Endemic species may have complex histories: within-refugium phylogeography of an endangered Iberian vole.

Glacial refugia protected and promoted biodiversity during the Pleistocene, not only at a broader scale, but also for many endemics that contracted and expanded their ranges within refugial areas. Understanding the evolutionary history of refugial endemics is especially important in the case of endangered species to recognize the origins of their genetic structure and thus produce better informed conservation practices. The Iberian Peninsula is an important European glacial refugium, rich in endemics of conservation concern, including small mammals, such as the Cabrera vole (Microtus cabrerae). This near-threatened rodent is characterized by an unusual suite of genetic, life history and ecological traits, being restricted to isolated geographic nuclei in fast-disappearing Mediterranean subhumid herbaceous habitats. To reconstruct the evolutionary history of the Cabrera vole, we studied sequence variation at mitochondrial, autosomal and sex-linked loci, using invasive and noninvasive samples. Despite low overall mitochondrial and nuclear nucleotide diversities, we observed two main well-supported mitochondrial lineages, west and east. Phylogeographic modelling in the context of the Cabrera vole's detailed fossil record supports a demographic scenario of isolation of two populations during the Last Glacial Maximum from a single focus in the southern part of the Iberian Peninsula. In addition, our data suggest subsequent divergence within the east, and secondary contact and introgression of the expanding western population, during the late Holocene. This work emphasizes that refugial endemics may have a phylogeographic history as rich as that of more widespread species, and conservation of such endemics includes the preservation of that genetic legacy.

Toward a genome-wide approach for detecting hybrids: informative SNPs to detect introgression between domestic cats and European wildcats (Felis silvestris).

Endemic gene pools have been severely endangered by human-mediated hybridization, which is posing new challenges in the conservation of several vertebrate species. The endangered European wildcat is an example of this problem, as several natural populations are suffering introgression of genes from the domestic cat. The implementation of molecular methods for detecting hybridization is crucial for supporting appropriate conservation programs on the wildcat. In this study, genetic variation at 158 single-nucleotide polymorphisms (SNPs) was analyzed in 139 domestic cats, 130 putative European wildcats and 5 captive-bred hybrids (N=274). These SNPs were variable both in wild (HE=0.107) and domestic cats (HE=0.340). Although we did not find any SNP that was private in any population, 22 SNPs were monomorphic in wildcats and pairwise FCT values revealed marked differences between domestic and wildcats, with the most divergent 35 loci providing an average FCT>0.74. The power of all the loci to accurately identify admixture events and discriminate the different hybrid categories was evaluated. Results from simulated and real genotypes show that the 158 SNPs provide successful estimates of admixture, with 100% hybrid individuals (two to three generations in the past) being correctly identified in STRUCTURE and over 92% using the NEWHYBRIDS' algorithm. None of the unclassified cats were wrongly allocated to another hybrid class. Thirty-five SNPs, showing the highest FCT values, provided the most parsimonious panel for robust inferences of parental and first generations of admixed ancestries. This approach may be used to further reconstruct the evolution of wildcat populations and, hopefully, to develop sound conservation guidelines for its legal protection in Europe.

Home-loving boreal hare mitochondria survived several invasions in Iberia: the relative roles of recurrent hybridisation and allele surfing.

Genetic introgression from a resident species into an invading close relative can result from repeated hybridisation along the invasion front and/or allele surfing on the expansion wave. Cases where the phenomenon is massive and systematic, such as for hares (genus Lepus) in Iberia, would be best explained by recurrent hybridisation but this is difficult to prove because the donor populations are generally extinct. In the Pyrenean foothills, Lepus europaeus presumably replaced Lepus granatensis recently and the present species border is parallel to the direction of invasion, so that populations of L. granatensis in the contact zone represent proxies of existing variation before the invasion. Among three pairs of populations sampled across this border, we find less differentiation of mitochondrial DNA (mtDNA) across than along it, as predicted under recurrent hybridisation at the invasion front. Using autosomal microsatellite loci and X- and Y-linked diagnostic loci, we show that admixture across the border is quasi-absent, making it unlikely that lack of interspecific mtDNA differentiation results from ongoing gene flow. Furthermore, we find that the local species ranges are climatically contrasted, making it also unlikely that ongoing ecology-driven movement of the contact account for mtDNA introgression. The lack of mtDNA differentiation across the boundary is mostly due to sharing of mtDNA from a boreal species currently extinct in Iberia (Lepus timidus) whose mitochondria have thus remained in place since the last deglaciation despite successive invasions by two other species. Home-loving mitochondria thus witness past species distribution rather than ongoing exchanges across stabilised contact zones.

Molecular and ecological signs of mitochondrial adaptation: consequences for introgression?

The evolution of the mitochondrial genome and its potential adaptive impact still generates vital debates. Even if mitochondria have a crucial functional role, as they are the main cellular energy suppliers, mitochondrial DNA (mtDNA) introgression is common in nature, introducing variation in populations upon which selection may act. Here we evaluated whether the evolution of mtDNA in a rodent species affected by mtDNA introgression is explained by neutral expectations alone. Variation in one mitochondrial and six nuclear markers in Myodes glareolus voles was examined, including populations that show mtDNA introgression from its close relative, Myodes rutilus. In addition, we modelled protein structures of the mtDNA marker (cytochrome b) and estimated the environmental envelopes of mitotypes. We found that massive mtDNA introgression occurred without any trace of introgression in the analysed nuclear genes. The results show that the native glareolus mtDNA evolved under past positive selection, suggesting that mtDNA in this system has selective relevance. The environmental models indicate that the rutilus mitotype inhabits colder and drier habitats than the glareolus one that can result from local adaptation or from the geographic context of introgression. Finally, homology models of the cytochrome b protein revealed a substitution in rutilus mtDNA in the vicinity of the catalytic fraction, suggesting that differences between mitotypes may result in functional changes. These results suggest that the evolution of mtDNA in Myodes may have functional, ecological and adaptive significance. This work opens perspective onto future experimental tests of the role of natural selection in mtDNA introgression in this system.

Recurrent introgression of mitochondrial DNA among hares (Lepus spp.) revealed by species-tree inference and coalescent simulations.

Understanding recent speciation history requires merging phylogenetic and population genetics approaches, taking into account the persistence of ancestral polymorphism and possible introgression. The emergence of a clear phylogeny of hares (genus Lepus) has been hampered by poor genomic sampling and possible occurrence of mitochondrial DNA (mtDNA) introgression from the arctic/boreal Lepus timidus into several European temperate and possibly American boreal species. However, no formal test of introgression, taking also incomplete lineage sorting into account, has been done. Here, to clarify the yet poorly resolved species phylogeny of hares and test hypotheses of mtDNA introgression, we sequenced 14 nuclear DNA and 2 mtDNA fragments (8205 and 1113 bp, respectively) in 50 specimens from 11 hare species from Eurasia, North America, and Africa. By applying an isolation-with-migration model to the nuclear data on subsets of species, we find evidence for very limited gene flow from L. timidus into most temperate European species, and not into the American boreal ones. Using a multilocus coalescent-based method, we infer the species phylogeny, which we find highly incongruent with mtDNA phylogeny using parametric bootstrap. Simulations of mtDNA evolution under the speciation history inferred from nuclear genes did not support the hypothesis of mtDNA introgression from L. timidus into the American L. townsendii but did suggest introgression from L. timidus into 4 temperate European species. One such event likely resulted in the complete replacement of the aboriginal mtDNA of L. castroviejoi and of its sister species L. corsicanus. It is remarkable that mtDNA introgression in hares is frequent, extensive, and always from the same donor arctic species. We discuss possible explanations for the phenomenon in relation to the dynamics of range expansions and species replacements during the climatic oscillations of the Pleistocene.

Cryptic speciation in the field vole: a multilocus approach confirms three highly divergent lineages in Eurasia.

Species are generally described from morphological features, but there is growing recognition of sister forms that show substantial genetic differentiation without obvious morphological variation and may therefore be considered 'cryptic species'. Here, we investigate the field vole (Microtus agrestis), a Eurasian mammal with little apparent morphological differentiation but which, on the basis of previous sex-linked nuclear and mitochondrial DNA (mtDNA) analyses, is subdivided into a Northern and a Southern lineage, sufficiently divergent that they may represent two cryptic species. These earlier studies also provided limited evidence for two major mtDNA lineages within Iberia. In our present study, we extend these findings through a multilocus approach. We sampled 163 individuals from 46 localities, mainly in Iberia, and sequenced seven loci, maternally, paternally and biparentally inherited. Our results show that the mtDNA lineage identified in Portugal is indeed a distinct third lineage on the basis of other markers as well. In fact, multilocus coalescent-based methods clearly support three separate evolutionary units that may represent cryptic species: Northern, Southern and Portuguese. Divergence among these units was inferred to have occurred during the last glacial period; the Portuguese lineage split occurred first (estimated at c. 70 000 bp), and the Northern and Southern lineages separated at around the last glacial maximum (estimated at c. 18 500 bp). Such recent formation of evolutionary units that might be considered species has repercussions in terms of understanding evolutionary processes and the diversity of small mammals in a European context.

Association between methylene tetrahydrofolate reductase and glutathione S-transferase M1 gene polymorphisms and chronic myeloid leukemia in a Brazilian population.

Chronic myeloid leukemia is a hematopoietic stem cell disorder that causes uncontrolled proliferation of white blood cells. Although the clinical and biological aspects are well documented, little is known about individual susceptibility to this disease. We conducted a case-control study analyzing the prevalence of the polymorphisms MTHFR C677T, MTHFR A1298C, del{GSTM1}, del{GSTT1}, and haptoglobin in 105 patients with chronic myeloid leukemia (CML) and 273 healthy controls, using PCR-based methods. A significant association with risk of developing CML was found for MTHFR 1298AA (odds ratio (OR) = 1.794; 95% confidence interval (CI) = 1.14-2.83) and GSTM1 non-null (OR = 1.649; 95%CI = 1.05-2.6) genotypes, while MTHFR 1298AC (OR = 0.630; 95%CI = 0.40-0.99) and GSTM1 null (OR = 0.606; 95%CI = 0.21-0.77) genotypes significantly decreased this risk. There appeared to be selection for heterozygosity at the MTHFR 1298 locus. The considerable range of variation in this and other human populations may be a consequence of distinctive processes of natural selection and adaptation to variable environmental conditions. The Brazilian population is very mixed and heterogeneous; we found these two loci to be associated with CML in this population.

Ex vivo expansion of CD56+ NK and NKT-like lymphocytes from peripheral blood mononuclear cells of patients with ovarian neoplasia.

Methods for ex vivo expansion of natural killer (NK) cells have allowed obtaining enough numbers of human NK cells for clinical trials. However, the evaluation of these methods has been mostly limited to haematological malignancies. This study aimed at evaluating a method for selective expansion of NK cells when applied in peripheral blood mononuclear cells (PBMC) of patients with ovarian neoplasia. PBMC from 13 volunteer patients with ovarian neoplasia, seven benign and six malignant tumours, were cultured in CellGro medium supplemented with anti-CD3 (9-10 initial days), IL-2 and foetal bovine serum for 21 days. The resulting effector cells were evaluated for their phenotype, cytotoxicity and cytokine secretion. PBMC cultures resulted in multiple populations (NK, NKT and T) of effector cells, enriched with CD56(+) lymphocytes. NK cells from patients with benign and malignant ovarian neoplasia were expanded 139.6 ± 63.4 and 82.7 ± 25.3-fold, respectively, being the largest lymphocyte subtype among CD56(+) population. Effector cells expanded from patients with malignant ovarian neoplasia had higher proportion of T lymphocytes and altered cytokine production patterns, characterized by lower INF-γ, TNF-α and higher IL-4, compared with patients with benign ovarian neoplasia. Effector cells were cytotoxic against K562 and OVCAR3 cell lines. Cytotoxicity was significantly higher (P < 0.05) using magnetically separated CD56(+) effector cell fractions compared with CD56-deprived ones. The present study demonstrates the feasibility of the culture system employed to generate effector cells, enriched with CD56(+) lymphocytes, from PBMC of patients with ovarian neoplasia. NK cells were the largest lymphocyte subtype among the CD56(+) population and the main variable among the final effector cell preparation affecting target cell killing.

Evidence for an association between haptoglobin and MnSOD (Val9Ala) gene polymorphisms in essential hypertension based on a Brazilian case-control study.

Essential hypertension is a complex and multifactorial trait; genetic and environmental factors interact to produce the final phenotype. Studies have demonstrated association of hypertension with varied gene polymorphisms. However, demonstration of common genetic causes in the general population remains elusive. We investigated a possible association between hypertension and haptoglobin, angiotensin I-converting enzyme (ACE), glutathione S-transferases GSTM1 and GSTT1, MnSOD (Val9Ala), CAT (-21A/T), and GPX1 (Pro198Leu) gene polymorphisms in an urban Brazilian population group from Brasília. Although ACE has been reported to be one of the main polymorphisms associated with hypertension, we found no association with ACE's specific genotypes. However, a possible association with Hp1-1 and MnSOD Val/Ala genotypes suggests that, at least for the Brazilian population, polymorphisms related to oxidative stress should be more deeply investigated.

The genomic legacy from the extinct Lepus timidus to the three hare species of Iberia: contrast between mtDNA, sex chromosomes and autosomes.

Extensive interspecific genetic introgression is often reported, and appraising its genomic impact can serve to determine whether it results from selection on specific loci or from demographic processes affecting the whole genome. The three species of hares present in the Iberian Peninsula harbour high frequencies of mitochondrial DNA (mtDNA) from Lepus timidus, an arctic/boreal species now extinct in the region. This could result from the invasive replacement of L. timidus by the temperate species during deglaciation but should then have left traces in the nuclear genome. We typed single nucleotide polymorphisms (SNPs) discovered by sequencing 10 autosomal loci, two X-linked and one Y-linked in species-wide samples of the four taxa. Based on lineage-diagnostic SNPs, we detected no trace of L. timidus sex chromosomes in Iberia. From the frequencies of inferred haplotypes, autosomal introgression into L. granatensis appeared mostly sporadic but always widespread instead of restricted to the north as mtDNA. Autosomal introgression into Iberian L. europaeus, inhabiting the Pyrenean foothills, was hardly detectable, despite quasi-fixation of L. timidus mtDNA. L. castroviejoi, endemic to the Cantabrian Mountains and fixed for L. timidus mtDNA, showed little traces of autosomal introgression. The absence of sex-chromosome introgression presumably resulted from X-linked hybrid male unfitness. The contrasting patterns between the autosomes and mtDNA could reflect general gender asymmetric processes such as frequency-dependent female assortative mating, lower mtDNA migration and higher male dispersal, but adaptive mtDNA introgression cannot be dismissed. Additionally, we document reciprocal introgression between L. europaeus and both L. granatensis in Iberia and L. timidus outside Iberia.

Evolution of rabbit haemorrhagic disease virus (RHDV) in the European rabbit (Oryctolagus cuniculus) from the Iberian Peninsula.

To date information on rabbit haemorrhagic disease virus (RHDV) in Spain and Portugal has been scarce, although the disease is endemic and continues to have a considerable impact on species conservation and hunting industry. We analysed RHDVs obtained between 1994 and 2007 at different geographic locations in Portugal (40 samples), Spain (3 samples) and France (4 samples) from wild European rabbits (Oryctolagus cuniculus) that succumbed to the disease. Phylogenetic analyses based on partial VP60 gene sequences allowed a grouping of these RHDVs into three groups, termed "Iberian" Groups IB1, IB2 and IB3. Interestingly, these three Iberian groups clustered separately, though not far from earlier RHDVs of Genogroup 1 (containing e.g., strain "AST89"), but clearly distinct from globally described RHDV strains of Genogroups 2-6. This result, supported by a bootstrap value of 76%, gives rise to the hypothesis that the virus evolved independently since its introduction to wild rabbit populations on the Iberian Peninsula, with the Pyrenees acting as a natural barrier to rabbit and hence to virus dispersal. No differences were observed in RHDV sequences obtained from geographic regions where the rabbit subspecies O. c. algirus prevails compared with those obtained from O. c. cuniculus.

Dietary carotenoid-rich pequi oil reduces plasma lipid peroxidation and DNA damage in runners and evidence for an association with MnSOD genetic variant -Val9Ala.

Physical training induces beneficial adaptations; however, exhausting exercise increases reactive oxygen species generation, resulting in damage to DNA and tissues. Pequi (Caryocar brasiliense), a fruit of the Brazilian Cerrado, contains a carotenoid-rich oil. We investigated whether pequi oil had antioxidant effects in runners. Evaluations were made after outdoor races before and after ingestion of 400 mg pequi-oil capsules for 14 days. Blood samples were taken after races and submitted to comet and TBARS assays and biochemical analyses of creatine kinase (CK), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). To determine if the protective effects of pequi-oil were influenced by antioxidant enzyme genotypes, MnSOD (-Val9Ala), CAT (-21A/T) and GPX1 (Pro198Leu) gene polymorphisms were also investigated. Pequi oil was efficient in reducing tissue injuries evaluated for AST and ALT, particularly in women, and in reducing DNA damages in both sexes. Except for CK levels, the results were influenced by MnSOD genotypes; heterozygous excess was related to less DNA damage, tissue injury and lipid peroxidation, besides presenting a better response to pequi oil against exercise-induced damage.

Insights into the evolution of the new variant rabbit haemorrhagic disease virus (GI.2) and the identification of novel recombinant strains.

Rabbit haemorrhagic disease (RHD) is a viral disease that affects the European rabbit. RHD was detected in 1984 in China and rapidly disseminated worldwide causing a severe decline in wild rabbit populations. The aetiological agent, rabbit haemorrhagic disease virus (RHDV), is an RNA virus of the family Caliciviridae, genus Lagovirus. Pathogenic (G1-G6 or variants GI.1a-GI.1d) and non-pathogenic strains (GI.4) have been characterized. In 2010, a new variant of RHDV, RHDV2/RHDVb/GI.2, was detected in France. GI.2 arrived to the Iberian Peninsula in 2011, and several recombination events were reported. Here, we sequenced full genomes of 19 samples collected in Portugal between 2014 and 2016. New GI.2 recombinant strains were detected, including triple recombinants. These recombinants possess a non-structural protein p16 related to a non-pathogenic strain. Evolutionary analyses were conducted on GI.2 VP60 sequences. Estimated time to the most recent common ancestor (tMRCA) suggests an emergence of GI.2 in July 2008, not distant from its first detection in 2010. This is the first study on GI.2 evolution and highlights the need of continued monitoring and characterization of complete genome sequences when studying lagoviruses' evolution.

Epidemiology of RHDV2 (Lagovirus europaeus/GI.2) in free-living wild European rabbits in Portugal.

As the detection of the first outbreak of a novel aetiological agent of rabbit haemorrhagic disease commonly called RHDV2 or RHDVb (Lagovirus europaeus/GI.2, henceforth GI.2) in France in 2010, the virus rapidly spread throughout continental Europe and nearby islands such as Great Britain, Sardinia, Sicily, the Azores and the Canary Islands among others. The outbreaks of this new lagovirus cause important economic losses in rabbitries, and ecological disruptions by affecting the conservation of rabbit-sensitive top predators. We analysed 550 rabbit carcasses collected in the field between May 2013 and March 2016, to investigate the epidemiology of GI.2 in free-living populations and to perform a comparative analysis with the epidemiology of classical rabbit haemorrhagic disease virus forms (RHDV, henceforth GI.1) in Portugal. Rabbits were sexed, aged and liver and blood samples were collected for subsequent RHDV screening and serology. A total of 172 samples were PCR-positive to GI.2, whereas GI.1 strains were not detected in any of the samples. The outbreaks of GI.2 revealed a marked seasonality, with peaks during the breeding season (November-May). We also found that approximately, one-third of free-ranging European rabbits in Portugal have seroconverted to GI.2. We demonstrate that the GI.2 lagovirus is currently widespread in wild populations in Portugal and is affecting a high proportion of adults and juveniles. Therefore, ongoing monitoring and surveillance are required to assess the effects of GI.2 on wild rabbit populations, its evolution, and to guide management actions aimed at mitigating the impacts of rabbit declines in the ecosystem and in rural economies.

Self-association of isolated large cytoplasmic domain of plasma membrane H+ -ATPase from Saccharomyces cerevisiae: role of the phosphorylation domain in a general dimeric model for P-ATPases.

Large cytoplasmic domain (LCD) plasma membrane H+ -ATPase from S. cerevisiae was expressed as two fusion polypeptides in E. coli: a DNA sequence coding for Leu353-Ileu674 (LCDh), comprising both nucleotide (N) and phosphorylation (P) domains, and a DNA sequence coding for Leu353-Thr543 (LCDDeltah, lacking the C-terminus of P domain), were inserted in expression vectors pDEST-17, yielding the respective recombinant plasmids. Overexpressed fusion polypeptides were solubilized with 6 M urea and purified on affinity columns, and urea was removed by dialysis. Their predicted secondary structure contents were confirmed by CD spectra. In addition, both recombinant polypeptides exhibited high-affinity 2',3'-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate (TNP-ATP) binding (Kd = 1.9 microM and 2.9 microM for LCDh and LCDDeltah, respectively), suggesting that they have native-like folding. The gel filtration profile (HPLC) of purified LCDh showed two main peaks, with molecular weights of 95 kDa and 39 kDa, compatible with dimeric and monomeric forms, respectively. However, a single elution peak was observed for purified LCDDeltah, with an estimated molecular weight of 29 kDa, as expected for a monomer. Together, these data suggest that LCDh exist in monomer-dimer equilibrium, and that the C-terminus of P domain is necessary for self-association. We propose that such association is due to interaction between vicinal P domains, which may be of functional relevance for H+ -ATPase in native membranes. We discuss a general dimeric model for P-ATPases with interacting P domains, based on published crystallography and cryo-electron microscopy evidence.

Pseudosubstrate hydrolysis by the erythrocyte plasma membrane Ca(2+)-ATPase: kinetic evidence for a modified E1 conformation in dimethylsulfoxide.

The purified Ca(2+)-ATPase of pig red cells displays a phosphatase activity towards p-nitrophenylphosphate which is inhibited by Ca2+ in the absence of solvents, and activated by calmodulin. This activity has been attributed to the E2 conformation of the enzyme. Here we show that the pNPPase activity in the absence of Ca2+ is stimulated 10-25-fold by the presence of the organic solvent dimethylsulfoxide (Me2SO). This is an activation that surpasses by severalfold that induced by calmodulin in the absence of the solvent. At 30% Me2SO, activation by calmodulin disappears. In the absence of calmodulin and at pH 7.2, the Ca2+ concentration needed for half-maximal inhibition of the pNPPase activity (K1) increases from 130 microM in the absence of Me2SO to 860 microM at 30% Me2SO. This effect of Me2SO is enhanced at pH 8.0: the K for Ca2+ increases from 2.7 microM in the absence of the solvent to 2.0 mM in its presence. However, the K0.5 for Ca2+ activation of the ATPase activity decreases from 8.3 to 2.6 microM following addition of the same Me2SO concentration. This indicates that, even in the presence of Me2SO, microM Ca2+ concentrations shift the equilibrium towards E1 but the decrease in activity that would be expected if pNPP hydrolysis were catalysed exclusively by the E2 conformation is not observed. The affinity for pNPP as a substrate increases from 2.6 mM in the absence of Me2SO to 1.6 mM in the presence of 20% Me2SO. These results suggest that Me2SO induces multiple effects in the Ca(2+)-ATPase that (i) increase the reactivity of E2 towards substrate: (ii) surpass the activation by calmodulin and, (iii) allow the enzyme to hydrolyze pNPP even when Ca2+ is bound to the high-affinity sites of the enzyme. The change in reactivity is attributed to an increase on substrate catalysis rather than on pNPP binding.

Iron-induced oxidative damage of corn root plasma membrane H(+)-ATPase.

The effect of iron on the activity of the plasma membrane H(+)-ATPase (PMA) from corn root microsomal fraction (CRMF) was investigated. In the presence of either Fe(2+) or Fe(3+) (100-200 microM of FeSO(4) or FeCl(3), respectively), 80-90% inhibition of ATP hydrolysis by PMA was observed. Half-maximal inhibition was attained at 25 microM and 50 microM for Fe(2+) and Fe(3+), respectively. Inhibition of the ATPase activity was prevented in the presence of metal ion chelators such as EDTA, deferoxamine or o-phenanthroline in the incubation medium. However, preincubation of CRMF in the presence of 100 microM Fe(2+), but not with 100 microM Fe(3+), rendered the ATPase activity (measured in the presence of excess EDTA) irreversibly inhibited. Inhibition was also observed using a preparation further enriched in plasma membranes by gradient centrifugation. Addition of 0.5 mM ATP to the preincubation medium, either in the presence or in the absence of 5 mM MgCl(2), reduced the extent of irreversible inhibition of the H(+)-ATPase. Addition of 40 microM butylated hydroxytoluene and/or 5 mM dithiothreitol, or deoxygenation of the incubation medium by bubbling a stream of argon in the solution, also caused significant protection of the ATPase activity against irreversible inhibition by iron. Western blots of CRMF probed with a polyclonal antiserum against the yeast plasma membrane H(+)-ATPase showed a 100 kDa cross-reactive band, which disappeared in samples previously exposed to 500 microM Fe(2+). Interestingly, preservation of the 100 kDa band was observed when CRMF were exposed to Fe(2+) in the presence of either 5 mM dithiothreitol or 40 microM butylated hydroxytoluene. These results indicate that iron causes irreversible inhibition of the corn root plasma membrane H(+)-ATPase by oxidation of sulfhydryl groups of the enzyme following lipid peroxidation.

Requirement of the hinge domain for dimerization of Ca2+-ATPase large cytoplasmic portion expressed in bacteria.

The large cytoplasmic domain of rabbit sarcoplasmic reticulum Ca2+-ATPase was overexpressed in Escherichia coli as a 48 kDa fusion protein, designated p48, containing an N-terminal hexa-His tag. Purification conditions were optimized, thus conferring long-term stability to p48. Circular dichroism spectroscopy and the pattern of limited trypsinolysis confirmed the proper folding of the domain. p48 retained 0.5 +/- 0.1 mol of high affinity 2',3'-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate (TNP-ATP) binding sites per mol of polypeptide chain with an apparent dissociation constant of about 8 microM. Size-exclusion FPLC using protein concentrations in the range 0.03 5 mg/ml showed that p48 was essentially monodisperse with apparent molecular mass and Stokes radius (Rs) values compatible with a dimer (100 kDa and 40 A, respectively). Analysis of p48 by small-angle X-ray scattering provided an independent second proof for a dimeric p48 particle with a radius of gyration (Rg) of 39 A, suggesting that the dimer was not spherical (Rs/Rg = 1.026). When digested by proteinase K, p48 was converted to a 30 kDa fragment, designated p30, which was very resistant to further proteolysis. p30 retained high affinity TNP-ATP binding (Kd = 8 microM) and eluted as a monomer (35 kDa) in size-exclusion FPLC. As opposed to p48, the p30 fragment did not react with monoclonal antibody A52 [Clarke et al., J. Biol. Chem. 264 (1989) 11246-11251] which recognizes region E657-R672 located upstream of the hinge domain of the Ca2+-ATPase. These results indicate a requirement of the hinge domain (670-728) region for self-association of the p48 large hydrophilic domain as a dimer. We propose that this behavior points to a possible role of the hinge domain in dimerization of sarcoplasmic reticulum Ca2+-ATPase in the native membrane.

Effect of microsatellite selection on individual and population genetic inferences: an empirical study using cross-specific and species-specific amplifications.

Although whole-genome sequencing is becoming more accessible and feasible for nonmodel organisms, microsatellites have remained the markers of choice for various population and conservation genetic studies. However, the criteria for choosing microsatellites are still controversial due to ascertainment bias that may be introduced into the genetic inference. An empirical study of red deer (Cervus elaphus) populations, in which cross-specific and species-specific microsatellites developed through pyrosequencing of enriched libraries, was performed for this study. Two different strategies were used to select the species-specific panels: randomly vs. highly polymorphic markers. The results suggest that reliable and accurate estimations of genetic diversity can be obtained using random microsatellites distributed throughout the genome. In addition, the results reinforce previous evidence that selecting the most polymorphic markers leads to an ascertainment bias in estimates of genetic diversity, when compared with randomly selected microsatellites. Analyses of population differentiation and clustering seem less influenced by the approach of microsatellite selection, whereas assigning individuals to populations might be affected by a random selection of a small number of microsatellites. Individual multilocus heterozygosity measures produced various discordant results, which in turn had impacts on the heterozygosity-fitness correlation test. Finally, we argue that picking the appropriate microsatellite set should primarily take into account the ecological and evolutionary questions studied. Selecting the most polymorphic markers will generally overestimate genetic diversity parameters, leading to misinterpretations of the real genetic diversity, which is particularly important in managed and threatened populations.

Oxidative damage to sarcoplasmic reticulum Ca2+-ATPase AT submicromolar iron concentrations: evidence for metal-catalyzed oxidation.

The sarcoplasmic reticulum (SR) calcium ATPase carries out active Ca2+ pumping at the expense of ATP hydrolysis. We have previously described the inhibition of SR ATPase by oxidative stress induced by the Fenton reaction (Fe2+ + H2O2 --> HO. + HO- + Fe3+). Inhibition was not related to peroxidation of the SR membrane nor to oxidation of ATPase thiols, and involved fragmentation of the ATPase polypeptide chain. The present study aims at further characterizing the mechanism of inhibition of the Ca2+-ATPase by oxygen reactive species at Fe2+ concentrations possibly found in pathological conditions of iron overload. ATP hydrolysis by SR vesicles was inhibited in a dose-dependent manner by micromolar concentrations of Fe2+, H2O2, and ascorbate. Measuring the rate constants of inactivation (k inact) at different Fe2+ concentrations in the presence of saturating concentrations of H2O2 and ascorbate (100 microM each) revealed a saturation profile with half-maximal inactivation rate at ca. 2 microM Fe2+. Inhibition was not affected by addition of 200 microM Ca2+ to the medium, indicating that it was not related to iron binding to the high affinity Ca2+ binding sites in the ATPase. Furthermore, inhibition was not prevented by the water-soluble hydroxyl radical scavengers mannitol or dimethylsulfoxide, nor by butylated hydroxytoluene (a lipid peroxidation blocker) or dithiothreitol (DTT). However, when Cu2+ was used instead of Fe2+ in the Fenton reaction, ATPase inhibition could be prevented by DTT. We propose that functional impairment of the Ca2+-pump may be related to oxidative protein fragmentation mediated by site-specific Fe2+ binding at submicromolar or low micromolar concentrations, which may occur in pathological conditions of iron overload.