Ae4 (Slc4a9) Anion Exchanger Drives Cl? Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells

Transcellular Cl⁻ movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na⁺-K⁺-2Cl⁻ cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl⁻ above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl⁻ uptake pathway concentrates Cl⁻ ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl⁻/HCO₃⁻ exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2^−/− mice. In contrast, saliva secretion was reduced by 35% in Ae4^−/− mice. The decrease in salivation was not related to loss of Na⁺-K⁺-2Cl⁻ cotransporter or Na⁺/H⁺ exchanger activity in Ae4^−/− mice but correlated with reduced Cl⁻ uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl⁻/HCO₃⁻ exchanger activity revealed that HCO₃⁻-dependent Cl⁻ uptake was reduced in the acinar cells of Ae2^−/− and Ae4^−/− mice. Moreover, Cl⁻/HCO₃⁻ exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl⁻/HCO₃⁻ exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.     

Efficacy of lime, biosolids, and mycorrhiza for the phytostabilization of sulfidic copper tailings in Chile: a greenhouse experiment

Inadequate abandonment of copper mine tailings under semiarid Mediterranean climate type conditions has posed important environmental risks in Chile due to wind and rain erosion. There are cost-effective technologies for tailings stabilization such as phytostabilization. However, this technology has not been used in Chile yet. This study evaluated in a greenhouse assay the efficacy of biosolids, lime, and a commercial mycorrhiza to improve adverse conditions of oxidized Cu mine tailings for adequate establishment and grow of Lolium perenne L. var nui. Chemical characterization of experimental substrates and pore water samples were performed; plant density, biomass production, chlorophyll content, and metal content in shoots was evaluated in rye grass plants after an eight-week growth period. Results showed that neutralization of tailings and superficial application of biosolids increased both aerial biomass production and chlorophyll content of rye grass. Increased Cu solubilization and translocation to shoots occurred after biosolids application (mixed), particularly on unlimed tailings, due to formation of soluble organometallic complexes with dissolved organic carbon (DOC) which can be readily absorbed by plant roots. Positive effects of mycorrhizal inoculation on rye grass growth were restricted to treatments with superficial application of biosolids, probably due to Cu toxicity effects on commercial mycorrhiza used (Glomulus intraradices).     

Biomarkers for screening of lung cancer and pre-neoplastic lesions in a high risk Chilean population

Background

The mortality of lung cancer (LC), increases each year in the world, in spite of any advances, in development of new drugs to advance stages of LC. The high incidence of LC has been associated with smoking habit, genetic diversity and environmental pollution. Antofagasta region has been reported to have the highest LC mortality rate in Chile and its inhabitants were exposed to arsenic in their drinking water in concentrations as high as 870 μg/L. Non-invasive techniques such as biomarkers (Automatic Quantitative Cytometry: AQC and DR70) and Auto Fluorescence Bronchoscopy (AFB) might be potentially useful as a supplementary diagnostic approach and early detection. Early detection is one of the most important factors to intervene and prevent cancer progression in LC. This is a work of an ongoing prospective bimodality cancer surveillance study in high risk LC volunteers. Enrolment was done in subjects from Antofagasta and Metropolitan regions. In addition, we enrolled subjects who were suspected of having lung cancer. AQC, DR70 and AFB were used as tools in the detection of pre-neoplastic (PNL) and neoplastic lesions (NL).

Results

Half of the samples, classified as suspicious by AFB, were confirmed as metaplasia or dysplasia by histopathology. For LC, DR70 showed a higher sensitivity (95.8%) and specificity (91.9%) than AQC. However, for PNL AQC showed a higher sensitivity (91.9%) than DR70 (27.3%), although both with low PPV values. As a pre screener, both biomarkers might be employed as complementary tools to detect LC, especially as serially combined tests, with a sensitivity of 60% and a PPV of 65.2%. Additionally, the use of parallel combined tests might support the detection of PNL (sensitivity 91.2%; PPV 49.1%).

Conclusion

This work adds information on cellular and molecular biomarkers to complement imaging techniques for early detection of LC in Latin America that might contribute to formulate policies concerning screening of LC. Supported by INNOVA-CORFO, Chile.     

Genomic evolution of the class Acidithiobacillia: deep-branching Proteobacteria living in extreme acidic conditions

Members of the genus Acidithiobacillus, now ranked within the class Acidithiobacillia, are model bacteria for the study of chemolithotrophic energy conversion under extreme conditions. Knowledge of the genomic and taxonomic diversity of Acidithiobacillia is still limited. Here, we present a systematic analysis of nearly 100 genomes from the class sampled from a wide range of habitats. Some of these genomes are new and others have been reclassified on the basis of advanced genomic analysis, thus defining 19 Acidithiobacillia lineages ranking at different taxonomic levels. This work provides the most comprehensive classification and pangenomic analysis of this deep-branching class of Proteobacteria to date. The phylogenomic framework obtained illuminates not only the evolutionary past of this lineage, but also the molecular evolution of relevant aerobic respiratory proteins, namely the cytochrome bo₃ ubiquinol oxidases.Subject terms: Bacterial evolution, Bacterial genomics, Phylogenetics     

Clcn2 encodes the hyperpolarization-activated chloride channel in the ducts of mouse salivary glands

Transepithelial Cl(-) transport in salivary gland ducts is a major component of the ion reabsorption process, the final stage of saliva production. It was previously demonstrated that a Cl(-) current with the biophysical properties of ClC-2 channels dominates the Cl(-) conductance of unstimulated granular duct cells in the mouse submandibular gland. This inward-rectifying Cl(-) current is activated by hyperpolarization and elevated intracellular Cl(-) concentration. Here we show that ClC-2 immunolocalized to the basolateral region of acinar and duct cells in mouse salivary glands, whereas its expression was most robust in granular and striated duct cells. Consistent with this observation, nearly 10-fold larger ClC-2-like currents were observed in granular duct cells than the acinar cells obtained from submandibular glands. The loss of inward-rectifying Cl(-) current in cells from Clcn2(-/-) mice confirmed the molecular identity of the channel responsible for these currents as ClC-2. Nevertheless, both in vivo and ex vivo fluid secretion assays failed to identify significant changes in the ion composition, osmolality, or salivary flow rate of Clcn2(-/-) mice. Additionally, neither a compensatory increase in Cftr Cl(-) channel protein expression nor in Cftr-like Cl(-) currents were detected in Clcn2 null mice, nor did it appear that ClC-2 was important for blood-organ barrier function. We conclude that ClC-2 is the inward-rectifying Cl(-) channel in duct cells, but its expression is not apparently required for the ion reabsorption or the barrier function of salivary ductal epithelium.     

Fluid Brain Glycolysis: Limits,Speed, Location,Moonlighting, and the Fates of Glycogen and Lactate

Glycolysis is the core of intermediate metabolism, an ancient pathway discovered in the heydays of classic biochemistry. A hundred years later, it remains a matter of active research, clinical interest and is not devoid of controversy. This review examines topical aspects of glycolysis in the brain, a tissue characterized by an extreme dependence on glucose. The limits of glycolysis are reviewed in terms of flux control by glucose transporters, intercellular lactate shuttling and activity-dependent glycolysis in astrocytes and neurons. What is the site of glycogen mobilization and aerobic glycolysis in brain tissue? We scrutinize the pervasive notions that glycolysis is fast and that catalysis is channeled through supramolecular assemblies. In brain tissue, most glycolytic enzymes are catalytically silent. What then is their function?

     

Imaging Mitochondrial Flux in Single Cells with a FRET Sensor for Pyruvate

Mitochondrial flux is currently accessible at low resolution. Here we introduce a genetically-encoded FRET sensor for pyruvate, and methods for quantitative measurement of pyruvate transport, pyruvate production and mitochondrial pyruvate consumption in intact individual cells at high temporal resolution. In HEK293 cells, neurons and astrocytes, mitochondrial pyruvate uptake was saturated at physiological levels, showing that the metabolic rate is determined by intrinsic properties of the organelle and not by substrate availability. The potential of the sensor was further demonstrated in neurons, where mitochondrial flux was found to rise by 300% within seconds of a calcium transient triggered by a short theta burst, while glucose levels remained unaltered. In contrast, astrocytic mitochondria were insensitive to a similar calcium transient elicited by extracellular ATP. We expect the improved resolution provided by the pyruvate sensor will be of practical interest for basic and applied researchers interested in mitochondrial function.