Characterizing exons 11 and 1 promoters of the mu opioid receptor (Oprm) gene in transgenic mice

BACKGROUND: The complexity of the mouse mu opioid receptor (Oprm) gene was demonstrated by the identification of multiple alternatively spliced variants and promoters. Our previous studies have identified a novel promoter, exon 11 (E11) promoter, in the mouse Oprm gene. The E11 promoter is located ~10 kb upstream of the exon 1 (E1) promoter. The E11 promoter controls the expression of nine splice variants in the mouse Oprm gene. Distinguished from the TATA-less E1 promoter, the E11 promoter resembles a typical TATA-containing eukaryote class II promoter. The aim of this study is to further characterize the E11 and E1 promoters in vivo using a transgenic mouse model. RESULTS: We constructed a ~20 kb transgenic construct in which a 3.7 kb E11 promoter region and an 8.9 kb E1 promoter region controlled expression of tau/LacZ and tau/GFP reporters, respectively. The construct was used to establish a transgenic mouse line. The expression of the reporter mRNAs, determined by a RT-PCR approach, in the transgenic mice during embryonic development displayed a temporal pattern similar to that of the endogenous promoters. X-gal staining for tau/LacZ reporter and GFP imaging for tau/GFP reporter showed that the transgenic E11 and E1 promoters were widely expressed in various regions of the central nervous system (CNS). The distribution of tau/GFP reporter in the CNS was similar to that of MOR-1-like immunoreactivity using an exon 4-specific antibody. However, differential expression of both promoters was observed in some CNS regions such as the hippocampus and substantia nigra, suggesting that the E11 and E1 promoters were regulated differently in these regions. CONCLUSION: We have generated a transgenic mouse line to study the E11 and E1 promoters in vivo using tau/LacZ and tau/GFP reporters. The reasonable relevance of the transgenic model was demonstrated by the temporal and spatial expression of the transgenes as compared to those of the endogenous transcripts. We believe that these transgenic mice will provide a useful model for further characterizing the E11 and E1 promoter in vivo under different physiological and pathological circumstances such as chronic opioid treatment and chronic pain models

Identification and differential regional expression of KOR-3/ORL-1 gene splice variants in mouse brain

AbstractKOR-3, also known as ORL-1, is a member of the opioid receptor family, encoding the murine receptor for orphanin FQ/nociceptin. In the current studies we have identified five different splice variants of KOR-3 in mouse brain, three of which have not been previously reported. In addition to variants with a 15 bp deletion at the 3′-end of the first coding exon (KOR-3d) and an 81 bp insertion between the second and third coding exons (KOR-3e), three new variants with insertions of 34 (KOR-3a), 98 (KOR-3b), and 139 bp (KOR-3c) between the first and second coding exons have been obtained. The expression of the three variants in mouse brain varies markedly among brain regions with a distribution which is quite distinct from KOR-3 itself. Of greatest interest was the presence of high levels of KOR-3a in the striatum, a region with no demonstrable KOR-3, and in the cortex. KOR-3c was seen in the periaqueductal gray and hypothalamus, regions where KOR-3 predominated. The brainstem had similar levels of KOR-3, KOR-3a, and KOR-3d. In contrast, KOR-3d was most prominent in the cerebellum. KOR-3b levels were very low throughout

Identification and characterization of seven new exon 11-associated splice variants of the rat mu opioid receptor gene, OPRM1

<p>Abstract</p> <p>Background</p> <p>The mouse mu opioid receptor (OPRM1) gene undergoes extensive alternative splicing at both the 3'- and 5'-ends of the gene. Previously, several C-terminal variants generated through 3' splicing have been identified in the rat OPRM1 gene. In both mice and humans 5' splicing generates a number of exon 11-containing variants. Studies in an exon 11 knockout mouse suggest the functional importance of these exon 11-associated variants in mediating the analgesic actions of a subset of mu opioids, including morphine-6β-glucuronide (M6G) and heroin, but not others such as morphine and methadone. We now have examined 5' splicing in the rat.</p> <p>Results</p> <p>The current studies identified in the rat a homologous exon 11 and seven exon 11-associated variants, suggesting conservation of exon 11 and its associated variants among mouse, rat and human. RT-PCR revealed marked differences in the expression of these variants across several brain regions, implying region-specific mRNA processing of the exon 11-associated variants. Of the seven rat exon 11-associated variants, four encoded the identical protein as found in rMOR-1, two predicted 6 TM variants, and one, rMOR-1H2, generated a novel N-terminal variant in which a stretch of an additional 50 amino acids was present at the N-terminus of the previously established rMOR-1 sequence. When expressed in CHO cells, the presence of the additional 50 amino acids in rMOR-1H2 significantly altered agonist-induced G protein activation with little effect on opioid binding.</p> <p>Conclusion</p> <p>The identification of the rat exon 11 and its associated variants further demonstrated conservation of 5' splicing in OPRM1 genes among rodents and humans. The functional relevance of these exon 11 associated variants was suggested by the region-specific expression of their mRNAs and the influence of the N-terminal sequence on agonist-induced G protein coupling in the novel N-terminal variant, rMOR-1H2. The importance of the exon 11-associated variants in mice in M6G and heroin analgesia revealed in the exon 11 knockout mouse implies that these analogous rat variants may also play similar roles in rat. The complexity created by alternative splicing of the rat OPRM1 gene may provide important insights of understanding the diverse responses to the various mu opioids seen in rats.</p

Identification and characterization of seven new exon 11-associated splice variants of the rat mu opioid receptor gene, OPRM1

<p>Abstract</p> <p>Background</p> <p>The mouse mu opioid receptor (OPRM1) gene undergoes extensive alternative splicing at both the 3'- and 5'-ends of the gene. Previously, several C-terminal variants generated through 3' splicing have been identified in the rat OPRM1 gene. In both mice and humans 5' splicing generates a number of exon 11-containing variants. Studies in an exon 11 knockout mouse suggest the functional importance of these exon 11-associated variants in mediating the analgesic actions of a subset of mu opioids, including morphine-6β-glucuronide (M6G) and heroin, but not others such as morphine and methadone. We now have examined 5' splicing in the rat.</p> <p>Results</p> <p>The current studies identified in the rat a homologous exon 11 and seven exon 11-associated variants, suggesting conservation of exon 11 and its associated variants among mouse, rat and human. RT-PCR revealed marked differences in the expression of these variants across several brain regions, implying region-specific mRNA processing of the exon 11-associated variants. Of the seven rat exon 11-associated variants, four encoded the identical protein as found in rMOR-1, two predicted 6 TM variants, and one, rMOR-1H2, generated a novel N-terminal variant in which a stretch of an additional 50 amino acids was present at the N-terminus of the previously established rMOR-1 sequence. When expressed in CHO cells, the presence of the additional 50 amino acids in rMOR-1H2 significantly altered agonist-induced G protein activation with little effect on opioid binding.</p> <p>Conclusion</p> <p>The identification of the rat exon 11 and its associated variants further demonstrated conservation of 5' splicing in OPRM1 genes among rodents and humans. The functional relevance of these exon 11 associated variants was suggested by the region-specific expression of their mRNAs and the influence of the N-terminal sequence on agonist-induced G protein coupling in the novel N-terminal variant, rMOR-1H2. The importance of the exon 11-associated variants in mice in M6G and heroin analgesia revealed in the exon 11 knockout mouse implies that these analogous rat variants may also play similar roles in rat. The complexity created by alternative splicing of the rat OPRM1 gene may provide important insights of understanding the diverse responses to the various mu opioids seen in rats.</p

Alternative pre-mRNA splicing of the mu opioid receptor gene, OPRM1: Insight into complex mu opioid actions

Most opioid analgesics used clinically, including morphine and fentanyl, as well as the recreational drug heroin, act primarily through the mu opioid receptor, a class A Rhodopsin-like G protein-coupled receptor (GPCR). The single-copy mu opioid receptor gene

GABA, progesterone and zona pellucida activation of PLA2 and regulation by MEK-ERK1/2 during acrosomal exocytosis in guinea pig spermatozoa

AbstractWe investigated whether GABA activates phospholipase A2 (PLA2) during acrosomal exocytosis, and if the MEK-ERK1/2 pathway modulates PLA2 activation initiated by GABA, progesterone or zona pellucida (ZP). In guinea pig spermatozoa prelabelled with [14C]arachidonic acid or [14C]choline chloride, GABA stimulated a decrease in phosphatidylcholine (PC), and release of arachidonic acid and lysoPC, during exocytosis. These lipid changes are indicative of PLA2 activation and appear essential for exocytosis since inclusion of aristolochic acid (a PLA2 inhibitor) abrogated them, along with exocytosis. GABA activation of PLA2 seems to be mediated, at least in part, by diacylglycerol (DAG) and protein kinase C since inclusion of the DAG kinase inhibitor R59022 enhanced PLA2 activity and exocytosis stimulated by GABA, whereas exposure to staurosporine decreased both. GABA-, progesterone- and ZP-induced release of arachidonic acid and exocytosis were prevented by U0126 and PD98059 (MEK inhibitors). Taken together, our results suggest that PLA2 plays a fundamental role in agonist-stimulated exocytosis and that MEK-ERK1/2 are involved in PLA2 regulation during this process

Prognostic value of neutrophil-to-lymphocyte ratio in breast cancer

AbstractInflammation is an essential component of pathogenesis and progression of cancer. A high neutrophil-to-lymphocyte ratio (NLR) is considered as a prognostic indicator for breast cancer. This meta-analysis was conducted to establish the overall accuracy of the NLR test in the diagnosis of breast cancer. A comprehensive search of the literature was conducted by using PubMed, Web of Science and China National Knowledge Infrastructure (CNKI). Published studies dating up to July 2014 and 4,293 patients were enrolled in the present study. In order to evaluate the association between NLR and overall survival (OS), disease-free survival (DFS), recurrence-free survival (RFS) or cancer specific survival (CSS), the hazard ratios (HRs) and their 95% confidence intervals (CIs) were extracted. OS was the primary outcome. The results suggested that increased NLR was a strong predictor for OS with HR of 2.28 (95% CI=1.08–4.80, Pheterogeneity<0.001). Stratified analyses indicated that a high NLR appeared to be a negative prognostic marker in Caucasian populations (HR=4.53, 95% CI=3.11–6.60, Pheterogeneity=0.096), multivariate analysis method (HR=2.10, 95% CI=1.52–2.89, Pheterogeneity=0.591), and mixed metastasis (HR=4.53, 95% CI=3.11–6.60, Pheterogeneity=0.096). Elevated NLR was associated with a high risk for DFS (HR=1.38, 95% CI=1.09–1.74, Pheterogeneity=0.050) and in subgroups of multivariate analysis (HR=1.64, 95% CI=1.25–2.14, Pheterogeneity=0.545) and mixed metastasis (HR=1.99, 95% CI=1.28–3.09, Pheterogeneity=0.992). In summary, NLR could be considered as a predictive factor for patients with breast cancer

Differential Expressions of the Alternatively Spliced Variant mRNAs of the µ Opioid Receptor Gene, OPRM1, in Brain Regions of Four Inbred Mouse Strains

The µ opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing in rodents and humans, with dozens of alternatively spliced variants of the OPRM1 gene. The present studies establish a SYBR green quantitative PCR (qPCR) assay to more accurately quantify mouse OPRM1 splice variant mRNAs. Using these qPCR assays, we examined the expression of OPRM1 splice variant mRNAs in selected brain regions of four inbred mouse strains displaying differences in µ opioid-induced tolerance and physical dependence: C56BL/6J, 129P3/J, SJL/J and SWR/J. The complete mRNA expression profiles of the OPRM1 splice variants reveal marked differences of the variant mRNA expression among the brain regions in each mouse strain, suggesting region-specific alternative splicing of the OPRM1 gene. The expression of many variants was also strain-specific, implying a genetic influence on OPRM1 alternative splicing. The expression levels of a number of the variant mRNAs in certain brain regions appear to correlate with strain sensitivities to morphine analgesia, tolerance and physical dependence in four mouse strains