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PubMed1992

The extracellular matrix of cartilage in the growth plate before and during calcification: changes in composition and degradation of type II collagen.

Calcification occurs in the extracellular matrix of the hypertrophic zone of the growth plate when the extracellular matrix volume is reduced to a minimum and alkaline phosphatase content is maximal. The present study shows that significant quantitative and qualitative changes occur in the composition and structure of macromolecules in the extracellular matrix before and during calcification in the proximal tibial growth plate of the bovine fetus. These were detected in part by using microchemical and microimmuno-chemical analyses of sequential transverse frozen sections at defined sites throughout the growth plate. Concentrations of matrix molecules in the extracellular matrix have not previously been determined biochemically. They were measured per unit matrix volume by using combined immunochemical/chemical-histomorphometric analyses. The concentrations within the extracellular matrix of the C-propeptide of type II collagen, aggregating proteoglycan (aggrecan), and hyaluronic acid all progressively increased in the maturing and hypertrophic zones, being maximal (or near maximal) at the time of initiation of mineralization. These results for proteoglycan are contrary to some earlier reports of a loss of proteoglycan are contrary to some earlier reports of a loss of proteoglycan prior to mineralization which measured the tissue content of proteoglycan rather than that present in the extracellular matrix, the volume of which is progressively reduced as the growth plate matures. The C-propeptide data provides a quantitative confirmation of previous immunohistochemical studies. Total collagen concentration (measured as hydroxyproline) in the extracellular matrix initially increased through the proliferating and maturing zones but then rapidly decreased in the hypertrophic zone.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1992

Fluoride binding by matrix proteins in rat mineralizing tissue.

Chronic fluoride exposure in vivo results in alterations in the formation of mineralizing tissues. One possible mechanism for the formation of fluorosed tooth enamel and bone is a binding of fluoride to matrix proteins, resulting in an alteration in their structure and function. Studies were designed to investigate fluoride binding to matrix proteins in vivo and their possible role in fluorosis. Rats were given either 0 or 100 parts/10(6) fluoride in drinking water for 6 weeks to allow the formation of fluorotic mineralizing tissues. The animals were killed by CO2 inhalation, and the enamel and bone were analysed for fluoride and calcium. Matrix binding by fluoride in enamel was determined after extraction of proteins from undemineralized matrix. In bone, the matrix was demineralized and F, Ca and P were determined in both ashed and unashed samples. The studies showed ionic binding of fluoride to the matrix in both enamel and bone, possibly associated with calcium binding by the matrix. There was no difference in the amount of matrix-bound fluoride in control as compared to fluorosed bone or maturation-stage enamel. This indicates that although matrix proteins can bind fluoride, it is not likely that this mechanism is important in the formation of fluorosed mineralizing tissues.

Animals↗
PubMed1990

Interaction of lipoprotein lipase with subendothelial extracellular matrix.

We have analyzed the binding of lipoprotein lipase (LPL) to the subendothelial extracellular matrix produced by cultured endothelial cells. Binding was linear up to a concentration of 0.5 microgram/ml (10 nM) enzyme used in this study, and equilibrium was achieved after 2 h of incubation with bovine 125I-LPL at 4 degrees C. Heparin and heparan sulfate effectively inhibited the binding of LPL to extracellular-matrix-coated plates; chondroitin sulfate had no effect, while high concentrations of dermatan sulfate or keratan sulfate inhibited binding of LPL to extracellular matrix by only 40%. Basic fibroblast growth factor (bFGF) did not affect LPL binding, while antithrombin-III (AT-III) caused up to a 50% inhibition of enzyme binding to extracellular matrix. alpha-Thrombin. 5.10(-6) M, and its esterolytically inactive derivative, DIP-alpha-thrombin, effectively inhibited binding of LPL to extracellular-matrix-coated plates. alpha-Thrombin was also able to release the extracellular-matrix-bound LPL in an active form. Extracellular-matrix-bound LPL detached into medium containing triolein emulsion and/or serum, and was catalytically active after being released. Extracellular-matrix-bound LPL lost 30% of its activity following incubation at 37 degrees C for 4 h. in contrast to soluble LPL which lost 75% of its activity. It is plausible to conclude from these data that in vivo the subendothelial basement membrane, similarly to extracellular matrix, sequesters and stabilizers LPL secreted into the subendothelial space by non-endothelial cells, and thus may play an important role in determining the route of LPL from its site of synthesis to its site of action.

Animals↗
PubMed1985

The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices.

Artificial extracellular matrices composed of collagen, glycosaminoglycans (GAG), proteoglycans (PG), plasma fibronectin (FN), and a hyaluronate-binding protein (HABP) have been prepared that morphologically resemble embryonic extracellular matrices in vivo at the light and electron microscope level. The effect of each of the above matrix molecules on the structure and "self-assembly" of these artificial matrices was delineated. (1) Matrix components assembled in vitro morphologically resemble their counterparts in vivo, for the most part. Scanning and transmission electron microscopy indicate that under our assembly and fixation conditions, collagen forms striated fibrils that are 125 nm in diameter, FN forms 30- to 60-nm granules, chondroitin sulfate proteoglycan (CSPG) forms 27- to 37-nm granules, chondroitin sulfate (CS) assembles into 100- to 250-nm spheres, and hyaluronate (HA) appears either as granular mats when fixed with cetylpyridinium chloride (CPC) or as 1.5- to 3-nm microfibrils when preserved with ruthenium red plus tannic acid. These molecules are known to assume the same configurations in embryonic matrices when the same preservation techniques are used with the exception of FN, which generally forms fibrillar arrays. (2) Addition of various matrix molecules can radically change the appearance of the collage gels. HA greatly expands the volume of the gel and increases the space between collagen fibrils. CSPG at low concentrations (less than 1 mg/ml) and CS at high concentrations (greater than 20 mg/ml) bundle the collagen fibrils into twisted ropes. (3) A variety of assays were used to examine binding between various matrix components and retention of these components in the hydrated collagen lattices. These assays included solid-phase binding assays, negative staining of spread mixtures of matrix components, cryostat sections of unfixed mixtures of matrix components, and retention of radiolabeled matrix molecules in fixed and washed gels. A number of these binding interactions may play a role in the assembly and stabilization of the matrix. (a) HA, CSPG, and FN bind to collagen. CS appears to only weakly bind to collagen, if at all. (b) FN promotes the increased retention of HA, CSPG, and to a very small degrees, CS, in collagen gels. Conversely, the GAG increase the retention of 3H-FN in the gels. Furthermore, FN binds to HA, CS, and CSPG as demonstrated by solid surface binding assays and morphological criteria. The increased retention of GAG and CSPG by the addition of FN may be due to both stabilization of binding to the collagen and trapping of matrix complexes within the gel. (c) HA binds to both CS and CSPG.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Electroelution for purification of influenza A matrix protein for use in immunoassay.

A new preparative method for isolation of matrix protein from type A influenza virus was developed. Commercially available whole virus or split virus vaccines were lysed, and the soluble proteins separated by electrophoresis on polyacrylamide gel. The matrix protein was located on the gel by precipitation with KCl, and recovered by electroelution. The method was technically simple and required little direct supervision during the two-step recovery process. Yields of A matrix were consistently high, averaging 68.1% in five trials with A/Brazil/X-71. The method was also successful with other A viruses, although not with influenza B virus. Isolated A matrix had less than 0.5% contamination by hemagglutinin or nucleoprotein, as determined by immunoblotting and ELISA. Matrix protein was immunoreactive in Western blots and was detectable in concentrations as low as 1 ng/ml with ELISA. The isolated matrix provided a suitable standard for detection of matrix protein in nasal washes from patients with influenza A virus infection, and could also be used to detect anti-matrix antibodies, including monoclonal antibodies in tissue culture supernatants. The advantages of electroelution for separation of matrix protein compared to other methods were its technical simplicity, applicability to formalin-fixed influenza virus in commercially available vaccines, its consistently high yield, and its very high level of purification.

Antibodies, Monoclonal↗
PubMed1992

Stimulation of matrix vesicle enzyme activity in osteoblast-like cells by 1,25(OH)2D3 and transforming growth factor beta (TGF beta).

After demonstrating the presence of matrix vesicles in three osteosarcoma cell lines, MG-63, ROS 17/2.8 and MC-3T3-E1, we sought to determine whether two major enzymes localized to matrix vesicles, alkaline phosphatase and phospholipase A2, could be regulated by 1,25(OH)2D3 and/or TGF beta. Intravesicular calcification is probably dependent on these two enzymes. Alkaline phosphatase is essential for hydrolysis of phosphate-containing substrates and phospholipase A2 hydrolyzes diacylphosphatides in a calcium-mediated manner at lipid-aqueous interfaces leading to changes in membrane fluidity and possibly breakdown of the matrix vesicle. The 1,25(OH)2D3 induced increase of alkaline phosphatase in bone cells is localized to the matrix vesicle. TGF beta also increased alkaline phosphatase activity in two of the cell lines, MG-63 and ROS 17/2.8 but to a greater degree than 1,25(OH)2D3. Matrix vesicle alkaline phosphatase activity exhibited a greater response than that in the plasma membrane. TGF beta increased phospholipase A2 activity in both matrix vesicles and plasma membranes, therefore, no targeting was observed with respect to this enzyme. When TGF beta was combined with 1,25(OH)2D3, 1,25(OH)2D3 had no effect on phospholipase A2 and did not interfere with TGF beta stimulation of phospholipase A2 activity. When 1,25(OH)2D3 and TGF beta were combined, a tremendous synergy was observed in alkaline phosphatase specific activity in both plasma membranes and matrix vesicles with targeting to matrix vesicles. Therefore, TGF beta not only plays an important role in matrix formation and differentiation, but works in conjunction with 1,25(OH)2D3 to greatly potentiate the effects seen with 1,25(OH)2D3 alone.

Alkaline Phosphatase↗
PubMed1986

Extracellular matrix proteoglycans and cell-substratum adhesion of human endothelial cells: the effect of methyl beta-D-xylopyranoside.

The influence of methyl beta-D-xylopyranoside on human endothelial cell proteoglycans isolated from the medium and extracellular matrix was investigated. Confluent cultures of human endothelial cells incorporate significant amounts of heparan sulfate (78%), chondroitin sulfate (10%), and dermatan sulfate (12%) into the extracellular matrix. Chondroitin sulfate (35%) and dermatan sulfate (37%) were the major glycosaminoglycans present in the medium. In the presence of methyl beta-D-xylopyranoside, incorporation of labeled proteoglycans into extracellular matrix was diminished by approximately 70%. Heparan sulfate comprised the major proteoglycan present in extracellular matrix (89%) in cells grown in the presence of methyl beta-D-xylopyranoside. In contrast to the incorporation of proteoglycan into extracellular matrix, methyl beta-D-xylopyranoside stimulated the secretion of labeled glycosaminoglycan chains into the medium 2.5-fold. In the presence of methyl beta-D-xylopyranoside, secretion of chondroitin sulfate into the medium was markedly stimulated, with a slight increase in secretion of heparan sulfate. Chondroitin sulfate (62%) and heparan sulfate (34%) were the major labeled glycosaminoglycans present in medium from methyl beta-D-xylopyranoside-treated cultures. The effect of methyl beta-D-xylopyranoside on cell adhesion and detachment was investigated. Cell detachment from extracellular matrix depleted of proteoglycan was significantly faster than detachment from normal matrix. Conversely, human endothelial cells adhered faster to normal matrix than to matrix depleted of proteoglycan.

Cell Adhesion↗
PubMed1989

Acellular vascular matrix: a natural endothelial cell substrate.

A preliminary assessment was made of the acellular vascular matrix graft as a substrate for endothelial cell seeding, with respect to surface pretreatment (none versus fibronectin and/or serum) and presence of exogenous growth factor. Arteries were harvested from greyhounds and exposed to a sequential detergent extraction process to produce the acellular vascular matrix. Human umbilical vein endothelial cells were grown in tissue culture, harvested in first passage, then seeded at 10(5) cells/cm2 on sections of acellular vascular matrix and on gel-coated polystyrene positive controls. After 18 hour incubation, endothelial cell-seeded acellular matrices were fixed and processed for histologic and planimetric analysis; control wells were fixed and endothelial cells were counted by planimetry. Pretreatment of the acellular vascular matrix was found to have no effect on the percentage of endothelial cell coverage of the matrix. There was significantly better endothelial cell coverage of the acellular matrix than on matched gel-treated polystyrene control wells. Withdrawal of growth factor resulted in a significant reduction in endothelial cell coverage for all acellular vascular matrix groups. Growth factor withdrawal also significantly reduced attachment of endothelial cells on gel-treated polystyrene. Cell surface area was significantly smaller when growth factor was withdrawn from all groups except from the acellular vascular matrix without pretreatment. We conclude that: (1) the acellular vascular matrix is conductive to endothelial cell adherence and spreading even without pretreatment; and (2) sudden withdrawal of exogenous growth factor may impair early coverage of substrates by endothelial cells due to an effect on their adherence or spreading.

Animals↗
PubMed1990

Progressive changes in the protein composition of the nuclear matrix during rat osteoblast differentiation.

Primary cultures of fetal rat calvarial osteoblasts undergo a developmental sequence with respect to the temporal expression of genes encoding osteoblast phenotypic markers. Based on previous suggestions that gene-nuclear matrix associations are involved in regulating cell- and tissue-specific gene expression, we investigated the protein composition of the nuclear matrix during this developmental sequence by using high-resolution two-dimensional gel electrophoresis. The nuclear matrix was isolated at times during a 4-week culture period that represent the three principal osteoblast phenotypic stages: proliferation, extracellular matrix (ECM) maturation, and mineralization. The most dramatic changes in the nuclear matrix protein patterns occurred during transitions from the proliferation to the ECM maturation stage and from ECM maturation to the mineralization period, with only minor variations in the profiles within each period. These stage-specific changes, corresponding to the major transition points in gene expression, indicate that the nuclear matrix proteins reflect the progressive differentiation of the bone cell phenotype. Subcultivation of primary cells delays mineralization, and a corresponding delay was observed for the nuclear matrix protein patterns. Thus, the sequential changes in protein composition of the nuclear matrix that occur during osteoblast differentiation represent distinct stage-specific markers for maturation of the osteoblast to an osteocytic cell in a bone-like mineralized ECM. These changes are consistent with a functional involvement of the nuclear matrix in mediating modifications of developmental gene expression.

Animals↗
PubMed1986

Schwann cell myelination: induction by exogenous basement membrane-like extracellular matrix.

Exposing rat Schwann cells co-cultured with nerve cells to a reconstituted basement membrane induced the formation of myelin segments by Schwann cells. This occurred in a serum-free culture medium in which, in the absence of this matrix, Schwann cells proliferate but fail to differentiate. This reconstituted basement membrane was prepared from solubilized extracellular matrix proteins synthesized by a basement membrane-producing murine tumor. The major constituents of this reconstituted matrix are collagen type IV, laminin, heparan sulfate proteoglycan, entactin, and nidogen. The matrix also elicited striking morphological changes in Schwann cells, inducing them to spread longitudinally along the nerve fibers (a necessary early step in the process of ensheathment of nerve fibers). Several observations indicated that the effect of the matrix was exerted directly on Schwann cells and not indirectly through an effect on nerve cells. First, the matrix-induced cell spreading occurred only in areas in which Schwann cells directly contacted the matrix; Schwann cells that were associated with the same nerve fibers but that did not themselves directly contact the matrix did not exhibit spreading. Second, the matrix-induced alteration in Schwann cell morphology was observed in cultures in which the nerve cells were removed. These results provide direct evidence that basement membrane contact induces normal Schwann cell differentiation, and support the idea that Schwann cell differentiation in vivo may be regulated by the appearance of the basement membrane, which normally envelops terminally differentiating Schwann cells.

Animals↗
PubMed1987

Perturbation of human endothelial cells by thrombin or PMA changes the reactivity of their extracellular matrix towards platelets.

In this study we have examined the influence of perturbation of endothelial cells on the amounts of fibronectin and von Willebrand factor in their extracellular matrix and the consequences of a changed composition of the matrix on platelet adhesion. For this purpose, we have used an in vitro perfusion system with which we can investigate the interactions of platelets in flowing blood with cultured endothelial cells and their extracellular matrix (Sakariassen, K. S., P. A. M. M. Aarts, P. G. de Groot, W. P. M. Houdgk, and J. J. Sixma, 1983, J. Lab. Clin Med. 102:522-535). Treatment of endothelial cells with 0.1-1.0 U/ml thrombin for 2 h increased the reactivity of the extracellular matrix, isolated after the thrombin treatment, towards platelets by approximately 50%. The increased reactivity did not depend on de novo protein synthesis but was inhibited by 3-deazaadenosine, an inhibitor of phospholipid methylation, which also inhibits the stimulus-induced instantaneous release of von Willebrand factor from endothelial cells. However, no changes in the amounts of von Willebrand factor and fibronectin in the matrix were detected. Thrombin may change the organization of the matrix proteins, not the composition. When endothelial cells were perturbed with the phorbol ester PMA or thrombin for 3 d, the adhesion of platelets to the extracellular matrix of treated cells was strongly impaired. This impairment coincided with a decrease in the amounts of von Willebrand factor and fibronectin present in the matrix. These results indicate that, after perturbation, endothelial cells regulate the composition of their matrix, and that this regulation has consequences for the adhesion of platelets.

Blood Platelets↗
PubMed1988

Metabolism of receptor-bound and matrix-bound basic fibroblast growth factor by bovine capillary endothelial cells.

Bovine capillary endothelial (BCE) cells were incubated at 4 degrees C with 5 ng/ml 125I-basic fibroblast growth factor (bFGF) to equilibrate 125I-bFGF with high affinity cell surface receptors and low affinity matrix binding sites. 67% of the added 125I-bFGF bound to the matrix and 7% bound to receptors. The fate of bound bFGF was followed after cells were incubated in bFGF-free medium and were shifted to 37 degrees C to restore cell metabolism. 125I-bFGF bound to receptors decreased rapidly while the amount of 125I-bFGF bound to matrix was reduced more slowly. The rapid decrease in receptor-bound 125I-bFGF appeared to be due to a down-regulation of bFGF receptors; cells that had been treated for 5 h with bFGF had 60% fewer high affinity receptors than untreated cells. Despite the initial high level of 125I-bFGF binding to matrix, most of this 125I-bFGF was mobilized and metabolized by the cells. 125I-bFGF was internalized by the cells at 37 degrees C, leading to a constant accumulation of 125I-bFGF within the cell. Internalized bFGF was rapidly cleaved from an 18-kD form to a 16-kD form. The 16-kD form was more slowly degraded with a half-life of approximately 8 h. Degradation of internalized 125I-bFGF was inhibited by chloroquine, suggesting that the digestion occurred in a lysosomal compartment. The role of matrix binding sites in the internalization process was investigated. Binding to matrix sites seemed not to be directly involved in the internalization process, since addition of heparin at a concentration that blocked 95% of the binding to matrix had no effect on the initial rate of internalization of bFGF. BCE cells also released a substance that competed for the binding of bFGF to matrix but not to receptors. This substance bound to DEAE-cellulose and was sensitive to heparinase treatment, suggesting that it was a heparinlike molecule. Thus, heparinlike molecules produced by BCE cells can modulate the cellular interaction with bFGF. Matrix-associated heparinlike molecules bind bFGF which can later be metabolized by the cell, and secreted heparinlike molecules release bFGF from matrices.

Animals↗
PubMed1989

The fibronectin receptor is organized by extracellular matrix fibronectin: implications for oncogenic transformation and for cell recognition of fibronectin matrices.

Cells interact with extracellular fibronectin (FN) via adhesive fibronectin receptors (FNRs) that are members of the very late antigens (VLAs) subgroup of the integrin family. In stationary fibroblasts, the FNR is highly organized and distributed identically to extracellular FN fibrils. However, in highly migratory neural crest cells and embryonic somatic fibroblasts, this organization is lost and the FNR appears diffuse. Similarly, oncogenic transformation typically leads to disorganization of the FN receptor and loss of matrix FN. Two models can account for these observations. First, the FN matrix may organize the FN receptor at extracellular matrix contacts on the cell surface. Motile cells not depositing FN matrices thus lack organized receptors. Alternatively, as the FNR is required for optimal FN matrix assembly, (McDonald, J. A., B. J. Quade, T. J. Broekelmann, R. LaChance, K. Forseman, K. Hasegawa, and S. Akiyama. 1987. J. Biol. Chem. 272:2957-2967; Roman, J. R. M. LaChance, T. J. Broekelmann, C. J. R. Kennedy, E. A. Wayner, W. G. Carter, J. A. McDonald. 1989. J. Cell Biol. 108:2529-2543) and has putative cytoskeletal links, it could be organized from within the cell helping to position newly forming FN fibrils. To study this question, we developed peptide antibodies specifically recognizing the alpha 5 subunit of the FNR. Using these antibodies, we examined the organization of FN and of the FNR in normal, matrix assembly inhibited, and SV40-transformed human fibroblasts. On FN-coated substrates, the FNR is found in focal contacts rather than diffusely on the basal cell surface, suggesting FNR interaction with intracellular components. However, when FN fibrils are deposited, the FNR is co-distributed with these fibrils. Preventing FN matrix assembly prevents organization of the FNR. Moreover, when fibroblasts with well established FN matrices and co-distributed FNR are incubated briefly with monoclonal antibodies that block FNR binding to FN, the FNR is no longer co-distributed with the FN matrix. Thus, the FN receptor is organized in fibrils on the cell surface in response to extracellular FN. Because exogenous FN restores a FN matrix and receptor organization to SV40-transformed cells, the diffuse FN receptor phenotype appears to be related to loss of the FN matrix rather than to impaired FNR function. These results explain diffusely distributed FNRs in migratory neural crest and embryonic fibroblasts lacking well organized FN matrices and emphasize the existence of separate but related systems controlling FN deposition and recognition by receptor-armed cells.

Cell Adhesion↗
PubMed1990

An instructive role for the interstitial matrix in tissue patterning: tissue segregation and intercellular invasion.

Intercellular invasion is the intrusion of the cells of one tissue into space occupied by a second tissue. The alternative situation to invasion, one characteristic of most coherent tissues, is segregation, with identifiable boundaries existing between contiguous tissues. The interfaces between mesenchymal and myocardial tissues in the developing avian heart show a profoundly different character in different regions of the heart: the interface between epicardial mesenchyme and heart wall myocardium is planar, without intermingling of the two cell types, whereas the interface between endocardial cushion mesenchyme and myocardium is diffuse, with extensive invasion of both tissue types across the border to produce intermingling of the two tissues. Thus, invasion and tissue segregation coexist in different regions of the mesenchyme-myocardium contact zone. Investigation of the involvement of the interstitial matrix in invasion and segregation has been conducted by maintaining the two tissues in mutual contact in organ culture. Investigation of the mechanisms by which the two cell types sort out in randomized chimeric tissue reaggregates has provided insight into the conditions for tissue segregation. We have modeled invasion in organ culture by fusing aggregates of myocardial cells with aggregates of cardiac mesenchymal cells. Cells of both tissues invaded the partner aggregate during a period of 1-3 d of coculture. Both invasion and segregation in the aggregates appear to depend on the presence or absence of a fibronectin-rich interstitial matrix elaborated by the cardiac mesenchyme. During sorting, the matrix appears selectively in regions occupied by the mesenchyme. Under conditions of culture that are nonpermissive for matrix deposition, sorting fails to occur. Stimulation of matrix deposition by addition of serum, transforming growth factor beta, or isolated matrix itself is accompanied by sorting out of the two tissues. Sorting out is blocked reversibly by inclusion of the fibronectin adhesion site peptide, GRGDSP. Invasion of fused aggregates is preceded by a redistribution of the fibronectin-containing matrix of the mesenchymal aggregate such that matrix-poor regions come to occupy the interface with the myocardial partner aggregate. The invasion that ensues involves mesenchymal cells emigrating from, and myocardial cells intruding into, matrix-poor regions of the mesenchymal aggregate.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Subendothelial proteins and platelet adhesion. von Willebrand factor and fibronectin, not thrombospondin, are involved in platelet adhesion to extracellular matrix of human vascular endothelial cells.

Endothelial cell matrix contained von Willebrand factor (VWF), fibronectin, and thrombospondin. The role of these proteins in the adhesion of platelets was investigated by preincubation of the matrix with specific antibodies and subsequent perfusion with human blood. When perfusions were performed with platelets in a human albumin solution (HAS) platelet adhesion was similar to that with normal plasma, indicating that proteins in the matrix can fully support adhesion. Preincubation of the matrix with a monoclonal antibody to VWF and perfusion with HAS showed a nearly complete inhibition of platelet adhesion at 1300 s-1, indicating a role for matrix-bound VWF at high shear rates and no requirement for VWF in plasma. Preincubation of the matrix with antihuman fibronectin F(ab')2 showed a slight inhibition of adhesion. The same result was obtained with perfusions with fibronectin-free plasma, and an untreated matrix. Preincubation with antifibronectin F(ab')2 and perfusion with fibronectin-free plasma showed a significant inhibition of platelet adhesion at all shear rates. These results indicate that fibronectin is required for adhesion at all shear rates. Preincubation of the matrix with different antibodies against human platelet thrombospondin showed no inhibition of platelet adhesion at all wall shear rates. Thrombospondin in the matrix is evidently not required for platelet adhesion.

Antibodies, Monoclonal↗
PubMed1991

The role of extracellular matrix components in dentin mineralization.

The extracellular matrix of dentin consists of mineral (hydroxyapatite), collagen, and several noncollagenous matrix proteins. These noncollagenous matrix proteins may be mediators of cell-matrix interactions, matrix maturation, and mineralization. This review describes the current knowledge of the chemistry of mineral crystal formation in dentin with special emphasis on the roles of the dentin matrix proteins. The functions of some of these matrix proteins in the mineralization process have been deduced based on in vitro studies. Functions for others have been postulated based on analogy with some of the bone matrix proteins. Evidence suggests that several of these matrix proteins may have multiple effects on nucleation, crystal growth, and orientation of dentin hydroxyapatite.

Crystallization↗
PubMed1991

Ultrastructural localization of nuclear matrix proteins in HeLa cells using silver-enhanced ultra-small gold probes.

We describe a method for immunogold staining of nuclear matrix proteins using ultra-small gold particles. The nuclear matrix of HeLa cells is obtained by two fractionation steps: (a) cell permeabilization with Triton X-100 to isolate the cytoskeleton, and (b) nuclease digestion followed by an incubation in 0.25 M ammonium sulfate to isolate the nuclear matrix. To prevent redistribution of internal matrix proteins during nuclear matrix preparation, pre-fixation with 0.1% acrolein was performed. Under this condition up to 80% of protein and 90% of DNA and RNA could be removed on nuclear matrix isolation, without redistribution of internal nuclear matrix proteins. For immunogold labeling, 1-nm gold probes appeared to be required to obtain optimal penetration into the nucleus. These particles can be visualized after silver enhancement. After gold labeling the matrices are stained, embedded in Epon, and ultra-thin sections are prepared for examination in the electron microscope. The applicability of this method is examplified by the localization of a 125 KD internal nuclear matrix protein and the lamins A and C in nuclear matrix preparations of HeLa cells.

Ammonium Sulfate↗
PubMed1990

Cell-free interaction of the estrogen receptor with mouse uterine nuclear matrix: evidence of saturability, specificity, and resistance to KCl extraction.

An integral part of the mechanism of estrogen action is the interaction of estrogen receptor (ER) complexes with specific nuclear acceptor sites to effect alterations in genomic expression. The localization of nuclear acceptor sites has been in question, but an increasing body of indirect evidence implicates the nuclear matrix. To assess the binding characteristics of [3H]estradiol-receptor complexes (3HER) to nuclear matrix, ER from ovariectomized mice was partially purified by ammonium sulfate precipitation and incubated under cell-free conditions with mouse uterine nuclear matrix at 4 C. The binding capacity of the nuclear matrix was determined to be 36.4 +/- 5.7 fmol/100 micrograms DNA, with a Kd of 0.23 +/- 0.03 nM. Binding to nuclear matrix sites was specific, as determined by the ability of increasing concentrations of unlabeled ER complexes to inhibit binding of 3HER. Spleen, used as a nontarget tissue, contained fewer binding sites (n = 4.07 fmol/100 micrograms DNA) than matrix from liver (n = 14.2). The binding affinity was the same in all three tissues. Injection of animals with estradiol before death was associated with loss of assayable nuclear matrix binding sites, implying occupancy of sites by ER in vivo. Unbound receptor (R) also demonstrated the ability to bind to uterine matrix (n = 40.2 +/- 2.7 fmol/100 micrograms DNA; Kd = 0.26 +/- 0.05 nM) as well as to competitively inhibit the binding of 3HER complexes. However, heat-inactivated receptor displayed no binding or competing activity, nor did the progesterone receptor. The two forms of the receptor can be functionally distinguished by extraction with 0.6 M KCl; 43% of ER, but no R, were resistant to KCl extraction. These results indicate that nuclear acceptor sites are associated with the nuclear matrix. Furthermore, these sites demonstrate the criteria expected of specific binding sites, i.e. high affinity, limited capacity, hormone receptor, and relative tissue specificity. The apparent association of uncomplexed receptor to nuclear acceptor sites may explain the uterine tissue nuclear localization of ER in the absence of hormone.

Animals↗