Differential expression of three Paralichthys olivaceus Hsp40 genes in responses to virus infection and heat shock

Heat shock proteins (Hsps) are a family of highly conserved cellular proteins present in all organisms, mediating a range of essential housekeeping and cytoprotective functions as well-known molecular chaperons and recently as regulators of the immune response. By subtractive suppression hybridization, three Hsp40 homologues have been identified in the flounder (Paralichthys olivaceus) embryonic cells (FEC) after treatment with UV-inactivated turbot (Scophthalmus maximus L.) rhabdovirus (SMRV), termed PoHsp40A4, PoHsp40B6 and PoHsp40B11, whose encoded proteins all possess the conserved DnaJ domain, a signature motif of the Hsp40 family. Based on different protein structure and phylogenetic analysis, they can be categorized into two subfamilies, PoHsp40A4 for Type I Hsp40, PoHsp40B6 and PoHsp40B11 for Type 11 Hsp40. Further expression analysis revealed two very different types of kinetics in response either to heat shock or to virus infection, with a marked induction for PoHsp4OA4 and a weak one for both PoHsp40B6 and PoHsp40B11. A very distinct tissue distribution of mRNA was also revealed among the three genes, even between PoHsp40B6 and PoHsp40B11. This is the first report on the transcriptional induction of Hsp40 in virally stimulated fish cells, and the differential expressions might reflect their different roles in unstressed and stressed cells. (c) 2005 Elsevier Ltd. All rights reserved.

Molecular identification and expression analysis of natural resistance associated macrophage protein (Nramp) cDNA from Japanese flounder (Paralichthys olivaceus)

Natural resistance associated macrophage protein (Nramp) controls partially innate resistance to intracellular parasites. Its function is to enhance the ability of macrophages to kill pathogens. However, little is known about the structure and function of Nramp in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding Nramp from Japanese flounder (Paratichthys olivaceus). The full-length cDNA of the Nramp is 3066 bp in length, including 224 bp 5' terminal UTR, 1662 bp encoding region and 1180 bp 3' terminal UTR. The 1662-nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of amino acid sequence indicated that Japanese flounder Nramp consists of 12 transmembrane (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of Japanese flounder had 77.30%, 82.71%, 82.67%, 79.64%, 80.72%, 90.97%, 91.16%, 60.14%, 71.48%, 61.69%, 72.37% identity with that of rainbow trout Nramp alpha and beta, channel catfish Nramp, fathead minnow Nramp, common carp Nramp, striped sea bass Nramp, red sea bream Nramp, mouse Nramp 1 and 2, human Nramp 1 and 2, respectively. RT-PCR indicated that Nramp transcripts were highly abundant in spleen, head kidney, abundant in intestine, liver and gill, and less abundant in heart. The level of Nramp mRNA in embryos gradually increases during embryogenesis from 4 h (8 cell stage) to 80 h (hatched stage) after fertilization. (c) 2005 Elsevier Ltd. All rights reserved.

Molecular cloning and characterisation of a fish PKR-like gene from cultured CAB cells induced by UV-inactivated virus

The double-stranded-RNA-dependent protein kinase (PKR) is an important component in an antiviral defence pathway that is mediated by interferon (IFN) in vertebrates. Previously, some important IFN system genes had been identified from an IFN-producing CAB (crucian carp Carassius auratus blastulae embryonic) cells after treatment with UV-inactivated GCHV (grass carp haemorrhage virus). Here, a fish PKR-like gene, named CaPKR-like, is cloned and sequenced from the same virally infected CAB cells. It has 2192 base pairs in length with a largest open reading frame (ORF) encoding a protein of 513 amino acid residues. BLAST search reveals that the putative CaPKR-like protein is most homologous to human PKR and also has a high-level homology with all members of a family of eIF2alpha kinases. Structurally, CaPKR-like possesses a conserved C-terminal catalytic domain of eIF2alpha kinase family and the most similarity to mammalian PKRs. Within its N-terminus, there are no dsRNA-binding domains conserved in mammalian PKRs instead of two putative Z-DNA binding domains (Zalpha). Like mammalian PKRs, CaPKR-like had a very low level of constitutive expression in normal CAB cells but was up-regulated in response to active GCHV, UV-inactivated GCHV and CAB IFN, implying that the transcriptional activation of CaPKR-like by viral infection is mediated possibly by newly produced CAB IFN, which was further supported by using cycloheximide, a potent inhibitor of protein synthesis. The results together suggested that CaPKR-like was the first identified fish gene most similar to mammalian PKRs. (C) 2004 Elsevier Ltd. All rights reserved.

Effect of replacement of fish meal by meat and bone meal and poultry by-product meal in diets on the growth and immune response of Macrobrachium nipponense

The potential use of poultry by-product meal (PBM) and meat and bone meal (MBM) as alternative dietary protein sources for juvenile Macrobrachium nipponense was studied by a 70-day growth trial. Triplicate groups of M. nipponense (initial body weight: 0.37 g) were fed at 20.7-22.4 degreesC on each of the five isoenergetic and isonitrogenous diets (protein content about 38%) with different replacement of fish meal by MBM or PBM. The control diet used white fish meal as the sole protein source, the other four diets were prepared with 15% or 50% fish meal protein substituted by either MBM (MBM15, MBM50) or PBM (PBM15, PBM50). The results showed that replacement of fish meal by MBM in diets did not affect growth performance of M. nipponense (P > 0.05), while specific growth rate in PBM15 was significantly higher than that in other groups (P < 0.05). Survival rates of shrimp fed with MBM15 diet were significantly higher than that in other groups (P < 0.05). No significant differences in immunological parameters, including total haemocyte count (THC), phenoloxidase activity (PO) and respiratory burst (O-2(-)), were observed between the shrimps that were fed five experimental diets, and all determined immunological parameters in control groups were slightly higher than those in replacement groups. In conclusion, either MBM or PBM investigated could replace up to 50% fish meal protein in diets for M. nipponense. (C) 2003 Elsevier Ltd. All rights reserved.

Genetic heterogeneity analysis and RAPD marker detection among four forms of Atrina pectinata Linnaeus

Pen shell (Atrina pectinata Linnaeus) can be distinguished into four forms based on the morphololgic characteristics. Genetic similarity, and heterogeneity were analyzed among the four forms by random amplified polymorphic DNA (RAPD) technique using 24 10-nucleotide-long primers. Of these primers, 22 pruners produced well-identifiable RAPD band patterns. Significant differences in RAPD band patterns were revealed among the four forms. A total of 198 polymorphic fragments were scored from 22 pruners. and they are specific for one form, shared by two or three forms. Several pruners, such as S451, S453 S463 S464, S470. S473 and S474, produced abundant band patterns and provided sufficient information for reliable discrimination of the four forms. The average genetic distances and phylogenetic relationships were calculated and analyzed according to the distinguishable fragments. The data indicate that pen shells of form G and form Y are similar not only among individuals within the same form, but also between individuals from the two forms, and that shells of form T and form S are highly divergent. The constructed phylogenetic free matches the average genetic distances. Three clusters were clearly distinguishable, in which two were corresponding to form S and form T respectively and one included forms G and Y. This Study will be benefit to further studies oil the taxonomy and selective breeding of Pinnid species. It is suggested that the four forms of pen shell should be categorized to at least two species taxonomically.

Molecular cloning and characterization of crucian carp (Carassius auratus L.) interferon regulatory factor 7

Interferon (IFN) can induce an antiviral state via interferon-regulatory transcription factors (IRFs), which bind to and control genes directed by the interferon-stimulated response element (ISRE). Here we describe a fish IRF, termed CaIRF7, cloned from a subtractive cDNA library which is constructed with mRNAs obtained from crucian carp (Carassius auratus L.) blastulae embryonic (CAB) cells infected by UV-inactivated GCHV and mock-infected cells. CaIRF7 cDNA was found to be 1816 bp in length, with a 42 bp 5' UTR and a 508 bp 3' UTR. The open reading frame translates into 421 amino acids in which a DNA-binding domain (DBD) containing the repeated tryptophan motif and IRFs association domain have been identified. Like chicken GgIRF3, CaIRF7 was most similar to mammalian IRF7 with 27 to 30% identity overall and some 37% identity in their DBDs. A single transcript of 1.9 kb was detected in virally induced CAB cells by virtual Northern blotting. RT-PCR analysis revealed a wide tissue distribution of CaIRF7 constitutive expression, with detectable transcript in non-infected CAB cells and various tissues of healthy crucian carp. In addition, CaIRF7 expression was differentially increased by stimulation of the CAB cells with active GCHV, UV-inactivated GCHV or CAB IFN, indicating that the activation of CaIRF7 was directly regulated by IFN. (C) 2003 Published by Elsevier Ltd.

Small fish communities in two regions of the Liangzi Lake, China, with or without submersed macrophytes

Small fish communities were compared between the vegetated and vegetation-free regions of the Liangzi Lake, a shallow lake along the middle reach of the Yangtze River, China. Fish were sampled using 10 x 10 m(2) block nets and poisoning. Three samples were taken from either the near shore area or lake centre of each region. A total of 19 fish species were collected; all species occurred in the vegetated region but only 12 occurred in the vegetation-free region. The dominant small fish were Carassius auratus auratus in the vegetated region and Ctenogobius giurinus in the vegetation-free region. Diversity, density and biomass of small fishes were significantly higher in the vegetated region than in the vegetation-free region in both near shore and lake centre areas. In the vegetated region, density and biomass of small fishes was significantly higher, while species diversity significantly lower in the near shore area than in the lake centre. In the vegetation-free region, density of small fishes was significantly higher in the near shore area than in the lake centre area, but species diversity or biomass was unaffected by location.

CYCLIC PEPTIDE HEPATOTOXINS FROM FRESH-WATER CYANOBACTERIAL (BLUE-GREEN-ALGAE) WATERBLOOMS COLLECTED IN CENTRAL CHINA

Toxic cyanobacteria (blue-green algae) waterblooms have been found in several Chinese water bodies since studies began there in 1984. Waterbloom samples for this study contained Anabaena circinalis, Microcystis aeruginosa and Oscillatoria sp. Only those waterblooms dominated by Microcystis aeruginosa were toxic by the intraperitoneal (i.p.) mouse bioassay. Signs of poisoning were the same as with known hepatotoxic cyclic peptide microcystins. One toxic fraction was isolated from each Microcystis aeruginosa sample. Two hepatotoxic peptides were purified from each of the fractions by high-performance liquid chromatography and identified by amino acid analysis followed by low and high resolution fast-atom bombardment mass spectrometry (FAB-MS). LD50 i.p. mouse values for the two toxins were 245-mu-g/kg (Toxin A) and 53-mu-g/g (Toxin B). Toxin content in the cells was 0.03 to 3.95 mg/g (Toxin A) and 0.18 to 3.33 mg/kg (Toxin B). The amino acid composition of Toxin A was alanine [1], arginine [2], glutamic acid [1] and beta-methylaspartic acid [1]; for Toxin B it was the same, except one of the arginines was replaced with a leucine. Low- and high-resolution FAB-MS showed that the molecular weights were 1,037 m/z (Toxin A) and 994 m/z (Toxin B), with formulas of C49H76O12N13 (Toxin A) and C49H75O12N10 (Toxin B). It was concluded that Toxin A is microcystin-RR and Toxin B is microcystin-LR, both known cyclic heptapeptide hepatotoxins isolated from cyanobacteria in other parts of the world. Sodium borohydride reduction of microcystin-RR yielded dihydro-microcystin-RR (m/z = 1,039), an important intermediate in the preparation of tritium-labeled toxin for metabolism and fate studies.

一种静态最少优先级分配算法

随着实时系统越来越多地应用于各种快速更新系统,尤其是各种片上系统,如PDA(personal digital assistant),PSP(play station portable)等,性价比已成为系统设计者的主要关注点.实际应用中,实时系统通常仅支持较少的优先级,常出现系统优先级数小于任务数的情况(称为有限优先级),此时,需将多个任务分配到同一系统优先级,RM(rate monotonic),DM(deadline monotonic)等静态优先级分配算法不再适用.为此,静态有限优先级分配是研究在任务集合静态优先级可调度的情况下,可否以及如何用较少或最少的系统优先级保持任务集合可调度.已有静态有限优先级分配可分为两类:固定数目优先级分配和最少优先级分配.给出了任意截止期模型下任务静态有限优先级可调度的充要条件以及不同静态有限优先级分配间转换时的几个重要性质,指出了系统优先级从低到高分配策略的优越性,定义了饱和任务组与饱和分配的概念,证明了在任务集合静态优先级可调度的情况下,最少优先级分配比固定数目优先级分配更具一般性.最后提出一种最少优先级分配算法LNPA(least-number priority assignment).与现有算法相比,LNPA适用范围更广,且复杂度较低.

一种个体软件过程能力度量方法

个体软件过程(PSP)是由卡内基×梅隆大学软件工程研究所的Humphrey领导开发的.它是一种可用于控制、管理和改进个人工作方式的自我持续改进过程.随着工业界对软件过程改进需求的日益增长,PSP成为了软件组织为达成完全(从宏观到微观)量化过程管理研究中的一个热点课题.软件过程研究表明,高水平的个体软件过程能力是软件项目成功的关键,如何进行有效的个体软件过程能力度量是PSP中的一个核心问题.现有方法不能同时有效处理个体软件过程能力度量中的可变规模收益、多变量输入/输出以及决策者偏好问题.提出了一种综合了数据包络分析(DEA)和层次分析法(AHP)的个体软件过程能力评价方法--PSPADA,介绍了PSPADA的个体软件过程能力评价模型和核心算法(集成决策者偏好和估计规模收益).实验结果显示,PSPADA能够在考虑决策者偏好的同时,有效地进行多指标、规模收益可变的量化评估.

基于软件仓库的个体软件过程能力度量技术研究

自60年代出现软件危机以来，世界各国政府、计算机软件研究机构和组织在软件工程化方法、技术和工具的研究、开发和实践方面投入了大量的人力、物力和资金。人们认识到，要高效率、高质量和低成本地开发软件，必须以改善软件生产过程为中心，实施过程指导的软件生产与质量管理。个体软件过程(PSP)是由卡内基·梅隆大学软件工程研究所的Humphrey领导开发的。它是一种可用于控制、管理和改进个人工作方式的自我持续改进过程。随着软件工业界对软件过程改进需求的日益增长，PSP的研究成为了软件组织为达成完全(从宏观到微观)量化过程管理研究中的一个热点课题。研究表明高水平的个体软件过程能力是软件项目成功的关键，如何进行有效的个体软件过程能力度量是PSP中的一个核心问题。 软件过程能力度量的准确度依赖于历史数据的积累，只有积累了大量客观充分的历史数据，软件过程度量所得到的结果才会更准确，对未来的过程改进才有指导意义。然而，工业生产中常见的协同软件开发，使得PSP能力指标的收集十分困难，例如，当一个软件系统由多人编码实现时，PSP能力的度量就面临着如何识别其中每个开发者所贡献的代码量，所引入的缺陷率以及所带来的程序复杂性等问题。同时，PSP能力度量问题本身具有多指标输入输出、规模收益可变以及需要考虑决策者偏好的特点，因此亟需一种面向PSP能力度量的量化分析方法，用于解决具有这类特点的量化度量问题。 由于软件仓库 (版本控制系统及缺陷跟踪系统等) 已经被广泛应用于大多数的软件项目开发之中，同时其中蕴含了丰富且极具价值的历史开发数据，这些数据和整个项目开发周期中开发人员的行为紧密相关，是个体软件活动的最直接反映，为PSP能力度量研究提供了大量客观的数据支持。因而本文提出了一种基于软件仓库的个体软件过程能力度量的新方法。该方法可分为两个步骤：基于软件仓库的PSP能力指标挖掘，以及支持PSP度量的量化分析模型。 首先本文通过充分研究当前常用的软件仓库数据挖掘技术，重点分析针对版本控制系统和缺陷跟踪系统的数据挖掘方法，提出了一种在协同工作环境中，基于软件仓库的PSP能力指标挖掘方法，并定义了四组指标进行详尽的分析，从理论和实践的角度，保证了PSP指标数据集的准确、客观和合理性 其次本文提出了一种基于数据包络分析(DEA)和层次分析法(AHP)的混合模型—PSPADA—用于PSP能力的度量分析，更进一步，还从理论上证明了PSPADA模型的正确性和可行性，并建立了与之相关的三个核心算法(综合决策者偏好，建立参考集和估计规模收益)。该模型能够同时解决多目标决策、可变规模收益以及主观决策者偏好的问题。应用该模型进行PSP指标数据的度量分析，其反馈的量化结果更为客观、更易理解，能有效地指导个体开发者实施个人软件过程改进。 然后，本文还实现了该度量方法的原型工具PSPstat。PSPstat实现了PSP指标收集和PSP能力度量分析的功能。它支持从软件仓库中自动挖掘多种PSP能力指标数据，使用PSPADA进行评价计算，并提供丰富的图形界面，展示指标数据和度量结果。PSPstat易于扩展，在设计上考虑了对多种版本控制系统、多种缺陷跟踪系统、多种程序语言、多种度量指标以及多种量化方法的支持，为进一步的研究和工作准备了必要的基础。 最后，在实例研究中，通过两个实验对本文提出的PSP能力度量模型及方法进行了验证。实验一的研究对象是一个标准的PSP数据集，侧重于从理论角度对PSP能力度量模型中的PSPADA方法进行有效性验证，证明PSPADA方法在结合决策者偏好的前提下，能有效度量个体软件过程的能力。实验二则以一个开源软件项目jEdit 为实验对象，获得了一个包含近百名个体开发者的大型工业数据集，因此在实验中，着重展示了该方法从工业软件仓库中挖掘个体软件过程能力指标的优势。 从本文的研究中可以看出，该基于软件仓库进行PSP能力指标挖掘的方法，可以保证度量指标的客观公正性，且将指标收集过程自动化，节省了大量的人力物力。同时其中的PSPADA度量模型能够在考虑决策者偏好的同时，有效的进行多指标、规模收益可变的量化评估，给出合理的度量结果，并指导未来的改进方向。因此该PSP能力度量方法对度量个体软件过程的能力，帮助软件企业建立IPRP薪资策略将有着显著的推动和促进作用。

一种个体软件过程能力度量方法

个体软件过程(PSP)是由卡内基?梅隆大学软件工程研究所的 Humphrey 领导开发的.它是一种可用于控制、管理和改进个人工作方式的自我持续改进过程.随着工业界对软件过程改进需求的日益增长,PSP 成为了软件组织为达成完全(从宏观到微观)量化过程管理研究中的一个热点课题.软件过程研究表明,高水平的个体软件过程能力是软件项目成功的关键,如何进行有效的个体软件过程能力度量是 PSP 中的一个核心问题.现有方法不能同时有效处理个体软件过程能力度量中的可变规模收益、多变量输入/输出以及决策者偏好问题.提出了一种综合了数据包络分析(DEA)和层次分析法(AHP)的个体软件过程能力评价方法——PSPADA,介绍了 PSPADA 的个体软件过程能力评价模型和核心算法(集成决策者偏好和估计规模收益).实验结果显示,PSPADA 能够在考虑决策者偏好的同时,有效地进行多指标、规模收益可变的量化评估.