Inhibition of synaptic transmission and G protein modulation by synthetic CaV2.2 Ca2+ channel peptides

Abstract: Modulation of presynaptic voltage-dependent Ca+ channels is a major means of controlling neurotransmitter release. The CaV 2.2 Ca2+ channel subunit contains several inhibitory interaction sites for Gβγ subunits, including the amino terminal (NT) and I–II loop. The NT and I–II loop have also been proposed to undergo a G protein-gated inhibitory interaction, whilst the NT itself has also been proposed to suppress CaV 2 channel activity. Here, we investigate the effects of an amino terminal (CaV 2.2[45–55]) ‘NT peptide’ and a I–II loop alpha interaction domain (CaV 2.2[377–393]) ‘AID peptide’ on synaptic transmission, Ca2+ channel activity and G protein modulation in superior cervical ganglion neurones (SCGNs). Presynaptic injection of NT or AID peptide into SCGN synapses inhibited synaptic transmission and also attenuated noradrenaline-induced G protein modulation. In isolated SCGNs, NT and AID peptides reduced whole-cell Ca2+ current amplitude, modified voltage dependence of Ca2+ channel activation and attenuated noradrenaline-induced G protein modulation. Co-application of NT and AID peptide negated inhibitory actions. Together, these data favour direct peptide interaction with presynaptic Ca2+ channels, with effects on current amplitude and gating representing likely mechanisms responsible for inhibition of synaptic transmission. Mutations to residues reported as determinants of Ca2+ channel function within the NT peptide negated inhibitory effects on synaptic transmission, Ca2+ current amplitude and gating and G protein modulation. A mutation within the proposed QXXER motif for G protein modulation did not abolish inhibitory effects of the AID peptide. This study suggests that the CaV 2.2 amino terminal and I–II loop contribute molecular determinants for Ca2+ channel function; the data favour a direct interaction of peptides with Ca2+ channels to inhibit synaptic transmission and attenuate G protein modulation. Non-technical summary: Nerve cells (neurones) in the body communicate with each other by releasing chemicals (neurotransmitters) which act on proteins called receptors. An important group of receptors (called G protein coupled receptors, GPCRs) regulate the release of neurotransmitters by an action on the ion channels that let calcium into the cell. Here, we show for the first time that small peptides based on specific regions of calcium ion channels involved in GPCR signalling can themselves inhibit nerve cell communication. We show that these peptides act directly on calcium channels to make them more difficult to open and thus reduce calcium influx into native neurones. These peptides also reduce GPCR-mediated signalling. This work is important in increasing our knowledge about modulation of the calcium ion channel protein; such knowledge may help in the development of drugs to prevent signalling in pathways such as those involved in pain perception.

Mechanisms of burst release from pH-responsive polymeric microparticles.

Microencapsulation of drugs into preformed polymers is commonly achieved through solvent evaporation techniques or spray drying. We compared these encapsulation methods in terms of controlled drug release properties of the prepared microparticles and investigated the underlying mechanisms responsible for the “burst release” effect. Using two different pH-responsive polymers with a dissolution threshold of pH 6 (Eudragit L100 and AQOAT AS-MG), hydrocortisone, a model hydrophobic drug, was incorporated into microparticles below and above its solubility within the polymer matrix. Although, spray drying is an attractive approach due to rapid particle production and relatively low solvent waste, the oil-in-oil microencapsulation method is superior in terms of controlled drug release properties from the microparticles. Slow solvent evaporation during the oil-in-oil emulsification process allows adequate time for drug and polymer redistribution in the microparticles and reduces uncontrolled drug burst release. Electron microscopy showed that this slower manufacturing procedure generated non-porous particles whereas thermal analysis and X-ray diffractometry showed that drug loading above the solubility limit of the drug in the polymer generated excess crystalline drug on the surface of the particles. Raman spectral mapping illustrated that drug was homogeneously distributed as a solid solution in the particles when loaded below saturation in the polymer with consequently minimal burst release.

Intra-ovarian roles of activins and inhibins

Granulosa cells are the main ovarian source of inhibins, activins and activin-binding protein (follistatin) while germ (oogonia, oocytes) and somatic (theca, granulosa, luteal) cells express activin receptors, signaling components and inhibin co-receptor (betaglycan). Activins are implicated in various intra-ovarian roles including germ cell survival and primordial follicle assembly; follicle growth from preantral to mid-antral stages; suppression of thecal androgen production; promotion of granulosa cell proliferation, FSHR and CYP19A1 expression; enhancement of oocyte developmental competence; retardation of follicle luteinization and/or atresia and involvement in luteolysis. Inhibins (primarily inhibin A) are produced in greatest amounts by preovulatory follicles (and corpus luteum in primates) and suppress FSH secretion through endocrine negative feedback. Together with follistatin, inhibins act locally to oppose auto-/paracrine activin (and BMP) signaling thus modulating many of the above processes. The balance between activin-inhibin shifts during follicle development with activin signalling prevailing at earlier stages but declining as inhibin and betaglycan expression rise.

Guanine specific binding at a DNA junction formed by d[CG(5-BrU)ACG]2 with a topoisomerase poison in the presence of Co2+Ions†,‡

The structure of the duplex d[CG(5-BrU)ACG]2 bound to 9-bromophenazine-4-carboxamide has been solved through MAD phasing at 2.0 Å resolution. It shows an unexpected and previously unreported intercalation cavity stabilized by the drug and novel binding modes of Co2+ ions at certain guanine N7 sites. For the intercalation cavity the terminal cytosine is rotated to pair with the guanine of a symmetry-related duplex to create a pseudo-Holliday junction geometry, with two such cavities linked through the minor groove interactions of the N2/N3 guanine sites at an angle of 40°, creating a quadruplex-like structure. The mode of binding of the drug is shown to be disordered, with the major conformations showing the side chain bound to the N7 position of adjacent guanines. The other end of the duplex exhibits a terminal base fraying in the presence of Co2+ ions linking symmetry-related guanines, causing the helices to intertwine through the minor groove. The stabilization of the structure by the intercalating drug shows that this class of compound may bind to DNA junctions as well as duplex DNA or to strand-nicked DNA (‘hemi-intercalated'), as in the cleavable complex. This suggests a structural basis for the dual poisoning of topoisomerase I and II enzymes by this family of drugs.

The EU, counter-terrorism and the protection of Muslims as European minorities

This article examines the EU’s promotion of the religious identity of Muslims within the context of European counter-terrorism measures. Counter-terrorism laws of the EU and its Member States impact on the religious identity of Muslims. They have an arguably disproportionate effect on the civil rights of individuals in the quest to combat terrorism and can be seen to increase Islamophobia in two ways: a rise in general discrimination against Muslims and a requirement on Muslims to distance their connection to Islamic practice and traditions. EU law dealing with terrorist offences speak little of this backlash that Muslims face in European countries. Although the EU has somewhat of a framework in place which concerns the protection of Islamic identity, the reluctance of the EU to take a determined stance on the issue of the protection of religious identity is illustrated through the ambiguous nature of its legislation.

Farm and socio-economic characteristics of smallholder milk producers and their influence on technology adoption in Central Mexico

In order to identify the factors influencing adoption of technologies promoted by government to small-scale dairy farmers in the highlands of central Mexico, a field survey was conducted. A total of 115 farmers were grouped through cluster analysis (CA) and divided into three wealth status categories (high, medium and low) using wealth ranking. Chi-square analysis was used to examine the association of wealth status with technology adoption. Four groups of farms were differentiated in terms of farms’ dimensions, farmers’ education, sources of incomes, wealth status, management of herd, monetary support by government and technological availability. Statistical differences (p < 0.05) were observed in the milk yield per herd per year among groups. Government organizations (GO) participated little in the promotion of the 17 technologies identified, six of which focused on crop or forage production and 11 of which were related to animal husbandry. Relatives and other farmers played an important role in knowledge diffusion and technology adoption. Although wealth status had a significant association (p < 0.05) with adoption, other factors including importance of the technology to farmers, usefulness and productive benefits of innovations together with farmers’ knowledge of them, were important. It is concluded that the analysis of the information per group and wealth status was useful to identify suitable crop or forage related and animal husbandry technologies per group and wealth status of farmers. Therefore the characterizations of farmers could provide a useful starting point for the design and delivery of more appropriate and effective extension.

The UK Department of National Heritage: a model for the development and integration of leisure policy in the new Europe?

Leisure is in the vanguard of a social and cultural revolution which is replacing the former East/West political bipolarity with a globalised economic system in which the new Europe has a central rôle. Within this revolution, leisure, including recreation, culture and tourism, is constructed as the epitome of successful capitalist development; the very legitimisation of the global transmogrification from a production to a consumption orientation. While acting as a direct encouragement to the political transformation in many eastern European states, it is uncertain how the issue of leisure policy is being handled, given its centrality to the new economic order. This paper therefore examines the experience of western Europe, considering in particular the degree to which the newly-created Department of National Heritage in the UK provides a potential model for leisure development and policy integration in the new Europe. Despite an official rhetoric of support and promotion of leisure activities, reflecting the growing economic significance of tourism and the positive relationship between leisure provision and regional economic development, the paper establishes that in the place of the traditional rôle of the state in promoting leisure interests, the introduction of the Department has signified a shift to the use of leisure to promote the Government's interests, particularly in regenerating citizen rights claims towards the market. While an institution such as the Department of National Heritage may have relevance to emerging states as a element in the maintenance of political hegemony, therefore, it is questionable how far it can be viewed as a promoter or protector of leisure as a signifier of a newly-won political, economic and cultural freedom throughout Europe.

Farmers’ attitudes to disease risk management in England: a comparative analysis of sheep and pig farmers

The UK Department for Environment, Food and Rural Affairs (Defra) identified practices to reduce the risk of animal disease outbreaks. We report on the response of sheep and pig farmers in England to promotion of these practices. A conceptual framework was established from research on factors influencing adoption of animal health practices, linking knowledge, attitudes, social influences and perceived constraints to the implementation of specific practices. Qualitative data were collected from nine sheep and six pig enterprises in 2011. Thematic analysis explored attitudes and responses to the proposed practices, and factors influencing the likelihood of implementation. Most feel they are doing all they can reasonably do to minimise disease risk and that practices not being implemented are either not relevant or ineffective. There is little awareness and concern about risk from unseen threats. Pig farmers place more emphasis than sheep farmers on controlling wildlife, staff and visitor management and staff training. The main factors that influence livestock farmers’ decision on whether or not to implement a specific disease risk measure are: attitudes to, and perceptions of, disease risk; attitudes towards the specific measure and its efficacy; characteristics of the enterprise which they perceive as making a measure impractical; previous experience of a disease or of the measure; and the credibility of information and advice. Great importance is placed on access to authoritative information with most seeing vets as the prime source to interpret generic advice from national bodies in the local context. Uptake of disease risk measures could be increased by: improved risk communication through the farming press and vets to encourage farmers to recognise hidden threats; dissemination of credible early warning information to sharpen farmers’ assessment of risk; and targeted information through training events, farming press, vets and other advisers, and farmer groups, tailored to the different categories of livestock farmer.

The effect of BREEAM on clients and construction professionals

The effects and influence of the Building Research Establishment’s Environmental Assessment Methods (BREEAM) on construction professionals are examined. Most discussions of building assessment methods focus on either the formal tool or the finished product. In contrast, BREEAM is analysed here as a social technology using Michel Foucault’s theory of governmentality. Interview data are used to explore the effect of BREEAM on visibilities, knowledge, techniques and professional identities. The analysis highlights a number of features of the BREEAM assessment process which generally go unremarked: professional and public understandings of the method, the deployment of different types of knowledge and their implication for the authority and legitimacy of the tool, and the effect of BREEAM on standard practice. The analysis finds that BREEAM’s primary effect is through its impact on standard practices. Other effects include the use of assessment methods to defend design decisions, its role in both operationalizing and obscuring the concept of green buildings, and the effect of tensions between project and method requirements for the authority of the tool. A reflection on assessment methods as neo-liberal tools and their adequacy for the promotion of sustainable construction suggests several limitations of lock-in that hinder variation and wider systemic change.

Antidepressant treatment and emotional processing: can we dissociate the roles of serotonin and noradrenaline?

The ability to match individual patients to tailored treatments has the potential to greatly improve outcomes for individuals suffering from major depression. In particular, while the vast majority of antidepressant treatments affect either serotonin or noradrenaline or a combination of these two neurotransmitters, it is not known whether there are particular patients or symptom profiles which respond preferentially to the potentiation of serotonin over noradrenaline or vice versa. Experimental medicine models suggest that the primary mode of action of these treatments may be to remediate negative biases in emotional processing. Such models may provide a useful framework for interrogating the specific actions of antidepressants. Here, we therefore review evidence from studies examining the effects of drugs which potentiate serotonin, noradrenaline or a combination of both neurotransmitters on emotional processing. These results suggest that antidepressants targeting serotonin and noradrenaline may have some specific actions on emotion and reward processing which could be used to improve tailoring of treatment or to understand the effects of dual-reuptake inhibition. Specifically, serotonin may be particularly important in alleviating distress symptoms, while noradrenaline may be especially relevant to anhedonia. The data reviewed here also suggest that noradrenergic-based treatments may have earlier effects on emotional memory that those which affect serotonin.

Provenance of chalk tesserae from a Roman town-house in Vine Street, Leicester

Fourth-century a.d. chalk tesserae from Roman Leicester (Ratae Corieltavorum) yield rich microfossil assemblages that identify a biostratigraphical age of Cretaceous Late Cenomanian to Early Turonian. The nearest chalk outcrops to Leicester lie in Hertfordshire, Lincolnshire, Yorkshire and north Norfolk, indicating that the material for the tesserae must have been sourced remotely and transported to Ratae. Superimposing the Roman road network onto a map of the relevant Chalk Group distribution provides a guide to possible sources. A process of evaluation identifies Baldock in Hertfordshire and Bridlington in Yorkshire as the most likely sources for the Leicester tesserae.

The supply and movement of Denarii in Roman Britain

Hoards of denarii are common in Britain and the number which have been recorded in detail means that it is now possible to suggest reasonably accurately what a ‘normal’ hoard of a particular date should look like. That being the case, we can then look for variation around that norm and both investigate and speculate what that variation means. A methodology is developed which suggests periods of faster and less rapid coin circulation which has implications for consideration of monetisation. The model also enables us to view where denarii entered circulation; unsurprisingly the army looms large in this picture. The methodology is directly transferable to other provinces and other periods where there are longlived, relatively stable monetary systems.