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PubMed1993

Adhesive interaction of hemopoietic progenitor cell membrane with the RGD domain of fibronectin.

The binding of two cloned hemopoietic progenitor cell lines, B6Sut (multipotential) and FDCP-1 (bipotential) to dishes coated with fibronectin or its chymotryptic fragments was studied by labeling the cells with 51Cr or [35S]methionine. Intact fibronectin molecule and its 120 kDa fragment, containing the Arg-Gly-Asp (RGD) sequence motif, as well as a synthetic RGD-containing peptide Peptite 2000 all bound progenitor cells. However, the 40 or 45 kDa fragments, containing the heparin-binding and CS-1 domains, failed to bind the cells in a comparable magnitude. The binding of intact fibronectin and its 120 kDa fragment was inhibited in a dose-dependent fashion with increasing concentration of RGD-containing Gly-Arg-Gly-Asp-Ser peptide, but not with Gly-Arg-Gly-Glu-Ser control peptide that does not contain the RGD sequence motif. To explore the nature of the receptor for this fragment of fibronectin, membrane proteins were labeled with 125I and subjected to affinity chromatography using a matrix to which the 120 kDa fragment of fibronectin was covalently bound. Specific competitive elution with RGD yielded two bands with molecular masses of 160 and 110 kDa, corresponding, respectively, to those of alpha 5 and beta 1 chains of integrin molecule. Western blotting of whole-cell-lysate proteins with a monospecific, polyclonal serum specific for vertebrate beta 1 integrins identified a beta 1 integrin in these cells. Thus, it appears that an interaction involving alpha 5 beta 1 integrin with 120 kDa fragment of fibronectin may be involved between hemopoietic progenitor cells and the fibronectin component of extracellular matrix.

3T3 Cells↗
PubMed1982

Patterns of matrix vesicle calcification in osteomalacia of Gyro mice.

Homozygous Gyro mice manifest widespread abnormalities in the inner ear and other parts of the skeleton. These disorders were attributed to a sex linked dominant gene. In the present study, tissue specimens were obtained from the inner ear and maxillary and tibial bones, and undecalcified samples were processed for light and transmission electron microscopy. Light microscopy revealed osseous changes consistent with osteomalacia. These alterations comprised broad bands of osteoid with partial or complete failure of calcification. Electron micrographs showed longitudinal calcifying fronts composed of hydroxyapatite crystals. Abundance of matrix vesicles could be demonstrated in the extracellular matrix. Many of the vesicles contained crystaline mineral. In addition, calcification alongside collagen fibers was recorded. Occasionally, apatite crystals were attached to plasma membranes. Female and hemizygous male mice showed normal patterns of calcification. The findings in the affected Gyro mice appear to be similar to previously reported changes in human osteomalacia. Further definition of the pathological process in this animal model may be valuable in defining mechanisms operative in the development of osteomalacia in man.

Animals↗
PubMed1997

Purification and characterization of a 31-kilodalton iron-regulated periplasmic protein from Pasteurella haemolytica A1.

A prominent iron-regulated periplasmic protein was purified from Pasteurella haemolytica grown in an iron-deficient chemically defined medium. The protein was purified by anion exchange chromatography and appeared as a single band by SDS-PAGE with a molecular weight of 32,000. A yield of five mg was obtained from 91 mg of protein extract. The iron-regulated protein existed as a monomer in the native state with an average molecular weight of 29,877 as determined by analytical ultracentrifugation. The protein had a molecular weight of 30,880 as determined by matrix-assisted laser desorption mass spectrometry, hence the protein is referred to as the 31 kDa protein. Isoelectric focusing showed four bands with pIs of 7.15, 6.8, 6.6, and 5.9. The secondary structure of the protein was determined by circular dichroism and contained 16% alpha-helical structure. The N-terminal sequence, EPFKVVTTFTVIQDIAQNVAGDKAT, showed a 95% identity with the 31 kDa iron-binding protein from Haemophilus influenzae. Isolation and characterization of iron-regulated proteins are of particular interest because of their potential roles in iron assimilation and microbial virulence.

Amino Acid Sequence↗
PubMed1996

Enhanced urokinase-type plasminogen activator activity by extracellular matrix protein obtained from highly metastatic human lung adenocarcinoma cell line.

A protein which enhanced urokinase-type plasminogen activator (u-PA) activity was purified from the extracts of extracellular matrix of highly metastatic cell line HAL-8 derived from human lung adenocarcinoma. The protein showed a single band with molecular weight of 65 kDa after the purification by Sephadex G-150 and diethylaminoethyl-cellulose followed by reversed phase separation in a high performance liquid chromatography system. The purified protein in the immobilized conditions enhanced u-PA activity in both plasminogen activation and S-2444 amidolysis by 4.6- and 2.8-fold increases in the second order rate constants (Kcat/K(m)), respectively. This protein was related to neither plasminogen nor single-chain u-PA by the immunological studies and with respect to retention time on reversed phase analysis. These results suggest that the purified material acts as an enhancer of u-PA in extracellular matrix of the cancer cells, inducing an effective tissue destruction and cell invasion and possessing a highly metastatic potential.

Adenocarcinoma↗
PubMed2003

Biomimetic mineralization of charged collagen matrices: in vitro and in vivo study.

Polyanionic collagen matrix prepared by hydrolysis side chain amides of asparagine and glutamine was mineralized in vivo, without inflammatory response, biodegradation, or resorption, with calcium phosphate deposited in close resemblance to the D-periodicity of collagen fibrils assembly. In vitro results with the same material produced mineralized collagen fibers with a similar morphology and chemical characteristics, suggesting that amide hydrolysis may have introduced into this matrix, signs for the controlled mineralization of collagen fiber. TEM indicated that amide hydrolysis occurred near the OVERLAP and GAP zones, as suggested by the significant reduction in inter-band distances in these regions. The lack of an inflammatory response associated to the similar mineralization pattern observed in vivo and in vitro suggests not only the biomimetic behavior of polyanionic collagen matrix, but also its potential uses as scaffold for bone tissue reconstruction. Based on these results, a model for the in vitro mineralization was also proposed.

Animals↗
PubMed2004

Experimental and theoretical study of line mixing in NH3 spectra. II. Effect of the perturber in infrared parallel bands.

In a previous paper [J. Chem. Phys. 116, 7544 (2002) (Paper I)] a model, based on the energy corrected sudden approximation, was proposed for the construction of the line-mixing relaxation matrix. It was successfully tested by comparisons with measured infrared spectra of ammonia-helium mixtures. The present paper extends this preliminary study by considering mixtures of NH3 with H2 and Ar. Measurements have been made at room temperature in the regions of the nu2 and nu1 bands for pressures up to several hundred atmospheres. As in Paper I, the relaxation operator is constructed, within the impact approximation, using the ECS approximation. The data required are dynamical factors (which can be predicted from the NH3-X potential energy surface) and a scaling length (adjusted using line broadening data). Comparisons between measured and calculated absorptions demonstrate the quality of the model which satisfactory corrects for the large deviations with respect to the purely Lorentzian behavior. Line-mixing effects for NH3-Ar and NH3-H2 are qualitatively similar to those observed for NH3-He but quantitative differences exist, particularly when intra- and interbranch couplings are considered. Finally, the proposed model leads to very satisfactory results in the wings of both the purely rotational and nu2 bands of NH3 diluted in H2, opening promising perspectives for the remote sensing study of planetary atmospheres.

Journal Article↗
PubMed2004

Studies on the microspheres comprised of reconstituted collagen and hydroxyapatite.

Microspheres comprised of hydroxyapatite particles, dispersed in reconstituted fibrous collagen, were prepared and characterized. The hydroxyapatite particles distributed evenly throughout the collagen matrix of the microsphere. Diameters of the reconstituted collagen fibers ranged from 30 to 90 nm, and exhibited a regular banding pattern with cross-striation of 50-60 nm under transmission electron microscope, suggesting that the reconstitution of collagen was not hindered by the hydroxyapatite particulates. When osteoblast cells isolated from newborn rat calvaria were seeded and cultured on the microspheres, the cell density increased from 2x10(4) to 3.2x10(4)cells/cm(2) in 8 days. Von Kossa staining exhibited spotty accumulation of mineral deposits on microspheres indicating matrix mineralization of the cultured cells. Analyses by electron microscopy and confocal microscopy showed that the osteoblast cells spread and attached to the microsphere via focal adhesion, while F-actin and DNA staining demonstrated the presence of stress fibers; moreover, mitotic cells could be observed. Together, these results indicate that osteoblast cells are capable of proliferating, differentiating and mineralizing in the matrix of the microspheres, and suggest that the microspheric composite is a potential grafting material for future clinical applications.

Animals↗
PubMed1992

Localization and cellular source of the extracellular matrix protein tenascin in normal and fibrotic rat liver.

The distribution and the cellular source of the novel extracellular matrix glycoprotein tenascin were studied in normal and fibrotic rat liver. Cryostat sections of normal rat livers, livers of rats treated with intraperitoneal injections of CCl4 and 4-day-old and 8-day-old primary fat-storing cell cultures were stained for tenascin and desmin using an immunoperoxidase procedure or a double-label immunofluorescence technique. Fat-storing cell cultures were metabolically labeled with 3H-proline. Radiolabeled proteins were immunoprecipitated from the supernatant with antitenascin antiserum and subjected to polyacrylamide gel electrophoresis. In normal rat livers, tenascin was detected discontinuously along the sinusoids, whereas portal tracts were devoid of staining. In fibrotic rat livers, tenascin was preferentially expressed in areas of cell damage, in slender septa or at connective tissue-parenchymal interfaces. The middle region of broad septa was negative. Desmin-positive fat-storing cells accumulated in areas strongly immunoreactive for tenascin, and double-label immunofluorescence showed cells positive for both tenascin and desmin. In fat-storing cell cultures, both intracellular positivity for tenascin and staining of extracellular fibers were seen. Gel electrophoresis of immunoprecipitated proteins revealed two major and three minor bands with molecular weights consistent with tenascin. We conclude that tenascin is a component of the extracellular matrix of both normal and fibrotic rat livers. The strong expression of tenascin in areas of cell damage, in "early" septa or at septal-parenchymal interfaces, in contrast to its absence from the middle region of mature septa, suggests a role in early matrix organization. Fat-storing cells synthesize and secrete tenascin.

Animals↗
PubMed2006

[Surface modification and microstructure of single-walled carbon nanotubes for dental composite resin].

In order to improve its dispersion condition in dental composite resin and enhance its interaction with the matrix, single-walled carbon nanotubes(SWNTs) were refluxed and oxidized, then treated by APTE. Their outer surface were coated by nano-SiO2 particles using sol-gel process, then further treated by organosilanes ATES. IR and TEM were used to analyze modification results. TEM pictures showed nano-particles were on the surface of SWNTs; IR showed characteristic adsorbing bands of SiO2. Composite resin specimen with modified SWNTs was prepared and examined by TEM. SWNTs were detected in composite resin matrix among other inorganic fillers.

Composite Resins↗
PubMed1981

The use of the 2-iminobiotin-avidin interaction for the selective retrieval of labeled plasma membrane components.

A general method for the selective retrieval of surface labeled plasma membrane components had been devised. The basis of the technique is the covalent attachment of compounds containing 2-iminobiotin, the cyclic guanidino analog of biotin, onto the cell surface proteins and the use of immobilized avidin to recover the labeled components uncontaminated by other cytosolic and membrane components. The pH-dependent interaction of 2-iminobiotin with avidin makes recovery possible. At high pH the free base form of 2-iminobiotin retains the high affinity specific binding to avidin characteristic of biotin, whereas at acidic pH values, the salt form of the analog interacts poorly with avidin. Model studies on the interaction of 2-iminobiotinylated proteins with avidin-Sepharose 4B show that for tight binding to the affinity matrix, the pH of the column must be 9.5 or higher, that a single 2-iminobiotin group is sufficient for binding, and that proteins with different extents of labeling behave similarly when the low pH buffer is applied. When intact human erythrocytes were sequentially labeled with periodate and 2-iminobiotin hydrazide and the Triton X-100-solubilized plasma membrane proteins were subjected to affinity isolation, the major sialoglycoproteins, periodic acid-Schiff (PAS) 1, PAS 2, and PAS 3, plus two proteins with apparent molecular weights higher than band 3 were retrieved. The recovery of these proteins is not due to a nonspecific adsorption to the affinity matrix.

Avidin↗
PubMed1983

Rapid isolation of bovine interphotoreceptor retinol-binding protein.

A large retinol-binding protein, interphotoreceptor retinol-binding protein, is found only in the interphotoreceptor matrix of the eye, and may function in vitamin A transport for the visual cycle. Interphotoreceptor retinol-binding protein is the major glycoprotein of this matrix, and can be isolated rapidly by affinity-adsorption onto concanavalin A-Sepharose. The yield is approx. 0.25 mg per bovine eye. Its apparent Mr is 250000 by gel-filtration chromatography, and 225000 by native polyacrylamide-gradient gel electrophoresis; this protein band displays endogenous retinol fluorescence on such gels. As measured by SDS-polyacrylamide gel electrophoresis, the apparent Mr is 140000. In the interphotoreceptor matrix most vitamin A-binding sites on this retinol-binding protein are unoccupied; however, addition of exogenous all-trans-retinol can saturate these sites. The apparent dissociation constant for retinol is 10(-6) M, as measured by fluorimetric titration.

Animals↗
PubMed1997

Precursor matrix proteins in the uterine fluid change with stages of eggshell formation in hens.

Organic constituents of the uterine fluid, the acellular milieu in which the eggshell is mineralized, were biochemically characterized at initial, mid and final stages of shell calcification in hens. The electrophoretic protein profiles changed at the different stages of shell mineralization. Two major bands (80-kDa and 43-kDa glycoproteins) with calcium affinity were specific to the initial stage. Four protein bands of 180, 150, 116 and 32 kDa, present at the phase of rapid shell formation, coprecipitated with calcium carbonate in vitro. At this stage were also present a calcium-binding glycoprotein of 36-kDa and a 20-kDa protein. Uterine fluid of the final stage was characterized by a darker intensity of the 66-kDa band, which showed calcium-binding ability and by the presence of three additional proteins (72, 13 and 6 kDa). At least seven bands of the uterine fluid showed similar migration patterns to those of eggshell extracts. Western blotting with ovocleidin and ovalbumin antisera demonstrated the presence of these matrix proteins in uterine fluid collected at initial and mid phase, respectively. Total uterine fluid collected at the end of calcification and dialyzed uterine fluid from the various stages delayed the rate of calcium precipitation in vitro. These observations demonstrate the presence of precursors of eggshell matrix in the uterine fluid and support the hypothesis of their involvement in the process of eggshell mineralization.

Animals↗
PubMed2000

Parameter-free quasiparticle calculations for YH3.

Electronic structure calculations for YH3 within the local density approximation result in a metallic ground state with the bands at the Fermi energy overlapping by more than 1 eV, whereas a band gap of 2.8 eV is deduced from optical experiments. Here, we report the results of parameter-free GW calculations which predict a fundamental gap of 1 eV. When we take into account electric dipole matrix elements a large optical gap of almost 3 eV is obtained. A combination of photoemission and inverse photoemission spectroscopy could test the prediction of a small fundamental band gap.

Journal Article↗
PubMed1998

Induction of matrix metalloproteinases and a collagen-degrading phenotype in fibroblasts and epithelial cells by secreted Porphyromonas gingivalis proteinase.

Periodontitis is characterized by advancement of a narrow band of epithelium (1-10 cells wide) through the collagenous periodontal ligament in response to bacterial accumulation and infection. A modulating role by epithelial cells in the progression of periodontitis was hypothesized due to the close proximity of the advancing epithelium to both the etiological bacteria and to the collagen fibers of the ligament. We demonstrate that rat mucosal epithelial cells and human fibroblasts are similarly stimulated to degrade a collagen type I cellular substrate by thiol-dependent activity released by the major periodontal pathogen Porphyromonas gingivalis. A purified, extracellular bacterial thiol-proteinase from P. gingivalis ATCC 33277 stimulated mucosal epithelial cells to upregulate expression of collagenase and stromelysin, and to degrade a collagen type I fibril matrix. Stimulation of the epithelial cells with this purified proteinase was associated with morphological changes in the cells and with accumulation of secreted latent procollagenase throughout the culture medium. Release of active collagenase was minimal and collagen degradation by the epithelial cells was discreet and localized subcellularly suggesting the possibility that activation of secreted procollagenase was cell-associated. We conclude that a collagen-degrading phenotype can be stimulated in relatively quiescent mucosal epithelial cells and fibroblasts by the presence of bacterial proteinase. These experiments suggest roles for the P. gingivalis thiol-proteinase and the epithelial cell in the pathogenesis of periodontal disease and demonstrate the potential for dysregulation of extracellular matrix remodeling events during healing of other bacterially infected wounds.

Adhesins, Bacterial↗
PubMed1999

Accumulation of matrilysin (MMP-7) and macrophage metalloelastase (MMP-12) in actinic damage.

Photodamage is characterized by degradation of collagen and accumulation of abnormal elastin in the superficial dermis and several matrix metalloproteinases have previously been implicated in this process. Using immunohistochemistry and in situ hybridization, we have studied the localization of two elastolytic matrix metalloproteinases, matrilysin (matrix metalloproteinase-7) and human macrophage metalloelastase (matrix metalloproteinase-12) in solar damage. Human macrophage metalloelastase protein was detected in the superficial dermis in areas of elastotic material. Matrix metalloproteinase-7 was seen in the mid-dermis in regions with less damaged elastic fibers and morphologically better preserved collagen as well as in a band-like pattern below basal keratinocytes in eight of 18 solar elastosis. In samples taken from healthy volunteers 3 d after repeated ultraviolet A or ultraviolet B photoprovocation, occasional immunopositive cells for human macrophage metalloelastase (stromal) or matrix metalloproteinase-7 (sweat gland epithelium) were detected. In samples taken 1 d after ultraviolet B exposure, however, basal keratinocytes were matrix metalloproteinase-7 immunopositive, explaining the linear immunostaining below basal keratinocytes noted particularly in ultraviolet B treated 3 d specimens. Upregulation of metalloelastase was also demonstrated in the skin of hairless mice after repeated ultraviolet exposure. In normal skin, no staining for human macrophage metalloelastase or matrix metalloproteinase-7 was observed in association with elastin. The amount of immunoreactivity for the substrates of matrix metalloproteinase-7, versican, and tenascin, was clearly increased in solar elastosis and photoprovocated skin; versican but not tenascin was detected in the same areas as matrix metalloproteinase-7. Our results suggest that both matrix metalloproteinase-7 and -12 may contribute to remodeling of elastotic areas in sun-damaged skin.

Adult↗
PubMed1994

Metalloproteinase-dependent neurite outgrowth within a synthetic extracellular matrix is induced by nerve growth factor.

In order to assess the requirement for matrix metalloproteinases in neuronal regeneration, in vitro neurite outgrowth by chick dorsal root ganglionic neurons (DRGn) was examined within a reconstituted extracellular matrix. For these studies, cultured neurons were treated with a synthetic peptide inhibitor of metalloproteinases (spIMP), LMHKPRCGVPDVGG. spIMP inhibited all neuronal metalloproteinase activities in zymography and substrate-release assays and was used to examine the role of metalloproteinases in neurite outgrowth by DRGn. Cultures of dissociated DRGn rapidly extended neurites on planar extracellular matrix substrates and this rate of outgrowth was not affected by adding NGF or spIMP. In contrast, neurite extension within a three-dimensional gel of extracellular matrix increased nearly threefold after adding NGF. The NGF-induced neurite penetration was negated in the presence of spIMP but not by control peptide. Similar results were obtained using explanted dorsal root ganglia. These findings suggested that NGF-induced neurite outgrowth within an extracellular matrix involves metalloproteinase activity. Zymographic analysis of media conditioned by NGF-treated DRGn revealed a pair of gelatinolytic bands with apparent molecular masses 72 and 66 kDa, which comigrated as a single 66-kDa band after activation with an organomercurial agent. The gelatinase activities were calcium- and zinc-dependent and were absent from zymograms developed in the presence of spIMP, indicating that NGF-treated DRGn release and activate a 72-kDa metalloproteinase. Samples from DRGn cultures treated with low levels of NGF contained similar amounts of latent and activated metalloproteinase, while high levels of NGF induced an apparent increase in total metalloproteinase secretion and a substantially greater proportion of activated enzyme. Western blot analysis showed this metalloproteinase was immunologically similar to 72-kDa type IV collagenase and immunoassays revealed that this matrix metalloproteinase was increased threefold by high NGF. Furthermore, after high NGF treatment, DRGn media contained sixfold more metalloproteinase activity in assays of matrix degradation. In summary, these results indicate that NGF enhanced metalloproteinase-dependent neurite outgrowth of DRGn within a reconstituted extracellular matrix. Also, NGF increased the expression and activation of 72-kDa type IV collagenase, suggesting a role for this matrix-degrading metalloproteinase in neuronal regeneration.

Amino Acid Sequence↗
PubMed1986

Mesangial cell hillocks. Nodular foci of exaggerated growth of cells and matrix in prolonged culture.

To examine the capability of glomerular mesangial cells (MCs) to produce extracellular matrix, the authors studied MCs in culture by light and electron microscopy as well as immunocytochemistry. MCs were obtained from isolated rat glomeruli and maintained up to 12 weeks in medium containing 20% fetal calf serum. MC outgrowth of primary culture and of up to three subcultures showed characteristic organization consisting of bands of elongated or stellate intertwined cells. After confluency at 10-16 days, MCs continued to grow in irregular multilayers. MCs produced extracellular matrix material within 2-4 days after plating, and large amounts of matrix accumulated with time. By 2-3 weeks, foci of exaggerated MC proliferation, matrix secretion, and necrotic cell debris formed nodular protrusions, which gradually produced large hillocks. Immunocytochemical studies of MC outgrowths were performed on culture plates or on sectioned material with the use of specific rabbit polyclonal antibodies to isolated matrix proteins and FITC-conjugated, affinity-purified second antibodies. Within 3 days of culture, MCs elaborated fibronectin and collagen Types I, III, IV, and V. With time, strands of matrix, notably in the central mass of hillocks, stained extensively for these constituents. Staining for laminin was less pronounced. Smooth muscle cell myosin was regularly found on distinct intracellular fibrils and in the extracellular material of hillocks. Electron microscopy revealed the hillocks to be composed of elongated cells on the surface and stellate cells intermingled with matrix and necrotic cell debris in the core. The results show that proliferating MCs can be maintained in homogeneous culture for a prolonged time period. MCs produce large amounts of the extracellular matrix proteins (Type IV and V collagen, fibronectin, laminin), which are found in normal glomeruli. Cultured MCs also produce interstitial collagen Types I and III. MC hillocks show the nodular accumulation of matrix similar to that seen in the mesangium of diseased glomeruli. It is concluded that the in vitro model of prolonged MC outgrowth may facilitate the investigation of factors that govern mesangial matrix production. Such a model could be used in examining the response of the mesangium to defined inflammatory or metabolic stimuli.

Animals↗
PubMed1985

Implementation of Rao's one-sample polynomial growth curve model using SAS.

Longitudinal data are frequently treated with the classic analysis of variance and regression models. However, these models assume independence of observations. Hoel (1964) demonstrated that the use of least-squares methods on intercorrelated serial observations results in the rejection of the null hypothesis much too frequently. Although appropriate models for analyzing longitudinal data have been available for quite some time, they have remained inaccessible due to cumbersome matrix manipulations. We implement Rao's (1959) one-sample polynomial growth curve model using the programming capability and matrix language of SAS, which involves testing the goodness-of-fit and calculation of confidence bands for polynomial growth curves fit to data at equally spaced time points. Confidence intervals for the parameters themselves are also computed. The method and program (presented in the Appendix) are illustrated with examples involving mandibular ramus height in 12 young male rhesus monkeys. The data set, which spans a 4 year period (yearly observations), is fit adequately by a quadratic equation. The data spanning a 2 year period (half-year observations) are fit adequately by the linear equation. These examples illustrate the considerable widening of confidence bands that occurs when polynomial equations having more terms than are needed to meet the goodness-of-fit requirement are considered.

Analysis of Variance↗