The analgesic and addictive properties of morphine and other opioid drugs are thought to result from their interaction with mu opioid receptors. Using a delta opioid receptor cDNA as a probe, we have isolated a murine mu opioid receptor cDNA clone (mMOR). Stable expression of mMOR in Chinese hamster ovary cells conferred high binding affinity for mu receptor ligands including morphine and [D-Ala2,N-methyl-Phe4,Gly5-ol]-enkephalin and low affinity for delta and kappa preferring ligands. Treatment of these cell lines with morphine and other mu agonists inhibited forskolin-induced cAMP accumulation, demonstrating a functional coupling of mMOR to the inhibition of adenylate cyclase. The predicted amino acid sequence of mMOR shares approximately 55% overall amino acid identity with the delta receptor and approximately 97% identity with the recently reported rat mu opioid receptor. Expression of the mu receptor in mouse brain as revealed by in situ hybridization parallels the reported pattern of distribution of mu-selective ligand binding sites. Chromosomal localization (to mouse chromosome 10) and Southern analysis are consistent with a single mu opioid receptor gene in the mouse genome, suggesting that the various pharmacologically distinct forms of the mu receptor arise from alternative splicing, post-translational events, or from a highly divergent gene(s).

Progesterone, an agonist for the progesterone receptor (PR), can be an efficacious and well-tolerated treatment in endometrial cancer. The clinical use of progesterone is limited due to the lack of biomarkers that predict hormone sensitivity. Despite its efficacy in cancer therapy, mechanisms and site of action for progesterone remain unknown. Using an in vivo endometrial cancer mouse model driven by clinically relevant genetic changes but dichotomous responses to hormonal therapy, we demonstrate that signaling through stromal PR is necessary and sufficient for progesterone anti-tumor effects. Endometrial cancers resulting from epithelial loss of PTEN (PTENKO) were hormone sensitive and had abundant expression of stromal PR. Stromal deletion of PR as a single genetic change in these tumors induced progesterone resistance indicating that paracrine signaling through the stroma is essential for the progesterone therapeutic effects. A hormone refractory endometrial tumor with low levels of stromal PR developed when activation of KRAS was coupled with PTEN-loss (PTENKO/Kras). The innate progesterone resistance in PTENKO/Kras tumors stemmed from methylation of PR in the tumor microenvironment. Add-back of stromal PR expressed from a constitutively active promoter sensitized these tumors to progesterone therapy. Results demonstrate that signaling through stromal PR is sufficient for inducing hormone responsiveness. Our findings suggest that epigenetic de-repression of stromal PR could be a potential therapeutic target for sensitizing hormone refractory endometrial tumors to progesterone therapy. Based on these results, stromal expression of PR may emerge as a reliable biomarker in predicting response to hormonal therapy.

Using a large consortium of undergraduate students in an organized program at the University of California, Los Angeles (UCLA), we have undertaken a functional genomic screen in the Drosophila eye. In addition to the educational value of discovery-based learning, this article presents the first comprehensive genomewide analysis of essential genes involved in eye development. The data reveal the surprising result that the X chromosome has almost twice the frequency of essential genes involved in eye development as that found on the autosomes.

Background Proton leak (H+ leak) dissipates mitochondrial membrane potential (mΔΨ) through the reentry of protons into the mitochondrial matrix independent of ATP synthase. Changes in H+ leak may affect reactive oxygen species (ROS) production. We measured H+ leak and ROS production during ischemia-reperfusion and ischemic preconditioning (IPC) and examined how changing mitochondrial respiration affected mΔΨ and ROS production. Materials/Methods Isolated rat hearts (n=6/group) were subjected to either Control-IR or IPC. Rate pressure product (RPP) was measured. Mitochondria were isolated at end reperfusion. Respiration was measured by polarography and titrated with increasing concentrations of malonate (0.5-2mM). mΔΨ was measured using a tetraphenylphosphonium electrode. H+ leak is the respiratory rate required to maintain membrane potential at -150mV in the presence of oligomycin-A Mitochondrial complex III ROS production was measured by fluorometry using Amplex-Red. Results IPC improved recovery of RPP at end reperfusion (63±4% vs. 21±2% in Control-IR, p<0.05). Ischemia-reperfusion caused increased H+ leak (94±12 vs. 31±1 nanomoles O/mg protein/min in Non-Ischemic Control, p<0.05). IPC attenuates these increases (55±9 nanomoles O/mg protein/min, p< 0.05 vs. Control-IR). IPC reduced mitochondrial ROS production compared to Control-IR (31±2 vs. 40±3 nanomoles/mg protein/min, p<0.05). As mitochondrial respiration decreased, mΔΨ and mitochondrial ROS production also decreased. ROS production remained lower in IPC than in Control-IR for all mΔΨ and respiration rates. Conclusions Increasing H+ leak is not associated with increased ROS production. IPC decreases both the magnitude of H+ leak and ROS production after ischemia-reperfusion.