Pharmacological profiles of opioid ligands at Kappa opioid receptors

BACKGROUND: The aim of the present study was to describe the activity of a set of opioid drugs, including partial agonists, in a human embryonic kidney cell system stably expressing only the mouse κ-opioid receptors. Receptor activation was assessed by measuring the inhibition of cyclic adenosine mono phosphate (cAMP) production stimulated by 5 μM forskolin. Intrinsic activities and potencies of these ligands were determined relative to the endogenous ligand dynorphin and the κ agonist with the highest intrinsic activity that was identified in this study, fentanyl. RESULTS: Among the ligands studied naltrexone, WIN 44,441 and dezocine, were classified as antagonists, while the remaining ligands were agonists. Intrinsic activity of agonists was assessed by determining the extent of inhibition of forskolin-stimulated cAMP production. The absolute levels of inhibition of cAMP production by each ligand was used to describe the rank order of intrinsic activity of the agonists; fentanyl = lofentanil ≥ hydromorphone = morphine = nalorphine ≥ etorphine ≥ xorphanol ≥ metazocine ≥ SKF 10047 = cyclazocine ≥ butorphanol > nalbuphine. The rank order of affinity of these ligands was; cyclazocine > naltrexone ≥ SKF 10047 ≥ xorphanol ≥ WIN 44,441 > nalorphine > butorphanol > nalbuphine ≥ lofentanil > dezocine ≥ metazocine ≥ morphine > hydromorphone > fentanyl. CONCLUSION: These results elucidate the relative activities of a set of opioid ligands at κ-opioid receptor and can serve as the initial step in a systematic study leading to understanding of the mode of action of these opioid ligands at this receptor

New insight into the role of the β3 subunit of the GABAA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout

<p>Abstract</p> <p>Background</p> <p>The β3 subunit of the γ-aminobutyric acid type A receptor (GABA_A-R) has been reported to be important for palate formation, anesthetic action, and normal nervous system function. This subunit has also been implicated in the pathogenesis of Angelman syndrome and autism spectrum disorder. To further investigate involvement of this subunit, we previously produced mice with a global knockout of β3. However, developmental abnormalities, compensation, reduced viability, and numerous behavioral abnormalities limited the usefulness of that murine model. To overcome many of these limitations, a mouse line with a conditionally inactivated β3 gene was engineered.</p> <p>Results</p> <p>Gene targeting and embryonic stem cell technologies were used to create mice in which exon 3 of the β3 subunit was flanked by loxP sites (i.e., floxed). Crossing the floxed β3 mice to a cre general deleter mouse line reproduced the phenotype of the previously described global knockout. Pan-neuronal knockout of β3 was achieved by crossing floxed β3 mice to Synapsin I-cre transgenic mice. Palate development was normal in pan-neuronal β3 knockouts but ~61% died as neonates. Survivors were overtly normal, fertile, and were less sensitive to etomidate. Forebrain selective knockout of β3 was achieved using α CamKII-cre transgenic mice. Palate development was normal in forebrain selective β3 knockout mice. These knockouts survived the neonatal period, but ~30% died between 15–25 days of age. Survivors had reduced reproductive fitness, reduced sensitivity to etomidate, were hyperactive, and some became obese.</p> <p>Conclusion</p> <p>Conditional inactivation of the β3 gene revealed novel insight into the function of this GABA_A-R subunit. The floxed β3 knockout mice described here will be very useful for conditional knockout studies to further investigate the role of the β3 subunit in development, ethanol and anesthetic action, normal physiology, and pathophysiologic processes.</p

A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries

Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol

Selective influence on contextual memory: Physiochemical properties associated with selectivity of benzodiazepine ligands at GABA\u3csub\u3eA\u3c/sub\u3e receptors containing the α5 subunit

Ligands that bind to the benzodiazepine binding site on the GABA receptor can attenuate or potentiate cognition. To investigate this property, the chemical determinants favoring selective binding or selective activation of the α5β2γ2 and α1β2γ2 GABA receptor isoforms were examined. A 3D-pharmacophore, developed from a diverse set of BDZR ligands, was used as an initial basis for multivariate discriminant, fragment, and 3D-quantitative structure-activity relationship analyses, which formed the criteria for selection of additional compounds for study. We found that the electrostatic potential near the ligands\u27 terminal substituent correlated with its binding selectivity toward the α5β2γ2 versus α1β2γ2 isoform; while the fragment length and frontier molecular orbital energetics correlated with a compounds influence on electrophysiological activity. Compounds with promising α5 profiles were further assessed for their ability to attenuate scopolamine-induced contextual memory impairment in mice. Surprisingly, both weak inverse agonist and antagonists that display binding selectivity toward the α5β2γ2 isoform were able to attenuate contextual memory impairment. © 2008 American Chemical Society. A

New insight into the role of the β3 subunit of the GABA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout-2

<p><b>Copyright information:</b></p><p>Taken from "New insight into the role of the β3 subunit of the GABA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout"</p><p>http://www.biomedcentral.com/1471-2202/8/85</p><p>BMC Neuroscience 2007;8():85-85.</p><p>Published online 10 Oct 2007</p><p>PMCID:PMC2100059.</p><p></p>d female pan-neuronal β3 knockouts were less sensitive to the sedative/hypnotic effects of etomidate (20 mg/kg) as evidenced by the reduced duration of the LORR compared to same sex controls. B. Ethanol-induced (3.5 g/kg) LORR did not differ between controls and pan-neuronal β3 knockouts. C. Etomidate-induced (20 mg/kg) LORR in forebrain selective β3 knockouts was not dependent on gender but knockouts were less sensitive compared to controls. D. Ethanol-induced (3.5 g/kg, i.p.) LORR of forebrain selective knockout mice did not differ compared to controls. *, p < 0.05; **, p ≤ 0.01

New insight into the role of the β3 subunit of the GABA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout-6

<p><b>Copyright information:</b></p><p>Taken from "New insight into the role of the β3 subunit of the GABA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout"</p><p>http://www.biomedcentral.com/1471-2202/8/85</p><p>BMC Neuroscience 2007;8():85-85.</p><p>Published online 10 Oct 2007</p><p>PMCID:PMC2100059.</p><p></p>(yellow boxes) that were used for Southern blot analysis. The targeting construct included a neomycin (neo) cassette that was flanked by two frt sites (blue triangles), two loxP sites (red triangles), a thymidine kinase (TK) cassette, and plasmid vector backbone (broken, wavy line). During vector construction, the BglII restriction sites shown in grey lettering were destroyed. Also shown is a correctly targeted locus (Fneo), a locus following FLPe mediated deletion of the neo cassette (F), and a locus following Cre mediated deletion of exon 3 (f). Abbreviations: BamHI, B; BglII, Bg; EcoRV, RV