Docosahexaenoic Acid, a Ligand for the Retinoid X Receptor in Mouse Brain
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Giguère V., Endocr. Rev. 20, 689 (1999).
Cells were transfected in triplicate in 24-well plates by the calcium phosphate method (20). The cells were harvested 24 hours later and the extracts were assayed for luciferase and reference β-galactosidase activity in a microplate luminometer/photometer reader (Lucy-1; Anthos Salzburg Austria).
Adult (6 to 8 weeks) wild-type (NMRI) male mice were perfused via the ascending aorta with cold phosphate-buffered saline. Organs were dissected cut into small pieces and placed in cell culture medium (MEM). Embryonal (E13.5) tissues were collected from wild-type (NMRI) embryos as above. Tissues were incubated with the medium overnight at 37°C. This conditioned medium was then separated from the tissue and frozen.
Supplemental Web material may be found at www.sciencemag.org/cgi/content/full/290/5499/2140/DC1.
Conditioned medium was acidified with hydrochloric acid mixed with an equal volume of hexane and shaken vigorously for 5 min. This mixture was centrifuged and the upper organic phase was recovered. Extraction of the aqueous phase was repeated once. The hexane extract was evaporated under nitrogen gas and for activity assays the residue was redissolved in 100 to 200 μl of ethanol. Hexane extract of conditioned medium was evaporated redissolved in hexane and injected onto a normal-phase HPLC column (Genesis Silica 4-μm particle size 4.6 mm by 25 cm Jones Chromatography Ltd. UK). Elution was performed using a linear gradient from hexane to hexane/dichloromethane/isopropanol (85:10:5 v/v) both containing 1% acetic acid in 30 min at a flow rate of 0.5 ml/min. Starting 17 min after injection 30 fractions containing 0.25 ml each were collected. Active fractions were pooled evaporated and redissolved in 50 μl 80% methanol. We injected 30 μl onto a reversed-phase HPLC column (Genesis C18 4-μm particle size 3.0 mm by 25 cm). A mobile phase of methanol/isopropanol/water (80:10:10 v/v) containing 1% acetic acid at a flow rate of 0.3 ml/min was used. Active fractions along with the preceding and subsequent fractions were analyzed by nano-ES mass spectrometry.
Negative-ion nano-ES spectra were recorded on a AutoSpec-OATOFFPD (Micromass Manchester UK) hybrid double focusing magnetic sector–orthogonal acceleration (OA) time-of-flight (TOF) tandem mass spectrometer focal plane detector (FPD). Mass spectra were recorded as magnet scans over an m/z range of 70 to 1000 at an instrument resolution of 3000 (10% valley definition). Collision-induced dissociation (CID) spectra were recorded by selecting the precursor [M-H] − ions with the double-focusing sectors of the instrument and focusing them into the fourth field–free region gas cell containing xenon collision gas. Undissociated precursor ions and fragment ions were pulsed into an OATOF mass analyzer.
All fatty acid ligands were purchased from Sigma except C21:1 cis 12 and C23:1 cis 14 which were purchased from Nu-Chek-Prep (Elysian MN).
A. Mata de Urquiza et al. data not shown.
Bacterially expressed glutathione S -transferase (GST) or GST fusion proteins were bound to glutathione-Sepharose 4B beads (Pharmacia Biotech). SRC-1 cDNA was used to generate [ 35 S]methionine-labeled proteins (49). The labeled proteins were incubated with beads containing GST or GST fusion proteins in the presence of dimethyl sulfoxide (DMSO) DHA or 1 μM estradiol. After an overnight incubation free proteins were washed away. Bound proteins were eluted in loading buffer separated by SDS–polyacrylamide gel electrophoresis and visualized by fluorography.
N. Salem Jr. H.-Y. Kim J. A. Yergey in Health Effects of Polyunsaturated Fatty Acids in Seafoods A. P. Simopoulos R. R. Kifer R. E. Martin Eds. (Academic Press New York 1986) chap. 15 pp. 263–317.
Sheaff Greiner R., Moriguchi T., Hutton A., Slotnick B. M., Salem N., Lipids 34, S239 (1999).
Bischoff E. D., Gottardis M. M., Moon T. E., Heyman R. A., Lamph W. W., Cancer Res. 58, 479 (1998).
Cavaillés V., Dauvois S., Danielian P. S., Parker M. G., Proc. Natl. Acad. Sci. U.S.A. 91, 10009 (1994).
We thank R. Heyman and R. Bissonnette for LG849 L. Foley for SR11237 D. Mangelsdorf for plasmids encoding RXRL436F and RXRF313I derivatives L. Solomin for helpful comments on this manuscript and members of the Perlmann lab for valuable discussion. Supported by the Human Frontiers Science Program the Göran Gustafssons Foundation and the Swedish Medical Research Council grant 03X-12551 (S.L. W.G. and J.S.) the Swedish Wennergren Foundation (M.S.) and the Swedish Cancer Society (M.S.).