2016 Fiscal Year Research-status Report
A new concept of salt handling by Na-binding proteins that immobilize excess Na+ to ease salt stress of seawater teleost fish
| Project/Area Number |
16K18575
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| Research Institution | The University of Tokyo |
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Principal Investigator |
黄 國成 東京大学, 大気海洋研究所, 特任助教 (40526901)
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Keywords | Na-binding protein / Osmoregulation / hypertension / Protein purification |
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| Outline of Annual Research Achievements |
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.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
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.
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| Strategy for Future Research Activity |
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.
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| Causes of Carryover |
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.
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| Expenditure Plan for Carryover Budget |
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.
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[Presentation] Sodium binding proteins in fish.2016
Author(s)
2.Wong MKS, Ogawa N, Pipil S, Ozaki H, Suzuki Y, Iwasaki W, Tsukada T, Takei Y
Organizer
41st Japanese Comparative Endocrinology Meeting
Place of Presentation
Kitasato University, Sagamihara, Kanagawa
Year and Date
2016-12-09 – 2016-12-11
