Dietary flavonoids with a catechol structure increase alpha-tocopherol in rats and protect the vitamin from oxidation in vitro

To identify dietary phenolic compounds capable of improving vitamin E status, male Sprague-Dawley rats were fed for 4 weeks either a basal diet (control) with 2 g/kg cholesterol and an adequate content of vitamin E or the basal diet fortified with quercetin (Q), (-)-epicatechin (EC), or (+)-catechin (C) at concentrations of 2 g/kg. All three catechol derivatives substantially increased concentrations of alpha-tocopherol (alpha-T) in blood plasma and liver. To study potential mechanisms underlying the observed increase of alpha-T, the capacities of the flavonoids to i) protect alpha-T from oxidation in LDL exposed to peroxyl radicals, ii) reduce alpha-tocopheroxyl radicals (alpha-T (.) ) in SDS micelles, and iii) inhibit the metabolism of tocopherols in HepG2 cells were determined. All flavonoids protected alpha-T from oxidation in human LDL ex vivo and dose-dependently reduced the concentrations of alpha-T (.) . None of the test compounds affected vitamin E metabolism in the hepatocyte cultures. In conclusion, fortification of the diet of Sprague-Dawley rats with Q, EC, or C considerably improved their vitamin E status. The underlying mechanism does not appear to involve vitamin E metabolism but may involve direct quenching of free radicals or reduction of the alpha-T (.) by the flavonoids.     

Oxidized derivatives of omega-3 fatty acids: identification of IPF3 alpha-VI in human urine

Isoprostanes (iPs) are prostaglandin-like molecules derived from autoxidation of polyunsaturated fatty acids (PUFAs). Urinary iP levels have been used as indices of in vivo lipid peroxidation. Thus far, it has only been possible to measure iPs derived from arachidonic acid in urine, because levels of iPs/neuroprostanes (nPs) derived from omega 3-PUFAs have been found to be below detection limits of available assays. Because of the interest in omega3-PUFA dietary supplementation, we developed specific methods to measure nPF4 alpha-VI and iPF3 alpha-VI [derived from 4,7,10,13,16,19-docosahexaenoic acid (DHA) and 5,8,11,14,17-eicosapentaenoic acid (EPA)] using a combination of chemical synthesis, gas chromatography/mass spectrometry (GC/MS), and liquid chromatography tandem mass spectrometry (LC/MS/MS). Although nPF4 alpha-VI was below the detection limit of the assay, we conclusively identified iPF3 alpha-VI in human urine by GC/MS and LC/MS/MS. The mean levels in 26 subjects were approximately 300 pg/mg creatinine. Our failure to detect nPF4 alpha-VI may have been due to its rapid metabolism by beta-oxidation to iPF3 alpha-VI, which we showed to occur in rat liver homogenates. In contrast, iPF3 alpha-VI is highly resistant to beta-oxidation in vitro. Thus iPF3 alpha-VI can be formed by two mechanisms: i) direct autoxidation of EPA, and ii) beta-oxidation of nPF4 alpha-VI, formed by autoxidation of DHA. This iP may therefore serve as an excellent marker for the combined in vivo peroxidation of EPA and DHA.     

Disturbance of brain energy and redox homeostasis provoked by sulfite and thiosulfate: Potential pathomechanisms involved in the neuropathology of sulfite oxidase deficiency

Sulfite oxidase (SO) deficiency is biochemically characterized by tissue accumulation and high urinary excretion of sulfite, thiosulfate and S-sulfocysteine. Affected patients present severe neurological symptoms and cortical atrophy, whose pathophysiology is still poorly established. Therefore, in the present work we investigated the in vitro effects of sulfite and thiosulfate on important parameters of energy metabolism in the brain of young rats. We verified that sulfite moderately inhibited the activity of complex IV, whereas thiosulfate did not alter any of the activities of the respiratory chain complexes. It was also found that sulfite and thiosulfate markedly reduced the activity of total creatine kinase (CK) and its mitochondrial and cytosolic isoforms, suggesting that these metabolites impair brain cellular energy buffering and transfer. In contrast, the activity of synaptic Na⁺,K⁺-ATPase was not altered by sulfite or thiosulfate. We also observed that the inhibitory effect of sulfite and thiosulfate on CK activity was prevented by melatonin, reduced glutathione and the combination of both antioxidants, as well as by the nitric oxide synthase N^ω-nitro-l-arginine methyl ester, indicating the involvement of reactive oxygen and nitrogen species in these effects. Sulfite and thiosulfate also increased 2′,7′-dichlorofluorescin oxidation and hydrogen peroxide production and decreased the activity of the redox sensor aconitase enzyme, reinforcing a role for oxidative damage in the effects elicited by these metabolites. It may be presumed that the disturbance of cellular energy and redox homeostasis provoked by sulfite and thiosulfate contributes to the neurological symptoms and abnormalities found in patients affected by SO deficiency.     

Production and Isolation of Dopastin,an Inhibitor of Dopamine β-Hydroxylase

A new inhibitor of dopamine β-hydroxylase, dopastin, has been isolated. The dopastin-producing strain was found in a mushroom culture, and after being separated, it was confirmed to be a member of Pseudomonas. Dopastin was obtained as colorless needles, mp 116~119°C, (c=0.5, С₂Н₅ОН), C₉H₁₇N₃O₃. The catalytic hydrogenation afforded dihydro-dopastin which also inhibits dopamine β-hydroxylase.     

Molecular identification of ω-amidase, the enzyme that is functionally coupled with glutamine transaminases, as the putative tumor suppressor Nit2

Our purpose was to identify the sequence of ω-amidase, which hydrolyses the amide group of α-ketoglutaramate, a product formed by glutamine transaminases. In the Bacillus subtilis genome, the gene encoding a glutamine transaminase (mtnV) is flanked by a gene encoding a putative ‘carbon-nitrogen hydrolase’. The closest mammalian homolog of this putative bacterial ω-amidase is ‘nitrilase 2’, whose size and amino acid composition were in good agreement with those reported for purified rat liver ω-amidase. Mouse nitrilase 2 was expressed in Escherichia coli, purified and shown to catalyse the hydrolysis of α-ketoglutaramate and other known substrates of ω-amidase. No such activity was observed with mouse nitrilase 1. We conclude that mammalian nitrilase 2 is ω-amidase.     

The biosynthesis of cutin and suberin as an alternative source of enzymes for the production of bio-based chemicals and materials

Oxygenated fatty acids such as ricinoleic acid and vernolic acid can serve in the industry as synthons for the synthesis of a wide range of chemicals and polymers traditionally produced by chemical conversion of petroleum derivatives. Oxygenated fatty acids can also be useful to synthesize specialty chemicals such as cosmetics and aromas. There is thus a strong interest in producing these fatty acids in seed oils (triacylglycerols) of crop species. In the last 15 years or so, much effort has been devoted to isolate key genes encoding proteins involved in the synthesis of oxygenated fatty acids and to express them in the seeds of the model plant Arabidopsis thaliana or crop species. An often overlooked but rich source of enzymes catalyzing the synthesis of oxygenated fatty acids and their esterification to glycerol is the biosynthetic pathways of the plant lipid polyesters cutin and suberin. These protective polymers found in specific tissues of all higher plants are composed of a wide variety of oxygenated fatty acids, many of which have not been reported in seed oils (e.g. saturated ω-hydroxy fatty acids and α,ω-diacids). The purpose of this mini-review is to give an overview of the recent advances in the biosynthesis of cutin and suberin and discuss their potential utility in producing specific oxygenated fatty acids for specialty chemicals. Special emphasis is given to the role played by specific acyltransferases and P450 fatty acid oxidases. The use of plant surfaces as possible sinks for the accumulation of high value-added lipids is also highlighted.     

Pullulan-sodium alginate based edible films: Rheological properties of film forming solutions

Rheological properties of pullulan, sodium alginate and blend solutions were studied at 20 °C, using steady shear and dynamic oscillatory measurements. The intrinsic viscosity of pure sodium alginate solution was 7.340 dl/g, which was much higher than that of pure pullulan (0.436 dl/g). Pure pullulan solution showed Newtonian behavior between 0.1 and 100 s⁻¹ shear rate range. However, increasing sodium alginate concentration in pullulan-alginate blend solution led to a shear-thinning behavior. The effect of temperature on viscosities of all solutions was well-described by Arrhenius equation. Results from dynamical frequency sweep showed that pure sodium alginate and blend solutions at 4% (w/w) polymer concentration were viscoelastic liquid, whereas the pure pullulan exhibited Newtonian behavior. The mechanical properties of pure sodium alginate and pullulan-alginate mixture were analyzed using the generalized Maxwell model and their relaxation spectra were determined. Correlation between dynamic and steady-shear viscosity was analyzed with the empirical Cox-Merz rule.     

Modulation of cytochrome P450 activity in the kidney of rats following long-term red wine exposure

Cytochrome P450 (CYP)-dependent oxidation of lauric acid, p-nitrophenol and ethanol by microsomal fractions of kidney were studied in control rats and in animals given either ethanol, red wine, or alcohol-free red wine for 10 weeks. Ethanol increased the total CYP content and specifically CYP 2E1, as well as p-nitrophenol and ethanol oxidation. The effects of ethanol treatment on the content and activity of CYP 2E1 were attenuated when red wine was administered, while the alcohol-free red wine values were similar to those of the control group. Although lauric acid hydroxylation was decreased by red wine treatment, the content of CYP 4A1 was not influenced by drinking fluids. We conclude that red wine administration attenuates the ethanol-induced enhancement of microsomal activities dependent on CYP 2E1 of rat kidney. Our results suggest that the non-alcoholic constituents of red wine could account for this modulation.     

Screening of oxylipins for control of oilseed rape (Brassica napus) fungal pathogens

Oxylipins are products of oxygenase-catalyzed reactions of fatty acids. Oxylipins have been found or implied to participate in a variety of different functions in or between organisms. In this report we investigated the potential of various naturally occurring oxylipins found in plants for their effects as fungicides on a number of fungal pathogens interfering with Brassica cultivation. The fungi investigated were Alternaria brassicae, Leptosphaeria maculans, Sclerotinia sclerotiorum and Verticillium longisporum. An in vitro growth inhibition assay was used, where the relative growth rate of the fungi were determined in the presence of various concentrations of oxylipins. While no universal fungicidic effect was found for the 10 compounds investigated there were examples of oxylipins having inhibitory effects. In certain cases the inhibitory effects was overcome by time, however. Since several of the oxylipins tested were found to be stable in the absence of the fungus this effect could be explained by induction of the degrading capacity of the fungus or increased tolerance. Several of the oxylipins also inhibited germination of L. maculans spores but the relative potency differed compared to the effects on hyphae. The study suggests that selected oxylipins may be used for disease control on Brassica plants.     

Exploring sequence space: profile analysis and protein-ligand docking to screen omega-aminotransferases with expanded substrate specificity

omega-Aminotransferase (omegaAT) is an interesting biocatalyst for the preparation of chiral amines, which are widely used as building blocks for pharmaceuticals, agrochemicals and fine chemicals. With the assumption that substrate and sequence spaces are in the process of co-evolution, we explored sequences space to screen the enzymes showing activity to new target compounds. Bacterial genome sequences (n=527) were analyzed by the profiles of subgroups in aminotransferase group II including ornithine aminotransferase (orAT), acetylornithine aminotransferase (aoAT), omegaAT, gamma-aminobutyrate aminotransferase (GABAAT) and 7,8-diaminopelargonate aminotransferase (DAPAAT). We selected the sequences having a Z score of 0-1.8 to guarantee the omegaAT reaction and to avoid the typical omegaAT sequences. Among the selected sequences, we filtered out the sequences with very low Z scores for the rest of four subgroups in aminotransferase group II to consider the diversity. For the selected sequences, we performed protein-ligand docking simulations to predict the docking pose of amino acceptor. Throughout all the analysis procedures, several candidate aminotransferase sequences for the asymmetric synthesis of chiral amines were obtained. An efficient procedure for virtual screening of novel enzymes was demonstrated.