Metal Fluoride Inhibition of a P-type H+ Pump: STABILIZATION OF THE PHOSPHOENZYME INTERMEDIATE CONTRIBUTES TO POST-TRANSLATIONAL PUMP ACTIVATION*

The plasma membrane H⁺-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H⁺/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H⁺-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H⁺-ATPases is labile in the basal state, which may provide an explanation for the low H⁺/ATP coupling ratio of these pumps in the basal state.     

Health benefits of microalgae and their microbiomes

Microalgae comprise a phylogenetically very diverse group of photosynthetic unicellular pro- and eukaryotic organisms growing in marine and other aquatic environments. While they are well explored for the generation of biofuels, their potential as a source of antimicrobial and prebiotic substances have recently received increasing interest. Within this framework, microalgae may offer solutions to the societal challenge we face, concerning the lack of antibiotics treating the growing level of antimicrobial resistant bacteria and fungi in clinical settings. While the vast majority of microalgae and their associated microbiota remain unstudied, they may be a fascinating and rewarding source for novel and more sustainable antimicrobials and alternative molecules and compounds. In this review, we present an overview of the current knowledge on health benefits of microalgae and their associated microbiota. Finally, we describe remaining issues and limitation, and suggest several promising research potentials that should be given attention.     

The Mycoplasma hominis vaa gene displays a mosaic gene structure

Mycoplasma hominis contains a variable adherence-associated ( vaa ) gene. To classify variants of the vaa genes, we examined 42 M. hominis isolates by PCR, DNA sequencing and immunoblotting. This uncovered the existence of five gene categories. Comparison of the gene types revealed a modular composition of the Vaa proteins. The proteins constituted a conserved N-terminal part followed by a varying number of interchangeable cassettes encoding approximately 110 amino acids with conserved sequence boxes flanking the cassettes. The interchangeable cassettes showed a high mutual homology and a conserved leucine zipper motif. The smallest product contained only one cassette and the largest five. Additionally, two types of stop mutations caused by substitutions resulting in the expression of truncated Vaa proteins were observed. Our results expand the known potential of the Vaa system in generating antigen variation.     

Instability of standard PCR reference genes in adipose-derived stem cells during propagation,differentiation and hypoxic exposure

Background  

For the accurate determination of gene expression changes during growth and differentiation studies on adipose-derived stem cells (ASCs), quantitative real-time RT-PCR has become a method of choice. The technology is very sensitive, however, without a proper selection of reference genes, to which the genes of interest are normalized, erroneous results may be obtained.     

Evaluation of the Redox Dye 5-Cyano-2,3-Tolyl-Tetrazolium Chloride for Activity Studies by Simultaneous Use of Microautoradiography and Fluorescence In Situ Hybridization

Three microscopic in situ techniques were used simultaneously to investigate viability and activity on a single-cell level in activated sludge. The redox dye 5-cyano-2,3-tolyl-tetrazolium chloride (CTC) was compared with microautoradiography (MAR) and fluorescence in situ hybridization (FISH) to indicate activity of cells in Thiothrix filaments and in single floc-forming bacteria. The signals from MAR and FISH correlated well, whereas only 65% of the active Thiothrix cells and 41% of all single cells were detectable by CTC reduction, which mainly targeted the most active cells.     

Quantification of lipids and protein in thin biofilms by fluorescence staining

The efficiency of removing unwanted biofilm from surfaces in industrial water systems was examined by fluorescence microscopy and image analysis. A quantitative assay for in situ determination of biofilm components was developed and tested on thin biofilms grown in reactors as well as real biofilms sampled from a fish processing factory. Different fluorescent dyes for in situ detection of protein, lipid and total organic matter were tested. It was possible to determine the approximate amounts, concentrations and coverage of the different components by correlating the fluorescent intensity of the biofilm components to standard solutions immobilised as a biofilm. The quantification methods were evaluated as a strategy for determining the efficiency of different disinfection/cleaning procedures, showing that quantification of these biofilm components was fast and reliable for optimisation of cleaning in place procedures. However, the approach also showed that bacterial cells, as investigated by culture-independent procedures, were killed but not removed by most disinfection procedures tested, potentially leading to surfaces which are easily recolonised.