DNA-protein conjugates can enter mitochondria via the protein import pathway

Mitochondria import most of their proteins and small molecules from the cytoplasm. There is some tentative evidence that they import some of their RNAs, but it is not known how nucleic acids could enter mitochondria. Here, we show that isolated yeast mitochondria can import a single-stranded or double-stranded 24-base pair piece of DNA whose 5' end is covalently linked to the C-terminus of a mitochondrial precursor protein.     

Conformational dynamics of individual DNA molecules during gel electrophoresis

Gel electrophoresis is widely used in molecular biology to separate DNA molecules according to their sizes. The physical basis of this size separation is, however, poorly understood. Here we report observations of individual, fluorescently stained DNA molecules as they migrate during various kinds of gel electrophoresis. Their movement, under the influence of either a steady electric field or a pulsed-field, is characterized by cycles of elongation and contraction. Initially relaxed coils of DNA lengthen into 'hook-shaped' configurations which temporarily 'hang-up' on obstacles in the gel matrix before sliding off, contracting and entering another cycle. The effects of a new electrophoresis technique, termed 'pulse-oriented electrophoresis', which allows the effective angle of the electric field, and hence the molecular orientation of DNA, to be varied without electrode rearrangement, are also studied. In this case the DNA adopts a 'staircase' configuration showing that the net orientation in a direction is given by the vector sum of the pulses used.     

Comparative Studies on Preparation of Large Plant DNA Fragments by Pulse Field Gel Electrophoresis

The preparation of large plant DNA fragments is extremely important to the construction of large insert DNA libraries (YAC, BAC, PAC and TAC). Although several techniques have been developed in each step of large plant DNA fragments preparation, the whole processing remains complicated and difficult. Based on authors‘ research experience and the recent worldwide development in this field, the following aspects are discussed in this paper: techniques of plant high molecular weight (HMW) DNA purification by pre-electrophoresis, the optimal conditions for the partial digestion of the HMW DNA by HindⅢ, the isolation effects of of large plant DNA fragments (100-400 kb) with different parameters of pulse field gel electrophoresis (PFGE), and the recovery of large DNA fragments. Through comparative studies, the advantages and disadvantages of each technique are discussed and some recommendations are proposed for preparing high quality large plant DNA fragments. The suggested techniques have been used in preparing the large DNA fragments of maize, rice, moss, laver, sea tangle and peach, and similar resuits are obtained among all the materials. This paper only reports the results using maize as material.     

利用低强度脉冲电磁场对家猪精子电穿孔的研究

用低强度脉冲电磁场对家猪精子细胞进行电击处理，发现台盼蓝染料能够进入细胞，而且通过扫描电镜可以观察到细胞膜表面有明显的孔洞。这证实了低强度瞬态电磁场能够引起猪精子细胞电穿孔和促使外源物质进入细胞内。     

聚晶金刚石的电火花磨削加工

为探讨在普通电参数下,电火花磨削人造聚晶金刚石(PCD)的加工性能,研究了电极轮转速、开路电压、峰值电流、脉冲宽度以及脉冲间隔对材料去除率、电极损耗以及加工表面粗糙度的影响.结果表明,采用电火花磨削方法,在较小的开路电压和峰值电流下,PCD的加工是可行的.使用电火花磨削方法在材料去除率和表面粗糙度方面要优于普通电火花加工.各个加工参数对加工表面粗糙度的影响不大,而与金刚石颗粒的粒度有关.加工时应选用大的开路电压,而峰值电流不宜取得过大,一般说来,不应超过32 A.脉冲宽度与脉冲间隔之间存在一个最优比例关系,在合理的脉冲宽度与脉冲间隔比例基础上,加大脉冲宽度可以提高加工性能.     

Effect of electric pulse on the crystal structure of solidified copper ingots

To obtain advanced quality pure copper, the microstructure of solidified copper was optimized by imposing electric pulse on liquid copper in this study. Experiments were performed to determine the effect of electric pulse voltage, arrangement mode of electrodes, and energy input on the microstructure of solidified copper. The results show that, when the energy input of electric pulse is bigger than 28.95 kJ per ton copper, the percent of fine grains increases noticeably with the increase of energy input; but when the energy input of electric pulse is smaller than 28.95 kJ per ton copper, the percent of fine grains decreases with the increase of energy input. The influence order of above factors on grain refinement is electric pulse voltage > arrangement mode of electrodes > energy input. According to the above experimental results, the optimum process conditions are chosen as the voltage being 400 V and the energy input greater than 28.95 kJ per ton copper. Meanwhile, the best arrangement mode of electrodes should be that, one electrode is immerged in the middle of liquid copper in the crystallizer, and the other is connected to the inner wall of the crystallizer, which is divided into two electrode poles for the symmetrical electric field distribution.     

电场强化旋流分离装置的内部流场分析

提出一种运用高压电场强化提高旋流分离效率的装置,实现工业废油资源化工艺环节中的高效破乳脱水处理;强化装置内部的流场分布情况直接影响了装置的分离效率,但比较复杂,目前尚不明确;通过建立电场强化旋流分离装置模型,联合电场控制方程和乳化油液滴粒径控制方程,分别对5种不同强化电压条件下的装置内部流场进行了数值模拟;计算结果表明,当强化电压为11 kV时,装置内部混合流体切向速度的最大增幅为15%,轴向速度最大降幅为20%,底流管段的压力梯度得到明显增大,有效提高了装置分离效率,试验结果表明:建立的装置数值模型,能够合理揭示电场强化旋流离心分离装置内部复杂流场的分布规律。     

静电分离器中电流体流场可视化研究

采用粒子成像测速技术(PIV)对线-板式静电分离器中微米级别油滴(2μm)的流动特征进行可视化研究,得到在充分发展的层流条件下(进口气流流速0.16 m/s)电流体随施加电压变化的典型流型及速度分布。结果表明:电流体流型和速度的分布随着施加电压的改变而变化;在外加电压低于起晕电压(小于8 kV)时,流场没有明显变化;随着电压的升高(8～12 kV),放电电极附近及分离器的下游油滴在静电力的作用下以喷嘴的形式向两个收集电极移动,径向运移速度和放电电极上游轴向速度随电压升高而升高;当施加电压大于12 kV时,在放电电极上游电流体二次流以两个对称的反向旋转涡的形式出现,涡的大小随着电压的升高而增大,径向运移速度最高可达0.4 m/s,同时在静电分离器下游出现回流,二次流和回流的出现阻碍了流体沿轴向运移;分离效率随着施加电压的增大而增加。     

电磁分离装置内油水分离特性实验研究

为研究电磁场油水分离过程中油水两相流的流动和分离特性,探究静态与动态条件下电磁分离装置内油水分离效果,在实验室内搭建了电磁场油水分离实验系统,并进行了实验研究。先后完成了电磁场油水分离静态实验与动态实验。静态实验表明:提高电压或增大磁场强度能有效提高油水两相流分离效果;动态实验表明:提高电压或增大磁场强度能有效提高油水两相流分离效果;达到相同油水处理效果的电压数值要大于静态实验;且存在一个临界电磁力数值,只有大于这个临界值,电磁力才能有效促进油水两相流的分离过程。     

Selection of single-stranded DNA molecules that bind and inhibit human thrombin.

Aptamers are double-stranded DNA or single-stranded RNA molecules that bind specific molecular targets. Large randomly generated populations can be enriched in aptamers by in vitro selection and polymerase chain reaction. But so far single-stranded DNA has not been investigated for aptamer properties, nor has a target protein been considered that does not interact physiologically with nucleic acid. Here we describe the isolation of single-stranded DNA aptamers to the protease thrombin of the blood coagulation cascade and report binding affinities in the range 25-200 nM. Sequence data from 32 thrombin aptamers, selected from a pool of DNA containing 60 nucleotides of random sequence, displayed a highly conserved 14-17-base region. Several of these aptamers at nanomolar concentrations inhibited thrombin-catalysed fibrin-clot formation in vitro using either purified fibrinogen or human plasma.     

Folding DNA to create nanoscale shapes and patterns

'Bottom-up fabrication', which exploits the intrinsic properties of atoms and molecules to direct their self-organization, is widely used to make relatively simple nanostructures. A key goal for this approach is to create nanostructures of high complexity, matching that routinely achieved by 'top-down' methods. The self-assembly of DNA molecules provides an attractive route towards this goal. Here I describe a simple method for folding long, single-stranded DNA molecules into arbitrary two-dimensional shapes. The design for a desired shape is made by raster-filling the shape with a 7-kilobase single-stranded scaffold and by choosing over 200 short oligonucleotide 'staple strands' to hold the scaffold in place. Once synthesized and mixed, the staple and scaffold strands self-assemble in a single step. The resulting DNA structures are roughly 100 nm in diameter and approximate desired shapes such as squares, disks and five-pointed stars with a spatial resolution of 6 nm. Because each oligonucleotide can serve as a 6-nm pixel, the structures can be programmed to bear complex patterns such as words and images on their surfaces. Finally, individual DNA structures can be programmed to form larger assemblies, including extended periodic lattices and a hexamer of triangles (which constitutes a 30-megadalton molecular complex).     

Domain of E. coli DNA polymerase I showing sequence homology to T7 DNA polymerase

Escherichia coli contains three DNA polymerases that differ in their size, ability to interact with accessory proteins and biological function. Monomeric DNA polymerase I (Pol I) has a relative molecular mass (Mr) of 103,000 (103K) and is involved primarily in the repair of damaged DNA and the processing of Okazaki fragments; polymerase II is of Mr 120K, and polymerase III has a Mr of 140K, is responsible for the replication of the DNA chromosome and is just one of several proteins that are required for replication. DNA polymerases from bacteriophage as well as those of eukaryotic viral and cellular origin also differ with respect to their size and the number of associated proteins that are required for them to function in replication. However, the template-directed copying of DNA is identical in all cases. The crystal structure of the large proteolytic fragment of Pol I shows that it consists of two domains, the larger of which contains a deep crevice whose dimensions are such that it can bind duplex DNA. The T7 polymerase consists of two subunits, the 80K gene 5 protein and the host-encoded 12K thioredoxin of E. coli. We show here that there is an amino acid sequence homology between at least eight polypeptide segments that form the large cleft in the Klenow fragment and polypeptides in T7 DNA polymerase gene 5 protein, suggesting that this domain evolved from a common precursor. The parts of the Pol I and T7 DNA polymerase molecules that bind the DNA substrate appear to share common structural features, and these features may be shared by all of these varied DNA polymerases.     

复乳法及其改良法制备的干扰素PLGA微球载药释药特性的对比

改良法在复乳法基础上结合了海藻酸钠与Ca2+螯合形成缓释凝胶的原理。分别用上述两法制备蛋白药物干扰素α-1b的聚乳酸聚乙醇酸(PLGA)微球,以包封率、载药量等为指标,评价其理化性质;扫描电镜观察微球内外形态,软件计算其结构参数;用表面蛋白染色、激光共聚焦显微镜观察蛋白在微球表面及骨架内的分布情况;考察微球体外释药行为。与复乳法相比,改良法所得微球有较高的包封率和载药量(61.85%,0.724 3%),表面孔洞少、孔隙率低;内部孔洞大而少,孔隙率无显著差异;蛋白更多地分布在微球内部孔洞壁上而非表面;突释显著降低,缓释时间延长至20 d以上,30 d累积释药约70%。表明改良法所得微球载药释药性能较好,释药符合缓释制剂标准。     

脉冲星旋转磁偶极场的线偏振位置角曲线

1967年发现第一颗脉冲星以来,在用于解释线偏振位置角随脉冲相位变化曲线的旋转矢量模型中,脉冲星磁场通常被近似为静态磁偶极场.但静态磁偶极场假设过于简化,未考虑推迟势效应.文章采用从推迟势导出的旋转真空磁偶极场,研究了在脉冲星磁场为旋转磁偶极场位形下的线偏振位置角曲线.在计算辐射的相位、余纬角和偏振位置角过程中考虑了光行差效应和延迟效应.相比旋转矢量模型的结果,旋转磁偶极场偏振位置角曲线在大磁倾角、碰撞角,或靠近磁轴的内层磁力线上有显著的变形.其与磁力线层等参数的依赖关系避免了传统旋转矢量模型的简并问题--在给定磁倾角和碰撞角情况下所有层磁力线的偏振位置角曲线都一样,这为通过拟合射电偏振观测数据来分辨不同层的辐射,从而限定脉冲星辐射区部位提供了一条有用的途径.     

高压脉冲作用下水中硬岩的电场特性研究

高压脉冲液相放电由于其众多优势,广泛应用在岩石的破碎、石油开采、污水治理等领域.本文通过推导等效放电回路方程,得到放电电流及达到峰值电流的时间表达式,从而在COMSOL Multiphysics中定义脉冲电压,并将其加载在仿真模型的高压电极上.模拟仿真硬岩矿物组成、粒径、电极距离对电场强度和分布特征的影响.结果表明电场强度高度依赖矿石的电学性质、进料粒径、以及矿粒的位置.矿粒的介电常数差异越大,电场越强,分布越不均匀;电场强度向矿石边界转移,体现了电破碎的选择性破碎的机理,以及最大场强随着矿粒尺寸呈指数减小.     

甲壳三糖-g-壳聚糖/累托石纳米复合材料作为基因载体的研究

本文合成了甲壳三糖-g-壳聚糖（TCS），并与累托石插层复合制备纳米复合材料，采用XRD和TEM对其进行了表征。粒径分析结果表明，两种分子量的TCS与pDNA复合物的粒径大小都在75nm左右，而累托石加入后，其粒径都不同程度的增加，最大达到191nm。在PBS中的聚集动力学及琼脂糖凝胶电泳实验发现，高分子量TCS /pDNA复合物较低分子量复合物稳定，而累托石的加入降低了其稳定性。体外转染实验初步表明，甲壳三糖-g-壳聚糖/累托石-DNA复合物能介入肝癌细胞中并表达荧光。该纳米复合材料可作为潜在的非病毒基因载体。     

Covalently closed circles of adenovirus 5 DNA

The genome of adenoviruses is a double-stranded linear DNA molecule with inverted terminal repeats about 100 base pairs (bp) in length and a terminal protein covalently linked to the 5' nucleotide of each strand. Both of these features permit the formation of DNA circles, the inverted repeats allowing the circularization of single-stranded DNA and the terminal protein the joining of one or more molecules to yield double-stranded circles or concatemers. However, although the existence of covalently closed circles has been postulated, double-stranded viral DNA purified from virions or infected cells by conventional methods (that is, using proteases and phenol or chloroform) has always been obtained in a linear form. Here, we present evidence for the existence in adenovirus 5 (Ad5) infected cells of novel structures resulting from covalent head-to-tail joining of viral DNA molecules and show that these structures are due at least in part to the formation of covalently closed circles.     

基于Comsol Multiphysics的新型高压直流继电器电弧仿真分析

传统直流电磁继电器采用电磁驱动方式，分断间距为2~3 mm，在工作负载增大时需增加线圈匝数，导致继电器质量增加、灭弧难度增大。为解决上述问题，采用微电机蜗轮蜗杆作为驱动机构、分断间距相比传统电磁继电器大数倍的新型高压直流继电器设计方案，并利用多物理场耦合软件Comsol Multiphysics，基于磁流体动力学（MHD）理论建立了直流继电器的二维电弧仿真模型。考虑到分断间距、电流、磁场强度以及分断速度等条件对电弧的影响，分析了不同上述条件下电弧的运动特性，得到了适定参数下较为合理的间距、工作电流、磁场强度和分断速度，为新型高压直流继电器灭弧系统的结构设计提供参考。     

Genome linking with yeast artificial chromosomes

The haploid genome of Caenorhabditis elegans consists of some 80 x 10(6) base pairs of DNA contained in six chromosomes. The large number of interesting loci that have been recognized by mutation, and the accuracy of the genetic map, mean that a physical map of the genome is highly desirable, because it will facilitate the molecular cloning of chosen loci. The first steps towards such a map used a fingerprinting method to link cosmid clones together. This approach reached its practical limit last year, when 90-95% of the genome had been cloned into 17,500 cosmids assembled into some 700 clusters (contigs), but the linking clones needed were either non-existent or extremely rare. Anticipating this, we had planned to link by physical means--probably by hybridization to NotI fragments separated by pulse field gel electrophoresis. NotI recognizes an eight base sequence of GC pairs; thus the fragments should be large enough to bridge regions that clone poorly in cosmids, and, with no selective step involved, would necessarily be fully representative. However, with the availability of a yeast artificial chromosome (YAC) vector, we decided to use this alternative source of large DNA fragments to obtain linkage. The technique involves the ligation of large (50-1,000 kilobase) genomic fragments into a vector that provides centromeric, telomeric and selective functions; the constructs are then introduced into Saccharomyces cerevisiae, and replicate in the same manner as the host chromosomes.