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34447313153
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note
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125I]NDP-α-MSH was separated by filtration through GF/C glass fibre filters presoaked with 0.1% (w/v) PEI. Filters were washed three times with 50 mM Tris-HCl, pH 7.4, at 0-4 °C and assayed for radioactivity using Perkin-Elmer Topcount counter. Binding data were analysed by computer-assisted non-linear regression analysis (XL fit; IDBS).
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15
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0032752856
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Evans, D.N.3
Feilden, H.4
Smith, P.W.5
Sollis, S.L.6
Taylor, N.7
Wonacott, A.J.8
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17
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34447344256
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®) was used instead of N,N′-dicyclohexylcarbodimide to facilitate by-products removal.
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18
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0022394724
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Janssens F., Torremans J., Janssen M., Stokbroekx R.A., Luyckx M., and Janssen P.A.J. J. Med. Chem. 28 (1985) 1925
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20
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0021343603
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Vickery, B.H.5
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34447298118
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note
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Cyclic AMP assay: Intracellular cyclic AMP (cAMP) levels were determined by an electro-chemiluminescence (ECL) assay (Meso Scale Discovery, MSD). CHO-K1 cells stably expressing the human MC4 receptors were suspended in RMPI 1640 containing 0.5 mM IBMX and 0.2% protein cocktail (MSD). They were dispensed (7000 cells/well) in multi-array plates containing integrated carbon electrodes and coated with anti-cAMP antibody. Concentration-response experiments of NDP-α-MSH were carried out in the presence of increasing concentrations of the tested compound by incubating the cells for 40 min at 37 °C. Then, the cells were lysed and 2.5 nM TAG ruthenium-labelled cAMP was added. After 90 min, cAMP levels were determined by ECL detection using sector imager 6000 reader (MSD). cAMP data were analysed by computer-assisted non-linear regression analysis (XL fit; IDBS). The Kb values were determined by Schild regression analysis.
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22
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34447334582
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Fed male Sprague-Dawley rats were dosed in groups of three animals (for each kinetic point). A solution of 4.5 μmol/kg in 1 ml/kg of a 10% DMA/90% propanediol solution was given by intravenous administration via the penile vein. Then, rats were deeply anaesthetised under Isoflurane® and blood was taken from the vena cava (at 1, 15, 30, 60, 90 and 120 min) and centrifuged (2000g, 15 min, 4 °C). The supernatants were mixed with cold acetonitrile to precipitate the proteins. After a further centrifugation (2000g, 15 min, 4 °C), the samples were analysed by HPLC (UV detection) or frozen at - 80° pending analysis. After blood withdrawal, tissues were rapidly washed by intracardiac perfusion with 50 ml of saline. The brains were then removed, frozen in liquid nitrogen and weighed. After mixing the brains with methanol for 40 s with an Ultraturax®, brain homogenates were centrifuged (25,000g, 15 min, 4 °C) and the supernatants were analysed by HPLC.
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