LC-MS/MS method for the determination of erianin in rat plasma: Application to a pharmacokinetic study.

Erianin is one of the bibenzyl ingredients isolated from Dctidrobium chrysotoxum Lindl. In recent years, erianin has attracted attention owing to its antitumor activity. In this study, an LC-MS/MS method was established to measure erianin in rat plasma. Gigantol was used as the internal standard. A Waters Acquity UPLC BEH C18 column was employed for chromatographic separation. The mobile phase consisted of water containing 0.1% formic acid and acetonitrile with a gradient elution at the flow rate of 0.4 ml/min. Selective reaction monitoring mode was used for quantitative analysis of erianin in positive electrospray ionization. In the concentration range of 0.1-1200 ng/ml, erianin in rat plasma was linear with correlation coefficient >0.999. The lowest limit of quantification was 0.1 ng/ml. The intra- and inter-day RSDs were <9.69%, while the RE was in the range of -8.59-11.24%. The mean recovery was >85.37%. Erianin was stable in rat plasma after storage under certain conditions. The validated method was demonstrated to be selective, sensitive and reliable, and has been successfully applied to pharmacokinetic study of erianin in rat plasma. Erianin was rapidly eliminated from rat plasma with a short half-life (〜1.5 h) and low oral bioavailability (8.7%).

Cloning, expression and antioxidant activity of a thioredoxin peroxidase from Branchiostoma belcheri tsingtaunese.

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes that catalyze the thioredoxin- dependent reduction of hydroperoxides. In this study, a novel thioredoxin peroxidase (Bbt-TPx1), a member of the peroxiredoxin superfamily, was found by EST sequence analysis of a cDNA library of Branchiostoma belcheri tsingtaunese ovary. The sequence of a full-length cDNA clone contained an open reading frame encoding a polypeptide of 198 amino acid residues, with a calculated molecular weight of 22,150 Da. The expression patterns of the protein at different developmental stages and adult amphioxus tissues indicate that this enzyme may play important roles in anti-oxidation and innate immunity. The recombinant Bbt-TPx1 protein was expressed with a polyhistidine-tag in Escherichia coli and purified using Ni chromatography followed by SP cation exchange chromatography. The rBbt-TPx1 protein existed as a dimer under non-reducing conditions, and was dissociated into monomers by dithiothreitol (DTT); it might predominantly exist in oligomeric form. The rBbt-TPx1 protein showed a significant thiol-dependent peroxidase activity, removing hydrogen peroxide in the presence of dithiothreitol (DTT), but not glutathione (GSH). Protection of plasmid DNA and the thiol-protein from damage by metal-catalyzed oxidation (MCO) in vitro was also revealed.

Integrase-deficient lentivirus: opportunities and challenges for human gene therapy.

Lentiviruses are powerful tools for gene delivery and have been widely used for the dissection of gene functions in both replicating and quiescent cells. Recently, lentiviruses have also been used for delivering target sequences in gene therapy. Although the lentiviral system provides sustained exogenous gene expression, serious concerns have been raised due to its unfavorable insertion-mediated mutagenesis effect, thereby resulting in the silencing or activation of some unexpected genes. Thus, an array of modifications of the original vectors may reduce risks. Here, we briefly review the structure of the integrase protein, which is an essential protein for viral insertion and integration; the mechanisms of integrase-mediated integration; and the effects of the modifications of integrase. Moreover, we discuss the advantages resulting from integrase modifications and their future applications. Taken together, the generation of integrase-deficient lentivirus (IDLV) not only provides us with an opportunity to reduce the risk of virus-mediated insertions, which would improve the safety of gene therapy, but also favors gene correction and vaccine development.