Spatial and temporal variation of genetic diversity and estimation of effective population sizes in Atlantic salmon (Salmo salar, L.) populations from Asturias (Northern Spain) using microsatellites

Rivers in Asturias (northern Spain) constitute the southern limit of the distribution of Atlantic salmon (Salmo salar L.) in Europe, a biological resource facing one of the more serious challenges for conservation today. In this work, eight microsatellite loci have been used to analyse samples collected in 1993 and 1999 from four Asturian rivers (Esva, Narcea, Sella, and Cares), obtaining information about the temporal and the spatial genetic variation in these populations and, in addition, estimations of their effective population sizes. The temporal analysis revealed a general decrease in all the estimated genetic variability parameters when samples from 1993 (mean A ₍₁₉₉₃₎ = 6.47, mean H _O(1993) = 0.472, mean H _E(1993) = 0.530) were compared with those obtained in 1999 (mean A ₍₁₉₉₉₎ = 6.16, mean H _O(1999) = 0.460, mean H _E(1999) = 0.490). This reduction was particularly notable for the case of the Esva river. Our results pointed to a pattern of spatial genetic differentiation inside the Asturian region (F _{ST (1993)} = 0.016 P < 0.01; F _{ST (1999)} = 0.023 P < 0.01). Using the standard Temporal Method we found estimates of N _e^{^} _(Esva) = 75.1 (33.2–267.2); N _e^{^} _(Cares) = 96.6 (40.0–507.5), N _e^{^} _(Sella) = 106.5 (39.1–9396.4) and N _e^{^} _(Narcea) = 113.9 (42.0–3693.3). The use of likelihood-based methods for the N _e^{^} estimations improved the results (smaller CIs) for the Esva and Cares rivers (N _e^{^} _(Esva) = 63.9 (32.3–165.3); N _e^{^} _(Cares) = 76.4 (38.8–202.0) using a Maximum likelihood approach) and suggested the presence of larger populations for the Sella and Narcea rivers (N _e^{^}≈200). These results showed that the Asturian Atlantic salmon populations (in particular Esva and Cares river populations) could be close to the conservation genetic borderline for avoiding inbreeding depression although we discuss some implications of the analysis of temporal genetic change in populations with overlapping generations.     

Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia

Background  

Studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. The normalization of relative qRT-PCR data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. No validated housekeeping genes have been reported for the relative quantification of the mRNA expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used.     

A role for protein kinase CK2 in plant development: evidence obtained using a dominant-negative mutant

Protein kinase CK2 is an evolutionary conserved Ser/Thr phosphotransferase composed of two distinct subunits, α (catalytic) and β (regulatory), that combine to form a tetrameric complex. Plant genomes contain multiple genes for each subunit, the expression of which gives rise to different active holoenzymes. In order to study the effects of loss of function of CK2 on plant development, we have undertaken a dominant-negative mutant approach. We generated an inactive catalytic subunit by site-directed mutagenesis of an essential lysine residue. The mutated open reading frame was cloned downstream of an inducible promoter, and stably transformed Arabidopsis thaliana plants and tobacco BY2 cells were isolated. Continuous expression of the CK2 kinase-inactive subunit did not prevent seed germination, but seedlings exhibited a strong phenotype, affecting chloroplast development, cotyledon expansion, and root and shoot growth. Prolonged induction of the transgene was lethal. Moreover, dark-germinated seedlings exhibited an apparent de-etiolated phenotype that was not caused by disruption of the light-signalling pathways. Short-term induction of the CK2 kinase-inactive subunit allowed plant survival, but root growth and lateral root formation were significantly affected. The expression pattern of CYCB1;1::GFP in the root meristems of mutant plants demonstrated an important decrease of mitotic activity, and expression of the CK2 kinase-inactive subunit in stably transformed BY2 cells provoked perturbation of the G1/S and G2 phases of the cell cycle. Our results are consistent with a model in which CK2 plays a key role in cell division and cell expansion, with compelling effects on Arabidopsis development.     

Synthesis of new platinum(II) compounds with several bidentate and tridentate nitrogen-donor ligands. Structural analyses by H, C{H} and Pt{H} NMR spectroscopy and X-ray crystal structure

The reaction of the N-alkylaminopyrazole (NN′) ligands 1-[2-(ethylamino)ethyl]-3,5-dimethylpyrazole (deae), 1-[2-(tert-butylamino)ethyl]-3,5-dimethylpyrazole (deat), or (NN′N) ligands bis[(3,5-dimethylpyrazolyl)methyl]ethylamine (bdmae) and bis[(3,5-dimethylpyrazolyl)ethyl]ethylamine (ddae) with [PtCl₂(CH₃CN)₂] affords a series of square-planar Pt(II) complexes with formula [PtCl₂(NN′)] (NN′ = deae (1); deat (2)), [PtCl₂(bdmae)] (3), or [PtCl(ddae)]Cl (4). Treatment of complex 4 in the presence of AgBF₄ in CH₂Cl₂/methanol (3:1) gives [PtCl(ddae)](BF₄) (5). These Pt(II) complexes have been characterised by elemental analyses, conductivity measurements and IR, ¹H, ¹³C{¹H}, and ¹⁹⁵Pt{¹H} NMR spectroscopies. The ¹H NMR spectroscopic studies of the complexes prove the rigid conformation of the ligands when they are complexed. The solid-state structure of complex 1 was determined by single crystal X-ray diffraction methods. The deae ligand is coordinated through the N_pz and N_amino atoms to the metallic centre, which completes its coordination with two chlorine atoms in cis disposition.     

Can sister chromatid intercrossings be considered as prelesions?

Summary In a study of the expression of folate-sensitive fragile sites in five normal individuals using RPMI medium containing methotrexate (MTX), we observed a high frequency of sister chromatid intercrossings (SCI) that is, the intersection of sister chromatids. The location of SCIs corresponded to fragile sites in 54.2% of the cases. Of the SCIs observed in each individual, 43%–53% were located at the same bands as their expressed fragile sites. Furthermore when RPMI+MTX medium was used instead of F-10 medium, the incidence of SCIs increased tenfold. We suggest that SCIs could indicate the existence of a pre-lesion.     

Postglacial climate changes and rise of three ecotypes of harbour porpoises,Phocoena phocoena,in western Palearctic waters

Despite no obvious barriers to gene flow in the marine realm, environmental variation and ecological specializations can lead to genetic differentiation in highly mobile predators. Here, we investigated the genetic structure of the harbour porpoise over the entire species distribution range in western Palearctic waters. Combined analyses of 10 microsatellite loci and a 5085 base‐pair portion of the mitochondrial genome revealed the existence of three ecotypes, equally divergent at the mitochondrial genome, distributed in the Black Sea (BS), the European continental shelf waters, and a previously overlooked ecotype in the upwelling zones of Iberia and Mauritania. Historical demographic inferences using approximate Bayesian computation (ABC) suggest that these ecotypes diverged during the last glacial maximum (c. 23–19 kilo‐years ago, kyrbp ). ABC supports the hypothesis that the BS and upwelling ecotypes share a more recent common ancestor (c. 14 kyrbp ) than either does with the European continental shelf ecotype (c. 28 kyrbp ), suggesting they probably descended from the extinct populations that once inhabited the Mediterranean during the glacial and post‐glacial period. We showed that the two Atlantic ecotypes established a narrow admixture zone in the Bay of Biscay during the last millennium, with highly asymmetric gene flow. This study highlights the impacts that climate change may have on the distribution and speciation process in pelagic predators and shows that allopatric divergence can occur in these highly mobile species and be a source of genetic diversity.     

Hydroxide and proton migration in aquaporins

Hypothetical hydroxide and proton migration along the linear water chain in Aquaporin GlpF from Escherichia coli are studied by ab initio Car-Parrinello molecular dynamics simulations. It is found that the protein stabilizes a bipolar single file of water. The single file features a contiguous set of water-water hydrogen bonds in which polarization of the water molecules vary with position along the channel axis. Deprotonation of the water chain promotes the reorientation of water molecules while the hydroxide ion rapidly migrates by sequentially accepting protons from the neighboring water molecules. The hydroxide ion is not attracted by a conserved, channel-lining arginine residue, but is immobilized at two centrally located, conserved Asparagine-Proline-Alanine motifs where fourfold coordination stabilizes the ion. Hydroxide transition from the channel vestibules into the channel lumen is strongly influenced by electrostatic coupling to two conserved oppositely aligned macrodipoles. This suggests that the macrodipole's negative poles play a role in preventing hydroxide ions from entering into the channel's inner vestibules. Water protonation within the lumen facilitates water reorientation and subsequent proton expelling occurs. In the periplasmic half-channel, expelling occurs via the Grotthuss mechanism. Protonation within the cytoplasmic half-channel implies wire-breakage at the Asn-Pro-Ala motifs. The proton is here diffusively rejected as (H(5)O(2))(+).     

Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia

The absence of adenosine A2A receptors, or its pharmacological inhibition, has neuroprotective effects. Experimental data suggest that glial A2A receptors participate in neurodegeneration induced by A2A receptor stimulation. In this study we have investigated the effects of A2A receptor stimulation on control and activated glial cells. Mouse cortical mixed glial cultures (75% astrocytes, 25% microglia) were treated with the A2A receptor agonist CGS21680 alone or in combination with lipopolysaccharide (LPS). CGS21680 potentiated lipopolysaccharide-induced NO release and NO synthase-II expression in a time- and concentration-dependent manner. CGS21680 potentiation of lipopolysaccharide-induced NO release was suppressed by the A2A receptor antagonist ZM-241385 and did not occur on mixed glial cultures from A2A receptor-deficient mice. In mixed glial cultures treated with LPS + CGS21680, the NO synthase-II inhibitor 1400W abolished NO production, and NO synthase-II immunoreactivity was observed only in microglia. Binding experiments demonstrated the presence of A2A receptors on microglial but not on astroglial cultures. However, the presence of astrocytes was necessary for CGS21680 potentiating effect. In light of the reported neurotoxicity of microglial NO synthase-II and the neuroprotection of A2A receptor inhibition, these data suggest that attenuation of microglial NO production could contribute to the neuroprotection afforded by A2A receptor antagonists.