128 research outputs found

    Gi- and Gs-coupled GPCRs show different modes of G-protein binding.

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    More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand-receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily. Nevertheless, GPCRs have selectivity for distinct G-protein family members, but the mechanism of selectivity remains elusive. Structures of GPCRs in complex with the stimulatory G protein, Gs, and an accessory nanobody to stabilize the complex have been reported, providing information on the intermolecular interactions. However, to reveal the structural selectivity filters, it will be necessary to determine GPCR-G protein structures involving other G-protein subtypes. In addition, it is important to obtain structures in the absence of a nanobody that may influence the structure. Here, we present a model for a rhodopsin-G protein complex derived from intermolecular distance constraints between the activated receptor and the inhibitory G protein, Gi, using electron paramagnetic resonance spectroscopy and spin-labeling methodologies. Molecular dynamics simulations demonstrated the overall stability of the modeled complex. In the rhodopsin-Gi complex, Gi engages rhodopsin in a manner distinct from previous GPCR-Gs structures, providing insight into specificity determinants

    Equine lamellar energy metabolism studied using tissue microdialysis

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    Failure of lamellar energy metabolism may contribute to the pathophysiology of equine laminitis. Tissue microdialysis has the potential to dynamically monitor lamellar energy balance over time. The objectives of this study were to develop a minimally invasive lamellar microdialysis technique and use it to measure normal lamellar energy metabolite concentrations over 24h. Microdialysis probes were placed (through the white line) into either the lamellar dermis (LAM) (n=6) or the sublamellar dermis (SUBLAM) (n=6) and perfused continuously over a 24h study period. Probes were placed in the skin dermis (SKIN) for simultaneous comparison to LAM (n=6). Samples were collected every 2h and analysed for glucose, lactate, pyruvate, urea and glycerol concentrations. LAM was further compared with SUBLAM by simultaneous placement and sampling in four feet from two horses over 4h. Horses were monitored for lameness, and either clinically evaluated for 1month after probe removal (n=4) or subjected to histological evaluation of the probe site (n=10).There were no deleterious clinical effects of probe placement and the histological response was mild. Sample fluid recovery and metabolite concentrations were stable for 24h. Glucose was lower (and lactate:glucose ratio higher) in LAM compared with SUBLAM and SKIN (

    Intraosseous infusion of the distal phalanx compared to systemic intravenous infusion for marimastat delivery to equine lamellar tissue

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    No validated laminitis drug therapy exists, yet pharmaceutical agents with potential for laminitis prevention have been identified. Many of these are impractical for systemic administration but may be effective if administered locally. This study compared intraosseous infusion of the distal phalanx (IOIDP) with systemic intravenous constant rate infusion (CRI) to determine which was more effective for lamellar marimastat delivery. Ultrafiltration probes were placed in both forefeet of five horses to collect lamellar interstitial fluid as lamellar ultrafiltrate (LUF). Marimastat solution (3.5 mg/mL) containing lidocaine (20 mg/mL) was infused by IOIDP at 0.15 mL/min for 12 h. After a 12 h wash-out, marimastat (3.5 mg/mL) and lidocaine were infused by constant rate infusion (CRI) at 0.15 mL/min for 12 h. LUF, plasma and lamellar tissue marimastat concentrations were quantified using UPLC-MS. Zymography was used to establish the inhibitory concentrations of marimastat for equine lamellar matrix metalloproteinases (MMPs). Data were analysed non-parametrically. There was no difference between the steady-state marimastat concentration in lamellar ultrafiltrate (LUF[M]) during IOIDP (139[88-497] ng/mL) and CRI (136[93-157] ng/mL). During IOIDP, there was no difference between marimastat concentrations in the treated foot (139[88-497] ng/mL), the untreated foot (91[63-154] ng/mL) and plasma (101[93-118] ng/mL). LUF[M] after IOIDP and CRI were >IC50 of lamellar MMP-2 and 9, but below the concentration considered necessary for in vivo laminitis prevention. Lamellar drug delivery during IOIDP was inconsistent and did not achieve higher lamellar marimastat concentrations than CRI. Modification or refinement of the IOIDP technique is necessary if it is to be consistently effective

    Beyond the 'Grid-Lock' in Electricity Interconnectors: The Case of Germany and Poland

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    The common European electricity market requires both market integration and transmission grid expansion, including trans-border interconnectors. Although the benefits of increased interconnectivity are widely acknowledged, expansion of interconnectors is often very slow. This paper gathers insights on the reasons behind this grid-lock drawing on the study of the German-Polish border. Although two interconnectors already exist, the trade is blocked by unplanned electricity loop flows. A third interconnector has been discussed for years, but saw little progress in spite of declarations of support on both sides. Drawing on the existing literature on the topic of grid expansion we identify four hypotheses for the grid-lock: inadequate financing; diverging interests; governance and administration problems; and different actors' motivations, trust and security perceptions. We evaluate them using the empirical material gathered through document analysis and stakeholder interviews conducted in Germany and Poland. None of the hypotheses on its own can explain the gridlock. However, while financing has not been a major obstacle, divergent interests had an impact on the project delay, administrative and governance problems are a great hindrance on the technical level, while motivations influence interstate political relations and policy shaping. EU support and closer bilateral cooperation provide opportunities to address these challenges

    Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

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    G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

    Cardiac and vascular structure and function parameters do not improve with alternate nightly home hemodialysis: An interventional cohort study

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    Background: Nightly extended hours hemodialysis may improve left ventricular hypertrophy and function and endothelial function but presents problems of sustainability and increased cost. The effect of alternate nightly home hemodialysis (NHD) on cardiovascular structure and function is not known

    Chemogenomic Analysis of G-Protein Coupled Receptors and Their Ligands Deciphers Locks and Keys Governing Diverse Aspects of Signalling

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    Understanding the molecular mechanism of signalling in the important super-family of G-protein-coupled receptors (GPCRs) is causally related to questions of how and where these receptors can be activated or inhibited. In this context, it is of great interest to unravel the common molecular features of GPCRs as well as those related to an active or inactive state or to subtype specific G-protein coupling. In our underlying chemogenomics study, we analyse for the first time the statistical link between the properties of G-protein-coupled receptors and GPCR ligands. The technique of mutual information (MI) is able to reveal statistical inter-dependence between variations in amino acid residues on the one hand and variations in ligand molecular descriptors on the other. Although this MI analysis uses novel information that differs from the results of known site-directed mutagenesis studies or published GPCR crystal structures, the method is capable of identifying the well-known common ligand binding region of GPCRs between the upper part of the seven transmembrane helices and the second extracellular loop. The analysis shows amino acid positions that are sensitive to either stimulating (agonistic) or inhibitory (antagonistic) ligand effects or both. It appears that amino acid positions for antagonistic and agonistic effects are both concentrated around the extracellular region, but selective agonistic effects are cumulated between transmembrane helices (TMHs) 2, 3, and ECL2, while selective residues for antagonistic effects are located at the top of helices 5 and 6. Above all, the MI analysis provides detailed indications about amino acids located in the transmembrane region of these receptors that determine G-protein signalling pathway preferences
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