Epidermal fatty acid‐binding protein protects nerve growth factor‐differentiated PC12 cells from lipotoxic injury

Epidermal fatty acid‐binding protein (E‐FABP/FABP5/DA11) binds and transport long‐chain fatty acids in the cytoplasm and may play a protecting role during neuronal injury. We examined whether E‐FABP protects nerve growth factor‐differentiated PC12 cells (NGFDPC12 cells) from lipotoxic injury observed after palmitic acid (C16:0; PAM) overload. NGFDPC12 cells cultures treated with PAM/bovine serum albumin at 0.3 mM/0.15 mM show PAM‐induced lipotoxicity (PAM‐LTx) and apoptosis. The apoptosis was preceded by a cellular accumulation of reactive oxygen species (ROS) and higher levels of E‐FABP. Antioxidants MCI‐186 and N‐acetyl cysteine prevented E‐FABP's induction in expression by PAM‐LTx, while tert‐butyl hydroperoxide increased ROS and E‐FABP expression. Non‐metabolized methyl ester of PAM, methyl palmitic acid (mPAM), failed to increase cellular ROS, E‐FABP gene expression, or trigger apoptosis. Treatment of NGFDPC12 cultures with siE‐FABP showed reduced E‐FABP levels correlating with higher accumulation of ROS and cell death after exposure to PAM. In contrast, increasing E‐FABP cellular levels by pre‐loading the cells with recombinant E‐FABP diminished the PAM‐induced ROS and cell death. Finally, agonists for PPARβ (GW0742) or PPARγ (GW1929) increased E‐FABP expression and enhanced the resistance of NGFDPC12 cells to PAM‐LTx. We conclude that E‐FABP protects NGFDPC12 cells from lipotoxic injury through mechanisms that involve reduction of ROS.

     

鸡胚内抗氧化物质的分布与变化

生物体内存在多种内源性抗氧化物质,在生命过程中发挥着基本的防御功能,是人们十分关注的研究领域。本文综述了近年来鸡胚内抗氧化物质的形成与来源等研究成果,分析了鸡胚孵育过程中维生素(A、C、E)、类胡萝卜素、硒、过氧化氢酶和超氧化物歧化酶等主要抗氧化物质的分布与变化,及内源性抗氧化系统的形成,旨在为今后的研究提供有益的科学依据。     

The first genetic engineered system for ovothiol biosynthesis in diatoms reveals a mitochondrial localization for the sulfoxide synthase OvoA

Diatoms represent one of the most abundant groups of microalgae in the ocean and are responsible for approximately 20% of photosynthetically fixed CO₂ on Earth. Due to their complex evolutionary history and ability to adapt to different environments, diatoms are endowed with striking molecular biodiversity and unique metabolic activities. Their high growth rate and the possibility to optimize their biomass make them very promising ‘biofactories’ for biotechnological applications. Among bioactive compounds, diatoms can produce ovothiols, histidine-derivatives, endowed with unique antioxidant and anti-inflammatory properties, and occurring in many marine invertebrates, bacteria and pathogenic protozoa. However, the functional role of ovothiols biosynthesis in organisms remains almost unexplored. In this work, we have characterized the thiol fraction of Phaeodactylum tricornutum, providing the first evidence of the presence of ovothiol B in pennate diatoms. We have used P. tricornutum to overexpress the 5-histidylcysteine sulfoxide synthase ovoA, the gene encoding the key enzyme involved in ovothiol biosynthesis and we have discovered that OvoA localizes in the mitochondria, a finding that uncovers new concepts in cellular redox biochemistry. We have also obtained engineered biolistic clones that can produce higher amount of ovothiol B compared to wild-type cells, suggesting a new strategy for the eco-sustainable production of these molecules.     

Anticarcinogenic antioxidants as inhibitors against intracellular oxidative stress

Oxidative stress has been implicated in the pathogenesis of numerous diseases, including cancer. In the present study, the protective effect of natural antioxidants, such as quercetin and tea polyphenols, on intracellular oxidative stress was studied. Here we report a novel function of quercetin and tea polyphenols, as potential inhibitors of 4-hydroxy-2-nonenal (HNE)-induced intracellular oxidative stress and cytotoxicity. In rat liver epithelial RL34 cells, a potent electrophile HNE dramatically induced the productions of reactive oxygen species (ROS), which correlated well with the reduction in cell viability. We found that quercetin and tea polyphenols, such as epigallocatechin gallate and theaflavins and their gallate esters, significantly inhibited the HNE-induced ROS production and cytotoxicity. In addition, HNE induced a transient decrease in the mitochondrial membrane potential (Δψ), which was also retarded by the antioxidants. These data suggest that the antioxidants, such as quercetin and tea polyphenols, are inhibitors against mitochondrial ROS production.     

茶树品种‘金牡丹’自然杂交后代遗传鉴定

为了对‘金牡丹’茶树自然杂交后代进行遗传鉴定,利用EST-SSR毛细管电泳荧光标记技术对65个金牡丹自然杂交后代进行研究。结果表明,28对SSR标记共扩增出192个等位片段,平均等位基因数(Na)、平均观测杂合度(Ho)及遗传多态信息量(PIC)分别为6.86、0.540、0.532。单亲基因型已知时的累积排除概率为0.999,说明选择的28对SSR标记位点具有高度的多态性和较高的排除概率,适用于遗传分析和个体的亲子鉴定。15个‘金牡丹’自然杂交后代的遗传鉴定结果表明,MD44、JMD45、JMD47、JMD32为早生绿茶类型;JMD51、JMD53为闽北肉桂乌龙茶类型;MD2、JMD56为闽南‘铁观音’乌龙茶类型;JMD24、JMD26、JMD29、JMD55、JMD59、JMD27、JMD61为‘黄棪’乌龙茶类型。     

The effect of oxidants on biomembranes and cellular metabolism

During the reductive process in the tissues, the aerobes generate a number of oxidants. Unless these oxidants are reduced, oxidative damage and cell death would occur. Oxidation of plasma membrane lipids leads to autocatalytic chain reactions which eventually alter the permeability of the cell. The role of oxidative damage in the pathophysiology of diabetic complications and ischemic reperfusion injury of myocardium, especially the changes in the channel activity which may lead to arrhythmia have been studied. Hyperglycemia activates aldose reductase which could efficiently reduce glucose to sorbitol in the presence of NADPH. Since NADPH is also aldose required by glutathione reductase for reducing oxidants, its diversion would lead to membrane lipid oxidation and permeability changes which are probably responsible for diabetic complications such as cataractogenesis, retinopathy, neuropathy etc. Antioxidants such as butylated hydroxy toluene (BHT) and also reductase inhibitors prevent or delay some of these complications. By using patch-clamp technique in isolated frog myocytes, we have shown that hydroxy radicals generated by ferrous sulfate and ascorbate as well as lipid peroxides such as t-butyl hydroperoxide facilitate the entry of Na⁺ by oxidizing Na⁺-channels. Increased intracellular Na⁺ leads to an increase in Na⁺/Ca²⁺ exchange. The increased Na⁺ concentration by itself may produce electrical disturbance which would result in arrhythmia. Increased Ca²⁺ may affect proteases and may help in the conversion of xanthine dehydrogenase to xanthine oxidase, consequently increased production of super oxide radicals. Increased membrane lipid peroxidation and other oxygen free-radical associated membrane damage in myocytes has been demonstrated.     

Induction of Haem Oxygenase as a Defence Against Oxidative Stress

Cells respond to metabolic perturbations by producing specific stress proteins. Exposure of mammalian cells to various forms of oxidative stress induces haem oxygenase, the rate-limiting enzyme in haem degradation. This response is proposed to represent an antioxidant defence operating at two different stages simultaneously. It (i) decreases the levels of the potential pro-oxidants haem and haem proteins such as cytochrome P-450 and protoporphyrinogen oxidase, and (ii) increases the tissue concentrations of antio-xidatively active bile pigments.     

Avoidance of oxidative-stress perturbation in yeast bioprocesses by proteomic and genomic biostrategies?

AIMS: Bioprocess oxidative stress caused by many reactive oxygen species (ROS) can lead to largely irreversible perturbation of yeast bioprocesses. These include the production of proteins derived from recombinant DNA yeast technology (aerobically grown Saccharomyces cerevisiae). These proteins include rennin, amyloglucosidases (glucamylases), interferons, interleukins, insulin, monoclonal antibodies, tissue plasminogen activators (t-PA), sexually transmitted disease antigens, and measles, mumps and rubella antigens, growth hormones, somatotropin, blood clotting factors VIII and XIII. In addition, there may be a demand for severe acute respiratory syndrome-coronavirus antigens, hepatitis A, B and C viral-selected antigens, HIV retroviral antigens, influenza antigens, trypanosomal antigens, and foot and mouth disease antigens. Prevention of oxidative stress has been achieved by application of antioxidant redox metalloenzymes such as superoxide dismutases (containing Cu/Zn cytosolic, Mn mitochondrial and Fe bacterial) glutathione peroxidases (and other Se-containing proteins and enzymes such as the thioredoxins), catalases (Fe-containing), cytochrome c peroxidases (Fe-containing), ceruloplasmins (Cu-containing), metallothionines (these cysteine thiol-rich proteins bind ions of cadmium and mercury) and tyrosinases(Cu-containing). METHODS AND RESULTS: ROS are generated inadvertently by single metal valency couples such as FeII/FeIII and by FeIII/FeV present in 2700 (including 57 human) isoforms in cytochromes P450 mixed-function oxidases (EC 1.14.14.1; O2 : mono-oxygenase NADPH/NADH requiring). In addition, mixed-metal couples such as valency unmatched forms in CuI/FeII and FeIII/MnIV can recycle electrons. Moreover, proteins/protein chaperone couples can recycle electrons, often where futile-recycling systems have been instigated. Furthermore, oxidized membrane phospholipids (R) can form ROOH (lipid hydroperoxides) and ROH (lipid alkoxides) that can generate ROS through Fenton chemistry (iron-catalysed) chain reactions. Utilization of chain-breaking antioxidants such as vitamin E (alpha-tocopherol) in the lipid phase and vitamin C (ascorbate) in the aqueous phase can terminate these ROS-producing reactions. CONCLUSIONS: The main significance of the study is that proteomic strategies of relief from bioprocess perturbation by ROS of yeast fermentations (used to manufacture proteins required in the food and therapeutic bioindustries) may become possible through addition of selected proteins (including metalloenzymes). The main impact of the study is that the utilization of genetically modified (GM) yeast produced by recombinant DNA technology genomic strategies could circumvent the bioprocessing problems that otherwise result from the bioprocess perturbations: this is as a result of oxidative stress caused by ROS, which is avoidable by deployment of appropriate antioxidants such as vitamins E, C and D (and antioxidant proteins and enzymes often of microbial origin via recombinant DNA technology).     

HPLC coupled on-line to ESI-MS and a DPPH-based assay for the rapid identification of anti-oxidants in Butea superba

A reversed-phase HPLC coupled on-line to a radical scavenging detection system and MS/MS was developed in order to combine separation, activity determination and structural identification of anti-oxidants in complex mixtures in one run. The sample was separated by HPLC and the eluate split into two flows. The major portion was fed into an electrospray ionisation MS/MS system, while the minor part was mixed with a free radical, 2,2'-diphenyl-1-picrylhydrazyl (DPPH), and the reaction determined spectrophotometrically. The negative peaks, which indicated the presence of anti-oxidant activity, were monitored by measuring the decrease in absorbance at 517 nm. The developed method was successfully applied to the identification of anti-oxidant compounds in a fraction, obtained by solid-phase extraction, of an extract of a Thai medicinal plant, Butea superba Roxb. The anti-oxidant compounds were separated and identified as procyanidin B2, (-)-epicatechin and procyanidin B5.     

Expression and regulation of adrenomedullin in renal glomerular podocytes

Adrenomedullin (AM) is postulated to exert organ-protective effects. It is expressed in the renal glomeruli, but its roles in the glomerular podocytes have been poorly elucidated. In the present study, we investigated the expression and regulation of AM in recently established conditionally immortalized mouse podocyte cell line in vitro and podocyte injury model in vivo. The cultured differentiated podocytes expressed AM mRNA and secreted measurable amount of AM. AM secretion from the podocytes was increased by H(2)O(2), hypoxia, puromycin aminonucleoside (PAN), albumin overload, and TNF-alpha. Real-time RT-PCR analysis revealed that AM mRNA expression in the podocytes was enhanced by PAN and TNF-alpha, both of which were suppressed by mitochondrial antioxidants. Furthermore, AM expression was upregulated in the glomerular podocytes of PAN nephrosis rats. These results indicated that AM expression in the podocytes was upregulated by stimuli or condition relevant to podocyte injury, suggesting its potential role in podocyte pathophysiology.