稀土离子与牛血Cu(Zn)-SOD的作用

采用ESR、CD谱和荧光光谱研究了pH=6.3时六次甲基四胺-HCl缓冲溶液中LaCl_3和TbCl_3与Cu(Zn)-SOD的配位作用和结构。Cu(Zn)-SOD可增强Tb~(3+)的荧光发射,Tb~(3+)与Cu(Zn)-SOD有多个配位位置,其中有2个强结合位点,La~(3+)与Tb~(3+)可竞争Cu(Zn)…SOD上相同结合位点,77K下La~(3+)与Tb~(3+)使Cu(Zn)-SOD的Cu~(2+)活性中心的配位环境由菱形对称结构向轴对称结构转变,使Cu(Zn)-SOD的局部结构变松散,但对SOD酶活性基本无影响,表明稀土离子主要与酶蛋白分子中的酸性氨基酸羧基配位,对酶蛋白二级结构仅产生微弱扰动,对活性中心空间结构影响较小,基本不影响Cu(Zn)-SOD酶活性。

Purification and partial characterization of Mn superoxide dismutase from muscle tissue of the shrimp Macrobrachium nipponense

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that protects against oxidative stress from superoxide radicals in living cells. This enzyme had been isolated, purified and partially characterized from muscle tissue of the shrimp Macrobrachium nipponense. The purification was achieved by heat treatment, ammonium sulfate fractionated precipitation and column chromatograph on DEAE-cellulose 32. Some physiological and biochemical characterization of it was tested. The molecular weight of it was about 21.7 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had an absorption peak of 278 nm in ultraviolet region, and the enzyme remained stable at 25-45 degreesC within 90 min. However, it was rapidly inactivated at higher temperature. Treatment of the enzyme with 1 mM ZnCl2, SDS and 1 mM or 10 mM mercaptoethanol showed some increasing activity. However, the enzyme activity was obviously inhibited by 10 mM CaCl2, CuSO4, ZnCl2 and 1 mM CaCl2 and 10 mM K2Cr2O7. SOD activity did not show significantly variation after incubated with 1 mM CaCl2, EDTA and 10 MM SDS. The enzyme was insensitive to cyanide and contained 1.03 +/- 0.14 atoms of manganese per subunit shown in atomic absorption spectroscopy, which revealed that purified SOD was Mn superoxide dismutase. (C) 2004 Elsevier B.V. All rights reserved.

The mitochondrial manganese superoxide dismutase gene in Chinese shrimp Fenneropenaeus chinensis: Cloning, distribution and expression

Manganese superoxide dismutase (MnSOD) plays an important role in crustacean immune defense reaction by eliminating oxidative stress. Knowledge on MnSOD at molecular level allows us to understand its regulatory mechanism in crustacean immune system. A novel mitochondrial manganese superoxide dismutase (mMnSOD) was cloned from hepatopancreas of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1185 bp with a 660 bp open reading frame, encoding 220 amino acids. The deduced amino acid sequence contains a putative signal peptide of 20 amino acids. Sequence comparison showed that the mMnSOD of F. chinensis shares 88% and 82% identity with that of giant freshwater prawn Macrobrachium rosenbergii and blue crab Callinectes sapidus, respectively. mMnSOD transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill by Northern blotting. RT-PCR analysis indicated that mMnSOD showed different expression profiles in shrimp hemocytes and hepatopancreas after artificial infection with while spot syndrome virus (WSSV). In addition, a fusion protein containing mMnSOD was produced in vitro. LC-ESI-MS analysis showed that two peptide fragments (-GDVNTVISLAPALK- and -NVRPDYVNAIWK-) of the recombinant protein were identical to the corresponding sequence of M. rosenbergii mMnSOD, and the enzyme activity of the refolded recombinant protein was also measured. (c) 2006 Elsevier Ltd. All rights reserved.

Cloning, characterization, and expression analysis of extracellular copper/zinc superoxide dismutase gene from bay scallop Argopecten irradians

Extracellular superoxide dismutase (ECSOD) is a major extracellular antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned a novel ECSOD from the bay scallop Argopecten irradians (AiECSOD) by 3' and 5' RACE. The full-length cDNA of AiECSOD was 893 bp with a 657 bp open reading frame encoding 218 amino acids. The deduced amino acid sequence contained a putative signal peptide of 20 amino acids, and sequence comparison showed that AiECSOD had low degree of homology to ECSODs of other organisms. The genomic length of the AiECSOD gene was about 5276 bp containing five exons and six introns. The promoter region contained many putative transcription factor binding sites such as c-Myb, Oct-1, Sp1, Kruppel-like, c-ETS, NF kappa B, GATA-1, AP-1, and Ubx binding sites. Furthermore, tissue-specific expressions of AiECSOD and temporal expressions of AiECSOD in haemocytes of bay scallops challenged with bacteria Vibrio anguillarum were quantified using qRT-PCR. High levels of expression were detected in haemocytes, but not in gonad and mantle. The expression of AiECSOD reached the highest level at 12 h post-injection with V. anguillarum and then returned to normal between 24 h and 48 h post-injection. These results indicated that AiECSOD was an inducible protein and that it may play an important role in the immune responses against V anguillarum. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved.

Intracellular copper/zinc superoxide dismutase from bay scallop Argopecten irradians: Its gene structure, mRNA expression and recombinant protein

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Two unique and highly compartmentalized bay scallop Argopecten irradians superoxide dismutases: MnSOD and ecCuZnSOD, have been molecularly characterized in our previous study. To complete characterize the SOD family in A. irradians, a novel intracellular copper/zinc SOD from the A. irradians (Ai-icCuZnSOD) was obtained and characterized. The full-length cDNA of Ai-icCuZnSOD was 1047 bp with a 459 bp open reading frame encoding 152 amino acids. The genomic length of the Ai-icCuZnSOD gene was about 4279 bp containing 4 exons and 3 introns. The promoter region containing many putative transcription factor binding sites were analyzed. Furthermore, quantitative reverse transcriptase real-time PCR (qRT-PCR) analysis indicated that the highest expression of the Ai-icCuZnSOD was detected in gill and the expression profiles in hemocytes of bay scallops challenged with bacteria Vibrio anguillarum and lipopolysaccharide (LPS) were different. The result presented an increased expression after injection with LPS whereas no significant changes were observed after V. anguillarum injection. A fusion protein containing Ai-icCuZnSOD was produced in vitro. The rAi-icCuZnSOD is a stable enzyme, retaining more than 80% of its activity between 10 and 60 degrees C and keeping above 88% of its activity at pH values between 5.8 and 9. Ai-icCuZnSOD is more stable under alkaline than acidic conditions. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

The manganese superoxide dismutase gene in bay scallop Argopecten irradians: Cloning, 3D modelling and mRNA expression

A novel manganese superoxide dismutase (MnSOD) was cloned from bay scallop Argopecten irradians by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of MnSOD was of 1207 bp with a 678 bp open reading frame encoding 226 amino acids. The deduced amino acid sequence contained a putative signal peptide of 26 amino acids. Sequence comparison showed that the MnSOD of A. irradians shared high identity with MnSOD in invertebrates and vertebrates, such as MnSOD from abalone Haliotis discus discus (ABG88843) and frog Xenopus laevis (AAQ63483). Furthermore, the 3D structure of bay scallop MnSOD was predicted by SWISS-MODEL Protein Modelling Server and compared with those of other MnSODs. The overall structure of bay scallop MnSOD was similar to those of zebrafish Danio rerio, fruit fly Drosophila melanogaster, Chinese shrimp Fenneropenaeus chinensis, human Homo sapiens, and had the highest similarity to scallop Mizuhopecten yessoensis and abalone H. discus discus. A quantitative real-time PCR (qRT-PCR) assay was developed to detect the mRNA expression of MnSOD in different tissues and the temporal expression in haemocytes following challenge with the bacterium Vibrio anguillarum. A higher-level of mRNA expression of MnSOD was detected in gill and mantle. The expression of MnSOD reached the highest level at 3 h post-injection with V. anguillarum and then slightly recovered from 6 to 48 h. The results indicated that bay scallop MnSOD was a constitutive and inducible protein and thus could play an important role in the immune responses against V anguillarum infection. (c) 2008 Elsevier Ltd. All rights reserved.

钠原子激光增强电离光谱研究—以石墨杯为原子化器 原子吸收光谱法对发样中Zn、Cu、Mn、Ap的测定

、I钠原子激光增强电离光谱(LEIS)方法的研究-以石墨杯为原子化器在LEIS方法中，最常见的原子化器是火焰。但由于火焰背景噪声严重且难以克服，在火焰原子化过程中，雾化和热离解不充分，仅有10~(-2)%的分析溶液参与吸收以及火焰气体使测定元素受到高度稀释等不利因素的影响，火焰原子化限制了LEIS方法灵敏度的进一步提高。考虑到石墨炉原子化器较火焰具有取样量少，绝对灵敏度高；样品（包括固态、液态）可直接引入石墨炉内；不会发生如同火焰中所存在的干扰效应；蒸发效率和原子化效率较高，几乎全部样品都能参与吸收等优点，本工作在已建立火焰LEIS方法的实验基础上，将原子化器改换为石墨杯进行了钠原子LEIS方法的研究。到目前为止，国内外仅有的几篇有关石黑炉LEIS的研究报告中，都报导了该方法对钠原子的检出限估计可达到10~(-14)-10~(-15)克，由于此项研究尚处于探索性研究阶段，故有关方法性的系统研究几乎还未见报导。本工作在未使用任何放大器的情况下（实验条件限制）对影响钠原子LEIS信号强度的诸因互进行了实验观察。主要包括：钠原子化条件；激光束位置、阳极电压、激光输出能量、电极位置以及激光脉冲重复率对LEIS信号强度的影响等。并绘制了校准曲线，统计方法的相对标准偏差分别为11%（高浓度）18.2%（低浓度），在现有仪器条件下，还不能测出检出限，测定下限为3*10~(-9)克。对固体粉末直接进行了尝试，检测下限为5*10~(-8)克，进样是为5毫克。在进一步的研究工作中，如有条件使用低噪声的放大器及Bxear积分器，选择门检时间窗，或采用分步激发等手段，估计本方法定会达到预想的高灵敏度，检敏度至少提高了个数量级。对石墨炉原子化LEIS法来说，似比较详细的研究报告，截至实验停止时还未见报导。II原子吸收光谱法对发样中Zn、Cu、Mn、Al的测定发中微量元素ZN、Cu、Mn均属人体必需元素，与人体的生长发育和多种生理功能，临床医学等方面有着极为密切的关系，而Al则被认为是异致某种疾病的元素之一。本工作报告了用火焰法测定Zn、Cu；石墨炉法测定Al、Mn的结果，其中，对Al的测定，为摆脱基体干扰，加入改进剂Mg(NO_3)_2，并采用平台石墨炉进行试验，得到了线性较好的工作曲线，但在实际测定时，由于实验条件的限制，只能采用一般石墨管加基体改进剂对少娄样品中Al含量进行测定。Zn、Cu、Al三种元素由标准曲线法测定；而Mn由于Fe的干扰无法消除而采用标准加入法测定，并因此限制了测定样品数。Cu、ZN、Mn三种元素的回收率分别为102.8%, 99.7%, 102.5%，变异系数为9.6%, 11.3%, 9.7%，对本地居民发中（30个发样）Zn、Cu含量进行测定，Zn、Cu的含量范围为148-318ppm，7.2-15ppm，并计算了Zn/Cu比。本方法对发样中四种元素的测定结果与ICP法进行对照。两种方法测定结果吻合得较好。

Theoretical studies on the reaction mechanism of oxidation of primary alcohols by Zn/Cu(II)-phenoxyl radical catalyst

The catalytic mechanism for the oxidation of primary alcohols catalyzed by the two functional models of galactose oxidase (GOase), M-II L (M = Cu, Zn; L = N,N'-bis(3,5-di-tert-butyl-2-hydroxyphenyl)1-2-diiminoquinone)), has been studied by use of the density functional method B3LYP The catalytic cycle of Cu- and Zn-catalysts consists of two parts, namely, substrate oxidation (primary alcohol oxidation) and O-2 reduction (catalyst regeneration). The catalytic mechanisms have been studied for the two reaction pathways (route 1 and route 2). The calculations indicate that the hydrogen atom transfer within the substrate oxidation part is the rate-determining step for both catalysts, in agreement with the experimental observation.

Physiological and immune responses of zhikong scallop Chlamys farreri to the acute viral necrobiotic virus infection

The Zhikong Scallop, Chlamys farreri, is one of the most Important bivalve mollusks cultured in northern China However, mass mortality of the cultured C farreri has posed a serious threat to the maricultural Industry in recent years. Acute Viral Necrobiotic Virus (AVNV) is believed as an important etiological agent causing the scallop mass mortalities To understand the mechanism behind the AVNV associated scallop disease and mortality, we assessed the physiological and immune responses of C farreri to the virus infection using oxygen consumption rate, ammonium-nitrogen excretion rate, hemocyte copper, zinc superoxide dismutase gene expression, and plasma superoxide dismutase activity and alkaline phosphatase activity as indicators Scallops challenged by AVNV at 25 C developed typical disease signs 2 days after virus injection Before the disease manifested, scallop oxygen consumption and NH4+-N excretion rates rose and then fell back. Real-time PCR revealed that the hemocyte cytosol Cu, Zn SOD gene expression was upregulated followed by recovery The plasma SOD activity, however, augmented consistently following virus injection Moreover, plasma AKP activity first lowered and then elevated gradually to the highest level at 24 h post virus injection Scallops challenged by AVNV at 17 degrees C neither developed notable disease nor showed obvious responses that could be associated with the virus infection. While the results suggested a correlation between the elevated seawater temperature and the AVNV infection associated C farreri mortalities, they also indicated that the viral infection provoked multiple physiological and immune responses in the host scallops (C) 2010 Elsevier Ltd All rights reserved

栉孔扇贝抗氧化酶基因的克隆与表达分析

扇贝是我国海水养殖的重要品种，但自1994年以来，养殖扇贝陆续爆发的大规模死亡，不但造成了巨大的经济损失，而且直接威胁到现有产业的生存和发展。扇贝病害的不断爆发以及病因的多样性迫切要求制定新的疾病防治措施和开发新型的抗菌物质。因此，深入研究扇贝免疫防御机制，探讨提高机体抗病力的有效途径和方法，改良种质和培育抗病品系，无疑是解决目前困扰扇贝养殖业健康可持续发展的必经之路。 抗氧化酶可以清除活性氧，是维持机体内氧环境平衡，抵抗外界环境影响的重要免疫因子。本研究采用大规模 EST 测序方法和同源克隆的方法，结合 cDNA 末端快速扩增（RACE）技术，从栉孔扇贝中克隆到了超氧化物岐化酶(Superoxide dismutase，SOD)、过氧化氢酶(Catalase，CAT)、谷胱甘肽过氧化物酶（Glutathione peroxidase，GPX）等抗氧化酶基因的全长 cDNA序列，并对其基因结构进行了分析。同时，用实时定量PCR方法对这三个基因在健康扇贝血淋巴细胞、肾、鳃、肌肉、性腺等组织和在分别用鳗弧菌，溶壁微球菌和假丝酵母处理扇贝后不同时间段的表达差异情况进行了研究。 超氧化物歧化酶基因CfSOD的cDNA 全长为1022 bp，其中开放阅读框（Open Reading Frame, ORF）含有 459 bp，编码 153个氨基酸残基，无信号肽，为胞内蛋白。经BLASTP分析发现，CfSOD与其它动物具有较高的同源性。CfSOD中存在两个Cu/Zn-SOD的签名序列；另外Cu结合必须氨基酸(His-45，-47，-62 和-119)和Zn结合必须氨基酸(His-62，-70，-79和Asp-82)在CfSOD中保守。实时定量PCR 检测发现，CfSOD在鳃、血细胞和肾中有较高的表达。在鳗弧菌和溶壁微球菌刺激后，CfSOD的相对表达量逐渐下降，然后分别在32小时和16小时的时候恢复到刺激前的表达水平。在假丝酵母刺激后，CfSOD的mRNA表达没有显著差异。 栉孔扇贝过氧化氢酶基因CfCAT的cDNA全长为3146 bp，其中开放阅读框含有1521bp，编码507个氨基酸残基，无信号肽，为胞内蛋白。经BLASTP分析发现，CfCAT与其它动物具有较高的同源性。 CfCAT中存在过氧化氢酶近端活性位点和过氧化氢酶近端血红素配体签名序列，另外存在两个糖基化位点 NFS和 NFT，同时在CfCAT 的C末端存在过氧化物酶体定位信号AQL，为典型过氧化氢酶。实时定量PCR 检测发现，健康的扇贝中CfCAT在鳃和性腺中有较高的表达。CfCAT基因在鳗弧菌刺后表达升高，在4小时达到最高，约是刺激前表达量的6.8倍（P<0.05），后随着时间的变化而逐渐下降。在8小时表达量达到为刺激前表达量的1.3倍（P<0.05），在16和32小时略高于刺激前的水平。在溶壁微球菌刺激后CfCAT基因表达量也呈上升趋势，在刺激后4小时达到刺激前表达量的约2倍，然后有所下降，在16 小时又上升到刺激前表达量的2.9倍。CfCAT基因在假丝酵母刺激后的表达略有升高，4小时约是刺激前的1.2倍（P<0.05），在其他时间段变化不明显。 栉孔扇贝谷胱甘肽过氧化物酶基因CfGPX的cDNA 全长为1290 bp，其中开放阅读框含有705bp，编码235个氨基酸残基，有一个24核苷酸的信号肽序列。经BLASTP 分析发现，CfSOD与其它动物具有较高的同源性。CfGPX中发现谷胱甘肽过氧化物酶活性位点的签名序列， 另外发现硒半胱氨酸和硒半胱氨酸插入序列，为含硒型谷胱甘肽过氧化物酶。实时定量PCR 检测发现，未经处理的扇贝中CfGPX在性腺、肌肉、血和肾中有较高的表达。CfGPX基因在鳗弧菌刺后表达量快速上升，在6 小时的时候表达量达到最高，为刺激前的4.0倍（P>0.05），后随着时间的变化而逐渐下降。在溶壁微球菌刺激后CfGPX在前6小时表达略有降低，在6小时的时候表达量为刺激前的0.5倍（P<0.05），后随着时间的变化而逐渐升高。在16小时的时候表达量为刺激前的2.1倍（P<0.05）。在假丝酵母刺激后， CfGPX的表达量略有下降在8小时的时候表达量为刺激前的0.8倍（P<0.05）。 实验证明栉孔扇贝的超氧化物歧化酶基因CfSOD，过氧化氢酶基因CfCAT，谷胱甘肽过氧化物酶基因CfGPX基因在机体抵抗外界微生物刺激中起到了重要的作用。

Concentrations of Cadmium and Zinc in Seawater of Bohai Bay and Their Effects on Biomarker Responses in the Bivalve Chlamys farreri

Both in-field chemical investigation and in the laboratory toxic tests were carried out to systematically understand the pollution status of cadmium (Cd) and zinc (Zn) in Bohai Bay. Samples collected from surface seawater were determined to describe the distributions of Cd and Zn in Bohai Bay. The average values in our study of Cd and Zn were 0.15 mu g/L and 19.68 mu g/L, respectively. Both of them were lower than the first class limit of seawater quality standard in China. In the laboratory, antioxidant enzymes [SOD (Cu/Zn-SOD, Mn-SOD), CAT], lipid peroxidation (MDA), phase I and phase II enzymes (CYP4501A and GST) were investigated in the bivalves Chlamys farreri exposed to Cd and Zn at the concentration levels of Bohai Bay seawater, which were obtained from our in-field investigation. The reduced SOD, CAT, and EROD (7-ethoxyresorufin-O-deethylase) activities (with the inhibitory rate of 16.8%, 31.5%, and 51.6%, respectively) in Cd treatment were observed and resulted in obvious lipid peroxidation damage. However, treatment of Zn showed elevations in SOD and GST by 13.3% and 29.9%, respectively, and with no influence on lipid peroxidation. In summary, seawater quality in Bohai Bay seawater was ranked as good in general, but it seemed that Cd might possess a potential environmental risk by effecting pro-oxidant/antioxidant balance and phase I detoxification in C. farreri.

On-line voltammetric monitoring of dissolved Cu and Ni in the Gulf of Cadiz, south-west Spain

Geochemical processes in estuarine and coastal waters often occur on temporally and spatially small scales, resulting in variability of metal speciation and dissolved concentrations. Thus, surveys, which are aimed to improve our understanding of metal behaviour in such systems, benefit from high-resolution, interactive sampling campaigns. The present paper discusses a high-resolution approach to coastal monitoring, with the application of an automated voltammetric metal analyser for on-line measurements of dissolved trace metals in the Gulf of Cadiz, south-west Spain. This coastal sea receives metal-rich inputs from a metalliferous mining area, mainly via the Huelva estuary. On-line measurements of dissolved Cu, Zn, Ni and Co were carried out on-board ship during an eight-day sampling campaign in the study area in June 1997. A pumping system operated continuously underway and provided sampled water from a depth of ca. 4 m. Total dissolved metal concentrations measured on-line in the Gulf of Cadiz ranged between <5 nM Cu (<3 nM Ni) ca. 50 km off-shore and 60–90 nM Cu (5–13 nM Ni) in the vicinity of the Huelva estuary. The survey revealed steep gradients and strong tidal variability in the dissolved metal plume extending from the Huelva estuary into the Gulf of Cadiz. Further on-line measurements were carried out with the automatic metal monitor from the bank of the Odiel estuary over a full tidal cycle, at dissolved metal concentrations in the μM range. The application confirmed the suitability of the automated metal monitor for coastal sampling, and demonstrated its adaptability to a wide range of environmental conditions in the dynamic waters of estuaries and coastal seas. The near-real time acquisition of dissolved metal concentrations at high resolution enabled an interactive sampling campaign and therefore the close investigation of tidal variability in the development of the Huelva estuary metal plume.

Thermodynamics of the displacive mechanism of alpha(1) transformation in a beta

Thermodynamics of the displacive mechanism of plate-shaped phase alpha(1) was analyzed in beta'Cu-Zn alloys. It was proposed that the displacive transformation of the alpha(1) plate took place in the solute-depleted region formed in the parent phase during the incubation period. The thermodynamic analysis indicated that the driving force of alpha(1) transformation, Delta G, increased with the reduction of x(d), the solute concentration of the depleted region. And, Delta G could overcome-the transformation barrier with solute depletion to a certain degree. In addition, x(d) was higher than the equilibrium concentration in the phase diagram. Therefore, the shear formation of alpha(1) plate in the solute-depleted region was thermodynamically supported.