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At least 973 records · Page 54Linked to original sources
PubMed1989

Freeze-substitution staining of rat growth plate cartilage with alcian blue for electron microscopic study of proteoglycans.

To selectively stain polyanionic macromolecules of growth plate cartilage and to prevent artifacts induced by aqueous fixation, proximal tibial growth plates were excised from rats, slam-frozen, and freeze-substituted in 100% methanol containing the cationic dye Alcian blue. Electron microscopic examination showed the tissue stained with Alcian blue to be comparable in ultrastructural preservation to tissues slam-frozen and freeze-substituted in the absence of Alcian blue. The extracellular matrix exhibited a characteristic staining pattern when stained by this method. The pericellular rim was identified as a band of varying width encircling the chondrocyte and its cell processes. Peripheral to the pericellular rim the heterogeneity of staining within the extracellular matrix increased, taking the form of polymorphic densities. X-ray microanalysis showed that the visual interpretation of electron density was related to the concentration of copper present, and that the concentration of sulfur was variable in the pericellular rim and in the interterritorial matrix. The difficulties associated with aqueous fixation and staining procedures are discussed in contrast to the improved preservation achieved by cryogenic methods.

Alcian Blue↗
PubMed1988

Effect of salt on the performance of immobilized pH gradient isoelectric focusing gels.

The effect of salt and buffer ions in the sample or in an immobilized pH gradient (IPG) on sample entry into the gel and on the final focused pattern are presented. During the initial phase of electrofocusing, ions present in the gel, either as counter ions to the immobilized charge groups of the IPG gel or added to the gel matrix during the rehydration process, are transported toward the electrodes. For ions present at a concentration exceeding approximately 1 mM the transport can be followed by the refractile line marking the trailing edge of an ion-containing zone. Gradual sample entry may be achieved by applying the sample at a site (near the anode or cathode) opposite to that from which the sharpest refractile line, marking the ion present in the highest concentration, approaches the sample. Additionally, lateral band spreading of the sample is avoided. Thus, sample applied at the cathode for IPG gels rehydrated with 1-2 mM Tris base, or at the anode for gels rehydrated with 1-2 mM acetic acid or sodium acetate, enters the gel matrix gradually without lateral band spreading. In contrast, sample applied at the anode, for Tris-containing gels, or at the cathode, for acetate-containing gels, enters rapidly in a sharp zone when the refractile line reaches the sample zone. This results in a high local protein concentration in the zone immediately behind the boundary with lateral band spreading.(ABSTRACT TRUNCATED AT 250 WORDS)

Gels↗
PubMed1997

Direct Measurements of Line-Mixing Coefficients in the nu1 + nu2 Q Branch of CO2

High-resolution measurements of the (nu1 + nu2) Q branch of pure CO2 were made using a difference frequency spectrometer with resolution of 5 x 10(-5) cm-1 and a signal-to-noise ratio of 2000:1. Lines Q(2) through Q(32) were measured with up to 14 lines in a single spectrum. The analysis of the branch has been performed on data taken at 301 K and pressures less than 11 kPa. The spectra were analyzed on a line-by-line basis within the Rosenkranz approximation of weak overlap [P. W. Rosenkranz, IEEE Trans. Antennas Propagation AP-23, 498 (1975)]. The lineshape profile included Doppler broadening and Dicke narrowing [R. H. Dicke, Phys. Rev. 89, 472 (1953)] using a modified hard collision model [S. G. Rautian and I. I. Sobel'man, Sov. Phys. Uspekh. 9, 701 (1967)] with line mixing. For each line the broadening, Dicke narrowing, and line-mixing coefficients were determined. The broadening coefficients are in good agreement with measurements of lines belonging to different CO2 bands. Our measured line-mixing parameters are compared to those predicted by a relaxation matrix which was calculated from an exponential power gap (EPG) law [L. L. Strow, D. C. Tobin, and S. E. Hannon, J. Quant. Spectrosc. Radiat. Transfer 52, 281 (1994)]. The vibrational band intensity and the linear pressure shift of the branch were also measured. Copyright 1997Academic Press

Journal Article↗
PubMed2001

Influence of static and dynamic disorder on the visible and infrared absorption spectra of carbonmonoxy horseradish peroxidase.

Spectroscopy of horseradish peroxidase with and without the substrate analog, benzohydroxamic acid, was monitored in a glycerol/water solvent as a function of temperature. It was determined from the water infrared (IR) absorption that the solvent has a glass transition at 170-180 K. In the absence of substrate, both the heme optical Q(0,0) absorption band and the IR absorption band of CO bound to heme broaden markedly upon heating from 10-300 K. The Q(0,0) band broadens smoothly in the whole temperature interval, whereas the IR bandwidth is constant in the glassy matrix and increases from 7 to 16 cm(-1) upon heating above the glass transition. Binding of substrate strongly diminishes temperature broadening of both the bands. The results are consistent with the view that the substrate strongly reduces the amplitude of motions of amino acids forming the heme pocket. The main contribution to the Q(0,0) bandwidth arises from the heme vibrations that are not affected by the phase transition. The CO band thermal broadening stems from the anharmonic coupling with motions of the heme environment, which, in the glassy state, are frozen in. Unusually strong temperature broadening of the CO band is interpreted to be caused by thermal population of a very flexible excited conformational substrate. Analysis of literature data on the thermal broadening of the A(0) band of Mb(CO) (Ansari et al., 1987. Biophys. Chem. 26:337-355) shows that such a state presents itself also in myoglobin.

Heme↗
PubMed2003

Ultrasonic wave interaction with multidirectional composites: modeling and experiment.

Phenomena of wave transmission through a multidirectional composite laminate immersed in a fluid have been investigated. Based on a recently-developed recursive stiffness matrix method time-domain beam models have been developed to simulate the problem. Experimental and theoretical results at frequency 2.25 MHz show that the transmission amplitude is highly dependent on lamina orientation and angle of incidence. Large transmission amplitude appears at small (<10 degrees) and large incident angles (45 degrees-60 degrees). At intermediate incident angles (16 degrees-40 degrees) the transmission amplitude is almost zero. At high frequency, the residue epoxy layers between each lamina become important and corresponding resonances may be observed. These transmission phenomena have been interpreted in terms of Floquet waves. It shows that the pass and stop bands of the three Floquet waves obtained from the unit cell stiffness matrix determine the transmission amplitude distribution in frequency, incident angle and rotation angle domains. The effect of random deviation of the laminated structure periodicity has also been assessed. At normal incidence, the variation of the thickness of the epoxy residual layer between composite lamina has little effect on the overall stop and pass band structures but introduces random reverberation and scattering in the pass bands. It is shown that for oblique incidence the transmittivity spectrum of a composite with random lamina lay-up converges with increase of randomness to that of a [0/-45/90/45]2s quasi-isotropic composite. Randomization of lamina lay-up produces a small effect in the transmittivity spectrum for a quasi-isotropic composite.

Journal Article↗
PubMed1989

Identification of a cell-surface protein involved in PC12 cell-substratum adhesion and neurite outgrowth on laminin and collagen.

On substrata coated with laminin or native collagen (Types I/III), PC12 cells employ an active adhesion mechanism (i.e., one inhibited at low temperature, by azide or in the absence of divalent cations) to attach and extend neurites; on substrata coated with wheat germ agglutinin (WGA) or polylysine, by contrast, PC12 cells attach via a passive mechanism and fail to extend neurites (Turner et al., 1987). This paper reports the isolation of 2 monoclonal antibodies (3A3 and 1B1) that promote retraction of neurites extended on laminin and collagen. In studies of initial cell attachment, 3A3 inhibited active attachment to laminin or collagen but not passive attachment to WGA or polylysine, whereas 1B1 inhibited both active and passive attachment. The more potent of the antibodies, 3A3, precipitates 2 radioactive protein bands (of approximately 185 and 125 kDa) from 1% Nonidet P-40 extracts of metabolically labeled PC12 cells. The properties of these proteins suggest that the antigen recognized by 3A3 is a member of the integrin family of matrix receptors. The other monoclonal antibody, 1B1, reacts with many PC12 proteins, including both bands precipitated by 3A3. The available data strongly suggest that an integrin with specificity for both laminin and collagen mediates PC12 adhesion to the substratum at both the cell body and the neurite growth cone.

Adrenal Gland Neoplasms↗
PubMed2004

The cellular responses of articular cartilage to sharp and blunt trauma.

OBJECTIVE: To determine the response of immature articular cartilage to both sharp and blunt trauma in terms of cell death, cell proliferation and matrix synthesis. DESIGN: Blunt wounds were made with a trephine in full depth immature bovine articular cartilage explants which were cut in half through the center of the trephine wound with a sharp scalpel to produce blunt and sharp trauma on the same explant. Explants were maintained in culture for up to 10 days. Prior to fixation at days 2, 5 and 10, medium was supplemented with 10 microCi ml-1 35S-sulphate, [3H]-proline or [3H]-thymidine for 24h to assess matrix synthesis and cell proliferation. Cell death was assessed using a Live/Dead label. RESULTS: In the case of blunt wounds, a band of cell death was observed adjacent to the lesion edge. Microautoradiography demonstrated little radiolabel incorporation and, therefore, no new matrix synthesis or cell proliferation within this region. In contrast, wounds made with a sharp scalpel showed restricted cell death, with radiolabel incorporation adjacent to the lesion edge at all time points. This demonstrated not only chondrocyte proliferation and new matrix synthesis at the wound margin, but also an up-regulation of matrix synthesis adjacent to the lesion edge. CONCLUSIONS: In terms of clinical relevance, the use of sharp precise instruments during the surgical management of cartilage defects may be necessary to reduce cell death and promote matrix elaboration at the lesion edge in order to facilitate successful integration.

Animals↗
PubMed1998

Regulation of endothelin-B receptor mRNA expression in human endothelial cells by cytokines and growth factors.

The regulation of endothelin-B receptor (ETB) mRNA expression in human endothelial cells (ECs) by cytokines and growth factors may play an important role in the response of the endothelium to inflammatory and angiogenic stimuli. Using quantitative RT-PCR, we studied ETB expression in human umbilical vein ECs (HUVECs) grown in culture on either plastic or fibrin matrix for 24 h in the presence or absence of tumor necrosis factor-alpha (TNF-alpha, 100 U/ml) or basic fibroblast growth factor (bFGF, 30 ng/ml). In addition, the effect of the nitric oxide (NO) donor S-nitrosyl-acetylpenicillamine (SNAP, 0.4 mM) was examined directly on ETB expression or on the response to bFGF. Under control conditions, ETB mRNA was detected after 35 cycles of amplification as a band of the expected size (553 bp). In the absence of fibrin matrix, ETB was downregulated by bFGF and TNF-alpha and could barely be detected by PCR. Southern analysis of the RT-PCR products after 25 cycles revealed that bFGF reduced ETB mRNA expression by 2.7 +/- 0.4-fold (p < 0.01) and TNF-alpha tended to reduce its expression by 1.8 +/- 0.9-fold of control, although this did not reach statistical significance (p < 0.20). In contrast, on fibrin matrix both bFGF and TNF-alpha increased ETB mRNA expression by 25 +/- 9-fold (p < 0.05) and 68 +/- 19-fold (p < 0.05) of control, respectively, suggesting a role for ETB in the vascular tube formation that occurs under these conditions. Pharmacologic addition of NO mimicked the effect of fibrin, converting the response to bFGF from down- to upregulation of ETB, raising the possibility that NO acts as a molecular switch modulating the response to angiogenic factors.

Blotting, Northern↗
PubMed2003

Soluble glucocorticoid-induced tumor necrosis factor receptor (sGITR) increased MMP-9 activity in murine macrophage.

Glucocorticoid induced tumor necrosis factor receptor (GITR), a new TNFR family, increased production of matrix matalloproteinase (MMP-9) in murine macrophages. Murine macrophages produced a band of gelatinolytic activity at 100 kDa when stimulated for 18 h with soluble GITR. MMP-9 was identified by gelatin zymography and Western blot. Previous results demonstrated that murine macrophages express GITR and GITR ligand constitutively. Induction of MMP-9 was synergistic with co-treatment of INF-gamma. MMPs could play a critical role in progression and promotion of tissue injury after inflammation stimulated by GITR/ligand system.

Animals↗
PubMed1997

Interaction of matrix metalloproteinases-2 and -9 with pregnancy zone protein and alpha2-macroglobulin.

The binding of matrix metalloproteinases-2 and -9 to pregnancy zone protein and alpha2-macroglobulin was studied. The binding was demonstrated by formation of dimeric as well as tetrameric complexes of pregnancy zone protein and by the formation of alpha2-macroglobulin complexes with fast and intermediate mobility in native gel electrophoresis. The complex formation was confirmed by the use of 125I-labeled matrix metalloproteinase-2. The cleavage sites in the "bait" regions following formation of high-molecular-weight complexes of matrix metalloproteinases with the alpha-macroglobulins were determined by protein sequence analysis. Pregnancy zone protein was cleaved at Thr693-Tyr694 and alpha2-macroglobulin at Gly679-Leu680 and Arg696-Leu697 by matrix metalloproteinase-2. Matrix metalloproteinase-9 cleaved alpha2-macroglobulin at the same site as matrix metalloproteinase-2, but cleavage of pregnancy zone protein was at Leu753-Ser754. The sequences of the bands, visualized in the SDS gel, of approximately 90 and 165 kDa or higher molecular weight complexes were the same. This indicates that the matrix metalloproteinases cleaved the inhibitors with or without binding to them. The present results suggest that matrix metalloproteinases-2 and -9 may interact with pregnancy zone protein and alpha2-macroglobulin in vivo.

Animals↗
PubMed2002

Optical and IR absorption as probe of dynamics of heme proteins.

The spectroscopy of horseradish peroxidase with and without the substrate analogue benzohydroxamic acid (BHA) was monitored in different solvents as a function of the temperature in the interval from 10 to 300 K. Thermal broadening of the Q(0,0) optical absorption band arises mainly from interaction of the electronic pi --> pi transition with the heme vibrations. In contrast, the width of the IR absorption band of CO bound to heme is controlled by the coupling of the CO transition moment to the electric field of the protein matrix. The IR bandwidth of the substrate free enzyme in the glycerol/H2O solvent hardly changes in the glassy matrix and strongly increases upon heating above the glass transition. Heating of the same enzyme in the trehalose/H2O glass considerably broadens the band. The binding of the substrate strongly diminishes the temperature broadening of the CO band. This result is consistent with the view that the BHA strongly reduces the amplitude of vibrations of the heme pocket environment. Unusually strong thermal broadening of the CO band above the glass transition is interpreted to be caused by thermal population of a very flexible excited conformational substate. The thermal broadening of the same band in the trehalose glass is caused by an increase of the protein vibrational amplitude in each of the conformational substates, their population being independent of the temperature in the glassy matrix.

Heme↗
PubMed1994

Intramembranous bone matrix is osteoinductive.

All known bone-derived osteoinductive factors have been isolated from endochondral (EC) bones and all initiate bone induction via EC ossification. However, to date no attempt has been made to isolate comparable factors from bones which form initially and completely via intramembranous (IM) ossification. The purpose of this work was to isolate osteoinductive proteins from IM bones. To accomplish this, we extracted proteins from bovine frontal bone matrix (intramembranous origin) using methods previously described for endochondral (EC) bone matrix (i.e., femur). Bone powder (< 1 mm) was decalcified and proteins extracted with 4 M guanidine hydrochloride. Ultrafiltration was used to isolate and concentrate a 10-100 kilodalton (kDa) fraction, upon which heparin-Sepharose (HS) affinity chromatography was performed. HS-binding (HS-B) and non-binding proteins (HS-NB) were lyophilized with bovine type I collagen (Vitrogen) to form pellets which were implanted subcutaneously in rats. Radiology as well as brightfield, fluorescent, and polarizing microscopy were used to assess the formation of ectopic bone at the site of pellet implantation. In this report we demonstrate that a heparin-Sepharose binding, osteoinductive factor can be extracted and partially purified from bovine intramembranous bone matrix. This factor has a different sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) banding pattern than a comparable osteoinductive/chondroinductive factor isolated from EC bone.

Animals↗
PubMed1985

Intercellular network in articular cartilage of pigs with experimentally induced arthritis. I. Fact or artifact?

Porcine articular cartilage from cases of experimentally induced Erysipelas arthritis was examined with different histological techniques and by electron microscopy. A small percentage of specimens of osteoarthritic cartilage revealed an interlacunar network in the extracellular matrix. These "matrix-streaks" showed characteristic features of densely packed fibrillar elements and collagenous fibrils, as narrow bands connecting adjacent chondrocytes. In a given cartilage site the network had a constant pattern. It is hypothesized that the network formation is correlated with altered joint metabolism and represents regressive changes in cartilage matrix as a result of external mechanical forces. Using a variety of methods, the possibility of the network being an artifact of histological processing could virtually be ruled out. As there are so far only few and contradictory reports on the interlacunar network of articular cartilage, its precise biological significance remains to be established.

Animals↗
PubMed1994

MspI8, the repetitive sequence specifically interacting with nuclear matrix of rat testis cells.

The nuclear matrix bound DNA fraction of rat testis showed enrichment in repetitive sequences found in the 450 bp band after gel electrophoresis of the MspI digested rat DNA. DNA fragments isolated from this band were cloned. DNA of the clone pMspI8 showed homology to some representatives of rat LINE sequence family, and complexed in vitro more efficiently with testes nuclear matrix proteins than with yeast ARS1 sequence containing the matrix association region (MAR) or DNA from an other clone, MspI19. Western blot analysis showed that MspI8 sequence interacts with testes matrix protein of about 120 kDa.

Animals↗
PubMed1999

Abnormal collagen assembly, though normal phenotype, in alginate bead cultures of chick embryo chondrocytes.

The collagens produced by chick embryo chondrocytes cultured in alginate beads were investigated both biochemically and ultrastructurally. The cartilage phenotype is maintained for at least 14 days, as indicated by the production of the cartilage-specific collagens II, IX, and XI and the absence of collagen I. There were differences in the distributions of collagens among the three different compartments analyzed (cells and their associated matrix, further-removed matrix (released by alginate solubilization), and culture medium), with large amounts of collagen IX (mainly in proteoglycan form) in the culture medium. Inhibition of lysyl oxidase activity by beta-aminopropionitrile led to an overall decrease in collagen production. In contrast to the biochemical observations, collagen ultrastructure in the extracellular matrix of alginate cultures was not in the form of the expected 64-nm banded fibrils, but rather in the form of segment-long-spacing-like crystallites. This abnormal structure is likely to be a result of alginate disrupting normal assembly. We conclude that, in this system, the native fibrillar structure of the collagenous matrix is not essential for the maintenance of the differentiated phenotype of chondrocytes.

Alginates↗
PubMed2004

Electron transfer within 2,7-dinitronaphthalene radical anion.

The optical spectrum of 2,7-dinitronaphthalene radical anion generated by Na(Hg) reduction in acetonitrile containing a large excess of cryptand[2.2.2] exhibits a Hush-type intervalence charge-transfer band at 1070 nm, estimated to correspond to an off-diagonal matrix coupling element of 310 cm(-)(1). The interpolated rate constant for intramolecular electron transfer at 293 K measured by ESR between 225 and 320 K for this solution is 3.1(+/-0.2) x 10(9) s(-)(1). Rate constants estimated in two ways from the optical parameters using the Marcus-Hush assumption that the diabatic surfaces should be parabolae are 1.0 and 0.11 x 10(9) s(-1), and those using diabatic surfaces that fit the observed charge-transfer band are 9.6 and 3.4 x 10(9) s(-)(1), when used with an electron-transfer distance on the adiabatic surfaces of 6.42 A. Similar measurements and comparisons were also carried out using dimethylformamide and butyronitrile as solvents. The success of simple, classical two-state Marcus-Hush theory precludes an electron-hopping mechanism. UHF calculations predict a planar unsymmetrical gas-phase structure for 1,3-dinitrobenzene radical anion but give serious spin contamination. Semiempirical AM1 calculations using singles excitation configuration interaction with an active space of 70 orbitals and the COSMO solvent model also give a planar unsymmetrical structure. These calculations make the internal vibrational component of the reorganization energy nearly constant, and much smaller than the solvent reorganizational component, and predict the transition energy to lie between that observed in acetonitrile (9360 cm(-1)) and those observed in dimethylformamide (8100 cm(-1)) and butyronitrile (8040 cm(-1)).

Journal Article↗
PubMed2002

Methodologies in cancer cytogenetics and molecular cytogenetics.

Various types of cytogenetic and molecular cytogenetic approaches, including conventional banding, fluorescence in situ hybridization (FISH), fiber-FISH, comparative genomic hybridization (CGH), matrix array CGH, chromosome microdissection, and microcell-mediated chromosome transfer are summarized. The rationale, advantage, and limitations of each approach are discussed with respect to research and clinical applications in human neoplasia.

Chromosome Banding↗
PubMed1956

Electron microscopy of cartilage and bone matrix at the distal epiphyseal line of the femur in the newborn infant.

An examination of the fine structure of cartilage and bone matrix at the distal epiphyseal line of the femur of a newborn infant has revealed the following information. Cartilage matrix is composed of a network of widely spaced fibers without obvious periodic banding. Calcification is first seen about the level of the third chondrocyte capsule distal to the furthest penetration of the capillaries. It starts as a haphazard deposition of crystals which have no obvious relationship to the location of the fibers. The process of calcification is completed before ossification commences but the central zone of matrix remains only partly mineralized. Bone matrix is formed over a bar of calcified cartilage. Fibers, recognizable as collagen, are deposited in a loose network in a narrow zone between the osteoblasts and cartilage. These fibers are 2 to 5 times as wide as the fibers in epiphyseal cartilage. Calcification then begins in the osteoid, crystals being first laid down irregularly on or close to the fibers. As they increase in number, the crystals tend to line up along the fibers and eventually are arranged so that the periodicity of the underlying collagen is emphasized. In such an area the fibers are more tightly packed than when uncalcified. There is no change observed in the calcified cartilage at this level. The extracellular matrices of this epiphyseal cartilage and bone can be distinguished from one another in the electron microscope.

Bone Matrix↗