The natural polyphenolic compound resveratrol (trans-3,4 H ,5-trihydroxystilbene) is shown to prevent apoptosis (programmed cell death) induced in human erythroleukemia K562 cells by hydrogen peroxide and other unrelated stimuli. Resveratrol reversed the elevation of leukotriene B 4 (from 6.40^0.65 to 2.92^0.30 pmol´mg protein 21 ) and prostaglandin E 2 (from 11.46^1.15 to 8.02^0.80 nmol´mg protein 21 ), induced by H 2 O 2 challenge in K562 cells. The reduction of leukotriene B 4 and prostaglandin E 2 correlated with the inhibition of the 5-lipoxygenase activity, and the cyclooxygenase and peroxidase activity of prostaglandin H synthase, respectively. Resveratrol also blocked lipoperoxidation induced by hydrogen peroxide in K562 cell membranes. Resveratrol was found to act as a competitive inhibitor of purified 5-lipoxygenase and 15-lipoxygenase and prostaglandin H synthase, with inhibition constants of 4.5^0.5 mm (5-lipoxygenase), 40^5.0 mm (15-lipoxygenase), 35^4.0 mm (cyclooxygenase activity of prostaglandin H synthase) and 30^3.0 mm (peroxidase activity of prostaglandin H synthase). Altogether, the results reported here suggest that the anti-apoptotic activity of resveratrol depends on the direct inhibition of the main arachidonate-metabolizing enzymes.Keywords: apoptosis; cyclooxygenase; lipoxygenase; oxidative stress; resveratrol.Resveratrol (trans-3,4 H ,5-trihydroxystilbene) occurs naturally in grapes and a variety of medicinal plants, where it functions as a phytoalexin that protects against infections and other stress factors [1]. Because of its high concentration in grape skin, a significant amount of resveratrol is present in red wine [2] and is thought to be responsible for the reduced risk of cardiovascular disease associated with moderate consumption of this beverage [2]. Resveratrol has been reported to have antiplatelet [1], anti-inflammatory [3,4] and anticarcinogenic [5] effects. Recently, it has also been reported to act as an agonist for the estrogen receptor [6] and to inhibit phorbolester-mediated activation of protein kinase C and AP-1-mediated gene expression in human mammary epithelial cells [7]. The various activities of resveratrol have been attributed to its antioxidant properties [8], which contribute to control the intracellular redox balance by inhibiting the formation of reactive oxygen species (ROS; [5]). Moreover, this polyphenolic compound interferes with the arachidonate metabolism, by reducing the levels of leukotrienes (generated by the`l ipoxygenase pathway' of the arachidonate cascade) and prostanoids (generated by the`cyclooxygenase pathway'), and this activity is also considered crucial for its biological effects [3,5,7]. Although the cyclooxygenase activity [5] and expression [7] of prostaglandin H synthase (PHS) have been shown to be reduced by resveratrol in cellular systems, a direct interaction of this compound with the enzyme has not been characterized to date. The effect of resveratrol on the other arachidonate-metabolizing enzyme, lipoxygenase, has not been inve...

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The relationships between tertiary structure and copper binding in Pseudomonas fiuorescens azurin have been studied by fluorescence, absorption, and electron paramagnetic resonance spectra. The fluorescence spectrum at neutral

Anandamide (AEA), a prominent member of the endogenous ligands of cannabinoid receptors (endocannabinoids), is known to affect several functions of brain and peripheral tissues. A potential role for AEA in skin pathophysiology has been proposed, yet its molecular basis remains unknown. Here we report unprecedented evidence that spontaneously immortalized human keratinocytes (HaCaT) and normal human epidermal keratinocytes (NHEK) have the biochemical machinery to bind and metabolize AEA, i.e. a functional type-1 cannabinoid receptor (CB1R), a selective AEA membrane transporter (AMT), an AEA-degrading fatty acid amide hydrolase (FAAH), and an AEA-synthesizing phospholipase D (PLD). We show that, unlike CB1R and PLD, the activity of AMT and the activity and expression of FAAH increase while the endogenous levels of AEA decrease in HaCaT and NHEK cells induced to differentiate in vitro by 12-O-tetradecanoylphorbol 13-acetate (TPA) plus calcium. We also show that exogenous AEA inhibits the formation of cornified envelopes, a hallmark of keratinocyte differentiation, in HaCaT and NHEK cells treated with TPA plus calcium, through a CB1R-dependent reduction of transglutaminase and protein kinase C activity. Moreover, transient expression in HaCaT cells of the chloramphenicol acetyltransferase reporter gene under control of the loricrin promoter, which contained a wild-type or mutated activating protein-1 (AP-1) site, showed that AEA inhibited AP-1 in a CB1R-dependent manner. Taken together, these data demonstrate that human keratinocytes partake in the peripheral endocannabinoid system and show a novel signaling mechanism of CB1 receptors, which may have important implications in epidermal differentiation and skin development.