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At least 1,045 records · Page 58Linked to original sources
PubMed1998

Irreversible binding of poly(ADP)ribose polymerase cleavage product to DNA ends revealed by atomic force microscopy: possible role in apoptosis.

During apoptosis, DNA undergoes fragmentation and caspase-3 cleaves poly(ADP-ribose) polymerase (PARP) into both a 24-kDa fragment containing the DNA binding domain and an 89-kDa fragment containing the catalytic and automodification domains. Atomic force microscopy revealed that recombinant full-length PARP bound to plasmid DNA fragments and linked them into chainlike structures. Automodification of PARP in the presence of NAD+ resulted in its dissociation from the DNA fragments, which, nevertheless, remained physically aligned. A recombinant 28-kDa fragment of PARP containing the DNA binding domain but lacking the automodification domain irreversibly bound to and linked DNA fragments in the absence or presence of NAD+. Identical results were obtained on incubation of internucleosomal DNA fragments from apoptotic cells with the products of cleavage of recombinant PARP by purified caspase-3. The 24-kDa product of PARP cleavage by caspase-3 may contribute to the irreversibility of apoptosis by blocking the access of DNA repair enzymes to DNA strand breaks.

Animals↗
PubMed2001

Visualization by atomic force microscopy of tobacco mosaic virus movement protein-RNA complexes formed in vitro.

The structure of complexes formed in vitro by tobacco mosaic virus (TMV)-coded movement protein (MP) with TMV RNA and short (890 nt) synthetic RNA transcripts was visualized by atomic force microscopy on a mica surface. MP molecules were found to be distributed along the chain of RNA and the structure of MP-RNA complexes depended on the molar MP:RNA ratios at which the complexes were formed. A rise in the molar MP:TMV RNA ratio from 20:1 to 60-100:1 resulted in an increase in the density of the MP packaging on TMV RNA and structural conversion of complexes from RNase-sensitive 'beads-on-a-string' into a 'thick string' form that was partly resistant to RNAse. The 'thick string'-type RNase-resistant complexes were also produced by short synthetic RNA transcripts at different MP:RNA ratios. The 'thick string' complexes are suggested to represent clusters of MP molecules cooperatively bound to discrete regions of TMV RNA and separated by protein-free RNA segments.

Aluminum Silicates↗
PubMed2003

Self-assembly and recrystallization of bacterial S-layer proteins at silicon supports imaged in real time by atomic force microscopy.

The self-assembly of bacterial surface-layer (S-layer) proteins (SbpA of Bacillus sphaericus CCM 2177) at silicon supports (hydrophobic, non-plasma-treated and hydrophilic, O2 plasma-treated silicon supports) was imaged in real time by atomic force microscopy (AFM). A closed mosaic layer consisting of small crystals (less than 200 nm in diameter) was formed at a hydrophobic silicon support, whereas a coherent crystalline lattice consisting of large domains (2-10 micro m in size) was generated at O2 plasma-treated, hydrophilic silicon wafers. The structure of the formed layers was a monolayer (9 nm in height) at the hydrophobic silicon and a bilayer (15 nm in height) at the hydrophilic silicon. In situ AFM measurements confirmed the importance of ionic bonds in the formation of crystalline SbpA layers at silicon supports. Rupture of the protein subunits with a metal chelator from the crystalline lattice of SbpA was visualized in situ by AFM. The stability of solid-supported SbpA layers could be enhanced by cross-linking the S-layers with amino-amino or amino-carboxyl group directed cross-linkers.

Bacillus↗
PubMed2001

Atomic force microscopy for the characterization of immobilized enzyme molecules on biosensor surfaces.

The development of biosensors has been one of the key areas in biotechnology and biomedical studies. Often it is difficult to investigate the immobilized biomolecules on the surfaces for biosensor optimization. Atomic force microscopy (AFM) should provide an ideal means for the visualization of biosensor surface and for the investigation of biomolecule activities. Therefore, AFM has been employed to study the surface topography of immobilized glutamate dehydrogenase (GDH) on two-dimensional glutamate biosensor surfaces. Correlation between the surface topography and the activity of the biosensor was investigated. Surface analysis has revealed that the enzymatic activity of the immobilized GDH molecules on the biosensor surface is linked to surface roughness, as measured by the peak-to-valley distance. Fractal dimension of the immobilization sensor surface was found to be a good parameter for judging the quality of the immobilized biosensors. As enzyme immobilization time increases, the biosensor has its maximum activity with around 18 h of immobilization in 10(-6) M GDH solution. Various biosensors prepared under different experimental conditions have been studied by AFM. This technique is shown to be an effective tool to characterize biosensor surfaces.

Animals↗
PubMed2006

Isolating recombinant antibodies against specific protein morphologies using atomic force microscopy and phage display technologies.

Isolation of antibodies to antigens that are either unstable, exist in multiple morphologies or have very limited availability can be prohibitively difficult. Here we describe a novel technique combining the capabilities of phage display antibody technology and atomic force microscopy (AFM) that is used to isolate antibody fragments that bind to a specific morphology of the target antigen, alpha-synuclein. AFM imaging allows us to both visualize the presence and morphology of the target antigen as well as to monitor the efficiency of each step in the bio-panning process. We demonstrate that phage displayed antibodies specific to the target antigen morphology can be isolated after only two rounds of selection. The target antigen, alpha-synuclein, has been correlated with the Parkinson's disease (PD). Accumulation of alpha-synuclein fibrillar aggregates into Lewy body inclusions is a hallmark feature of PD. While alpha-synuclein can form several different aggregate morphologies including oligomers, protofibrils and fibrils, the role of these morphologies in the progression of PD is not known. The successful selection of the recombinant antibody described here can have potential therapeutic value since the single-chain fragment variable (scFv) can be expressed intracellularly to control folding and toxicity of the specific protein aggregates.

Antibodies↗
PubMed2002

Segmented nanofibers of spider dragline silk: atomic force microscopy and single-molecule force spectroscopy.

Despite its remarkable materials properties, the structure of spider dragline silk has remained unsolved. Results from two probe microscopy techniques provide new insights into the structure of spider dragline silk. A soluble synthetic protein from dragline silk spontaneously forms nanofibers, as observed by atomic force microscopy. These nanofibers have a segmented substructure. The segment length and amino acid sequence are consistent with a slab-like shape for individual silk protein molecules. The height and width of nanofiber segments suggest a stacking pattern of slab-like molecules in each nanofiber segment. This stacking pattern produces nano-crystals in an amorphous matrix, as observed previously by NMR and x-ray diffraction of spider dragline silk. The possible importance of nanofiber formation to native silk production is discussed. Force spectra for single molecules of the silk protein demonstrate that this protein unfolds through a number of rupture events, indicating a modular substructure within single silk protein molecules. A minimal unfolding module size is estimated to be around 14 nm, which corresponds to the extended length of a single repeated module, 38 amino acids long. The structure of this spider silk protein is distinctly different from the structures of other proteins that have been analyzed by single-molecule force spectroscopy, and the force spectra show correspondingly novel features.

Amino Acid Sequence↗
PubMed2005

Interaction analysis of chimeric metal-binding green fluorescent protein and artificial solid-supported lipid membrane by quartz crystal microbalance and atomic force microscopy.

Non-specific adsorption and specific interaction between a chimeric green fluorescent protein (GFP) carrying metal-binding region and the immobilized zinc ions on artificial solid-supported lipid membranes was investigated using the quartz crystal microbalance technique and the atomic force microscopy (AFM). Supported lipid bilayer, composed of octanethiol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid)succinyl] (NTA-DOGS)-Zn2+, was formed on the gold electrode of quartz resonator (5 MHz). Binding of the chimeric GFP to zinc ions resulted in a rapid decrease of resonance frequency. Reversibility of the process was demonstrated via the removal of metal ions by EDTA. Nanoscale structural orientation of the chimeric GFP on the membrane was imaged by AFM. Association constant of the specific binding to metal ions was 2- to 3-fold higher than that of the non-specific adsorption, which was caused by the fluidization effect of the metal-chelating lipid molecules as well as the steric hindrance effect. This infers a possibility for a further development of biofunctionalized membrane. However, maximization is needed in order to attain closer advancement to a membrane-based sensor device.

Adsorption↗
PubMed2004

Atomic force microscopy investigation of wild-type Moloney murine leukemia virus particles and virus particles lacking the envelope protein.

Moloney murine leukemia virus (M-MuLV) lacking the gene for the envelope glycoprotein (env(-)) was produced in NIH 3T3 cells and investigated using atomic force microscopy (AFM). The particles were compared with similarly produced wild-type virions, some of which had been exposed to a monoclonal antibody against the surface component of the envelope protein (SU protein). The env(-) particles generally exhibit a distinctly different external appearance suggesting only a low density of associated proteins that have an almost fluid, mechanically unstable character. The weakly associated proteins may be host cell membrane proteins that are incorporated into the viral membrane in place of or in addition to virus envelope protein. The amount of this non-viral protein on virion surfaces appears to vary from negligible in most cases to a substantial complement in others. It seems clear that the presence of the envelope protein, in a mechanical sense, significantly strengthens and stabilizes the virion envelope. Binding of monoclonal antibody to wild-type virions indicates that some particles expose a significant amount of antigen while adjacent virions may not. This suggests that the conformation of the envelope glycoprotein or the disposition of oligosaccharides may be different among particles, on some virions exposing the specific epitope, and others little or none.

Animals↗
PubMed2001

Atomic force microscopy as a novel pharmacological tool.

With the advent of the atomic force microscope (AFM), the study of biological samples has become more realistic because, in most cases, samples are not covered or fixed, which makes it possible to observe them while the cells are alive. This advantage of the AFM allowed the advent of a new invention: nanobiosensors using the cantilever (probe) of the AFM and, in this case, it is possible to observe the entering or exiting of specific molecules (including medications) from living cells. This is the smallest biosensor in the world, measuring about 100 microm long (about the width of a hair). Beyond sensing the area of interest with this biosensor, it is also possible to see the area and exactly what is occurring on it, in real time. This new tool will be very useful for several areas: molecular pharmacology, enzymology, physiology, molecular biology, biotechnology, biophysics, physical chemistry, analytical chemistry, and organic chemistry. This article discusses, mainly, the applications of this new technique to the field of pharmacology.

Biosensing Techniques↗
PubMed2006

Atomic force microscopy investigation of Turnip Yellow Mosaic Virus capsid disruption and RNA extrusion.

Turnip Yellow Mosaic Virus (TYMV) was subjected to a variety of procedures which disrupted the protein capsids and produced exposure of the ssRNA genome. The results of the treatments were visualized by atomic force microscopy (AFM). Both in situ and ex situ freeze-thawing produced RNA emission, though at low efficiency. The RNA lost from such particles was evident, in some cases in the process of exiting the virions. More severe disruption of TYMV and extrusion of intact RNA onto the substrate were produced by drying the virus and rehydrating with neutral buffer. Similar products were also obtained by heating TYMV to 70 -75 degrees C and by exposure to alkaline pH. Experiments showed the nucleic acid to have an elaborate secondary structure distributed linearly along its length.

Capsid↗
PubMed2002

Growth of beta-amyloid(1-40) protofibrils by monomer elongation and lateral association. Characterization of distinct products by light scattering and atomic force microscopy.

Amyloid plaques in brain tissue are a hallmark of Alzheimer's disease. Primary components of these plaques are 40- and 42-residue peptides, denoted A beta(1-40) and A beta(1-42), that are derived by proteolysis of cellular amyloid precursor protein. Synthetic A beta(1-40) and A beta(1-42) form amyloid fibrils in vitro that share many features with the amyloid in plaques. Soluble intermediates in A beta fibrillogenesis, termed protofibrils, have been identified previously, and here we describe the in vitro formation and isolation of A beta(1-40) protofibrils by size exclusion chromatography. In some experiments, the A beta(1-40) was radiomethylated to better quantify various A beta species. Mechanistic studies clarified two separate modes of protofibril growth, elongation by monomer deposition and protofibril-protofibril association, that could be resolved by varying the NaCl concentration. Small isolated protofibrils in dilute Tris-HCl buffers were directed along the elongation pathway by addition of A beta(1-40) monomer or along the association pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrils with initial molecular masses M(w) of (7-30) x 10(3) kDa grew to M(w) values of up to 250 x 10(3) kDa by these two growth processes. However, the mass per unit length of the associated protofibrils was about 2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer deposition with the elongated, associated, and initial protofibril pools were identical when equal number concentrations of original protofibrils were compared, indicating that the original number of protofibril ends had not been altered by the elongation or association processes. Atomic force microscopy revealed heterogeneous initial protofibrils that became more rodlike following the elongation reaction. Our data indicate that protofibril elongation in the absence of NaCl results from monomer deposition only at the ends of protofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril association occurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactions that result in a relatively disordered fibril structure.

Alzheimer Disease↗
PubMed1998

Atomic force microscopy: influence of air drying and fixation on the morphology and viscoelasticity of cultured cells.

The influence of fixation, air-drying and liquid-imaging on the morphology as well as on the viscoelasticity of malignant mesothelioma cells was studied by atomic force microscopy. In this study, dehydrated cells were more easily scanned and offered faster data recording than hydrated cells. However, the influence of fixation strength was more noticeable. Strong fixation induced flattening of the cytoplasm and loss of nuclear structure, resulting in a clearly visible cytoskeleton which could be easily seen as fibres orientated in the direction of the cell growth. By contrast, the morphology of hydrated cells was influenced to a lesser degree on fixation and showed an overall 'rounding' of the surface with vague, ill-defined structures. Nuclear areas of these samples were difficult to image. Viscoelasticity measurements also exhibited large differences. Dehydrated cells were much harder and showed a uniform indentation profile over the whole cell that was independent of fixation. Indentation on hydrated cells was large and depended on the height of the measuring spot, the submembranous structure and, to a lesser extent, on fixation. To calculate an overall 'cellular' viscoelasticity, different methods were tested on these samples. Indentations of multiple, randomly chosen points, covering the whole cell, were measured and averaged to yield a mean indentation score. We avoided the thin and shadowed areas since it was shown that these regions were less suited for measuring. Using this design, large viscoelasticity differences were found, on which the influence of the external parameters could be shown. In another set-up, layered imaging was tried. However, long data acquisition times caused cellular activation and rearrangement, making this scanning mode unsatisfactory.

Cells, Cultured↗
PubMed2000

Atomic force microscopy to study the effects of ITIM-bearing FcgammaRIIB on the activation of RBL-2H3 cells.

We have studied here the effects of ITIM-bearing FcgammaRIIB2 on the FcvarepsilonRI-dependent activation of rat basophilic leukemia (RBL-2H3) cells by atomic force microscopy (AFM). AFM images showed that ruffling and degranulation of RBL-2H3 cells were significantly reduced but the cell spreading was not by the co-clustering of FcgammaRIIB2 and FcvarepsilonRI. From the results it was shown that the co-clustering of ITIM-bearing negative co-receptors in a single RBL cell hardly induce the degranulation though the [Ca(2+)](i) increased transiently in the cell. This suggested that secretory responses and the membrane ruffling which were induced by the crosslinkng of FcgammaRII should be negatively controlled by the co-clustering of FcgammaRII.

Animals↗
PubMed2003

Real-time analysis of the effects of cholesterol on lipid raft behavior using atomic force microscopy.

Cholesterol plays a crucial role in cell membranes, and has been implicated in the assembly and maintenance of sphingolipid-rich rafts. We have examined the cholesterol-dependence of model rafts (sphingomyelin-rich domains) in supported lipid monolayers and bilayers using atomic force microscopy. Sphingomyelin-rich domains were observed in lipid monolayers in the absence and presence of cholesterol, except at high cholesterol concentrations, when separate domains were suppressed. The effect of manipulating cholesterol levels on the behavior of these sphingomyelin-rich domains in bilayers was observed in real time. Depletion of cholesterol resulted in dissolution of the model lipid rafts, whereas cholesterol addition resulted in an increased size of the sphingomyelin-rich domains and eventually the formation of a single raftlike lipid phase. Cholesterol colocalization with sphingomyelin-rich domains was confirmed using the sterol binding agent filipin.

Cholesterol↗
PubMed2002

Atomic force microscopy imaging of novel type of polymeric colloidal nanostructures.

Polymeric nanoparticles were prepared by the interfacial poly-condensation of the lipophilic monomer, phtaloyldichloride and the hydrophilic monomer, diethylenetriamine, in the presence and absence of the surfactant Pluronic F68. The colloidal systems were analysed by dynamic light scattering and atomic force microscopy, the structures formed have two populations (150 and 350 nm) in the presence of the surfactant and one population (450 nm) in the absence of the surfactant. The results can be interpreted in terms of the formation of hollow nanocapsules that collapse on deposition and drying.

Colloids↗
PubMed1999

Watching amyloid fibrils grow by time-lapse atomic force microscopy.

Late-onset diabetes is typically associated with amyloid deposits of fibrillar amylin in the pancreatic islets. Aqueous synthetic human amylin spontaneously forms polymorphic fibrils in vitro, and this system was used to examine the dynamics of fibril assembly. By time-lapse atomic force microscopy (AFM), the growth of individual amylin fibrils on a mica surface was observed over several hours. Prominent was the assembly of a protofibril with an elongation rate in these experiments of 1.1(+/-0.5) nm/minute. The assembly of higher order polymorphic fibrils was also observed. Growth of the protofibrils was bidirectional, i.e. it occurred by elongation at both ends. This ability of AFM to continuously monitor growth, directionality, and changes in morphology for individual fibrils, provides a significant advantage over spectroscopy-based bulk methods which average the growth of many fibrils and typically require 100 to 1000-fold more protein. The time-lapse AFM procedure used for human amylin here is thus likely to be applicable to fibril formation from other amyloid proteins and peptides.

Aluminum Silicates↗
PubMed2000

Probing protein-peptide-protein molecular architecture by atomic force microscopy and surface plasmon resonance.

We demonstrate the creation of a protein multilayer which utilises the high affinity interaction between streptavidin and biotin and incorporates a peptidic spacer. Surface plasmon resonance measurements enabled us to monitor the construction of the multilayer in real time. Atomic force microscopy was utilised to determine surface functionality at each stage of the multilayer construction, allowing us to investigate the associated mechanical properties. In this context we observed an increase in biomolecular stretching on the formation of the multilayer. We demonstrate, utilising circular dichroism, that variations in the solvent can affect the secondary structure of the peptide linker and hence its mechanical properties. Trifluoroethanol titrations on the assembled system indicate that the multilayer properties are also stimuli responsive with regard to solvent conditions. These results indicate that the multilayer stretch before cleavage is increased in the presence of trifluoroethanol. This was not expected from the study of the individual linker alone, indicating the need to study the system as a whole as opposed to the isolated components.

Biosensing Techniques↗
PubMed2004

Organization of telomeric nucleosomes: atomic force microscopy imaging and theoretical modeling.

Telomeric chromatin has peculiar features with respect to bulk chromatin, which are not fully clarified to date. Nucleosomal arrays, reconstituted on fragments of human telomeric DNA and on tandemly repeated tetramers of 5S rDNA, have been investigated at single-molecule level by atomic force microscopy and Monte Carlo simulations. A satisfactory correlation emerges between experimental and theoretical internucleosomal distance distributions. However, in the case of telomeric nucleosomal arrays containing two nucleosomes, we found significant differences. Our results show that sequence features of DNA are significant in the basic chromatin organization, but are not the only determinant.

Animals↗