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Biomedical subjects

Y Kuboki

Publications and source records attributed to Y Kuboki.

At least 73 records · Page 4Linked to original sources
PubMed1996

Time-dependent expression of bone sialoprotein fragments in osteogenesis induced by bone morphogenetic protein.

Bone sialoprotein is unique to bone and dentin, but its precise role in these tissues is still unknown, although several hypotheses have been presented. We chose ectopic chondro- and osteogenesis induced by bone morphogenetic protein (BMP) as a model system to examine the role of this protein. Partially purified bovine BMP obtained by a three-step chromatographic procedure contained all the active BMPs (natural BMP cocktail). It was combined with insoluble bone matrix and subcutaneously implanted into rats. Expression of bone sialoprotein (BSP) in the implants was followed by using a monoclonal antibody as previously reported. Immunostaining studies showed BSP in the osteoblasts lining the new bone surface at 5 weeks. Western blotting showed 53 and 30 kDa bands, instead of the 57 kDa band normally found in rat femur. These two fragments were metabolically labeled with [3H]proline. The total amount of the fragments rapidly increased after 3 weeks, and at 5 weeks was 3 times as high as that at 2 weeks and still increasing. This time-dependent change was almost parallel to that of osteocalcin. The amount of bone estimated in terms of calcium content increased until 3 weeks and was remained at a plateau thereafter. Alkaline phosphatase activity was prominent only in the first 3 weeks. It was concluded that the 53 and 30 kDa BSP fragments might contribute to maintenance or remodeling in BMP-induced ectopic bone formation.

Aging↗
PubMed1996

Promotion of the osteogenetic activity of recombinant human bone morphogenetic protein by basic fibroblast growth factor.

In this study, we investigated the promotive effect of basic fibroblast growth factor (bFGF) on the osteogenetic activity of bone morphogenetic protein (BMP) used in conjunction with porous hydroxyapatite ceramic pellets. Results showed that there was extensive bone formation as early as 3 weeks after insertion in the group that received pellets treated with a relatively large amount of recombinant human (rh) BMP alone as well as in the group in which pellets were treated with a small amount of rhBMP combined with rhbFGF. Subsequently, osteogenesis within the pores of the pellets of the two groups slowly progressed over time; by 9 weeks after insertion, most of the pellet pores were filled with newly generated bone. In contrast, the group that received pellets treated with rhbFGF alone showed little osteogenesis, as was the case with the control group. The group that received the pellets treated with the small amount of rhBMP as well as the rhbFGF group exhibited significantly greater bone induction than the group that received the pellets treated with the small amount of rhBMP alone. These results indicated that a very small amount of rhbFGF has a strong promotive effect on the osteogenetic activity of rhBMP.

Analysis of Variance↗
PubMed1996

Electrophoretic gels of dentin matrix proteins as diffusion media for in vitro mineralization.

Non-collagenous proteins of dentin and bone have important effects on mineralization which have been studied by various in vitro systems. We developed an in vitro mineralization system using electrophoretic gels as diffusion media of calcium and phosphate ions. Calcium and phosphate ions were diffused naturally or propelled by electric potential. Calcium phosphate was precipitated in the gel, and the precipitation was affected by proteins in the gel which had been separated by electrophoresis. We applied this system to analysis of non-collagenous proteins of dentin. Among the proteins, phosphophoryns promoted calcium phosphate precipitation in the natural-diffusion system. A non-collagenous protein having a molecular mass of 60 kDa inhibited precipitation. The results were different, however, in the electric-diffusion system, in which phosphophoryns had a negative effect. The present system enabled us to compare the effects of plural proteins rapidly, even using unpurified material.

Animals↗
PubMed1996

Purification of a noncollagenous high molecular weight protein from bovine articular cartilage.

A noncollagenous high molecular weight protein (HMW) was isolated from bovine articular cartilage by CsCl density gradient centrifugation followed by DEAE-cellulose chromatography and gel filtration chromatography. The molecular weight was estimated to be 320 kDa and the reduced HMW had a molecular weight of 105 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid composition of HMW showed a high content of aspartic acid, but no hydroxylysine and hydroxyproline were detected, indicating that the protein was not a collagen. Affinity purified rabbit antibody against HMW reacted specifically with HMW, but there was no immunoreaction with cartilage-specific proteoglycan, type II collagen and type I collagen. HMW showed dose-dependent attachment to types I, II, III and V collagens. However, HMW did not bind to fibronectin, decorin and heparin. Furthermore, the binding of HMW to heat-denatured collagens was lower than native collagens. In these observation, HMW may be recognized the triple helix structure of collagen for the interaction.

Animals↗
PubMed1995

Developmental and differential regulations in gene expression of Xenopus pleiotrophic factors-alpha and -beta.

Using conserved nucleotide sequences in mammalian osteoblast specific factor-1 (OSF-1) coding regions, we isolated two kinds of cDNA clones from the Xenopus brain library. The encoded proteins, named Xenopus pleiotrophic factors (X-PTFs)-alpha and -beta, were 65 and 87% homologous to human midkine and OSF-1, respectively. In the adult frog, X-ptf-alpha was expressed in the ovary, brain, eye, bone, heart and lung, whereas X-ptf-beta was expressed in the brain, eye and bone. By in situ hybridization of the tailbud embryo, X-ptf-alpha mRNA was detected rather broadly in the head/tail regions including the central nervous system (CNS), whereas X-ptf-beta mRNA was restricted to the CNS, particularly in the hind-brain. During embryogenesis, X-ptf-alpha mRNA was detected in the one-cell stage embryo, whereas only zygotic expression was observed in X-ptf-beta. X-ptf-beta mRNAs contained approximately 79 bp tandem repeats in the 3'-untranslated region, complementary to those found in retinoic acid cellular receptor mRNA and in the sense strand of short interspersed repeat transcripts in X. laevis.

Aging↗
PubMed1995

TGF-beta accelerated the osteogenic differentiation of bone marrow cells induced by collagen matrix.

Bone marrow cells have multipotency for differentiation. Recently we found that type I collagen matrix induced the osteogenic differentiation of bone marrow cells. In this study we showed that TGF-beta enhanced the effect of collagen matrix on bone marrow cells and accelerated the osteogenic differentiation. These results imply that TGF-beta might play a crucial role in the osteogenic differentiation of bone marrow cells during embryogenesis and the repair of bone.

Alkaline Phosphatase↗
PubMed1995

Modulated expression of type X collagen in Meckel's cartilage with different developmental fates.

Mammalian Meckel's cartilage undergoes regionally diverse histodifferentiation: the caudal end of Meckel's cartilage extends to the developing ear and gives rise to malleus and incus through endochondral ossification while its major distal region differentiates into sphenomandibular ligament and the anterior ligament of the malleus tympanic plate through fibrous transformation. Since the entire Meckel's cartilage develops up to chondrocyte hypertrophy, the regional extracellular matrix components in the hypertrophic Meckel's cartilage may differ in association with the diverse developmental fates. In this project, the expressions of cartilage collagens were investigated in developing rat Meckel's cartilage and particular interest was given to type X collagen. A cDNA, HP114, encoding the NC1 domain of rat alpha 1(X) collagen was cloned, and a synthetic peptide based on the sequence deduced from HP114 was used to generate a monospecific antibody. In situ hybridization of newborn rat condylar and angular cartilages undergoing endochondral ossification showed restricted labeling with the alpha 1(X) collagen probe in the hypertrophic chondrocyte layer. In contrast, the alpha 1(X) collagen probe totally failed to label the major distal portion of Meckel's cartilage even in the hypertrophic cartilage zone. Immunohistochemistry using the anti-type X collagen monospecific antibody consistently failed to recognize the epitope in the corresponding portion of Meckel's cartilage throughout the experimental periods of gestational Day 17, newborn, and Postnatal Day 7, while the strictly localized positive staining was found in the posterior part of Meckel's cartilage which gave rise to malleus and incus. Since major cartilage collagens type II and type IX were found to be present throughout Meckel's cartilage, we postulate that the regulatory molecular mechanism of type X collagen expression may be closely associated with the developmental fates of fibrous transformation and endochondral ossification in mammalian Meckel's cartilage.

Amino Acid Sequence↗
PubMed1995

Further characterization of interaction between bone sialoprotein (BSP) and collagen.

Bone sialoprotein (BSP) has an affinity to collagen fibrils [25]. A role of carbohydrate chains in the affinity was examined by removing sialic acids of BSP. Neuraminidase treatment of the BSP increased the binding to collagen. Binding sites of BSP on collagen were examined by biochemical and electron-microscopic methods. Purified bovine BSP was labeled with biotin. Collagen alpha chains or CNBr peptides were separated by electrophoresis and transfered to nitrocellulose membranes. The membranes were incubated with the biotin-labeled BSP, and the bound BSP was visualized with avidin conjugated with alkaline phosphatase. The labeled BSP was preferentially bound to the alpha 2 chain, and peptides derived from alpha 2 chain. In another experiment, the labeled BSP was incubated with reconstituted native collagen fibrils. The mixture was put on a copper grid, reacted with avidin conjugated with gold particles, and observed with an electron microscope. The gold particles were seen mainly within hole zones of the fibrils. BSP bound to the alpha 2 chain within the hole zones may regulate the onset of calcification at hole zones and the cell binding to collagen fibrils.

Animals↗
PubMed1995

Conformational changes of bovine bone osteonectin induced by interaction with calcium.

To clarify calcium-induced conformational changes in bovine bone osteonectin, the protein was labeled with fluorescein isothiocyanate (FITC) in the presence and absence of calcium. By calcium titration using fluorescence spectrometry, it was demonstrated that FITC-osteonectin labeled in the presence of 2 mM CaCl2 showed a much higher affinity for calcium ions than did that labeled in the absence of calcium ions. The midpoint for completion of the increase in the intrinsic fluorescence (K0.5) of the two were 1 x 10(-7) M and 5 x 10(-7) M, respectively. By tryptic digestion and isolation of the fluorescent peptide of both FITC-osteonectins, the site of FITC-labeling was determined to be Lys174. Furthermore, it was found that the efficacy of labeling in this specific binding site was three times higher in the FITC-osteonectin labeled in the presence of 2 mM CaCl2 than in that labeled in the absence of calcium. The results indicate that in the presence of 2 mM CaCl2 the microenvironment around Lys174 of osteonectin was more open to modification than in the absence of calcium.

Amino Acid Sequence↗
PubMed1995

Stimulation by bone sialoprotein of calcification in osteoblast-like MC3T3-E1 cells.

Bone sialoprotein (BSP) containing an Arg-Gly-Asp cell-binding sequence was purified from bovine bone 4 M guanidine-HCl extract after HCl demineralization by a series of chromatographic procedures. When this protein was coated on culture dishes in the presence of type I collagen, it increased both DNA content and alkaline phosphatase (ALP) activity in osteoblast-like MC3T3-E1 cells, and stimulated calcification in the cells, whereas fibronectin, another cell-binding protein, showed a marked increase in the DNA content but had little effect on the ALP activity. These findings suggest that BSP is mitogenic for preosteoblasts and differentiating the cells into osteoblasts, thereby stimulating bone calcification.

3T3 Cells↗
PubMed1995

Extracellular processing of dentin matrix protein in the mineralizing odontoblast culture.

Odontoblasts that we prepared from bovine incisors produced a dentin-specific protein, phosphophoryn, and accumulated it in mineralized nodules. The time course of mineralization was detected by measuring osteocalcin and mineral in the nodules. The sequence of developmental expression of proteins in this mineralizing dentin cell culture is very similar to that in bone cells, suggesting a common mechanism for matrix mineralization in bone and dentin. Casein kinase II, which phosphorylates bone phosphoproteins and dentin phosphorylates bone phosphoproteins and dentin phosphophoryn, also emerges coinciding with the initiation of mineralization. Furthermore, we have detected extracellular phosphorylation by casein kinase II of a dentin protein of M(r) 60,000, which we recovered from the phosphophoryn fraction in CaCl2 precipitate.

Animals↗
PubMed1995

Efficacy of hydroxyapatite ceramic as a carrier for recombinant human bone morphogenetic protein.

In this study, we investigated the efficacy of hydroxyapatite ceramic (HAP) as a carrier of bone morphogenetic protein (BMP) using porous HAP pellets artificially fabricated from limestone. After treatment with recombinant human BMP-2, the pellets were inserted beneath the cranial periosteum of rabbits, and the degree of osteogenesis was examined histologically. The degree of osteogenesis was also evaluated using image-analyzing procedures. Results showed that extensive bone formation had occurred around the pellets 3 weeks after insertion in the group that received pellets treated with recombinant human BMP alone as well as in the group that received recombinant human BMP in addition to type I collagen-treated HAP pellets. Subsequently, osteogenesis within the pellets slowly progressed over time, and by 9 weeks after insertion most of the pellet pores in both groups were filled with newly generated bone. The recombinant human BMP-collagen group, however, exhibited a significantly greater bone induction. These results indicated that if recombinant human BMP is used clinically in the future, artificially fabricated HAP would be a suitable carrier.

Analysis of Variance↗
PubMed1995

Bone induction of hydroxyapatite combined with bone morphogenetic protein and covered with periosteum.

Using a rabbit model, we evaluated the role of the periosteum in bone induction using hydroxyapatite ceramic pellets, some of which had been coated with bone morphogenetic protein. Eighteen rabbits were divided into two groups, a control group that received pellets soaked in phosphate-buffered saline alone and another group for which the pellets had been supplemented with bone morphogenetic protein. After making a skin incision on the head of each rabbit, two caudally pedicled periosteal flaps measuring 1 cm in width and 2.5 cm in length were elevated. These flaps were wrapped around hydroxyapatite pellets manufactured from limestone and fixed with sutures. After implantation, both groups of rabbits were returned to their cages, maintained for 3, 6, or 9 weeks [every group consisted of 3 rabbits (6 pellets)], and then sacrificed. In this study, the extent of bone induction, which was measured with a dual x-ray densitometer, that resulted from covering the hydroxyapatite pellet with periosteum alone (control group) was minimal. On the contrary, since osteogenesis from the periosteum toward the pores of the pellet was observed in the bone morphogenetic protein group much more than in the control group, the usefulness of our technique was confirmed. However, active osteogenesis was observed with subperiosteal implantation of the hydroxyapatite-bone morphogenetic protein complex in the bone morphogenetic protein group, but the osteogenesis observed was not distributed over the entire pellet.

Animals↗
PubMed1995

Two distinctive BMP-carriers induce zonal chondrogenesis and membranous ossification, respectively; geometrical factors of matrices for cell-differentiation.

A partially purified BMP preparation was combined with a fibrous glass membrane (FGM) or porous particles of hydroxyapatite (PPHAP), and then implanted subcutaneously into the backs of rats. As a control of these new carriers, a conventional carrier of insoluble bone matrix (IBM) was also used. These new geometrically different solid-state carriers induced tissues in quite different manners. FGM/BMP implants induced cartilage formation within the entire inner area of the membrane accompanied by a small amount of bone formation on the surface of the membrane. In contrast, PPHAP/BMP implants induced only bone within the pores of PPHAP without any detectable cartilage formation. Enzyme-linked immunosorbent assay revealed that the type II collagen content in FGM/BMP was six times higher than that in IBM/BMP, while there was no detectable type II collagen in PPHAP/BMP. The results were explained by the geometric properties of the two distinctive carriers.

Animals↗
PubMed1995

A new method for in vitro calcification using acrylamide gel and bovine serum.

To investigate the mechanism of biological calcification in vitro, a model system consisting of an acrylamide gel block (1 x 3 x 3 mm) and fetal bovine serum was developed. Mineral deposition was induced in gel blocks which were immersed in 300 microliters of fetal bovine serum at 37 degrees C for 7 days in a CO2 incubator. X-ray diffraction indicated that the mineral was hydroxyapatite with low crystallinity. Effects of the concentration of acrylamide gel, the partial pressure of CO2 and matrix proteins within the gel on the mineral formation were investigated. In the gel concentration range of 10-60%, the largest amount of crystal grew in 40% acrylamide gel, where the serum protein did not penetrate. With an increase in the partial pressure of CO2 the Ca content in the gel block increased, reached the highest level at about 3.5% CO2 and then began to decrease. In 40% gel and at 5% CO2, the mineral formation was enhanced by phosvitin, phosphophoryn, demineralized dentin powder and alkaline phosphatase. Mineral deposition occurred around the collagen fibers immobilized in 40% acrylamide gel. These results indicate that 1) a putatively serum-derived inhibitor of calcification with high-molecular weight was prevented from penetrating into the 40% acrylamide gels, 2) immobilized polyanionic proteins and alkaline phosphatase were able to increase mineral deposition and 3) the partial pressure of CO2 greatly influenced the mineral deposition. It was concluded that this gel system is useful to investigate the mechanism of biological calcification in vitro.

Acrylamides↗
PubMed1995

Oriented growth of octacalcium phosphate on and inside the collagenous matrix in vitro.

To investigate the factors which regulate the growth of apatite in a collagenous matrix, a calcification experiment was carried out in a model system, where Ca2+ and PO43- ions diffused into a slice of Achilles tendon from mutually opposite grew on the Ca-side of the collagenous matrix, while small plate-like OCP crystals (< 1 micron) grew inside the matrix. The major part of crystals grew with the c-axis parallel to the collagen fibers. Others grew with the c-axis parallel to the collagen bands or perpendicular to the fibrils. Crystals grew with orientation on collagen fibrils from the beginning. It was concluded that collagen fibrils and property of the collagenous matrix played a regulatory role in the deposition and growth of OCP in the collagenous matrix.

Achilles Tendon↗
PubMed1994

Biochemical properties of collagen from ligaments and periarticular tendons of the human knee.

The biochemical properties of collagens from the periarticular connective tissues of the human knee (ligaments, semitendinosus and gracilis tendons, and the iliotibial band) were investigated in subjects ranging from 20 to 70 years of age. Although the total collagen content of most tissues was more than 90% of the dry weight, the anterior cruciate ligament and the iliotibial band had relatively low collagen contents. There were no significant changes in the tissue collagen content with aging. However, the anterior cruciate ligament and the patellar tendon of the 20-year-old subject had a higher content of soluble collagen than the other ligaments and tendons. The iliotibial band of the 20-year-old subject contained more collagen that was soluble by a combination of salt, sodium citrate extractions and pepsin digestion. Dihydroxylysinonorleucine was the major reducible cross-link of collagen from all the ligaments. The amount of dihydroxylysinonorleucine in the anterior cruciate ligament of the 20-year-old subject was much higher than that in the other ligaments. In contrast, the tendons and the iliotibial band contained a large amount of histidino-hydroxymerodesmosine and hydroxylysinonorleucine, while the patellar tendon was the only tendon with a significant content of dihydroxylysinonorleucine. Hydroxypyridinium non-reducible cross-links were more abundant in collagens from ligaments than in collagens from the other tissues. The cross-link study and the analysis of collagen solubility showed that patellar tendon collagen more closely resembled the collagen from the anterior cruciate ligament than that from periarticular tendons. It was also shown that the anterior cruciate ligament contains relatively immature collagen compared with the other ligaments.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗