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PubMedDate not supplied

Imaging of RNA-protein interactions in splicing complexes with dark-field STEM.

Splicing complexes were assembled in vitro on pre-messenger RNA. These complexes represent stages in the processing of pre-mRNA by snRNPs and accessory factors to remove the introns, thus forming messenger RNA. Complexes were purified using biotinylated oligonucleotide tags together with streptavidin-gold and simple centrifugation. The oligonucleotide tags with attached streptavidin-gold also serve as unambiguous labels of the splicing complexes for EM observation. The complexes were observed using dark-field scanning transmission electron microscopy (STEM). The high contrast and the high efficiency in detecting scattered electrons make it possible to visualize specimens at high resolution with low radiation dose. STEM visualization of the complexes allows a unique view of the events occurring in the splicing process. Three different classes of complex were identified. These complexes support the currently postulated steps in RNA splicing and demonstrate the dynamic nature of splicing complexes. This approach has broad application in high-resolution structural studies of nucleic acids and nucleic acid-protein interactions.

Bacterial Proteins↗
PubMed1999

Ni-NTA-gold clusters target His-tagged proteins.

Addition of six histidines to recombinant proteins has proved useful in their purification by nickel-affinity columns. This technology was adapted by synthesizing the chelator for nickel (nitrilotriacetic acid, NTA) onto the surface of gold clusters. These Ni-NTA-gold clusters were shown to specifically target the 6His region of tagged proteins. Results were verified by column chromatography, dot and overlay blots, UV-Vis spectroscopy, and scanning transmission electron microscopy. A 6His-tagged adenovirus "knob" protein was also shown to maintain receptor binding activity after gold labeling. Two types of gold clusters were used: 1.4-nm Nanogold and a new 1.8-nm "PeptideGold" coated with an NTA-dipeptide-thiol. These novel labels should be useful in site-specific high-resolution EM labeling, as well as in metallographic development, detection in the light microscope, or direct visualization.

Adenoviridae↗
PubMed1999

Giant platinum clusters: 2-nm covalent metal cluster labels.

A new class of covalently linkable platinum cluster reagents with core diameters close to 2 nm has been prepared. The new label offers the performance advantages and versatility of the 1.4-nm Nanogold in a larger label which is more clearly visualized against electron-dense regions of a specimen. Large platinum clusters were prepared by the reduction of platinum(II) acetate in the presence of 1,10-phenanthroline ligands which had been synthetically modified to include solubilizing and reactive cross-linkable functional groups. These were then conjugated site-specifically to antibody IgG molecules and to Fab' fragments and visualized by scanning transmission electron microscopy. The resulting conjugates may be autometallographically enhanced and show sensitivity similar to that of Nanogold conjugates in immunoblotting experiments. In preliminary experiments, they have also exhibited labeling of tissue antigens and penetrate to access nuclear targets.

Affinity Labels↗
PubMed1999

Quantitative energy-dispersive X-ray microanalysis of calcium dynamics in cell suspensions during stimulation on a subsecond time scale: preparative and analytical aspects as exemplified with paramecium cells.

We analyzed preparative and analytical aspects of the dynamic localization of Ca(2+) during cell stimulation, using a combination of quenched flow and energy-dispersive X-ray microanalysis (EDX). Calcium (or Sr, as a substitute) was retained as fluorides during freeze-substitution, followed by epoxide embedding. The quenched-flow used allowed analyses, during stimulation, in the subsecond time range. Sections of 500 nm were analyzed and no artificial Ca or Sr leakage was recognizable. We calculated a primary beam spread from 63 to 72 nm that roughly indicated the resolution of EDX/structure correlation. These values are quite compatible with the size of potential structures of interest, e.g., Ca stores (approximately 100-nm thickness) or cilia (approximately 250-nm diameter). We used widely different standards to calibrate the ratio of CaK(alpha) net counts in relation to actual ¿Ca. Calibration curves showed a linear relationship and a detection limit of ¿Ca = 2 mM, while ¿Ca in cytosol was 3 mM and in stores was 43 mM, both in nonactivated cells. Eventually Sr(2+) can rapidly be substituted for Ca(2+) in the medium before and during stimulation, thus allowing one to determine Me(2+) fluxes. With our "model" cell, Paramecium, we showed that, upon stimulation (causing rapid Ca(2+) mobilization from subplasmalemmal stores), Ca was immediately exchanged for Sr in stores.

Animals↗
PubMed1983

Siliceous deposits in human urinary calculi--an E. M. study.

Silica calculi in man are extremely rare; only 12 cases have been reported. In an electron microscopic study of human urinary stones, siliceous deposits were encountered in three out of 180 stones as a discrete component of stones. There were no histories of magnesium trisilicate administration in these cases. Morphology of the deposits was distinctive from any other components of the stones and bore some resemblance to animal silica calculi. Siliceous deposits in human urinary stones appear to be more common than generally recognized.

Electron Probe Microanalysis↗
PubMed1994

Effect of delay in cryofixation on the elemental composition of biopsies and post mortem specimens of the thyroid gland.

The time between the excision and cryofixation of a biopsy is most important regarding its elemental composition as demonstrated by an investigation of the thyroid glands of rats and pigs. Biopsies taken and cryofixed immediately served as control specimens. Biopsies that were allowed to stand at room temperature for 20 min before cryofixation and specimens cryofixed at 1 h post mortem were also investigated. Significant changes in the ion concentration of the cells and colloid were apparent in biopsies in which cryofixation was delayed for 20 min and in thyroids cryofixed 1 h post mortem. It was demonstrated that redistribution of electrolytes occurs within 1h post mortem and that similar changes occur in biopsies allowed to stand for 20 min at room temperature before cryofixation. The results stress the importance of immediate cryofixation after surgical excision of a biopsy. This is especially important since numerous elemental changes due to delayed cryofixation resemble those which occur in pathological processes.

Animals↗
PubMed1978

Electron probe X-ray microanalysis of human muscle biopsies.

The elemental composition of human muscle fibres have been determined by electron probe microanalysis. In order to distinguish between different types of fibres, two approaches were used. In one approach individual fibres were isolated, portions of them used for a typing by histochemical methods and the main part used for X-ray microanalysis. In the other approach the muscle biopsy was serial-sectioned, some sections used for a histochemical typing and the others (16 micrometer thick cryosections) used for X-ray microanalysis in th eelectron microscopy. The comparison of the ratios between P, S and K in Study No. 1 and 2 indicates different concentrations of sulphur in the subsarcolemmal zone and in the interior of the fibre. Both routes give information on all elements (except the ten lightest ones) contained in the fibres or in sections of them, provided the concentration is high enough. In order to obtain quantitative data, expressed as mmol/kgw, the spectra of the specimens were compared to those of standards of known composition and the data subjected to a so called ZAF-correction (corrections for the atomic number effect, absorption of X-rays in the specimen and secondary fluorescence). Quantitative data concerning phosphorus, sulphur, chlorine and potassium were obtained in Study No. 2. A significantly higher sulphur concentration was found in type IIA muscle fibres as compared to those of type I.

Chlorine↗
PubMed1994

Measurement of BBN-induced alterations in rat urothelium by electron microscopic X-ray microanalysis.

The aim of this study was to analyse N-butyl-n-butanol-4-nitrosamine (BBN)-induced alterations of the urothelium in rats concerning its content of phosphorus, sulphur, chlorine, potassium and calcium using electron microscopic X-ray microanalysis (REM analysis). The following histopathological findings of the bladder mucosa were discovered after exposure to BBN: normal urothelium (n = 36); focal epithelial proliferations (n = 12) following 6-12 week's exposure; epithelial hyperplasia (n = 8) after urothelial carcinoma (n = 4) following 12 weeks' exposure. The observed phosphorus/sulphur and phosphorus/calcium ratios based on REM analysis did not show any statistical correlation with the morphological changes classified by light microscopy. Our data do not support the hypothesis raised by other investigators that an increase in phosphorus content or phosphorus/sulphur or phosphorus/calcium ratio could indicate early neoplastic transformations of urothelial cells as "tumor markers".

Animals↗
PubMed1988

Na transport compartment in rabbit urinary bladder.

Electron microprobe analysis was used to determine cellular electrolyte concentrations in rabbit urinary bladder. Under control conditions the mean cellular electrolyte concentrations were for Na 11.6 +/- 2.0, for K 124.1 +/- 15.3, and for Cl 26.0 +/- 5.1 mmol/kg wet weight. The dry weight content was 19.0 +/- 2.0 g/100 g. Inhibition of the Na/K-pump with ouabain resulted in drastic changes of the cellular element concentrations. Similar changes also occurred when in addition to ouabain the apical side was kept Na-free. In all epithelial layers the Na and Cl concentrations increased by 90 and 30 mmol/kg wet weight, whereas the K concentration and the dry weight content decreased by 90 mmol/kg wet weight and 6 g/100 g wet weight, respectively. With Na-free choline-Ringer's solution on the basal side ouabain led to a decrease in the K concentration by about 60 mmol/kg wet weight while the Na and Cl concentrations remained unchanged. These data indicate that the basolateral membrane is permeable to Na, choline, Cl, and K. Nystatin produced drastic changes in the cellular electrolyte concentrations when Na- or Rb-sulfate Ringer's solutions were present on the apical side. With Na-sulfate Ringer's solution the Na concentration increased by about 25, the Cl concentration by 30 mmol/kg wet weight and the dry weight content decreased by 4.5 g/100 g, respectively. With Rb-Ringer's solution about 20 mmol/kg wet weight of the cellular K was exchanged against Rb. The concentration changes were identical in all epithelial layers supporting the idea that the rabbit urinary bladder represents a functional syncytium with regard to the transepithelial Na transport.

Animals↗
PubMed1984

Fe-rich corpuscles in Diplogonoporus grandis detected using X-ray microanalysis.

An energy dispersive X-ray microanalysis of the individual corpuscles in the parenchyma of Diplogonoporus grandis was performed for the chemical identification of their inorganic composition. Besides the corpuscles showing distinct peaks for calcium, a major inorganic element, a type of corpuscle showing distinct peaks for iron was observed. It is suggested that it is necessary to examine not only the whole corpuscles isolated but also individual corpuscles in the tissue when studying the nature and roles of corpuscles in cestodes.

Animals↗
PubMed2001

Trace metal and mineral speciation of remediated wastes using electron microscopy.

Electron microscopic techniques, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), and electron probe microanalyses (EPMA), were used to evaluate metal species and mineralogical phases associated with metal-bearing contaminated soil and industrial wastes that have been solidified and stabilized with Portland cement. Metals present in the wastes included arsenic, barium, cadmium, chromium, copper, lead, nickel, and zinc. In addition, mineral alterations and weathering features that affect the durability and containment of metals in aged remediated wastes were analyzed microscopically. Physical and chemical alteration processes identified included: freeze-thaw cracking; cracking caused by the formation of expansive minerals, such as ettringite and thaumasite; carbonation; and the movement of metals from waste aggregates into the surrounding cement matrix. Preliminary results show that although the extent of degradation after 6 years is considered slight to moderate, evaluations of durability and permanence of metals containment cannot be based on leaching and bulk chemistry analyses alone. The use of electron microscopic analyses is vital in studies that evaluate trace metal and mineral species and that attempt to predict the long-term performance of metal containment in solidified and stabilized wastes.

Industrial Waste↗
PubMed2003

The influence of fluoride exposure on dentin mineralization using an in vitro organ culture model.

This study aimed to characterize fluoride-induced alterations in dentin mineralization within a dentin-pulp organ culture system. Tooth sections derived from male Wistar rat incisors were cultured in Trowel-type culture for 14 days, in the presence of 0 mM, 1 mM, 3 mM and 6 mM sodium fluoride. Tooth sections were processed and analyzed for uptake of fluoride, its subsequent effect on dentin mineralization by tetracycline hydrochloride incorporation and mineral composition, expressed as calcium/phosphorous (Ca/P) ratios. Tetracycline hydrochloride incorporation was demonstrated to decrease with increased fluoride exposure, accompanied by significant increases in both Ca/P ratios and fluoride incorporation. These findings provide further evidence that the established alterations in dentin formation during fluorosis are a consequence of disruption to the mineralization process, and provide a model system with which to investigate further the potential role the extracellular matrix plays in inducing the apparent changes in mineral composition.

Animals↗
PubMed2007

Mechanism of calcium accumulation in acetate-fed aerobic granule.

High calcium content has been widely reported in acetate-fed aerobic granules, but the reason behind this is unclear yet. By SEM-energy dispersive X-ray mapping analysis, this study showed that the majority of calcium was presented in the central part of the acetate-fed aerobic granule, and the granule shell part was nearly calcium-free. The elemental analysis of calcium ions coupled with the chemical titration of carbonate further revealed that the calcium ions that accumulated in the acetate-fed aerobic granule mainly existed in the form of calcium carbonate (CaCO3). The formation of the CaCO3 appeared to be highly dependent on the size of the aerobic granule, i.e., the CaCO3 precipitation was found only in aerobic granules with radiuses larger than 0.5 mm. These experimental observations with regard to the formation of CaCO3 in the acetate-fed aerobic granule were further confirmed by the model simulation, which was based on the principles of mass diffusion and carbonate dissociation in liquid phase. This study for the first time showed that the size of the acetate-fed aerobic granule would indeed play an essential role in the CaCO3 formation, and provided experimental evidence that a crystal CaCO3 core was not necessarily required for granulation.

Acetates↗
PubMed2000

The endocytic pathway in Entamoeba histolytica.

We studied the endocytic pathway of Entamoeba histolytica using (a) confocal laser scanning microscopy to observe living cells labeled with fluorescent probes and (b) transmission electron microscopy of cells incubated in the presence of gold-labeled proteins or in a cytochemical medium designed for the localization of acid phosphatase activity. Images of acridine-orange-labeled cells showed that most of the intracellular vacuoles were acidic and were also labeled with Lucifer yellow, a fluorescent dye widely used for labeling of compartments of the endocytic pathway. A similar labeling pattern was observed when the cells were incubated in the present of fluorescein-labeled bovine albumin. However, no labeling was observed when fluorescein-labeled transferrin was used. Gold-labeled proteins (albumin, transferrin, horseradish peroxidase, and lactoferrin) were used for further characterization of the endocytic pathway. With the exception of transferrin, all the proteins bound to the protozoan surface and were subsequently internalized, appearing in small peripheral vesicles and some tubular structures. The ingested molecules accumulated in large vesicles located in the more central portion of the cell, which also presented acid phosphatase activity.

Animals↗
PubMed1991

Characterization of the enamel ultrastructure and mineral content in hypoplastic amelogenesis imperfecta.

Amelogenesis imperfecta (AI) comprises a diverse group of hereditary enamel disorders that are characterized by hypoplastic and in some cases hypomineralized defects. The specific biochemical abnormalities remain unknown for all AI types, making histologic and chemical analyses of affected dentitions essential for resolving the etiology of AI. The purpose of this investigation was to characterize the ultrastructure and mineral content of smooth hypoplastic AI enamel. The AI enamel showed no evidence of surface pitting and was uniformly reduced in thickness by approximately 60% compared with control enamel. Imbibition studies indicated that the AI enamel was generally porous. The first 30 microns of AI enamel adjacent to the dentinoenamel junction was translucent with poorly formed prisms. Abnormal prism structure was seen throughout the AI enamel. Amorphous, presumably organic material that may have been retained enamel protein was also seen. Although the crystallite widths were similar in both AI and normal enamel, the AI teeth showed areas where the crystallite order and continuity appeared disrupted. The mean mineral content was similar for all variables measured except sodium, which was significantly lower in the AI teeth. The calcium concentration was very low in the AI enamel directly adjacent to the dentinoenamel junction and showed a steeper concentration gradient moving from the dentin to the surface compared with control teeth. It may be concluded that the ameloblasts in smooth hypoplastic AI produce a tissue of reduced thickness, which is excessively porous and displays alterations in its ultrastructural organization.

Ameloblasts↗
PubMed1993

Auger electron spectroscopy of dentin: elemental quantification and the effects of electron and ion bombardment.

Auger electron spectroscopy was used in this investigation to quantify the elemental composition of various dentin surfaces. The surfaces included the "smeared layer," produced by abrasion, as well as surfaces prepared by fracturing in vacuum and in air. Quantification was aided by spectra obtained from standard samples of the two main components of dentin, hydroxyapatite, and collagen. Experimental conditions were found whereby the samples could be analyzed without conductive coatings. Ion sputter etch rates were measured for dentin, hydroxyapatite, and collagen. Under the conditions used here, it was observed that collagen etched at a rate approximately five times greater than hydroxyapatite. Auger spectra of the sputtered surfaces indicated significant ion beam-induced sample changes in collagen and dentin; no significant changes were found with hydroxyapatite. The results of this investigation demonstrate that Auger electron spectroscopy can be used to characterize dentinal surfaces and that ion sputtering can cause changes in the surface composition of dentin.

Collagen↗
PubMed1986

Electrochemical study of endodontic silver cones used in root canal therapy.

In previous studies it was observed that endodontic silver cones placed in fine canals became dislodged as a result of corrosion. To investigate the corrosion of high purity silver, potentiostatic and potentiodynamic electrochemical techniques were used. Triangular potential sweeps made in physiological solutions and human plasma showed similar potential-current relationships. However, in human plasma, peak currents were lower and peak potentials were more anodic than those observed in the physiological solutions. The electron microprobe analysis and the EDAX of the film formed in the biological fluids revealed the presence of silver and chloride and a certain amount of carbon. The addition of small quantities of Na2S to the physiological solutions favoured metal dissolution and promoted the formation of a mixed film of AgCL and Ag2S. According to these results chloride and sulphide anions seem to be particularly aggressive towards the metal surface in implanted silver cones. Precautions to avoid direct contact of the cones with saliva and tissue fluids must be taken. Fractures and discontinuities present in the cement considerably increase the corrosion risks.

Electrochemistry↗
PubMed1993

Expanded polytetrafluoroethylene arterial prostheses in humans: chemical analysis of 79 explanted specimens.

The expanded polytetrafluoroethylene (ePTFE) vascular prostheses are widely used as small and medium diameter blood conduits when an autologous venous material is not available or is not suitable. The long-term performance of a prosthesis is dependent on several factors, including its healing characteristics and its stability in vivo. This study was undertaken to assess whether chemical degradation of ePTFE occurs when such arterial substitutes are implanted in humans. Seventy-nine ePTFE grafts excised for complications were analysed using the following techniques: measurement of the contact angle (theta), electron spectroscopy for chemical analysis (ESCA or XPS), Fourier transform infra-red spectroscopy (FTIR) and differential scanning calorimetry (DSC). The results were compared with those obtained from virgin ePTFE and virgin ePTFE washed prostheses. The measurement of the contact angle (theta) permits the comparison of the level of hydrophobicity of material after in vivo residency. The contact angles of explanted ePTFE grafts are greater than those of virgin ones but remain close to those of washed virgin prostheses. The ESCA method allowed investigation of the chemical changes which occur on the surface of ePTFE prostheses after implantation because of the low penetration of the X-ray (about 50 A). This study did not reveal any chemical degradation of the ePTFE with time of implantation for periods up to 6.5 yr. Changes in the surface composition were probably related to lipid and/or protein uptake. The FTIR spectroscopy provides information about the chemical composition of material. Compared with the virgin ePTFE prostheses, the FTIR spectra of explanted prostheses showed specific bands which are characteristic of lipid and/or protein absorptions. The bulk properties of ePTFE studied by DSC did not show any significant changes with time of implantation. It is concluded that ePTFE grafts remain stable in vivo for periods up to 6.5 yr.

Biocompatible Materials↗