Intraprotein radical transfer during photoactivation of DNA photolyase

Amino-acid radicals play key roles in many enzymatic reactions. Catalysis often involves transfer of a radical character within the protein, as in class I ribonucleotide reductase where radical transfer occurs over 35 A, from a tyrosyl radical to a cysteine. It is currently debated whether this kind of long-range transfer occurs by electron transfer, followed by proton release to create a neutral radical, or by H-atom transfer, that is, simultaneous transfer of electrons and protons. The latter mechanism avoids the energetic cost of charge formation in the low dielectric protein, but it is less robust to structural changes than is electron transfer. Available experimental data do not clearly discriminate between these proposals. We have studied the mechanism of photoactivation (light-induced reduction of the flavin adenine dinucleotide cofactor) of Escherichia coli DNA photolyase using time-resolved absorption spectroscopy. Here we show that the excited flavin adenine dinucleotide radical abstracts an electron from a nearby tryptophan in 30 ps. After subsequent electron transfer along a chain of three tryptophans, the most remote tryptophan (as a cation radical) releases a proton to the solvent in about 300 ns, showing that electron transfer occurs before proton dissociation. A similar process may take place in photolyase-like blue-light receptors.     

Specific inhibition of herpesvirus ribonucleotide reductase by synthetic peptides

Ribonucleotide reductase is an essential enzyme for DNA synthesis in all prokaryotic and eukaryotic cells; it catalyses the reductive conversion of ribonucleotides to deoxyribonucleotides. Several herpesviruses including herpes simplex virus type 1 (HSV-1), HSV-2, pseudorabies virus (PRV), equine herpesvirus type 1 (EHV-1) and Epstein-Barr virus (EBV) have been found to induce novel ribonucleotide reductase activities. There is evidence that the HSV-1 ribonucleotide reductase activity is virus-encoded and essential for virus replication. This makes herpesvirus ribonucleotide reductases potential targets for antiviral chemotherapy. The HSV-1-encoded enzyme consists of two subunits: V136, the large subunit of relative molecular mass (Mr) 136,000 (136K) (RR1), which has been shown to be essential for enzyme activity, and V38, the small subunit (RR2) which forms a complex with the large subunit and is also likely to be essential for enzyme activity. Two particular features of the enzyme make it an attractive antiviral target. First, there is evidence for a common, highly conserved herpesvirus ribonucleotide reductase and second, the interaction between the large and small subunits may itself be exploitable. Here we identify a synthetic peptide which specifically inhibits the activity of virus-induced enzyme. We deduce that the mechanism of inhibition involves interference with the normal interaction between the two types of subunit.     

A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage

The p53 gene is frequently inactivated in human cancers. Here we have isolated a p53-inducible gene, p53R2, by using differential display to examine messenger RNAs in a cancer-derived human cell line carrying a highly regulated wild-type p53 expression system. p53R2 contains a p53-binding sequence in intron 1 and encodes a 351-amino-acid peptide with striking similarity to the ribonucleotide reductase small subunit (R2), which is important in DNA synthesis during cell division. Expression of p53R2, but not R2, was induced by ultraviolet and gamma-irradiation and adriamycin treatment in a wild-type p53-dependent manner. Induction of p53R2 in p53-deficient cells caused G2/M arrest and prevented cells from death in response to adriamycin. Inhibition of endogenous p53R2 expression in cells that have an intact p53-dependent DNA damage checkpoint reduced ribonucleotide reductase activity, DNA repair and cell survival after exposure to various genotoxins. Our results indicate that p53R2 encodes a ribonucleotide reductase that is directly involved in the p53 checkpoint for repair of damaged DNA. The discovery of p53R2 clarifies a relationship between a ribonucleotide reductase activity involved in repair of damaged DNA and tumour suppression by p53.     

Specific inhibition of herpesvirus ribonucleotide reductase by a nonapeptide derived from the carboxy terminus of subunit 2

Ribonucleotide reductase, an essential enzyme for the synthesis of deoxyribonucleotides, is formed by the association of two nonidentical subunits in almost all prokaryotic and eukaryotic cells. The same model probably holds for the herpes simplex virus (HSV)-encoded ribonucleotide reductase; two polypeptides of relative molecular mass 136,000 (136K; H1) and 40K (H2) (referred to elsewhere as RR1 and RR2; see for example, Dutia et al.) have been associated with the viral enzyme by both genetic and immunological studies. Furthermore, DNA sequence analyses have shown significant stretches of amino-acid homology between these viral polypeptides and those of, respectively, subunit 1 (ref. 12) and subunit 2 (ref. 13) of the Escherichia coli and mammalian enzymes. To assess the involvement of the 40K polypeptide in reductase activity, we synthesized a nonapeptide corresponding to the sequence of its carboxy terminus with the intention of raising neutralizing antibodies specific for the viral activity (E.A.C. et al., in preparation). We report here the unexpected finding that the nonapeptide itself specifically inhibits the HSV ribonucleotide reductase activity in a reversible, non-competitive manner, and we suggest that it does this by impairment of the correct association of the two subunits. This phenomenon emphasizes the potential usefulness of synthetic peptides in probing critical sites involved in macromolecular interactions.     

Is ribonucleotide reductase the transforming function of herpes simplex virus 2?

Transformation of cells by herpes simplex virus 2 (HSV-2) can be induced by the BglII C (0.43-0.58 map units) or N (0.58-0.625) fragments of the viral genome. Sequences partially overlapping both fragments (0.566-0.602) encode two 3' coterminal mRNAs; these in turn direct the synthesis of two related polypeptides of molecular weight 140,000 (140K) and 35K (refs 4, 7), which may be involved in transformation. Recently, a temperature-sensitive (ts) mutation affecting HSV-induced ribonucleotide reductase has been mapped within this common region (B.M. Dutia, personal communication). We have partially purified the induced reductase and raised a rabbit antiserum to it which inhibits the enzyme activity and immunoprecipitates from infected cells a 144K polypeptide and minor species including a 38K polypeptide. Here we show that a monoclonal antibody to the putative transforming proteins competes with the rabbit serum for the 144K and 38K antigens and also immunoprecipitates specifically the induced reductase activity. These results suggest a possible role for ribonucleotide reductase in HSV-2-induced transformation.     

韭菜叶及韭菜籽醇提物的抗氧化活性研究

采用乙醇浸润萃取制备韭菜叶及韭菜籽醇提物,测定提取物对DPPH自由基和羟基自由基的清除能力。结果表明韭菜籽及韭菜叶醇提物对DPPH自由基和羟基自由基的都有较强的清除能力。韭菜籽醇提液对DPPH自由基的清除率的IPC50为0.501 mg/mL;对羟基自由基的清除率的IPC50为0.794 mg/mL。韭菜叶醇提液对DPPH自由基的清除率的IPC50为0.540 mg/mL;对羟基自由基的清除率的IPC50为0.496 mg/mL。     

多相催化氧化降解苯酚过程的研究

利用紫外可见光谱和高效液相色谱研究了催化氧化降解苯酚的过程.结果表明,非晶铁氧化物催化剂在催化氧化降解苯酚时具有较高的催化活性,苯酚降解的中间产物有:对苯二酚、邻苯二酚、苯醌和低级脂肪酸,其降解机理是羟基自由基攻击苯环上OH的邻位或对位而使苯环开环,生成低级脂肪酸,并最终转化成二氧化碳和水,从而使苯酚得以降解.     

4种植物多酚对生物大分子的保护作用

为了研究多酚类化合物对生物大分子氧化损伤的保护作用,采用MDA测定、SDSPAGE、琼脂糖电泳,检测了4种多酚对由自由基引起的脂质过氧化、蛋白质氧化降解、DNA断裂的保护作用.结果表明:槲皮素在抑制脂质过氧化中作用最突出,在保护蛋白质氧化降解中香草醛作用最强;芦丁在保护DNA的氧化性损伤中最有效.说明4种化合物在不同的抗氧化体系中有不同的活性.     

维生素E与运动能力研究的新进展

长时间剧烈运动的生物体，会产生大量自由基，从而对生物膜结构的完整性和某些酶活性产生伤害．维生素Ｅ是体内重要的自由基清除剂，对生物膜有保护作用，还可保持酶的正常活性，维持人体正常的运动能力和消除运动后的疲劳．本文论述了维生素Ｅ的抗氧化、消除自由基的作用机制，分析了维生素与运动能力的关系以及补充维生素对运动能力的重要作用．     

DNA sequence and expression of the B95-8 Epstein-Barr virus genome

The complete (172,282 base pairs) nucleotide sequence of the B95-8 strain of Epstein-Barr virus has been established using the dideoxynucleotide/M13 sequencing procedure. Many RNA polymerase II promoters have been mapped and the mRNAs from these promoters have been assigned to the latent or early/late productive virus cycles. Likely protein-coding regions have been identified and three of these have been shown to encode a ribonucleotide reductase, a DNA polymerase and two surface glycoproteins.     

香椿叶水溶性多糖初步纯化及清除自由基活性研究

对香椿叶粗多糖进行了提取,测定了其蛋白质含量,并对其进行了初步纯化,然后对粗多糖和初步纯化多糖进行了抗氧化实验,测定了不同质量浓度的香椿粗多糖和初步纯化多糖对自由基的清除作用．纸层析、GC分析结果表明：香椿叶初步纯化多糖的单糖组成为Xyl,Ara,Rha,Gal,Glc,Gal—A,Man;摩尔比为1．0：3．7：1．3：9．0：2．7：10．0：0．7．抗氧化实验结果表明：不同质量浓度的粗多糖和初步纯化多糖都能有效地清除羟自由基和超氧阴离子,而且初步纯化多糖比粗多糖的清除效率要高,香椿粗多糖对R·有清除作用,而初步纯化多糖在实验规定的浓度范围内对R·却没有清除作用．     

NH2自由基电子基态及激发态解离通道的静力学分析

本文利用原子分子反应静力学的基本原理,研究了NH2自由基体系的可能解离通道,得到了NH2自由基电子基态2B1及第一激发态2A1合理的解离通道。     

细菌铁(Ⅲ)结合蛋白:结构与机理(英文)

铁是生命体必需的微量元素,在大多数环境中铁是有限的资源.高效摄取铁是入侵属主的微生物生存和毒力的关键.许多致病细菌进化出一个特异的铁吸收系统,利用特定的外膜受体和周质铁离子结合蛋白(FBP或FbpA)从属主偷取铁离子.FBP在高效摄取铁的过程中,无论从自由铁源摄取铁或从属主体内转铁蛋白和乳铁蛋白摄取结合的铁,都起着至关重要的作用.FBP的铁结合机制在不同物种间高度保守.结构数据显示,FBP的三维折叠类似于哺乳动物转铁蛋白(TF)的一叶,铁(Ⅲ)在FBP的两个结构域之间的间隙结合但其铁结合位点较其在转铁蛋白中更暴露于溶剂.该小综述总结了FBP铁转运系统,主要讨论了铁结合蛋白的结构和配位化学特征以及铁转运及调控机理.     

Iron-catalyzed benzylation of 1,3-dicarbonyl compounds by simple toluene derivatives

An iron-catalyzed oxidative C-C bond formation by the reactions of simple toluene derivatives with 1,3-dicarbonyl compounds is developed.A benzylic radical addition to a benzoylmethanato iron species is proposed for the transformation.     

A ferric-chelate reductase for iron uptake from soils

Iron deficiency afflicts more than three billion people worldwide, and plants are the principal source of iron in most diets. Low availability of iron often limits plant growth because iron forms insoluble ferric oxides, leaving only a small, organically complexed fraction in soil solutions. The enzyme ferric-chelate reductase is required for most plants to acquire soluble iron. Here we report the isolation of the FRO2 gene, which is expressed in iron-deficient roots of Arabidopsis. FRO2 belongs to a superfamily of flavocytochromes that transport electrons across membranes. It possesses intramembranous binding sites for haem and cytoplasmic binding sites for nucleotide cofactors that donate and transfer electrons. We show that FRO2 is allelic to the frd1 mutations that impair the activity of ferric-chelate reductase. There is a nonsense mutation within the first exon of FRO2 in frd1-1 and a missense mutation within FRO2 in frd1-3. Introduction of functional FRO2 complements the frd1-1 phenotype in transgenic plants. The isolation of FRO2 has implications for the generation of crops with improved nutritional quality and increased growth in iron-deficient soils.     

诱导效应指数与烷基衍生物某些热力学参数的关系

利用诱导效应指数，分别建立了计算烷基衍生物的标准生成热、键裂能的方法，并且得到自由基的生成热的计算方法。计算了一些烷基衍生物的标准生成热、键裂能及自由基的标准生成热，平均偏差分别为２．１８ｋＪ·ｍｏｌ－１，２．３９ｋＪ·ｍｏｌ－１，１．８７ｋＪ·ｍｏｌ－１。     

针刺对胸内肿瘤化学疗法中自由基等指标的影响

恶性肿瘤的化学疗法会产生自由基升高和白细胞数目降低等副作用,临床表现恶心、呕吐、厌食、疲乏等症状。 本文应用针刺“足三里”、“阴陵泉”、“内关”等穴位配合化学疗法,结果血中脂质过氧化物(LPO)降低,白细胞增多、消化道症状和疲乏感降低,这说明针刺疗法可作为化学疗法的重要支持手段。 针刺降低自由基的原理估计是激发了体内降解酶的作用。     

水解度对蚕蛹蛋白酶解物的物化特性和抗氧化能力的影响

对蛋白质进行适度酶解是改善动植物蛋白原料的物化性质和提高其生理活性的常用方法.研究了水解度对蚕蛹蛋白的溶解性、乳化性、起泡性等物化性质的影响,以及水解度对蚕蛹蛋白的还原力,对DPPH自由基、超氧阴离子自由基和羟基自由基的清除能力等抗氧化特性的影响.实验结果表明,蚕蛹蛋白经适度酶解后溶解性得到显著改善,且水解度越大溶解性越好.蚕蛹蛋白经酶解后乳化性与乳化稳定性均降低,但起泡性和起泡稳定性增强,水解度增至5%时,起泡性和起泡稳定性达到最高值,继续增大水解度,反而下降.蚕蛹蛋白经酶解后抗氧化性显著提高,水解度为5%时,还原力和对DPPH自由基和羟基自由基的清除能力最高,水解度为15%时,对超氧阴离子自由基的清除能力最高,继续增加水解度,抗氧化能力反而下降.这说明,对蛋白质原料进行酶解改性时,不能简单的追求高水解度,而应根据产品的要求控制合适的水解度,过度水解是无益的.     

白花丹提取物抗氧化活性研究

采用电子自旋共振法(ESR)、光敏化合物微生物法、光度法对白花丹提取物的抗氧化性能进行了检测.结果:白花丹提取物对单线态氧自由基、混合自由基、羟自由基及香烟烟气自由基的清除率分别是53.3%、59.1%、40.2%、26.0.0%,白花丹提取物具有较好的抗氧化性能,其抗氧化能力与其质量浓度呈正相关.     

维生素C抗氧化作用及其在运动中的应用

运动可使体内自由基含量升高．自由基与生物膜中的不饱和脂肪酸之间有高度的亲和力,二者一旦结合,就会生成不饱和脂肪酸的过氧化物,使生物膜的结构、功能受到破坏,对组织造成伤害,影响运动能力．维生素Ｃ作为抗氧化剂和自由基清除剂,在自由基清除过程中可发挥重要作用．本文论述了维生素抗氧化和清除自由基的机制,分析了维生素Ｃ与运动能力之间的关系以及维生素在运动实践中的应用．