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PubMed1990

Expression of GlN1-immunoreactive glycoproteins during development and regeneration in avian peripheral nerve.

Reactivity of the monoclonal antibody GlN1 was examined by immunocytochemistry and immunoblotting in quail and chick developing and regenerating wing nerves; its distribution is compared to that of HNK-1 which detects a carbohydrate epitope widely distributed in the nervous system. Reactivity was detected by immunofluorescence in cryostat sections, by a postembedding electron-microscopic immunogold technique and in immunoblots of nerve homogenates. From E11-E16, reactivity was detected in several large (120-260 kD) glycoprotein bands; and thereafter, principally, in 3 myelin-related glycoproteins (100, 26.5, and 21.5/ 19.5 kD). The HNK-1 carbohydrate epitope was detected in all these and in other bands permitting identification of the 100-kD moiety as the myelin-associated glycoprotein and the 26.5-kD protein as the P0 protein; the myelin-related 21.5/19.5-kD doublet appears as distinct, probable adhesion molecule(s). During development and regeneration, GlN1 reactivity detected by immunogold labeling of sections appeared first over the extracellular matrix, and later over thicker myelin sheaths. After transection, immunoreactivity in immunoblots was lost in distal stumps but reappeared with time; first in the larger (greater than 120 kD) moieties and then in the myelin-related bands, the same sequence observed in development. Electron-microscopic detection of both GlN1 and HNK-1 carbohydrate epitopes by immunogold labeling of resin-embedded sections is localized most consistently in the thicker (greater than 0.3 microns) myelin sheaths of nerves from chicks at or after hatching. Immunoblots of mature fowl nerve tissues homogenized at various stages of preparation for electron microscopy (after fixation or after fixation and dehydration) show sustained immunoreactivity in the 21.5/19.5-kD bands, reduction or complete suppression in others, and evidence of immunoreactivity in high molecular weight, presumably cross-linked, constituents that remain in the stacking gel portion of the blots.

Animals↗
PubMed1998

Application of the Effective Operator Approach to the Calculation of 12C16O2 Line Intensities

The effective operator approach has been applied to the calculation of line intensities of 12C16O2. Using the eigenfunctions of our reduced effective Hamiltonian (1992, J. Mol. Spectrosc. 156, 48-64) the experimental line intensities of cold and hot bands lying in the regions of 15, 10, and 5 μm have been fitted to parameters of the corresponding effective dipole moment matrix elements. The quality of fittings and the extrapolational properties of the fitted parameters are discussed. Formulas in terms of basic molecular properties for some of the parameters of the effective dipole moment matrix elements are derived. Values of these parameters calculated from the theoretical formulas using the force field and dipole moment expansions of Wattson and Rothman (1992, J. Quant. Spectrosc. Radiat. Transfer 48, 763-780) have been compared with corresponding fitted values. Copyright 1998 Academic Press. Copyright 1998Academic Press

Journal Article↗
PubMed1985

Electron-microscopic study of the collagen fibrils of the rat tail tendon as revealed by freeze-fracture and freeze-etching techniques.

The ultrastructure of the collagen of rat tail tendon was investigated by the freeze-fracture technique. Collagen fibers were pretreated with the digestive enzymes, alpha-amylase, elastase and collagenase to remove matrix substances. Some of the samples were etched for 20 min. Fibrils had an average diameter of 318 +/- 12 nm and a banded structure with a mean periodicity of 64.2 +/- 0.9 mm; the banding was most marked in alpha-amylase/elastase-treated specimens, although the periodicity was independent of pretreatment. Microfibrils were well-displayed following alpha-amylase/elastase and collagenase pretreatments. A difference in the diameters of microfibrils was, however, observed between etched specimens (8.3 +/- 0.3 nm) and those prepared by other experimental methods (11.4 +/- 0.5 nm). In replicas of collagenase-treated and etched specimens, the interconnecting filaments in the interfibrillar region formed a network that was continuous with the microfibrils of collagen fibrils. The diameter of the interconnecting filaments was the same as that of microfibrils. Microfibrillar bundles were observed in the interfibrillar region.

Amylases↗
PubMed1987

A centrosomal inclusion (striped nebulous body) in pinealocytes of the golden hamster.

Electron microscopy has revealed the presence of a special cytoplasmic body in the centrosomal area of pinealocytes of the golden hamster, Mesocricetus auratus. It consists of branching electron-dense ribbons embedded in a flocculent matrix of moderate electron density, and is tentatively named the 'striped nebulous body' (SNB). The dense ribbon usually appears as a thick band accompanied by a parallel thin one to one side. The SNB is not bounded by a membrane, and is roughly round or kidney-shaped. Closely associated with the SNB are centrioles, microtubular sheaves and striated fibrils, the latter two being centriolar derivatives. The observations suggest that the SNB might be formed by the amalgamation of these microtubular sheaves and striated fibrils. In golden hamsters, pinealocytes often have multiple centrioles and centriolar derivatives. The SNB of adult hamsters is seen in the pinealocytes of both sexes, irrespective of the season of the year and the activity of the pineal gland. The functional significance of this body remains to be studied.

Animals↗
PubMed1983

Silver stain for detecting 10-femtogram quantities of protein after polyacrylamide gel electrophoresis.

A rapid and highly sensitive silver stain and color stain were developed for visualizing proteins. The procedure is simple and the bands were clear. This silver stain detects 100 pg quantities of proteins. In order to stain quickly, sensitively, and sharply a protein matrix in a gel, the repeated shrinkage and swelling gel was developed with a hyper- and hypotonic solution to remove the sodium dodecyl sulfate (SDS) from SDS-protein complex and to generate influx of staining solution into the gel. We have found that the silver staining method with the repeated exposure to hyper- and hypotonic solution and a narrow well produced 10 fg order of proteins.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

A new class of absorption feature in Io's near-infrared spectrum.

We report our discovery of an absorption feature in the infrared spectrum of Io centered at 2.1253 micrometers (4705.2 cm-1). This band is marginally resolved at resolving power 1200 with a deconvolved full width at half-maximum (FWHM) of about 4 cm-1. This contrasts with the 30- to 50-cm-1 widths of the broad absorption features previously detected at longer wavelengths which arise from mixtures of SO2 with H2S and H2O. This newly discovered feature is relatively weak, having a core only 5% below the continuum at this resolving power. Our survey from 1.98 to 2.46 micrometers (5050-4065 cm-1) at this same resolving power revealed no other feature greater than 1% of the continuum level shortward of 2.35 micrometers, and 3% elsewhere. The feature does not correspond to any gas- or solid-phase absorption that might be expected from previously identified constituents of Io's surface. No temporal or longitudinal variation has been detected in the course of 18 nights of observation over the past year and no significant variation in the strength of the feature was seen during an emergence from eclipse. These observations indicate that the source material of the feature is reasonably stable, and is more uniformly distributed in longitude than Io's hot spots. These characteristics all indicate that the feature belongs to a class different from those characterizing other known absorption features in Io's spectrum. Consequently, it should reveal important new information about Io's atmosphere-surface composition and interaction. A series of laboratory experiments of plausible surface ices indicates that (i) the band does not arise from overtones or combinations of any of the molecular vibrations associated with species already identified on Io (SO2, H2S, H2O) or from chemical complexes of these molecules, (ii) the band does not arise from H2 trapped in SO2, and (iii) the band may arise from the 2 nu3 mode of CO2. If the band arises from CO2, it is clear from its detailed shape and position that the molecules are not embedded in an SO2 matrix, as are H2S and H2O, but may be present as multimers or "clusters."

Atmosphere↗
PubMed1979

Odontogenic myxoma. Surgical management and an ultrastructural study.

Odontogenic myxoma is a tumor of primitive mesenchyme, probably derived from the dental apparatus. Surgical management of this benign but locally aggressive tumor is either by curettage or en bloc resection. Two cases of odontogenic myxoma of the mandible are reported. In one case, en bloc resection. Two cases of odontogenic myxoma of the mandible are reported. In one case, en block resection with immediate reconstruction was undertaken. In the second case, the neoplasm was thoroughly curetted. The first case was studied by electron microscopy. Six ultrastructural studies of seven odontogenic myxomas have been previously reported. The ultrastructural features of odontogenic epithelium that may be present in myxomas of the jaws have been examined by electron microscopy once before. Our observations are in agreement with those of other workers who suggest that the connective tissue cell is the proliferating component of the tumor and is very similar to a fibroblast but has enough ultrastructural and functional features to deserve the term myxoblast. The majority of the tumor cells are metabolically active and secretory and there is adequate evidence that the tumor matrix consists of large amounts of mucopolysaccharides. It is reasonable, therefore, to conclude that myxoblasts secrete the mucoid matrix that is so characteristic of odontogenic myxomas. Unlike normal or neoplastic fibroblasts, the myxoblasts are not engaged in abundant synthesis of banded collagen throughout the entire lesion.

Adult↗
PubMed2003

Heterodimer formation is essential for heparanase enzymatic activity.

Heparanase is an endo-beta-D-glucuronidase involved in cleavage of heparan sulfate residues and hence participates in extracellular matrix degradation and remodeling. The heparanase cDNA encodes for a polypeptide of 543 amino acids that appears as a approximately 65 kDa band in SDS-PAGE analysis. The protein undergoes a proteolytic cleavage that is likely to occur at two potential cleavage sites, Glu(109)-Ser(110) and Gln(157)-Lys(158), yielding an 8 kDa polypeptide at the N-terminus, a 50 kDa polypeptide at the C-terminus, and a 6 kDa linker polypeptide that resides in-between. The active form of heparanase has long been thought to be a 50 kDa polypeptide isolated from cells and tissues. However, attempts to obtain heparanase activity after expression of the 50 kDa polypeptide failed, suggesting that the N-terminal region is important for heparanase enzymatic activity. It has been hypothesized that heterodimer formation between the 8 and 50 kDa heparanase subunits is important for heparanase enzymatic activity. By individually or co-expressing the 8 and 50 kDa heparanase subunits in mammalian cells, we demonstrate specific association between the heparanase subunits by means of co-immunoprecipitation and pull-down experiments. Moreover, a region in the 50 kDa heparanase subunit that mediates interaction with the 8 kDa subunit was identified. Altogether, our results clearly indicate that heterodimer formation is necessary and sufficient for heparanase enzymatic activity in mammalian cells.

Amino Acids↗
PubMed2002

Trace determination of priority pesticides in water by means of high-speed on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry using turbulent-flow chromatography columns for enrichment and a short monolithic column for fast liquid chromatographic separation.

An integrated on-line SPE-HPLC-MS/MS system has been developed for the rapid analysis of various trace level priority pesticides in surface and drinking water. Eleven pesticides were included in this study, with various phenylureas, triazines and organophosphorous species among them. Use of turbulent-flow chromatography columns (TFC, 50 x 1 mm, 30-50 microm particle size) as extraction cartridges enables fast on-line SPE at high sampling flow-rate (5 ml/min). Polymeric and carbon based TFC columns (Oasis HLB, Cyclone, Hypercarb) allow complete extraction with good recoveries from water volumes up to 50 ml. On-line coupling to HPLC is performed with re-mixing of the organic TFC eluate with water in front of the analytical column to ensure efficient band focussing. For fast HPLC analysis, a short monolithic column is applied in combination with highly selective API-MS/MS detection. Matrix effects on the APCI-MS/MS signal were found to be reduced by the system to an acceptable minimum. Limits of detection, determined for 10-ml samples of river water were in the range between 0.4 and 13 ng/l typically, except trifluralin (approximately 280 ng/l), which is less susceptible to ionization under atmospheric pressure conditions. At an enriched water volume of 10 ml, the whole SPE-HPLC-MS/MS procedure requires less than 14 min. The method was successfully applied to the analysis of drinking and surface water samples taken from several sampling sites around the city of Leipzig, Germany. Concentrations measured (maximum: 16 ng/l simazine in river water) were far below the concentration limits scheduled by law.

Chromatography, High Pressure Liquid↗
PubMed2002

Effect of lead on dental enamel formation.

In this work the effects of lead toxicity on dental enamel formation were studied. Epidemiological data and animal studies show an association between lead exposure and higher caries prevalence, but the mechanism underlying this association is still unknown. Here we present data on enamel formation in rats exposed to lead for 70 days in the drinking water. Enamel matrix was used for protein analysis and dry weight determination, while mature enamel was used for microhardness testing. Enamel matrix was scraped from the teeth and analyzed by electrophoresis in bulk or according to developing stage. Increased amounts of protein were observed in animals exposed to lead when the same weight of matrix was electrophoresed by protein electrophoresis. When extracts were prepared according to developing stages, no differences in the amount of protein or band pattern were observed. Upper incisors were cut longitudinally for Knoop enamel microhardness determination in four regions of the teeth. Microhardness analysis revealed statistically significant (P<0.05) decrease in the microhardness values of enamel from rats exposed to lead in regions of maturation but not of fully mature enamel. These results indicate a delay in enamel mineralization in incisor teeth from animals exposed to lead, highlighting a potentially important effect of lead toxicity not yet explored.

Animals↗
PubMed2002

Transfer-matrix simulations of field emission from a metallic (5,5) carbon nanotube.

We present three-dimensional simulations of field emission from an open (5,5) carbon nanotube without adsorption, by using a transfer-matrix methodology. By introducing pseudopotentials for the representation of carbon atoms and by repeating periodically a basic unit of the nanotube, band-structure effects are manifested in the distributions of energies. A representation of the band structure of the (5,5) nanotube is presented. The total-energy distributions of both the incident and field-emitted electrons contain peaks, which are related to discontinuities in the band structure or to standing waves in the carbon nanotube (a total length of 5.657 nm is considered). These peaks move to lower energies when the extraction field is increased. Such peaks should be observable in field-emission experiments.

Journal Article↗
PubMed2004

Surface-enhanced Raman spectroscopy for bacterial discrimination utilizing a scanning electron microscope with a Raman spectroscopy interface.

Surface-enhanced Raman scattering (SERS) utilizing colloidal silver has already been shown to provide a rapid means of generating "whole-organism fingerprints" for use in bacterial identification and discrimination. However, one of the main drawbacks of the technique for the analysis of microbiological samples with optical Raman microspectroscopy has been the inability to acquire pre-emptively a region of the sample matrix where both the SERS substrate and biomass are both present. In this study, we introduce a Raman interface for scanning electron microscopy (SEM) and demonstrate the application of this technology to the reproducible and targeted collection of bacterial SERS spectra. In secondary electron mode, the SEM images clearly reveal regions of the sample matrix where the sodium borohydride-reduced silver colloidal particles are present, Stokes spectra collected from these regions are rich in vibrational bands, whereas spectra taken from other areas of the sample elicit a strong fluorescence response. Replicate SERS spectra were collected from two bacterial strains and show excellent reproducibility both by visual inspection and as demonstrated by principal components analysis on the whole SERS spectra.

Bacillus subtilis↗
PubMed2000

A stable argon compound

The noble gases have a particularly stable electronic configuration, comprising fully filled s and p valence orbitals. This makes these elements relatively non-reactive, and they exist at room temperature as monatomic gases. Pauling predicted in 1933 that the heavier noble gases, whose valence electrons are screened by core electrons and thus less strongly bound, could form stable molecules. This prediction was verified in 1962 by the preparation of xenon hexafluoroplatinate, XePtF6, the first compound to contain a noble-gas atom. Since then, a range of different compounds containing radon, xenon and krypton have been theoretically anticipated and prepared. Although the lighter noble gases neon, helium and argon are also expected to be reactive under suitable conditions, they remain the last three long-lived elements of the periodic table for which no stable compound is known. Here we report that the photolysis of hydrogen fluoride in a solid argon matrix leads to the formation of argon fluorohydride (HArF), which we have identified by probing the shift in the position of vibrational bands on isotopic substitution using infrared spectroscopy. Extensive ab initio calculations indicate that HArF is intrinsically stable, owing to significant ionic and covalent contributions to its bonding, thus confirming computational predictions that argon should form a stable hydride species with properties similar to those of the analogous xenon and krypton compounds reported before.

Journal Article↗
PubMed1997

Heart rate variability spectral indices for haemodynamic classification of haemodialysis patients.

The usefulness of spectral indices extracted from the heart rate variability (HRV) in discriminating between hypotension-prone and hypotension-resistant haemodialysis patients was investigated. In 30 patients, classified as hypotension resistant (stable group) or hypotension prone (unstable group), beat-to-beat heart period was measured during haemodialysis sessions terminated without collapses. HRV was analysed in the frequency domain combining classic autoregressive spectral estimation with two eigen decomposition-based techniques: the reduced rank approximation (RRA) of the autocorrelation matrix and the Pisarenko harmonic decomposition (PHD). Five spectral indices were obtained: the ratio between the powers in the LF and HF bands (LF/HF), the same ratio calculated after application of RRA (LF/HFRRA), the frequency of the main oscillatory component of HRV estimated through PHD with a decomposition order equal to 1 (F1) and equal to 2 (F2) and the difference between the frequencies of the two oscillatory components resolved in the latter cas (Fd). The performances of these indices in discriminating between the two groups of patients were evaluated estimating the misclassification probability (Pm) of a Bayesian quadratic classifier. The HRV spectral pattern was markedly different: in the stable patients power was mainly in the low-frequency band, whereas in the unstable group it was mainly in the high-frequency band. The frequency of the main oscillatory component was significantly greater in the unstable group than in the stable one. Spectral indices displayed good discrimination power, increasing with the length of the dialysis interval. Best performances were achieved by LF/HFRRA both over short dialysis periods (Pm approximately 12% over 20 min intervals) and over longer periods (Pm = 3.3% over 160 min); similar results were obtained with Fd over short periods and LF/HF over long periods. Spectral HRV indices demonstrate, therefore, a diagnostic value in discriminating between hypotension-resistant and hypotension-prone patients.

Bayes Theorem↗
PubMedDate not supplied

A unique mosaic in the visual cortex of the reeler mutant mouse.

Numerous studies have revealed abnormal cytoarchitectonics in the reeler mouse brain. In the present study, acetylcholinesterase (AChE) histochemistry has revealed a distinctive mosaic within the occipital cortex of the reeler mouse. The mosaic does not appear until after the second postnatal week, perhaps in association with eye opening. AChE staining in the visual cortex of normal littermates does not exhibit a mosaic pattern, but rather, is present within bands or laminae. The AChE mosaic in reeler persists into adulthood. Immunocytochemical staining of the tenascin glycoprotein, an astrocyte-derived extracellular matrix molecule that is concentrated in boundaries around emerging functional patterns in the CNS, reveals a boundary-mosaic pattern in the first postnatal week. Dil axonal tracing in normal versus reeler mice indicates that the thalamocortical projections may also be associated with the AChE mosaic. The observation that a mosaic is unique to the occipital cortex of reeler mice suggests that it may evolve through abnormal cell and molecular interactions in the mutant cortex that normally lead to the development of functional visual representations.

Acetylcholinesterase↗
PubMed1976

The effect of endogenous phosphate on the H+/Mn2+ ratio and the state of Mn2+ in the mitochondrial matrix.

1. Kinetics and stoichiometry of H+ extrusion and reuptake and of Mn2+ uptake and release have been measured in respiring liver mitochondria in the absence of external added Pi. H+ and Mn2+ fluxes are parallel during aerobic cation uptake but not during uncoupler induced cation release. The H+/Mn2+ is 1.24. Addition of SH reagents, in concentrations inhibiting the Pi carrier, modifies the kinetics of H+ extrusion and of Mn2+ uptake and release. The slow phase of uncoupler induced Mn2+ release is diminished. The H+/Mn2+ is increased to 1.72. Addition of SH reagents, after the phase of aerobic uptake is completed, results in a significant reduction of the extent of uncoupler-induced Mn2+ release. The extent of reuptake of endogenous Pi during aerobic uptake of Mn2+ is about 8 nmol x mg protein-1. 2. Aerobic uptake of Mn2+ in the absence of external Pi results in an electron spin resonance spectrum which is the sum of two components. One, denoted as S, corresponds to Mn(H2O)2+(6). Another denoted as E, reflects spin exchange narrowing. In contrast to previous claims the following evidence suggests that the spin exchange component is due to Mn3(PO4)2 precipitate: (a) the dimension of the spin exchange spectrum is markedly reduced by abolition of Pi transport; (b) the spin exchange spectrum is released very slowly by addition of uncouplers under conditions where uncouplers cause a rapid deenergization of mitochondria, reuptake of H+ and release of cations; (c) the free matrix Mn2+ is released slowly after addition of uncoupler if there is a large spin exchange signal; howeover the free matrix Mn2+ is abolished rapidly by uncoupler when formation of the spin exchange signal is prevented by pretreatment with Ca2+; (d) the band width of the spin exchange fraction is independent of the Mn2+/protein ratio either under kinetic or steady state conditions; (e) the experimental spectrum recalls closely that obtained by computer simulation by assuming it as a combination of Mn(H2O)2+(6) and Mn3(PO4)2. 3. It is concluded that endogenous Pi affects the process of aerobic divalent cation uptake. A part of Mn2+ uptake in the absence of externally added anions, consists of a Mn3(PO4)2 precipitate. This accounts for a H+/Mn2+ ratio lower than 2.

Animals↗
PubMed1993

Gly85 to Val substitution in pro alpha 1(I) chain causes mild osteogenesis imperfecta and introduces a susceptibility to protease digestion.

In this paper we describe a mild moderate form of osteogenesis imperfecta caused by a point mutation in COL1A1 which converted glycine 85 to valine. The valine substitution introduced into the triple-helical domain of type-I collagen a conformational perturbation causing susceptibility to digestive proteases. In fact, SDS/PAGE of pepsin-treated collagen showed the presence of a faint band, migrating between alpha 1(I) and alpha 2(I), both in the medium and in the cell layer. On trypsin digestion the band, a shortened form of alpha 1(I), had a melting temperature of 39.5 degrees C. If the triple-helical collagen was obtained after trypsin or chymotrypsin digestion of procollagen, two shortened bands were identified; the enzymes cleaved about 40% of the trimers. The mutant procollagen was normally secreted and processed in the extracellular matrix at a normal rate. When native type-I collagen was formed after dextran-sulfate incubation, only chains of normal length were found, suggesting that the fibroblast proteases did not recognize the alteration introduced by the mutation. The effects of glycine 85 to valine substitution are compared with those produced by a previously described arginine substitution of the same residue (Deak et al., 1991).

Adult↗
PubMed1987

Direct visualization of IgM antibodies bound to tissue antigens using a monoclonal anti-type III collagen IgM as a model system.

A mouse monoclonal IgM antibody directed against human Type III collagen was utilized to immunolocalize Type III collagen by transmission and scanning electron microscopy without the use of an electron-dense conjugate. Because bound IgM can be directly visualized, primary or secondary antibody conjugates, such as ferritin, HRP, colloidal gold, etc., are unnecessary in this method. Immunolocalization to Type III collagen in the matrix of human skin and to fibrils formed in vitro using only IgM antibody reveals uninterrupted IgM binding which exactly matches the banding period of the collagen fibrils. In contrast, colloidal gold-conjugated secondary antibody complexes directed against primary IgM binding sites reveal less precise labeling. The data suggest that direct visualization of primary monoclonal IgM antibodies may be useful in a wide variety of highly specific ultrastructural immunolocalization studies without requiring the use of electron-dense conjugates.

Animals↗