Phenolic acid amides of phenolic benzylamines against UVA-induced oxidative stress in skin

UVA-radiation is suspected to be a main cause for extrinsic skin ageing in humans. Recent studies showed that topically administered molecular antioxidants are able to protect skin against UVA-generated oxidative stress. Therefore, new phenolic acid amides of phenolic benzylamines were synthesized as potential antioxidants by reaction of (if necessary protected) N-succinimidylesters of ferulic acid, sinapic acid, caffeic acid, 3-(4-hydroxy-3-methoxyphenyl)propanoic acid, 3-(3,4-dihydroxyphenyl)propanoic acid, 2-(4-hydroxy-3-methoxyphenyl)acetic acid and 2-(3,4-dihydroxyphenyl) acetic acid with different phenolic benzylamines in moderate to good yields. The radical scavenging activities of the compounds were determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay and by a superoxide radical anion trapping assay. Antioxidant activities in bulk lipids were tested by accelerated peroxidation with or without test compounds performed in the Rancimat apparatus. Stripped soybean oil and the skin sebum component squalene were used as lipids. The synthesized compounds were found to be efficient radical scavengers and antioxidants, especially the hydroxyphenylacetic amides and the hydroxydihydrocinnamic acid amides of polyhydroxybenzylamines, which are superior to the standards alpha-tocopherol and l-ascorbic acid. A topically applied 0.1% 1,3-butyleneglycol solution of N-(3,4-dihydroxybenzyl)-2-(4-hydroxy-3-methoxyphenyl)acetic acid amide 19 inhibits the UVA-induced sebum peroxidation in human skin significantly, by 39%.     

Svf1 inhibits reactive oxygen species generation and promotes survival under conditions of oxidative stress in Saccharomyces cerevisiae

Aberrant regulation of apoptosis, or programmed cell death, contributes to the aetiology of several diseases, including cancers, immunodeficiencies and neurodegenerative illnesses. We hypothesized that key features of mammalian cell death regulation may be conserved in single celled organisms such as the budding yeast Saccharomyces cerevisiae. We previously identified the yeast gene SVF1 in a screen for mutations that could be functionally complemented by exogenous expression of the human anti-apoptotic gene Bcl-x(L). Anti-apoptotic Bcl-2 family members have been shown to promote redox stability through upregulation of antioxidant pathways in mammalian cells. Here we demonstrate that the Svf1 protein is required for yeast survival under conditions of oxidative stress, including cold stress. Cells lacking SVF1 are hypersensitive to conditions associated with increased reactive oxygen species (ROS) generation and to direct chemical precursors of ROS, and demonstrate increased levels of ROS under these conditions. Hypersensitivity to oxidative stress can be reversed by treatment with the antioxidant N-acetylcysteine or expression of exogenous SVF1, although exogenous expression of Bcl-x(L) did not protect cells from cold stress. Exogenous SVF1 expression in mammalian cells confers resistance to H(2)O(2) exposure. Our data are consistent with previous observations suggesting a key role of oxidative stress response in mammalian apoptotic regulation and validate the use of S. cerevisiae as a model for studying programmed cell death.     

Effects of Graptopetalumparaguayense extract on tert‐ butylhydroperoxide‐induced oxidative stress

BACKGROUND: Graptopetalum paraguayense E. Walther is used in folk medicine to lower blood pressure, protect liver function and relieve pain and infection. The effect and mechanism of its 50% ethanolic extract (GE50) were investigated in tert‐butylhydroperoxide (t‐BHP)‐induced Wistar rats. The serum was analysed for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities while the tissue cytosols were analysed for lipid peroxidation, antioxidant level and enzyme activities. RESULTS: The liver was the primary target organ while the heart appeared to be the least responsive organ for t‐BHP treatment among the three tissues investigated. t‐BHP treatment increased serum AST and ALT activities, which are indicators of liver toxicity, while GE50 pretreatment reduced t‐BHP‐induced liver damage. t‐BHP treatment induced lipid peroxidation in the liver and brain but not in the heart. GE50 pretreatment prevented t‐BHP‐induced lipid peroxidation by maintaining superoxide dismutase (SOD) activity as well as glutathione (GSH) and vitamin C levels in the liver and vitamin E level in the brain. Although t‐BHP treatment did not induce lipid peroxidation in the heart, it caused a decrease in antioxidant level. GE50 pretreatment prevented the t‐BHP‐induced decrease in vitamin C level in the heart. CONCLUSION: GE50 pretreatment in rats protects the liver and brain from possible damage by t‐BHP‐induced lipid peroxidation. Copyright © 2007 Society of Chemical Industry     

Nrf2-ARE介导的黄酮类化合物抗氧化应激的研究进展

氧化应激与人体健康密切相关,而黄酮类化合物因能激活人体内调节众多抗氧化基因表达的关键性防御通道——Nrf2-ARE信号通道,对抗氧化应激有着十分显著的作用。文章介绍了Nrf2-ARE信号通道的激活、调控Nrf2-ARE激活的相关信号通路、受Nrf2-ARE调控的下游基因以及Nrf2-ARE介导的黄酮类化合物对炎症和凋亡反应的影响,同时对Nrf2-ARE介导的八大类主要黄酮化合物在抗氧化应激方面的研究进行综述,并提出现阶段研究存在的主要科学问题及解决策略,以期为黄酮类化合物在预防和治疗氧化应激相关性疾病等领域的研究提供参考。     

Effect of coffee melanoidin on human hepatoma HepG2 cells. Protection against oxidative stress induced by tert-butylhydroperoxide

Soluble high-molecular weight fraction (named melanoidin) from coffee brew was isolated by ultrafiltration, subsequently digested by simulating a gastric plus pancreatic digestive condition and partly characterized by CZE, gel-filtration and browning. The objective of the present study was to investigate the potential protective effect of the coffee melanoidin submitted to gastrointestinal digestion on cell viability (lactate dehydrogenase leakage) and redox status of cultured human hepatoma HepG2 cells submitted to oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Concentration of reduced glutathione (GSH) and malondialdehyde (MDA), generation of reactive oxygen species (ROS) and activity of antioxidant enzymes glutathione peroxidase (GPx) and reductase (GR) were used as markers of cellular oxidative status. Pretreatment of cultured HepG2 cells with 0.5-10 microg/mL digested coffee melanoidin (DCM) for 2 or 20 h completely prevented the increase in cell damage and GR and partly prevented the decrease of GSH and the increase of MDA and GPx evoked by t-BOOH in HepG2 cells. In contrast, increased ROS generation induced by t-BOOH was not prevented when cells were pretreated with DCM. The results show that treatment of HepG2 cells with concentrations of DCM within the expected physiological range confers the cells a significant protection against an oxidative insult.     

Detection of Proteinases in Saccharomyces cerevisiae by Flow Cytometry

The physiological state of a yeast population used for inoculation determines how rapidly the cells adapt to new environmental conditions, begin proliferating and utilising extract. The decision as to whether a yeast culture is suitable for re‐pitching should not be based only on viability determinations since this can be misleading. Increased proteolytic activity in a yeast population indicates the onset of senescence. A flow cytometric method has been developed for measuring a wide variety of proteinases in Saccharomyces cerevisiae employing a commercially available casein‐dye conjugate. The detection of intracellular proteinase activity gives an early indication of apoptotic events and allows improved assessment of the physiological state of a yeast population. This knowledge will assist the industry to optimize the selection of yeast and its subsequent fermentation performance. Yeast cell autolysis with all its negative consequences for beer quality and stability will thus be minimised.     

Bacterial plasmid pBR322 sequences serve as upstream activating sequences in Saccharomyces cerevisiae

The expression of acid phosphatase (APase) from PHO5 and MF alpha-PHO5 hybrid genes is regulated by inorganic phosphate and mating type locus respectively, as well as the PHO4 and MAT alpha 1 gene products respectively. When PHO5 and MF alpha-PHO5 hybrid genes were cloned in the BamHI site of the pBR322 sequence of the yeast shuttle vectors (YRp7 or YEp9T), in one orientation they were regulated normally but in the other orientation their expression was not regulated but expressed constitutively. The pBR322 sequences present upstream of the inserted genes are responsible for the constitutive expression. By replacing the PHO5 upstream activating sequences (UAS) element with pBR322 fragments, we have identified three pBR322 sequences, from nucleotides 376 to 650, 2068 to 2116 and 2136 to 2247, which were able to promote expression of APase. A comparison of these three pBR322 fragments revealed 5' ATCGCGCGAG 3' and 5' CGGTGATGNCGG 3' to be the common sequences likely to act as UASs in Saccharomyces cerevisiae. By using synthetic oligonucleotides, it was found that both sequences are required for maximum expression of APase activity.     

Cytoprotective activity of extract of roasted coffee residue on mouse embryonic fibroblasts cells against apoptosis induced by oxidative stress

This study was conducted to evaluate the cytoprotective activity of roasted coffee residues (RCRs) extract on mouse embryonic fibroblast (MEF) cells. RCRs originated from Colombia and Honduras are relatively nontoxic to cell growth and even stimulate cell proliferation. Colombian RCRs showed most efficient protective effects on MEF cells against oxidative damage induced by H₂O₂ compared among the extracts prepared under the same roasting time. The most significant radical scavenging activity was measured in RCR with roasting time of 8.5 min. Phenolic and nonphenolic compounds in RCRs were chlorogenic acid, caffeine, caffeic acid, nicotinic acid, trigonelline, and 5-(hydroxymethyl)furfuralolehyde. Effect of Colombian RCRs on apoptosis occurred by oxidative damage was evaluated by morphological and flow cytometric analysis. Apoptotic cell accumulation was decreased by cotreatment of MEF cells with Colombian RCRs. These results suggested that antioxidant potency of RCRs suppresses the cytotoxicity which is induced by H₂O₂ and has a protective effect on MEF cell against oxidative stress.     

Expression and in vivo determination of firefly luciferase as gene reporter in Saccharomyces cerevisiae

The LUC gene coding for Photinus pyralis firefly luciferase was cloned in different yeast episomal plasmids in order to assess its possibilities as an in vivo reporter gene. Activity of the enzyme in transformed cells in vivo was measured by following light emission and assay conditions optimized in intact cells, with regard to oxygen concentration, temperature, cell concentration in assay mixtures and external ATP concentration. Among the factors tested, light emission was drastically influenced by the external pH in the assay (which resulted in a ten-fold amplification signal) and by substrate permeability. The growth phase of the cells was also important for the level of activity detected. Cloning of firefly luciferase gene under the control of different yeast-regulated promoters (ADH1, GAL1–10) enabled us to measure their strength which correlated well with previously described data. We conclude that firefly luciferase is an adequate gene reporter for the in vivo sensitive determination of gene expression and promoter strength in yeast.     

Antioxidant effect of grapevine leaf extract on the oxidative stress induced by a high-fat diet in rats

This study was conducted to investigate the effect of grapevine leaf extract (GLE) on the oxidative stress of rats fed a high-fat diet. Rats were divided into six groups: a normal diet with 0% (NC), 1.0% (NG1.0), and 1.5% GLE (NG1.5); and a high-fat diet with 0% (HFC), 1.0% (HFG1.0), and 1.5% GLE (HFG1.5). Treatment with GLE to the high-fat diet reduced lipid peroxide concentrations in plasma and liver compared to those of HFC rats. Total glutathione and GSH/GSSG of the groups with GLE were higher than those of untreated groups. Hepatic glutathione peroxidase, glutathione-S-transferase, and glutathione reductase activities along with catalase and glutathione peroxidase activities in erythrocytes increased after GLE treatment to high-fat diet compared with HFC rats. Hepatic retinol and tocopherol increased in HFG1.5 group compared to those of HFC group. These results indicate that GLE can protect against oxidative stress induced by a high-fat diet in rats.     

FPG1, a gene involved in foam formation in Saccharomyces cerevisiae

Foam formation in fermentations conducted by Saccharomyces cerevisiae, either at the beginning of the fermentation process or at the end in the case of sparkling wines, is due, to a large extent, to cell wall mannoproteins, which provide hydrophobicity to the yeast cells and favour their floating index as well as stabilization of the foam. The foam may be an undesirable by-product if it accumulates on top of the fermentation tanks, but its formation is a good property in either beer or sparkling wines. It is therefore important to know the yeast genes involved in foam formation, in order to suppress or potentiate their expression according to the end product to be obtained. The present study identified and characterized, for the first time in an oenological S. cerevisiae strain, a gene involved in foam formation, named FPG1 (foam-promoting gene). The protein encoded by FPG1 is a mannoprotein precursor present in the cell wall and somewhat homologous to Awa1p, a foaming protein described in a sake S. cerevisiae strain. A foamless strain was prepared by FPG1 deletion, and a foam hyper-producing strain was also constructed, thus allowing the conclusion that Fpg1p is a mannoprotein involved in yeast frothing.     

黄酮对AAPH诱导的HepG2细胞氧化应激的作用

研究部分黄酮对AAPH诱导的HepG2细胞氧化应激的作用。建立AAPH诱导HepG2细胞氧化应激的模型,比较对照组和实验组细胞内的氧化自由基ROS的含量。结果表明,用不同浓度的黄酮处理HepG2细胞1h后,经过相同浓度的氧化剂作用后,细胞内自由基ROS的含量相对空白对照组均发生变化。这些选择性的黄酮对于AAPH诱导的HepG2细胞氧化应激具有促进或者抑制作用,其作用机制可能清除HepG2细胞内ROS以及与细胞内氧化酶和抗氧化酶系统的作用有关。     

Relatedness of medically important strains of Saccharomyces cerevisiae as revealed by phylogenetics and metabolomics

Ten medically important Saccharomyces strains, comprising six clinical isolates of Saccharomyces cerevisiae and four probiotic strains of Saccharomyces boulardii, were characterized at the genetic and metabolic level and compared with non-medical, commercial yeast strains used in baking and wine-making. Strains were compared by genetic fingerprinting using amplified fragment length polymorphism (AFLP) analysis, by ribosomal DNA ITS1 sequencing and by metabolic footprinting using both direct injection mass spectrometry (DIMS) and gas chromatography-time of flight-mass spectrometry (GC-ToF-MS). Overall, the clinical isolates fell into different groupings when compared with the non-medical strains, with good but not perfect correlation amongst strains at both the genetic and metabolic levels. Probiotic strains of S. boulardii that are used therapeutically to treat human gastro-intestinal tract disorders showed tight clustering both genetically and metabolically. Metabolomics was found to be of value both as a taxonomic tool and as a means to investigate anomalous links between genotype and phenotype. Key discriminatory metabolites were identified when comparing the three main groups of clinical, probiotic and non-medical strains and included molecules such as trehalose, myo-inositol, lactic acid, fumaric acid and glycerol 3-phosphate. This study confirmed the link between a subset of clinical isolates and baking or probiotic strains but also highlighted that in general the clinical strains were more diverse at both the genomic and metabolic levels.     

Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae

The widely used pESC vector series (Stratagene, La Jolla, CA, USA) with the bidirectional GAL1/GAL10 promoter provides the possibility of simultaneously expressing two different genes from a single vector in Saccharomyces cerevisiae. This system can be induced by galactose and is repressed by glucose. Since S. cerevisiae prefers glucose as a carbon source, and since its growth rate is higher in glucose than in galactose‐containing media, we compared and evaluated seven different promoters expressed during growth on glucose (pTEF1, pADH1, pTPI1, pHXT7, pTDH3, pPGK1 and pPYK1) with two strong galactose‐induced promoters (pGAL1 and pGAL10), using lacZ as a reporter gene and measuring LacZ activity in batch and continuous cultivation. TEF1 and PGK1 promoters showed the most constant activity pattern at different glucose concentrations. Based on these results, we designed and constructed two new expression vectors which contain the two constitutive promoters, TEF1 and PGK1, in opposite orientation to each other. These new vectors retain all the features from the pESC–URA plasmid except that gene expression is mediated by constitutive promoters. Copyright © 2010 John Wiley & Sons, Ltd.