Membrane composition and ion-permeability in extremophiles

Abstract: Protons and sodium ions are the only used coupling ions in energy transduction in Bacteria and Archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high as compared to sodium ions. In some thermophiles, therefore, sodium is the sole energy coupling ion. Comparison of the proton- and sodium permeability of the membranes of variety of bacterial and archaeal species that differ in their optimal growth temperature reveals that the permeation processes of protons and sodium ions must occur by different mechanisms. The proton permeability increases with the temperature, and has a comparable value for most species at their respective growth temperatures. The sodium permeability is lower than the proton permeability and increases also with the temperature, but is lipid independent. Therefore, it appears that for most bacteria the physical properties of the cytoplasmic membrane are optimised to ensure a low proton permeability at the respective growth temperature.     

Localization of membrane proteins in membrane vesicles of Bacillus subtilis.

Electrons can be transferred to the respiratory chain in whole cells and in membrane vesicles of Bacillus subtilis W 23 by the membrane impermeable electron donor reduced 5-N-methyl-phenazonium-3-sulfonate as efficiently as by the membrane permeable electron donor reduced 5-N-methyl-phenazonium methyl-sulfate, indicating that the respiratory chain is accessible from the outside of the membrane.Succinate is oxidized by whole cells and membrane vesicles at a low rate and does not energize transport of l-glutamate. In the presence of 5-N-methyl-phenazonium-3-sulfonate or 5-N-methyl-phenazonium methyl-sulfate, the oxidation rate and the rate of l-glutamate transport are increased considerably. The electrons are transferred directly from succinic dehydrogenase to these acceptors. Succinic dehydrogenase must therefore be exposed to the outside surface of the membrane in both membrane vesicles and whole cells. The exposure of succinic dehydrogenase to the outside is also indicated by the observations that only a 5% increase in the oxidation rates of succinate-5-N-methyl-phenazonium methylsulfate and succinate-5-N-methyl-phenazonium-3-sulfonate is observed upon solubilization of the membrane with the nonionic detergent Brij-58. Furthermore, treatment of membrane vesicles with trypsin decreases by more than 95% these oxidation rates.NADH is oxidized at a high rate and energizes transport of l-glutamate in whole cells and membrane vesicles effectively. The NADH-oxidation is not effected by trypsin treatment of the vesicles indicating that the oxidation occurs at the inside-surface of the membrane. Trypsin treatment of the vesicles, however, significantly decreases the rate of l-glutamate transport driven by NADH. Therefore component(s) of the transport system for l-glutamate must be effected by trypsin treatment. No apparent differences could be observed in the localization of membrane-bound functions between membrane vesicles and whole cells. This strongly supports the contention that the vesicle membrane of B. subtilis has the same orientation as the cytoplasmic membrane of whole cells.     

Competence of general practitioners in requesting and interpreting ECGs - a case vignette study

Background

Performing electrocardiography is common in general practice, but the quality of indication setting and diagnostic accuracy have been disputed.

Objectives

To assess the competence of general practitioners (GPs) in their decision-making process with regard to recording and interpreting an electrocardiogram (ECG) and evaluating the relevance of the result for management.

Methods

An online case vignette survey was performed among GPs and cardiologists (in 2015). Nine cases describing situations for which Dutch clinical guidelines recommend or advise against recording an ECG were presented. In each case, the participant had to make choices on recording an ECG, interpreting it, and using the result in a management decision. The reference standard for each ECG diagnosis was set by the expert author team.

Results

Fifty GPs who interpret ECGs themselves, eight GPs who do not and 12 cardiologists completed the survey. Adherence to guidelines recommending an ECG was high for suspected atrial fibrillation, suspected arrhythmia present during consultation, including bradycardia, but much lower for progressive heart failure and stable angina. Diagnostic accuracy of GPs was best in atrial fibrillation (96%), sick sinus syndrome (85%) and old myocardial infarction (82%), but poor in left anterior fascicular block (16%) and incomplete right bundle branch block (10%). GPs often acknowledged the low relevance of the results of a non-indicated ECG.

Conclusion

GPs do not fully adhere to Dutch cardiovascular guidelines on indications for recording ECGs. Diagnostic accuracy was high for atrial fibrillation, sick sinus syndrome and old myocardial infarction and poor for left anterior fascicular block and incomplete right bundle branch block.

     

S-decyl-glutathione nonspecifically stimulates the ATPase activity of the nucleotide-binding domains of the human multidrug resistance-associated protein, MRP1 (ABCC1).

The human multidrug resistance-associated protein(MRP1) is an ATP-dependent efflux pump that transports anionic conjugates, and hydrophobic compounds in a glutathione dependent manner. Similar to the other, well-characterized multidrug transporter P-gp, MRP1 comprises two nucleotide-binding domains (NBDs) in addition to transmembrane domains. However, whereas the NBDs of P-gp have been shown to be functionally equivalent, those of MRP1 differ significantly. The isolated NBDs of MRP1 have been characterized in Escherichia coli as fusions with either the glutathione-S-transferase (GST) or the maltose-binding domain (MBP). The nonfused NBD1 was obtained by cleavage of the fusion protein with thrombin. The GST-fused forms of NBD1 and NBD2 hydrolyzed ATP with an apparent K(m) of 340 microm and a V(max) of 6.0 nmol P(I) x mg-1 x min-1, and a K(m) of 910 microm ATP and a V(max) of 7.5 nmol P(I) x mg-1 x min-1, respectively. Remarkably, S-decyl-glutathione, a conjugate specifically transported by MRP1 and MRP2, was able to stimulate the ATPase activities of the isolated NBDs more than 2-fold in a concentration-dependent manner. However,the stimulation of the ATPase activity was found to coincide with the formation of micelles by S-decyl-glutathione. Equivalent stimulation of ATPase activity could be obtained by surfactants with similar critical micelle concentrations.     

Specificity of the transhydrogenase factor for chromatophores of Rhodopseudomonas spheroides and Rhodospirillum rubrum

Extensive washing of chromatophores of Rhodospirillum rubrum and Rhodopseudomonas spheroides with dilute buffer results in a complete loss of the energylinked transhydrogenase activities of Rsp. rubrum but only a partial loss of the light-driven reaction in chromatophores of Rps. spheroides. It was not possible to reactivate the Rps. spheroides transhydrogenation with the Rsp. rubrum transhydrogenase factor nor with a protein fraction of Rps. spheroides isolated by procedures identical to that used for the isolation of the Rsp. rubrum transhydrogenase factor. The Rsp. rubrum factor is highly specific and cannot be replaced by a number of sulfhydryl compounds tested for reconstitution of Rsp. rubrum transhydrogenation. A published procedure for the isolation of a “transhydrogenase factor” from Rps. spheroides chromatophores yields a preparation having energy-dependent transhydrogenation when supplemented with dithiothreitol in the absence of added chromatophores.     

Effects of acetate and other short-chain fatty acids on sugar and amino acid uptake of Bacillus subtilis

Acetate and other short chain n-fatty acids (C(1)-C(6)) inhibit strongly the uptake of l-serine or other l-amino acids but inhibit only weakly that of alpha-methylglucoside or fructose, whether measured in whole cells of Bacillus subtilis or in membrane vesicles that have been energized with reduced nicotinamide adenine dinucleotide (NADH), l-alpha-glycerol phosphate, or ascorbate plus phenazine methosulfate. The acetate inhibition is noncompetitive, as was shown for l-alpha-aminoisobutyric acid uptake by whole cells and for l-serine uptake by membrane vesicles. In membrane preparations, neither NADH oxidation nor the reduction of cytochromes by NADH are affected by fatty acids. All of these effects are similar to those of 2, 4-dinitrophenol. It is concluded that the fatty acids "uncouple" the amino acid carrier proteins from the cytochrome-linked electron transport system (to which they may be coupled via protein interaction or via a cation gradient).     

New Procedure for the Isolation of Membrane Vesicles of Bacillus subtilis and an Electron Microscopy Study of Their Ultrastructure

A rapid procedure for the isolation of membrane vesicles of Bacillus subtilis is described that minimizes the action of proteolytic enzymes, excreted by this organism, on the membrane proteins. The membrane vesicles obtained have, in addition to a low endogenous respiration rate, a low endogenous activity for transport of amino acids and carboxylic acids. In the presence of the electron donor, ascorbate-phenazine methosulfate, the transport activities for these compounds were comparable to the activities of intact cells. In addition, these activities were retained for a prolonged period of time. Electron microscopy examination of thin sections of the vesicles showed that the preparation consisted almost exclusively of membrane vesicles which were not contaminated with other cell components. The membrane vesicles, which are six to seven times smaller in diameter than protoplasts, often enclosed smaller vesicles. Freeze-etching of intact cells, protoplasts, and membrane vesicles showed that the orientation of the membrane of the vesicles was identical to the orientation of the plasma membrane in intact cells and protoplasts. This also held for the majority of the membranes of the enclosed vesicles, only 15% having the opposite orientation.