Functionality of Melatonin Receptors: Internalization.

The main step of classical desensitization of a receptor, by mean of its disappearance from the plasma membrane, is its internalization. This is a key factor in the regulation of agonist-mediated signaling pathways, as it most of the time stops the activation of the receptor. Internalization is thus important to evaluate, as a complementary information for a natural ligand or an alternative synthetic agonist. Enzyme fragment complementation is an elegant but delicate way to measure this phenomenon, by fusing two complementary parts of an enzyme to two partners, and to measure the activity of the reconstituted enzyme upon complexation of the partners. In the present chapter, using two parts of ß-galactosidase, one fused to the C-terminus of the MT1 receptor, the other to an endosomal protein, one can measure the formation of the complex; thus, the transfer of the receptor to the endosome from which MT1 will be recirculated.

Gene expression profiling during hibernation in the European hamster.

Hibernation is an exceptional physiological response to a hostile environment, characterized by a seasonal period of torpor cycles involving dramatic reductions of body temperature and metabolism, and arousal back to normothermia. As the mechanisms regulating hibernation are still poorly understood, here we analysed the expression of genes involved in energy homeostasis, torpor regulation, and daily or seasonal timing using digital droplet PCR in various central and peripheral tissues sampled at different stages of torpor/arousal cycles in the European hamster. During torpor, the hypothalamus exhibited strongly down-regulated gene expression, suggesting that hypothalamic functions were reduced during this period of low metabolic activity. During both torpor and arousal, many structures (notably the brown adipose tissue) exhibited altered expression of deiodinases, potentially leading to reduced tissular triiodothyronine availability. During the arousal phase, all analysed tissues showed increased expression of the core clock genes Per1 and Per2. Overall, our data indicated that the hypothalamus and brown adipose tissue were the tissues most affected during the torpor/arousal cycle, and that clock genes may play critical roles in resetting the body's clocks at the beginning of the active period.

Natural Inhibitors of the RhoA-p115 Complex from the Bark of Meiogyne baillonii.

In an effort to find potent natural inhibitors of RhoA and p115 signaling G-proteins, a systematic in vitro evaluation using enzymatic and plasmonic resonance assays was undertaken on 11 317 plant extracts. The screening procedure led to the selection of the New Caledonian endemic species Meiogyne baillonii for a chemical investigation. Using a bioguided isolation procedure, three enediyne-γ-butyrolactones (1-3) and two enediyne-γ-butenolides (4 and 5), named sapranthins H-L, respectively, two enediyne carboxylic acid (6 and 7), two depsidones, stictic acid (8) and baillonic acid (9), aristolactams AIa and AIIa (10 and 11), and two aporphines, dehydroroemerine (12) and noraristolodione (13), were isolated from the ethyl acetate extract of the bark. The structures of the new compounds (1-6, 9, and 11) and their relative configurations were established by NMR spectroscopic analysis and by X-ray diffraction analysis for compound 9. Only stictic acid (8) exhibited a significant inhibiting activity of the RhoA-p115 complex, with an EC50 value of 0.19 ± 0.05 mM. This is the first time that a natural inhibitor of the complex RhoA-p115's activity was discovered from an HTS performed over a collection of higher plant extracts. Thus, stictic acid (8) could be used as the first reference compound inhibiting the interaction between RhoA and p115.

Piceatannol and resveratrol share inhibitory effects on hydrogen peroxide release, monoamine oxidase and lipogenic activities in adipose tissue, but differ in their antilipolytic properties.

Piceatannol is a hydroxylated derivative of resveratrol. While both dietary polyphenols coexist in edible plants and fruits, and share equivalent concentrations in several wines, the influence of piceatannol on adiposity has been less studied than that of resveratrol. Though resveratrol is now recognized to limit fat deposition in various obesity models, the benefit of its dietary supplementation remains under debate regarding human obesity treatment or prevention. The research for more potent resveratrol analogs is therefore still undergoing. This prompted us to compare various effects of piceatannol and resveratrol directly on human adipose tissue (hAT). Hydrogen peroxide release was measured by Amplex Red-based fluorescence in subcutaneous hAT samples from obese patients. Interactions of stilbenes with human amine oxidases and quinone reductase were assessed by radiometric methods, computational docking and electron paramagnetic resonance. Influences on lipogenic and lipolytic activities were compared in mouse adipocytes. Resveratrol and piceatannol inhibited monoamine oxidase (MAO) with respective IC50 of 18.5 and 133.7 µM, but not semicarbazide-sensitive amine oxidase (SSAO) in hAT. For both stilbenes, the docking scores were better for MAO than for SSAO. Piceatannol and resveratrol similarly hampered hydrogen peroxide detection in assays with and without hAT, while they shared pro-oxidant activities when incubated with purified quinone reductase. They exhibited similar dose-dependent inhibition of adipocyte lipogenic activity. Only piceatannol inhibited basal and stimulated lipolysis when incubated at a dose ≥100 µM. Thus, piceatannol exerted on fat cells dose-dependent effects similar to those of resveratrol, except for a stronger antilipolytic action. In this regard, piceatannol should be useful in limiting the lipotoxicity related to obesity when ingested or administered alone - or might hamper the fat mobilization induced by resveratrol when simultaneously administered with it.

Expression of the orphan GPR50 protein in rodent and human dorsomedial hypothalamus, tanycytes and median eminence.

The melatonin receptor family is composed of three members, MT(1) and MT(2) receptors that bind melatonin with high affinity and the orphan GPR50 that does not bind melatonin but shares significant sequence homology with the two other subtypes. In the absence of any known ligand for this orphan receptor, little is still known about its function. We recently reported the development of the first anti-GPR50 antibodies that reliably recognized the recombinant human GPR50. We here used these antibodies to study the expression of GPR50 in mouse, rat and human hypothalamus, a region reported to express GPR50 mRNA. GPR50 immunoreactivity (ir) was observed in dorsomedial hypothalamic (DMH) cells co-stained with the neuronal marker HuC/D. GPR50-ir was also observed in cells of the ependymal layer of the third ventricle that co-stained with vimentin. More specifically, its localization in the lower region of the third ventricle and along the long basal processes contacting portal blood vessels in the median eminence (ME) suggested expression of GPR50 in tanycytes. Consistent staining patterns were observed in all three species with two different antibodies. Taken together, our study validates two GPR50-specific antibodies for the use in rodent and human tissue. Evolutionary conserved expression of GPR50 in DMH neurons and tanycytes, together with previously reported expression of the receptor in the pituitary, support the potentially important role of GPR50 in key hypothalamic functions, including regulation of the hypothalamo-pituitary axes.

Cytotoxic pentacyclic triterpenoids from Combretum sundaicum and Lantana camara as inhibitors of Bcl-xL/BakBH3 domain peptide interaction.

In an effort to discover potent inhibitors of the antiapoptotic protein Bcl-xL, a systematic in vitro evaluation was undertaken on extracts prepared from various parts of Vietnamese plants. The ethyl acetate extracts obtained from the leaves and flowers of Combretum sundaicum and the leaves of Lantana camara were selected for their interaction with the Bcl-xL/Bak association. Bioassay-guided purification of these species led to the isolation of 15 pentacyclic triterpenoids (1-15) possessing olean-12-en-28-oic acid and olean-12-en-29-oic acid aglycons, of which compounds 1-6 and 8-10 are new. Five compounds exhibited binding activity with K(i) values between 5.3 and 17.8 microM. The cytotoxic activity of 1-15 was also evaluated on various cancer cell lines.

A Dimeric sesquiterpenoid from a Malaysian Meiogyne as a new inhibitor of Bcl-xL/BakBH3 domain peptide interaction.

In an effort to find potent inhibitors of the antiapoptotic protein Bcl-xL, a systematic in vitro evaluation was undertaken on 1470 Malaysian plant extracts. The ethyl acetate extract obtained from the bark of Meiogyne cylindrocarpa was selected for its interaction with the Bcl-xL/Bak association. Bioassay-guided purification of this species led to the isolation of two new dimeric sesquiterpenoids (1 and 2) possessing an unprecedented substituted cis-decalin carbon skeleton. Meiogynin A (1) showed the strongest activity with a K(i) of 10.8 +/- 3.1 microM.

High-throughput screening of novel antagonists on melanin-concentrating hormone receptor-1.

AIM: To find new antagonists on human melanin-concentrating hormone receptor-1 (MCHR-1) through high-throughput screening (HTS) of a diverse compound library. METHODS: MCHR-1, [3H]SNAP7941, and FlashBlue G-protein-coupled receptor beads were used to measure the receptor-binding activities of various compounds based on scintillation proximity assay (SPA) technology. The guanosine 5'(gamma-[35S]thio) triphosphate ([35S]GTPgammaS) binding assay was subsequently applied to functionally characterize the "hits" identified by the HTS campaign. RESULTS: Of the 48,240 compounds screened with the SPA method, 12 hits were confirmed to possess MCHR-1 binding activities, 8 were functionally studied subsequently with the [35S]GTPgammaS binding assay, and only 1 compound (NC127816) displayed moderate human MCHR-1 binding affinity (Ki=115.7 nmol/L) and relatively potent antagonism (KB=23.8 nmol/L). This compound shares a novel scaffold (1-ethoxy-2H-2-aza-1-phospha-naphthalene 1-oxide) with 3 other analogs in the group. CONCLUSION: Considering the marked difference in molecular shape and electrostatic status between NC127816 and the structures reported elsewhere, we anticipate that its derivatives may represent a new class of potent MCHR-1 modulators.