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。     

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。     

原子力显微镜技术在细胞力学和病理学研究中的应用

原子力显微镜(AFM)由于具有纳米量级的空间分辨率和皮牛(pN)量级的力分辨率已经在活细胞和细胞组织超微结构的研究中取得重大进展,该技术为细胞生物力学的研究提供了新方法。通过力曲线可以得到与单个细胞的力学性质相关的信息。细胞弹性的变化是生物细胞发生病变的特征之一。利用AFM研究各种细胞的弹性特性,为疾病的早期诊断和治疗以及病理机制的研究提供了一种强有力的工具。本文主要综述了近些年用AFM技术研究疾病相关的细胞弹性特性的应用新进展,如发现多种类型的癌细胞都比健康细胞软,以及在相关血液性疾病(如冠状动脉疾病、高血压和糖尿病)中红细胞的弹性也发生了变化。这些特性可对疾病的辅助诊断提供参考,为病理学和临床医学研究提供了新依据。     

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。     

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。     

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.     

Oligomerization states of Bowman-Birk inhibitor by atomic force microscopy and computational approaches

Several methods have been applied to study protein-protein interaction from structural and thermodynamic point of view. The present study reveals that atomic force microscopy (AFM), molecular modeling, and docking approaches represent alternative methods offering new strategy to investigate structural aspects in oligomerization process of proteinase inhibitors. The topography of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) was recorded by AFM and compared with computational rigid-bodies docking approaches. Multimeric states of BTCI identified from AFM analysis showed globular-ellipsoidal shapes. Monomers, dimers, trimers, and hexamers were the most prominent molecular arrays observed in AFM images as evaluated by molecular volume calculations and corroborated by in silico docking and theoretical approaches. We therefore propose that BTCI adopts stable and well-packed self-assembled states in monomer-dimer-trimer-hexamer equilibrium. Although there are no correlation between specificity and packing efficiency among proteinases and proteinase inhibitors, the AFM and docked BTCI analyses suggest that these assemblies may exist in situ to play their potential function in oligomerization process.     

A combined atomic force microscopy imaging and docking study to investigate the complex between p53 DNA binding domain and Azurin

The tumor suppressor p53 interacts with the redox copper protein Azurin (AZ) forming a complex which is of some relevance in biomedicine and cancer therapy. To obtain information on the spatial organization of this complex when it is immobilized on a substrate, we have used tapping mode‐atomic force microscopy (TM‐AFM) imaging combined with computational docking. The vertical dimension and the bearing volume of the DNA binding domain (DBD) of p53, anchored to functionalized gold substrate through exposed lysine residues, alone and after deposing AZ, have been measured by TM‐AFM. By a computational docking approach, a three‐dimensional model for the DBD of p53, before and after addition of AZ, have been predicted. Then we have calculated the possible arrangements of these biomolecular systems on gold substrate by finding a good agreement with the related experimental distribution of the height. The potentiality of the approach combining TM‐AFM imaging and computational docking for the study of biomolecular complexes immobilized on substrates is briefly discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

肌动蛋白的原子力显微镜研究

原子力显微镜 (AFM )是一种能够在生理条件下对生物大分子、活细胞表面以及细胞膜下结构进行在体或离体研究的强有力的新型工具 ,具有原子级的成像分辨率和纳牛顿级的力测定功能。目前原子力显微镜已被广泛地应用于生物大分子、超分子体系的结构解析、动力学过程观察 ,分子力学研究及细胞功能鉴定。原子力显微镜能够通过尖锐探针扫描待测样品表面 ,收集被测样品表面地貌坐标数据从而对单分子或细胞进行成像或操作 ,并能通过移动探针、记录探针与样品之间的作用力 ,对生物大分子 (蛋白质、核酸和多糖等 )的结构力学特性进行分析以获取分子构象、功能及其相互关系的有用信息。肌动蛋白是一种细胞内普遍存在 ,具有广泛、复杂生理功能的重要蛋白质 ,原子力显微镜的各项功能已广泛地用于肌动蛋白结构、功能及动力学研究。通过综述原子力显微镜在肌动蛋白研究中的应用 ,阐明了原子力显微镜在现代生命科学研究中的重要意义及巨大应用前景。     

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。     

Visualization of enzymatic hydrolysis of cellulose using AFM phase imaging

Complete cellulase, an endoglucanase (EGV) with cellulose-binding domain (CBD) and a mutant endoglucanase without CBD (EGI) were utilized for the hydrolysis of a fully bleached reed Kraft pulp sample. The changes of microfibrils on the fiber surface were examined with tapping mode atomic force microscopy (TM–AFM) phase imaging. The results indicated that complete cellulase could either peel the fibrillar bundles along the microfibrils (peeling) or cut microfibrils into short length across the length direction (cutting) during the process. After 24 h treatment, most orientated microfibrils on the cellulose fiber surface were degraded into fragments by the complete cellulase. Incubation with endoglucanase (EGV or EGI) also caused peeling action. But no significant size reduction of microfibrils length was observed, which was probably due to the absence of cellobiohydrolase. The AFM phase imaging clearly revealed that individual EGV particles were adsorbed onto the surface of a cellulose fiber and may be bound to several microfibrils.     

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.     

Direct Force Measurement of the Interaction Between Liposome and the C2A Domain of Synaptotagmin I using Atomic Force Microscopy

The binding force between a liposome and the C2A domain of synaptotagmin I was determined by an atomic force microscopy (AFM). Liposomes were immobilized on the surface of the L1 sensor chip and the C2A domains, which recognize phosphatidylserine, were chemically conjugated onto a gold-coated cantilever tip. The average interaction force between the C2A domain and the liposome was 306 (±57) pN while the force between untreated cantilever and the liposome was 58 (±16) pN. This work helps understand the physicochemical interactions between proteins and lipid vesicles for the design of high affinity protein probes against the apoptotic cell surface. Revisions requested 13 December 2005; Revisions received 9 January 2006     

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.     

Conformational Plasticity of Human Protease-Activated Receptor 1 upon Antagonist- and Agonist-Binding

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Two-dimensional intraventricular flow pattern visualization using the image-based computational fluid dynamics

The image-based computational fluid dynamics (IB-CFD) technique, as the combination of medical images and the CFD method, is utilized in this research to analyze the left ventricle (LV) hemodynamics. The research primarily aims to propose a semi-automated technique utilizing some freely available and commercial software packages in order to simulate the LV hemodynamics using the IB-CFD technique. In this research, moreover, two different physiological time-resolved 2D models of a patient-specific LV with two different types of aortic and mitral valves, including the orifice-type valves and integrated with rigid leaflets, are adopted to visualize the process of developing intraventricular vortex formation and propagation. The blood flow pattern over the whole cardiac cycle of two models is also compared to investigate the effect of utilizing different valve types in the process of the intraventricular vortex formation. Numerical findings indicate that the model with integrated valves can predict more complex intraventricular flow that can match better the physiological flow pattern in comparison to the orifice-type model.     

AFM study of the colonisation of stainless steel by Aquabacterium commune

Aquabacterium commune, a member of the beta proteobacteria family that is a recently isolated, predominant member of various European drinking water distribution system biofilms, was selected as a test organism in this study. Attachment of A. commune cells onto two increasingly popular pipe materials, stainless steel EN1.4307 and medium density polyethylene (MDPE) was studied at 15 °C, 150 rpm, and a hydraulic retention time of 10.5 h in a rotating annular biofilm (RAB) reactor. Planktonic and sessile growth was monitored by spread plate technique. Atomic force microscopy (AFM) was used to obtain information about surface topography and biofilm formation pattern. Our study has shown that: (i) Steady-state conditions were reached after ca. 100 h for both materials; (ii) biofilm cell density on MDPE slides is four times greater than on stainless; (iii) the primary colonization of MDPE and stainless steel occurred at the edge of the slides; and (iv) no preferential attachment to stainless steel grain boundaries was observed. Stainless-steel manufacturers and suppliers, researchers, and companies working in the drinking-water sector will benefit from this paper. It is suggested that electropolishing of stainless-steel pipes for drinking water installations is not necessary to remove specific biofilm formation sites (i.e. grain boundaries). Furthermore, this paper provides, for the first time, some fundamental information for the continuous cultivation of the recently isolated drinking water microorganism, A. commune.