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Biomedical subjects

A Ichikawa

Publications and source records attributed to A Ichikawa.

At least 163 records · Page 9Linked to original sources
PubMed1995

[Two patients with aplastic anemia successfully treated with G-CSF and erythropoietin].

Two patients with severe aplastic anemia received combination therapy consisting of granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO) by subcutaneous injection. During the first 8 weeks, we administered only G-CSF, but subsequently we administered EPO with G-CSF. Administration of G-CSF caused rapid increase in neutrophil counts in each case. In the first case, a 23-year-old woman, the first sign of improvement of anemia appeared in the 12th week but 50 more weeks elapsed before improvement of platelet count. In the second case, a 59-year-old woman, marked increase of reticulocytes appeared in the 32nd week, and the RBC count become normal in the 46th week. Minor improvement of platelet count was obtained in the 40th week. After 53 weeks of treatment, we stopped administration of G-CSF and EPO, and all of peripheral blood cell counts decreased. Therefore G-CSF and EPO were given to her again, and she showed the same response as first administration. It showed that she was dependent upon G-CSF and EPO. Finally she obtained normalization of all blood cell counts. In both cases, no serious side effects were observed. Combination therapy consisting of G-CSF and EPO may be beneficial for patients with aplastic anemia.

Adult↗
PubMed1995

De Novo synthesis and posttranslational processing of L-histidine decarboxylase in mice.

We purified L-histidine decarboxylase from mouse mastocytoma cells and cloned mouse HDC cDNA, and found that the primary translated product (74 kD) is posttranslationally processed in its C-terminal region to yield a native HDC subunit (53 kD). Recombinant 74-kD, but not 53-kD HDC species was present mainly in the particulate fraction of Sf9 cells. The particulate 74-kD recombinant HDC was cleaved by porcine pancreatic elastase, and a homodimer of a 53-kD subunit having the identical catalytic properties to those of native HDC was solubilized. The particulate HDC from mouse stomach was partially purified and it was solubilized by porcine pancreatic elastase to yield the 53-kD subunit of HDC. We identified endogenous proteolytic activity, which converts the particulate recombinant 74-kD HDC to the soluble 53-kD HDC in the supernatant of mouse stomach. In mastocytoma cells, we demonstrated that the induction of HDC activity and HDC mRNA synergistically occurred upon treatment with dexamethasone + TPA, and also with cAMP + Ca2+. On a genomic DNA cloning, we found that two upregulations occurred via the involvement of the regulatory elements binding to the sequences from -132 to -53 and -267 to -53, respectively.

Animals↗
PubMed1995

Effect of repeated cold stress on mouse stomach histidine decarboxylase.

The effect of repeated cold stress (RCS) on both mRNA level and enzyme activity of stomach histidine decarboxylase (HDC) was studied in ddY mice. Following 1-day treatment of RCS, stomach HDC activity, but not its mRNA level, increased two fold. Following 3-day treatment of RCS, which is the essential period for the induction of hyperalgesia in mice, HDC mRNA level and enzyme activity increased in the stomach. After cessation of RCS treatment, HDC mRNA level decreased and reached the level of non-RCS treated mice, but HDC activity did not. The stomach from the 1-day RCS-treated mouse contained proteolytic activity, which converts the in vitro-translated 74 kD HDC species into the 53 kD HDC species. These data demonstrate that RCS-treated mouse stomach induces both the de novo synthesis of the 74 kD HDC species and its proteolytic cleavage to 53 kD HDC species.

Animals↗
PubMed1994

The MDM2 oncogene overexpression in chronic lymphocytic leukemia and low-grade lymphoma of B-cell origin.

The expression of the murine double minute-2 (MDM2) gene, the product of which binds to and inactivates p53, was studied in 60 patients with B-cell chronic lymphocytic leukemia (B-CLL) or non-Hodgkin's lymphoma (B-NHL). Northern blot analysis showed that the level of MDM2 gene expression was low in normal human B-cells, whereas 17 of the patients (28.3%) with B-CLL or NHL had more than 10-fold higher levels of MDM2 gene expression than that observed in normal B cells. Immunohistochemical analysis confirmed MDM2 overexpression at the cellular protein level. MDM2 gene overexpression was found more frequently in patients with the low-grade type of lymphoma (56.5%) than in those with intermediate-/high-grade types (10.8%) (P = .001). Moreover, MDM2 overexpression was found significantly more frequently in patients at advanced clinical stages. Simultaneous analysis of p53 gene mutation showed that three patients had both MDM2 gene overexpression and p53 gene mutation. The results of the present study suggest that MDM2 gene overexpression may play an important role in the tumorigenicity and/or disease progression of CLL and low-grade lymphomas of B-cell origin.

Adult↗
PubMed1994

Mouse prostaglandin E receptor EP3 subtype mediates calcium signals via Gi in cDNA-transfected Chinese hamster ovary cells.

We recently cloned the mouse prostaglandin (PG) E receptor EP3 subtype that is coupled to adenylate cyclase inhibition through Gi and identified three isoforms which are produced through alternative splicing. In Chinese hamster ovary cells expressing each EP3 isoform, PGE2 induced an immediate increase in the intracellular Ca2+ concentration ([Ca2+]i) due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. This increase was abolished by prior treatment with pertussis toxin (PT). PGE2 also stimulated an accumulation of inositol trisphosphate (IP3) in a PT-sensitive manner. Both the PGE2-induced increase in [Ca2+]i and accumulation of IP3 were blocked by the phospholipase C inhibitor U-73122. Thus, EP3 is linked to phospholipase C activation via Gi, and this activation leads to Ca2+ mobilization from internal stores and influx from the extracellular medium.

Adenylate Cyclase Toxin↗
PubMed1994

The C-terminus of the prostaglandin-E-receptor EP3 subtype is essential for activation of GTP-binding protein.

Three isoforms of the mouse prostaglandin-E-receptor EP3 subtype (EP3), EP3 alpha, EP3 beta and EP3 gamma, with different C-termini, which are produced through alternative splicing, showed different efficiencies with respect to heterotrimeric GTP-binding protein activation and adenylate cyclase inhibition [Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, T., Honda, A., Watabe, A., Hirata, M., Narumiya, S. & Ichikawa, A. (1993) J. Biol. Chem. 268, 2712-2718; Irie, A., Sugimoto, Y., Namba, T., Harazono, A., Honda, A., Watabe, A., Negishi, M., Narumiya, S. & Ichikawa, A. (1993) Eur. J. Biochem. 217, 313-318]. To assess the role of the C-terminus in GTP-binding protein coupling, we truncated the C-terminus of EP3 at an alternative splicing site and expressed the mutant receptor. The truncated receptor retained the ability to physically associate with Gi2, forming an agonist/receptor/Gi2 ternary complex, and to undergo the characteristic conversion of its agonist-binding affinity, mediated by a guanine nucleotide from a low-affinity state to a high-affinity state. However, sulprostone, an EP3 agonist, failed not only to inhibit the forskolin-induced cAMP accumulation in the mutant receptor-expressing cells but also to stimulate the GTPase activity in the mutant receptor-expressing cell membrane. These results indicated that the C-terminus of EP3 is essential for the activation of GTP-binding protein.

Adenylyl Cyclase Inhibitors↗
PubMed1994

Enhancement of adenylate cyclase stimulation by prostaglandin E receptor EP3 subtype isoforms with different efficiencies.

We recently cloned the mouse prostaglandin (PG) E receptor EP3 subtype that is coupled to adenylate cyclase inhibition through Gi and identified two isoforms of EP3, EP3 alpha and EP3 beta, which are produced through alternative splicing and differ only in the carboxyl-terminal domain. Preincubation of Chinese hamster ovary cells expressing each isoform with PGE2 concentration-dependently enhanced both the basal and forskolin-stimulated cAMP formation, but two orders higher concentrations of PGE2 were required for EP3 beta than EP3 alpha for 50% enhancement of both formations. This enhancement by EP3 isoforms was completely blocked by pertussis toxin treatment, indicating that it is mediated through Gi activation. Thus, the two EP3 isoforms with different carboxyl-terminal tails induce enhancement of adenylate cyclase stimulation with different efficiencies.

Adenylate Cyclase Toxin↗
PubMed1994

Molecular cloning and expression of rat prostaglandin E receptor EP2 subtype.

A cDNA clone encoding the rat prostaglandin (PG) E receptor EP2 subtype was cloned from a rat lung cDNA library. It encodes 488 amino acid residues with putative seven-transmembrane domains. Specific binding of [3H]PGE2 was found in COS-7 cells transfected with the cDNA and was displaced with unlabeled prostaglandins in the order of PGE2 = PGE1 >> iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the significant expression being observed in the thymus, lung, spleen, heart stomach, and pancreas.

Amino Acid Sequence↗
PubMed1994

Cloning and expression of a cDNA for the human prostacyclin receptor.

A functional cDNA for the human prostacyclin receptor was isolated from a cDNA library of CMK cells, a human megakaryocytic leukaemia cell line. The cDNA encodes a protein consisting of 386 amino acid residues with seven putative transmembrane domains and a deduced molecular weight of 40,956. [3H]Iloprost specifically bound to the membrane of CHO cells stably expressing the cDNA with a Kd of 3.3 nM. This binding was displaced by unlabelled prostanoids in the order of iloprost = cicaprost >> carbacyclin > prostaglandin E1 (PGE1) > STA2. PGE2, PGD2 and PGF 2 alpha did not inhibit it. Iloprost in a concentration-dependent manner increased the cAMP level and generated inositol trisphosphate in these cells, indicating that this human receptor can couple to multiple signal transduction pathways.

Amino Acid Sequence↗
PubMed1994

cDNA cloning of a mouse prostacyclin receptor. Multiple signaling pathways and expression in thymic medulla.

A functional cDNA for a mouse prostacyclin receptor was isolated from a mouse cDNA library by reverse transcription polymerase chain reaction and hybridization screening. The cDNA encodes a polypeptide of 417 amino acid residues with putative seven transmembrane domains and an calculated molecular weight of 44,722. The amino acid sequence is 30-40% identical in the transmembrane domains to those of the mouse prostaglandin (PG) E receptor subtypes and thromboxane A2 receptor. [3H]Iloprost, a specific prostacyclin receptor radioligand, specifically bound to the membrane of Chinese hamster ovary cells permanently expressing the cDNA with Kd of 4.6 nM. This binding was displaced with unlabeled prostanoids in the order of cicaprost > iloprost, both prostacyclin agonists > PGE1 > carbacyclin >> PGD2 approximately STA2, a thromboxane A2 agonist approximately PGE2 > PGF2 alpha. Iloprost in a concentration-dependent fashion increased cAMP level and generated inositol phosphates in these cells, indicating that the receptor couples to multiple signal transduction pathways. Northern blot analysis revealed that the mRNA is expressed most abundantly in thymus, followed by spleen, heart, and lung. In situ hybridization of thymus showed that it is expressed exclusively in medulla and not in cortex.

Amino Acid Sequence↗
PubMed1994

Peripheral blood cells are predominantly chimeric of affected and normal cells in patients with paroxysmal nocturnal hemoglobinuria: simultaneous investigation on clonality and expression of glycophosphatidylinositol-anchored proteins.

To investigate clonal compositions of hematologic cells in paroxysmal nocturnal hemoglobinuria (PNH), we analyzed peripheral blood (PB) cells of 12 female patients with PNH, by clonality analysis using X-chromosome inactivation and assessment of expression of glycophosphatidylinositol-anchored proteins (GPI-APs) by flow cytometry. Southern hybridization showed that granulocytes were monoclonal in three and polyclonal in eight patients, respectively, whereas lymphocytes were polyclonal in all nine patients examined. Expressions of CD16 and CD59 on granulocytes varied greatly in seven patients examined. Clonality analysis of granulocytes by the polymerase chain reaction showed that CD59- and CD59low+ cells were monoclonal, whereas CD59+ cells were polyclonal. It was shown that PB cells are predominantly chimeric of clonal (GPI-AP- or GPI-APlow+) and nonclonal (GPI-AP+) cells in PNH, and that degrees of chimerism differ greatly from patient to patient.

Adolescent↗
PubMed1994

Cloning and expression of a cDNA for mouse prostaglandin F receptor.

A functional cDNA clone for mouse prostaglandin (PG) F receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of cloned prostanoid receptors, and cross-hybridization screening. The mouse PGF receptor consists of 366 amino acid residues with putative seven transmembrane domains. The sequence revealed the highest homology to the EP1 subtype of PGE receptor and thromboxane (TX) A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGF2 alpha binding. The binding was displaced with unlabeled PGs in the order of PGF2 alpha = 9 alpha, 11 beta PGF2 > PGF 1 alpha > PGD2 > STA2 (a stable TXA2 agonist) > PGE2 > iloprost (a stable PGI2 agonist). PGF2 alpha increased inositol trisphosphate formation in a concentration-dependent manner in COS cells expressing PGF receptor. RNA blot and in situ hybridization analyses demonstrated that the PGF receptor transcripts are abundantly expressed in luteal cells of corpus luteum and in a lesser amount in kidney, heart, stomach, and lung.

Amino Acid Sequence↗
PubMed1994

Mapping of the genes encoding mouse thromboxane A2 receptor and prostaglandin E receptor subtypes EP2 and EP3.

Thromboxane A2 (TXA2) and prostaglandin E2 (PGE2) are two of the most representative eicosanoids that are formed from arachidonic acid. They produce a broad spectrum of biological effects mediated through specific cell surface receptors. We have mapped genetic loci for TXA2 receptor, Tbxa2r, and for PGE2 receptor subtypes EP2 and EP3, Ptgerep2 and Ptgerep3, respectively, using restriction fragment length variants in interspecific backcross mice. None of the three loci cosegregated with each other. Tbxa2r mapped to Chr 10, Ptgerep2 mapped to the distal end of Chr 3. Possible human loci for these receptors are predicted based on the homology between mouse and human chromosomes.

Animals↗
PubMed1994

Gastrin receptor genes are expressed in gastric parietal and enterochromaffin-like cells of Mastomys natalensis.

Although gastric enterochromaffin-like (ECL) carcinoid tumors are known to develop in patients with long-standing hypergastrinemia, the expression of the gastrin receptor gene in ECL cells has not yet been demonstrated. Therefore, this study was designed to examine gastrin receptor gene expression in ECL cells. Mastomys gastric mucosal cells isolated by enzyme dispersion were separated into 10 fractions (F1-10) by centrifugal elutriation. Each fraction was examined histologically to determine whether they contained ECL and/or parietal cells and Northern blot analysis was used to confirm the presence of histidine decarboxylase and H+, K(+)-ATPase gene expression. ECL cells were found only in fractions 1 and 2, whereas parietal cells were detected in fractions 6-10. Gastrin receptor gene expression was demonstrated in both parietal cell-rich and ECL cell-rich fractions. In addition, the gastrin receptor cDNA sequences obtained from the two of the fractions (F1 and 8) were identical. These results suggest that gastrin receptor genes are expressed in ECL cells as well as in parietal cells and that these receptors are identical.

Animals↗
PubMed1994

TEI-3356, a highly selective agonist for the prostaglandin EP3 receptor.

Recently, we cloned cDNAs for the three mouse PGE receptor subtypes, EP1, EP2 and EP3, and the prostacyclin receptor, and established cells that stably express each receptor. We examined the selectivity of TEI-3356, an isocarbacyclin analogue, compared with other EP agonists, sulprostone and misoprostol, using Chinese hamster ovary cells expressing each cloned receptor. TEI-3356 selectively displaced the [3H]PGE2 binding to EP3-expressing cell membranes, but showed very low affinity for both EP1 and EP2. Although TEI-3356 is an isocarbacyclin analogue, it showed low affinity for the prostacyclin receptor. On the other hand, sulprostone strongly displaced the [3H]PGE2 binding to EP1 and EP3, but not to EP2. Misoprostol weakly bound to the three subtypes without selectivity. TEI-3356 decreased the forskolin-induced cAMP formation in a concentration-dependent manner in the EP3-expressing cells, the half-maximal concentration for the inhibition being similar to that of sulprostone but lower than that of PGE2. These results demonstrate that TEI-3356 is a potent and highly selective agonist for the EP3 receptor.

Animals↗
PubMed1994

Cloning and expression of a cDNA for rat prostaglandin F2 alpha receptor.

We have cloned a cDNA for rat prostaglandin (PG) F2 alpha receptor from cultured rat astrocytes. The cDNA encodes a polypeptide of 366 amino acids with seven putative transmembrane domains. Specific binding of [3H]PGF2 alpha in membranes of COS-7 cells transfected with the cDNA was displaced with unlabeled PGs in the order of PGF2 alpha > PGD2 > PGE2 > PGI2. In the cDNA-transfected LLC-PK1 cells, PGF2 alpha stimulated phosphoinositide hydrolysis. A significant 4.7-kb mRNA transcript was detected in cultured rat astrocytes and whole brain and pregnant ovary of adult rats by Northern blot analysis.

Amino Acid Sequence↗
PubMed1994

Distribution of the messenger RNA for the prostaglandin E receptor subtype EP3 in the mouse nervous system.

Distribution of the messenger RNA for the prostaglandin E receptor subtype EP3 was investigated by in situ hybridization in the nervous system of the mouse. The hybridization signals for EP3 were widely distributed in the brain and sensory ganglia and specifically localized to neurons. In the dorsal root and trigeminal ganglia, about half of the neurons were labeled intensely. In the brain, intensely labeled neurons were found in Ammon's horn, the preoptic nuclei, lateral hypothalamic area, dorsomedial hypothalamic nucleus, lateral mammillary nucleus, entopeduncular nucleus, substantia nigra pars compacta, locus coeruleus and raphe nuclei. Moderately labeled neurons were seen in the mitral cell layer of the main olfactory bulb, layer V of the entorhinal and parasubicular cortices, layers V and VI of the cerebral neocortex, nuclei of the diagonal band, magnocellular preoptic nucleus, globus pallidus and lateral parabrachial nucleus. In the thalamus, moderately labeled neurons were distributed in the anterior, ventromedial, laterodorsal, paraventricular and central medial nuclei. Based on these distributions, we suggest that EP3 not only mediates prostaglandin E2 signals evoked by blood-borne cytokines in the areas poor in the blood-brain barrier, but also responds to those formed intrinsically within the brain to modulate various neuronal activities. Possible EP3 actions are discussed in relation to the reported neuronal activities of prostaglandin E2 in the brain.

Animals↗