Suppression of p75 Neurotrophin Receptor Surface Expression with Intrabodies Influences Bcl-xL mRNA Expression and Neurite Outgrowth in PC12 Cells

Background: Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies. Results: Three monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells. Conclusion: The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but als

The role of impulsivity in the aetiology of drug dependence: reward sensitivity versus automaticity

Journal ArticleResearch Support, Non-U.S. Gov'tCopyright © The Author(s) 2011.RATIONALE: Impulsivity has long been known as a risk factor for drug dependence, but the mechanisms underpinning this association are unclear. Impulsivity may confer hypersensitivity to drug reinforcement which establishes higher rates of instrumental drug-seeking and drug-taking behaviour, or may confer a propensity for automatic (non-intentional) control over drug-seeking/taking and thus intransigence to clinical intervention. METHOD: The current study sought to distinguish these two accounts by measuring Barratt Impulsivity and craving to smoke in 100 smokers prior to their completion of an instrumental concurrent choice task for tobacco (to measure the rate of drug-seeking) and an ad libitum smoking test (to measure the rate of drug-taking-number of puffs consumed). RESULTS: The results showed that impulsivity was not associated with higher rates of drug-seeking/taking, but individual differences in smoking uptake and craving were. Rather, nonplanning impulsivity moderated (decreased) the relationship between craving and drug-taking, but not drug-seeking. CONCLUSIONS: These data suggest that whereas the uptake of drug use is mediated by hypervaluation of the drug as an instrumental goal, the orthogonal trait nonplanning impulsivity confers a propensity for automatic control over well-practiced drug-taking behaviour.MR

Safety out of control: dopamine and defence

We enjoy a sophisticated understanding of how animals learn to predict appetitive outcomes and direct their behaviour accordingly. This encompasses well-defined learning algorithms and details of how these might be implemented in the brain. Dopamine has played an important part in this unfolding story, appearing to embody a learning signal for predicting rewards and stamping in useful actions, while also being a modulator of behavioural vigour. By contrast, although choosing correct actions and executing them vigorously in the face of adversity is at least as important, our understanding of learning and behaviour in aversive settings is less well developed. We examine aversive processing through the medium of the role of dopamine and targets such as D2 receptors in the striatum. We consider critical factors such as the degree of control that an animal believes it exerts over key aspects of its environment, the distinction between 'better' and 'good' actual or predicted future states, and the potential requirement for a particular form of opponent to dopamine to ensure proper calibration of state values

A roadmap to generate renewable protein binders to the human proteome

Despite the wealth of commercially available antibodies to human proteins, research is often hindered by their inconsistent validation, their poor performance and the inadequate coverage of the proteome. These issues could be addressed by systematic, genome-wide efforts to generate and validate renewable protein binders. We report a multicenter study to assess the potential of hybridoma and phage-display technologies in a coordinated large-scale antibody generation and validation effort. We produced over 1,000 antibodies targeting 20 SH2 domain proteins and evaluated them for potency and specificity by enzyme-linked immunosorbent assay (ELISA), protein microarray and surface plasmon resonance (SPR). We also tested selected antibodies in immunoprecipitation, immunoblotting and immunofluorescence assays. Our results show that high-affinity, high-specificity renewable antibodies generated by different technologies can be produced quickly and efficiently. We believe that this work serves as a foundation and template for future larger-scale studies to create renewable protein binders

Trends and Quality of Care in Outpatient Visits to Generalist and Specialist Physicians Delivering Primary Care in the United States, 1997–2010

BACKGROUND: Although many specialists serve as primary care physicians (PCPs), the type of patients they serve, the range of services they provide, and the quality of care they deliver is uncertain. OBJECTIVE: To describe trends in patient, physician, and visit characteristics, and compare visit-based quality for visits to generalists and specialists self-identified as PCPs. DESIGN: Cross-sectional study and time trend analysis. DATA: Nationally representative sample of visits to office-based physicians from the National Ambulatory Medical Care Survey, 1997–2010. MAIN MEASURES: Proportions of primary care visits to generalist and specialists, patient characteristics, principal diagnoses, and quality. KEY RESULTS: Among 84,041 visits to self-identified PCPs representing an estimated 4.0 billion visits, 91.5 % were to generalists, 5.9 % were to medical specialists and 2.6 % were to obstetrician/gynecologists. The proportion of PCP visits to generalists increased from 88.4 % in 1997 to 92.4 % in 2010, but decreased for medical specialists from 8.0 % to 4.8 %, p = 0.04). The proportion of medical specialist visits in which the physician self-identified as the patient’s PCP decreased from 30.6 % in 1997 to 9.8 % in 2010 (p < 0.01). Medical specialist PCPs take care of older patients (mean age 61 years), and dedicate most of their visits to chronic disease management (51.0 %), while generalist PCPs see younger patients (mean age 55.4 years) most commonly for new problems (40.5 %). Obstetrician/gynecologists self-identified as PCPs see younger patients (mean age 38.3 p < 0.01), primarily for preventive care (54.0 %, p < 0.01). Quality of care for cardiovascular disease was better in visits to cardiologists than in visits to generalists, but was similar or better in visits to generalists compared to visits to other medical specialists. CONCLUSIONS: Medical specialists are less frequently serving as PCPs for their patients over time. Generalist, medical specialist, and obstetrician/gynecologist PCPs serve different primary care roles for different populations. Delivery redesign efforts must account for the evolving role of generalist and specialist PCPs in the delivery of primary care. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11606-014-2808-y) contains supplementary material, which is available to authorized users