| Budget Amount *help |
¥173,160,000 (Direct Cost: ¥133,200,000、Indirect Cost: ¥39,960,000)
Fiscal Year 2018: ¥37,960,000 (Direct Cost: ¥29,200,000、Indirect Cost: ¥8,760,000)
Fiscal Year 2017: ¥37,960,000 (Direct Cost: ¥29,200,000、Indirect Cost: ¥8,760,000)
Fiscal Year 2016: ¥52,260,000 (Direct Cost: ¥40,200,000、Indirect Cost: ¥12,060,000)
Fiscal Year 2015: ¥28,340,000 (Direct Cost: ¥21,800,000、Indirect Cost: ¥6,540,000)
Fiscal Year 2014: ¥16,640,000 (Direct Cost: ¥12,800,000、Indirect Cost: ¥3,840,000)
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| Outline of Final Research Achievements |
We have established the in-cell NMR methodology that enables the observation of the intracellular biological events in real-time manner. To observe the intracellular molecular response against oxidative stresses, we prepared the HeLa cells, in which both thioredoxin and glutathione were isotopically labeled, and monitored the redox status of Trx and glutathione by alternate in-cell NMR measurements. This study demonstrated the redox status of Trx is regulated not only by the intracellular redox potential but also by various endogenous regulatory molecules, such as Trx reductases. We also performed the in-cell NMR observation of small GTPase Ras and its oncogenic variants. We demonstrated that the GTP-bound ratio of Ras in the steady state is decreased in the cell for all Ras constructs, due to the increase of khy and the decrease of kex. We also succeeded in quantifying the contribution of the specific regulatory proteins by in-cell NMR experiments using the knockout cells.
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