Trends of Superoxide Dismutase and Soluble Protein of Aquatic Plants in Lakes of Different Trophic Levels in the Middle and Lower Reaches of the Yangtze River, China

A limnological study was carried out to determine the responses of superoxide dismutase (SOD) activities and soluble protein (SP) contents of 11 common aquatic plants to eutrophication stress. Field investigation in 12 lakes in the middle and lower reaches of the Yangtze River was carried out from March to September 2004. Our results indicated that non-submersed (emergent and floating-leafed) plants and submersed plants showed different responses to eutrophication stress. Both SOD activities of the non-submersed and submersed plants were negatively correlated with their SP contents (P < 0.000 1). SP contents of non-submersed plants were significantly correlated with all nitrogen variables in the water (P < 0.05), whereas SP contents of submersed plants were only significantly correlated with carbon variables as well as ammonium and Secchi depth (SD) in water (P < 0.05). Only SOD activities of submersed plants were decreased with decline of SD in water (P < 0.001). Our results indicate that the decline of SOD activities of submersed plants were mainly caused by light limitation, this showed a coincidence with the decline of macrophytes in eutrophic lakes, which might imply that the antioxidant system of the submersed plants were impaired under eutrophication stress.

Molecular cloning and characterization of the copper/zinc and manganese superoxide dismutase genes from the human parasite Clonorchis sinensis

A copper/zinc superoxide dismutase (Cu/ZnSOD) gene and a manganese superoxide dismutase (MnSOD) gene of the human parasite Clonorchis sinensis have been cloned and their gene products functionally characterized. Genes Cu/ZnSOD and MnSOD encode proteins of 16 kDa and 25.4 kDa, respectively. The deduced amino acid sequences of the two genes contained highly conserved residues required for activity and secondary structure formation of Cu/ZnSOD and MnSOD, respectively, and show up to 73.7% and 75.4% identities with their counterparts in other animals. The genomic DNA sequence analysis of Cu/ZnSOD gene revealed this as an intronless gene. Inhibitor studies with purified recombinant Cu/ ZnSOD and MnSOD, both of which were functionally expressed in Escherichia coli, confirmed that they are copper/zinc and manganese-containing SOD, respectively. Immunoblots showed that both C. sinensis Cu/ZnSOD and MnSOD should be antigenic for humans, and both, especially the C. sinensis MnSOD, exhibit extensive cross-reactions with sera of patients infected by other trematodes or cestodes. RT-PCR and SOD activity staining of parasite lysates indicate that there are no significant differences in mRNA level or SOD activity for both species of SOD, indicating cytosolic Cu/ZnSOD and MnSOD might play a comparatively important role in the C. sinensis antioxidant system.

Toxicity of cypermethrin on growth, pigments, and superoxide dismutase of Scenedesmus obliquus

Cypermethrin is a synthetic pyrethroid that is particularly toxic to crustaceans. It is therefore applied as a chemotherapeutant in farms for the treatment of pests. The effective concentrations of cypermethrin on the inhibition of Scenedesmus ohliquus growth at 96h (96h EC50) were determined to be 50, 100, 150, 200, and 250mg/L. Algal growth, pigment fractions, and the activity of superoxide dismutase (SOD) in the algal cells were measured in the exponential phase after exposure to cypermethrin. The results show that higher concentration of cypermethrin is inhibitory for growth and other metabolic activities and the 96h EC50 of cypermethrin to S. ohliquus is 112 +/- 9 mg/L; the potential application of SOD activity in S. ohliquus as a sensitive biomarker for cypermethrin exposure is also discussed. (C) 2004 Elsevier Inc. All rights reserved.

Effects of microcystins on the growth and the activity of superoxide dismutase and peroxidase of rape (Brassica napus L.) and rice (Oryza sativa L.)

Microcystins are naturally occurring hepatotoxic cyclic heptapeptides produced by some toxic freshwater cyanobacterial species. In this study, crude extract of toxic cyanobacterial blooms from Dianchi Lake in southwestern China was used to determine the effects of microcystins on rape (Brassica napus L.) and rice (Oryza sativa L.). Experiments were carried out on a range of doses of the extract (equivalent to 0, 0.024, 0.12, 0.6 and 3 mug MC-LR/ml). Investigations showed that exposure to microcystins inhibited the growth and development of both rice and rape seedlings, however, microcystins had more powerful inhibition effect on rape than rice in germination percentage of seeds and seedling height. Microcystins significantly inhibited the elongation of primary roots of rape and rice seedlings. Determination of the activities of peroxidase and superoxide dismutase demonstrated that microcystin stress was manifested as an oxidative stress. Using ELISA, microcystins were examined from the extract of exposed rape and rice seedlings, indicating that consumption of edible plants exposed to microcystins via irrigation route may have health risks. Significantly different levels of recovered microcystins between exposed rice and rape seedlings Suggested that there might be different tolerant mechanisms toward microcystins. (C) 2004 Elsevier Ltd. All rights reserved.

EFFECTS OF LOW-TEMPERATURE AND PHYSIOLOGICAL AGE ON SUPEROXIDE-DISMUTASE IN WATER HYACINTH (EICHHORNIA-CRASSIPES SOLMS)

Superoxide dismutase activity in water hyacinth leaves was not sensitive to small changes in environmental pH, but declined markedly with greater pH changes. KCN inhibited superoxide dismutase activity, suggesting that the enzyme was mainly composed of the Cu-Zn form. Low temperature (2-degrees-C) treatment caused a decline in superoxide dismutase activity. This effect became more pronounced as the treatment time was prolonged. Furthermore, the decline was much more significant than reductions of glucose-6-phosphate dehydrogenase activity or respiration under comparable conditions. With increasing physiological age, superoxide dismutase activity declined and was significantly lower in old than in young leaves. Therefore, superoxide dismutase activity might be employed as one of physiological parameters in studying leaf senescence.

INTERACTIONS OF RARE-EARTH LENS WITH BOVINE BLOOD CU(ZN)-SUPEROXIDE DISMUTASE

The interactions of lanthanium trichloride and terbium trichloride with bovine blood Cu (Zn)-superoxide dismutase [Cu(Zn)-SOD] in the aqueous solution of hexamethylenetetrarnine buffer (pH = 6.3) have been studied by using fluorescece, CD and ESR spectra. The results indicated that rare earth ions were coordinated to the carboxyl groups of acidic amino acid residues which were far from active center of the Cu(Zn)-SOD molecule and only lightly disturbed the secondary structure of the enzyme protien, and made the coordination structure of enzyme-bound CU2+ come from the rhombchedron to the axial shape at 77 K and the activity of Cu(Zn)-SOD enzyme was not nearly changed at room temperature.

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 cDNA cloning and mRNA expression of cytoplasmic Cu, Zn superoxide dismutase (SOD) gene in scallop Chlamys farreri

Cu, Zn superoxide dismutases (SODs) are rnetalloenzymes that represent one important line of defence against reactive oxygen species (ROS). A cytoplasmic Cu. Zn SOD cDNA sequence was cloned from scallop Chlamys farreri by the homology-based cloning technique. The full-length cDNA of scallop cytoplasmic Cu, Zn SOD (designated CfSOD) was 1022 bp with a 459 bp open reading frame encoding a polypeptide of 153 amino acids. The predicted amino acid sequence of CfSOD shared high identity with cytoplasmic Cu. Zn SOD in molluscs, insects, mammals and other animals, such as cytoplasmic Cu, Zn SOD in oyster Crassostrea sostrea gigas (CAD42722), mosquito Aedes aegypti (ABF18094), and cow Bos taurus (XP_584414). A quantitative reverse transcriptase real-time PCR (qRT-PCR) assay was developed to assess the mRNA expression of CfSOD in different tissues and the temporal expression of CfSOD in scallop challenged with Listonella anguillarum, Micrococcus luteus and Candida lipolytica respectively. Higher-level mRNA expression of CfSOD was detected in the tissues of haemocytes, gill filaments and kidney. The expression of CfSOD dropped in the first 8-16 h and then recovered after challenge with L. anguillarum and M. litteus, but no change was induced by the C. lipolytica challenge. The results indicated that CfSOD was a constitutive and inducible acute-phase protein, and could play an important role in the immune responses against L. anguillarum and M. luteus infection. (C) 2007 Elsevier Ltd. 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.

果实成熟衰老与抗病性的调控机制研究

　　果实为开花植物所特有的发育器官，在种子的成熟和传播过程中发挥着重要作用。同时，肉质果实中含有丰富的营养物质，包括纤维素、维生素、抗氧化剂等，成为人们饮食的重要组成部分。由于果实的成熟衰老和抗病性直接影响果品的质量和市场价值，因此，研究果实成熟衰老和抗病性的调控机制具有重要的理论意义和应用前景。本文主要利用蛋白质组学的方法，探讨外源化学物质抑制果实成熟衰老和诱导抗病性的调控机制。 1. 硅对果实的抗病性诱导：用硅酸钠（1%）处理采后的甜樱桃果实，再接种褐腐病原菌（Molinilia fracticola），置于20C下，观测贮藏期间果实的发病率，并分析硅处理后诱导的主要蛋白质及调控机制。研究结果表明：硅酸钠处理可显著抑制贮藏期间褐腐病的发生，其抑病机理与硅诱导PR-蛋白的表达，提高果实的抗氧化水平，减轻由病原菌侵染造成的氧化胁迫相关。同时，硅处理还能保护细胞骨架结构，有利于增强果实对病原菌入侵的抵抗力。 2. 水杨酸对果实的抗病性诱导：用水杨酸（SA，2mM）在果园处理三种成熟度的甜樱桃果实，然后接种青霉病原菌（Penicillium expansum）观察其发病情况，并取样分析参与抗病性应答的主要蛋白质及调控机制。试验结果表明：SA处理能显著降低青霉病的发病率和抑制病斑扩展，而且SA对低成熟度甜樱桃果实的抗性诱导效果更好。在八成熟的果实中，有5个热激蛋白和4个脱氢酶蛋白被SA诱导，这些蛋白参与了糖酵解和三羧酸循环。抗氧化蛋白和PR蛋白主要参与较低成熟度果实的抗性应答，而热激蛋白和脱氢酶在较高成熟度果实的抗性应答中更明显，SA诱导的抗性与代谢途径相关。 　　3. 草酸对果实的抗性诱导：用5mM的草酸处理冬枣果实后，接种青霉菌（P. expansum），观察果实发病情况，测定果实相关的生理指标，分析参与果实抗性应答的主要蛋白质及调控机制。结果表明：草酸能明显延缓冬枣果实的衰老，提高果实对青霉菌的抗性。草酸处理能抑制果实乙烯的释放量和呼吸强度，延缓叶绿素的降解，减少乙醇积累。利用蛋白质组学的研究方法证实了在25个参与了草酸处理应答的蛋白中，胱硫醚-β-合酶结构域包含蛋白（CBB domain-containing protein）和3个与光合作用相关蛋白[二磷酸核酮糖羧化酶/加氧酶（Ribulose bisphosphate carboxylase/oxygenase activase, chloroplast precursor），二磷酸核酮糖羧化酶/加氧酶大亚基结合蛋白（RuBisCO large subunit-binding protein subunit beta, chloroplast precursor），植物光系统Ⅱ放氧复合蛋白2（PSII oxygen-evolving complex protein 2）]的表达量上调，乙醇脱氢酶的表达量出现下调。草酸处理还提高了与乙烯合成前体相关蛋白的表达，抑制了ACC合成酶的活性。草酸提高果实抗病的机制与延缓果实成熟衰老和保持果实抗性有关。 4. 果实衰老的调控机制：采用高氧（100%）和低氧（2-3%）处理苹果果实，观察果实衰老的进程，并基于蛋白质组学的研究方法，探讨苹果果实衰老与线粒体蛋白质组的关系。结果表明，在苹果衰老过程中有22个蛋白的表达量发生变化，这些蛋白主要参与了三羧酸循环，电子传递，碳代谢和胁迫应答。高氧处理能诱导氧化胁迫，加速了果实的衰老。质谱鉴定结果证明：在高氧胁迫下，超氧化物歧化酶（manganese superoxide dismutase，MnSOD）和线粒体外膜通道蛋白（porin) 的表达量降低，MnSOD的活性受到抑制，由此提高了线粒体中超氧阴离子的含量，增加了蛋白质的氧化损伤。 此外，高氧处理改变了porin的功能，导致了线粒体膜的透势发生变化，从而引起外膜损伤。由此阐明了活性氧在果实的成熟衰老调控中的重要作用。