The genes of N. pharaonis SRII and the carboxy terminal truncated transducer (1-114) were cloned into a pET27bmod expression vector 24 with a C-terminal £ 7 His tag, respectively. Proteins were expressed in Escherichia coli strain BL21 (DE3), and purified as described 25,26. After removal of imidazol by diethyl-aminoethyl chromatography, SRII-His and HtrII 114-His were mixed in a 1:1 ratio, followed by reconstitution into purple membrane (the bacteriorhodopsin containing membrane patches of H. salinarum) lipids 7 (protein to lipid ratio 1:35). After filtration, the reconstituted proteins were pelleted by centrifugation at 100,000g. For resolubilization, the samples were resuspended in a buffer containing 2% n-octyl-b-D-glucopyranoside and shaken for 16 h at 4 8C in the dark. The resolubilized complex was isolated by centrifugation at 100,000g. Crystallization, structure determination and refinement We added the solubilized complex in crystallization buffer (150 mM NaCl, 25 mM Na/KPi, pH 5.1, 0.8% n-octyl-b-D-glucopyranoside) to the lipidic phase, formed from monovaccenin (Nu-Chek Prep). Precipitant was 1 M salt Na/KPi, pH 5.6. Crystals were grown at 22 8C. X-ray diffraction data were collected at beamline ID14-1 of the European Synchrotron Radiation Facility (ESRF), Grenoble, France, using a Quantum ADSC Q4R CCD (charge-coupled device) detector. Data were integrated using MOSFILM 27 and SCALA 28. Molecular replacement using MOLREP 28 to phase a polyalanine model (from Protein Data Bank accession number 1JGJ (ref. 12)) gave a unique solution (R ¼ 0.568, correlation coefficient C ¼ 0.357) at 2.9 A ˚. After inserting side chains for SRII, the helices of HtrII were found (R ¼ 0.329, C ¼ 0.711). Simulated annealing, positional refinement and temperature factor refinement were performed in CNS 29 ; model rebuilding was carried out in O 30 (Table 1).

Dielectrophoresis was employed to distinguish the electroporated from non-electroporated cells. It was found that the electric field frequency at which cells change the direction of their movement (the crossover frequency f(CO)) is higher when cells are electroporated. The contribution to the cell dielectrophoretic behavior of four electric and geometrical cell parameters was analyzed using a single shell model. f(CO) measurements were performed in media with conductivities of 0.001-0.09S/m, on B16F10 cells which were electroporated in a Mannitol solution (0.001S/m), using rectangular or exponential pulses. The control cells' f(CO) was found in a domain of 2 to 105 kHz, while the electroporated cells' f(CO) was in a domain of 5 to 350 kHz, depending on the external media conductivities. At exterior conductivities above ~0.02S/m, f(CO) of electroporated cells became significantly higher compared to controls. Even though the possible contribution of membrane permittivity to explain the observed f(CO) shift toward higher values cannot be excluded, the computations highlight the fact that the variation of cytosol conductivity might be the major contributor to the dielectrophoretic behavior change. Our experimental observations can be described by considering a linear dependence of electroporated cells' cytosol conductivity against external conductivity.

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The present study concerns the in vitro oxidative stress responses of non-malignant murine cells exposed to surfactant-tailored ZnO nanoparticles (NPs) with distinct morphologies and different levels of manganese doping. Two series of Mn-doped ZnO NPs were obtained by coprecipitation synthesis method, in the presence of either polyvinylpyrrolidone (PVP) or sodium hexametaphosphate (SHMTP). The samples were investigated by powder X-ray Diffraction, Transmission Electron Microscopy, Fourier-Transform Infrared and Electron Paramagnetic Resonance spectroscopic methods, and N2 adsorption–desorption analysis. The observed surfactant-dependent effects concerned: i) particle size and morphology; ii) Mn-doping level; iii) specific surface area and porosity. The relationship between the surfactant dependent characteristics of the Mn-doped ZnO NPs and their in vitro toxicity was assessed by studying the cell viability, intracellular reactive oxygen species (ROS) generation, and DNA fragmentation in NIH3T3 fibroblast cells. The results indicated a positive correlation between the specific surface area and the magnitude of the induced toxicological effects and suggested that Mn-doping exerted a protective effect on cells by diminishing the pro-oxidative action associated with the increase in the specific BET area. The obtained results support the possibility to modulate the in vitro toxicity of ZnO nanomaterials by surfactant-controlled Mn-doping.