Detection and enumeration of sulphate-reducing bacteria in estuarine sediments by competitive PCR

The distribution of sulphate-reducing bacteria (SRB) in the sediments of the Colne River estuary, Essex, UK covering different saline concentrations of sediment porewater was investigated by the use of quantitative competitive PCR. Here, we show that a new PCR primer set and a new quantitative method using PCR are useful tools for the detection and the enumeration of SRB in natural environments. A PCR primer set selective for the dissimilatory sulphite reductase gene (dsr) of SRB was designed. PCR amplification using the single set of dsr-specific primers resulted in PCR products of the expected size from all 27 SRB strains tested, including Gram-negative and positive species. Sixty clones derived from sediment DNA using the primers were sequenced and all were closely related with the predicted dsr of SRB. These results indicate that PCR using the newly designed primer set are useful for the selective detection of SRB from a natural sample. This primer set was used to estimate cell numbers by dsr selective competitive PCR using a competitor, which was about 20% shorter than the targeted region of dsr. This procedure was applied to sediment samples from the River Colne estuary, Essex, UK together with simultaneous measurement of in situ rates of sulphate reduction. High densities of SRB ranging from 0.2 - 5.7 × 108 cells ml-1 wet sediment were estimated by the competitive PCR assuming that all SRB have a single copy of dsr. Using these estimates cell specific sulphate reduction rates of 10-17 to 10-15 mol of SO42- cell-1 day-1 were calculated, which is within the range of, or lower than, those previously reported for pure cultures of SRB. Our results show that the newly developed competitive PCR technique targeted to dsr is a powerful tool for rapid and reproducible estimation of SRB numbers in situ and is superior to the use of culture-dependent techniques.

Bacterial iron homeostasis

Iron is essential to virtually all organisms, but poses problems of toxicity and poor solubility. Bacteria have evolved various mechanisms to counter the problems imposed by their iron dependence, allowing them to achieve effective iron homeostasis under a range of iron regimes. Highly efficient iron acquisition systems are used to scavenge iron from the environment under iron-restricted conditions. In many cases, this involves the secretion and internalisation of extracellular ferric chelators called siderophores. Ferrous iron can also be directly imported by the G protein-like transporter, FcoB. For pathogens, host-iron complexes (transferrin, lactoferrin, haem, haemoglobin) are directly used as iron sources. Bacterial iron storage proteins (ferritin, bacterioferritin) provide intracellular iron reserves for use when external supplies are restricted, and iron detoxification proteins (Dps) are employed to protect the chromosome from iron-induced free radical damage. There is evidence that bacteria control their iron requirements in response to iron availability by downregulating the expression of iron proteins during iron-restricted growth. And finally, the expression of the iron homeostatic machinery is subject to iron-dependent global control ensuring that iron acquisition, storage and consumption are geared to iron availability and that intracellular levels of free iron do not reach toxic levels. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Global iron-dependent gene regulation in Escherichia coli - A new mechanism for iron homeostasis

Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by consuming intracellular iron stores. Escherichia coli, in which iron metabolism is particularly well understood, contains at least 7 iron-acquisition systems encoded by 35 iron-repressed genes. This Fe-dependent repression is mediated by a transcriptional repressor, Fur ( ferric uptake regulation), which also controls genes involved in other processes such as iron storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress resistance. Our macroarray-based global analysis of iron- and Fur-dependent gene expression in E. coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron- transport pathways. Interestingly, a large group of energy metabolism genes was found to be iron and Fur induced. Many of these genes encode iron- rich respiratory complexes. This iron- and Fur-dependent regulation appears to represent a novel iron-homeostatic mechanism whereby the synthesis of many iron- containing proteins is repressed under iron- restricted conditions. This mechanism thus accounts for the low iron contents of fur mutants and explains how E. coli can modulate its iron requirements. Analysis of Fe-55-labeled E. coli proteins revealed a marked decrease in iron- protein composition for the fur mutant, and visible and EPR spectroscopy showed major reductions in cytochrome b and d levels, and in iron- sulfur cluster contents for the chelator-treated wild-type and/or fur mutant, correlating well with the array and quantitative RT-PCR data. In combination, the results provide compelling evidence for the regulation of intracellular iron consumption by the Fe2+-Fur complex.

Dietary alpha-tocopherol affects differential gene expression in rat testes

Gene-chip technology was employed to study the effect of dietary vitamin E (VE) on gene expression in rat testes. Male albino rats were fed with either a diet deficient in VE or a standard diet containing VE. Differential gene expression was monitored at five individual time-points over a period of 14 months with all animals individually pro. led. Low VE intake resulted in the consistent upregulation of 7-dehydrocholesterol reductase and GATA binding protein 4, both involved in testosterone synthesis. Cyclin D3, important in cell cycle progression and Wilms tumor 1, related to cancer development, were also up-regulated in the vitamin E deficient animals. This study demonstrates that low dietary VE intake has long-term effects on gene expression in the testes. Our data provides insights into the possible molecular mechanisms underlying the beneficial effects of vitamin E on the male reproductive organ.

Critical stages of the brewing process for changes in antioxidant activity and levels of phenolic compounds in ale

Samples were taken at each stage of brewing (malt, milling, mashing, wort separation, hop addition, boiling, whirlpool, dilution, fermentation, warm rest, chill-lagering, beer filtration, carbonation and bottling, pasteurization, and storage). The level of antioxidant activity of unfractionated, low-molecular-mass (LMM) and high-molecular-mass (HMM) fractions was measured by the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfortic acid) radical cation (ABTS(.+)) and ferric-reducing antioxidant power (FRAP) procedures. Polyphenol levels were assessed by HPLC. The LMM fraction (<5 kDa) was responsible for similar to80% of the level of antioxidant activity of the unfractionated malt and beer samples. In the unfractionated samples, significant decreases (P < 0.001) in antioxidant activity levels were observed after milling and beer filtration, with the decrease after beer filtration being accompanied by a significant decrease (P > 0.001) in catechin and ferulic acid levels. Increases in antioxidant activity levels were observed after mashing, boiling, fermentation, chill-lagering, and pasteurization, in line with previous studies on lager. Additionally, increases in the level of antioxidant activity occurred after wort separation and carbonation and bottling and were accompanied by increases in levels of most monitored polyphenols. Data from the ABTS(.-) and FRAP assays indicated that the compounds contributing to the levels of antioxidant activity responded differently in the two procedures. Levels of ferulic, vanillic, and chlorogenic acids and catechin accounted for 45-61% of the variation in antioxidant activity levels.

Antioxidant properties of Teaw (Cratoxylum formosum Dyer) extract in soybean oil and emulsions

The antioxidant activity of an extract from Teaw (Cratoxylum formosum Dyer) leaves was studied in soybean oil and soybean oil-in-water emulsions. Samples containing the extract or reference antioxidants including chlorogenic acid, which comprises 60% of the Teaw extract, were stored at 60 degrees C and analyzed periodically for peroxide value (PV) and thiobarbituric acid reactive substances (TBARS) to allow both hydroperoxides and hydroperoxide degradation products to be monitored. Chlorogenic acid and the Teaw extract were more effective than a-tocopherol in inhibiting lipid oxidation in bulk oil but were less effective in an oil-in-water emulsion in accordance with the polar paradox. The PV/TBARS ratio for oil samples containing chlorogenic acid was higher than for alpha-tocopherol and BHT because chlorogenic acid inhibits both hydroperoxide formation by radical scavenging and hydroperoxide decomposition by metal chelation. The importance of the metal-chelating activity in retarding hydroperoxide decomposition was confirmed by studying the decomposition of oil samples containing added ferric ions. The PV/TBARS ratio was higher for citric acid than for (x-tocopherol in the presence of added ferric chloride, but the order was reversed in samples lacking ferric chloride. Samples containing added chlorogenic acid gave the highest PV/TBARS ratios both in the presence and absence of ferric ions. The PV/TBARS ratios for the samples containing antioxidants fell rapidly to lower values in a soybean oil-in-water emulsion than in the soybean oil. This was due to increased hydroperoxide decomposition in the emulsion at the same PV. The Teaw extract contained 12% oil-soluble components, which contributed to a slightly higher oil-water partition coefficient than that of chlorogenic acid. The antioxidant activity of the aqueous phase of the Teaw extract was reduced more than that of chlorogenic acid by partitioning of the oil-soluble components into oil, which showed that the less-polar components contributed to the antioxidant activity of the Teaw extract in aqueous media.

Antioxidant and tyrosinase inhibitory activity of mango seed kernel by product

The antioxidant and tyrosinase inhibitory properties of extracts of mango seed kernel (Mangifera indica L.), which is normally discarded when the fruit is processed, were studied. Extracts contained phenolic components by a high antioxidant activity, which was assessed in homogeneous solution by the 2,2-diphenyt-1-picrylhydrazyl radical and 2,2'-azinobis (3-ethylbenzothialozinesulfonic acid) radical cation-scavenging assays and in an emulsion with the ferric thiocyanate test. The extracts also possessed tyrosinase inhibitory activity. Drying conditions and extraction solvent were varied, and optimum conditions for preparation of mango seed kernel extract were found to be sun-drying with ethanol extraction at room temperature. Refluxing in acidified ethanol gave an increase in yield and the obtained extract had the highest content of total phenolics, and also was the most effective antioxidant with the highest radical-scavenging, metal-chelating and tyrosinase inhibitory activity. The extracts did not cause acute irritation of rabbit skins. Our study for the first time reveals the high total phenol content, radical-scavenging, metal-chelating and tyrosinase inhibitory activities of the extract from mango seed kernel. This extract may be suitable for use in food, cosmetic, nutraceutical and pharmaceutical applications. (C) 2009 Elsevier Ltd. All rights reserved.

Effect of a cellulase treatment on extraction of antioxidant phenols from black currant (Ribes nigrum L.) pomace

The effect of a commercial cellulase preparation on phenol liberation and extraction from black currant pomace was studied. The enzyme used, which was from Trichoderma spp., was an effective "cellulase-hemicellulase" blend with low P-glucosidase activity and various side activities. Enzyme treatment significantly increased plant cell wall polysaccharide degradation as well as increasing the availability of phenols for subsequent methanolic extraction. The release of anthocyanins and other phenols was dependent on reaction parameters, including enzyme dosage, temperature, and time. At 50 degrees C, anthocyanin yields following extraction increased by 44% after 3 h and by 60% after 1.5 h for the lower and higher enzyme/substrate ratio (E/S), respectively. Phenolic acids were more easily released in the hydrolytic mixture (supernatant) and, although a short hydrolysis time was adequate to release hydroxybenzoic acids (HBA), hydroxycinnamic acids (HCA) required longer times. The highest E/S value of 0.16 gave a significant increase of flavonol yields in all samples. The antioxidant capacity of extracts, assessed by scavenging of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation, the oxygen radical absorbance capacity, and the ferric reducing antioxidant potential depended on the concentration and composition of the phenols present.

Influence of apolipoprotein E genotype and dietary alpha-tocopherol on redox status and C-reactive protein levels in apolipoprotein E3 and E4 targeted replacement mice

The molecular basis of the positive association between apoE4 genotype and CVD remains unclear. There is direct in vitro evidence indicating that apoE4 is a poorer antioxidant relative to the apoE3 isoform, with some indirect in vivo evidence also available. Therefore it was hypothesised that apoE4 carriers may benefit from alpha-tocopherol (alpha-Toc) supplementation. Targeted replacement mice expressing the human apoE3 and apoE4 were fed with a diet poor (0 mg/kg diet) or rich (200 mg/kg diet) in alpha-Toc for 12 weeks. Neither apoE genotype nor dietary alpha-Toc exerted any effects on the antioxidant defence system, including glutathione, catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase activities. In addition, no differences were observed in mitogen-induced lymphocyte proliferation. alpha-Toc concentrations were modestly higher in plasma and lower in tissues of apoE4 compared with apoE3 mice, with the greatest differences evident in the lung, suggesting that an apoE4 genotype may reduce alpha-Toc delivery to tissues. A tendency towards increased plasma F-2-isoprostanes in apoE4 mice was observed, while liver thiobarbituric acid-reactive substances did not differ between apoE3 and apoE4 mice. In addition, C-reactive protein (CRP) concentrations were reduced in apoE4 mice indicating that this positive effect on CRP may in part negate the increased CVD risk associated with an apoE4 genotype.

Effect of a simulated kilning regime on the profile and antioxidant activity of the free phenolic acids extracted from green malt

Free phenolic acids were extracted from a laboratory-produced sample of green malt. Aliquots of the phenolic acid extract were heated from 25 to 110°C over 27 h, representative of a commercial kilning regime. Samples were taken at regular intervals throughout heating and were assessed for changes in antioxidant activity by both the 2,2(prime)-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical-cation scavenging (ABTS(^•+)) and the ferric-reducing antioxidant potential (FRAP) assays. Changes in the profile of the phenolic acids of the extracts were determined by HPLC. Overall, there was a decrease in both antioxidant activity level and the level of phenolic acids, but as the temperature increased from 80 to 100°C, there was an increase in both the antioxidant activity level and the level of detected phenolic acids.

Influence of habitual diet on antioxidant status: a study in a population of vegetarians and omnivores

Background: Antioxidant status can be used as a biomarker to assess chronic disease risk and diet can modulate antioxidant defence. Objective: To examine effects of vegetarian diet and variations in the habitual intakes of foods and nutrients on blood antioxidants. Subjects and Setting: Thirty-one vegetarians (including six vegans) and 58 omnivores, non-smokers, in Northern Ireland. Design: A diet history method was used to assess habitual diet. Antioxidant vitamins, carotenoids, uric acid, zinc-and ferric-reducing ability of plasma (FRAP) were measured in fasting plasma and activities of glutathione peroxidase (GPX), superoxide dismutase ( SOD) and glutathione S-transferase (GST) and level of reduced glutathione (GSH) were measured in erythrocytes. Results: Vegetarians had approximately 15% higher levels of plasma carotenoids compared with omnivores, including lutein (P <= 0.05), a-cryptoxanthin (P <= 0.05), lycopene (NS), alpha-carotene (NS) and beta-carotene (NS). The levels/activities of all other antioxidants measured were similar between vegetarians and omnivores. Total intake of fruits, vegetables and fruit juices was positively associated with plasma levels of several carotenoids and vitamin C. Intake of vegetables was positively associated with plasma lutein, alpha-cryptoxanthin, alpha-carotene and beta-carotene, whereas intake of fruits was positively associated with plasma beta-cryptoxanthin. Intake of tea and wine was positively associated with FRAP value, whereas intake of herbal tea associated positively with plasma vitamin C. Intakes of meat and fish were positively associated with plasma uric acid and FRAP value. Conclusions: The overall antioxidant status was similar between vegetarians and omnivores. Good correlations were found between intakes of carotenoids and their respective status in blood.

Watercress supplementation in diet reduces lymphocyte DNA damage and alters blood antioxidant status in healthy adults

Background: Cruciferous vegetable (CV) consumption is associated with a reduced risk of several cancers in epidemiologic studies. Objective: The aim of this study was to determine the effects of watercress (a CV) supplementation on biomarkers related to cancer risk in healthy adults. Design: A single-blind, randomized, crossover study was conducted in 30 men and 30 women (30 smokers and 30 nonsmokers) with a mean age of 33 y (range: 19-55 y). The subjects were fed 85 g raw watercress daily for 8 wk in addition to their habitual diet. The effect of supplementation was measured on a range of endpoints, including DNA damage in lymphocytes (with the comet assay), activity of detoxifying enzymes (glutathione peroxidase and superoxide dismutase) in erythrocytes, plasma antioxidants (retinol, ascorbic acid, a-tocopherol, lutein, and beta-carotene), plasma total antioxidant status with the use of the ferric reducing ability of plasma assay, and plasma lipid profile. Results: Watercress supplementation (active compared with control phase) was associated with reductions in basal DNA damage (by 17%; P = 0.03), in basal plus oxidative purine DNA damage (by 23.9%; P = 0.002), and in basal DNA damage in response to ex vivo hydrogen peroxide challenge (by 9.4%; P = 0.07). Beneficial changes seen after watercress intervention were greater and more significant in smokers than in nonsmokers. Plasma lutein and P-carotene increased significantly by 100% and 33% (P < 0.001), respectively, after watercress supplementation. Conclusion: The results support the theory that consumption of watercress can be linked to a reduced risk of cancer via decreased damage to DNA and possible modulation of antioxidant status by increasing carotenoid concentrations.

alpha-tocopherol affects androgen metabolism in male rat

The Alpha-Tocopherol Beta-Carotene Cancer Prevention Study has provided the first evidence implicating vitamin E in hormone synthesis. The effect of vitamin E on stereoidogenesis in testes and adrenal glands was assessed in growing rats using Affymetrix gene-chip technology. Dietary supplementation of rats with vitamin E (60 mg/kg feed) for a period of 429 days caused a significant repression of genes encoding for proteins centrally involved in the uptake (low-density lipoprotein receptor) and de novo synthesis (for example, 7-dehydrocholesterol reductase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, isopentenyl-diphosphate delta-isomerase, and farnesyl pyrophosphate synthetase) of cholesterol, the precursor of all steroid hormones. The present investigation indicates that dietary vitamin E may induce changes in stereoidogenesis by affecting cholesterol homeostasis.

Identification of hepatic molecular mechanisms of action of alpha-tocopherol using global gene expression profile analysis in rats

The recent discovery that vitamin E (VE) regulates gene activity at the transcriptional level indicates that VE may exert part of its biological effects by mechanisms which may be independent of its well-recognised antioxidant function. The objective of this study was the identification of hepatic vitamin E-sensitive genes and examination of the effects of VE on their corresponding biological endpoints. Two groups of male rats were randomly assigned to either a VE-sufficient diet or to a control diet deficient in VE for 290 days. High-density oligonucleotide microarrays comprising over 7000 genes were used to assess the transcriptional response of the liver. Differential gene expression was monitored over a period of 9 months, at four different time-points, and rats were individually profiled. This experimental strategy identified several VE-sensitive genes, which were chronically altered by dietary VE. VE supplementation down-regulated scavenger receptor CD36, coagulation factor IX and 5-alpha-steroid reductase type 1 mRNA levels while hepatic gamma glutamyl-cysteinyl synthetase was significantly up-regulated. Measurement of the corresponding biological endpoints such as activated partial thromboplastin time, plasma dihydrotestosterone and hepatic glutathione substantiated the gene chip data which indicated that dietary VE plays an important role in a range of metabolic processes within the liver. (C) 2004 Elsevier B.V. All rights reserved.

Changes in the antioxidant properties of protein solutions in the presence of epigallocatechin gallate

beta-Casein and alpha-casein showed radical-scavenging activities in aqueous solution, whereas bovine serum albumin (BSA), alpha-lactalbumin and P-lactoglobulin showed much weaker antioxidant activity, when assessed by the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical-scavenging assay. However, beta-casein and alpha-casein showed reduced antioxidant activity after storage at 30 degrees C. An increase in radical- scavenging activity and a fall in fluorescence of the protein component were evident after 6 h, when BSA, beta-lactoglobulin or casein were mixed with EGCG, and excess EGCG was removed, indicating the formation of a complex with this protein on mixing. Storage of all the proteins with EGCG at 30 degrees C caused an increase in the antioxidant activity of the isolated protein component after separation from excess EGCG. This showed that EGCG was reacting with the proteins and that the protein-bound catechin had antioxidant properties. The reaction of EGCG with BSA, casein and beta-lactoglobulin was confirmed by the loss of fluorescence of the protein on storage, and the increase in UV absorbance between 250 and 400 nm. The increase in antioxidant activity of BSA after storage with EGCG was confirmed by the ferric reducing antioxidant potential (FRAP) and the oxygen radical antioxidant capacity (ORAC) assays. (c) 2006 Elsevier Ltd. All rights reserved.