Antioxidant and prooxidant effects of polyphenol compounds on copper-mediated DNA damage

Inhibition of copper-mediated DNA damage has been determined for several polyphenol compounds. The 50% inhibition concentration values (IC₅₀) for most of the tested polyphenols are between 8 and 480 μM for copper-mediated DNA damage prevention. Although most tested polyphenols were antioxidants under these conditions, they generally inhibited Cu^I-mediated DNA damage less effectively than Fe^II-mediated damage, and some polyphenols also displayed prooxidant activity. Because semiquinone radicals and hydroxyl radical adducts were detected by EPR spectroscopy in solutions of polyphenols, Cu^I, and H₂O₂, it is likely that weak polyphenol-Cu^I interactions permit a redox-cycling mechanism, whereby the necessary reactants to cause DNA damage (Cu^I, H₂O₂, and reducing agents) are regenerated. The polyphenol compounds that prevent copper-mediated DNA damage likely follow a radical scavenging pathway as determined by EPR spectroscopy.     

Oxidative coupling of the pyrogallol B-ring with a galloyl group during enzymatic oxidation of epigallocatechin 3-O-gallate

In order to clarify the mechanism for formation of catechin oligomers during the fermentation stage of black tea manufacture, epigallocatechin-3-O-gallate, the most abundant tea flavanol in fresh tea leaves, was enzymatically oxidized and the resulting unstable quinone metabolites were converted to phenazine derivatives by treatment with o-phenylenediamine. In addition to formation of monomeric and dimeric derivatives, four trimeric derivatives were isolated whose structures were determined by application of spectroscopic methods. The derivatives differed from each other in the location of the phenazine moieties and in the atropisomerism of the biphenyl bond. The results suggested that oxidative coupling of the galloyl group with the B-ring proceeds by a quinone dimerization mechanism similar to that for production of theasinensins.     

Immobilization of polyphenol oxidase on chitosan–SiO<Subscript>2</Subscript> gel for removal of aqueous phenol

A partially purified potato polyphenol oxidase (PPO) was immobilized in a cross-linked chitosan–SiO₂ gel and used to treat phenol solutions. Under optimized conditions (formaldehyde 20 mg/ml, PPO 4 mg/ml and pH 7.0), the activity of immobilized PPO was 1370 U/g and its K _m value for catechol was 12 mm at 25°C. The highest activity of immobilized enzyme was at pH 7.4. Immobilization stabilized the enzyme with 73 and 58% retention of activity after 10 and 20 days, respectively, at 30°C whereas most of the free enzyme was inactive after 7 days. The efficiency of removing phenol (10 mg phenol/l) by the immobilized PPO was 86%, and about 60% removal efficiency was retained after five recycles. The immobilized PPO may thus be a useful for removing phenolic compounds from industrial waste-waters.     

Rosmarinic acid and its esters inhibit membrane cholesterol domain formation through an antioxidant mechanism based,in nonlinear fashion,on alkyl chain length

BackgroundUnder conditions of oxidative stress, cholesterol aggregates into discrete membrane bilayer domains that precipitate the formation of extracellular crystals, a feature of advanced atheroma in cardiovascular disease. Therapeutic interventions using membrane-directed antioxidants, such as polyphenolic esters, may reduce cholesterol domains and crystal formation. In this study, the effects of rosmarinic acid (RC0) and rosmarinic esters, with alkyl chain lengths ranging from 4 to 16?carbons (RC4-RC16), on membrane lipid oxidation and cholesterol domain formation were investigated.MethodsModel membranes were prepared with 1,2-dilinoleoyl-sn-glycero-3-phosphocholine and cholesterol at different cholesterol-to-phospholipid mole ratios (0.3:1, 0.9:1, and 1.2:1), in the absence or presence of each molecule and exposed to 72 h of oxidation. Changes in lipid hydroperoxide (LOOH) and cholesterol domain formation were measured using iodometric and small angle x-ray diffraction approaches, respectively.ResultsRosmarinic acid and its esters had differential effects on LOOH formation based on alkyl chain length. RC8 exhibited the greatest antioxidant effect, reducing LOOH levels by 82%, and inhibited cholesterol domain formation. By contrast, RC0 and RC16 failed to inhibit either LOOH formation or cholesterol domain formation.ConclusionThese data indicate that the membrane antioxidant and cholesterol domain inhibition activities of rosmarinic acid esters are dependent, nonlinearly, on alkyl chain length. The mechanism for this effect is attributed to the influence of alkyl chain length on the optimal depth of the polyphenols into the lipid bilayer for trapping free radicals.General significanceThese findings provide insight into novel atheroprotective benefits of polyphenol esters that are dependent on their membrane location.     

Ericoid mycorrhizal root fungi and their multicopper oxidases from a temperate forest shrub

Ericoid mycorrhizal fungi (ERM) may specialize in capturing nutrients from their host's litter as a strategy for regulating nutrient cycles in terrestrial ecosystems. In spite of their potential significance, we know little about the structure of ERM fungal communities and the genetic basis of their saprotrophic traits (e.g., genes encoding extracellular enzymes). Rhododendron maximum is a model ERM understory shrub that influences the nutrient cycles of montane hardwood forests in the southern Appalachians (North Carolina, USA). We sampled ERM roots of R. maximum from organic and mineral soil horizons and identified root fungi by amplifying and sequencing internal transcribed spacer (ITS) ribosomal DNA (rDNA) collected from cultures and clones. We observed 71 fungal taxa on ERM roots, including known symbionts Rhizoscyphus ericae and Oidiodendron maius, putative symbionts from the Helotiales, Chaetothyriales, and Sebacinales, ectomycorrhizal symbionts, and saprotrophs. Supporting the idea that ERM fungi are adept saprotrophs, richness of root-fungi was greater in organic than in mineral soil horizons. To study the genetic diversity of oxidative enzymes that contribute to decomposition, we amplified and sequenced a portion of genes encoding multicopper oxidases (MCOs) from ERM ascomycetes. Most fungi possessed multiple copies of MCO sequences with strong similarities to known ferroxidases and laccases. Our findings indicate that R. maximum associates with a taxonomically and ecologically diverse fungal community. The study of MCO gene diversity and expression may be useful for understanding how ERM root fungi regulate the cycling of nutrients between the host plant and the soil environment.     

The use of diversity profiling to characterize chemical modulators of the histone deacetylases

Target specificity and off-target liabilities are routinely monitored during the early phases of drug discovery for most kinase projects. Typically these criteria are evaluated using a profiling panel comprised of a diverse collection of in vitro kinase assays and relates compound structure to potency and selectivity. The success of these efforts has led to the design of similar panels for phosphatase, protease, and epigenetic targets. Here the implementation of an epigenetic profiling panel, comprised of eleven histone deacetylases (HDACs) and one histone acetyltransferase (HAT), was used to evaluate chemical modulators of these enzymes. HDAC inhibitors (HDACi) such as sodium butyrate and trichostatin A demonstrate diverse biological effects which have led to broad speculation about their therapeutic potential in multiple disease states. Some HDACi have demonstrated tumor suppression in vivo and recently Zolinza was the first HDACi approved by the FDA for the treatment of cutaneous T-cell lymphoma. While HDACi have demonstrated therapeutic utility, many of the first generation compounds are pan-inhibitors. Thus, use of an HDAC profiling panel will be essential in achieving isoform specificity of the next generation of inhibitors. To this end, twenty-one compounds, twelve of which are known to have activities against the HDACs, were tested to evaluate the utility of the epigenetic panel. Additionally, these compounds were tested against a larger 72 member enzyme panel comprised of kinase, phosphatase and protease activities. This effort represents the first time these compounds have been profiled with such a broad range of biochemical activities.     

Polyphenol oxidases in plants and fungi: going places? A review

The more recent reports on polyphenol oxidase in plants and fungi are reviewed. The main aspects considered are the structure, distribution, location and properties of polyphenol oxidase (PPO) as well as newly discovered inhibitors of the enzyme. Particular stress is given to the possible function of the enzyme. The cloning and characterization of a large number of PPOs is surveyed. Although the active site of the enzyme is conserved, the amino acid sequence shows very considerable variability among species. Most plants and fungi PPO have multiple forms of PPO. Expression of the genes coding for the enzyme is tissue specific and also developmentally controlled. Many inhibitors of PPO have been described, which belong to very diverse chemical structures; however, their usefulness for controlling PPO activity remains in doubt. The function of PPO still remains enigmatic. In plants the positive correlation between levels of PPO and the resistance to pathogens and herbivores is frequently observed, but convincing proof of a causal relationship, in most cases, still has not been published. Evidence for the induction of PPO in plants, particularly under conditions of stress and pathogen attack is considered, including the role of jasmonate in the induction process. A clear role of PPO in a least two biosynthetic processes has been clearly demonstrated. In both cases a very high degree of substrate specificity has been found. In fungi, the function of PPO is probably different from that in plants, but there is some evidence indicating that here too PPO has a role in defense against pathogens. PPO also may be a pathogenic factor during the attack of fungi on other organisms. Although many details about structure and probably function of PPO have been revealed in the period reviewed, some of the basic questions raised over the years remain to be answered.     

Effects of salicylic acid and cold treatments on protein levels and on the activities of antioxidant enzymes in the apoplast of winter wheat leaves

The effects of salicylic acid (SA) and cold on apoplastic protein levels and activities of apoplastic catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) were investigated in winter wheat (Triticum aestivum cv. Dogu-88) leaves. The plants were grown with and without 10 microM SA treatment at both control (20/18 degrees C for 30 and 45-day) and cold (10/5 degrees C for 30-day and 5/3 degrees C for 45-day) acclimatisations. Molecular masses of the apoplastic polypeptides were shown ranging in size from 20 to 66 kDa on SDS-PAGE. Accumulation and pattern of the polypeptides were changed by both SA and cold. It is observed that CAT, POX and PPO activities at 45-day control leaves were higher than at 30-day. When the activities with SA and cold treatments are compared to their controls, CAT activities were decreased while POX and PPO activities were increased by both the treatments. When the activities with cold+SA treatment are compared to their cold treatments, CAT and POX activities were decreased while PPO activity was increased by SA. It is concluded that exogenous SA can be involved in cold tolerance by regulating apoplastic proteins and antioxidant enzyme activities.     

Accumulation of gentisic acid as associated with systemic infections but not with the hypersensitive response in plant-pathogen interactions

In the present work we have studied the accumulation of gentisic acid (2,5-dihydroxybenzoic acid, a metabolic derivative of salicylic acid, SA) in the plant-pathogen systems, Cucumis sativus and Gynura aurantiaca, infected with either prunus necrotic ringspot virus (PNRSV) or the exocortis viroid (CEVd), respectively. Both pathogens produced systemic infections and accumulated large amounts of the intermediary signal molecule gentisic acid as ascertained by electrospray ionization mass spectrometry (ESI-MS) coupled on line with high performance liquid chromatography (HPLC). The compound was found mostly in a conjugated (β-glucoside) form. Gentisic acid has also been found to accumulate (although at lower levels) in cucumber inoculated with low doses of Pseudomonas syringae pv. tomato, producing a nonnecrotic reaction. In contrast, when cucumber was inoculated with high doses of this pathogen, a hypersensitive reaction occurred, but no gentisic-acid signal was induced. This is consistent with our results supporting the idea that gentisic-acid signaling may be restricted to nonnecrotizing reactions of the host plant (Bellés et al. in Mol Plant-Microbe Interact 12:227–235, 1999). In cucumber and Gynura plants, the activity of gentisic acid as inducing signal was different to that of SA, thus confirming the data found for tomato. Exogenously supplied gentisic acid was able to induce peroxidase activity in both Gynura and cucumber plants in a similar way as SA or pathogens. However, gentisic-acid treatments strongly induced polyphenol oxidase activity in cucumber, whereas pathogen infection or SA treatment resulted in a lower induction of this enzyme. Nevertheless, gentisic acid did not induce other defensive proteins which are induced by SA in these plants. This indicates that gentisic acid could act as an additional signal to SA for the activation of plant defenses in cucumber and Gynura plants.     

Tyrosinase activity in ionic liquids

Activity of mushroom tyrosinase was studied in three ionic liquids, 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF₆]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]) and 1-butyl-3-methylimidazolium methylsulfate ([BMIm][MeSO₄]), and was compared to that in chloroform. Kinetic parameters of the enzyme were determined and the results indicate that the enzyme in ionic liquids basically follows the same catalytic mechanism as in water, and that the ionic liquids may affect the enzyme activity by direct interacting with the enzyme and thus hindering the E–S binding due to their high hydrophilicity and polarity.