Cloning and characterization of two ferritin subunit genes from bay scallop, <Emphasis Type="Italic">Argopecten irradians</Emphasis> (Lamarck 1819)

We have cloned two full-length cDNAs from two ferritin genes (Aifer1 and Aifer2) of the bay scallop, Argopecten irradians (Lamarck 1819). The cDNAs are 1,019 and 827 bp in length and encode proteins of 171 and 173 amino acids, respectively. The 5′ UTR of each contains a conserved iron response element (IRE) motif. Sequence analyses reveal that both proteins belong to the H-ferritin family with seven conserved amino acids in the ferroxidase center. Highest expression of Aifer1 is found in the mantle and adductor muscle, while that of Aifer2 is only in the latter tissue. These Aifer genes are differentially expressed following bacterial challenge of the scallop. The expression level of Aifer1 was acutely up-regulated (over 10 fold) at 6 h post-bacteria injection, whereas Aifer2 expression was not significantly changed by bacterial challenge. Both genes were effectively expressed in E. coli BL21 (DE3), producing proteins of similar molecular weight, approximately 23 kDa. Purified Aifer1 and Aifer2 proteins exhibited iron-chelating activity of 33.1% and 30.4%, respectively, at a concentration of 5 mg/ml. Cations, Mg²⁺, Zn²⁺ and Ca²⁺, depressed iron-chelating activity of both proteins. Additionally, the E. coli cells expressing recombinant Aifer1 and Aifer2 showed tolerance to H₂O₂, providing a direct evidence of the antioxidation function of ferritin. The results presented in this study suggest important roles of Aifer1 and Aifer2 in the regulation of iron homeostasis, immune response, and antioxidative stress in A. irradians.     

A new long terminal repeat (LTR) sequence allows to identify J genome from J<Superscript>S</Superscript> and St genomes of <Emphasis Type="Italic">Thinopyrum intermedium</Emphasis>

A repetitive sequence of 491 bp, named pMD232-500, was isolated from S. cereale cv. Kustro using wheat SSR marker Xgwm232. GenBank BLAST search revealed that the sequence of pMD232-500 was highly similar to a part of retrotransposon Nusif-1. Fluorescence in situ hybridization (FISH) analysis using pMD232-500 as probe indicated that only 14 Thinopyrum intermedium chromosomes and all the chromosomes of S. cereale cv. Kustro bear FISH signals, however, no FISH signals were observed on Dasypyrum villosum chromosomes. In addition, the FISH signals were distributed on whole arms except their terminal regions. Further genomic in situ hybridization (GISH) analysis using genomic DNA from Pseudoroegneria spicata indicated that the 14 Th. intermedium chromosomes bearing FISH signals should belong to J genome. Thereafter, the repetitive elements pMD232-500 showed the unambiguous features of genomic constitution of Th. intermedium. In addition, the results in the present study have indicated the similarity of genomes from Th. intermedium and S. cereale.     

成人降主动脉置换术中三种体外循环灌注技术的应用

目的总结分析成人降主动脉置换术中各种体外循环灌注的技术特点和管理方法。方法 2006年1月至2009年12月,共有60例患者行降主动脉置换手术。按体外循环灌注技术类型分为3组：左心转流组4例,股动静脉转流组37例,上下半身分别灌注组19例。三组预充均采用勃脉力A和胶体,常规加入白蛋白和激素,监测混合静脉氧饱和度和血细胞压积,积极应用超滤技术和自体血液回收技术。结果术中转流平稳,血流动力学稳定,监测指标均在正常范围,仅出现9例并发症（截瘫、偏瘫、谵妄、苏醒延迟、低氧血症）。结论成人降主动脉置换术中根据不同的手术方式,正确选择和熟练应用相应的灌注技术是决定手术成功的重要因素。     

运用分子生物学技术监测水环境中产毒蓝藻

有毒蓝藻在形成水华破坏水体环境的同时,也对人畜产生危害。运用分子生物学技术对水环境中产毒蓝藻进行监测,由于其简单、快速和经济等优点,逐渐成为各国学者关注的热点。本文从以下3个方面对其进行了综述:1)早期分子生物学技术在产毒蓝藻诊断中的运用;2)以产毒基因为靶标进行的产毒蓝藻诊断;3)运用基因芯片技术对产毒蓝藻进行检测。与传统的分子生物学技术相比,生物芯片技术具有体积小、重量轻、便于携带、分析自动快速、高通量等许多传统方法所不能比拟的优势。因此,该技术在蓝藻毒素检测中的运用必将给目前的产毒蓝藻的检测带来一场新的革命。     

水牛MyD88cDNA的克隆与原核表达

采用RT-PCR方法从水牛外周血白细胞总RNA中扩增出髓样分化因子88 (mydoid differentiation factor 88,MyD88) cDNA序列,PCR产物分离纯化后,与pMD20-T载体连接,重组质粒经PCR、酶切鉴定后测序,并进行生物信息学分析;构建pET28a-MyD88表达载体,并将其转化至E.coli BL21 (DE3),经IPTG诱导表达后,进行SDS-PAGE、镍柱亲和层析纯化和Western blotting分析.结果显示,克隆到的水牛MyD88 cDNA全长为1 189 bp,含有1个891 bp的开放阅读框,编码296个氨基酸,理论等电点为5.65.经IPTG诱导表达后,得到一个带His·Tag的约39 kD的重组融合蛋白.用抗His单克隆抗体进行Western blotting,得到1条约39 kD特异性抗体结合带,表明水牛MyD88原核表达载体成功构建并表达.本研究为进一步开展水牛MyD88的结构功能分析奠定了基础.     

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) receptor specific peptide analogues for PET imaging of breast cancer: In vitro/in vivo evaluation

Vasoactive intestinal peptide and pituitary adenylate cyclase activating peptide have high affinity for VPAC1, VPAC2 and PAC1 receptors overexpressed on human cancer cells. Four potent analogues of these peptides, TP3939, TP3982, TP4200 and TP3805 were labeled with (64)Cu and evaluated ex vivo and in vivo to asses their biological activity and receptor specificity. The ultimate goal is to utilize (64)Cu analogues for positron emission tomography (PET) imaging of breast cancers in humans. Radiochemical purity of each analogue was >92%. The muscle relaxivity assay revealed IC(50) to be 5.3x10(-8) M, 4.4x10(-8) M, 8.1x10(-8) M, 8.1x10(-9) M and Kd values determined by receptor specific cell binding assays were 3.3 nM, 0.33 nM, 0.2 nM and 0.72 nM for TP3805, TP3939, TP3982, and TP4200 respectively. The receptor affinity, using human breast cancer tissues, was 10.93 times greater than normal breast tissues. RT-PCR confirmed increased VPAC1 receptor expression on human breast tumor cells over normal cells and corroborated with autoradiography data. The blood clearance was rapid and in vivo translocation of (64)Cu to plasma protein was <15%. Data demonstrate that these analogues are potent, have uncompromised biological activity and are worthy of further evaluation for accurate PET imaging of human breast cancers and in determining malignant and benign lesions.     

A new family of antimicrobial peptides from skin secretions of Rana pleuraden

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Guizhou region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the Yunnan frog, Rana pleuraden. Members of the new peptide family named pleurain-As are composed of 26 amino acids with a unique N-terminal sequence (SIIT) and a disulfide-bridged heptapeptide sequence (CRLYNTC). By BLAST search, pleurain-As had no significant similarity to any known peptides. Native and synthetic peptides showed antimicrobial activities against tested microorganisms including Gram-negative and Gram-positive bacteria and fungi. Twenty different cDNAs encoding pleurain-As were cloned from the skin cDNA library of R. pleuraden. The precursors of pleurain-As are composed of 69 amino acid residues including predicted signal peptides, acidic propieces, and cationic mature antimicrobial peptides. The preproregion of pleurain-A precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature pleurain-As are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. Furthermore, pleurain-As could exert antimicrobial capability against Helicobacter pylori. This is the first report of naturally occurring peptides with anti-H. pylori activity from Rana amphibians.