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Biomedical subjects

A Ichikawa

Publications and source records attributed to A Ichikawa.

At least 199 records · Page 11Linked to original sources
PubMed1993

Functional interaction of prostaglandin E receptor EP3 subtype with guanine nucleotide-binding proteins, showing low-affinity ligand binding.

The functional interaction of prostaglandin E (PGE) receptor EP3 subtype with GTP-binding proteins (G proteins) was characterized in the membranes prepared from mouse EP3 receptor cDNA-transfected Chinese hamster ovary cells. PGE2 inhibited forskolin-stimulated adenylate cyclase activity in CHO cells expressing EP3 receptor and this inhibition was abolished by pertussis toxin (PT) treatment. The PGE2 binding to the membranes was increased by GTP gamma S, and PT treatment also increased the binding activity to the same level as that increased by GTP gamma S, but the sensitivity of GTP gamma S was lost. Reconstitution with PT-sensitive G proteins into the ADP-ribosylated membranes reduced the PGE2 binding activity with the following preference: Gi1 = Gi2 > Gi3 > GO, but GTP gamma S completely blocked the reduction by G proteins. The G-protein-induced reduction of the binding was due to the increase in Kd without the change of Bmax, and due to suppression of association rate. [3H]PGE2-bound EP3 receptor solubilized from the ADP-ribosylated membranes in the presence or absence of GTP gamma S was eluted at the position of M(r) = approx. 60 kDa, similar to the relative molecular mass of EP3 receptor deduced from its amino acid sequence. In contrast, [3H]PGE2-bound receptor solubilized from Gi2-reconstituted membranes was eluted at the position of M(r) = approx. 130 kDa, corresponding to the M(r) of the complex of EP3 receptor and Gi2, but GTP gamma S shifted the position of its elution from M(r) = 130 to 60 kDa. Furthermore, addition of PGE2 stimulated the GDP release from G proteins reconstituted into the ADP-ribosylated membranes, and PGE2 inhibited forskolin-stimulated adenylate cyclase activity in G-protein-reconstituted membranes with a selectivity order of Gi1 = Gi2 > Gi3 > GO. These results indicate that EP3 receptor can functionally couple to PT-sensitive G proteins and unusually the complex form with G proteins has low affinity for the ligand but the form not associated with G proteins has high affinity.

Adenosine Diphosphate↗
PubMed1993

Two isoforms of the EP3 receptor with different carboxyl-terminal domains. Identical ligand binding properties and different coupling properties with Gi proteins.

Functional cDNA clones for two isoforms of the mouse prostaglandin E receptor EP3 subtype derived from alternative RNA splicing were obtained. The two isoforms are only different in the sequence of the putative cytoplasmic carboxyl-terminal tail and their hydrophobicity; one isoform, named EP3 alpha, has a hydrophilic tail, and the other, named EP3 beta, has a hydrophobic tail. When expressed, the two receptors displayed identical ligand binding properties but different responses to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Without a change in the Bmax value, GTP gamma S increased Kd for prostaglandin E2 of EP3 beta and decreased that of EP3 alpha. These effects were abolished by the treatment of membranes with pertussis toxin and restored by the addition of Gi2. Although both isoforms exerted inhibition of forskolin-induced cAMP accumulation, three orders lower concentrations of agonists were required for EP3 alpha than EP3 beta for 50% inhibition of cAMP formation. A similar difference in agonist potency was observed also for agonist-induced stimulation of GTPase activity in membranes. Thus, the two receptors with different carboxyl-terminal tails show different coupling to the Gi protein, leading to the opposite responses to GTP in the ligand binding affinity and to different affinities of the agonist-occupied receptors to the G proteins.

Adenylyl Cyclases↗
PubMed1993

Enhancement by protein kinase C of prostacyclin receptor-mediated activation of adenylate cyclase through a calmodulin/myristoylated alanine-rich C kinase substrate (MARCKS) system in IC2 mast cells.

The addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly enhanced cAMP formation induced by carbacyclin, a stable prostacyclin analogue, in cultured mast cells (IC2 cells), but did not enhance basal or NaF plus AlCl3-induced cAMP formation. On the other hand, W-7, a calmodulin (CaM) inhibitor, almost completely suppressed the enhancing activity of TPA, suggesting the involvement of CaM in the enhancement by TPA of carbacyclin-induced cAMP formation. The enhancing activity of TPA disappeared in TPA-treated cells permeabilized with saponin in the presence of Ca2+, but reconstitution with CaM in the permeable cells resulted in remarkable restoration of the action of TPA. On the other hand, TPA treatment induced the phosphorylation and translocation of myristoylated alanine-rich C kinase substrate (MARCKS) from the membrane to the cytosol. Exogenously added protein kinase C (PKC) also phosphorylated MARCKS and induced its translocation in the cells permeabilized with saponin. Whereas the addition of CaM did not enhance the carbacyclin-stimulated GTPase activity and adenylate cyclase activity in the control permeable cells, in which MARCKS bound to the membrane, CaM markedly enhanced those activities in the PKC-treated permeable cells, which lost endogenous membrane-bound MARCKS. When MARCKS was added to the PKC-treated permeable cells, MARCKS bound to the membrane and inhibited the effects of CaM. These results suggest that activation of PKC enhances the prostacyclin-activated adenylate cyclase through a CaM/MARCKS system.

Adenylyl Cyclases↗
PubMed1993

Nucleotide sequence of the cDNA encoding nucleoside diphosphate kinase II from spinach leaves.

The primary structure of nucleoside diphosphate (NDP) kinase II, one of the two isozymes found in spinach leaves, has been deduced from its cDNA sequence. NDP kinase II comprises 233 amino acid residues and has a molecular mass of 26,107 Da, which is larger than that of the purified NDP kinase II subunits (18 kDa) by about 8 kDa, suggesting that NDP kinase II might be post-translationally processed. Homology was found between the sequence of spinach NDP kinase II, and the sequences of spinach NDP kinase I, rat NDP kinases alpha and beta, Dictyostelium discoideum NDP kinase, the human Nm23-H1 and Nm23-H2 proteins and the awd protein of Drosophila melanogaster.

Amino Acid Sequence↗
PubMed1993

Proliferative activity of gastric cancer assessed by immunostaining for proliferating cell nuclear antigen.

The growth activity of 107 gastric carcinomas was assessed by immunohistochemical staining for formalin-fixed, paraffin-embedded tissue with a monoclonal antibody against proliferating cell nuclear antigen (PCNA). When the tumor doubling times (Tds) of 10 patients were estimated from the serum levels of carcinoembryonic antigen and carbohydrate antigen 19-9, there was an inverse correlation between the Tds and PCNA labeling index (LI) at P = 0.055. Flow-cytometric analysis was carried out by double staining for PCNA and DNA using fresh materials from 14 patients. The PCNA-positive cell fraction revealed by flow cytometry showed a good linear correlation with PCNA LI in routinely stained tissue. The LI of well-differentiated adenocarcinoma was significantly higher than that of the poorly differentiated type. When the LI was analyzed in well- or poorly differentiated adenocarcinoma, the value was significantly higher in the well-differentiated type with hepatic metastasis and in the poorly differentiated type with lymph node metastasis.

Adenocarcinoma↗
PubMed1993

The close relationship between DNA replication and the selection of differentiation lineages of human erythroleukemia cell lines K562, HEL, and TF1 into either erythroid or megakaryocytic lineages.

The selection of differentiation lineages into either erythroid or megakaryocytic series was analyzed with human erythroleukemia cell lines K562, HEL, and cytokine-dependent TF1. A tumor promoter, TPA, induced a megakaryocyte marker, glycoprotein IIb/IIIa (GP IIb/IIIa) or IIIa (GP IIIa), but suppressed erythroid differentiation. On the other hand, aphidicolin, which is a potent inhibitor of DNA replication, inhibited GP IIb/IIIa or IIIa expression, but induced the expression of erythroid phenotypes. These phenomena were observed in all erythroleukemia cell lines tested. The bromodeoxyuridine labeling experiments indicated that de novo DNA synthesis was completely suppressed by aphidicolin treatment but was well preserved in TPA-treated cells. Among these three cell lines, erythropoietin (EPO) treatment induced erythroid differentiation of TF1 cells, which was dependent on GM-CSF or IL-3. In this case, EPO functioned as the survival factor and mild stimulator for cell proliferation as well as the inducer of erythroid differentiation. However, when either GM-CSF or IL-3 was depleted from the culture medium, TF1 ceased cell growth; concomitantly, hemoglobin-positive cells appeared, which is consistent with the results obtained with aphidicolin. The incubation of K562 cells for 48 h with either TPA or aphidicolin induced the irreversible commitment of cells to megakaryocytic and erythroid lineages, respectively. Our results using three different erythroleukemia cell lines suggest that a possible linkage between the DNA replication system and the selection of a differentiation lineage is the common feature of human erythroleukemia cell lines, and that these culture systems provide a suitable model for the analysis of the signal transduction system for differentiation lineage selection.

Aphidicolin↗
PubMed1993

Two types of prostacyclin receptor coupling to stimulation of adenylate cyclase and phosphatidylinositol hydrolysis in a cultured mast cell line, BNu-2cl3 cells.

Prostacyclin (PGI2)-mediated signal transduction was examined in interleukin 3 (IL-3)-dependent BNu-2cl3 mast cells. Iloprost, a stable PGI2 analogue, induced the accumulation of intracellular cAMP and IP3, and an increase in the intracellular Ca2+ concentration. Pretreatment of the cells with a protein kinase C activator, 12-O-tetradecanoyl phorbol 13-acetate, suppressed the iloprost-induced IP3 accumulation and Ca2+ mobilization, but inversely potentiated the cAMP accumulation, suggesting that neither of these signal transduction pathways of iloprosts is the result of a secondary effect of activation of the other. Removal of IL-3 from the culture medium reduced the iloprost-induced IP3 accumulation and Ca2+ mobilization, while it had no effect on the iloprost-induced cAMP accumulation at all. These results taken together suggest that BNu-2cl3 cells express two types of PGI2 receptor; one couples to stimulation of adenylate cyclase, its expression being independent of IL-3, while the other couples to phosphatidylinositol hydrolysis, its expression being dependent on IL-3.

Adenylyl Cyclases↗
PubMed1993

[Thirteen-week repeated dose toxicity by oral administration of trandolapril (RU44570) with 4-week recovery test in beagles].

Trandolapril (RU44570) was orally administered to dogs at a daily dose of 2.5, 25 or 250 mg/kg for 13 weeks. After the administration period, 25 and 250 mg/kg groups were observed for recovery for 4 weeks. The results obtained are as follows: 1. One male in the 250 mg/kg group showed decrease of food consumption and body weight, stomatitis, hematemesis, decumbence, hypothermia, and finally loss of reactivity to stimuli. This animal was killed because of these severe changes on the 39th day of administration. Among the surviving animals, a temporary loss of body weight was observed in a few animals of the 25 and 250 mg/kg groups, and a decreased food consumption was sporadically seen in a few animals of the 250 mg/kg group during the administration period. No abnormal changes were found in the clinical observation and water intake in the surviving animals. 2. The changes attributable to the pharmacological effect of RU44570 were a decreased activity of the angiotensin-converting enzyme, increases in plasma renin activity and urine volume, and decreases in specific gravity and concentrations of Na, K and Cl in the urine of every administration group. A decrease in blood pressure and an increase in the PAS and Bowie positive granules in the juxtaglomerular cells were also found in the 25 and 250 mg/kg groups. In addition, thickening of the afferent arteriolar wall of the glomeruli, a basophilic change of the renal tubular epithelial cells, and localized atrophy and hypertrophy of the renal tubules were observed in the 25 and 250 mg/kg groups, and increases in BUN, ALP and creatinine, and a slight dilation of the renal tubules were seen in the 250 mg/kg group. These observations indicated that RU44570 affected renal structure at a dose of 25 mg/kg or more renal function at a dose of 250 mg/kg. The animal killed in a moribund state showed nephrosis which consisted mainly of a moderate dilation of the renal tubules and vacuolation of the renal tubular epithelial cells, stomatitis, severe hemorrhage and necrosis with neutrophil infiltration in the fundus of the glandular stomach, atrophy of the hemopoietic system, and ectopic calcification in the heart, kidneys, stomach, trachea and alveolar wall. Changes in the kidneys similar to those observed in other animals were also detected. These changes suggested that this animal lapsed into a moribund state due to renal dysfunction and the resultant uremia.(ABSTRACT TRUNCATED AT 400 WORDS)

Administration, Oral↗
PubMed1993

Mutations of the p53 gene in B-cell lymphoma.

Mutations of p53 gene have been recognized to be the most common genetic changes in human cancers. Recently, p53 gene mutations have been found in some patients with common subtypes of B-cell lymphoma (9/48:18.8%), Burkitt lymphoma (9/27:33.3%) and chronic lymphocytic leukemia (6/40:15%). Evidences to suggest that p53 gene mutations are associated with the disease progression in B-cell lymphoma have emerged. Functions of wild-type p53 and its mutant's probable role in B-cell lymphomagenesis are described in this review.

Genes, p53↗
PubMed1993

Induction of neutrophil alkaline phosphatase gene expression by granulocyte colony-stimulating factor in chronic myelogenous leukemia.

The mechanism underlying defective neutrophil alkaline phosphatase (NAP) activity in chronic myelogenous leukemia (CML) was examined using Northern blotting analysis and NAP staining. Fourteen patients with Ph1-positive CML in the chronic phase were included in this study. Polymorphonuclear cells (PMN), containing more than 80% of stab and segmented neutrophils, were collected from peripheral blood (PB) by methylcellulose sedimentation after density gradient centrifugation. Neutrophil alkaline phosphatase mRNA (NAPmRNA) expression was very weak in all patients. When incubated with granulocyte colony-stimulating factor (G-CSF) for 24 h, the PMN of all patients expressed a detectable level of NAPmRNA. NAP score, which semiquantitatively reflects the activity of gene products, was also elevated after incubation with G-CSF in 12 patients. However, a higher dose of G-CSF was required for the maximum response than was the case for normal bone marrow PMN, which also manifested increases of NAPmRNA and its products on stimulation by G-CSF. The present study suggests that the PMN of CML express NAPmRNA and produce its products in response to G-CSF, but that they are less sensitive to G-CSF than normal bone marrow PMN.

Adolescent↗
PubMed1992

Interaction of alpha subunit of GTP-binding protein Go with a 20-kDa Triton-insoluble membrane protein in bovine brain.

Heterotrimeric Go bound to the membranes of bovine brain, but Go alpha remained bound to the membranes even after activation with GTP gamma S. Furthermore, Go alpha bound to a Triton X-100-insoluble fraction of the membranes in a saturable manner. However, the 37-kDa Go alpha eliminated by trypsin at the amino-terminus could not bind to the fraction. Using a blot overlay approach of the Triton-insoluble fraction, only a 20-kDa protein was identified that interacts with Go alpha. These results indicate that Go alpha binds to a 20-kDa Triton-insoluble protein in the bovine brain membranes.

Animals↗
PubMed1992

Glycyrrhetinic acid bound to 11 beta-hydroxysteroid dehydrogenase in rat liver microsomes.

A binding protein which exhibits high affinity to [3H]glycyrrhetinic-acid in the rat liver microsomal fraction was solubilized with 0.2% Triton DF-18 and then purified to homogeneity. The equilibrium dissociation constant of the [3H]glycyrrhetinic-acid binding reaction and the maximal concentration for the binding of the purified protein, as determined by Scatchard plot analysis, were 27.6 nM and 7.79 nmol/mg protein, respectively. The molecular mass of the subunit (34 kDa) and 30 amino acids of N-terminal sequence of the purified protein were entirely the same as those of the reported 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). In each purification step, the recovery and purification (fold) of the glycyrrhetinic-acid binding activity corresponded to the values of 11 beta-HSD activity. These results show that the purified [3H]glycyrrhetinic-acid binding protein is 11 beta-HSD. From the molecular mass of 11 beta-HSD (135 kDa) and the maximal concentration of the binding site, it was calculated that one glycyrrhetinic acid molecule binds to one 11 beta-HSD molecule. The inhibitory effects of various glycyrrhetinic-acid derivatives on [3H]glycyrrhetinic acid binding and 11 beta-HSD activity indicate that the C30-carboxyl and C11-carbonyl groups of glycyrrhetinic acid are the principal structures for the 11 beta-HSD inhibition.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1992

Inhibitory effect of an intracellular glutathione on delta 12-prostaglandin J2-induced protein syntheses in porcine aortic endothelial cells.

Delta 12-Prostaglandin (PG) J2 caused porcine aortic endothelial cells to synthesize a 31,000-dalton heme oxygenase and a 67,000-dalton protein (p67). Treatment of the cells with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, depleted intracellular GSH, and enhanced the induction of heme oxygenase and p67 syntheses by delta 12-PGJ2. In contrast, treatment with GSH increased the intracellular GSH level and reduced the induction. There was a reciprocal relationship between the level of intracellular GSH, and that of the induction of heme oxygenase and p67 syntheses by delta 12-PGJ2. An increase in the intracellular GSH level caused an increase in the ethyl acetate-unextractable form of delta 12-PGJ2 in the cytosol, but suppressed the accumulation of delta 12-PGJ2 in the nuclei. Furthermore, GSH strongly inhibited the in vitro binding of delta 12-PGJ2 to isolated nuclei, which is N-ethylmaleimide sensitive. Moreover, the induction of heme oxygenase and p67 syntheses by the thiol-reactive agents arsenite and diethylmaleate was also inhibited by GSH treatment and enhanced by BSO treatment. These results demonstrate that intracellular GSH suppresses delta 12-PGJ2-induced heme oxygenase and p67 syntheses by inhibiting the binding of delta 12-PGJ2 to nuclei.

Animals↗
PubMed1992

Identification of the prostacyclin receptor by use of [15-3H1]19-(3-azidophenyl)-20-norisocarbacyclin, an irreversible specific photoaffinity probe.

The prostaglandin I (PGI2) receptor of mouse mastocytoma P-815 cells was characterized by photo-affinity labeling with the stable PGI2 analogue [15-3H1]-19-(3-azidophenyl)-20-norisocarbacyclin ([3H] APNIC) used as a potential photoaffinity probe for the receptor. [3H]APNIC bound to the mastocytoma membrane with high affinity and in a saturable manner. Scatchard plot analysis indicated a single binding site with a Kd of 4.7 nM and a Bmax of 0.58 pmol/mg protein. The binding of [3H]APNIC was dose dependently inhibited by APNIC and iloprost, another stable PGI2 agonist, and to a much lesser extent by PGE2. The binding of the radioligand showed sensitivity to the guanine nucleotide guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S). Photolysis of [3H]APNIC-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 43 kDa. Photolabeling was inhibited by PGI2 agonists and other prostaglandins with specificity for the PGI2 receptor and was modulated by GTP gamma S. A protein of approximately 45 kDa was also labeled by [3H]APNIC in the membrane of porcine platelets, membranes that are known to be abundant in PGI2 receptors. These results demonstrate that [3H]APNIC specifically labels a protein that may represent the PGI2 receptor and that this radioprobe will be a useful reagent for further characterization and purification of the PGI2 receptor.

Affinity Labels↗
PubMed1992

The amino acid sequence of nucleoside diphosphate kinase I from spinach leaves, as deduced from the cDNA sequence.

The primary structure of nucleoside diphosphate (NDP) kinase from spinach leaves has been deduced from its cDNA sequence. A lambda gt 11 cDNA library derived from spinach leaves was screened using an antibody against NDP kinase I, which we previously purified to electrophoretic homogeneity (T. Nomura, T. Fukui, and A. Ichikawa, 1991, Biochim. Biophys. Acta 1077, 47-55). The cDNA sequences of positive clones contained the amino acid coding region (444 base pairs) for NDP kinase I as well as 5' and 3' noncoding regions of 33 and 361 base pairs, respectively. The cDNAs hybridized to a 1.1-kb mRNA. NDP kinase I contains 148 amino acid residues with a molecular mass of 16,305, which is in excellent agreement with that of the purified enzyme (16 kDa). Homology was found between the sequence of spinach NDP kinase I and those of the rat, Myxococcus xanthus, and Dictyostelium discoideum NDP kinases, as well as the human Nm23-gene product and the awd protein of Drosophila melanogaster.

Amino Acid Sequence↗
PubMed1992

Activation of DNA cleavage by dynemicin A in a B-Z conformational junction.

We report here that the DNA strand scission by dynemicin A is not only sequence-specific but also conformation-specific. The salt-induced B----Z conformational transition dramatically enhanced the cleavage by dynemicin A in a B-Z junction region. By contrast, the bleomycin-Fe(II) complex, the elsamicin A-Fe(II) complex, and esperamicin A1 did not induce any preferential DNA cutting in such a DNA structure. The characteristic hyperreactivity of dynemicin A is observed in (dC-dG)8- and (dC-dG)12-inserted DNAs, but not in (dC-dG)5-inserted DNA. These results suggest value in the use of dynemicin A as proof of the existence of a B-Z junction in vivo and also may aid in understanding the structure of B-Z junctions.

Aminoglycosides↗