Structural basis for allosteric regulation of GPCRs by sodium ions

Pharmacological responses of G protein-coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A(2A) adenosine receptor by replacing its third intracellular loop with apocytochrome b(562)RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp(2.50). Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.     

Enzyme-linked immunosorbent assay (ELISA) measurement of vitellogenin in plasma and liver histopathology in barfin plaice <Emphasis Type="Italic">Liopsetta pinnifasciata</Emphasis> from Amursky Bay,Sea of Japan

Vitellogenin (Vg) of the barfin plaice Liopsetta pinnifasciata was isolated and purified. In native polyacrylamide gel electrophoresis, Vg appeared as one band. After being subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE), Vg fraction produced several polypeptides with molecular masses of 180, 98, 70, 52, 41 and 37 kDa. MALDI–TOF mass spectrometry (MS) of the 180- and 98-kDa Vg polypeptides from the SDS–PAGE gel and de novo sequencing of their four peptide fragments based on MS/MS analysis confirmed that the purified proteins were vitellogenins, which shared high similarity with the Vgs of the barfin flounder Verasper moseri and Atlantic halibut Hippoglossus hippoglossus. The most part of the predicted sequences obtained from the L. pinnifasciata 180-kDa polypeptide has previously been found in the V. moseri vitellogenin type B, the sequences obtained from the 98-kDa polypeptide were found in V. moseri vitellogenin type A, so these findings allow us to propose that L. pinnifasciata has at least two different forms of Vg. Rabbit polyclonal antibodies against Vg were produced, and a quantitative enzyme-linked immunosorbent assay was developed. The concentration of Vg in barfin plaice from the moderately contaminated area of Amursky Bay in the Sea of Japan was detected based on the maturity stage of their gonads. In November 2008, the Vg concentration in the plasma of females with advanced oogenesis varied from 5.295 to 28.367 mg/ml (mean 16.38 ± 6.73 mg/ml, CV = 41.1%); in the plasma of males, the concentration ranged from non-detectable to 0.957 mg/ml (0.29 ± 0.42 mg/ml, CV = 127.9%). In October 2009, the Vg concentration in female plasma was lower than in November 2008 (2.21–13.87 mg/ml). High individual variability of plasma Vg was characteristic for maturing males (CV = 200.3%) and immature females (CV = 255.5%), and there was no significant difference between plasma Vg concentrations in males captured in November 2008 and October 2009 or in maturing males and immature females. Vacuolisation of hepatocytes was more typical for males with low plasma Vg concentrations and females with high plasma Vg concentrations. Necrosis and pyknosis of hepatocyte nuclei were more frequent in males with high Vg concentrations and in females with low plasma Vg concentrations.     

SSR fingerprinting of a German <Emphasis Type="Italic">Rubus</Emphasis> collection and pedigree based evaluation on trueness-to-type

Eighty-two genotypes of Rubus available in germplasm collections, nurseries and home gardens were collected and evaluated using a set of 16 simple sequence repeat (SSR) markers to estimate the level of genetic diversity and relatedness of the germplasm and for testing them on trueness-to-type. Each of the 16 SSRs was successful in amplifying alleles from most genotypes. Fifteen of the markers produced polymorphic bands, whereas marker RhM023 was monomorphic. The polymorphic information content among genotypes varied from 0.056 to 0.83 with an average of 0.348. A neighbor-joining analysis allocated the genotypes to four major clusters containing 11, 24, 39 and eight genotypes, respectively. Cluster I consists of floricane-fruiting cultivars originating from the Scottish and/or British breeding programs or cultivars which have those cultivars in their pedigree. Cluster II included cultivars that have ‘Autumn Bliss’ or ‘Tulameen’ in their pedigree. Cluster III consists of summer-bearing raspberry cultivars, some primocane-fruiting cultivars, and a few intermediate summer-fall-bearing types. Cluster IV consists of the blackberry ‘Navaho’ (R. fruticosus L.), the interspecific hybrid ‘Dorman Red’ and a few other raspberry varieties. A number of yellow fruited varieties was dispersed on three different clusters suggesting a convergent evolution of this trait. The pedigree of several genotypes could be confirmed using a Pedimap based approach, whereas other cultivars were found to be genetically identical. The results disclose the alarming narrow genetic base of Rubus resources in Germany. Broadening of this base is urgently needed.     

Thermonuclear supernovae: simulations of the deflagration stage and their implications

Large-scale, three-dimensional numerical simulations of the deflagration stage of a thermonuclear supernova explosion show the formation and evolution of a highly convoluted turbulent flame in the gravitational field of an expanding carbon-oxygen white dwarf. The flame dynamics are dominated by the gravity-induced Rayleigh-Taylor instability that controls the burning rate. The thermonuclear deflagration releases enough energy to produce a healthy explosion. The turbulent flame, however, leaves large amounts of unburned and partially burned material near the star center, whereas observations that imply these materials are present only in outer layers. This disagreement could be resolved if the deflagration triggers a detonation.     

Thinning and flow of Tibetan crust constrained by seismic anisotropy

Intermediate-period Rayleigh and Love waves propagating across Tibet indicate marked radial anisotropy within the middle-to-lower crust, consistent with a thinning of the middle crust by about 30%. The anisotropy is largest in the western part of the plateau, where moment tensors of earthquakes indicate active crustal thinning. The preferred orientation of mica crystals resulting from the crustal thinning can account for the observed anisotropy. The middle-to-lower crust of Tibet appears to have thinned more than the upper crust, consistent with deformation of a mechanically weak layer that flows as if confined to a channel.     

The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist

The adenosine class of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A2A adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.