Regulation of mesangial cell ion channels by insulin and angiotensin II. Possible role in diabetic glomerular hyperfiltration

We used patch clamp methodology to investigate how glomerular mesangial cells (GMC) depolarize, thus stimulating voltage-dependent Ca2+ channels and GMC contraction. In rat GMC cultures grown in 100 mU/ml insulin, 12% of cell-attached patches contained a Ca(2+)-dependent, 4-picosiemens Cl- channel. Basal NPo (number of channels times open probability) was < 0.1 at resting membrane potential. Acute application of 1-100 nM angiotensin II (AII) or 0.25 microM thapsigargin (to release [Ca2+]i stores) increased NPo. In GMC grown without insulin, Cl- channels were rare (4%) and unresponsive to AII or thapsigargin in cell-attached patches, and less sensitive to [Ca2+]i in excised patches. GMC also contained 27-pS nonselective cation channels (NSCC) stimulated by AII, thapsigargin, or [Ca2+]i, but again only when insulin was present. In GMC grown without insulin, 15 min of insulin exposure increased NPo (insulin > or = 100 microU/ml) and restored AII and [Ca2+]i responsiveness (insulin > or = 1 microU/ml) to both Cl- and NSCC. GMC AII receptor binding studies showed a Bmax (binding sites) of 2.44 +/- 0.58 fmol/mg protein and a Kd (binding dissociation constant) of 3.02 +/- 2.01 nM in the absence of insulin. Bmax increased by 86% and Kd was unchanged after chronic (days) insulin exposure. In contrast, neither Kd nor Bmax was significantly affected by acute (15-min) exposure. Therefore, we concluded that: (a) rat GMC cultures contain Ca(2+)-dependent Cl- and NSCC, both stimulated by AII. (b) Cl- efflux and cation influx, respectively, would promote GMC depolarization, leading to voltage-dependent Ca2+ channel activation and GMC contraction. (c) Responsiveness of Cl- and NSCC to AII is dependent on insulin exposure; AII receptor density increases with chronic, but not acute insulin, and channel sensitivity to [Ca2+]i increases with both acute and chronic insulin. (d) Decreased GMC contractility may contribute to the glomerular hyperfiltration seen in insulinopenic or insulin-resistant diabetic patients.     

Thin film preparation and characterization of wide band gap Cu3TaQ4 (Q = S or Se) p-type semiconductors

The structural, optical, and electronic properties of thin films of a family of wide band gap (E_g > 2.3 eV) p-type semiconductors Cu₃TaQ₄ (Q = S or Se) are presented. Thin films prepared by pulsed laser deposition of ceramic Cu₃TaQ₄ targets and ex-situ annealing of the as-deposited films in chalcogenide vapor exhibit mixed polycrystalline/[100]-directed growth on amorphous SiO₂ substrates and strong (100) preferential orientation on single-crystal yttria-stabilized zirconia substrates. Cu₃TaS₄ (E_g = 2.70 eV) thin films are transparent over the entire visible spectrum while Cu₃TaSe₄ (E_g = 2.35 eV) thin films show some absorption in the blue. Thin film solid solutions of Cu₃TaSe_4 − xS_x and Cu₃TaSe_4 − xTe_x can be prepared by annealing Cu₃TaSe₄ films in a mixed chalcogenide vapor. Powders and thin films of Cu₃TaS₄ exhibit visible photoluminescence when illuminated by UV light.     

The DNA-binding and tau2 transactivation domains of the rat glucocorticoid receptor constitute a nuclear matrix-targeting signal

Using an ATP-depletion paradigm to augment glucocorticoid receptor (GR) binding to the nuclear matrix, we have identified a minimal segment of the receptor that constitutes a nuclear matrix targeting signal (NMTS). While previous studies implicated a role for the receptor's DNA-binding domain in nuclear matrix targeting, we show here that this domain of rat GR is necessary, but not sufficient, for matrix targeting. A minimal NMTS can be generated by linking the rat GR DNA-binding domain to either its tau2 transactivation domain in its natural context, or a heterologous transactivation domain derived from the Herpes simplex virus VP16 protein. The transactivation and nuclear matrix-targeting activities of tau2 are separable, as transactivation mutants were identified that either inhibited or had no apparent effect on matrix targeting of tau2. A functional interaction between the NMTS of rat GR and the RNA-binding nuclear matrix protein hnRNP U was revealed in cotransfection experiments in which hnRNP U overexpression was found to interfere with the transactivation activity of GR derivatives that possess nuclear matrix-binding capacity. We have therefore ascribed a novel function to a steroid hormone transactivation domain that could be an important component of the mechanism used by steroid hormone receptors to regulate genes in their native configuration within the nucleus.     

Comparative behaviour of yeast strains for ethanolic fermentation of culled apple juice

The culled apple juice contained (% w/v): nitrogen, 0.036; total sugars, 11.6 and was of pH 3.9. Saccharomyces cerevisiae NCIM 3284, Pichia kluyeri and Candida krusei produced more ethanol from culled apple juice at its optimum initial pH 4.5, whereas S. cerevisiae NCIM 3316 did so at pH 5.0. An increase in sugar concentration of apple juice from natural 11.6% to 20% exhibited enhanced ethanol production and improved fermentation efficiency of both the S. cerevisiae strains, whereas P. kluyveri and C. krusei produced high ethanol at 11.6% and 16.0% sugar levels, respectively. Urea was stimulatory for ethanol production as well as fermentation efficiency of the yeast strains under study.     

Isotopic analysis of the reaction catalyzed by glycerol dehydrogenase

Glycerol dehydrogenase catalyzes the reversible NAD(+)-dependent oxidation of glycerol to form dihydroxyacetone. Initial velocity, product, and dead-end inhibition studies performed for the forward and reverse reactions support an ordered kinetic mechanism with NAD+ binding first and NADH released last. A monovalent cation is required for enzymatic activity and glycerol binding, with K+ having the highest activity as measured by V. The pH dependence of the kinetic parameters V and V/Kglycerol, as well as the temperature dependence of the V pH profile, suggested that an enzymic carboxylate group functions as a base in catalysis. The pH dependence of the primary deuterium kinetic isotope effect shows that DV/Kglycerol increases from a pH-independent value of 1.15 at high pH values to a pH-independent value of 2.44 at low pH values. DV exhibits a similar pH dependence, increasing from a pH-independent value of 2.57 at high pH values to a pH independent value of 4.88 at low pH values. A chemical mechanism for enzymatic glycerol oxidation is proposed based on the data.