Simplified ultraviolet liquid chromatographic method for determination of sertindole, dehydrosertindole and norsertindole, in human plasma

An ultra-violet high-performance liquid chromatographic method was developed for the determination of sertindole, an atypical antipsychotic drug and its main metabolites dehydrosertindole and norsertindole, in human plasma. With a small sample volume, after a single-step liquid-liquid extraction, the compounds were separated on a reversed-phase XTerra RP(18) column, eluted with 45% of acetonitrile and 55% of ammonium acetate buffer (0.05 M, adjusted pH 8) and detected at 256 nm within 11 min. This method shows a good linearity for plasma concentration between 5-100 ng/ml and 100-1000 ng/ml, a good precision (inter and intra day CV < 11%) and a good inter-assay accuracy (bias < 11%). The limit of quantification concentration was 5 ng/ml. The absolute recovery of sertindole was higher than 99%. This rapid and sensitive method could be used for therapeutic drug monitoring as well as for overdose management.     

Genome-wide microRNA expression analysis of clear cell renal cell carcinoma by next generation deep sequencing

MicroRNAs (miRNAs), non-coding RNAs regulating gene expression, are frequently aberrantly expressed in human cancers. Next-generation deep sequencing technology enables genome-wide expression profiling of known miRNAs and discovery of novel miRNAs at unprecedented quantitative and qualitative accuracy. Deep sequencing was performed on 11 fresh frozen clear cell renal cell carcinoma (ccRCC) and adjacent non-tumoral renal cortex (NRC) pairs, 11 additional frozen ccRCC tissues, and 2 ccRCC cell lines (n?=?35). The 22 ccRCCs patients belonged to 3 prognostic sub-groups, i.e. those without disease recurrence, with recurrence and with metastatic disease at diagnosis. Thirty-two consecutive samples (16 ccRCC/NRC pairs) were used for stem-loop PCR validation. Novel miRNAs were predicted using 2 distinct bioinformatic pipelines. In total, 463 known miRNAs (expression frequency 1-150,000/million) were identified. We found that 100 miRNA were significantly differentially expressed between ccRCC and NRC. Differential expression of 5 miRNAs was confirmed by stem-loop PCR in the 32 ccRCC/NRC samples. With respect to RCC subgroups, 5 miRNAs discriminated between non-recurrent versus recurrent and metastatic disease, whereas 12 uniquely distinguished non-recurrent versus metastatic disease. Blocking overexpressed miR-210 or miR-27a in cell line SKCR-7 by transfecting specific antagomirs did not result in significant changes in proliferation or apoptosis. Twenty-three previously unknown miRNAs were predicted in silico. Quantitative genome-wide miRNA profiling accurately separated ccRCC from (benign) NRC. Individual differentially expressed miRNAs may potentially serve as diagnostic or prognostic markers or future therapeutic targets in ccRCC. The biological relevance of candidate novel miRNAs is unknown at present.     

Interaction between complement subcomponent C1q and bacterial lipopolysaccharides.

The heptose-less mutant of Escherichia coli, D31m4, bound complement subcomponent C1q and its collagen-like fragments (C1qCLF) with Ka values of 1.4 x 10(8) and 2.0 x 10(8) M-1 respectively. This binding was suppressed by chemical modification of C1q and C1qCLF using diethyl pyrocarbonate (DEPC). To investigate the role of lipopolysaccharides (LPS) in this binding, biosynthetically labelled [14C]LPS were purified from E. coli D31m4 and incorporated into liposomes prepared from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) [PC/PE/LPS, 2:2:1, by wt.]. Binding of C1q or its collagen-like fragments to the liposomes was estimated via a flotation test. These liposomes bound C1q and C1qCLF with Ka values of 8.0 x 10(7) and 2.0 x 10(7) M-1; this binding was totally inhibited after chemical modification of C1q and C1qCLF by DEPC. Liposomes containing LPS purified from the wild-strain E. coli K-12 S also bound C1q and C1qCLF, whereas direct binding of C1q or C1qCLF to the bacteria was negligible. Diamines at concentrations which dissociate C1 into C1q and (C1r, C1s)2, strongly inhibited the interaction of C1q or C1qCLF with LPS. Removal of 3-deoxy-D-manno-octulosonic acid (2-keto-3-deoxyoctonic acid; KDO) from E. coli D31m4 LPS decreases the binding of C1qCLF to the bacteria by 65%. When this purified and modified LPS was incorporated into liposomes, the C1qCLF binding was completely abolished. These results show: (i) the essential role of the collagen-like moiety and probably its histidine residues in the interaction between C1q and the mutant D31m4; (ii) the contribution of LPS, particularly the anionic charges of KDO, to this interaction.     

A collagen-like surface glycoprotein is a structural component of the Bacillus anthracis exosporium

Bacillus anthracis, the aetiological agent of anthrax, is a Gram-positive spore-forming bacterium. The exosporium is the outermost integument surrounding the mature spore. Here, we describe the purification and the characterization of an immunodominant protein of the spore surface. This protein was abundant, glycosylated and part of the exosporium. The amino-terminal sequence was determined and the corresponding gene was identified. It encodes a protein of 382 amino acid residues, the central part of which contains a region of GXX motifs presenting similarity to mammalian collagen proteins. Thus, this collagen-like surface protein was named BclA (for Bacillus collagen-like protein of anthracis). BclA was absent from vegetative cells; it was detected only in spores and sporulating cells. A potential promoter, dependent on the sigma factor sigma(K), which is required for a variety of events late in sporulation, was found upstream from the bclA gene. A bclA deletion mutant was constructed and analysed. Electron microscopy studies showed that BclA is a structural component of the filaments covering the outer layer of the exosporium.     

The water-soluble matrix fraction from the nacre of Pinctada maxima produces earlier mineralization of MC3T3-E1 mouse pre-osteoblasts

Nacre or mother of pearl is a calcified structure that forms the lustrous inner layer of some shells. We studied the biological activity of the water-soluble matrix (WSM) extracted from powdered nacre from the shell of the pearl oyster, Pinctada maxima, on the MC3T3-E1 pre-osteoblast cell line from mouse calvaria. This cell line has the ability to differentiate into osteoblasts and to mineralize in the presence of beta-glycerophosphate and ascorbic acid. Cell proliferation and alkaline phosphatase activity were measured as markers of osteoblast differentiation, and mineralization was analyzed. These studies revealed that WSM stimulates osteoblast differentiation and mineralization by day 6 instead of the 21-day period required for cells grown in normal mineralizing media. We compared the activity of WSM with that of dexamethasone on this cell line. WSM can inhibit alkaline phosphatase (ALP) activity and the activity of dexamethasone on MC3T3-E1 cells. This study shows that nacre WSM could speed up the differentiation and mineralization of this cell line more effectively than dexamethasone.     

A maurotoxin with constrained standard disulfide bridging: innovative strategy of chemical synthesis,pharmacology, and docking on K+ channels

Maurotoxin (MTX) is a 34-residue toxin that has been isolated initially from the venom of the scorpion Scorpio maurus palmatus. It presents a large number of pharmacological targets, including small conductance Ca2+-activated and voltage-gated K+ channels. Contrary to other toxins of the alpha-KTx6 family (Pi1, Pi4, Pi7, and HsTx1), MTX exhibits a unique disulfide bridge organization of the type C1-C5, C2-C6, C3-C4, and C7-C8 (instead of the conventional C1-C5, C2-C6, C3-C7, and C4-C8, herein referred to as Pi1-like) that does not prevent its folding along the classic alpha/beta scaffold of scorpion toxins. Here, we developed an innovative strategy of chemical peptide synthesis to produce an MTX variant (MTXPi1) with a conventional pattern of disulfide bridging without any alteration of the toxin chemical structure. This strategy was used solely to address the impact of half-cystine pairings on MTX structural properties and pharmacology. The data indicate that MTXPi1 displays some marked changes in affinities toward the target K+ channels. Computed docking analyses using molecular models of both MTXPi1 and the various voltage-gated K+ channel subtypes (Shaker B, Kv1.2, and Kv1.3) were found to correlate with MTXPi1 pharmacology. A functional map detailing the interaction between MTXPi1 and Shaker B channel was generated in line with docking experiments.     

The structural and functional organization of H-NS-like proteins is evolutionarily conserved in Gram-negative bacteria

The structural gene of the H-NS protein, a global regulator of bacterial metabolism, has been identified in the group of enterobacteria as well as in closely related bacteria, such as Erwinia chrysanthemi and Haemophilus influenzae . Isolated outside these groups, the BpH3 protein of Bordetella pertussis exhibits a low amino acid conservation with H-NS, particularly in the N-terminal domain. To obtain information on the structure, function and/or evolution of H-NS, we searched for other H-NS-related proteins in the latest databases. We found that HvrA, a trans -activator protein in Rhodobacter capsulatus , has a low but significant similarity with H-NS and H-NS-like proteins. This Gram-negative bacterium is phylogenetically distant from Escherichia coli . Using theoretical analysis (e.g. secondary structure prediction and DNA binding domain modelling) of the amino acid sequence of H-NS, StpA (an H-NS-like protein in E. coli ), BpH3 and HvrA and by in vivo and in vitro experiments (e.g. complementation of various H-NS-related phenotypes and competitive gel shift assay), we present evidence that these proteins belong to the same class of DNA binding proteins. In silico analysis suggests that this family also includes SPB in R. sphaeroides , XrvA in Xanthomonas oryzae and VicH in Vibrio cholerae . These results demonstrate that proteins structurally and functionally related to H-NS are widespread in Gram-negative bacteria.     

Genetic determinism of anatomical and hydraulic traits within an apple progeny

The apple tree is known to have an isohydric behaviour, maintaining rather constant leaf water potential in soil with low water status and/or under high evaporative demand. However, little is known on the xylem water transport from roots to leaves from the two perspectives of efficiency and safety, and on its genetic variability. We analysed 16 traits related to hydraulic efficiency and safety, and anatomical traits in apple stems, and the relationships between them. Most variables were found heritable, and we investigated the determinism underlying their genetic control through a quantitative trait loci (QTL) analysis on 90 genotypes from the same progeny. Principal component analysis (PCA) revealed that all traits related to efficiency, whether hydraulic conductivity, vessel number and area or wood area, were included in the first PC, whereas the second PC included the safety variables, thus confirming the absence of trade-off between these two sets of traits. Our results demonstrated that clustered variables were characterized by common genomic regions. Together with previous results on the same progeny, our study substantiated that hydraulic efficiency traits co-localized with traits identified for tree growth and fruit production.