Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets.

Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinase-dependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cgamma2 are early events in collagen-induced activation. We recently proposed that collagen-signaling in platelets involves a receptor or a receptor-associated protein containing an immunoreceptor tyrosine-based activation motif (ITAM) enabling interaction with Syk. In this report we show that collagen stimulation of platelets causes rapid tyrosine phosphorylation of the ITAM containing Fc receptor gamma-chain and that this is precipitated by the tandem Src homology 2 (SH2) domains of Syk expressed as a fusion protein. In addition we demonstrate an association between the Fc receptor gamma-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor gamma-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin alpha2beta1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, FcgammaRIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor gamma-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin alpha2beta1 and involves phosphorylation of the Fc receptor gamma-chain, its association with Syk and subsequent phosphorylation of phospholipase Cgamma2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.

Characterization of Grb2-binding proteins in human platelets activated by Fc gamma RIIA cross-linking.

Glutathione-S-transferase (GST)-Grb2 fusion proteins have been used to identify the potential role of Grb2-binding proteins in platelet activation by the platelet low-affinity IgG receptor, Fc gamma RIIA. Two tyrosine phosphoproteins of 38 and 63 kD bind to the SH2 domain of Grb2 following Fc gamma RIIA stimulation of platelets. Both are located in the particulate fraction following platelet activation and are also able to bind to a GST-construct containing the SH2 and SH3 domains of phospholipase C gamma 1. p38 also forms a complex with the tyrosine kinase csk in stimulated cells and is a substrate for the kinase. The SH3 domains of Grb2 form a stable complex with SOS1 and two proteins of 75 kD and 120 kD, which undergo tyrosine phosphorylation in Fc gamma RIIA stimulated cells. The 75-kD protein is recognized by antibodies to SLP-76, which has recently been isolated from T cells and sequenced. Tyrosine phosphorylation of p38 and p63 is also observed in platelets stimulated by the tyrosine kinase-linked receptor agonist collagen and by the G protein-coupled receptor agonist thrombin, although phosphorylation of SLP-76 is only observed in collagen-stimulated platelets. p38 and p63 may provide a docking site for Grb2, thereby linking Grb2 SH3-binding proteins SOS1, SLP-76, and p120 to downstream signalling events.

Affinity purification of 61- and 65-kDa rat brain corticotropin-releasing factor receptors and receptor-associated G proteins.

Corticotropin-releasing factor (CRF) has been shown to have a central role in physiological adaptation to stress. It is recognized for stimulating the release of adrenocorticotropin from the anterior pituitary gland, and has more recently been implicated as a regulator of autonomic and immunological responses to stress. Much confusion has surrounded the characterization of CRF receptors, with proteins of varying molecular weights having been identified but never purified and characterized. Recently, two CRF receptors have been cloned from brain and pituitary gland, but evidence from in-situ hybridization studies suggests that further CRF receptor types exist. We therefore developed two techniques which enable the isolation of CRF receptors from whole rat brain. The use of a solid-phase CRF analogue affinity column and elution using a competing ligand resulted in the purification of a single protein of 61 kDa. A second technique was devised which allowed the co-isolation of associated signalling proteins and the identification of CRF bound species following purification. CRF was covalently cross-linked to receptors and the complex purified using antibodies specific for the ligand. This enabled the purification of a CRF receptor of approximately 65 kDa and associated alpha and beta gamma G protein subunits. This study demonstrates the successful isolation of CRF receptors which are of different molecular weights to those previously observed from affinity cross-linking studies or predicted from cloned genes. In addition, we confirm the involvement of G proteins in CRF stimulated cell signalling by demonstrating their association with purified CRF receptor.

Elevated levels of corticotrophin-releasing factor binding protein in the blood of patients suffering from arthritis and septicaemia and the presence of novel ligands in synovial fluid.

In view of the reported inflammatory effects of corticotrophin-releasing factor (CRF) and the associated regulatory elements in the gene of its binding protein (BP), we postulate that both BP as well as novel BP-ligands other than CRF may be involved in inflammatory disease. We have investigated BP in the blood of patients with arthritis and septicaemia and have attempted to identify CRF and other BP-ligands in synovial fluid. The BP was found to be significantly elevated in the blood of patients with rheumatoid arthritis and septicaemia. There was less BP-ligand and CRF in synovial fluid from patients with rheumatoid arthritis that from those with osteo- or psoriatic arthritis. There was at least 10-fold more BP-ligand than CRF in the fluid of all three groups of patients. A small amount of immunoreactive human (h)CRF, eluting in the expected position of CRF-41, was detected after high-pressure liquid chromatography of arthritic synovial fluid; however, the bulk of material with BP-ligand binding activity eluted earlier, suggesting that synovial fluid contained novel peptides that interacted with the BP. These results would suggest that the BP and its ligands could play an endocrine immunomodulatory role in inflammatory disease.

Corticotropin-releasing factor binding protein dimerizes after association with ligand.

We have suggested recently that the fall in plasma CRF-binding protein (BP) during the last few weeks of pregnancy is a direct effect of association with its ligand because of the rapid decrease in plasma BP concentration seen in normal males reaching a nadir some 15 min after a bolus injection of synthetic CRF. In the present study, we have investigated the physicochemical properties of both natural and recombinant BP by gel filtration under physiological conditions and have shown that association of human CRF to this BP results in an increase in molecular weight consistent with the formation of a dimer form of the BP ligand complex. The dimer is more stable when the interaction occurs in the presence of serum or if a peptide with a higher affinity for the BP is substituted as ligand. Experimental evidence would also suggest that the dimer BP has a higher affinity for ligand than the monomeric form. We suggest that this dimerization occurs in vivo when CRF is released into the bloodstream and provides the trigger that causes the uptake of the complex at specific receptor sites.

Comment on “Multiyear prediction of monthly mean Atlantic meridional overturning circulation at 26.5°N”

Matei et al. (Reports, 6 January 2012, p. 76) claim to show skillful multiyear predictions of the Atlantic Meridional Overturning Circulation (AMOC). However, these claims are not justified, primarily because the predictions of AMOC transport do not outperform simple reference forecasts based on climatological annual cycles. Accordingly, there is no justification for the “confident” prediction of a stable AMOC through 2014.

‘New World and Old Europe: American Screen Images of Britain and France‘

Contrary to the dearly held belief by Britons that among the nations of the world, they are the favourites of the Americans, Holliwood movies show that even today, judging by the accents of "baddies", the English incarnate the arch-enemy. French villains come a close second. Britain and France are the reactionary, corrupt "old Europe" from whom the Americans tried to cut away ever since 1775, and it is actually the Central-East European countries who as "new Europe" enjoy greater popularity as bearers of hope.

Playing with history: negotiating subjectivity in contemporary lens based work

Contemporary artists exploring Jewish identity in the UK are caught between two exclusions, broadly speaking: an art community that that sees itself as ‘post –identity’ and a ‘black’ art scene that revolves around the organizations that emerged out of the Identity debates of the 1980s and 1990s, namely Iniva, Third Text, Autograph. These organizations and those debates, don’t usually include Jewish identity within their remit as Jewish artists are considered to be well represented in the British art scene and, in any case, white. Out of these assumptions, questions arise in relation to the position of Jews in Britain and what is at stake for an artist in exploring Jewish Identity in their work. There is considerable scholarship, relatively speaking on art and Jewish Identity in the US (such as Lisa Bloom; Norman Kleeblatt; Catherine Sousslouf), which inform the debates on visual culture and Jews. In this chapter, I will be drawing out some of the distinctions between the US and the UK debates within my analysis, building on my own writing over the last ten years as well as the work of Juliet Steyn, Jon Stratton and Griselda Pollock. In short, this chapter aims to explore the problematic of what Jewish Identity can offer the viewer as art; what place such art inhabits within a wider artistic context and how, if at all, it is received. There is a predominance of lens based work that explores Identity arising out of the provenance of feminist practices and the politics of documentary that will be important in the framing of the work. I do not aim to consider what constitutes a Jewish artist, that has been done elsewhere and is an inadequate and somewhat spurious conversation . I will also not be focusing on artists whose intention is to celebrate an unproblematised Jewishness (however that is constituted in any given work). Recent artworks and scholarship has in any case rendered the trumpeting of attachment to any singular identity anachronistic at best. I will focus on artists working in the UK who incorporate questions of Jewishness into a larger visual enquiry that build on Judith Butler’s notion of identity as process or performative as well as the more recent debates and artwork that consider the intersectionality of identifications that co-constitute provisional identities (Jones, Modood, Sara Ahmed, Braidotti/Nikki S Lee, Glenn Ligon). The case studies to think through these questions of identity, will be artworks by Susan Hiller, Doug Fishbone and Suzanne Triester. In thinking through works by these artists, I will also serve to contextualise them, situating them briefly within the history of the landmark exhibition in the UK, Rubies and Rebels and the work of Ruth Novaczek, Lily Markewitz, Oreet Ashery and myself.

Insights into dietary flavonoids as molecular templates for the design of anti-platelet drugs

Flavonoids are low-molecular weight, aromatic compounds derived from fruits, vegetables, and other plant components. The consumption of these phytochemicals has been reported to be associated with reduced cardiovascular disease (CVD) risk, attributed to their anti-inflammatory, anti-proliferative, and anti-thrombotic actions. Flavonoids exert these effects by a number of mechanisms which include attenuation of kinase activity mediated at the cell-receptor level and/or within cells, and are characterized as broad-spectrum kinase inhibitors. Therefore, flavonoid therapy for CVD is potentially complex; the use of these compounds as molecular templates for the design of selective and potent small-molecule inhibitors may be a simpler approach to treat this condition. Flavonoids as templates for drug design are, however, poorly exploited despite the development of analogues based on the flavonol, isoflavonone, and isoflavanone subgroups. Further exploitation of this family of compounds is warranted due to a structural diversity that presents great scope for creating novel kinase inhibitors. The use of computational methodologies to define the flavonoid pharmacophore together with biological investigations of their effects on kinase activity, in appropriate cellular systems, is the current approach to characterize key structural features that will inform drug design. This focussed review highlights the potential of flavonoids to guide the design of clinically safer, more selective, and potent small-molecule inhibitors of cell signalling, applicable to anti-platelet therapy.

Food and Health Forum meeting - nutritional approaches to cardiovascular health: workshop report

This report summarises the proceedings of a meeting held by the Food and Health Forum at the Royal Society of Medicine, London, on 12 October 2011. The objective of the meeting was to highlight nutritional strategies targeted at cardiovascular health. This included a review of the effects of various foods, nutrients and ingredients on maintenance of healthy cholesterol levels, endothelial function and blood pressure

Have aerosols caused the observed Atlantic multidecadal variability?

Identifying the prime drivers of the twentieth-century multidecadal variability in the Atlantic Ocean is crucial for predicting how the Atlantic will evolve in the coming decades and the resulting broad impacts on weather and precipitation patterns around the globe. Recently, Booth et al. showed that the Hadley Centre Global Environmental Model, version 2, Earth system configuration (HadGEM2-ES) closely reproduces the observed multidecadal variations of area-averaged North Atlantic sea surface temperature in the twentieth century. The multidecadal variations simulated in HadGEM2-ES are primarily driven by aerosol indirect effects that modify net surface shortwave radiation. On the basis of these results, Booth et al. concluded that aerosols are a prime driver of twentieth-century North Atlantic climate variability. However, here it is shown that there are major discrepancies between the HadGEM2-ES simulations and observations in the North Atlantic upper-ocean heat content, in the spatial pattern of multidecadal SST changes within and outside the North Atlantic, and in the subpolar North Atlantic sea surface salinity. These discrepancies may be strongly influenced by, and indeed in large part caused by, aerosol effects. It is also shown that the aerosol effects simulated in HadGEM2-ES cannot account for the observed anticorrelation between detrended multidecadal surface and subsurface temperature variations in the tropical North Atlantic. These discrepancies cast considerable doubt on the claim that aerosol forcing drives the bulk of this multidecadal variability.

Genome-wide association mapping to candidate polymorphism resolution in the unsequenced barley genome

Although commonplace in human disease genetics, genome-wide association (GWA) studies have only relatively recently been applied to plants. Using 32 phenotypes in the inbreeding crop barley, we report GWA mapping of 15 morphological traits across ∼500 cultivars genotyped with 1,536 SNPs. In contrast to the majority of human GWA studies, we observe high levels of linkage disequilibrium within and between chromosomes. Despite this, GWA analysis readily detected common alleles of high penetrance. To investigate the potential of combining GWA mapping with comparative analysis to resolve traits to candidate polymorphism level in unsequenced genomes, we fine-mapped a selected phenotype (anthocyanin pigmentation) within a 140-kb interval containing three genes. Of these, resequencing the putative anthocyanin pathway gene HvbHLH1 identified a deletion resulting in a premature stop codon upstream of the basic helix-loop-helix domain, which was diagnostic for lack of anthocyanin in our association and biparental mapping populations. The methodology described here is transferable to species with limited genomic resources, providing a paradigm for reducing the threshold of map-based cloning in unsequenced crops.

Association mapping of partitioning loci in barley

Background: Association mapping, initially developed in human disease genetics, is now being applied to plant species. The model species Arabidopsis provided some of the first examples of association mapping in plants, identifying previously cloned flowering time genes, despite high population sub-structure. More recently, association genetics has been applied to barley, where breeding activity has resulted in a high degree of population sub-structure. A major genotypic division within barley is that between winter- and spring-sown varieties, which differ in their requirement for vernalization to promote subsequent flowering. To date, all attempts to validate association genetics in barley by identifying major flowering time loci that control vernalization requirement (VRN-H1 and VRN-H2) have failed. Here, we validate the use of association genetics in barley by identifying VRN-H1 and VRN-H2, despite their prominent role in determining population sub-structure. Results: By taking barley as a typical inbreeding crop, and seasonal growth habit as a major partitioning phenotype, we develop an association mapping approach which successfully identifies VRN-H1 and VRN-H2, the underlying loci largely responsible for this agronomic division. We find a combination of Structured Association followed by Genomic Control to correct for population structure and inflation of the test statistic, resolved significant associations only with VRN-H1 and the VRN-H2 candidate genes, as well as two genes closely linked to VRN-H1 (HvCSFs1 and HvPHYC). Conclusion: We show that, after employing appropriate statistical methods to correct for population sub-structure, the genome-wide partitioning effect of allelic status at VRN-H1 and VRN-H2 does not result in the high levels of spurious association expected to occur in highly structured samples. Furthermore, we demonstrate that both VRN-H1 and the candidate VRN-H2 genes can be identified using association mapping. Discrimination between intragenic VRN-H1 markers was achieved, indicating that candidate causative polymorphisms may be discerned and prioritised within a larger set of positive associations. This proof of concept study demonstrates the feasibility of association mapping in barley, even within highly structured populations. A major advantage of this method is that it does not require large numbers of genome-wide markers, and is therefore suitable for fine mapping and candidate gene evaluation, especially in species for which large numbers of genetic markers are either unavailable or too costly.

Direct evidence for attention-dependent influences of the frontal eye-fields on feature-responsive visual cortex

Voluntary selective attention can prioritize different features in a visual scene. The frontal eye-fields (FEF) are one potential source of such feature-specific top-down signals, but causal evidence for influences on visual cortex (as was shown for "spatial" attention) has remained elusive. Here, we show that transcranial magnetic stimulation (TMS) applied to right FEF increased the blood oxygen level-dependent (BOLD) signals in visual areas processing "target feature" but not in "distracter feature"-processing regions. TMS-induced BOLD signals increase in motion-responsive visual cortex (MT+) when motion was attended in a display with moving dots superimposed on face stimuli, but in face-responsive fusiform area (FFA) when faces were attended to. These TMS effects on BOLD signal in both regions were negatively related to performance (on the motion task), supporting the behavioral relevance of this pathway. Our findings provide new causal evidence for the human FEF in the control of nonspatial "feature"-based attention, mediated by dynamic influences on feature-specific visual cortex that vary with the currently attended property.

The challenge of validation: assessing the performance of a test of productive vocabulary

This paper assesses the performance of a vocabulary test designed to measure second language productive vocabulary knowledge.The test, Lex30, uses a word association task to elicit vocabulary, and uses word frequency data to measure the vocabulary produced. Here we report firstly on the reliability of the test as measured by a test-retest study, a parallel test forms experiment and an internal consistency measure. We then investigate the construct validity of the test by looking at changes in test performance over time, analyses of correlations with scores on similar tests, and comparison of spoken and written test performance. Last, we examine the theoretical bases of the two main test components: eliciting vocabulary and measuring vocabulary. Interpretations of our findings are discussed in the context of test validation research literature. We conclude that the findings reported here present a robust argument for the validity of the test as a research tool, and encourage further investigation of its validity in an instructional context