A novel N-terminal isoform of the neuron-specific K-Cl cotransporter KCC2

The neuronal K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the hyperpolarizing actions of inhibitory neurotransmitters gamma-aminobutyric acid and glycine in the central nervous system. This study shows that the mammalian KCC2 gene (alias Slc12a5) generates two neuron-specific isoforms by using alternative promoters and first exons. The novel KCC2a isoform differs from the only previously known KCC2 isoform (now termed KCC2b) by 40 unique N-terminal amino acid residues, including a putative Ste20-related proline alanine-rich kinase-binding site. Ribonuclease protection and quantitative PCR assays indicated that KCC2a contributes 20-50% of total KCC2 mRNA expression in the neonatal mouse brain stem and spinal cord. In contrast to the marked increase in KCC2b mRNA levels in the cortex during postnatal development, the overall expression of KCC2a remains relatively constant and makes up only 5-10% of total KCC2 mRNA in the mature cortex. A rubidium uptake assay in human embryonic kidney 293 cells showed that the KCC2a isoform mediates furosemide-sensitive ion transport activity comparable with that of KCC2b. Mice that lack both KCC2 isoforms die at birth due to severe motor defects, including disrupted respiratory rhythm, whereas mice with a targeted disruption of the first exon of KCC2b survive for up to 2 weeks but eventually die due to spontaneous seizures. We show that these mice lack KCC2b but retain KCC2a mRNA. Thus, distinct populations of neurons show a differential dependence on the expression of the two isoforms: KCC2a expression in the absence of KCC2b is presumably sufficient to support vital neuronal functions in the brain stem and spinal cord but not in the cortex.     

H-Type Bovine Spongiform Encephalopathy: Complex Molecular Features and Similarities with Human Prion Diseases

We previously reported that some cattle affected by bovine spongiform encephalopathy (BSE) showed distinct molecular features of the protease-resistant prion protein (PrP^res) in Western blot, with a 1–2 kDa higher apparent molecular mass of the unglycosylated PrP^res associated with labelling by antibodies against the 86–107 region of the bovine PrP protein (H-type BSE). By Western blot analyses of PrP^res, we now showed that the essential features initially described in cattle were observed with a panel of different antibodies and were maintained after transmission of the disease in C57Bl/6 mice. In addition, antibodies against the C-terminal region of PrP revealed a second, more C-terminally cleaved, form of PrP^res (PrP^res #2), which, in unglycosylated form, migrated as a ≈ 14 kDa fragment. Furthermore, a PrP^res fragment of ≈7 kDa, which was not labelled by C-terminus-specific antibodies and was thus presumed to be a product of cleavage at both N- and C-terminal sides of PrP protein, was also detected. Both PrP^res #2 and ≈7 kDa PrP^res were detected in cattle and in C57Bl/6 infected mice. These complex molecular features are reminiscent of findings reported in human prion diseases. This raises questions regarding the respective origins and pathogenic mechanisms in prion diseases of animals and humans.Key Words: prion, BSE, Creutzfeldt-Jakob, Gerstmann-Sträussler-Scheinker, Western blot, amyloid     

Host origin of plastid solute transporters in the first photosynthetic eukaryotes

Background  

It is generally accepted that a single primary endosymbiosis in the Plantae (red, green (including land plants), and glaucophyte algae) common ancestor gave rise to the ancestral photosynthetic organelle (plastid). Plastid establishment necessitated many steps, including the transfer and activation of endosymbiont genes that were relocated to the nuclear genome of the 'host' followed by import of the encoded proteins into the organelle. These innovations are, however, highly complex and could not have driven the initial formation of the endosymbiosis. We postulate that the re-targeting of existing host solute transporters to the plastid fore-runner was critical for the early success of the primary endosymbiosis, allowing the host to harvest endosymbiont primary production.     

Molecular evolution of mammalian aquaporin-2: further evidence that elephant shrew and aardvark join the paenungulate clade

A 328-bp sequence from exon 1 of the gene for aquaporin-2 (AQP2) was compared in 12 mammalian species, representing as many eutherian orders. This sequence encodes the N-terminal half of this kidney- specific water channel protein. Most amino acid replacements, as well as an insertion, have occurred in extracellular loops connecting the transmembrane helices, in agreement with a lower functional importance of these loops. Phylogenetic analyses were performed with parsimony, distance, and maximum-likelihood methods. The AQP2 data set, alone as well as in combination with previously published alpha A-crystallin protein sequences, strongly supports a clade consisting of elephant, hyrax, aardvark, and elephant shrew, reaching bootstrap values of 99%. This finding fully agrees with the only other presently available sequence data sets that include these taxa, those of von Willebrand factor and interphotoreceptor retinoid-binding protein, and suggests that this extended paenungulate clade is one of the most conspicuous superordinal groupings in eutherian phylogeny. Some support was obtained for an artiodactyl/perissodactyl clade, while the grouping of pholidotes with edentates was contradicted.      

Diet of bluegill Lepomis macrochirus in a South African reservoir during winter and summer

Alien fishes are considered a major threat to aquatic biodiversity in South Africa, yet relatively little regional information on their biology and ecology is available for many of these species. Seasonal changes in the diet of the bluegill Lepomis macrochirus in Howieson’s Poort Dam, Grahamstown, were assessed during summer and winter in 2014–2015, using stomach content analysis. In winter, juvenile and adult fish diets were dominated by crustacean zooplankton and insects, respectively. In summer, juvenile fish fed on crustaceans and insects, whereas adults consumed mostly fish eggs, indicating a potential impact by these invasive fish on native fish through oophagy.     

"Silent" sites in Drosophila genes are not neutral: evidence of selection among synonymous codons

The patterns of synonymous codon usage in 91 Drosophila melanogaster genes have been examined. Codon usage varies strikingly among genes. This variation is associated with differences in G+C content at silent sites, but (unlike the situation in mammalian genes) these differences are not correlated with variation in intron base composition and so are not easily explicable in terms of mutational biases. Instead, those genes with high G+C content at silent sites, resulting from a strong "preference" for a particular subset of the codons that are mostly C- ending, appear to be the more highly expressed genes. This suggests that G+C content is reduced in sequences where selective constraints are weaker, as indeed seen in a pseudogene. These and other data discussed are consistent with the effects of translational selection among synonymous codons, as seen in unicellular organisms. The existence of selective constraints on silent substitutions, which may vary in strength among genes, has implications for the use of silent molecular clocks.