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Biomedical subjects

K Ohishi

Publications and source records attributed to K Ohishi.

At least 91 records · Page 5Linked to original sources
PubMed1996

Cloning and characterization of the murine GPI anchor synthesis gene Pigf, a homologue of the human PIGF gene.

Many eukaryotic proteins are bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Its core backbone, which is conserved in different organisms, is synthesized in the endoplasmic reticulum by the sequential addition of glycan components to phosphatidylinositol. One of the human GPI synthesis genes, PIGF (phosphatidylinositol glycan complementation class F), which is involved late in the synthesis pathway, has been cloned. In this study, we isolated complementary and genomic clones of Pigf, a murine counterpart of PIGF. Pigf encodes a 219 amino acid protein that complements a class F mutation. The Pigf gene consists of six exons spanning 30 kb and was mapped to chromosome 17 at 17E4-E5. These features are very similar to PIGF, thus demonstrating the interspecies conservation of structure, function, gene organization, and genetic locus between these GPI synthesis genes. The results also extend a region in murine distal chromosome 17 that is syntenic to human chromosome 2p16-p22.

Amino Acid Sequence↗
PubMed1996

Accelerated cell-cycling of hematopoietic progenitors by the flt3 ligand that is modulated by transforming growth factor-beta.

Although hematopoietic growth factors have been extensively studied as to their roles in recruitment of hematopoietic progenitors from quiescence state to cell division state, little is known of their effects on cell-cycling of progenitors that have already transited from quiescence into active cell-cycling. We examined the effects of the flt3 ligand (FL) on cell-cycling of hematopoietic progenitors in serum-free culture. Results from our serial observations of colony formation and replating experiments suggest that FL enhances the rate of growth of interleukin-3 (IL-3)-dependent colonies by shortening the time for each progenitor in the colonies to divide. Cell-cycle analysis showed that shortening of cell-cycle time induced by FL is mainly because of alteration in the G1 phase that hematopoietic progenitors go through. We next investigated the role of transforming growth factor-beta (TGF-beta) in cell-cycling of progenitors, using TGF-beta protein and TGF-beta antisense oligonucleotides, because mRNA of TGF-beta was detected by reverse transcriptase polymerase reaction in blast cells that we used as a source of progenitors. TGF-beta lengthened the time required for IL-3-dependent progenitors to become two daughter cells, whereas the effects of TGF-beta antisense oligonucleotides were opposite to those of TGF-beta. The addition of TGF-beta neutralizing monoclonal antibodies to the cultures resulted in effects similar to those seen with TGF-beta antisense oligonucleotides. DNA studies indicated that both TBF-beta and TGF-beta antisense oligonucleotides change the length of G1 phase of the cell-cycle. TGF-beta abrogated the effects of FL on the growth rate of hematopoietic progenitors, whereas the combination of FL with TGF-beta antisense oligonucleotides exerted additive effects. These data show that FL has the potential to accelerate cell-cycling of hematopoietic progenitors, which is susceptible to the modulation by TGF-beta.

Animals↗
PubMed1996

Potentiation of antitumor and antimetastatic activities of adriamycin by a novel N-alkylated dihydropyridine, AC394, and its enantiomers in colon cancer-bearing mice.

PURPOSE: We have previously shown that a series of N-alkylated 1,4-dihydropyridines potentiate the therapeutic efficacy of vincristine in vincristine-resistant P388 leukemia. The purpose of this study was to investigate the ability of one of the compounds, AC394, and its enantiomers to potentiate the antitumor activity of adriamycin against colon cancer cells in vitro and in vivo. METHODS: The effects of AC394 on potentiation of adriamycin cytotoxicity and enhancement of its accumulation were evaluated using colon 26, HCT-15 and MCF-7 cells. Furthermore, the activities of AC394 and its enantiomers were compared. We also studied the combined effects of (+)-AC394 and adriamycin on subcutaneously (s.c.)-implanted and liver metastasis tumor models. RESULTS: AC394 potentiated the cytotoxicity of adriamycin and enhanced its accumulation in colon cancer cells (colon 26 and HCT-15), which are known to express P-GP (P-glycoprotein) intrinsically. Enhancement of adriamycin accumulation by AC394 was found in s.c.-implanted colon 26 cells in vivo. Although both enantiomers of AC394 showed equal activity in vitro, (+)-AC394 was more effective than (-)-AC394 given orally. (-)-AC394 was found to be cleared more rapidly from the plasma than (+)-AC394. Thus, (+)-AC394 was evaluated for further study. Administration of (+)-AC394 significantly potentiated the antitumor activities of adriamycin in human colon cancer HCT-15 cells implanted s.c. Furthermore, in the liver metastasis model using colon 26 cells, a model completely resistant to adriamycin, the combination therapy of adriamycin with (+)-AC394 produced superior antitumor effects over adriamycin alone. CONCLUSIONS: A newly synthesized N-alkylated 1,4-dihydropyridine derivative, (+)-AC394, showed superior effects on the potentiation of adriamycin antitumor and antimetastatic activities in vivo. These results suggest that this combination may have therapeutic efficacy not only against primary colon cancers but also against metastatic liver cancer.

Animals↗
PubMed1996

Induction of bovine leukaemia virus Env-specific Th-1 type immunity in mice by vaccination with short synthesized peptide-liposome.

Cell-mediated immunity (CMI) is considered to be effective in controlling retrovirus infection and replication. To develop a peptide-based vaccine capable of inducing CMI, mannan-coated liposome encapsulating 20-mer synthetic peptide, spanning the 98-117 amino acids of bovine leukaemia virus (BLV) envelope glycoprotein (Env) gp51 was constructed. The liposome induced specific delayed-type hypersensitivity, lymphocyte proliferative responses and a weak cytotoxic lymphocyte response in mice. By stimulation with the peptide and BLV virion, the spleen cells from the immunized mice produced a large amount of IFN-gamma and IL-2, whereas they released neither IL-4 or IL-10. These results indicate the augmentation of Th-1 type immunity in mice by the T-cell epitope synthetic peptide-liposome.

Amino Acid Sequence↗
PubMed1996

An effective peptide vaccine to eliminate bovine leukaemia virus (BLV) infected cells in carrier sheep.

Protective effects of the gp51 of bovine leukaemia virus (BLV) expressed by a recombinant baculovirus (rgp51) and synthetic multiple antigenic peptides (MAP) of T-helper, T-cytotoxic, and B-cell epitopes of gp51 were investigated against BLV challenge. Two and three sheep were immunized with rgp51 and a mixture of peptides with Freund's complete adjuvant, respectively. BLV was detected from all the immunized sheep at 2 weeks and showed peak levels at 4 weeks after the challenge. However, in two sheep immunized with the mixed peptides, the titer of BLV gradually decreased and one sheep eliminated BLV completely at 28 weeks after the challenge. These two sheep showed higher lymphocyte proliferative responses against the immunized peptides than the other sheep. One of the sheep also showed the specific cytotoxic lymphocyte activity against the BLV gp51-expressing target in vitro. These results suggest the possibility of the peptide vaccine for elimination of BLV in carrier animals in vivo.

Amino Acid Sequence↗
PubMed1996

Mitral valve repair for mitral regurgitation with ventricular septal defect in children.

BACKGROUND: We examined the results of intermediate and long-term follow-up of 25 patients aged 3 months to 11 years (mean, 2.6 +/- 2.3 years) who initially underwent conservative mitral valve repair for mitral regurgitation associated with ventricular septal defect between April 1973 and March 1991. METHODS: The preoperative degree of mitral regurgitation was 2+ in 3, 3+ in 17, and 4+ in 5 patients, and the major causes of mitral regurgitation were annular dilatation and prolapse of the anterior leaflet. Annuloplasty was performed in all except 2 patients, suturing of the cleft was done in 3 patients, and posterior mitral leaflet advancement was done in 2 patients. In addition, the papillary muscle was incised and adhesive chordae were removed in 1 patient, and adhesive fused chordae were detached from a leaflet in 1 other patient. RESULTS: There were no early deaths. Two patients with residual mitral regurgitation with or without mitral stenosis underwent reoperation for mitral valve replacement 2 months and 6 years after the mitral repair, respectively. Late death occurred in 2 patients, and the actuarial survival rate was 92.0% at 15 years after operation. The freedom from reoperation was 91.3% at both 10 and 15 years after the initial operation. Postoperative color Doppler flow imaging was performed in 22 of the 23 survivors, and results showed no mitral regurgitation in 4, mild regurgitation in 14, and moderate regurgitation in 4 patients. Four patients presently have mitral stenosis, with a mean transmitral pressure gradient greater than 10 mm Hg. The residual lesion of moderate mitral regurgitation with or without mitral stenosis developed in 6 of 11 patients in whom bilateral mitral annuloplasty was applied after the initial operation. Nineteen of the 22 survivors without reoperation were in New York Heart Association class I, and 3 were in class II. CONCLUSIONS: Clinical improvement was observed after conservative mitral repair in most pediatric patients with ventricular septal defect. However, careful follow-up for growth potential still appears to be needed to detect changes in mitral regurgitation and the development of mitral stenosis after valve repair, especially after bilateral annuloplasty.

Child↗
PubMed1996

A possible change in doubling time of haemopoietic progenitor cells with stem cell development.

We separated haemopoietic progenitors derived from marrow cells of 5-fluorouracil (5-FU)-treated mice into three groups, based on the stages of stem cell development and studied doubling time, using a serum-free clonal culture system. Stage I progenitors were those present in primary marrow cells from 5-FU-treated mice. Stages II and III progenitors were early and late progenies in culture of stage I progenitors respectively. The morphological analysis of colonies derived from stage I, II and III progenitors demonstrated an association of progression of stages with loss of multipotentiality. The doubling time of haemopoietic progenitors was estimated by sequential analysis of colony formation and studies of growth fraction. The time required for haemopoietic progenitors to double shortened as their stage of development progressed. Alteration in one doubling time of haemopoietic progenitors at progressive stages of stem cell development was seen in cultures supported by various combinations of growth factors, including interleukin-3 (IL-3), IL-11. and steel factor (SF), Cell-cycle analysis suggested that reduction of the doubling time of haemopoietic progenitors is probably due to a decrease in the time spent in the G1 phase of the cell cycle. Our results suggest that in early haemopoiesis the doubling time of haemopoietic progenitors may change with stem cell development.

Animals↗
PubMed1996

Activity of the ligand for c-mpl, thrombopoietin, in early haemopoiesis.

We examined the role of the ligand for c-mpl. thrombopoietin (TPO). in murine early haemopoiesis. using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) supported colony formation by marrow cells of 5-fluorouracil (5-FU)-treated mice whereas TPO alone yielded no colony. When blast cell colonies grown in the presence of TPO plus SF or TPO plus IL-3 were individually replated in suspension cultures containing serum and several growth factors, various combinations of myeloid lineages were seen, indicating that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of haemopoietic progenitors. We then studied the effects of TPO on proliferative kinetics of cycling progenitors. TPO hastened IL-3-dependent growth of progenitors by shortening the time required for cell cycling. These results suggest that TPO as a single factor, can support the survival of haemopoietic progenitors and TPO synergizes with SF or IL-3 to act on early multipotential haemopoietic progenitors.

Animals↗
PubMed1996

Endotoxin-induced enhancement of glucose influx into murine peritoneal macrophages via GLUT1.

Hypoglycemia is among the most injurious metabolic disorders caused by endotoxemia. In experimental endotoxemia with lipopolysaccharide (LPS) in animals, a marked glucose consumption is observed in macrophage-rich organs. However, the direct effect of LPS on the uptake of glucose by macrophages has not been fully understood, and the present study was undertaken to shed light on this point. The consumption and uptake of glucose, as measured with 2-deoxy-D-[3H]glucose, by murine peritoneal exudate macrophages in culture were accelerated two- to threefold by stimulation with 3 ng of LPS per ml. The rate of glucose uptake reached a plateau after 20 min of stimulation and remained at the maximum as long as LPS was present. Northern (RNA) blot analysis with cDNA probes for five known isoforms of glucose transporter (GLUT) revealed that the expression of GLUT by macrophages was restricted to the GLUT1 isoform during LPS stimulation and the amount of GLUT1 mRNA was increased by the stimulation. These results suggest that macrophage responses to LPS are supported by a rapid and sustained glucose influx via GLUT1 and that this is a participating factor in the development of systemic hypoglycemia when endotoxemia is prolonged.

Amino Acid Sequence↗
PubMed1996

Autoantibodies and immunoglobulins in atomic bomb survivors with human T-lymphotropic virus type I.

The association of human T-lymphotropic virus type I (HTLV-I) with autoimmune disorders was investigated on the basis of prevalence of antinuclear antibody (ANA), rheumatoid factor and anti-thyroglobulin antibody as well as immunoglobulin (Ig) serum level (IgG, IgA, and IgM). The subjects, all atomic bomb survivors, were 59 HTLV-I-seropositive people without HTLV-I-associated myelopathy or adult T-cell leukemia and 149 HTLV-I-seronegative persons. The mean serum level of IgM was higher in HTLV-I-seropositive subjects than in HTLV-I-seronegative subjects, and a significant association with HTLV-I and sex was indicated in the IgM serum level. No association with HTLV-I was indicated in the prevalence of the autoantibodies except for ANA. These results suggest some clear humoral immunity differences between HTLV-I-infected and noninfected subjects, but whether HTLV-I infection can lead to autoimmune disorders remains uncertain.

Adult↗
PubMed1996

Very low incidence rates of community-acquired hepatitis C virus infection in company employees, long-term inpatients, and blood donors in Japan.

To assess the contemporary rate of hepatitis C virus (HCV) propagation in Japanese community, we conducted a sentinel study on three groups of people: company employees, long-term inpatients, and blood donors. A total of 3079 company employees negative for anti-HCV were followed thereafter, and seroconversion to anti-HCV was found in 5 per 5786 person-years. None of them was positive for HCV RNA. In the group of 703 long-term inpatients (25 of whom were initially positive for anti-HCV) none showed seroconversion to anti-HCV per 2712 person-years. As for 114,266 repeated blood donors who were initially antibody-negative, 227 became anti-HCV positive later. Of these seroconverted donors, 83 were found to have anti-HCV with titers of 2(6) or greater. HCV RNA was positive in only 3 of them. Thus, the incidence rates of acquired HCV viremia in these three groups were 0 for both company employees and long-term inpatients, and 1.78 (95% C.I.: 0.37-5.19) per 100,000 person-years in blood donors. These results suggest that community-acquired HCV infection is now rare in Japan, and that even if it occurred it hardly leads to persistent viremia.

Adolescent↗
PubMed1995

Structure and chromosomal localization of the GPI-anchor synthesis gene PIGF and its pseudogene psi PIGF.

Posttranslational modification by the GPI glycolipid anchor is essential for the surface expression of many membrane proteins. Defect of GPI biosynthesis due to somatic mutation in the hematopoietic stem cell is the basis for an acquired genetic disease, paroxysmal nocturnal hemoglobinuria (PNH). Previously, an X-linked gene PIGA (phosphatidylinositol glycan class A), which participates in the first step of the biosynthesis, was shown to be mutated in abnormal cells from all 60 patients with PNH. The cDNA of another GPI synthesis gene PIGF was previously cloned, but it is not involved in pathogenesis of PNH. In the present study, we have analyzed PIGF genomic clones. The PIGF gene contained six exons spanning about 40 kb and was located to the short arm of chromosome 2 at 2p16-p21. The frequency of mutations on both alleles of PIGF should be much lower than that of mutation in the X-linked PIGA, accounting for a lack of involvement of PIGF in PNH. We also identified the processed pseudogene of PIGF (psi PIGF) and mapped it to 5q35.

Base Sequence↗
PubMed1995

Accelerated cell-cycling of hematopoietic progenitor cells by growth factors.

Recent advances in molecular biology have led to the identification of hematopoietic growth factors that support and influence the proliferation of hematopoietic progenitor cells in vitro and in vivo. Although these factors have been extensively studied, little is known of their role in the regulation of cell-cycling of hematopoietic progenitors, especially in the early stage of hematopoiesis. In the present study, we examined the effects of early acting growth factors on proliferative kinetics of hematopoietic progenitors by monitoring the number of cells in individual developing colonies, using an in vitro clonal assay. Interleukin-11 (IL-11) or steel factor (SF), alone or in combination, shortened the time for the size of IL-3-dependent colonies to double. Consecutive replating experiments provided evidence for direct action of growth factors on the growth rate of hematopoietic progenitor cells. Shortening of the time for the total cell number in the colonies to double was due to a reduction in time for each single cell within the respective colonies to become two daughter cells, and there was no alteration in the incidence of cells with a proliferative capacity. Cell-cycle analysis demonstrated that IL-11 has the potential to induce a shortened time for cell-cycle of hematopoietic progenitor cells without affecting distribution of each fraction of the cell-cycle, whereas SF has the potential to reduce cell-cycle time mainly by decreasing the time required for hematopoietic progenitor cells to go through the G1 phase. These results suggest that growth factors may modulate cell-cycling of hematopoietic progenitor cells.

Animals↗
PubMed1995

Oligomannose-coated liposomes as an adjuvant for the induction of cell-mediated immunity.

The effect of the coating of ovalbumin-reconstituted liposomes with various oligosaccharides on their immunogenicity was investigated in mice. The coating of liposomes with oligomannose or yeast mannan drastically enhanced their ability to induce an ovalbumin-specific delayed-type footpad swelling response with a peak at 24 to 48 h post-challenge. Among various oligosaccharides tested, only those with mannose residue at the nonreducing termini manifested the activity when applied to liposomes. Since such oligosaccharides are ubiquitously found in the body, these results suggested the usefulness of oligomannose-coated liposomes as a safe adjuvant for the induction of cell-mediated immunity.

Adjuvants, Immunologic↗
PubMed1995

Complement activation by cross-linked B cell-membrane IgM.

The B cell membrane IgM (mIgM) occurs in a monomeric form incapable of activating C. However, when cross-linked by a polyvalent ligand, mIgM may activate C by assuming a polymeric structure like secreted IgM. This possibility was tested with CR2- lymphoma cells, which did not activate C spontaneously. When CR2-deficient mIgM(lambda)+ Ramos cells were treated with F(ab')2 goat anti-lambda, then exposed to human serum, a marked C3 deposition took place, as examined by the flow cytometry. Similarly, C3 deposition on mIgM(kappa)+, mIgD(kappa)+ P32 cells was induced by F(ab')2 of either anti-kappa or anti-mu. Anti-delta was without effect, but the C3 deposition resulting from anti-kappa was markedly enhanced after mIgD was modulated by anti-delta. The mIgM-cross-linked cells bound C1q, and C3 deposition on these cells was abrogated by depletion of C1q, but not Factor B nor D, from serum. The C1-binding step of the mIgM-mediated C activation was inhibited by monomeric Fab' of polyclonal anti-mu containing a blocking Ab to the hemolytic activity of human IgM Forssman Ab. A large proportion of C3 deposits on mIgM-cross-linked cells was found to be associated with mIgM in the form of C3dg or C3d. These results demonstrate that cross-linked mIgM indeed triggers the classical pathway of C.

B-Lymphocytes↗
PubMed1995

Exaggerated messenger RNA expression of inflammatory cytokines in human T-cell lymphotropic virus type I-associated myelopathy.

OBJECTIVE: To investigate the expression of inflammatory cytokine messenger RNA (mRNA) in peripheral blood mononuclear cells of patients with human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM). PATIENTS: Seventeen patients with HAM, 18 HTLV-I-seropositive carriers, and 10 seronegative individuals were studied. MAIN OUTCOME MEASURE: We compared the expression of tumor necrosis factor alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha (IFN-alpha), IFN-beta, and IFN-gamma, and interleukin 1 alpha (IL-1 alpha) and IL-1 beta by reverse transcriptase-polymerase chain reaction. RESULTS: In patients with HAM, the reverse transcriptase-polymerase chain reaction products of TNF-alpha, GM-CSF, IFN-gamma, and IL-1 alpha were detected in significantly higher incidences than in HTLV-I-seropositive carriers and seronegative controls. Furthermore, simultaneous mRNA expression of three or more of these four cytokines was detected in all patients with HAM compared with only 21.4% of HTLV-I-seropositive carriers. By contrast, there was no significant difference in mRNA expression of IFN-alpha, IFN-beta, and IL-1 beta among patients with HAM, HTLV-I-seropositive carriers, and HTLV-I-seronegative controls. CONCLUSIONS: An exaggerated mRNA expression of several inflammatory cytokines, including TNF-alpha, GM-CSF, IFN-gamma, and IL-1 alpha, was demonstrated in peripheral blood mononuclear cells of patients with HAM. Moreover, transcripts of these cytokines were simultaneously up-regulated in patients with HAM, suggesting that an inflammatory state in the central nervous system may be related to the pathogenesis of HAM.

Adult↗