产紫杉醇内生真菌EFY-21的鉴定与生物学特性研究

利用PCR技术克隆了产紫杉醇内生真菌EFY-21的18S rDNA序列,通过同源性分析,初步确定该菌与拟盘多毛孢属(Pestalotiopsis)有较高的同源性,相似性为99%。为了进一步了解EFY-21的有关生物学特性,分别选用PDA、PSA、查氏、玉米粉琼胶、牛肉膏蛋白胨5种培养基,按照常规方法培养,用十字法测量菌落直径;同时选用查氏培养基为基本培养基,分别观察不同碳源葡萄糖、甘露醇、麦芽糖、果糖、可溶性淀粉,不同氮源KNO3、Ca(N03)2、(NH4)2SO4、NH4N03、(NH4)2HPO4、蛋白胨、尿素,不同培养温度10,15,20,25,28,30,37℃,不同pH值4,5,6,7,8,9对内生真菌菌丝的影响。试验结果表明:EFY-21在PDA培养基上生长最快,生长状况最好;供试的碳氮源中,对EFY-21菌丝生长影响的大小顺序为葡萄糖甘露醇果糖麦芽糖可溶性淀粉;蛋白胨KNO3Ca(N03)2NH4N03(NH4)2HPO4(NH4)2SO4尿素;最适培养温度为25～30℃;最适pH为5～7。     

越香竹——香竹属一新种

报导了从云南省金平县用种子(种子来源于越南莱州省的燕汤)繁殖的幼苗引入四川长宁县栽培的香竹属一新种越香竹(Chimonoca lamus peregrinus Yiet L.S.Ma)。新种与马关香竹(Ch.makuanensis Hsuehet Yi)相近似[1,3～5],但节间幼时微被灰粉,密被紫色小点,在分枝一侧中下部或基部扁平,并具纵脊和沟槽,无毛,刺状气生根较短,长约1～3mm,箨鞘短于节间长度,无不同颜色的纵条纹,箨舌三角形、"山"字形或稀近于截平形,高1～2mm,小枝具叶(4)6～8枚,叶鞘口繸毛2～4枚,长3～4mm,叶片下面淡绿色;也近似角香竹(Ch.bicorniculatusS.F.LietZ.P.Wang)[2,4,5],不同在于节间无毛,在分枝一侧中下部或基部扁平,并具纵脊与沟槽,节上气生根刺离生,箨鞘顶端两侧低于或远低于中部,箨舌高1～2mm,小枝具叶(4)6～8枚,叶片长8.5～13cm,宽6～10mm,次脉3(4)对,笋期在春季和秋季,易于区别。     

Cardioprotection in ischemia/reperfusion injury: Spotlight on sphingosine-1-phosphate and bradykinin signalling

Complex signal-transduction cascades are known to be involved in regulating cardiomyocyte function, death and survival during acute cardiac ischemia-reperfusion process, but detailed survival signalling pathways are not clear. This review presents and discusses the recent findings bearing upon the evidence on the cardioprotective effect of sphingosine-1-phosphate (S1P) and bradykinin in acute cardiac ischemia-reperfusion and underlying signalling mechanisms, particularly, through activation of P21 activated kinase.     

Determining the genetic diversity of lactobacilli from the oral cavity

Several methods for determining the diversity of Lactobacillus spp were evaluated with the purpose of developing a realistic approach for further studies. The patient population was comprised of young children with an oral disease called severe early childhood caries. The ultimate goal of these studies was to ascertain the role of lactobacilli in the caries process. To accomplish that goal, we evaluated several methods and approaches for determining diversity including AP-PCR, chromosomal DNA fingerprinting, denaturing gradient gel electrophoresis, and 16S rRNA gene sequencing. Central to these methods was the gathering and screening of isolates from cultivation medium. Using various estimates of diversity, we addressed the question as to how many isolates represent the overall diversity and how cultivation compares to non-cultivation techniques. Finally, we proposed a working approach for achieving the goals outlined framed by both practical constraints in terms of time, effort and efficacy while yielding a reliable outcome.     

拆分获得(S)-酮基布洛芬脂肪酶基因在枯草芽孢杆菌中的克隆与表达

【目的】筛选具有不对称拆分消旋酮基布洛芬氯乙酯能力的脂肪酶基因,构建其表达分泌型工程菌,并进一步提高该脂肪酶的立体选择性。【方法】以自筛选出的一株具有不对称拆分消旋酮基布洛芬氯乙酯能力的菌株NK13为材料,通过构建其基因组文库,筛选具有不对称拆分消旋酮基布洛芬氯乙酯能力的脂肪酶基因。通过构建该脂肪酶基因的分泌型诱导表达载体pHY300-plk-sacR-gene,将其转入枯草芽孢杆菌WB600,获得基因重组菌WB600(pHY300-plk-sacR-gene)。用SDS-PAGE检测其表达和转化情况,采用非变性聚丙烯酰胺凝胶电泳的方法纯化脂肪酶;并利用TLC和HPLC检测该酶的立体选择专一性。【结果】得到了具有专一性拆分获得(S)-酮基布洛芬能力、长度为633bp的脂肪酶基因(GenBank登录号为:EU381317)。该脂肪酶在枯草芽孢杆菌WB600中得到了分泌表达。TLC和HPLC检测结果显示,纯化的脂肪酶对底物转化40h时转化率为30%,生成(S)-酮基布洛芬的e.e.%值最高,达60.02%,与未加Tween-80的枯草芽孢杆菌转化子体系相同。而在含Tween-80的环境下,枯草芽孢杆菌表达重组菌对底物转化36h时转化率约为45%,生成(S)-酮基布洛芬的e.e.%值最高,达93.64%,是野生菌NK13的16倍。【结论】从NK13号菌株中筛选得到的新的脂肪酶具有很高的不对称拆分获得(S)-酮基布洛芬的能力,实现了NK13菌中633bp脂肪酶基因在枯草芽孢杆菌中的分泌表达,研究证明Tween-80能提高该脂肪酶的拆分专一性。     

Prevention of a systematic underestimation of antioxidant activity in competition assays. The impact of unspecific reactions of the reactive species

In antioxidant competition assays, an antioxidant (A) and a detector compound (D) compete for a reactive species (R). In the evaluation of these assays, it is tacitly assumed that all of R is captured by either D or A. Due to the - by definition - high reactivity of R, unspecific reactions of R are likely to occur and neglecting these reactions will result in a systematic underestimation of antioxidant activity. It was shown that in the standard hydroxyl radical scavenging assay this was indeed the case; the inaccurate mathematical evaluation resulted in an underestimation of antioxidant activity of 25% in this competition assay. The systematic underestimation of antioxidant activity can be prevented by using an adjusted Stern-Volmer equation that takes into account that only part of R is captured by D or A.     

Regulation of survivin by PI3K/Akt/p70S6K1 pathway

PI3K activation is commonly observed in many human cancer cells. Survivin expression is elevated in cancer cells, and induced by some growth factors through PI3K activation. However, it is not clear whether PI3K activation is sufficient to induce survivin expression. To investigate the role of PI3K pathway in the regulation of survivin, we expressed an active form of PI3K, v-P3k in chicken embryonic fibroblast cells (CEF), and found that overexpression of PI3K-induced survivin mRNA expression. Forced expression of wild-type but not mutant tumor suppressor PTEN in CEF decreased survivin mRNA levels. PI3K regulates survivin expression through Akt activation. To further investigate downstream target of PI3K and Akt in regulating the expression of survivin mRNA, we found that PI3K and Akt-induced p70S6K1 activation and that overexpression of p70S6K1 alone was sufficient to induce survivin expression. The treatment of CEF cells by rapamycin decreased the survivin mRNA expression. This result demonstrated that p70S6K1 is an important target downstream of PI3K and Akt in regulating suvivin mRNA expression. The knockdown of survivin mRNA expression by its specific siRNA induced apoptosis of cancer cells when the cells were treated with LY294002 or taxol. Taken together, these results demonstrated that PI3K/Akt/p70S6K1 pathway is essential for regulating survivin mRNA expression.     

5S rRNA-assisted DnaK refolding

Although accumulating evidence has revealed that most proteins can fold without the assistance of molecular chaperones, little attention has been paid to other types of chaperoning macromolecules. A variety of proteins interact with diverse RNA molecules in vivo, suggesting a potential role of RNAs for folding of their interacting proteins. Here we show that the in vitro refolding of a representative molecular chaperone, DnaK, an Escherichia coli homolog of Hsp70, could be assisted by its interacting 5S rRNA. The folding enhancement occurred in RNA concentration and its size dependent manner whereas neither the RNA with the reverse sequence of 5S rRNA nor the RNase pretreated 5S rRNA stimulated the folding in vitro. Based on our results, we propose that 5S rRNA could exert the chaperoning activity on DnaK during the folding process. The results suggest an interesting possibility that the folding of RNA-interacting proteins could be assisted by their cognate RNA ligands.     

对Bt蛋白敏感敏感和敏感的棉铃虫中肠细菌群落的比较

摘要：【目的】通过比较Cry1Ac蛋白抗性及敏感棉铃虫中肠细菌群落的结构组成,研究中肠微生物是否与棉铃虫Bt抗性产生有关。【方法】首先提取了棉铃虫中肠微生物基因组DNA,通过PCR扩增获得了16S rDNA全长片段及V3区。采用基于16S rDNA 的免培养技术—16S rDNA文库建立和变性梯度凝胶电泳（DGGE）研究了国内特有的Bt抗性和敏感品系棉铃虫中肠细菌群落组成,并对其进行分析和比较。【结果】16S rDNA文库测序结果表明,抗性品系与敏感品系棉铃虫中肠细菌群落特别是优势菌群非常相似,但在部分劣势菌群上存在差异。抗性品系中主要优势菌有：不可培养微生物（Uncultured bacterium）占56.4%,鹑鸡肠球菌（Enterococcus gallinarum）占17.0%,铅黄肠球菌（Enterococcus casseliflavus）占17.0%;敏感品系中主要优势菌为不可培养微生物（Uncultured bacterium）60.2%,鹑鸡肠球菌（Enterococcus gallinarum）占19.3%,铅黄肠球菌（Enterococcus casseliflavus）占14.7%。随后进行的PCR验证表明,部分有差异的劣势菌在两种品系虫体都存在。DGGE图谱分析表明,这两个品系棉铃虫中肠菌群相似性达到92.3%。【结论】敏感品系与抗性品系棉铃虫肠道菌群组成极其相似,推测抗性的产生与肠道微生物无直接关系。