immunoreactive with the antibody. It is likely that these immunoreactive cells correspond to gill chloride cells. These data indicate that this antibody is a useful tool for studying changes in and the function of Na+, K+-ATPase during osmoregulatory adaptation in a variety of fish species.
  • T IKEUCHI, K MOCHIDA, K URA, S ADACHI, K YAMAUCHI
    ZOOLOGICAL SCIENCE 12 3 317 - 323 1995年06月 [査読有り][通常論文]
     
    Specific polyclonal antibodies against the two salmonid gonadotropins (GTHs) were developed using two synthetic oligopeptides corresponding to GTH I beta-(96-113) and GTH II beta-(107-119), respectively. By Western blot analysis under reducing conditions, anti-GTH I beta-(96-113) detected one specific band corresponding to GTH I beta (17 kDa) and anti-GTH II beta-(107-119) detected one specific band corresponding to GTH II beta (20 kDa). Neither antibody recognized bands under nonreducing conditions. By immunohistochemical analysis, immunoreactive (ir-) I beta-(96-113)and ir-II beta-(107-119)-cells had different regional distributions among the aldehyde fuchsin positive cells and were not stained with an anti-human thyrotropin beta antibody. In salmonid fishes (chum salmon, rainbow trout, brook trout, whitespotted char, dolly varden and huchen), each antibody reacted with distinctly separate hypophyseal cells. However, in nonsalmonid species, and even in the salmoniform fish, the ayu (Plecoglossus altivelis), no pituitary cell was stained with these antibodies. These data indicate that these antibodies are useful tools for studying the change and the function of GTHs in salmonid fishes.
  • M NAGAE, H FUDA, K URA, H KAWAMURA, S ADACHI, A HARA, K YAMAUCHI
    FISH PHYSIOLOGY AND BIOCHEMISTRY 13 1 41 - 48 1994年05月 [査読有り][通常論文]
     
    Immunoglobulin M (IgM) is known as a main factor in the humoral immune system of teleosts. In the present study, the effect of cortisol on plasma IgM concentrations was investigated using a specific antibody to IgM in masu salmon (Oncorhynchus masou). Cortisol was orally administered each day for 2 weeks at a dose of 1 mg g-1 in the diet, and for the following week the fish were fed a non-treated diet. Blood plasma samples were collected at 0, 1, 2 and 3 weeks after the initiation of treatment. Oral administration of cortisol elevated plasma cortisol concentrations to about 40 ng/ml for 2 weeks after administration and slightly reduced plasma IgM concentration; the suppression was statistically significant one week after the period of hormone administration. However, treatment with cortisol did not affect plasma concentrations of total protein or alpha1-protein, one of the major serum proteins, during the experimental period. These results indicate that cortisol specifically suppresses plasma IgM concentrations.
  • K URA, A HARA, K YAMAUCHI
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-PHYSIOLOGY 107 4 607 - 612 1994年04月 [査読有り][通常論文]
     
    Changes in serum thyroid hormones (thyroxine:T-4, triiodothyronine:T-3), skin and serum guanine, total serum protein and albumin-like protein (alpha 1-protein) concentrations were investigated during smoltification in yearling masu salmon, Oncorhynchus masou. Moreover, the effects of T-4 on their concentrations were examined in the underyearling fish. Skin and serum guanine levels increased as smoltification progressed. T-4, and T-5 levels followed skin and serum guanine profiles. At least four guanine-binding proteins were found in the serum. One of them was identified immunologically as alpha 1-protein. T-4 treatment caused increase in guanine levels in skin and serum. However, T-4 led to no change of alpha 1-protein concentrations. The present results suggest a possible pathway whereby guanine is induced by T-4 and transported to the skin by guanine-binding protein in the blood.
  • K URA, A HARA, H KAWAMURA, K YAMAUCHI
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 107 2 225 - 229 1994年02月 [査読有り][通常論文]
     
    Serum protein profiles of parr and smelt in masu salmon (Oncorhynchus mason) were analyzed by crossed immunoelectrophoresis (CIE) and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D-SDS-PAGE) combined with Western blotting. Comparing with parr serum and smelt serum by CIE, three proteins were identified to be changed quantitatively. By ZD-SDS-PAGE combined with Western blotting, two proteins with molecular weights of 43,000 and 80,000, were identified as possible smolt-specific serum proteins, since they were not detected in parr.
  • 浦 和寛, 原 彰彦, 山内 晧平
    日本水産学会誌 59 7 1225 - 1229 日本水産學會 1993年07月 [査読無し][通常論文]
     
    Changes in concentrations of serum thyroxine, skin and serum guanine, and total serum protein were measured in hatchery-reared masu salmon Oncorhynchus masou during smoltification. In June, the condition factor was at its lowest value while the serum thyroxine was at its highest level, showing the occurrence of the peak of smoltification in the stock used in the present study. Total serum protein decreased significantly in May, gradually increasing thereafter. Serum guanine increased from April and reached a maximum in May. In contrast, skin guanine dropped to a minimum in May but rose in June to reach a peak in July, suggesting that guanine was transported to the skin from the circulation. By measuring the serum guanine concentrations, it is possible to analogize the degree of the development of smolting.
  • Purification and quantification of albumin-like protein (a1-protein) from
    K. Ura, A. Hara, K. Yamauchi
    Bull. Fac. Fish. Hokkaido Univ. 44 95 - 104 1993年 [査読無し][通常論文]
  • 浦 和寛, 原 彰彦, 山内晧平
    日本水産学会誌 59 7 1225 - 1229 1993年 [査読有り][通常論文]
     
    Changes in concentrations of serum thyroxine, skin and serum guanine, and total serum protein were measured in hatchery-reared masu salmon Oncorhynchus masou during smoltification. In June, the condition factor was at its lowest value while the serum thyroxine was at its highest level, showing the occurrence of the peak of smoltification in the stock used in the present study. Total serum protein decreased significantly in May, gradually increasing there-after. Serum guanine increased from April and reached a maximum in May. In contrast, skin guanine dropped to a minimum in May but rose in June to reach a peak in July, suggesting that guanine was transported to the skin from the circulation. By measuring the serum guanine concentrations, it is possible to analogize the degree of the development of smolting. © 1993, The Japanese Society of Fisheries Science. All rights reserved.

その他活動・業績

  • 小林 豊, 高村 巧, 下野 功, 浦 和寛, 都木 靖彰 水産増殖 = The aquiculture 57 (2) 271 -278 2009年06月20日 [査読無し][通常論文]
     
    岩手県大槌産、岩手県山田産、北海道鹿部産、北海道知内産の2年貝養殖ホタテガイの貝殻中のLi、Mn、SrおよびBa濃度を誘導結合プラズマ質量分析装置を用いて測定したところ、産地により微量元素組成に有意な差が認められた。MANOVAの判別分析を用いて産地識別を試みたところ、48個体中9個体で誤判別が発生し、判別的中率は81.3％であった。大槌産と他の産地との間で誤判別される例が多かったが、そのほかの産地間の誤判別は少なかった。同様の方法を用いて岩手県八幡平産および北海道七飯産の養殖ニジマスの耳石中の微量元素濃度を定量した結果、その組成にも産地により有意な差があった。判別的中率は95.5％であった。生産年による微量元素組成の変動が小さければ、貝殻および耳石微量元素組成による産地識別が可能であると考えられる。
  • 浦 和寛, 田中 恵梨, 東藤 孝, 後藤 孝弘, 清水 幹博, 尾島 孝男, 都木 靖彰 北海道大学水産科学研究彙報 58 (3) 21 -28 2009年02月 [査読無し][通常論文]
     
    We purified a subtilase from digestive system of short spined sea urchin Strongylocentortus intermedius by a combination of ion-exchange chromatography and gel filtration. The molecular weight of purified subtilase on SDS-PAGE under both reducing and nonreducing conditions was 35,000. Antiserum against subtilase specifically immunostained its antigen. By Western blot analysis, immunoreactive with this antibody were observed in stomach and intestine.
  • 澤田 好史, 樋口 和宏, 芳賀 穣, 浦 和寛, 石橋 泰典, 倉田 道雄, 宮武 弘史, 片山 茂和, 瀬岡 学 日本水産学会誌 74 (2) 144 -151 2008年03月 [査読無し][通常論文]
     
    体節形成期シマアジ胚への低酸素・高二酸化炭素環境の影響を明らかにした。溶存酸素(DO)濃度O%の極端な低酸素,溶存二酸化炭素(DCD)濃度120mg/Lの高二酸化炭素は,体節分節異常を誘導した。DO 25%の中庸の低酸素のみでは体節分節異常誘導はなかったが,DCD 120mg/Lの高二酸化炭素部併発すると分節異常部誘導され,その誘導率はD0 100%,DCD 120 mg/Lの場合より高い傾向にあった。したがって,低酸素環境と高二酸化炭素環境部併発する場合,体節分節異常誘導の相乗的効果が認められる。
  • Kurin Iimura, Hidekazu Tohse, Kazuhiro Ura, Yasuaki Takagi ZOOLOGICAL SCIENCE 23 (12) 1180 -1181 2006年12月 [査読無し][通常論文]
  • Y Sawada, M Hattori, M Iteya, Y Takagi, K Ura, M Seoka, K Kato, M Kurata, H Mitatake, S Katayama, H Kumai FISHERIES SCIENCE 72 (2) 364 -372 2006年04月 [査読無し][通常論文]
     
    Artificially hatched Seriola species have the problem of malformation, mainly in their vertebrae, head, and mouth parts. To clarify the cause of vertebral malformation, the effects of hypoxia during embryogenesis on the induction of centrum defects was investigated in artificially hatched amberjack Seriola dumerili. Firstly, 7-somite stage embryos were exposed to waters of 0, 12.5, 25, 50, and 100% dissolved oxygen (DO) for 0.5, 1, 2, 3, 4, and 5 h to confirm the effective dose (DO concentration and duration of exposure) of hypoxia that induces somitic disturbances in newly hatched larvae. Exposure of embryos to 12.5% DO concentration for longer than 0.5 h induced somitic disturbances. Following this result, centrum defects in juveniles were investigated by an induction experiment with embryos exposed to 12.5% DO for 2 h at the gastrula, 1- or 2-somite, 10-somite, 15-somite, or heart beating stage. This experiment revealed that centrum defects were induced only during somitogenesis, and somitic disturbances were the premonitory symptom of centrum defects. These results indicate hypoxia during somitogenesis as a possible cause of centrum defects in amberjack.
  • 持田和彦, 松原孝博, 安藤忠, 浦和寛, 足立伸次, 山内こう平 日本水産学会大会講演要旨集 2001 2001年
  • Ura K., Onodera Y., Mizuno S., Hara A., Adachi S., Yamauchi K. Zoological science 16 105 -105 1999年
  • 浦 和寛 日本比較内分泌学会ニュース = Newsletter of Japan Society for Comparative Endocrinology 88 41 -42 1998年02月 [査読無し][通常論文]

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年04月 -2025年03月 
    代表者 : 都木 靖彰, 浦 和寛, 東藤 正浩
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 浦 和寛
     
    本研究の最終目標は、低コストウニ用配合飼料の開発を目指している。この最終目標を実現するために、本研究では、ウニ生殖巣の肥大に伴う栄養の合成・蓄積に最も関与している核内受容体COUP-TFのリガンドを特定することを第一目的とする。COUP-TFは、無脊椎動物から脊椎動物において存在し、タンパク質・糖・脂質類の合成を制御しているが、未だ生体内のリガンドは不明である。ウニ生殖巣の肥大時にタンパク質、糖、脂質類の合成・蓄積が核内受容体の制御により成されている科学的根拠を基にして、本研究で明らかにするCOUP-TFのリガンドを含む低コスト天然素材を用いウニ用配合飼料を設計に応用する。 MYPプロモーター領域DNAと蛍光タンパク質を組み込んだプラスミドベクター(pGL4.10)およびキタムラサキウニCOUP-TFの発現ベクター(pcDNA3.1)を動物細胞に導入し共発現させるレポーターアッセイ系を構築した。マウスCOUP-TFのレポーターアッセイ系では、レチノイン酸で活性が認められることが報告されている。ウニ生殖巣から総脂質を抽出し、総脂質を添加した結果、濃度依存的に活性が上昇した。このことから、ウニ生殖巣の総脂質中にウニCOUP-TFのリガンドが存在することが示された。さらに、ウニ生殖巣総脂質をシリカゲルクロマトグラフィーにより４つの分画に分けた (Fraction1-4)。各フラクションをレポーターアッセイ系で活性を評価した結果、Fraction1で他の分画に比べ最も高い活性が認められた。また、Fraction1の活性度合いは、レチノイン酸添加群より活性が高かった。Fracation1を薄層クロマトグラフィーで解析した結果、Fracation1はレチノイン酸を含まないことが明らかになった。これらのことから、レチノイン酸以外がCOUP-TFのリガンドである可能性が示された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年07月 -2023年03月 
    代表者 : 都木 靖彰, 浦 和寛
     
    小課題1．チョウザメ脊索Ⅱ型コラーゲン（NC）を用いたⅡ型コラーゲン線維コート技術の開発： 本研究室で開発されたチョウザメ浮袋コラーゲン（SBC）を用いた線維コート技術 (Moroi et al., DOI: 10.1016/j.msec.2019.109925) を基盤としてNCの線維コート技術を開発した（線維コートとは、コラーゲン原線維を培養容器底面に一様にコーティングする技術で、生体組織のコラーゲン性基質と細胞とのインタラクションの研究を可能にする新技術である）。Ⅱ型コラーゲン溶液の濃度、線維化バッファー（リン酸バッファー）の濃度、ｐH、インキュベーション温度などを最適化することで、世界で初めてⅡ型コラーゲンの線維コートに成功した。また、マウス軟骨前駆細胞ATDC5を用いた培養試験を実施し、Ⅱ型コラーゲン分子コートと比較して線維コート上では細胞増殖速度が低下する一方で軟骨基質産生を示すアルシアンブルー染色が強陽性となる（＝前軟骨細胞から成熟軟骨細胞への分化が促進される）ことを確認した。今後軟骨細胞遺伝子発現量を定量する。 小課題2．NCを用いた3次元足場ゲルの開発： 市販のブタⅠ型コラーゲンのゲル化法を参考に、細胞毒性の低い架橋剤ゲニピンを添加することで、NCを用いてNC原線維から成るゲルとNC分子からなるゲルの合成に成功した。今後ゲルの粘弾性や細胞培養への応用技術を開発する。 小課題3．SBCもしくはNCを用いた軟骨細胞spheroid作成技術の開発： SBCを用いてマウス由来前骨芽細胞MC3T3-E1をスフェロイド化する技術を応用し、ATDC5細胞のスフェロイド化に挑戦した。しかし、MC3T3-E1がスフェロイド化する条件ではATDC5細胞はスフェロイド化しなかった。本技術開発には別のアイディアが必要であると思われる。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2019年06月 -2022年03月 
    代表者 : 都木 靖彰, 柚木 俊二, 成田 武文, 畑山 博哉, 浦 和寛
     
    本研究では魚類コラーゲン（SBC）を用いることで，ミクロンレベルの大直径コラーゲン線維の成長機構を直接観察するためのAFM観察，デジタルマイクロスコープ観察技術，NaCl濃度やPB濃度によるバンドル形成と細い線維の形成の制御技術を開発した。またSBC線維，分子上の細胞の遺伝子発現を変化させる情報の入り口が細胞接着部にある可能性を示した。今後これらの技術を用いることでバンドル形成機構をさらに詳細に検証できる。また，細胞とコラーゲン線維との接着装置であるインテグリンとその後の情報伝達経路を線維と分子で比較することで，線維への接着が遺伝子発現を制御する機構の解明が実現する可能性がある。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 都木 靖彰, 浦 和寛, 東藤 孝
     
    ウニ生殖巣の肥大化メカニズムを研究するツールとして，生殖巣器官培養系を確立し，これを用いて生殖巣の肥大に重要な主要卵黄タンパク質（MYP）の合成を誘起する因子を探索した。仮説としてウニ体腔液中の因子と核内受容体に結合する脂質を設定し，ウニ生殖巣が肥大した時期の体腔液および肥大したウニ生殖巣から抽出した総脂質を添加して生殖巣を培養し，MYP mRNA量を定量ＰＣＲにより測定したが，どちらも優位な変動は認められなかった。本研究期間において，ウニ生殖巣中のMYP mRNAの発現を誘起する因子の決定はできなかった。今後，MYPの合成に関与する核内受容体を特定しそのリガンドを添加する実験が必要である。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2013年04月 -2016年03月 
    代表者 : 浦 和寛, 都木 靖彰, 井尻 成保
     
    ウニの生殖巣肥大にステロイドホルモンが関与しているか解明するために、生殖巣が小さいキタムラサキウニに給餌し生殖巣を人為的に肥大させた。給餌開始前後のウニから生殖巣を摘出し、次世代シークエンス解析によりトランスクリプトーム解析を行い、22種類の核内受容体および21種類のP450を同定した。脊椎動物に見られるステロイドホルモンをリガンドとする核内受容体は認められなかった。一方、脊椎動物においてステロイドホルモンの合成に必須のP450と相同性を持つP450遺伝子は認められなかった。さらに、生殖巣ではEcR/FXRが認められたことからウニ特有のステロイドホルモンを合成・代謝している可能性が示された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2011年 -2013年 
    代表者 : 都木 靖彰, 浦 和寛, 東藤 孝
     
    これまで、ウニの生殖巣の発達につれて生殖巣で合成・蓄積されるタンパク質（主要卵黄タンパク質 Major Yolk Protein :MYP）の遺伝子構造・発現部位が明らかにされていたが、生殖巣の発達を統御機構に関しては不明なまま残されていた。本研究では、MYPおよびGAPDH mRNA発現定量系の確立をおこなうとともに、MYPの合成を誘起する生殖腺刺激ホルモンの探索を行うための生殖巣器官培養の確立を試み、短期間の器官培養系を確立した。これにより、今後 MYP mRNA 発現量を指標に生殖の内分泌統御に関する研究が可能になる。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2010年 -2012年 
    代表者 : 浦 和寛, 都木 靖彰
     
    北海道はウニの主要な生産地であり、従来天然ウニを漁獲すると共に、生殖巣の発達の悪いウニを良漁場へ移植するなどの対処がなされてきた。そこで、これらのウニを養殖することで生殖巣の増大を図り、商品として有効利用する試みが行われ生殖巣の品質向上に適した餌の開発が必要とされている。養殖事業や中間育成事業を効率良く行うためには、量や品質を管理しやすい人工餌料の開発が重要である。人工餌料の開発にはウニの消化吸収能力に関する基礎的知見の集積が必須であるが、これまで殆ど行われてきていなかった。本研究では、ウニ消化酵素を精製し生化学的特性を明らかにすることを目的とした。その結果、エゾバフンウニの消化酵素はキタムラサキウニに比べ高温に弱いということが明らかになった。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2009年 -2012年 
    代表者 : 都木 靖彰, 生駒 俊之, 浦 和寛
     
    質量分析を用いて,鱗コラーゲンの高次配向制御に関わる可能性が高いSLRPsを同定するとともに,その遺伝子クローニングをおこなって発現部位を明らかにした。また,コラーゲン変性温度がα3鎖の量とプロリン残基水酸化率とで制御される可能性を示した。さらに,磁場内でコラーゲン線維配向構造創出をおこなって粘弾性特性,引張強度を明らかにするとともに,再線維化コラーゲンを用いて高密度の新規メソ多孔材料の開発に成功した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2006年 -2008年 
    代表者 : 都木 靖彰, 浦 和寛, 生駒 俊之
     
    ウロコのコラーゲンを用いて再生医療用人工基質の合成をめざして、生物学と材料科学の両面から研究を推進した。生物学的アプローチにより鱗形成細胞分化の分子機構、コラーゲン配向を制御する候補分子、組織ごとのコラーゲンα鎖組成を明らかにした。また、材料科学的アプローチにより、ブタ及びティラピアコラーゲン線維配向ゲルを高磁場内で実現するとともに、湿潤環境下による乾燥により10~20wt%の高密度化に成功した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2006年 -2007年 
    代表者 : 都木 靖彰, 浦 和寛
     
    我が国の水産業において、水産無脊椎動物は重要な増養殖対象動物としての位置を占めており、種苗生産・放流や養殖が盛んに行われている種も多い。近年、日本沿岸域における水産生物資源、特に無脊椎動物の資源量は減少傾向にあることから、今後は優良品種の育種や高度で効率的な増養殖技術の開発が必要になると考えられるが、水産無脊椎動物においてそのような試みは著しく立ち遅れている。その大きな理由として、水産無脊椎動物の成長や成熟を制御する内分泌系に関する基礎的知見が極めて少ないといえる。比較的研究されているウニでも、糖質・脂質・タンパク質の代謝を制御する内的因子(ホルモンなど)の存在の有無や、それらの作用機序について正確に把握されていないのが現状である。本研究は、ウニをモデル生物として選定し、無脊椎動物の内分泌機構の解明に着手するものである。 今年度は、エゾバフンウニ消化器官において、モルモット抗ブタインスリン抗血清を用いて免疫組織化学的解析を行った。その結果、胃において消化管内腔側の上皮細胞の小さな顆粒に陽性反応が認められた。また、卵巣、精巣、放射神経にも免疫陽性反応が認められた。このことからエゾバフンウニにインスリン様物質が局在している可能性が示された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2006年 -2007年 
    代表者 : 長江 真樹, 原 彰彦, 征矢野 清, 浦 和寛
     
    環境ホルモンの生物影響評価は、女性ホルモン様作用検出においては研究が進展しているが、男性ホルモン様作用に関しては立ち後れている。それは、化学物質の持つ男性ホルモン作用を評価するツールの開発が遅遅として進んでいないためである。イトヨはその特殊な生物学的特徴から、男性ホルモンおよび女性ホルモン作用の両方を効果的に検出するバイオマーカーを備えている。本研究課題では、それらバイオマーカーを定量するための測定系の構築ならびに曝露試験法の開発を実施した。 平成18年度は、男性ホルモンバイオマーカーであるスピギンの超高感度測定系の開発を行い、正確かつ鋭敏な測定系を確立した。また、女性ホルモンバイオマーカーであるビテロゲニンの精製ならびに抗体作成を行った。 平成19年度には、特異抗体を用いたビテロゲニンの化学発光測定系を確立した。本測定法は、発光検出系の導入により極めて感度の高い測定系となった。また、イトヨ個体を用いて曝露試験を実施する際の曝露条件(曝露日数および曝露水温)の検討も行った。その結果、合成男性ホルモンであるMT0.1,1および10μg/Lの濃度曝露では3日間の曝露で十分な生物学的評価が実施可能であると裏付けられた。さらに曝露試験時の水温影響についても5,10および15℃での曝露を実施し、スピギンmRNA濃度変化を解析したところ、15℃が最も高感度にホルモン作用を発現させることが明らかとなった。 以上本研究課題では、イトヨの優良バイオマーカーを用いた環境ホルモン影響評価法を開発し、極めて実用性の高い測定法ならびに曝露試験法を確立した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2005年 -2006年 
    代表者 : 浦 和寛
     
    北海道において経済的価値の高いウニは養殖事業の最適種の一つであり、ウニの種苗生産技術は、ほぼ確立さているものの、食品的価値の高いサイズまで効率的に飼育する中間育成技術の確立には至っていない。本研究では、ウニ類の新たな養殖技術に応用するための第一歩として、ウニ類の生殖生理学の基礎的知見の集積を目的としている。本年度では、エゾバフンウニをモデル生物とし、生殖巣の発達に関与するタンパク質の同定と遺伝子配列の決定を行った。多くの卵生動物では、卵黄中に個体発生に必要な栄養源として卵黄タンパク質を蓄積している。ウニ類においても卵黄タンパク質は存在するが、それらの機能などは詳細にされていない。ウニ類において興味深いのは、魚類では主要な卵黄タンパク質は雌特異的に存在するが、ウニ類においては雌雄生殖巣内に共通に存在することが知られている。本研究では、雌特異タンパク質として分子量約33kDaのタンパク質(YP33kDa)を免疫生化学手法により同定し、粗精製を行った後にN末端アミノ酸配列を解読した。解読したアミノ酸配列をもとにプライマーを作製し、RACE-PCR法によりcDNAのクローニングを行い、アミノ酸348残基をコードする全長1044bpのcDNAを得た。アミノ酸配列の解析の結果、脊椎動物の神経細胞に発現している細胞接着機能を有する。 Fasciclinタンパク質と相同性を持っていた。これらのことから、YP33kDaは受精時の精子との細胞接着に関与している可能性が示唆された。次に、YP33kDaの遺伝子の発現解析を行った。YP33kDaについては、雌生殖巣のみで発現が認められた。これらの結果から、今回同定されたタンパク質はエゾバフンウニ生殖巣の発達機構解明のための分子マーカーとして有用であると示された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2004年 -2005年 
    代表者 : 都木 靖彰, 浦 和寛
     
    1.昨年にひきつづき、キンギョの鱗再生にともない発現するRunx2(Cbfa1)およびBMP2,4遺伝子の塩基配列決定をおこなった。Runx2は4種のアイソフォームの全塩基配列を決定した。BMP2はほぼ70%程度の配列の決定を終えた。キンギョRunx2遺伝子はこれまでに報告されたゼブラフィッシュRunx2bと最も高い相同性(約90%)を示した。BMP2および4のcDNA断片はゼブラフィッシュBMP2および4と高い相同性を示した。 2.鱗有機基質の主成分であるキンギョI型コラーゲンα鎖cDNA塩基配列を決定した。3種のα1(I)cDNA(α1(I)-A,B,C)を得、α1(I)-Aはその全塩基配列を、α1(I)-B,Cはその70%を決定した。α2(I)も3種のcDNAを得、それらの全塩基配列を決定した。α3(I)は1種類の全塩基配列を決定した。キンギョα鎖は、ゼブラフィッシュα鎖と約90%、哺乳類のα鎖と約70%の相同性を示した。また、分子系統樹ではα3(I)がα1(I)から分岐していることが確認された。さらに、既報の脊椎動物I型コラーゲンα鎖のPro残基数とコラーゲン変性温度の関係を近似式で表し、Y=0.3853X-44.712(r=0.9916)を得た。ここで、Y:変成温度(℃)、X:アミノ酸1000残基あたりのPro残基数である。この式を用いてキンギョのコラーゲンの変成温度を推定したところ、変成温度は三量体を構成するα鎖の組成で異なり、α3(I)鎖を多く含むと変成温度が高くなることが示された。 3.低リン・低カルシウム条件下におかれたキンギョの再生鱗の有機基質の微細構造解析をおこなった。リン酸カルシウム結晶は、非コラーゲン性有機基質の電子密度の高い物質中で形成され、成長することを明らかにした。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2004年 -2005年 
    代表者 : 清水 幹博, 都木 靖彰, 尾島 孝男, 浦 和寛
     
    ウニは北海道において重要な漁業資源であるが、それらの天然資源は減少している。そのため、養殖システムの開発が重要となってきている。そのためには栄養吸収機構を理解する必要があるが、基礎的な生理学的知見が少ない。そこで本研究では、ウニの栄養吸収機構の基礎的知見得ることを目的とし、以下の知見を得た。 1.エゾバフンウニを用いて、消化器官(咽頭、食道、胃、腸、直腸)における組織観察を行った。その結果、咽頭と食道ではこれまで1種類の顆粒細胞の存在が報告されていたが、本研究により少なくとも2種類の顆粒細胞が存在していることが明らかとなった。 2.キタムラサキウニ消化管由来のアルカリセリンプロテアーゼ(SUPase)のcDNAおよび構造遺伝子をクローニングした。SUPaseのアミノ酸配列は、スブチラーゼに属する他の酵素の触媒領域と33-46%の同一性を示すこと、さらに、Asp-His-Serからなる荷電リレーを構成するアミノ酸も良く保存されていることから、SUPaseはスブチラーゼに分類されるプロテアーゼであることが明らかになった。 3.キタムラサキウニ消化管由来のセルラーゼ(SnEG54)のcDNAをクローニングした。SnEG54のアミノ酸配列をGHF9に属するセルラーゼである、シロアリ、ホヤ、アワビの触媒ドメインのアミノ酸配列と比較したところ、51-57%の相同性を示した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 1998年 -1999年 
    代表者 : 浦 和寛

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