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Biomedical subjects

A Ichikawa

Publications and source records attributed to A Ichikawa.

At least 217 records · Page 12Linked to original sources
PubMed1992

Mouse thromboxane A2 receptor: cDNA cloning, expression and northern blot analysis.

A cDNA clone for the mouse thromboxane A2 receptor was isolated from mouse lung cDNA library. The cDNA has a 1,023 base pair open reading frame which encodes a protein of 341 amino acid residues. STA2 and U-46619 induced inward current in Xenopus laevis oocytes injected with the transcript of the clone. Specific binding of [3H]S-145 was found in membranes of COS-1 cells transfected with the cDNA (Kd = 3.3 nM) and was displaced with unlabeled prostaglandins and thromboxane analogues in the order of S-145 greater than STA2 greater than U-46619 greater than PGD2 greater than PGF2 alpha = PGE2. Northern blot analysis demonstrated that thromboxane A2 receptor mRNA is expressed abundantly in thymus, spleen and lung.

Amino Acid Sequence↗
PubMed1992

Mutations of p53 gene and their relation to disease progression in B-cell lymphoma.

The alteration of p53 tumor suppressor gene was studied in 48 patients with B-cell lymphoma. A sequential combined technique of polymerase chain reaction-mediated single-strand conformational polymorphism (PCR-SSCP) or reverse transcription (RT)-PCR-SSCP and direct sequencing were used as a simple and sensitive approach to analyze nucleotide changes. By these methods, we identified 8 missense point mutations and 2 codon deletions in 9 of the 48 patients. These mutations were located in or close to the evolutionally highly conserved regions of the p53 gene. Eight of nine patients having p53 gene alterations were in advanced clinical stage (IV). It is the first report of p53 gene mutations in follicular and diffuse lymphoma. These observations suggest that the p53 gene alteration may play an important role in lymphomagenesis and/or disease progression in some types of B-cell lymphoma.

Base Sequence↗
PubMed1992

Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype.

A functional cDNA clone for mouse EP3 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of the human thromboxane A2 receptor and cross-hybridization screening. The mouse EP3 receptor consists of 365 amino acid residues with putative seven-transmembrane domains. The sequence revealed significant homology to the human thromboxane A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGE2 binding. The binding was displaced with unlabeled PGs in the order of PGE2 = PGE1 greater than iloprost greater than PGF2 alpha greater than PGD2. The EP3-selective agonists, M&B 28,767 or GR 63799X, potently competed for the [3H]PGE2 binding, but no competition was found with EP1- or EP2-selective ligands. PGE2 and M&B 28,767 decreased forskolin-induced cAMP formation in a concentration-dependent manner in Chinese hamster ovary cells permanently expressing the cDNA. Northern blot analysis demonstrated that the EP3 mRNA is expressed abundantly in kidney, uterus, and mastocytoma P-815 cells and in a lesser amount in brain, thymus, lung, heart, stomach, and spleen.

Alprostadil↗
PubMed1992

Involvement of protein kinase C in thrombin-induced translocation of Gi2 alpha from the membrane to the cytosol in mouse mastocytoma P-815 cells.

Recently, we reported that in mouse mastocytoma P-815 cells the cytosol contains some factor(s) which promotes the release of GTP-activated Gi2 alpha from the membrane, and that thrombin induces the translocation of Gi2 alpha from the membrane to the cytosol (Takahashi, S., Negishi, M. and Ichikawa, A. (1991) J. Biol. Chem. 266, 5367-5370). Here we investigated the mechanism underlying the thrombin-induced translocation of Gi2 alpha in mastocytoma cells. Thrombin induced a rapid and transient increase in the intracellular Ca2+ concentration ([Ca2+]i) within 1 min, attenuated pertussis toxin-catalyzed ADP-ribosylation of Gi2 in the membrane, and caused the subsequent translocation of Gi2 alpha. Thrombin induced the translocation of protein kinase C from the cytosol to the membrane, and a protein kinase C inhibitor, staurosporine, completely inhibited the thrombin-induced translocation of Gi2 alpha. When cells were treated with thrombin, the ability of the cytosol to release Gi2 alpha from the membrane in the presence of GTP gamma S markedly increased. This stimulatory effect of thrombin on the ability of the cytosol was mimicked by 12-O-tetradecanoylphorbol 13-acetate (TPA), but not by the Ca2+ ionophore, ionomycin. The thrombin- and TPA-induced potentiation of the ability of the cytosol to release Gi2 alpha was completely abolished by staurosporine. Furthermore, phosphorylation of the cytosol by protein kinase C markedly potentiated the ability of the cytosol to release Gi2 alpha. These results together demonstrate that the thrombin-induced translocation of Gi2 alpha is due to enhancement of the ability of the cytosol to release Gi2 alpha via activation of protein kinase C.

Adenosine Diphosphate Ribose↗
PubMed1992

Translocation of alpha subunits of stimulatory guanine nucleotide-binding proteins through stimulation of the prostacyclin receptor in mouse mastocytoma cells.

The translocation of the alpha subunits of Gs from the membrane to the cytosol by iloprost, a stable prostacyclin analogue, was studied in mouse mastocytoma P-815 cells. In the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S), iloprost stimulated the adenylate cyclase activity, caused the release of both 42- and 45-kDa proteins reactive with the anti Gs alpha carboxyl-terminal antibody, RM/1, from the membrane and attenuated cholera toxin-catalyzed ADP-ribosylation of the 42- and 45-kDa proteins in the membrane. The iloprost-stimulated adenylate cyclase activity and release of Gs alpha from the membrane were markedly suppressed by RM/1. Cholera toxin treatment also stimulated the adenylate cyclase activity and release of Gs alpha from the membrane, and iloprost synergistically potentiated these actions of cholera toxin. In mastocytoma cells, iloprost induced the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol, 45-kDa Gs alpha remaining in the cytosol for a longer time than 42- kDa Gs alpha. Whereas 42-kDa Gs alpha in the cytosol was eluted at the position of Mr = approximately 40,000 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 from a Superose 12 gel filtration column. In contrast, both 42- and 45-kDa Gs alpha released in vitro from the membrane by iloprost plus GTP gamma S were eluted at the position of Mr = approximately 40,000, but only 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 when it was incubated with cytosol. These results taken together demonstrate that iloprost induces the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol and that only the 45-kDa Gs alpha released exists in the cytosol as a soluble complex with unidentified component(s) in mastocytoma cells.

Adenosine Diphosphate Ribose↗
PubMed1992

Purification and properties of L-histidine decarboxylase from mouse stomach.

L-Histidine decarboxylase was purified to electrophoretic homogeneity from mouse stomach according to a procedure described previously [Ohmori E, Fukui T, Imanishi N, Yatsunami K and Ichikawa A, J Biochem (Tokyo) 107: 834-839, 1990]. The purified enzyme exhibited a specific activity of 750 nmol histamine formed per min per mg protein, which constituted a 37,500-fold purification compared to the crude extract, with a 1.6% yield. The molecular mass of the enzyme was estimated to be 54 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 100 kDa by gel filtration. The isoelectric point of the enzyme was determined to be pH 5.4. The Km value for L-histidine was estimated to be 0.29 mM. The single mRNA encoding the amino acid sequence of the mouse stomach enzyme was examined and its size was found to be 2.7 kb. These molecular and catalytic property values of the L-histidine decarboxylase of mouse stomach are quite similar to those of the enzyme from mouse mastocytoma P-815 cells.

Animals↗
PubMed1992

Synergistic effects of 12-O-tetradecanoylphorbol-13-acetate and dexamethasone on de novo synthesis of histidine decarboxylase in mouse mastocytoma P-815 cells.

12-O-Tetradecanoylphorbol-13-acetate (TPA) markedly enhanced the increase in L-histidine decarboxylase (HDC) activity induced by dexamethasone in mouse mastocytoma P-815 cells, even with a concentration of the latter that had the maximal effect, whereas it induced a rapid and transient increase in HDC activity, which peaked after 3 h in the absence of dexamethasone. The synergistic effect of TPA on HDC activity induced by dexamethasone was detected after 4 h, a plateau level being reached by 6 h, which was similar to the time course with dexamethasone alone. TPA enhanced the induction of HDC activity by various glucocorticoids, but had no effect on the induction by dibutyryl cAMP, prostaglandin E2 or sodium butyrate. Both 1-oleoyl-2-acetylglycerol, a protein kinase C activator, and okadaic acid, a protein phosphatase inhibitor, enhanced the increase in HDC activity induced by dexamethasone, but 4 alpha-phorbol-12,13-didecanoate, an inactive derivative of TPA, did not. Protein kinase C inhibitors, such as staurosporin, H-7 and K255a, suppressed the increase in HDC activity induced by TPA with or without dexamethasone. The enhancement of HDC activity by dexamethasone was completely suppressed by cycloheximide or actinomycin D. Furthermore, TPA markedly enhanced the accumulation of HDC mRNA due to dexamethasone (5 to 10-fold, from 6 to 12 h after). TPA did not cause a significant increase in the level of either [3H]dexamethasone binding capacity or preformed HDC activity in cells. These results taken together suggest that dexamethasone-induced de novo synthesis of HDC in mastocytoma P-815 cells is up-regulated by TPA-activated protein kinase C through the mechanism involving an increased rate of transcription.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Synergistic effects of cyclic AMP and Ca2+ ionophore A23187 on de novo synthesis of histidine decarboxylase in mastocytoma P-815 cells.

In the preceding paper (Kawai, H. et al. (1992) Biochim. Biophys. Acta 1133, 172-178), we reported that in mastocytoma P-815 cells dexamethasone and 12-O-tetradecanoylphorbol-13-acetate (TPA) synergistically enhanced the de novo synthesis of L-histidine decarboxylase (HDC). Here we found that Ca2+ acted synergistically with cAMP in the induction of HDC mRNA and HDC activity in mastocytoma P-815 cells, and that the mechanism underlying the enzyme induction by Ca2+ plus cAMP was distinguishable from that by dexamethasone plus TPA. Ca2+ ionophore A23187, itself having no significant activity, markedly enhanced the induction of HDC activity by N6,O2'-dibutyryl cAMP (db cAMP) or cAMP-inducible prostaglandins such as PGE1, PGE2 and PGI2 in the presence of the phosphodiesterase inhibitor, Ro201724. However, A23187 had little effect on increases in HDC activity induced by other known stimulants, such as TPA, dexamethasone and sodium butyrate. These results suggest that A23187 has a specific effect on the induction of HDC activity due to an increased level of cAMP. The finding that both A23187 and cAMP enhanced HDC activity suggests that both Ca2+/calmodulin and cyclic nucleotide dependent protein kinase play essential roles in the process of enhancement of HDC activity. To examine this possibility, we studied the effects of W-7, an inhibitor of calmodulin, removal of extracellular Ca2+, and H-8, an inhibitor of cAMP-dependent protein kinase, on the enhancing activity of A23187 plus db cAMP. The enhancement of HDC activity by A23187 plus db cAMP was inhibited by W-7, removal of extracellular Ca2+, and H-8. The increase in HDC activity was due to the de novo synthesis of the enzyme, since it was suppressed by the addition of cycloheximide or actinomycin D, and was well correlated with the marked accumulation of a 2.7 kilobase HDC mRNA. Furthermore, the mechanism underlying the induction of HDC by db cAMP plus A23187 is distinguishable from that in the case of dexamethasone plus TPA, since preexposure to dexamethasone plus TPA for 12 h, for a plateau level to be reached, did not affect the subsequent increase in HDC activity due to db cAMP plus A23187.

Animals↗
PubMed1992

Induction of heme oxygenase by delta 12-prostaglandin J2 in porcine aortic endothelial cells.

delta 12-Prostaglandin (PG)J2 stimulated the synthesis of a 31,000-dalton protein (termed p31) and the induction of cellular heme oxygenase activity in porcine aortic endothelial cells. A good correlation was observed between the time courses and dose dependencies of the induction of p31 synthesis and that of heme oxygenase activity by delta 12-PGJ2. Hemin, a known inducer of heme oxygenase, also induced p31 synthesis as well as heme oxygenase activity in the cells. On two-dimensional gel electrophoresis, p31 induced by delta 12-PGJ2 exhibited an isoelectric point of 5.4, which coincided exactly with that induced by hemin. These results indicate that the p31 induced by delta 12-PGJ2 in porcine aortic endothelial cells is heme oxygenase.

Animals↗
PubMed1992

Subcellular localization of xanthine oxidase in rat hepatocytes: high-resolution immunoelectron microscopic study combined with biochemical analysis.

Xanthine oxidase (XO), a molybdo-flavoprotein enzyme involved in purine degradation, was localized immunocytochemically in rat hepatocytes by high-resolution immunoelectron microscopy. XO was isolated from rat liver and a 150 KD polypeptide was purified. Antibodies were raised in rabbits. Small pieces of fresh liver were quickly frozen by contact with a copper block pre-cooled with liquid helium and were freeze-substituted with either 2.5% OsO4 or 0.2% glutaraldehyde in acetone. They were then warmed and embedded in Epon-Araldite or Araldite 6005. Resin sections were treated by indirect immunostaining using anti-rat liver XO antibody and protein A-gold. The labeling pattern was clearly over the cytosol and not on cell organelles. A few gold particles were found over the mitochondrial matrix, but not over the endoplasmic reticulum, Golgi apparatus, lysosomes, or peroxisomes, including their crystalloid core. These results are consistent with those of the biochemical assay of XO in this study. The significance of the occasional immunolabeling of the mitochondrial matrix remains obscure, since biochemical determinations in this study indicate no XO activity in the mitochondrial fraction.

Animals↗
PubMed1992

Effect of adenosine 3',5'-cyclic monophosphate derivatives on acute liver injury induced by carbon tetrachloride.

The inhibitory effects of 12 synthetic N6-alkyl cAMPs, 8-substituted cAMPs and cAMP alkylphosphoramidate derivatives (50 or 100 mg/kg, bolus, i.p.) on serum GOT and GPT activities and hepatocyte cytoplasmic vacuolation were examined in male Fischer 344 rats, which were exposed to CCl4 (0.5 mg/kg, p.o.) 30 min prior to the administration of cAMP derivatives. In CCl4-treated rats 6 h later, serum GOT and GPT levels were elevated 10- and 12-fold higher than those of vehicle rats, respectively. Treating CCl4-exposed rats with all cAMP derivatives, except those of alkylphosphoramidate, significantly decreased the levels of serum enzymes. Based on the effects of both serum GOT and GPT elevation, N6-butyl- and N6-heptyl-cAMP were the most potent. It was also observed histopathologically, that both compounds inhibited the occurrence of cytoplasmic vacuolation in CCl4-treated liver cells. This is the first report that cAMP derivatives possess a protective effect in the liver injury model induced by CCl4.

Alanine Transaminase↗
PubMed1992

Inhibition of salvage synthesis of nucleic acid by adenosine 3',5'-cyclic decylphosphoramidate in mastocytoma P-815 cells.

Constant exposure of mastocytoma P-815 cells to adenosine 3',5'-cyclic decylphosphoramidate (1), which is permeable to the cell membrane and resistant to the action of phosphodiesterase, caused a dose-dependent (1 to 50 microM) inhibition in the synthesis of DNA and cell proliferation. Pretreating the cells with compound 1 (20 microM, 4 h) caused considerable inhibition of the incorporation of [3H]thymidine ([3H]TdR) into [3H]deoxythymidine 5'-triphosphate ([3H]dTTP) and that of [14C]hypoxanthine into nucleic acid, but not the synthesis of [14C]dTTP from [U-14C]aspartate. These results indicate that compound 1 preferentially inhibits the salvage synthesis of intracellular nucleotides and nucleic acids. Thymidine kinase, a key enzyme in salvage synthesis of nucleotides, was almost undetectable in cells pretreated with compound 1 at 20 microM for 4 h or at 5 microM for 15 h. On the other hand, compound 1 activated partially purified cAMP-dependent protein kinase A from bovine heart. Judging from these observations, it is likely that compound 1 readily permeates the cell membrane, activates cAMP-dependent protein kinase, then inhibits the salvage synthesis of nucleotides and nucleic acids by inhibiting thymidine kinase, which results in the inhibition of cell growth.

Animals↗
PubMed1992

Acute subdural hematoma in young patient with moyamoya disease--case report.

A 17-year-old boy with known moyamoya disease developed an acute subdural hematoma after a mild head trauma. He had been confined to a wheelchair with contracture in the upper and lower extremities due to juvenile rheumatoid arthritis since age 1 year. He had undergone encephalo-duro-arterio-synangiosis (EDAS) on the right and encephalo-myo-synangiosis (EMS) on the left at 13 years of age. He was admitted with headache, nausea, and vomiting after a fall from his wheelchair at age 17. Computed tomography on admission showed a large acute subdural hematoma in the right fronto-temporal region but no bleeding at the EDAS or EMS sites. Cerebral angiography 12 weeks after the head trauma revealed a remarkable reduction in the spontaneous transdural external-internal carotid anastomoses in the right frontal region. The acute subdural hematoma was probably caused by rupture of the spontaneous transdural anastomoses.

Acute Disease↗
PubMed1992

Remission induction of acute promyelocytic leukemia by all-trans-retinoic acid: molecular evidence of restoration of normal hematopoiesis after differentiation and subsequent extinction of leukemic clone.

Sequential molecular and cellular changes during remission induction were investigated in a case of acute promyelocytic leukemia (APL) treated with all-trans-retinoic acid (ATRA). By means of clonality analysis, the assessment of the retinoic acid receptor alpha gene alterations, as well as conventional cytologic studies, it was demonstrated that remission induction of APL by ATRA proceeds in two steps: first, the differentiation of the leukemic clone to mature granulocytes and its subsequent extinction; second, proliferation/differentiation of the residual normal clones to restore polyclonal hematopoiesis.

Adult↗
PubMed1992

[Flow cytometric techniques for measurement of proliferating cell nuclear antigen (PCNA)].

Flow cytometric techniques have been developed for measurement of proliferating cell nuclear antigen (PCNA), which allows studies on the proliferative capacity of cells and tissues. PCNA-DNA dual staining procedures, for both fixed and unfixed cells, and analytical method by FCM are presented. We recommend performing either the fixed or unfixed method. Our studies using the MCF7 human breast adenocarcinoma cell line and the A549 human lung squamous cell carcinoma cell line, in the exponential growth phase, revealed that both ethanol and acetone were satisfactory fixatives, in contrast to methanol and paraformaldehyde, and PI with a concentration of 25 micrograms/ml was most suitable compared with 50 and 100 micrograms/ml.

Adenocarcinoma↗
PubMed1991

Prostaglandin E1 receptor from mouse mastocytoma P-815 cells couples to 60 kDa GTP-binding protein.

The stable [3H]prostaglandin E1 (PGE1)-bound receptor, which couples to 60 kDa GTP-binding protein, from membranes of mouse mastocytoma P-815 cells has been purified and characterized. When the membranes were preincubated with [3H]PGE1 for 60 min at 37 degrees C, the dissociation of the ligand from the receptor was remarkably decreased, even in the presence of GTP gamma S. The stable [3H]PGE1-bound receptor complex was solubilized with 6% digitonin. The solubilized [3H]PGE1 receptor was eluted with [35S]GTP gamma S bindings activity from an Ultrogel AcA44 column. The fractions containing activities of both [3H]PGE1 and [35S]GTP gamma S bindings were further purified by column chromatographies on wheat germ agglutinin (WGA)-agarose and phenyl-Sepharose CL-4B. The partially purified [3H]PGE1-bound receptor was affinity-labeled with [14C]5'-p-fluorosulfonylbenzoylguanosine and a protein with a molecular mass of 60 kDa was detected. These results suggest that the ligand-bound PGE1 receptor of P-815 cells associates with a novel GTP-binding protein with a molecular mass of 60 kDa.

Adenylyl Cyclases↗
PubMed1991

Induction of a 31,000-dalton stress protein by prostaglandins D2 and J2 in porcine aortic endothelial cells.

Prostaglandin (PG) D2 and PGJ2 stimulated porcine aortic endothelial cells to synthesize a 31,000-dalton protein (termed p31) in a time- and concentration-dependent manner. The induction of p31 synthesis was specific for PGD2, PGJ2 and PGA1 among the various PGs tested. p31 was also synthesized in response to the thiol-reactive agent diethylmaleate and heavy metal sodium arsenite but not to high temperature treatment, platelet-derived growth factor, and 12-O-tetradecanoylphorbol 13-acetate. Using two-dimensional polyacrylamide gel electrophoresis, p31 induced by PGJ2 had an isoelectric point of 5.4, which overlapped exactly with that induced by by arsenite. These results taken together indicate that p31 represents one of the stress proteins whose expression is regulated primarily by thio-active compounds but not by hyperthermia. Furthermore, it was induced by PGD2 and PGJ2 in rat capillary endothelial cells, rat skin fibroblasts, and rat hepatocytes. The data obtained from this study suggest that p31 induced by PGD2 and PGJ2 may play a role in the metabolic regulation of many mammalian cells.

Animals↗