Purification and characterization of vanilla bean (Vanilla planifolia Andrews) beta-D-glucosidase

Vanilla bean beta-D-glucosidase was purified to apparent homogeneity by successive anion exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme is a tetramer (201 kDa) made up of four identical subunits (50 kDa). The optimum pH was 6.5, and the optimum temperature was 40 degrees C at pH 7.0. K(m) values for p-nitrophenyl-beta-D-glucopyranoside and glucovanillin were 1.1 and 20.0 mM, respectively; V(max) values were 4.5 and 5.0 microkat.mg(-1). The beta-D-glucosidase was competitively inhibited by glucono-delta-lactone and 1-deoxynojirimycin, with respective K(i) values of 670 and 152 microM, and not inhibited by 2 M glucose. The beta-D-glucosidase was not inhibited by N-ethylmaleimide and DTNB and fully inhibited by 1.5-2 M 2-mercaptoethanol and 1,4-dithiothreitol. The enzyme showed decreasing activity on p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, and p-nitrophenyl-beta-D-xylopyranoside. The enzyme was also active on prunasin, esculin, and salicin and inactive on cellobiose, gentiobiose, amygdalin, phloridzin, indoxyl-beta-D-glucopyranoside, and quercetin-3-beta-D-glucopyranoside.     

Fatty acid and tocopherol patterns of variation within the natural range of the shea tree (Vitellaria paradoxa)

The shea tree, Vitellaria paradoxa, is one of the most economically and culturally important indigenous tree species in the Sudano-Sahelian region. Its seeds contain a vegetable fat, internationally known as shea butter, which is widely used in edible, cosmetic and pharmaceutical sectors. Based on samples from 456 trees distributed in 17 locations across the species natural range from Senegal to Uganda, the fatty acid and tocopherol variation, and its relationship with geographic and climatic variables, was assessed in order to address the pattern and the origin of this variation across the natural range. Significant differences between Western and Eastern regions for oleic, stearic acid, saturated–unsaturated acid ratio and γ-tocopherol were identified that it is postulated maybe a result of genetic drift due to the evolutionary history of shea tree populations. Within regions the difference among stands was significant for most constituents; however the major part of the variation was observed among trees within stand (53–90 %). Relationships with climatic variables were not verified, weakening evidence for clinal variation hypotheses suggested by previous studies.     

A potential biomarker of androgen exposure in European bullhead (Cottus sp.) kidney

The aim of this study was to identify a signal that could be used as an androgen exposure indicator in the European bullhead (Cottus sp.). For this purpose, the ultra-structure of the kidney was characterized to identify normal structure of this organ, and histological changes previously described in the kidney of breeding male bullheads were quantified using the kidney epithelium height (KEH) assay previously developed and validated for the stickleback. In the next step, the effect of trenbolone acetate (TbA), a model androgen, was assessed to identify potential androgenic regulation of bullhead kidney hypertrophy. Measurement of KEH performed on adult non-breeding male and female bullheads exposed for 14 and 21 days to 0, 1.26 and 6.50 μg/L showed that kidney hypertrophy is induced in a dose-dependent manner, confirming the hypothesis that the European bullhead possesses a potential biomarker of androgen exposure. Combined with the wide distribution of the European bullhead in European countries and the potential of this fish species for environmental toxicology studies in field and laboratory conditions, the hypothesis of a potential biomarker of androgen exposure offers interesting perspectives for the use of the bullhead as a relevant sentinel fish species in monitoring studies. Inducibility was observed with high exposure concentrations of TbA. Further studies are needed to identify molecular signals that could be more sensitive than KEH.     

Immunological detection of Phaeoacremonium aleophilum,a fungal pathogen found in esca disease

Phaeoacremonium aleophilum is a fungal pathogen implicated in esca, a devastating disease affecting grapevines around the world. We have previously reported that the fungus secreted in its culture medium a variety of polypeptides, which allowed us to develop a serological method of detection. In this method, rabbit antibodies raised against the polypeptide fraction recognized secreted fungal proteins with high sensitivity (commonly 1 ng). These antibodies were specific since they cross-reacted with polypeptides secreted by various strains of Pm. aleophilum, but not with the proteins secreted by many other fungal pathogens implicated in other grapevine infections. Importantly, as shown by ELISA tests and immunolocalization, they did not cross-react with the secreted polypeptides of Eutypa lata (agent of Eutypa dieback), Neofusicoccum parvum (found in Black Dead Arm), and Phaeomoniella chlamydospora (also found in the fungal complex inducing esca). This serological approach permitted the development of a reliable dot-blot method to detect the presence in woody members of Pm. aleophilum in selectively infected trunk and canes. This method of diagnosis is rapid, easy to perform and non destructive for grapevines. Advantages and drawbacks of the method are discussed.     

Trends in additive,dominance and environmental effects with age for growth traits in <Emphasis Type="Italic">Eucalyptus</Emphasis> hybrid populations

Expression of additive and dominance effects during development of forest trees species is poorly documented. A set of 10 subpopulations, generated from factorial mating amongst subsets of 88 females and 107 males representing 684 families and 37,206 individual trees, was used to estimate variance components between 4 and 65 months in a Eucalyptus urophylla * grandis breeding population. The variance components were significantly different from zero for female, male and female-by-male interaction effects for height and circumference. The results, from the set of experiments, stressed the huge dispersion of variance component estimates among subpopulations. The trends were modelled using linear and nonlinear models. The trends in additive, dominance and environmental variances with age showed three phases corresponding to the stage of competition during stand development. No significant trend was observed for the coefficients of variation of these three effects. The dominance to additive variance ratio did not show specific age-related trends and was close to 1.2 during the growth period. The additive, dominance and environmental correlations between height and circumference were higher than 0.8 after 24 months. A similar age-related trend was observed for the additive and dominance effects. These results emphasise the significant role of dominance in the genetic control of growth traits. They differ from the majority of studies of pines, where additive variance is preponderant, but are similar to those of other eucalyptus studies. In the context of Congolese breeding strategy, this can be explained by the phenotypic selection of parents constituting the breeding population and/or the expression of overdominance in eucalyptus hybrid populations planted in marginal zones.     

Do elevation of CO(2) concentration and nitrogen fertilization alter storage and remobilization of carbon and nitrogen in pedunculate oak saplings?

Soil nitrogen can alter storage and remobilization of carbon and nitrogen in forest trees and affect growth responses to elevated carbon dioxide concentration ([CO(2)]). We investigated these effects in oak saplings (Quercus robur L.) exposed for two years to ambient or twice ambient [CO(2)] in combination with low- (LN, 0.6 mmol N l(-1)) or high-nitrogen (HN, 6.1 mmol N l(-1)) fertilization. Autumn N retranslocation efficiency from senescing leaves was less in HN saplings than in LN saplings, but about 15% of sapling N was lost to the litter. During the dormant season, nonstructural carbohydrates made up 20 to 30% of the dry mass of perennial organs. Starch was stored mainly in large roots where it represented 35-46% of dry mass. Accumulation of starch increased in large roots in response to LN but was unaffected by elevated [CO(2)]. The HN treatment resulted in high concentrations of N-soluble compounds, and this effect was reduced by elevated [CO(2)], which decreased soluble protein N (-17%) and amino acid N (-37%) concentrations in the HN saplings. Carbon and N reserves were labeled with (13)C and (15)N, respectively, at the end of the first year. In the second year, about 20% of labeled C and 50% of labeled N was remobilized for spring growth in all treatments. At the end of leaf expansion, 50-60% of C in HN saplings originated from assimilation versus only 10-20% in LN saplings. In HN saplings only, N uptake occurred, and some newly assimilated N was allocated to new shoots. Through effects on the C and N content of perennial organs, elevated [CO(2)] and HN increased remobilization capacity, thereby supporting multiple shoot flushes, which increased leaf area and subsequent C acquisition in a positive feedback loop.     

Plasticity of animal genome architecture unmasked by rapid evolution of a pelagic tunicate

Genomes of animals as different as sponges and humans show conservation of global architecture. Here we show that multiple genomic features including transposon diversity, developmental gene repertoire, physical gene order, and intron-exon organization are shattered in the tunicate Oikopleura, belonging to the sister group of vertebrates and retaining chordate morphology. Ancestral architecture of animal genomes can be deeply modified and may therefore be largely nonadaptive. This rapidly evolving animal lineage thus offers unique perspectives on the level of genome plasticity. It also illuminates issues as fundamental as the mechanisms of intron gain.     

Resistance to metalaxyl in isolates of the sunflower pathogen Plasmopara halstedii

Plasmopara halstedii isolates showing an atypical reaction to metalaxyl were collected in France, in 1995 and 1996, and tested in the laboratory for their level of sensitivity to this fungicide. Primary and secondary infections caused by one of these isolates were not controlled by the metalaxyl concentration registered for seed treatment. The EC₅₀ of this isolate was 12 800 mg a.i. kg^-1 compared with 22 mg a.i. kg^-1 for sensitive isolate, indicating a 582-fold decrease in sensitivity to the compound. There was no reduction in the agressiveness of the resistant isolate. Using other anti-oomycete fungicides, it appeared that propamocarb, contact fungicides (fluazinam, folpet, mancozeb) and the mixed formulations dimethomorph + mancozeb, cymoxanil + mancozeb and ofurace + folpet were effective against primary infections made with metalaxyl resistant and sensitive isolates, but not against secondary infections. Metalaxyl mixed with fluazinam, folpet or mancozeb was more effective against primary infections with the resistant isolate than metalaxyl alone. The EC₅₀ of five other isolates ranged from 5 800 to 32 900 mg a.i. kg^-1, indicating a variability in metalaxyl sensitivity of resistant sunflower downy mildew isolates. This is the first report of physiological resistance to metalaxyl in Plasmopara halstedii.     

Hydrolysis of the amyloid prion protein and nonpathogenic meat and bone meal by anaerobic thermophilic prokaryotes and streptomyces subspecies

Transmissible spongiform encephalopathies are caused by accumulation of highly resistant misfolded amyloid prion protein PrPres and can be initiated by penetration of such pathogen molecules from infected tissue to intact organism. Decontamination of animal meal containing amyloid prion protein is proposed thanks to the use of proteolytic enzymes secreted by thermophilic bacteria Thermoanaerobacter, Thermosipho, and Thermococcus subsp. and mesophilic soil bacteria Streptomyces subsp. Keratins alpha and beta, which resemble amyloid structures, were used as the substrates for the screening for microorganisms able to grow on keratins and producing efficient proteases specific for hydrolysis of beta-sheeted proteic structures, hence amyloids. Secretion of keratin-degrading proteases was evidenced by a zymogram method. Enzymes from thermophilic strains VC13, VC15, and S290 and Streptomyces subsp. S6 were strongly active against amyloid recombinant ovine prion protein and animal meal proteins. The studied proteases displayed broad primary specificities hydrolyzing low molecular mass peptide model substrates. Strong amyloidolytic activity of detected proteases was confirmed by experiments of hydrolysis of PrPres in SAFs produced from brain homogenates of mice infected with the 6PB1 BSE strain. The proteases from Thermoanaerobacter subsp. S290 and Streptomyces subsp. S6 are the best candidates for neutralization/elimination of amyloids in meat and bone meal and other protein-containing substances and materials.