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、...     

Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.     

放牧对牧草光合作用、呼吸作用和氮、碳吸收与转运的影响

研究放牧对草地植物生理活动的影响,对于揭示草地放牧演替的生理机制有重要意义.大量研究表明,家畜放牧对牧草光合作用、呼吸作用以及C和N吸收与转运的影响,可以分为生理伤害和生理恢复2个阶段.放牧通过改变草地冠层结构影响牧草光合作用,净光合作用速率短期内迅速下降,随着叶面积指数增加又逐渐上升,呼吸作用有相似的变化趋势.牧草放牧后再生长所需的C和N最初主要来自根系和留茬中的贮藏物质,此后随着牧草生长恢复逐渐由同化作用供给,C代谢与土壤N水平负相关.放牧后牧草生理活动变化与牧草遗传特性、种间竞争、家畜放牧特征、非生物环境等因素密切相关.     

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。     

The septomaxilla of fossil and recent synapsids and the problem of the septomaxilla of monotremes and armadillos

The septomaxilla is a paired intramembranous ossification in the external nares that occurs in Lepidosauria among Recent Sauropsida and is purported to be present in Monotremata and Dasypodidae (armadillos) among Recent Mammalia. A review of neontological and palaeontological evidence regarding this element in mammals supports the following conclusions: (1) monotremes have a true septomaxilla resembling that known for non-mammalian therapsids and some Mesozoic mammals; (2) the element in dasypodids is a neomorph; it neither resembles the septomaxilla of other synapsids nor does it exhibit the same relationship to the developing nasal-floor cartilage as the septomaxilla of lepidosaurs and monotremes; (3) a septomaxilla is lacking in all Recent therians, and there is no evidence that this bone is fused to the premaxilla in Recent therians, as has been suggested by previous authors.     

颧骨颧弓骨折的分类及诊治

颧骨颧弓骨折是最常见的面中部骨折之一,迄今为止,在其分类及诊治的选择上仍然存在一定争议,本文对目前颧骨颧弓骨折的分类及诊治的现状和进展作一综述,以期对临床工作提供参考。     

Dogs and Practices of Community and Neighboring

ABSTRACT

Dogs are important facilitators of social interaction. However, little attention has been given to the specific mechanisms through which these relations proceed, or to the ways that dogs help to broker, maintain, and even disrupt social relations. This paper addresses this absence through an indepth qualitative analysis of the everyday experiences of 24 dog owning households who live in apartments in Sydney, Australia. It shows that dogs encourage people to spend more time outside, make people recognizable within their neighborhood, provide a topic of conversation, and actively solicit the attention of strangers. Dogs help make people recognizable and identifiable to others, while also creating social distance. The paper connects to broader literature on neighboring and community practice to show that community relations shaped by dogs involve practices of inclusion as well as exclusion. Exclusion provides an important motivation for new community formation.     

Effect of soil salinity and water stresses on growth and content of nitrogen,chloride and phosphate of wheat and triticale

Summary Three wheats and one triticale were grown, up to flowering stage, in pots on calcareous soil adjusted to a range of salinities (S₁=3.5, S₂=6, S₃=8.5, and S₄=11 mmhos/cm, 20°C, soilpaste extract) by adding solution consisting of 3∶2∶1 of Na-, Ca- and Mg chlorides in chemical equivalent amounts. Moisture in the pots was kept at 100% (W₁), 40% (W₂) and 20% (W₃) of the available water. The vegetative growth, nitrogen and phosphate were affected by S and W treatments, chloride was affected only by S. The interaction S×W affected only dry weight. Varietal effect was observed between wheat as a group and triticale. Multiple quadratic regression equations of these properties on salinity and water revealed that the higher the available water the wider the range of tolerable salinity. Triticale was relatively more tolerant to water stress. Salinity increases Cl and decreases N, whereas water stress enhances N accumulation to a certain extent. However, in triticale at S₃ and S₄ the effect of water stress on N was overshadowed by the excessive salinity. This did not occur for the wheat (Florence). P trends were described. R² for P was low (0.7435–0.3603) which made interpretations rather difficult.     

Non-linear effects of temperature and urea on the thermodynamics and kinetics of folding and unfolding of hisactophilin

Extensive measurements and analysis of thermodynamic stability and kinetics of urea-induced unfolding and folding of hisactophilin are reported for 5-50 degrees C, at pH 6.7. Under these conditions hisactophilin has moderate thermodynamic stability, and equilibrium and kinetic data are well fit by a two-state transition between the native and the denatured states. Equilibrium and kinetic m values decrease with increasing temperature, and decrease with increasing denaturant concentration. The betaF values at different temperatures and urea concentrations are quite constant, however, at about 0.7. This suggests that the transition state for hisactophilin unfolding is native-like and changes little with changing solution conditions, consistent with a narrow free energy profile for the transition state. The activation enthalpy and entropy of unfolding are unusually low for hisactophilin, as is also the case for the corresponding equilibrium parameters. Conventional Arrhenius and Eyring plots for both folding and unfolding are markedly non-linear, but these plots become linear for constant DeltaG/T contours. The Gibbs free energy changes for structural changes in hisactophilin have a non-linear denaturant dependence that is comparable to non-linearities observed for many other proteins. These non-linearities can be fit for many proteins using a variation of the Tanford model, incorporating empirical quadratic denaturant dependencies for Gibbs free energies of transfer of amino acid constituents from water to urea, and changes in fractional solvent accessible surface area of protein constituents based on the known protein structures. Noteworthy exceptions that are not well fit include amyloidogenic proteins and large proteins, which may form intermediates. The model is easily implemented and should be widely applicable to analysis of urea-induced structural transitions in proteins.     

Engineering of caseins and modulation of their structures and interactions

β-Casein (β-CN) is a milk protein widely used in food industries because of its mild emulsifying properties due to its amphiphilicity. However, the elements determining its micellization behavior in solution and interfacial behavior at the air–water interface are not well known. In order to study how the forced dimerisation influences functional properties of β-CN, recombinant wild-type β-CN was produced and distal cysteinylated forms of recombinant β-CN were engineered. We show that 1) cysteinylated β-CN formed mainly dimers bridged by disulfide bonds; 2) the process of dimerization adds to the micellization process with temperature and is poorly reversible; 3) covalent disulfide linkage forms at the air–water interface at a lower temperature than in bulk. In conclusion, the location of the cysteinylation in the C-terminus or N-terminus or both is of importance for the properties of β-CN.     

昆虫及其病毒基因启动子的研究及应用

分子生物学研究领域中,基因结构、功能和表达调控的研究逐渐成为热点.启动子作为基因表达调控的重要元件,深入研究其结构和功能,对于阐明真核细胞基因表达调控和基因调节机制意义重大,同时也为揭示病毒的致病机理从而防治相关疾病提供依据,另外,也为病毒新型表达载体鉴定和构建奠定基础.综述了启动子克隆方法及应用,阐述了启动子的一般特点,介绍了研究启动子功能的常用方法及在科研中的应用前景,着重介绍了该领域的最新进展.     

Identity and stereochemistry of allopurpurin and Tedaniaxanthin

The identity of the carotenoids tedaniaxanthin and allopurpurin from marine sponges has been demonstrated by a direct quantitative comparison of their I₂-catalysed stereomutation mixtures (HPLC and visible spectra). Studies on the geometrical isomerism are reported. The 2R-configuration is assigned on the basis of a CD-correlation of the HPLC-purified all-trans isomer and (3R,3′R)-aloxanthin.     

Biology and conservation of sturgeon and paddlefish

The Acipenseriformes (sturgeon and paddlefish)live in the Northern Hemisphere; half of thesespecies live in Europe, mostly in thePonto-Caspian region, one third in NorthAmerica, and the rest in East Asia and Siberia.They reproduce in freshwater and most of themmigrate to the sea, either living in brackishwater (Caspian, Azov, Black and Baltic Seas) orin full seawater on the oceanic continentalshelf. Most species feed on benthic organisms.Puberty usually occurs late in life (5–30 yearsof age) and adult males and females do notspawn on an annual basis. Adults continue togrow and some species such as the beluga (Huso huso) have reached 100 years of age andmore than 1,000 kg weight. Stocks of sturgeonsare dramatically decreasing, particularly inEurasia; the world sturgeon catch was nearly28,000 t in 1982 and less than 2,000 t by 1999.This decline resulted from overfishing andenvironmental degradation such as: accumulationof pollutants in sediments, damming of rivers,and restricting water flows, which becomeunfavorable to migration and reproduction.Several protective measures have beeninstituted; for example, fishing regulation,habitat restoration, juvenile stocking, and theCITES listing of all sturgeon productsincluding caviar. In addition, sturgeon farmingpresently yields more than 2,000 t per year(equivalent to wild sturgeon landings) andabout 15 t of caviar. Hopefully, thisartificial production will contribute to areduction of fishing pressure and lead to therehabilitation of wild stocks.