Selective and antagonist-dependent µ-opioid receptor activation by the combination of 2-{[2-(6-chloro-3,4-dihydro-1(2H)-quinolinyl)-2-oxoethyl]sulfanyl}-5-phenyl-4,6-(1H,5H)-pyrimidinedione and naloxone/naltrexone.

We identified, via high-throughput screening using a FLIPR® calcium assay, compound 1, which incorporated a dihydroquinolinyl-2-oxoethylsulfanyl-(1H,5H)-pyrimidinedione core and activated the µ-opioid receptor (MOR) in the presence of naloxone or naltrexone. A structure-activity relationship study of the analogs of 1 led to the design of compound 21, which activated MOR in the presence of naloxone with an EC50 of 3.3 ± 0.2 µM. MOR activation by the compound 21-antagonist pair was antagonist-dependent. Compound 21 did not affect the potency of the orthosteric agonist, morphine, toward MOR, indicating that it affected the function of MOR antagonists rather than that of the agonists. Computer modeling of the compound 21-MOR-naloxone complex revealed major interactions between compound 21 and MOR, including hydrogen bonding with Ser196, π-π stacking with Tyr149, and sulfur-aromatic interaction with Trp192. This study may pave the way for developing agents capable of safe and effective MOR modulation.

Kappa opioid receptor controls neural stem cell differentiation via a miR-7a/Pax6 dependent pathway.

Although the roles of opioid receptors in neurogenesis have been implicated in previous studies, the mechanism by which κ-opioid receptor (OPRK1) regulates adult neurogenesis remains elusive. We now demonstrate that two agonists of OPRK1, U50,488H and dynorphin A, inhibit adult neurogenesis by hindering neuronal differentiation of mouse hippocampal neural stem cells (NSCs), both in vitro and in vivo. This effect was blocked by nor-binaltorphimine (nor-BNI), a specific antagonist of OPRK1. By examining neurogenesis-related genes, we found that OPRK1 agonists were able to downregulate the expression of Pax6, Neurog2, and NeuroD1 in mouse hippocampal NSCs, in a way that Pax6 regulates the transcription of Neurog2 and Neurod1 by directly interacting with their promoters. Moreover, this effect of OPRK1 was accomplished by inducing expression of miR-7a, a miRNA that specifically targeted Pax6 by direct interaction with its 3'-UTR sequence, and thereby decreased the levels of Pax6, Neurog2, and NeuroD1, thus resulted in hindrance of neuronal differentiation of NSCs. Thus, by modulating Pax6/Neurog2/NeuroD1 activities via upregulation of miR-7a expression, OPRK1 agonists hinder the neuronal differentiation of NSCs and hence inhibit adult neurogenesis in mouse hippocampus.

Oxycodone in the Opioid Epidemic: High 'Liking', 'Wanting', and Abuse Liability.

It is estimated that nearly a third of people who abuse drugs started with prescription opioid medicines. Approximately, 11.5 million Americans used prescription drugs recreationally in 2016, and in 2018, 46,802 Americans died as the result of an opioid overdose, including prescription opioids, heroin, and illicitly manufactured fentanyl (National Institutes on Drug Abuse (2020) Opioid Overdose Crisis. https://www.drugabuse.gov/drugs-abuse/opioids/opioid-overdose-crisis . Accessed 06 June 2020). Yet physicians will continue to prescribe oral opioids for moderate-to-severe pain in the absence of alternative therapeutics, underscoring the importance in understanding how drug choice can influence detrimental outcomes. One of the opioid prescription medications that led to this crisis is oxycodone, where misuse of this drug has been rampant. Being one of the most highly prescribed opioid medications for treating moderate-to-severe pain as reflected in the skyrocketed increase in retail sales of 866% between 1997 and 2007, oxycodone was initially suggested to be less addictive than morphine. The false-claimed non-addictive formulation of oxycodone, OxyContin, further contributed to the opioid crisis. Abuse was often carried out by crushing the pills for immediate burst release, typically by nasal insufflation, or by liquefying the pills for intravenous injection. Here, we review oxycodone pharmacology and abuse liability as well as present the hypothesis that oxycodone may exhibit a unique pharmacology that contributes to its high likability and abuse susceptibility. We will discuss various mechanisms that likely contribute to the high abuse rate of oxycodone including clinical drug likability, pharmacokinetics, pharmacodynamics, differences in its actions within mesolimbic reward circuity compared to other opioids, and the possibility of differential molecular and cellular receptor interactions that contribute to its selective effects. We will also discuss marketing strategies and drug difference that likely contributes to the oxycodone opioid use disorders and addiction.

Naloxone Facilitates Contextual Learning and Memory in a Receptor-Independent and Tet1-Dependent Manner.

Opioids, like morphine and naloxone, regulate the proliferation and neuronal differentiation of neural stem cells (NSCs) in a receptor-independent and ten-eleven translocation methylcytosine dioxygenase (TET1)-dependent manner in vitro. Whether naloxone regulates hippocampal NSCs and contextual learning in vivo in a similar manner was determined. Naloxone infusion increased the Ki67 and Doublecortin positive cells in subgranular zone of wild type mice, which suggested the increased proliferation and differentiation of hippocampal NSCs in vivo and was consistent with the in vitro functions of naloxone. In addition, naloxone infusion also facilitated the contextual learning and memory of wild type mice. To determine the contribution of µ-opioid receptor (OPRM1) and TET1 to these functions of naloxone, several types of knockout mice were used. Since Tet1-/- mice have high deficiency in contextual learning and memory, Tet1+/- mice were used instead. The abilities of naloxone to regulate NSCs and to facilitate contextual learning were significantly impaired in Tet1+/- mice. In addition, these abilities of naloxone were not affected in Oprm1-/- mice. Therefore, naloxone facilitates contextual learning and memory in a receptor-independent and Tet1-dependent manner, which provides new understanding on the receptor-independent functions of opioids.

Publisher Correction: Manufacturing and preclinical validation of CAR T cells targeting ICAM-1 for advanced thyroid cancer therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Naloxone regulates the differentiation of neural stem cells via a receptor-independent pathway.

The abilities of opioids to activate downstream signaling pathways normally depend on the binding between opioids and their receptors. However, opioids may also function in a receptor-independent manner, especially in neural stem cells (NSCs) in which the expression of opioid receptors and endogenous opioid agonists is low. When two opioids, morphine and naloxone, were used during the early stage of NSC differentiation, increased neurogenesis was observed. However, naloxone methiodide, a membrane impenetrable analog of naloxone, did not affect the NSC differentiation. The abilities of morphine and naloxone to facilitate neurogenesis were also observed in opioid receptor-knockout NSCs. Therefore, morphine and naloxone promote neurogenesis in a receptor-independent manner at least during the early stage. In addition, the receptor-independent functions of opioids were not observed in methylcytosine dioxygenase ten-eleven translocation 1 (Tet1) knockout NSCs. When the expression of opioid receptors increased and the expression of Tet1 decreased during the late stage of NSC differentiation, morphine, but not naloxone, inhibited neurogenesis via traditional receptor-dependent and miR181a-Prox1-Notch-related pathway. In summary, the current results demonstrated the time-dependent effects of opioids during the differentiation of NSCs and provided additional insight on the complex functions of opioids.

Morphine and Naloxone Facilitate Neural Stem Cells Proliferation via a TET1-Dependent and Receptor-Independent Pathway.

Normally, opioids function in a receptor-dependent manner. They bind to opioid receptors, activate or inhibit receptor activation, and subsequently modulate downstream signal transduction. However, the complex functions of opioids and the low expression of opioid receptors and their endogenous peptide agonists in neural stem cells (NSCs) suggest that some opioids may also modulate NSCs via a receptor-independent pathway. In the current study, two opioids, morphine and naloxone, are demonstrated to facilitate NSC proliferation via a receptor-independent and ten-eleven translocation methylcytosine dioxygenase 1 (TET1)-dependent pathway. Morphine and naloxone penetrate cell membrane, bind to TET1 protein via three key residues (1,880-1,882), and subsequently result in facilitated proliferation of NSCs. In addition, the two opioids also inhibit the DNA demethylation ability of TET1. In summary, the current results connect opioids and DNA demethylation directly at least in NSCs and extend our understanding on both opioids and NSCs.

Manufacturing and preclinical validation of CAR T cells targeting ICAM-1 for advanced thyroid cancer therapy.

While the majority of thyroid cancer patients are easily treatable, those with anaplastic or poorly differentiated recurrent thyroid carcinomas have a very poor prognosis with a median survival of less than a year. Previously, we have shown a significant correlation between ICAM-1 overexpression and malignancy in thyroid cancer, and have pioneered the use of ICAM-1 targeted CAR T cells as a novel treatment modality. For clinical translation of this novel modality, we designed CAR T cells possessing micromolar rather than nanomolar affinity to ICAM-1 to avoid cytotoxicity in normal cells with basal levels of ICAM-1 expression. Herein, we report the automated process of CAR T cell manufacturing with CliniMACS Prodigy (Miltenyi Biotec) using cryopreserved peripheral blood leukocytes from apheresis collections. Using Prodigy, thawed leukopak cells were enriched for CD4+ and CD8+ T cells, subjected to double transduction using lentiviral vector, and expanded in culture for a total of 10 days with a final yield of 2-4 × 109 cells. The resulting CAR T cells were formulated for cryopreservation to be used directly for infusion into patients after thawing with no further processing. We examined cross-reactivity of CAR T cells toward both human and murine ICAM-1 and ICAM-1 expression in human and mouse tissues to demonstrate that both efficacy and on-target, off-tumor toxicity can be studied in our preclinical model. Selective anti-tumor activity in the absence of toxicity provides proof-of-concept that micromolar affinity tuned CAR T cells can be used to target tumors expressing high levels of antigen while avoiding normal tissues expressing basal levels of the same antigen. These studies support the initiation of a phase I study to evaluate the safety and potential efficacy of micromolar affinity tuned CAR T cells against newly diagnosed anaplastic and refractory or recurrent thyroid cancers.

Delta-opioid receptor antagonist naltrindole reduces oxycodone addiction and constipation in mice.

Oxycodone, a widely prescribed and very potent oral opioid analgesic agent, is highly addictive and has many side effects, including troublesome constipation. Our studies in mice indicated that pretreatment of naltrindole did not significantly affect the analgesic efficacy of oxycodone but attenuated the tolerance and withdrawal induced by chronic oxycodone administration. Naltrindole also attenuated the oxycodone-induced rewarding and re-instatement behaviors, as shown by the conditioned place preference test. Further, oxycodone-induced decrease in intestinal transit (i.e., constipation) was reduced by naltrindole. However, naltrindole did not block the respiratory depression produced by oxycodone. Taken together, these data suggest that naltrindole can attenuate some major side effects while retaining the analgesic efficacy of oxycodone in mice. Naltrindole and oxycodone may have the potential to be a potent analgesic combination with much lower levels of oxycodone's side effects of addictive liability and constipation.

Convallatoxin enhance the ligand-induced mu-opioid receptor endocytosis and attenuate morphine antinociceptive tolerance in mice.

Morphine is a unique opioid analgesic that activates the mu-opioid receptor (MOR) without efficiently promoting its endocytosis that may underlie side effects. Our objective was to discover a novel enhancer of ligand-induced MOR endocytosis and determine its effects on analgesia, tolerance and dependence. We used high-throughput screening to identify convallatoxin as an enhancer of ligand-induced MOR endocytosis with high potency and efficacy. Treatment of cells with convallatoxin enhanced morphine-induced MOR endocytosis through an adaptor protein 2 (AP2)/clathrin-dependent mechanism, attenuated morphine-induced phosphorylation of MOR, and diminished desensitization of membrane hyperpolarization. Furthermore, co-treatment with chronic convallatoxin reduced morphine tolerance in animal models of acute thermal pain and chronic inflammatory pain. Acute convallatoxin administration reversed morphine tolerance and dependence in morphine-tolerant mice. These findings suggest convallatoxin are potentially therapeutic for morphine side effects and open a new avenue to study MOR trafficking.

M1 muscarinic receptors regulate the phosphorylation of AMPA receptor subunit GluA1 via a signaling pathway linking cAMP-PKA and PI3K-Akt.

M1 muscarinic acetylcholine receptors are highly expressed in key areas that control cognition, such as the cortex and hippocampus, representing one potential therapeutic target for cognitive dysfunctions of Alzheimer's disease and schizophrenia. We have reported that M1 receptors facilitate cognition by promoting membrane insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor AMPA receptor subunit 1 (GluA1) through phosphorylation at Ser845. However, the signaling pathway is still unclear. Here we showed that adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and PKA inhibitor KT5720 inhibited enhancement of phosphorylation of Ser845 and membrane insertion of GluA1 induced by M1 receptor activation. Furthermore, PI3K inhibitor LY294002 and protein kinase B (Akt) inhibitor IV blocked the effects of M1 receptors as well. Remarkably, the increase of the activity of PI3K-Akt signaling induced by M1 receptor activation could be abolished by cAMP-PKA inhibitors. Moreover, inhibiting the mammalian target of rapamycin (mTOR) complex 1, an important downstream effector of PI3K-Akt, by short-term application of rapamycin attenuated the effects of M1 receptors on GluA1. Furthermore, such effect was unrelated to possible protein synthesis promoted by mTOR. Taken together, these data demonstrate that M1 receptor activation induces membrane insertion of GluA1 via a signaling linking cAMP-PKA and PI3K-Akt-mTOR pathways but is irrelevant to protein synthesis.-Zhao, L.-X., Ge, Y.-H., Li, J.-B., Xiong, C.-H., Law, P.-Y., Xu, J.-R., Qiu, Y., Chen, H.-Z. M1 muscarinic receptors regulate the phosphorylation of AMPA receptor subunit GluA1 via a signaling pathway linking cAMP-PKA and PI3K-Akt.

The in vivo antinociceptive and µ-opioid receptor activating effects of the combination of N-phenyl-2',4'-dimethyl-4,5'-bi-1,3-thiazol-2-amines and naloxone.

Morphine is widely used for the treatment of severe pain. This analgesic effect is mediated principally by the activation of µ-opioid receptors (MOR). However, prolonged activation of MOR also results in tolerance, dependence, addiction, constipation, nausea, sedation, and respiratory depression. To address this problem, we sought alternative ways to activate MOR - either by use of novel ligands, or via a novel activation mechanism. To this end, a series of compounds were screened using a sensitive CHO-K1/MOR/Gα15 cell-based FLIPR® calcium high-throughput screening (HTS) assay, and the bithiazole compound 5a was identified as being able activate MOR in combination with naloxone. Structural modifications of 5a resulted in the discovery of lead compound 5j, which could effectively activate MOR in combination with the MOR antagonist naloxone or naltrexone. In vivo, naloxone in combination with 100 mg/kg of compound 5j elicited antinociception in a mouse tail-flick model with an ED50 of 17.5 ±â€¯4 mg/kg. These results strongly suggest that the mechanism by which the 5j/naloxone combination activates MOR is worthy of further study, as its discovery has the potential to yield an entirely novel class of analgesics.

Non-nociceptive roles of opioids in the CNS: opioids' effects on neurogenesis, learning, memory and affect.

Mortality due to opioid use has grown to the point where, for the first time in history, opioid-related deaths exceed those caused by car accidents in many states in the United States. Changes in the prescribing of opioids for pain and the illicit use of fentanyl (and derivatives) have contributed to the current epidemic. Less known is the impact of opioids on hippocampal neurogenesis, the functional manipulation of which may improve the deleterious effects of opioid use. We provide new insights into how the dysregulation of neurogenesis by opioids can modify learning and affect, mood and emotions, processes that have been well accepted to motivate addictive behaviours.

Chronic granulomatous skin lesions leading to a diagnosis of TAP1 deficiency syndrome.

Transporter associated with antigen processing (TAP) is essential for the stabilization and surface expression of major histocompatibility complex class I molecules of all nucleated cells. TAP deficiency syndrome, also known as bare lymphocyte syndrome type I, is a rare primary immunodeficiency disorder. We report a case of TAP1 deficiency revealed by skin lesions long before the occurrence of respiratory infectious manifestations.

Morphine regulates adult neurogenesis and contextual memory extinction via the PKCε/Prox1 pathway.

We have previously reported that the miR-181a/Prox1/Notch1 pathway mediates the effect of morphine on modulating lineage-specific differentiation of adult neural stem/progenitor cells (NSPCs) via a PKCε-dependent pathway, whereas fentanyl shows no such effect. However, the role of the PKCε/Prox1 pathway in mediating drug-associated contextual memory remains unknown. The current study investigated the effect of PKCε/Prox1 on morphine-induced inhibition of adult neurogenesis and drug-associated contextual memory in mice, while the effect of fentanyl was tested simultaneously. By using BrdU labeling, we were able to examine the lineages of differentiated NSPCs in adult DG. PKCε knockout blocked morphine's effects on inducing in vivo astrocyte-preferential differentiation of NSPCs, but did not alter NSPC lineages upon fentanyl treatment. Inhibited adult neurogenesis further resulted in prolonged extinction and enhanced reinstatement of morphine-induced CPP, as well as prolonged extinction of space reference memory indicated by the Morris water maze paradigm. However, after fentanyl administration, no significant changes were found between wild-type and PKCε knockout mice, during either CPP or water maze tasks. When the lentivirus encoding Nestin-promoter-controlled Prox1 cDNA was injected into hippocampi of wildtype and PKCε knockout adult mice to modulate PKCε/Prox1 activity, similar effects were discovered in adult mice injected with lentivirus encoding Prox1, and more dramatic effects were found in PKCε knockout mice with concurrent Prox1 overexpression. In conclusion, morphine mediates lineage-specific NSPC differentiation, inhibits adult neurogenesis and regulates contextual memory retention via the PKCε/Prox1 pathway, which are implicated in the eventual context-associated relapse.

M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

M1 muscarinic acetylcholine receptors (M1 mAChRs) are the most abundant muscarinic receptors in the hippocampus and have been shown to have procognitive effects. AMPA receptors (AMPARs), an important subtype of ionotropic glutamate receptors, are key components in neurocognitive networks. However, the role of AMPARs in procognitive effects of M1 mAChRs and how M1 mAChRs affect the function of AMPARs remain poorly understood. Here, we found that basal expression of GluA1, a subunit of AMPARs, and its phosphorylation at Ser845 were maintained by M1 mAChR activity. Activation of M1 mAChRs promoted membrane insertion of GluA1, especially to postsynaptic densities. Impairment of hippocampus-dependent learning and memory by antagonism of M1 mAChRs paralleled the reduction of GluA1 expression, and improvement of learning and memory by activation of M1 mAChRs was accompanied by the synaptic insertion of GluA1 and its increased phosphorylation at Ser845. Furthermore, abrogation of phosphorylation of Ser845 residue of GluA1 ablated M1 mAChR-mediated improvement of learning and memory. Taken together, these results show a functional correlation of M1 mAChRs and GluA1 and the essential role of GluA1 in M1 mAChR-mediated cognitive improvement.-Zhao, L.-X., Ge, Y.-H., Xiong, C.-H., Tang, L., Yan, Y.-H., Law, P.-Y., Qiu, Y., Chen, H.-Z. M1 muscarinic receptor facilitates cognitive function by interplay with AMPA receptor GluA1 subunit.

Drug survival and postdrug survival of first-line immunosuppressive treatments for atopic dermatitis: comparison between methotrexate and cyclosporine.

INTRODUCTION: Cyclosporine and methotrexate are the two preferred first-line immunosuppressive treatments in atopic dermatitis. The aim of this study was to compare the treatment profiles of methotrexate and cyclosporine in daily practice as the first-line immunosuppressive treatment in atopic dermatitis, using two survival analyses, 'drug survival' (time on the drug) and 'postdrug survival' (time between two drugs). METHODS: Retrospective study including patients with moderate-to-severe atopic dermatitis treated with methotrexate or cyclosporine as the first-line immunosuppressive treatment. The reasons for discontinuation of treatment were collected as follows: controlled disease, treatment failure, side event pregnancy and non-compliance. 'Drug survival' and 'postdrug survival' analyses were performed using the Kaplan-Meier method and predictive factors were analysed using uni- and multivariate Cox regression analyses. RESULTS: Fifty-six patients, among whom 25 patients treated with cyclosporine and 31 with methotrexate (median age: 34 ± 15 years), were included between 2007 and 2016. Reasons for discontinuation were not significantly different between 'controlled disease' and other reasons (P = 0.11). The median 'drug survival' was significantly longer for methotrexate (23 months) than for cyclosporine (8 months) (P < 0.0001). Six months from baseline, 93% of patients treated with methotrexate were still being treated vs 63% among patients treated with cyclosporine. The median of 'postdrug survival' was significantly longer for methotrexate (12 months) than for cyclosporine (2 months). Only treatment with CYC was a predictive factor for decreased 'drug survival' and 'postdrug survival'. CONCLUSION: This is the first direct comparison between methotrexate and cyclosporine as first-line immunosuppressive treatments for moderate-to-severe atopic dermatitis in daily practice. We evidenced two different treatment profiles: the duration of methotrexate administration is longer than that of cyclosporine. 'Postdrug survival' could be a new tool to assess the maintenance of effect of a drug after withdrawal in atopic dermatitis, and more broadly in chronic skin disease.

Temporal effect of manipulating NeuroD1 expression with the synthetic small molecule KHS101 on morphine contextual memory.

The treatment of opioid addiction is challenging because addicts are highly prone to relapse when the memory of the former drug experience is triggered by emotional or environmental cues. An emerging and promising concept in addiction biology is that by manipulating adult hippocampal neurogenesis, a phenomenon involved in learning and memory, drug reward-like behaviors and relapse can be attenuated. We tested a new synthetic compound, KHS101, in an animal model of drug-associated contextual memory. KHS101 has been reported to increase the expression of neurogenic differentiation 1 (NeuroD1), a transcription factor involved in adult neurogenesis, and to specifically induce neuronal differentiation both in vitro and in vivo. Our results indicated that the subcutaneous injection of 3 mg/kg KHS101 for 7 days before conditioned place preference (CPP) training prolonged CPP extinction, while the same treatment after training accelerated extinction. This effect paralleled that observed following temporally controlled, tetracycline-induced NeuroD1 overexpression. Furthermore, the effect of KHS101 may occur via its induction of NeuroD1 expression as demonstrated by the abolition of the KHS101-mediated modulation of morphine-induced CPP extinction after the stereotaxic injection of lentiviral NeuroD1 small interfering RNA into the dentate gyrus (DG) of the hippocampus. These results suggest that the KHS101-mediated modulation of neurogenesis at a critical stage of the conditioning or the extinction of an opioid-associated experience may disrupt the memory trace of the existing opioid-associated experience to facilitate the extinction of drug-associated contextual memory. This implies that KHS101 has therapeutic potential for the treatment of opioid addiction.

Src-dependent phosphorylation of µ-opioid receptor at Tyr336 modulates opiate withdrawal.

Opiate withdrawal/negative reinforcement has been implicated as one of the mechanisms for the progression from impulsive to compulsive drug use. Increase in the intracellular cAMP level and protein kinase A (PKA) activities within the neurocircuitry of addiction has been a leading hypothesis for opiate addiction. This increase requires the phosphorylation of µ-opioid receptor (MOR) at Tyr336 by Src after prolonged opiate treatment in vitro Here, we report that the Src-mediated MOR phosphorylation at Tyr336 is a prerequisite for opiate withdrawal in mice. We observed the recruitment of Src in the vicinity of MOR and an increase in phosphorylated Tyr336 (pY336) levels during naloxone-precipitated withdrawal. The intracerebroventricular or stereotaxic injection of a Src inhibitor (AZD0530), or Src shRNA viruses attenuated pY336 levels, and several somatic withdrawal signs. This was also observed in Fyn-/- mice. The stereotaxic injection of wild-type MOR, but not mutant (Y336F) MOR, lentiviruses into the locus coeruleus of MOR-/- mice restored somatic withdrawal jumping. Regulating pY336 levels during withdrawal might be a future target for drug development to prevent opiate addictive behaviors.

Differential regulation of mouse and human Mu opioid receptor gene depends on the single stranded DNA structure of its promoter and α-complex protein 1.

The Mu opioid receptor (MOR) mediates various functions of opioid-induced analgesia, euphoria and respiratory depression, and is a major target of opioid analgesics. Understanding of MOR gene expression among species is important for understanding its analgesic function in humans. In the current study, the polypyrimidine/polypurine (PPy/u) region, a key element of MOR gene expression, was compared in humans and mice. The mouse PPy/u element is highly homologous to its human element (84%), and the mouse MOR (mMOR) reporter drives luciferase activity 35-fold more effectively than the human MOR (hMOR) reporter. The structural study of reporter plasmids using S1 nuclease indicates that the mouse PPy/u element has a particular conformational structure, namely a single-stranded DNA (ssDNA) region that promotes strong promoter activity. DNA electrophoretic mobility shift assays demonstrated that the recombinant α-complex protein 1 (α-CP1) is capable of binding to a single-stranded mouse PPy/u sequence. Furthermore, plasmid-expressing α-CP1 activated the expression of a luciferase reporter when cotransfected with a single-stranded (p336/306) construct. In addition, the α-CP1 gene induced the mMOR gene in mouse neuronal cells and did not induce the human neuronal MOR gene. The current study demonstrates that α-CP1 functions as a transcriptional activator in the mMOR gene, but does not function in the hMOR gene due to species-specific structural differences. The differences in human and mouse MOR gene expression are based on α-CP1 and the ssDNA structure of the MOR promoter. The MOR gene is species-specifically regulated, as the PPy/u element adopts a unique species-specific conformation and α-CP1 recruitment.