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Biomedical subjects

K Ohya

Publications and source records attributed to K Ohya.

At least 19 recordsLinked to original sources
PubMed2007

Molecular characterization of infectious bursal disease virus (IBDV): diversity of very virulent IBDV in Tanzania.

Nucleotide sequences of the VP2 hypervariable region (VP2-HVR) of 14 infectious bursal disease viruses (IBDVs) isolated in Tanzania from 2001 to 2004 were determined. Phylogenetic analysis showed that the isolates diverged into two genotypes and belonged to the very virulent (VV) type. In the phylogenetic tree, strains in one genotype clustered in a distinct group and were closely related to some strains isolated in western Africa, with nucleotide similarities of 96.1-96.8%, while strains in another genotype were clustered within the European/Asian VV type with nucleotide similarities ranging from 97.5 to 99.3%. Both genotypes were widely distributed throughout Tanzania, and had conserved putative virulence marker amino acids (aa) at positions 222(A), 242(I), 256(I), 294(I) and 299(S). Our findings demonstrate for the first time the existence of both African and European/Asian VV-IBDV variants in Tanzania.

Amino Acid Sequence↗
PubMed2006

Medullary sponge kidney in a 10-year-old Shih Tzu dog.

Medullary sponge kidney was diagnosed in a 10-year-old male Shih Tzu dog with a long history of hyposthenuria, but with no other findings indicating renal failure or hormonal aberration. At the dog's death from heart failure, an autopsy was performed. On gross morphology, bilateral kidneys were normal size and had many cysts ranging from the corticomedullary junction to renal papillae. Histopathologic findings showed that almost all of the cysts were lined by monolayered or multilayered and columnar or cuboidal epithelium with chilium similar to epididymis. Immunohistochemically, all of these cells were strongly positive for AE1/AE3 and negative for vimentin. Many of these cells were positive for cytokeratin 7 (CK7), and only a few cells were positive for desmin. The results of staining are the same as those for epithelium of the collecting duct of normal canine kidney. This is the first report of this pathologic entity in the canine kidney.

Animals↗
PubMed2005

Contribution of Va24Vb11 natural killer T cells in Wilsonian hepatitis.

Wilson disease (WD) is an autosomal recessive disorder of copper transport, resulting in copper accumulation and toxicity to the liver and brain. There is no evidence that the WD patient's immune system attacks copper accumulated hepatocytes. Here we describe that the frequency and absolute number of Valpha24+Vbeta11+ natural killer T (NKT) cells were significantly increased in 3 cases of WD, whereas those of CD3+CD161+ NKT cells were within the normal range. Patients no. 1 and 2 had a presymptomatic form of WD. Their tissue specimens showed pathological changes of mild degeneration of hepatocytes with a few infiltrating mononuclear cells and a low degree of fatty change. Patient no. 3 displayed fulminant hepatitis with Coombs-negative haemolytic anaemia. The tissue specimens of patient no. 3 showed macronodular cirrhosis with thick fibrosis, inflammatory infiltrates and spotty necrosis. Human Valpha24+Vbeta11+ NKT cells are almost equal to CD1d-restricted NKT cells. Therefore we investigated CD1d-restricted NKT cells in the LEC rat as an animal model of WD. In LEC rats before hepatitis onset, the number and phenotype of liver NKT cells were normal. At about 4 months of age all LEC rats developed acute hepatitis accompanied by acute jaundice, and CD161high NKT cells developed in their livers. CD161highalphabetaTCRbright NKT cells developed in some of them. Their hepatitis was severe. CD161highalphabetaTCRbright NKT cells expressed an invariant rat Valpha14-Jalpha281 chain, which is CD1d-restricted. Furthermore, liver lymphocytes in the acute jaundiced LEC rats with CD161highalphabetaTCRbright NKT cells had significant and CD1d-specific cytotoxic activity.

Adolescent↗
PubMed2004

The deficiency of immunoregulatory receptor PD-1 causes mild osteopetrosis.

Recently, the involvement of immune responses in metabolic bone disease and/or local bone destruction has received much attention. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), a member of the immunoglobulin (Ig) superfamily, negatively regulates T cell activation. The deficiency of CTLA-4 induces profound osteopenia with an increase in osteoclastogenesis, suggesting the important role of activated T cells in osteoclastogenesis. Programmed death-1 (PD-1) is the newly identified immunoregulatory receptor, which also belongs to the Ig superfamily. Both CTLA-4 and PD-1 are induced on activated T cells, however, there are no reports linking PD-1 with osteoclasts. In the present study, we have examined the bone phenotype in PD-1-deficient mice PD-1-/- and the role of PD-1 in osteoclastogenesis and osteoclast function. Both trabecular and cortical bone mineral densities of tibia were significantly increased, as observed in peripheral quantitative computed tomography (pQCT), at 12 weeks of age in PD-1-/- mice. Histomorphometric analysis of the PD-1-/- mice and the age-matched controls at 12 weeks of age showed a 2-fold increase in bone volume (BV/TV) with a 55% decrease in osteoclast number (N.Oc/BS). Bone formation indices were similar in both groups. The number of soluble receptor activator of nuclear factor kappaB ligand (sRANKL)-induced osteoclast-like cells (OCLs) derived from the PD-1-deficient splenocytes was significantly decreased (by 25%). On the other hand, PD-1 deficiency did not affect the bone-resorbing activity of mature osteoclasts. Our results suggest that PD-1 deficiency reduces osteoclastogenesis resulting in an osteopetrotic phenotype. Identical members of the Ig superfamily, CTLA-4 and PD-1, which negatively regulate immune responses, may differentially affect osteoclastogenesis and bone remodeling.

Animals↗
PubMed2003

Bone mineral density of the mandible in ovariectomized rats: analyses using dual energy X-ray absorptiometry and peripheral quantitative computed tomography.

INTRODUCTION: Although previous studies have shown that maxillary molar extraction in ovariectomized (OVX) animals causes mandibular loss of bone, it is still questionable as to whether estrogen deficiency affects mandibles with functional occlusion. MATERIALS AND METHODS: To answer this question, 13-week-old female Sprague-Dawley rats were bilaterally OVX or sham-operated. After 109 days, the bone mineral density (BMD) of the femurs and mandibles was measured using dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). RESULTS: In DEXA analysis, although the BMD of the total mandible of the OVX rats was similar to that of the sham-operated rats, the BMD of the condylar region in the OVX rats had decreased by 12.5%. In pQCT analysis, decrease in trabecular BMD of the mandibular bone was detectable but low in the molar region (maximal 13%), whereas no difference was seen in cortical BMD. In the femurs, the trabecular bone prominently decreased in OVX rats (30% decrease in pQCT analysis) as previously reported. CONCLUSION: This study revealed regional differences in the mandibular bone decrease in OVX rats. Although the mechanism of low susceptibility of the mandible to estrogen-deficient conditions remains unknown, it is likely that mechanical stress derived from functional occlusion is preventing bone loss in this pathological condition. Furthermore, this study demonstrated the advantage of pQCT in analyzing rat mandibular bone.

Absorptiometry, Photon↗
PubMed2002

Localization of cathepsin K in bovine odontoclasts during deciduous tooth resorption.

Cathepsin K is a cysteine proteinase, which is abundantly and selectively expressed in osteoclasts. It is believed to play an important role in the proteolysis of bone resorption by osteoclasts. The objectives of this study were to investigate the association of cathepsin K in the physiological root resorption of deciduous teeth and to identify the cathepsin K-producing cells in deciduous root resorption. RT-PCR and Northern blot analysis of the total RNAs extracted from bovine active and resting root-resorbing tissues, which lie between the root of deciduous tooth and its permanent successor, were performed. The active root-resorbing tissue, which has a high population of odontoclasts on its surface that is attached to resorbing root surface, showed an extremely high expression of cathepsin K in comparison with the resting root-resorbing tissue. By in situ hybridization, cathepsin K mRNA was highly and selectively expressed in multinucleated odontoclasts that aligned along the surface of the tissue and apposed to the resorbing root surface of the deciduous tooth. Western blot analysis of the active root-resorbing tissue was used to characterize the anti-cathepsin K antibody. A band of 27 kDa, corresponding with the predicted size for mature cathepsin K, was demonstrated. Immunohistochemistry confirmed the specific localization of cathepsin K protein to the odontoclasts. These results demonstrate that odontoclasts in the deciduous root resorption express cathepsin K mRNA and protein that may participate in the proteolysis of root resorption of the deciduous tooth.

Animals↗
PubMed2002

Enhancement of osteogenesis on hydroxyapatite surface coated with synthetic peptide (EEEEEEEPRGDT) in vitro.

Some dental implants are coated with hydroxyapatite (HA), which preferentially binds to bone. Several matrix proteins have an arginine-glycine-aspartic acid (RGD) sequence where cells attach via an integrin receptor. We hypothesized that coating an HA surface with an RGD-containing peptide might enhance the attachment and differentiation of osteoblasts. The HA disks (diameter 34 mm, thickness 1 mm) were treated with a solution (50 mM Tris/HCl and 150 mM NaCl, pH 7.4) containing the peptide EEEEEEEPRGDT, in which the E repetition exerts a high affinity to HA. After washing with phosphate-buffered saline, KUSA/A1 mouse osteoblastic cells were inoculated onto the HA surface and cultured. After 30 min, the number of cells attached to the surface was counted. The DNA content and alkaline phosphatase (ALP) activity were measured after 10 days in culture. Expression of bone matrix proteins was also examined by means of reverse transcriptase-polymerase chain reaction at 7 days; the mineralized area of the culture was also evaluated by staining with Alizarin Red S after 10 days. Treatment with the peptide stimulated cell attachment and increased DNA content and ALP activity. Furthermore, matrix protein expression and mineralized nodule formation were enhanced to a greater extent on the peptide-treated surface than on the nontreated surface. Our results indicate that coating an HA surface with RGD-containing peptide enhances osteoblast attachment and differentiation. This peptide treatment of HA-coated implants may stimulate the osseointegration of the implants.

Animals↗
PubMed2002

Hypergravity modulates behavioral nociceptive responses in rats.

Hypergravity (2G) exposure elevated the nociceptive threshold (pain suppression) concomitantly with evoked neuronal activity in the hypothalamus. Young Wistar male rats were exposed to 2G by centrifugal rotation for 10 min. Before and after 2G exposure, the nociceptive threshold was measured as the withdrawal reflex by using the von Frey type needle at a total of 8 sites of each rat (nose, four quarters, upper and lower back, tail), and then rats were sacrificed. Fos expression was examined immunohistochemically in the hypothalamic slices of the 2G-treated rats. When rats were exposed to 2G hypergravity, the nociceptive threshold was significantly elevated to approximately 150 to 250% of the 1G baseline control levels in all the examination sites. The 2G hypergravity remarkably induced Fos expression in the paraventricular and arcuate nuclei of the hypothalamus. The analgesic effects of 2G hypergravity were attenuated by naloxone pretreatment. Data indicate that hypergravity induces analgesic effects in rats, mediated through hypothalamic neuronal activity in the endogenous opioid system and hypothalamo-pituitary-adrenal axis.

Animals↗
PubMed2002

Synergistic effect of fibroblast growth factor-4 in ectopic bone formation induced by bone morphogenetic protein-2.

Bone morphogenetic protein family members (BMPs) are essential signaling molecules during limb development and, in this process, fibroblast growth factor family members (FGFs) cooperate with BMPs. FGFs also exert anabolic effects in bone when systemically or locally applied. Thus, it is likely that the cooperation with FGFs also occurs in BMP-induced ectopic bone formation and that the exogenous FGF application would promote this bone formation. In the present study, after subcutaneously implanting recombinant human BMP-2 (rhBMP-2) in rats, we examined the expression of FGF-4 and FGF receptors (FGFRs) mRNAs and the effect of exogenous recombinant human FGF-4 (rhFGF-4) on bone formation. Three days after implantation, the pellets containing rhBMP-2 were surrounded by fibroblastic mesenchymal cells; on day 7, cartilage tissue appeared; on day 10, hypertrophic chondrocytes and a small amount of mineralized tissue were observed; and, on day 14, the amount of mineralized tissue increased. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that FGF-4 expression appeared at early stages (days 3 and 7) and its expression decreased at later stages (days 10, 14, and 21), whereas FGFRs were expressed continuously. In situ hybridization revealed that, on days 3 and 7, FGF-4, and FGFR subtypes 1 and 2 (FGFR-1 and FGFR-2) were expressed in mesenchymal cells and chondrocytes, and in the area of alkaline phosphatase (ALP) expression. On day 10, FGF-4 was not detected, whereas the expression of FGFR-1 and FGFR-2 was detectable in the area of alkaline phosphatase (ALP) expression. Injection of rhFGF-4 on days 2, 3, and 4 enhanced the mineralized tissue formation induced by rhBMP-2; however, neither rhFGF-4 treatment on days 6, 7, and 8 nor rhFGF-4 treatment on days 9, 10, and 11 influenced the amount of rhBMP-2-induced mineralization. Our results indicate that FGF-4 and FGFR signals play important roles during rhBMP-2-induced bone formation. We further suggest that the combination of rhBMP-2 and rhFGF-4 would be useful for bone augmentation.

Animals↗
PubMed2002

Expression of matrix metalloproteinase-9 mRNA and protein during deciduous tooth resorption in bovine odontoclasts.

Matrix metalloproteinase-9 (MMP-9, or gelatinase B) is an extracellular proteinase that is highly expressed in osteoclasts and has been postulated to play an important role in their resorptive activity. Although MMP-9 has been reported to play a role in bone resorption, the association of this enzyme during deciduous tooth resorption has not yet been clarified. The purpose of the present study was to increase our understanding of the role of MMP-9 during deciduous tooth resorption. Reverse transcription-polymerase chain reaction (RT-PCR) and northern blot analysis of total RNAs extracted from bovine root-resorbing tissues, which lie between the root of a deciduous tooth and its permanent successor, revealed the expression of mRNA for MMP-9 in the tissue. These results indicate that MMP-9 may be involved in the process of deciduous tooth resorption. In addition, in situ hybridization and immunohistochemistry were also performed to identify the cells that produced MMP-9 in bovine root-resorbing tissue. MMP-9 mRNA was highly expressed in odontoclasts that were aligned along the surface of the tissue. Immunohistochemistry confirmed the predominant localization of MMP-9 in odontoclasts. The present data demonstrate that odontoclasts in deciduous root resorption express MMP-9, which may participate in proteolysis during root resorption of deciduous tooth.

Animals↗
PubMed2002

Efficient selection of genomic clones from a female chicken bacterial artificial chromosome library by four-dimensional polymerase chain reactions.

A bacterial artificial chromosome (BAC) library consisting of 49,152 genomic clones was constructed from partially HindIII-digested female chicken embryo genomic DNA using the pBAC-Lac vector and maintained in 512 96-well plates. The mean insert size was approximately 150 kb, and the total library was estimated to contain about 3.2 times coverage of the diploid genome. In order to screen this library by the PCR, 296 BAC clone DNA samples were prepared: one sample each from 8 superpools (64 plates per superpool) and 36 samples of four-dimensionally (4-D) mixed clones from each superpool. A BAC clone of interest was selected by two-step PCR. First, 8 DNA samples representing superpools were subjected to PCR with a set of primers to amplify a part of the genomic sequence of interest. Second, 36 4-D DNA samples from the superpool that contained BAC clone(s) of interest were subjected to PCR with the same set of primers. The second step identified a plate and a well containing the BAC clone of interest. Selection of target BAC clone(s) from the whole library with the above procedure can be achieved within 1 to 4 d without using a radioactive probe. This procedure was applied successfully in the selection of BAC clones for Wpkci, chPKCI/HINT, ZOV3, and 17beta-HSD genes.

Amino Acid Sequence↗
PubMed2001

A comparative study of clinical features between pure checkers and pure washers categorized using a lifetime symptom rating method.

The current study assessed lifetime obsessive-compulsive disorder (OCD) symptoms in 156 Japanese patients with OCD in order to investigate clinical differences between pure lifetime checkers and pure lifetime washers. Fifty subjects (32%), who had no lifetime history of washing or checking compulsions, or who had a principal symptom other than washing or checking compulsions, were initially excluded. The remaining 106 subjects were divided into three groups: 43 pure lifetime washers (W), 33 pure lifetime checkers (C) and 30 subjects who had experienced both checking and washing compulsions over their lifetime (WC). No differences in clinical characteristics were observed between the W and C groups, suggesting that the lifetime washer-checker distinction may not be useful in subtyping OCD. However, subjects in the WC group differed from other subjects in a number of respects including poorer level of insight, more severe psychopathology and global dysfunction. Thus, they can be generally distinguished by more pervasive and severe psychopathological features, and may constitute a valid subgroup of OCD. Further work from a multidimensional perspective is required to verify the clinical significance of a typology based on lifetime symptoms.

Adolescent↗
PubMed2001

Isoform-dependent interaction of BRDG1 with Tec kinase.

Tec is the prototype of an emerging family of protein-tyrosine kinases. Tec and Btk, another member of this family, together participate in the development of B-cell immune system. We previously identified one of the downstream messengers for human Tec kinase, BRDG1. BRDG1 is associated with Tec and becomes tyrosine-phosphorylated in B-cells by the engagement of B-cell antigen receptor (BCR). Here we show that overexpression of BRDG1 strongly augments BCR-mediated activation of cAMP-response element binding protein (CREB) but not that of c-Jun and the promoters of c-MYC and BCL-xL genes. Furthermore, we isolated the murine orthologue of BRDG1. Three isoforms of BRDG1 are generated by alternative splicing of the message. Two of them have a deletion of 33 amino acids in a Pleckstrin homology (PH) domain of BRDG1. Both the tyrosine-phosphorylation and CREB-activating ability of BRDG1 were isoform-dependent, suggesting a role of the PH domain of BRDG1. These data have identified a novel regulatory mechanism of CREB family of transcriptional factors.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Sak serine-threonine kinase acts as an effector of Tec tyrosine kinase.

The murine sak gene encodes a putative serine-threonine kinase which is homologous to the members of the Plk/Polo family. Although Sak protein is presumed to be involved in cell growth mechanism, efforts have failed to demonstrate its kinase activity. Little has been, therefore, elucidated how Sak is regulated and how Sak contributes to cell proliferation. Tec is a cytoplasmic protein-tyrosine kinase (PTK) which becomes activated by the stimulation of cytokine receptors, lymphocyte surface antigens, heterotrimeric G protein-linked receptors, and integrins. To clarify the in vivo function of Tec, we have tried to isolate the second messengers of Tec by using the yeast two-hybrid screening. One of such Tec-binding proteins turned out to be Sak. In human kidney 293 cells, Sak became tyrosine-phosphorylated by Tec, and the serine-threonine kinase activity of Sak was detected only under the presence of Tec, suggesting Sak to be an effector molecule of Tec. In addition, Tec activity efficiently protects Sak from the "PEST" sequence-dependent proteolysis. Internal deletion of the PEST sequences led to the stabilization of Sak proteins, and expression of these mutants acted suppressive to cell growth. Our data collectively supports a novel role of Sak acting in the PTK-mediated signaling pathway.

Amino Acid Sequence↗
PubMed2001

Interaction between monocytes and vascular endothelial cells induces adrenomedullin production.

Adrenomedullin (AM), a potent vasodilator peptide, has natriuretic effects, and its plasma concentration is elevated in cardiovascular diseases. In the present study, we investigated the induction of AM expression due to interactions between THP-1 cells (human monocytic cell line) and human umbilical cord vein endothelial cells (HUVECs). AM levels in the culture medium were measured by radioimmunoassay. The luciferase vector containing the 5'-flanking region of the human AM gene was transfected into either HUVECs or THP-1 cells. Addition of THP-1 cells to HUVECs for 48 h induced marked increases in AM levels, which were 16-fold higher than those of HUVECs alone. Luciferase vectors containing the 5'-flanking region of human AM gene (pLCF-1534) were transferred into THP-1 cells or HUVECs. Addition of THP-1 cells to pLCF-1534-transfected HUVECs induced an increase in luciferase activity in cell lysates, which was 5-fold higher than that of the transfected HUVECs alone. In contrast, the luciferase activity of lysates from pLCF-1534-transfected THP-1 cells was not affected by coculture with HUVECs. A separate coculture experiment revealed that direct contact of THP-1 cells and HUVECs contributed to enhanced AM production in the cocoulture. Co-incubation of the cell membrane fraction from THP-1 cells augmented AM production by HUVECs. Both anti-interleukin (IL)-1alpha antibody and IL-1 receptor antagonist significantly inhibited AM production in the cocultures. The cell-to-cell interaction between monocytes and HUVECs induces AM production by HUVECs, which may play an important role in the pathogenesis of vascular disorders.

Adrenomedullin↗
PubMed2001

Expression of peripheral myelin protein zero in sural nerve of patients with Charcot-Marie-Tooth disease 1B.

A Charcot-Marie-Tooth disease 1B (CMT1B) family with a mutation of the Po gene is presented. A to G substitution of nucleotide 389 in exon 3 resulted in Lys 131 Arg substitution. Immunostaining for Po in biopsied sural nerve from one family member with CMT1B was expressed in a small number of myelinated fibers. Immunoblot analysis for Po revealed that it was of normal molecular weight (29 kDa) although significantly reduced in amount. This heterozygous mutation could lead to a reduction in the total amount of normal protein in peripheral nerves through a mechanism of loss of function.

Amino Acid Substitution↗
PubMed2001

Predominant IgG4 subclass in autoantibodies of pemphigus vulgaris and foliaceus.

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune skin diseases caused by autoantibodies against desmoglein (Dsg) 3 and Dsg1. We have previously developed ELISAs using recombinant Dsg3 and Dsg1 expressed by baculovirus as a diagnostic tool for pemphigus. In this study, we determined the frequency of coexistence of IgA class as well as distribution of IgG subclass. Two out of 49 PV and PF sera tested had anti-Dsg1 IgA in addition to anti-Dsg1 IgG. Interestingly, one of them showed prominent pustular formation. Among IgG subclass, IgG4 was predominant and found in all of the 30 PV and 19 PF sera tested, followed by IgG1, detected in 25 out of 30 PV and 12 out of 19 PF sera. Even though IgG2 and IgG3 were detected in 13 and one PV and 6 and 4 PF sera, respectively, the ELISA titers had barely exceeded the cutoff value in most of the cases. There was no IgG subclass shift during the course of the disease in seven cases examined. These findings indicate that IgG4 subclass is the predominant autoantibodies in both PV and PF, while IgG1 is also frequently found.

Antibody Specificity↗