Linkage arrangement of Na,K-ATPase genes in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss)

As part of our efforts to characterize Na,K-ATPase isoforms in salmonid fish, we investigated the linkage arrangement of genes coding for the alpha and beta-subunits of the enzyme complex in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss). Genetic markers were developed from four of five previously characterized alpha-subunit isoforms (alpha1b, alpha1c, alpha2 and alpha3) and four expressed sequence tags derived from yet undescribed beta-subunit isoforms (beta1a, beta1b, beta3a and beta3b). Sex-specific linkage analysis of polymorphic loci in a reference meiotic panel revealed that Na,K-ATPase genes are generally dispersed throughout the rainbow trout genome. A notable exception was the colocalization of two alpha-subunit genes and one beta-subunit gene on linkage group RT-12, which may thus share a conserved orthologous segment with linkage group 1 in zebrafish (Danio rerio). Consistent with previously reported homeologous relationships among the chromosomes of the rainbow trout, primers designed from the alpha3-isoform detected a pair of duplicated genes on linkage groups RT-27 and RT-31. Similarly, the evolutionary conservation of homeologous regions on linkage groups RT-12 and RT-16 was further supported by the map localization of gene duplicates for the beta1b isoform. The detection of homeologs within each gene family also raises the possibility that novel isoforms may be discovered as functional duplicates.     

Thermal acclimation is not necessary to maintain a wide thermal breadth of aerobic scope in the common killifish (Fundulus heteroclitus)

Loss of aerobic scope at high and low temperatures is a physiological mechanism proposed to limit the thermal performance and tolerance of organisms, a theory known as oxygen- and capacity-limited thermal tolerance (OCLTT). Eurythermal organisms maintain aerobic scope over wide ranges of temperatures, but it is unknown whether acclimation is necessary to maintain this breadth. The objective of this study was to examine changes in aerobic scope in Fundulus heteroclitus, a eurythermal fish, after acclimation and acute exposure to temperatures from 5° to 33°C. The range of temperatures over which aerobic scope was nonzero was similar in acclimated and acutely exposed fish, suggesting that acclimation has modest effects on the thermal breadth of aerobic scope. However, in acclimated fish, there was a clear optimum temperature range for aerobic scope between 25° and 30°C, whereas aerobic scope was relatively constant across the entire temperature range with acute temperature exposure. Therefore, the primary effect of acclimation was to increase aerobic scope between 25° and 30°C, which paradoxically resulted in a narrower temperature range of optimal performance in acclimated fish compared to acutely exposed fish. There was only weak evidence for correlations between the thermal optimum of aerobic scope and the thermal optimum of measures of performance (specific growth rate and gonadosomatic index), and indicators of anaerobic metabolism (lactate accumulation and lactate dehydrogenase activity) only increased at high temperatures. Together these data fit many, but not all, of the predictions made by OCLTT.     

Profiling the phospho-status of the BKCa channel alpha subunit in rat brain reveals unexpected patterns and complexity

Molecular diversity of ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and non-excitable cells. Protein phosphorylation and alternative splicing of pre-mRNA are two important mechanisms to generate structural and functional diversity of ion channels. However, systematic mass spectrometric analyses of in vivo phosphorylation and splice variants of ion channels in native tissues are largely lacking. Mammalian large-conductance calcium-activated potassium (BK(Ca)) channels are tetramers of alpha subunits (BKalpha) either alone or together with beta subunits, exhibit exceptionally large single channel conductance, and are dually activated by membrane depolarization and intracellular Ca(2+). The cytoplasmic C terminus of BKalpha is subjected to extensive pre-mRNA splicing and, as predicted by several algorithms, offers numerous phospho-acceptor amino acids. Here we use nanoflow liquid chromatography tandem mass spectrometry on BK(Ca) channels affinity-purified from rat brain to analyze in vivo BKalpha phosphorylation and splicing. We found 7 splice variations and identified as many as 30 Ser/Thr in vivo phosphorylation sites; most of which were not predicted by commonly used algorithms. Of the identified phosphosites 23 are located in the C terminus, four were found on splice insertions. Electrophysiological analyses of phospho- and dephosphomimetic mutants transiently expressed in HEK-293 cells suggest that phosphorylation of BKalpha differentially modulates the voltage- and Ca(2+)-dependence of channel activation. These results demonstrate that the pore-forming subunit of BK(Ca) channels is extensively phosphorylated in the mammalian brain providing a molecular basis for the regulation of firing pattern and excitability through dynamic modification of BKalpha structure and function.     

Signalling from adenosine receptors to mitogen-activated protein kinases

The purine nucleoside adenosine acts via four distinct adenosine receptor subtypes: the adenosine A(1), A(2A), A(2B), and A(3) receptor. They are all G protein-coupled receptors (GPCR) coupling to classical second messenger pathways such as modulation of cAMP production or the phospholipase C (PLC) pathway. In addition, they couple to mitogen-activated protein kinases (MAPK), which could give them a role in cell growth, survival, death and differentiation. Although each of the adenosine receptors can activate one or more of the MAPKs, the mechanisms appear to differ substantially, both between receptor subtypes in the same cell type and between the same receptor in different cell types.     

Metabolic alteration of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cell suspension cultures overexpressing <Emphasis Type="Italic">geraniol synthase</Emphasis> in the plastids or cytosol

Previous studies showed that geraniol could be an upstream limiting factor in the monoterpenoid pathway towards the production of terpenoid indole alkaloid (TIA) in Catharanthus roseus cells and hairy root cultures. This shortage in precursor availability could be due to (1) limited expression of the plastidial geraniol synthase resulted in a low activity of the enzyme to catalyze the conversion of geranyl diphosphate to geraniol; or (2) the limitation of geraniol transport from plastids to cytosol. Therefore, in this study, C. roseus’s geraniol synthase (CrGES) gene was overexpressed in either plastids or cytosol of a non-TIA producing C. roseus cell line. The expression of CrGES in the plastids or cytosol was confirmed and the constitutive transformation lines were successfully established. A targeted metabolite analysis using HPLC shows that the transformed cell lines did not produce TIA or iridoid precursors unless elicited with jasmonic acid, as their parent cell line. This indicates a requirement for expression of additional, inducible pathway genes to reach production of TIA in this cell line. Interestingly, further analysis using NMR-based metabolomics reveals that the overexpression of CrGES impacts primary metabolism differently if expressed in the plastids or cytosol. The levels of valine, leucine, and some metabolites derived from the shikimate pathway, i.e. phenylalanine and tyrosine were significantly higher in the plastidial- but lower in the cytosolic-CrGES overexpressing cell lines. This result shows that overexpression of CrGES in the plastids or cytosol caused alteration of primary metabolism that associated to the plant cell growth and development. A comprehensive omics analysis is necessary to reveal the full effect of metabolic engineering.     

Gating of inward-rectifier K+ channels by intracellular pH.

Inward rectifier K+ channels of the Kir1.1 (ROMK) and Kir4.1 subtype are predominantly expressed in epithelial cells where they are responsible for K+ transport across the plasma membrane. Uniquely among the members of the Kir family, these channels are gated by intracellular pH in the physiological range. pH-gating involves structural rearrangements in cytoplasmic domains and the P-loop of the Kir protein. The energy for the gating transition is delivered by protonation of a lysine residue that is located prior to the first transmembrane segment and serves as a 'pH sensor'. The anomalous titration required for lysine operating in the neutral pH range results from its close interaction with two positively charged arginines from the distant N- and C-termini termed the R/K/R triad. Disturbance of this triad as results from a number of point mutations found in patients with hyperprostaglandin E syndrome (HPS) increases the pKa of the pH sensor and results in channels being permanently inactivated under physiological conditions. This article will focus on the mechanism of pH-gating, its implications for the tertiary structure of Kir proteins and on its significance for the pathogenesis of HPS.     

Lectin histochemistry of complex carbohydrates in human cervix

Summary Complex carbohydrates in the human cervix were studied histochemically using lectins, conjugated to horseradish peroxidase and correlated procedures. Stratified squamous epithelium of the exocervix and columnar epithelium of the endocervix in some, but not all specimens showed staining for terminal -N-acetyl-d-galactosamine, -d-galactose, -d-galactose and -l-fucose. the staining for -N-acetylgalactosamine and -galactose, the terminal sugars in blood group A and B antigens respectively, corresponded to a large extent with ABO blood type. One exception was the lack of staining for terminal -N-acetylgalactosamine in endocervical secretions in three of nine blood type A patients. A second exception was the staining for terminal -galactose in endocervical secretions in about half of blood type O and A specimens. The type and amount of glycoprotein formed by endocervical columnar cells differed according to location in superficial compared with deep portions of the glands and according to location at the junction with exocervix compared with the more internal regions. Staining of endothelial cells for blood group A and B antigens was confined to subjects of blood type A and B respectively, although three of nine type A specimens showed no lectin reactivity for group, A antigen. Endothelial cells evidenced affinity forUlex europeus I agglutinin demonstrative of fucose in all specimens. Mast cells disclosed lectin affinity consistent with the presence of terminal or internal mannose orN-acetylglucosamine residues. Two blood type O specimens were examined with conjugated lectins at the ultrastructural level. Secretory granules stained for content of terminal -galactose, -galactose and fucose. These results support and concur with biochemical studies of complex carbohydrates in human cervical tissues. They reveal, in addition, the location of the blood group antigens in the human exocervix and endocervix and the marked heterogeneity among endocervical columnar cells in glycoprotein production.