PI3K signaling is required for prostaglandin-induced mucosal recovery in ischemia-injured porcine ileum

We have previously shown that PGE(2) and PGI(2) induce recovery of transepithelial resistance (TER) in ischemia-injured porcine ileal mucosa, associated with initial increases in Cl(-) secretion. We believe that the latter generates an osmotic gradient that stimulates resealing of tight junctions. Because of evidence implicating phosphatidylinositol 3-kinase (PI3K) in regulating tight junction assembly, we postulated that this signaling pathway is involved in PG-induced mucosal recovery. Porcine ileum was subjected to 45 min of ischemia, after which TER was monitored for a 180-min recovery period. Endogenous PG production was inhibited with indomethacin (5 microM). PGE(2) (1 microM) and PGI(2) (1 microM) stimulated recovery of TER, which was inhibited by serosal application of the osmotic agent urea (300 mosmol/kgH(2)O). The PI3K inhibitor wortmannin (10 nM) blocked recovery of TER in response to PGs or mucosal urea. Immunofluorescence imaging of recovering epithelium revealed that PGs restored occludin and zonula occludens-1 distribution to interepithelial junctions, and this pattern was disrupted by pretreatment with wortmannin. These experiments suggest that PGs stimulate recovery of paracellular resistance via a mechanism involving transepithelial osmotic gradients and PI3K-dependent restoration of tight junction protein distribution.     

Characterisation of a novel high-purity, double virus inactivated von Willebrand Factor and Factor VIII concentrate (Wilate)

This study summarises the biochemical and functional properties of a new generation plasma-derived, double virus inactivated von Willebrand Factor/Factor VIII (VWF/FVIII) concentrate, Wilate, targeted for the treatment of both von Willebrand disease (VWD) and haemophilia A. The manufacturing process comprises two chromatographic steps based on different performance principles, ensuring a high purity of the concentrate (mean specific activity in 15 consecutive production batches: 122 IU FVIII:C/mg total protein) and, thus, minimising the administered protein load to the patient (specification: < or = 15 mg total protein per 900 IU Wilate). The optimised solvent/detergent (S/D) treatment and prolonged terminal dry-heat (PermaHeat) treatment of the lyophilised product at a specified residual moisture (RM) provide two mechanistically independent, effective and robust virus inactivation procedures for enveloped viruses and one step for non-enveloped viruses. These process steps are aggressive enough to inactivate viruses efficiently, but yet gentle enough to maintain the structural integrity and function of the VWF and FVIII molecules, as proven by state-of-the-art assays covering the diverse features of importance. The VWF multimeric pattern is close to the one displayed by normal plasma, with a consistent content of more than 10 multimers, but a relatively lower portion of the very high multimers. The multimeric triplet structure is normal, underlining the gentle and effective manufacturing process, which does not require the addition of protein stabilisers at any step. The balanced activity ratio of VWF to FVIII is close to that of plasma from healthy subjects, rendering Wilate suitable also for the safe and effective treatment of patients with VWD.     

Robust Selection of Cancer Survival Signatures from High-Throughput Genomic Data Using Two-Fold Subsampling

Identifying relevant signatures for clinical patient outcome is a fundamental task in high-throughput studies. Signatures, composed of features such as mRNAs, miRNAs, SNPs or other molecular variables, are often non-overlapping, even though they have been identified from similar experiments considering samples with the same type of disease. The lack of a consensus is mostly due to the fact that sample sizes are far smaller than the numbers of candidate features to be considered, and therefore signature selection suffers from large variation. We propose a robust signature selection method that enhances the selection stability of penalized regression algorithms for predicting survival risk. Our method is based on an aggregation of multiple, possibly unstable, signatures obtained with the preconditioned lasso algorithm applied to random (internal) subsamples of a given cohort data, where the aggregated signature is shrunken by a simple thresholding strategy. The resulting method, RS-PL, is conceptually simple and easy to apply, relying on parameters automatically tuned by cross validation. Robust signature selection using RS-PL operates within an (external) subsampling framework to estimate the selection probabilities of features in multiple trials of RS-PL. These probabilities are used for identifying reliable features to be included in a signature. Our method was evaluated on microarray data sets from neuroblastoma, lung adenocarcinoma, and breast cancer patients, extracting robust and relevant signatures for predicting survival risk. Signatures obtained by our method achieved high prediction performance and robustness, consistently over the three data sets. Genes with high selection probability in our robust signatures have been reported as cancer-relevant. The ordering of predictor coefficients associated with signatures was well-preserved across multiple trials of RS-PL, demonstrating the capability of our method for identifying a transferable consensus signature. The software is available as an R package rsig at CRAN (http://cran.r-project.org).     

Natural products and their derivatives as inhibitors of glycogen phosphorylase: potential treatment for type 2 diabetes

Glycogen phosphorylase (GP) (EC 2.4.1.1) is an important therapeutic target for the potential treatment of type 2 diabetes. The search for potent, selective and drug-like GP inhibitors which may eventually lead to hypoglycaemic agents has to date uncovered a number of natural product inhibitors with both pharmaceutical and nutraceutical potential. GP is an allosteric protein with at least six different ligand binding sites that modulate its enzymatic activity. Hence, inhibitors with considerable structural diversity can be designed. This review is focused on advances in the discovery of natural products and their derivatives as GP inhibitors.     

Scanning electron microscope studies of the surface of the sea urchin egg

Summary The surface of the egg of the two sea urchin species,Strongylocentrotus droebachiensis andS. pallidus, was studied with SEM. Clear differences were observed between the two species, in both the number and form of the microvilli. The changes in the egg surface which follow insemination are described.     

Determination of 2-isopropoxyphenol in urine by capillary gas chromatography and mass-selective detection

An analytical method for the assessment of the exposure of workers to the pesticide propoxur through biological monitoring has been developed. This study was part of a survey of occupational exposure to pesticides used in greenhouses for the growing of ornamental plants. In order to assess the actual absorbed amount of propoxur in the body, an analytical method for its metabolite 2-isopropoxyphenol in urine was required. This led to the development of a gas chromatographic—mass spectrometric assay involving hydrolysis and solvent extraction. A mass-selective detector, operated in single-ion mode, provides a selective and sensitive quantification of 2-isopropoxyphenol with a detection limit of 6 μg/l. The method has been validated with respect to the hydrolysis of urine samples, analytical recovery of 2-isopropoxyphenol, stability of its conjugates, limit of detection, background and precision. The analytical recovery from spiked urine was over 95%. 2-Isopropoxyphenol was excreted in urine as a conjugate and was stable for at least seven months when stored at − 20°C. It was not detected in urine from non-exposed persons. Between-day coefficients of variation were 20, 10, 7 and 4% for concentrations of 15, 29, 150 and 213 μg/l, respectively. Measured as 2-isopropoxyphenol, ca. 80% of an orally administered dose of propoxur was excreted in urine within 10 h.     

Enhanced in vitro growth of murine fibroblast cells and preimplantation embryos cultured in medium supplemented with an amphipathic peptide.

Preliminary studies on the proliferative effects of lytic peptides were carried out using NIH 3T3 murine fibroblast cells and human lymphocytes. Cells were cultured in various concentrations of three different amphipathic peptides (SB-37, Shiva-1, and Vishnu), and enhanced proliferation was determined by uptake of 3H-thymidine with treated cells compared with control cultures. Enhanced proliferation of 3T3 cells was observed in cultures containing 50 microM or less SB-37. The primary study consisted of 263 four-cell- to eight-cell-stage mouse embryos from naturally bred mice and incubated in Whitten's medium containing 0.2, 1, or 10 microM of the amino terminus of an amphipathic cecropin B analog (Vishnu) or in Whitten's medium alone. Embryos were cultured to the hatched blastocyst stage, and effect of treatment was determined by the rate of growth to that stage of development. Statistical analysis revealed that culture in all three levels of Vishnu significantly accelerated in vitro growth of these stages of preimplantation embryos compared with controls. These results indicate that Vishnu promotes increased cleavage rates of embryos in vitro. A growth factor receptor clustering mechanism of action is proposed. This peptide may have some potential as an embryo culture medium additive to enhance in vitro growth rate.