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Biomedical subjects

A Ichikawa

Publications and source records attributed to A Ichikawa.

At least 253 records · Page 14Linked to original sources
PubMed1990

Identification of a prostacyclin receptor coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mouse mastocytoma P-815 cells.

A stable analogue of prostacyclin, iloprost, specifically bound to 30,000 x g pellet (the membrane fraction) prepared from mouse mastocytoma P-815 cells. The binding was dependent on time, temperature and pH, and absolutely required a divalent cation. The equilibrium dissociation constant and the maximal concentration of the binding site as determined by Scatchard plot analysis were 10.4 nM and 1.12 pmol/mg of protein, respectively. The Hill coefficient was 1.0, indicating a single entity of binding site and no cooperativity. The binding site was highly specific for iloprost among PGs tested (iloprost much greater than PGE1 greater than carbacyclin greater than PGE2). In contrast, the membrane fraction had the binding site specific for PGE2 and PGE1, which was distinct from the prostacyclin receptor. The dissociation of bound [3H]iloprost from the membrane fraction was specifically enhanced by guanine nucleotides. Furthermore, iloprost dose-dependently enhanced the activity of adenylate cyclase in a GTP-dependent manner. These results indicate that a specific prostacyclin receptor is coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mastocytoma cells.

Adenine Nucleotides↗
PubMed1990

Purification and characterization of l-histidine decarboxylase from mouse mastocytoma P-815 cells.

Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.

Animals↗
PubMed1990

Detection of human T-cell leukemia virus type I (HTLV-I) provirus in an infected cell line and in peripheral mononuclear cells of blood donors by the nested double polymerase chain reaction method: comparison with HTLV-I antibody tests.

Human T-cell leukemia virus type I (HTLV-I) provirus DNA from the cultured cell line HUT 102 and from peripheral mononuclear cells (PBMC) of anti-HTLV-I antibody-positive Japanese blood donors was detected by the nested double polymerase chain reaction (PCR) method. This procedure consists of a first amplification and a second amplification with the products of the first amplification and primers interior to the first primers. Using this method, we demonstrated that it is possible to detect single-template DNA. Polyacrylamide gel electrophoresis of the nested double PCR products, with our primers, revealed three bands with excess amounts of template DNA, two bands with moderate amounts, and a single band with limited amounts. The amount of provirus in PBMC was roughly estimated from the results of the nested double PCR. Particle agglutination (PA) assays and indirect immunofluorescence testing (IF) with mixed MT-2 cells and Molt-4 cells as targets to detect anti-HTLV-I antibody were performed, and the results were compared with those of the nested double PCR of the pX region. None of the 101 PA-negative samples were positive in either the IF or PCR test. Of the 155 samples that were antibody positive by the PA assay, 57 were positive by both PCR and IF. Furthermore, the results of the IF and PCR tests coincided completely. It was therefore concluded that the IF method is most appropriate for confirmation of the PA assay currently used in most diagnostic laboratories and blood centers.

Base Sequence↗
PubMed1990

Identification of hypoxanthine and inosine in brain dialyzable fraction as stimulators for growth of porcine aortic endothelial cells in response to fibroblast growth factor in either dialyzed serum media or low serum media.

The rate of proliferation of porcine aortic endothelial cells (PAEC) in response to fibroblast growth factor (FGF) was largely retarded when incubated in Dulbecco's modified Eagle's medium (DMEM) supplemented with either 1% fetal bovine serum (FBS) or 10% dialyzed FBS in place of 10% FBS. Proliferation of endothelial cells in low serum media in response to FGF was enhanced to the level of media containing FGF plus 10% FBS by the addition of the dialyzable fraction from bovine brain homogenates. From the bovine brain dialyzable fraction, two active components were purified and identified as hypoxanthine and inosine. Either hypoxanthine or inosine, at a dose of 5 microM in DMEM with 1% FBS, maximally increased the incorporation of [3H]thymidine into DNA of PAEC in low serum media in the presence of FGF. However, no additive effect was observed when hypoxanthine and inosine were added simultaneously. The present data indicate that the proliferative action of FGF on PAEC can be potentiated by hypoxanthine and inosine.

Animals↗
PubMed1990

[Chronic cytopenia associated with T8 lymphocytosis successfully treated with glucocorticoids].

We reported a prednisolone effective case of T 8 lymphocytosis. The patient was 83 years old male, who had suffered from hypoplastic anemia for more than 5 years before he admitted to our hospital. His blood picture was peculiar because adding to granulocytopenia, CD8 (+) large granular lymphocytes were dominant among his lymphocytes. A few days after administration of bolus methylprednisolone, prominent reticulocytosis and rapid improvement of anemia occurred and the latter continued for more than 1 year. We performed in vitro study on the mechanism of his cytopenias. The result of Southern blot analysis showed that his blood lymphocytes had a unique rearranged pattern of T cell receptor beta chain gene. Mononuclear cells had an inhibitory effect on autologous and allogenic CFU-E colony formation of bone marrow cells. These facts suggest that colony proliferating CD8 (+) lymphocytes had an inhibitory effect on erythropoiesis in vivo, and prednisolone blocked this inhibitory pathway in this patient.

Aged↗
PubMed1989

The monocytic differentiation of HL60 induced by rat kidney NADPH-linked high-Km aldehyde reductase protein.

The human promyelocytic leukemia cell line HL60 differentiates to monocyte/macrophage cells when incubated with NADPH-linked high-Km aldehyde reductase (EC 1.1.1.2) purified from the cytosol of rat kidney. Differentiation was assessed by cell growth, morphology, adhesiveness, nitro blue tetrazolium reduction, and nonspecific esterase activity. The extent of differentiation induced by the reductase and measured at 4 days by nitro blue tetrazolium reduction is dose-dependent with an ED50 (dose required for half-maximal effect) of 71 nM. In the presence of 10 nM retinoic acid the ED50 for reductase is reduced to 18 nM and an isobologram analysis of this effect indicates that the combination is synergistic. Inactivation of the enzymatic activity is not associated with a decrease in differentiation-induced activity. These results suggest that the structure of the enzyme protein and not its enzymatic activity is involved with induction of differentiation. This view is supported by the demonstration that aldehyde reductase binds specifically to HL60 cells with a KD of 70 nM and that there are 13,000 binding sites/cell. Thus, the extent of differentiation induced by various concentrations of aldehyde reductase are directly related to the expected level of receptor occupancy.

Animals↗
PubMed1989

Effects of glycyrrhizin and glycyrrhetinic acid on dexamethasone-induced changes in histamine synthesis of mouse mastocytoma P-815 cells and in histamine release from rat peritoneal mast cells.

The effects of glycyrrhizin and its aglycone, glycyrrhetinic acid, on dexamethasone-induced changes in the histamine synthesis of mastocytoma P-815 cells and in the histamine release from antigen-stimulated rat peritoneal mast cells were investigated. Glycyrrhetinic acid but not glycyrrhizin, at concentrations from 20 to 35 microM, almost completely inhibited the dexamethasone-induced increases in both the histamine content and histidine decarboxylase activity of cultured mastocytoma P-815 cells. Glycyrrhetinic acid, however, showed practically no inhibition of [3H]dexamethasone binding to the cytoplasmic receptor. On the other hand, glycyrrhetinic acid but not glycyrrhizin markedly inhibited the release of histamine from antigen-stimulated rat mast cells, and intensified the inhibitory activity induced by dexamethasone. Glycyrrhetinic acid inhibited the antigen-induced release and incorporation of [3H]arachidonic acid in immunized rat mast cells. The administration of glycyrrhizin into rats, in contrast to the in vitro treatment of the cells with glycyrrhizin, markedly inhibited histamine release from antigen-stimulated rat mast cells. These results suggest that glycyrrhetinic acid inhibited dexamethasone-induced changes in the histamine synthesis of mastocytoma P-815 cells, and in the histamine release from rat mast cells. On the other hand, glycyrrhizin may exert its effect after conversion to glycyrrhetinic acid in vivo.

Animals↗
PubMed1989

Prostaglandin D2 receptor of mastocytoma P-815 cells--possible regulation by phosphorylation and dephosphorylation.

The 3H-labeled prostaglandin D2 [( 3H]PGD2) binding protein in the membrane fraction of mastocytoma P-815 cells was characterized. The specific binding of [3H]PGD2 to the cells or the membranes reached a maximum at pH 5.6, and was saturable, displaceable and of high affinity when incubated at 0 or 37 degrees C. The Bmax values for [3H]PGD2 binding in the two preparations at pH 5.6 were much higher at 0 degrees C than at 37 degrees C, whereas the Kd values were almost equal (85.3 nM for the cells and 80.5 nM for the membranes, respectively). High specific [3H]PGD2 binding activity in the mildly acid-treated cells was still observed when the external pH was raised from 5.6 to 7.2. Furthermore, specific [3H]PGD2 binding to the membranes (at 0 degrees C, pH 5.6) increased on addition of phosphatase inhibitors (NaF and molybdate) in the presence of 10 microM ATP, but practically disappeared on pretreatment of the membranes with phosphatase. On incubation of the membrane with [gamma-32P]ATP and molybdate, the stimulated incorporation of the [32P]phosphate into several peptides, including ones having an Mr of around 100,000-120,000, was observed. These results suggest that [3H]PGD2 binding in the mastocytoma P-815 cell membrane is controlled through phosphorylation-dephosphorylation of the receptor itself.

Acid Phosphatase↗
PubMed1989

Optimal preparatory procedures of cryofixation for immunocytochemistry.

To examine the optimal preparatory procedures of cryofixation for immunocytochemistry, the labeling density over the antigenic sites in cells processed by various protocols of freeze-substitution and embedding was quantitatively evaluated. Fresh tissue blocks of gerbil parotid gland were quickly frozen by a metal contact method using liquid helium and freeze-substituted with one of the following media: 4% OsO4 in acetone or 0.4% OsO4 in acetone or 0.3% glutaraldehyde in acetone. They were then embedded in either an Epon-Araldite mixture or Araldite 6005, which were polymerized at 60 degrees C and 50 degrees C, respectively. Some frozen samples substituted with aldehyde-containing acetone were embedded in Lowicryl K4M (polymerized at -30 degrees C). Immunocytochemical localization of amylase was examined by indirect immunostaining by using antigerbil parotid amylase antibody and protein A/gold complex. Thin sections of epoxyresin-embedded materials were treated with oxidizing agents before immunostaining. The central dense core of heterogeneous secretory granules in the acinar cells was heavily labeled with immunogold, regardless of substitution media and embedding resins employed. The labeling density on thin sections of all the cryofixed materials examined was about 1.5 times or more as high as in those processed by conventional chemical fixation. The highest value of the labeling density was obtained from material which was substituted with 0.3% glutaraldehyde in acetone and embedded in Araldite 6005. Substitution with osmium-containing acetone appeared not to seriously affect immunoreactivity of the antigenic sites and was advantageous because of the distinctive images of membranes. Advantages and disadvantages of the individual protocols employed are discussed.

Amylases↗
PubMed1989

Fibroblast growth factor-stimulated growth of porcine aortic endothelial cells depends on hypoxanthine in fetal bovine serum in culture media.

The rate of proliferation of porcine aortic endothelial cells (PAEC) in response to stimulation of fibroblast growth factors (FGFs) was largely retarded in media supplemented with 10% dialyzed fetal bovine serum (FBS) in place of nondialyzed FBS. This inhibition was overcome by supplement of dialyzable fraction, and hypoxanthine was purified from the dialyzable fraction as the active compound which stimulated the basal and FGF-dependent growth rates of dialyzed FBS-treated PAEC. Addition of hypoxanthine (5 microM) to media with 10% dialyzed FBS containing FGFs (10 ng/ml) markedly increased the rate of both cell proliferation and DNA synthesis of PAEC, and their maximal levels were comparable to those attained by cells in media with 10% nondialyzed FBS. Hypoxanthine changed the spindle-like morphology of dialyzed FBS-treated PAEC even in the presence of FGFs into the cobblestone-like morphology of regular PAEC in media with 10% FBS.

Animals↗
PubMed1989

Immunocytochemical localization of amylase in gerbil salivary gland acinar cells processed by rapid freezing and freeze-substitution fixation.

We examined the immunocytochemical localization of amylase in cryofixed serous acinar cells of gerbil major salivary glands by indirect immunostaining, using anti-gerbil parotid amylase antibody and protein A-gold complex. Fresh tissue blocks were quickly frozen by the metal-contact method, using liquid helium, and were freeze-substituted with either osmium-acetone solution or glutaraldehyde-containing acetone. They were then embedded in an epoxy resin mixture which was polymerized at 60 degrees C. Some tissue blocks substituted with aldehyde-acetone solution were embedded in Lowicryl K4M, polymerized at -30 degrees C. Thin sections of epoxy resin-embedded materials were treated with an oxidizing agent before immunostaining. The labeling density on the materials processed by various protocols for preparatory procedures was quantitatively compared to examine the usefulness of application of cryofixation to immunocytochemistry. The central dense core of heterogeneous secretory granules in the serous acinar cells of the parotid and sublingual glands was heavily labeled with immunogold, regardless of substitution media and embedding resins employed. The immunolabeling pattern clearly distinguished between the dense core and the surrounding matrix. Labeling density in the cryofixed materials was about 1.5 times greater than in those processed by conventional chemical fixation. Seromucous secretory granules in the submandibular gland acinar cells were only faintly labeled. The results obtained indicate that application of immunostaining to quick-frozen, substitution-fixed tissues is useful for high-resolution immunocytochemistry.

Amylases↗
PubMed1989

[Reconstructive vascular surgery in the territory of the anterior cerebral artery in children with moyamoya disease].

The hemodynamics of the anterior cerebral arteries (ACAs) were studied in 28 children with moyamoya disease. In 39% of 56 cerebral hemispheres, antegrade filling of the ACAs was not verified by preoperative angiography. On the other hand, adequate collateral pathways to the ACA territory were generally not achieved by encephalomyosynangiosis (EMS) or superficial temporal artery-middle cerebral artery (STA-MCA) anastomosis. The authors have devised new surgical methods for the revascularization of the ACA territory, which they term "encephaloarteriosynangiosis" (EAS) and "encephalogaleosynangiosis" (EGS). In these procedures, one or several burr holes are made in the frontal skull, the dura mater is incised, and either the frontal branch of the STA or the pedicled galea aponeurotica stump is placed on the surface of the frontal cortex. In addition to EMS or STA-MCA anastomosis, these methods were applied to 23 cerebral hemispheres in 14 pediatric patients. In 14 cerebral hemispheres (61%) of 10 patients, postoperative external carotid angiograms showed some cortical branches in the ACA territory, and clinical improvement was achieved in patients with transient ischemic attacks affecting the lower extremities. The results of this study demonstrate that EAS and EGS are simple and useful techniques for revascularization of the ACA territory. In addition, EGS can be applied to the territory of the posterior cerebral artery.

Adolescent↗
PubMed1989

[Salvage chemotherapy with a combination of VP-16, ifosfamide, procarbazine, prednisolone, bleomycin and methotrexate (VIPP-BM) for refractory malignant lymphoma].

Twenty patients with refractory malignant lymphoma were treated with a combination of VP-16, ifosfamide, procarbazine, prednisolone, bleomycin and methotrexate (VIPP-BM) as salvage chemotherapy. These patients were either resistant to front-line therapy or refractory in their relapses. Two patients (10%) achieved a complete remission and eleven patients (55%) attained a partial remission. An overall response rate was 65%. Major toxicities were myelosuppression, nausea and vomiting, and mucositis. However they were well tolerated. This regimen has been effective in the treatment for the patients with refractory lymphoma.

Adolescent↗
PubMed1989

[Cell kinetic analysis with flow cytometry as a disdifferentiation mechanism in human neuroblastoma cells in vitro].

The established cell line NB6 (human neuroblastoma) was perturbed in vitro, in order to evaluate the disdifferentiation mechanism of malignant neoplastic cells during cultivation. The methods for perturbation were as follows: 1) 50%-cell removal from dishes; 2) 0.1 mg/ml of vincristine (VCR) contact; and 3) 10Gy of beta-radiation. Specimens were harvested every 24 hours until 72 hours, simultaneously stained for DNA and RNA with acridine orange (AO), and cell kinetically analyzed for DNA/RNA/cell-distribution with the aid of flow cytometry (FCM). Thus disdifferentiated cells and/or newly cell cycle traversing cells, were evaluated. The new subpopulations were considered to be recruited from G1A and/or Gq (Darzynkieowicz et al.) into the cell cycle after perturbations. Cells of the new subpopulations had smaller volume compared with the control cells. Furthermore, in the current study, newly cell cycle traversing cells of NB6 appeared to have a disturbed cell cycle traverse in G2B phase from the clonological changing patterns of FCM cytograms. These new subpopulations were revealed to be able to passage up to five or six times. Though it was impossible to make them appear, these new cycling subpopulations might be capable of remaining as permanent clones in vitro.

Beta Particles↗
PubMed1989

Freeze-substitution for thin-section study of biological specimens.

Freeze-substitution as a preparation technique for frozen specimens is briefly reviewed. Recent advance in cryotechniques, especially quick freezing followed by freeze-substitution, has made it possible to preserve the ultrastructural features of cells and tissues to reflect more closely their living state and capture transient physiological processes as evanescent as fusion and fission of biological membranes. The technique is also advantageous to retain soluble substances of the cellular components. Thus, histochemical, autoradiographic and/or immunocytochemical analyses of quick-frozen, substitution-fixed samples allow us to obtain more precise localization of chemical components in cells than in the case of conventional chemical fixation. Several examples indicating the superiority of the preparatory method are presented.

Animals↗