Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine

Ferric uptake regulator (Fur) is a global transcription regulator that is ubiquitous to Gram-negative bacteria and regulates diverse biological processes, including iron uptake, cellular metabolism, stress response, and production of virulence determinants. As a result, for many pathogenic bacteria, Fur plays a crucial role in the course of infection and disease development. In this study, the fur gene was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased Japanese flounder cultured in a local farm. TSS Fur can partially complement the defective phenotype of an Escherichia coli fur mutant. A TSS fur null mutant, TFM, was constructed. Compared to TSS, TFM exhibits reduced growth ability, aberrant production of outer membrane proteins, decreased resistance against host serum bactericidal activity, impaired ability to disseminate in host blood and tissues, and drastic attenuation in overall bacterial virulence in a Japanese flounder infection model. When used as a live vaccine administered via the injection, immersion, and oral routes, TFM affords high levels of protection upon Japanese flounder against not only P.fluorescens infection but also Aeromonas hydrophila infection. Furthermore, a plasmid, pJAQ, was constructed, which expresses the coding element of the Vibrio harveyi antigen AgaV-DegQ. TFM harboring pJAQ can secret AgaV-DegQ into the extracellular milieu. Vaccination of Japanese flounder with live TFM/pJAQ elicited strong immunoprotection against both V. harveyi and A. hydrophila infections. (C) 2009 Elsevier Ltd. All rights reserved.

Expressed sequence tags from the zhikong scallop (Chlamys farreri): Discovery and annotation of host-defense genes

A high-quality cDNA library was constructed from whole body tissues of the zhikong scallop, Chlamys farreri, challenged by Listonella anguillarum. A total of 5720 clones were sequenced, yielding 5123 expressed sequence tags (ESTs). Among the 3326 unique genes identified, 2289 (69%) genes had no significant (E-value < 1e-5) matches to known sequences in public databases and 194 (6%) matched proteins of unknown functions. The remaining 843 (25%) genes that exhibited homology with genes of known functions, showed broad involvement in metabolic processes (31%), cell structure and motility (20%), gene and protein expression (12%), cell signaling and cell communication (8%), cell division (4%), and notably, 25% of those genes were related to immune function. They included stress response genes, complement-like genes, proteinase and proteinase inhibitors, immune recognition receptors and immune effectors. The EST collection obtained in this study provides a useful resource for gene discovery and especially for the identification of host-defense genes and systems in scallops and other molluscs. (C) 2009 Elsevier Ltd. All rights reserved.

中华卤虫滞育卵发育和重金属刺激相关蛋白质组学研究

卤虫(Artemia)是一种广温、耐高盐的小型甲壳动物，广泛分布于内陆盐湖和沿海盐田中。卤虫的无节幼体作为重要的蛋白优质饵料，被广泛的应用于水产养殖生产。卤虫具有特殊的生物学特性，是研究甲壳动物胚胎发育的良好的实验材料，同时也是一种研究动物抗逆机制的模式动物。卤虫有卵生和卵胎生两种繁殖后代的方式，当环境条件适宜时，卤虫倾向于采取卵胎生方式，即直接产生无节幼体；而在恶劣的环境条件下，卵生方式占主要地位，产生处于滞育状态的、具有复杂外壳的休眠卵。卤虫的滞育卵具有独特的生物学特性和特殊的生理生化特点。其发育停滞，细胞分裂停止，酶活力下降，代谢活动受到抑制并可耐受各种极端恶劣环境，如缺氧、低温、紫外线、干燥等。即使在最适的环境中滞育卵的孵化率也很低，只有受到某些特定的非生物信号的刺激才自能终止这种滞育状态，恢复生理代谢；当环境条件适宜时，能够继续发育孵化成无节幼体。因此，卤虫的滞育卵在卤虫的整个生活史中占有重要的地位。另一方面，卤虫是极端环境生物，能够抵抗各种恶劣环境胁迫刺激，因此是研究抗逆机理的良好的实验动物。 本论文利用蛋白质组学技术，研究了卤虫滞育卵及滞育卵发育过程中的蛋白质组表达情况，并研究了卤虫幼体在重金属刺激后蛋白表达的变化情况。得到如下结果： 建立了中华卤虫滞育卵可溶性总蛋白的双向凝胶电泳对照图谱。在pH 4–7、分子量10-100 kDa范围内，检测到约 233个蛋白点，并利用高效液相色谱-质谱联用（LC-ESI-MS/MS）技术鉴定了其中的48个丰度较大及感兴趣的蛋白点，根据这些蛋白的生物学功能进行分类，功能类别包括细胞防御蛋白、抗氧化蛋白、细胞骨架蛋白、代谢相关蛋白等。在卤虫滞育卵中共分离鉴定到6个分子量和等电点存在差异的小热休克蛋白p26的异构体，生物信息学分析表明该蛋白有三种不同的功能位点，分别是蛋白激酶C磷酸化位点，Casein 激酶II磷酸化位点及 N-myristoylation 位点。 采用低温脱水的方法对滞育卵进行激活刺激，并对活化卵和滞育卵蛋白表达图谱进行了对比分析。结果表明对卤虫滞育卵的激活刺激引起了其蛋白表达的明显变化。活化卵图谱中蛋白点总数比滞育卵中明显增多，特别是在pI<5.5范围内。约70个蛋白点在激活刺激后上调表达，包括部分只在激活卵中表达的蛋白；25个下调表达，包括部分只在滞育卵中表达的蛋白；其余约60％（占滞育卵蛋白点数目百分比）的蛋白点表达量基本恒定。热休克蛋白家族、抗氧化蛋白家族成员等蛋白变化明显，小热休克蛋白p26、小热休克蛋白ArHsp21蛋白以及过氧化物还原酶异构体在激活卵中特异表达。 活化卵孵化过程中不同发育时期的蛋白表达又呈现出不同的特点，分别在孵化后6h、12h、18h和24h的蛋白质组学图谱上检测到267、285、195和210个蛋白点。孵化后6h和12h休眠卵蛋白表达个数相对较多，与胚胎发育过程中的器官发生和剧烈的形态变化相适应；孵化后18h和24h休眠卵蛋白表达明显下降，部分蛋白的表达关闭，部分蛋白开始富集表达。 利用双向凝胶电泳技术分析了中华卤虫幼体受到急性硫酸铜刺激后的蛋白表达变化情况。通过图谱对比分析，检测到了5mM硫酸铜刺激24h后，卤虫幼体中14个差异表达的蛋白点。利用LC-ESI-MS/MS技术鉴定了其中的7个蛋白，其中3个蛋白上调表达，分别是热休克蛋白70（7.5倍）, 肌动蛋白（2.3倍）和伴侣分子亚基1（3.0倍）。3个蛋白下调表达，分别是：精氨酸激酶（2.8倍）, 延伸因子2 (2.0倍） 和富含甘氨酸蛋白(2.0倍）。硫酸铜刺激后特异表达的一个蛋白被鉴定为过氧化物还原酶(Peroxiredoxin，Prx)。根据质谱检测提供的蛋白肽段信息和其他生物过氧化物还原酶保守氨基酸序列设计简并引物，结合RACE技术，从中华卤虫幼体中克隆到了过氧化物还原酶基因，该基因的cDNA全长为756个碱基，其中开放阅读框为594个碱基，编码198个氨基酸，其蛋白理论分子量为22.0 kDa，理论等电点为6.98。多序列比对结果显示中华卤虫Prx基因的推导氨基酸序列与美国卤虫和中国对虾的同源性高达98%和94%。实时荧光定量PCR结果显示，硫酸铜刺激后，该基因在卤虫无节幼体中的转录水平明显升高，在24h达到正常水平的3.0倍。

我国部分地区中学生的学校回避倾向研究

The aim of this research is to explore the relationships between school avoidance tendency and school stressors, stress response, social support, coping style of secondary school students. Three studies were carried out. School avoidance tendency scale, mental health scale, coping style scale of secondary school students from Japan were revised in the first research. In the second research, the difference in gender, cities, grades of five variables were discussed. In the third research, the relationships of five variables were discussed. A total of 883 students from grade one of middle school to grade 3 of high school in Beijing, Taiyuan and Wulanchabu, completed three scales. Analysis showed that: 1. The main effects of city, gender, and grade were present significantly on School avoidance tendency, and the inter-effects were not significant. The differences were significant between three grades of middle schools, and not significant between three grades of high schools. The high school students were significant than middle school students on school avoidance tendency. 2. The main effect of city was not significant, but gender, and grade were present significantly on stressor, and the inter-effects were not significant. The most stressor was study one, than the relationship stressor from teachers. The relationship stressor from friends was at least. 3. The main effects of city and grade were present significantly on stress responses, but the main effect of gender was not significant, and the inter-effect was not significant. The most stress response was the physical response. 4. The main effects of city and gender, the inter-effect were not present significantly on social supports. The main effect of grade was present significantly. The most social support was from mother, than from friends, father, and the teachers. 5. The differences between cites on coping styles were not significant. Positive coping style was used mostly, than the style of help requirement, cognitive coping style. 6. Stressor and stress responses had significant positive predictive role on school avoidance tendency. Social supports had negative predictive role. Social supports had not moderator role between stressor, stress responses and schools avoidance tendency. Stress responses had a part of mediator between stressor and school avoidance tendency. 7. In the coping styles, positive coping style and help requirement style had negative predictive role, but cognitive coping style had positive predictive role on school avoidance tendency. Coping styles had negative predictive role. Coping styles had not moderator role between stressor, stress responses and school avoidance tendency.

Increasing Critical Sensitivity of the Load/Unload Response Ratio before Large Earthquakes with Identified Stress Acclumulation Pattern

The Load/Unload Response Ratio (LURR) method is proposed for short-to-intermediate-term earthquake prediction [Yin, X.C., Chen, X.Z., Song, Z.P., Yin, C., 1995. A New Approach to Earthquake Prediction — The Load/Unload Response Ratio (LURR) Theory, Pure Appl. Geophys., 145, 701–715]. This method is based on measuring the ratio between Benioff strains released during the time periods of loading and unloading, corresponding to the Coulomb Failure Stress change induced by Earth tides on optimally oriented faults. According to the method, the LURR time series usually climb to an anomalously high peak prior to occurrence of a large earthquake. Previous studies have indicated that the size of critical seismogenic region selected for LURR measurements has great influence on the evaluation of LURR. In this study, we replace the circular region usually adopted in LURR practice with an area within which the tectonic stress change would mostly affect the Coulomb stress on a potential seismogenic fault of a future event. The Coulomb stress change before a hypothetical earthquake is calculated based on a simple back-slip dislocation model of the event. This new algorithm, by combining the LURR method with our choice of identified area with increased Coulomb stress, is devised to improve the sensitivity of LURR to measure criticality of stress accumulation before a large earthquake. Retrospective tests of this algorithm on four large earthquakes occurred in California over the last two decades show remarkable enhancement of the LURR precursory anomalies. For some strong events of lesser magnitudes occurred in the same neighborhoods and during the same time periods, significant anomalies are found if circular areas are used, and are not found if increased Coulomb stress areas are used for LURR data selection. The unique feature of this algorithm may provide stronger constraints on forecasts of the size and location of future large events.

Response of Microcystis to copper stress - Do phenotypes of Microcystis make a difference in stress tolerance?

To elucidate the role of phenotype in stress-tolerant bloom-forming cyanobacterium Microcystis, two phenotypes of M. aeruginosa-unicellular and colonial strains were selected to investigate how they responded to copper stress. Flow cytometry (FCM) examination indicated that the percents of viable cells in unicellular and colonial Microcystis were 1.92-2.83% and 72.3-97.51%, respectively, under 0.25 mg l(-1) copper sulfate treatment for 24 h. Upon exposure to 0.25 mg l(-1) copper sulfate, the activities of antioxidative enzyme, such as superoxide dismutase (SOD) and catalase (CAT), were significantly increased in colonial Microcystis compared to unicellular Microcystis. Meanwhile, the values of the photosynthetic parameters (F-v/F-m, ETRmax and oxygen evolution rate) decreased more rapidly in unicellular Microcystis than in colonial Microcystis. The results indicate that colonial Microcystis has a higher endurance to copper than unicellular Microcystis. This suggests that the efficient treatment concentration of copper sulfate as algaecides will be dependent on the phenotypes of Microcystis. (C) 2006 Elsevier Ltd. All rights reserved.

The involvement of HSP22 from bay scallop Argopecten irradians in response to heavy metal stress

Heat shock protein 22 (HSP22) is an important member of small heat shock protein (sHSP) subfamily which plays a key role in the process of protecting cells, facilitating the folding of nascent peptides, and responding to stress. In the present study, the cDNA of HSP22 was cloned from Argopecten irradians (designated as AiHSP22) by rapid amplification cDNA end (RACE) based on the expressed sequence tags (ESTs). The full-length cDNA of AiHSP22 was of 1,112 bp, with an open reading frame of 588 bp encoding a polypeptide of 195 amino acids. The deduced amino acid sequence of AiHSP22 showed high similarity to previously identified HSP22s. The expression patterns of AiHSP22 mRNA in different tissues and in haemocytes of scallops exposed to Cd2+, Pb2+ or Cu2+ were investigated by real-time quantitative RT-PCR. The mRNA of AiHSP22 was constitutively expressed in all examined tissues, including haemocyte, muscle, kidney, gonad, gill and heart. The expression level in heart and muscle was higher than that in other tissues. The mRNA level of AiHSP22 in haemocytes was up-regulated after a 10 days exposure of scallops to Cu2+, Pb2+ and Cd2+. However, the expression of AiHSP22 did not increase linearly along with the rise of heavy metal concentration. Different concentrations of the same metal resulted in different effects on AiHSP22 expression. The sensitive response of AiHSP22 to Cu2+, Pb2+ and Cd2+ stress indicated that it could be developed as an indicator of exposure to heavy metals for the pollution monitoring programs in aquatic environment.

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.