Intermediate Formation via Proton Release during the Photoassembly of the Water-Oxidizing Mn4CaO5 Cluster in Photosystem II.

The early stages of the photoassembly of the water-oxidizing Mn4CaO5 cluster in spinach photosystem II (PSII) were monitored using rapid-scan time-resolved Fourier transform infrared (FTIR) spectroscopy. Carboxylate stretching and the amide I bands, which appeared upon the flash-induced oxidation of a Mn2+ ion, changed their features during the subsequent dark rearrangement process, indicating the relocation of the Mn3+ ion concomitant with protein conformational changes. Monitoring the isotope-edited FTIR signals of a Mes buffer estimated that nearly two protons are released upon the Mn2+ oxidation. Quantum chemical calculations for models of the Mn binding site suggested that the proton of a water ligand is transferred to D1-H332 through a hydrogen bond upon the Mn3+ formation and then released to the bulk as the Mn3+ shifts to bind to this histidine. Another Mn2+ ion may be inserted to form a binuclear Mn3+Mn2+ complex, whose structure was calculated to be stabilized by a µ-hydroxo bridge hydrogen-bonded with deprotonated D1-H337. Nearly one additional proton can thus be released from this histidine, assuming that it is mostly protonated before illumination. Alternatively, a proton could be released by further insertion of Ca2+, forming a Mn3+Mn2+Ca2+ complex with another hydroxo ligand connecting Ca2+ to the Mn3+Mn2+ complex.

Viability variation of T-cells under ultrasound exposure according to adhesion condition with bubbles.

PURPOSE: Although cellular immunotherapy is expected as a new cancer treatment, its therapeutic efficiency is limited in solid tumors, because most cells return to the bloodstream rather than adhere to the target site. Therefore, we are motivated to develop a technique to concentrate the cells in the blood flow using active control of bubble-surrounded cells under ultrasound exposure considering both aspects of cell controllability and viability. METHODS: We prepared a lipid bubble conjugating ligand to adhere to the surface of the T-cells. First, we evaluated the cell controllability by retaining the cells on a wall of an artificial blood vessel through continuous ultrasound exposure. Next, we investigated the cell viability under ultrasound exposure in a suspension with various bubble concentrations. RESULTS: We estimated the concentration of bubbles when the adhesion to the cell surface was saturated. Then, we evaluated the cell viability with various conditions of ultrasound exposure and bubble concentrations. However, it was confirmed that cell damage occurred under conditions that achieved proper control of the cells. Therefore, we exposed the cells to burst waves to reduce the applied ultrasound intensity. Consequently, the significant increase in cell viability was confirmed to be inversely proportional to the duty ratio. CONCLUSION: To retain cells on a vessel wall, determining the appropriate ultrasound condition including sound pressure and waveform is important to maintain cell viability.