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Biomedical subjects

K Kimata

Publications and source records attributed to K Kimata.

At least 181 records · Page 10Linked to original sources
PubMed1984

Complex carbohydrates in cartilaginous and other tissues of cartilage matrix deficiency (cmd/cmd) mice as studied by light microscopic histochemical methods.

Mice homozygous for the autosomal recessive gene, cartilage matrix deficiency (cmd/cmd) are characterized by disproportionate dwarfism and cleft palate and are suffered from genetic failure in biosynthesis of cartilage-type proteoglycans. To investigate histochemical aspects of the defects in cartilaginous and other tissues of the mutant mice, complex carbohydrates in the tissues have been studied by methods of light microscopy. As a result of the present investigations, the intercellular matrix of cartilaginous tissues from the mutant was shown to exhibit weaker positive reactions for sulfated and acidic complex carbohydrates, as compared with the corresponding tissues from the control. In certain muscular and nervous tissues from the mutant, the reactions for sulfated complex carbohydrates tended to be weaker in intensity than those in the control. Furthermore, the dermal tissues of the mutant showed apparent mastocytosis. All these results indicate that there exist accessory effects in non-cartilaginous tissues in addition to the defects of cartilaginous tissues in the mutant mice.

Animals↗
PubMed1983

Presence of link protein in cartilage from cmd/cmd (cartilage matrix deficiency) mice.

Immunohistochemical and biochemical evidence that the cartilage from cmd/cmd mice, who have an autosomal recessive lethal mutation causing cartilage matrix deficiency, synthesizes link protein nearly at a normal level is provided. Since cartilage-characteristic proteoglycan is not synthesized in this mutant mouse (K. Kimata, H-J. Barrach, K. S. Brown, and J. P. Pennypacker (1981) J. Biol. Chem. 256, 6961-6968), link proteins are apparently not in conventional proteoglycan aggregate. However, the link proteins are functional and able to interact with exogenous cartilage-characteristic proteoglycan monomer and hyaluronic acid to form aggregates.

Animals↗
PubMed1983

The occurrence of three different proteoglycan species in chick embryo cartilage. Isolation and characterization of a second proteoglycan (PG-Lb) and its precursor form.

Three different molecular species of proteoglycan (designated PG-H, PG-Lb, and PG-Lt) have been isolated from chick embryo epiphyseal cartilage. PG-H is a major proteoglycan of the tissue and identical, or nearly identical, with so-called cartilage-characteristic proteoglycan previously described in mammalian and avian cartilages. The third proteoglycan, PG-Lt, differs from the other two in containing disulfide-bonded collagenous polypeptides (Noro, A., Kimata, K., Oike, Y., Shinomura, T., Maeda, N., Yano, S., Takahashi, N., and Suzuki, S. (1983) J. Biol. Chem. 258, 9323-9331). The second proteoglycan, PG-Lb, consists of a core protein with Mr congruent to 52,000 dermatan sulfate copolymer chains with glucuronic acid/iduronic acid residues. Upon chondroitinase ABC digestion, the proteoglycan yields a protein-enriched core fraction of Mr congruent to 43,000. Its amino acid composition, tryptic peptide profile, and immunochemical properties indicate that PG-Lb is distinctly different from PG-H and PG-Lt in core protein structure. PG-Lb shows no specific binding with hyaluronic acid. Pulse-chase experiments with [3H]serine indicate that PG-Lb is first synthesized as a precursor form (pro-PG-Lb) that can be distinguished from PG-Lb by the production of a core molecule of Mr congruent to 52,000 after chondroitinase ABC digestion. This core molecule is labeled when [2-3H]mannose is used as a precursor, suggesting that it contains a glycoprotein type oligosaccharide. Since the core molecule from pro-PG-Lb is significantly larger in molecular weight than that from PG-Lb, the conversion of pro-PG-Lb to PG-Lb should involve scission of the polypeptide or possibly removal of mannose-containing oligosaccharide chain.

Amino Acids↗
PubMed1983

Isolation and characterization of a third proteoglycan (PG-Lt) from chick embryo cartilage which contains disulfide-bonded collagenous polypeptide.

Chick embryo epiphyseal cartilage has been shown to contain three different proteoglycan species (PG-H, PG-Lb, and PG-Lt). This report is concerned with the purification and characterization of the third proteoglycan, PG-Lt. The proteoglycan can be separated from the other two by virtue of its low buoyant density in a CsCl density gradient and further purified by consecutive ion exchange and gel chromatography. The final preparation is composed of PG-Lt monomer and PG-Lt oligomer. The amino acid composition of PG-Lt is quite different from that of PG-H and PG-Lb and rather resembles that of collagens with respect to high content of glycine and high degrees of hydroxylation of proline and lysine. PG-Lt monomer is composed of disulfide-bonded subunits of Mr congruent to 120,000 and 190,000 as demonstrated by its gel electrophoretic behavior after reduction with 2-mercaptoethanol. The latter, but not the former, contains dermatan sulfate chains with glucuronic acid/iduronic acid residues and yields a protein-enriched core molecule of Mr congruent to 100,000 after digestion with chondroitinase ABC. Both of the protein subunits are completely digestible with bacterial collagenase. Immunofluorescence microscopic examination of cartilage tissues, using an antibody against PG-Lt, shows that this proteoglycan exists in both the cartilage matrix and perichondrial noncartilagenous region. When chondrocytes are plated onto tissue culture dishes, the antibody stains strands found on the cell surfaces and in the intercellular space of substrate-attached cell layers, suggesting that PG-Lt mediates cell-to-cell and cell-to-substrate contacts.

Amino Acids↗
PubMed1983

Increased synthesis of hyaluronic acid by mouse mammary carcinoma cell variants with high metastatic potential.

Variant subpopulations of FM3A mouse mammary carcinoma cells that have increased lung-colonizing potential were obtained previously by sequentially harvesting pulmonary metastases, culturing their cells in vitro, and reestablishing the metastases in vivo. In the present study, glycosaminoglycan production by the parental and variant cells was studied after metabolic labeling of cultures by [14C]glucosamine for 24 hr. Analysis of the products indicated that the rate of incorporation of the labeled precursor into hyaluronic acid in the high-metastatic variant cells was 27 to 54 times the rate in the low-metastatic variant cells and that the increase in hyaluronic acid synthesis was not associated with an increase in the rate of synthesis of other glycosaminoglycans. Both the cell layers and media of high-metastatic variants contained a much higher proportion of radioactivity in hyaluronic acid than did the corresponding fractions of low-metastatic cell lines. The results provide a basis for further investigation of the potential role of hyaluronic acid in control of the behavior of epithelial tumor cells during metastasis.

Animals↗
PubMed1982

A mapping technique for probing the structure of proteoglycan core molecules.

Our previous work showed that treatment of chick embryo cartilage proteoglycan (PG-H) with chondroitinase-AC II and keratanase yielded a protein-rich core fraction having enzymatically modified linkage oligosaccharides. The core sample has now been analyzed by tryptic peptide mapping, in which the isolated core sample contained in a single Coomassie blue-staining band from a dried slab gel is radioiodinated and treated with trypsin, and the resultant tryptic peptides are displayed two-dimensionally on a silica gel thin layer plate. The map thus obtained exhibited 22 major peptide spots, the resolution and location of which were reproducible. In order to identify regions of the core polypeptide from which the tryptic peptides are derived, PG-H was cleaved with clostripain under conditions that yield a hyaluronic acid-binding fragment with an apparent Mr = 150,000 and chondroitin sulfate-peptide clusters of smaller molecular sizes. Although the peptide maps of the two size classes of clostripain fragments differed significantly from each other, the patterns of spots, as a whole, were extensively similar to those observed with the intact core molecule. These results have provided additional evidence that PG-H has a single, nonvariable core protein structure. In addition, the technique used here will provide a versatile method for the identification of genetic types in this increasingly complex family of matrix macromolecules.

Animals↗
PubMed1982

Mechanisms for dissociating proteoglycan aggregates.

Preformed Cs2SO4 zonal gradients were used to purify aggregates from proteoglycan preparations derived from associative extracts of the Swarm rat chondrosarcoma. Zonal gradients were used in solvents with different concentrations of guanidine HCl and different solvent pH values to study the mechanisms for dissociating the aggregates. Aggregates are stable in concentrations of guanidine HCl up to 1.5 M at pH 6.6. At 2 M guanidine HCl, partial dissociation occurs over 20 h in which a link protein is completely dissociated for every monomer proteoglycan dissociated from the aggregate structure. This suggests that in this solvent disaggregation occurs concurrent with complete separation of link protein from monomer. At solvent pH 2.7 to 3.3 in ionic conditions which normally promote aggregation, dissociation occurs by a mechanism in which the link protein remains associated with monomer. Thus, link protein-monomer complexes dissociate as bimolecular units from hyaluronic acid; such complexes then exhibit physical properties indistinguishable from pure monomers. The link protein-monomer complexes reassociate with hyaluronic acid to form link-stabilized aggregates when the solvent pH is raised to pH 7, i.e. to associative conditions. The study provides additional evidence for the role that link protein-monomer interactions have in proteoglycan aggregate structures.

Animals↗
PubMed1982

Altered proteoglycan synthesis by epiphyseal cartilages in culture at low SO4(2-) concentration.

Proteoglycan Type H formed by incubation of chick embryo epiphyseal cartilages with [14C]glucosamine altered its buoyant density as the concentration of SO4(2-) in medium was lowered from 300 to 0 microM. At the highest SO4(2-) concentration, the labeled product was essentially identical with unlabeled proteoglycan Type H (i.e. the molecules already preformed in ovo) with respect to the buoyant density and hyaluronic acid-binding activity. With decreasing the SO4(2-) concentration in medium, the products showed a gradual decrease in the buoyant density with no apparent change in the hyaluronic acid-binding activity. This change of buoyant density was directly related to a decrease in average sulfation degree of the chondroitin sulfate moiety, which in turn reflected an increase in the ratio of low sulfated chondroitin chains (Chn) to high sulfated chondroitin sulfate chains (CS) per proteoglycan molecule. At intermediate SO4(2-) concentrations, 50 and 100 microM, the labeled products were of hybrid type containing both Chn and CS chains. Treatment of such hybrids with trypsin yielded, in each case, a mixture of CS- and Chn-carrying peptide fragments with an average buoyant density slightly lower than that of the parent proteoglycan. In no case were these fragments separated into two discrete groups corresponding to Chn-rich peptides and CS-rich peptides, respectively, suggesting that the two types of polysaccharide chain are rather evenly distributed along the core protein.

Animals↗
PubMed1982

Immunofluorescence localization of fibronectin in chondrosarcoma cartilage matrix.

In this study, we have compared the extracellular matrix components and the in vitro adhesion characteristics of normal rat epiphysial chondrocytes with those from the Swarm rat chondrosarcoma, which has many of the biochemical characteristics of normal cartilage. With the use of immunofluorescence techniques, tissue slices and chondrocytes in culture were tested for the presence of collagen types I and II, cartilage-characteristic proteoglycan, and fibronectin. Both normal and tumor matrix contained type II collagen and cartilage proteoglycan, but only the tumor matrix contained fibronectin. In culture, tumor-derived chondrocytes continued to accumulate fibronectin in their matrix, even after deposition of type II collagen and proteoglycans, while normal chondrocytes did not. When the attachment characteristics of both types of chondrocytes were compared, tumor chondrocytes required fibronectin for attachment, while normal chondrocytes used another attachment factor that had been identified previously as chondronectin. These studies suggest that, although biochemically similar to normal chondrocytes, tumor chondrocytes are no longer able to express the regulatory mechanisms for fibronectin accumulation.

Animals↗
PubMed1981

Absence of proteoglycan core protein in cartilage from the cmd/cmd (cartilage matrix deficiency) mouse.

Mice homozygous for the autosomal recessive gene, cartilage matrix deficiency (cmd/cmd), are characterized by disproportionate dwarfism and cleft palate. The collagen and proteoglycan of fetal limb cartilage was examined by biochemical and immunofluorescent techniques. While a normal amount of type II collagen was found, the amount of proteoglycan was reduced as determined by chemical analysis and incorporation of labeled precursors. Analyses of labeled proteoglycans by glycerol density gradient centrifugation under dissociative conditions and by gel filtration showed that the major high molecular weight proteoglycan characteristic of cartilage was absent, but smaller proteoglycans were present in normal amounts. Antibodies directed against proteoglycan core protein failed to stain the cmd/cmd cartilage while antibodies to type II collagen stained the cartilage without hyaluronidase pretreatment. Addition of beta-D-xyloside, an exogenous substrate for chondroitin sulfate synthesis, and direct assay for beta-D-xylosyltransferase activity indicated that cmd/cmd cartilage cells contained normal levels of the enzymes required for chondroitin sulfate synthesis. The data suggest that cmd/cmd is defective in the synthesis of the cartilage proteoglycan core protein.

Animals↗
PubMed1980

Structural analysis of chick-embryo cartilage proteoglycan by selective degradation with chondroitin lyases (chondroitinases) and endo-beta-D-galactosidase (keratanase).

Digestion of chick-embryo cartilage proteoglycan (type H) with chondroitin AC II lyase or keratanase, in the presence of EDTA, N-ethylmaleimide, phenylmethanesulphonyl fluoride and pepstatin, resulted in the removal of the bulk of the chondroitin sulphate or keratan sulphate chains respectively, without altering the protein portion of the macromolecule. An exhaustive treatment of the proteoglycan with chondroitin AC II lyase followed by digestion with keratanase yielded a core fraction having the enzymically modified linkage oligosaccharides. Zonal sedimentation of this core preparation on a sucrose gradient in 0.5% SDS resulted in a single narrow band with a sedimentation coefficient of 6S. In 4 M-guanidinium chloride, the core preparation showed a tendency to aggregate to multiple-molecular-weight forms which could dissociate in the presence of Triton X-100. The results indicate that the preponderance of glycosaminoglycans in the proteoglycan molecule is a main reason for both polydispersity and hydrophilicity of the proteoglycan preparation, and further suggest that the enzymic procedures could prove useful as a method to obtain new information about the structure and properties of proteoglycan core molecules.

Animals↗
PubMed1980

Proteinase activity in chondroitin lyase (chondroitinase) and endo-beta-D-galactosidase (keratanase) preparations and a method to abolish their proteolytic effect on proteoglycan.

Significant amounts of proteinase activity have been found in chondroitin ABC lyase (EC 4.2.2.4), chondroitin AC II lyase and endo-beta-D-galactosidase (keratanase) from commercial sources. It would appear, therefore, that certain earlier biochemical and histochemical studies, which employed these commercial enzyme preparations for their presumed ability to degrade only glycosaminoglycans, may require re-evaluation. A mixture of EDTA, N-ethylmaleimide, phenylmethanesulphonyl fluoride and pepstatin abolishes the effect of the contaminating proteinases on proteoglycan with less significant effect on the chondroitin lyase or keratanase activity.

Animals↗
PubMed1980

Characterization of fibronectin interactions with glycosaminoglycans and identification of active proteolytic fragments.

Fibronectin is a major cell-surface glycoprotein which has been reported to interact with glycosaminoglycans. A nitrocellulose filter-binding assay was developed to quantitate these interactions at physiological pH and ionic strength. Fibronectin isolated from chick embryo fibroblasts binds both hyaluronic acid and heparin; heparan sulfate is bound less efficiently, and chondroitin sulfate and glycopeptides are bound minimally. The binding of hyaluronic acid and heparin to fibronectin is saturable and reversible and occurs at separate binding sites. The binding of both molecules to fibronectin is not blocked by EDTA or by other glycosaminoglycans, and is only moderately inhibited by elevated ionic strength. Scatchard analyses revealed nonlinear, high affinity binding to fibronectin with a KD of approximately 10(-7) to 10(-8) M for these glycosaminoglycans. The affinity for heparin was utilized for the isolation of heparin-binding domains of fibronectin on heparin-agarose affinity columns. Heparin-binding proteolytic fragments with apparent molecular weights of 160,000 and 50,000 were isolated following hydrolysis of fibronectin by chymotrypsin or pronase, respectively. The possible involvement of such high affinity binding sites of fibronectin in the binding of glycosaminoglycans to the cell surface or in the organization of extracellular matrices is discussed.

Animals↗