Open Channel Structure of MscL: A Patch-Clamp and Spectroscopic Study

MscL, the bacterial mechanosensitive (MS) channel of large conductance, has become a prototype channel to study structure–function relationship of the MS class of ion channels. Within a few years of cloning the mscL gene, the three-dimensional structure of the MscL protein was determined by X-ray crystallography, which allowed for detailed molecular studies of its structure and function. A combination of the (1) patch-clamp recording examining the function of MscL channels reconstituted into artificial liposomes, and (2) electron paramagnetic resonance (EPR) and fluorescence resonance energy transfer (FRET) spectroscopy examining structural changes occurring during gating of the reconstituted channels, provided a plausible molecular mechanism of gating of MS channels. This experimental approach helped to establish two physical mechanisms as triggers of the MscL channel gating by lipid bilayer deformation forces: (1) the energetic cost of protein-bilayer hydrophobic mismatch and (2) the geometric consequences of bilayer intrinsic curvature. The results of the EPR and FRET spectroscopic studies showed that the open state of MscL is highly dynamic, supporting a water-filled pore of at least 25 Å in diameter corresponding to an overall change in diameter of the channel protein of about 15 Å.     

On Spectral Polynomials of the Heun Equation. II

The well-known Heun equation has the form

Potential of capillary zone electrophoresis for estimation of humate acid-base properties

Capillary zone electrophoresis (CZE) has been applied for fractionation and characterization of soil-derived humic acids (HAs). Humic acids from soddy-podzolic (HA(s)) and chernozem (HA(ch)) soils were studied as well as hydrophobic high-molecular-weight (HMW) and hydrophilic low-molecular-weight (LMW) HA(s) fractions obtained by salting-out with ammonium sulfate at a saturation of 0-40% and >70%, respectively. The possibility of CZE partial fractionation of HAs has been demonstrated. The shape of "humic hump" was shown to depend on the pH of running electrolyte. Almost the whole peak overlapping occurred if alkaline solutions were used for fractionation, but the peak resolution was improved at pH 5-7. Under appropriate fractionation conditions (pH 7), at least three humic acid subfractions with different electrophoretic mobilities were distinguished in the electropherograms of initial HA and HA(s) fractions. Such a high peak resolution has never been achieved for humic acids before. The presence of three subfractions in the HA is in agreement with gel-filtration analysis and was confirmed by comparison of the electrophoretic behavior of HA(s) with those of its HMW (hydrophobic) and the LMW (hydrophilic) fractions. The potentiometric titration of HA and its fractions was performed and the pK(a) of the functional groups were calculated. An attempt was made for the first time to relate the variation of electrophoretic mobility values with acid-base properties of humic acids. It was shown that changes in the humate charge resulting from the variation of the ionization degree of its functional groups as a function of pH can be estimated on the basis of electrophoretic mobility values. Potential of CZE in estimation of HA isoelectric point was demonstrated. The pH value corresponding to the lowest absolute electrophoretic mobility value of about 20 x 10(-5) cm(2) V(-1) s(-1) can be used for approximate estimation of HA isoelectric point. The data were discussed and agreement with the random coil structural model has been shown.     

Synthesis, molecular structure, and reactivity of the isolable silylenoid with a tricoordinate silicon

The first tricoordinate fluorosilylenoid, (t-Bu2MeSi)2SiFLi.3THF (1), was synthesized, and its X-ray molecular structure was determined. 1 was synthesized in 40% yield by a bromine-lithium exchange reaction in THF of the corresponding fluorobromosilane with t-Bu2MeSiLi. 1 is best described as an R2SiF- anion attracted to a (Li.3THF)+ cation with a small contribution of resonance structure that consists of a silylene fragment and FLi.3THF. 1 reacts as a nucleophile with MeCl, PhH2SiCl, H2O, and MeOH, as an electrophile with MeLi, and as a silylene with Li (or t-BuLi) and Na, yielding alpha-lithium and alpha-sodium silyl radicals, respectively. Either photolysis or thermolysis of 1 yields the corresponding disilene R2Si=SiR2 (R = t-Bu2MeSi), probably via dimerization of R2Si:.     

Corrosion studies and interfacial bonding of urea/poly(dimethylsiloxane) sol/gel hydrophobic coatings on AA 2024 aluminum alloy

Bis[(ureapropyl)triethoxysilane] bis(propyl)-terminated-polydimethylsiloxane 1000 (PDMSU), an organic-inorganic hybrid, diluted in either EtOH or a mixture of EtOH-PrOH, was used in thin film form (<200 nm) to inhibit the corrosion of AA 2024 alloy. Potentiodynamic, time-dependent cyclovoltammetric measurements and salt spray tests showed that the corrosion inhibition of the latter was 10 times higher than that of the former films. This was correlated with the higher degree of hydrolysis and the formation of more open polyhedral silsesquioxane species (T2) in the bulk heat-treated PDMSU/EtOH-PrOH xerogels (29Si NMR spectra). The structure of the coatings deposited on AA 2024 Al alloy was deduced from the infrared reflection-absorption (IR RA) spectra, which revealed more extensive urea-urea interactions and more efficient silane-Al interface bonding for the PDMSU/EtOH-PrOH coatings with higher corrosion inhibition. Ex situ IR RA potentiodynamic spectroelectrochemical measurements of PDMSU coatings revealed that their degradation did not proceed via the formation of silanol groups and consequent hydration of the coatings but that they decomposed above E(corr) by forming fragments composed of -CH2- segments in an all-trans conformation.     

Utilization and transport of l-arabinose by non-Saccharomyces yeasts

l-Arabinose is one of the sugars found in hemicellulose, a major component of plant cell walls. The ability to convert l-arabinose to ethanol would improve the economics of biomass to ethanol fermentations. One of the limitations for l-arabinose fermentation in the current engineered Saccharomyces cerevisiae strains is poor transport of the sugar. To better understand l-arabinose transport and use in yeasts and to identify a source for efficient l-arabinose transporters, 165 non-Saccharomyces yeast strains were studied. These yeast strains were arranged into six groups based on the minimum time required to utilize 20 g/L of l-arabinose. Initial transport rates of l-arabinose were determined for several species and a more comprehensive transport study was done in four selected species. Detailed transport kinetics in Arxula adeninivorans suggested both low and high affinity components while Debaryomyces hansenii var. fabryii, Kluyveromyces marxianus and Pichia guilliermondii possessed a single component, high affinity active transport systems.     

In vivo electrical impedance measurements during and after electroporation of rat liver

Electroporation is used for in vivo gene therapy, drug therapy and minimally invasive tissue ablation. Applying electrical pulses across cells can have a variety of outcomes; from no effect to reversible electroporation to irreversible electroporation. Recently, it has been proposed that measuring the passive electrical properties of electroporated tissues could provide real time feedback on the outcome of the treatment. Here we describe the results from the impedance characterization (single dispersion Cole model) for up to 30 min of the electroporation process in in vivo rat livers (n=8). The electroporation sequence consisted of 8 pulses of 100 micros with a period of 100 ms. Half of the animals were subjected to field magnitudes considered to have reversible effects (R group, E=450 V/cm) whereas for the other half irreversible field amplitudes were applied (I group, E=1500 V/cm). As expected, there was an immediate increase of conductivity (R group Deltasigma/sigma(t=0)=9+/-3%; I group Deltasigma/sigma(t=0)=43+/-1%). However, the overall long term pattern of change in conductivity after electroporation is complex and different between reversible and irreversible groups. This suggests the superposition of different phenomena which together affect the electrical properties.     

Characterization and quantification of Sm(III)/ and Cm(III)/clay mineral outer-sphere species by TRLFS in D2O and EXAFS studies

In order to assess the long-term safety of deep radioactive waste repositories, a precise characterization of the different sorption processes on a molecular basis and the exact definition of geochemical boundary conditions for their relevance are of immense importance. Through sorption on various minerals the migration of radionuclides will be hindered and their retention will be ensured. Using time-resolved laser fluorescence spectroscopy (TRLFS) and extended X-ray absorption fine structure (EXAFS) spectroscopy, it was possible to identify outer-sphere sorbed trivalent lanthanides and actinides onto different montmorillonites and illite. Furthermore, the quantification of Cm(III)/clay outer-sphere sorption in D(2)O at different ionic strengths was shown. The results were confirmed by ion exchange model calculations. Finally, the structural parameters of a Sm(III)/clay outer-sphere complex were obtained by EXAFS measurements.     

Development of a method for the determination of 93Zr and 94Nb in radioactive waste using TEVA® resin

The behaviour of ⁹⁵Zr and ⁹⁵Nb isotopes on TEVA^® resin column was studied. The influence of hydrofluoric, hydrochloric, oxalic and sulphuric acids on the separation and chemical yield of zirconium and niobium was tested. The method is suitable for the separation of ⁹³Zr and ⁹⁴Nb isotopes from hydrofluoric, hydrochloric and sulphuric acid by using different concentrations of the acid for the load or elutes solutions. Samples from reactor and fuel assemblies such as the control-rod connection rod, shielding/absorber parts of fuel rods, neutron in-core measurement channels, pressure vessel basic construction material and internal cladding, core barrel and reactor protection tube unit were analysed.