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Biomedical subjects

K Ohya

Publications and source records attributed to K Ohya.

At least 73 records · Page 4Linked to original sources
PubMed1998

Grb10/GrbIR as an in vivo substrate of Tec tyrosine kinase.

BACKGROUND: Tec is a member of the recently emerging subfamily among nonreceptor protein-tyrosine kinases (PTKs). Although many members of this family have been shown to be involved in a wide range of cytokine-mediated signalling systems, the molecular mechanism by which they exert in vivo effects remains obscure. To gain insights into the downstream pathways of Tec, we here looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid screening. RESULTS: One of TIPs turned out to be Grb10/GrbIR, which carries one pleckstrin homology domain and one Src homology 2 domain. Grb10/GrbIR was known to bind receptor PTKs in a ligand-dependent fashion, but not to be phosphorylated on tyrosine residues. In a transient expression system in human kidney 293 cells, however, Grb10/GrbIR becomes profoundly tyrosine-phosphorylated by Tec, but not by Syk, Jak2 or insulin receptor. We also reveal that expression of Grb10/GrbIR suppresses the cytokine-driven and Tec-driven activation of the c-fos promoter. CONCLUSION: Our results indicate a novel role of Grb10/GrbIR as an effector molecule to a subset of nonreceptor PTKs.

Amino Acid Sequence↗
PubMed1998

Anti-Jo-1 antibodies in polymyositis or dermatomyositis: evaluation by ELISA using recombinant fusion protein Jo-1 as antigen.

We evaluated an enzyme-linked immunosorbent assay (ELISA) for detecting anti-Jo-1 antibodies in patients with polymyositis (PM) or dermatomyositis (DM) by use of the recombinant fusion protein Jo-1. Sera from 64 patients with PM or DM, from 80 patients with other connective tissue diseases, and from 64 healthy subjects matched for age, sex and race, were studied by the ELISA and by the double immunodiffusion (DID) method. Eight patients with myositis (six PM, one DM and one DM with malignancy) with positive anti-Jo-1 by DID also showed positive results by the ELISA method, whereas five patients with positive anti-Jo-1 by this ELISA showed negative results on DID. One of the five had non-specific results. The incidence of positive results for anti-Jo-1 with the ELISA (18.8%) was greater than that for DID (12.5%), but the difference was not statistically significant. All patients with positive results for anti-Jo-1 by DID were also positive by the ELISA. The ELISA system with the recombinant Jo-1 antigen was useful in the detection of anti-Jo-1 antibodies in patients with PM/DM.

Adolescent↗
PubMed1998

Demonstration of putative Ca-binding domains in dentin matrix of rat incisors after daily injections of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP).

In order to clarify the initial process of dentin mineralization, the inhibitory effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) on dentin mineralization was investigated. Rats (100 g) were subcutaneously injected with HEBP (8 mg P/kg) for 7 or 14 d, and the incisors were processed for Ca histochemistry and/or electron microscopy. HEBP-treated incisors demonstrated ladder-like alternate rows of mineralized and non-mineralized dentin at the apical end. GBHA revealed moderate Ca reactions in the non-mineralized circumpulpal dentin matrix where electron microscopy revealed rich distribution of fine mesh-like electron-dense material. Non-mineralized mantle dentin matrix was negative for Ca but contained numerous matrix vesicles (MVs) filled with crystalline and/or amorphous mineral deposits. Mineralization of circumpulpal dentin occurred independently of MV-rich mantle dentin layer in affected specimens. Our data provide histochemical evidence of possible Ca-binding property of the circumpulpal dentin matrix and its absence in the mantle dentin where MV-mediated mineralization occurs. In the mantle dentin, HEBP does not interfere with crystal growth in MVs but inhibits its outgrowth after membrane rupture. It is proposed that circumpulpal dentin matrix has a potential to mineralize independently of MV-mediated mineralization of mantle dentin, although MVs determine the initial site and timing of dentin mineralization.

Aminophenols↗
PubMed1998

cDNA cloning of S100 calcium-binding proteins from bovine periodontal ligament and their expression in oral tissues.

The periodontal ligament (PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. To characterize PDL cells at the molecular level, we constructed a cDNA library from bovine PDL tissue. We then focused on the isolation of S100 calcium-binding proteins (CaBPs), because they mediate Ca2+ signaling and control important cellular processes such as differentiation and metabolism. We screened the PDL cDNA library with a mouse S100A4 cDNA, and cloned the bovine cDNAs of two S100 CaBPs (S100A4 and S100A2). In northern blotting analysis, the highest expression of S100A4 was detected in PDL from erupted teeth (PDLE). PDL from teeth under eruption (PDLU) showed a lower expression of S100A4, and its expression in gingiva was faintly detectable. S100A4 expression was also high in the pulp tissue followed by the dental papilla of the tooth germ. S100A2 expression was high in PDLE and gingiva. Interestingly, only PDLE exhibited a high expression of both S100A4 and S100A2. PDLE also expressed the highest level of beta-actin, a target cytoskeletal protein for S100A4. It is conceivable that the high expression of S100A4 in PDLE is a result of the maturation of the PDL and/or a response to mechanical stress generated by mastication. Since there was a marked difference of S100A4 expression between PDL and gingiva, we propose that S100A4 could be a useful marker for distinguishing cells from these two tissues.

Amino Acid Sequence↗
PubMed1997

Tomaculous neuropathy in Charcot-Marie-Tooth disease with myelin protein zero gene mutation.

Mutation of the myelin protein zero (MPZ) gene is associated with a small number of Charcot-Marie-Tooth (CMT) patients. We present a patient with Lys 130 Arg substitution in the extracellular domain who showed tomacula formation in biopsied sural nerve. CMT patients with mutations Ly 96 Glu, Lys 130 Arg and Ile 135 Leu showed tomaculous neuropathy. Present and previously reported investigations suggest that the pathological phenotypes of peripheral nerve are probably related to the mutations of the MPZ gene.

Amino Acid Substitution↗
PubMed1997

Congenital muscular dystrophy with partial deficiency of merosin.

We present a Japanese patient who has congenital muscular dystrophy, with partial merosin deficiency. The patient had characteristic findings of clinical features and brain MRI. Muscle biopsy showed advanced muscular dystrophy, with greatly reduced muscle fibers and massive infiltration of interstitial connective and fatty tissues. On immunostaining for cytoskeletal proteins, merosin was greatly reduced. The other cytoskeletal proteins, including dystrophin and 50 kDa alpha-sarcoglycan were normally expressed around all muscle fibers.

Child, Preschool↗
PubMed1997

Selective inhibitors of cyclic AMP-specific phosphodiesterase: heterocycle-condensed purines.

To reverse the adverse reactions of alkylxanthines and to develop novel inhibitors of cyclic AMP-specific phosphodiesterase (PDE IV), a series of heterocycle-condensed purines were designed and synthesized. Some of these new compounds had similar or more potent and selective inhibitory activity against PDE IV than known PDE IV inhibitors. The tracheal-relaxant activity of these compounds was closely correlated with their PDE IV-inhibitory activity. Moreover, these purine analogues did not have any positive-chronotropic action or adenosine-antagonistic action on isolated heart preparations, which are the particular adverse reactions of alkylxanthines. Among them, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (1c), which was the most selective and potent PDE IV inhibitor, did not cause emesis in Suncus murinus at a dosage range of 10-100 mg/kg (po), while an imidazole analogue of 1c (4c) and known PDE IV inhibitors such as rolipram and denbufylline caused emesis even at 10 or 30 mg/kg.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Reduction of bone loss by denbufylline, an inhibitor of phosphodiesterase 4.

The effects of denbufylline, a xanthine derivative with selective inhibitory activity on the phosphodiesterase (PDE) 4 isoenzyme, on bone loss in Walker 256/S-bearing rats and on mineralized nodule formation and osteoclastlike cell formation in bone marrow culture systems were examined. Serial oral administrations of denbufylline inhibited the decrease in the bone mineral density of femurs from Walker 256/S-bearing rats, without influence on the healthy rats. Denbufylline restored the bone mass and the number of osteoclasts and osteoblasts per trabecular surface in the femur metaphysis. Among PDE inhibitors, only PDE4-selective inhibitors increased the number of mineralized nodules and decreased the number of osteoclastlike cells in the in vitro bone marrow culture systems, and dibutyryl cyclic AMP mimicked these effects in the in vitro systems. These results suggest that the PDE4 isoenzyme may play an important role in bone turnover through cyclic AMP and that its inhibitors are candidates for therapeutic drugs for the bone loss diseases.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Detection of the CTG repeat expansion in congenital myotonic dystrophy.

Myotonic dystrophy (DM) is caused by an abnormal expansion of an unstable CTG trinucleotide repeat in the 3' untranslated region of mRNA encoding a putative serine/threonine protein kinase. We analyzed 59 patients with DM (28 congenital DM families: 27 families with maternal transmission and 1 paternal transmission) and 27 normal control subjects to evaluate their CTG repeat size between DM patients and the normal controls, and to search for a correlation between the clinical characteristics of congenital DM (CDM) and CTG repeat expansions. Analysis was on the basis of the Southern blot and polymerase chain reaction (PCR) methods, and by direct sequencing of PCR amplified CTG repeats. Analysis of intergenerational differences in the CTG repeat size for mother-child pairs showed a positive correlation (y = 1.0384x + 1265.2, r2 = 0.311). In addition to the strong parental bias, this group showed genetic anticipation. There was a significant correlation of the CTG repeat expansion with disease severity. The largest CTG repeat expansion (2,293 CTG repeats) on average belonged to the severe CDM group, and the smallest (129 CTG repeats) to the subclinical DM group. The mutant allele of an asymptomatic father in the paternally transmitted pedigree revealed 75 CTG repeats, demonstrating that he was a DM protomutation carrier.

Blotting, Southern↗
PubMed1997

Adenosine stimulates nitric oxide synthesis in vascular smooth muscle cells.

OBJECTIVE: The aim was to investigate the effects of adenosine on nitric oxide (NO) synthesis in vascular smooth muscle cells. METHODS: NO and cAMP synthesis was measured in confluent rat vascular smooth muscle cells in culture at passage 5-10, using Griess reagent and an enzyme immunoassay kit, respectively. The expression of inducible NO synthase mRNA was assayed by Northern blotting. RESULTS: Incubation of cultures with interleukin-1 beta (10 ng/ml) for 24 h caused a significant increase in nitrite production. The interleukin-1 beta-induced nitrite production by vascular smooth muscle cells was significantly increased by adenosine or its stable analogue, 2-chloroadenosine, in a dose-dependent manner. The adenosine A2a receptor antagonist, KF17837, but not the A1 receptor antagonist, DPCPX, significantly inhibited 2-chloroadenosine-mediated nitrite production. The 2-chloroadenosine-mediated nitrite production by interleukin-1 beta-stimulated cells was accompanied by increased inducible NO synthase mRNA accumulation. In the presence of dibutyryl-cAMP (1 mM), interleukin-1 beta-induced nitrite accumulation was further increased, but the effect of 2-chloroadenosine was not additive or synergistic. Addition of 2-chloroadenosine dose-dependently increased intracellular cAMP levels of vascular smooth muscle cells. CONCLUSIONS: These results indicate that adenosine acts on A2 receptors and augments NO synthesis in interleukin-1 beta-stimulated vascular smooth muscle cells, at least partially through a cAMP-dependent pathway.

2-Chloroadenosine↗
PubMed1997

Molecular and pathological studies in Charcot-Marie-Tooth disease 1A.

We analyzed a 1.5-Mb duplication of the p11.2-12 region of chromosome 17, including the PMP-22 gene (CMT1A duplication), seven families with Charcot-Marie-Tooth disease type I (CMT I) and six sporadic patients with suspected CMT I by Southern blot analysis. In order to detect the CMT 1A duplication, probe pVAW409R3a, probe PMP-22 cDNA and reference probe SF85 were used for Southern hybridization. In six out of seven families with CMT I, CMT1A duplication was identified. One of six sporadic CMT patients had CMT1A duplication. The probe pVAW4O9R3a was more informative than PMP-22 cDNA and SF85 for detecting CMT1A duplication. In pathological study of biopsied sural nerve, thickened myelin sheath was observed in some myelinated fibers in patients with CMT1A duplication.

Adolescent↗
PubMed1997

Haplotype analysis of congenital myotonic dystrophy patients from asymptomatic DM father.

We report a family with congenital myotonic dystrophy (CDM) transmitted from an asymptomatic DM father; we analyzed the haplotype of this family by using polymorphism within and by flanking the DM protein kinase locus. One patient with congenital DM was homozygous for all markers studied, except for the expanded CTG repeats. Two other patients with congenital DM were heterozygous. One patient with congenital DM who was homozygous had greater clinical severity and more expanded CTG repeats than other CDM patients who were heterozygous. The asymptomatic father had a DM protomutation.

Child↗
PubMed1997

De novo mutation of Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT 1A) is an autosomal dominant demyelinating polyneuropathy associated with a 1.5-Mb duplication of the p11.2-p12 region of chromosome 17, including the peripheral myelin protein-22 (PMP-22) gene (CMT 1A duplication). We report a male patient with a de novo CMT 1A diagnosed on clinical, electrophysiologic, and molecular grounds. Motor nerve conduction velocity (MCV) of the patient was 10.9 m/s in the ulnar nerve. The MCV of both his parents was within the normal range. Southern blot analysis of BamHI digestion showed reduced intensity rate of SF85/PMP-22, indicating CMT 1A duplication. Haplotype analysis with pVAW4093a, demonstrated that the de novo CMT 1A duplication was of paternal origin.

Blotting, Southern↗
PubMed1997

Effects of surface roughness of titanium implants on bone remodeling activity of femur in rabbits.

We investigated the bone remodeling activity on titanium implants with different surface roughnesses using a confocal laser scanning microscope (CLSM). Two kinds of implants were used, the machined smooth-surfaced titanium and the plasma-sprayed rough-surfaced titanium. These implants were randomly inserted in a rabbit's femur from the lateral aspect of the diaphysis bicortically. Rabbits were killed at 6, 16, and 42 weeks after surgery. The implant-bone blocks were embedded in polyester resin, and were prepared to make undecalcified ground sections. Histomorphometric analyses were performed at the cortical bone-implant interface using the image obtained by CLSM. Percentages of direct bone-implant contact and bone volume (BV/TV) around the implant was greater in rough-surfaced titanium compared with the smooth-surfaced titanium at 42 weeks after implantation. On the contrary, the eroded surface (ES/BS) appeared to be less in the rough-surfaced titanium than in the smooth-surfaced titanium at 6 weeks after implantation, but thereafter, no difference was found between the two kinds of implants. Mineralizing surface (MS/BS) and mineral apposition rate (MAR) showed no significant differences throughout the experimental period. These results indicate that increased bone volume in the rabbits of rough-surfaced titanium implants is due to less remodeling activity during the early stage after implantation compared with the smooth-surfaced implants. The surface roughness of titanium is one factor which helps in determining the balance between bone formation and resorption of remodeling at the interface of the bone implants.

Animals↗
PubMed1997

Detection of the mutation in facioscapulohumeral muscular dystrophy patients.

The gene responsible for facioscapulohumeral muscular dystrophy (FSHD) was mapped to chromosome 4q35 by linkage analyses. Recently, the probe p13E-11 derived from the cosmid clone 13E, which has been mapped to 4qter, detected a polymorphic EcoRI fragment, usually greater than 28 kb in normal individuals. In sporadic and familial FSHD patients, a specific shorter fragment, usually smaller than 28 kb, was found to cosegregate with FSHD. Two FSHD patients are presented here. Patient 1 is a sporadic case of FSHD with healthy parents. He had a de novo mutation identified by Southern blot analysis using the above-mentioned probe. Patient 2 is a typical familial FSHD patient clinically and histologically. A rearranged and shortened EcoRI fragment was identified by molecular analysis. Southern blot analysis using the probe p13E-11 also indicated a rearranged EcoRI fragment in both patients.

Alleles↗
PubMed1997

Deficiency of syntrophin, dystroglycan, and merosin in a female infant with a congenital muscular dystrophy phenotype lacking cysteine-rich and C-terminal domains of dystrophin.

Primary deficiency of merosin is the cause of the classic form of congenital muscular dystrophy (CMD) accompanied by brain white matter abnormalities. We report a female infant with dystrophinopathy who was deficient in merosin in skeletal muscle. The patient had a phenotype of typical CMD and white matter abnormalities on brain MRI. Merosin was greatly reduced in the biopsied skeletal muscle. However, the expression of dystroglycan and syntrophin was also greatly reduced, and the immunoreactivity for the antibodies against the cysteine-rich/C-terminal domains of dystrophin was absent in the sarcolemma. Reverse transcriptase polymerase chain reaction analysis of the dystrophin gene revealed a complete lack of exons 71 through 74. In skeletal muscle, only the mutant gene was expressed. These results suggest that the patient is a symptomatic Duchenne muscular dystrophy carrier with skewed X-inactivation. This patient illustrates for the first time that a dystrophin abnormality can cause a secondary deficiency of merosin in dystrophinopathy. The reduction of merosin may account for the clinical phenotype of CMD and correlate with the white matter abnormalities in our patient.

Base Sequence↗