New PDF release: Nuclear Receptors

By David W. Russell

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Casserly, D. P. Via, L. C. Smith, J. St. Pyrek, and G. J. , J. Lipid Res. 30, 1097 (1989). 36 ANALYSIS OF NUCLEAR RECEPTOR LIGANDS [2] Breuer and Bjo¨rkhem23 have performed careful experiments to determine which oxysterols are formed in vivo in humans and rats, respectively. Whereas Kudo et al. analyzed oxysterols under a controlled atmosphere where air was excluded, Breuer and Bjo¨rkhem metabolically labeled oxysterols in vivo by keeping rats in an [18O]-containing atmosphere, permitting the identification of oxysterols formed in vivo by their incorporation of 18O.

00 38 ANALYSIS OF NUCLEAR RECEPTOR LIGANDS [3] the pharmacological character of ER ligands based on their stabilization of ER dimers, assessed using site-specific fluorescent-labeled receptors. 6 Our site-specific fluorescent labeled NR lacks a large GFP fluorophore (about 25,000 Da7) and is instead covalently labeled with a small fluorophore (about 400–800 Da) at a single cysteine residue. Site-specific fluorescent NRs have at least three distinct advantages. First, the relatively small fluorophore can be site-specifically attached to a reactive-cysteine residue at a conformationally sensitive region of the receptor (while retaining the native functionality of the NR) as a sensor of its environment, to monitor ligand- and coregulator-induced conformational changes in the NR.

The temperature program for the gas chromatograph is as follows: 180 C for 1 min, 20 /min to 250 C, 5 /min to 300 C, 300 C for 8 min. 8 ml helium/min. Injector (splitless mode) and transfer line are kept at 270 C and 280 C, respectively. The mass selective detector is run in the selective ion monitoring (SIM) mode using ions m/z 456 (7 -, 7b-, and 27hydroxycholesterol), 474 (cholesterol-5 ,6 -epoxide and cholesterol-5b,6bepoxide), 546 (cholestane-3b,5 ,6b-triol), 413 (24-hydroxycholesterol), 131 (25-hydroxycholesterol), 472 (7-oxocholesterol) for the analytes, and m/z 462, 460 ([2H4]27-hydroxycholesterol), 480, 552, 416, 134, 478 for the corresponding deuterium labeled internal standards.

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Nuclear Receptors by David W. Russell


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