Phylogeography of the brown hare (Lepus europaeus) in Europe: a legacy of south‐eastern Mediterranean refugia?

Aim We analysed the population genetics of the brown hare (Lepus europaeus) in order to test the hypothesis that this species migrated into central Europe from a number of late glacial refugia, including some in Asia Minor. Location Thirty‐three localities in Greece, Bulgaria, Italy, Croatia, Serbia, Poland, Switzerland, Austria, France, Germany, the Netherlands, Spain, the United Kingdom, Turkey and Israel. Methods In total, 926 brown hares were analysed for mitochondrial DNA (mtDNA) variation by restriction fragment length polymorphism (RFLP) performed on polymerase chain reaction‐amplified products spanning cytochrome b (cyt b)/control region (CR), cytochrome oxidase I (COI) and 12S–16S rRNA. In addition, sequence analysis of the mtDNA CR‐I region was performed on 69 individuals, and the data were compared with 137 mtDNA CR‐I sequences retrieved from GenBank. Results The 112 haplotypes detected were partitioned into five phylogeographically well‐defined major haplogroups, namely the ‘south‐eastern European type haplogroup’ (SEEh), ‘Anatolian/Middle Eastern type haplogroup’ (AMh), ‘European type haplogroup, subgroup A’ (EUh‐A), ‘European type haplogroup, subgroup B’ (EUh‐B) and ‘Intermediate haplogroup’ (INTERh). Sequence data retrieved from GenBank were consistent with the haplogroups determined in this study. In Bulgaria and north‐eastern Greece numerous haplotypes of all five haplogroups were present, forming a large overlap zone. Main conclusions The mtDNA results allow us to infer post‐glacial colonization of large parts of Europe from a late glacial/early Holocene source population in the central or south‐central Balkans. The presence of Anatolian/Middle Eastern haplotypes in the large overlap zone in Bulgaria and north‐eastern Greece reveals gene flow from Anatolia to Europe across the late Pleistocene Bosporus land‐bridge. Although various restocking operations could be partly responsible for the presence of unexpected haplotypes in certain areas, we nevertheless trace a strong phylogeographic signal throughout all regions under study. Throughout Europe, mtDNA results indicate that brown hares are not separated into discernable phyletic groups.     

Cytogenetic analysis of Anaecypris hispanica and its relationship with the paternal ancestor of the diploid-polyploid Squalius alburnoides complex.

The karyotype of the endangered fish Anaecypris hispanica was revisited using advanced cytogenetic techniques to elucidate its putative relationship with the paternal ancestor of the hybrid complex Squalius alburnoides and to clarify some of the recently described cytogenetic patterns of the complex. The results of chromomycin A3 and Ag staining, as well as fluorescent in situ hybridization with 28S and 5S rDNA and the (TTAGGG)n telomeric probes, were compared with the patterns observed in specimens of the all-male nonhybrid lineage of S. alburnoides complex, which is considered to reconstitute the nuclear genome of the probably extinct paternal ancestor. Several cytogenetic features observed in A. hispanica specimens were indeed shared by S. alburnoides nuclear nonhybrid males, supporting the hypothesis of a close evolutionary link between A. hispanica and the paternal ancestor of the complex. The genomic rearrangements involving 28S rDNA sites previously described in the S. alburnoides complex and in its maternal ancestor (S. pyrenaicus) were not detected in A. hispanica; they are, therefore, probably due to mechanisms related to hybridization and polyploidy.     

Inhibition of doxorubicin-induced mutagenicity by Baccharis dracunculifolia

Baccharis dracunculifolia DC (Asteraceae), a native plant from Brazil, have been used as an antipyretic, stomachic and health tonic in Brazil. The objective of the present study was to investigate the potential mutagenic effect of B. dracunculifolia ethyl acetate extract (Bd-EAE) and its influence on the mutagenicity induced by the chemotherapeutic agent doxorubicin (DXR) using the rat bone marrow and peripheral blood micronucleus test. Wistar rats were divided into 10 treatment groups. Five groups received DXR (90 mg/kg body weight, b.w., intraperitoneally) to induce mutagenicity and three of these groups received a single oral dose of Bd-EAE at a concentration of 6, 12 or 24 mg/kg b.w. prior to DXR administration. A vehicle-treated control group and Bd-EAE control groups were also included. The results showed that Bd-EAE itself was not mutagenic, in the rat micronucleus assay. In animals treated with Bd-EAE and DXR, the number of MNPCEs was significantly decreased compared to animals receiving DXR alone. HPLC analysis of the extract obtained permitted the identification of the following phenolic compounds: caffeic acid, p-coumaric acid, aromadendrin-4'O-methyl ether, 3-prenyl-p-coumaric acid (drupanin), 3,5-diprenyl-p-coumaric acid (artepillin C) and baccharin. The putative antioxidant activity or the interference of one or more of the active compounds of Bd-EAE with mutagenic metabolic pathways may explain its effect on DXR mutagenicity.     

Genome of the opportunistic pathogen Streptococcus sanguinis

The genome of Streptococcus sanguinis is a circular DNA molecule consisting of 2,388,435 bp and is 177 to 590 kb larger than the other 21 streptococcal genomes that have been sequenced. The G+C content of the S. sanguinis genome is 43.4%, which is considerably higher than the G+C contents of other streptococci. The genome encodes 2,274 predicted proteins, 61 tRNAs, and four rRNA operons. A 70-kb region encoding pathways for vitamin B(12) biosynthesis and degradation of ethanolamine and propanediol was apparently acquired by horizontal gene transfer. The gene complement suggests new hypotheses for the pathogenesis and virulence of S. sanguinis and differs from the gene complements of other pathogenic and nonpathogenic streptococci. In particular, S. sanguinis possesses a remarkable abundance of putative surface proteins, which may permit it to be a primary colonizer of the oral cavity and agent of streptococcal endocarditis and infection in neutropenic patients.     

Evaluation of oxacillin and cefoxitin disks for detection of resistance in coagulase negative staphylococci

Coagulase-negative Staphylococcus spp. was considered nonpathogenic until the emergence of multiresistance and the demonstration of their participation as infectious agents. In Brazil, oxacillin resistance may be present in over 80% of isolates, and the Clinical and Laboratory Standards Institute standardized a disk-diffusion method to predict this resistance in Staphylococcus. The aim of this study was to evaluate the variability among commercial disks of oxacillin (1 microg) and cefoxitin (30 microg) widely used in clinical laboratories of microbiology, compared with mecA gene and minimum inhibitory concentration (MIC) of oxacillin. The use of oxacillin and cefoxitin disks simultaneously allowed the detection of important differences, particularly, in less frequent species such as S. cohnii, S. haemolyticus, S. saprophyticus, and S. sciuri. Disks of cefoxitin of the brand 2 displayed good correlation with the mecA gene (98.7%) and oxacillin MIC (97.8%), while major discrepancies were observed using disks of brand 1. One of the critical points in the diffusion disk test is the quality of the disks: the use of better quality disks associated with molecular methods lead to better results to define the best antibiotic therapy.     

Assessment of a DNA vaccine encoding an anchored-glycosylphosphatidylinositol tegumental antigen complexed to protamine sulphate on immunoprotection against murine schistosomiasis

Protamine sulphate/DNA complexes have been shown to protect DNA from DNase digestion in a lipid system for gene transfer. A DNA-based vaccine complexed to protamine sulphate was used to induce an immune response against Schistosoma mansoni anchored-glycosylphosphatidylinositol tegumental antigen in BALB/c mice. The protection elicited ranged from 33 to 44%. The spectrum of the elicited immune response induced by the vaccine formulation without protamine was characterized by a high level of IgG (IgG1> IgG2a). Protamine sulphate added to the DNA vaccine formulation retained the green fluorescent protein encoding-plasmid longer in muscle and spleen. The experiments in vivo showed that under protamine sulphate effect, the scope of protection remained unchanged, but a modulation in antibody production (IgG1= IgG2a) was observed.     

Effect of sperm numbers and concentration on sperm transport and uterine inflammatory response in the mare

Our objective was to determine whether the concentration of cooled sperm inseminated influenced sperm transport and intensity of the uterine inflammatory reaction 2, 4 and 24h after insemination. Experimental subjects were 189 estrous mares with a dominant follicle > or =35 mm in diameter and no bacterial growth or neutrophils detected in uterine smears. Each mare was randomly assigned to receive one of the following intrauterine treatments (volume, 20 mL): insemination with 5x10(6) mL(-1) or 25x10(6) mL(-1) or 50x10(6) mL(-1) sperm diluted in 3 mL seminal plasma (SP) and 17 mL skim milk; seminal plasma or skim milk extender. Mares in a control group received no intrauterine treatment. Mares were slaughtered 2, 4 or 24h after insemination or infusion. Oviducts were separated from the uterus, and uterus and oviducts were then flushed with phosphate-buffered saline (PBS). After flushing, an endometrial sample was collected for further histopathological examination. The grade of uterine fibrosis and the amount of neutrophils in the stratum compactum were evaluated. A sample of each tubal flushing was examined for sperm count, and a sample of each uterine flushing was examined for PMN count. It was concluded that compounds in the insemination dose provoked a uterine inflammatory response, which was more rapid and intense as sperm concentration increased. In contrast, sperm transport through 4h after insemination was not influenced by sperm concentration.     

The correction of reaction rates in continuous fluorometric assays of enzymes

The kinetic data obtained from the action of a cathepsin D-like enzyme from Biomphalaria glabrata hepatopancreas (digestive gland) on MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNp)-D-Arg-NH(2), was studied as a data prototype, generated by means of a fluorogenic substrate. An initial fluorescence, due to incomplete energy transfer, of about 8% of the values attained after complete substrate hydrolysis; a non-linear standard curve even at microM concentrations and an exponential decay of the steady state fluorescence of reaction product of the order of 10(-4) x s(-1) were the main analytical problems encountered. The standard curves for fluorescence of the substrate reaction product after 48 h of hydrolysis, and the reference compound MOCAc-Pro-Leu-Gly-NH(2), were fitted by polynomial approximation and the point derivates used as calibration factors. Time dependence of the calibration factor for the reaction product was -2.96 x 10(-4) a.u microM(-1) x s(-1) that is, in the same order of observed enzymic reaction rates. A mathematical treatment was devised for obtaining rates corrected for errors derived from the three analytical problems indicated. The method is of general application in continuous fluorometric assays, irrespective of the particular enzyme used, but of special value for substrates that present significant initial fluorescence. The reaction rates were 11% higher; as calculated by means of the calibration factor [substrate]/(final-initial fluorescence intensities), which is the prevalent procedure in the literature; leading to underestimation of K(m) and overestimation of V(max).     

Downstream processing of triple layered rotavirus like particles

Rotavirus like particles (RLPs) constitute a potential vaccine for the prevention of rotavirus disease, responsible for the death of more than half a million children each year. Increasing demands for pre-clinical trials material require the development of reproducible, scaleable and cost-effective purification strategies as alternatives to the traditional laboratory scale CsCl density gradient ultracentrifugation methods commonly used for the purification of these complex particles. Self-assembled virus like particles (VLPs) composed by VP2, VP6 and VP7 rotavirus proteins (VLPs 2/6/7) were produced in 5l scale using the insect cells/baculovirus expression system. A purification process using depth filtration, ultrafiltration and size exclusion chromatography as stepwise unit operations was developed. Removal of non-assembled rotavirus proteins, concurrently formed particles (RLP 2/6), particle aggregates and products of particle degradation due to shear was achieved. Particle stability during storage was studied and assessed using size exclusion chromatography as an analytical tool. Formulations containing either glycerol (10% v/v) or trehalose (0.5 M) were able to maintain 75% of intact triple layered VLPs, at 4 degrees C, up to 4 months. The overall recovery yield was 37% with removal of 95% of host cell proteins and 99% of the host cell DNA, constituting a promising strategy for the downstream processing of other VLPs.