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.     

‘Who’s in the Network?’ When Stakeholders Influence Data Analysis

Environmental applications of social network analysis (SNA) are just beginning to emerge, and so far have focussed on understanding the characteristics of social networks that increase the likelihood of collective action and successful natural resource management. We move beyond this discussion to demonstrate how knowledge gained from analysing the social networks of stakeholders can be harnessed for selecting stakeholders, and further, how these analyses can be influenced by the expressed wishes and concerns of stakeholders. Although we began our SNA using concepts derived from the resource-management literature, stakeholder involvement in the interpretation of the results led to the use of SNA techniques that had not previously been applied in the context of resource management. We thus re-analysed our data and modified our selection of research participants. Re-analysis led to the selection of research participants who (1) had unique positions in the network, thus occupying non-redundant communication roles in the network, (2) came from different stakeholder categories and (3) were relatively well-connected to others and tended to broker across different segments of the network. By combining insights from researchers and stakeholders in this way, it was possible to use SNA in an innovative and sensitive way to better meet the needs of the stakeholders and the research project.

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.     

Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits.

Agonist-bound receptors activate heterotrimeric (alpha beta gamma) G proteins by catalysing replacement by GTP of GDP bound to the alpha subunit, resulting in dissociation of alpha-GTP from the beta gamma subunits. In most cases, alpha-GTP carries the signal to effectors, as in hormonal stimulation and inhibition of adenylyl cyclase by alpha s and alpha i respectively. By contrast, genetic evidence in yeast and studies in mammalian cells suggest that beta gamma subunits of G proteins may also regulate effector pathways. Indeed, of the four recombinant mammalian adenylyl cyclases available for study, two, adenylyl cyclases II and IV, are stimulated by beta gamma. This effect of beta gamma requires costimulation by alpha s-GTP. This conditional pattern of effector responsiveness led to the prediction that receptors coupled to many G proteins will mediate elevation of cellular cyclic AMP, provided that Gs is also active. We now confirm this prediction. Coexpression of mutationally active alpha s with adenylyl cyclase II converted agonists that act through 'inhibitory' receptors (coupled to Gi) into stimulators of cAMP synthesis. Experiments using pertussis toxin and a putative scavenger of beta gamma, the alpha subunit of transducin, suggest that beta gamma subunits of the Gi proteins mediated this stimulation. These findings assign a new signalling function to beta gamma subunits of Gi proteins, the conditional stimulation of cAMP synthesis by adenylyl cyclase II.