Evidence for Introduction Bottleneck and Extensive Inter-Gene Pool (Mesoamerica x Andes) Hybridization in the European Common Bean (Phaseolus vulgaris L.) Germplasm

Common bean diversity within and between Mesoamerican and Andean gene pools was compared in 89 landraces from America and 256 landraces from Europe, to elucidate the effects of bottleneck of introduction and selection for adaptation during the expansion of common bean (Phaseolus vulgaris L.) in Europe. Thirteen highly polymorphic nuclear microsatellite markers (nuSSRs) were used to complement chloroplast microsatellite (cpSSRs) and nuclear markers (phaseolin and Pv-shatterproof1) data from previous studies. To verify the extent of the introduction bottleneck, inter-gene pool hybrids were distinguished from “pure” accessions. Hybrids were identified on the basis of recombination of gene pool specific cpSSR, phaseolin and Pv-shatterproof1 markers with a Bayesian assignments based on nuSSRs, and with STRUCTURE admixture analysis. More hybrids were detected than previously, and their frequency was almost four times larger in Europe (40.2%) than in America (12.3%). The genetic bottleneck following the introduction into Europe was not evidenced in the analysis including all the accessions, but it was significant when estimated only with “pure” accessions, and five times larger for Mesoamerican than for Andean germplasm. The extensive inter-gene pool hybridization generated a large amount of genotypic diversity that mitigated the effects of the bottleneck that occurred when common bean was introduced in Europe. The implication for evolution and the advantages for common bean breeding are discussed.     

Silver colloidal nanoparticles: antifungal effect against adhered cells and biofilms of Candida albicans and Candida glabrata

The aim of this study was to evaluate the effect of silver nanoparticles (SN) against Candida albicans and Candida glabrata adhered cells and biofilms. SN (average diameter 5 nm) were synthesized by silver nitrate reduction with sodium citrate and stabilized with ammonia. Minimal inhibitory concentration (MIC) tests were performed for C. albicans (n = 2) and C. glabrata (n = 2) grown in suspension following the Clinical Laboratory Standards Institute microbroth dilution method. SN were applied to adhered cells (2 h) or biofilms (48 h) and after 24 h of contact their effect was assessed by enumeration of colony forming units (CFUs) and quantification of total biomass (by crystal violet staining). The MIC results showed that SN were fungicidal against all strains tested at very low concentrations (0.4–3.3 μg ml^?1). Furthermore, SN were more effective in reducing biofilm biomass when applied to adhered cells (2 h) than to pre-formed biofilms (48 h), with the exception of C. glabrata ATCC, which in both cases showed a reduction ～90%. Regarding cell viability, SN were highly effective on adhered C. glabrata and respective biofilms. On C. albicans the effect was not so evident but there was also a reduction in the number of viable biofilm cells. In summary, SN may have the potential to be an effective alternative to conventional antifungal agents for future therapies in Candida-associated denture stomatitis.     

Sequence dependence of translational positioning of core nucleosomes

The basis for the choice of translational position of a histone octamer on DNA is poorly understood. To gain further insights into this question we have studied the translational and rotational settings of core particles assembled on a simple repeating 20 bp positioning sequence. We show that the translational positions of the core particles assembled on this sequence are invariant with respect to the DNA sequence and occur at 20 bp intervals. Certain modifications of the original sequence reduce the spacing of possible dyads to 10 bp. At least one of these alters both the translational and rotational settings. We conclude that the translational position of a core particle is specified by sequence determinants additional to those specifying rotational positioning. The rotational settings on either side of the dyads of core particles assembled on the wild-type and a mutant sequence differ by +2 bp, corresponding to an overall helical periodicity of approximately 10.15 bp. The average helical periodicity of the central two to four turns is 10.5-11 bp whilst that of the flanking DNA is closer to 10 bp. The DNA immediately flanking the dyad is also characterised by a more extensive susceptibility to cleavage by hydroxyl radical.     

Differential involvement of DNases in HeLa cell apoptosis induced by etoposide and long term-culture

We have applied to human HeLa cells two different stimuli of apoptosis: the antitumoral drug etoposide, and a more 'physiological' death condition, obtained by growing cells in the same medium for long time periods, for up to 10 days. Analysis of different parameters demonstrated that in both experimental systems the same apoptotic features are visible. However, the DNA degradation pattern appeared to be different, suggesting the involvement of different DNases. In this view, we have analyzed the activity and expression of Ca2+-Mg2+-dependent and acid DNases. We have observed that DNase I is not modulated during apoptosis. In contrast, the acid L-DNase II (derived from Leukocyte Elastase Inhibitor by post-translational modification), recently identified in our laboratory, is mainly active in the apoptotic pathway induced by long term-culture. Furthermore, we have provided evidence that while caspase 3 is activated by both inducers, caspase 1 is essential only for the etoposide-induced apoptosis.     

Metamorphosis of a scleractinian coral in response to microbial biofilms

Microorganisms have been reported to induce settlement and metamorphosis in a wide range of marine invertebrate species. However, the primary cue reported for metamorphosis of coral larvae is calcareous coralline algae (CCA). Herein we report the community structure of developing coral reef biofilms and the potential role they play in triggering the metamorphosis of a scleractinian coral. Two-week-old biofilms induced metamorphosis in less than 10% of larvae, whereas metamorphosis increased significantly on older biofilms, with a maximum of 41% occurring on 8-week-old microbial films. There was a significant influence of depth in 4- and 8-week biofilms, with greater levels of metamorphosis occurring in response to shallow-water communities. Importantly, larvae were found to settle and metamorphose in response to microbial biofilms lacking CCA from both shallow and deep treatments, indicating that microorganisms not associated with CCA may play a significant role in coral metamorphosis. A polyphasic approach consisting of scanning electron microscopy, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) revealed that coral reef biofilms were comprised of complex bacterial and microalgal communities which were distinct at each depth and time. Principal-component analysis of FISH data showed that the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Cytophaga-Flavobacterium of Bacteroidetes had the largest influence on overall community composition. A low abundance of Archaea was detected in almost all biofilms, providing the first report of Archaea associated with coral reef biofilms. No differences in the relative densities of each subdivision of Proteobacteria were observed between slides that induced larval metamorphosis and those that did not. Comparative cluster analysis of bacterial DGGE patterns also revealed that there were clear age and depth distinctions in biofilm community structure; however, no difference was detected in banding profiles between biofilms which induced larval metamorphosis and those where no metamorphosis occurred. This investigation demonstrates that complex microbial communities can induce coral metamorphosis in the absence of CCA.     

Antinociceptive activity of a novel buprenorphine analogue

HS-599 is a didehydroderivative of buprenorphine that displays high affinity and good selectivity for mu-opioid receptors. We studied its antinociceptive properties after s.c. injection in mice with the tail-flick and hot-plate tests. In the tail-flick test HS-599 (AD50 = 0.2801 micromol/kg s.c.) behaved as a full agonist and was twice as potent as buprenorphine (AD50=0.4569 micromol/kg s.c.) and 50 times more potent than morphine (AD50 = 13.3012 micromol/kg s.c.). Whereas the mu-opioid receptor antagonists naloxone (1-10 mg/kg s.c.) and naltrexone (5-15 mg/kg s.c.) antagonized HS-599 induced analgesia, the delta-opioid receptor antagonist naltrindole (20 mg/kg s.c.) and the kappa-opioid receptor antagonist nor-binaltorphimine (20 mg/kg s.c.) did not. With the hot-plate test at 50 degrees C, HS-599 (AD50 = 0.0359 micromol/kg s.c.) was a full agonist about 130 times more potent than morphine (AD50 = 4.8553 micromol/kg s.c.). With a high intensity nociceptive stimulus (55 degrees C) HS-599 (AD50 = 1.0382 micromol/kg s.c.) remained 7 times more potent than morphine (AD50 = 7.0210 micromol/kg s.c.) but never exceeded the 55% of the maximum possible effect, behaving as a partial agonist able to antagonize morphine antinociception in a dose-dependent manner. HS-599 promises to be a potent and safe new analgesic, preferentially acting at spinal level.