Biomedical subjects

Toshio Yamamoto

Publications and source records attributed to Toshio Yamamoto.

At least 19 recordsLinked to original sources

PubMed2006

Simultaneous gene transfer of bone morphogenetic protein (BMP) -2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression.

BACKGROUND: Transcutaneous in vivo electroporation is expected to be an effective gene-transfer method for promoting bone regeneration using the BMP-2 plasmid vector. To promote enhanced osteoinduction using this method, we simultaneously transferred cDNAs for BMP-2 and BMP-7, as inserts in the non-viral vector pCAGGS. METHODS: First, an in vitro study was carried out to confirm the expression of BMP-2 and BMP-7 following the double-gene transfer. Next, the individual BMP-2 and BMP-7 plasmids or both together were injected into rat calf muscles, and transcutaneous electroporation was applied 8 times at 100 V, 50 msec. RESULTS: In the culture system, the simultaneous transfer of the BMP-2 and BMP-7 genes led to a much higher ALP activity in C2C12 cells than did the transfer of either gene alone. In vivo, ten days after the treatment, soft X-ray analysis showed that muscles that received both pCAGGS-BMP-2 and pCAGGS-BMP-7 had better-defined opacities than those receiving a single gene. Histological examination showed advanced ossification in calf muscles that received the double-gene transfer. BMP-4 mRNA was also expressed, and RT-PCR showed that its level increased for 3 days in a time-dependent manner in the double-gene transfer group. Immunohistochemistry confirmed that BMP-4-expressing cells resided in the matrix between muscle fibers. CONCLUSION: The simultaneous transfer of BMP-2 and BMP-7 genes using in vivo electroporation induces more rapid bone formation than the transfer of either gene alone, and the increased expression of endogenous BMP-4 suggests that the rapid ossification is related to the induction of BMP-4.

Animals↗

PubMed2005

Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems.

Regeneration of plant organs is often the essential step in genetic transformation; however, the regeneration ability of a plant varies depending on the genetic background. By conventional crosses of low-regeneration rice strain Koshihikari with high-regeneration rice strain Kasalath, we identified some quantitative trait loci, which control the regeneration ability in rice. Using a map-based cloning strategy, we isolated a main quantitative trait loci gene encoding ferredoxin-nitrite reductase (NiR) that determines regeneration ability in rice. Molecular analyses revealed that the poor regeneration ability of Koshihikari is caused by lower expression than in Kasalath and the specific activity of NiR. Using the NiR gene as a selection marker, we succeeded in selectively transforming a foreign gene into rice without exogenous marker genes. Our results demonstrate that nitrate assimilation is an important process in rice regeneration and also provide an additional selectable marker for rice transformation.

Amino Acid Sequence↗

PubMed2005

Cytokinin oxidase regulates rice grain production.

Most agriculturally important traits are regulated by genes known as quantitative trait loci (QTLs) derived from natural allelic variations. We here show that a QTL that increases grain productivity in rice, Gn1a, is a gene for cytokinin oxidase/dehydrogenase (OsCKX2), an enzyme that degrades the phytohormone cytokinin. Reduced expression of OsCKX2 causes cytokinin accumulation in inflorescence meristems and increases the number of reproductive organs, resulting in enhanced grain yield. QTL pyramiding to combine loci for grain number and plant height in the same genetic background generated lines exhibiting both beneficial traits. These results provide a strategy for tailormade crop improvement.

Alleles↗

PubMed2005

Immunohistochemical analysis of Sox9 expression in periosteum of tibia and calvaria after surgical release of the periosteum.

Sox9 is a transcription factor belonging to the SRY family of high-mobility box proteins, and plays a major role in endochondral ossification. Sox9 is a potent activator of the type-2 collagen pheno-type marker of articular cartilage. Regulation of osteogenic molecular signals in periosteal bone formation has not yet been elucidated yet. The purpose of the present study was to analyze histologically the bone formation in surgically released and repositioned periosteum, and to determine expression of Sox9 and type-2 collagen in periosteal bone formation of tibia and calvaria. After surgery, the released tibial periosteum formed ectopic cartilage. At 7 days, a combination of endochondral and intramembranous ossification was apparent. Some fibroblasts derived from the released periosteum showed Sox9 expression. Chondrocytes and cartilage matrix both displayed type-2 collagen expression. At 7 days, an additional new bone was formed on the calvaria. Osteoblasts and fibroblasts derived from released calvarial periosteum did not express Sox9 or type-2 collagen. Sox9 was not expressed throughout the process periosteal bone formation on the calvaria. It is concluded that we revealed Sox9 and type-2 collagen expression in periosteal cells after periosteum release and that the generative potential of periosteal cells of calvaria is different from that of tibia.

Animals↗

PubMed2005

Human BMP-2 gene transfer using transcutaneous in vivo electroporation induced both intramembranous and endochondral ossification.

It has been generally accepted that bone morphogenetic protein-2 (BMP-2) can induce osteogenesis in skeletal muscles via endochondral ossification. However, it is not clear how the ossification process occurs after the BMP-2 gene transfer to skeletal muscles in rats using in vivo electroporation. In this study, we evaluated the ossification process by BMP-2 gene transfer using in vivo electroporation. The gastrocnemius muscles of Wistar rats were injected with human BMP-2 gene expression vector (pCAGGS-BMP-2), followed by electroporation under the condition of 100 V, 50 msec per 1 sec, x8. Light and electron microscopic and radiographic analyses were performed at 1, 3, 5, 7, and 10 days after treatment. At 7 days, no sign of cartilage and/or bone formation was detected. However, at 10 days after in vivo electroporation, soft X-ray analysis revealed small lucent areas around the plasmid-injected region. Clusters of both cartilage tissues, leading to endochondral ossification and intramembranous bones of various sizes, were observed between muscle fibers. RT-PCR detected osteocalcin mRNA, showing bone formation at 10 days. Our findings strongly suggest that BMP-2 gene transfer using in vivo electroporation induces not only endochondral ossification but also intramembranous ossification.

Animals↗

PubMed2005

Ultrastructural and immunohistochemical studies of medullary bone calcification, with special reference to sulphated glycosaminoglycans.

Histochemical, immunohistochemical and electron energy-loss spectroscopic studies were performed to examine the relationship between sulphated glycosaminoglycans and medullary bone calcification using oestrogen-injected male Japanese quail. Sulphated glycosaminoglycans, detected by high iron diamine (HID) or HID-thiocarbohydrazide-silver protein (HID-TCH-SP) methods, were distributed throughout the matrix of medullary bone, some periphery and extending tips of the trabeculae stained weakly, and the globular structures at osteoid areas were exclusively positive for HID-TCH-SP stain. Immunohistochemistry identified keratan sulphate located in the globular structures at osteoid areas and calcified matrix, but chondroitin-4 sulphate and chondroitin-6 sulphate were not detected in the matrix. Using electron spectroscopic imaging, sulphur was determined to be localized in the globular structures. These results demonstrate that medullary bone matrix accumulates keratan sulphate in the globular structures, which are the foci for calcification, and eventually in the calcified areas. This suggests that keratan sulphate containing sulphur is maintained in the calcified matrix. These results indicate a unique process of calcification exists in medullary bone.

Animals↗

PubMed2005

Localization of CD44 (hyaluronan receptor) and hyaluronan in rat mandibular condyle.

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.

Animals↗

PubMed2005

Distribution of actin filaments, non-muscle myosin, M-Ras, and extracellular signal-regulated kinase (ERK) in osteoclasts after calcitonin administration.

Scanning electron microscopy (SEM) was employed to study the effect of calcitonin on the distribution of actin filaments in osteoclasts obtained from rat tibiae. Fluorescent microscopy was also applied to examine calcitonin-induced changes in the distribution of actin filaments, non-muscle myosin, M-Ras, and extracellular signal-regulated kinase (ERK) to clarify the role of ERK in the cytoskeleton of osteoclasts. SEM of control osteoclasts revealed a ring-like structure in the peripheral region. Labeled actin filaments and non-muscle myosin were detected in the peripheral region and exhibited a ring-like pattern. Immunoreactivity indicating M-Ras and ERK was also detected in the vicinity of the actin ring. After calcitonin treatment, many osteoclasts exhibited a retracted appearance and lacked a discernible actin ring. Numerous retraction fibers were found at the edge of calcitonin-treated osteoclasts. Actin filaments and non-muscle myosin were concentrated in the cytoplasm of calcitonin-treated osteoclasts, and exhibited a filamentous pattern. Labeled M-Ras and ERK also accumulated in the central region of these osteoclasts. These findings suggest that actin-myosin interaction plays an essential role in the retraction of osteoclasts induced by calcitonin. ERK may play a role in this interaction by activating myosin light chain kinase, as previously observed in smooth muscle cells.

Actins↗

PubMed2005

In vivo effect of fluoride-substituted apatite on rat bone.

Different types of calcium phosphate compounds are commercially available for medical and dental applications as bone substitute materials. Biological apatites contain several kinds of minor elements such as carbonate (CO3), magnesium (Mg), and fluoride (F) in enamel, dentin, and bone. It has been shown that F ion and F-substituted apatite promoted osteoblast proliferation and inhibited osteoclast cell activity. The purpose of this study was to investigate the in vivo rat tibia activity on F-substituted apatite (FAp). Apatites of unsintered calcium deficient apatite (CDA), and FAps, with low, medium, and high F concentrations, were implanted in rat tibia for 1 and 2 weeks. Implanted tissues were embedded in paraffin blocks, stained with hematoxylin-eosin and histomorphometrically observed. Results showed that low F concentration induced better and faster new bone formation in vivo compared to CDA. Therefore the results suggested that F as a minor element in bone rendered a suitable effect on bone formation in vivo.

Animals↗

PubMed2004

In vitro response of osteoblast-like and odontoblast-like cells to unsubstituted and substituted apatites.

Different types of calcium phosphate compounds [calcium-deficient apatite (CDA); beta-tricalcium phosphate (beta-TCP); biphasic calcium phosphate (BCP)] are commercially available for medical and dental applications as bone substitute materials. Most of the reported in vitro studies on cell-material interactions have used osteoblast-like cells. The purpose of this study was to investigate the in vitro response of osteoblast-like (MC3T3-E1) and odontoblast-like (MDPC23) cells on unsubstituted (HA) and substituted (F-substituted) apatites. MC3T3-E1 and MDPC23 were cultured in alpha-modified medium containing 10% fetal bovine serum, ascorbic acid (50 microg/mL) and beta-glycerophosphate (2 mM). The cells were seeded on pellets made from HA, and FAp (with low, medium, and high F concentrations). Cell morphology was observed after 7 and 14 days using scanning electron microscopy (SEM). Cell attachment and differentiation were determined from the DNA content, alkaline phosphatase (ALP) activity, and total collagen content. Pellet surface composition was characterized by using Fourier Transform infrared spectroscopy. MC3T3-E1 and MDPC23 cells on HA were normal in shape and in fusion but not on FAp. Results of this study showed that the pattern of cell proliferation of osteoblast-like cells was different from that of the odontoblast-like cells. This study suggests that cell morphology, fusion, and proliferation on biomaterial surfaces depend on cell type (osteoblast-like vs odontoblast-like cell) and biomaterial composition (unsubstituted vs substituted F-apatites).

Alkaline Phosphatase↗

PubMed2004

Different type and localization of CD44 on surface membrane of regenerative renal tubular epithelial cells in vivo.

BACKGROUND: CD44 is a transmembrane glycoprotein comprising an extracellular domain, a transmembrane domain, and a cytoplasmic tail. Previous studies demonstrated that CD44 was generally restricted to lateral-basal plasma membrane (PM) of epithelial cells, whether it localized on apical PM in vivo has not been clarified. METHODS: In this study, we used a gentamicin-induced acute tubular necrosis (ATN) and spontaneous recovery model in rats and two distinct antibodies, an anti-rat distal extracellular domain (OX49) of standard CD44 (CD44-OX49) and an anti-rat CD44 cytoplasmic tail (CD44CPT), to survey the localization of CD44-OX49 and CD44CPT on the PM in renal tubular epithelial cells in different recovery stages after ATN with immunohistochemistry and immunoelectron-microscopic examinations. RESULTS: CD44-OX49 was localized not only on the lateral-basal PM in tubular epithelial cells, but also on the apical surface membrane in PCNA-positive newly regenerative tubular epithelial cells in early recovery stages after ATN. However, CD44CPT was only localized on the lateral-basal PM. The immunoelectron-microscopic results showed that CD44-OX49 localization was changed from the apical to lateral to basal surface membrane in renal tubular epithelial cells during the recovery process after ATN, finally disappearing from basal PM when normal polarized epithelial cells formed. CONCLUSIONS: These results suggest that there were two types of CD44 including CD44 without a cytoplasmic tail localizing on the apical surface membrane related to newly regenerative epithelial cells, and CD44 with a cytoplasmic tail localizing on the lateral-basal PM related to establishment of tubular epithelial cell polarity after ATN in vivo.

Animals↗

PubMed2004

Expression of the Ellis-van Creveld (Evc) gene in the rat tibial growth plate.

Ellis-van Creveld (EvC) syndrome is an autosomal recessive chondrodysplasia characterized by short limbs, postaxial polydactyly, natal teeth, and dysplastic nails. The Ellis-van Creveld (EVC) gene, which is mutated in patients with EvC syndrome, has been identified by positional cloning. However, the physiological roles of the EVC gene have not been elucidated. Histopathological analyses of EvC syndrome have shown disturbed chondrocytic phenotypes during cartilage development. We therefore postulated that the EVC gene is a critical factor for chondrocytes during endochondral ossification. The present study focuses on the relationship between the Evc gene and chondrocytes, and examines Evc gene expression in the rat tibial growth plate at the mRNA and protein levels. Evc mRNA in tibial epiphyseal cartilage was expressed at postnatal day (P) 1, P28, and P56 by RT-PCR. Immunohistochemical analyses localized the Evc protein mainly in prehypertrophic and hypertrophic chondrocytes of the epiphyseal growth plate in the tibia during the embryonic and postnatal periods. Evc mRNA was also detected in prehypertrophic and hypertrophic chondrocytes by in situ hybridization. These results indicate that the Evc gene functions mainly in the prehypertrophic and hypertrophic chondrocytes of the epiphyseal growth plate. The data presented here are important for future studies of the underlying mechanism of chondrodysplasia in EvC syndrome.

Animals↗

PubMed2004

Immunolocalization of matrix metalloproteinase-13 on bone surface under osteoclasts in rat tibia.

Matrix metalloproteinase (MMP)-13 (an interstitial collagenase also called collagenase 3) is involved in degradation of extracellular matrix in various tissues. Using immunohistochemistry and Western blotting, we investigated localization of MMP-13 in rat tibia, to clarify the role of MMP-13 in bone resorption. MMP-13 reactivity was mainly seen on bone surfaces under osteoclasts, and in some osteocytes and their lacunae near osteoclasts. However, immunoreactivity was not seen in chondrocytes or osteoclasts. MMP-13 was also localized on cement lines in the epiphysis. In the growth plate erosion zone, perivascular cells showed MMP-13 reactivity. Immunoelectron microscopy revealed that MMP-13 was localized on the bone surfaces, under the ruffled borders and some clear zones of osteoclasts. Gold-labeled MMP-13 was closely associated with collagen fibrils. Gold labeling was also detected in Golgi apparatus of osteocytes adjacent to osteoclasts and bone lining cells. Western blotting showed that MMP-13 was mainly associated with mineralized bone matrix. These findings suggest that MMP-13 synthesized and secreted by osteoblast-lineage cells is localized under the ruffled borders of osteoclasts. MMP-13 may play an important role in degradation of type I collagen in bone matrix, acting in concert with cathepsin K and MMP-9 produced by osteoclasts. MMP-13 in perivascular cells may be involved in removal of cartilage matrix proteins such as type II collagen and aggrecan.

Animals↗

PubMed2004

Immunolocalization of vascular endothelial growth factor during heterotopic bone formation induced from grafted periosteum.

Vessel invasion is an important step in cartilage replacement that leads to bone formation, and vascular endothelial growth factor (VEGF) has been implicated as a key player in this process. Although grafted periosteum undergoes endochondral ossification, little is known about the role of VEGF in this process. In the current study the authors investigated by immunohistochemical, histochemical, and ultrastructural techniques the localization of VEGF during bone formation in periosteal grafts. At day 14 after grafting the tibias of Japanese white rabbits, periosteal cells in the grafted tissue had differentiated into chondrocytes to form cartilage. Some chondrocytes were immunopositive for VEGF expression, and subsequent vessel invasion occurred predominantly in these VEGF-positive areas. At day 45, the cartilage invaded by blood vessels had been replaced by newly formed bone. These findings suggest that VEGF is associated with the process of blood vessel invasion into cartilage before bone replacement in endochondral ossification from grafted periosteum.

Animals↗

PubMed2003

Immunohistochemical observations of cellular differentiation and proliferation in endochondral bone formation from grafted periosteum: expression and localization of BMP-2 and -4 in the grafted periosteum.

PURPOSE: To clarify the involvement of bone morphogenetic proteins (BMPs) in the proliferation and differentiation of osteo/chondrogenic cells during the process of bone formation from grafted periosteum. MATERIAL AND METHODS: Tibial periosteum of young Japanese white rabbits was grafted into suprahyoid muscles and removed after 7, 9, 14 or 21 days. BMP-2, -4, proliferative cell nucleus antigen (PCNA) immunoreaction and Alcian blue staining in grafted periosteum was then sought microscopically. RESULTS: PCNA positive cells in the grafted periosteum expressed BMP-2 at 7 days. These cells differentiated into chondroblasts that expressed BMP-2 and Alcian blue at 9 days. After 14 days, cartilage formation was seen, and BMP-2 and -4 expressions were observed in mature and hypertrophic chondrocytes. Endochondral ossification was observed at 21 days and osteoblasts showed both BMP-2 and -4 expression. CONCLUSION: Both BMP-2 and -4 appear to play regulatory roles in the process of endochondral ossification from grafted periosteum, due to their involvement in the proliferation and differentiation into chondrogenic and osteogenic cells.

Alcian Blue↗

PubMed2003

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein in the silkworm, Bombyx mori.

We have cloned the full length of a novel cDNA named Bombyx mori cuticle protein that contains an AlaAlaProAla/Val-repeat (BMCPA) from a cDNA library of integument in the larval silkworm. Both a typical tandem repeat (A-A-P-A/V) for cuticle protein and a unique tandem repeat with Ser, Ala, Gly, Pro, Val, Tyr and Thr were observed in the predicted amino acid sequence of the cDNA encoding BMCPA. Approximately 80% of the amino acids in BMCPA were composed of Ser, Ala, Gly, Pro, Val and Tyr. Northern-hybridization analysis indicated that BMCPA mRNA is expressed only in the larval epidermis and that the expression pattern of the BMCPA gene in the developmental stage was observed mainly at the larval stage. We propose BMCPA may be a novel component of cuticle, and may play an important role in the integument of the larval silkworm.

Amino Acid Sequence↗

PubMed2003

Regeneration of the mandibular head from grafted periosteum.

Grafted periosteum has a rich potential to induce heterotopic bone formation. In the current study the authors investigate whether autogenous periosteal grafts can regenerate the mandibular head in a rabbit model. They removed the mandibular head of Japanese white rabbits and grafted tibial periosteum to the cut surface of the mandible. Grafted periosteum was observed histologically and radiographically at day 7, 14, 21, and 45 after surgery. At day 7 after grafting, grafted tissue showed remarkable cell proliferation. By 14 days these cells had differentiated into chondrocytes to form cartilage, and endochondral ossification took place after 21 days. At 45 days after surgery, soft X-ray findings showed a newly formed mandibular head, which was similar histologically to that of a normal mandibular head. The cut mandible without periosteal graft showed no regeneration. These findings indicate that grafted periosteum can regenerate the mandibular head without special procedures such as bone fixation in a rabbit model, and suggest that this technique may be useful clinically.

Acid Phosphatase↗

PubMed2003

Role of osteoclast extracellular signal-regulated kinase (ERK) in cell survival and maintenance of cell polarity.

UNLABELLED: Morphological changes of osteoclasts by a MEK1 inhibitor, PD98059, were investigated to clarify a role of ERK. PD98059 promoted apoptosis of osteoclasts and the loss of ruffled borders. This study supports the importance of ERK in survival and polarity of osteoclasts. INTRODUCTION: Extracellular signal-regulated kinase (ERK) is a mitogen activated protein kinase (MAPK) that has been reported to play a role in the survival and apoptosis of osteoclasts. However, the precise signal transduction mechanism is not fully understood. The aim of this study was to clarify the role of ERK in osteoclasts by histological analysis. MATERIALS AND METHODS: Using a rat calvarial organ culture system, the inhibition of ERK phosphorylation by PD98059, a MAPK/ERK kinase 1 (MEK1) inhibitor, was assayed by immunoblotting. Morphological changes in osteoclasts induced by PD98059 were elucidated by light and electron microscopy. The cellular localization of ERK was also determined by immunoelectron microscopy. RESULTS: PD98059 inhibited phosphorylated ERK after a 1-h incubation. Ultrastructural study demonstrated that PD98059 induced the accumulation of vesicles and vacuoles in osteoclasts and the loss of ruffled border at 1 h. At 3 h, some osteoclasts showed apoptosis with nuclear condensation, and at 6 h after PD98059 treatment, many osteoclasts were detached from the bone surface and had lost their cell polarity. Electron microscopic immunohistochemistry revealed that ERK was mainly localized in the cytoplasm of clear zones in control osteoclasts, but apoptotic osteoclasts also showed immunoreactivity in clear zone-like structures in contact with osteoblast-lineage cells. CONCLUSION: These findings indicate that ERK in osteoclasts is involved in their survival and may be involved in the formation of a ruffled border and the maintenance of cell polarity.

Animals↗