蚕豆和玉米叶绿体atpE基因表达产物的生物活性差异 …

编码蚕豆和玉米叶绿体ＡＴＰ合酶ε亚基的ａｔｐＥ基因分别在大肠杆菌中获得了高效表达,两种表达的ε亚基蛋白在抑制ＣＦ１－ＡＴＰ酶水解ＡＴＰ、阻塞类囊体膜质子通道以及它促进光合磷酸化等方面均明显地强于蚕豆的ε亚基蛋白。该结果表明：（１）ε亚基对ＡＴＰ合酶活性的调节作用与基同ＡＴＰ合酶其他亚基间的亲和力大小密切相关;（２）ε亚基抑制ＣＦ１水解ＡＴＰ和阻塞质子通道两个功能是呈正相关的。圆二色性（ｃｉｒｃｕｌ     

高等植物叶绿体ATP合酶ε亚基的结构与功能

ε亚基是叶绿体ATP合酶最小的一个亚基,有阻塞ATP合酶的质子通道和抑制其水解ATP活力的两种功能.用定点突变和缺失等分子生物学方法对ε亚基的结构功能进行了研究,结果表明:ε亚基42位上的苏氨酸(Thr42)对维持其结构和功能都很重要.与大肠杆菌ATP合酶相比,叶绿体ATP合酶ε亚基C端和N端的氨基酸残基缺失对其结构功能的影响更为敏感.     

高等植物叶绿体ATP合酶ε亚基的结构与功能

ε亚基是叶绿体ATP合酶最小的一个亚基,有阻塞ATP合酶的质子通道和抑制其水解ATP活力的两种功能。用定点突变和缺失等分子生物学方法对ε亚基的结构功能进行了研究,结果表明：ε亚基42位上的苏氨酸(Thr42)对维持其结构和功能都很重要。与大肠杆菌ATP合酶相比,叶绿体ATP合酶ε亚基C端和N端的氨基酸残基缺失对其结构功能的影响更为敏感。     

用酵母双杂交系统检测菠菜叶绿体ATP合酶CF1各亚基间的相互作用

将菠菜叶绿体ATP合酶CF1的5种亚基基因分别插入到质粒pGBT9和pGAD424,双转化入酵母菌株,用酵母双杂交系统检测菠菜叶绿体CF1各亚基间的相互作用.结果显示CF1的5种亚基中,γ与ε亚基有较强的相互作用;α与β,α与ε,β与ε,β与δ亚基间也有稳定的相互作用;γ与δ,δ与ε亚基间有微弱的或短暂的相互作用;而α与γ,α与δ,β与γ等亚基间在酵母双杂交系统中没有相互作用.这些结果有助于研究ATP合酶在催化过程中的结构变化和亚基间的相互关系.     

结核分枝杆菌H37Rv 色氨酸合成酶α亚基编码基因的克隆、表达及酶学性质分析

目的 克隆表达色氨酸合成酶α亚基编码基因并对其进行功能分析。方法 以结核分枝杆菌( 简称结核杆菌)H37Rv 基因组为模板, 扩增trpA 基因, 构建pET30a-trpA 重组质粒; 转化重组质粒到大肠埃希菌DH5α并在BL21( DE3) 诱导表达, 纯化可溶性的结核杆菌重组色氨酸合成酶α亚基( His-rMtTrpA) 。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳( SDS-PAGE) 和质谱分析测定相对分子质量( Mr) 后, 用圆二色光谱( CD) 分析和同源模建方法检测二级和三级结构。研究不同浓度HisrMtTrpA对β亚基酶活反应的影响。结果 成功克隆了813 bp 的目的基因trpA, 并获得了高纯度的His-rMtTrpA 蛋白。重组蛋白Mr 为33. 151 ×10³ ( 含载体蛋白) 。25 ℃时His-rMtTrpA 的二级结构包括31. 8% α 螺旋、31. 8% β 折叠、8. 4% β转角和27. 9%无规则卷曲, 它的三维模型显示为( β/ α) 8 桶状结构。酶学性质研究表明, 在His-rMtTrpA 与MtTrpB 的摩尔比为2. 2时, 色氨酸合成酶α亚基可以最大限度促进β亚基酶活反应。结论 成功得到高纯度的重组目的蛋白His-rMtTrpA, 其功能分析为针对色氨酸合成酶的药物筛选设计提供理论基础。     

猪β2m二级结构预测及同源模建

目的:研究猪β2m的结构.方法:应用EXPASY服务器(http://www.expasy.ch/tools)上的SOPMA法对β2m进行二级结构的预测,并与人的β2m的氨基酸和二级结构成分比较,在此基础上同源模建β2m的三级结构.结果:β2m二级结构成分α螺旋、β折叠、转角和无规则卷曲的数目分别为5、36、5和52,与人β2m相应二级结构的符合率分别达到100%(α螺旋)、92.3%(B折叠)、71.7%(转角)和92.3%(无规则卷曲),且二级结构的同源率要大于氨基酸的同源率.同源模建分析表明猪和人的3D结构也非常相似,只是存在个别氨基酸的差异.结论:猪和人β2m空间结构相似.     

菠菜和蚕豆叶绿体CF_1结构与功能的比较

比较了菠菜和蚕豆叶绿体的光合磷酸化活力以及由不同活化方法活化的叶绿体及可溶CF1的Mg2＋－ATPase和Ca2＋－ATPase的活力,观测到两种叶绿体ATPase的合成和水解ATP的功能有明显差异。从两种叶绿体CF1的SDS－PAGE图谱上可见蚕豆CF1的ε亚基分子量明显小于菠菜的,蚕豆CF1的α和β亚基间分子量的差别也比菠菜的小。     

猪肾果糖1,6-二磷酸酯酶N-端60肽的溶液构象

猪肾果糖1,6-二磷酸酯酶N-端60个氨基酸是结合AMP的别构部位的重要组成部分.这一段肽在天然酶中具有2段α-螺旋,不含β-折叠,螺旋含量占这段肽的60%左右.经枯草杆菌蛋白酶对果糖1,6-二磷酸酯酶限制性酶解可以专一地作用在这一位点.得到S-肽和S-蛋白,虽然S-肽与S-蛋白的肽键已经分开,但是它们依然缔合在酶的4个亚基之中.在9%的甲酸溶液中,用SephadexG-75柱(1.3×195cm)可以把S-肽与S-蛋白分开.除去甲酸后,用CD测定二级结构表明,S-肽有30%的α-螺旋和38.5%的转角或类似转角的二级结构,没有β-折叠.蛋白质的生物合成是从N-端开始向C-端延伸,新生肽在生物合成的过程中,它的构象由一级结构决定它自发形成某种结构的趋势,但是,其它部分的相互作用可以在较大程度上改变它的结构,使它最后形成天然状态的空间构象.用6mol/L盐酸胍破坏S-肽链的溶液构象,然后逐步透析去掉盐酸胍,重新测定CD的数值,所得结果与胍变性前的二级结构含量相同,用加入乙醇,环己烷,少量十二烷基磺酸钠等扰动方法都不能增加α-螺旋的含量.相反,随着加入量的增加,有序结构减少,说明当S-肽从酶上被分离下来后,在水溶液中只能形成其在该天然酶蛋白中的一半的螺旋,而另一半却变成转角或类似转角的有序结构的结果并不是由于分离过程产生的假象.     

菠菜叶绿体CF1-ATP合酶的分离纯化及其结构的原子力显微镜研究

用纤维素DE-52柱层析和硫酸铵分级分离法快速分离、纯化获得CF1-ATP合酶,聚丙烯酰胺凝胶电泳技术对其纯度和凝胶内酶活性进行检测,研究了温度、pH值、盐浓度对CF1水解酶活性的影响,用原子力显微镜对CF1的形态结构进行了观察.结果发现,当pH值为8.0时,CF1可在0℃存放数天并保持其50%的活性,20%的甲醇溶液能够有效地保护酶分子的结构;实验还通过低温盐解作用获得了具有活性的亚基;用原子力显微镜观测显示CF1-ATP合酶分子平均大小约为18nm,按其直径大小可以分为三类,推测其分别对应着ATP合酶的三种状态,即空置态、疏松结合态和紧密结合态.     

α-天门冬氨酰二肽酶及其进化酶的FT-IR研究

用分子定向进化技术,在酶活力和热稳定性双重选择压力下,筛选到了一株Kcat/KM是天然酶47倍的进化酶.用FT-IR方法,测定了α-天门冬氨酰二肽酶及其进化酶的酰胺I带图谱,定量估算了天然酶和进化酶的各种二级结构含量.天然酶中,β折叠结构含量为28.5%,α螺旋结构含量为33%,这与园二色谱测量α螺旋结构为33%的结果有很好的一致,剩余的残基形成不同类型的转角和无规结构,其总含量为38.5%.在进化酶中,β折叠结构含量为26.8%,α螺旋结构含量为31%,其它结构为不同类型的转角和无规结构,含量为42.2%.     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Comparison of BMI and percentage of body fat of Indian and German children and adolescents

Today, serious health problems as overweight and obesity are not just constricted to the developed world, but also increase in the developing countries (Prentice 2006, Ramachandram et al. 2002). Focusing on this issue, BMI and percentage of body fat were compared in 2094 schoolchildren from two cross-sectional studies from India and Germany investigated in 2008 and 2009. The German children are in all age groups significantly taller, whereas the Indian children show higher values in BMI (e.g. 12 years: Indian: around 22 kg/m2; German: around 19 kg/m2) and in the percentage of body fat (e.g. 12 years: Indian: around 27%; German: around 18-20%) in most of the investigated age groups. The Indian children have significantly higher BMI between 10 and 13 (boys) respectively 14 years (girls). Indian children showed significant higher percentage of body fat between 10 and 15 years (boys) and between 8 and 16 years (girls). The difference in overweight between Indian and German children was strongest at 11 (boys) and 12 (girls) years: 70% of the Indian but 20% of the German children were classified as overweight. In countries such as India that undergo nutritional transition, a rapid increase in obesity and overweight is observed. In contrast to the industrialized countries, the risk of overweight in developing countries is associated with high socioeconomic status. Other reasons of the rapid increase of overweight in the developing countries caused by different environmental or genetic factors are discussed.     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent.faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium -like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum , shared characteristics with Ent. columbae.
The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol.
Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.     

The synthesis and evaluation of flavone and isoflavone chimeras of novobiocin and derrubone

The natural products novobiocin and derrubone have both demonstrated Hsp90 inhibition and structure–activity relationships have been established for each scaffold. Given these compounds share several key structural features, we hypothesized that incorporation of elements from each could provide insight to structural features important for Hsp90 inhibition. Thus, chimeric analogues of novobiocin and derrubone were constructed and evaluated. These studies confirmed that the functionality present at the 3-position of the isoflavone plays a critical role in determining Hsp90 inhibition and suggests that the bicyclic ring system present in both novobiocin and derrubone do not share similar modes of binding.