The motor protein myosin-I produces its working stroke in two steps

Many types of cellular motility, including muscle contraction, are driven by the cyclical interaction of the motor protein myosin with actin filaments, coupled to the breakdown of ATP. It is thought that myosin binds to actin and then produces force and movement as it 'tilts' or 'rocks' into one or more subsequent, stable conformations. Here we use an optical-tweezers transducer to measure the mechanical transitions made by a single myosin head while it is attached to actin. We find that two members of the myosin-I family, rat liver myosin-I of relative molecular mass 130,000 (M(r) 130K) and chick intestinal brush-border myosin-I, produce movement in two distinct steps. The initial movement (of roughly 6 nanometres) is produced within 10 milliseconds of actomyosin binding, and the second step (of roughly 5.5 nanometres) occurs after a variable time delay. The duration of the period following the second step is also variable and depends on the concentration of ATP. At the highest time resolution possible (about 1 millisecond), we cannot detect this second step when studying the single-headed subfragment-1 of fast skeletal muscle myosin II. The slower kinetics of myosin-I have allowed us to observe the separate mechanical states that contribute to its working stroke.     

莱茵衣藻外源基因整合特征分析及蛋白表达

将衣藻表达载体pSP108转化莱茵衣藻细胞壁缺陷型藻株CC-400,通过接头PCR获得并分析阳性转化子中ble基因的侧翼序列发现:外源基因的插入位点呈现随机性,并且有些转化子中衣藻基因组和质粒序列发生断裂和重排.通过间接ELISA分析外源蛋白表达量,并结合外源基因整合状态时发现:在ble基因启动子核心区域缺失的阳性转化子中,ble可以利用自身基因启动子进行蛋白表达,但表达量相比使用质粒载体PSP108上RBCS2启动子的表达量要低;在部分启动子完整的阳性转化子中蛋白表达量低下,可能与衣藻基因组大片段的缺失相关.     

YidC mediates membrane protein insertion in bacteria

The basic machinery for the translocation of proteins into or across membranes is remarkably conserved from Escherichia coli to humans. In eukaryotes, proteins are inserted into the endoplasmic reticulum using the signal recognition particle (SRP) and the SRP receptor, as well as the integral membrane Sec61 trimeric complex (composed of alpha, beta and gamma subunits). In bacteria, most proteins are inserted by a related pathway that includes the SRP homologue Ffh, the SRP receptor FtsY, and the SecYEG trimeric complex, where Y and E are related to the Sec61 alpha and gamma subunits, respectively. Proteins in bacteria that exhibit no dependence on the Sec translocase were previously thought to insert into the membrane directly without the aid of a protein machinery. Here we show that membrane insertion of two Sec-independent proteins requires YidC. YidC is essential for E. coli viability and homologues are present in mitochondria and chloroplasts. Depletion of YidC also interferes with insertion of Sec-dependent membrane proteins, but it has only a minor effect on the export of secretory proteins. These results provide evidence for an additional component of the translocation machinery that is specialized for the integration of membrane proteins.     

家蚕浓核病毒BmDNV-1(伊那株)结构蛋白基因VP4的克隆、表达及抗体制备

为研发新的日本血吸虫(Schistosoma japonicum)疫苗候选抗原基因,为血吸虫诊断和疫苗研制提供依据,提取和纯化S.japonicum成虫DNA,PCR扩增相关基因,然后将其克隆入pBST载体,对菌落PCR阳性克隆子测序和分析.菌落PCR获得一条与PCR产物一致的DNA片段,PCR产物与S.japonicum 22 600抗原分子DNA序列具有高度同源性,636 bp大小,oRF189 bp,ORF小是全长的蒯读框,只获得部分编码区,能编码63个氨基酸,表达产物含有一个包含9个氨基酸残基的潜在螺旋跨膜片段(25～33位)和一个酪氨酸激酶磷酸化位点(38～39位).本研究所获取的阳性克隆是与S.japonicum 22 600抗原编码基因有高度同源性的基因.通过Blastn和BLASTP分析,预测该基因为体膜相关蛋白基因,可编码血吸虫体壁相关蛋白,可能是信号传递分子.     

基于虚拟样机技术的织机引纬机构研究

为了研究共轭凸轮引纬机构的动力学、运动学特性,采用ADAMS软件建立虚拟样机的方法,分别对共轭凸轮引纬机构进行了运动学、动力学仿真分析,得到了连续运转周期的动力学、运动学曲线,通过改进共轭凸轮轮廓曲线,仿真出新的引纬运动曲线,得到改进轮廓曲线有助于稳定和提高引纬质量的结论.提出的研究方法为设计开发新型织机,以及对现有织机进行改进,提供了一种有效的分析和设计手段.     

城建档案的利用应做好四个环节

城建档案的利用是城建档案工作的重要环节，是实现城建档案信息资源转化为社会、经济效益的重要途径。新形势下，为做好城建档案的综合利用工作，应着重做好城建档案利用的推介与宣传、创新、保密与保护及反馈四个方面的工作。     

论计算机取证的原则和步骤

由于计算机证据具有不同于一般证据的特殊性,其获取、传输、存储和分析都需要遵循一定的原则和步骤,否则,无法保证证据的关联性、客观性和合法性。针对计算机证据的特点论述了计算机取证应该遵循的原则和计算机取证的步骤。     

英语精读课堂教学步骤谈

在大学英语精读课程的教学中,不少教师认为应该在字、词、句、篇等各方面精讲细练,此做法容易导致教师难讲、学生厌听的现象。按照自由发言、导读、课文讲解和教学反馈的教学步骤能调动学生学习的积极性、主动性和创造性。     

Synchronised transmembrane insertion and glycosylation of a nascent membrane protein.

Studies of the synthesis and incorporation of the vesicular stomatitis virus glycoprotein into membranes in a synchronised cell-free system demonstrate a tight coupling between polypeptide synthesis and membrane insertion, as a result of which the nascent chain crosses the membrane. The studies reveal a surprisingly precise sequence by which the nascent chain of this membrane glycoprotein is glycosylated in two steps. These findings have important implications for the mechanisms of membrane assembly.     

Density functional theory study on the insertion reaction mechanism of dibromocarbene with formaldehyde

The insertion reaction mechanism of CBr2 with CH3CH2O has been studied by using the B3LYP/6-311G(d) and CCSD(T)/6-311G(d) at single point. The geometries of reactions,transition state and products were completely optimized. All the transition state is verified by the vibrational analysis and the internal re-action coordinate (IRC) calculations. The results show that reaction (1) is the dominant reaction path,which proceeds via two steps: i) two reactants form an intermediate (IM1),which is an exothermal re-action of 8.62 kJ·mol?1 without energy barrier; ii) P1 is obtained via the TS1 and the H-shift,in which the energy barrier is 44.53 kJ·mol?1. The statistical thermodynamics and Eyring transition state theory with Wigner correction are used to study the thermodynamic and kinetic characters of this reaction in temperature range from 100 to 2200 K. The results show that the appropriate reaction temperature ranges from 200 to 1900 K at 1.0 atm,in which the reaction has a bigger spontaneity capability,equi-librium constant (K) and higher rate constant (k).     

T细胞蛋白p80调控c-Myc表达的机理

为了解p80在正常T细胞以及T细胞癌变过程中的作用,在缺失p80的T淋巴瘤细胞中重新表达p80,其结果显示:原癌基因c-Myc的表达受到显著抑制,且细胞周期在G1期出现了停滞.定量PCR的结果表明:p80对c-Myc表达的抑制是转录水平的抑制.双荧光素酶报告基因试验表明:p80对c-Myc的抑制作用定位于c-Myc启动子上相对于转录起始位点的-1042bp～-630bp区域.运用TFSEARCH软件对这一区域的分析发现存在多个c-Myb结合位点.在稳定表达p80的T淋巴癌细胞中敲低c-Myb,表明p80对c-Myc的转录抑制是通过c-Myb来实现的.免疫共沉淀实验表明p80和c-Myb在细胞中存在相互作用.进一步的研究显示p80对c-Myc的转录抑制依赖于组蛋白去乙酰化酶的活性.研究结果表明p80有可能通过与c-Myb的相互作用将组蛋白去乙酰化酶募集至c-Myc启动子区域,并通过组蛋白的去乙酰化抑制c-Myc的表达.     

Detection of refolding conformers of complement protein C9 during insertion into membranes

Human complement protein C9 is a hydrophilic serum glycoprotein responsible for efficient expression of the cytotoxic and cytolytic functions of complement. It assembles on the surface of a target cell together with C5, C6, C7 and C8 to form the membrane attack complex (MAC) and therefore has to change structure to become an integral membrane protein. As the protein assumes a stable structure in an aqueous environment, the question arises as to how it can enter the hydrophobic interior of a membrane. During MAC assembly C9 polymerizes into a circular structure, termed poly(C9) (ref. 8), which is responsible for the cylindrical electron microscopic appearance of the MAC. The suggestion has been made that C9 must at least partly unfold in order to enter a membrane and also that polymerization of the molecule is intimately linked to insertion and cytotoxicity. The extent of unfolding and the mechanism of polymerization are not understood, nor is it known precisely which parts of the molecule participate in the proposed structural changes. We have been able to capture refolding C9 conformers during membrane insertion with the help of sequence-specific anti-peptide antibodies. Some of these antibodies inhibit C9-mediated haemolysis but not C9 polymerization, while others have the opposite effect. This suggests that the two processes are independent.     

HCC细胞膜相关蛋白的快速鉴定

为快捷、特异地鉴定HCC（hepatocellular carcinoma, 人肝细胞癌）细胞的膜相关蛋白,用DOC（脱氧胆酸钠）对贴壁培养的HCC细胞系HepG2进行处理,使细胞皱缩并脱离培养面,收集未脱离的HepG2膜相关蛋白,经过聚丙烯酰胺凝胶电泳分离,最后应用ESI-QUAD-TOF MS(electrospray ionization quadrupole timeofflight mass spectrometry),成功鉴定出包括热休克蛋白90α、玻璃体粘连蛋白前体、层粘连蛋白α5亚单位     

计算机视觉系统的二步法颜色恒常性

理论研究表明 ,三色视觉系统在单视野条件下 ,三维颜色恒常性的计算问题只有在一些约束下才能解决。约束选择的合理与否和后续的任务密切相关。针对不同环境光照条件下的人体肤色分割的具体任务 ,提出了一个新的关于三色系统颜色恒常性问题的物理模型 :1 )将视觉系统所获得的原始信息分裂为亮度信息和颜色信息。 2 )将传统的颜色恒常性问题分解为基于亮度的亮度恒常性和基于颜色信息的颜色恒常性 ,并用二维描述子描述等亮度彩色图像中物理表面的颜色特征。该描述子具有颜色恒常的特性。基于颜色的图像分割实验结果表明 ,二步法颜色恒常性技术是一种非常有效的方法。