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Biomedical subjects

H Ohashi

Publications and source records attributed to H Ohashi.

At least 199 records · Page 11Linked to original sources
PubMed1998

Plasma soluble fas and soluble fas ligand in chronic glomerulonephritis.

It has been reported that glomerular cells with apoptosis and positive Fas immunoreactivity are seen in proliferative glomerulonephritis (PGN). Fas induces apoptosis when it binds to Fas ligand (Fas-L) or soluble Fas-L (sFas-L). However, soluble Fas (sFas) blocks apoptosis by inhibiting binding between Fas and Fas-L or sFas-L. That is, Fas, Fas-L, and sFas-L are inducers of apoptosis, but sFas is an inhibitor of apoptosis. We studied the relationship between the plasma levels of sFas and sFas-L in 32 patients with various types of adult chronic glomerulonephritis. Patients with serum creatinine levels >1.5 mg/dl (132.6 micromol/l) were excluded. The plasma levels of sFas-L were within the normal limits in all patients. The plasma levels of sFas in the patients with minimal-change (n = 8) and membranous nephropathy (n = 7) were similar to the age- and sex-matched controls. However, the plasma sFas levels were significantly elevated in patients with mesangial PGN (n = 10) and membranoproliferative glomerulonephritis (n = 7)(3. 4 +/- 0.9 and 3.9 +/- 1.5 ng/ml, respectively) as compared with the age- and sex-matched controls (controls: 2.1 +/- 0.4 and 2.2 +/- 0.6 ng/ml, respectively). In PGN, according to increase of histological grade and decrease of creatinine clearance, the number of TUNEL-positive cells in glomeruli is decreased in spite of an increase of the Fas positivity, and plasma sFas is increased. The degree of proliferative change is determined by the balance between proliferation and apoptosis and/or necrosis. Therefore, increased plasma sFas in PGN may inhibit apoptosis in glomeruli and may be one of the progressing factors in PGN. Thus, we conclude that an increase in plasma sFas levels is important to the protection of apoptosis in PGN.

Adult↗
PubMed1998

M2 and M3 muscarinic receptors couple, respectively, with activation of nonselective cationic channels and potassium channels in intestinal smooth muscle cells.

Smooth muscle cells of guinea pig ileum express both M2 and M3 subtypes of muscarinic receptors. Under voltage clamp, activation of the muscarinic receptors with carbachol (CCh) induces Ca2+-activated K+ current (I[K-Ca]) and nonselective cationic current (Icat). Receptor subtypes mediating the current responses were characterized by using pirenzepine, AF-DX116, 4-DAMP and atropine, which have different profiles of the affinity constants for muscarinic receptor subtypes. The muscarinic antagonists inhibited either CCh-evoked I(K-Ca) or Icat with different potencies. Their relative potencies for I(K-Ca) and Icat inhibition resembled the relative affinity constants for M3 and M2 subtypes, respectively. Thus, the I(K-Ca) is mediated via the M3 subtype and the Icat via the M2 subtype.

Animals↗
PubMed1998

Potentiation by neurotensin of carbachol-induced tension development in beta-escin-skinned smooth muscle of guinea-pig ileum.

Effect of neurotensin (NT) on carbachol(CCh)-induced tension development due to Ca2+ release from intracellular stores was investigated in beta-escin-skinned smooth muscle of guinea-pig ileum. NT (10 nM) increased the tension development in response to CCh. NT also increased the tension response to caffeine, another store-Ca2+ releaser. NT did not exert such an effect in pertusis toxin (PTX)-treated preparations. Treatment with isoprenaline to elevate endogenous cyclic AMP levels or with dibutyryl cyclic AMP did not affect the effect of NT. A nonpeptide NT antagonist, SR 48692, failed to block the effect of NT. NT shifted the pCa-tension relationship in the lower direction of Ca2+ concentration. NT was incapable of releasing Ca2+ from intracellular stores. The results suggest that NT may cause an increase in Ca2+ sensitivity of contractile elements to potentiate the CCh-induced tension development due to release of stored Ca2+ and that the effect is mediated by SR 48692-insensitive NT receptors linked to a PTX-sensitive G protein which works with no relation to a change in cytosolic cyclic AMP levels.

Animals↗
PubMed1998

Wear and osteolysis in total joint replacements.

UNLABELLED: This presentation summarizes the results of our recent studies on the pathogenesis of osteolysis around total joint arthroplasties. First, interface tissues with adjacent bone were retrieved and histopathologically investigated with reference to the cells on the bone surface. Secondly, polyethylene particles were extracted with the tissue digestion method and characterized with scanning electron microscopy. Finally, an animal model for osteolysis was created and various interface conditions were compared concerning their resistance to particle migration. Histopathological examinations demonstrated that active bone formation, regarded as a repair process, was the commonest feature, even in revised cases. They also highlighted the role played by macrophages, not as cells producing inflammatory mediators which could activate osteoclasts, but as cells primarily responsible for the bone loss in osteolytic lesions. Among the particle species present, only polyethylene particles were shown to play a significant role in macrophage recruitment and subsequent osteolysis. A quantitative extraction of polyethylene particles showed a significant difference in the "number" of particles between osteolysis positive and negative cases whereas the "sizes" of particles were similar in these two groups. The critical number of particles for osteolysis was around 1 x 10(10) particles/g tissue and the cellular reaction against phagocytosable particles accumulated over this concentration may be the prerequisite for progression of osteolysis. The animal model for osteolysis indicated that the progression of osteolysis depends on the integrity of the bone-implant interface. We suggest that the solid fixation of the prosthesis performed by current techniques (e.g., improved cementing technique, hydroxyapatite coating) is beneficial for preventing particle migration and subsequent osteolysis. CLINICAL RELEVANCE: Osteolysis induced by particulate wear debris from implant materials has been recognized as the major cause of long-term failure in total joint replacements. However, the development of preventive measures for this phenomenon has not been successful because the mechanism in which wear particles cause osteolysis is not quite clear. On the basis of results obtained in this study, we believe that the basic strategy for addressing the problem of osteolysis is to reduce the "number" of accumulated wear particles in the interface tissues. This could be achieved either by improving the materials or the geometry of the articulating counterface. Another possibility is to increase the integrity of the bone-implant interface to prevent particle migration. It is important to note that pre-clinical testing of materials and prosthetic designs should include an analysis of the characteristics of the particle generated (e.g., size and number). The widespread bone formation, even in revised cases, is encouraging in view of "conservative treatment" of aseptic loosening. Assuming that bone loss in aseptic loosening is not a remorseless process, some form of intervention, whether mechanical or pharmacological, might be possible to tip the balance more in favour of bone formation than resorption. A comprehensive understanding of the bone reactions in osteolysis, including the basic mechanisms of bone loss, shown in this study, are decisive for the development of preventive measures that may minimize the clinical impact of this phenomenon.

Animals↗
PubMed1998

[Participation of apoptosis in renal amyloidosis].

Renal amyloidosis shows symptoms of renal dysfunction due to the deposition of amyloid protein in the kidney. Recently, it was reported that apoptosis plays an important role in the pathogenesis of type-2 diabetes mellitus and Alzheimer's disease of which amyloid deposition is seen in the tissue. We investigated whether or not apoptosis and related factors are observed in renal amyloidosis. In situ nick end labeling (TUNEL) was performed in seven autopsied renal tissues with primary and secondary amyloidosis and 10 autopsied renal tissues without renal disease as the control. The number of TUNEL-positive cells was significantly increased in both the glomeruli and tubulus of the kidney with amyloidosis than in the control. Electron microscopic analysis was performed on one biopsied renal tissue with amyloidosis and six biopsied renal tissues with minor abnormalities as the control. Typical apoptotic cells were observed only in the former. Bax product, an inducer of apoptosis, and Bcl-2 protein, an inhibitor of apoptosis, were examined immunohistochemically in the seven autopsied renal tissues with amyloidosis and 10 autopsied control tissues. Bax was overexpressed in the tubulus and glomeruli of subjects with renal amyloidosis, compared to the normal controls. However, Bcl-2 protein was not detected in the glomeruli in any of the subjects examined. These results indicate that apoptotic cells are increased in number in renal amyloidosis and Bax overexpression may play an important role in this increase.

Aged↗
PubMed1998

Minute chromosomal rearrangements detected prenatally by fluorescence in situ hybridization.

Initial approaches to prenatal diagnosis from fetal karyotyping involved application of standard cytogenetic techniques. However, when fetal samples, such as chorionic villus cells or amniocytes are used, small chromosome rearrangements cannot be easily identified because they lack a distinct banding pattern. We report here two cases with minute chromosome rearrangements detected prenatally by fluorescence in situ hybridization. The use of this technique allowed precise identification of fetal chromosome abnormalities, demonstrating its usefulness for characterizing conditions that would be difficult to diagnose correctly with conventional banding methods alone.

Adult↗
PubMed1997

HDR syndrome (hypoparathyroidism, sensorineural deafness, renal dysplasia) associated with del(10)(p13).

A combination of hypoparathyroidism, sensorineural deafness, and renal dysplasia has been considered to be a new syndrome inherited in an autosomal dominant fashion; we name the condition "HDR syndrome." We describe a Japanese girl who has HDR syndrome associated with de novo del(10)(p13). The chromosome deletion suggests that the putative gene(s) responsible for HDR syndrome is located at a 10pter-->p13 region.

Adult↗
PubMed1997

Genome organization of human 48-kDa oligosaccharyltransferase (DDOST).

The enzyme oligosaccharyltransferase (dolichyl-diphosphooligosaccharide-protein glycosyltransferase; EC 2. 4.1.119) (DDOST) catalyzes the transfer of a high-mannose oligosaccharide (GlcNac2Man9Glc3) from a dolichol-linked oligosaccharide donor (dolichol-P-GlcNac2Man9Glc3) onto the asparagine acceptor site within an Asn-X-Ser/Thr consensus motif in nascent polypeptide chains across the membrane of the endoplasmic reticulum. We isolated mouse and human DDOST cDNAs from retinoic acid-treated mouse P19 EC cells and human NT-2 cells, respectively. DDOST mRNA is expressed intensely in heart and pancreas, but at lower levels in brain. Here we show that the human DDOST 48-kDa subunit gene (HGMW-approved symbol DDOST) is organized into 11 exons expanding about 9 kb. This DDOST subunit gene is localized on chromosome 1p36.1 by fluorescence in situ hybridization analysis.

Amino Acid Sequence↗
PubMed1997

An autocatalytic expression system for regulated production of recombinant protein in mammalian cells.

An autocatalytic inducible mammalian expression system was established. This system is composed of two sets of vectors: one carries a selectable marker gene and the other carries a bicistronic expression unit consisting of a target gene, an internal ribosomal entry site, and a tetracycline-controlled transactivator gene. Both the selectable marker and the bicistronic unit are controlled by the tetracycline-responsive promoter (PhCMV*-1). When the two vectors are cotransfected into host cells, only a single selection round in the absence of tetracycline is necessary to generate clones expressing the target gene. The expression level is high and easily regulated by tetracycline. Combination with a gene amplification system may allow further enhance the foreign gene expression. Because the PhCMV*-1 promoter is relatively independent of cellular regulation signals, this system is expected to work in a wide variety of cell types.

Animals↗
PubMed1997

A 1.2-megabase BAC/PAC contig spanning the 14q13 breakpoint of t(2; 14) in a mirror-image polydactyly patient.

We previously assigned a 14q13 breakpoint of t(2; 14) in a patient with mirror-image polydactyly to a segment between two loci, AFM200ZH4 and D14S306, within a genetic distance of 0.6 cM. In the present study, we constructed a 1.2-Mb high-resolution physical map with a contig composed of 16 bacterial artificial chromosomes (BACs) and 6 P1-derived artificial chromosomes (PACs) at a region around the breakpoint, extending from D14S75 to D14S728 loci. Thirty-four novel sequence-tagged sites (STSs) were also characterized at this region. Of nine ESTs that had been mapped between D14S75 and D14S288, T99065 was confirmed to be in two BAC clones, B102 and B319. This BAC/PAC contig with STSs is useful for further genomic sequencing, for construction of a transcription map, and for the isolation of the putative gene for mirror-image polydactyly.

Chromosome Mapping↗
PubMed1997

Selective inhibition of platelet-derived growth factor (PDGF) receptor autophosphorylation and PDGF-mediated cellular events by a quinoline derivative.

This report describes the biological effects of our original compound, Ki6783 ((3,4-dimethoxy)-4-phenoxy-6,7-dimethoxyquinoline), a potent and selective inhibitor of platelet-derived growth factor (PDGF) receptor autophosphorylation. This compound strongly inhibited autophosphorylation of the PDGF beta-receptor in cultured rat glomerular mesangial cells (MC) bearing this receptor (IC50 0.1 microM), although it did not inhibit autophosphorylation of other growth factor receptors even at 100 microM. In a cell-free kinase experiment, it showed selective inhibition of PDGF beta-receptor tyrosine kinase. A kinetic study of the compound to this tyrosine kinase revealed a competitive mode of action to ATP. [3H]Thymidine incorporation and cell proliferation of MC were inhibited by Ki6783 in a dose-dependent manner after Ki6783 and PDGF-BB were added to the culture medium. Furthermore, this compound normalized the fibrotic cell shape of v-sis-transformed NIH3T3 cells, which grow in an autocrine manner via the PDGF receptor. These effects could be explained by the inhibition of intracellular signal transduction triggered by PDGF receptor autophosphorylation, in which activation of mitogen-activated protein kinase occurs. These results suggest that Ki6783 is one of the more potent and selective inhibitors of PDGF receptor autophosphorylation and that it may be useful in ameliorating cell abnormalities due to excess action of PDGF and its receptor systems in several diseases.

3T3 Cells↗
PubMed1997

Thrombin cleaves recombinant human thrombopoietin: one of the proteolytic events that generates truncated forms of thrombopoietin.

A heterogeneity in the molecular weight (Mr) of thrombopoietin (TPO) has been reported. We found several thrombin cleavage sites in human, rat, murine, and canine TPOs, and also found that human TPO undergoes selective proteolysis by thrombin. Recombinant human TPO (rhTPO) was incubated with human platelets in the presence of calcium ions to allow the generation of thrombin, and was cleaved into low Mr peptide fragments. The cleavage was completely inhibited by hirudin, indicating that the proteolysis was mediated by thrombin. In a platelet-free system, analyses of thrombin cleavage by immunoblotting using anti-human TPO peptide antibodies revealed that the four major thrombin-cleaved peptide fragments were selectively generated depending on the digestion time. The amino acid sequences of the thrombin-polypeptides were further analyzed, and two major thrombin cleavage sites were determined. One of them was at AR191-T192 in the C-terminal domain of TPO, and thrombin cleaved first at this site. The other site at GR117-T118 in the N-terminal domain was subsequently cleaved by prolonged thrombin digestion. As a result, the biological activity of TPO was modulated. The generation of truncated forms of TPO by thrombin may be a notable event in view of the platelet-related metabolism of TPO.

Amino Acid Sequence↗
PubMed1997

Thrombopoietin and thrombin induce tyrosine phosphorylation of Vav in human blood platelets.

Thrombopoietin has an essential role in megakaryopoiesis and thrombopoiesis. To investigate the signaling processes induced by thrombopoietin, we have employed human platelets and recently demonstrated that thrombopoietin induces rapid tyrosine phosphorylation of Jak-2, Tyk2, Shc, Stat3, Stat5, p120(c-cbl) and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine phosphorylated protein in platelets stimulated by thrombopoietin is approximately 85 to 95 kD, we examined the possibility that this could be Vav, a 95-kD proto-oncogene product. Specific antisera against Vav recognized the same 95 kD protein in lysates of Jurkat cells, which are known to express Vav, and platelets, indicating that platelets have Vav. Thrombopoietin induced rapid tyrosine phosphorylation of Vav in platelets without an elevation in cytosolic free calcium concentration or activation of protein kinase C. Vav was also tyrosine phosphorylated upon treatment of platelets with thrombin, collagen, or U46619, which activate phospholipase C, leading to an increased ionized calcium concentration and activation of protein kinase C. Ionomycin or phorbol 12-myristate 13-acetate (PMA) also induces tyrosine phosphorylation of Vav, suggesting that an increase in ionized calcium concentration or activation of protein kinase C may lead to phosphorylation of Vav. Thrombopoietin also induced tyrosine phosphorylation of Vav in FDCP-2 cells, genetically engineered to express human c-Mpl (FDCP-hMpl5). However, neither ionomycin nor PMA induced an increase in tyrosine phosphorylation of Vav in FDCP-hMpl5 cells, suggesting that the calcium and protein kinase C pathways of Vav phosphorylation may be unique to platelets. Further, Vav became incorporated into the Triton X-100 insoluble 10,000 g sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. Vav was constitutively associated with a 28-kD adapter protein, Grb2, which is also incorporated into the cytoskeleton in an aggregation-dependent fashion. Lastly, we found that Vav is cleaved when there is activation of calpain, a protease that may have a role in postaggregation signaling processes. Our data suggest that thrombopoietin and other agonists may induce tyrosine phosphorylation of Vav by different mechanisms and Vav may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.

Animals↗
PubMed1997

Locations of crossover breakpoints within the CMT1A-REP repeat in Japanese patients with CMT1A and HNPP.

The crossover breakpoints for Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are located in the CMT1A-REP repeat flanking a 1.5-Mb region of chromosome 17p11.2-12. The precise locations of the breakpoints are heterogeneous, and we analyzed the relative frequency distribution of breakpoints in 33 unrelated Japanese CMT1A and 3 unrelated HNPP families. The CMT1A-REP repeat region was divided into five regions, A, B, C, D and E, based on restriction site differences between the proximal and distal CMT1A-REP repeats. The frequency distribution of breakpoints within the CMT1A-REP repeat in the Japanese patients was 3% in region A, .78% in B/C and 19% in D, which is similar to that in Caucasian patients. This result also indicates that an 8-kb region defined by region B/C is a recombinational hotspot within the CMT1A-REP repeat in Japanese patients.

Asian People↗
PubMed1997

Molecular mapping of a translocation breakpoint at 14q13 in a patient with mirror-image polydactyly of hands and feet.

Mirror hands and feet (MIM, 135750) is a rare congenital anomaly, and mirror-image polydactyly is considered to be a variant of mirror hands and feet. To our knowledge, seven patients with the disorder have been reported in the literature. Parent-to-child transmission was reported in two families, which may indicate a single-gene defect inherited in an autosomal dominant fashion. We had previously encountered a boy with mirror-image polydactyly whose karyotype showed 46,XY,t(2;14) (p23.3;q13) de novo. We hypothesized that at least one of the putative genes responsible for the determination of an anterior-posterior limb pattern is disrupted by a translocation breakpoint. In this study, we identified a yeast artificial chromosome clone spanning a translocation breakpoint at 14q13, and the breakpoint was confirmed to be located between two loci, AFM200ZH4 and D14S306, within a genetic distance of 0.6 cM.

Chromosome Mapping↗
PubMed1997

New p57KIP2 mutations in Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is characterized by numerous growth abnormalities and an increased risk of childhood tumors. The gene for BWS is localized in the 11p15.5 region, as determined by linkage analysis of autosomal dominant pedigrees. The increased maternal transmission pattern seen in the autosomal dominant-type pedigrees and the findings of paternal uniparental disomy reported for a subgroup of patients indicate that the gene for BWS is imprinted. Previously, we found p57KIP2, which is a Cdk-kinase inhibitor located at 11p15, is mutated in two BWS patients. Here, we screened for the mutation of the gene in 15 BWS patients.

Austria↗