HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants

G protein-coupled receptors (GPCRs) are notoriously difficult to detect in native tissues. In an effort to resolve this problem, we have developed a novel mouse model by fusing the hemagglutinin (HA)-epitope tag sequence to the amino-terminus of the µ-opioid receptor (MOP). Although HA-MOP knock-in mice exhibit reduced receptor expression, we found that this approach allowed for highly efficient immunodetection of low abundant GPCR targets. We also show that the HA-tag facilitates both high-resolution imaging and immunoisolation of MOP. Mass spectrometry (MS) confirmed post-translational modifications, most notably agonist-selective phosphorylation of carboxyl-terminal serine and threonine residues. MS also unequivocally identified the carboxyl-terminal 387LENLEAETAPLP398 motif, which is part of the canonical MOP sequence. Unexpectedly, MS analysis of brain lysates failed to detect any of the 15 MOP isoforms that have been proposed to arise from alternative splicing of the MOP carboxyl-terminus. For quantitative analysis, we performed multiple successive rounds of immunodepletion using the well-characterized rabbit monoclonal antibody UMB-3 that selectively detects the 387LENLEAETAPLP398 motif. We found that >98% of HA-tagged MOP contain the UMB-3 epitope indicating that virtually all MOP expressed in the mouse brain exhibit the canonical amino acid sequence

Proteome analysis of human gastric cardia adenocarcinoma by laser capture microdissection

<p>Abstract</p> <p>Background</p> <p>The incidence of gastric cardiac adenocarcinoma (GCA) has been increasing in the past two decades in China, but the molecular changes relating to carcinogenesis have not been well characterised.</p> <p>Methods</p> <p>In this study, we used a comparative proteomic approach to analyse the malignant and nonmalignant gastric cardia epithelial cells isolated by navigated laser capture microdissection (LCM) from paired surgical specimens of human GCA.</p> <p>Results</p> <p>Twenty-seven spots corresponding to 23 proteins were consistently differentially regulated. Fifteen proteins were shown to be up-regulated, while eight proteins were shown to be down-regulated in malignant cells compared with nonmalignant columnar epithelial cells. The identified proteins appeared to be involved in metabolism, chaperone, antioxidation, signal transduction, apoptosis, cell proliferation, and differentiation. In addition, expressions of HSP27, 60, and Prx-2 in GCA specimens were further confirmed by immunohistochemical and western blot analyses.</p> <p>Conclusion</p> <p>These data indicate that the combination of navigated LCM with 2-DE provides an effective strategy for discovering proteins that are differentially expressed in GCA. Such proteins may contribute in elucidating the molecular mechanisms of GCA carcinogenesis. Furthermore, the combination provides potential clinical biomarkers that aid in early detection and provide potential therapeutic targets.</p

A Citrus based sensory functional food ingredient induces antidepressant-like effects: possible involvement of an interplay between the olfactory and the serotonergic systems

International audienceIn the present study, we examined the neurobehavioral effects of a sensory functional food ingredient mainly based on Citrus sinensis extracts (D11399) using a battery of tests recapitulating various endophenotypes of depression such as anxiety in the open field (OF), the elevated plus-maze (EPM), and the novelty suppressed feeding (NSF), self-care in the splash test (ST), despair in the forced swimming task (FST) but also anhedonia in the sucrose preference test (SPT) in mice. A one-week oral administration of D11399 promoted anxiolytic- and antidepressant-like responses in naïve mice subjected to the NSF and FST. In a marked contrast, the administration of D11399 by oral gavage or the inhibition of olfaction by methimazole prevented such beneficial effects. We further investigated the neurobehavioral properties of a ten-week oral administration of D11399 in the corticosterone (CORT) mouse model of depression. Interestingly, D11399 also elicited anxiolytic- and antidepressant-like effects in various paradigms. To characterize the putative underpinning neurobiological mechanisms in CORT mice, we investigated whether cellular and molecular processes commonly associated with antidepressant responses such as monoaminergic neurotransmission and neuronal maturation in the hippocampus were impacted. Although D11399 did not modify the hippocampal extracellular levels of monoamines (i.e. serotonin and norepinephrine), it reversed the ability of CORT to decrease serotonin neurons firing rate in the dorsal raphe and neuronal maturation in the hippocampus. These findings suggest that the anxiolytic- and antidepressant-like effects of this sensory functional food ingredient are closely related with olfaction and likely a concomitant change in the activity of the central serotonergic system. Further experiments are warranted to precise the neuronal circuits linking sensorial and emotional modalities and identify innovative therapeutic strategies aimed to relieve depressive endophenotypes

Involvement of Protein Degradation by the Ubiquitin Proteasome System in Opiate Addictive Behaviors

Plastic changes in the nucleus accumbens (NAcc), a structure occupying a key position in the neural circuitry related to motivation, are among the critical cellular processes responsible for drug addiction. During the last decade, it has been shown that memory formation and related neuronal plasticity may rely not only on protein synthesis but also on protein degradation by the ubiquitin proteasome system (UPS). In this study, we assess the role of protein degradation in the NAcc in opiate-related behaviors. For this purpose, we coupled behavioral experiments to intra-accumbens injections of lactacystin, an inhibitor of the UPS. We show that protein degradation in the NAcc is mandatory for a full range of animal models of opiate addiction including morphine locomotor sensitization, morphine conditioned place preference, intra-ventral tegmental area morphine self-administration and intra-venous heroin self-administration but not for discrimination learning rewarded by highly palatable food. This study provides the first evidence of a specific role of protein degradation by the UPS in addiction