天花粉蛋白对红细胞损伤作用的AFM研究

目的：利用原子力显微镜（atomic force microscopy,简称AFM）观察红细胞（red blood cells,简称RBC）与天花粉蛋白（trichosanthin,简称TCS）作用后形态上的变化以及细胞膜的损伤情况。方法：将1.2mg/ml的TCS溶液与红细胞的PBS缓冲溶液（pH7.4)按1：4的比例混合,在35℃温度下作用2h后,用原子力显微镜观察受损的红细胞,与正常红细胞进行对比。结果：（1）与正常红细胞相比,与TCS作用后的红细胞的高度明显降低,凹陷部分更加明显。（2）对红细胞上小范围扫描成像的结果显示,受损后的红细胞膜表面结构发生了变化,膜表面颗粒排列的特征依然存在,但颗粒之间开始产生连接。结论：TCS能损伤红细胞膜,改变细胞膜的微结构,引起红细胞的溶血作用。     

女贞和珊瑚树叶片表面特征的AFM观察

应用原子力显微镜观察了女贞(Ligustrum lucidum)、珊瑚树(Viburnum odoratissimum)幼叶和成熟叶的表面特征,并探讨了叶面微结构对滞尘能力的可能影响以及抵抗干旱、污染物等胁迫的能力。女贞幼叶和成熟叶正背面的粗糙度Ra分别为417.8、794.5,1069、957.4 nm;珊瑚树幼叶和成熟叶正背面的粗糙度Ra分别为471.3、469.6,291.1、865.9 nm。和幼叶相比,成熟叶表面的粗糙度发生变化,但2个物种的变化趋势不同,这种变化可能与气孔的发育以及外界环境条件对叶片表面形态结构、蜡质含量和成分的影响不同有关。叶片表面存在大量的沟状、孔状峰谷区域和直径约为10 μm的凹陷,有利于PM₁₀的滞留。女贞和珊瑚树成熟叶气孔只分布在叶下表皮且下陷。这些特征均说明女贞和珊瑚树具有较强的滞尘能力和抵抗干旱、污染物胁迫的能力,作为绿篱植物对消减城市大气颗粒物污染和提高空气质量具有重要的意义。     

原子力显微镜 (AFM) 在细菌生物被膜研究中的应用

原子力显微镜(AFM)作为一项重要的表面可视化技术,以其独特的优势(纳米级的空间分辨率、皮牛级力灵敏度、免标记、可在溶液环境下工作)被广泛应用于生物被膜的研究。AFM不仅可以在近生理环境下对生物被膜表面超微形貌进行可视化表征,同时还可以通过纳米压痕对生物被膜的机械特性(弹性和粘性)进行定量测量,利用AFM单细胞和单分子力谱技术可以获得生物被膜形成过程中细胞-基底以及细胞-细胞之间的相互作用力,为生物被膜的实时原位系统研究提供了可行性。本文简述了AFM的基本操作原理,综述了近年来AFM用于生物被膜表面超微结构成像、机械特性测量以及相互作用力研究方面的进展,并对AFM在生物被膜研究中面临的问题和未来的发展方向进行了讨论。     

Identification of P2X2/P2X4/P2X6 heterotrimeric receptors using atomic force microscopy (AFM) imaging

Seven P2X purinergic receptor subunits have been identified: P2X1–P2X7. The overlapping expression of P2X2, P2X4 and P2X6 subunits has been shown in different cell types, and functional analysis of P2X receptors in Leydig cells suggests that the three subunits might interact. Here, His₆-tagged P2X2, HA-tagged P2X4 and FLAG-tagged P2X6 subunits were co-expressed in tsA 201 cells. After sequential co-immunoprecipitation using anti-HA and anti-FLAG beads, all three subunits were present, demonstrating their interaction. Atomic force microscopy (AFM) imaging revealed receptors that were specifically decorated by both an anti-His₆ antibody and an anti-HA Fab fragment, indicating the presence of a P2X2/4/6 heterotrimer. To our knowledge, this is the first report of a P2X receptor containing three different subunits.     

北京西山绿化树种秋季滞纳PM2.5能力及其与叶表面AFM特征的关系

以北京西山6种绿化树种白皮松、油松、柳树、五角枫、银杏、山杨为对象,应用气溶胶再发生器对植物叶片秋季PM_2.5吸附量进行定量研究,同时应用原子力显微镜(AFM)观察叶表面微形态特征,分析了叶表面粗糙度等参数,阐释了各树种叶片吸附PM_2.5的机制.结果表明: 不同树种单位叶面积PM_2.5吸附量排序为白皮松(2.44±0.22 μg·cm^-2)＞油松(2.40±0.23 μg·cm^-2)＞柳树(1.62±0.09 μg·cm^-2)＞五角枫(1.23±0.01 μg·cm^-2)＞银杏(1.00±0.07 μg·cm^-2)＞山杨(0.97±0.03 μg·cm^-2);从秋季不同月份来看,不同树种单位叶面积PM_2.5吸附量表现为11月(2.33±0.43 μg·cm^-2)＞10月(1.62±0.64 μg·cm^-2)＞9月(1.51±0.50 μg·cm^-2).白皮松和油松有大量凹陷和突起,相对高差较大,粗糙度较大,吸滞PM_2.5能力强;柳树和五角枫叶片有褶皱,粗糙度相对较高,分布有大量的突起和凹陷,吸滞PM_2.5能力居中;银杏和山杨因其叶表面平滑、气孔多为长圆形,粗糙度较小,吸滞PM_2.5能力较弱.不同树种正背面粗糙度平均值为白皮松(149.91±16.38 nm)＞油松(124.47±10.52 nm)＞柳树(98.85±5.36 nm)＞五角枫(93.74±21.75 nm)＞银杏(80.84±0.88 nm)＞山杨(67.72±8.66 nm),这与不同树种单位叶面积PM_2.5吸附量排序完全一致,叶片粗糙度与单位叶面积PM_2.5吸附量呈显著正相关(R²=0.9498).为提高城市植被的环境效应,可选择叶表面形态有利于吸滞PM_2.5等颗粒物的树种.     

人工操纵病毒的原子力显微镜研究

人工操纵生物大分子是目前科学研究的一个前沿领域, 我们利用改进的“分子梳”方法,首次实现了复杂的体系——一种线性噬菌体病毒的人工拉直与定向. 这种操纵是在大面积平整的固体表面实现的, 并利用原子力显微镜对拉直前后的病毒进行了观察与测量.     

钝顶螺旋藻表面微观形貌的原子力显微镜研究

该文应用原子力显微镜(AFM)纳米级的分辨率对钝顶螺旋藻(Spirulina platensis)表面微观形貌进行了研究,获得了扫描范围为5.000×5.000μm、1.000×1.000μm和400.0×400.0nm三组清晰、稳定的图像,并对其进行了线性分析。结果表明:螺旋藻表面由紧密且无序堆积的突起结构组成,其高度小于20nm;突起结构高度从3nm~15nm不等,平均高度约为8~9nm。此法用于生物体表面,操作简单、快速、灵敏度好且样品无损伤,结果令人满意。     

Atomic force microscopy study of the collagen fibre structure

Observations of intact reconstituted and native collagen fibres were performed with the atomic force microscope. The results are compared between the two types of fibres and with those obtained previously with the electron microscope on freeze-etched or negative stained samples. Some of the findings presented here indicate that the specimens observed in air with the atomic force microscope were still in a hydrated state.     

原子力显微镜研究UV-B对I型胶原结构的影响

利用原子力显微镜(AFM)成像技术观察胶原蛋白溶液在UV-B照射前后形态的变化,发现UV-B引起胶原纤维交联度的增加,当交联达一定程度后,照射时间的增加对交联度增加的影响不明显。AFM作为一种高分辨的表面分析仪器,为分子生物学领域的研究提供了一种新的手段。是探讨胶原光作用机理直观、有效的方法。     

杜仲抗真菌蛋白晶体生长的原子力显微成像研究——快速生长与晶面生长速率

杜仲抗真菌蛋白(Eucommiaantifungalprotein,EAFP)的单晶体具有在几小时内就可长大的快速生长特性.用原子力显微成像(atomicforcemicroscope,AFM)技术,原位实时观测了EAFP单斜晶体生长过程中的｛10 0｝表面形貌动态变化,并分别在不同的过饱和度下测量了其生长速率.结果表明,EAFP晶体生长的速率与蛋白质溶液的过饱和度相关,在过饱和度高时(σ =1 78)晶面生长极快;在中等过饱和度(σ =1 5)下,其晶面台阶的生长速率沿b,c方向分别为 12nm/s和 2 4 2nm/s,比溶菌酶生长速率(6～ 7nm/s)快很多;在蛋白质浓度很低的情况下,其生长速率仍与其他蛋白质相当.EAFP晶体快速生长可能与该分子尺寸较小,内部结构紧凑,分子骨架呈刚性和分子表面性质等其固有特性密切相关.沉淀剂浓度对EAFP晶体生长也有影响.过饱和度很低时,提高沉淀剂浓度会干扰晶体生长.     

利用原子力显微镜对A型流感病毒的形态学研究

利用透射电子显微镜(TEM)和原子力显微镜(AFM)观察流感病毒(H1N1),探讨AFM在病毒形态研究中的应用,为病毒形态学研究提供一种新型、简便、快捷的工具.TEM采用磷钨酸负染方法,AFM采用轻敲模式在大气常温下扫描成像,并对主要指标长度(直径)、Ra、Rq等进行测量.两种方法最终得到相似的形态学结果,流感病毒呈球状、丝状,并有一些形状介于两者之间.TEM提供了流感病毒二维图像,可见钉状突起,AFM则呈现了流感病毒三维图像,且可见病毒表面有凹凸不平的特征和边缘有齿轮状的突起,同时获得表面粗糙度等可以量化指标.与TEM观察相比,原子力显微镜是一种制样简单、观察直观的新型病毒形态学研究工具,其表征参数可以作为病毒形态学研究的量化指标.     

Fine mapping of inherent flexibility variation along DNA molecules. Validation by atomic force microscopy (AFM) in buffer

Curvature and flexibility are structural properties of central importance to genome function. However, due to the difficulties in finding suitable experimental conditions, methods for studying one without the interference of the other have proven to be difficult. We propose a new approach that provides a measure of inherent flexibility of DNA by taking advantage of two powerful techniques, X-ray crystallography and nuclear magnetic resonance. Both techniques are able to detect local curvature on DNA fragments but, while the first analyzes DNA in the solid state, the second works on DNA in solution. Comparison of the two data sets allowed us to calculate the relative contribution to flexibility of the three rotations and three translations, which relate successive base pair planes for the ten different dinucleotide steps. These values were then used to compute the variation of flexibility along a given nucleotide sequence. This allowed us to validate the method experimentally through comparisons with maps of local fluctuations in DNA molecule trajectory constructed from atomic force microscopy imaging in solution. We conclude that the six dinucleotide-step parameters defined here provide a powerful tool for the exploration of DNA structure and, consequently will make an important contribution to our understanding of DNA-sequence-dependent biological processes.     

原子力显微镜研究小鼠免疫球蛋白G自组装

应用原子力显微镜观察小鼠免疫球蛋白G样品,结果不管是IgG1还是IgG1与IgG2的混合样品,在制样2-3d后,均发现有圆环状聚集区,也有部分圆面状聚集区域,一般环中心有核,核为IgG分子多聚体的再聚集,多聚体中亚基（单体）数目因环而异,在圆环和贺湎状区域外,也有零星分散的聚集体,不参与成环,另外,IgG1和IgG1 IgG2聚集情况有所不同,后者更倾向于形成圆面状聚集,另外,在聚集发生前,亚基已自组装形成各种形状的多聚体,然后多聚体进一步聚集形成各种圆环或圆面状,对免疫球蛋白G先自组装而后聚集的过程,从生物学,物理学角度作了初步解释。     

AFM study of potato virus X disassembly induced by movement protein

Recently we have reported that a selective binding of potato virus X (PVX)-coded movement protein (termed TGBp1 MP) to one end of a polar coat protein (CP) helix converted viral RNA into a translatable form and induced a linear destabilization of the whole helical particle. Here, the native PVX virions, RNase-treated (PVX(RNA-DEG)) helical particles lacking intact RNA and their complexes with TGBp1 (TGBp1-PVX and TGBp1-PVX(RNA-DEG)), were examined by atomic force microscopy (AFM). When complexes of the TGBp1 MP with PVX were examined by means of AFM in liquid, no structural reorganization of PVX particles was observed. By contrast, the products of TGBp1-dependent PVX degradation termed "beads-on-string" were formed under conditions of AFM in air. The AFM images of PVX(RNA-DEG) were indistinguishable from images of native PVX particles; however, the TGBp1-dependent disassembly of the CP-helix was triggered when the TGBp1-PVX(RNA-DEG) complexes were examined by AFM, regardless of the conditions used (in air or in liquid). Our data supported the idea that binding of TGBp1 to one end of the PVX CP-helix induced linear destabilization of the whole helical particle, which may lead to its disassembly under conditions of AFM.     

利用激光AFM和TEM探索肌动蛋白体外自装配聚合结构

细胞内肌动蛋白(actin)通过与actin结合蛋白(actin binding proteins,ABPs)相互作用,形成以F-actin为基础多种ABPs参与装配的高度有序的超分子聚合结构,行使各种重要生理功能。在体外聚合条件下,不存在F-actin稳定剂时纯化的actin主要通过自装配形成大尺度的聚集堆积结构;这种表观无序的结构体系由于被认为不具备细胞功能活性而受到忽视。利用激光原子力显微镜(atomic force microscope,AFM)和透射电子显微镜(transmission electron microscope,TEM)技术,对actin体外通过自装配过程形成的大尺度聚集结构进行了细致的观察和分析。研究发现,actin在体外通过自装配过程除了形成无序的蛋白堆积物之外,还能够聚合形成复杂的离散结构,包括树状分支的纤维丛、无规卷曲的纤维簇以及具有不同直径的长纤维等;这些大尺度纤维复合物明显不同于在ABPs或过量F-actin稳定剂参与下形成的由单根微丝和微丝束构成的聚合结构。表明无ABPs或F-actin稳定剂存在的情况下,体外聚合的F-actin在一定条件下可进一步聚集缠绕形成复杂的纤维结构或无序的蛋白堆积物。事实上,actin自装配过程反映了其固有的聚合热力学特性,深入探索将有助于理解ABPs在体内actin超分子聚合结构体系装配中的调控作用及其分子机制。     

原子力显微镜对人羊膜上皮细胞的观察

目的:在单细胞水平上分析人羊膜上皮细胞的超微结构及其机械性能(粘弹力、杨氏模量、硬度等),为进一步认识细胞结构与功能的关系奠定基础.方法:应用原子力显微镜(AFM)高分辨率、高灵敏度的特点,对人的羊膜上皮细胞进行观察.结果:人羊膜上皮细胞呈椭圆形,由原子力显微镜力位移曲线测量系统,可得粘弹力:1034.375±294.21 pN.硬度:1.1815±0.326mN/m,杨氏模量:16.44±4.67Kpa.结论:AFM能对人羊膜上皮细胞表面超微结构清晰地成像及提供更多更确切的表面信息及机械性能,从而增加对羊膜上皮细胞的认识.     

Atomic Force Microscopic Observation of Escherichia coli Ribosomes in Solution

Ribosomes of Escherichia coli were visualized in buffer solution by atomic force microscopy (AFM). A series of time-dependent AFM images showed that ribosomes spontaneously adsorb on mica. Although ribosomes observed in air are forced to flatten on the surface, the height of ribosomal particles obtained under a physiological buffer solution is 21.8±0.5 nm, which is consistent with the ideal diameter. We succeeded in observing single ribosomes in a near-native condition.     

alpha-Synuclein misfolding: single molecule AFM force spectroscopy study

Protein misfolding and aggregation are the very first and critical steps in development of various neurodegenerative disorders, including Parkinson’s disease, induced by misfolding of α-synuclein. Thus, elucidating properties of proteins in misfolded states and understanding the mechanisms of their assembly into the disease prone aggregates are critical for the development of rational approaches to prevent protein misfolding-mediated pathologies. To accomplish this goal and as a first step to elucidate the mechanism of α-synuclein misfolding, we applied single-molecule force spectroscopy capable of detecting protein misfolding. We immobilized α-synuclein molecules at their C-termini at the atomic force microscope tips and substrate surfaces, and measured the interaction between the proteins by probing the microscope tip at various locations on the surface. Using this approach, we detected α-synuclein misfolded states by enhanced interprotein interaction. We used a dynamics force spectroscopy approach to measure such an important characteristic of dimers of misfolded α-synuclein as their lifetimes. We found that the dimer lifetimes are in the range of seconds and these values are much higher than the characteristics for the dynamics of the protein in monomeric state. These data show that compared to highly dynamic monomeric forms, α-synuclein dimers are much more stable and thus can serve as stable nuclei for the formation of multimeric and aggregated forms of α-synuclein. Importantly, two different lifetimes were observed for the dimers, suggesting that aggregation can follow different pathways that may lead to different aggregated morphologies of α-synuclein.     

An in situ study of the nanomechanical properties of barnacle (Balanus amphitrite) cyprid cement using atomic force microscopy (AFM)

Abstract

Cyprids are the final planktonic stage in the larval dispersal of barnacles and are responsible for surface exploration and attachment to appropriate substrata. The nanomechanical properties of barnacle (Balanus amphitrite) cyprid permanent cement were studied in situ using atomic force microscopy (AFM). Force curves were recorded from the cement disc continually over the course of its curing and these were subsequently analysed using custom software. Results showed a narrowing of the pull-off force distribution with time, as well as a reduction in molecular stretch length over time. In addition, there was a strong correlation between maximum pull-off force and molecular stretch length for the cement, suggesting ‘curing’ of the adhesive; some force curves also contained a ‘fingerprint’ of modular protein unfolding. This study provides the first direct experimental evidence in support of a putative ‘tanning’ mechanism in barnacle cyprid cement.     

Characterisation of the polysaccharide produced by Acetobacter xylinum strain CR1/4 by light scattering and atomic force microscopy

The molecular weight of the extracellular polysaccharide (CR1/4) produced by Acetobacter xylinum strain CR1/4 has been shown to be dependent upon growth conditions. Under normal growth conditions a high molecular weight polysaccharide (>1×10⁶ Da) is produced. Maintaining the pH at 5 results in an order of magnitude increase in the total yield of polysaccharide, but also an order of magnitude decrease in molecular weight. Analysis of the CR1/4 polysaccharides by the techniques of atomic force microscopy and static light scattering suggests that they are double helices. In solution the molecules behave as stiff coils with a Kuhn statistical segment length of 325 nm.