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At least 1,063 records · Page 59Linked to original sources
PubMed2003

Atomic force microscopy studies of interaction of the 20S proteasome with supported lipid bilayers.

The 20S proteasome plays important roles in degradation of intracellular proteins. Mechanisms of its activation, its localization in cells, and its binding to biomembranes are not well understood. In this study, we used atomic force microscopy (AFM) to investigate interactions between the 20S proteasome and supported bilayers of various lipids in a buffer. We found that the 20S proteasome specifically bound to supported bilayers containing phosphatidylinositol (PI), but did not bind to supported bilayers containing phosphatidylcholine, phosphatidic acid or dioleoyltrimethylammonium propane. Binding of the 20S proteasomes had a high orientation; almost all were in a top view position. The specific and orientational binding of the 20S proteasome with PI may play important roles inside cells such as endoplasmic reticulum (ER) membrane. Use of AFM to study supported bilayers provides new information on ligand-receptor interactions.

Animals↗
PubMed1999

An atomic force microscopy study on the effect of bleaching agents on enamel surface.

OBJECTIVES: The aim of the present study was to evaluate the effect of three peroxide-containing bleaching agents, Opalescence, Nite White and a 30% hydrogen peroxide solution, on enamel surfaces using Atomic Force Microscopy (AFM). METHODS: Fifteen non-carious human incisors (ten maxillary and five mandibular, extracted for periodontal reasons) were used. The teeth were divided randomly into three groups of five, according to the bleaching agents. The labial surface of each tooth was imaged by AFM before and after treatment. Each bleaching agent was applied for a total of 28 h (in individual 4 h treatments). The specimens were examined only after 28 h of treatment. RESULTS: On comparing the AFM images of untreated and treated enamel, surface alterations were observed after 28 h of treatment with Opalescence, Nite White and 30% hydrogen peroxide solution. Several grooves present in the enamel surface of untreated teeth became deeper after the bleaching procedure. The depths of the grooves increased in each case. The increase in the depth of grooves was more pronounced in the case of the 30% H2O2 solution. CONCLUSION: Home-use bleaching agents are capable of causing enamel surface alterations. It is hypothesized that the peroxide-containing bleaching agents affect the organic phase of enamel. Peroxides can affect not only the surface but also the inner structure of enamel. As a result of its low molecular weight, hydrogen peroxide can penetrate into the enamel. Thus, inner oxidative effects are more likely to occur in the subsurface enamel where more organic material is present and oxidation is capable of altering the outer enamel and the surface.

Acrylic Resins↗
PubMed2004

Analysis of solid-phase immobilized antibodies by atomic force microscopy.

Antibody adsorption to solid surfaces creates a number of constraints that may interfere with epitope recognition and ligand-antibody interaction. By optimizing the conditions of adsorption, one may minimize these constraints. We have studied several factors that affect the antibody adsorption using atomic force microscopy (AFM) as a readout mechanism. AFM provides a highly sensitive, label-free method for detecting and analyzing molecular interactions. In this report, AFM was used to study antibody properties, the efficiency of particle capture and ligand-antibody interaction using anti-bacteriophage fd antibodies in a solid phase assay format. The capture efficiencies of anti-fd preparations adsorbed onto gold surfaces under various conditions including pH and antibody concentration were determined and compared. The relative sensitivities of each antibody for the capture of phage fd as a function of applied phage concentrations was evaluated. The collective data indicates that AFM is effective as an analytical instrument for studying the functionality of surface adsorbed antibodies in particle capture assays. This method of analysis can be extended to rapidly screen and select antibodies or other ligands with a specific set of characteristics. As the number and complexity of chip-based analytical platforms in proteomics increases, rapid selection/screening processes such as that described here will become invaluable.

Animals↗
PubMed2000

Adsorbed Layers of Ferritin at Solid and Fluid Interfaces Studied by Atomic Force Microscopy.

The adsorption of the iron storage protein ferritin was studied by liquid tapping mode atomic force microscopy in order to obtain molecular resolution in the adsorbed layer within the aqueous environment in which the adsorption was carried out. The surface coverage and the structure of the adsorbed layer were investigated as functions of ionic strength and pH on two different charged surfaces, namely chemically modified glass slides and mixed surfactant films at the air-water interface, which were transferred to graphite substrates after adsorption. Surface coverage trends with both ionic strength and pH indicate the dominance of electrostatic effects, with the balance shifting between intermolecular repulsion and protein-surface attraction. The resulting behavior is more complex than that seen for larger colloidal particles, which appear to follow a modified random sequential adsorption model monotonically. The structure of the adsorbed layers at the solid surfaces is random, but some indication of long-range order is apparent at fluid interfaces, presumably due to the higher protein mobility at the fluid interface. Copyright 2000 Academic Press.

Journal Article↗
PubMed2006

In situ adsorption studies of a 14-amino acid leucine-lysine peptide onto hydrophobic polystyrene and hydrophilic silica surfaces using quartz crystal microbalance, atomic force microscopy, and sum frequency generation vibrational spectroscopy.

The adsorption of a 14-amino acid amphiphilic peptide, LK14, which is composed of leucine (L, nonpolar) and lysine (K, charged), on hydrophobic polystyrene (PS) and hydrophilic silica (SiO2) was investigated in situ by quartz crystal microbalance (QCM), atomic force microscopy (AFM), and sum frequency generation (SFG) vibrational spectroscopy. The LK14 peptide, adsorbed from a pH 7.4 phosphate-buffered saline (PBS) solution, displayed very different coverage, surface roughness and friction, topography, and surface-induced orientation when adsorbed onto PS versus SiO2 surfaces. Real-time QCM adsorption data revealed that the peptide adsorbed onto hydrophobic PS through a fast (t < 2 min) process, while a much slower (t > 30 min) multistep adsorption and rearrangement occurred on the hydrophilic SiO2. AFM measurements showed different surface morphologies and friction coefficients for LK14 adsorbed on the two surfaces. Surface-specific SFG spectra indicate very different ordering of the adsorbed peptide on hydrophobic PS as compared to hydrophilic SiO2. At the LK14 solution/PS interface, CH resonances corresponding to the hydrophobic leucine side chains are evident. Conversely, only NH modes are observed at the peptide solution/SiO2 interface, indicating a different average molecular orientation on this hydrophilic surface. The surface-dependent difference in the molecular-scale peptide interaction at the solution/hydrophobic solid versus solution/hydrophilic solid interfaces (measured by SFG) is manifested as significantly different macromolecular-level adsorption properties on the two surfaces (determined via AFM and QCM experiments).

Adsorption↗
PubMed2006

Membrane resistance to Triton X-100 explored by real-time atomic force microscopy.

Lateral segregation of lipids and proteins in biological membranes leads to the formation of detergent-resistant domains, also called "rafts". Understanding the mechanisms governing the biomembrane's resistance to solubilization by detergents is crucial in biochemical research. Here, we used real-time atomic force microscopy (AFM) imaging to visualize the behavior of a model supported lipid bilayer in the presence of different Triton X-100 (TX-100) concentrations. Mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) supported bilayers were prepared by vesicle fusion. Real-time AFM imaging revealed that, at concentrations below the critical micelle concentration (CMC), TX-100 did not solubilize the bilayer, but the DPPC domains were eroded in a time-dependent manner. This effect was attributed to the DPPC molecular packing disorganization by the detergent starting from the DOPC/DPPC interface. Just above the CMC, the detergent led to a complete solubilization of the DOPC matrix, leaving the DPPC domains unaltered. At higher TX-100 concentrations, the DOPC was also immediately removed just after detergent addition, and the DPPC domains remaining on the mica surface appeared to be more swollen and were gradually solubilized. This progressive solubilization of the DPPC remaining phase did not start at the edge of the domains but from holes appearing and expanding at the center of DPPC patches. The swelling of the DPPC domains was directly correlated with TX-100 concentration above the CMC and with detergent intercalation between DPPC molecules. We are convinced that this approach will provide a key system to elucidate the physical mechanisms of membrane solubilization by nonionic detergents.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2003

Atomic force microscopy analysis of rolling circle amplification of plasmid DNA.

Rolling circle amplification (RCA) of plasmid DNA using random hexamers and bacteriophage phi29 DNA polymerase is an increasingly applied technique for amplifying template DNA for DNA sequencing. We analyzed this RCA reaction at a single-molecular level by atomic force microscopy (AFM) and found that multibranched amplified products containing tandem repeats of a circle unit are formed within 1 h. We also used the RCA product of a GFP expression vector for the protein expression in cells, and found that the crude RCA product from one bacterial colony is sufficient for the GFP expression. Thus, the RCA reaction is useful in amplifying DNA for both DNA sequencing and protein expression.

Animals↗
PubMed2005

Atomic force microscopy study of the effect of antimicrobial peptides on the cell envelope of Escherichia coli.

The influences of the antibacterial magainin 2 and PGLa from the African clawed frog (Xenopus laevis) and the hemolytic bee venom melittin on Escherichia coli as the target cell were studied by atomic force microscopy (AFM). Nanometer-scale images of the effects of the peptides on this gram-negative bacterium's cell envelope were obtained in situ without the use of fixing agents. These high-resolution AFM images of the surviving and intact target cells before and after peptide treatment showed distinct changes in cell envelope morphology as a consequence of peptide action. Although all three peptides are lytic to E. coli, it is clear from this AFM study that each peptide causes distinct morphological changes in the outer membrane and in some cases the inner membrane, probably as a consequence of different mechanisms of action.

Animals↗
PubMed2005

Visualization of single Escherichia coli FtsZ filament dynamics with atomic force microscopy.

FtsZ, the prokaryotic homologue of tubulin, is an essential cell division protein. In the cell, it localizes at the center, forming a ring that constricts during division. In vitro, it binds and hydrolyzes GTP and polymerizes in a GTP-dependent manner. We have used atomic force microscopy to study the structure and dynamics of FtsZ polymer assembly on a mica surface under buffer solution. The polymers were highly dynamic and flexible, and they continuously rearranged over the surface. End-to-end joining of filaments and depolymerization from internal zones were observed, suggesting that fragmentation and reannealing may contribute significantly to the dynamics of FtsZ assembly. The shape evolution of the restructured polymers manifested a strong inherent tendency to curve. Polymers formed in the presence of non-hydrolyzable nucleotide analogues or in the presence of GDP and AlF(3) were structurally similar but showed a slower dynamic behavior. These results provide experimental evidence supporting the model of single-strand polymerization plus cyclization recently proposed to explain the hydrodynamic behavior of the polymers in solution.

Adsorption↗
PubMed2004

Evaluation of the ordering of membranes in multilayer stacks built on an ATR-FTIR germanium crystal with atomic force microscopy: the case of the H(+),K(+)-ATPase-containing gastric tubulovesicle membranes.

Polarized attenuated total reflection Fourier transform infrared spectra were recorded on multilayer stacks of native gastric tubulovesicle membranes. The spectral intensity and linear dichroism were measured for average thicknesses ranging between 0 and 100 bilayers. Atomic force microscopy was used to investigate the orientation of the membranes at the top of the stack. Height profiles were obtained along randomly drawn lines and slopes were computed over various distances. Orientation distribution functions were obtained from the slopes and decomposed into Legendre polynomials. It was found that the second Legendre polynomials coefficient characterizing the membrane orientation was always larger than 0.9. It could therefore be concluded that the membrane tilt does not significantly contribute to the infrared dichroism, even for the largest thicknesses tested.

Animals↗
PubMed2004

Dynamic elastic modulus of porcine articular cartilage determined at two different levels of tissue organization by indentation-type atomic force microscopy.

Cartilage stiffness was measured ex vivo at the micrometer and nanometer scales to explore structure-mechanical property relationships at smaller scales than has been done previously. A method was developed to measure the dynamic elastic modulus, |E(*)|, in compression by indentation-type atomic force microscopy (IT AFM). Spherical indenter tips (radius = approximately 2.5 microm) and sharp pyramidal tips (radius = approximately 20 nm) were employed to probe micrometer-scale and nanometer-scale response, respectively. |E(*)| values were obtained at 3 Hz from 1024 unloading response curves recorded at a given location on subsurface cartilage from porcine femoral condyles. With the microsphere tips, the average modulus was approximately 2.6 MPa, in agreement with available millimeter-scale data, whereas with the sharp pyramidal tips, it was typically 100-fold lower. In contrast to cartilage, measurements made on agarose gels, a much more molecularly amorphous biomaterial, resulted in the same average modulus for both indentation tips. From results of AFM imaging of cartilage, the micrometer-scale spherical tips resolved no fine structure except some chondrocytes, whereas the nanometer-scale pyramidal tips resolved individual collagen fibers and their 67-nm axial repeat distance. These results suggest that the spherical AFM tip is large enough to measure the aggregate dynamic elastic modulus of cartilage, whereas the sharp AFM tip depicts the elastic properties of its fine structure. Additional measurements of cartilage stiffness following enzyme action revealed that elastase digestion of the collagen moiety lowered the modulus at the micrometer scale. In contrast, digestion of the proteoglycans moiety by cathepsin D had little effect on |E(*)| at the micrometer scale, but yielded a clear stiffening at the nanometer scale. Thus, cartilage compressive stiffness is different at the nanometer scale compared to the overall structural stiffness measured at the micrometer and larger scales because of the fine nanometer-scale structure, and enzyme-induced structural changes can affect this scale-dependent stiffness differently.

Animals↗
PubMed1999

Monitoring the assembly of Ig light-chain amyloid fibrils by atomic force microscopy.

Aggregation of Ig light chains to form amyloid fibrils is a characteristic feature of light-chain amyloidosis, a light-chain deposition disease. A recombinant variable domain of the light chain SMA was used to form amyloid fibrils in vitro. Fibril formation was monitored by atomic force microscopy imaging. Single filaments 2.4 nm in diameter were predominant at early times; protofibrils 4.0 nm in diameter were predominant at intermediate times; type I and type II fibrils 8.0 nm and 6.0 nm in diameter, respectively, were predominant at the endpoints. The increase in number of fibrils correlated with increased binding of the fluorescent dye thioflavin T. The fibrils and protofibrils showed a braided structure, suggesting that their formation involves the winding of protofibrils and filaments, respectively. These observations support a model in which two filaments combine to form a protofibril, two protofibrils intertwine to form a type I fibril, and three filaments form a type II fibril.

Amyloid↗
PubMed1998

Structures and dynamic motion of laminin-1 as observed by atomic force microscopy.

Laminins are a family of multifunctional extracellular matrix glycoproteins that play important roles in the development and maintenance of tissue organization via their interactions with cells and other extracellular matrix proteins. To understand the structural basis of laminins' functions, we examined the motion of laminin-1 (Ln-1) in physiological buffers using atomic force microscopy. While many Ln-1 molecules assumed the expected cruciform structure, unexpected dynamic movements of the Ln-1 arms were observed in aqueous environments. These dynamic movements of the Ln-1 arms may contribute to the diversity of laminin functions.

Animals↗
PubMed2003

Hierarchical chromatin structure of Schizosaccharomyces pombe revealed by atomic force microscopy.

Many structural studies on higher eukaryotic chromatin have been carried out, but chromatin structure in fungi remains unclear. Schizosaccharomyces pombe has been used for investigations of chromosome function; however, the structural details of S. pombe chromatin have not been clarified owing to its small nucleus. We used atomic force microscopy for nano-scale imaging of chromatin isolated from S. pombe. Topographic images indicated that nuclear chromatin contained at least three hierarchical structures: large-scale chromatin fibers, spherical domains in the fibers, and nodules in the domains. The average diameters of the domain and the nodule were 363 +/- 85.2 nm and 46.2 +/- 9.30 nm. Each structure comprising the hierarchy was similar to higher eukaryotic chromatin thus far observed, despite definite differences in chromatin organization at the nucleosomal level. The presence of histone H1 suggested that there might be an alternative to compensate for histone H1 lacking in S. pombe.

Cell Cycle↗
PubMed2003

Ca2+ dependency of N-cadherin function probed by laser tweezer and atomic force microscopy.

This study was undertaken to provide a biophysical basis for the hypothesis that activity-dependent modulation of cadherin-mediated adhesion by transient changes of extracellular calcium ([Ca2+]e) is causally involved in coordination of synaptic plasticity. Characterization of homophilic N-cadherin binding by atomic force microscopy and laser tweezer trapping of N-cadherin-coated microbeads attached to the cell surface of cultured neuronal cells showed that adhesive activity of N-cadherin is effectively regulated between 0.3 and 0.8 mm [Ca2+]e. Furthermore, we show that an increase of [Ca2+]i, which is known to be essential for induction of synaptic plasticity, causes significant reduction of cadherin-mediated bead adhesion that could be completely suppressed by inhibition of actin depolymerization. The results of this study show that N-cadherin has ideal biophysical properties to serve as a Ca2+-dependent sensor for synaptic activity and, at the same time, is strategically located to control synaptic adhesion. A drop of [Ca2+]e and a concomitant increase of [Ca2+]i may act in concert to modulate N-cadherin-based adhesive contacts at synaptic sites.

Actins↗
PubMed2002

Imaging real-time aggregation of amyloid beta protein (1-42) by atomic force microscopy.

Amyloid beta protein (AbetaP) is the major fibrillar constituent of senile plaques. However, no causative role for AbetaP-fibers in Alzheimer's disease (AD) pathology is established. Globular AbetaPs are continuously released during normal cellular metabolism at pico- to nano-molar concentration. We used atomic force microscopy (AFM) to examine aggregation of freshly prepared AbetaP(1-42) and to examine the role of AbetaP concentration, imaging medium (air, water, or PBS) and agonists/antagonists on AbetaP-fibrillogenesis. At even very high and non-physiological AbetaP concentrations, 24-48 h of real-time AFM imaging (a) in water show only multiple layers of globular aggregates and no fibrils and (b) in PBS show mainly the globular structures and some short fibrils. On-line addition of Zn, an agonist for AbetaP-fibrillogenesis, induced a slow but non-fibrillar aggregation of globular AbetaPs. EDTA, a chelator of Zn and calcium (a modulator of AbetaP-mediated toxicity) induced a reversible change in the Zn-mediated aggregation. These results strongly suggest that no AbetaP-fibers are formed for the physiologically relevant concentration and thus the plaque-associated fibers may not account for the AD pathophysiology.

Air↗
PubMed2000

Atomic force microscopy analysis of intermediates in cobalt hexammine-induced DNA condensation.

The packaging pathway of cobalt hexammine-induced DNA condensation on the surface of mica was examined by varying the concentration of Co(NH3)6(3+) in a dilute DNA solution and visualizing the condensates by atomic force microscopy (AFM). Images reveal that cobalt hexammine-induced DNA condensation on mica involves well-defined structures. At 30 microM Co(NH3)6(3+), prolate ellipsoid condensates composed of relatively shorter rods with linkages between them are formed. At 80 microM Co(NH3)6(3+), the condensed features include toroids with average diameter of approximately 240 nm as well as U-shaped and rod-like condensates with nodular appearances. The results imply that the condensates, whether toroids, U-shaped or rod-like structures have similar intermediate state which includes relatively shorter rod-like segments. The average size of the condensed toroids after incubated at room temperature for 5 h (approximately 240 nm) is much larger than that incubated for 0.5 h (approximately 100 nm). The results indicate that the condensation of DNA by Co(NH3)6(3+) is a kinetic-controlled process.

Adsorption↗
PubMed2000

Atomic force microscopy proposes a 'kiss and pull' mechanism for enhancer function. off.

The DNase I-hyper-sensitive sites (HS2-HS4) in the beta-globin gene enhancer region (locus control region; LCR) have been known as the target of Bach1/MafK heterodimers. We have demonstrated previously by utilizing atomic force microscopy (AFM) that Bach1/MafK mediates the formation of a looped-DNA structure in the LCR fragment. Here we perform further detailed analyses of the loop structure formed between each HSs by AFM, and propose a novel model for the enhancer/protein interaction: the Bach1/MafK heterodimer preferentially binds to HS2 with highest affinity and to HS3 with lower affinity. However, they assemble to each other to form a stable complex of four heterodimers and mediate a DNA-loop formation. Once the DNA loop is formed between HS2 and HS3, the Bach1/MafK complex at the HS3 side leaves the HS3 and starts to slide along the DNA strand towards HS2 with the other side of the complex fixed at the HS2 region. This 'kiss and pull' model will contribute to understand the function of regulatory proteins at enhancer regions in terms of higher-order structure of DNA, e.g. nucleosomes and chromatin.

Basic-Leucine Zipper Transcription Factors↗