复合与合成培养基对大肠杆菌胞外生产α-环糊精葡萄糖基转移酶的影响

为实现来源于Paenibacillus macerans JFB05-01的α-环糊精葡萄糖基转移酶（α-CGT酶）的高效胞外表达,以含分泌型信号肽OmpA的大肠杆菌E.coli BL21（DE3）{pET-20b（+）/α-cgt}为研究对象,比较了其在不同诱导条件下复合与合成培养基中生长产酶的规律。结果表明在添加甘氨酸的条件下采用合成培养基,以0.8 g/L/h的乳糖进行流加诱导所得的胞外酶活和生产强度最高。在该条件下发酵30小时后胞外α-CGT酶的环化活性达113.0U/ml（水解活性为79 100.0IU/ml）,是复合培养基胞外产酶的2.3倍,完全满足工业化生产的需求。     

不同株型芝麻种质湿害后产量性状研究及耐湿性评价

于盛花期对66份种质进行湿害处理,结果表明:湿害对单秆型和分枝型种质的产量性状影响差异明显,对单秆型芝麻产量性状的影响(湿害指数值)大小依次为单株种子干重(69.18%)蒴果数(67.48%)有效果节数(49.10%)有效果轴长度(45.69%)株高(16.40%),对分枝性芝麻的影响依次为分枝有效果节数(65.96%)分枝蒴果数(64.73%)总蒴果数(52.01%)单株种子干重(49.92%)主茎蒴果数(41.66%)主茎有效果轴长度(37.57%)有效分枝数(34.21%)主茎有效果节数(20.12%)株高(15.43%);湿害对三蒴型芝麻的侧位蒴果影响较大,对单秆三蒴型芝麻的影响为侧位蒴果数(92.25%)中位蒴果数(50.25%),对分枝三蒴型芝麻的影响为分枝侧位蒴果数(92.86%)主茎侧位蒴果数(69.14%)分枝中位蒴果数(44.17%)主茎中位蒴果数(32.97%)。根据相对湿害产量可以将供试种质聚为耐湿与不耐湿二大类,不耐湿类型种质61份,占92.42%;耐湿类型种质5份,占7.58%,为竹山白芝麻、西平二郎花、阜南芝麻、嘉兴紧口黑和麻城黑芝麻,可作为耐湿种质加以利用。     

超滤法提纯万古霉素的应用

采用Ultra-flo超滤系统提纯未经任何预处理的万古霉素发酵液。结果表明:万古霉素收率由传统工艺(板框加压过滤)的87%提高到94%,滤液的透光度比传统工艺提高了25%;系统平均膜通量可达120L/(m2.h);被污染的膜经清洗后与新膜没有明显差异;合理加水量为投料量的2.5倍。Ultra-flo超滤系统完全能代替传统工艺。     

VersusSNP:一种单碱基突变的筛选及分类工具

单碱基突变的筛选和分类是SNP分析的基础。为解决手工进行突变位点挖掘工作的困难,编写了VersusSNP软件。它可以解析并过滤序列比对结果,并根据突变类型将位点加以分类,以图形界面呈现给用户。使用VersusSNP,用户可以直观地了解基因组中单碱基突变的情况。其程序及源代码可以从http://sourceforge.net/projects/versussnp下载。     

Java AWT和JAI技术在生物数据库图像显示上的应用

利用Java AWT和JAI技术绘制生物图像的方法可以使在实验室中得到的生物实验数据自动转化为需要的图谱,并且图像的精确度高。绘制成的图像既可以保存在本地硬盘中,也可以在浏览器中以网页的形式展示出来供研究人员分析使用。该方法目前已在上海生物信息技术研究中心开发的可视化工具MapViewer和主题数据库H2H,SRA中得到应用。     

Response of Solenopsis invicta (Hymenoptera: Formicidae) to potassium oleate water solution

Abstract In contrast to floating on the surface of distilled water, ants were immediately submerged after being placed in the potassium oleate (PO) water solution, which led to immobilization within minutes. However, some workers survived after being immersed in 0.03% PO water solution at 25°C for up to 640 min. Elevated temperature of the PO water solution is needed to kill ants within a shorter time frame. At 50°C and 0.13% PO concentration, total mortality was achieved with 10-min immersion for all ants, including brood and adult ants. Soil has a negative effect on the effectiveness of potassium oleate; however, such negative effect can be overcome by increasing either treatment temperature or duration of the treatment. In addition to immobilizing and lethal effect, PO repels ants. PO may have the potential to be incorporated into immersion treatment for the quarantine of imported fire ants to reduce the use of synthetic contact insecticides.     

计算机模拟技术结合多媒体教学法在心肺复苏培训中的应用

目的:探讨和评价计算机模拟技术结合多媒体教学法在心肺复苏培训中的应用价值。方法:对62名急诊医生进行心肺复苏理论和技能测试,应用算机模拟技术结合多媒体教学模式,进行综合模拟演练并再次考核,比较两次成绩的差异;并发放调查问卷,分析对教学方法的评价及满意度。结果:培训后再次考核理论成绩、技能成绩和团队合作能力较初步测试成绩有显著提高,P0.05;98.3%喜欢该教学法,95.1%认为该教学法可以提高团队合作能力。结论:计算机模拟技术结合多媒体教学法在心肺复苏培训中具有重要的应用价值,可以显著提高团队合作能力。     

严重多发创伤失血性休克患者限制性液体复苏的临床应用评价

目的:评价限制性液体复苏在严重多发失血性休克救治中的作用.方法:对88例多发创伤合并失血性休克的患者随机分为两组,观察组(n=48)在创伤失血性休克出血未控制前行限制性液体复苏,对照组(n=40)则进行常规行充分液体复苏.比较两组的液体摄入量、输血量、收缩压、术前复苏时间;住院期间急性呼吸窘迫综合征(ARDS)、急性肾功能衰竭(ARF)、弥漫性血管内凝血(DIC)、脓毒血症的发生率和总死亡率.结果:观察组的液体输入量和术前复苏时间均低于对照组,差异有显著性(P＜0.05);两组输血量和收缩压差异无显著性.观察组ARDS、脓毒血症的发生率和总死亡率均明显低于对照组,差异有显著性(P＜0.05).结论:在创伤性休克术前未控制性出血条件下,限制性液体复苏可缩短术前复苏时间,明显降低并发症的发生率和死亡率.     

Wide distribution among halophilic archaea of a novel polyhydroxyalkanoate synthase subtype with homology to bacterial type III synthases

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular carbon and energy storage polymers by various bacteria and a few haloarchaea. In this study, 28 strains belonging to 15 genera in the family Halobacteriaceae were investigated with respect to their ability to synthesize PHAs and the types of their PHA synthases. Fermentation results showed that 18 strains from 12 genera could synthesize polyhydroxybutyrate (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). For most of these haloarchaea, selected regions of the phaE and phaC genes encoding PHA synthases (type III) were cloned via PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and were sequenced. The PHA synthases were also examined by Western blotting using haloarchaeal Haloarcula marismortui PhaC (PhaC(Hm)) antisera. Phylogenetic analysis showed that the type III PHA synthases from species of the Halobacteriaceae and the Bacteria domain clustered separately. Comparison of their amino acid sequences revealed that haloarchaeal PHA synthases differed greatly in both molecular weight and certain conserved motifs. The longer C terminus of haloarchaeal PhaC was found to be indispensable for its enzymatic activity, and two additional amino acid residues (C143 and C190) of PhaC(Hm) were proved to be important for its in vivo function. Thus, we conclude that a novel subtype (IIIA) of type III PHA synthase with unique features that distinguish it from the bacterial subtype (IIIB) is widely distributed in haloarchaea and appears to be involved in PHA biosynthesis.     

Characterization of a Novel LysM Domain from Lactobacillus fermentum Bacteriophage Endolysin and Its Use as an Anchor To Display Heterologous Proteins on the Surfaces of Lactic Acid Bacteria

The endolysin Lyb5, from Lactobacillus fermentum temperate bacteriophage φPYB5, showed a broad lytic spectrum against Gram-positive as well as Gram-negative bacteria. Sequence analysis revealed that the C terminus of the endolysin Lyb5 (Ly5C) contained three putative lysin motif (LysM) repeat regions, implying that Ly5C was involved in bacterial cell wall binding. To investigate the potential of Ly5C for surface display, green fluorescent protein (GFP) was fused to Ly5C at its N or C terminus and the resulting fusion proteins were expressed in Escherichia coli. After being mixed with various cells in vitro, GFP was successfully displayed on the surfaces of Lactococcus lactis, Lactobacillus casei, Lb. brevis, Lb. plantarum, Lb. fermentum, Lb. delbrueckii, Lb. helveticus, and Streptococcus thermophilus cells. Increases in the fluorescence intensities of chemically pretreated L. lactis and Lb. casei cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for Ly5C. Moreover, the pH and concentration of sodium chloride were optimized to enhance the binding capacity of GFP-Ly5C, and high-intensity fluorescence of cells was observed under optimal conditions. All results suggested that Ly5C was a novel anchor for constructing a surface display system for lactic acid bacteria (LAB). To demonstrate the applicability of the Ly5C-mediated surface display system, β-galactosidase (β-Gal) from Paenibacillus sp. strain K1, replacing GFP, was functionally displayed on the surfaces of LAB cells via Ly5C. The success in surface display of GFP and β-Gal opened up the feasibility of employing the cell wall anchor of bacteriophage endolysin for surface display in LAB.Surface display of heterologous proteins or peptides on bacteria is potentially important in several areas of biotechnology, including development of live vaccine delivery systems, diagnostics, whole-cell absorbents, and novel biocatalysts (11). Lactic acid bacteria (LAB) have the status of being generally recognized as safe (GRAS), making them certainly more useful in food and medical applications than other bacterial species. The development of cell surface display systems for LAB has recently become one of the most active research areas. Most of the cell surface display systems for LAB reported thus far have made use of the C terminus of a cell wall-anchoring protein via an LPXTG motif (8, 12, 19, 24). This anchoring mechanism requires processing by a sortase for covalent anchoring of the protein to the cell wall peptidoglycan (15). Various anchoring proteins, such as membrane-spanning protein PgsA (16) and S-layer protein (3), have also been exploited for surface display. However, heterologous proteins have been anchored to the producer cells, and the use of genetically modified organisms is less desirable or at least still being debated. Surface display of heterologous proteins on genetically unmodified Gram-positive bacteria has been successfully carried out using the peptidoglycan binding lysin motif (LysM) domain of the major autolysin AcmA of Lactococcus lactis (1, 2, 4, 18, 28).LysM was first discovered in the lysozyme of Bacillus phage φ29 as a C-terminal repeat composed of 44 amino acids separated by 7 amino acids (6). LysM is a common module found in more than 4,000 proteins of both prokaryotes and eukaryotes (6). Many bacterial proteins containing LysM are peptidoglycan hydrolases, such as p60 (20), Sep (26), LytF (31), AcmA (5), and Mur (7). The best-characterized LysM-containing protein is the N-acetylglucosaminidase AcmA of L. lactis subsp. cremoris MG1363. AcmA is the major autolysin and is required for cell separation and cell lysis during the stationary phase of L. lactis (5). It contains three domains: the N-terminal signal peptide, an active domain, and a C-terminal peptidoglycan anchor (cA) which consists of three LysM repeats (22). Several functional proteins, including malaria parasite surface antigen, β-lactamase, α-amylase, and viral capsid proteins, have been noncovalently bound to cell walls of AcmA-producing and non-AcmA-producing L. lactis as well as several other Gram-positive bacteria via cA (4, 17, 18, 23, 25).Endolysins from bacteriophages are cell wall hydrolases involved in cell lysis to release the progeny particles from the host cells (9, 30). Most endolysins lack a signal peptide and are translocated across the membrane by the aid of the holin protein. This protein typically contains an N-terminal catalytic domain and a C-terminal cell wall binding domain (33). The endolysins Ply118 and Ply500 of a Listeria monocytogenes phage share a unique C-terminal cell wall binding domain which establishes specific recognition of and high-affinity binding to bacterial cell wall carbohydrates (13). The temperate bacteriophage φPYB5, isolated from the Lactobacillus fermentum YB5 strain, has a hexagonal head, noncontractile tails, and several fibers and belongs to Bradley''s group B as defined by the International Committee on Taxonomy of Viruses (32). The sequence of the endolysin gene lyb5 from the genome of φPYB5 has been deposited in GenBank under accession number {"type":"entrez-nucleotide","attrs":{"text":"EF531306","term_id":"145687952","term_text":"EF531306"}}EF531306, and the gene product has been successfully expressed in Escherichia coli and has shown a broad lytic spectrum (30).Here, we generated a fusion of green fluorescent protein (GFP) to the C terminus of Lyb5 (Ly5C) to construct a surface display system for LAB. The GFP was bound to the surfaces of various LAB cells by the aid of Ly5C. Moreover, by using the system constructed, β-galactosidase (β-Gal) was functionally displayed on the surfaces of LAB cells and retained its activity.