We have developed a new in-cell NMR method that is applicable to any type of cell and does not require target protein modification or specialized equipment. The stable-isotope-labeled target protein, thymosin beta4 (Tbeta4), was delivered to 293F cells, which were permeabilized by a pore-forming toxin, streptolysin O, and resealed by Ca(2+) after Tbeta4 uptake. As a result, we successfully observed (1)H-(15)N HSQC signals originating from the Tbeta4, including those from the N-terminal acetylation, which had occurred inside the cell as a post-translational modification.

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Pyrolysis and oxidation of acetaldehyde were studied behind reflected shock waves in the temperature range 1000-1700 K at total pressures between 1.2 and 2.8 atm. The study was carried out using the following methods, (1) time-resolved IR-laser absorption at 3.39 µm for acetaldehyde decay and CH-compound formation rates, (2) time-resolved UV absorption at 200 nm for CH 2 CO and C 2 H 4 product formation rates, (3) time-resolved UV absorption at 216 nm for CH 3 formation rates, (4) time-resolved UV absorption at 306.7 nm for OH radical formation rate, (5) time-resolved IR emission at 4.24 µm for the CO 2 formation rate, (6) time-resolved IR emission at 4.68 µm for the CO and CH 2 CO formation rate, and (7) a single-pulse technique for product yields. From a computer-simulation study, a 178-reaction mechanism that could satisfactorily model all of our data was constructed using new reactions, CH 3 CHO (+M) → CH 4 + CO (+M), CH 3 CHO (+M) → CH 2 CO + H 2 (+M),

To understand how intracellular proteins respond to oxidative stresses, the redox status of the target protein, as well as the intracellular redox potential ( E), which is defined by the concentrations of reduced and oxidized glutathione, should be observed simultaneously within living cells. In this study, we developed a method that can monitor the redox status of thioredoxin (Trx) and E by direct NMR observation of Trx and glutathione within living cells. Unlike the midpoint potential of Trx measured in vitro (∼ -300 mV), the intracellular Trx exhibited the redox transition at E between -250 and -200 mV, the range known to trigger the oxidative stress-mediated signalings. Furthermore, we quantified the contribution of Trx reductase to the redox status of Trx, demonstrating that the redox profile of Trx is determined by the interplay between the elevation of E and the reduction by Trx reductase and other endogenous molecules.

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