Steps and fluctuations of Listeria monocytogenes during actin-based motility

The actin-based motility of the bacterium, Listeria monocytogenes, is a model system for understanding motile cell functions involving actin polymerization. Although the biochemical and genetic aspects of Listeria motility have been intensely studied, biophysical data are sparse. Here we have used high-resolution laser tracking to follow the trailing ends of Listeria moving in the lamellae of COS7 cells. We found that pauses during motility occur frequently and that episodes of step-like motion often show pauses spaced at about 5.4 nm, which corresponds to the spatial periodicity of F-actin. We occasionally observed smaller steps (<3 nm), as well as periods of motion with no obvious pauses. Clearly, bacteria do not sense cytoplasmic viscoelasticity because they fluctuate 20 times less than adjacent lipid droplets. Instead, bacteria bind their own actin 'tails, and the anchoring proteins can 'step' along growing filaments within the actin tail. Because positional fluctuations are unusually small, the forces of association and propulsion must be very strong. Our data disprove the brownian ratchet models and limit alternative models, such as the 'elastic' brownian ratchet or the 'molecular' ratchet.     

The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization.

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen capable of rapid movement through the host cell cytoplasm. The biophysical basis of the motility of L. monocytogenes is an interesting question in its own right, the answer to which may shed light on the general processes of actin-based motility in cells. Moving intracellular bacteria display phase-dense 'comet tails' made of actin filaments, the formation of which is required for bacterial motility. We have investigated the dynamics of the actin filaments in the comet tails using the technique of photoactivation of fluorescence, which allows monitoring of the movement and turnover of labelled actin filaments after activation by illumination with ultraviolet light. We find that the actin filaments remain stationary in the cytoplasm as the bacterium moves forward, and that length of the comet tails is linearly proportional to the rate of movement. Our results imply that the motile mechanism involves continuous polymerization and release of actin filaments at the bacterial surface and that the rate of filament generation is related to the rate of movement. We suggest that actin polymerization provides the driving force for bacterial propulsion.     

The dynamics of actin-based motility depend on surface parameters

In cells, actin polymerization at the plasma membrane is induced by the recruitment of proteins such as the Arp2/3 complex, and the zyxin/VASP complex. The physical mechanism of force generation by actin polymerization has been described theoretically using various approaches, but lacks support from experimental data. By the use of reconstituted motility medium, we find that the Wiskott Aldrich syndrome protein (WASP) subdomain, known as VCA, is sufficient to induce actin polymerization and movement when grafted on microspheres. Changes in the surface density of VCA protein or in the microsphere diameter markedly affect the velocity regime, shifting from a continuous to a jerky movement resembling that of the mutated 'hopping' Listeria. These results highlight how simple physical parameters such as surface geometry and protein density directly affect spatially controlled actin polymerization, and play a fundamental role in actin-dependent movement.     

Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfoleded state depends on two chaperonin proteins and Mg-ATP.

In vitro reconstitution of active ribulose bisphosphate carboxylase (Rubisco) from unfolded polypeptides is facilitated by the molecular chaperones: chaperonin-60 from Escherichia coli (groEL), yeast mitochondria (hsp60) or chloroplasts (Rubisco sub-unit-binding protein), together with chaperonin-10 from E. coli (groES), and Mg-ATP. Because chaperonins are ubiquitous, a conserved Mg-ATP-dependent mechanism exists that uses the chaperonins to facilitate the folding of some other proteins.     

New antiviral strategy using capsid-nuclease fusion proteins.

Overexpression of dominant-negative mutants of various viral proteins can result in 'intracellular immunization'. Here we describe a new approach to interfering with viral replication in which a nuclease is fused to a capsid component so that the nuclease is encapsidated inside the virion where it can inactivate viral nucleic acid. We used Ty1, a yeast retrotransposon whose transposition closely parallels retroviral replication mechanisms and serves as an easily manipulated model for the retroviral infection process. We constructed fusion genes consisting of the region encoding the N-terminal portion of the TYA/TYB open reading frames of retrotransposon Ty1 and either of two different nuclease genes. Ty1-nuclease fusion proteins are targeted to Ty1 virus-like particles, and are active in degrading nucleic acids. A Ty1-barnase fusion protein causes 98-99% reduction in the efficiency of Ty1 transposition in vivo, presumably by degrading encapsidated Ty1 RNA. This strategy, referred to as capsid-targeted viral inactivation, may be useful for interfering with the replication of retroviruses and other viruses.     

Efficacy of chalcopyrite bioleaching using a pure and a mixed bacterium

To determine the efficacy of chalcopyrite bioleaching using pure cultures of Thiobacillus ferrooxidans or Thiobacillus thiooxidans and a mixed culture composed of Thiobacillus ferrooxidans and Thiobacillus thiooxidans, experiments were carried out in shake flasks with [Fe2+] 4 g·L-1 and S 1 g·L-1 at pH=1.80, 130 r/min and 30℃. The tests showed that the copper extraction in a mixed culture composed of Thiobacillus ferrooxidans and Thiobacillus thiooxidans is higher than that in a pure culture. On the other hand, an important potential of Thiobacillus thiooxidans to leaching chalcopyrite was indicated. Thiobacillus thiooxidans can prevent jarosites accumulating on the substrate and allow further copper to dissolute through the action of ferric ion. The selection of the suitable pH in a leaching solution would be significant. Thiobacillus thiooxidans and Thiobacillus ferrooxidans play an important role in the bioleaching process. Finally, the mechanism and the reason for iron precipitation were also discussed in detail.     

氯化血红素作为过氧化氢模拟酶制备高纯度低聚果糖

为了探索以树脂作为载体、以戊二醛作为交联剂制备的固定化血红素作为过氧化氢酶模拟酶,用于制备高纯度低聚果糖,以1.0 g树脂在0.05%(W/V)戊二醛溶液中与10 mg氯化血红素交联,在35℃下反应8 h,可得到53 U/g的固定化血红素作为过氧化氢模拟酶,然后以3.5 g(185 U)固定化血红素、2.0 g(160 U)固定化果糖基转移酶和2.5 g(100 U)固定化葡萄糖氧化酶作用于250 mL 20%(W/V)的蔗糖溶液,结果表明,反应24 h后得到含量>80%的高纯度低聚果糖。实验证明,氯化血红素可以作为过氧化氢模拟酶,用于高纯度低聚果糖的制备,明显降低低聚果糖的生产成本。     

以纯烷烃为基质的石油发酵体系动力学分析

石油发酵体系动力学的研究是石油发酵过程工业化及工艺条件优化之基础。本文首先系统地介绍了以纯烷烃为基质的石油发酵体系中菌体生长的动力学模型，然后对该体系的动力学作了全面的分析，在此基础上提出了石油发酵体系动力学研究的一些初步想法。     

广西食蟹猴群中志贺氏菌的检出、分型以及对抗菌药物的敏感性试验

采用国际（ＧＢ４７８９．２８－８４）的检验方法，对４４５０份猴粪便检测，检出志贺氏菌１７２株，阳性检出率３．９％，取其中５０株做血清学分型，皆属于Ｂ群志贺氏菌，主要血清型为３ｃ型３２株（占６４％），Ｙ变种１２株（占２４％）其次４ｂ型３株，Ｘ变种２株２ａ型１株（分别占６％，４％，２％），以此５０株福氏志贺氏菌对１０种抗菌素作了敏感度及抑菌效果评价，结果丁胺卡那霉素及先锋霉素Ｖ敏感度为１００％（高度敏     

用分子相互作用模型预测纯液体的均匀核化过热极限

采用液滴上升法测定纯液体的气泡均匀核化过热极值值，并用分子相互作用模型计算了９种纯液体的过热极限值；计算结果与实验，文献值均符合良好。     

Improvements in the microstructure and fatigue behavior of pure copper using equal channel angular extrusion

In this study, annealed pure copper was extruded using equal channel angular extrusion (ECAE) for a maximum of eight passes. The fatigue resistance of extruded specimens was evaluated for different passes and applied stresses using fatigue tests, fractography, and metallography. The mechanical properties of the extruded material were obtained at a tensile test velocity of 0.5 mm/min. It was found that the maximum increase in strength occurred after the 2nd pass. The total increase in ultimate strength after eight passes was 94%. The results of fatigue tests indicated that a significant improvement in fatigue life occurred after the 2nd pass. In subsequent passes, the fatigue life continued to improve but at a considerably lower rate. The improved fatigue life was dependent on the number of passes and applied stresses. For low stresses (or high-cycle fatigue), a maximum increase in fatigue resistance of approximately 500% was observed for the extruded material after eight passes, whereas a maximum fatigue resistance of 5000% was obtained for high-applied stresses (or low-cycle fatigue). Optical microscopic examinations revealed grain refinements in the range of 32 to 4 μm. A maximum increase in impact energy absorption of 100% was achieved after eight passes. Consistent results were obtained from fractography and metallography examinations of the extruded material during fatigue tests.     

利用杨树原生质体瞬时表达系统筛选杨生褐盘二孢菌效应因子蛋白

效应因子蛋白在病原真菌侵染宿主植物过程中发挥着重要作用,效应因子的筛选和鉴定是目前研究的难点。笔者以杨生褐盘二孢菌全基因组测序结果为基础,根据真菌效应因子蛋白结构特征,预测出106个候选效应因子,并成功克隆其中25个。利用杨树原生质体瞬时表达系统对这25个候选效应因子蛋白进行细胞内功能筛选,鉴定出4个效应因子蛋白可以抑制杨树转录因子WRKY29启动子的活性,表明这4个效应因子蛋白在干扰植物免疫系统的信号传导途径中发挥重要作用。     

Silver staining of proteins and DNA.

Silver stains offer high sensitivity for the detection of proteins and DNA separated on gels and membranes. These stains depend on the reduction of ionic to metallic silver.     

面心纯铝轧制织构的晶体塑性有限元模拟

基于率相关晶体塑性本构模型,分别将Taylor模型和有限单元模型两种多晶模型嵌入大型有限元程序ABAQUS,实现了晶体塑性学有限元模拟. 直接将电子背散射衍射(EBSD)获取的晶粒初始取向输入晶体塑性有限元模型,预测了两种不同应变情况下面心1 050纯铝轧制织构的演化. 模拟结果与EBSD实验测得的织构演化结果有较好的一致性,随着变形程度的增加,预测织构与实测织构变得更加锋锐. 经过比较,Taylor型模型预测出了{4 4 11}〈11 11 8〉的Dillamore取向,而有限单元模型预测出了铜型织构取向,比Taylor模型预测结果更接近实验验证结果. 两种模型并不能预测出{011}〈211〉黄铜取向、{123}〈523〉S取向、{011}〈100〉Goss取向及其他理想取向.     

福氏志贺菌ipaB基因的克隆及序列测定

目的:克隆福氏志贺菌(Shigella flexneri,S.flexne)的ipaB基因。方法:以福氏志贺菌2a 2457T(Nal)r为模板,用PCR扩增ipaB目的基因片段,克隆至pQE-30表达载体中,构建含目的基因的表达质粒pQE-ipaB。结果:构建了重组质粒pQE-ipaB,ipaB基因全长1 743 bp,经测序分析与预期相符。结论:成功克隆了ipaB基因。