Platelet signal transduction defect with G�� subunit dysfunction and diminished G��q in a patient with abnormal platelet responses

G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca2+ mobilization. Present studies demonstrated that platelet phospholipase A2 (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca2+ release induced by guanosine 5′-[γ-thio]triphosphate (GTP[γS]) was diminished in the patient’s platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of α-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A2 analog U46619. Binding of 35S-labeled GTP[γS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I2 analog) -induced rise in cAMP (mediated by G��s) and its inhibition (mediated by G��i) by thrombin in the patient’s platelet membranes were normal. Immunoblot analysis of G�� subunits in the patient’s platelet membranes showed a decrease in G��q (<50%) but not G��i, G��z, G��12, and G��13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein α-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of G��q in thrombin-induced responses.

RGS proteins reconstitute the rapid gating kinetics of G��γ-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of G��(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20–40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of “regulators of G protein signaling��� (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor → GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent “basal” GIRK current was suppressed by ≈40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of “slow” postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.

Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein α subunit G��13

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein α subunits were characterized in transfection systems. G��q, G��12, and G��13, but not G��i, activate SRF through RhoA. When G��q, α12, or α13 were coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, G��13, but not G��q or G��12, showed synergistic activation of SRF with GEF115. The synergy between G��13 and GEF115 depends on the N-terminal part of GEF115, and there was no synergistic effect between G��13 and another Rho-specific exchange factor Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibited G��13- and G��12-induced, but not GEF115 itself- or G��q-induced, SRF activation. The DH-domain-deletion mutant also suppressed thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of G��12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both G��12 and G��13. Thus, the inhibition of G��12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between G��13 and GEF115 indicates that GEF115 mediates G��13-induced activation of Rho and SRF.

The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families.

Thyrotropin is the primary hormone that, via one heptahelical receptor, regulates thyroid cell functions such as secretion, specific gene expression, and growth. In human thyroid, thyrotropin receptor activation leads to stimulation of the adenylyl cyclase and phospholipase C cascades. However, the G proteins involved in thyrotropin receptor action have been only partially defined. In membranes of human thyroid gland, we immunologically identified alpha subunits of the G proteins Gs short, Gs long, Gi1, Gi2, Gi3, G(o) (Go2 and another form of Go, presumably Go1), Gq, G11, G12, and G13. Activation of the thyrotropin (TSH) receptor by bovine TSH led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha subunits of all G proteins detected in thyroid membranes. This effect was receptor-dependent and not due to direct G protein stimulation because it was mimicked by TSH receptor-stimulating antibodies of patients suffering from Grave disease and was abolished by a receptor-blocking antiserum from a patient with autoimmune hypothyroidism. The TSH-induced activation of individual G proteins occurred with EC50 values of 5-50 milliunits/ml, indicating that the activated TSH receptor coupled with similar potency to different G proteins. When human thyroid slices were pretreated with pertussis toxin, the TSH receptor-mediated accumulation of cAMP increased by approximately 35% with TSH at 1 milliunits/ml, indicating that the TSH receptor coupled to Gs and G(i). Taken together, these findings show that, at least in human thyroid membranes, in which the protein is expressed at its physiological levels, the TSH receptor resembles a naturally occurring example of a general G protein-activating receptor.

Istorīi���a︡ politicheskoĭ literatury XIX-go stoli���e︡tīi���a︡ /

Mode of access: Internet.

God v sel skokhozi a i stvennom otnosheni i po otvi e tam, poluchennym ot khozi a ev /

Description based on: 1894 g.

O narodowości polskiej z punktu widzenia katolicyzmu i postępu /

Errata slip inserted.

Vospominani��i���a︡ zhizni /

Mode of access: Internet.

Investigating G protein signalling bias at the glucagon-like peptide-1 receptor in yeast

Background and Purpose The glucagon-like peptide 1 (GLP-1) receptor performs an important role in glycaemic control, stimulating the release of insulin. It is an attractive target for treating type 2 diabetes. Recently, several reports of adverse side effects following prolonged use of GLP-1 receptor therapies have emerged: most likely due to an incomplete understanding of signalling complexities. Experimental Approach We describe the expression of the GLP-1 receptor in a panel of modified yeast strains that couple receptor activation to cell growth via single G��/yeast chimeras. This assay enables the study of individual ligand-receptor G protein coupling preferences and the quantification of the effect of GLP-1 receptor ligands on G protein selectivity. Key Results The GLP-1 receptor functionally coupled to the chimeras representing the human G��s, G��i and G��q subunits. Calculation of the dissociation constant for a receptor antagonist, exendin-3 revealed no significant difference between the two systems. We obtained previously unobserved differences in G protein signalling bias for clinically relevant therapeutic agents, liraglutide and exenatide; the latter displaying significant bias for the G��i pathway. We extended the use of the system to investigate small-molecule allosteric compounds and the closely related glucagon receptor. Conclusions and Implications These results provide a better understanding of the molecular events involved in GLP-1 receptor pleiotropic signalling and establish the yeast platform as a robust tool to screen for more selective, efficacious compounds acting at this important class of receptors in the future. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

An investigation of the molecular pharmacology of G protein-coupled receptor 35

G protein-coupled receptors (GPCRs) are seven-pass integral membrane proteins that act as transducers of extracellular signals across the lipid bilayer. Their location and involvement in basic and pathological physiological processes has secured their role as key targets for pharmaceutical intervention. GPCRs are targeted by many of the best-selling drugs on the market and there are a substantial number of GPCRs that are yet to be characterised; these could offer interest for therapeutic targeting. GPR35 is one such receptor that, as a result of gene knockout and genome wide association studies, has attracted interest through its association with cardiovascular and gastrointestinal disease. Elucidation of the basic physiological function of GPR35 has, however, been difficult due a paucity of potent and selective ligands in addition to a lack of consensus on the endogenous ligand. Herein, a focussed drug discovery effort was carried out to identify agonists of GPR35. Various in vitro cellular assays were employed in conjunction with N- or C-terminally manipulated forms of the receptor to investigate GPR35’s signalling profile and to provide an assay format suitable for the characterisation of newly identified ligands. Although GPR35 associates with both G��i/o and G��13 families of small heterotrimeric G proteins, the G protein-independent β-arrestin-2 recruitment format was found to be the most suited to drug screening efforts. Small molecule compound screening, carried out in conjunction with the Medical Research Council Technology, identified compound 1 as the most potent ligand of human GPR35 reported at that time. However, the lower efficacy and potency of compound 1 at the rodent species orthologues of GPR35 prevented its use in in vivo studies. A subsequent effort, carried out with Novartis, focused on mast cell stabilisers as putative agonists of GPR35, revealed lodoxamide and bufrolin as highly potent agonists that activated human and rat GPR35 with equal potency. This finding offered–for the first time–the opportunity to employ the same GPR35 ligand between species at a similar concentration, an important factor to consider when translating rodent in vivo functional studies to those in man. Additionally, using molecular modelling and site directed mutagenesis studies, these newly identified compounds were used to aid characterisation of the ligand binding pockets of human and rat GPR35 to reveal the molecular basis of species selectivity at this receptor. In summary, this research effort presents GPR35 tool compounds that can now be used to dissect the basic biology of GPR35 and investigate its contribution to disease.

Ledningens ansvar för icke-finansiell information och dess rapportering i aktiebolag.

Syftet med avhandlingen är att redog��ra för ledningens ansvar för icke-finansiell information och dess rapportering i aktiebolag. Avhandlingen utg��r från finsk bolagsrätt och särskiljer mellan små, medelstora och stora samt mellan onoterade och noterade aktiebolag. Ledningen definieras som aktiebolagets styrelse och VD. Avhandlingen inleds med att redog��ra för vad icke-finansiell information är, vad det innebär för aktiebolag och vad som reglerar rapporteringen av informationen. Avhandlingen diskuterar bolagets intressen för att utreda vinstsyftet ur ett aktieägarperspektiv och som en del av ett större företagsansvar. Detta för att klarg��ra hur långt ledningen kan g�� i sitt arbete med icke-finansiell information och hållbarhet. Avhandlingen avslutas med en redog��relse för ledningens skadeståndsrättsliga och straffrättsliga ansvar. Rapportering av icke-finansiell information har tre tydliga problemområden. För det första skapar den oklara definitionen förvirring. Informationen är företagsspecifik vilket försvårar en granskning av kvaliteten. För det andra så finns det olika tolkningar av vad som är bolagets intressen och hur verksamhetens syfte ska tolkas. Ett kortsiktigt vinstsyfte prioriterar ett aktieägarperspektiv medan en långsiktig värdeökning tenderar att se till flera sidogrupper som mottagare av icke-finansiell information. För det tredje så är gränsen mellan frivillig och obligatorisk rapportering diffus. Dessa tre problemområden innebär att ledningens ansvar är tolkningsbart beroende på den aktuella situationen och det specifika aktiebolaget. Regleringen måste g��ras tydligare och de olika begreppen inom icke-finansiell information definieras. Ledningen har en rättighet att arbeta för hållbarhet och rapportera icke-finansiell information, dock så har de ingen uttrycklig skyldighet att g��ra det. Sanktionsmöjligheterna vid bristfällig eller felaktig informationsutgivning är i praktiken svaga.

Receptor activity-modifying protein-directed G protein signaling specificity for the calcitonin gene-related peptide family of receptors

The calcitonin gene-related peptide (CGRP) family of G protein- coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in aG��s-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/G�� subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both G��s and G��q but also identify a G��i component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMPdependent signaling bias among the G��s, G��i, and G��q/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology.

Reconstructing the event stratigraphy from the complex structural - stratigraphic architecture of an Archaean volcanic – intrusive – sedimentary succession : the Boorara Domain, Eastern Goldfields Superterrane, Western Australia

Two main deformational phases are recognised in the Archaean Boorara Domain of the Kalgoorlie Terrane, Eastern Goldfields Superterrane, Yilgarn Craton, Western Australia, primarily involving southover- north thrust faulting that repeated and thickened the stratigraphy, followed by east northeast – west-southwest shortening that resulted in macroscale folding of the greenstone lithologies. The domain preserves mid-greenschist facies metamorphic grade, with an increase to lower amphibolite metamorphic grade towards the north of the region. As a result of the deformation and metamorphism, individual stratigraphic horizons are difficult to trace continuously throughout the entire domain. Volcanological and sedimentological textures and structures, primary lithological contacts, petrography and geochemistry have been used to correlate lithofacies between faultbounded structural blocks. The correlated stratigraphic sequence for the Boorara Domain comprises quartzo-feldspathic turbidite packages, overlain by high-Mg tholeiitic basalt (lower basalt), coherent and clastic dacite facies, intrusive and extrusive komatiite units, an overlying komatiitic basalt unit (upper basalt), and at the stratigraphic top of the sequence, volcaniclastic quartz-rich turbidites. Reconstruction of the stratigraphy and consideration of emplacement dynamics has allowed reconstruction of the emplacement history and setting of the preserved sequence. This involves a felsic, mafic and ultramafic magmatic system emplaced as high-level intrusions, with localised emergent volcanic centres, into a submarine basin in which active sedimentation was occurring.

An integrated approach to supporting land-use decisions in site redevelopment for urban renewal in Hong Kong

Urban renewal is a significant issue in developed urban areas, with a particular problem for urban planners being redevelopment of land to meet demand whilst ensuring compatibility with existing land use. This paper presents a geographic information systems (GIS)-based decision support tool (called LUDS) to quantitatively assess land-use suitability for site redevelopment in urban renewal areas. This consists of a model for the suitability analysis and an affiliated land-information database for residential, commercial, industrial, G/I/C (government/institution/community) and open space land uses. Development has occurred with support from interviews with industry experts, focus group meetings and an experimental trial, combined with several advanced techniques and tools, including GIS data processing and spatial analysis, multi-criterion analysis, as well as the AHP method for constructing the model and database. As demonstrated in the trial, LUDS assists planners in making land-use decisions and supports the planning process in assessing urban land-use suitability for site redevelopment. Moreover, it facilitates public consultation (participatory planning) by providing stakeholders with an explicit understanding of planners' views.

A comparative analysis of the integration of SOA elements in widely-used enterprise architecture frameworks

In recent years, enterprise architecture (EA) has captured increasing interest as a means to systematically consolidate and manage various enterprise artefacts in order to provide holistic decision support for business/IT alignment and business/IT landscapes management. To provide a holistic perspective on the enterprise over time, EA frameworks need to co-evolve with the changes in the enterprise and its IT over time. In this paper we focus on the emergence of Service-Oriented Architecture (SOA). There is a need to integrate SOA with EA to keep EA relevant and to use EA products to help drive successful SOA. This paper investigates and compares the integration of SOA elements in five widely used EA frameworks: Archimate, The Open Group Architecture Framework (TOGAF), Federal Enterprise Architecture Framework (FEAF), Department of Defence Architecture Framework (DoDAF) and the Ministry of Defence Architecture Framework (MODAF). It identifies what SOA elements are considered and their relative position in the overall structure. The results show that services and related elements are far from being well-integrated constructs in current EA frameworks and that the different EA frameworks integrated SOA elements in substantially different ways. Our results can support the academic EA and SOA communities with a closer and more consistent integration of EA and SOA and support practitioners in identifying an EA framework that provides the SOA support that matches their requirements.