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PubMed2006

Lipid-induced beta-amyloid peptide assemblage fragmentation.

Alzheimer's disease is the most common cause of dementia and is widely believed to be due to the accumulation of beta-amyloid peptides (Abeta) and their interaction with the cell membrane. Abetas are hydrophobic peptides derived from the amyloid precursor proteins by proteolytic cleavage. After cleavage, these peptides are involved in a self-assembly-triggered conformational change. They are transformed into structures that bind to the cell membrane, causing cellular degeneration. However, it is not clear how these peptide assemblages disrupt the structural and functional integrity of the membrane. Membrane fluidity is one of the important parameters involved in pathophysiology of disease-affected cells. Probing the Abeta aggregate-lipid interactions will help us understand these processes with structural detail. Here we show that a fluid lipid monolayer develop immobile domains upon interaction with Abeta aggregates. Atomic force microscopy and transmission electron microscopy data indicate that peptide fibrils are fragmented into smaller nano-assemblages when interacting with the membrane lipids. Our findings could initiate reappraisal of the interactions between lipid assemblages and Abeta aggregates involved in Alzheimer's disease.

Amyloid beta-Peptides↗

PubMed2004

Complex molecular architecture of beet yellows virus particles.

Closteroviruses possess exceptionally long filamentous virus particles that mediate protection and active transport of the genomic RNA within infected plants. These virions are composed of a long "body" and short "tail" whose principal components are the major and minor capsid proteins, respectively. Here we use biochemical, genetic, and ultrastructural analyses to dissect the molecular composition and architecture of particles of beet yellows virus, a closterovirus. We demonstrate that the virion tails encapsidate the 5'-terminal, approximately 650-nt-long, part of the viral RNA. In addition to the minor capsid protein, the viral Hsp70-homolog, 64-kDa protein, and 20-kDa protein are also incorporated into the virion tail. Atomic force microscopy of virions revealed that the tail possesses a striking, segmented morphology with the tip segment probably being built of 20-kDa protein. The unexpectedly complex structure of closterovirus virions has important mechanistic and functional implications that may also apply to other virus families.

Blotting, Northern↗

PubMed2006

Self-assembly of synthetic collagen triple helices.

Collagen is the most abundant protein in animals and the major component of connective tissues. Although collagen isolated from natural sources has long served as the basis for some biomaterials, natural collagen is difficult to modify and can engender pathogenic and immunological side effects. Collagen comprises a helix of three strands. Triple helices derived from synthetic peptides are much shorter (<10 nm) than natural collagen (approximately 300 nm), limiting their utility. Here, we describe the synthesis of short collagen fragments in which the three strands are held in a staggered array by disulfide bonds. Data from CD spectroscopy, dynamic light scattering, analytical ultracentrifugation, atomic force microscopy, and transmission electron microscopy indicate that these "sticky-ended" fragments self-assemble via intermolecular triple-helix formation. The resulting fibrils resemble natural collagen, and some are longer (>400 nm) than any known collagen. We anticipate that our self-assembly strategy can provide synthetic collagen-mimetic materials for a variety of applications.

Amino Acid Sequence↗

PubMedDate not supplied

Preparation, characterization and protein loading of hexanoyl-modified chitosan nanoparticles.

Hexanoyl chitosan was synthesized through a coupling reaction between chitosan and hexanoic anhydride. Proton nuclear magnetic resonance (1HNMR) and fourier-transform infrared (FTIR) spectroscopy studies showed the formation of hexanoyl chitosan. The nanoparticles of hexanoyl chitosan were prepared through ionotropic gelation with tripolyphosphate (TPP) followed by sonication. The hexanoyl chitosan-TPP nanoparticles exhibited uniform spherical shape with smooth surface as observed by atomic force microscopy and transmission electron microscopy. The particle size of nanoparticles was between 54.1 to 724 nm with a mean diameter of 324 nm. At 0.2, 0.4, and 0.6 mg/mL bovine serum albumin initial concentration, the encapsulation efficiency and loading capacity of hexanoyl-chitosan-TPP nanoparticles were 58.2, 44.5, and 28.1%, and 14.1, 23.4, and 30.3%, respectively.

Caproates↗

PubMed2006

Chemomechanical nanolithography: nanografting on silicon and factors impacting linewidth.

We present a two-fold extension of previous work on Atomic Force Microscope-based chemomechanical functionalization: (1) chemomechanical nanografting, which extends chemomechanical functionalization to a more stable initial surface, and (2) linewidth studies that show the impact of force and Atomic Force Microscope probe tip wear on patterning resolution. Alkene, alcohol, and alkyl halide molecules were nanografted to silicon and imaged with in situ atomic force microscopy, time-of-flight secondary ion mass spectrometry with Automated eXpert Spectrum Image Analysis, and scanning electron microscopy. Chemomechanical nanografting demonstrated linewidths down to 50 nm. Lines written on hydrogen-terminated silicon were used to explore the impact of tip radius and tip wear on linewidth when using Si3N4 coated tips.

Coated Materials, Biocompatible↗

PubMed2005

Influence of autoclave sterilization on the surface parameters and mechanical properties of six orthodontic wires.

Orthodontic wires are frequently packaged in individual sealed bags in order to avoid cross-contamination. The instructions on the wrapper generally advise autoclave sterilization of the package and its contents if additional protection is desired. However, sterilization can modify the surface parameters and the mechanical properties of many types of material. The aim of this research was to determine the influence of one of the most widely used sterilization processes, autoclaving (18 minutes at 134 degrees C, as recommended by the French Ministry of Health), on the surface parameters and mechanical properties of six wires currently used in orthodontics (one stainless steel alloy: Tru-Chrome RMO; two nickel-titanium shape memory alloys: Neo Sentalloy and Neo Sentalloy with Ionguard GAC; and three titanium-molybdenum alloys: TMA(R) and Low Friction TMA Ormco and Resolve GAC). The alloys were analysed on receipt and after sterilization, using surface structure observation techniques, including optical, scanning electron and atomic force microscopy and profilometry. The mechanical properties were assessed by three-point bending tests. The results showed that autoclave sterilization had no adverse effects on the surface parameters or on the selected mechanical properties. This supports the possibility for practitioners to systematically sterilize wires before placing them in the oral environment.

Chromium Alloys↗

PubMed2005

Nanodispersion of single-walled carbon nanotubes using dichloroethane.

In this paper we present a simple approach of nanodispersing single-walled carbon nanotubes (SWCNTs) in a non-polar 1,2-dichloroethane (DCE) solvent. After filtration with isopropanol and acetone, the purified SWCNTs were immersed in DCE, followed by sonication for about 15 hours. The samples were further centrifuged at 17,000 revolutions per minute for about 3 hours. Atomic force microscopy (AFM) demonstrated that the spin-coated nanotubes were mostly individual nanotubes with an average diameter of 1.6 nm and a length of about 250 nm. We also found that the presence of water, and the dry process during DCE treatment, prevented nanotubes from being nanodispersed.

2-Propanol↗

PubMed1997

The bacteriophage phi29 head-tail connector imaged at high resolution with the atomic force microscope in buffer solution.

The surfaces of two- and three-dimensional phi29 connector crystals were imaged in buffer solution by atomic force microscopy (AFM). Both topographies show a rectangular unit cell with dimensions of 16.5 nm x 16.5 nm. High resolution images of connectors from the two-dimensional crystal surface show two connectors per unit cell confirming the p42(1)2 symmetry. The height of the connector was estimated to be at least 7.6 nm, a value close to that found in previous studies using different techniques. The 12 subunits of the wide connector domain were clearly resolved and showed a right-handed vorticity. The channel running along the connector had a diameter of 3.7 nm in the wide domain, while it was 1.7 nm in the narrow domain end, thus suggesting a tronco-conical channel shape. Moreover, the narrow connector end appears to be rather flexible. When the force applied to the stylus was between 50 and 100 pN, the connector end was fully extended. At forces of approximately 150 pN, these ends were pushed towards the crystal surface. The complementation of the AFM data with the three-dimensional reconstruction obtained from electron microscopy not only confirmed the model proposed, but also offers new insights that may help to explain the role of the connector in DNA packing.

Bacillus Phages↗

PubMed2001

Invasive forms of Toxoplasma gondii, Leishmania amazonensis and Trypanosoma cruzi have a positive charge at their contact site with host cells.

We examined the surface charges of invasive forms of Toxoplasma gondii, Leishmania amazonensis, and Trypanosoma cruzi by atomic force microscopy and surface potential spectroscopy. We found that the specific part of the protozoan which makes initial contact with the host cell is positively charged. This indicates that the positive charge at the site of contact facilitates binding of the invasive protozoan to negatively charged host cells.

Animals↗

PubMed2006

Specific vectorial immobilization of oligonucleotide-modified yeast cytochrome C on carbon nanotubes.

Iso-1-cytochrome c from the yeast Saccharomyces cerevisiae (YCC) contains a surface cysteine residue, Cys102, that is located opposite to the lysine-rich side containing the exposed heme edge, which is the docking site for enzymes. Site-specific vectorial immobilization of YCC via Cys102 on single-walled carbon nanotubes (SWNT) thus provides a selective interface between nanoscopic electronic devices and complex enzymes. We have achieved this by modification of Cys102 with an oligonucleotide (dT(18)). Atomic force microscopy, fluorescence imaging, and cyclic voltammetry show the specific adsorption of YCC, modified with dT(18), on the SWNT sidewall with retention of its native properties. Pretreatment of the SWNT with Triton-X405 blocks the nonspecific binding of untreated YCC but does not interfere with binding of the oligonucleotide-modified YCC.

Adsorption↗

PubMed2006

A novel approach to the micropatterning of proteins using dewetting of polymer bilayers.

The ability to control protein and cell positioning on a microscopic scale is crucial in many biomedical and bioengineering applications, such as tissue engineering and the development of biosensors. We propose here a novel, simple, and versatile method for the micropatterning of proteins. Micropatterned substrates are produced by the dewetting of a metastable polymer film on top of another polymer film. Selective adsorption, or micropatterning, of proteins can be achieved on such substrates by choosing pairs of polymers which differ in protein affinity. In this study, patterns were produced in bilayers of poly(methylmethacrylate) (PMMA) and polystyrene (PS), and of PMMA and octadecyltrichlorosilane (OTS). Fluorescence microscopy and atomic force microscopy (AFM) provide evidence that model proteins adsorb preferentially on isolated bio-adhesive (PS and OTS) micropatches in a protein-resistant (PMMA) matrix. "Inverse" protein patterns, containing non-adhesive (PMMA) islands in a protein-adhesive (PS) matrix can also be produced. Such micropatterned substrates could potentially be used in the development of biosensors and bioassays, and in the study of cell growth and motility.

Animals↗

PubMed2005

Electrospun protein fibers as matrices for tissue engineering.

Electrospinning has recently emerged as a leading technique for generating biomimetic scaffolds made of synthetic and natural polymers for tissue engineering applications. In this study, we compared collagen, gelatin (denatured collagen), solubilized alpha-elastin, and, as a first, recombinant human tropoelastin as biopolymeric materials for fabricating tissue engineered scaffolds by electrospinning. In extending previous studies, we optimized the shape and size (diameter or width) of the ensuing electrospun fibers by varying important parameters of the electrospinning process, such as solute concentration and delivery rate of the polymers. Our results indicate that the average diameter of gelatin and collagen fibers could be scaled down to 200-500 nm without any beads, while the alpha-elastin and tropoelastin fibers were several microns in width. Importantly, and contrary to any hitherto reported structures of electrospun polymers, fibers composed of alpha-elastin, especially tropoelastin, exhibited "quasi-elastic" wave-like patterns at increased solution delivery rates. The periodicity of these wave-like tropoelastin fibers was partly affected by the delivery rate. Atomic force microscopy was utilized to profile the topography of individual electrospun fibers and microtensile testing was performed to measure their mechanical properties. Cell culture studies confirmed that the electrospun engineered protein scaffolds support attachment and growth of human embryonic palatal mesenchymal (HEPM) cells.

Animals↗

PubMed2005

Nanoscale operation of a living cell using an atomic force microscope with a nanoneedle.

We have developed a tool for performing surgical operations on living cells at nanoscale resolution using atomic force microscopy (AFM) and a modified AFM tip. The AFM tips are sharpened to ultrathin needles of 200-300 nm in diameter using focused ion beam etching. Force-distance curves obtained by AFM using the needles indicated that the needles penetrated the cell membrane following indentation to a depth of 1-2 microm. The force increase during the indentation process was found to be consistent with application of the Hertz model. A three-dimensional image generated by laser scanning confocal microscopy directly revealed that the needle penetrated both the cellular and nuclear membranes to reach the nucleus. This technique enables the extended application of AFM to analyses and surgery of living cells.

Cell Membrane↗

PubMedDate not supplied

AFM observation of band-like cellulose assemblies produced by Acetobacter xylinum.

We succeeded in estimating the thickness of band-like cellulose assemblies by combined use of atomic force microscopy (AFM) and transmission electron microscopy. The thickness of "dense" band-like cellulose assemblies was estimated at 20-30 nm from their AFM height profiles, which was several times greater than that of "coarse" band-like and ribbonlike cellulose assemblies. On the basis of these results, the folded- chain model previously proposed was discussed, and a different organization of TC subunits was suggested for the "dense" band-like cellulose assembly.

Cellulose↗

PubMedDate not supplied

Imaging of lactic acid bacteria with AFM--elasticity and adhesion maps and their relationship to biological and structural data.

The adhesion of lactic acid bacteria to the intestinal epithelium is one of the most important factors determining probiotic ability of a bacterial strain. Studying bacterial adhesion requires knowledge of the structure and properties of the bacterial surface, which can be studied by atomic force microscopy under native conditions. The observation of the surface topography of bacteria from the species Lactobacillus crispatus, L. helveticus and L. johnsonii shows major differences between bacteria having a crystalline-like protein layer as part of the cell wall and those without such layers. Force volume images calculated into elasticity and adhesion force maps of different bacterial strains show that L. crispatus and L. helveticus have a surface with a homogeneous stiffness with no adhesion events. This is most likely caused by the S-layer, which completely covers the surface of the bacteria. We infer that the absence of adhesion peaks is caused by the semi-crystalline character of such protein layers, in agreement with the results obtained from electron microscopy. Analysis of a number of L. johnsonii strains shows that these bacteria have surface properties which strongly differ from the L. crispatus and L. helveticus strains. For L. johnsonii DMS20533 and L. johnsonii ATCC33200 high adhesion forces are observed, which can be related to a surface rich in polysaccharides. L. johnsonii ATCC332 has lower adhesion forces compared to the other two and, furthermore, the surface topography shows depressions. We suppose that this strain has a surface pattern consisting of crystalline-like proteins alternating with polysaccharide-rich domains. The wide variety in surface properties of lactobacilli could well have wide-ranging implications for food processing and for health benefits.

Bacterial Adhesion↗

PubMed2002

The Saccharomyces cerevisiae Fin1 protein forms cell cycle-specific filaments between spindle pole bodies.

The FIN1 gene from the yeast Saccharomyces cerevisiae encodes a basic protein with putative coiled-coil regions. Here we show that in large-budded cells a green fluorescent protein-Fin1 fusion protein is visible as a filament between the two spindle pole bodies. In resting cells the protein is undetectable, and in small-budded cells it is localized in the nucleus. During late mitosis it localizes on the spindle pole bodies. Filaments of cyano fluorescent protein-tagged Fin1 colocalize with filaments of green fluorescent protein-tagged Tub1 only in large-budded cells. By electron and atomic force microscopy we showed that purified recombinant Fin1p self-assembles into filaments with a diameter of approximately 10 nm. Our results indicate that the Fin1 protein forms a cell cycle-specific filament, additional to the microtubules, between the spindle pole bodies of dividing yeast cells.

Cell Cycle↗

PubMed2002

Padlock oligonucleotides as a tool for labeling superhelical DNA.

Labeling of a covalently closed circular double-stranded DNA was achieved using a so-called 'padlock oligonucleotide'. The oligonucleotide was targeted to a sequence which is present in the replication origin of phage f1 and thus in numerous commonly used plasmids. After winding around the double-stranded target DNA sequence by ligand-induced triple helix formation, a biotinylated oligonucleotide was circularized using T4 DNA ligase and in this way became catenated to the plasmid. A gel shift assay was developed to measure the extent of plasmid modification by the padlock oligonucleotide. A similar assay showed that a modified supercoiled plasmid was capable of binding one streptavidin molecule thanks to the biotinylated oligonucleotide and that this binding was quantitative. The catenated complex was visualized by electron and atomic force microscopies using streptavidin conjugates or single strand-binding proteins as protein tags for the padlock oligonucleotide. This method provides a versatile tool for plasmid functionalization which offers new perspectives in the physical study of supercoiled DNA and in the development of improved vectors for gene therapy.

Base Sequence↗

PubMed2005

Crystal surface adhesion explains the pathological activity of calcium oxalate hydrates in kidney stone formation.

Renal tubular fluid in the distal nephron of the kidney is supersaturated with calcium oxalate (CaOx), which crystallizes in the tubules as either calcium oxalate monohydrate (COM) or calcium oxalate dihydrate (COD). Kidney stones are aggregates, most commonly containing microcrystals of COM as the primary inorganic constituent. Stones also contain small amounts of embedded proteins, which are thought to play an adhesive role in these aggregates, and they often are found attached to the tip of renal papilla, presumably through adhesive contacts. Voided urine, however, often contains COD in the form of single micron-sized crystals. This suggests that COD formation protects against stone disease because of its reduced capacity to form stable aggregates and strong adhesion contacts to renal epithelial cells. Using atomic force microscopy configured with tips modified with biologically relevant functional groups, we have compared the adhesion strengths of the morphologically important faces of COM and COD. These measurements provide direct experimental evidence, at the near molecular level, for poorer adhesion at COD crystal faces, which explains the benign character of COD and has implications for resolving one of the mysteries of kidney stone formation.

Adhesiveness↗