A Functional Screen Provides Evidence for a Conserved, Regulatory, Juxtamembrane Phosphorylation Site in Guanylyl Cyclase A and B

Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ∼20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation – processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200–1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor 32PO4 co-migration studies detected phospho-Ser-473 or phospho-Ser-489 in cells. We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods

Homologous and heterologous desensitization of guanylyl cyclase-B signaling in GH3 somatolactotropes

The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells. Primary rat pituitary and GH3 somatolactotropes expressed functional GC-A and GC-B receptors that had similar EC50 properties in terms of cGMP production. Interestingly, GC-B signaling underwent rapid homologous desensitization in a protein phosphatase 2A (PP2A)-dependent manner. Chronic exposure to either CNP or ANP caused a significant down-regulation of both GC-A- and GC-B-dependent cGMP accumulation in a ligand-specific manner. However, this down-regulation was not accompanied by alterations in the sub-cellular localization of these receptors. Heterologous desensitization of GC-B signaling occurred in GH3 cells following exposure to either sphingosine-1-phosphate or thyrotrophin-releasing hormone (TRH). This heterologous desensitization was protein kinase C (PKC)-dependent, as pre-treatment with GF109203X prevented the effect of TRH on CNP/GC-B signaling. Collectively, these data indicate common and distinct properties of particulate guanylyl cyclase receptors in somatotropes and reveal that independent mechanisms of homologous and heterologous desensitization occur involving either PP2A or PKC. Guanylyl cyclase receptors thus represent potential novel therapeutic targets for treating growth-hormone-associated disorders

Composites Curriculum Development: tackling the skills gap in UK advanced composites

The aim of this project is to generate an industrially relevant and academically rigorous curriculum which could be deployed to tackle the significant skills gap in composites professionals, vital for delivering on the UK’s National Composite Strategy and allowing the industry to grow to its full potential, forecast by the Composites Leadership Forum to grow by a factor of 5 by 2030. A Masters’ level curriculum of short, industrially focused units has been specified and a small number of trial units developed. Engagement of academics in this novel collaborative curriculum development, utilising each institution’s expertise, has been very good and feedback from industry and participants in pilot units has been positive. Consortium participants are investigating numerous options for developing this further and have begun to put plans in place for the next stage

Mendelian randomization study of B-type natriuretic peptide and type 2 diabetes: evidence of causal association from population studies

<p>Background: Genetic and epidemiological evidence suggests an inverse association between B-type natriuretic peptide (BNP) levels in blood and risk of type 2 diabetes (T2D), but the prospective association of BNP with T2D is uncertain, and it is unclear whether the association is confounded.</p> <p>Methods and Findings: We analysed the association between levels of the N-terminal fragment of pro-BNP (NT-pro-BNP) in blood and risk of incident T2D in a prospective case-cohort study and genotyped the variant rs198389 within the BNP locus in three T2D case-control studies. We combined our results with existing data in a meta-analysis of 11 case-control studies. Using a Mendelian randomization approach, we compared the observed association between rs198389 and T2D to that expected from the NT-pro-BNP level to T2D association and the NT-pro-BNP difference per C allele of rs198389. In participants of our case-cohort study who were free of T2D and cardiovascular disease at baseline, we observed a 21% (95% CI 3%-36%) decreased risk of incident T2D per one standard deviation (SD) higher log-transformed NT-pro-BNP levels in analysis adjusted for age, sex, body mass index, systolic blood pressure, smoking, family history of T2D, history of hypertension, and levels of triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. The association between rs198389 and T2D observed in case-control studies (odds ratio = 0.94 per C allele, 95% CI 0.91-0.97) was similar to that expected (0.96, 0.93-0.98) based on the pooled estimate for the log-NT-pro-BNP level to T2D association derived from a meta-analysis of our study and published data (hazard ratio = 0.82 per SD, 0.74-0.90) and the difference in NT-pro-BNP levels (0.22 SD, 0.15-0.29) per C allele of rs198389. No significant associations were observed between the rs198389 genotype and potential confounders.</p> <p>Conclusions: Our results provide evidence for a potential causal role of the BNP system in the aetiology of T2D. Further studies are needed to investigate the mechanisms underlying this association and possibilities for preventive interventions.</p&gt

Natriuretic peptide activation of extracellular regulated kinase 1/2 (ERK1/2) pathway by particulate guanylyl cyclases in GH3 somatolactotropes.

The natriuretic peptides, Atrial-, B-type and C-type natriuretric peptides (ANP, BNP, CNP), are regulators of many endocrine tissues and exert their effects predominantly through the activation of their specific guanylyl cyclase receptors (GC-A and GC-B) to generate cGMP. Whereas cGMP-independent signalling has been reported in response to natriuretic peptides, this is mediated via either the clearance receptor (Npr-C) or a renal-specific NPR-Bi isoform, which both lack intrinsic guanylyl cyclase activity. Here, we report evidence of GC-B-dependent cGMP-independent signalling in pituitary GH3 cells. Stimulation of GH3 cells with CNP resulted in a rapid and sustained enhancement of ERK1/2 phosphorylation (P-ERK1/2), an effect that was not mimicked by dibutryl-cGMP. Furthermore, CNP-stimulated P-ERK1/2 occurred at concentrations below that required for cGMP accumulation. The effect of CNP on P-ERK1/2 was sensitive to pharmacological blockade of MEK (U0126) and Src kinases (PP2). Silencing of the GC-B1 and GC-B2 splice variants of the GC-B receptor by using targeted short interfering RNAs completely blocked the CNP effects on P-ERK1/2. CNP failed to alter GH3 cell proliferation or cell cycle distribution but caused a concentration-dependent increase in the activity of the human glycoprotein α-subunit promoter (αGSU) in a MEK-dependent manner. Finally, CNP also activated the p38 and JNK MAPK pathways in GH3 cells. These findings reveal an additional mechanism of GC-B signalling and suggest additional biological roles for CNP in its target tissues

Advising overweight persons about diet and physical activity in primary health care: Lithuanian health behaviour monitoring study

BACKGROUND: Obesity is a globally spreading health problem. Behavioural interventions aimed at modifying dietary habits and physical activity patterns are essential in prevention and management of obesity. General practitioners (GP) have a unique opportunity to counsel overweight patients on weight control. The purpose of the study was to assess the level of giving advice on diet and physical activity by GPs using the data of Lithuanian health behaviour monitoring among adult population. METHODS: Data from cross-sectional postal surveys of 2000, 2002 and 2004 were analysed. Nationally representative random samples were drawn from the population register. Each sample consisted of 3000 persons aged 20–64 years. The response rates were 74.4% in 2000, 63.4% in 2002 and 61.7% in 2004. Self-reported body weight and height were used to calculate body mass index (BMI). Information on advising in primary health care was obtained asking whether GP advised overweight patients to change dietary habits and to increase physical activity. The odds of receiving advice on diet and physical activity were calculated using multiple logistic regression analyses according to a range of sociodemographic variables, perceived health, number of visits to GPs and body-weight status. RESULTS: Almost a half of respondents were overweight or obese. Only one fourth of respondents reported that they were advised to change diet. The proportion of persons who received advice on physical activity was even lower. The odds of receiving advice increased with age. A strong association was found between perceived health and receiving advice. The likelihood of receiving advice was related to BMI. GPs were more likely to give advice when BMI was high. More than a half of obese respondents (63.3%) reported that they had tried to lose weight. The association between receiving advice and self-reported attempt to lose weight was found. CONCLUSION: The low rate of dietary and physical activity advice reported by overweight patients implies that more lifestyle counselling should be provided in primary health care. There is an obvious need for improved training and education of GPs in counselling of overweight patients focusing on methods of giving dietary and physical activity advice

Methods for specifying the target difference in a randomised controlled trial : the Difference ELicitation in TriAls (DELTA) systematic review

Peer reviewedPublisher PD

The behaviour of inositol 1,3,4,5,6-pentakisphosphate in the presence of the major biological metal cations

The inositol phosphates are ubiquitous metabolites in eukaryotes, of which the most abundant are inositol hexakisphosphate (InsP6) and inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P5)]. These two compounds, poorly understood functionally, have complicated complexation and solid formation behaviours with multivalent cations. For InsP6, we have previously described this chemistry and its biological implications (Veiga et al. in J Inorg Biochem 100:1800, 2006; Torres et al. in J Inorg Biochem 99:828, 2005). We now cover similar ground for Ins(1,3,4,5,6)P5, describing its interactions in solution with Na+, K+, Mg2+, Ca2+, Cu2+, Fe2+ and Fe3+, and its solid-formation equilibria with Ca2+ and Mg2+. Ins(1,3,4,5,6)P5 forms soluble complexes of 1:1 stoichiometry with all multivalent cations studied. The affinity for Fe3+ is similar to that of InsP6 and inositol 1,2,3-trisphosphate, indicating that the 1,2,3-trisphosphate motif, which Ins(1,3,4,5,6)P5 lacks, is not absolutely necessary for high-affinity Fe3+ complexation by inositol phosphates, even if it is necessary for their prevention of the Fenton reaction. With excess Ca2+ and Mg2+, Ins(1,3,4,5,6)P5 also forms the polymetallic complexes [M4(H2L)] [where L is fully deprotonated Ins(1,3,4,5,6)P5]. However, unlike InsP6, Ins(1,3,4,5,6)P5 is predicted not to be fully associated with Mg2+ under simulated cytosolic/nuclear conditions. The neutral Mg2+ and Ca2+ complexes have significant windows of solubility, but they precipitate as [Mg4(H2L)]·23H2O or [Ca4(H2L)]·16H2O whenever they exceed 135 and 56 μM in concentration, respectively. Nonetheless, the low stability of the [M4(H2L)] complexes means that the 1:1 species contribute to the overall solubility of Ins(1,3,4,5,6)P5 even under significant Mg2+ or Ca2+ excesses. We summarize the solubility behaviour of Ins(1,3,4,5,6)P5 in straightforward plots