Antibody-independent localization of the electroneutral Na+-HCO3- cotransporter NBCn1 (slc4a7) in mice

The expression pattern of the electroneutral Na(+)-HCO(3)(-)cotransporter NBCn1 (slc4a7) was investigated by beta-galactosidase staining of mice with a LacZ insertion into the NBCn1 gene. This method is of particular value because it is independent of immunoreactivity. We find that the NBCn1 promoter is active in a number of tissues where NBCn1 has previously been functionally or immunohistochemically identified, including a broad range of blood vessels (vascular smooth muscle cells and endothelial cells), kidney thick ascending limb and medullary collecting duct epithelial cells, the epithelial lining of the kidney pelvis, duodenal enterocytes, choroid plexus epithelial cells, hippocampus, and retina. Kidney corpuscles, colonic mucosa, and nonvascular smooth muscle cells (from the urinary bladder, trachea, gastrointestinal wall, and uterus) were novel areas of promoter activity. Atrial but not ventricular cardiomyocytes were stained. In the brain, distinct layers of the cerebral cortex and cerebellar Purkinje cells were stained as was the dentate nucleus. No staining of skeletal muscle or cortical collecting ducts was observed. RT-PCR analyses confirmed the expression of NBCn1 and beta-galactosidase in selected tissues. Disruption of the NBCn1 gene resulted in reduced NBCn1 expression, and in bladder smooth muscle cells, reduced amiloride-insensitive Na(+)-dependent HCO(3)(-) influx was observed. Furthermore, disruption of the NBCn1 gene resulted in a lower intracellular steady-state pH of bladder smooth muscle cells in the presence of CO(2)/HCO(3)(-) but not in its nominal absence. We conclude that NBCn1 has a broad expression profile, supporting previous findings based on immunoreactivity, and suggest several new tissues where NBCn1 may be important.     

Microautoradiographic Study of Rhodocyclus-Related Polyphosphate-Accumulating Bacteria in Full-Scale Enhanced Biological Phosphorus Removal Plants

The ecophysiology of uncultured Rhodocyclus-related polyphosphate-accumulating organisms (PAO) present in three full-scale enhanced biological phosphorus removal (EBPR) activated sludge plants was studied by using microautoradiography combined with fluorescence in situ hybridization. The investigations showed that these organisms were present in all plants examined and constituted 5 to 10, 10 to 15, and 17 to 22% of the community biomass. The behavior of these bacteria generally was consistent with the biochemical models proposed for PAO, based on studies of lab-scale investigations of enriched and often unknown PAO cultures. Rhodocyclus-related PAO were able to accumulate short-chain substrates, including acetate, propionate, and pyruvate, under anaerobic conditions, but they could not assimilate many other low-molecular-weight compounds, such as ethanol and butyrate. They were able to assimilate two substrates (e.g., acetate and propionate) simultaneously. Leucine and thymidine could not be assimilated as sole substrates and could only be assimilated as cosubstrates with acetate, perhaps serving as N sources. Glucose could not be assimilated by the Rhodocyclus-related PAO, but it was easily fermented in the sludge to products that were subsequently consumed. Glycolysis, and not the tricarboxylic acid cycle, was the source that provided the reducing power needed by the Rhodocyclus-related PAO to form the intracellular polyhydroxyalkanoate storage compounds during anaerobic substrate assimilation. The Rhodocyclus-related PAO were able to take up orthophosphate and accumulate polyphosphate when oxygen, nitrate, or nitrite was present as an electron acceptor. Furthermore, in the presence of acetate growth was sustained by using oxygen, as well as nitrate or nitrite, as an electron acceptor. This strongly indicates that Rhodocyclus-related PAO were able to denitrify and thus played a role in the denitrification occurring in full-scale EBPR plants.     

Internalization Mechanisms of the Epidermal Growth Factor Receptor after Activation with Different Ligands

The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.     

High and stable substrate specificities of microorganisms in enhanced biological phosphorus removal plants

Microbial communities are typically characterized by conditions of nutrient limitation so the availability of the resources is likely a key factor in the niche differentiation across all species and in the regulation of the community structure. In this study we have investigated whether four species exhibit any in situ short‐term changes in substrate uptake pattern when exposed to variations in substrate and growth conditions. Microautoradiography was combined with fluorescence in situ hybridization to investigate in situ cell‐specific substrate uptake profiles of four probe‐defined coexisting species in a wastewater treatment plant with enhanced biological phosphorus removal. These were the filamentous ‘Candidatus Microthrix’ and Caldilinea (type 0803), the polyphosphate‐accumulating organism ‘Candidatus Accumulibacter’, and the denitrifying Azoarcus. The experimental conditions mimicked the conditions potentially encountered in the respective environment (starvation, high/low substrate concentration, induction with specific substrates, and single/multiple substrates). The results showed that each probe‐defined species exhibited very distinct and constant substrate uptake profile in time and space, which hardly changed under any of the conditions tested. Such niche partitioning implies that a significant change in substrate composition will be reflected in a changed community structure rather than the substrate uptake response from the different species.     

Quantifying training load and intensity in elite male ice hockey according to game-related contextual variables

We aimed to quantify training load (TL) and intensity during practice sessions according to game-related contextual variables (game outcome, opponent standard, game location) in an elite male ice hockey team. Practice data were collected using a wearable 200-Hz accelerometer, heart rate (HR) recording, and session-rating of perceived exertion (s-RPE) throughout 23 sessions (n = 306 files). The reference team performed a greater number of accelerations, decelerations, spent longer time > 85% maximum HR (t85%HR_max) and reported greater s-RPE after losing a game compared to a win (r = 0.13–0.19). Moreover, a lower number of accelerations, decelerations, t85%HR_max and s-RPE (r = 0.15–0.45) were found before playing against a top-ranked opponent. In contrast, more accelerations, decelerations, longer t85%HR_max and greater s-RPE were observed after playing against a top-ranked team opponent (r = 0.15–0.41). The players performed more accelerations/min, spent more t85%HR_max and reported greater s-RPE before playing an away game (r = 0.13–0.22). Weekly TL seems to slightly increase after losing a game, when preparing a game against a weaker opponent, after playing against a stronger opponent, and when preparing an away game. On the other hand, training intensity seems not to be affected by game-related contextual variables. Thus, ice hockey practitioners involved with TL monitoring should consider the interplay of the numerous variables that influence the volume of prescribed training and the actual training responses in each individual player.     

High diversity and abundance of putative polyphosphate-accumulating Tetrasphaera-related bacteria in activated sludge systems

The diversity of the putative polyphosphate-accumulating genus Tetrasphaera in wastewater treatment systems with enhanced biological phosphorus removal (EBPR) was investigated using the full-cycle rRNA approach combined with microautoradiography and histochemical staining. 16S rRNA actinobacterial gene sequences were retrieved from different full-scale EBPR plants, and the sequences belonging to the genus Tetrasphaera (family Intrasporangiaceae) were found to form three clades. Quantitative FISH analyses of the communities in five full-scale EBPR plants using 10 new oligonucleotide probes were carried out. The results showed that the probe-defined Tetrasphaera displayed different morphologies and constituted up to 30% of the total biomass. It was shown that active uptake of orthophosphate and formation of polyphosphate took place in most of the probe-defined Tetrasphaera populations. However, aerobic uptake of orthophosphate only took place after uptake of certain carbon sources under anaerobic conditions and these were more diverse than hitherto assumed: amino acids, glucose, and for some also acetate. Tetrasphaera seemed to occupy a slightly different ecological niche compared with 'Candidatus Accumulibacter' contributing to a functional redundancy and stability of the EBPR process.