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PubMed2005

Fabrication and evaluation of nanoporous alumina membranes for osteoblast culture.

An understanding of osteoblast response to surface topography is essential for successful bone tissue engineering applications. Alumina has been extensively used as a substrate for bone tissue constructs. However, current techniques do not allow precise surface topography and orientation of the material. In this research, a two-step anodization process was optimized for the fabrication of nanoporous alumina membranes with uniform pore dimension and distribution. The anodization voltage can be varied to create nanoporous alumina membranes with pore sizes ranging from 30 to 80 nm in diameter. The impact of the nanoscale pores on osteoblast response was studied by evaluating cell adhesion, morphology, and matrix production. Scanning electron microscopy and atomic force microscopy were used to characterize the nanoporous alumina membranes. Osteoblast adhesion and morphology were investigated using scanning electron microscopy images and matrix production was characterized using energy dispersive spectroscopy. This research combined the advantages of using alumina, a material with proven biocompatibility and current orthopedic clinical applications, and incorporated porous features on the nanoscale which have been reported to improve osteoblast response.

Aluminum Oxide↗
PubMed1996

[Complementarity of microscopies in the structural analysis of DNA minicircles associated to protein MC1].

Electron microscopy of DNA, either free or complexed with ligands, allows the analysis of local conformational variations along individual molecules. Electron microscopy is unique, in that it has the capacity to determine the average behaviour of a population of molecules observed individually, and can thus provide a better appreciation of variability within the series of molecules than biophysical or biochemical methods. Very encouraging results have been obtained by cryoelectron and near-field microscopies, especially atomic force microscopy, in parallel with traditional techniques for visualizing DNA molecules adsorbed onto a support film. Differences in sample processing procedures and image formation modes render these 3 types of microscopies complementary. The torsional stress of a DNA molecule together with a local curvature induced by the protein MC1 from archaebacteria, can be detected within minicircles comprising 207 base pairs.

Adenosine Triphosphatases↗
PubMedDate not supplied

Photocurable liquid biodegradable copolymers: in vitro hydrolytic degradation behaviors of photocured films of coumarin-endcapped poly(epsilon-caprolactone-co-trimethylene carbonate).

Coumarin-endcapped tetrabranched liquid copolymers composed of epsilon-caprolactone and trimethylene carbonate (TMC), prepared using pentaerythritol or four-branched poly(ethylene glycol) (PEG) as an initiator, were ultraviolet irradiated to produce photocured solid biodegradable copolymers. The hydrolytic degradation behaviors of photocured films were determined from the weight loss of the films. The initial hydrolysis rate (determined for up to 24 h using a quartz crystal microbalance) was enhanced with aqueous solutions of higher pH. The hydrolysis rate in the early period of immersion was increased with an increase in TMC content, whereas that in the later period (week order) decreased with a increase in TMC content. This inverse relation of composition dependence on the hydrolysis rate between the early and late periods was discussed. Topological measurements using scanning electron microscopy and atomic force microscopy as well as depth profiles of the fluorescein-stained hydrolyzed layer showed that for the pentaerythritol-initiated copolymer, irrespective of copolymer composition, hydrolysis occurred at surface regions and surface erosion proceeded with immersion time. For PEG-based copolymers, both surface erosion and bulk degradation occurred simultaneously. The hydrolyzed surfaces became highly wettable with water and exhibited noncell adhesivity.

Adhesives↗
PubMed2005

Structural characterization of artificial self-assembling porphyrins that mimic the natural chlorosomal bacteriochlorophylls c, d, and e.

We report two crystal structures of a synthetic porphyrin molecule which was programmed for self-assembly. The same groups which ensure that bacteriochlorophylls c, d, and e can self-assemble into the chlorosomal nanorods, the photosynthetic antenna system of some green bacteria, have been engineered into desired positions of the tetrapyrrolic macrocycle. In the case of the 5,15-meso-substituted anchoring groups, depending upon the concentration, by using the same crystallization solvents, either a tetragonal or a layered structure of porphyrin stacks were encountered. Surprisingly, pi-pi interactions combined with extensive dispersive interactions, which also encompass cyclohexane, one of the crystallization solvents, win over putative hydrogen bonding. We are aware that our compounds differ considerably from the natural bacteriochlorophylls, but based upon our findings, we now question the hydrogen-bonding network, previously proposed to organize stacks of bacteriochlorophylls. Transmission electron microscopy (TEM), atomic force microscopy (AFM), and small-angle X-ray scattering (SAXS) on various isomeric compounds support our challenge of current models for the chlorosomal antenna as these show structures, astonishingly similar to those of chlorosomes.

Bacterial Proteins↗
PubMed2004

Changes in morphology of starch-based prothestic thermoplastic material during enzymatic degradation.

This work evaluates the structural changes of an interpenetrated starch thermoplastic blend withstanding different enzymatic alpha-amylase degradation periods (up to 200 days), and establishes the relationships between the kinetics degradation rate and the structure of the material. It characterises the different stages of the enzymatic degradation process on starch/ethylenevinyl-alcohol blends, based on the attack of the connected starch domains that can be accessed by the enzymatic solution. The completely encapsulated starch particles remain practically unchanged. Furthermore, it was also found that the enzymatic degradation process was limited after 100 days of immersion. In order to understand such phenomenon several techniques were used, namely differential scanning calorimetry, contact-angle measurements, high-performance liquid chromatography, Fourier transform infrared spectrometry, scanning electron microscopy and atomic force microscopy. The materials were evaluated with respect to the enzymatic degradation rate, surface morphology and degradation behaviour. The results show that the ethylene-vinylalcohol phase wraps the starch domains, preventing the respective degradation. Consequently, the degraded material in the solution comes only from the starch particles that could be reached by the enzyme.

Alcohols↗
PubMed2005

Amyloid-beta aggregates formed at polar-nonpolar interfaces differ from amyloid-beta protofibrils produced in aqueous buffers.

The deposition of aggregated amyloid-beta (Abeta) peptides in the brain as senile plaques is a pathological hallmark of Alzheimer's disease (AD). Several lines of evidence indicate that fibrillar and, in particular, soluble aggregates of these 40- and 42-residue peptides are important in the etiology of AD. Recent studies also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we review our recent reports that Abeta(1-40) in vitro can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta(1-40) in low ionic strength buffers. These aggregates were quite stable and disaggregated to only a limited extent on dilution. A second class of soluble Abeta aggregates was generated at polar-nonpolar interfaces. Aggregation in a two-phase system of buffer over chloroform occurred more rapidly than in buffer alone. In buffered 2% hexafluoroisopropanol (HFIP), microdroplets of HFIP were formed and the half-time for aggregation was less than 10 minutes. Like Abeta protofibrils, these interfacial aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. However, electron microscopy and atomic force microscopy revealed very different morphologies. The HFIP aggregates formed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP these aggregates initially were very unstable and disaggregated completely within 2 minutes. However, their stability increased as they progressed to fibers. It is important to determine whether similar interfacial Abeta aggregates are produced in vivo.

Amyloid beta-Peptides↗
PubMed2005

Formation of morphologically similar globular aggregates from diverse aggregation-prone proteins in mammalian cells.

Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine repeat expansion in the first exon of the huntingtin (Htt) protein. N-terminal Htt peptides with polyglutamine tracts in the pathological range (51-122 glutamines) form high-molecular-weight protein aggregates with fibrillar morphology in vitro, and they form discrete inclusion bodies in a cell-culture model. However, in some studies, formation of discrete Htt inclusions does not correlate well with cell death. We coexpressed N-terminal Htt fragments containing 91 glutamines fused to different affinity tags in HEK293 cells, and we isolated small aggregates by double sequential-affinity chromatography to assure the isolation of multimeric molecules. Transmission electron microscopy and atomic force microscopy revealed the isolated aggregates as globules or clusters of globules 4-50 nm in diameter without any detectable fibrillar species. Because small nonfibrillar oligomers, not mature fibrils, recently have been suggested to be the principal cytotoxic species in neurodegenerative disease, these Htt globular aggregates formed in cells may represent the pathogenic form of mutant Htt.

Affinity Labels↗
PubMed2005

New insights into the structural organization of the plant polymer lignin.

The organizational features of lignin structure and the mechanism of its synthesis have significant implications for the response of the plant to stress. It was unknown whether the enzymic formation of lignin in the cell wall is an uncontrolled process or finely regulated in time and space. In vitro scanning tunneling microscopy (STM), atomic force microscopies (AFM), near-field scanning optical microscopy (NSOM). and the novel environmental scanning electron microscopy (ESEM) imaging studies of the lignin model compounds have directly shown its highly ordered structure and elucidated its modular and fractal organization. Direct evidence was presented for the existence of strong intermolecular forces responsible for holding lignin globules together in highly ordered structures. Fractal analysis was applied as a theoretical approach, to show regularity and modular organization of lignin. Surface chemistry studies of the lignin monolayer reveal intrinsic properties that may be a key to osmotic pressure and cell size control mechanism in the higher plant cells. The obtained data contribute to the explanation of the mechanisms of cell wall synthesis in vivo.

Cell Size↗
PubMed2005

Stability of lipid films formed on gamma-aminopropyl monolayers.

The stability of supported lipid membranes (SLMs) deposited on planar substrates derivatized with (gamma-aminopropyl)silane (GAPS) was examined. Ellipsometry, fluorescence microscopy, and atomic force microscopy were used to characterize SLMs exposed to repeated drying and rehydration. Vesicle fusion on GAPS-coated substrates produced SLMs with a thickness significantly greater than that of a single lipid bilayer. Exposure to even one cycle of drying/rehydration significantly decreased the thickness of a SLM on GAPS, and repeated drying/rehydration resulted in near quantitative lipid desorption. Thus SLMs on GAPS do not appear to be significantly more stable than the single bilayer SLM that is formed on bare glass or SiO2 under equivalent conditions.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

Chain length of the polylysine in receptor-targeted gene transfer complexes affects duration of reporter gene expression both in vitro and in vivo.

Complexes composed of peptide ligand for the serpin enzyme complex receptor covalently coupled to poly-L-lysine condensed by charge interaction with plasmid DNA direct gene transfer into receptor bearing cells. We compared intensity and duration of reporter gene expression in vitro and in vivo from serpin-enzyme receptor-directed gene transfer complexes prepared with poly-L-lysine of different chain lengths. When substituted with linker and ligand to comparable extents, DNA complexes containing short chain poly-L-lysine were larger and gave higher peak expression but significantly shorter duration of expression than those containing long chain poly-L-lysine. Both peak expression and duration of expression exceeded that observed with Lipofectin. Neither naked DNA nor DNA complexed with unsubstituted polylysine was effective in gene transfer. For in vivo experiments, complexes containing optimal ligand and degree of substitution (based on in vitro data, peptide C105Y, 11 ligands/plasmid DNA molecule) were prepared with either short chain or long chain polylysine and a beta-galactosidase expression plasmid. Following injection into the tail veins of mice, longer chain complexes gave significantly higher expression of reporter gene in lung and spleen that lasted for a significantly longer period of time than the shorter chain complexes. The short chain poly-L-lysine-DNA complexes were larger in diameter, as assessed by electron microscopy or atomic force microscopy, and gave less protection against DNase digestion in vitro than longer chain complexes. Thus, for gene transfer complexes directed at the serpin enzyme complex receptor, longer chain poly-L-lysine gave a much longer duration of expression both in vitro and in vivo. We speculate that this may be due to protection against degradation afforded the plasmid DNA by the tighter compaction produced by long chain poly-L-lysine.

Amino Acid Sequence↗
PubMedDate not supplied

Solid lipid nanoparticles (SLN/Lipopearls)--a pharmaceutical and cosmetic carrier for the application of vitamin E in dermal products.

Solid lipid nanoparticles (SLN, Lipopearls) are nanoparticles made from solid lipids by high pressure homogenization. Incorporation of chemically labile active ingredients into the solid lipid matrix protects against chemical degradation, which is shown for vitamin E. The SLN are physically stable in aqueous dispersions and also after incorporation into a dermal cream as proven by photon correlation spectroscopy and differential scanning calorimetry. Electron microscopy and atomic force microscopy data reveal the spherical shape of the SLN and the detailed structure of the particle surface. Ultrafine particles form an adhesive film leading to an occlusive effect on the skin. The occlusion promotes the penetration of vitamin E into the skin, as shown by the stripping test. In addition to chemical stabilization of active ingredients, occlusive effects on the skin and subsequent enhanced penetration of compounds, the SLN also possess a pigment effect covering undesired colours leading to an increased aesthetic acceptance by the customer.

Calorimetry, Differential Scanning↗
PubMed2006

Vitamin A potently destabilizes preformed alpha-synuclein fibrils in vitro: implications for Lewy body diseases.

Alpha-synuclein (alphaS) is the major component of the filamentous inclusions that constitute defining characteristics of Lewy body diseases (LBD) and multiple system atrophy (MSA). Clinically, antioxidant vitamins, such as vitamin E and the vitamin-like substance coenzyme Q10, have been used in the treatment of LBD with some efficacy. Using fluorescence spectroscopy with thioflavin S, electron microscopy and atomic force microscopy, here we examined the effects of ten antioxidant vitamins and vitamin-like substances, vitamin A (retinol, retinal and retinoic acid), beta-carotene, vitamins B2, B6, C, E, coenzyme Q10 and alpha-lipoic acid, on the formation of alphaS fibrils (falphaS) and on preformed falphaS. Among them, vitamin A, beta-carotene and coenzyme Q10 dose-dependently inhibited the formation of falphaS. Moreover, they also dose-dependently destabilized preformed falphaS. With such potent anti-fibrillogenic as well as fibril-destabilizing activities, these compounds could be useful in the treatment and prevention of LBD and MSA.

Antioxidants↗
PubMed2004

Amyloid-beta protofibrils differ from amyloid-beta aggregates induced in dilute hexafluoroisopropanol in stability and morphology.

The brains of Alzheimer's disease (AD) patients contain large numbers of amyloid plaques that are rich in fibrils composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the etiology of AD. Recent reports also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we demonstrate that Abeta-(1-40) can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta-(1-40) at low ionic strength. Dilution of these aggregation reactions induced disaggregation to monomers as measured by size exclusion chromatography. Protofibril concentrations monitored by thioflavin T fluorescence decreased in at least two kinetic phases, with initial disaggregation (rate constant approximately 1 h(-1)) followed by a much slower secondary phase. Incubation of the reactions without agitation resulted in less disaggregation at slower rates, indicating that the protofibrils became progressively more stable over time. In fact, protofibrils isolated by size exclusion chromatography were completely stable and gave no disaggregation. A second class of soluble Abeta aggregates was generated rapidly (<10 min) in buffered 2% hexafluoroisopropanol (HFIP). These aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. Electron microscopy and atomic force microscopy revealed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP, these aggregates initially were very unstable and disaggregated completely within 2 min. However, their stability increased as they progressed to fibers. Relative to Abeta protofibrils, the HFIP-induced aggregates seeded elongation by Abeta monomer deposition very poorly. The techniques used to distinguish these two classes of soluble Abeta aggregates may be useful in characterizing Abeta aggregates formed in vivo.

Alzheimer Disease↗
PubMed2004

Molecular architecture of photosynthetic membranes in Rhodobacter sphaeroides: the role of PufX.

The effects of the PufX polypeptide on membrane architecture were investigated by comparing the composition and structures of photosynthetic membranes from PufX+ and PufX- strains of Rhodobacter sphaeroides. We show that this single polypeptide profoundly affects membrane morphology, leading to highly elongated cells containing extended tubular membranes. Purified tubular membranes contain helical arrays composed solely of dimeric RC-LH1-PufX (RC, reaction centre; LH, light harvesting) complexes with apparently open LH1 rings. PufX- cells contain crystalline membranes with a pseudo-hexagonal packing of monomeric core complexes. Analysis of purified complexes by electron microscopy and atomic force microscopy shows that LH1 and PufX form a continuous ring of protein around each RC. A model of the tubular membrane is presented with PufX located adjacent to the stained region created by a vacant LH1beta. This arrangement, coupled with a flexible ring, would give the RC QB site transient access to the interstices in the lattice, which might be of functional importance. We discuss the implications of our data for the export of quinol from the RC, for eventual reduction of the cytochrome bc1 complex.

Bacterial Proteins↗
PubMed2006

Fibronectin organization under and near cells.

Polymerization of soluble fibronectin molecules results in fibres that are visible as networks using fluorescently labelled fibronectin protomers or by antibody labelling. Displacement of fibres composed of modified protomers in living cells provides information regarding matrix structure, organization, and movement. A static analysis of fibronectin structures and patterns of organization provide insight into their reorganization during adhesion and motility. Confocal microscopy and atomic force microscopy (AFM) reveal fibronectin-containing networks aligned in arrays perpendicular to the retracting cell edge and in apparently disordered networks of fibres under the cell. The change in patterns suggests a reorganization of fibronectin from disordered arrays used for adhesion into ordered arrays during movement of the cell. Comparison of confocal images with corresponding AFM images confirms that the fibres left on the surface as the cell moves away do contain fibronectin. The orientation of these fibres relative to the tail (uropod) and the receding edges of the cell leads us to propose that cells generate a force on the fibres that exceeds the adhesion force of the fibres to the surface causing them to pull fibronectin fibres into straight arrays. However, when the fibres are parallel to the direction of pull, the fibres remain attached to the surface. The data supports the hypothesis that disorganized, linear fibres are the product of Fn polymerization induced by the cell beneath it and serve to adhere the cell to the substrate as the cell spreads, whereas arrays of fibres found outside the cell are formed as existing fibrils and reorganize during cell motility.

Animals↗
PubMed2006

Characterizing nanoscale topography of the aortic heart valve basement membrane for tissue engineering heart valve scaffold design.

A fully effective prosthetic heart valve has not yet been developed. A successful tissue-engineered valve prosthetic must contain a scaffold that fully supports valve endothelial cell function. Recently, topographic features of scaffolds have been shown to influence the behavior of a variety of cell types and should be considered in rational scaffold design and fabrication. The basement membrane of the aortic valve endothelium provides important parameters for tissue engineering scaffold design. This study presents a quantitative characterization of the topographic features of the native aortic valve endothelial basement membrane; topographical features were measured, and quantitative data were generated using scanning electron microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM), and light microscopy. Optimal conditions for basement membrane isolation were established. Histological, immunohistochemical, and TEM analyses following decellularization confirmed basement membrane integrity. SEM and AFM photomicrographs of isolated basement membrane were captured and quantitatively analyzed. The basement membrane of the aortic valve has a rich, felt-like, 3-D nanoscale topography, consisting of pores, fibers, and elevations. All features measured were in the sub-100 nm range. No statistical difference was found between the fibrosal and ventricular surfaces of the cusp. These data provide a rational starting point for the design of extracellular scaffolds with nanoscale topographic features that mimic those found in the native aortic heart valve basement membrane.

Animals↗
PubMed2004

Supramolecular amyloid-like assembly of the polypeptide sequence coded by exon 30 of human tropoelastin.

Elastin is known to self-aggregate in twisted-rope filaments. However, an ultrastructural organization different from the fibrils typical of elastin, but rather similar to those shown by amyloid networks, is shown by the polypeptide sequence encoded by exon 30 of human tropoelastin. To better understand the molecular properties of this sequence to give amyloid fibers, we used CD, NMR, and FTIR (Fourier transform infrared spectroscopy) to identify the structural characteristics of the peptide. In this study, we have demonstrated, by FTIR, that antiparallel beta-sheet conformation is predominant in the exon 30 fibers. These physical-chemical studies were combined with transmission electron microscopy and atomic force microscopy to analyze the supramolecular structure of the self-assembled aggregate. These studies show the presence of fibrils that interact side-by-side probably originating from an extensive self-interaction of elemental cross beta-structures. Similar sequences, of the general type XGGZG(X, Z = V, L, A, I), are widely found in many proteins such as collagens IV and XVII, major prion protein precursor, amyloid beta A4 precursor protein-binding family, etc., thus suggesting that this sequence could be involved in contributing to the self-assembly of amyloid fibers even in other proteins.

Amino Acid Sequence↗
PubMed2000

Antibacterial activity of human prostasomes.

BACKGROUND: Prostasomes are prostate-derived organelles in semen exhibiting pluripotent properties. The present study deals with their possible antibacterial effects. METHODS: Antibacterial activity was assessed by growth inhibition of bacteria in an incubation medium containing prostasomes, after which the incubate was inoculated on cystine lactose electrolyte deficient agar (CLED) plates. In cases involving Bacillus megaterium, the effects were also documented ultrastructurally with scanning electron microscopy and atomic force microscopy. RESULTS: A dose-dependent growth inhibition was apparent, and a complete inhibition of growth was seen at a prostasome protein concentration of 30 microg/ml with Bacillus megaterium. Ultrastructurally, increasingly irregular contours and a loosening of the smooth surface were observed, combined with a fragmentation of the bacteria. Among 9 other bacterial strains tested, a complete growth inhibition by prostasomes was attained in 3 strains, while the other 6 were unaffected. CONCLUSIONS: Our data suggest that prostasomes, or prostasome-derived proteins, are responsible for the antibacterial effects on Bacillus megaterium and some other bacterial strains. The results may serve as a basis of development of a new class of antibacterial drugs.

Bacillus megaterium↗