MOR Is Not Enough: Identification of Novel Mu-Opioid Receptor Interacting Proteins Using Traditional and Modified Membrane Yeast Two-Hybrid Screens.

MOR Is Not Enough: Identification of Novel mu-Opioid Receptor Interacting Proteins Using Traditional and Modified Membrane Yeast Two-Hybrid Screens.

PLoS One. 2013; 8(6): e67608
Petko J, Justice-Bitner S, Jin J, Wong V, Kittanakom S, Ferraro TN, Stagljar I, Levenson R

The mu-opioid receptor (MOR) is the G-protein coupled receptor primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. We have utilized a combination of traditional and modified membrane yeast two-hybrid screening methods to identify a cohort of novel MOR interacting proteins (MORIPs). The interaction between the MOR and a subset of MORIPs was validated in pulldown, co-immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta and kappa opioid receptors, suggesting that they may represent general opioid receptor interacting proteins (ORIPS). Expression of several MORIPs was altered in specific mouse brain regions after chronic treatment with morphine, suggesting that these proteins may play a role in response to opioid agonist drugs. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction. HubMed – addiction


Gene transfer of mutant mouse cholinesterase provides high lifetime expression and reduced cocaine responses with no evident toxicity.

PLoS One. 2013; 8(6): e67446
Geng L, Gao Y, Chen X, Hou S, Zhan CG, Radic Z, Parks RJ, Russell SJ, Pham L, Brimijoin S

Gene transfer of a human cocaine hydrolase (hCocH) derived from butyrylcholinesterase (BChE) by 5 mutations (A199S/F227A/S287G/A328W/Y332G) has shown promise in animal studies for treatment of cocaine addiction. To predict the physiological fate and immunogenicity of this enzyme in humans, a comparable enzyme was created and tested in a conspecific host. Thus, similar mutations (A199S/S227A/S287G/A328W/Y332G) were introduced into mouse BChE to obtain a mouse CocH (mCocH). The cDNA was incorporated into viral vectors based on: a) serotype-5 helper-dependent adenovirus (hdAD) with ApoE promoter, and b) serotype-8 adeno-associated virus with CMV promoter (AAV-CMV) or multiple promoter and enhancer elements (AAV-VIP). Experiments on substrate kinetics of purified mCocH expressed in HEK293T cells showed 30-fold higher activity (U/mg) with (3)H-cocaine and 25% lower activity with butyrylthiocholine, compared with wild type BChE. In mice given modest doses of AAV-CMV-mCocH vector (0.7 or 3×10(11) particles) plasma hydrolase activity rose 10-fold above control for over one year with no observed immune response. Under the same conditions, transduction of the human counterpart continued less than 2 months and antibodies to hCocH were readily detected. The advanced AAV-VIP-mCocH vector generated a dose-dependent rise in plasma cocaine hydrolase activity from 20-fold (10(10) particles) to 20,000 fold (10(13) particles), while the hdAD vector (1.7×10(12) particles) yielded a 300,000-fold increase. Neither vector caused adverse reactions such as motor weakness, elevated liver enzymes, or disturbance in spontaneous activity. Furthermore, treatment with high dose hdAD-ApoE-mCocH vector (1.7×10(12) particles) prevented locomotor abnormalities, other behavioral signs, and release of hepatic alanine amino transferase after a cocaine dose fatal to most control mice (120 mg/kg). This outcome suggests that viral gene transfer can yield clinically effective cocaine hydrolase expression for lengthy periods without immune reactions or cholinergic dysfunction, while blocking toxicity from drug overdose. HubMed – addiction


PTEN loss defines a PI3K/AKT pathway-dependent germinal center subtype of diffuse large B-cell lymphoma.

Proc Natl Acad Sci U S A. 2013 Jul 9;
Pfeifer M, Grau M, Lenze D, Wenzel SS, Wolf A, Wollert-Wulf B, Dietze K, Nogai H, Storek B, Madle H, Dörken B, Janz M, Dirnhofer S, Lenz P, Hummel M, Tzankov A, Lenz G

Diffuse large B-cell lymphoma (DLBCL) represents a heterogeneous diagnostic category with distinct molecular subtypes that can be defined by gene expression profiling. However, even within these defined subtypes, heterogeneity prevails. To further elucidate the pathogenesis of these entities, we determined the expression of the tumor suppressor phosphatase and tensin homolog (PTEN) in 248 primary DLBCL patient samples. These analyses revealed that loss of PTEN was detectable in 55% of germinal center B-cell-like (GCB) DLBCLs, whereas this abnormality was found in only 14% of non-GCB DLBCL patient samples. In GCB DLBCL, the PTEN status was inversely correlated with activation of the oncogenic PI3K/protein kinase B (AKT) pathway in both DLBCL cell lines and primary patient samples. Reexpression of PTEN induced cytotoxicity in PTEN-deficient GCB DLBCL cell line models by inhibiting PI3K/AKT signaling, indicating an addiction to this pathway in this subset of GCB DLBCLs. PI3K/AKT inhibition induced down-regulation of the transcription factor MYC. Reexpression of MYC rescued GCB DLBCL cells from PTEN-induced toxicity, identifying a regulatory mechanism of MYC expression in DLBCL. Finally, pharmacologic PI3K inhibition resulted in toxicity selectively in PTEN-deficient GCB DLBCL lines. Collectively, our results indicate that PTEN loss defines a PI3K/AKT-dependent GCB DLBCL subtype that is addicted to PI3K and MYC signaling and suggest that pharmacologic inhibition of PI3K might represent a promising therapeutic approach in these lymphomas. HubMed – addiction


Internet-based interventions for smoking cessation.

Cochrane Database Syst Rev. 2013 Jul 10; 7: CD007078
Civljak M, Stead LF, Hartmann-Boyce J, Sheikh A, Car J

The Internet is now an indispensable part of daily life for the majority of people in many parts of the world. It offers an additional means of effecting changes to behaviour such as smoking.To determine the effectiveness of Internet-based interventions for smoking cessation.We searched the Cochrane Tobacco Addiction Group Specialized Register. There were no restrictions placed on language of publication or publication date. The most recent search was conducted in April 2013.We included randomized and quasi-randomized trials. Participants were people who smoked, with no exclusions based on age, gender, ethnicity, language or health status. Any type of Internet intervention was eligible. The comparison condition could be a no-intervention control, a different Internet intervention, or a non-Internet intervention.Two authors independently assessed and extracted data. Methodological and study quality details were extracted using a standardized form. We extracted smoking cessation outcomes of six months follow-up or more, reporting short-term outcomes where longer-term outcomes were not available. We reported study effects as a risk ratio (RR) with a 95% confidence interval (CI). Clinical and statistical heterogeneity limited our ability to pool studies.This updated review includes a total of 28 studies with over 45,000 participants. Some Internet programmes were intensive and included multiple outreach contacts with participants, whilst others relied on participants to initiate and maintain use.Fifteen trials compared an Internet intervention to a non-Internet-based smoking cessation intervention or to a no-intervention control. Ten of these recruited adults, one recruited young adult university students and two recruited adolescents. Seven of the trials in adults had follow-up at six months or longer and compared an Internet intervention to usual care or printed self help. In a post hoc subgroup analysis, pooled results from three trials that compared interactive and individually tailored interventions to usual care or written self help detected a statistically significant effect in favour of the intervention (RR 1.48, 95% CI 1.11 to 2.78). However all three trials were judged to be at high risk of bias in one domain and high statistical heterogeneity was detected (I² = 53%), with no obvious clinical explanation. Pooled results from two studies of an interactive, tailored intervention involving the Internet and automated phone contacts also detected a significant effect (RR 2.05, 95% CI 1.42 to 2.97, I² = 42%). Results from a sixth study comparing an interactive but non-tailored intervention to control did not detect a significant effect, nor did the seventh study, which compared a non-interactive, non-tailored intervention to control. Three trials comparing Internet interventions to face-to-face or phone counselling also did not detect evidence of an effect, nor did two trials evaluating Internet interventions as adjuncts to other behavioural interventions. A trial in college students increased point prevalence abstinence after 30 weeks but had no effect on sustained abstinence. Two small trials in adolescents did not detect an effect on cessation compared to control.Fourteen trials, all in adult populations, compared different Internet sites or programmes. Pooled estimates from three trials that compared tailored and/or interactive Internet programmes with non-tailored, non-interactive Internet programmes did not detect evidence of an effect (RR 1.12, 95% CI 0.95 to 1.32, I² = 0%). One trial detected evidence of a benefit from a tailored email compared to a non-tailored one, whereas a second trial comparing tailored messages to a non-tailored message did not detect evidence of an effect. Trials failed to detect a benefit of including a mood management component (three trials), or an asynchronous bulletin board.Results suggest that some Internet-based interventions can assist smoking cessation at six months or longer, particularly those which are interactive and tailored to individuals. However, the trials that compared Internet interventions with usual care or self help did not show consistent effects and were at risk of bias. Further research is needed despite 28 studies on the subject. Future studies should carefully consider optimising the interventions which promise most effect such as tailoring and interactivity. HubMed – addiction