Fiona Mitchell

Widespread distribution of Gqα/Gllα detected immunologically by an antipeptide antiserum directed against the predicted C-terminal decapeptide

Antisera were raised to a synthetic peptide which represents the predicted C‐terminal decapeptide of the α subunit of the G‐proteins Gq and Gll. Competitive ELISA indicated that antiserum CQ2 displayed strong reactivity against this peptide. Antiserum CQ2 identified an apparently single polypeptide of 42 kDa which was expressed widely. The mobility of this polypeptide in SDS‐PAGE was not modified by pretreatment of cells with pertussis toxin, indicating that it was not a substrate for this toxin. Furthermore, the levels and mobility of this polypeptide were unaltered by treatment of cells with cholera toxin, defining that it was not related to Gsα.

The human muscarinic M1 acetylcholine receptor, when expressed in CHO cells, activates and downregulates both Gqα and G11α equally and non-selectively

CHO cells express both of the phosphoinositidase C‐linked G‐proteins Gq and G11 G11α is some 2.5‐fold more highly expressed than Gqα in membranes of these cells. Following transfection and stable expression of CHO cells with DNA encoding the human muscarinic M1 acetylcholine (HM1) receptor, chronic treatment of the cells with the cholinergic agonist carbachol resulted in down‐regulation of membrane levels of both Gqα and G11α. Dose‐response curves to carbachol produced identical EC50 values for agonist‐induced down‐regulation of the two G‐proteins and both were down‐regulated with the same time course. These data indicate that the HM1 receptor interacts with and activates both Gqα and G11α equivalently and non‐selectively in a whole cell system in which the receptor has access to both G‐proteins.

Immunohistochemical distribution of neurons containing the G-proteins Gqα/G11α in the adult rat brain

A new class of G-proteins, the Gq family, has been recently identified and found to be involved in phospholipase C activation. The alpha subunits of the Gq and G11 members of this family are separate polypeptides but appear to have the same function. In this study, the cellular distribution in the adult rat brain of these G-proteins, Gq alpha/G11 alpha, was determined by immunohistochemistry using an antipeptide antiserum directed against the predicted C-terminal decapeptide which is conserved between these polypeptides. The specificity of the antiserum was verified by Western blot analysis using rat brain homogenates. Immunoreactivity was detected in neurons, where it was localized in the dendrites and at the periphery of the cell bodies. The staining was abundant in the dendrites of cerebellar Purkinje cells and hippocampal CA1 pyramidal cells. Staining was also found in neurons in the olfactory bulb, minor and major islets of Calleja, anterior olfactory nuclei and piriform cortex; the different cortical areas especially in their superficial layers; caudate-putamen, accumbens and olfactory tubercle; lateral septum and amygdala; hippocampal CA2-4 sectors of Ammons horn, dentate gyrus and hilus; hypothalamic supraoptic nucleus; cerebellar granular layer; colliculi and superficial layers of the dorsal horn of the spinal cord. In conclusion, the brain neuronal localizations of Gq alpha/G11 alpha match that of phospholipase C, 1,4,5-triphosphate receptor and, to a lesser extent 1,4,5-triphosphate-3-kinase.

Immunological identification of the α subunit of G13, a novel guanine nucleotide binding protein

An antiserum (13CB) was generated against a synthetic peptide, HDNLKQLMLQ, which is predicted to represent the C‐terminal decapeptide of the α subunit of the novel G‐protein, G13. Competitive ELISA indicated that the antiserum reacted with this peptide but that it showed minimal ability to recognize peptides which represent the equivalent regions of the pertussis toxin‐insensitive G‐proteins, Gq + G11, G12, G15 + G16, GL1 (also called G14) as Gz, and well as other G‐proteins. Immunoblots of human platelet membranes with antiserum 13CB identified a single 43‐kDa polypeptide, and while this immunoreactivity was abolished by the presence of the cognate peptide it was not modified by the presence of peptides corresponding to the equivalent region of other G‐proteins. Immunoreactivity corresponding to G13α was detected in a range of cell types with human platelets having the highest levels of this polypeptide.

Process evaluation of the Walk Well study: a cluster-randomised controlled trial of a community based walking programme for adults with intellectual disabilities.

BackgroundWalking interventions can be effective in encouraging sedentary populations to become more active; however, limited research has explored the effectiveness of walking interventions for adults with intellectual disabilities. This process evaluation explored the delivery of a community based walking intervention for adults with intellectual disabilities.MethodsWalk Well was a single-blind cluster randomised controlled trial of a 12-week physical activity consultation-led walking intervention. 102 participants were randomised to the Walk Well intervention or a waiting list control group. Participants in the intervention group received three physical activity consultations with a walking advisor at baseline, 6 & 12-weeks. They were encouraged to use a pedometer to set goals and monitor their daily step count. Primary outcome was change in daily step count at 12-weeks. Process evaluation measures included qualitative interviews with key stakeholders (n = 6) and quantifiable data collected as part of the intervention. Additional process data were extracted from a sub-set of qualitative interviews with participants and carers (n = 20). Data were analysed for process information related to context, recruitment and retention, reach, implementation, and fidelity.ResultsWalk Well was not effective in significantly increasing levels of physical activity. The process evaluation did, however, highlight several important areas for consideration in future studies, including: a successful recruitment and retention strategy reaching a representative sample of adults with intellectual disabilities in the community; feasible and (for most) enjoyable methods of engaging adults with intellectual disabilities in activities to support behaviour change; potential need for greater intervention duration and frequency of contact; advantages and disadvantages of using pedometers as a behaviour change tool; the need for strategies which engage carers in supporting participants; and the complex issue of ‘freedom of choice’ in relation to lifestyle behaviours and study participation.ConclusionsWalking interventions for adults with intellectual disabilities can be feasibly delivered in the community in relation to reach, recruitment, retention and intervention fidelity. More intensive intervention methods need to be explored as well as strategies to engage and motivate carers in their support of participants.Trial registrationCurrent Controlled Trials ISRCTN50494254 (3rd April 2012).

Development and feasibility testing of an intervention to support active lifestyles in youths with Type 1 diabetes - the ActivPals programme: a study protocol

BackgroundThe global incidence of type 1 diabetes is rising, and youths with type 1 diabetes continue to suffer poorer health than peers without diabetes. Evidence suggests youths with type 1 diabetes have physical activity (PA) levels well below the recommendations for health and have high levels of sedentary behaviour. An active lifestyle is therefore recommended to improve health. There is limited research showing effective lifestyle behaviour change in this population; therefore, an evidence gap exists between the need to promote physical activity in type 1 diabetes care and lack of understanding on how to do this. This protocol paper describes a feasibility and pilot study of the ActivPals programme—an intervention to support active lifestyles in youths with type 1 diabetes.Methods/designKey intervention components have been identified from preliminary work (individual and family focus, peer mentoring, technology integration and improved communication and understanding) and are being developed into a pragmatic randomised controlled trial (RCT) supported by recruitment pathways. A steering group of health care professionals and managers will refine the intervention to patient needs. A pilot trial is providing data on intervention implementation, acceptability and feasibility. Twenty youths with type 1 diabetes are being recruited and randomised into an intervention or control group. Physical activity is being measured objectively using the Actigraph GT3X+ monitor at baseline and 1-month follow-up. Contextual factors associated with intervention delivery are being explored.DiscussionThis study will contribute to the development of evidence-based, user-informed and pragmatic interventions leading to healthier lifestyles in youths with type 1 diabetes.

Correlates of objectively measured sedentary time in adults with intellectual disabilities

Sedentary behaviour is an independent risk factor for adverse health conditions. Adults with intellectual disabilities spend a high proportion of their day engaged in sedentary behaviour, however, there is limited evidence on potential correlates of objectively measured sedentary behaviour in this population group. In Glasgow, UK from July to September 2017, a secondary analysis of pooled baseline accelerometer data from two randomised controlled trials of lifestyle behaviour change programmes was conducted. Backwards linear regression was used to investigate the associations between demographic, biological, and environmental correlates and objective measure of sedentary behaviour (percentage of time spent sedentary). One-hundred and forty-three participants provided valid accelerometer data. Mean percentage time spent sedentary (adjusted for wear time) was 72.9% [Standard Deviation (SD) = 8.7] per day. In the final model, physical and mental health problems were significantly (p < 0.05) associated with increased percentage time spent sedentary. This is the first study to provide evidence on multi-level, demographic, biological, and environmental correlates of objectively measured sedentary behaviour in adults with intellectual disabilities. To inform the development of interventions to modify sedentary behaviours in adults with intellectual disabilities, further research is required including a wide range of socio-ecological correlates.

A qualitative exploration of participants’ experiences of taking part in a walking programme: Perceived benefits, barriers, choices and use of intervention resources

BACKGROUND Adults with intellectual disabilities (ID) experience significant inequalities and tend to be more sedentary and less physically active than the wider population. Walking programmes are an effective way to increase physical activity (PA) but have not been used in studies involving adults with intellectual disabilities. METHOD Nineteen adults with intellectual disabilities participated in semistructured interviews or focus groups exploring their experiences of taking part in a walking programme (Walk Well). Data were coded using thematic analysis. RESULTS Four overarching themes emerged: perceived benefits of taking part in the programme, perceived drawbacks/ barriers, walking choices and using the Walk Well resources. While there was not a significant increase in walking for all, the participants reported positive experiences of taking part in the programme. Self-monitoring proved difficult for some, particularly reading the daily step count recorded on the pedometer and writing it in the diary. Carers also played an important role in facilitating and preventing behaviour change in adults with intellectual disabilities. CONCLUSION Additional barriers prevent many adults with intellectual disabilities from participating in PA. Capturing participant experiences provides important information for designing effective and equitable health improvement programmes.

Motivations for physical activity in youth with type 1 diabetes participating in the ActivPals project: a qualitative study

Around two‐thirds of 5–18 year olds fail to meet physical activity (PA) recommendations. Children with type 1 diabetes tend to be less active and more sedentary than non‐diabetic peers. Research into motivations for PA in this population is lacking.

An arginine residue is the site of receptor-stimulated, cholera toxin-catalysed ADP-ribosylation of pertussis toxin-sensitive G-proteins

Cholera toxin-catalysed [32P]ADP-ribosylations were performed in the absence of guanine nucleotides on membranes of a clone (1C) of Rat 1 fibroblasts which express high levels of the alpha 2-C10 adrenergic receptor. As well as incorporation of radioactivity into 45,000 and 42,000 M(r) polypeptides which represent forms of Gs alpha, there was weak labelling of a 40,000 M(r) polypeptide(s). Addition of the alpha 2 adrenergic agonist UK14304 to such assays enhanced markedly the incorporation of radioactivity into the 40,000 M(r) polypeptide(s) but did not alter labelling of the forms of Gs. We have previously demonstrated that the 40,000 M(r) polypeptide(s) labelled in such a manner represents a combination of the alpha subunits of Gi2 and Gi3 [Milligan et al. (1991) J. biol. Chem. 266, 6447-6455]. Mercuric acetate treatment of membranes prelabelled with [32P]ADP-ribose by exposure to pertussis toxin and [32P]NAD removed totally the radiolabel from both Gi2 and Gi3. However, cholera toxin-catalysed [32P]ADP-ribosylation of either the alpha subunits of the Gi-subtypes or of forms of Gs was unaffected by such treatment. By contrast, prolonged, but not short-term, exposure to neutral hydroxylamine removed radiolabel incorporated by cholera toxin from the Gi-subtypes and from Gs but did not remove [32P]ADP-ribose incorporated by pertussis toxin from the Gi-subtypes. It is concluded that ADP-ribosylation of pertussis toxin-sensitive G-proteins by cholera toxin, which can be induced by exposure of membranes to agonists for receptors which interact with that G-protein, is at an arginine residue. It is suggested that this residue may be Arg 179 in Gi2 alpha and Arg 178 in Gi3 alpha.