Unusual Low Proton Permeability of Liposomes Prepared from the Endogenous Myelin Lipids

Abstract: In contrast with most other lipid substrates, in this article we show that liposomes prepared from the total myelin lipids exhibited a negligible proton permeability. Neither the generation of valinomycin-induced potassium diffusion potentials as high as -177 mV nor the imposition of large pH gradients (up to three units) was able to produce a substantial flux of protons through liposomal membranes, as determined by the distribution of [¹⁴C]-methylamine, or the changes in the fluorescence of the probes 9-aminoacridine, acridine orange, and pyranine. The presence of cations (Na⁺, K⁺, Ca²⁺) did not alter this behavior. Voltage clamping did not increase the trans-membrane ApH-driven proton permeability. However, II-posome diameter was found to be critical because small unilamellar vesicles displayed a much higher proton permeability than large unilamellar or multilamellar vesicles. This abnormally low proton permeability is interpreted by virtue of the characteristic biochemical composition of myelin lipid matrix, with a high content of cholesterol and sphingolipids and a very low level of free fatty acids. These results could be important for elucidating the role of myelin in the regulation of pH in the brain. In addition, the myelin lipid extract could be useful for reconstituting proteins that participate in the transport of H⁺ through the membrane.     

Regulation of principal cell pH by Na/H exchange in rabbit cortical collecting tubule

Summary Changes in intracellular pH (pH _i ) were measured using the pH indicator, BCECF, in principal cells from split opened cortical collecting tubules (CCTs) derived from rabbits maintained on a normal diet. This monolayer preparation has the advantage of allowing us to visualize the morphological differences in the two major cell types in this nephron segment under transmitted light. The visual identification of the cell types was verified using emission measurements taken from single principal and intercalated cells in the opened tubule which had been exposed to fluorescein isothiocyanate (FITC)-labeled peanut lectin. We confirmed the existence of an amiloride-sensitive Na/H exchange process activated during intracellular acidosis in principal cells. In addition, the exchanger was active under basal conditions and over a wide range of pH _i . Because the exchanger was active under basal conditions we tested the hypothesis that changes in intracellular Na (Na _i ) would alter pH _i in a predictable way. Maneuvers designed to alter Na _i were without significant effects within a 10-min time frame. Specifically, addition of 100 m ouabain to increase Na _i or exposure of the tubules to 10^–5 m amiloride to decrease luminal Na entry and reduce Na _i did not have an effect on pH _i . In some experiments we did observe however, after a 30-min exposure to ouabain, a small decrease in pH _i . These results suggest that Na/H exchange is a major regulator of pH _i in principal cells. However, regulation of Na transport by changes in pH _i in principal cells of rabbit CCT via the activity of a Na/H exchanger do not seem to contribute to the feedback control of Na transport.This work was supported by U.S. Public Health Service grants DK27847 to L.G. Palmer and DK11489 to E.E. Windhager.     

Visualizing cytoskeleton dynamics in mammalian cells using a humanized variant of monomeric red fluorescent protein

Fluorescent proteins are versatile tools for live cell imaging studies. In particular, recent progress was achieved in the development of monomeric red fluorescent proteins (mRFPs) that show improved properties in respect to maturation and intracellular fluorescence. mRFPmars, a red fluorescent protein designed especially for the use in Dictyostelium, proved to be a brilliant label for different cytoskeletal elements. Here we report on the synthesis of a humanized version of a monomeric RFP, mRFPruby, which differs in sequence from mRFPmars in four amino acids and has a codon usage that is optimized for the application in mammalian cells. In order to demonstrate the usefulness of this new mRFP variant, mRFPruby fused to beta-actin was expressed in different mouse cell lines and used to visualize actin cytoskeleton dynamics by live cell microscopy.     

Evaluation of effective diffusion coefficient and intrinsic kinetic parameters on azo dye biodegradation using PVA-immobilized cell beads

An immobilized mixed culture (Aeromonas hydrophila, Comamonas testosteroni, and Acinetobacter baumannii) was prepared by entrapment into phosphorylated polyvinyl alcohol (PVA) gel beads. The unsteady-state diffusion mechanism in a gel bead was applied to estimate the effective diffusion coefficients (D(e)) and the partition coefficients (K(p)) of azo dye. In addition, a simple method was developed to determine the intrinsic kinetic parameters of immobilized cells from observed reaction rates and the intrinsic kinetic parameters were then verified by fitting the experimental data into the reaction-diffusion model in a batch reactor running at a well-stirred state. The calculated effectiveness factor (eta(cal)) approached unity at Thiele modulus (Phi) < 0.3 (i.e., d(p) < 0.475 mm). The experimental effectiveness factor (eta(exp)) was in the range of 0.71-0.45 for a corresponding sphere diameter (d(p)) of 1.91 +/- 0.16 to 4.43 +/- 0.07 mm at an initial dye concentration of 200 mg/L. The results show that intraparticle diffusion resistance has a significant effect on the azo dye biodegradation rate.     

Purification and partial characterization of azoreductase from Enterobacter agglomerans

Azoreductase, an enzyme catalyzing the reductive cleavage of the azo bond of methyl red (MR) and related dyes, was purified to electrophoretic homogeneity from Enterobacter agglomerans. This bacterial strain, isolated from dye-contaminated sludge, has a higher ability to grow, under aerobic conditions, on culture medium containing 100mg/L of MR. The enzyme was purified approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity). The purified enzyme is a monomer with a molecular weight of 28,000 Da. The maximal azoreductase activity was observed at pH 7.0 and at 35 degrees C. This activity was NADH dependent. The K(m) values for both NADH and MR were 58.9 and 29.4 microM, respectively. The maximal velocity (V(max)) was 9.2 micromol of NADH min(-1)mg(-1). The purified enzyme is inhibited by several metal ions including Fe(2+) and Cd(2+).     

Fully Plastic Dye Solar Cell Devices by Low‐Temperature UV‐Irradiation of both the Mesoporous TiO2 Photo‐ and Platinized Counter‐Electrodes

The application of UV irradiation processes are successfully proposed for the first time in the fabrication of both of the two plastic electrodes in flexible dye solar cells (DSCs) and modules. For the realization of the photo‐electrode, a customized TiO₂ paste formulation and UV processing method was developed which yields 134% (48%) performance enhancement with respect to the same (binder‐free) paste treated at 120 °C. UV treatment induces both complete removal of organic media and more efficient charge collection. Significantly, highly catalytic platinized flexible counter‐electrodes are also obtained via UV photo‐induced reduction of screen‐printed platinum precursor pastes based on hexachloroplatinic acid. Using both UV‐processed electrodes, a fully plastic DSC is fabricated with a conversion efficiency of 4.3% under 1 Sun (semitransparent) and 5.3% under 0.2 Sun (opaque). Performance is within 10% of the efficiency of a glass‐based DSC prepared with the same materials but with conventional high temperature processes. The material formulations and processes are simple, and easily up‐scaled over large areas, even directly and simultaneously applicable to the preparation of both the photo‐and counter‐electrode on the same substrate which enabled us to demonstrate the first module on plastic realized with a W series interconnection.     

Identification and Growth Characteristics of α-Galactosidase-producing Microorganisms

Two kinds of α-galactosidase-producing microorganisms, strain No. 31–2 and strain No. 7–5, have been isolated from soil and subjected to a determinative study. On the basis of the morphological and physiological characters, the strain No. 31–2 was identified to be belonged to genus Micrococcus and the strain No. 7–5 to genus Bacillus. The former strain, Micrococcus sp. No. 31–2, produced exclusively an intracellular α-galactosidase, and the latter one, Bacillus sp. No. 7–5, secreted the enzyme into culture medium. The cell growth and enzyme production of both strains were observed to reach the maximum under an alkaline culture condition. The intracellular α-galactosidase of Micrococcus sp. No. 31–2 was inducible by galactose, melibiose, and raffinose, while the α-galactosidase of Bacillus sp. No. 7–5 was produced constitutively.     

Artificial Feeding Systems for Plant-Parasitic Nematodes

Feeding of an "obligate" plant-parasitic nematode (nonfungal feeder), Pratylenchus scribneri, in the absence of plant tissue was demonstrated in an artificial system consisting of liquid media and indicator dyes including amaranth and various nontoxic food colors. Among the compounds tested, sucrose, dextrose, Gamborg''s B5 medium, and DL-methionine stimulated a small percentage of feeding (12-36%). A high percentage of feeding (90-100%) occurred in a filtrate from excised corn roots cultured in Gamborg''s B5 medium. This feeding system has the potential to develop an artificial medium for plant-parasitic nematodes and to screen novel nematicides that are stomach poisons.     

A near-infrared fluorescence dye for sensitive detection of hydrogen sulfide in serum

Cy-Cl, a cationic near-infrared cyanine dye, readily reacts with hydrogen sulfide (H₂S) via nucleophilic thiolation to give dose-dependent ‘turn-off’ fluorescence and colorimetric read-out, allowing selective detection of low levels of H₂S in serum and imaging of mitochondrial H₂S in living cells.     

A new fluorescent pH probe for imaging lysosomes in living cells

A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5–4.1 with the pK_a value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no ‘alkalizing effect’ on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells.