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The expression of melanosomal matrix protein in the transdifferentiation of pigmented epithelial cells into lens cells.

A monoclonal antibody (MC/1) was constructed against melanosomes purified from the chicken pigmented epithelial cells (PECs) in order to characterize the differentiative phenotypes of PEC in the process of transdifferentiation into lens cells. Immunofluorescent studies revealed that MC/1 antibody specifically stains both retinal PECs in the eye and melanocytes in the skin, of chicken embryos. Immunoelectron microscopy showed that the antigen molecules are located on the peripheral region of the melanosomal matrix. A single protein band with an apparent molecular weight of 115,000 was labelled by MC/1 in Western blotting. The 115 kDa polypeptide identified by MC/1 is considered to be a member of the melanosomal matrix proteins. The maintenance of specificity of pigment cell nature is followed in the system of transdifferentiation of PEC into lens in vitro, utilizing 115 kDa protein as a marker. In the dedifferentiated PECs, this protein was undetectable.

Animals↗

Computer-assisted pattern recognition model for the identification of slowly growing mycobacteria including Mycobacterium tuberculosis.

We present a computerized pattern recognition model used to speciate mycobacteria based on their restriction fragment length polymorphism (RFLP) banding patterns. DNA fragment migration distances were normalized to minimize lane-to-lane variability of band location both within and among gels through the inclusion of two internal size standards in each sample. The computer model used a library of normalized RFLP patterns derived from samples of known origin to create a probability matrix which was then used to classify the RFLP patterns from samples of unknown origin. The probability matrix contained the proportion of bands that fell within defined migration distance windows for each species in the library of reference samples. These proportions were then used to compute the likelihood that the banding pattern of an unknown sample corresponded to that of each species represented in the probability matrix. As a test of this process, we developed an automated, computer-assisted model for the identification of Mycobacterium species based on their normalized RFLP banding patterns. The probability matrix contained values for the M. tuberculosis complex, M. avium, M. intracellulare, M. kansasii and M. gordonae species. Thirty-nine independent strains of known origin, not included in the probability matrix, were used to test the accuracy of the method in classifying unknowns: 37 of 39 (94.9%) were classified correctly. An additional set of 16 strains of known origin representing species not included in the model were tested to gauge the robustness of the probability matrix. Every sample was correctly identified as an outlier, i.e. a member of a species not included in the original matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Bacterial↗

Analysis of trichothecene mycotoxins in contaminated grains by gas chromatography/matrix isolation/Fourier transform infrared spectroscopy and gas chromatography/mass spectrometry.

Gas chromatography/matrix isolation/Fourier transform infrared (GC/MI/FTIR) spectroscopy and GC/mass spectrometry (MS) were used to confirm the identities of trimethylsilyl (TMS) derivatives of trichothecene mycotoxins in naturally contaminated grains. Infrared spectral bands observed in the fingerprint region were unique for 10 trichothecene standards. Characteristic absorption bands were observed for the ester (near 1750 cm-1) and ketone (near 1700 cm-1) carbonyl stretching vibrations, the acetate CH3 symmetric bend (1370 cm-1), the epoxide ring (1262 cm-1), the trimethylsilyl CH3 in-plane deformation (1253 cm-1), the ester (O)C-O asymmetric stretching vibration (near 1244 cm-1), and several other bands including intense features due to the TMS function. Infrared bands observed under cryogenic matrix isolation conditions were compared with those found at room temperature in a potassium bromide matrix for 5 of these standards. Identities of deoxynivalenol (DON) from barley and mixed feed, nivalenol from wheat and barley, and DON and fusarenon-x from sweet corn were confirmed by comparison of their infrared spectral bands with those of standards. The identity of DON in the same test samples of sweet corn was confirmed further by GC/MS. GC/MS was also used to quantitate the levels of DON (67-455 ppm) in sweet corn test samples.

Chromatography, Gas↗

SPACR, a novel interphotoreceptor matrix glycoprotein in human retina that interacts with hyaluronan.

SPACR (sialoprotein associated with cones and rods), is the major 147-150-kDa glycoprotein present in the insoluble interphotoreceptor matrix of the human retina. Immunocytochemistry localizes SPACR to the matrix surrounding rods and cones (Acharya, S., Rayborn, M. E., and Hollyfield, J. G. (1998) Glycobiology 8, 997-1006). From affinity-purified SPACR, we obtained seven peptide sequences showing 100% identity to the deduced sequence of IMPG1, a purported chondroitin 6-sulfate proteoglycan core protein, which binds peanut agglutinin and is localized to the interphotoreceptor matrix. We show here that SPACR is the most prominent 147-150-kDa band present in the interphotoreceptor matrix and is the gene product of IMPG1. SPACR is not a chondroitin sulfate proteoglycan, since it is not a product of chondroitinase ABC digestion and does not react to a specific antibody for chondroitin 6-sulfate proteoglycan. Moreover, the deduced amino acid sequence reveals no established glycosaminoglycan attachment site. One hyaluronan binding motif is present in the predicted sequence of SPACR. We present evidence that SPACR has a functional hyaluronan binding domain, suggesting that interactions between SPACR and hyaluronan may serve to form the basic macromolecular scaffold, which comprises the insoluble interphotoreceptor matrix.

Amino Acid Sequence↗

Myosin heavy chain regulation and myocyte contractile depression after LV hypertrophy in aortic-banded mice.

Using an adult mouse aortic-banded model of pressure-overload hypertrophy and isolated cardiomyocyte mechanics studies, we examined the hypothesis that contractile depression is due to altered cardiac contractile proteins rather than changes in left ventricular (LV) geometry, loading, or the extracellular matrix. FVB mice were banded at the transverse aortic arch or sham operated and studied after 7 days. In nine animals the gradient across the aortic band averaged 47 +/- 4 mmHg. Compared with sham-operated controls, banded animals had increased LV weight-to-body weight ratio (2.8 +/- 0.1 and 3.5 +/- 0.1, respectively; P = 0.035). Left ventricles from additional age-matched groups of mice that underwent identical surgical procedures were examined for altered transcriptional control of myosin heavy chains (MHCs). beta-MHC protein content increased (15 +/- 2%) vs. shams (3.8 +/- 2%; P = 0.004). Dot blots of LV RNA showed a corresponding increase in beta-MHC transcripts in banded animals (15.8 +/- 2%) vs. controls (5.7 +/- 2%; P = 0.012). Contractile performance was assessed using enzymatically disaggregated isolated LV myocytes paced at 0.5 Hz. There was no difference in percentage myocyte shortening between banded (8.6 +/- 0.5%) and control (9.1 +/- 0.5%) animals. However, maximal velocity of contraction was depressed after aortic banding (129 +/- 11 vs. 233 +/- 28 microns/s; P = 0.007), as was velocity of relaxation (105 +/- 11 vs. 188 +/- 22 microns/s; P = 0.007). These results suggest that depressed myocyte contractility after induction of pressure-overload hypertrophy in aortic-banded mice may be, in part, a consequence of transcriptional upregulation of the beta-MHC.

Animals↗

Porcine dentin matrix protein 1: gene structure, cDNA sequence, and expression in teeth.

Dentin matrix protein 1 (DMP1) is an acidic non-collagenous protein that is necessary for the proper biomineralization of bone, cartilage, cementum, dentin, and enamel. Dentin matrix protein 1 is highly phosphorylated and potentially glycosylated, but there is no experimental data identifying which specific amino acids are modified. For the purpose of facilitating the characterization of DMP1 from pig, which has the advantage of large developing teeth for obtaining protein in quantity and extensive structural information concerning other tooth matrix proteins, we characterized the porcine DMP1 cDNA and gene structure, raised anti-peptide immunoglobulins that are specific for porcine DMP1, and detected DMP1 protein in porcine tooth extracts and histological sections. Porcine DMP1 has 510 amino acids, including a 16-amino acid signal peptide. The deduced molecular weight of the secreted, unmodified protein is 53.5 kDa. The protein has 93 serines and 12 threonines in the appropriate context for phosphorylation, and four asparagines in a context suitable for glycosylation. Dentin matrix protein 1 protein bands with apparent molecular weights between 30 and 45 kDa were observed in partially purified dentin extracts. In developing teeth, immunohistochemistry localized DMP1 in odontoblasts and the dentinal tubules of mineralized dentin and in ameloblasts, but not in the enamel matrix.

Amino Acid Sequence↗

Identification of phototrophic sulfur bacteria through the analysis of lmwRNA band patterns

Several phototrophic sulfur bacteria were identified preliminarily through the analysis of the low-molecular-weight RNA fraction (lmwRNA) of bacterial cells. This fraction includes the ribosomal 5S RNA and several transfer RNAs. These molecules were separated by high-resolution electrophoresis in polyacrylamide gels, and the resulting band patterns were used as fingerprints for the identification of the organisms. We examined a large number of well-characterized reference strains together with a broad range of purple sulfur bacterial isolates from freshwater and marine environments. A cluster analysis was run using the similarity matrix calculated from the band patterns. Despite the shortcomings of the method, close relatives were clustered together yielding a number of groups consistent with the phylogenetic arrangement established through the analyses of a few available 16S rRNA gene sequences. Thus, the classification obtained gives further support to rearrangement of the group as the analyses of 16S rRNA gene sequences had previously suggested. We conclude that the analysis of lmwRNA band patterns is a rapid and simple tool for grouping and preliminarily identifying new isolates of phototrophic sulfur bacteria.

Journal Article↗

[Study on the spectral characteristics of tobacco peroxidase I with high purity].

Tobacco peroxidase (TOPI) has been purified by ammonium sulfate fractionation and column chromatography, involving ion exchange on DE-52 cellulose, gelfiltration on Sephadex G-75 and ion exchange on DEAE-sephadex A-50. The specific activity of peroxidase purified was 4,826 U/mg. It has been demonstrated by SDS-PAGE as a single band. Its molecular weight (matrix assisted laser desorption/ionization time of flight mass spectra) and isoelectric point (pI) have been determined to be 21,888.5 and 3.5 respectively. The native enzyme is one of a haemachrome-containing acidic protein and has its soret maximum at 402 nm and alpha and beta bands at 636 nm and 498 nm, respectively. Its characteristic absorption spectra and fluorescence spectra changed in different pH and denaturant. It has its own characteristic absorption spectra and fluorescence spectra.

Isoelectric Point↗

Jet-discharge cavity ring-down spectroscopy of ionized polycyclic aromatic hydrocarbons: progress in testing the PAH hypothesis for the diffuse interstellar band problem.

Naphthalene cations (C10H+8) were produced in a slit jet coupled with an electronic discharge, and cavity ring down was used to obtain its absorption spectrum in the region 645-680 nm. Two of the strongest C10H+8 bands previously characterized by matrix isolation spectroscopy were found, both with a fractional blue shift of about 0.5%. This is the first gas-phase electronic absorption spectrum of an ionized polycyclic aromatic hydrocarbon (PAH). This work opens the way for a direct comparison of laboratory PAH spectra with the diffuse interstellar bands (DIB), the origin of which still constitutes an open problem in astrophysics.

Astronomical Phenomena↗

Perineuronal nets ensheath fast spiking, parvalbumin-immunoreactive neurons in the medial septum/diagonal band complex.

Perineuronal nets, composed of extracellular matrix material, have previously been associated with parvalbumin-immunoreactive neurons in the medial septum/diagonal band (MS/DB) complex of the rat. The aim of this study was to correlate the presence of perineuronal nets with electrophysiological properties and parvalbumin immunoreactivity in MS/DB neurons. Intracellular recordings were made from cells in a brain slice preparation maintained in vitro, and neurons were characterized into four populations: (i) slow-firing neurons, (ii) burst-firing neurons, (iii) fast spiking neurons with narrow action potentials and a small degree of spike frequency adaptation, and (iv) regular spiking neurons with broader action potentials and a high degree of spike frequency adaptation. Following electrophysiological characterization, neurons were filled with biocytin, processed for parvalbumin immunoreactivity and stained for perineuronal nets using Wisteria floribunda lectin. The three substances were viewed with triple fluorescence. Fast spiking, nonadapting neurons, shown previously to contain parvalbumin immunoreactivity, were nearly all ensheathed by perineuronal nets. There was a population of small parvalbumin-immunoreactive neurons which did not possess perineuronal nets, and which were not encountered with the intracellular electrodes. The other three neuron types in the MS/DB did not contain parvalbumin immunoreactivity or perineuronal nets. In keeping with this neurochemical profile for electrophysiologically identified neurons, burst-firing neurons had action potential parameters more similar to those of regular spiking than of fast spiking neurons. We conclude that fast spiking neurons, presumed to be GABAergic septohippocampal projection neurons, are surrounded by supportive structures to enable the high level of neuronal discharge required for producing disinhibition of hippocampal pyramidal neurons.

Action Potentials↗

Infrared absorption spectrum of matrix-isolated noble-gas hydride molecules: fingerprints of specific interactions and hindered rotation.

Noble-gas hydride molecules with the general formula HNgY (Ng denotes noble-gas atom and Y denotes electronegative fragment) are usually prepared in solid noble gases. In many cases, the matrix-isolated HNgY molecules show a characteristic structure of the H-Ng stretching absorption: A close doublet as the main spectral feature and a weaker satellite at higher energy. This characteristic band structure is studied here for matrix-isolated HXeBr and HKrCl molecules. Based on the experimental and theoretical results, we suggest a model explaining the common features of the band structure of the HNgY molecules in noble-gas matrices. In this model, the main doublet bands are attributed to matrix sites where the splitting is caused by specific interactions of the embedded molecule with noble-gas matrix atoms in certain local morphology. The weaker blueshifted band is probably a fingerprint of hindered rotation (libration) of the embedded molecule in the lattice. This librational band has a mirror counterpart at lower energies appearing at higher matrix temperatures. Our present ab initio calculations for the one-to-one Xe...HXeBr complexes and the simulation of hindered rotation in a matrix support this image.

Journal Article↗

Resolution-enhanced Fourier transform infrared spectroscopy study of the environment of phosphate ions in the early deposits of a solid phase of calcium-phosphate in bone and enamel, and their evolution with age. I: Investigations in the upsilon 4 PO4 domain.

In order to investigate the possible existence in biological and poorly crystalline synthetic apatites of local atomic organizations different from that of apatite, resolution-enhanced, Fourier transform infrared spectroscopy studies were carried out on chicken bone, pig enamel, and poorly crystalline synthetic apatites containing carbonate and HPO4(2-) groups. The spectra obtained were compared to those of synthetic well crystallized apatites (stoichiometric hydroxyapatite, HPO4(2-)-containing apatite, type B carbonate apatite) and nonapatitic calcium phosphates which have been suggested as precursors of the apatitic phase [octacalcium phosphate (OCP), brushite, and beta tricalcium phosphate and whitlockite]. The spectra of bone and enamel, as well as poorly crystalline, synthetic apatite in the upsilon 4 PO4 domain, exhibit, in addition to the three apatitic bands, three absorption bands that were shown to be independent of the organic matrix. Two low-wave number bands at 520-530 and 540-550 cm-1 are assigned to HPO4(2-). Reference to known calcium phosphates shows that bands in this domain also exist in HPO4(2-)-containing apatite, brushite, and OCP. However, the lack of specific absorption bands prevents a clear identification of these HPO4(2-) environments. The third absorption band (610-615 cm-1) is not related to HPO4(2-) or OH- ions. It appears to be due to a labile PO4(3-) environment which could not be identified with any phosphate environment existing in our reference samples, and thus seems specific of poorly crystalline apatites. Correlation of the variations in band intensities show that 610-615 cm-1 band is related to an absorption band at 560 cm-1 superimposed on an apatite band. All the nonapatitic phosphate environments were shown to decrease during aging of enamel, bone, and synthetic apatites. Moreover, EDTA etching show that the labile PO4(3-) environment exhibited a heterogeneous distribution in the insoluble precipitate.

Aging↗

Isolation of matrix vesicles by isoelectric focusing in Pevikon-Sephadex.

We have investigated the use of an isoelectric focusing (IEF) technique for isolating and characterizing matrix vesicles. Focusing was performed on crude preparations of matrix vesicles isolated from collagenase digests of chick epiphyseal cartilage and purified by discontinuous sucrose gradient centrifugation. Crude and partially purified vesicle preparations were subjected to flat bed IEF in a slurry of Pevikon-Sephadex. Partially purified matrix vesicles focused as a narrow band (pI congruent to to 6.5). Alkaline phosphatase, solubilized from matrix vesicles, focused with a pl of 4.0-4.5. The IEF profile of matrix vesicles also differed from that of chondrocyte membranes. Thus, the membrane pls were congruent to to 5.4 and 6.6-7.8, respectively. The latter peak probably corresponded to the pl of the matrix vesicle preparation. This observation lends support to the view that vesicles originate from distinct regions of the chondrocyte membrane.

Alkaline Phosphatase↗

Enhancement of the thermal lens signal induced by sample matrix absorption of the probe laser beam.

The effect of the absorption of the probe laser beam by the sample matrix on the thermal lens signal of a solute was investigated for aqueous solutions of Tb(III), Yb(III), and Nd(III). The measurements were performed with a thermal lens instrument in which the pump and the probe beam were derived from a tunable Ti:sapphire laser. Thermal lens signals were found to be enhanced in the region where the probe beam overlapped with the absorption band of the sample matrix. The observed enhancement was confirmed further with samples of the same solutes (lanthanide ions) but in D20, which does not absorb in the same spectral region as water. The enhancement may be due to the fact that absorption by the sample matrix led to a change in its refractive index and the production of a temperature gradient. In addition to fundamental importance, the observed enhancement can be used to improve the sensitivity of the thermal lens measurements by judiciously selecting a solvent that absorbs the probe laser beam.

Journal Article↗

[Local rotational flap of the nail].

An original pattern of nail flap is described. It consists of a longitudinal band of lateral nail matrix and bed associated with its lateral wall. A vascular anatomic study with latex neopren infusion has been performed. The dorsal branch of the medial phalangeal artery could be shown to be the arterial pedicle of the flap. The venous system was shown to be concomitant. Eight nail autoplasties have been performed in eight cases, proving the reliability of the flap. It provides an interesting way of reconstruction of the nail in tumors, black longitudinal nail bands, posttraumatic and also post-infectious lesions of the nail.

Adult↗

Novel Ti-base nanostructure-dendrite composite with enhanced plasticity.

Single-phase nanocrystalline materials undergo inhomogeneous plastic deformation under loading at room temperature, which results in a very limited plastic strain (smaller than 0-3%). The materials therefore display low ductility, leading to catastrophic failure, which severely restricts their application. Here, we present a new in situ-formed nanostructured matrix/ductile dendritic phase composite microstructure for Ti-base alloys, which exhibits up to 14.5% compressive plastic strain at room temperature. The new composite microstructure was synthesized on the basis of the appropriate choice of composition, and by using well-controlled solidification conditions. Deformation occurs partially through dislocation movement in dendrites, and partially through a shear-banding mechanism in the nanostructured matrix. The dendrites act as obstacles restricting the excessive deformation by isolating the highly localized shear bands in small, discrete interdendritic regions, and contribute to the plasticity. We suggest that microscale ductile crystalline phases might therefore be used to toughen nanostructured materials.

Alloys↗

Chorioallantoic placenta formation in the rat: I. Luminal epithelial cell death and extracellular matrix modifications in the mesometrial region of implantation chambers.

On days 7 and 8 of pregnancy, mesometrial regions of rat gestation sites were examined by light microscopy and transmission electron microscopy to determine what changes occur before the chorioallantoic placenta forms in that region. By day 7, gestation sites contained a uterine lumen mesometrially and an antimesometrial extension of the uterine lumen, the implantation chamber. The implantation chamber consisted of a mesometrial chamber between the uterine lumen and the conceptus, an antimesometrial chamber that contained the conceptus, and a decidual crypt antimesometrial to the conceptus. Stromal cells that formed the walls of the implantation chamber were closely packed decidual cells, while those that surrounded the uterine lumen were loosely arranged. Late on day 7, a portion of the epithelium lining the mesometrial chamber was degenerating, but this area of initial degeneration was never adjacent to the antimesometrial chamber. By early day 8, most of the epithelial cells lining the mesometrial chamber were degenerating and were being sloughed into the chamber lumen. Although degeneration of these epithelial cells morphologically resembled necrosis, it was precisely controlled, since adjacent epithelial cells lining the uterine lumen remained healthy. The space that separated the denuded luminal surface of the mesometrial chamber from underlying decidual cells became wider and was occupied by an extracellular matrix rich in cross-banded collagen fibrils. Decidual cell processes, that earlier had penetrated the basal lamina beneath healthy epithelial cells, protruded into this matrix and penetrated the basal lamina at the luminal surface. By late day 8, large areas of denuded chamber wall were covered with decidual cell processes, little remained of the basal lamina, and cross-banded collagen fibrils were scarce in the area occupied by decidual cell processes. During the times studied, uterine tissues that formed the walls of the mesometrial chamber were not in direct contact with the conceptus. This study indicates that trophoblast does not play a direct role in epithelial degeneration, basal lamina penetration, or extracellular matrix modifications in the mesometrial region of implantation chambers where part of the chorioallantoic placenta forms, although trophoblast may be required to trigger or modulate some of the changes.

Animals↗

Localization of the undulin gene (UND) to human chromosome band 8q23.

Undulin, a large extracellular matrix (ECM) mosaic glycoprotein related to collagen type XIV, is associated with dense collagen matrices in soft tissues and is likely to be involved in the supramolecular organization of interstitial collagens. By fluorescence in situ hybridization (FISH), we have assigned the undulin locus (UND) to band q23 of human chromosome 8.

Chromosome Mapping↗