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2016 年度 実施状況報告書

A new concept of salt handling by Na-binding proteins that immobilize excess Na+ to ease salt stress of seawater teleost fish

研究課題

研究課題/領域番号 16K18575
研究機関東京大学

研究代表者

黄 國成  東京大学, 大気海洋研究所, 特任助教 (40526901)

研究期間 (年度) 2016-04-01 – 2019-03-31
キーワードNa-binding protein / Osmoregulation / hypertension / Protein purification
研究実績の概要

The research of the first year was performed as planned. In the eel, I have discovered the special cells (club cells in the esophagus) producing putative Na-binding proteins. This protein is high in freshwater environment and could be important for the retention of Na in ion-poor environment. I have also developed other easier methods to detect protein-bounded Na using Coro Na-green and Na-22 as markers. This simplifies the work-flow and paves the way to identify the protein structure as electron-probe microanalysis is time consuming. I used different traditional stains, and new polysaccharide markers to distinguish various types of mucus cells in the esophagus and their abundance on the course from freshwater to seawater.
The new discoveries were due to a change in fixative from phosphate buffered paraformaldehyde to Carnoy’s fluid. The paraformaldehyde stimulated the secretion of mucus in the esophagus, thus reduced the number and variety of mucus cell, resulting in an underestimation. The Carnoy’s fluid prevents the secretion of mucus and preserves the mucus layers as the mucus proteins are not cross-linked, which is closer to the instantly fixed state. The new findings on the various mucus cells and their Na-binding activities are in preparation for further publications.
Native, denatured, and 2D gels were used to analyze the proteins from esophagus and initial Western blots indicated specific bands that are positive to Na-green.

現在までの達成度 (区分)
現在までの達成度 (区分)

3: やや遅れている

理由

I have established the basics of Na-binding phenomenon in eel during the first year as planned. The protein puriication was slightly delay as I encountered technical difficulties to develop the Na-binding assay. Recently, I have overcomed the difficulties by using radioactive Na-22 and new version of Na-green (Coro Na-green)as Na-marker. The protein purificaion process continue in next year.

今後の研究の推進方策

As the detection method for Na-binding proteins was simplified, I am planning to expand this studies to a comparative studies according to the research plan, using stenohaline species such as goldfish and euryhaline species such as salmons. I continue to establish the Na-binding assay and use various protein purification methods to purify the Na-binding proteins from eels. The purified proteins will be analyzed by MS/MS to identify the sequence and finally mapped on the assembled transcriptome. This will be the first described Na-binding protein known to science.

次年度使用額が生じた理由

As the protein purification was delayed as mentioned, the cost of mass spectrometry analysis was not used. Furthermore, I have developed a newer method to visualize the bound Na using Na-green instead of EPMA, thus the cost of EPMA consumables decreased.

次年度使用額の使用計画

For the identfication of Na-binding protein, I will spend the funding on the mass spectrometry analysis and also raising antibodies on the newly identified proteins early this year. I will also attend international conference to present the results of this projects.

  • 研究成果

    (7件)

すべて 2017 2016

すべて 雑誌論文 (5件) (うち国際共著 1件、 査読あり 5件、 オープンアクセス 1件) 学会発表 (2件)

  • [雑誌論文] Development of an animal-borne blood sample collection device and its deployment for the determination of cardiovascular and stress hormones in submerged phocid seals.2017

    • 著者名/発表者名
      Takei Y, Suzuki I, Wong MKS, Milne R, Moss S, Sato K, Hall A.
    • 雑誌名

      Am J Physiol Regul Integr Comp Physiol

      巻: in press ページ: in press

    • DOI

      doi: 10.1152/ajpregu.00211.2016.

    • 査読あり / 国際共著
  • [雑誌論文] Molecular mechanisms underlying active desalination and low water permeability in the esophagus of eels acclimated to seawater.2017

    • 著者名/発表者名
      Takei Y, Wong MKS, Pipil S, Ozaki H, Suzuki Y, Iwasaki W, Kusakabe M.
    • 雑誌名

      Am J Physiol Regul Integr Comp Physiol

      巻: 312 ページ: R231-R244

    • DOI

      doi: 10.1152/ajpregu.00465.2016.

    • 査読あり
  • [雑誌論文] Molecular and evolutionary perspectives of the renin-angiotensin system from lamprey.2017

    • 著者名/発表者名
      Wong MKS, Takei Y.
    • 雑誌名

      Gen Comp Endocrinol

      巻: in press ページ: in press

    • DOI

      doi: 10.1016/j.ygcen.2017.01.031.

    • 査読あり
  • [雑誌論文] Duplicated CFTR isoforms in eels diverged in regulatory structures and osmoregulatory functions.2016

    • 著者名/発表者名
      Wong MKS, Pipil S, Kato A, Takei Y.
    • 雑誌名

      Comp Biochem Physiol A Mol Integr Physiol

      巻: 199 ページ: 130-141

    • DOI

      doi: 10.1016/j.cbpa.2016.06.018.

    • 査読あり
  • [雑誌論文] Flexible selection of diversified Na(+)/K(+)-ATPase α-subunit isoforms for osmoregulation in teleosts.2016

    • 著者名/発表者名
      Wong MKS, Pipil S, Ozaki H, Suzuki Y, Iwasaki W, Takei Y.
    • 雑誌名

      Zoological Lett

      巻: 2 ページ: 15

    • DOI

      doi: 10.1186/s40851-016-0050-7

    • 査読あり / オープンアクセス
  • [学会発表] Japanese eels regulate osmolality by uncharacterized sodium-binding proteins: identification via transcriptome2017

    • 著者名/発表者名
      1.Wong MKS, Tsukada T, Ogawa N, Pipil S, Ozaki H, Suzuki Y, Iwasaki W, Takei Y
    • 学会等名
      Advanced Genome Science International Symposium "The Start of New Genomics"
    • 発表場所
      University of Tokyo, Bunkyo, Tokyo
    • 年月日
      2017-01-09 – 2017-01-10
  • [学会発表] Sodium binding proteins in fish.2016

    • 著者名/発表者名
      2.Wong MKS, Ogawa N, Pipil S, Ozaki H, Suzuki Y, Iwasaki W, Tsukada T, Takei Y
    • 学会等名
      41st Japanese Comparative Endocrinology Meeting
    • 発表場所
      Kitasato University, Sagamihara, Kanagawa
    • 年月日
      2016-12-09 – 2016-12-11

URL: 

公開日: 2018-01-16  

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