In situ atomic force microscopy study of Alzheimer's beta-amyloid peptide on different substrates: new insights into mechanism of beta-sheet formation.
We have applied in situ atomic force microscopy to directly observe the aggregation of Alzheimer's beta-amyloid peptide (Abeta) in contact with two model solid surfaces: hydrophilic mica and hydrophobic graphite. The time course of aggregation was followed by continuous imaging of surfaces remaining in contact with 10-500 microM solutions of Abeta in PBS (pH 7.4). Visualization of fragile nanoscale aggregates of Abeta was made possible by the application of a tapping mode of imaging, which minimizes the lateral forces between the probe tip and the sample. The size and the shape of Abeta aggregates, as well as the kinetics of their formation, exhibited pronounced dependence on the physicochemical nature of the surface. On hydrophilic mica, Abeta formed particulate, pseudomicellar aggregates, which at higher Abeta concentration had the tendency to form linear assemblies, reminiscent of protofibrillar species described recently in the literature. In contrast, on hydrophobic graphite Abeta formed uniform, elongated sheets. The dimensions of those sheets were consistent with the dimensions of beta-sheets with extended peptide chains perpendicular to the long axis of the aggregate. The sheets of Abeta were oriented along three directions at 120 degrees to each other, resembling the crystallographic symmetry of a graphite surface. Such substrate-templated self-assembly may be the distinguishing feature of beta-sheets in comparison with alpha-helices. These studies show that in situ atomic force microscopy enables direct assessment of amyloid aggregation in physiological fluids and suggest that Abeta fibril formation may be driven by interactions at the interface of aqueous solutions and hydrophobic substrates, as occurs in membranes and lipoprotein particles in vivo. (+info)
Ethanol-induced structural transitions of DNA on mica.
The effect of ethanol on the structure of DNA confined to mica in the presence of Mg2+was examined by varying the ethanol concentration and imaging the DNA by atomic force microscopy. Contour length measurements of the DNA show a transition from all-B-form at 0% ethanol to all-A-form at >25% ethanol. At intermediate ethanol concentrations, contour lengths suggest that individual molecules of air-dried DNA are trapped with mixed compositions of A-form and B-form. The relative composition depends on the ethanol concentration. Fitting the length distributions at intermediate ethanol concentrations to a simple binomial model results in an upper bound estimate for the A-form and B-form domains of approximately 54 bp in the individual molecules. In addition to length changes, the apparent persistence length of DNA decreases with increasing ethanol concentration. At high concentrations of ethanol (>20%), DNA formed several higher order structures, including flower shaped condensates and toroids. (+info)
Microstructure and homogeneity of dental porcelain frits.
The microstructure and homogeneity of three commercial dentin and incisal unfired porcelain frits (one conventional and two ultra-low fusing types, fused-to metal were analyzed by X-ray diffractometry, scanning electron microspectroscopy, and wavelength- and energy dispersive X-ray microspectroscopy. The average contents of tetragonal and cubic leucite for the conventional and one of the ultra-low fusing type frits were 20.1-22.6 wt% and 0-2.6 wt%, respectively, whereas those of another of the ultra-low fusing type frits were about 11.5-11.6 wt% and 2.9-4.6 wt%, respectively. The conventional type frits seemed to be admixtures of three kinds of glass frits. One of the ultra-low fusing type frits seemed to be an admixture of four kinds of glass frits. Another ultra-low fusing frits seemed to be only one kind of glass frit dispersed with small size, less than 1 micron, leucite crystals. There were no remarkable differences in microstructure and homogeneity between dentin and incisal porcelain frits in each brand. (+info)
Phase imaging by atomic force microscopy: analysis of living homoiothermic vertebrate cells.
Atomic force microscope-based phase imaging in air is capable of elucidating variations in material properties such as adhesion, friction, and viscoelasticity. However, the interpretation of phase images of specimens in a fluid environment requires clarification. In this report, we systematically analyzed atomic force microscope-derived phase images of mica, glass, and collagen under the same conditions as used for living cells at various tapping forces; the resulting data provide critical information for the interpretation of phase images of living cells. The peripheral regions of COS-1 cells consistently show a more negative phase shift than the glass substrate in phase images at set-point amplitude: free amplitude (Asp/A0) = 0.6-0.8. In addition, at all Asp/A0 values suitable for phase imaging, tapping frequency appears to be high enough to ensure that phase shifts are governed primarily by stiffness. Consequently, phase imaging is capable of high resolution studies of the cellular surface by detecting localized variations in stiffness. We demonstrate that phase imaging of a bifurcating fiber in COS-1 cell cytoplasm is readily capable of a lateral resolution of approximately 30 nm. (+info)
Atomic force microscopy imaging of DNA covalently immobilized on a functionalized mica substrate.
A procedure for covalent binding of DNA to a functionalized mica substrate is described. The approach is based on photochemical cross-linking of DNA to immobilized psoralen derivatives. A tetrafluorphenyl (TFP) ester of trimethyl psoralen (trioxalen) was synthesized, and the procedure to immobilize it onto a functionalized aminopropyl mica surface (AP-mica) was developed. DNA molecules were cross-linked to trioxalen moieties by UV irradiation of complexes. The steps of the sample preparation procedure were analyzed with x-ray photoelectron spectroscopy (XPS). Results from XPS show that an AP-mica surface can be formed by vapor phase deposition of silane and that this surface can be derivatized with trioxalen. The derivatized surface is capable of binding of DNA molecules such that, after UV cross-linking, they withstand a thorough rinsing with SDS. Observations with atomic force microscopy showed that derivatized surfaces remain smooth, so DNA molecules are easily visualized. Linear and circular DNA molecules were photochemically immobilized on the surface. The molecules are distributed over the surface uniformly, indicating rather even modification of AP-mica with trioxalen. Generally, the shapes of supercoiled molecules electrostatically immobilized on AP-mica and those photocross-linked on trioxalen-functionalized surfaces remain quite similar. This suggests that UV cross-linking does not induce formation of a noticeable number of single-stranded breaks in DNA molecules. (+info)
Computer-assisted morphometry of cell-substratum contacts.
AIM: Quantitative analysis of size and shape of the cell-substratum contacts in Dictyostelium and comparison of these parameters between wild-type cells and the cells bearing cytoskeletal protein mutations. METHODS: Reflection interference contrast microscopy (RICM) was used to image the areas of contact between aggregation-competent Dictyostelium cells and weakly adhesive mica surfaces. The cell-substratum contact areas were automatically identified in RICM micrographs by digital image processing. Information about the size and shape of the contact areas was obtained by using the shape descriptors based on two-dimensional geometrical moment invariants. RESULTS: Lack of either of the two actin-crosslinking proteins, a-actinin and 120 kDa gelation factor, similarly affects the cell-substratum interactions of Dictyostelium cells. The shape descriptors, elongation and dispersion, of the contact areas were reduced by 10% to 30% in mutant cells when compared to the wild type, but the size of the contacts was not affected. CONCLUSION: Video microscopy combined with digital image processing and quantitative image analysis is capable of revealing small phenotypic effects of cytoskeletal protein mutations on the level of single cells. Such automated microscopic methods are expected to gain importance and find a widespread use in biomedicine. (+info)
Risks of respiratory disease in the heavy clay industry.
OBJECTIVES: Little information is available on the quantitative risks of respiratory disease from quartz in airborne dust in the heavy clay industry. Available evidence suggested that these risks might be low, possibly because of the presence in the dust of other minerals, such as illite and kaolinite, which may reduce the harmful effects of quartz. The aims of the present cross sectional study were to determine among workers in the industry (a) their current and cumulative exposures to respirable mixed dust and quartz; (b) the frequencies of chest radiographic abnormalities and respiratory symptoms; (c) the relations between cumulative exposure to respirable dust and quartz, and risks of radiographic abnormality and respiratory symptoms. METHODS: Factories were chosen where the type of process had changed as little as possible during recent decades. 18 were selected in England and Scotland, ranging in size from 35 to 582 employees, representing all the main types of raw material, end product, kilns, and processes in the manufacture of bricks, pipes, and tiles but excluding refractory products. Weights of respirable dust and quartz in more than 1400 personal dust samples, and site histories, were used to derive occupational groups characterised by their levels of exposure to dust and quartz. Full size chest radiographs, respiratory symptoms, smoking, and occupational history questionnaires were administered to current workers at each factory. Exposure-response relations were examined for radiographic abnormalities (dust and quartz) and respiratory symptoms (dust only). RESULTS: Respirable dust and quartz concentrations ranged from means of 0.4 and 0.04 mg.m-3 for non-process workers to 10.0 and 0.62 mg.m-3 for kiln demolition workers respectively. Although 97% of all quartz concentrations were below the maximum exposure limit of 0.4 mg.m-3, 10% were greater than this among the groups of workers exposed to most dust. Cumulative exposure calculations for dust and quartz took account of changes of occupational group, factory, and kiln type at study and non-study sites. Because of the importance of changes of kiln type additional weighting factors were applied to concentrations of dust and quartz during previous employment at factories that used certain types of kiln. 85% (1934 employees) of the identified workforce attended the medical surveys. The frequency of small opacities in the chest radiograph, category > or = 1/0, was 1.4% (median reading) and seven of these 25 men had category > or = 2/1. Chronic bronchitis was reported by 14.2% of the workforce and breathlessness, when walking with someone of their own age, by 4.4%. Risks of having category > or = 0/1 small opacities differed by site and were also influenced by age, smoking, and lifetime cumulative exposure to respirable dust and quartz. Although exposures to dust and to quartz were highly correlated, the evidence suggested that radiological abnormality was associated with quartz rather than dust. A doubling of cumulative quartz exposure increased the risk of having category > or = 0/1 by a factor of 1.33. Both chronic bronchitis and breathlessness were significantly related to dust exposure. CONCLUSIONS: Although most quartz concentrations at the time of this study were currently below regulatory limits in the heavy clay industry, high exposures regularly occurred in specific processes and occasionally among most occupational groups. However, there are small risks of pneumoconiosis and respiratory symptoms in the industry, although frequency of pneumoconiosis is low in comparison to other quartz exposed workers. (+info)
Polymer-cushioned bilayers. II. An investigation of interaction forces and fusion using the surface forces apparatus.
We have created phospholipid bilayers supported on soft polymer "cushions" which act as deformable substrates (see accompanying paper, Wong, J. Y., J. Majewski, M. Seitz, C. K. Park, J. N. Israelachvili, and G. S. Smith. 1999. Biophys. J. 77:1445-1457). In contrast to "solid-supported" membranes, such "soft-supported" membranes can exhibit more natural (higher) fluidity. Our bilayer system was constructed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles onto polyethylenimine (PEI)-supported Langmuir-Blodgett lipid monolayers on mica. We used the surface forces apparatus (SFA) to investigate the long-range forces, adhesion, and fusion of two DMPC bilayers both above and below their main transition temperature (T(m) approximately 24 degrees C). Above T(m), hemi-fusion activation pressures of apposing bilayers were considerably smaller than for solid-supported bilayers, e.g., directly supported on mica. After separation, the bilayers naturally re-formed after short healing times. Also, for the first time, complete fusion of two fluid (liquid crystalline) phospholipid bilayers was observed in the SFA. Below T(m) (gel state), very high pressures were needed for hemi-fusion and the healing process became very slow. The presence of the polymer cushion significantly alters the interaction potential, e.g., long-range forces as well as fusion pressures, when compared to solid-supported systems. These fluid model membranes should allow the future study of integral membrane proteins under more physiological conditions. (+info)