Combining isotopic signatures of n(Sr)/n(Sr) and light stable elements (C,N, O,S) with multi-elemental profiling for the authentication of provenance of European cereal samples

The aim of this work (from the FP6 project TRACE) was to develop methods based on the use of geochemical markers for the authentication of the geographical origin of cereal samples in Europe (cf. EC regulations 2081/92 and 1898/06). For the first time, the potential usefulness of combining n(⁸⁷Sr)/n(⁸⁶Sr) and δ¹³C, δ¹⁵N, δ¹⁸O and δ³⁴S isotopic signatures, alone or with key element concentrations ([Na], [K], [Ca], [Cu] and [Rb], progressively identified out of 31 sets of results), was investigated through multiple step multivariate statistics for more than 500 cereal samples collected over 2 years from 17 sampling sites across Europe representing an extensive range of geographical and environmental characteristics.     

Multiple Seasonal Modifications in Enzymatic Flavour Formation in Tea

Carotenoid cleavage enzymes isolated from Japanese Camellia sinensis leaves （cultivar Yabukita） were used for investigating the structural patterns of carotenoid cleavage enzymes.Fresh tea leaves were used for the isolation of active enzymes and purified to single band stage in SDS PAGE gels after isoelectric focusing.The specific activity of the carotenoid cleavage enzymes was tested and the active fractions selected for further analysis.The sugar content and the amount of phosphate present in the purified enzymes were elucidated by the following methods：Phosphates were detected by phosphatase assays,fluorescence marker kits and ammoniumheptamolybdate complex measurements after incineration of the samples.Sugars were detected in gels using PAS reagent （periodide acid/ Schiff reagent） staining and by GC-MS after hydrolysation of the proteins with trifluoric acid.Phosphorylations as well as glycosylations of the samples could be detected in all cases,thus giving evidence for an increasing phosphorylation level of proteins in Camellia sinensis from spring （1.84 g/mg） to autumn （2.39 g/rg） as well as the presence of at least four different sugars （arabinose,xylose,galactose and ribose）.These secondary modifications of the carotenoid cleavage enzymes and their dependency on the harvesting season may well correspond to the changes on the functional level which were detected between spring （Michaelis Constant （Km） =9.45 mol/1） and autumn （K =17.16 mol/l） harvests.     

Autonomic innervation of the ovary of the Atlantic cod,Gadus morhua

The autonomic innervation of the ovary of the Atlantic cod was investigated using histochemical and physiological/pharmacological methods. The paired ovary receives autonomic innervation via branches of the posterior splanchnic nerve (vesicular nerve).Histochemical studies demonstrated vasoactive intestinal polypeptide (VIP)-immunoreactive, 5-hydroxytryptamine-immunoreactive and adrenergic nerve fibers, but a number of antisera raised against other peptides failed to reveal any specific reaction in the tissue preparations. It is concluded that the cod ovary receives a double antagonistic autonomic innervation of excitatory cholinergic fibers and non-adrenergic inhibitory fibers. The nature of the inhibitory neurotransmitter is not known.     

Screening and characterization of sex-specific DNA fragments in the freshwater fish matrinchã, Brycon amazonicus (Teleostei: Characiformes: Characidae)

The matrinch? Brycon amazonicus, a commercially important freshwater fish resource, has no heteromorphic sex chromosomes so far described. In the present study, we performed a screening of sex-associated DNA markers in this species, through the use of a random amplified polymorphic DNA (RAPD) assay and a genomic DNA restriction digestion analysis. DNA digestions evidenced no differences between sexes. Sixty-six random primers were used in pooled and individual DNA samples of males and females, and the analysis of the RAPD fingerprints revealed one female sex-associated band. Cloning and sequencing of this band led to the identification of two distinct DNA segments. While one of the isolated fragments showed a significant identity with a described protein gene (phosphatidylinositol glycan anchor biosynthesis, class W), the other fragment, composed of 535?bp, corresponds to a novel DNA marker. Further experiments were performed with this second DNA fragment in order to verify its sex-specificity. Data on dot blot hybridization, using total DNA of both sexes, confirmed its female-specificity in B. amazonicus. A primer set was designed based on its sequence data and used in PCR with DNA samples of this species, leading to diagnose the animals' sexes with a 100?% overall accuracy through a sequence characterized amplified region approach. No amplification results were found for two other species of the genus-B. orbignyanus and B. lundii. The obtained data can lead to the hypothesis that B. amazonicus may present heteromorphic sex chromosomes that should be in an early phase of differentiation.     

Effects of L-carnitine supplementation in pregnant sows on plasma concentrations of insulin-like growth factors, various hormones and metabolites and chorion characteristics

Previous studies have shown that supplementation of sow diets with L-carnitine increases the body weight of piglets at birth. This study was conducted to elucidate the reasons for this phenomenon. Three experiments with 24 (experiment 1), 40 (experiment 2) and 12 (experiment 3) sows were conducted. In all three experiments, sows were allotted to two groups which had free access to a nutritionally adequate diet. Sows of one group were supplemented with 125 mg L-carnitine/day during pregnancy; sows of the other group (control group) did not receive L-carnitine. In experiment 1, plasma samples were collected at day 95 of pregnancy, in experiment 2 plasma samples were collected at days 80 and 100 of pregnancy. In experiment 3, chorions of the sows were collected at parturition. L-carnitine-treated sows had higher plasma concentrations of total L-carnitine than control sows (p < 0.05). The number of piglets born and weights of litter and individual piglets at birth were not different between both groups in all three experiments. L-carnitine-treated sows had higher plasma concentrations of insulin-like growth factor-I (IGF-I) on day 80 of pregnancy (experiment 2, p < 0.05) and on day 95 (experiment 1, p < 0.10), and a higher plasma concentration of IGF-II on day 80 (experiment 2, p < 0.05) than control sows. Moreover, sows supplemented with L-carnitine had heavier chorions (+22%, p =0.10) with greater amounts of protein (+45%, p < 0.05) and DNA (+38%, p < 0.10) and a higher protein concentration of glucose transporter-1 (+62%, p < 0.05). Plasma concentrations of 17beta-oestradiol, progesterone and thyroid hormones as well as concentrations of urea and total free amino acids were not different between both groups of sows. Plasma concentrations of non-esterified fatty acids, ketone bodies, triacylglycerols and cholesterol were also largely indifferent between both groups of sows. In conclusion, this study shows that L-carnitine has less influence on lipid metabolism and utilization of nitrogen in pregnant sows but increases their plasma concentrations of IGFs. This in turn may enhance development of the placentae and the intrauterine nutrition of the fetuses. This may be the reason for increased birth weights observed in recent studies in sows supplemented with L-carnitine.     

Estimating tree species diversity across geographic scales

The relationship between number of species and area observed has been described using numerous approaches and has been discussed for more than a century. The general objectives of our study were fourfold: (1) to evaluate the behaviour of species–area curves across geographic scales, (2) to determine sample sizes necessary to produce acceptably precise estimates of tree species diversity, (3) to evaluate relationships among estimates of tree species diversity for local to large geographic scales, and (4) to determine whether the proportion of native tree species may be precisely estimated using sample sizes smaller than those necessary to estimate total tree species diversity. Such investigations are necessary to improve biodiversity monitoring at large scales. The analytical approaches included Monte Carlo bootstrap simulations and two geospatial methods and relied on a database populated with data for more than 12,000 national forest inventory plots (NFI) from 16 regional units, 13 European countries and three ecoprovinces of the United States of America (USA). Four primary results were obtained. First, tree species diversity may be precisely estimated using observations for a random subsample of 2,000–4,000 NFI plots. Second, large sample sizes are necessary to estimate tree species diversity for regional units or forest categories, except possibly for boreal forests for which the number of tree species is small. Third, estimates of the proportion of native tree species may be precisely estimated using tree species information for a random sample of approximately 30 NFI plots. Finally, our estimated species–area curves show that the curve shapes and relationships among estimates of tree species diversity at large scales clearly depend on the relative geographic location of the anchor regional unit (a European country or ecoprovince of the USA) relative to the other regional units. None of the well-known models for species–area curves adequately describes our results. The conclusion was that the uncertainties of the estimates reflect the unfavourable state of global biodiversity monitoring of species groups. The total numbers of tree species in our regional units, which cannot be adequately estimated, are small relative to other tree species-rich regions. Consequently, monitoring of tree species diversity is currently a highly uncertain enterprise at large scales.     

A fine-scale chimpanzee genetic map from population sequencing

To study the evolution of recombination rates in apes, we developed methodology to construct a fine-scale genetic map from high-throughput sequence data from 10 Western chimpanzees, Pan troglodytes verus. Compared to the human genetic map, broad-scale recombination rates tend to be conserved, but with exceptions, particularly in regions of chromosomal rearrangements and around the site of ancestral fusion in human chromosome 2. At fine scales, chimpanzee recombination is dominated by hotspots, which show no overlap with those of humans even though rates are similarly elevated around CpG islands and decreased within genes. The hotspot-specifying protein PRDM9 shows extensive variation among Western chimpanzees, and there is little evidence that any sequence motifs are enriched in hotspots. The contrasting locations of hotspots provide a natural experiment, which demonstrates the impact of recombination on base composition.     

Characterisation of novel <Emphasis Type="Italic">Fusarium graminearum</Emphasis> microsatellite markers in different <Emphasis Type="Italic">Fusarium</Emphasis> species from various countries

Fifteen novel microsatellite markers were isolated from Fusarium graminearum. The level of polymorphism at these novel and 13 previously published microsatellite markers was analysed in 33 F. graminearum strains from Europe, North America, and Nepal. The number of alleles for each of the novel markers ranged from 4 to 20 and gene diversity from 0.417 to 0.962. In comparison with the previously published markers, the resolution for distinguishing among different strains was slightly increased. Twenty-seven markers were also detectable in three F. culmorum strains and one F. crookwellense strain. None of the markers was detected in three F. avenaceum and four F. poae strains, underlining the potential use of these microsatellite markers for species differentiation.     

Somatic embryogenesis in a broad spectrum of grape genotypes

Somatic embryogenesis is the preferred method for cell-to-plant regeneration of grapevine. In this study, we tested the embryogenic capacity of anther-derived calli from 59 grape genotypes, representing a diverse group of Vitis vinifera and hybrid varieties, and hybrids and accessions of non-vinifera Vitis species. Most genotypes were tested on two types of media: MST1 medium, which contained plant growth regulators (PGRs) 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), and MSE medium, which contained 2,4-D and 6-benzylaminopurine (BAP). Twenty-four of the grape genotypes produced embryogenic callus on one or both of these media, eighteen of which have not been reported to form somatic embryos before. The results also suggested that the various PGR combinations are differentially effective at inducing somatic embryos in various classes of grape genotypes. For example, seven of the eight V. vinifera conv. occidentalis varieties brought forth somatic embryos on MSE medium, and three out of four American Vitis genotypes produced somatic embryos on MST1 medium. We could not observe any apparent association between frequency of callus formation and embryogenic capacity of the anthers.