优化玉米CMS-S双向电泳体系与差异蛋白比较

利用自主创建的Rf3近等基因系不育群体（P）s和可育恢复系群体（Pf）为试材,采用改进的双向（二维）聚丙烯酰胺凝胶电泳（2D-PAGE）技术,对苗期、成株期叶片细胞总蛋白进行分析,分离到在Pf遗传背景下一特异蛋白质RRPⅥ,分子量为30.8kD,等电点为5.0,该特异蛋白质点的出现与消失可能与玉米CMS-S的育性恢复有一定关系,对其纯化分离将为Rf3基因的克隆奠定基础和进一步了解玉米质核互作不育表达模式的分子机制。讨论了采用2D-PAGE技术在玉米分子生物学研究中的优化以及基因组学与蛋白组学研究的衔接。     

石油降解细菌降解条件优化研究

采用富集培养法从工业油污土壤中分离到1株能以石油为惟一碳源而生长的细菌菌株,采用正交设计实验对该菌株的降解条件进行了初步研究。结果表明,最佳降解条件为NH_4Cl 4.0 g/LL,K_2HPO_4 1.5 g/L,pH 8.0,NaCl 15.0 g/L。在最佳条件下,浓度为1 mL/L的原油可在4 d内降解50%以上。     

从鼠黑色素瘤细胞系中分离和鉴定癌干细胞样细胞

目的：从鼠黑色素瘤BL6F10细胞系中分离与鉴定癌干细胞（CSC）样细胞,为今后对CSC的鉴定及靶向治疗奠定基础。方法：用不同免疫磁珠标记的单克隆抗体,从BL6F10细胞系中分离有特征性CD表型的瘤细胞,体外观察不同CD表型瘤细胞在软琼脂培养基上形成克隆的能力;将这些瘤细胞皮下注射到C57BL/6小鼠,比较其致瘤性。结果：从BL6F10细胞系中分离出不同CD表型的特征性瘤细胞;在软琼脂培养基上,CD133^＋、CD44^＋和CD44^＋CD133^＋细胞克隆形成率分别高于CD133^-、CD44^-和CD44^＋CD133^-细胞;CD133^＋、CD44^＋、CD44^＋CD133^＋和CD44^＋CD133^＋CD24＋细胞在小鼠体内的致瘤性分别强于CD133^-、CD44^-、CD44^＋CD133^-和CD44^＋CD133^＋CD24^-细胞。结论：CD44^＋CD133^＋CD24＋表型的BL6F10细胞的某些生物学特性与CSC样细胞相似,具有CSC特征,这些实验结果为进一步鉴定BL6F10细胞系中的CSC提供了重要的实验资料。     

Gene cloning, expression, and characterization of a novel beta-mannanase from Bacillus circulans CGMCC 1416

A DNA fragment containing 2,079 base pairs from Bacillus circulans CGMCC 1416 was cloned using degenerate PCR and inverse PCR. An open reading frame containing 981 bp was identified that encoded 326 amino acids residues, including a putative signal peptide of 31 residues. The deduced amino acid sequence showed the highest identity (68.1%) with endo-beta-1,4-D-mannanase from Bacillus circulans strain K-1 of the glycoside hydrolase family 5 (GH5). The sequence encoding the mature protein was cloned into the pET-22b(+) vector and expressed in Escherichia coli as a recombinant fusion protein containing an N-terminal hexahistidine sequence. The fusion protein was purified by Ni2+ affinity chromatography and its hexahistidine tag cleaved to yield a 31-kDa beta-mannanase having a specific activity of 481.55 U/mg. The optimal activity of the purified protein, MANB48, was at 58 degrees C and pH 7.6. The hydrolysis product on substrate locust bean gum included a monosaccharide and mainly oligosaccharides. The recombinant MANB48 may be of potential use in the feed industry.     

螯合细胞外钙离子对体外培养的大鼠卵巢颗粒细胞成熟的影响

在发现利用钙离子螯合剂EGTA螯合细胞外钙离子(Ca 2 )后,可以显著抑制促滤泡(激)素(FSH)刺激体外培养的颗粒细胞合成和分泌雌激素,并且该抑制作用呈剂量依赖性.假设该特异性反应是通过Ca2 影响细胞内腺苷酸环化酶(AC)发挥作用的,因为Ca 2 具有激活ACVIII的作用.通过RT-PCR和Northern印迹检测大鼠不同阶段卵巢组织中ACVIII的表达.结果表明,虽然Ca2 可以调控颗粒细胞类固醇激素的合成,但不同阶段的卵巢组织中均检测不到ACVIII的mRNA.实验间接提示了Ca2 促进颗粒细胞成熟的作用不是通过ACVIII发挥作用的,而可能是通过其他AC同工酶或其他Ca2 信号通路发挥作用.     

Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitro

Members of the RNase III family are the primary cellular agents of dsRNA (double-stranded RNA) processing. Bacterial RNases III function as homodimers and contain two dsRBDs (dsRNA-binding domains) and two catalytic sites. The potential for functional cross-talk between the catalytic sites and the requirement for both dsRBDs for processing activity are not known. It is shown that an Escherichia coli RNase III heterodimer that contains a single functional wt (wild-type) catalytic site and an inactive catalytic site (RNase III[E117A/wt]) cleaves a substrate with a single scissile bond with a k(cat) value that is one-half that of wt RNase III, but exhibits an unaltered K(m). Moreover, RNase III[E117A/wt] cleavage of a substrate containing two scissile bonds generates singly cleaved intermediates that are only slowly cleaved at the remaining phosphodiester linkage, and in a manner that is sensitive to excess unlabelled substrate. These results demonstrate the equal probability, during a single binding event, of placement of a scissile bond in a functional or nonfunctional catalytic site of the heterodimer and reveal a requirement for substrate dissociation and rebinding for cleavage of both phosphodiester linkages by the mutant heterodimer. The rate of phosphodiester hydrolysis by RNase III[E117A/wt] has the same dependence on Mg(2+) ion concentration as that of the wt enzyme, and exhibits a Hill coefficient (h) of 2.0+/-0.1, indicating that the metal ion dependence essentially reflects a single catalytic site that employs a two-Mg(2+)-ion mechanism. Whereas an E. coli RNase III mutant that lacks both dsRBDs is inactive, a heterodimer that contains a single dsRBD exhibits significant catalytic activity. These findings support a reaction pathway involving the largely independent action of the dsRBDs and the catalytic sites in substrate recognition and cleavage respectively.