By David W. Russell
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Additional resources for Nuclear Receptors
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  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  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.
Nuclear Receptors by David W. Russell