Hydrogen Peroxide Metabolism in Yeasts

A catalase-negative mutant of the yeast Hansenula polymorpha consumed methanol in the presence of glucose when the organism was grown in carbon-limited chemostat cultures. The organism was apparently able to decompose the H₂O₂ generated in the oxidation of methanol by alcohol oxidase. Not only H₂O₂ generated intracellularly but also H₂O₂ added extracellularly was effectively destroyed by the catalase-negative mutant. From the rate of H₂O₂ consumption during growth in chemostat cultures on mixtures of glucose and H₂O₂, it appeared that the mutant was capable of decomposing H₂O₂ at a rate as high as 8 mmol · g of cells⁻¹ · h⁻¹. Glutathione peroxidase (EC 1.11.1.9) was absent under all growth conditions. However, cytochrome c peroxidase (CCP; EC 1.11.1.5) increased to very high levels in cells which decomposed H₂O₂. When wild-type H. polymorpha was grown on mixtures of glucose and methanol, the CCP level was independent of the rate of methanol utilization, whereas the level of catalase increased with increasing amounts of methanol in the substrate feed. Also, the wild type decomposed H₂O₂ at a high rate when cells were grown on mixtures of glucose and H₂O₂. In this case, an increase of both CCP and catalase was observed. When Saccharomyces cerevisiae was grown on mixtures of glucose and H₂O₂, the level of catalase remained low, but CCP increased with increasing rates of H₂O₂ utilization. From these observations and an analysis of cell yields under the various conditions, two conclusions can be drawn. (i) CCP is a key enzyme of H₂O₂ detoxification in yeasts. (ii) Catalase can effectively compete with mitochondrial CCP for hydrogen peroxide only if hydrogen peroxide is generated at the site where catalase is located, namely in the peroxisomes.     

外源H2O2处理对唐古特白刺愈伤组织脯氨酸代谢的影响

以唐古特白刺（Nitraria tangutorum Bobr.）愈伤组织为材料,研究外源H₂O₂（2和10 μmol·L^-1）处理下其脯氨酸含量及相关代谢酶活性的变化,试图从细胞水平揭示H₂O₂影响脯氨酸代谢的生理机制。结果显示,2和10 μmol·L^-1 H₂O₂处理24 h使唐古特白刺愈伤组织脯氨酸含量分别变为对照的112%和92%,而处理72 h后,脯氨酸含量增加为对照的141%和119%;与对照相比,外源H₂O₂处理诱导愈伤组织脯氨酸脱氢酶活性降低,而谷氨酸激酶活性升高,但鸟氨酸转氨酶活性无显著变化;此外,H₂O₂处理使唐古特白刺愈伤组织内源性H₂O₂含量升高。结果表明,外源H₂O₂诱导了唐古特白刺愈伤组织H₂O₂含量的增高和脯氨酸的积累,且H₂O₂处理下脯氨酸脱氢酶活性的降低及谷氨酸激酶的升高与愈伤组织脯氨酸的积累有关。     

家蚕胚胎发育中过氧化氢的代谢(英文)

过氧化氢（Ｈ_２Ｏ_２）是生物体内主要的活性氧来源之一。在超氧化物歧化酶（ＳＯＤ）、过氧化氢酶（ＣＡＴ）等的催化作用下,Ｈ_２Ｏ_２。被降解,释放出活性氧。所以,生物个体发育过程中体内Ｈ_２Ｏ_２、ＳＯＤ和ＣＡＴ含量的变化反映着Ｈ_２Ｏ_２的代谢水平。另外,家蚕是蚕卵滞育昆虫,实验设计考虑到了滞育前后可能会有的差别。取产后１０分钟内的卵为供试材料。采用即时浸酸法解除卵滞育。采用比色法和氧电极法测定并比较家蚕胚胎滞育形成与解除过程中过氧化氢的代谢。结果表明：（１）受精初期（０～４ｈ）,Ｈ_２Ｏ_２含量在２．５ｈ时达到峰值（Ｆｉｇ．１）,相应地ＳＯＤ活性处于较高水平,而ＣＡＴ活性处于最低水平（Ｆｉｇ．２）;（２）胚胎发育过程中（即时浸酸解除滞育）,Ｈ_２Ｏ_２含量除１６８～２１６ｈ处于低水平外均显著高于滞育卵（Ｆｉｇ．３）,ＳＯＤ活性分别在７２ｈ、１６８ｈ,形成小大两峰,后期显著高于滞育卵（Ｆｉｇ．４）,而ＣＡＴ活性７２－１９２ｈ保持平稳,随后急剧上升,前期显著低于滞育卵,后期相反（Ｆｉｇ．５）;（３）滞育形成过程中Ｈ_２Ｏ_２水平变化平缓（Ｆｉｇ．６）,ＳＯＤ活性前期剧烈变动,但后期保持平稳（Ｆｉｇ．７）,ＣＡＴ活性逐步升     

Formation of Hydrogen Peroxide by Group N Streptococci and Its Effect on Their Growth and Metabolism

The formation of hydrogen peroxide by group N streptococci was found to occur through the action of a reduced nicotinamide adenine dinucleotide (NADH) oxidase which catalyzed the oxidation of NADH by molecular oxygen. The enzyme was activated by flavine adenine dinucleotide. Whereas some of the hydrogen peroxide formed was removed through the action of an NADH peroxidase, sufficient accumulated in media to inhibit the growth, respiration, and viability of these organisms. The amount of hydrogen peroxide which accumulated varied among strains, and this variation could be related to differences in the properties of the NADH oxidase present.     

Bactericidal Effect of Hydrogen Peroxide on Spacecraft Isolates

Solutions of 3, 10, and 15% hydrogen peroxide were found to have pronounced bactericidal effects, as a function of time of exposure, on sporeformers and non-sporeformers isolated from spacecraft.     

Cytotoxic Power of Hydrogen Peroxide Effect on Endothelial Cells in vitro

The currently existing terms “apoptotic index” and “apoptotic potential” are insufficient for the adequate qualitative evaluation and analysis of the proapoptotic activity of chemical agents within the wide range of therapeutic and toxic doses. Herein we introduce a new concept of cytotoxic power as an alternative to these terms related to apoptosis and other types of cell death on the basis of experimental data of hydrogen peroxide cytotoxic activity towards the human umbilical vein endothelial cells (HUVEC). We also propose an algorithm for computing cytotoxic power. Correlations between the percentage of early apoptotic and late apoptotic/necrotic cells and the expression or concentration of endothelial markers such as CD143, CD309, von Willebrand factor, phosphatase PTEN, have been revealed.     

过氧化氢对培养心肌细胞损伤作用的研究

氧化应激时产生大量的自由基,造成心肌细胞的损伤.过氧化氢(H₂O₂)是有机体氧化代谢产物,同时是一种活性氧.应用不同浓度的H₂O₂,分别于不同作用时间,动态观察其对心肌细胞的损伤作用.从实验结果看到,低浓度的H₂O₂（＜0.1 mmol/L）作用2 h,使心肌细胞产生早期的生物化学的改变,如MDA产生堆积和细胞周期时相改变（G1期细胞增加,G2期细胞减少）,此时心肌酶基本无泄漏,心肌细胞的死亡率很低,HE形态学观察基本无改变;随着H₂O₂浓度的增加（1～5 mmol/L）和作用时间的延长,进一步诱导细胞损伤加剧,LDH释放和MDA积累明显升高,细胞死亡率也明显增加,已具有统计学意义.同时可观察到其病理形态学的坏死性改变;当10 mmol/L H₂O₂作用时,细胞大量死亡,形态学可见细胞极度收缩、脱落,形成大面积的细胞脱失区.因此,H₂O₂作为一种活性氧自由基,依其浓度和作用时间不同可造成不同程度的心肌细胞的损伤.辣根过氧化物酶作为一种自由基清除剂,可明显减少H₂O₂活性氧自由基对心肌细胞的损伤作用.     

Effects on Liver Hydrogen Peroxide Metabolism Induced by Dietary Selenium Deficiency or Excess in Chickens

To determine the relationship between dietary selenium (Se) deficiency or excess and liver hydrogen peroxide (H₂O₂) metabolism in chickens, 1-day-old chickens received insufficient Se (0.028 mg Se per kg of diet) or excess Se (3.0 or 5.0 mg Se per kg of diet) in their diets for 8 weeks. Body and liver weight changes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, H₂O₂ content, and activities and mRNA levels of enzymes associated with H₂O₂ metabolism (catalase (CAT) and superoxide dismutase (SOD) 1–3) were determined in the liver. This study showed that Se deficiency or excess Se intake elicited relative severe changes. Se deficiency decreased growth, while Se excess promoted growth in chickens. Both diets vastly altered the liver function, but no obvious histopathological changes were observed in the liver. Se deficiency significantly lowered SOD and CAT activities, and the H₂O₂ content in the liver and serum increased. Se excess (3.0 mg/kg) decreased SOD and CAT activities with changes in their mRNA levels, and the H₂O₂ content increased. The larger Se excess (5.0 mg/kg) showed more serious effects but was not fatal. These results indicated that the H₂O₂ metabolism played a destructive role in the changes in bird liver function induced by Se deficiency or excess.     

Some Enzymes of Hydrogen Peroxide Metabolism in Leaves and Root Nodules of Medicago sativa

Leaves and nodules (bacteroids and cytosol) of alfalfa (Medicago sativa L. cv Aragon) plants inoculated with Rhizobium meliloti strain 102F51 have been analyzed for the presence of the enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (EC 1.11.1.6), and peroxidase (EC 1.11.1.7). All three fractions investigated (leaves, bacteroids, and nodular cytosol) show Cu,Zn-SOD activity. Besides, the bacteroids and cytosol of nodules possess CN⁻-insensitive SOD activities. Studies of SOD inactivation with H₂O₂ indicate that, very likely, a Mn-SOD is present in the bacteroids, and suggest that the cytosol contain both Mn-SOD and Fe-SOD. Bacteroids show high catalase activity but lack peroxidase. By contrast, the nodule cytosol exhibits an elevated peroxidase activity as compared with the foliar tissue; this activity was completely inhibited by 50 to 100 micromolar KCN. The significantly lower contents of H₂O₂ and malondialdehyde (a product of lipid peroxidation) in nodules with respect to those in leaves reveal that the above-mentioned bacteroid and cytosol enzymes act in an efficient and combined manner to preserve integrity of nodule cell membranes and to keep leghemoglobin active.     

A Role of Hydrogen Peroxide in the Metabolism of Phenolics in Mesophyll Cells of Viciafaba L.

3,4-Dihydroxyphenylalanine (DOPA) and flavonols were oxidizedby externally added H₂O₂and the oxidation was inhibited by KCN(5 mM) in protoplasts of mesophyll cells of Viciafaba. DOPAwas also oxidized by light in the presence of methyl viologen(MV), which can stimulate formation of O^–₂ and H₂O₂ invivo, both in the light and in the dark, in isolated mesophyllcells. The light-dependent oxidation of DOPA was partially inhibitedby removal of MV or addition of NaN₃ (10 mM), an inhibitor ofperoxidases, suggesting the participation of H₂O₂, generatedin vivo, in the oxidation. The effects of light on the levelof flavonols in isolated mesophyll cells were rather complicated.Level of flavonols increased by about 10–20% in the darkin the presence of MV. The levels in the light in the presenceof MV were lower than those in the dark. The data suggest thatflavonols can be oxidized by O^–₂ and/or H₂O₂ generatedin cells. Based on the data, the role of H₂O₂ in the metabolismof phenolics in mesophyll cells is discussed. (Received June 8, 1988; Accepted January 13, 1989)     

过氧化氢对内皮祖细胞凋亡相关蛋白表达的影响

目的:比较不同浓度过氧化氢(hydrogen peroxide,H2O2)对人外周血来源的内皮祖细胞(endothelial progenitor cells,EPCs)生存能力的改变及其对凋亡相关蛋白表达的影响。方法:采用密度梯度离心法和差速贴壁法从人外周静脉血中分离培养内皮祖细胞。选取传代后第3代EPCs作为研究对象,以终浓度分别为50μmol/L、100μmol/L、150μmol/L、200μmol/L、300μmol/L和400μmol/L的过氧化氢处理内皮祖细胞12 h,同时设立正常处理对照组。CCK-8法检测各组内皮祖细胞生存能力的差异;Western blot分析各组内皮祖细胞中凋亡相关蛋白Bax、Bcl-2和p53的蛋白表达情况。结果:与对照组相比,在浓度为50μmol/L、100μmol/L和150μmol/L过氧化氢处理组中细胞存活能力逐渐增强,促凋亡蛋白Bax、p53随之下调,抗凋亡蛋白Bcl-2则显著上调;在浓度为200μmol/L、300μmol/L和400μmol/L过氧化氢处理组中细胞生存能力相对于正常对照组逐渐减弱,促凋亡蛋白Bax、p53表达水平逐渐增加,抗凋亡蛋白Bcl-2表达水平下降。结论:过氧化氢对内皮祖细胞存活能力和凋亡相关蛋白Bax、Bcl-2、p53表达的影响均呈双相性变化。     

外源NO供体对小麦离体叶片过氧化氢代谢的影响

分析了外源一氧化氮 (nitricoxide ,NO)供体硝普钠 (sodiumnitroprusside ,SNP)对离体小麦 (TriticumaestivumL .)叶片过氧化氢 (H2 O2 )含量及其清除酶活力的调节作用。不同浓度的SNP (1mmol/L和 5mmol/L)处理 3 0min内 ,离体小麦叶片H2 O2 含量均有一个显著上升的过程 ,同时过氧化物酶 (POD)活力受到显著抑制 ,而过氧化氢酶 (CAT)活力则轻微下降 ;处理 3 0min到 2 4 0min时 ,POD活力的抑制状态基本维持不变 ,而CAT活力开始恢复上升 ,H2 O2 含量也相应地开始下降。粗酶液的体外实验也表明 ,SNP对POD和CAT的抑制类型不同 ,前者可能是不可逆抑制 ,后者则可能是可逆抑制。因此NO可通过对POD和CAT的不同抑制作用来调节小麦叶片内源H2 O2 含量     

The Effect of Hydrogen Peroxide and Ultraviolet Irradiation on Non-sporing Bacteria

A kill of 99.99% was obtained in cell suspensions of Escherichia coli and Streptococcus faecalis by incubation with hydrogen peroxide 1.0% (w/v) for 75 and 180 min respectively. The same kill was produced by 30 s irradiation with ultraviolet (u.v.) light in the presence of hydrogen peroxide 1.0% (w/v). This simultaneous treatment with u.v. and hydrogen peroxide produced a synergistic kill at least 30-fold greater than that produced by irradiation of cell suspensions of Esch. coli with or without subsequent incubation with hydrogen peroxide.     

The Effect of Lipid Peroxide on the Lipid and Carbohydrate Metabolism in Rat Liver

Feeding tests were carried out on rats to clarify the mechanisms of fatty liver formation induced by autoxidized methyl linoleate. Lipid peroxides prepared by autoxidation of highly purified methyl linoleate were given orally to rats. Triglyceride and glycogen contents in liver were determined and enzyme activities including triglyceride synthetase and α-glycerophosphate dehydrogenase were also examined. The following results were obtained. 1. Triglyceride accumulation in rat liver fed autoxidized methyl linoleate was observed. 2. Increase in triglyceride content in rat liver was soon followed by the decrease of hepatic glycogen. 3. When rats were starved prior to introduction of autoxidized methyl linoleate, hepatic triglyceride accumulation did not occur. 4. The activities of α-glycerophosphate dehydrogenase and triglyceride synthetase in liver, and those of glutamic oxalacetic transaminase and leucine aminopeptidase in plasma were practically similar among the rats of test groups fed fresh or autoxidized methyl linoleate and the control fed diet without methyl linoleate. 5. The addition of l-carnitine which is a stimulator of fatty acid oxidation retarded the accumulation of the hepatic triglyceride mentioned above.     

Effect of Catalase on Hydrogen Peroxide Penetration into Pseudomonas aeruginosa Biofilms

The penetration of hydrogen peroxide into biofilms formed by wild-type and catalase-deficient Pseudomonas aeruginosa strains was measured using microelectrodes. A flowing stream of hydrogen peroxide (50 mM, 1 h) was unable to penetrate or kill wild-type biofilms but did penetrate and partially kill biofilms formed by an isogenic strain in which the katA gene was knocked out. Catalase protects aggregated bacteria by preventing full penetration of hydrogen peroxide into the biofilm.     

Hydrogen Peroxide in Human Blood

Blood and plasma of humans and rats were analyzed for hydrogen peroxide. The samples were analyzed after deproteinization with trichloroacetic acid, immediately after they were withdrawn from human volunteers or rats. A radio-isotopic technique based on peroxide-dependent decarboxylation of 1-¹⁴C-alpha-ketoacids and consequent liberation of ¹⁴CO₂ was used. The results demonstrate the presence ofmicromolar levels of H₂O₂, both, in the plasma as well as in the whole blood. The values in the whole blood were substantially greater than the plasma. This was true for rats as well as humans. The presence of such significant quantities of H₂O₂ in the blood have been demonstrated for the first time. The investigation, therefore, opens a newer avenue of research on diseases purported to be related to the generation of oxygen radicals in vivo.     

小剂量过氧化氢对大鼠心肌细胞钙瞬变及凋亡的作用

目的:探讨小剂量过氧化氢导致的氧化应激对大鼠心肌细胞钙瞬变及细胞凋亡的作用。方法:解剖取出成年大鼠心脏,应用langendorff方法分离心肌细胞,加入fluo-3荧光指示剂后,应用不同浓度的过氧化氢作用于心肌细胞,在共聚焦显微镜下测定心肌细胞内钙瞬变。分离并培养新生大鼠心肌细胞,观察过氧化氢处理心肌细胞后其形态的变化,从而评价小剂量过氧化氢对心肌细胞的凋亡作用。结果:应用0.125 mmol/L、0.25 mmol/L及0.375 mmol/L的过氧化氢作用于心肌细胞后,心肌细胞内钙瞬变幅度明显升高,并呈时间剂量依赖性。在培养的大鼠原代心肌细胞中加入0.25 mmol/L的过氧化氢后,心肌细胞发生凋亡的形态变化。结论:小剂量过氧化氢可开放心肌细胞L-钙通道,明显增加心肌细胞内钙瞬变,并导致心肌细胞凋亡。     

过氧化氢对铜锌超氧化物歧化酶活性及某些理化性质的影响

用H_2O_2作用于牦牛红细胞铜锌超氧化物歧化酶。观察到酶活性随H_2O_2浓度升高及作用时间增加而下降;酶分子连接的铜和锌有所丢失;PAGE图谱中三条酶活性带成为四条酶活性带;等电点下降;680nm处表征二价铜光学性质的可见光吸收减弱;紫外吸收增加,表现为增色效应;内源性荧光减弱;在含有3.0mol/LKCl的PH3.8—5.4琥珀酸缓冲液中溶解度下降;酶对胰蛋白酶水解的敏感性增加。     

Sirt3对过氧化氢诱导的骨髓间充质干细胞凋亡的保护作用

目的:通过调控骨髓间充质干细胞(mesenchymal stem cells,MSCs)中的Sirtuin-3(Sirt3)蛋白的表达水平,阐明Sirt3对过氧化氢(H2O2)诱导MSCs凋亡的保护作用及其机制。方法:将大鼠MSCs分为正常对照组、H2O2刺激组、H2O2+Sirt3转染组、H2O2+Sirt3 si RNA转染组。利用Western blot法检测Sirt3及cyclophilin d蛋白的表达水平、利用免疫沉淀法检测cyclophilin d乙酰化水平、利用流式细胞仪检测细胞凋亡。结果:1H2O2刺激使MSCs中Sirt3表达水平降低。2转染Sirt3可减少H2O2诱导的MSCs的凋亡,而转染Sirt3 si RNA可增加H2O2诱导的MSCs的凋亡。3Sirt3蛋白的含量并不影响cyclophilin d的表达,但影响cyclophilin d的乙酰化水平。结论:Sirt3可能通过减少线粒体通透性转换孔(mitochondrial permeablity transition pore,m PTP)中的关键蛋白cyclophilin d的乙酰化水平,进一步抑制m PTP的开放,从而减少H2O2诱导的MSCs的凋亡。