Biomedical subjects

K Ohya

Publications and source records attributed to K Ohya.

At least 91 records · Page 5Linked to original sources

PubMed1997

Temporal changes of mRNA expression of matrix proteins and parathyroid hormone and parathyroid hormone-related protein (PTH/PTHrP) receptor in bone development.

Expression of parathyroid hormone and parathyroid hormone-related protein (PTH/PTHrP) receptor is one of the osteoblastic phenotypes; however, it has not been clear whether this phenotype expression is a marker of immature or mature osteoblasts. We examined the temporal expression pattern of PTH/PTHrP receptor in bone development in vivo and in vitro compared with the expression of other osteoblastic phenotypes: osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC), alkaline phosphatase (ALP), and mineralization. Total RNA was extracted from rat calvariae, and cell culture of rat bone marrow at different developmental stages and then Northern blot hybridization were performed. Mineralization was detected with contact microradiography (CMR) in calvaria or with Alizarin Red S staining in bone marrow cell culture. Both in calvaria and in marrow cell culture, extensive expression of OPN, BSP, type I collagen (COL I), and ALP coincided with the onset of mineralization, and OC expression was observed after mineralized tissue formation. Notably, PTH/PTHrP receptor was expressed at an early developmental stage (prenatal day 14 in calvaria, day 5 in culture) when mineralized tissue was not formed and other osteoblastic phenotypes were scarcely detected. Further study in cell culture revealed that the fold increase in cyclic adenosine monophosphate (cAMP) in response to PTH was elevated with the advance in the culture stage. These results indicate that mRNA expression of PTH/PTHrP receptor could be the early differentiation marker in osteoblastic lineage and that the levels of cAMP production in response to PTH represent the stage of osteoblastic differentiation.

Alkaline Phosphatase↗

PubMed1997

Expression of cyclooxygenase genes and involvement of endogenous prostaglandin during osteogenesis in the rat tibial bone marrow cavity.

Many researchers have demonstrated that the administration of prostaglandins (PGs), especially PGE2, increases the bone volume in vivo. It is still not clear how endogenous PG is associated with such bone formation. Cyclooxygenase (COX), which acts in the synthesis of PG from arachidonic acid, has been recently revealed to have two subtypes, a constitutive type (COX-1) and an inducible one (COX-2). We examined the expression of each COX subtype during osteogenesis with the established model of the rat tibial medullary cavity in which osteogenesis can be induced by the injection of colchicine. The expression of both COX-1 and COX-2 genes was enhanced after colchicine injection in the early stage before the start of bone formation. Only the COX-2 gene expression was elevated again later during the beginning of bone formation. Furthermore, the daily administration of indomethacin, COX inhibitor, could reduce the bone mass induced by colchicine in the rat tibiae. These data indicate that endogenous PGs are associated with osteogenesis in this model. Moreover, the present study suggests that COX-2 and COX-1 would both be involved in the early stage of osteogenesis, and COX-2 is likely to be more associated with the maturation of osteoblasts in the later stage.

Animals↗

PubMed1997

[Localization of DM-PK in normal and myotonic dystrophy muscles].

Myotonic dystrophy (DM) is an autosomal-dominant, multi-system disorder characterized by myotonia, progressive muscleatrophy and weakness. DM is also associated with smooth muscle, cardiac muscle, lens, endocrine and central nervous system abnormalities. The phenotypic expression of DM varies from asymptomatic adults to severely affected neonates. The genetic basis for DM is the expansion of a CTG repeat in the 3' end of a transcript that encodes a protein with putative serine/threonine protein kinase (myotonic dystrophy protein kinase, DM-PK). The predicted molecular weight of the full-length human DM-PK is about 69 kDa, while it may have some isoforms. DM-PK expression is observed in neuromuscular junction, muscle spindle, and sarcoplasm on both normal and DM muscles. Other muscular dystrophies, such as Duchenne and Becker type, DM-PK is intensively expressed in cytoplasm on immature regenerating fibers.

Adult↗

PubMed1996

A new mutation of the Po gene in patients with Charcot-Marie-Tooth disease type 1B: screening of the Po gene by heteroduplex analysis.

Most of Charcot-Marie-Tooth (CMT) 1 families are associated with a duplication in chromosome 17p11.2-p12, which includes the gene encoding peripheral myelin protein-22 (PMP-22). Point mutations of the Po gene have been identified in a few of the CMT 1 families in whom no duplication was found. We investigated a new mutation of the Po gene in one of those families. A to G substitution of nucleotide 389 in exon 3 resulted in Lys 131 Arg substitution. This structural change of extracellular domain of Po would alter the function of Po and result in an impairment of peripheral myelin compaction.

Adult↗

PubMed1996

Therapy-related leukemia with a novel 21q22 rearrangement.

We present a case of a 59-year-old Japanese man with therapy-related acute myeloblastic leukemia (AML) after the chemotherapy for non-Hodgkin's lymphoma (NHL). Accumulated doses of cyclophosphamide, procarbazine, doxorubicin, mitoxantrone, and etoposide were 18,300 mg, 3000 mg, 580 mg, 100 mg, and 4150 mg, respectively, which had been administered for the treatment of NHL. Myeloblasts in the peripheral blood increased 43 months after the onset of NHL. He was diagnosed as having AML (M2; FAB classification). The karyotype of the bone marrow cells in the present case contained the following abnormalities: t(2;21)(q21;q22), t(8;21)(q22;q22), and add(13)(q34). In the present case, 645 base pairs of chimeric mRNA were detected by reverse transcription-polymerase chain reaction, indicating the presence of AML1/MTG8 rearrangement. Translocation (2;21)(q21;q22) has not been described previously to our knowledge. It is interesting that the breakpoint of 21q22 existed both in t(2;21) and t(8;21). The disrupted AML1 gene resulting from two 21q22 rearrangements may be involved in the pathogenesis of AML in the present case. The clinical importance of therapy-related AML having the 21q22 rearrangement remains to be examined.

Aclarubicin↗

PubMed1996

Toxicity of camphorated phenol and camphorated parachlorophenol in dental pulp cell culture.

The toxicity of phenol, parachlorophenol, camphorated phenol, camphorated parachlorophenol, and camphor was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay on established rat dental pulp cells (RPC-C2A). RPC-C2A cells at the confluent stage were incubated for 24 h in an experimental medium containing each compound at different concentrations. All tested drugs showed cytotoxicity in the MTT assay in a concentration-dependent manner. It is believed that camphor is a vehicle, and it reduces the toxicity of phenol and parachlorophenol. However, camphor itself showed cytotoxicity, and the addition of camphor increased the toxicity of phenol and parachlorophenol, reconfirming the cytotoxicity of these classical antiseptics.

Analysis of Variance↗

PubMed1996

Insulin enhancement of in vitro wound healing in fetal rat parietal bones.

PURPOSE: This study evaluated the effect of insulin on fracture in fetal rat intramembranous bone using an organ culture system in which bone mineralizes in vitro. MATERIALS AND METHODS: Twenty-day-old fetal rat parietal bones were fractured parallel to the frontal plane and cultured in a serum-free medium with or without insulin for 4 to 12 days. They were then processed for light microscopy. RESULTS: In the specimens not treated with insulin, bony bridges were observed in less than 10% on day 4 and 8, and only 45% on day 12. In bones treated with insulin at 10(-6) mol/L, bony bridges were observed in 73% on day 4 and 100% on day 8 and 12. No callus or cartilage formation was observed during the wound repair process, indicating that intramembranous ossification occurred. CONCLUSION: These findings suggest that insulin has a direct effect on bone cells, which enhances the healing of fractures in intramembranous bone.

Animals↗

PubMed1996

Deletion of Src homology 3 domain results in constitutive activation of Tec protein-tyrosine kinase.

Tec protein-tyrosine kinase (PTK) is the prototype of a new subfamily of non-receptor type PTKs, and is abundantly expressed in hematopoietic tissues. We have revealed that Tec is inducibly tyrosine-phosphorylated and activated by stimulation with a wide range of cytokines. To get more insight into the signaling mechanism through Tec, we have generated a constitutively active form of Tec PTK. Deletion of the Src homology (SH) 3 domain gave rise to a hyperphosphorylated and activated Tec kinase (Tec deltaSH3). The activity of Tec deltaSH3 was confirmed in 293 cells, as well as in cytokine-dependent hematopoietic cells (BA/F3). Tec deltaSH3 should be a useful tool to study the in vivo substrates of Tec PTK.

Animals↗

PubMed1996

A new murine lymphocytotoxic monoclonal antibody recognizing HLA-A2, -A28 and -A9.

Monoclonal antibodies recognizing polymorphic as well as monomorphic epitopes on HLA antigens are important tools for understanding the immunobiology of HLA molecules. We immunized BALB/c mice with a HLA-A2 transfectant and screened for hybridomas which reacted with a HLA-A2 transfectant but not with a HLA-B75 transfectant. After subcloning by limiting dilution four times, a hybridoma secreting a monoclonal antibody (mAb) (IgG 2a, kappa) designated 1-145 was established. 1-145 reacted with Epstein-Barr virus transformed B lymphoblastoid cell lines (B cell lines) which expressed HLA-A2, -A28, -A23 and -A24. The titer of 1-145 in culture supernatant against HLA-A2 and -A28 antigens was similar and the titer against HLA-A23 was lower. 1-145 reacted with cells expressing HLA-A24 but the titer against HLA-A24 antigens was even lower than that against HLA-A23 antigens. The HLA-A24 antigens on the peripheral blood lymphocytes were not detected by 1-145 possibly due to the lower expression compared to the B cell lines. These differences of the titers were reflected to microlymphocytotoxicity assay in which 1-145 culture supernatant lysed all PBLs expressing HLA-A2,-A28 and -A23 but did not lyse PBLs expressing HLA-A24. Published deduced amino acid sequence data of HLA class 1 molecules indicate that Lys in position 127 may be critical for 1-145 binding.

Amino Acid Sequence↗

PubMed1996

CTG repeat size and histologic findings of skeletal muscle from patients with congenital myotonic dystrophy.

An approximate correlation has been demonstrated between the degree of CTG repeat expansion and clinical severity among myotonic dystrophy patients. Congenital myotonic dystrophy, which is the most severe form of the disease, has the largest size of CTG repeat. Muscle immaturity is a characteristic finding in congenital myotonic dystrophy muscle. We compared the CTG repeat size and histologic findings of skeletal muscle from patients with congenital myotonic dystrophy. An 8.6 kb or 9.8 kb plus an expanding band ranging from 15 kb to 17.5 kb was observed in muscle from five patients with congenital myotonic dystrophy by Southern blot analysis using EcoRI-digested DNAs probed with p5B1.4. There was no correlation between immaturity of skeletal muscle and the degree of CTG repeat expansion on skeletal muscle. Undetermined maternal factors may have an important role in the cause of immaturity of muscle in congenital myotonic dystrophy patients.

Blotting, Southern↗

PubMed1996

Immunocytochemical localization of myotonin protein kinase on muscle from patients with congenital myotonic dystrophy.

Using a polyclonal anti myotonin-protein kinase (M-PK) antibody against synthetic M-PK peptides corresponding to part of the amino acid sequence, and the immunohistochemical analysis of indirect immunoperoxidase, we have investigated localization of M-PK on muscle from patients with congenital myotonic dystrophy. In congenital myotonic dystrophy (MD) patients, one month and 3 months old, M-PK was weakly expressed at sarcolemma of muscle fibers. In congenital MD patients from 2 to 9 years of age, M-PK was clearly expressed at sarcolemma of muscle fibers. M-PK of immature muscle is weakly expressed at sarcolemma. With aging, M-PK is clearly expressed at sarcolemma of muscle from MD patient and normal control.

Adult↗

PubMed1995

Expression of bone sialoprotein mRNA during bone formation and resorption induced by colchicine in rat tibial bone marrow cavity.

In the rat tibial bone marrow cavity, following colchicine injection, there is a phase of osteogenesis in which bone trabeculae replace the necrotic bone marrow tissues and fill the marrow cavity. The newly formed bone is subsequently resorbed by osteoclasts and normal bone marrow is restored. In this study, we correlated these morphologic events with the pattern of gene expression of bone sialoprotein (BSP), an extracellular matrix protein in mineralized tissues, to elucidate the possible functions of BSP in bone formation and resorption in vivo. The expressions of osteopontin (OPN) and type I collagen were also examined. Northern hybridization of the tibia demonstrated that OPN mRNA was gradually increased and expressed at a maximal level 10 days after colchicine injection (during the bone resorption process), while BSP mRNA expression already reached a maximal level at day 6 (during the initial process of bone formation). Its expression was, thus, quite temporary at the beginning of bone formation and different from that of type I collagen, which was continually elevated from days 6 to 10. In situ hybridization of the newly formed bone induced in the tibia revealed that BSP mRNA was evenly expressed in most osteoblasts and osteocytes, moreover in interconnecting colonies of spindle-shaped cells, possibly preosteoblasts, at day 6. At day 10, however, its expression became restricted to some cells on the bone surfaces, some osteoblasts, and most osteoclasts. These observations suggest that BSP may play an important role mainly in the initiation of bone formation and is also associated with the functions of osteoclast in vivo.

Amino Acid Sequence↗

PubMed1995

MRI of peripheral nerves and pathology of sural nerves in hereditary motor and sensory neuropathy type III.

We investigated two patients with hereditary motor and sensory neuropathy type III, one with Déjérine-Sottas disease and the other with congenital hypomyelination neuropathy based on nerve pathology and MRI of the sciatic nerve. On biopsy of the sural nerve of the patient with Déjérine-Sottas disease, myelin debris, indicating demyelination, was observed in an onion-bulb pattern surrounding myelinated fibres. In the patient with congenital hypomyelination neuropathy, onion bulbs were formed of two parallel layers of basement membrane. There was no evidence of myelin breakdown. On axial T2-weighted MRI, a severely hypertropied sciatic nerve containing multiple rounded lesions, suggesting inflammation or demyelination, was observed in the patient with Déjérine-Sottas disease. In contrast, the sciatic nerve of the patient with congenital hypomyelination neuropathy showed slight hypertrophy without demyelination. MRI of the sciatic nerve may represent a useful tool for characterisation of demyelinating disease and its prognosis.

Adolescent↗

PubMed1995

Somatic cell heterogeneity between DNA extracted from lymphocytes and skeletal muscle in congenital myotonic dystrophy.

Myotonic dystrophy (DM) results from the expansion of an unstable CTG trinucleotide repeat in the 3' untranslated region of mRNA encoding a putative serine/threonine protein kinase. The degree of the CTG repeat amplification in genomic DNAs extracted from lymphocytes correlates with disease severity. We have analyzed the amplification of the CTG repeat of DNAs extracted from skeletal muscles and lymphocytes in five congenital DM patients. The amplification from skeletal muscles showed an increase of about 1.5 kb to 3.5 kb larger than that from lymphocytes in all patients. Furthermore, we have investigated the somatic instability of the CTG repeat in various tissues from a severe congenital DM patient.

Adolescent↗

PubMed1995

Expression of myotonic dystrophy protein kinase in biopsied muscles.

We present expression of myotonic dystrophy protein kinase (DM-PK) on biopsied muscles by immunocytochemistry using antibody against synthetic DM-PK peptide antigen. Immunolocalization of DM-PK was observed in neuromuscular junctions, muscle spindle, and sarcoplasm on both normal and DM muscles. DM-PK expression of sarcoplasm was present in adult normal and DM muscles. In Duchenne and Becker muscular dystrophies, DM-PK was intensively expressed in cytoplasm on immature regenerating fibers. DM-PK is initially produced in cytoplasm of regenerating fibers and migrates toward sarcoplasm with maturity of muscle cell.

Adult↗

PubMed1995

Minimal somatic instability of CTG repeat in congenital myotonic dystrophy.

The molecular basis of myotonic dystrophy was identified as an unstable trinucleotide (CTG) repeat in the 3' untranslated region of an mRNA encoding a member of the protein kinase family. Unstable DNA was analyzed from various tissue samples of a patient with severe congenital myotonic dystrophy. In BamHI- or BglI-digested DNA, a normal band plus differently expanded ones were identified in various tissue samples. These observations demonstrated somatic instability of the repeat in a congenital myotonic dystrophy patient.

Blotting, Southern↗

PubMed1995

Muscle involvement in congenital insensitivity to pain with anhidrosis.

A patient with congenital insensitivity to pain with anhidrosis, who had characteristic clinical features and biopsied sural nerve, is presented. Nerve pathology findings indicated a loss of the small myelinated and unmyelinated fibers. Biopsied muscle disclosed a marked variation in fiber size, some small fibers with central nuclei, and a small number of small angulated fibers, consistent with neurogenic and myogenic changes. Many patients with congenital insensitivity to pain with anhidrosis had muscle weakness and absent or decreased deep tendon reflexes with normal nerve conduction velocity. We confirmed that lack of small myelinated fibers in motor neurons resulted in a striking change of muscle in our patient.

Biopsy↗

PubMed1995

Expression of prostaglandin E receptor subtypes in bone: expression of EP2 in bone development.

Prostaglandin (PG) E2 displays physiological and pharmacological action in various tissues including bone. It increases intracellular Ca, and stimulates or inhibits cAMP production through the PGE receptor subtypes EP1, EP2, and EP3, respectively. These receptor subtypes have been recently cloned. In the present study, we investigate the expression of these receptor subtypes in bone tissue. RT-PCR revealed that EP1, EP2, and EP3 were expressed in rat calvariae and that osteoblastic cells (MC3T3-E1) expressed EP1 and EP2. In situ hybridization analysis using cryosection of neonatal calvariae revealed that EP2 was expressed by osteoblasts and cells not in contact with bone, probably including preosteoblasts. EP2 expression was observed at an early stage in calvarial development, at 14 days prenatal. EP2 expression was also observed at day 3 in rat bone marrow cell culture in which bone-like mineralized nodules are formed at day 8. It has been established that PGE2 response accompanying cAMP production is one of the characteristics of osteoblasts. The present results indicate that this phenotype appears at an early stage of osteoblastic differentiation and bone development.

3T3 Cells↗