Melanin-concentrating hormone (MCH) is a cyclic nona- Melanin-concentrating hormone (MCH)1 has been initially described in fish as a heptadecapeptide (Asp-Thr-Met-Arg-CysMet-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val (1)). Its structure was relatively conserved throughout evolution, although in mammals the sequence of MCH is a nonadecapeptide with differences mainly in the N terminus (Asp-Phe-Asp-MetLeu-Arg-Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-GlnVal (2)). In rodents, there are now several lines of evidence for the involvement of MCH in the central regulation of feeding behavior as reviewed by Tritos and Maratos-Flier (3). The MCH peptide and its receptor are expressed in the hypothalamus, a region involved in energy balance and food intake (4 -7). In this particular brain area, MCH mRNA is overexpressed and up-regulated during fasting in ob/ob mice as well as in rats (8, 9). Intra-cerebroventricular injections of MCH promote feeding in mice and rats (9 -12). Finally, transgenic mice lacking the MCH gene are lean and hypophagic (13). Interestingly, in peripheral tissues, MCH also stimulates the release of leptin from isolated rat adipocytes (14). The lack of suitable binding conditions, mainly due to the hydrophobic and sticky nature of MCH itself or derivatives (15, 16), was probably a limitation for expression cloning of the receptor. The MCH receptor was nevertheless recently identified by several groups using reverse pharmacology (17-21). The MCH function was assigned to the previously described orphan receptor SLC-1 (22, 23), using inhibition of forskolin-stimulated cAMP production and induction of calcium rise.Receptor cloning and association of functional tests open the way to the search for pharmacological tools, especially receptor antagonists that are needed to study receptor functions. One of the possible strategies to this goal is the chemical modification of the natural peptide including peptide shortening, amino acid substitution, and conformation restriction with the help of structure-activity relationships and modeling studies toward optimized nonpeptide ligands. Such a strategy has been successful for the design of subtype-specific antagonists of neuropeptide Y receptors (24 -27).In the case of MCH, only two sets of data have been published on the pharmacological action and binding affinity of MCH analogues in vitro. A first series of experiments with fish MCH on fish, frog, or other batrachian skin assays were reported (28 -32), showing that fish MCH could be shortened at * This work was supported by a a Convention CIFRE between the Association Nationale de la Recherche Technique, the Institut de Recherches SERVIER and the Centre National de la Recherche Scientifique (to T. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.*

Bedut S, Seminatore-Nole C, Lamamy V, Caignard S, Boutin JA, Nosjean O, Stephan JP, Coge F. High-throughput drug profiling with voltage-and calcium-sensitive fluorescent probes in human iPSC-derived cardiomyocytes. Am J Physiol Heart Circ Physiol 311: H44 -H53, 2016. First published May 3, 2016 doi:10.1152/ajpheart.00793.2015.-Cardiomyocytes derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) are increasingly used for in vitro assays and represent an interesting opportunity to increase the data throughput for drug development. In this work, we describe a 96-well recording of synchronous electrical activities from spontaneously beating hiPSC-derived cardiomyocyte monolayers. The signal was obtained with a fast-imaging plate reader using a submillisecond-responding membrane potential recording assay, FluoVolt, based on a newly derived voltagesensitive fluorescent dye. In our conditions, the toxicity of the dye was moderate and compatible with episodic recordings for Ͼ3 h. We show that the waveforms recorded from a whole well or from a single cell-sized zone are equivalent and make available critical functional parameters that are usually accessible only with gold standard techniques like intracellular microelectrode recording. This approach allows accurate identification of the electrophysiological effects of reference drugs on the different phases of the cardiac action potential as follows: fast depolarization (lidocaine), early repolarization (nifedipine, Bay K8644, and veratridine), late repolarization (dofetilide), and diastolic slow depolarization (ivabradine). Furthermore, the data generated with the FluoVolt dye can be pertinently complemented with a calcium-sensitive dye for deeper characterization of the pharmacological responses. In a semiautomated plate reader, the two probes used simultaneously in 96-well plates provide an easy and powerful multiparametric assay to rapidly and precisely evaluate the cardiotropic profile of compounds for drug discovery or cardiac safety.

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The development of cell-based assays for high-throughput screening (HTS) approaches often requires the generation of stable transformant cell lines. However, these cell lines are essentially created by random integration of a gene of interest (GOI) with no control over the level and stability of gene expression. The authors developed a targeted integration system in Chinese hamster ovary (CHO) cells, called the cellular genome positioning system (cGPS), based on the stimulation of homologous gene targeting by meganucleases. Five different GOIs were knocked in at the same locus in cGPS CHO-K1 cells. Further characterization revealed that the cGPS CHO-K1 system is more rapid (2-week protocol), efficient (all selected clones expressed the GOI), reproducible (GOI expression level variation of 12%), and stable over time (no change in GOI expression after 23 weeks of culture) than classical random integration. Moreover, in all cGPS CHO-K1 targeted clones, the recombinant protein was biologically active and its properties similar to the endogenous protein. This fast and robust method opens the door for creating large collections of cell lines of better quality and expressing therapeutically relevant GOIs at physiological levels, thereby enhancing the potential scope of HTS. (Journal of Biomolecular Screening 2010:956-967)

The neuropeptide Y Y5 receptor gene generates two splice variants, referred to here as Y5(L) (long isoform) and Y5(S) (short isoform). Y5(L) mRNA differs from Y5(S) mRNA in its 5' end, generating a putative open reading frame with 30 additional nucleotides upstream of the initiator AUG compared with the Y5(S) mRNA. The purpose of the present work was to investigate the existence of the Y5(L) mRNA. The authenticity of this transcript was confirmed by isolating part of its 5' untranslated region through 5' rapid amplification of cDNA ends and analysing its tissue distribution. To study the initiation of translation on Y5(L) mRNA, we cloned the Y5(L) cDNA and two Y5(L) cDNA mutants lacking the first or the second putative initiation start codon. Transient expression of the three plasmids in COS-7 cells and saturation binding experiments using (125)I-labelled polypeptide YY (PYY) as a ligand showed that initiation of translation on Y5(L) mRNA could start at the first AUG, giving rise to a Y5(L) receptor with an N-terminal 10-amino-acid extension when compared with the Y5(S) receptor. The human Y5(L) and Y5(S) receptor isoforms displayed similar affinity constants (1.3 nM and 1.5 nM respectively). [(125)I]PYY binding to COS-7 cells expressing either the Y5(L) or the Y5(S) isoform was inhibited with the same rank order of potency by a selection of six chemically diverse compounds: PYY>neuropeptide Y>pancreatic polypeptide>CGP71683A>Synaptic 34>Banyu 6. Comparison of the tissue distribution of Y5(L) and Y5(S) mRNAs, as determined by reverse transcription-PCR analysis, indicated that expression of Y5(L) mRNA occurs in a tissue-specific manner. Finally, we have shown that the two AUG triplets contained in the 5' untranslated region of Y5(L) mRNA did not affect receptor expression.