Responses of gill mitochondria-rich cells in Mozambique tilapia exposed to acidic environments (pH 4.0) in combination with different salinities.

On exposure to hyposmotic acidic water, teleost fish suffer from decreases in blood osmolality and pH, and consequently activate osmoregulatory and acid-base regulatory mechanisms to restore disturbed ion and acid-base balances. In Mozambique tilapia Oreochromis mossambicus exposed to acidic (pH 4.0) or neutral (pH 7.4-7.7) freshwater in combination with 0mM or 50mM NaCl, we examined functional and morphological changes in gill mitochondria-rich (MR) cells. We assessed gene expression of Na(+)/H(+) exchanger-3 (NHE3), Na(+)/Cl(-) cotransporter (NCC), vacuolar-type H(+)-ATPase (V-ATPase) and Na(+)/HCO(3)(-) cotransporter-1 (NBC1) in the gills. The mRNA expression of NHE3 and NCC in tilapia gills were higher in acidic freshwater than in that supplemented with 50mM NaCl, while there was no significant difference in mRNA levels of V-ATPase and NBC1. In addition, immunocytochemical observations showed that apical-NHE3 MR cells were enlarged, and frequently formed multicellular complexes with developed deep apical openings in acidic freshwater with 0mM and 50mM NaCl. These findings suggest that gill MR cells respond to external salinity and pH treatments, by parallel manipulation of osmoregulatory and acid-base regulatory mechanisms.

Ion transporters for fluid reabsorption in the rooster (Gallus domesticus) epididymal region.

Testicular fluid is highly condensed during its passage through the epididymal region in the avian species. In the present study, major ion transporters that are responsible for condensation mainly by water resorption in the reproductive tract as identified in the mammalian epididymis were localized within the rooster (Gallus domesticus) epididymis by immunohistochemistry. The results show that the efferent ductule epithelium expressed sodium-potassium ATPase (Na(+),K(+)-ATPase), carbonic anhydrase II (CAII) and sodium hydrogen exchanger isoform 3 (NHE3) and that the connecting ductule and epididymal duct epithelia expressed Na(+),K(+)-ATPase and CAII. These data suggest that a model proposed for reabsorption in mammalian efferent ductules can be applied to avian efferent ductules.

Arsenate respiratory reductase gene (arrA) for Desulfosporosinus sp. strain Y5.

Desulfosporosinus sp. strain Y5 is a spore-forming bacterium capable of dissimilatory arsenate reduction coupled to the oxidation of aromatic compounds. In arsenate respiration, the arsenate respiratory reductase (ARR) catalyzes the reduction of arsenate to arsenite. Our objective is to characterize the arrA gene, encoding the ARR, for Desulfosporosinus sp. strain Y5. Oligonucleotide primers were designed based on the few arrA gene sequences available at the time and validated against positive and negative controls. The resulting arrA-amplicon of approximately 2.0kb was cloned and sequenced. The arrA from Desulfosporosinus sp. Y5 is closely related to Desulfitobacterium hafniense (similarity of 77% and 81% at the nucleotide and amino acid levels, respectively). Phylogenetic topology based on the arrA gene was partially congruent with that of 16S rRNA-based analysis. This arrA sequence will support the development of specific tracking probes for Desulfosporosinus sp. Y5 and the molecular characterization and monitoring of dissimilatory arsenate reducing bacteria.

[Protein profiling of human dendritic cells infected with mycobacterium tuberculosis].

The response of dendritic cells (DCs) plays an essential role in the initiation of immune responses following Mycobacterium tuberculosis (MTB) challenge. Two-dimensional electrophoresis (2-DE) was employed to compare the global protein patterns between human DCs infected and that uninfected with MTB H37Rv ATCC 27294 strains, and 45 protein spots were found to express differentially. Four protein spots which remarkably changed in DCs infected with MTB H37Rv ATCC 27294 strains were measured by matrix assisted laser desorption/ionization tandem time-of-flight (TOF/TOF) mass spectrometry. The data obtained from peptide mass fingerprinting were used in protein database search. Four protein spots in gel were identified as Human Arsenite-stimulated ATPase (hASNA-I), Annexin IV, gamma-actin and Heat shock protein27 (HSP27). These data provide insight into the changed global protein patterns of the DCs after infection and may prove useful for further study in the interaction between MTB and host.

Specific potassium binding stabilizes pI258 arsenate reductase from Staphylococcus aureus.

Arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 catalyzes the reduction of arsenate to arsenite and plays a role in bacterial heavy metal resistance. The high resolution x-ray structure of ArsC reveals the atomic details of the K+ binding site situated next to the catalytic P-loop structural motif of this redox enzyme. A full thermodynamic study of the binding characteristics of a series of monovalent cations (Li+, Na+, K+, Rb+, and Cs+) and their influence on the thermal stability of ArsC was performed with isothermal titration calorimetry, circular dichroism spectroscopy, and differential scanning calorimetry. Potassium has the largest affinity with a Ka of 3.8 x 10(3) m(-1), and the effectiveness of stabilization of ArsC by monovalent cations follows the binding affinity order: K+ > Rb+ > Cs+ > Na+ > Li+. A mutagenesis study on the K+ binding side chains showed that Asn-13 and Asp-65 are essential for potassium binding, but the impact on the stability of ArsC was the most extreme when mutating Ser-36. Additionally, the thermal stabilization by K+ is significantly reduced in the case of the ArsC E21A mutant, showing the importance of a Glu-21-coordinated water molecule in its contact with K+. Although potassium is not essential for catalysis, in its presence the kcat/KM increases with a factor of 5. Altogether, the interaction of K+ with specific residues in ArsC is an enthalpydriven process that stabilizes ArsC and increases the specific activity of this redox enzyme.

Characterization and molecular localization of anion transporters in colonic epithelial cells.

This study describes the identification and characterization of anion transporters in apical membrane (APM) and basolateral membrane (BLM) of rat distal colon. Cl-HCO3, Cl-OH, Cl-butyrate, and butyrate-HCO3 exchanges and Na-HCO3 cotransporter are present in rat distal epithelial cells. Cl-HCO3 exchange (1) is present only in APM from surface, but not from crypt cells; (2) is also present in BLM; and (3) of surface cell is encoded by anion exchange (AE)-1 isoform, whereas BLM Cl-HCO3 is encoded by AE2 isoform. Cl-OH exchange is present only in APM, but not in BLM from surface and crypt cells, and is responsible for regulation of cell functions (i.e., cell pH and cell volume regulation). Butyrate-HCO3 exchange (1) is also present in apical membrane vesicles (AMV) from surface, but not from crypt cells; (2) is present in BLM; and (3) is responsible for SCFA-dependent HCO3 secretion. By contrast, Cl-butyrate exchange: (1) is present in APM from both surface and crypt cells; (2) is not present in BLM; and (3) recycles butyrate by absorbing Cl. Na-HCO3 cotransport: (1) is present only in BLM; (2) is expressed predominantly in midcrypt regions; and (3) may be linked to HCO3 secretion. A mechanism for HCO3 movement across the crypt apical membrane has not as yet been identified.