N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP

N-Glycosylation affects the function of ion channels at the level of multisubunit assembly, protein trafficking, ligand binding and channel opening. Like the majority of membrane proteins, ionotropic P2X receptors for extracellular ATP are glycosylated in their extracellular moiety. Here, we used site-directed mutagenesis to the four predicted N-glycosylation sites of P2X(3) receptor (Asn(139), Asn(170), Asn(194) and Asn(290)) and performed comparative analysis of the role of N-glycans on protein stability, plasma membrane delivery, trimer formation and inward currents. We have found that in transiently transfected HEK293 cells, Asn(170) is apparently the most important site for receptor stability, since its mutation causes a primary loss in protein content and indirect failure in membrane expression, oligomeric association and inward current responses. Even stronger effects are obtained when mutating Thr(172) in the same glycosylation consensus. Asn(194) and Asn(290) are the most dispensable, since even their simultaneous mutation does not affect any tested receptor feature. All double mutants containing Asn(170) mutation or the Asn(139)/Asn(290) double mutant are instead almost unable to assemble into a functional trimeric structure. The main emerging finding is that the inability to assemble into trimers might account for the impaired function in P2X(3) mutants where residue Asn(170) is replaced. These results improve our knowledge about the role of N-glycosylation in proper folding and oligomeric association of P2X(3) receptor.     

Multitargeted drugs discovery: balancing anti-amyloid and anticholinesterase capacity in a single chemical entity

Memoquin (1) is a lead compound multitargeted against Alzheimer’s disease (AD). It is an AChE inhibitor, free-radical scavenger, and inhibitor of amyloid-β (Aβ) aggregation. A new series of 1 derivatives was designed and synthesized by linking its 2,5-diamino-benzoquinone core with motifs that are present in the structure of known amyloid binding agents like curcumin, the benzofuran derivative SKF64346, or the benzothiazole bearing compounds KHG21834 and BTA-1. The weaker AChE inhibitory potencies and the concomitant nearly equipotent anti-amyloid activities of the new compounds with respect to 1 resulted in a more balanced biological profile against both targets. Selected compounds turned out to be effective Aβ aggregation inhibitors in a cell-based assay. By properly combining two or more distinct pharmacological properties in a molecule, we can achieve greater effectiveness compared to single-targeted drugs for investigating AD.     

Greater effort boosts the affective taste properties of food

Actions can create preferences, increasing the value ascribed to commodities acquired at greater cost. This behavioural finding has been observed in a variety of species; however, the causal factors underlying the phenomenon are relatively unknown. We sought to develop a behavioural platform to examine the relationship between effort and reinforcer value in mice trained under demanding or lenient schedules of reinforcement to obtain food. In the initial experiment, expenditure of effort enhanced the value of the associated food via relatively lasting changes in its hedonic attributes, promoting an acquired preference for these reinforcers when tested outside of the training environment. Moreover, otherwise neutral cues associated with those reinforcers during training similarly acquired greater reinforcing value, as assessed under conditioned reinforcement. In a separate experiment, expenditure of effort was also capable of enhancing the value of less-preferred low-caloric reinforcers. Analysis of licking microstructure revealed the basis for this increased valuation was, in part, due to increased palatability of the associated reinforcer. This change in the hedonic taste properties of the food can not only serve as a basis for preference, but also guide decision-making and foraging behaviour by coordinating a potentially adaptive repertoire of incentive motivation, goal-directed action and consumption.     

Analysis of the rpn11-m1 proteasomal mutant reveals connection between cell cycle and mitochondrial biogenesis

The proteasomal lid subunit Rpn11 is essential for maintaining a correct cell cycle and mitochondrial morphology in Saccharomyces cerevisiae. In this paper, we show that the rpn11-m1 mutant has a peculiar cell cycle defect reminiscent of mutants defective in the FEAR pathway that delay the release of the Cdc14 protein phosphatase from the nucleolus. We analyzed the rpn11-m1 phenotypes and found that overexpression of Cdc14 suppresses all the rpn11-m1 defects, including the mitochondrial ones. Suppression by Cdc14 of the rpn11-m1 mitochondrial morphology defect reveals an uncharacterized connection between mitochondrial and cell cycle events. Interestingly, the overexpression of Cdc14 also partially restores the tubular network in an Δmmm2 strain, which lacks a mitochondrial protein belonging to the complex necessary to anchor the mitochondrion to the actin cytoskeleton. Altogether our findings indicate, for the first time, a cross-talk between the cell cycle and mitochondrial morphology.     

The endangered white-clawed crayfish <Emphasis Type="Italic">Austropotamobius pallipes</Emphasis> (Decapoda,Astacidae) east and west of the Maritime Alps: a result of human translocation?

The genetic variability among Italian populations of the white-clawed crayfish (Austropotamobius pallipes) was examined to determine their phylogeography and to assess their conservation status as a management unit. A fragment of the mitochondrial DNA COI gene of 107 specimens from ten populations was sequenced, and the phylogenetic relationships were established. Two out of three haplotypes sampled in two French populations from the Rhône basin were shared with Italian populations. Despite a moderate level of genetic variability within the Italian populations of A. pallipes, no genetic structure was revealed. It has been suggested that there have been translocation events throughout the Alpine barrier between the North-Western Italian basins and the Rhône basin. Genetic exchangeability of the French and Italian populations was demonstrated in this study, and a shift of conservation efforts towards the native, congeneric Austropotamobius italicus is recommended.     

Genetic diversity of the genus Malus and implications for linkage mapping with SNPs

Knowledge about the sequence-based genetic diversity of a crop species is important in order to develop highly informative genotyping assays, which will eventually positively impact breeding practice. Diversity data were obtained from two pools of 185 and 75 accessions each, representing most of the species belonging to the genus Malus, by re-sequencing 27 gene-specific amplicons and by screening 237 Malus × domestica SNPs using the multiplex genotyping technology SNPlex™. Nucleotide diversity and insertion/deletion rates in M. × domestica were estimated as π = 0.0037 and 1/333 bp, respectively. The SNP frequency was estimated as 0.0194 (1 SNP/52 bp) while within a single apple cultivar an average of one SNP in every 455 bp was found. We also investigated transferability (T _SNP) of the heterozygous state of SNPs across the species M. × domestica and the genus Malus. Raw re-sequencing showed that 12–15% of M. × domestica SNPs are transferable to a second M. × domestica cultivar, however T _SNP rose to ∼41% with SNPs selected for high minor allele frequency. T _SNP of chosen SNPs averaged ∼27% in the two M. × domestica-related species, Malus sieversii and Malus sylvestris, but was much lower in more distantly related species. On the basis of T _SNP, simulations, and empirical results, we calculated that a close-design, multiplexed genotyping array with at least 2,000 SNPs is required for building a highly saturated linkage maps within any M. × domestica cross. The same array would gradually lose informativeness in increasingly phylogenetically distant Malus species.     

The skeletal organic matrix from Mediterranean coral Balanophyllia europaea influences calcium carbonate precipitation

Scleractinian coral skeletons are made mainly of calcium carbonate in the form of aragonite. The mineral deposition occurs in a biological confined environment, but it is still a theme of discussion to what extent the calcification occurs under biological or environmental control. Hence, the shape, size and organization of skeletal crystals from the cellular level through the colony architecture, were attributed to factors as diverse as mineral supersaturation levels and organic mediation of crystal growth. The skeleton contains an intra-skeletal organic matrix (OM) of which only the water soluble component was chemically and physically characterized. In this work that OM from the skeleton of the Balanophyllia europaea, a solitary scleractinian coral endemic to the Mediterranean Sea, is studied in vitro with the aim of understanding its role in the mineralization of calcium carbonate. Mineralization of calcium carbonate was conducted by overgrowth experiments on coral skeleton and in calcium chloride solutions containing different ratios of water soluble and/or insoluble OM and of magnesium ions. The precipitates were characterized by diffractometric, spectroscopic and microscopic techniques. The results showed that both soluble and insoluble OM components influence calcium carbonate precipitation and that the effect is enhanced by their co-presence. The role of magnesium ions is also affected by the presence of the OM components. Thus, in vitro, OM influences calcium carbonate crystal morphology, aggregation and polymorphism as a function of its composition and of the content of magnesium ions in the precipitation media. This research, although does not resolve the controversy between environmental or biological control on the deposition of calcium carbonate in corals, sheds a light on the role of OM, which appears mediated by the presence of magnesium ions.     

Functional magnetic resonance imaging reveals different neural substrates for the effects of orexin-1 and orexin-2 receptor antagonists

Orexins are neuro-modulatory peptides involved in the control of diverse physiological functions through interaction with two receptors, orexin-1 (OX1R) and orexin-2 (OX2R). Recent evidence in pre-clinical models points toward a putative dichotomic role of the two receptors, with OX2R predominantly involved in the regulation of the sleep/wake cycle and arousal, and the OX1R being more specifically involved in reward processing and motivated behaviour. However, the specific neural substrates underlying these distinct processes in the rat brain remain to be elucidated. Here we used functional magnetic resonance imaging (fMRI) in the rat to map the modulatory effect of selective OXR blockade on the functional response produced by D-amphetamine, a psychostimulant and arousing drug that stimulates orexigenic activity. OXR blockade was produced by GSK1059865 and JNJ1037049, two novel OX1R and OX2R antagonists with unprecedented selectivity at the counter receptor type. Both drugs inhibited the functional response to D-amphetamine albeit with distinct neuroanatomical patterns: GSK1059865 focally modulated functional responses in striatal terminals, whereas JNJ1037049 induced a widespread pattern of attenuation characterised by a prominent cortical involvement. At the same doses tested in the fMRI study, JNJ1037049 exhibited robust hypnotic properties, while GSK1059865 failed to display significant sleep-promoting effects, but significantly reduced drug-seeking behaviour in cocaine-induced conditioned place preference. Collectively, these findings highlight an essential contribution of the OX2R in modulating cortical activity and arousal, an effect that is consistent with the robust hypnotic effect exhibited by JNJ1037049. The subcortical and striatal pattern observed with GSK1059865 represent a possible neurofunctional correlate for the modulatory role of OX1R in controlling reward-processing and goal-oriented behaviours in the rat.     

Insulin in methionine and homocysteine kinetics in healthy humans: plasma vs. intracellular models

Methionine is a sulfur-containing amino acid that is reversibly converted into homocysteine. Homocysteine is an independent cardiovascular risk factor frequently associated with the insulin resistance syndrome. The effects of insulin on methionine and homocysteine kinetics in vivo are not known. Six middle-aged male volunteers were infused with L-[methyl-2H3,1-13C]methionine before (for 3 h) and after (for 3 additional hours) an euglycemic hyperinsulinemic (150 mU/l) clamp. Steady-state methionine and homocysteine kinetics were determined using either plasma (i.e., those of methionine) or intracellular (i.e., those of plasma homocysteine) enrichments. By use of plasma enrichments, insulin decreased methionine rate of appearance (Ra; both methyl- and carbon Ra) by 25% (P < 0.003 vs. basal) and methionine disposal into proteins by 50% (P < 0.0005), whereas it increased homocysteine clearance by approximately 70% (P < 0.025). With intracellular enrichments, insulin increased all kinetic rates, mainly because homocysteine enrichment decreased by approximately 40% (P < 0.001). In particular, transmethylation increased sixfold (P < 0.02), transsulfuration fourfold (P = 0.01), remethylation eightfold (P < 0.025), and clearance eightfold (P < 0.004). In summary, 1) physiological hyperinsulinemia stimulated homocysteine metabolic clearance irrespective of the model used; and 2) divergent changes in plasma methionine and homocysteine enrichments were observed after hyperinsulinemia, resulting in different changes in methionine and homocysteine kinetics. In conclusion, insulin increases homocysteine clearance in vivo and may thus prevent homocysteine accumulation in body fluids. Use of plasma homocysteine as a surrogate of intracellular methionine enrichment, after acute perturbations such as insulin infusion, needs to be critically reassessed.