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At least 1,171 records · Page 65Linked to original sources
PubMed1998

Retrieval and amplification of single-copy genomic DNA from a nanometer region of chromosomes: a new and potential application of atomic force microscopy in genomic research.

Using a modified tip of the atomic force microscope (AFM), we harvested several strands of genomic DNA from a nanometer region of mouse chromosomes. We have also co-developed a random PCR method to amplify the recovered genomic DNA, in which a single DNA molecule of several kilobasepairs could be amplified efficiently. A subsequent fluorescence in situ hybridization (FISH) indicated that the amplified DNA originally came from the tip-manipulated regions of mouse chromosomes. Several fragments containing unique sequences were identified using Southern hybridization after subcloning the PCR products into pUC18 plasmid. The present results showed a potential application of AFM to genomic analysis.

3T3 Cells↗
PubMed2005

Monolayer-multilayer transitions in a lung surfactant model: IR reflection-absorption spectroscopy and atomic force microscopy.

A hydrophobic pulmonary surfactant protein, SP-C, has been implicated in surface-associated activities thought to facilitate the work of breathing. Model surfactant films composed of lipids and SP-C display a reversible transition from a monolayer to surface-associated multilayers upon compression and expansion at the air/water (A/W) interface. The molecular-level mechanics of this process are not yet fully understood. The current work uses atomic force microscopy on Langmuir-Blodgett films to verify the formation of multilayers in a dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, cholesterol, and SP-C model system. Isotherms of SP-C-containing films are consistent with exclusion and essentially complete respreading during compression and expansion, respectively. Multilayer formation was not detected in the absence of SP-C. Most notable are the results from IR reflection-absorption spectroscopy (IRRAS) conducted at the A/W interface, where the position and intensity of the Amide I band of SP-C reveal that the predominantly helical structure changes its orientation in monolayers versus multilayers. IRRAS measurements indicate that the helix tilt angle changed from approximately 80 degrees in monolayers to a transmembrane orientation in multilayers. The results constitute the first quantitative measure of helix orientation in mixed monolayer/multilamellar domains at the A/W interface and provide insight into the molecular mechanism for SP-C-facilitated respreading of surfactant.

Lipid Bilayers↗
PubMed2004

Susceptibility of deciduous and permanent enamel to dietary acid-induced erosion studied with atomic force microscopy nanoindentation.

The initial stages of dental enamel demineralization by dietary acids are of particular clinical relevance. Some authors have reported a difference in the susceptibility of deciduous and permanent enamel to erosion, although other studies could not confirm these findings. The aim of the present study was therefore to investigate whether there are differences in the susceptibility of deciduous and permanent enamel to dietary acid induced erosion (demineralization). Nanoindentation combined with atomic force microscopy was used to investigate the erosive effect of four different drinks (cola, lemonade, juice drink, and water) on enamel at early stages in vitro. Surface nanohardness and reduced elastic modulus were obtained for the soft drink-treated enamel samples as a function of the exposure time. The results obtained showed that the four drinks had widely differing erosive effects due to their diverse compositions. Water did not show statistically significant changes in nanomechanical properties for all exposure times compared with untreated enamel. Exposure of enamel to cola, lemonade and the juice drink led to a statistically significant surface softening of the enamel with cola and the lemonade having a much stronger softening effect than the juice drink. For longer exposure times enamel softening increased for cola and lemonade and stayed unchanged for the juice drink. Deciduous enamel was not found to be more susceptible to erosion than permanent enamel under the conditions of the present study.

Acids↗
PubMed2001

Mechanical unfolding of single filamin A (ABP-280) molecules detected by atomic force microscopy.

Filamin A (ABP-280), which is an actin-binding protein of 560 kDa as a dimer, can, together with actin filaments, produce an isotropic cross-linked three-dimensional network (actin/filamin A gel) that plays an important role in mechanical responses of cells in processes such as maintenance of membrane stability and translational locomotion. In this study, we investigated the mechanical properties of single filamin A molecules using atomic force microscopy. In force-extension curves, we observed sawtooth patterns corresponding to the unfolding of individual immunoglobulin (Ig)-fold domains of filamin A. At a pulling speed of 0.37 microm/s, the unfolding interval was sharply distributed around 30 nm, while the unfolding force ranged from 50 to 220 pN. This wide distribution of the unfolding force can be explained by variation in values of activation energy and the width of activation barrier of 24 Ig-fold domains of the filamin A at the unfolding transition. This unfolding can endow filamin A with great extensibility. The refolding of the unfolded chain of filamin A occurred when the force applied to the protein was reduced to near zero, indicating that its unfolding is reversible. Based on these results, we discuss here the physiological implications of the mechanical properties of single filamin A molecules.

Contractile Proteins↗
PubMed2005

Visualization of inositol 1,4,5-trisphosphate receptor by atomic force microscopy.

Inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) acts as a ligand-gated channel that mediates neuronal signals by releasing Ca(2+) from the endoplasmic reticulum. The three-dimensional (3D) structure of tetrameric IP(3)R has been demonstrated by using electron microscopy (EM) with static specimens; however, the dynamic aspects of the IP(3)R structure have never been visualized in a native environment. Here we attempt to measure the surface topography of IP(3)R in solution using atomic force microscopy (AFM). AFM revealed large protrusions extending approximately 4.3 nm above a flat membrane prepared from Spodoptera frugiperda (Sf9) cells overexpressing mouse type 1 IP(3)R (Sf9-IP(3)R1). The average diameter of the large protrusions was approximately 32 nm. A specific antibody against a cytosolic epitope close to the IP(3)-binding site enabled us to gold-label the Sf9-IP(3)R1 membrane as confirmed by EM. AFM images of the gold-labeled membrane revealed 7.7-nm high protrusions with a diameter of approximately 30 nm, which should be IP(3)R1-antibody complexes. Authentic IP(3)R1 immuno-purified from mouse cerebella had approximately the same dimensions as those of the IP(3)R-like protrusions on the membrane. Altogether, these results suggest that the large protrusions on the Sf9-IP(3)R1 membrane correspond to the cytosolic domain of IP(3)R1. Our study provides the first 3D representation of individual IP(3)R1 particles in an aqueous solution.

Calcium Channels↗
PubMed2000

Translocation-independent dimerization of the EcoKI endonuclease visualized by atomic force microscopy.

Bacterial type I restriction/modification systems are capable of performing multiple actions in response to the methylation pattern on their DNA recognition sequences. The enzymes making up these systems serve to protect the bacterial cells against viral infection by binding to their recognition sequences on the invading DNA and degrading it after extensive ATP-driven translocation. DNA cleavage has been thought to occur as the result of a collision between two translocating enzyme complexes. Using atomic force microscopy (AFM), we show here that EcoKI dimerizes rapidly when bound to a plasmid containing two recognition sites for the enzyme. Dimerization proceeds in the absence of ATP and is also seen with an EcoKI mutant (K477R) that is unable to translocate DNA. Only monomers are seen when the enzyme complex binds to a plasmid containing a single recognition site. Based on our results, we propose that the binding of EcoKI to specific DNA target sequences is accompanied by a conformational change that leads rapidly to dimerization. This event is followed by ATP-dependent translocation and cleavage of the DNA.

DNA Restriction Enzymes↗
PubMed2005

Membrane binding of beta2-glycoprotein I can be described by a two-state reaction model: an atomic force microscopy and surface plasmon resonance study.

Complexes formed between beta2GPI (beta2-glycoprotein I), a human plasma protein, and biological membranes are considered to be targets of macrophages and antiphospholipid autoantibodies involved in autoimmune diseases, such as antiphospholipid syndrome or systemic lupus erythematosus. The positively charged lysine-rich fifth domain of beta2GPI facilitates its interaction with phospholipid membranes containing acidic phospholipids, which normally become exposed by apoptotic processes. In the present study, atomic force microscopy was applied to visualize the binding of beta2GPI to a mixed phospholipid model membrane at physiological ionic strength. On supported lipid bilayers the formation of supramolecular assemblies of the protein with a height of approx. 3.3 nm was observed, suggesting a lateral agglomeration of beta2GPI. Detailed analysis of kinetic constants using surface plasmon resonance revealed that the binding can be described by a two-state reaction model, i.e. a very fast interaction step, depending on the content of acidic phospholipids in the bilayer, and a second step with significantly lower k(on) and k(off) values. Taken together, our results suggest a biphasic interaction mechanism: a fast step of beta2GPI binding to negatively charged lipids, mainly based on electrostatic interactions, and a slower phase of agglomeration of the protein on the bilayer surface accompanied by a protein-induced rigidification of the membrane, as revealed by electron paramagnetic resonance.

Cell Membrane↗
PubMed2004

Heterogeneous nanostructural and nanoelastic properties of pericellular and interterritorial matrices of chondrocytes by atomic force microscopy.

Hyaline cartilage consists of sparse chondrocytes and abundant extracellular matrix. There is a paucity of experimental data in support of the notion of conceivable regional differences in the mechanical properties of chondral matrices. Upon visual differentiation of the pericellular and interterritorial matrices in each of 19 fresh growth plate samples with toluidine blue and alizarin red labels, nanoindentation was applied separately to the pericellular matrix and interterritorial matrix to using fluid-phase atomic force microscopy and real-time imaging. The interterritorial matrix demonstrated elongated parallel ridges, whereas the pericellular matrix showed irregular, short-range elevations with characteristic pores and canals. Analysis of surface contours at 600nm(2) scan size revealed that the interterritorial matrix had significantly greater surface roughness (71+/-18nm; mean+/-SE) than the pericellular matrix (24+/-4nm) ( P< 0.001). The average Young's modulus of the interterritorial matrix was 636+/-123 (kPa), significantly greater than the pericellular matrix (265+/-53kPa) (P< 0.001 ). Thus, the interterritorial matrix appears to possess not only distinct microtopographic contours in comparison with the pericellular matrix, but also significantly greater mechanical stiffness. These distinctive nanostructural and nanomechanical properties may have implications in nutrient diffusion and fluid dynamics, both of which are of vital importance for cartilage health and function.

Animals↗
PubMed2004

Oxidative dissolution of chalcopyrite by Acidithiobacillus ferrooxidans analyzed by electrochemical impedance spectroscopy and atomic force microscopy.

The microbiological leaching of chalcopyrite (CuFeS(2)) is of great interest because of its potential application to many CuFeS(2)-rich ore materials. However, the efficiency of the microbiological process is very limited because this mineral is one of the most refractory to bacterial attack. Knowledge of bacterial role during chalcopyrite oxidation is very important in order to improve the efficiency of bioleaching operation. The oxidative dissolution of a massive chalcopyrite electrode by Acidithiobacillus ferrooxidans was evaluated by electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). A massive chalcopyrite electrode was utilized in a Tait-type electrochemical cell in acid medium for different immersion times in the presence or absence of bacterium. The differences observed in the impedance diagrams were correlated with the adhesion process of bacteria on the mineral surface.

Acidithiobacillus↗
PubMed2003

Revealing the mode of action of DNA topoisomerase I and its inhibitors by atomic force microscopy.

In this study, we used, for the first time, atomic force microscope (AFM) images to investigate the mode of action of DNA topoisomerase I (topo I) in the presence and absence of its inhibitors: camptothecin (CPT) and tyrphostin AG-1387. The results revealed that in the absence of the inhibitors, the enzyme relaxed supercoiled DNA starting from a certain point in the DNA molecules and proceeded in one direction towards one of the edges of the DNA molecule. In addition, the relaxation of the supercoiled DNA is subsequently followed by a knotting event. In the presence of CPT, enzyme-supercoiled DNA complexes in which the enzyme is locked inside a relaxed region of the supercoiled DNA molecule were observed. Tyrphostin AG-1387 altered the DNA relaxation process of topo I producing unique shapes of DNA molecules. AFM images of the topo I protein provided a picture of the enzyme, which resembles its known crystallographic structure. Thus, AFM images provide new information on the mode of action of topo I in the absence and presence of its inhibitors.

Animals↗
PubMed2001

Investigation of the morphology of the lacunocanalicular system of cortical bone using atomic force microscopy.

Mechanical loading has been implicated as a powerful driving mechanism for interstitial fluid flow through bone. However, little information is available with regard to the morphology of bone fluid spaces, e.g., the canalicular wall, which would be expected to dictate the type of flow regime developing in the lacunocanalicular system under mechanical loads. The purpose of this study was to examine the fine structure of the lacunocanalicular system in cortical bone using atomic force microscopy (AFM), resin casting methods, and selective etching of the specimen surface. A resin-cast replica of the canalicular wall was produced and surface morphology and dimensions were observed using AFM in tapping mode. Material contrast was obtained using surface potential measurements. A striped pattern perpendicular to the canaliculus long axis with a periodicity of 125 nm dominated the structure of the canalicular wall; it is likely that this was caused by the imprint of collagen fibrils arranged in parallel, lining the canaliculus wall. The largest dimension measured for canalicular diameter was on the order of 500 nm. The regular dips and ridges caused by the collagen that lines the wall are a source of roughness which may influence shear stresses imparted by the fluid on the cell surface as well as mixing of solutes within the lacunocanalicular system. In addition, the lacunocanalicular wall lining is likely to affect physicochemical interactions between the fluid and bone matrix. This has important implications for modeling and understanding the microfluid mechanics and rheology of the fluid-filled lacunocanalicular network.

Animals↗
PubMed2004

Atomic force microscopy of human metaphase chromosomes after differential staining of sister chromatids.

Human metaphase chromosomes, in which 5-bromo-deoxyuridine (BrdU) had been incorporated into the DNA, were treated with the fluorescent plus Giemsa (FPG) method. Use of this method distinctly stained one of the paired sister chromatids with the Giemsa solution due to the difference in content of BrdU in the two chromatids. These chromosomes with their differential staining of sister chromatids were observed by atomic force microscopy (AFM). In the air-dried specimens, one of the paired chromatids that was stained strongly with Giemsa solution was about two times higher than the counterpart that was stained faintly with Giemsa solution. In the critical point dried chromosomes, the height of the Giemsa positive chromatid roughly matched that of the Giemsa negative counterpart. These findings imply that the arrangement of the Giemsa negative chromatid after FPG staining is fragile and easily collapses due to the surface tension of water during air-drying. At higher magnifications, the surface structure differed between Giemsa positive and negative chromatids; the Giemsa positive chromatid (i.e., unifilarly BrdU-incorporated chromatid) was composed of fibrous structures while the Giemsa negative chromatid (i.e., bifilarly BrdU-incorporated chromatid) exhibited a fine granular appearance. These structural changes in the sister chromatids are thought to arise from the ultraviolet irradiation and heating of the chromosomes during FPG staining.

Antimetabolites↗
PubMed1996

Atomic force microscopy studies of solid lipid nanoparticles.

PURPOSE: Solid Lipid Nanoparticles (SLN) are an alternative carrier system for the controlled delivery of drugs. In most cases prednisolone loaded SLN show a biphasic release behaviour. The initial phase is characterised by a fast drug release, which is followed by a sustained drug release over several weeks. METHODS: The particles are produced by high pressure homogenisation of a lipid (e.g. compritol, cholesterol) dispersed in an aqueous surfactant solution. In this study atomic force microscopy was used to image the original unaltered shape and surface properties of the particles. The crystallinity of the nanoparticles was investigated by differential scanning calorimetry. RESULTS: The AFM investigations revealed the disc like shape of the particles. From differential scanning calorimetry data it can be concluded that the particle core is in the crystalline state. Additionally it was proven that the particles are surrounded by a soft layer. CONCLUSIONS: Thus it is conceivable that the fast initial drug release during in vitro dissolution tests takes place by drug release of the outer noncrystalline layers of the particles. The following sustained drug release can be assigned to the predisolone release of the inner crystalline particle layers.

Calorimetry, Differential Scanning↗
PubMed2005

Calcium-dependent human erythrocyte cytoskeleton stability analysis through atomic force microscopy.

Erythrocytes affected by age and diseases such as sickle cell anemia, hypertension, diabetes, etc., exhibit abnormally high intracellular Ca2+ ion levels, and appear to have altered cytoskeleton properties. It has been proposed that extra binding of Ca2+ to membrane-associated calmodulin attenuates the spectrin-ankyrin-Band 3 tether of the cytoskeleton to the cytoplasmic membrane and might change the cytoskeleton structure. Due to the close apposition of the network, direct observation of such a structural change in vivo is restricted. In this study, atomic force microscopy and quantitative image analysis were applied to investigate the structural change of young healthy erythrocyte cytoskeletons upon extra Ca2+ binding to the cytoplasmic membrane in vitro. The results show that extra Ca2+ binding increased the cytoskeleton rigidity and prevented spectrin aggregation during sample preparation. The cytoskeleton morphology observed in Ca2+ -incubated healthy young cell were similar to the glutaraldehyde-fixed healthy young cells.

Anion Exchange Protein 1, Erythrocyte↗
PubMed2005

Adhesiveness of dental resin-based restorative materials investigated with atomic force microscopy.

This study aimed to show that the polymerization contraction of dental methacrylate-based materials, when used as adhesives on hard substrate, produces voids at the material-substrate interface. This phenomenology is closely related with the nanoleakage and the sealing ability of these materials. One prime/bond system, three restorative composite resins, and one orthodontic bonding system were cured by using mirror-like glass slides as a compliance-free reference substrate. The adhesive surface was analyzed by atomic force microscopy, and the polymerization contraction of bulk material was tested by laser beam-scanning method. Nanoperiodic structure of three-dimensional (3D) images, section analysis, and roughness characteristics (R(a) and R(z)) indicated that polymerization contraction produced voids at the interface. When the adhesive surface was exposed to oral simulating fluids (water, ethanol, and lactic acid solutions), hydrolytic degradation involved some hundreds of nanometers in depth. In visible light-cured (VLC) materials, the interface porosity decreased when an irradiation pause ( approximately 2 min) was carried out during gelation.

Adhesiveness↗
PubMed2002

Interplay between nonlinearity, scan speed, damping, and electronics in frequency modulation atomic-force microscopy.

Numerical simulations of the frequency modulation atomic force microscope, including the whole dynamical regulation by the electronics, show that the cantilever dynamics is conditionally stable and that there is a direct link between the frequency shift and the conservative tip-sample interaction. However, a soft coupling between the electronics and the nonlinearity of the interaction may significantly affect the damping. A resonance between the scan speed and the response time of the system can provide a simple explanation for the spatial shift and contrast inversion between topographical and damping images, and for the extreme sensitivity of the damping to a tip change.

Journal Article↗
PubMed2005

Fine structure study of Abeta1-42 fibrillogenesis with atomic force microscopy.

One of the hallmarks of Alzheimer's disease is the self-aggregation of the amyloid beta peptide (Abeta) in extracellular amyloid fibrils. Among the different forms of Abeta, the 42-residue fragment (Abeta1-42) readily self-associates and forms nucleation centers from where fibrils can quickly grow. The strong tendency of Abeta1-42 to aggregate is one of the reasons for the scarcity of data on its fibril formation process. We have used atomic force microscopy (AFM) to visualize in liquid environment the fibrillogenesis of synthetic Abeta1-42 on hydrophilic and hydrophobic surfaces. The results presented provide nanometric resolution of the main structures characteristic of the several steps from monomeric Abeta1-42 to mature fibrils in vitro. Oligomeric globular aggregates of Abeta1-42 precede the appearance of protofibrils, the first fibrillar species, although we have not obtained direct evidence of oligomer-protofibril interconversion. The protofibril dimensions deduced from our AFM images are consistent with a model that postulates the stacking of the peptide in a hairpin conformation perpendicular to the long axis of the protofibril, forming single beta-sheets ribbon-shaped. The most abundant form of Abeta1-42 fibril exhibits a nodular structure with a ~100-nm periodicity. This length is very similar 1) to the length of protofibril bundles that are the dominant feature at earlier stages in the aggregation process, 2) to the period of helical structures that have been observed in the core of fibrils, and 3) to the distance between regularly spaced, structurally weak fibril points. Taken together, these data are consistent with the existence of a ~100-nm long basic protofibril unit that is a key fibril building block.

Amyloid↗
PubMed1999

Calcium-mediated structural changes of native nuclear pore complexes monitored by time-lapse atomic force microscopy.

Nuclear pore complexes (NPCs) are large macromolecular assemblies embedded in the double membrane nuclear envelope. They are the major gateways mediating transport of ions, small molecules, proteins, RNAs, and ribonucleoprotein particles in and out of the nucleus in interphase cells. Understanding structural changes at the level of individual pores will be a prerequisite to eventually correlate the molecular architecture of the NPC with its distinct functional states during nucleocytoplasmic transport. Toward this goal, we have employed time-lapse atomic force microscopy of native NPCs kept in buffer, and recorded calcium-mediated structural changes such as the opening (i.e. +Ca2+) and closing (i.e. -Ca2+) of individual nuclear baskets. Most likely, this structural change of the nuclear basket involves its distal ring which may act as an iris-like diaphragm. In order to directly correlate distinct structural features with corresponding functional states and dynamic aspects, we also addressed the question of whether the "central plug" or "transporter" actually represents a calcium-sensitive component of the NPC involved in mediating nucleocytoplasmic transport. Our data indicate that in the absence of ATP, cytoplasmic plugging/unplugging of the NPC is insensitive to calcium.

Animals↗