Characterization of ovarian follicular fluids of sheep, pigs and cows using proton nuclear magnetic resonance spectroscopy

Proton NMR spectra were produced for Graafian follicular fluids obtained by aspiration from sheep, pig and cow ovaries. The following low molecular mass, non-protein-bound metabolites were detected at concentrations exceeding 0.1 mM: acetate, alanine, creatinine/creatine, glycine, D-3-hydroxybutyrate, lactate, valine. Glucose was difficult to quantify and N-acetyl sugars gave a broad resonance at 2.06 ppm, presumably representing side-chains of glycoproteins. Ethanol was detected at up to millimolar concentrations in some specimens, though the physiological significance of this finding was not clear. The concentrations of all metabolites were comparable to those of plasma. These results have therefore shown that NMR spectroscopy is useful for gaining a broad and semiquantitative impression of the more abundant metabolites in the fluids of preovulatory Graafian follicles.     

Characterization of ovarian follicular fluids of sheep,pigs and cows using proton nuclear magnetic resonance spectroscopy

Summary Proton NMR spectra were produced for Graafian follicular fluids obtained by aspiration from sheep, pig and cow ovaries. The following low molecular mass, non-protein-bound metabolites were detected at concentrations exceeding 0.1 mM: acetate, alanine, creatinine/creatine, glycine, D-3-hydroxybutyrate, lactate, valine. Glucose was difficult to quantify and N-acetyl sugars gave a broad resonance at 2.06 ppm, presumably representing side-chains of glycoproteins. Ethanol was detected at up to millimolar concentrations in some specimens, though the physiological significance of this finding was not clear. The concentrations of all metabolites were comparable to those of plasma. These results have therefore shown that NMR spectroscopy is useful for gaining a broad and semiquantitative impression of the more abundant metabolites in the fluids of preovulatory Graafian follicles.     

An inhibitor of Bcl-2 family proteins induces regression of solid tumours

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.     

Antiproliferative effect of β-elemene in chemoresistant ovarian carcinoma cells is mediated through arrest of the cell cycle at the G2-M phase

Elemene is a natural antitumor plant drug. However, the effect of elemene on cell growth in ovarian cancer is unknown. In this study, we show that -elemene inhibited the proliferation of cisplatin-resistant human ovarian cancer cells and their parental cells, but had only a marginal effect in human ovary cells, indicating differential inhibitory effects on cell growth between ovarian cancer cells and normal ovary cells. We also demonstrated for the first time that -elemene markedly enhanced cisplatin-induced growth inhibition in resistant cells compared to sensitive cells. In addition, cell cycle analysis revealed a synergistic effect of -elemene and cisplatin on the induction of cell cycle G2-M arrest in our resistant ovarian carcinoma cells. Furthermore, we showed that treatment of these cells with both drugs downregulated cyclin B1 and Cdc2 expression, but elevated the levels of p53, p21waf1/cip1, p27kip1 and Gadd45. Finally, the combination of -elemene and cisplatin was found to increase the phosphorylation of Cdc2 and Cdc25C, which leads to a reduction in Cdc2-cyclin B1 activity. These novel findings suggest that -elemene sensitizes chemoresistant ovarian carcinoma cells to cisplatin-induced growth suppression partly through modulating the cell cycle G2 checkpoint and inducing cell cycle G2-M arrest, which lead to blockade of cell cycle progression.Received 19 January 2005; accepted 5 February 2005     

Pre-mRNA splicing and mRNA export linked by direct interactions between UAP56 and Aly.

Recent studies indicate that splicing of pre-messenger RNA and export of mRNA are normally coupled in vivo. During splicing, the conserved mRNA export factor Aly is recruited to the spliced mRNA-protein complex (mRNP), which targets the mRNA for export. At present, it is not known how Aly is recruited to the spliced mRNP. Here we show that the conserved DEAD-box helicase UAP56, which functions during spliceosome assembly, interacts directly and highly specifically with Aly. Moreover, UAP56 is present together with Aly in the spliced mRNP. Significantly, excess UAP56 is a potent dominant negative inhibitor of mRNA export. Excess UAP56 also inhibits the recruitment of Aly to the spliced mRNP. Furthermore, a mutation in Aly that blocks its interaction with UAP56 prevents recruitment of Aly to the spliced mRNP. These data suggest that the splicing factor UAP56 functions in coupling the splicing and export machineries by recruiting Aly to the spliced mRNP.     

Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface.

Activated platelets bind numerous adhesive and procoagulant proteins by receptor-mediated processes. Although there is little evidence to suggest that these processes are heterogeneous in platelets, we previously found that platelets co-stimulated with collagen and thrombin express functional alpha-granule factor V only on a subpopulation of cells. Here we show that these cells, referred to as 'COAT-platelets', bind additional alpha-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, fibronectin and alpha2-antiplasmin. These proteins are all transglutaminase substrates, and inhibitors of transglutaminase prevent the production of COAT-platelets. A synthetic transglutaminase substrate (CP15) also binds to COAT-platelets, and analysis by high performance liquid chromatography/mass spectrometry shows that a product is formed with a relative molecular mass (Mr) equal to CP15 plus 176. Serotonin, an abundant component of platelet-dense granules, has an Mr of 176, and fibrinogen isolated from COAT-platelets contains covalently linked serotonin. Synthetic bovine serum albumin-(serotonin)6 binds selectively to COAT-platelets and also inhibits the retention of procoagulant proteins on COAT-platelets. These data indicate that COAT-platelets use serotonin conjugation to augment the retention of procoagulant proteins on their cell surface through an as yet unidentified serotonin receptor.