Cloning of cytoplasmic heat shock protein 90 (FcHSP90) from Fenneropenaeus chinensis and its expression response to heat shock and hypoxia

Heat shock protein 90 (HSP90) works as a multi-functional chaperone and is involved in the regulation of many essential cellular pathways. In this study, we have identified a full-length complementary DNA (cDNA) of HSP90 (FcHSP90) from Chinese shrimp Fenneropenaeus chinensis. FcHSP90 full-length cDNA comprised 2,552 bp, including a 2,181-bp open reading frame encoding 726 amino acids. Both homology analyses using alignment with previously identified HSP90 and a phylogeny tree indicated that FcHSP90 was a cytoplasmic HSP90. Real-time reverse transcription polymerase chain reaction analysis revealed that FcHSP90 was ubiquitously expressed in all the examined tissues but with highest levels in ovary of F. chinensis. FcHSP90 mRNA levels were sensitively induced by heat shock (from 25A degrees C to 35A degrees C) and reached the maximum at 6 h during heat shock treatment. Under hypoxia conditions, FcHSP90 mRNA levels, in both hemocytes and gill, were induced at 2 h and depressed at 8 h during hypoxia stress. The assessment of FcHSP90 mRNA levels under heat shock and hypoxia stresses indicated that the transcription of FcHSP90 was very sensitive to heat shock and hypoxia, so we deduced that FcHSP90 might play very important roles for shrimp to cope with environmental stress.

Photosynthetic parameters of sexually different parts of Porphyra katadai var. hemiphylla (Bangiales, Rhodophyta) during dehydration and re-hydration

Physiological data from extreme habitat organisms during stresses are vital information for comprehending their survival. The intertidal seaweeds are exposed to a combination of environmental stresses, the most influential one being regular dehydration and re-hydration. Porphyra katadai var. hemiphylla is a unique intertidal macroalga species with two longitudinally separated, color distinct, sexually different parts. In this study, the photosynthetic performance of both PSI and PSII of the two sexually different parts of P. katadai thalli during dehydration and re-hydration was investigated. Under low-grade dehydration the variation of photosystems of male and female parts of P. katadai were similar. However, after the absolute water content reached 42%, the PSI of the female parts was nearly shut down while that of the male parts still coordinated well and worked properly with PSII. Furthermore, after re-hydration with a better conditioned PSI, the dehydrated male parts were able to restore photosynthesis within 1 h, while the female parts did not. It is concluded that in P. katadai the susceptibility of photosynthesis to dehydration depends on the accommodative ability of PSI. The relatively lower content of phycobiliprotein in male parts may be the cause for a stronger PSI after severe dehydration.

Cloning and characterization of beta-carotene ketolase gene promoter in Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis accumulates a highly valuable ketocarotenoid, astaxanthin, under various environmental stresses. beta-carotene ketolase (BKT) plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, an approximate 700 bp 5'-flanking region of the bkt gene containing a putative promoter was cloned through walking upstream. The results of the sequence analysis showed that this bkt 5'-flanking region might have cis-acting elements such as sterol regulatory element (SRE-1)-like motifs, the C-repeat/dehydration responsive element (DRE) and al-3 proximal element (APE)-like motifs, except for typical TATA and CCAAT boxes. The results of the P-galactosidase assay and the transient expression of lacZ driven by a series of sequential deletions revealed that a minimal promoter-like region might exist from -630 to -408 bp, and the highest promoter activity was observed to span the positions from -630 to -308 bp. The results of the site-directed mutagenesis of a C-repeat/DRE and two APE-like motifs in a promoter-like region (-630 to -308 bp) suggested that two APE-like motifs might be essential for transcriptional control of the bkt gene.

cDNA cloning and mRNA expression of heat shock protein 90 gene in the haemocytes of Zhikong scallop Chlamys farreri

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. The full-length cDNA of Zhikong scallop Chlamysfarreri HSP90 (designated CfHSP90) was cloned by EST and rapid RACE techniques. It was of 2710 bp, including an open reading frame (ORF) of 2181 bp encoding a polypeptide of 726 amino acids with all the five HSP90 family signatures. BLAST analysis revealed that the CfHSP90 gene shared high similarity with other known HSP90 genes. Fluorescent real-time quantitative RT-PCR was used to examine the expression pattern of CfHSP90 mRNA in haemocytes of scallops exposed to Cd2+, Pb2+ and Cu2+ for 10 and 20 days, respectively. All the three heavy metals could induce CfHSP90 expression. There was a clear dose-dependent expression pattern of CfHSP90 after heavy metals exposure for 10 days or 20 days. Different concentrations of the same metal resulted in different effects on CfHSP90 expression. The results indicated that CfHSP90 responded to various heavy metal stresses with a dose-dependent expression pattern as well as exposure time effect, and could be used as a molecular biomarker in a heavy metal polluted environment. (c) 2007 Elsevier Inc. All rights reserved.

cDNA cloning, characterization and expression analysis of the antioxidant enzyme gene, catalase, of Chinese shrimp Fenneropenaeus chinensis

Catalase is an important antioxidant protein that protects organisms against various oxidative stresses by eliminating hydrogen peroxide. The full-length catalase cDNA of Chinese shrimp Fenneropenaeus chinensis was cloned from the hepatopancreas using degenerate primers by the method of 3' and 5' rapid amplification of cDNA ends PCR. The cDNA sequence consists of 1892 bp with a 1560 bp open reading frame, encoding 520 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases. The sequence includes the catalytic residues His71, Asn144, and Tyr354. The molecular mass of the predicted protein is 58824.04 Da with an estimated pl of 6.63. Sequence comparison showed that the deduced amino acid sequence of F. chinensis catalase shares 96%, 73%, 71% and 70% identity with that of Pacific white shrimp Litopenaeus vannamei, Abalone Haliotis discus hannai, Zhikong scallop Chlamys farreri and Human Homo sapiens, respectively. Catalase transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill. by real-time PCR. The variation of catalase mRNA transcripts in hemocytes and hepatopancreas was also quantified by real-time PCR and the result indicated that the catalase showed up-regulated expression trends in hemocytes at 14 h and in hepatopancreas at 37 h after injection with white spot syndrome virus (WSSV). (c) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning, expression of a peroxiredoxin gene in Chinese shrimp Fenneropenaeus chinensis and the antioxidant activity of its recombinant protein

Peroxiredoxin (Prx) is known to be an antioxidant protein that protects the organisms against various oxidative stresses and functions in intracellular signal transduction. A Prx gene was firstly isolated in the crustacean, Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA consists of 942 bp with a 594 bp open reading frame, encoding 198 amino acids. The molecular mass of the deduced amino acid is 22041.17 Da with an estimated pI of 5.17. Sequence comparison showed that Prx of F. chinensis shares 76%, 73% and 72% identity with that of Aedes aegypti, Branchiostoma belcheri tsingtaunese and Drosophila melanogaster, respectively. Northern blot analysis revealed the presence of Prx transcripts of F chinensis in all tissues examined. Real-time PCR analysis indicated that the Prx showed different expression profiles in shrimp hemocytes and hepatopancreas after artificial infection with Vibrio anguillarum. In addition, a fusion protein containing Prx was produced in vitro. LC-ESI-MS analysis showed that four peptide fragments of the recombinant protein were identical to the corresponding sequence of F. chinensis Prx. And the purified recombinant proteins were shown to reduce H2O2 in the presence of dithiothreitol. (c) 2007 Elsevier Ltd. All rights reserved.

Identification and molecular analysis of a stress-inducible Hsp70 from Sciaenops ocellatus

Hsp70 proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In this study, an Hsp70 homologue (SoHsp70) was identified from red drum Sciaenops ocellatus and analyzed at molecular level. The open reading frame of SoHsp70 is 1920 bp and intronless, with a 5'-untranslated region (UTR) of 399 bp and a 3'-UTR of 241 bp. The deduced amino acid sequence of SoHsp70 shares 84-92% overall identities with the Hsp70s of a number of fish species. In silico analysis identified in SoHsp70 three conserved Hsp70 domains involved in nucleotide and substrate binding. The coding sequence of SoHsp70 was subcloned into Escherichia coli, from which recombinant SoHsp70 was purified and, upon ATPase assay, found to exhibit apparent ATPase activity. Expressional analysis showed that constitutive expression of SoHsp70 was detectable in heart, liver, spleen, kidney, brain, blood, and gill. Experimental challenges with poly(I:C) and bacterial pathogens of Gram-positive and Gram-negative nature induced SoHsp70 expression in kidney to different levels. Stress-responsive analysis of SoHsp70 expression in primary cultures of red drum hepatocytes showed that acute heat shock treatment elicited a rapid induction of SoHsp70 expression which appeared after 10 min and 30 min of treatment. Exposure of hepatocytes separately to iron, copper, mercury, and hydrogen peroxide significantly unregulated SoHsp70 expression in time-dependent manners. Vaccination of red drum with a Streptococcus iniae bacterin was also found to induce SoHsp70 expression. Furthermore, recombinant SoHsp70 enhanced the immunoprotective effect of a subunit vaccine. Taken together, these results suggest that SoHsp70 is a stress-inducible protein that is likely to play a role in immunity and in coping with environmental and biological stresses. (C) 2010 Elsevier Ltd. All rights reserved.

Simulating a typhoon storm surge in the East Sea of China using a coupled model

A coupled numerical model with a 2' x 2' resolution grid has been developed and used to simulate five typical typhoon storm surges (5612, 7413, 7910, 8114, and 9711) in the East Sea of China. Three main driving forces have been considered in this coupled model: wave radiation stress, combined wave-current bottom shear stress and wave-state-dependent surface wind stress. This model has then been compared with in situ measurements of the storm set-up. The effect of different driving force components on the total storm surge has also been investigated. This study has found that the coupled model with high resolution is capable of simulating the five typical typhoons better than the uncoupled models, and that the wave-dependent surface wind stress plays an important role in typhoon storm surge-wave coupling in this area and can increase the storm set-up by 1 m. The study of the five typhoon cases has shown that the general coupling effects could increase storm set-up by 20-32%. Thus, it is suggested that to predict typhoon storm surges in the East Sea of China, a storm surge-wave coupled model be adopted. (C) 2008 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

中国明对虾在胁迫条件下肝胰脏的差异蛋白质组学研究

对虾养殖业的发展面临着病害严重和环境恶化的严重挑战。对虾细菌和病毒性疾病的频发已给对虾的养殖业造成了严重损失。养殖环境恶化造成的环境胁迫，不但影响对虾的生长性状，而且导致对虾的抵抗力下降，更容易引发病害的发生。研究对虾在外界胁迫（环境胁迫、病原感染）后蛋白表达模式的变化有助于弄清对虾的抗病抗逆机理，为抗病抗逆对虾的培育提供理论依据。 肝胰脏是对虾的重要器官，参与各种重要的生理活动包括消化、吸收、储存和代谢，并且在启动对虾免疫反应起重要作用，其蛋白质表达的变化与对虾的健康状况密切相关。 本研究运用蛋白质组学技术平台（双向电泳、串联质谱、数据库搜索），对中国明对虾肝胰脏蛋白进行研究，获得了一些重要进展，结果如下： 1.初步构建了覆盖范围广、分辨率高、重复性好的中国明对虾肝胰脏双向电泳参考图谱，并利用质谱分析和多种数据库搜索方法成功鉴定了122个（68种）蛋白质点，包括14个（9种）能量代谢蛋白、15个（9种）氨基酸代谢蛋白、10个（5种）碳水化合物代谢蛋白、2个（1种）多聚糖代谢蛋白、11个（5种）辅酶和维生素代谢蛋白、1个（1种）脂类代谢蛋白、5个（3种）核酸代谢蛋白、7个（3种）消化酶、3个（1种）细胞骨架蛋白、14个（8种）免疫相关蛋白、9个（6种）抗氧化蛋白、19个（7种）伴侣蛋白、4个（3种）信号转导蛋白、3个（3种）翻译功能蛋白和5个（4种）未分组蛋白。 2.在参考图谱的基础上，研究了缺氧胁迫下中国明对虾肝胰脏蛋白质表达模式的变化，成功鉴定了52个（15个上调、37个下调）差异表达的蛋白，包括11个能量产生相关蛋白、13个免疫相关蛋白、11个代谢相关蛋白、6个抗氧化蛋白、7个分子伴侣、2个细胞骨架蛋白和2个未分类蛋白，利用实时定量PCR技术，研究了缺氧胁迫下10个差异蛋白的基因在转录水平上的变化；同时研究了金属镉胁迫后中国明对虾肝胰脏蛋白质表达模式的变化，成功鉴定了29个（4个上调、25个下调）差异表达的蛋白，包括9个能量产生相关蛋白、3个免疫相关蛋白、9个代谢相关蛋白、5个抗氧化蛋白、2个分子伴侣和1个未分类蛋白。 3.分别进行了灭活鳗弧菌和WSSV感染后中国明对虾肝胰脏的差异蛋白质组学研究。灭活鳗弧菌刺激后，45个差异表达的蛋白质（4个上调、41个下调）被成功鉴定出，包括6个能量产生相关蛋白、4个免疫相关蛋白、22个代谢相关蛋白、4个抗氧化蛋白、7个分子伴侣、1个翻译相关蛋白和2个未分类蛋白；WSSV感染后， 48个差异表达的蛋白质（11个上调、37个下调）被成功鉴定出，包括7个能量产生相关蛋白、9个免疫相关蛋白、20个代谢相关蛋白、5个抗氧化蛋白和7个分子伴侣。 通过对上述实验数据的分析，初步探讨了中国明对虾对虾对环境胁迫和病原感染的应答机理。为下一步筛选中国明对虾环境胁迫及免疫应答关键蛋白并研究其功能奠定了基础。

Effects of acute temperature or salinity stress on the immune response in sea cucumber, Apostichopus japonicus

Invertebrates are increasingly raised in mariculture, where it is important to monitor immune function and to minimize stresses that could suppress immunity. The activities of phagocytosis, superoxide dismutase (SOD), catalase (CAT), myeloperoxiclase (MPO), and lysozyme (LSZ) were measured to evaluate the immune capacities of the sea cucumber, Apostichopus japonicus, to acute temperature changes (from 12 degrees C to 0 degrees C, 8 degrees C, 16 degrees C, 24 degrees C, and 32 degrees C for 72 h) and salinity changes (from 30 parts per thousand to 20 parts per thousand, 25 parts per thousand, and 35 parts per thousand for 72 h) in the laboratory. Phagocytosis was significantly affected by temperature increases in 3 h, and by salinity (25 parts per thousand and 35 parts per thousand) changes in 1 h. SOD activities decreased significantly in 0.5 h to 6 h samples at 24 degrees C. At 32 degrees C, SOD activities decreased significantly in 0.5 h and 1 h exposures, and obviously increased for 12 h exposure. CAT activities decreased significantly at 24 degrees C for 0.5 h exposure, and increased significantly at 32 degrees C in 3 h to 12 h exposures. Activities of MPO increased significantly at 0 degrees C in 0.5 h to 6 In exposures and at 8 degrees C for 1 h. By contrast, activities of MPO decreased significantly in 24 degrees C and 32 degrees C treatments. In elevated-temperature treatments, activities of LSZ increased significantly except at 32 degrees C for 6 h to 12 h exposures. SOD activity was significantly affected by salinity change. CAT activity decreased significantly after only 1 h exposure to salinity of 20 parts per thousand.. Activities of MPO and LSZ showed that A. japonicus tolerates limited salinity stress. High-temperature stress had a much greater effect on the immune capacities of A. japonicus than did low-temperature and salinity stresses. Crown Copyright (C) 2008 Published by Elsevier Inc. All rights reserved.

塑料压力管道热板焊接接头瞬时应力有限元分析

基于热粘弹性积分型本构关系,考虑材料性能依赖于温度变化及相变潜热的影响,利用有限元软件ANSYS热-力耦合及载荷步功能模拟结晶型高密度聚乙烯塑料压力管道热板焊接过程。并对焊接接头的应力分布进行有限元分析,得到了环向、轴向及径向瞬态应力分布规律。采用盲孔法和锯切法测量焊后残余应力,实测结果与数值分析基本吻合。

高密度聚乙烯塑料压力管道热板焊接头应力分布有限元分析

基于热粘弹性积分型本构关系,考虑材料性能依赖于温度变化及相变潜热的影响,利用AN SYS热-力耦合及载荷步功能模拟结晶型高密度聚乙烯(HDPE)塑料压力管道热板焊接过程,并对焊接接头的应力分布进行有限元分析,得到了环向、轴向以及径向瞬态应力分布的基本规律。采用盲孔法和锯切法测量焊后残余应力,实测结果与数值分析基本符合

数字图像处理中多窗口下的自适应中值滤波

根据心理物理学、神经生理学、认知神经学等学科在视觉认知领域的部分研究成果，结合机器视觉、图像处理领域在图像增强方面已经提出的一些方法，提出了结合先验知识的多窗口结构下的分块中值滤波方法，在每一个窗口内单独进行处理与分析，突出了视觉处理目的，减少了运算量， 节省数据存储空间，达到了令人满意的滤波效果，能够在原始图像比较复杂的情况下，较好地对其进行预处理，可以改善、提高后期图像处理过程，如图像分割、图像分析的正确性和有效性。

不同基板预热温度对激光金属沉积成形过程热应力影响的研究

研究不同基板预热温度对激光金属沉积成形过程热应力的影响,对于降低成形过程的热应力,抑制成形过程裂缝的产生,减小成形过程试样和基板的翘曲变形具有非常重要的意义。根据有限元分析中的"单元生死"技术,编程建立了基板预热对激光金属沉积成形过程热应力影响的三维多道多层数值模拟模型,详细分析了基板未预热和分别预热到200℃、300℃、400℃、500℃、600℃时对沉积成形过程VonMise’s热应力、X方向、Y方向以及Z方向热应力的影响。在与模拟过程相同的参数下,利用镍基合金粉末分别在基板未预热和分别预热到300℃、400℃、500℃、600℃时进行了成形试验,试验的结果跟数值模拟结果吻合较好。

小湾电站坝基岩体卸荷工程地质力学研究

Dam is the key main works in the construction of water power. The success or failure of the construction of the dam mainly depends on the stability of the dam foundation. The double curvature arch dam-XiaoWan Dam is the highest one among the dams with the same type in the world, and the water thrust acted on it reaches ton, so the rock bearing capacity of dam foundation becomes more important. Because of the high and steep valley-side slope, the large scale of excavation and the complex body type of excavation, it is prominent that the problem of stress release of the rock mass in dam foundation. More great attentions should be paid for the stability and the degraded of rock properties of rock mass induced by the stress release. In this paper, the phenomena of stress release of rock mass in XiaoWan Dam foundation and its mechanisms were analyzed based on the collection of data, the detailed field engineering investigations, measurement of the rock mass and the 2D numerical calculations. The rock mass under the foundation is weak-weathered to intact, the quality of which is good. After excavation of the foundation, the rock mass near the slope surface occurred extend, stretch and stick-slip along original textures till the new fracture surface formed. Then platy structure of the rock mass takes on. The rock mass in the dam foundation occur resilience due to stress release towards free faces with the characteristics of time effect and localized deformation. In-situ measurements show that the rock mass near the surface are degraded. The stress release induced by excavation is a process of the interaction between engineering structures and geologic body. The stress release of rock mass in dam foundation is related to the changed degree of geometrical conditions. The rock near excavation surface failed nearly under uniaxial stresses. The bending-breaking mechanism of plate girder can interpret the failure model of the rock mass with platy structure in dam foundation slope. In essence, the stress release is the change of stress field including the change of directions and magnitudes of stress induced by excavation, which can induce the comedown of the safety margin. In this paper, the inducing conditions of stress release were calculated by numerical analyses. Moreover, from the point of view that the change of stress field, the coefficient of K, i.e. the variable load coefficient was proposed. Then the law of the change of it is interpreted. The distributional characteristics of fracture zone were expressed by the coefficient. The stress release of hard rock has the characteristic of localization. The measuring technique of sound wave can not reflect the small cracks in this kind of rock mass due to stress release. So, the spectral analysis method was proposed. At the same time, the application foreground in engineering of the Stockwell Time-Frequency- Spectrum method was discussed with a view to the limitation of it.