The adenosine A2A receptor interacts with the actin-binding protein alpha-actinin

Recently, evidence has emerged that heptaspanning membrane or G protein-coupled receptors may be linked to intracellular proteins identified as regulators of receptor anchoring and signaling. Using a yeast two-hybrid screen, we identified alpha-actinin, a major F-actin-cross-linking protein, as a binding partner for the C-terminal domain of the adenosine A2A receptor (A2AR). Colocalization, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between A2AR and alpha-actinin in transfected HEK-293 cells and also in rat striatal tissue. A2AR activation by agonist induced the internalization of the receptor by a process that involved rapid beta-arrestin translocation from the cytoplasm to the cell surface. In the subsequent receptor traffic from the cell surface, the role of actin organization was shown to be crucial in transiently transfected HEK-293 cells, as actin depolymerization by cytochalasin D prevented its agonist-induced internalization. A2ADeltaCTR, a mutant version of A2AR that lacks the C-terminal domain and does not interact with alpha-actinin, was not able to internalize when activated by agonist. Interestingly, A2ADeltaCTR did not show aggregation or clustering after agonist stimulation, a process readily occurring with the wild-type receptor. These findings suggest an alpha-actinin-dependent association between the actin cytoskeleton and A2AR trafficking.     

七姊妹山自然保护区蕨类植物区系地理及资源开发研究

运用植物区系地理分析方法对七姊妹山自然保护区的蕨类植物区系特征进行研究。科的地理区系分析表明,热带、亚热带成分高达58.3%,占主导地位。属的地理区系分析也表明了类似的趋势。种的地理区系分析表明,除了热带、亚热带成分占有较高比例外,温带分布的种也具有较高的比例,占非世界广布种的1.2%。特别是世界温带分布的种占较高比例,达24.2%,这反映了温带成分的重要地位。七姊妹山蕨类植物资源丰富,可区分为药用、观赏、食用、指示类植物等几大类。各种蕨类植物资源显示了广阔的利用前景。     

Binding of Glutathione to Enterovirus Capsids Is Essential for Virion Morphogenesis

Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis.     

Determination of the characteristics, properties and homogeneity of rat brain microsomes. Binding of lactate dehydrogenase, malate dehydrogenase and 5' nucleotidase to microsomal membranes

Quantitatively, the amount of microsomes obtained using dimethyl sulfoxide is larger than that obtained from sucrose solutions (Centelles, Franco & Bozal (1986) Biol. Chem. Hoppe Seyler 367, 461-475). In this paper it is demonstrated that from a qualitative point of view they appeared to be indistinguishable with respect to molecular characteristics. Thus, both types of microsomes had the same behaviour in experiments of isopicnic ultracentrifugation with Percoll, isoelectric focusing and gel permeation. In these experiments, the 5'-nucleotidase, lactate dehydrogenase and malate dehydrogenase activities bound to the microsomal fraction were also studied. Lactate and malate dehydrogenase activities were always found in free and membrane-bound form. In contrast, 5'-nucleotidase activity was always encountered bound to microsomal membranes.     

An in vitro system for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation

We have developed an assay based on rice embryogenic callus for rapid functional characterization of metabolic genes. We validated the assay using a selection of well‐characterized genes with known functions in the carotenoid biosynthesis pathway, allowing rapid visual screening of callus phenotypes based on tissue color. We then used the system to identify the functions of two uncharacterized genes: a chemically synthesized β–carotene ketolase gene optimized for maize codon usage, and a wild‐type Arabidopsis thaliana ortholog of the cauliflower Orange gene. In contrast to previous reports (Lopez, A.B., Van Eck, J., Conlin, B.J., Paolillo, D.J., O'Neill, J. and Li, L. ( 2008 ) J. Exp. Bot. 59, 213–223; Lu, S., Van Eck, J., Zhou, X., Lopez, A.B., O'Halloran, D.M., Cosman, K.M., Conlin, B.J., Paolillo, D.J., Garvin, D.F., Vrebalov, J., Kochian, L.V., Küpper, H., Earle, E.D., Cao, J. and Li, L. ( 2006 ) Plant Cell 18, 3594–3605), we found that the wild‐type Orange allele was sufficient to induce chromoplast differentiation. We also found that chromoplast differentiation was induced by increasing the availability of precursors and thus driving flux through the pathway, even in the absence of Orange. Remarkably, we found that diverse endosperm‐specific promoters were highly active in rice callus despite their restricted activity in mature plants. Our callus system provides a unique opportunity to predict the effect of metabolic engineering in complex pathways, and provides a starting point for quantitative modeling and the rational design of engineering strategies using synthetic biology. We discuss the impact of our data on analysis and engineering of the carotenoid biosynthesis pathway.     

Impact of Virgin Olive Oil and Phenol-Enriched Virgin Olive Oils on the HDL Proteome in Hypercholesterolemic Subjects: A Double Blind,Randomized, Controlled,Cross-Over Clinical Trial (VOHF Study)

The effects of olive oil phenolic compounds (PCs) on HDL proteome, with respect to new aspects of cardioprotective properties, are still unknown. The aim of this study was to assess the impact on the HDL protein cargo of the intake of virgin olive oil (VOO) and two functional VOOs, enriched with their own PCs (FVOO) or complemented with thyme PCs (FVOOT), in hypercholesterolemic subjects. Eligible volunteers were recruited from the IMIM-Hospital del Mar Medical Research Institute (Spain) from April 2012 to September 2012. Thirty-three hypercholesterolemic participants (total cholesterol >200mg/dL; 19 men and 14 women; aged 35 to 80 years) were randomized in the double-blind, controlled, cross-over VOHF clinical trial. The subjects received for 3 weeks 25 mL/day of: VOO, FVOO, or FVOOT. Using a quantitative proteomics approach, 127 HDL-associated proteins were identified. Among these, 15 were commonly differently expressed after the three VOO interventions compared to baseline, with specific changes observed for each intervention. The 15 common proteins were mainly involved in the following pathways: LXR/RXR activation, acute phase response, and atherosclerosis. The three VOOs were well tolerated by all participants. Consumption of VOO, or phenol-enriched VOOs, has an impact on the HDL proteome in a cardioprotective mode by up-regulating proteins related to cholesterol homeostasis, protection against oxidation and blood coagulation while down-regulating proteins implicated in acute-phase response, lipid transport, and immune response. The common observed protein expression modifications after the three VOOs indicate a major matrix effect.

Trial Registration

International Standard Randomized Controlled Trials ISRCTN77500181.     

Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB1 and Serotonin 5-HT2A Receptors

Activation of cannabinoid CB1 receptors (CB₁R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT_2A receptors (5-HT_2AR) revealed a remarkable 5-HT_2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT_2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB₁R and 5-HT_2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT_2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB₁R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB₁R and 5-HT_2AR mediating cognitive impairment. CB₁R-5-HT_2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties.