Evaluation of a protocol for metabolic profiling studies on human blood plasma by combined ultra-performance liquid chromatography/mass spectrometry: From extraction to data analysis

The investigation presented here describes a protocol designed to perform high-throughput metabolic profiling analysis on human blood plasma by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). To address whether a previous extraction protocol for gas chromatography (GC)/MS-based metabolic profiling of plasma could be used for UPLC/MS-based analysis, the original protocol was compared with similar methods for extraction of low-molecular-weight compounds from plasma via protein precipitation. Differences between extraction methods could be observed, but the previously published extraction method was considered the best. UPLC columns with three different stationary phases (C8, C18, and phenyl) were used in identical experimental runs consisting of a total of 60 injections of extracted male and female plasma samples. The C8 column was determined to be the best for metabolic profiling analysis on plasma. The acquired UPLC/MS data of extracted male and female plasma samples was subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, a strategy for compound identification was applied here, demonstrating the strength of high-mass-accuracy time-of-flight (TOF)/MS analysis in metabolic profiling.     

Turning of the receptor-binding domains opens up the murine leukaemia virus Env for membrane fusion

The activity of the membrane fusion protein Env of Moloney mouse leukaemia virus is controlled by isomerization of the disulphide that couples its transmembrane (TM) and surface (SU) subunits. We have arrested Env activation at a stage prior to isomerization by alkylating the active thiol in SU and compared the structure of isomerization-arrested Env with that of native Env. Env trimers of respective form were isolated from solubilized particles by sedimentation and their structures were reconstructed from electron microscopic images of both vitrified and negatively stained samples. We found that the protomeric unit of both trimers formed three protrusions, a top, middle and a lower one. The atomic structure of the receptor-binding domain of SU fitted into the upper protrusion. This was formed similar to a bent finger. Significantly, in native Env the tips of the fingers were directed against each other enclosing a cavity below, whereas they had turned outward in isomerization-arrested Env transforming the cavity into an open well. This might subsequently guide the fusion peptides in extended TM subunits into the target membrane.     

The crystal structure of peroxymyoglobin generated through cryoradiolytic reduction of myoglobin compound III during data collection

Myoglobin has the ability to react with hydrogen peroxide, generating high-valent complexes similar to peroxidases (compounds I and II), and in the presence of excess hydrogen peroxide a third intermediate, compound III, with an oxymyoglobin-type structure is generated from compound II. The compound III is, however, easily one-electron reduced to peroxymyoglobin by synchrotron radiation during crystallographic data collection. We have generated and solved the 1.30 A (1 A=0.1 nm) resolution crystal structure of the peroxymyoglobin intermediate, which is isoelectric to compound 0 and has a Fe-O distance of 1.8 A and O-O bond of 1.3 A in accordance with a Fe(II)-O-O- (or Fe(III)-O-O2-) structure. The generation of the peroxy intermediate through reduction of compound III by X-rays shows the importance of using single-crystal microspectrophotometry when doing crystallography on metalloproteins. After having collected crystallographic data on a peroxy-generated myoglobin crystal, we were able (by a short annealing) to break the O-O bond leading to formation of compound II. These results indicate that the cryoradiolytic-generated peroxymyoglobin is biologically relevant through its conversion into compound II upon heating. Additionally, we have observed that the Xe1 site is occupied by a water molecule, which might be the leaving group in the compound II to compound III reaction.     

Pharmacological analysis of components of the change in transmural potential difference evoked by distension of rat proximal small intestine in vivo

The reflex response to distension of the small intestine in vivo is complex and not well understood. The aim of this study was to characterize the neural mechanisms contributing to the complex time course of the intestinal secretory response to distension. Transmucosal potential difference (PD) was used as a marker for mucosal chloride secretion, which reflects the activity of the secretomotor neurons. Graded distensions (5, 10, and 20 mmHg) of distal rat duodenum with saline for 5 min induced a biphasic PD response with an initial peak (rapid response) followed by a plateau (sustained response). The rapid response was significantly reduced by the neural blockers tetrodotoxin and lidocaine (given serosally) and by intravenous (iv) administration of the ganglionic blocker hexamethonium and the NK(1) receptor antagonist SR-140333. Serosal TTX and iv SR-140333 significantly reduced the sustained response, which was also reduced by the NK(3) receptor antagonist talnetant and by the vasoactive intestinal polypeptide (VPAC) receptor antagonist [4Cl-d-Phe(6), Leu(17)]-VIP. Serosal lidocaine and iv hexamethonium had no significant effect on this component. Inhibition of nitric oxide synthase had no effect on any of the components of the PD response to distension. The PD response to distension thus seems to consist of two components, a rapidly activating and adapting component operating via nicotinic transmission and NK(1) receptors, and a slow component operating via VIP-ergic transmission and involving both NK(1) and NK(3) receptors.     

Receptor-triggered but alkylation-arrested env of murine leukemia virus reveals the transmembrane subunit in a prehairpin conformation

A central feature of the prevailing model for retrovirus fusion is conversion of the transmembrane (TM) subunit from a prehairpin to a hairpin-like structure. The fusion inhibition of many retroviruses, except murine leukemia virus (MLV), with peptides corresponding to interacting regions in the hairpin supports the model. MLV fusion is controlled by isomerization of the intersubunit disulfide in Env. We show here that TM peptides bind to MLV Env that has been arrested at an intermediate stage of activation by alkylation of the isomerization-active thiol in the surface subunit. This inhibits fusion rescue by dithiothreitol-mediated reduction of the surface protein-TM disulfide.     

Activation of the phosphatidylinositol 3-kinase Vps34 by a G protein alpha subunit at the endosome

In the yeast Saccharomyces cerevisiae, the G protein beta gamma subunits are essential for pheromone signaling. The Galpha subunit Gpa1 can also promote signaling, but the effectors in this pathway are not well characterized. To identify candidate Gpa1 effectors, we expressed the constitutively active Gpa1(Q323L) mutant in each of nearly 5000 gene-deletion strains and measured mating-specific responses. Our analysis reveals a requirement for both the catalytic (Vps34) and regulatory (Vps15) subunits of the sole phosphatidylinositol 3-kinase in yeast. We demonstrate that Gpa1 is present at endosomes, where it interacts directly with both Vps34 and Vps15 and stimulates increased production of phosphatidylinositol 3-phosphate. Notably, Vps15 binds to GDP-bound Gpa1 and is predicted to have a seven-WD repeat structure similar to that of known G protein beta subunits. These findings reveal two new components of the pheromone signaling pathway. More remarkably, these proteins appear to comprise a preformed effector-G beta subunit assembly and function at the endosome rather than at the plasma membrane.     

Molecular insights into ligand recognition and G protein coupling of the neuromodulatory orphan receptor GPR139

Dear Editor, The G protein-coupled receptor GPR139 is involved in neuro-modulation,and one of its agonists is in clinical trials for the treatment of cognitive ...     

Biomass production, symbiotic nitrogen fixation and inorganic N use in dual and tri-component annual intercrops

The interspecific complementary and competitive interactions between pea (Pisum sativum L.), barley (Hordeum vulgare L.) and oilseed rape (Brassica napus L.), grown as dual and tri-component intercrops were assessed in a field study in Denmark. Total biomass production and N use at two levels of N fertilisation (0.5 and 4.0 g N/m²), were measured at five harvests throughout a growing season. All intercrops displayed land equivalent ratio values close to or exceeding unity, indicating complementary use of growth resources. Whereas both rape and barley responded positively to increased N fertilisation, irrespective of whether they were grown as sole- or intercrops, pea was strongly suppressed when grown in intercrop. Of the three crops barley was the strongest competitor for both soil and fertiliser N, rape intermediate and pea the weakest. Faster initial growth of barley than pea and rape gave barley an initial competitive advantage, an advantage that in the two dual intercrops was strengthened by the addition of N. Apparently the competitive superiority of barley was less strong in the tri-component intercrop, indicating that the impact of the dominantmay, through improved growth of both rape and pea, have been diminished through indirect facilitation. Interspecific competition had a promoting effect on the percent of nitrogen derived from N₂ fixation of pea, and most so at the low N fertilisation level. Results indicate that the benefits achieved from the association of a legume and nonlegume, in terms of N₂ fixed were greatest when pea was grown in association with rape as opposed to barley which could indicate that the benefits achieved from the association of a legume and nonlegume are partly lost if the nonlegume is too strong a competitor.     

Distinct patterns in alpine vegetation around dens of the Arctic fox

The arctic fox Alopex lagopus excavates its dens in gravely ridges and hillocks, and creates a local environment quite distinct from the surrounding tundra or heath landscape. In northern Sweden, the vegetation of 18 dens of the arctic fox was investigated, as well as reference areas off the dens but in geologically and topographically similar locations. The species composition showed considerable differences between den and reference areas, with grasses and forbs occurring more abundantly on the dens, and evergreen dwarf-shrubs occurring more in reference areas. The effect of the foxes' activities is thought to be either through mechanical soil disturbance, or through nutrient enrichment via scats, urine, and carcasses. This was expected to result in differences in plant traits with key functional roles in resource acquisition and regeneration, when comparing dens with reference areas. We hypothesised that the community mean of specific leaf area (SLA) would differ if nutrient enrichment was the more important effect, and that seed weight, inversely proportional to seed number per ramet and hence dispersal ability, would differ if soil disturbance was the more important effect. Specific leaf area showed a significant difference, indicating nutrient enrichment to be the most important effect of the arctic fox on the vegetation on its dens. Arctic foxes act as ecosystems engineers on a small scale, maintaining niches for relatively short-lived nutrient demanding species on their dens in spite of the dominance of long-lived ericaceous dwarf-shrubs in the landscape matrix. Thus, foxes contribute to the maintenance of species richness on the landscape level.