双链聚乙二醇与超氧化物歧化酶共价结合物的理化和免疫学性质

用三聚氯氰活化单甲氧基聚乙二醇,得到了双链聚乙二醇(PEGm₂).以PEGm₂修饰牛血Cu,Zn-SOD,得到了氨基修饰率为30％,残余活力为80％的纯PEGm₂-SOD.修饰酶的荧光光谱及圆二色谱均有改变;盐酸胍变性及胃蛋白酶水解实验结果表明,修饰酶的稳定性高于天然酶;免疫学实验表明,与天然酶比较,修饰酶的免疫原性及抗原抗体反应性大为降低.     

鉴别超氧化物歧化酶类型的定位染色法

介绍一种鉴别SOD类型的方法─—聚丙烯酰胺凝胶电泳的定位染色法. 由于不同类型的SOD对抑制剂的表现各异, 电泳后的凝胶经不同的抑制剂处理, 染色, 结果展示在凝胶上, CuZn-SOD酶带在H₂O₂或CN^-的作用下消失, Mn-SOD在CHCl₃-CH₃OH作用下消失, Fe-SOD在H₂O₂或. CHCl₃-CH₃CH₂OH作用下失活, 从酶带消失或存活的情况, 可以判断SOD的类型.     

S1核酸酶作用与微环DNA分子的克隆策略

米曲霉来源的S1核酸酶具有降解单链DNA或RNA的作用。在适当的条件下, 该酶能将不同的环形DNA分子从超螺旋转变成开环和线形结构,对质粒Puc19的实验证明, S₁核酸酶的这种转变作用与加入的酶量呈正相关。在25μL总反应体积中,按100ng DNA加入5u至17u的S1核酸酶,能获得较高比例的线形DNA。由于微环DNA分子太小,单酶切位点的出现率较低,很难用常规方式进行克隆,以S₁核酸酶进行线形化是微环DNA克隆的途径。pC3是已知最小的真核生物线粒体DNA类质粒（537bp）,经S₁核酸酶线形化后,成功地克隆到pMD18-T载体上。     

玉米跨代干旱胁迫记忆生理机制及DNA甲基化变化分析

为研究亲代干旱锻炼对后代玉米生理特性和DNA甲基化修饰的影响,以亲代(G₀代)经干旱锻炼的玉米自交系B73和H99自交后代(G₁代)为材料,利用20% PEG 6000模拟干旱胁迫条件,检测G₁和G₀代叶片相对含水量(RWC)与丙二醛(MDA)、可溶性糖、脯氨酸含量以及超氧化物歧化酶(SOD)、过氧化物酶(POD)活性变化,并利用甲基化敏感扩增多态性技术(MSAP)检测G₁和G₀代基因组DNA甲基化状况,分析2个世代玉米生理指标和基因组DNA甲基化修饰的变异规律。结果表明：(1)在相同干旱胁迫条件下,玉米B73和H99 自交系G₁代叶片的RWC、可溶性糖与脯氨酸含量以及SOD和POD活性均高于G₀代,其G₁代MDA含量则低于G₀代;G₁代叶片的RWC减少量和MDA增加量小于G₀代,G₁代可溶性糖和脯氨酸含量以及SOD和POD活性增加量均大于G₀代。(2)干旱胁迫诱发了B73和H99 自交系G₁和G₀代DNA甲基化水平和甲基化模式的改变;在相同干旱胁迫条件下,两自交系G₁代DNA甲基化修饰变化均大于G₀代。(3)B73和H99 自交系DNA甲基化修饰变异规律不同,随胁迫时间延长,B73 自交系2个世代CG、CHG甲基化水平均呈上升趋势,H99 自交系2个世代CG甲基化水平呈上升趋势,CHG甲基化水平呈下降趋势;B73 自交系2个世代均以CG hypo和CHG hypo变化为主,H99 自交系2个世代均以CHG hypo和CG hyper变化为主。研究发现,B73和H99玉米自交系G₁代植株的抗氧化和渗透调解能力以及DNA甲基化修饰变化均大于G₀代,其抗旱性也强于G₀代,从而证明玉米存在跨代干旱胁迫记忆。     

SelS高表达保护人脐静脉内皮细胞免于H2O2诱导的细胞损伤

采用以下方法探讨SelS在内皮细胞中的表达和作用：将SelS基因克隆到真核表达载体pLNCX2，RT-PCR、XhoⅠ/ClaⅠ双酶切以及DNA序列分析验证目的基因；利用脂质体转染技术将pLNCX2-SelS或pLNCX2转染至人脐静脉内皮细胞(ECV₃₀₄细胞)，RT-PCR检测重组基因SelS的表达；MTT方法检测转染后过氧化氢(H₂O₂)对内皮细胞增殖能力的影响；硫代巴比妥酸法测定暴露于H₂O₂中不同转染组细胞脂质过氧化产物丙二醛含量. 结果表明：成功构建真核表达载体pLNCX2-SelS；转染后重组SelS mRNA表达水平是内源性水平的1.76倍；H₂O₂对ECV₃₀₄细胞损伤后，高表达SelS组细胞活性增强、H₂O₂诱导产生的丙二醛减少. 上述结果表明，高表达SelS可保护内皮细胞免于H₂O₂诱导的细胞损伤，其作用机制与抗氧化有关.     

H2O2－Fe3+所致人淋巴细胞DNA双链断裂损伤

采用脉冲电场凝胶电泳法检测H₂O₂－Fe³⁺体系产生的OH^·对人淋巴细胞DNA的双链断裂损伤．H₂O₂－Fe³⁺浓度与DNA双链断裂呈明显量效关系;随OH^·作用时间延长,细胞DNA双链断裂加重;过氧化氢酶对OH^·损伤有明显抑制作用．脉冲电场凝胶电泳法可检测到的H₂O₂和FeCl₃引起细胞DNA双链断裂的最低浓度为0.3 mmol/L和6 μmol/L．     

高温和H2O2诱导酵母细胞产生活性衍生物的研究*

对高温和H₂O₂ 应激条件下产生活性酵母细胞衍生物 (Live Yeast Cell Derivative ,简称LYCD)进行了研究。结果表明 :低剂量的预处理 ( 37℃和 0.2mmol LH₂O₂ )能够增加细胞内谷胱甘肽 (GSH)含量 ,提高超氧化物歧化酶 (SOD)、过氧化氢酶 (CAT)活性。两种预处理均可以诱导对致死浓度H₂O₂ 的抗性。通过 37℃和 0.     

P2-DNA的转染及提纯

P₂-DNA即Phage 2型噬菌体的染色体DNA,是一条线状双股螺旋的DNA分子,分子量为2．2×10⁷Da。有19个碱基的牯性末端．可以连接成环状^[1]．1970年Bertani L.E. 和 Bertani G分离纯化得到P₂噬苗体并对其遗传学及理化特性进行了研究^[2],发现它在DNA复制,溶源性的控制以及基因重组等方面与温和性λ菌体不同;1979年Saint R B 等和Westoo A等先后研究了P₂-DNA的几种限制酶的切割图谱^[3]。P₂-DNA可望成为分子生物学研究的重要工具和实验材料。目前国内尚无这种材料．我们试图将少量的P₂-DNA转染获得P₂噬菌体,加以扩增纯化．抽提得到大量的P₂-DNA。为分子克隆和限制酶的研究提供有实用价值的材料和研究工具。     

螺旋藻POD、CAT和SOD同工酶的研究*

采用聚丙烯酰胺凝胶电泳法对毛乌素沙地碱湖的钝顶螺旋藻S3和鄂尔多斯螺旋藻S4与国外引进的钝顶螺旋藻S₁和极大螺旋藻S₂ 的POD、CAT和SOD同工酶进行了比较研究。结果表明 :4个样品的 3种酶同工酶带数目不同 ,依次是S₄ >S₃>S₁>S₂ ;S₃和S₄ 酶带数多 ,对环境适应性强 ,进化程度较高。螺旋藻不同种间的酶谱相似系数     

棉铃虫核型多角体病毒sod基因在大肠杆菌中的表达

用PCR方法从棉铃虫(Helicoverpa armigera)单粒包埋型核型多角体病毒(HaSNPV) C1株基因组中扩增sod基因编码区,克隆到pGEM-T-easy vector,测定了核苷酸序列.将基因编码区克隆到原核表达载体pETblue2,构建了含表达质粒pETblue2/HaSNPV SOD,转化大肠杆菌DE3(BL21)进行IPTG诱导表达. SDS-PAGE分析表明SOD的表达量约为细胞总蛋白的37%.邻苯三酚法测定表达蛋白活性,结果表明每毫克菌体可溶性总蛋白中表达产物校正酶活力单位为694U/mg.     

Divalent-metal-dependent nucleolytic activity of Cu, Zn superoxide dismutase

The known action of Cu, Zn superoxide dismutase (holo SOD) that converts O₂ ⁻ to O₂ and H₂O₂ plays a crucial role in protecting cells from toxicity of oxidative stress. However, the overproduction of holo SOD does not result in increased protection but rather creates a variety of unfavorable effects, suggesting that too much holo SOD may be injurious to the cells. In the in vitro study, we report a finding that the holo SOD from bovine erythrocytes and its apo form possess a divalent-metal-dependent nucleolytic activity, which was confirmed by UV–vis absorption titration of calf thymus DNA (ctDNA) with the holo SOD, quenching of holo SOD intrinsic fluorescence by ctDNA, and by gel electrophoresis monitoring conversion of DNA from the supercoiled DNA to nicked and linear forms, and fragmentation of a linear λDNA. Moreover, the DNA cleavage activity was examined in detail under certain reaction conditions. The steady-state study indicates that DNA cleavage supported by both forms of SOD obeys Michaelis–Menten kinetics. On the other hand, the assays with some other proteins indicate that this new function is specific to some proteins including the holo SOD. Therefore, this study reveals that the divalent-metal-dependent DNA cleavage activity is an intrinsic property of the holo SOD, which is independent of its natural metal (copper and zinc) sites, and may provide an alternative insight into the link between SOD enzymes and neurodegenerative disorders.     

Strand scission in DNA by Gossypol and Cu(II): Role of Cu(I) and oxygen-free radicals

Gossypol, a polyphenolic binaphthyl dialdehyde found in cotton seeds, is a dietary mutagen and a potential male contraceptive. In the presence of Cu(II), gossypol caused breakage of supercoiled plasmid pBR322 DNA. The products were relaxed circles or a mixture of these and linear molecules. Other metal ions tested [Ni(II), Co(II), Mn(II), and Fe(II)] were ineffective or less effective in the DNA breakage reaction. In the case of gossypol-Cu(II) mediated cleavage, (Cu(I) was shown to be an essential intermediate by using the Cud) sequestering reagent bathocuproine. By using job plots, it was established that in the absence of DNA, eight Cu(II) ions can be reduced by one gossypol molecule. The involvement of active oxygen species, such as singlet oxygen and H₂O₂, was established by the inhibition of DNA breakage by catalase and by sodium azide. It was further shown that gossypol is capable of directly producing H₂O₂.     

Chemically and photochemically initiated DNA cleavage by an insulin-mimeticbisperoxovanadium complex

Chemically and photochemically induced cleavage of DNA by the insulin-mimetic compound NH₄[VO(O₂)₂-(1,10-phenanthroline)], bpV(phen), have been studied.⁵¹V NMR and absorption indicate that photoirradiation with low energy UV light of aqueous solutions containing bpV(phen) leads to the conversion of the compound to simple vanadates. Photoillumination of the compound in the presence of supercoiled pBR322 DNA results in cutting of the plasmid to produce nicked circular and linear DNA. Quantitative analysis of agarose gel data shows that bpV(phen) is a single strand nicking agent exhibiting sequence and/or base specificity.     

DNA and RNA strand scission by copper, zinc and manganese superoxide dismutases

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O _inf2 ^sup– , to O₂ and H₂O₂, play a major role in protecting cells from toxicity of oxidative stress. However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may he injurious to the cell. To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied. High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage X174 supercoiled double-stranded DNA to open circular and linear forms. Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme. The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD. Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H₂O₂. Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide. It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA. Both types of SOD were shown to effectively cleave RNA. These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene.     

Mitochondria-derived Hydrogen Peroxide Selectively Enhances T Cell Receptor-initiated Signal Transduction

T cell receptor (TCR)-initiated signal transduction is reported to increase production of intracellular reactive oxygen species, such as superoxide (O₂^⨪) and hydrogen peroxide (H₂O₂), as second messengers. Although H₂O₂ can modulate signal transduction by inactivating protein phosphatases, the mechanism and the subcellular localization of intracellular H₂O₂ as a second messenger of the TCR are not known. The antioxidant enzyme superoxide dismutase (SOD) catalyzes the dismutation of highly reactive O₂^⨪ into H₂O₂ and thus acts as an intracellular generator of H₂O₂. As charged O₂^⨪ is unable to diffuse through intracellular membranes, cells express distinct SOD isoforms in the cytosol (Cu,Zn-SOD) and mitochondria (Mn-SOD), where they locally scavenge O₂^⨪ leading to production of H₂O₂. A 2-fold organelle-specific overexpression of either SOD in Jurkat T cell lines increases intracellular production of H₂O₂ but does not alter the levels of intracellular H₂O₂ scavenging enzymes such as catalase, membrane-bound peroxiredoxin1 (Prx1), and cytosolic Prx2. We report that overexpression of Mn-SOD enhances tyrosine phosphorylation of TCR-associated membrane proximal signal transduction molecules Lck, LAT, ZAP70, PLCγ1, and SLP76 within 1 min of TCR cross-linking. This increase in mitochondrial H₂O₂ specifically modulates MAPK signaling through the JNK/cJun pathway, whereas overexpressing Cu,Zn-SOD had no effect on any of these TCR-mediated signaling molecules. As mitochondria translocate to the immunological synapse during TCR activation, we hypothesize this translocation provides the effective concentration of H₂O₂ required to selectively modulate downstream signal transduction pathways.     

l-Histidyl-glycyl-glycyl-l-histidine. Amino-acid structuring of the bleomycin-type pentadentate metal-binding environment capable of efficient double-strand cleavage of plasmid DNA

A tetrapeptide, l-histidyl-glycyl-glycyl-l-histidine (HGGH), was synthesized and the pUC19 plasmid DNA cleaving activity by copper(II) complex of HGGH (Cu(II)−HGGH) was investigated. Cu(II)−HGGH showed bleomycin-like DNA cleaving activity and, at 50 nM, converted a supercoiled DNA efficiently to a linear DNA in the presence of 500 μM H₂O₂/sodium ascorbate through an oxidative pathway.     

Molecular Characterization of <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Brassica juncea</Emphasis> Cu/Zn-Superoxide Dismutases Reveals Their Regulation of Shoot Regeneration

Superoxide dismutases (SODs) are ubiquitous metalloenzymes that catalyze the dismutation of superoxide radicals (O₂^-) to molecular oxygen (O₂) and hydrogen peroxide (H₂O₂). In this study we characterized an Arabidopsis thaliana CuZnSOD (CSD1), a close ortholog of a previously identified Brassica juncea CuZnSOD (MSOD1). CSD1 and other two homologs CSD2 and CSD3 were spatially regulated in Arabidopsis, and CSD1 exhibited distinct expression patterns in response to different stress treatments. To investigate the in vivo function of SOD, transgenic Arabidopsis plants, expressing sense and antisense MSOD1 RNAs, were generated and those with altered SOD activity were selected for further characterization. Although SOD transgenic plants exhibited normal phenotypes, the shoot regeneration response in transgenic explants was significantly affected by the modulated SOD activity and the corresponding H₂O₂ levels. Transgenic explants with downregulated SOD activity were poorly regenerative, whereas those with upregulated SOD activity were highly regenerative. These results suggest that shoot regeneration in vitro is regulated by the SOD activity.     

Phage-inactivating Effect of Iron(II)-Ascorbate Complex

The effect of iron(II)-ascorbate complex on various phages was investigated. At 10- ⁶ M, the complex inactivated all nine phages examined. The mechanism of the inactivation was studied with phage J1, the most sensitive to the complex. The addition of H₂O₂ or Cu²⁺ to the reaction mixture increased the inactivation. Bubbling of nitrogen through the reaction mixture and the addition of Fe³⁺, a reducing agent, a chelating agent, or a radical scavenger prevented inactivation. These findings suggest the involvement of oxygen radicals in the inactivation. The complex had no effects on the SDS-PAGE pattern or amino acid composition of bovine serum albumin, or the structural protein of phage J1. The complex nicked the supercoiled form of pUC18 DNA, giving first single-stranded breaks (the open circular form) and then double-stranded breaks (the linear form). Strands of M13mp8 DNA, λDNA, and J1 DNA were also broken. The breaks could account for the inactivation.     

Identification and characterization of superoxide dismutase in Phytophthora cinnamomi

Superoxide dismutase (SOD) activities of the oomycete Phytophthora cinnamomi were examined. Five polypeptides with manganese superoxide dismutase (MnSOD) activity were found in mycelium growing in liquid culture with relative molecular weights ranging from approximately 25 to 100 kDa. Comparison with characterized avocado SODs showed no evidence for the presence of either iron or copper/zinc SODs in P. cinnamomi. The level of activity of the MnSOD polypeptides decreased in the presence of avocado root or cell wall components. Growth of P. cinnamomi, measured as dry weight, increased when the mycelium was grown in the presence of superoxide anion (O₂ ^?), which was added exogenously. Our results suggest that the metabolism of O₂ ^? has an important role in the development of P. cinnamomi.     

Production of active oxygen species (1O2 and O2⨪) by psoralens and ultraviolet radiation (320–400 nm)

Furocoumarins (psoralens) are potent skin photosensitizing agents that are used in combination with long-wavelength ultraviolet radiation (320–400 nm) in the treatment of psoriasis and other skin diseases. Twelve linear and angular psoralens, capable of forming monofunctional and bifunctional adducts with DNA, were examined with a view to elucidate the role of ¹O₂ and O₂^? in evoking skin photosensitization reactions and skin carcinogenesis. The results showed that both linear psoralens (capable of forming interstrand cross-links) and isopsoralens (angular, monofunctional type) and 3-carbethoxypsoralen (a linear and monofunctional type) produced ¹O₂ and O₂^?, although at varying degrees. Psoralen and 3-carbethoxypsoralen produced ¹O₂ greater than isopsoralens (angelicins). However, nonphotosensitizing angelicin, 5-methyl-angelicin, and 4,8-dimethyl-5′-carboxypsoralen produced ¹O₂ greater than 8-methoxypsoralen and 5-methoxypsoralen. The three monofunctional angelicin derivatives (isopsoralens) produced more O₂^? than 8-methoxypsoralen, 5-methoxypsoralen, and 3,4′-dimethyl-8-methoxypsoralen. 3-Carbethoxypsoralen, a potent generator of ¹O₂ and a moderate producer of O₂^?, was highly photolabile. Until recently, skin photosensitization reactions (erythema, edema, damage to DNA or the membrane of cutaneous cells, the inhibition of scheduled DNA synthesis and skin carcinogenesis, etc.) were believed to involve photocyclo-addition of psoralens to DNA mediated by a type-I or anoxic reaction (a sensitizer-substrate interaction through the transfer of hydrogen atoms or electrons, but no direct involvement of molecular oxygen). Oxygen-dependent sensitized photodynamic reactions of type-II, involving the production of reactive oxygen (¹O₂ and O₂^?), were believed not to mediate psoralen photosensitization reactions. We suggest that ¹O₂ and O₂^? may also participate in skin photosensitization and cell membrane-damaging reactions. The fact that certain monofunctional isopsoralens produce ¹O₂ and O₂^? at rates comparable to or better than bifunctional psoralens suggests that these reactive moieties of oxygen could play a major role in explaining their recently observed carcinogenic property and cell membrane-damaging reactions (e.g., edema or inflammation, etc.).