Biomedical subjects

Y Kuboki

Publications and source records attributed to Y Kuboki.

At least 91 records · Page 5Linked to original sources

PubMed1994

In vitro and in vivo association of dentin phosphophoryn with alpha1CB6 peptide of type I collagen.

A small number of molecules of phosphophoryns, dentin phosphoproteins, are associated to collagen tightly with the maturation of dentin. As an in vitro model of the process, we examined non-covalent association of phosphophoryns and collagen CNBr peptides. Cyanogen bromide peptides of type I collagen were separated with electrophoresis and transferred to a membrane, which was probed with labeled phosphophoryns. Phosphophoryns were bound preferentially to alpha1CB6 peptide. Another experiment using a cleavable crosslinking agent also demonstrated the affinity between phosphophoryns and the alpha1CB6 region of collagen. The matrix-bound fraction of phosphophoryns was solubilized by CNBr digestion of bovine dentin, and was partially purified. Compositional analysis revealed that the fraction was composed of association products of phosphophoryns and collagen at the ratio of 2:3. Considering the high molecular weight of the fraction, one phosphophoryn molecule should be associated with several collagen peptides. The fraction reacted with the antibody against alphalCB6 peptide. When the fraction was digested with lysyl endopeptidase, several peptides that coincided with peptides from alpha1CB6 were released. One of the peptides was sequenced and identified to be a peptide with Asp 975 of the alpha1(I) chain as an amino terminal residue. The alpha1CB6 peptide may be involved in the matrix-bound fraction, although involvement of other collagen peptides cannot be ruled out. Phosphophoryns may associate with collagen at the alpha1CB6 region at first, and then be immobilized on it, possibly by covalent crosslinking, with maturation of dentin. Binding of several alpha1CB6 peptides to a single phosphophoryn molecule is possible in current 3-dimensional models of collagen packing in mineralized tissue, which allows alignment of several hole zones in register.

Amino Acid Sequence↗

PubMed1994

A new porous hydroxyapatite ceramic prepared by cold isostatic pressing and sintering synthesized flaky powder.

A new porous hydroxyapatite ceramic was prepared by cold isostatic pressing and sintering of the flaky powder, that was synthesized through two-stage hydrolysis of brushite; (1) a structural change into the apatite structure and (2) a compositional increase in Ca/P ratio, according to the method of Monma and Kamiya. The appearance of the synthesized powder resembled the flaky shape of the starting materials and its average particle size was about 15 microns. This powder consisted of fine needle crystals, which had a tendency to grow into the larger grains, but the powder was highly resistant to sintering under the usual heating conditions at 1200 degrees C. Porous hydroxyapatite blocks and granules were prepared by cold isostatic pressing and sintering a pellet consisting of the hydroxyapatite powder and spherical polymer beads. The product showed a 70% apparent porosity with spherical pores, ranging from 100-200 microns in size, and most pores were interconnected. These properties were ascribed to the effect of cold isostatic pressing on the hydroxyapatite powders with the flaky shape.

Ceramics↗

PubMed1993

BMPs induce direct bone formation in ectopic sites independent of the endochondral ossification in vivo.

Bone formation in vivo occurs via two major processes, one of which depends on pre-existing cartilage, and the other does not. Bone morphogenetic proteins (BMPs) have been suggested to induce cartilage formation from non-skeletogenic mesenchymal cell population, which results in osteogenesis through the endochondral sequence. In the present study we examined if BMPs could cause direct bone formation independent of pre-existing cartilage using bovine fibrous collagen membrane (FCM) as a carrier for BMPs. Bovine metatarsal bone was extracted in 4 M guanidine HC1 and BMPs were partially purified through the hydroxyapatite chromatography and the Heparin-Sepharose CL6B chromatography. The carrier was loaded with BMPs and then implanted in Wistar rats subcutaneously. The implants were fixed together with surrounding tissue every week after implantation and processed for von Kossa stain, immunohistochemistry, and electron microscopy. The phenotypes of bone and cartilage were identified histologically and immunohistochemically using antibodies against type I and type II collagen. Cartilage and bone were independently induced by 2 weeks. The bone formed directly on the collagen substrate of FCM without pre-existing cartilage. Calcification occurred in the carrier as well as the cartilage and bone matrix. The present study suggests that the BMPs induce osteogenesis in vivo independent of the endochondral sequence.

Animals↗

PubMed1993

Differences in composition of cell-attachment sialoproteins between dentin and bone.

Matrices of dentin and bone were compared with respect to the content of cell-attachment sialoproteins. The levels of two sialoproteins, osteopontin (OPN) and bone sialoprotein (BSP), were determined in dentin and bone by immunochemical procedures. Polyclonal antibodies against bovine BSP and an antibody against the amino-terminal decapeptide of rat OPN were used. The relative levels of OPN and BSP in dentin were less than one-tenth of the levels in bone. The differences between dentin and bone levels of OPN and BSP were thus larger than those for osteonectin or bone Gla protein in the two tissues. The scarcity of the cell-attachment proteins in dentin may reflect the metabolic inactivity of dentin.

Animals↗

PubMed1993

Amino-terminal location of pyridinoline in dentin collagen.

Cross-linking is believed to be one of the major factors that characterize the calcifiability of dentin and bone collagens. Dehydro-dihydroxylysinonorleucine and pyridinoline which constitute the principal cross-links of dentin collagen have so far been located only in the carboxy terminal telopeptide of the molecules [alpha 1(I)-chain 87 x alpha 1(I)-chain 16C]. This situation suggested that the amino terminal telopeptide portion might be "open" without intermolecular cross-linking in hard tissue collagen fibrils. However, the present study provided evidence that pyridinoline is also located in amino-terminal telopeptides (alpha 1-chain 9N or alpha 2-chain 5N) and alpha 1-chain 930. Bovine dentin collagen was digested with trypsin followed by heating at 60 degrees C before and after the digestion. This method gave complete trypsin peptides of dentin collagen. Fluorescent pyridinoline peptides with a smaller molecular size were isolated by Sephadex G-50 superfine, DEAE-cellulose and reverse-phase HPLC. Automatic Edman analysis of several isolated peptides revealed the five-residue sequence, Gly-Ile-X-Gly-His-Arg, the only assignment of which was alpha 1-chain 928-933. The above evidence together with the amino acid compositions of the peptides led to the conclusion that pyridinoline is located not only in the carboxy-terminal but also in the amino-terminal telopeptide in dentin collagen.

Amino Acid Sequence↗

PubMed1993

Localization of bone morphogenetic protein-induced bone and cartilage formation on a new carrier: fibrous collagen membrane.

A fibrous collagen membrane (FCM) made of crosslinked reconstituted collagen fibers was applied as a carrier of BMP. The effectiveness of FCM as a BMP carrier was compared with conventionally used insoluble bone matrix (IBM). Partially purified BMP was obtained from a guanidine HCl extract of bovine bone after a three-step chromatographic procedure. The BMP fraction was combined with FCM or IBM and implanted subcutaneously in the back of rats. Bone and cartilage formation were determined by radiographic, histologic, and biochemical analyses after removal at 1, 2, 3 and 4 weeks. After 2 weeks, bone and cartilage formation were evident histologically in IBM-BMP and FCM-BMP. In IBM-BMP, cartilage was formed in the area of presumptive bone. On the other hand, in FCM-BMP, cartilage formation occurred in the space between the fibers of fiber-bundles, while on the surface of the fibers bone formation started independently. Biochemically, the most noteworthy difference between FCM-BMP and IBM-BMP was the marked increase in type II collagen content in FCM-BMP, which contrasted with its decrease in IBM-BMP at 3 weeks. This study demonstrated that FCM definitely functioned as a BMP carrier that led to bone and cartilage formation at distinct sites. Therefore, we conclude that BMP could induce differentiation of immature cells preferentially into either osteogenic cells or chondrocytes depending upon the nature of the carrier which provides the environment for cell differentiation.

Alkaline Phosphatase↗

PubMed1992

Effects of a bone lysine-rich 18 kDa protein on osteoblast-like MC3T3-E1 cells.

A lysine-rich 18 kDa protein was isolated from bovine bone and examined for its effects on osteoblast-like MC3T3-E1 cells. This protein is homologous to a heparin-binding protein in brain and uterus. This protein enhanced cell attachment independent of the Arg-Gly-Asp cell-binding sequence and stimulated proliferation during the growth phase. Addition of this protein to cell cultures on days 11, 12, and 13 after confluency resulted in a 1.6-2.0-fold increase in the alkaline phosphatase activity and little increase in the DNA content. These findings suggest that the 18 kDa protein may be functional in promoting the proliferation and differentiation of osteoblasts.

3T3 Cells↗

PubMed1992

Affinity of bone sialoprotein and several other bone and dentin acidic proteins to collagen fibrils.

Bone and dentin contain several kinds of mineral-binding proteins and cell-attachment proteins. The authors examined the affinity of these proteins to type I collagen, a major matrix protein of the tissue. Bone sialoprotein (BSP), bone Gla protein (BGP), bone small proteoglycan II (PG II), osteonectin (ON), and dentin phosphophoryn (DPP) were labeled with fluorescein isothiocyanate and incubated with reconstituted type I collagen fibril. DPP, BGP, BSP, and PG II were absorbed significantly to the collagen fibril at physiological ionic strength with dissociation constants of 10(-6)-10(-7) M. BSP and PG II enhanced the fibrillogenesis of collagen. These acidic proteins can affect the surface properties of collagen fibril, and BSP, having the cell-attachment sequence Arg-Gly-Asp, possibly mediates interaction between collagen fibril and cells.

Amino Acid Sequence↗

PubMed1992

Time-dependent changes of collagen cross-links and their precursors in the culture of osteogenic cells.

The early stage of cross-link formation in bone collagen was studied in a cell culture system. An osteogenic cell line that produces and accumulates a remarkably high amount of collagen, and that eventually forms bone-like structures, was used in this study for its time-dependent development of reducible cross-links. It was found that precursors of the cross-link, dehydro-dihydroxynorleucine and dehydro-hydroxynorleucine became detectable as soon as the cells attained a confluent state. They showed maximal amounts at days 3-5 after confluence, but substantially disappeared at day 10 after confluence. In contrast, two characteristic cross-links of bone collagen, dehydro-dihydroxylysinorleucine (dehydro-DHLNL) and dehydro-hydroxylysinorleucine (dehydro-HLNL), which were present in trace amounts at the stage of cell confluence, gradually increased in amount and reached a plateau at day 10, just when their precursors disappeared. Thus, it was found that there was a time lag of about a week between the maximal formations of precursors and cross-links of bone collagen in this system. The significance of this time lag was interpreted in terms of the minimum essential accumulation of collagen for the precursor-product transition. The ratio of dehydro-DHLNL to dehydro-HLNL was as low as 0.7 at day 3 after confluency, increased to 4.2 at day 20, the period just before mineralization began, and decreased thereafter, suggesting a qualitative change in bone collagen associated with mineralization.

Animals↗

PubMed1992

An osteonectin-like protein in the matrix of cultured osteogenic cell-line MC3T3-E1, which is associated with calcification.

Time-dependent changes of the [3H]-proline-labeled noncollagenous proteins synthesized by the osteogenic cell-line MC3T3-E1 were analyzed over a range starting from cell confluency to 13 days after confluency during which time cells formed a bone-like structure. It was found that the 40 kDa protein on SDS-polyacrylamide gel (SDS-PAGE) remarkably increased in the cell-matrix layer at about 9 days after cell confluence, just before calcification. This protein was highly purified and was found to contain high amounts of glutamic acid, glycine, and serine. An internal amino acid sequence of this protein was revealed to be K-X-M-A-P-E-E-X-P, which showed homology with the sequence of the EF-hand domain in osteonectin/SPARC (secreted protein, acidic, and rich in cysteine). This protein co-migrated with collagen in gel filtration, and ion-exchange chromatography. Furthermore, it showed high affinity to type I collagen.

Amino Acid Sequence↗

PubMed1992

A study on bone induction in hydroxyapatite combined with bone morphogenetic protein.

Our present study consisted of an implantation of artificially made hydroxyapatite (HAP) ceramic pellets under the periosteum of the rabbit skull with subsequent inspection of further progress of bone formation and also of an evaluation of the effects of bone morphogenetic protein (BMP). The results revealed that the alkali phosphatase (AL-P) activity of the pellets was elevated only in those of the bone morphogenetic protein group. The results of determination of bone mineral density at the site of the pellets revealed that the increase in bone mineral density was the most remarkable in the bone morphogenetic protein group rather than the control group. The results of the histopathologic examinations revealed that marginal bone formation was found in the pores on the surface between the pellets and the skull in the control group and in the collagen group, whereas in the bone morphogenetic protein group very active bone formation was found not only on the interface in contact with the skull but also surrounding the whole pellet. It also was noted in the animals in the bone morphogenetic protein group that the pellets were corrupted from the peripheries and then absorbed into the newly formed bone. From these results, the efficacy of the hydroxyapatite-collagen-bone morphogenetic protein complex was made clear, and applications in clinical practice are expected in the near future.

Alkaline Phosphatase↗

PubMed1991

Preferential adsorption of dentin and bone acidic proteins on the (100) face of hydroxyapatite crystals.

Interaction of bone and dentin proteins with minerals is an elementary step in the regulation of mineralization in these tissues. Adsorption of acidic non-collagenous proteins on hydroxyapatite was examined using fluorescence-labeled protein and synthetic hydroxyapatite. Phosphophoryn, bone Gla protein, osteonectin and bone small proteoglycan II were prepared and labeled with fluorescein. All of these proteins were adsorbed on hydroxyapatite with a dissociation constant on the order of 10(-7) M. The more acidic proteins had lesser binding capacities. Hydroxyapatite single crystals were incubated with labeled proteins and observed with a fluorescence microscope. Phosphophoryn and other acidic proteins were adsorbed preferentially on the (100) face of the crystal. This preferential adsorption of the acidic proteins may be responsible for the morphogenesis of biological hydroxyapatite.

Animals↗

PubMed1991

Salivary histatin as an inhibitor of a protease produced by the oral bacterium Bacteroides gingivalis.

We examined the effect of histatin 5 from human parotid saliva on various proteases. Histatin 5 strongly inhibited a trypsin-like protease produced by Bacteroides gingivalis with an IC50 value of 55 nM. Clostripain was also inhibited (IC50 = 800 nM). Activities of other proteases were not affected significantly. Because B. gingivalis is a suspected periodontal pathogen and its proteolytic enzymes have been considered to be associated with periodontal tissue destruction, it is suggested that salivary histatins play a role as a preventive against periodontal disease.

Amino Acid Sequence↗

PubMed1991

Flow effects on cultured vascular endothelial and smooth muscle cell functions.

Cultured vascular endothelial cells were exposed to fluid shear stress by means of a rotary-disc shear-loading device, and the physiological effects of the conditioned medium (CM) and the homogenate (HM) of the cells on migration, adhesion and growth of endothelial cells (EC) or smooth muscle cells (SMC) were studied. Effects of shear stress on the production and secretion of collagen, one of the extracellular matrices of EC, were also studied. CM stimulated the adhesion and growth of SMC, but not of EC themselves. The ability to stimulate SMC adhesion and growth was similar in CM obtained from the static and shear-loaded cells. HM of the shear-loaded EC stimulated SMC migration. Further, HM of the shear-loaded EC contained increased amounts of collagen compared with the static EC. These results suggest that: 1) EC produce and secrete accelerators for the adhesion and growth of SMC, 2) EC react to the physical stimulus of fluid shear stress to produce stimulators of SMC migration, and 3) EC produce collagen, the production of which is enhanced by fluid shear stress.

Animals↗

PubMed1990

High-resolution solid-state nuclear magnetic resonance spectra of dentin collagen.

Insoluble collagen of bovine dentin was characterized by high-resolution solid-state 13C nuclear magnetic resonance (NMR) spectroscopy using a cross-polarization magic angle spinning procedure. A downfield shift was observed in the signal of hydroxyproline C beta compared with that in skin collagen, indicating a distortion in the hydroxyproline structure. A signal of 31P NMR was detected in dentin collagen that was compatible with the presence of matrix-associated phosphoprotein.

Animals↗

PubMed1990

[Longitudinal observation of condylar position in anterior crossbite at the mixed dentition during orthodontic treatment].

We investigated the condylar position by transcranial radiography which can be put daily clinical use easily. The patients are 8 anterior crossbite at the mixed dentition (4 functional crossbite and 4 skeletal crossbite). We observed their condylar positions before treatment (average age is 9.3 years old), after correction of overbite (average age is 10.0 years old) and 3-4 years after correction of overbite (average age is 14.0 years old). The results were as follows: 1. Condylar position of 7 cases changed after correction of overbite. The differences between right and left condylar position of six in seven cases had been recognized before treatment decreased after correction of overbite. Their condylar position were stable during the observation period of 3-4 years after correction of overbite. The condylar position of the other one case closed to fossa and then relapsed 3-4 years later. 2. Condylar position of only one case hasn't changed during the observation. 3. In functional crossbite cases, there were some differences between right and left condylar position before treatment, but no differences after crossbite correction. In skeletal crossbite cases, there was no tendency of the changing on their condylar position through treatment. 4. The changing to same direction was observed in the decrease of differences on condylar position and in the result of frontal cephalogram analysis. 5. There wasn't special relationship between changing of condylar position and orthodontic appliances. 6. TMJ dysfunction was observed in the case which has significant difference in its condylar positions before treatment and in the other case in which difference was not corrected after treatment. We recognized the usefulness of transcranial radiography for orthodontic treatment.

Child↗

PubMed1989

Separation of bone matrix proteins by calcium-induced precipitation.

It was found that significant precipitation occurred immediately after calcium, at a concentration as low as 2 mM, was added to a desalted solution of EDTA extract of adult bovine femur. The maximal yield of the precipitates was observed at a calcium concentration of 30 mM. These precipitates were dissolved in 0.5 M EDTA, desalted, and characterized by Sepharose CL-6B gel filtration chromatography and high performance gel-exclusion chromatography. Results revealed that the precipitates were enriched in a 40 K protein and a higher molecular weight fraction as compared with the original extract of bone proteins. The 40 K fraction was isolated and identified as osteonectin, as judged from amino acid analysis, electrophoresis, and immunodetection. The supernatant after calcium-induced precipitation predominantly contained osteocalcin and a 50 K protein that was tentatively identified as alpha 2HS protein. Osteonectin was purified from the calcium-induced precipitates from the EDTA extract of bovine bone. By calcium titration using fluorescence spectrometry, the isolated osteonectin showed high affinity to calcium ions with an apparent dissociation constant (K0.5) of 8 x 10(-7) M. Thus, the use of calcium to separate bone proteins, especially osteonectin, was proved to be a useful technique. In addition, calcium-induced precipitation of osteonectin suggested a possible in vivo mechanism via which osteonectin might interact with calcium ions and participate in the initial immobilization of calcium to induce the nucleation of calcification in bone tissue.

Animals↗

PubMed1989

Changes in levels of osteonectin in bovine dentine during tooth development.

Bovine incisors were classified into three developmental stages and non-collagenous proteins extracted from them. Sodium dodecyl sulphate gel electrophoresis of the extracts showed a reduction in osteonectin with the various stages. The reduction was confirmed by enzyme immunoassay using antiserum against bone osteonectin. This change is in contrast to dentine phosphoprotein, indicating functional differences between these two proteins.

Animals↗