Construction of dextrin and isomaltose-assimilating brewer’s yeasts for production of low-carbohydrate beer

Most Saccharomyces spp. cannot degrade or ferment dextrin, which is the second most abundant carbohydrate in wort for commercial beer production. Dextrin-degrading brewer’s bottom and top yeasts expressing the glucoamylase gene (GAM1) from Debaryomyces occidentalis were developed to produce low-carbohydrate (calorie) beers. GAM1 was constitutively expressed in brewer’s yeasts using a rDNA-integration system that contained yeast CUP1 gene coding for copper resistance as a selective marker. The recombinants secreted active glucoamylase, displaying both α-1,4- and α-1,6-debranching activities, that degraded dextrin and isomaltose and consequently grew using them as sole carbon source. One of the recombinant strains expressing GAM1 hydrolyzed 96 % of 2 % (w/v) dextrin and 98 % of 2 % (w/v) isomaltose within 5 days of growth. Growth, substrate assimilation, and enzyme activity of these strains were characterized.     

细叶黄芪叶肉原生质体发育早期细胞壁再生的研究

采用透射电镜术、电镜多糖细胞化学染色、细胞壁荧光染色以及香豆素抑制细胞壁再生等方法,对细叶黄芪（Ａｓｔｒａｇａｌｕｓｍ ｅｌｉｌｏｔｏｉｄｅｓ ｖａｒ．ｔｅｎｕｉｓ）叶肉原生质体细胞壁的再生及其化学特点进行了研究。结果表明,离体培养２４ 小时的原生质体表面产生一些突起小泡,有时可见少量纤维组分的形成。培养３ 天时这种纤维组分明显增多。至５ 天时可清楚看到再生壁是由纤维和颗粒构成。六亚甲四胺银染色证明它们都是由多糖组分组成的。另外,培养３６ 小时的原生质体有相互粘连的现象。电镜观察、荧光染色及香豆素处理的研究表明粘连与再生壁的形成有关。根据上述观察结果,对原生质体再生壁的结构及其化学性质等问题进行了讨论     

Expression and purification of Mycobacterium tuberculosis ESAT-6 and MPT64 fusion protein and its immunoprophylactic potential in mouse model

The completion of Mycobacterium tuberculosis genome sequence has opened a new way for the identification and characterization of bacterial antigens, such as ESAT-6, CFP10, MPT64, and Ag85 complex, which are helpful for tuberculosis control. In this work, genes of ESAT-6 and MPT64 were fused and expressed in Escherichia coli in form of inclusion bodies with a histidine tag. The expressed fusion protein was purified by nitrilotriacetic acid (Ni-NTA) affinity chromatography under denaturing conditions, and the yield was 18mg/L of culture. In mice, the purified ESAT-6-MPT64 fusion protein elicited stronger humoral response, greater splenic lymphocyte stimulated index, and higher levels of IFN-gamma and IL-12 production than that of the single MPT64 inoculation group, and rendered modest protection on the experimental tuberculosis mouse models. In short, the ESAT-6-MPT64 fusion protein might be a potential candidate vaccine for tuberculosis.     

Photoaffinity labeling identifies the substrate-binding site of mammalian squalene epoxidase

Squalene epoxidase (SE) catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) in order to identify the location of the substrate-binding site and the roles of key residues in catalysis. Truncated 50-kDa rrSE was purified and photoaffinity labeled by competitive SE inhibitor (Ki=18.4 microM), [(3)H]TNSA-Dza. An 8-kDa CNBr/BNPS-skatole peptide was purified and the first 24 amino acids were sequenced by Edman degradation. The sequence PASFLPPSSVNKRGVLLLGDAYNL corresponded to residues 388-411 of the full-length rat SE. Three nucleophilic residues (Lys-399, Arg-400, and Asp-407) were labeled by [(3)H]TNSA-Dza. Triple mutants were prepared in which bulky groups were used to replace the labeled charged residues. Purified mutant enzymes showed lower enzymatic activity and reduced photoaffinity labeling by [(3)H]TNSA-Dza. This constitutes the first evidence as to the identity of the substrate-binding site of SE.     

3A蛋白104–115位氨基酸缺失口蹄疫A型标记病毒的构建

【目的】构建一株含3A非结构蛋白104–115位氨基酸缺失的口蹄疫A型标记病毒,分析其生物学特性和发展标记疫苗的潜力。【方法】采用融合PCR技术,在当前流行毒株A/Sea-97/CHA/2014全长感染性克隆p QAHN中引入3A104–115位氨基酸的缺失,构建全长重组质粒。全长质粒经NotI线化后转染表达T7RNA聚合酶的稳定细胞系,拯救标记病毒。RT-PCR、序列分析、间接免疫荧光和Western blotting鉴定标记病毒。噬斑表型和一步生长曲线分析标记病毒的生物学特性,并用实验室开发的针对3A优势表位(AEKNPLE)的阻断ELISA方法分析其区分亲本和标记病毒感染的动物。【结果】成功拯救到一株含3A 104–115位氨基酸缺失的口蹄疫A型标记病毒,3A表位的缺失没有影响标记病毒的噬斑表型和一步生长曲线。3A单抗阻断ELISA可以明显区分标记病毒和亲本病毒感染的动物。【结论】本研究构建的3A蛋白104–115位氨基酸缺失的标记病毒可以作为发展口蹄疫鉴别诊断疫苗的候选毒株,用于我国未来口蹄疫A型的有效防控。     

DNA damage in the early primordial anther is closely correlated with stamen arrest in the female flower of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

To investigate the regulatory mechanisms of sex expression in cucumber, morphological observations and biochemical analyses were carried out on inappropriate stamen development of female flowers of cucumber. It was found that developmental arrest of the inappropriate stamen mainly occurs at the anther primordium. This arrest is closely correlated with DNA damage, as detected by TUNEL assay, and might result from anther-specific DNase activation. It was also found that the DNA damage does not lead to cell degeneration, although chromatin condensation is observed in the anther primordia.Abbreviations DAPI 4,6-diamidine-2-phenylindole dihydrochloride - MTT 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - PCD programmed cell death - TUNEL TdT-mediated dUTP nick-end labeling     

HSP90 mRNA在胃癌和大肠癌中表达的研究

目的为探讨胃癌和大肠癌细胞HSP90 mRNA表达的特点.方法利用核酸原位杂交技术,对79例胃肠癌组织进行检测.结果表明HSP90 mRNA在胃癌和大肠癌中的阳性率分别为55.56%(15/27)和69.23%(36/52).mRNA表达与病理类型、分化程度和有否淋巴结转移有相关性.结论 HSP90 mRNA在胃肠癌中有较高表达,检测HSP90 mRNA可以作为提示预后的重要临床指标.