Synaptic Depression and Cortical Gain Control

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The ethyl acetate extract (90 g) (88% inhibition of COX activity at 69 !g/ml) was separated into 28 fractions with chloroform-methanol (0 to 30% methanol) as eluent over a silica gel chromatographic column. Fractions 13 to 17 (66 to 85% inhibition of COX activity at 69 !g/ml) were combined (4 g), and this material was subjected to further column chromatography. The column was developed with chloroform-methanol (1 to 15% methanol) and hexaneethyl acetate (3 :1 to 1:1) to afford an active compound (30 mg, 0.003%) that was determined to be C14H12O3 by high-resolution mass spectral (MS) analysis. This compound was identified as resveratrol by comparison of its physical data and 1H–nuclear magnetic resonance (NMR), 13C-NMR, and MS data with those of an authentic sample [E. Mannila, A. Talvitie, E. Kolehmainen, Phytochemistry 33, 813 (1993); G. S. Jayatilake et al., J. Nat. Prod. 56, 1805 (1993)]. Additional resveratrol for the studies here was purchased from Sigma (St. Louis, MO). The commercial material was evaluated by high-pressure liquid chromatography coupled with MS analysis and found to be pure. 6. L. W. Wattenberg, Cancer Res. 53, 5890 (1993). 7. J. K. Gierse et al., Biochem. J. 305, 479 (1995). 8. S. Sharma, J. D. Stutzman, G. J. Kelloff, V. E. Steele, Cancer Res. 54, 5848 (1994). HL-60 cells were maintained in RPMI 1640 medium (Gibco BRL, Grand Island, NY ) supplemented with 5% heat-inactivated calf serum, penicillin G sodium (100 U/ml), and streptomycin sulphate (100 !g/ml) (Gibco BRL) at 37°C in a humidified atmosphere at 5% CO2 in air. Differentiation was induced by a 7-day treatment with 1.3% dimethyl sulfoxide (DMSO), and the cells were cultured in 96-well plates (1" 106 cells per well) in Hanks’ balanced salt solution containing 30 mM Hepes, pH 7.6. After the addition of TPA (8 !M) to induce free radical formation, cytochrome c (160 !M) and resveratrol were added. The cells were incubated for 1 hour at 37°C, and antioxidant activity was determined by monitoring absorbance at 550 nm. The same reaction mixture, without the HL-60 cells, was used as a blank control. 9. L. A. Shamon et al., Anticancer Res. 14, 1775 (1995). A reaction mixture was prepared containing Salmonella typhimurium strain TM677, S9 liver homogenate derived from Aroclor 1254-pretreated rats, a NADPH-generating system (NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate), and various concentrations of resveratrol, which were added 1 min before the addition of 80 !M DMBA. After incubation for 2 hours at 37°C, the bacteria were recovered by centrifugation, resuspended, and plated (in triplicate) on minimal agar in the presence or absence of 8-azaguanine. The plates were then incubated for 48 hours at 37°C, and the results were expressed as mutant fractions, that is, the average number of colonies capable of growing in the presence of 8-azaguanine divided by the average number of colonies capable of growing in the absence of 8-azaguanine, after correcting for dilution factors. The percent inhibition was calculated relative to control plates that were treated with DMSO only. 10. H. J. Prochaska and A. B. Santamaria, Anal. Biochem. 169, 328 (1988). 11. Y. Zhang, T. W. Kensler, C.-G. Cho, G. H. Posner, P. Talalay, Proc. Natl. Acad. Sci. U.S.A. 91, 3147 (1994). 12. N. Suh, L. Luyengi, H. H. S. Fong, A. D. Kinghorn, J. M. Pezzuto, Anticancer Res. 15, 233 (1995). 13. R. C. Moon and R. G. Mehta, in Chemistry and Biology of Retinoids, M. I. Dawson and W. H. Okamura, Eds. (CRC Press, Boca Raton, FL, 1990), pp. 501– 518. 14. W. Dercks and L. L. Creasy, Physiol. Mol. Plant Pathol. 34, 289 (1989). 15. P. Jeandet, R. Bessis, B. Gautheron, Am. J. Enol. Vitic. 42, 41 (1991); E. H. Siemann and L. L. Creasy, ibid. 43, 49 (1992); D. M. Goldberg et al., ibid. 46, 159 (1995). 16. A. I. Romero-Pérez, R. M. Lamuela-Raventós, A. L. Waterhouse, M. C. de la Torre-Boronat, J. Agric. Food Chem. 44, 2124 (1996). 17. E. B. Rimm et al., Lancet 338, 464 (1991); A. L. Klatsky, M. A. Armstrong, G. D. Friedman, Ann. Intern. Med. 117, 646 (1992); A. L. Klatsky, Clin. Exp. Res. 18, 88 (1994). 18. D. M. Goldberg, S. E. Hahn, J. G. Parkes, Clin. Chim. Acta 237, 155 (1995). 19. H. Arichi et al., Chem. Pharm. Bull. 30, 1766 (1982); Y. Kimura, H. Lkuda, S. Arichi, Biochim. Biophys. Acta 834, 275 (1985); C. R. Pace-Asciak et al., Clin. Chim. Acta 235, 207 (1995). 20. Daily consumption of two to five glasses (or a maximum of 375 ml/day) of red wine may deliver a sufficient amount of resveratrol to alter arachidonic acid metabolism or other physiological responses, depending on absorption, metabolism, and residence time within the blood circulation and relevant tissues [D. M. Goldberg, Clin. Chem. 41, 14 (1995)]. Resveratrol concentrations in other food products, such as grape juice, pomace, and purees are provided in B. J. Ector, J. B. Magee, C. P. Hegwood, and M. J. Coign [Am. J. Enol. Vitic. 47, 57 (1996)]. 21. F. J. G. Van der Ouderaa and M. Muytenhek, Methods Enzymol. 86, 60 (1982); R. J. Kulmacz and E. M. Lands, in Prostaglandins and Related Substances. A Practical Approach, C. Benedetto, R. G. McDonaldGibson, S. Nigam, T. F. Slater, Eds. (IRL Press, Oxford, 1987), pp. 209–227. 22. K. Slowing, E. Carretero, A. Villar, J. Ethnopharmacol. 43, 9 (1994). Female Wistar rats (150 to 200 g body weight) were divided into groups of seven animals each. All rats received 0.1 ml of Freund’s complete adjuvant (Difco; Sigma) by intradermal injection into the tail. Animals were used 7 days after injection of adjuvant. One hour after oral administration of resveratrol (3 or 8 mg per kilogram of body weight) or reference drugs including phenylbutazone (80 mg/ kg) and indomethacin (5 mg/kg), the rats were injected with 0.1 ml of a 2% (w/v) suspension of carrageenan in saline solution into the left hind paw. For the control group, a 1:1 mixture of Tween 80 and water (0.2/3.3, v/v) and 1% (w/v) methylcellulose was used as a vehicle. The left hind paw volume of each rat was measured by water plethysmography (Letica, model L17500) before the adjuvant injection and, again, 6 days later, before the injection of carrageenan. Paw volumes were determined within 3 to 144 hours after injection of carrageenan. Inhibition of edema was calculated relative to the mean edema of the vehicle-treated control group. 23. C. Gerhäuser et al., Nature Med. 1, 260 (1995). 24. We thank N. Suh, S. K. Lee, C. Gerhäuser, L. A. Shamon, M. Hawthorne, and D. D. Lantvit for technical assistance; W. G. Thilly for S. typhimurium strain TM677; J. P. Whitlock for Hepa 1c1c7 and BPrC1 cells; H. Constant for HPLC-MS analysis of resveratrol; and J. K. Gierse for COX-2. Supported by grant P01 CA48112 from the National Cancer Institute.