Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with beta-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of beta-arrestin1 and PP2A with noninternalized NK(1)R. beta-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that beta-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping beta-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires beta-arrestin1. ECE-1 promotes this process by releasing beta-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.

Ubiquitin-dependent down-regulation of the neurokinin-1 receptor

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.

Mapping the platelet profile for functional genomic studies and demonstration of the effect size of the GP6 locus

BACKGROUND: Evidence suggests the wide variation in platelet response within the population is genetically controlled. Unraveling the complex relationship between sequence variation and platelet phenotype requires accurate and reproducible measurement of platelet response. OBJECTIVE: To develop a methodology suitable for measuring signaling pathway-specific platelet phenotype, to use this to measure platelet response in a large cohort, and to demonstrate the effect size of sequence variation in a relevant model gene. METHODS: Three established platelet assays were evaluated: mobilization of [Ca(2+)](i), aggregometry and flow cytometry, each in response to adenosine 5'-diphosphate (ADP) or the glycoprotein (GP) VI-specific crosslinked collagen-related peptide (CRP). Flow cytometric measurement of fibrinogen binding and P-selectin expression in response to a single, intermediate dose of each agonist gave the best combination of reproducibility and inter-individual variability and was used to measure the platelet response in 506 healthy volunteers. Pathway specificity was ensured by blocking the main subsidiary signaling pathways. RESULTS: Individuals were identified who were hypo- or hyper-responders for both pathways, or who had differential responses to the two agonists, or between outcomes. 89 individuals, retested three months later using the same methodology, showed high concordance between the two visits in all four assays (r(2) = 0.872, 0.868, 0.766 and 0.549); all subjects retaining their phenotype at recall. The effect of sequence variation at the GP6 locus accounted for approximately 35% of the variation in the CRP-XL response. CONCLUSION: Genotyping-phenotype association studies in a well-characterized, large cohort provides a powerful strategy to measure the effect of sequence variation in genes regulating the platelet response.

CalDAG-GEFI is at the nexus of calcium-dependent platelet activation.

The importance of the second messengers calcium (Ca(2+)) and diacylglycerol (DAG) in platelet signal transduction was established more than 30 years ago. Whereas protein kinase C (PKC) family members were discovered as the targets of DAG, little is known about the molecular identity of the main Ca(2+) sensor(s). We here identify Ca(2+) and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) as a critical molecule in Ca(2+)-dependent platelet activation. CalDAG-GEFI, through activation of the small GTPase Rap1, directly triggers integrin activation and extracellular signal-regulated kinase-dependent thromboxane A(2) (TxA(2)) release. CalDAG-GEFI-dependent TxA(2) generation provides crucial feedback for PKC activation and granule release, particularly at threshold agonist concentrations. PKC/P2Y12 signaling in turn mediates a second wave of Rap1 activation, necessary for sustained platelet activation and thrombus stabilization. Our results lead to a revised model for platelet activation that establishes one molecule, CalDAG-GEFI, at the nexus of Ca(2+)-induced integrin activation, TxA(2) generation, and granule release. The preferential activation of CalDAG-GEFI over PKC downstream of phospholipase C activation, and the different kinetics of CalDAG-GEFI- and PKC/P2Y12-mediated Rap1 activation demonstrate an unexpected complexity to the platelet activation process, and they challenge the current model that DAG/PKC-dependent signaling events are crucial for the initiation of platelet adhesion.

A comparison of the Thlaspi caerulescens and Thlaspi arvense shoot transcriptomes

Whole-genome transcriptome profiling is revealing how biological systems are regulated at the transcriptional level. This study reports the development of a robust method to profile and compare the transcriptomes of two nonmodel plant species, Thlaspi caerulescens, a zinc (Zn) hyperaccumulator, and Thlaspi arvense, a nonhyperaccumulator, using Affymetrix Arabidopsis thaliana ATH1-121501 GeneChip (R) arrays (Affymetrix, Santa Clara, CA, USA). Transcript abundance was quantified in the shoots of agar- and compost-grown plants of both species. Analyses were optimized using a genomic DNA (gDNA)-based probe-selection strategy based on the hybridization efficiency of Thlaspi gDNA with corresponding A. thaliana probes. In silico alignments of GeneChip (R) probes with Thlaspi gene sequences, and quantitative real-time PCR, confirmed the validity of this approach. Approximately 5000 genes were differentially expressed in the shoots of T. caerulescens compared with T. arvense, including genes involved in Zn transport and compartmentalization. Future functional analyses of genes identified as differentially expressed in the shoots of these closely related species will improve our understanding of the molecular mechanisms of Zn hyperaccumulation.

Variation among Attic fine wares: the case of the Pan Painter's pelikai

This paper is a case study of the continuum between standardization and variation in the production of red-figure Athenian fine wares in the first half of the 5th century BC. An investigation of the Pan Painter's pelikai reveals that they fall into 3 distinct groups, according to size. While the pelikai in each group are also distinguishable from each other by shape, pattern, and iconography, the next clearest distinction between the groups (after size) is in their style of decoration. The pelikai in the largest group, which is comprised of small pelikai, are particularly distinct from the Pan Painter's broader oeuvre of ca. 220 vases insofar as they exemplify a lackadaisical painting style, which I have termed banausic, on account of its frequent use for images of craftsmen, women at work, and other such genre images. While this casual style is antithetical to the Pain Painter's refined style, for which he is better known, and which he employs for his large pelikai, affinities between the 2 styles—as judged by his confident line, anatomical details, and other technical features—permit the conclusion that this group of pelikai were executed by one and the same craftsman as the others. As with all of the vases attributed to this talented painter, however, the pelikai—whether large or small—are decorated with a great bariety of images. While most painted Athenian vases are understood to have been individually created, not mass-produced, the Pan Painter's coherent group of small pelikai seem to have been created en masse, in a uniform size and shape and with a distinct decorative style. This group of standardized vases represents a body of work executed under the influence or at the behest of a specific vase workshop. The form of the small pelikai in fact allows us to associate them with the Geras Painter. With his work on these small pelikai, perhaps in the latter part of his career, the Pan Painter may have intentionally minimized variability in favour of standardization, to meet market demands.

The research productivity of the AIB Farmer Award finalists

Purpose – This paper aims to analyze the research productivity and impact of the finalists of the AIB best dissertation award, now titled the Buckley and Casson Award, but from 1987 to 2012 the Farmer Award. Specifically, this paper examines whether there is a relationship between winning the best dissertation award and subsequent publication productivity and impact. Relationships between academic institution and institutional geographic location and finalists are also examined. Design/methodology/approach – The paper examines 25 years of citation counts and the number of publications in Google Scholar of Farmer Award winners and finalists of the AIB best dissertation award from inception in 1987 to 2009, with cited publications as a measure of productivity and citations as a measure of impact. Top performers in productivity and impact are identified, and the averages of winners and non-winners are analyzed in aggregate, over time and per year. Data on finalists' institution and geographic location of institution are analyzed to describe the importance of location and institution to the award. Findings – It is found that the overall average citations of the winners of the award is less than that of the non-winners, and that in the large majority of years the non-winners have an average citation count higher than that of the winners. However, taking averages in five year increments shows more mixed results, with non-winners performing better in two periods and winners performing better in two periods, with the remaining period being split as to research productivity and impact. Originality/value – Aggarwal et al. in this journal summarized a variety of data on Farmer Award finalists from the 1990s to gain insights on institutions represented by finalists, the publication record of finalists, and content of dissertations, among other characteristics. This paper updates some of the insights from that paper by examining data on award winners from 1987 to 2013, and adds further insight by examining for the first time cited publications and citation counts winners and non-winners for the same period excluding the last two years.

The timing and causes of the Neolithic elm decline: new evidence from the Lower Thames Valley (London, UK)

Two new multi-proxy records of environmental change are provided from Horton Kirby Paper Mill and Old Seager Distillery in the Lower Thames Valley. Each site has evidence for a decline in elm woodland, which at Horton Kirby Paper Mill is recorded earlier than any other published record from the British Isles: sometime between 7320 and 7240 cal BP. Scolytus scolytus/S. multistriatus (the vectors for Dutch elm disease) are recorded after the decline in both sequences, adding to the number of sites with such evidence in the British Isles. Evidence of paludification and human activity are also recorded at the time of the elm decline reinforcing the multi-causal hypothesis. Integration of these results with 21 palaeoenvironmental records has produced a large number of well-dated, multiproxy records of the elm decline in this part of the UK. On the basis of this dataset, a classification system for categorising the relationships between the causal factors of the elm decline is proposed and recommended for future studies.

Cobalt incorporation in calcite: Thermochemistry of (Ca,Co)CO3 solid solutions from density functional theory simulations

The incorporation of cobalt in mixed metal carbonates is a possible route to the immobilization of this toxic element in the environment. However, the thermodynamics of (Ca,Co)CO3 solid solutions are still unclear due to conflicting data from experiment and from the observation of natural ocurrences. We report here the results of a computer simulation study of the mixing of calcite (CaCO3) and spherocobaltite (CoCO3), using density functional theory calculations. Our simulations suggest that previously proposed thermodynamic models, based only on the range of observed compositions, significantly overestimate the solubility between the two solids and therefore underestimate the extension of the miscibility gap under ambient conditions. The enthalpy of mixing of the disordered solid solution is strongly positive and moderately asymmetric: calcium incorporation in spherocobaltite is more endothermic than cobalt incorporation in calcite. Ordering of the impurities in (0001) layers is energetically favourable with respect to the disordered solid solution at low temperatures and intermediate compositions, but the ordered phase is still unstable to demixing. We calculate the solvus and spinodal lines in the phase diagram using a sub-regular solution model, and conclude that many Ca1-xCoxCO3 mineral solid solutions (with observed compositions of up to x=0.027, and above x=0.93) are metastable with respect to phase separation. We also calculate solid/aqueous distribution coefficients to evaluate the effect of the strong non-ideality of mixing on the equilibrium with aqueous solution, showing that the thermodynamically-driven incorporation of cobalt in calcite (and of calcium in spherocobaltite) is always very low, regardless of the Co/Ca ratio of the aqueous environment.

Global forecasting of thermal health hazards: the skill of probabilistic predictions of the Universal Thermal Climate Index (UTCI)

Although over a hundred thermal indices can be used for assessing thermal health hazards, many ignore the human heat budget, physiology and clothing. The Universal Thermal Climate Index (UTCI) addresses these shortcomings by using an advanced thermo-physiological model. This paper assesses the potential of using the UTCI for forecasting thermal health hazards. Traditionally, such hazard forecasting has had two further limitations: it has been narrowly focused on a particular region or nation and has relied on the use of single ‘deterministic’ forecasts. Here, the UTCI is computed on a global scale,which is essential for international health-hazard warnings and disaster preparedness, and it is provided as a probabilistic forecast. It is shown that probabilistic UTCI forecasts are superior in skill to deterministic forecasts and that despite global variations, the UTCI forecast is skilful for lead times up to 10 days. The paper also demonstrates the utility of probabilistic UTCI forecasts on the example of the 2010 heat wave in Russia.

"Just as fragments are part of a vessel": a translation into medieval Occitan of the life of Alexander the Great

This article examines a 14th-c. translation into Old Occitan prose of a late-antique life of Alexander the Great: Justin’s Epitome of the 'Historia Philippicae' of Pompeius Trogus. The article argues that it is the work of translators whose knowledge of pagan Latin materials was incomplete and whose use of their native tongue rested on non-literary bases. This text has not been edited before, and examining its uneven treatment of its source provides important new insights into the work of translators in the later Middle Ages. In conclusion, the article suggests some new approaches to the understanding of translation as a process of reconstruction and adaptation.

An evaluation of the simulated water balance of Eurasia and northern Africa at 6000 yr B.P. using lake status data

Changes in the water balance of Eurasia and northern Africa in response to insolation forcing at 6000 y BP simulated by five atmospheric general circulation models have been compared with observations of changes in lake status. All of the simulations show enhancement of the Asian summer monsoon and of the high pressure cells over the Pacific and Central Asia and the Middle East, causing wetter conditions in northern India and southern China and drier conditions along the Chinese coast and west of the monsoon core. All of the models show enhancement of the African monsoon, causing wetter conditions in the zone between ca 10–20 °N. Four of the models show conditions wetter than present in southern Europe and drier than present in northern Europe. Three of the models show conditions similar to present in the mid-latitude continental interior, while the remaining models show conditions somewhat drier than present. The extent and location of each of the simulated changes varies between the models, as does the mechanism producing these changes. The lake data confirm some features of the simulations, but indicate discrepancies between observed and simulated climates. For example, the data show: (1) conditions wetter than present in central Asia, from India to northern China and Mongolia, indicating that the simulated Asian monsoon expansion is too small; (2) conditions wetter than present between ca. 10–30 °N in Africa, indicating that the simulated African monsoon expansion is too small; (3) that northern Europe was drier, but the area of significantly drier conditions was more localized (around the Baltic) than shown in the simulations; (4) that southern Europe was wetter than present, apparently consistent with the simulations, but pollen data suggest that this reflects an increase in summer rainfall whereas the models show winter precipitation, and (5) that the mid-latitude continental interior was generally wetter than present.

Spontaneous local symmetry breaking: a conformational study of glycine on Cu{311}

Understanding the interplay between intrinsic molecular chirality and chirality of the bonding footprint is crucial in exploiting enantioselectivity at surfaces. As such, achiral glycine and chiral alanine are the most obvious candidates if one is to study this interplay on different surfaces. Here, we have investigated the adsorption of glycine on Cu{311} using reflection-absorption infrared spectroscopy, low-energy electron diffraction, temperature-programmed desorption and first-principles density-functional theory. This combination of techniques has allowed us to accurately identify the molecular conformations present under different conditions, and discuss the overlayer structure in the context of the possible bonding footprints. We have observed coverage-dependent local symmetry breaking, with three-point bonded glycinate moieties forming an achiral arrangement at low coverages, and chirality developing with the presence of two-point bonded moieties at high coverages. Comparison with previous work on the self-assembly of simple amino acids on Cu{311} and the structurally-similar Cu{110} surface has allowed us to rationalise the different conditions necessary for the formation of ordered chiral overlayers.

Middle and Late Pleistocene humid periods recorded in palaeolake deposits of the Nafud desert, Saudi Arabia

Present climate in the Nafud desert of northern Saudi Arabia is hyper-arid and moisture brought by north-westerly winds scarcely reaches the region. The existence of abundant palaeolake sediments provides evidence for a considerably wetter climate in the past. However, the existing chronological framework of these deposits is solely based on radiocarbon dating of questionable reliability, due to potential post-depositional contamination with younger 14C. By using luminescence dating, we show that the lake deposits were not formed between 40 and 20 ka as suggested previously, but approximately ca 410 ka, 320 ka, 200 ka, 125 ka, and 100 ka ago. All of these humid phases are in good agreement with those recorded in lake sediments and speleothems from southern Arabia. Surprisingly, no Holocene lake deposits were identified. Geological characteristics of the deposits and diatom analysis suggest that a single, perennial lake covered the entire south-western Nafud ca 320 ka ago. In contrast, lakes of the 200 ka, 125 ka, and 100 ka humid intervals were smaller and restricted to interdune depressions of a pre-existing dune relief. The concurrent occurrence of humid phases in the Nafud, southern Arabia and the eastern Mediterranean suggests that moisture in northern Arabia originated either from the Mediterranean due to more frequent frontal depression systems or from stronger Indian monsoon circulation, respectively. However, based on previously published climate model simulations and palaecolimate evidence from central Arabia and the Negev desert, we argue that humid climate conditions in the Nafud were probably caused by a stronger African monsoon and a distinct change in zonal atmospheric circulation.