stilbenes and esculetin

Hydroxylated phenylpropanoid compounds (e.g., esculetin, piceatannol, and eriodictyol) have been proved to possess important biological activities and pharmacological properties. These compounds exist at low abundance in nature, which hampers their cost-effective isolation, and broad application. Meanwhile, regiospecific hydroxylation of complex aromatic compounds is still quite challenging for chemical synthesis. In past decades, biocatalytic hydroxylation of plant phenylpropanoids was achieved due to the identification and engineering of some cytochrome P450 hydroxylases; however, the conversion efficiency was still too low for scale-up production use. In this work, we identify a non-P450 monooxygenase (HpaBC) from Escherichia coli, which is able to catalyze the efficient ortho-hydroxylation towards plant phenylpropanoids umbelliferone and resveratrol; meanwhile it also exhibits activity towards naringenin. On this basis, whole-cell biocatalysis enables the production of esculetin and piceatannol at high titers (2.7 and 1.2 g/L, respectively, in shake flasks) and high yields (close to 100%). To our knowledge, this work reports the highest titers and yields for biotechnological production of esculetin and piceatannol, representing a promising hydroxylation platform.

Topics: Escherichia coli; Metabolic Engineering; Resveratrol; Stilbenes; Umbelliferones

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease, characterized by progressive dysfunction and death of motor neurons. Although evidence for oxidative stress in ALS pathogenesis is well described, antioxidants have generally shown poor efficacy in animal models and human clinical trials. We have developed an in vitro screening cascade to identify antioxidant molecules capable of rescuing NSC34 motor neuron cells expressing an ALS-associated mutation of superoxide dismutase 1. We have tested known antioxidants and screened a library of 2000 small molecules. The library screen identified 164 antioxidant molecules, which were refined to the 9 most promising molecules in subsequent experiments. Analysis of the in silico properties of hit compounds and a review of published literature on their in vivo effectiveness have enabled us to systematically identify molecules with antioxidant activity combined with chemical properties necessary to penetrate the central nervous system. The top-performing molecules identified include caffeic acid phenethyl ester, esculetin, and resveratrol. These compounds were tested for their ability to rescue primary motor neuron cultures after trophic factor withdrawal, and the mechanisms of action of their antioxidant effects were investigated. Subsequent in vivo studies can be targeted using molecules with the greatest probability of success.

Topics: Amyotrophic Lateral Sclerosis; Animals; Antioxidants; Apoptosis; Blood-Brain Barrier; Caffeic Acids; Cell Culture Techniques; Cell Line; Central Nervous System; Humans; Mice; Mice, Inbred C57BL; Motor Neurons; Mutation; Oxidative Stress; Resveratrol; Small Molecule Libraries; Stilbenes; Superoxide Dismutase; Superoxide Dismutase-1; Transgenes; Umbelliferones

1alpha,25-(OH)(2)D(3) mediates its effects on growth zone chondrocytes via rapid membrane-associated events as well as through traditional nuclear receptor mechanisms. The membrane-associated signaling pathways include rapid production of diacylglycerol and activation of protein kinase C (PKC), as well as activation of phospholipase A(2) (PLA(2)), increased production of arachidonic acid, and increased production of prostaglandins. This study examined the roles of PLA(2) and cyclooxygenase (Cox) in the mechanism of action of 1alpha,25-(OH)(2)D(3) in these cells to determine whether one or both enzymes catalyze the rate limiting step and whether constitutive or inducible Cox is involved. Cultures were incubated with 1alpha,25-(OH)(2)D(3) for 9 min to measure PKC or for 24 h to measure physiological responses ([(3)H]-thymidine incorporation, alkaline phosphatase specific activity, [(35)S]-sulfate incorporation). Based on RT-PCR and Northern blot analysis, growth zone chondrocytes expressed mRNAs for both Cox-1 and Cox-2 and neither Cox was modulated by 1alpha,25-(OH)(2)D(3). To examine the role of Cox, the cultures were also treated with resveratrol (a specific inhibitor of Cox-1), NS-398 (a specific inhibitor of Cox-2), or indomethacin (a general Cox inhibitor). The results showed that Cox-1 inhibition reduced the 1alpha,25-(OH)(2)D(3)-dependent effects on proliferation, differentiation, and matrix production, whereas inhibition of Cox-2 only had an effect on proliferation. The effects of Cox inhibition were not rate limiting, based on experiments in which PLA(2) was activated with melittin or inhibited with quinacrine. However, at least part of the action of 1alpha,25-(OH)(2)D(3) was regulated by metabolism of arachidonic acid to prostaglandins. This supports the hypothesis that 1alpha,25-(OH)(2)D(3) exerts its effects via more than one signaling pathway and that these pathways are interrelated via the modulation of PLA(2) as a rate-limiting step. PKC regulation may occur at multiple stages in the signal transduction cascade. J. Cell. Biochem. Suppl. 36: 32-45, 2001.

Topics: Alkaline Phosphatase; Animals; Arachidonic Acid; Blotting, Northern; Calcitriol; Cells, Cultured; Chondrocytes; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Growth Plate; Indomethacin; Isoenzymes; Lipoxygenase; Lipoxygenase Inhibitors; Masoprocol; Membrane Proteins; Nitrobenzenes; Phospholipases A; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Proteoglycans; Rats; Resveratrol; Reverse Transcriptase Polymerase Chain Reaction; Stilbenes; Sulfonamides; Thymidine; Umbelliferones

The potential activities of butylated hydroxyanisole (BHA), myo-inositol, curcumin, esculetin, resveratrol and lycopene-enriched tomato oleoresin (LTO) as chemopreventive agents against lung tumor induction in A/J mice by the tobacco smoke carcinogens benzo[a]pyrene (BaP) and 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were evaluated. Groups of 20 A/J mice were treated weekly by gavage with a mixture of BaP and NNK (3 micromol each) for 8 weeks, then sacrificed 26 weeks after the first carcinogen treatment. Mice treated with BHA (20 or 40 micromol) by gavage 2 h before each dose of BaP and NNK had significantly reduced lung tumor multiplicity. Treatment with BHA (20 or 40 micromol) by gavage weekly or with dietary BHA (2000 ppm), curcumin (2000 ppm) or resveratrol (500 ppm) from 1 week after carcinogen treatment until termination had no effect on lung tumor multiplicity. Treatment with dietary myo-inositol (30,000 ppm) or esculetin (2000 ppm) from 1 week after carcinogen treatment until termination significantly reduced lung tumor multiplicity, with the effect of myo-inositol being significantly greater than that of esculetin. Treatment with dietary LTO (167, 1667 or 8333 ppm) from 1 week before carcinogen treatment until termination had no effect on lung tumor multiplicity. The results of this study demonstrate that BHA is an effective inhibitor of BaP plus NNK-induced lung tumorigenesis in A/J mice when administered during the period of carcinogen treatment and that, among the compounds tested, myo-inositol is most effective after carcinogen treatment.

Topics: Animals; Anticarcinogenic Agents; Antioxidants; Benzo(a)pyrene; Butylated Hydroxyanisole; Carotenoids; Curcumin; Female; Inositol; Lung Neoplasms; Lycopene; Mice; Mice, Inbred A; Nitrosamines; Resveratrol; Stilbenes; Umbelliferones