单分子化学与物理

单分子科学的概念是在扫描探针显微镜、激光镊子和其他相关光谱法在纳米级或分子级研究中的应用中发展起来的。我们在此对近来单分子化学和物理的进展作一评论性的回顾，并主要介绍单分子化学和物理的概念以及单分子表面研究、单分子光谱检测、单原子和/蜮分子操纵、单分子荧光法、激光摄子、生物分子的力学研究，以及所谓单电子效应和单电子器件的进展情况。本文最后强调了单分子科学和技术的重要性。     

分子水平的仿生科学

本文讲座了在分子水平进行的模仿生物大分子生物功能的研究现状，研究技术，及其在基础研究和生物制品生产中的应用。     

介电泳技术在生物信号检测和相分离阻变材料中的应用

介电泳是操纵微纳米级粒子的强大工具,已经在生物细胞和无机微粒的分离、检测、操控方面得到了广泛的应用.本文突破对介电泳技术的传统定位,简要介绍介电泳效应的两例新的应用.首先是和新兴的纳米孔技术结合,利用介电泳的富集效应,在微纳环境下对单分子行为进行操控,解决目前纳米孔基因测序面临的通量低等难题.其次在某些相分离固体材料中,介电泳可以通过调节电子相的几何结构引起渗流,从而实现电致阻变效应.这些研究不仅扩大了介电泳技术的应用范围,且具有多学科技术交叉融合的特点,为生物检测技术的开发创新以及新型功能材料的设计提供了新的思路.     

基于光诱导介电泳的微纳米生物粒子操纵平台关键技术

在分析介电泳的微纳米生物粒子操纵研究现状和存在问题基础上，研究了基于光诱导介电泳的微纳米生物粒子操纵的理论基础和建模仿真，给出了光诱导介电泳芯片在空间电场分布和不同高度介电泳力分布关系．在此基础上进行微操纵系统的核心部件——光电导层芯片的选材、制作工艺和性能分析测试，给出了悬浮液层分压和有效电压频谱关系图．最后，组合机器视觉检测与实时跟踪子系统，构建了基于光诱导介电泳的微纳米生物粒子操纵实验平台，完成了对微纳米生物粒子快速聚集、输运、分离等操纵实验，为建立以微流控芯片为基础的重大疾病的快速、准确、低成本的检测和早期诊断提供了基础．     

NIH未来医学研究领域及战略

美国国立卫生院（National Institutes of Health，NIH）是当今世界上最著名的医学领域研究和管理机构。2003年9月该机构公布了未来10年能导致生命科学原创发现的重点研究领域和研究战略。重点研究领域包括：1．生物学通道和网络研究，重点是蛋白组学和代谢组学研究。2．建立分子库和分子图像及筛选中心，开展化学信息学研究和分子图像探针合成关键设备研制。3．结构生物学研究领域。4．生物信息学和计算生物学研究领域。5．纳米医学研究领域。研究战略之一是加强未来医学研究队伍建设，重点支持高风险研究，建立多学科交叉研究队伍和建立政府和私人机构的合作联系。研究战略之二是重铸临床研究，重点加快科学发现向临床应用转化的研究，加强临床工作人员的培训和建立临床研究网络。NIH确定的研究领域和研究战略对加速分子医学革命的到来有重要意义，对我国生命科学研究和宏观管理具有极大的参考价值。     

中国科学院化学研究所

化学研究所是以开展基础研究和有重点地开展国家急需的、有重大战略目标的高技术创新研究为主，并与高新技术应用和转化工作相协调发展的多学科、综合性研究所。化学所的奋斗目标是建设具有世界一流水平的基础研究基地和优秀人才培养基地，为我国国民经济建设、国家安全和提高人民生活水平等方面做出重大贡献。重点研究领域为：化学反应动态学和结构化学、分子聚集体化学、合成与制备科学、高技术材料研究、纳米科技、化学生物学和理论化学等。     

RAPD技术在生物学研究中的应用

ＲＡＰＤ标记具有简易,快速,灵敏,经济的优点,本文介绍了该技术在生物物种鉴别,遗传多样性,基因定位,分子连锁图谱构建和外源导入基因的分子检测等生物学领域广泛应用的概况。由ＲＡＰＤ转化而来的ＲＡＰＤ－ＰＣＲ－ＳＣＡＲ标记稳定,灵敏,准确性高,在种质资源鉴别,分子标记辅助育种中有着潜在的应用前景。     

在硅表面上的单原子操纵

室温条件下Si表面的单原子操纵在基础研究和微电子应用两方面都具有十分重要的意义。本文详细介绍了利用STM在Si(111)×7、Si（100）－2×1：H表面进行单原子操纵和加工原子级人工微结构的基本原理和技术方法。综述了近年来这一领域研究工作的最新进展，并探讨单原子操纵技术和激光选健技术相结合从而实现选键化学反应或“分子手术”的可能性和前景。     

生物炭对土壤中农药的吸附-解吸行为和生物有效性的影响综述

生物炭是生物质在缺氧条件下热解产生的一种多孔富炭物质,由于其巨大的比表面积和表面理化性质,生物炭对水土环境介质中的有机污染物有较强的吸附能力。本文说明了生物炭的施用对土壤中农药吸附-解吸行为以及生物有效性的影响;由于自身疏松多孔、比表面积和表面能较大等性质,以及高度芳香化结构使其能够强烈的吸附土壤中残留的农药污染物;并且使解吸滞后现象增强,减少农药的解吸量。另外,生物炭的施用减弱了土壤中农药的生物有效性及药效。同时,总结了目前在生物炭对在农药迁移机理的影响与其在农药污染控制中的应用研究中存在的不足之处,提出需要解决的主要科学问题。最后,对生物炭在农药污染控制中的应用前景进行了展望。     

基于微纳制造的下一代基因测序系统研究现状与展望

基因是遗传的基本单元,是DNA或RNA分子上具有遗传信息的特定核苷酸序列,它不仅与生物体的生、老、病、死等一切生命现象直接相关,也是决定生命健康的内在因素,下一代基因测序仪的研制目标是希望能够将基因测序的成本大幅度降低,测序速度快速提高,推进基因组技术的个性化医学走向现实,基于固体纳米孔的基因测序是最有可能实现高通量的物理测序技术,但目前面临生物分子电信号的信噪比低、生物分子过孔速度太快以及生物分子在纳米孔内的输运规律缺失等挑战,设计多模式纳米孔传感器,通过多模式信号交叉对比,同时降低纳米孔长度等方法,是提高检测灵敏度的有效方法,借鉴生物纳米孔对DNA过孔速度的控制,利用磁场、声叉等外力场可以为单碱基的辨识奠定基础,当纳米通道的几何尺寸小于双电层厚度时,基于连续理论的泊松-玻尔兹曼方程和纳维-斯托克斯方程将面临挑战,建立纳通道内生物分子输运规律将丰富流体力学的研究内容。     

Study of molecular events in cells by fluorescence correlation spectroscopy

To understand processes in a living cell, sophisticated and creative approaches are required that can be used for gathering quantitative information about large number of components interacting across temporal and spatial scales without major disruption of the integral network of processes. A physical method of analysis that can meet these requirements is fluorescence correlation spectroscopy (FCS), which is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution. Since its introduction about 3 decades ago, this until recently emerging technology has reached maturity. As commercially built equipment is now available, FCS is extensively applied for extracting biological information from living cells unattainable by other methods, and new biological concepts are formulated based on findings by FCS. In this review, we focus on examples in the field of molecular cellular biology. The versatility of the technique in this field is illustrated in studies of single-molecule dynamics and conformational flexibility of proteins, and the relevance of conformational flexibility for biological functions regarding the multispecificity of antibodies, modulation of activity of C5a receptors in clathrin-mediated endocytosis and multiplicity of functional responses mediated by the p53 tumor suppressor protein; quantitative characterization of physicochemical properties of the cellular interior; protein trafficking; and ligand-receptor interactions. FCS can also be used to study cell-to-cell communication, here exemplified by clustering of apoptotic cells via bystander killing by hydrogen peroxide.Received 15 July 2004; received after revision 13 October 2004; accepted 12 November 2004     

Ein Mikro-Homogenisator

Summary Homogenation of very small amounts of biological material (e.g. single cells) can be carried out without losses by using a loop of stainless steel thread inside of a capillary. A useful device is described.     

Ultrastructural localization of 5-methylcytosine on DNA and RNA

DNA methylation is the major epigenetic modification and it is involved in the negative regulation of gene expression. Its alteration can lead to neoplastic transformation. Several biomolecular approaches are nowadays used to study this modification on DNA, but also on RNA molecules, which are known to play a role in different biological processes. RNA methylation is one of the most common RNA modifications and 5-methylcytosine presence has recently been suggested in mRNA. However, an analysis of nucleic acid methylation at electron microscope is still lacking. Therefore, we visualized DNA methylation status and RNA methylation sites in the interphase nucleus of HeLa cells and rat hepatocytes by ultrastructural immunocytochemistry and cytochemical staining. This approach represents an efficient alternative to study nucleic acid methylation. In particular, this ultrastructural method makes the visualization of this epigenetic modification on a single RNA molecule possible, thus overcoming the technical limitations for a (pre-)mRNA methylation analysis.     

Genetic studies of diseases

The biological system is a complex physicochemical system consisting of numerous dynamic networks of biochemical reactions and signaling interactions between cellular components. This complexity makes it virtually unanalyzable by traditional methods. Hence, biological networks have been developed as a platform for integrating information from high- to low-throughput experiments for analysis of biological systems. The network analysis approach is vital for successful quantitative modeling of biological systems. The numerous online pathway databases vary widely in coverage and representation of biological processes. An integrated network-based information system for querying, visualization and analysis promised successful integration of data on a large scale. Such integrated systems will greatly facilitate the understanding of biological interactions and experimental verification.     

AID and Apobec3G haphazard deamination and mutational diversity

Activation-induced deoxycytidine deaminase (AID) and Apobec 3G (Apo3G) cause mutational diversity by initiating mutations on regions of single-stranded (ss) DNA. Expressed in B cells, AID deaminates C → U in actively transcribed immunoglobulin (Ig) variable and switch regions to initiate the somatic hypermutation (SHM) and class switch recombination (CSR) that are essential for antibody diversity. Apo3G expressed in T cells catalyzes C deaminations on reverse transcribed cDNA causing HIV-1 retroviral inactivation. When operating properly, AID- and Apo3G-initiated mutations boost human fitness. Yet, both enzymes are potentially powerful somatic cell “mutators”. Loss of regulated expression and proper genome targeting can cause human cancer. Here, we review well-established biological roles of AID and Apo3G. We provide a synopsis of AID partnering proteins during SHM and CSR, and describe how an Apo2 crystal structure provides “surrogate” insight for AID and Apo3G biochemical behavior. However, large gaps remain in our understanding of how dC deaminases search ssDNA to identify trinucleotide motifs to deaminate. We discuss two recent methods to analyze ssDNA scanning and deamination. Apo3G scanning and deamination is visualized in real-time using single-molecule FRET, and AID deamination efficiencies are determined with a random walk analysis. AID and Apo3G encounter many candidate deamination sites while scanning ssDNA. Generating mutational diversity is a principal aim of AID and an important ancillary property of Apo3G. Success seems likely to involve hit and miss deamination motif targeting, biased strongly toward miss.     

Collective cell migration has distinct directionality and speed dynamics

When a constraint is removed, confluent cells migrate directionally into the available space. How the migration directionality and speed increase are initiated at the leading edge and propagate into neighboring cells are not well understood. Using a quantitative visualization technique—Particle Image Velocimetry (PIV)—we revealed that migration directionality and speed had strikingly different dynamics. Migration directionality increases as a wave propagating from the leading edge into the cell sheet, while the increase in cell migration speed is maintained only at the leading edge. The overall directionality steadily increases with time as cells migrate into the cell-free space, but migration speed remains largely the same. A particle-based compass (PBC) model suggests cellular interplay (which depends on cell–cell distance) and migration speed are sufficient to capture the dynamics of migration directionality revealed experimentally. Extracellular Ca²⁺ regulated both migration speed and directionality, but in a significantly different way, suggested by the correlation between directionality and speed only in some dynamic ranges. Our experimental and modeling results reveal distinct directionality and speed dynamics in collective migration, and these factors can be regulated by extracellular Ca²⁺ through cellular interplay. Quantitative visualization using PIV and our PBC model thus provide a powerful approach to dissect the mechanisms of collective cell migration.     

Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain

Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood–brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000? was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.     

Structure and dynamics of rotary V<Subscript>1</Subscript> motor

Rotary ATPases are unique rotary molecular motors that function as energy conversion machines. Among all known rotary ATPases, F₁-ATPase is the best characterized rotary molecular motor. There are many high-resolution crystal structures and the rotation dynamics have been investigated in detail by extensive single-molecule studies. In contrast, knowledge on the structure and rotation dynamics of V₁-ATPase, another rotary ATPase, has been limited. However, recent high-resolution structural studies and single-molecule studies on V₁-ATPase have provided new insights on how the catalytic sites in this molecular motor change its conformation during rotation driven by ATP hydrolysis. In this review, we summarize recent information on the structural features and rotary dynamics of V₁-ATPase revealed from structural and single-molecule approaches and discuss the possible chemomechanical coupling scheme of V₁-ATPase with a focus on differences between rotary molecular motors.     

Genetic visualization of notch signaling in mammalian neurogenesis

Notch signaling plays crucial roles in fate determination and the differentiation of neural stem cells in embryonic and adult brains. It is now clear that the notch pathway is under more complex and dynamic regulation than previously thought. To understand the functional details of notch signaling more precisely, it is important to reveal when, where, and how notch signaling is dynamically communicated between cells, for which the visualization of notch signaling is essential. In this review, we introduce recent technical advances in the visualization of notch signaling during neural development and in the adult brain, and we discuss the physiological significance of dynamic regulation of notch signaling.     

Binding of matrilysin-1 to human epithelial cells promotes its activity

Matrix metalloproteinase-7 (MMP-7, matrilysin- 1) modulates crucial biological events by processing many epithelial cell surface-associated effectors. We addressed MMP-7 interaction with human epithelial cells and its resulting activity. In human endometrium, a model of controlled tissue remodeling, proMMP-7 was diffusely immunolocalized inside epithelial cells, whereas MMP-7 delineated their entire plasma membrane. Endometrial explants preferentially retained active MMP-7, but not proMMP-7. Endometrial epithelial cells and carcinoma cells from various tissues bound active MMP-7. Endometrial carcinoma-derived Ishikawa cells showed high affinity (K_D of ~2.5 nM) and capacity (~260 000 sites per cell) for MMP-7. MMP-7 binding decreased by extracting membrane sterols or interfering with heparan sulfate proteoglycans, and was abrogated by tissue inhibitors of metalloproteinase-2 (TIMP-2) or synthetic MMP inhibitors. Bound MMP-7 not only remained fully active towards a macromolecular substrate but also became resistant to TIMP-2. We conclude that MMP-7-selective targeting to the plasma membrane of epithelial cells promotes its activity by conferring resistance to TIMP-2. A. Berton, C. Selvais: These authors contributed equally to this work. P. J. Courtoy, E. Marbaix, H. Emonard: These authors contributed equally to the supervision of this work. Received 20 September 2006; received after revision 30 November 2006; accepted 18 January 2007