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Biomedical subjects

J Kishi

Publications and source records attributed to J Kishi.

At least 37 records · Page 2Linked to original sources
PubMed1990

Tissue inhibitor of metalloproteinases from human bone marrow stromal cell line KM 102 has erythroid-potentiating activity, suggesting its possibly bifunctional role in the hematopoietic microenvironment.

In this study, we demonstrated that tissue inhibitor of metalloproteinases (TIMP) produced by human bone marrow stromal cell line KM-102 had erythroid-potentiating activity (EPA) which stimulates the proliferation of erythroid progenitor cells. We, then, propose a scheme for the bifunctional role of TIMP/EPA in hematopoietic microenvironment, that is, the maintenance of the integrity of bone marrow matrix and the proliferation of erythroid progenitor cells proceeding on the matrix.

Bone Marrow↗
PubMed1989

Characteristics of collagenase in experimental periapical granulomas from the teeth of dogs.

Periapical granulomas were induced, with a success rate of about 60% (66 granulomas produced out of 109 roots treated) in mandibular premolars. The average wet weight of the granulomas, 46.1 +/- 34.5 mg (mean +/- SD, n = 22), was sufficient to allow individual specimens to be used for most of the biochemical analyses. High collagenase activity was extracted directly from the granulomas with 4 M urea solution. The enzyme was a typical animal collagenase (EC 3.4.24.7) which clove native collagen molecules into three-quarter (alpha A) and one-quarter (alpha B) length fragments. The collagenase was activated by 1 mM p-aminophenylmercuric acetate. This activated enzyme broke down collagen I rather than collagen III preferentially, which is similar to the activity of human polymorphonuclear leucocyte collagenase. The molecular weights of the latent and activated collagenases were 67 and 49 K, respectively.

Animals↗
PubMed1989

A sandwich enzyme immunoassay for collagenase inhibitor using monoclonal antibodies.

A sandwich enzyme immunoassay for collagenase inhibitor was set up with a pair of monoclonal antibodies prepared against bovine dental pulp collagenase inhibitor. Two different combinations of the antibodies were found to be applicable to the immunoassay; one was for the determination of both bovine and human collagenase inhibitors, but the other was only for the bovine one. Minimum sensitivity was 1 pg/tube (3 pg/ml) for bovine collagenase inhibitor and 1.5 pg/tube (5 pg/ml) for human inhibitor. The levels of collagenase inhibitor in several normal human body fluids were measured by the immunoassay, and the values obtained were as follows: serum, 183 +/- 30 ng/ml (mean +/- SD); platelet-poor plasma, 68 +/- 13; cerebrospinal fluid, 60 +/- 13; amniotic fluid, 1,780 +/- 969; tear fluid, 418 +/- 145; mixed saliva, 316 +/- 166; urine, 7 +/- 8.

Animals↗
PubMed1989

Synthesis of latent collagenase and collagenase inhibitor by bovine aortic medial explants and cultured medial smooth muscle cells.

Bovine aortic medial tissue and medial smooth muscle cells were demonstrated for the first time to synthesize a latent collagenase together with collagenase inhibitor in culture. Molecular weights of the latent collagenase and its inhibitor derived from aortic medial tissue explant were estimated to be about 52 K by gel filtration and 26.5 K by electrophoresis, respectively. Activated aortic collagenases cleaved type I collagen in solution into 3/4 (alpha A) and 1/4 (alpha B) length cleavage fragments and were inhibited by EDTA, o-phenanthroline, dithiothreitol, bovine serum, and highly purified dental pulp and aortic collagenase inhibitors. The aortic inhibitors showed inhibitory activity against all the animal collagenases tested, except for bacterial collagenase. Double-immunodiffusion analysis using a monospecific antiserum prepared against dental pulp inhibitor showed that the aortic inhibitors are immunologically identical to the pulp inhibitor. Using the same antiserum, we found immunoreactive collagenase inhibitor protein to be localized along the collagen fibers between elastic membranes in aortic medial tissue.

Animals↗
PubMed1988

Inactivation of tissue inhibitor of metalloproteinases by neutrophil elastase and other serine proteinases.

Tissue inhibitor of metalloproteinases (TIMP) from cultured bovine dental pulp inhibits human rheumatoid synovial matrix metalloproteinase 3 (MMP-3) with a stoichiometry of 1:1 on a molar basis. Among the serine proteinases examined, human neutrophil elastase, trypsin and alpha-chymotrypsin destroyed the inhibitory activity of TIMP against MMP-3 by degrading the inhibitor molecule into small fragments. In contrast, the inhibitory activity of TIMP was not significantly reduced by the actions of cathepsin G, pancreatic elastase and plasmin. These data indicate that neutrophils which infiltrate tissues in various inflammatory conditions may play an important role in regulating TIMP activity in vivo through the action of neutrophil elastase.

Animals↗
PubMed1988

The role of interstitial collagens in cleft formation of mouse embryonic submandibular gland during initial branching.

An interstitial collagenase was purified from the explant medium of bovine dental pulp and was shown to degrade collagens I and III but not IV and V. The enzyme halted cleft initiation in the epithelium of 12-day mouse embryonic submandibular glands in vitro, indicating the active involvement of interstitial collagens in the branching morphogenesis. Transmission electron microscopic observation of the intact 12-day gland without any clefts showed the scattered localization of a few collagen fibrils at the epithelial-mesenchymal interface of the bulb and also revealed the presence of numerous microfibrils around the stalk. Collagen bundles were regularly seen close to the wavy basal lamina at the bottom of clefts of the intact 13-day gland and 12-day gland cultured for 17 h under normal conditions. Mesenchymal cells were found in the clefts together with the frequent localization of peripheral nerve fibres and capillary endothelial cells. The collagen bundles were more often observed in the 12-day gland cultured in the presence of bovine dental pulp collagenase inhibitor, which had been shown to enhance cleft formation. In contrast, collagen fibrils were rarely found at the epithelial-mesenchymal interface of the 12-day gland cultured in the presence of Clostridial or bovine dental pulp collagenase. The findings indicated that the formation of interstitial collagen bundles is essential to form clefts in the epithelium both in vivo and in vitro.

Animals↗
PubMed1988

Accumulation of collagen III at the cleft points of developing mouse submandibular epithelium.

The distribution of collagens I, III, IV and V was studied by immunoperoxidase staining of early developing mouse submandibular glands. Collagen I was always present in the extracellular matrices of the mesenchyme and at the epithelial-mesenchymal interfaces of the 12-day gland with no clefts and of the 13-day gland with a few definite clefts. Collagen III was found in a similar fashion to that of collagen I in the mesenchyme, but the distribution at the epithelial-mesenchymal interfaces was very different. In the mid 12-day gland with a round lobule, collagen III was distributed at every slightly indented site of basal epithelial surfaces. At the late 12-day stage, a few initial signs of cleft appeared on the surface, at which accumulation of collagen III became evident. Intense immunoreaction of collagen III in the early 13-day gland was seen at the bottom of every narrow cleft. No specific accumulation of collagens IV and V was observed in clefts of the late 12-day and early 13-day glands. Staining of collagen III in the 12-day gland cultured for 10 h in the presence of bovine dental pulp collagenase inhibitor, which has been shown to stimulate cleft initiation, was very prominent at the bottom of every narrow cleft. These observations suggest that collagen III works as a key substance for either in vitro or in vivo cleft initiation of the mouse embryonic submandibular epithelium.

Animals↗
PubMed1987

Monoclonal antibodies to bovine collagenase inhibitor.

Hybridoma antibodies against bovine collagenase inhibitor were produced by fusion of myeloma cells NS-1 (P3-NS1-1) with spleen cells from mice hyperimmunized with collagenase inhibitor purified from the explant medium of bovine dental pulps. Hybridomas positive by an enzyme-linked immunosorbent assay (ELISA) for bovine collagenase inhibitor were cloned by the dilution method. Seventeen hybridomas producing antibodies were isolated, four of which also recognized purified human collagenase inhibitor in the ELISA. Using a monoclonal antibody-Sepharose affinity column, we easily purified both bovine and human collagenase inhibitors to homogeneity. They showed the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to a molecular mass of 32,000 daltons.

Animals↗
PubMed1986

Collagenase inhibitor stimulates cleft formation during early morphogenesis of mouse salivary gland.

A collagenase inhibitor obtained from the culture medium of bovine dental pulp markedly enhanced the cleft formation of mouse embryonic salivary gland epithelium when the inhibitor was included in the culture medium for 12-day and 13-day salivary glands. Determination of collagenase activity using [3H]collagen as substrate indicated that there was a latent collagenase activity in 12-day glands. In addition, a highly purified Clostridial collagenase freed from protease and hyaluronidase activities, strongly inhibited initiation of the cleft formation of the 12-day epithelium. Scanning electron microscopic observation showed that abundant collagen-like fibrils were seen on the epithelium in the collagenase-inhibitor-treated glands compared to those in the control. These findings suggest that during early morphogenesis tissue collagenase may regulate the cleft formation in the epithelium.

Animals↗
PubMed1986

Immunoelectron microscopic localization of collagenase inhibitor in bovine dentin.

The localization of collagenase inhibitor in bovine dentin was investigated by immunohistochemistry and immunoelectron microscopy using a specific rabbit antiserum raised against the inhibitor purified from culture medium of bovine unerupted third molar pulps. Immunoreactive collagenase inhibitor was mainly localized as an amorphous or granular accumulation along the wall of dentinal tubules in decalcified bovine dentin. A part of the immunoreactive inhibitor appeared as two periodic bands on the collagen fibrils surrounding the dentinal tubules. Those two, a major and a minor band, corresponded to D-periodic fibril bands V-VII and I, respectively. As those two bands are located on both borders between overlap and hole zones of the collagen fibril, C- and N-terminal non-helical peptides and eight regions which are nD (n = 1-4) apart from each non-helical peptide along the triple helix are all equally possible binding sites of the collagenase inhibitor on the collagen molecule. It is interesting that the major band is located not exactly at, but fairly close to, the locus of collagenolytic cleavage, which lies between D-periodic fibril bands IV and V.

Animals↗
PubMed1986

Scanning electron microscopic observation of mouse embryonic submandibular glands during initial branching: preferential localization of fibrillar structures at the mesenchymal ridges participating in cleft formation.

Branching submandibular glands of 12-day mouse embryos and those cultured in the presence and absence of a collagenase inhibitor from the culture medium of bovine dental pulp or a Clostridial collagenase were examined with the scanning electron microscope. Fracturing of fixed and dried glands with the tip of a fine needle succeeded in exposing the surfaces of the lobules and of their mesenchymal replicas at different stages of branching. At the beginning of branching, corresponding parts of the mesenchyme formed ridges on or in which the fibrillar structures were often found. At the stage forming deeper clefts thicker fibres, 0.5-2.5 micron in diameter, were observed between two adjacent lobules. On the contrary, no apparent differences in the fibrillar structures on the epithelial surfaces were detected between the shallow cleft and noncleft regions at the initial phase of branching. These fibrillar structures were very abundant in glands cultured with collagenase inhibitor and were completely lost in glands cultured with bacterial collagenase, strongly indicating that these materials consisted of collagen. The possible involvement of mesenchyme in epithelial branching is discussed with special reference to mesenchymal traction forces that would be elicited by fibrillar collagens.

Animals↗
PubMed1985

Immunohistochemical localization of collagenase inhibitor in bovine dental pulp, pulp cells in monolayer culture, and in some oral connective tissues.

The development of an antiserum, monospecific to the collagenase inhibitor, from bovine dental pulps permitted localization of immunoreactive inhibitor protein, by means of both immunofluorescence and immunoperoxidase-staining techniques in sections of bovine dental pulps. The immunoreactive inhibitor protein in bovine dental pulps is present both in cells and extracellular matrices. When cultured in Eagle minimal essential medium, coronal pulps from bovine-unerupted teeth were shown, by assay of the medium, to produce only about 1/10 of the amount of inhibitor produced by the root pulps. When compared by immunohistochemical observation, however, essentially no differences in fluorescent activity was found between coronal and root pulps. Specific cytoplasmic staining was seen both in explanted root-pulp tissues and in immature fibroblast-like pulp cells from monolayer cell cultures of bovine root pulps, which indicate that the pulp cells are responsible for inhibitor production. Sections of dental follicle and gingiva from the same animal, showed a distribution of immunoreactive inhibitor protein similar to that in dental pulps.

Animals↗
PubMed1984

Purification and characterization of bovine dental pulp collagenase inhibitor.

Root pulps from bovine unerupted wisdom teeth produce a potent collagenase inhibitor together with latent collagenase when cultured in Eagle's minimal essential medium (Biochem. Int. 5, 763, 1982). The inhibitor was purified more than 700-fold from the explant medium using Con A-Sepharose, Ultrogel AcA 44 and DE-52 cellulose columns. It showed a single band (MW = 36,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but showed multiple bands on basic (pH 8.3) polyacrylamide gel electrophoresis and electrofocusing. The inhibitor is a sialo-glycoprotein containing approx. 20% carbohydrate by weight and its composition suggests that it contains complex-type oligosaccharides. The electrophoretic heterogeneity of the inhibitor was proved to be due to the attachment of different numbers of sialic acid residues. All the SH groups were demonstrated to exist as six disulfide linkages which might be involved in the inhibitory activity. The bovine pulp inhibitor does not combine with collagen. The addition of the inhibitor to activated collagenase resulted in dose-dependent inhibition of the enzyme activity, but the interaction between the inhibitor and activated collagenase is not tight enough for the complex to remain intact during gel filtration column chromatography. A rabbit antiserum was prepared against the inhibitor, and immunoglobulin purified from the antiserum can completely abolish the inhibitory activity of the inhibitor.

Amino Acids↗
PubMed1984

Dual regulation of collagenase activity in bovine dental pulps.

A monospecific rabbit anti-inhibitor serum did not cross-react with either intact latent collagenase or with the one pretreated by p-aminophenylmercuric acetate. Furthermore, immunoglobulin G purified from the antiserum quantitatively inhibited the anti-collagenolytic activity of the inhibitor in either the presence or absence of p-aminophenylmercuric acetate. But the immunoglobulin G did not affect latent collagenase at all. These facts, along with other lines of evidence, strongly support the possibility that the inhibitor may not be responsible for the latency of collagenase and allow us to propose a dual regulatory mechanism of collagenase activity; that is, an inactive form, per se, may thus be additionally kept in the inactive state by the existence of an inhibitor which is synthesized in the same pulp tissues.

Animals↗
PubMed1982

Extraction of latent-type collagenase from cultured bovine dental pulps with NaCl or urea or by collagen degradation.

All of the collagenase activity extracted from cultured bovine dental pulp tissue with NaCl or urea solutions was due to enzyme in a latent form and identified as a typical animal collagenase. Isotonic sucrose solution solubilized no detectable collagenase activity from cultured dental pulps. Also, no collagenase activity was extracted from either fresh bovine dental pulps or those cultured in Tyrode's solution containing 50 micrograms/ml cycloheximide. A two-day difference was observed between the appearance of collagenase activity in the cultured pulps and in the culture medium. The activity profiles in the culture media showed essentially no difference with or without the addition of cyclohexamide on and after the 10th day of culture, indicating that the biosynthesis of collagenase in the cultured pulp might have terminated around that time. About 80% of the total pulp collagenase activity was extracted by a bacterial collagenase procedure. Nearly half (43.5%) of the collagenase activity extracted from the cultured pulps with NaCl or urea solution was precipitated with collagen molecules in the presence NaCl, and most of the precipitated activity retained in the precipitate even after washing it with NaCl-buffer solution. These facts suggest a close association of collagenase with the collagen in cultured dental pulp tissue.

Animals↗