Hypertrophic cardiomyopathy (HCM) is frequently associated with sudden cardiac death, presumably due to the development of malignant arrhythmias. The risk of sudden cardiac death due to HCM has been reported to be predicted by assessing electrocardiographic (ECG) changes including frequencies and complexities of arrhythmias as well as heart rate variability (HRV) as an assessment of autonomic balance. Sudden cardiac death in association with naturally-occurring left ventricular hypertrophy (LVH) has been reported in a colony of rhesus macaques and is under investigation as a potential non-human primate model of human HCM. In the present study, 10 rhesus macaques with LVH and 10 without the signs of LVH confirmed by an echocardiographic examination were recruited for assessing ECG and HRV parameters. ECG morphology on 10-s, 6-lead ECG analysis, and the frequency and complexity of arrhythmias as well as HRV on 20-h ambulatory ECG Holter analyses were assessed. On the standard 10-s 6-lead ECG analysis, P wave and QRS complex duration as well as the QRS complex amplitude were significantly increased in the LVH-affected rhesus macaques compared to control rhesus macaques. Analysis of 20-h Holter monitoring revealed no statistically significant differences in the frequency or the complexity of arrhythmias between the LVH and the control groups. Several HRV parameters were smaller in the LVH group than the control group throughout the majority of Holter recordings showing periods of reduced variability, however, no statistically significant differences were achieved across groups and/or time points. These findings indicate that ECG analysis and Holter monitoring of rhesus macaques are feasible and that ECG morphological changes in association with LVH could be used as a possible component of an antemortem screening tool. The rhesus macaques of this study did not reveal clear indications of risk for sudden cardiac death. Further studies are necessary to determine the etiology of sudden cardiac death due in LVH affected rhesus macaques and identify if any parameters of arrhythmia assessment or HRV can be used to predict the development of sudden cardiac death.

ClinicalTrials.gov identifier no. NCT01475669; EudraCT trial no. 2011-002685-20.

Secreted frizzled related protein 1 (SFRP1) is an antagonist of the transmembrane frizzled receptor, a component of the Wnt signaling pathway, and has been suggested to be a candidate tumor suppressor in several human malignancies. Since SFRP1 is located at chromosome 8p11, where lung cancers also exhibit frequent allelic loss, we hypothesized that the inactivation of SFRP1 is also involved in lung carcinogenesis. To substantiate this, we performed mutational analysis of SFRP1 for 29 nonsmall-cell lung cancer (NSCLC) and 25 small-cell lung cancer (SCLC) cell lines, and expression analysis for the same cell lines. Although somatic mutations were not detected in the coding sequence, downregulation of SFRP1 was observed in 14 (48%) NSCLC and nine (36%) SCLC cell lines. We analysed epigenetic alteration of the SFRP1 promoter region and detected hypermethylation in 15 (52%) of 29 NSCLC cell lines, two (8%) of 25 SCLC cell lines, and 44 (55%) of 80 primary lung tumors. By comparing the methylation status with SFRP1 expression, we found a significant correlation between them. We also performed loss of heterozygosity (LOH) analysis and found that 15 (38%) of 40 informative surgical specimens had LOH in the SFRP1 gene locus. Furthermore, we performed colony formation assay of two NSCLC cell lines (NCI-H460 and NCI-H2009) and found the reduction of colony formation with SFRP1 transfection. In addition, we also detected that SFRP1 inhibits the transcriptional activity of b-catenin, which is thought to be a downstream molecule of SFRP1, with luciferase reporter assay. Our current studies demonstrated that the SFRP1 gene is frequently downregulated by promoter hypermethylation and suppresses tumor growth activity of lung cancer cells, which suggests that SFRP1 is a candidate tumor suppressor gene for lung cancer.Oncogene (

It is unclear how receptor/ligand families that are evolutionarily closely related achieve functional separation. To address this question, we focus here on the newly discovered Orphanin FQ, a peptide homologous to the opioid peptide Dynorphin, and its receptor, the Orphanin FQ receptor, which is highly homologous to the opioid receptors. In spite of this high degree of homology in terms of both ligands and receptors, there is little direct cross-talk between the Orphanin FQ system and the endogenous opioid system. Thus, the opioid peptides show either relatively low affinity or no affinity toward the Orphanin FQ receptor; conversely, Orphanin FQ has no affinity toward any of the opioid receptors. We sought to investigate the molecular basis of such discrimination by attempting to reverse it and endowing the Orphanin FQ receptor with the ability to bind opioids. We report that by mutating as few as four amino acids, we can produce a receptor that recognizes pro-Dynorphin products with very high affinity and yet still binds Orphanin FQ as well as the wild-type receptor. This suggests that the Orphanin FQ receptor has developed features that specifically exclude the opioids and that these features are distinct from those required for the high affinity binding of its own endogenous ligand.The three major types of opioid receptors, , ␦, and , have been cloned and shown to belong to the seven transmembrane domain, G protein-coupled family (1, 2). In addition, several laboratories have cloned a protein highly homologous to these opioid receptors but that, nevertheless, does not bind with high affinity any known opioid peptides or alkaloids (3). The endogenous ligand for this opioid-like orphan receptor has recently been isolated by two independent groups (3, 4). Interestingly, it is a 17-amino acid peptide with a significant degree of sequence homology to DynA-(1-17). This novel peptide has been termed Nociceptin by Meunier et al. (3) to denote its ability to increase pain responsiveness and Orphanin FQ by Civelli and coworkers (4). We shall refer here to the ligand as Orphanin FQ and to its receptor as the Orphanin FQ receptor.It is clear from ongoing anatomical studies that Orphanin FQ and its receptor represent a novel and distinct peptidergic system with a unique anatomical distribution within the central nervous system 1 and the gastrointestinal system 2 . Behavioral results have already demonstrated a novel profile for Orphanin FQ in pain and place preference tests and in patterns of tolerance development (5, 6). Orphanin FQ has at its amino terminus the sequence Phe-Gly-Gly-Phe, which only differs from the common opioid core (Tyr-Gly-Gly-Phe) by a single OH group. Yet, since the N-terminal tyrosine is critical for the binding of all opioid peptides (7), this change is sufficient to preclude Orphanin FQ from binding to any of the opioid receptors. Conversely, reciprocal events must have taken place to preclude the opioid peptides from being recognized by the Orphanin FQ receptor. Thus, this system offers a most...

Although much has been learned about the mechanisms of ligand selectivity between different opioid receptor subtypes, little is known about the common opioid binding pocket shared by all opioid receptors. The recently discovered orphanin system offers a good opportunity to study the mechanisms involved in the binding of opioid versus nonopioid ligands. In the current study, we adopt a "gain of function" approach aimed at shifting the binding profile of the orphanin FQ receptor toward that of the opioid receptors. After two rounds of mutagenesis, several orphanin FQ receptor mutants can be labeled with the opiate alkaloid [3H]naltrindole and show greatly increased affinities toward the opiate antagonists naltrexone, nor-binaltrophine HCl, and (-)-bremazocine. These orphanin FQ receptor mutants also display stereospecificity similar to that of opioid receptors. Furthermore, the orphanin FQ receptor mutant that has the best affinities toward the opioid alkaloids shows, in the presence of GTP and high salt concentration, an affinity-shift profile similar to that of the delta receptor. Most strikingly, the same mutant exhibits naltrindole-sensitive etorphine-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding, whereas the effect of etorphine on GTP binding cannot be inhibited by naltrindole in the wild-type receptor. Our results indicate that 1) several residues in the orphanin FQ receptor are critical to its selectivity against the opiate alkaloids, particularly antagonists; and 2) mutating these residues to those of the opioid receptor at the corresponding position preserves the agonist/antagonist nature of opiate alkaloids as they interact with the mutant receptor. It is reasonable to hypothesize that the corresponding residues in the opioid receptors may form a functional common binding pocket for opiate alkaloids. These findings may be helpful to medicinal chemists in designing ligands for the orphanin FQ receptor based on the structure of the opiate alkaloids.