基于蛋氨酸代谢调控的内源性抗氧化分子机制

蛋氨酸是一种含硫必需氨基酸,在蛋白质组成和新陈代谢中都发挥着独特的作用。蛋氨酸具有内源性抗氧化作用,一方面蛋氨酸在蛋氨酸亚砜还原酶(MSR)作用下通过自身氧化还原反应来发挥内源性抗氧化作用,另一方面蛋氨酸可通过代谢途径(GSH合成、Nrf2抗氧化通路等)来增强内源性抗氧化能力。然而目前缺乏对蛋氨酸抗氧化分子机制全面深入的研究报道。因此,本文在蛋氨酸代谢的基础上,对蛋氨酸促进GSH合成、激活MSR抗氧化系统以及调控Nrf2抗氧化通路的分子机制进行综述,并对GSH合成、MSR与Nrf2抗氧化体系之间的关系进行阐述,为全面解析蛋氨酸内源性抗氧化分子机制提供理论依据。     

含硫氨基酸的抗氧化作用

抗氧化剂是维持动物体内自由基平衡和稳定的重要物质。甲硫氨酸(Met)和半胱氨酸(Cys) 是组成蛋白质的2种含硫氨基酸,基于其独特的结构,在动物体内起着与动物营养与免疫相关的重要生理功能,而其抗氧化能力越来越受关注。综述了蛋白质Met残基的氧化还原作用和含硫氨基酸的代谢产物谷胱甘肽(GSH)的抗氧化作用。     

松栎柱锈菌春孢子多糖体外抗氧化活性

为了研究松栎柱锈菌（Cronartium orientale S.Kaneko）春孢子多糖的体外抗氧化活性,制备了该菌破壁春孢子精制多糖,并对所得春孢子多糖进行了总抗氧化能力、总还原力以及清除二苯代苦味酰基（DPPH·）、清除羟基自由基（·OH）、清除超氧阴离子（O₂^·）的活性进行了测定。体外抗氧化活性分析结果表明：松栎柱锈菌春孢子多糖具有较强的总抗氧化能力;虽然该菌多糖不具有清除多数氧自由基母体（O₂^·）的作用,也不具有清除人工合成自由基DPPH·的作用,但是其能够有效清除对机体破坏力最强的自由基·OH。这表明本文获得的松栎柱锈菌春孢子多糖具有对自由基选择性清除的能力,对其他自由基研究者具有参考价值,可为该菌的开发利用提供参考,也能够为森林有害生物开发利用提供理论依据。     

疏花水柏枝内生真菌QY-1的抗氧化活性分析

以从疏花水柏枝(Myricaria laxiflora)中分离的1株内生真菌QY-1为研究对象,为分析其体内及体外的抗氧化活性,对其发酵液及其提取物的活性进行了综合评价。对nr DNA内转录间隔区进行测序鉴定。分别采用总抗氧化力试剂盒、自由基清除试剂盒和还原力测定试剂盒评价发酵液及粗提物的体外抗氧化能力活性;对发酵液提取物及其主成分进行了大肠埃希菌、人神经母瘤细胞SH-SY5Y的体内抗氧化损伤保护作用分析。结果表明QY-1是1株球毛壳菌(Chaetomium globosum),其粗提物具有很高的抗氧化活性,总抗氧化活力、自由基清除率及总还原力均接近维生素C的50%,远高于国际报道水平。且其发酵液提取物有促进细胞增殖和抗氧化保护作用,显著提高大肠埃希菌细胞和神经细胞在H2O2胁迫下的存活率。其粗提物可保护神经细胞膜的完整性,有效减低乳酸脱氢酶(LDH)的渗漏率。从粗提物中分离到一种主成分SF2,可显著抑制凋亡蛋白Caspase 3和Caspase 9的活性。结果表明内生真菌球毛壳菌QY-1不管在体内还是体外都具备较强的抗氧化能力,是一种具有潜在开发价值的天然抗氧化剂资源。     

功能性氨基酸调控猪氧化应激的研究进展

氧化应激是指人和动物机体自由基产生急剧增多或清除自由基能力下降,从而打破了抗氧化-氧化平衡.在猪生产上,多种因素均能诱导产生氧化应激,其中包括出生应激、断奶应激、饲料霉菌毒素污染以及饲养环境和社会因素等.猪氧化应激发生往往伴随着其他病理因素,对猪生产性能和健康生长具有重要影响.目前,多种功能性氨基酸被报道具有抗氧化能力,包括含硫氨基酸、谷氨酸、精氨酸以及色氨酸等.因此,通过营养干预方法在饲料中补充或添加功能性氨基酸对猪氧化应激具有缓解效应.本文主要就猪出生、断奶、霉菌毒素以及饲养环境造成的氧化应激以及功能性氨基酸营养调控进行了探讨,以期为猪营养研究及功能性氨基酸科学应用提供参考.     

风干羊肉中乳酸菌的体内外抗氧化特性

【背景】机体的衰老和一些疾病多与氧化作用有关,随着对抗氧化制剂研究的深入,人工合成抗氧化剂的安全性受到质疑,因此寻找天然抗氧化制剂的研究已成为热点。【目的】从风干羊肉中筛选抗氧化活性较高的乳酸菌,分析肉源乳酸菌体内外抗氧化能力。【方法】以24株肉源乳酸菌为研究对象,以自由基清除能力、亚铁离子螯合能力、还原能力及抗脂质过氧化能力为体外抗氧化能力分析指标,筛选出体外抗氧化活性较强的乳酸菌,运用16S rRNA基因序列同源性分析进行菌种鉴定,通过小鼠试验研究其体内抗氧化能力。【结果】试验所测24株乳酸菌均具有一定的体外抗氧化能力,其中菌株TR13为24株乳酸菌中体外抗氧化能力较强的菌株,其超氧阴离子自由基(·O2-)清除率为54.29%,羟自由基(·OH)清除率为90.84%, 1,1-二苯基-2-三硝基苯肼自由基(1,1-diphenyl-2-picrylhydrazyl,DPPH)清除率为99.38%,亚铁离子螯合率为55.85%,还原能力达1.345,抗脂质过氧化率为39.99%。通过16S rRNA基因鉴定,菌株TR13为一株瑞士乳杆菌。通过给D-半乳糖诱导的衰老型小鼠饲喂TR13菌...     

层孔菌属真菌抗氧化活性物质测定及抗氧化能力分析

本文以层孔菌属真菌液体发酵产物作为研究材料,测定抗氧化活性物质含量,并利用多种抗氧化测定方法,分析不同菌株的体外抗氧化能力,评价活性物质含量间、抗氧化方法间及抗氧化方法与含量间的相关性。研究结果表明,供试菌株活性物质含量间的差异较大,其中菌株H2粗多糖和总酚含量较高,菌株S黄酮含量较高,且活性物质含量间相关性不大;针对供试菌株抗氧化能力测定结果表明,菌株H2具有较高的还原力、铁离子螯合能力,菌株H2和H9的羟自由基清除作用最强,菌株H2和L的DPPH自由基清除作用最强,菌株H2、H5、H8、H9具有较高的超氧自由基清除作用,且不同方法间存在较大差异,羟自由基与DPPH自由基、铁离子螯合能力及还原力,DPPH自由基与铁离子螯合能力和还原力,以及铁离子螯合能力与还原力这几种方法的相关性较高(P0.01);总酚含量与羟自由基、DPPH自由基、铁离子螯合能力及还原力方法具有较高的相关性(P0.01);总黄酮含量与DPPH自由基、铁离子螯合能力及还原力方法间相关系数分别为0.810(P0.01)、0.725、0.738(P0.05);粗多糖含量与各测定方法间的相关系数较低,以上结果说明多酚类物质和黄酮类物质是抗氧化作用的重要因子,对抗氧化能力具有重要贡献。     

连翘花黄色素对线虫在氧化应激下的保护作用

自由基过度引起的氧化应激是多种疾病发生的因素。连翘花黄色素(forsythia flower yellow pigment, FFYP)中含有大量的抗氧化活性物质,但其对氧化应激的抵抗性仍不清楚。本文首先通过化学方法检测FFYP的体外抗氧化活性;用细胞内抗氧化活性（cellular antioxidant activity,CAA）方法检测FFYP细胞内抗氧化活性;然后以秀丽隐杆线虫（Caenorhabditis elegans,C. elegans）为模型,检测FFYP对线虫氧化应激抵抗力及体内抗氧化指标的影响;用Daf 16和Skn 1突变体线虫和qRT PCR实验探究其作用机制。研究结果表明,FFYP具有1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基清除能力,铁离子还原能力和活性氧自由基(reactive oxygen species, ROS）清除能力,并且具有浓度依赖性。用500 μmol/L的胡桃醌提供氧化应激压力时,FFYP能显著提高线虫在氧化应激下的寿命,表明FFYP可以提高线虫对氧化应激的抵抗力。进一步研究发现,FFYP可显著降低线虫体内ROS自由基含量,提高超氧化物歧化酶(superoxide dismutase, SOD)和过氧化氢酶(catalase, CAT)活性,增加还原型谷胱甘肽(glutathione, GSH)含量,表明FFYP通过提高线虫体内抗氧化防御系统活性清除自由基来提高线虫对氧化应激的抵抗力。突变体线虫实验显示,FFYP对线虫延长氧化应激下寿命的效应在Skn-1突变体线虫中完全消失,在Daf-16突变体中效应被减弱。qRT-PCR实验也显示,Daf-16和Skn-1靶基因的表达量均被提高。表明FFYP对线虫氧化应激抵抗力提高的作用是通过Daf-16和Skn-1共同作用。这预示着FFYP具有很好的抗氧化及抗应激药用价值,有潜力成为一种新的有生物活性的天然色素。     

酶解骨胶原多肽的抗氧化特性研究

本文主要研究了酶解法制备的骨胶原多肽的总抗氧化能力、羟自由基清除作用和抑制超氧阴离子自由基的能力.通过与谷胱甘肽(GSH)的比较发现,溶液浓度在1130～150 mg/mL时,该骨胶原多肽的总抗氧化能力为GSH的71.92%.溶液浓度在2.5～20 mg/mL时,该多肽羟自由基清除作用为谷胱甘肽的1.36倍.溶液浓度为10～150 mg/mL时,多肽抑制超氧阴离子自由基的能力低于谷胱甘肽.     

忍冬木层孔菌多糖的提取工艺优化及其体外抗氧化活性研究

忍冬木层孔菌(Phellinus lonicerinus(Bond.) Bond.et Sing)为湖北等地习用的桑黄品种,也是土家族常用药,多糖是其主要活性成分。为了研究忍冬木层孔菌子实体多糖(Phellinus lonicerinus polysaccharide, PLP)的提取工艺及其体外抗氧化活性,采用冷凝回流提取法在单因素实验结果的基础上,进行Box-Behnken响应面分析,确定PLP的最佳提取工艺,并通过测定忍冬木层孔菌多糖(PLP30、PLP60、PLP85、PLPA1和PLPA2)的还原能力、对DPPH、ABTS、羟基自由基的清除能力、对酪氨酸酶活性的抑制能力。结果显示PLP最佳提取工艺的参数为：料液比1∶20 g/mL、提取时间120 min、提取温度100℃,此条件下PLP提取率为(3.16±0.05)%;五种多糖对DPPH、ABTS和羟基自由基均具有较强的清除作用,且具有较强的还原力,其对ABTS自由基清除作用和还原力均与浓度呈量效关系。表明忍冬木层孔菌子实体多糖具有良好的体外抗氧化和一定的酪氨酸酶活性抑制能力。     

A biochemical basis for induction of retina regeneration by antioxidants

The use of antioxidants in tissue regeneration has been studied, but their mechanism of action is not well understood. Here, we analyze the role of the antioxidant N-acetylcysteine (NAC) in retina regeneration. Embryonic chicks are able to regenerate their retina after its complete removal from retinal stem/progenitor cells present in the ciliary margin (CM) of the eye only if a source of exogenous factors, such as FGF2, is present. This study shows that NAC modifies the redox status of the CM, initiates self-renewal of the stem/progenitor cells, and induces regeneration in the absence of FGF2. NAC works as an antioxidant by scavenging free radicals either independently or through the synthesis of glutathione (GSH), and/or by reducing oxidized proteins through a thiol disulfide exchange activity. We dissected the mechanism used by NAC to induce regeneration through the use of inhibitors of GSH synthesis and the use of other antioxidants with different biochemical structures and modes of action, and found that NAC induces regeneration through its thiol disulfide exchange activity. Thus, our results provide, for the first time, a biochemical basis for induction of retina regeneration. Furthermore, NAC induction was independent of FGF receptor signaling, but dependent on the MAPK (pErk1/2) pathway.     

Arginine metabolism in developing soybean cotyledons : I. Relationship to nitrogen nutrition

The free and protein amino acid composition of Glycine max (L.) Merrill cotyledons was determined for the entire developmental period using high performance liquid chromatography. Arginine constituted 18% of the total protein nitrogen throughout development, and there was a linear arginine nitrogen accumulation rate of 1212 nanomoles per cotyledon per day between 16 and 58 days after anthesis. Arginine and asparagine were major constituents of the free amino acid pool, constituting 14 to 62% and 2 to 41% of the total free amino acid nitrogen, respectively. The urea cycle intermediates, citrulline, ornithine, and argininosuccinate were also detected in the free pool. A comparison of the amino acid composition of cotyledonary protein and of seedcoat exudate suggested that 72% of the cotyledon's arginine requirement is satisfied by in situ biosynthesis, and that 20% of the transformed nitrogen is incorporated into arginine. Also, [1-¹⁴C]glutamate and [U-¹⁴C]glutamine were fed to excised cotyledons. After 4 hours, ¹⁴C was incorporated into protein and released as ¹⁴CO₂, but none was incorporated into the C-1 and C-6 positions of free and protein arginine, determined using arginine-specific enzyme-linked assays. It is not currently known whether arginine biosynthesis in the cotyledon involves glutamate delivered from the mother plant or glutamate derived in situ.     

Metabolic regulation of manganese superoxide dismutase expression via essential amino acid deprivation

Organisms respond to available nutrient levels by rapidly adjusting metabolic flux, in part through changes in gene expression. A consequence of adaptations in metabolic rate is the production of mitochondria-derived reactive oxygen species. Therefore, we hypothesized that nutrient sensing could regulate the synthesis of the primary defense of the cell against superoxide radicals, manganese superoxide dismutase. Our data establish a novel nutrient-sensing pathway for manganese superoxide dismutase expression mediated through essential amino acid depletion concurrent with an increase in cellular viability. Most relevantly, our results are divergent from current mechanisms governing amino acid-dependent gene regulation. This pathway requires the presence of glutamine, signaling via the tricarboxylic acid cycle/electron transport chain, an intact mitochondrial membrane potential, and the activity of both the MEK/ERK and mammalian target of rapamycin kinases. Our results provide evidence for convergence of metabolic cues with nutrient control of antioxidant gene regulation, revealing a potential signaling strategy that impacts free radical-mediated mutations with implications in cancer and aging.     

Loblolly pine arginase responds to arginine in vitro

Following germination of loblolly pine (Pinus taeda L.) seeds, storage proteins in the embryo and megagametophyte are broken down to provide nitrogen, in the form of amino acids, to the developing seedling. A substantial portion of the free amino acids released in this process is arginine. Arginine is hydrolyzed in the cotyledons of the seedling by the enzyme arginase (EC 3.5.3.1), which is under developmental control. It has been shown previously that the seedling is able to initiate arginase gene expression in vitro in the absence of the megagametophyte, however, presence of the megagametophyte causes a greater accumulation of arginase protein and mRNA. Using an in vitro culture system we show that arginine itself may be responsible for up-regulating arginase activity. Application of exogenous arginine to cotyledons of seedlings germinated in the absence of the megagametophyte caused an increase in total shoot pole arginase activity as well as arginase specific activity. Arginine was also able to induce arginase mRNA accumulation in the same tissue.     

精氨酸代谢途径抗酸关键基因对乳酸乳球菌Lactococcus lactis NZ9000胁迫抗性的影响

【目的】寻找精氨酸代谢途径中与酸胁迫相关的关键作用因素。【方法】通过在Lactococcus lactis NZ9000中分别过量表达来源于Lactobacillus casei Zhang的精氨酰琥珀酸合成酶(ASS)和精氨酰琥珀酸裂解酶(ASL)改变精氨酸代谢提高酸胁迫抗性。【结果】与对照菌株对比,重组菌株在环境胁迫下表现了较高的生长性能、存活率和发酵性能。生理学分析发现,酸胁迫环境下,重组菌株细胞有较高的胞内NH4+、ATP含量和H+-ATPase活性,并显著提高了精氨酸脱亚胺酶(ADI)途径中的氨基酸浓度。进一步的转录分析发现,天冬氨酸合成、精氨酸代谢相关的基因转录水平上调。【结论】在L.lactis NZ9000中过量表达ASS或ASL可以引发精氨酸代谢流量的上调,进而提高了细胞的多种胁迫抗性。精氨酸合成途径广泛存在于多种微生物中,为微生物,尤其是工业微生物提高胁迫抗性提供了新思路。     

Protective effect of L-cysteine and glutathione on rat brain Na+,K+-ATPase inhibition induced by free radicals

The aim of this study was to investigate whether the preincubation of brain homogenates with L-phenylalanine (Phe), L-cysteine (Cys) or reduced glutathione (GSH) could reverse the free radical effects on Na+,K+-ATPase activity. Two well established systems were used for the production of free radicals: 1) FeSO4 (84 microM) plus ascorbic acid (400 microM) and 2) FeSO4, ascorbic acid and H2O2 (1 mM) for 10 min at 37 degrees C in homogenates of adult rat whole brain. Changes in brain Na+,K+-ATPase activity and total antioxidant status (TAS) were studied in the presence of each system separately, with or without Phe, Cys or GSH. TAS value reflects the amount of free radicals and the capacity of the antioxidant enzymes to limit the free radicals in the homogenate. Na+,K+-ATPase was inhibited by 35-50% and TAS value was decreased by 50-60% by both systems of free radical production. The enzymatic inhibition was completely reversed and TAS value increased by 150-180% when brain homogenates were preincubated with 0.83 mM Cys or GSH. However, this Na+,K+-ATPase inhibition was not affected by 1.80 mM Phe, which produced a 45-50% increase in TAS value. It is suggested that the antioxidant action of Cys and GSH may be due to the binding of free radicals to sulfhydryl groups of the molecule, so that free radicals cannot induce Na+,K+-ATPase inhibition. Moreover, Cys and GSH could regulate towards normal values the neural excitability and metabolic energy production, which may be disturbed by free radical action on Na+,K+-ATPase.     

Osmotic Stress-Induced Polyamine Accumulation in Cereal Leaves : II. Relation to Amino Acid Pools

Arginine decarboxylase activity increases 2- to 3-fold in osmotically stressed oat leaves in both light and dark, but putrescine accumulation in the dark is only one-third to one-half of that in light-stressed leaves. If arginine or ornithine are supplied to dark-stressed leaves, putrescine rises to levels comparable to those obtained by incubation under light. Thus, precursor amino acid availability is limiting to the stress response. Amino acid levels change rapidly upon osmotic treatment; notably, glutamic acid decreases with a corresponding rise in glutamine. Difluoromethylarginine (0.01-0.1 millimolar), the enzyme-activated irreversible inhibitor of arginine decarboxylase, prevents the stress-induced putrescine rise, as well as the incorporation of label from [¹⁴C]arginine, with the expected accumulation of free arginine, but has no effect on the rest of the amino acid pool. The use of specific inhibitors such as α-difluoromethylarginine is suggested as probes for the physiological significance of stress responses by plant cells.     

Studies on the Nitrogen Metabolism in Ectomycorrhizae

Concentrations of free and bound amino acids were determined in 1) the mycorrhizal fungus Boletus variegatus Fr. 2) nonmycorrhizal root systems of aseptically grown Pinus sylvestris L. seedlings, and 3) mycorrhizal root systems of seedlings developed aseptically using the two symbionts. Arginine (total) was the major amino acid constituent in the mycelium of B. variegatus (18%–22%) during the exponential phase of growth. While 59%–86% of the available arginine was bound during the acceleration phase of growth, in the logarithmic phase 63%–75% was in the free pool. There were differences in the proportions between the individual amino acids in the bound fraction at different stages of growth suggesting production of diverse proteins. Twenty per cent of the amino acid content of uninfected P. sylvestris root systems was arginine. Infection of the root systems by the fungal symbiont did not result in an increase but a slight decrease in the free arginine content of the composite structure. Almost all other amino acids in the free pool were found in higher concentrations in the mycorrhizal root system. It is suggested that arginine synthesis in B. variegatus is repressed by the arginine available in the host. The mycorrhizal fungus possibly metabolizes the host arginine pool ultimately resulting in more efficient protein synthesis in both the partners.