Quantitative determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cellular environment of a human colon adenocarcinoma cell line for pharmacokinetic studies

A simple, accurate, precise, specific and reproducible high-performance liquid chromatography (HPLC) method was developed for determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cell suspension. The assay procedure involved simple liquid-liquid extraction, the supernatant liquid was added an equal volume of water to avoid solvent effect. The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 303 nm. The analysis used a Hypersil ODS2 C18 column (5 microm, 4.6 mm x 250 mm) and methanol/distilled water as the mobile phase (flow rate=1 mL/min). A total analytical run was achieved within 6.0 min and calibration curve was linear over a wide concentration range of 0.25-40 microg/mL for plasma sample and 1.0-500 microM for cell suspension, the coefficients of correlation were 0.9997 and 0.9999 or better, respectively. There was 80.7+/-7.86%, 96.8+/-3.20% and 102.7+/-9.72% recovery from 0.5, 10, and 40 microg/mL plasma samples, respectively. Intra- and inter-batch accuracy and precision were acceptable for the both matrices. The RSD of intra- and inter-day assay variations were all less than 10%. Both analyte and IS were stable in the battery of stability studies, freeze-thaw cycles. The described assay method was applied to pharmacokinetic studies in rats and a human colon adenocarcinoma cell line (Caco-2) successfully. The application of the assay to determine the pharmacokinetic is described.     

Comparative Study and Mutational Analysis of Distinctive Structural Elements of Hyperthermophilic Enzymes

Comparison of the three-dimensional structure of hyperthermophilic and mesophilic β-glycosidases shows differences in secondary structure composition. The enzymes from hyperthermophilic archaea have a significantly larger number of β-strands arranged in supernumerary β-sheets compared to mesophilic enzymes from bacteria and other organisms. Amino acid replacements designed to alter the structure of the supernumerary β-strands were introduced by site directed mutagenesis into the sequence encoding the β-glycosidase from Sulfolobus solfataricus. Most of the replacements caused almost complete loss of activity but some yielded enzyme variants whose activities were affected specifically at higher temperatures. Far-UV CD spectra recorded as a function of temperature for both wild type β-glycosidase and mutant V349G, one of the mutants with reduced activity at higher temperatures, were similar, showing that the protein structure of the mutant was stable at the highest temperatures assayed. The properties of mutant V349G show a difference between thermostability (stability of the protein structure at high temperatures) and thermophilicity (optimal activity at high temperatures).     

Distribution dynamics of the Tnt1 retrotransposon in tobacco

Retrotransposons contribute significantly to the size, organization and genetic diversity of plant genomes. Although many retrotransposon families have been reported in plants, to this day, the tobacco Tnt1 retrotransposon remains one of the few elements for which active transposition has been shown. Demonstration that Tnt1 activation can be induced by stress has lent support to the hypothesis that, under adverse conditions, transposition can be an important source of genetic variability. Here, we compared the insertion site preference of a collection of newly transposed and pre-existing Tnt1 copies identified in plants regenerated from protoplasts or tissue culture. We find that newly transposed Tnt1 copies are targeted within or close to host gene coding sequences and that the distribution of pre-existing insertions does not vary significantly from this trend. Therefore, in spite of their potential to disrupt neighboring genes, insertions within or near CDS are not preferentially removed with age. Elimination of Tnt1 insertions within or near coding sequences may be relaxed due to the polyploid nature of the tobacco genome. Tnt1 insertions within or near CDS are thus better tolerated and can putatively contribute to the diversification of tobacco gene function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Using physiological tests on the selection of cut roses' varieties

We have observed that the evolution of the senescence in vase can be appreciated by the measures of pH, conductivity, or osmolarity of the petal cell sap of a rose variety. We wanted to check whether these physiological tests could be used to select the post-harvest quality of varieties in process of evaluation. Whereas the measures of pH and osmolarity do not seem to be correlated with the flower's vase life, those of conductivity seem to be linked to it (r=-0.74). As far as the pH is concerned, when the correlation is tested on varieties grouped in colours (white, yellow and ochre; pink and red; multicoloured) and not on the whole mixed varieties, then the correlation seems to be satisfactory, being characterized by r coefficients between -0.78 and -0.91.     

Malaria Plasmodium agent induces alteration in the head proteome of their Anopheles mosquito host

Despite increasing evidence of behavioural manipulation of their vectors by pathogens, the underlying mechanisms causing infected vectors to act in ways that benefit pathogen transmission remain enigmatic in most cases. Here, 2-D DIGE coupled with MS were employed to analyse and compare the head proteome of mosquitoes (Anopheles gambiae sensu stricto (Giles)) infected with the malarial parasite (Plasmodium berghei) with that of uninfected mosquitoes. This approach detected altered levels of 12 protein spots in the head of mosquitoes infected with sporozoites. These proteins were subsequently identified using MS and functionally classified as belonging to metabolic, synaptic, molecular chaperone, signalling, and cytoskeletal groups. Our results indicate an altered energy metabolism in the head of sporozoite-infected mosquitoes. Some of the up-/down-regulated proteins identified, such as synapse-associated protein, 14-3-3 protein and calmodulin, have previously been shown to play critical roles in the CNS of both invertebrates and vertebrates. Furthermore, a heat shock response (HSP 20) and a variation of cytoarchitecture (tropomyosins) have been shown. Discovery of these proteins sheds light on potential molecular mechanisms that underlie behavioural modifications and offers new insights into the study of intimate interactions between Plasmodium and its Anopheles vector.     

Stocking may increase mitochondrial DNA diversity but fails to halt the decline of endangered Atlantic salmon populations

Over the last 50 years, Spanish Atlantic salmon (Salmo salar) populations have been in decline. In order to bolster these populations, rivers were stocked with fish of northern European origin during the period 1974–1996, probably also introducing the furunculosis-inducing pathogen, Aeromonas salmonicida. Here we assess the relative importance of processes influencing mitochondrial (mt)DNA variability in these populations from 1948 to 2002. Genetic material collected over this period from four rivers in northern Spain (Cantabria) was used to detect variability at the mtDNA ND1 gene. Before stocking, a single haplotype was found at high frequency (0.980). Following stocking, haplotype diversity (h) increased in all rivers (mean h before stocking was 0.041, and 0.245 afterwards). These increases were due principally to the dramatic increase in frequency of a previously very low frequency haplotype, reported at higher frequencies in northern European populations proximate to those used to stock Cantabrian rivers. Genetic structuring increased after stocking: among-river differentiation was low before stocking (1950s/1960s Φ _ST = –0.00296–0.00284), increasing considerably at the height of stocking (1980s Φ _ST = 0.18932) and decreasing post-stocking (1990s/2002 Φ _ST = 0.04934–0.03852). Gene flow from stocked fish therefore seems to have had a substantial role in increasing mtDNA variability. Additionally, we found significant differentiation between individuals that had probably died from infectious disease and apparently healthy, angled fish, suggesting a possible role for pathogen-driven selection of mtDNA variation. Our results suggest that stocking with non-native fish may increase genetic diversity in the short term, but may not reverse population declines.     

Characterization of reemerging chikungunya virus

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.     

Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

Nine yeast strains were isolated from spontaneous fermentations in the Alsace area of France, during the 1997, 1998 and 1999 grape harvests. Strains were characterized by pulsed-field gel electrophoresis, PCR-restriction fragment length polymorphism (RFLP) of the MET2 gene, delta-PCR, and microsatellite patterns. Karyotypes and MET2 fragments of the nine strains corresponded to mixed chromosomal bands and restriction patterns for both Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum. They also responded positively to amplification with microsatellite primers specific to both species and were demonstrated to be diploid. However, meiosis led to absolute nonviability of their spores on complete medium. All the results demonstrated that the nine yeast strains isolated were S. cerevisiaexS. bayanus var. uvarum diploid hybrids. Moreover, microsatellite DNA analysis identified strains isolated in the same cellar as potential parents belonging to S. bayanus var. uvarum and S. cerevisiae.     

Proteomics demonstration that normal breast epithelial cells can induce apoptosis of breast cancer cells through insulin-like growth factor-binding protein-3 and maspin

Normal breast epithelial cells are known to exert an apoptotic effect on breast cancer cells, resulting in a potential paracrine inhibition of breast tumor development. In this study we purified and characterized the apoptosis-inducing factors secreted by normal breast epithelial cells. Conditioned medium was concentrated by ultrafiltration and separated on reverse phase Sep-Pak C18 and HPLC. The proapoptotic activity of eluted fractions was tested on MCF-7 breast cancer cells, and nano-LC-nano-ESI-MS/MS allowed the identification of insulin-like growth factor-binding protein-3 (IGFBP-3) and maspin as the proapoptotic factors produced by normal breast epithelial cells. Western blot analysis of conditioned media confirmed the specific secretion of IGFBP-3 and maspin by normal cells but not by breast cancer cells. Immunodepletion of IGFBP-3 and maspin completely abolished the normal cell-induced apoptosis of cancer cells, and recombinant proteins reproduced the effect of normal cell-conditioned medium on apoptosis of breast cancer cells. Together our results indicated that normal breast epithelial cells can induce apoptosis of breast cancer cells through IGFBP-3 and maspin. These findings provide a molecular hypothesis for the long observed inhibitory effect of normal surrounding cells on breast cancer development.