Molecular cloning and expression of interleukin 1beta (IL-1 beta) from red seabream (Pagrus major)

The interleukin 1beta (IL-1beta) cDNA was cloned from the red seabream (Pagrus major) by homology cloning strategy. A cDNA fragment was amplified by PCR using two degenerated primers, which were designed according to the conserved regions of other known IL-1beta sequences, and elongated by 3' ends and 5' ends RACE PCR to get the full length coding sequence of red seabream IL-1beta (RS IL-1beta). The sequence contained 1252 nucleotides that included a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 410 bp and an open reading frame (ORF) of 759 nucleotides which could be translated into a putative peptide of 253 amino acids with molecular weight of 28.6 kD and putative isoelectric point pI of 5.29. The deduced peptide contained two potential N-glycosylation sites and an identifiable IL1 family signature, but lacked the signal peptide and the clear ICE cut site, which were common in other nonmammalian IL-1beta genes. The RS IL-1beta had the highest homology with piscine IL-1beta according to phylogenetic tree analysis. The transcript expression was detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen in the pathogen challenged and healthy red seabream by RTPCR. Results showed that the RS IL-1beta mRNA was constitutively expressed in most of the tissues both in stimulated and un-stimulated fish, and the expression could be enhanced by pathogen challenging.

Molecular cloning and expression analysis of Ch-penaeidin, an antimicrobial peptide from Chinese shrimp, Fenneropenaeus chinensis

A new member of antimicrobial peptide genes of the penaeidin family, Ch-penaeidin, has been cloned from the haemocytes of Chinese shrimp, Fenneropenaeus chinensis, by reverse transcription PCR (RT-PCR), 3'-rapid amplification of cDNA end (3'-RACE) and smart cDNA methods. The Ch-penaeidin cDNA was 655 bp and the open reading frame of the cDNA encoded a 71 amino acid peptide. Ch-penaeidin contained a putative NH2-terminal signal Sequence (1-19) followed by a mature peptide (20-71). The sequence identify with other penaeidins from Litopenaeus vannamei and Litopenaeus setiferus is between 48% and 71%. The signal sequence of Ch-penaeidin is almost completely identical to that of other penaeidins, while differing relatively in the N-terminal domain of the mature peptide. Ch-penaeidin was designated as a novel member of class penaeidin 3 according to phylogenetic analysis. The Mature peptide. with a predicted molecular weight of 5589.32 Da, and a pI of 9.77, has eight positively charged amino acids and no negatively charged amino acids. The expression and distribution of Ch-penaeidin in Unchallenged shrimps were studied by RT-PCR, Northern blot and in situ hybridisation. The results showed that the Ch-penaeidin transcripts were detected in haemocytes (granular haemocytes), heart, gill, intestine, and subcuticular epithelia of the shrimp. and that Ch-penaeidin was constitutively expressed mainly in haemocytes. (C) 2003 Elsevier Ltd. All rights reserved.

Molecular biotechnology of marine algae in China

Molecular biotechnology of marine algae is referred to as the biotechnology on the identification, modification, production and utilization of marine algal molecules. It involves not only the manipulation of macromolecules such as DNA, RNA and proteins, but also deals with low molecular weight compounds such as secondary metabolites. In the last decade, molecular systematic researches to investigate the relationship and to examine the evolutionary divergence among Chinese marine algae have been carried out by Chinese scientists. For example, RAPD has been widely used in several laboratories to elucidate genetic variations of the reds, such as Porphyra, Gracilaria, Grateloupia and the greens such as Ulva and Enteromorpha. Some important data have been obtained. The study on molecular genetic markers for strain improvement is now in progress. In 1990s, genetic engineering of economic seaweeds such as Laminaria, Undaria, Porphyra, Gracilaria and Grateloupia has been studied in China. For Laminaria japonica, the successfully cultivated kelp in China, a model transformation system has been set up based on the application of plant genetic techniques and knowledge of the algal life history. Progress has been made recently in incorporating a vaccine gene into kelp genome. Evidence has been provided showing the expression of gene products as detectable vaccines. In the present paper, the progress of molecular biotechnological studies of marine algae in China, especially researches on elucidating and manipulating nucleic acids of marine algae, are reviewed.

Molecular cloning and characterization of a serine proteinase homolog prophenoloxidase-activating factor in the swimming crab Portunus trituberculatus

Serine proteinase homologues (SPHs), as one of prophenoloxiase-activating factors (PPAFs), play critical roles in innate immunity of crabs. Based on an EST from the eyestalk full length cDNA library, the complete cDNA (designated as PtSPH) and genomic DNA of SPH from the swimming crab Portunus trituberculatus were cloned in this study. The estimated molecular weight of mature PtSPH (354 amino acids) was 38.7 kDa and its isoelectric point was 5.08. Multiple sequence alignment revealed that PtSPH lacked a catalytic residue with a substitution of Ser in the active site triad to Gly. Phylogenetic analysis indicated PtSPH together with PPAFs of Callinectes sapidus (AAS60227), Eriocheir sinensis (ACU65942), Penaeus monodon (ABE03741, ACP19563) and Pacifastacus leniusculus (ACB41380), formed a distinct cluster which only included clip-SPHs. As the first analyzed genomic structure of PPAFs in crustaceans, two introns were found in the open reading frame region of this gene. The mRNA transcripts of PtSPH could be detected in all the examined tissues, and were higher expressed in the eyestalk than that in gill, hepatopancreas, haemocytes and muscle. Accompanied with the change in phenoloxidase (PO) activity and total haemocyte counts, the temporal expression of PtSPH gene in haemocytes after Vibrio alginolyticus challenge demonstrated a clear time-dependent expression pattern with two peaks within the experimental period of 32 h. These findings suggest that PtSPH is involved in the antibacterial defense mechanism of Portunus tritubercualtus crab. (C) 2010 Elsevier Ltd. All rights reserved.

Two different proteases produced by a deep-sea psychrotrophic bacterial strain, Pseudoaltermonas sp SM9913

A psychrotrophic bacterial strain, Pseudoaltermonas sp. SM9913, was isolated from deep-sea sediment collected at 1,855 m depth. Two proteases produced by Pseudoaltermonas sp. SM9913 were purified, MPC-01 and MCP-02. MCP-01 is a serine protease with a molecular weight of 60.7 kDa. It is cold-adapted with an optimum temperature of 30-35degreesC. Its K-m and E-a for the hydrolysis of casein were 0.18% and 39.1 kJ mol(-1), respectively. It had low thermostability, and its activity was reduced by 73% after incubation at 40degreesC for 10 min. MCP-02 is a mesophilic metalloprotease with a molecular weight of 36 kDa. Its optimum temperature for the hydrolysis of casein was 50-55degreesC. The K-m and E-a of MCP-02 for the hydrolysis of casein were 0.36% and 59.3 kJ mol(-1), respectively. MCP-02 had high thermostability, and its activity was reduced by only 30.5% after incubation at 60degreesC for 10 min. At low temperatures, Pseudoaltermonas sp. SM9913 mainly produced the psychrophilic protease MCP-01.

红枫湖、百花湖水中溶解有机物分子量分布特征及环境地球化学意义

溶解有机物（dissolved organio matter， DOM）通常是指能够通过0.45μm滤膜的有机物质。0.45μm的定义是根据传统的过滤滤膜直径来划分的，不是一个绝对的定义。DOM在水环境中起着重要的作用，它是水环境中重要的配位体和吸附载体。DOM在水化学和生态环境中的重要作用，主要表现在它不仅可以和基本的金属离子和有机化合物结合，还可以与毒性金属离子和除草剂、杀虫剂结合（wershow ond Goldberg，1972）。湖泊水中DoM的浓度及物理化学性质能够影响水生态环境的结构及初级生产力。溶解有机物按元素组成来分，主要含有C、O、H、N、P、S以及灰分。按重量计算，其中C占总有机物的50％，其次是O（科O％）、H（一5％）、N（0.5～6.5％）、P（＜1.0％）、S（＜1.0％）和灰分（1.2-5.0％）（Thurman，1985）。DOM一般由多种有机化合物组成，其中腐殖物质是溶解有机质的主要组成部分，一般占有机质总量的50-80％。目前通常可人为地将腐殖物质划分为胡敏素、胡敏酸和富里酸（Aiken et oL，1 985）。其余的非腐殖类物质主要包括碳水化合物、氨基酸、叶绿素、藻类分泌物、酚酮类化合物、脂肪酸和亲水性有机酸等（Thurman，1985）a在水环境中一些金属离子和微量元素通常和DOM相结合，而这些元素的作用和在水中的迁移都受DoM的分子量分布的影响（chiou etal．，1986）。 DOM的分子量及其分布是指示水质量的一个重要参数，同时在水处理过程中对于研究DOM的去处效率有重要作用（vuorio et oL，1998）。具有不同分子量分布的DOM与金属的络合能力也不同，则它们对金属的生物有效性的作用就不同（wuetal．，2002）。分子量变化的程度可以进一步理解DOM的自然形成过程，也可以在弓！用水处理过程中优化腐殖质的去除过程。红枫湖和百花湖是贵州地区两个人工水库，通过对两湖水文条件的测量，探讨了影响DOM分子量及分布变化的环境因素，包括溶解氧的含量、水体温度、pH值、叶绿素。的含量、光照强度、藻类生长情况、生物活动等，以及溶解有机物的来源，人为污染情况等。本文采样高效体积排阻色谱法（High PerformanceSizeExclusion cbromatograP场，HPsEC）测量了红枫湖、百花湖水中的DOM的含量及分子量分布，得出了以下几点认识：1、选择HPsEC实验的最佳条件：0.03M NaCl和0.002的磷酸盐缓冲液作为流动相，pH为6.8，离子强度为0．034mol几，紫外检测波长为254nm。2、红枫湖、百花湖水中DOM主要以分子量小于3500Da的组分为主，占总溶解有机物的90％以上。根据洗脱曲线的峰面积，可以把DOM分为3个不同 分子量的组分，其中大分子量组分（MW＞2000Da）的含量占总DOM的35％一68％；中等分子量组分（Z000Da＞MW>1000Da）的含量为27％-57％；小分子量组分（MW＜1000Da）占总DOM的3％一31％。 3、红枫湖、百花湖水中Doc含量受气候条件的影响。丰水期，水体上部DOC含量较大，下部较小‘平水期和枯水期，上下水体中DOC含量一致。各组分DOc含量变化与总DOC含量变化相似。DOC含量也表现出明显的季节性变化特征。丰水期DOC含量较高，而在平水期和枯水期含量较低。红枫湖和百花湖二者之间DOC含量没有明显的差别。4、红枫湖、百花湖水中DOM平均分子量也受气候条件的影响。丰水期，随深度的增大，DoM平均分子量增大；平水期和枯水期，上下水体DOM平均分子量一致。DO入左的季节性变化与DOC相反，丰水期，DOM平均分子量偏低；而在平水期和枯水期，DOM平均分子量偏高。5、重均分子量和数均分子量虽然变化趋势相似，但是它们的变化幅度不同，所以多分散系数也会发生变化。总的来说，在水体上部多分散系数偏大，水体下部偏小。丰水期多分散系数较大，平水期和枯水期多分散系数较小。6、随着气候条件的变化，各分子量组分的DOM含量及DOC浓度也在发生变化。大分子量组分随深度而增大，而中等和小分子量组分随深度而减小。丰水期，中等和小分子量组分含量较多，而大分子量组分较少；平水期和枯水期，大分子量组分含量较多，中等和小分子量组分含量较少。

Isospecific polymerization of styrene with modified ziegler-type catalysts

Effects of various kinds of additives as well as aging of the catalyst on the polymerization of styrene catalyzed by TiCl4/MgCl2-AlEt3 system have been studied. Experiments show that in toluene the isotacticity of polystyrene can be up to 83% for aged catalyst, whereas when the catalyst is not aged. non-stereospecific polymer is the main product. When PCl3 is used as an additive, the catalyst system gives high activity and isotacticity. The use of a mixture of AlEt3/H2O (1: 1 mole ratio) as a cocatalyst is also efficient. The catalyst [TiCl4-PCl3/MgCl2-AlEt3/H2O] displays high activity and product isotacticity (94%) with an average molecular weight up to 2 X 10(-6). When Co(acac)(3) is added to to [TiCl4/MgCl2-AlEt3] catalyst after it was aged, the isotacticity can be up to 97%. (C) 2001 Elsevier Science Ltd. All rights reserved.