Biomedical subjects

A Ichikawa

Publications and source records attributed to A Ichikawa.

At least 181 records · Page 10Linked to original sources

PubMed1994

Distinct cellular localization of mRNAs for three subtypes of prostaglandin E receptor in kidney.

Distribution of the mRNAs for three subtypes of prostaglandin E (PGE) receptors in the mouse kidney was investigated by in situ hybridization. The mRNA for EP1 subtype, which is coupled to Ca2+ mobilization, was specifically localized to the collecting ducts from the cortex to the papilla. The mRNA for EP2 subtype, which is linked to stimulation of adenylate cyclase, was localized to the glomeruli. The mRNA for EP3 subtype, which is coupled to inhibition of adenylate cyclase, was located densely in the tubules in the outer medulla and in the distal tubules in the cortex. These results exhibit distinct cellular localization of three subtypes of PGE receptor in the kidney and suggest that PGE2 exerts multiple functions via these subtypes expressed in different segments of the nephron.

Adenylyl Cyclases↗

PubMed1994

Prostacyclin (PGI) receptor binding and cyclic AMP synthesis activities of PGI1 analogues, SM-10906 and its methyl ester, SM-10902, in mastocytoma P-815 cells.

The prostacyclin I1 (PGI1) analogue, SM-10906 and its methyl ester, SM-10902, have been compared with the PGI2 analogue, iloprost, with respect to binding to the PGI2 receptor, stimulation of adenylate cyclase activity and inhibition of thrombin-induced Ca2+ mobilization in mastocytoma P-815 cells. SM-10906 displaced [3H]iloprost binding to the membrane fraction, the IC50 value being 100 nM, but showed very low affinity for the prostaglandin E (PGE) receptor. SM-10906 dose-dependently stimulated GTP-dependent adenylate cyclase activity in the membrane fraction, the EC50 value being 35 nM. Furthermore, SM-10906 prevented a thrombin-induced increase in the intracellular Ca2+ concentration, the IC50 value being 300 nM. These IC50 and EC50 values are much lower than those of SM-10902. These results demonstrate that SM-10906, a stable PGI1 derivative, is an agonist for the [3H]iloprost-binding (PGI2) receptor, and that it prevents thrombin-induced Ca2+ mobilization through stimulation of the adenylate cyclase system in mastocytoma cells. On the other hand, a methyl ester derivative of PGI1, SM-10902, was inactive in the binding assay, but it seems to be a partial agonist for adenylate cyclase activity [corrected].

Adenylyl Cyclases↗

PubMed1994

[Molecular biology of L-histidine decarboxylase].

L-Histidine decarboxylase (HDC) catalyzes the formation of histamine from L-histidine. This biogenic amine is known to exert various effects in physiological and pathological reactions. In contrast to the well-known mechanism of histamine action through its interaction with specific receptors, the mechanisms regulating HDC gene expression are not elucidated. We have purified HDC from mouse mastocytoma cells, and isolated mouse HDC cDNA, and found that the primary translated product is posttranslationally processed to yield a mature active enzyme. In mastocytoma cells, we demonstrated that the induction of HDC activity and HDC mRNA synergistically occurred on treatment with dexamethasone+TPA, and also cAMP+Ca2+. To clarify the mechanism of up-regulation by these stimuli of the transcription of the HDC gene, we have isolated a genomic DNA clone encoding 5'-flanking region sequence and the first two exons. The transcription start site and the nucleotide sequences of the promoter regions including TATA- and GC-boxes were determined. With mastocytoma cells transiently transfected with 5' deletion constructs of HDC-CAT fusion gene, it was found that the sequences from -132 to -53 and -267 to -53 are essential for the regulatory elements involved in the increased transcription of the HDC gene with dexamethasone+TPA and cAMP+Ca2+, respectively. Furthermore, we have isolated a genomic DNA from human basophilic cells, and analysed its structure to elucidate the mechanisms regulating the tissue specificity of HDC gene expression.

Amino Acid Sequence↗

PubMed1994

Tumor necrosis factor-beta gene expression and its relationship to the clinical features and histopathogenesis of peripheral T-cell lymphomas.

We investigated the levels of tumor necrosis factor-beta (TNF-beta) mRNA in the tumorous tissues of a series of 18 patients with peripheral T-cell lymphomas (PTCL), to assess the contribution of the expression of this gene to the features of the disease. Total RNA, extracted from diagnostic tissue specimens, was subjected to semiquantitative analysis by reverse transcription-coupled polymerase chain reaction (RT-PCR). The level of TNF-beta mRNA was semiquantified against that in MT-2 cells, a line of human T cells infected with human T cell leukemia virus type I (HTLV-I). Expression of TNF-beta in neoplastic T-cells was confirmed by immunohistochemistry. The extent of TNF-beta gene expression was correlated with the histopathological features of neovascularization. There was also a relationship between the extent of TNF-beta gene expression and the presence of B-symptoms. Results suggest that TNF-beta produced by neoplastic T-cells influences clinical features and is involved in histopathogenesis of PTCL.

Female↗

PubMed1994

Recovery of polyclonal hematopoiesis in patients with myelodysplastic syndromes following successful chemotherapy.

We performed X-linked restriction fragment length polymorphism (RFLP)-methylation analysis to study the clonality of hematopoiesis in five patients with myelodysplastic syndromes (MDS), who had responded to chemotherapy. Two patients had MDS in blast crisis, two had refractory anemia with an excess of blasts in transformation (RAEB-T), and one had acute myelogenous leukemia with trilineage myelodysplasia (AML-TMDS). Following the administration of low-dose cytarabine therapy or of conventional intensive chemotherapy, we observed a reversion to polyclonal hematopoiesis in three patients who achieved a complete remission (CR). Polyclonal hematopoiesis persisted in one patient in CR, and monoclonal hematopoiesis persisted in another patient of minor response. These results indicate that polyclonal hematopoiesis can be restored in some MDS patients who achieved CR. We are encouraged, therefore, to administer intensive consolidation chemotherapy to prolong the duration of remission.

Adult↗

PubMed1993

Opposite coupling of prostaglandin E receptor EP3C with Gs and G(o). Stimulation of Gs and inhibition of G(o).

We recently identified four isoforms of bovine prostaglandin E receptor EP3 subtype, which are coupled to different signaling pathways; EP3A is coupled to inhibition of adenylate cyclase, while EP3B and EP3C are coupled to its stimulation and EP3D is coupled to phosphatidylinositol turnover, in addition to the adenylate cyclase system (Namba, T., Sugimoto, Y., Negishi, M., Irie, A., Ushikubi, F., Kakizuka, Ito, S., A., Ichikawa, A., and Narumiya, S. (1993) Nature 365, 166-170). We examined here the identity of coupled G proteins and their regulation by one of the isoforms, EP3C, in the membranes of EP3C cDNA-transfected Chinese hamster ovary cells. M&B 28767, an EP3 agonist, stimulated the GTPase activity in the pertussis toxin (PT)-treated cell membrane, but inhibited it in the cholera toxin (CT)-treated cell membrane, while the agonist neither stimulated nor inhibited it in the both PT- and CT-treated cell membrane. In the PT- and CT-treated cell membrane reconstituted with various G proteins, M&B 28767 inhibited the GTPase activity of G(o), but stimulated that of Gs. On the other hand, M&B 28767 did not affect the GTPase activity of Gi1, Gi2, or Gi3. M&B 28767 increased the apparent affinity of G(o) for GDP without any change in that for GTP, as assessed by displacement of [35S]GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) binding to G(o). In contrast, M&B 28767 increased the apparent affinity of Gs for GTP but decreased that for GDP. These results demonstrated that the EP3 receptor isoform is coupled to two different G proteins, and oppositely regulates their activities, inhibition of G(o), and stimulation of Gs.

Alprostadil↗

PubMed1993

Expression and characterization of recombinant mouse mastocytoma histidine decarboxylase.

The possibility of post-translational processing of mouse mastocytoma histidine decarboxylase (HDC; EC 4.1.1.22) was investigated. The molecular mass of the recombinant HDC expressed in Sf9 cells using HDC cDNA from mouse mastocytoma cells was determined to be 74 kDa by SDS-PAGE. In contrast to the native HDC from mastocytoma cells, the recombinant 74 kDa HDC was essentially inactive and precipitable in Sf9 cells. On the other hand, deletion mutants of the recombinant HDC lacking a C-terminal region equivalent to 10 (64 kDa) or 20 kDa (54 kDa) in size were present as active forms in the soluble fraction of Sf9 cells. To examine the C-terminal deletion of the 74 kDa species yielding the 53 kDa species by means of the immunoblotting analysis, two peptides (corresponding to residues 323-337 and 572-586 of the recombinant 74 kDa HDC peptide) were synthesized, and rabbit antiserum specific for each peptide was prepared. On immunoblotting analysis, anti-peptide 323-337 antiserum recognized both the recombinant 74 kDa and native enzyme subunit peptides, but anti-peptide 572-586 antiserum recognized only the recombinant 74 kDa peptide, i.e., not the native enzyme subunit peptide. Furthermore, HDC activity in the crude extract from Sf9 cells was not precipitable with antipeptide 572-585 antiserum. These results strongly suggest that the 53 kDa subunit peptide of native mastocytoma HDC is derived from the unidentified inactive 74 kDa HDC peptide, probably by post-translational processing of HDC in its C-terminal region.

Amino Acid Sequence↗

PubMed1993

Enhanced expression of the mouse L-histidine decarboxylase gene with a combination of dexamethasone and 12-O-tetradecanoylphorbol-13-acetate.

We previously reported that the induction of L-histidine decarboxylase (HDC) in mouse mastocytoma cells was synergistically potentiated with a combination of dexamethasone and 12-O-tetradecanoylphorbol-13-acetate (TPA) [Biochim. Biophys. Acta, 1133, 172-178 (1992)]. To clarify the molecular mechanism of this synergistic action on HDC expression, we have isolated genomic DNA clone (MGH5), including 5'-flanking region of the mouse HDC gene. The transcription start site and the nucleotide sequences of the promoter regions were determined. We found that this clone contains a TATA-like box and a GC-box in the promoter region, and several putative binding sites for regulatory proteins in the 5'-flanking region. With mastocytoma cells transiently transfected with 5' deletion constructs of HDC-CAT fusion gene, it was found that the sequence from -267 to -43 is essential for the regulatory elements(s) involved in the increased transcription of the HDC gene with dexamethasone and TPA.

Amino Acid Sequence↗

PubMed1993

Conformation-selective DNA strand breaks by dynemicin: a molecular wedge into flexible regions of DNA.

We present evidence that the enediyne antitumor antibiotic dynemicin recognizes and cleaves conformationally flexible regions of DNA. This is based on specific strand breaks at mismatches, bulges, and nicks as determined by high-resolution sequencing gels. On the basis of the weak association constant of dynemicin for DNA, it is expected that these more flexible regions would be preferred sites. In addition, the DNA unwinding behavior of dynemicin and the absorption spectrum of the dynemicin-DNA complex are strongly indicative of its intercalative binding with DNA. The results allow us to propose dynemicin as a molecular wedge that binds to the DNA by intercalating into the minor-groove side of conformationally flexible regions of DNA. Presumably, DNA local flexibility is able to create an open pocket in the minor groove, permits facile intercalation of dynemicin, and then increases the chances of its DNA damaging event. Implications for the biological action of dynemicin have also been discussed.

Anthraquinones↗

PubMed1993

Third isoform of the prostaglandin-E-receptor EP3 subtype with different C-terminal tail coupling to both stimulation and inhibition of adenylate cyclase.

A functional cDNA clone for a third isoform of the mouse prostaglandin-E-receptor EP3 subtype, derived by alternative RNA splicing, named the EP3 gamma receptor, was obtained in addition to those for the two other isoforms, EP3 alpha and EP3 beta. The three isoforms are only different in the amino acid sequence of the putative cytoplasmic carboxy-terminal tail. When expressed, EP3 gamma shows identical ligand-binding properties to these of the other isoforms. The EP3-selective agonist, M&B 28767, increased the basal cAMP level and inhibited the forskolin-induced increase in the cAMP level in EP3 gamma, while it decreased both the basal and forskolin-elevated cAMP levels in EP3 alpha and EP3 beta. The M&B 28767-stimulated GTPase activity consisted of pertussis-toxin-sensitive and cholera-toxin-sensitive portions in the EP3 gamma-expressing cell membrane, suggested that EP3 gamma is coupled to both guanine nucleotide-binding inhibitory and stimulatory proteins. These results indicate that EP3 gamma is coupled to both stimulation and inhibition of adenylate cyclase, but that EP3 alpha and EP3 beta are exclusively coupled to inhibition of adenylate cyclase. Thus, alternative splicing produces a third isoform with a different carboxy-terminal tail, which differs from the other two isoforms in the specificity of coupling to a signal-transduction pathway.

Adenylyl Cyclase Inhibitors↗

PubMed1993

Cloning and expression of cDNA for a mouse EP1 subtype of prostaglandin E receptor.

A functional cDNA clone encoding a mouse EP1 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse thromboxane A2 receptor cDNA. The clone isolated encodes a protein consisting of 405 amino acid residues with putative seven-transmembrane domains. [3H]PGE2 specifically bound to the membrane of Chinese hamster ovary cells expressing this clone. The binding to the membrane was displaced with unlabeled PGs in the order of PGE2 > iloprost (a prostacyclin analogue) > PGE1 > PGF2 alpha > U-46619 (a thromboxane A2 analogue) > PGD2. The binding was also inhibited by 17-phenyl trinor PGE2 (an EP1 agonist) and sulprostone (an EP1 and EP3 agonist) but not by 11-deoxy PGE1 (an EP2 and EP3 agonist) and butaprost (an EP2 agonist). PGE2 induced a rapid increase in intracellular Ca2+ concentration in Chinese hamster ovary cells expressing the receptor. These results suggest that this receptor belongs to EP1 subtype of PGE receptor. Northern blot analysis demonstrated that the mRNA of this receptor is expressed abundantly in kidney and in a lessor amount in lung.

Amino Acid Sequence↗

PubMed1993

Alternative splicing of C-terminal tail of prostaglandin E receptor subtype EP3 determines G-protein specificity.

Peptide hormones, neurotransmitters, and autacoids activate a family of seven-transmembrane-domain receptors. Each of these receptors specifically couples to one of several G proteins, Gs, Gi, G(o) and Gp, to activate a specific second messenger system. Cell surface receptors for prostanoids have been characterized pharmacologically and the complementary DNAs for thromboxane A2 receptor and the EP3 subtype of the prostaglandin (PG)E receptor reveal that they belong to the seven-transmembrane-domain receptor family. The EP3 receptor mediates the diverse physiological actions of PGE2 (ref. 3). Although most of them occur through coupling of the EP3 receptor to Gi and inhibition of adenylyl cyclase, the EP3-mediated contraction of uterine muscle can only occur by activation of another second messenger pathway. In chromaffin cells, two different second messenger pathways are activated by PGE2 binding to an apparently single EP3 receptor class. Here we show that at least four isoforms of the EP3 receptor, which differ only at their C-terminal tails and are produced by alternative splicing, couple to different G proteins to activate different second messenger systems.

Adrenal Medulla↗

PubMed1993

Characterization of the prostaglandin E receptor expressed on a cultured mast cell line, BNu-2cl3.

Interleukin 3-dependent BNu-2cl3 mast cells, mucosal type-like mast cells, exhibited a specific high-affinity binding site for [3H]prostaglandin (PG) E2. The binding was completely displaced by M&B 28767, an EP3-selective agonist, but not by EP1- or EP2-selective ligands, indicating that the PGE2 binding site is of the EP3 subtype PGE receptor. Whereas the EP3 subtype is presumed to be coupled to inhibition of adenylate cyclase in various tissues and cells, in BNu-2cl3 cells PGE2 had no ability to inhibit adenylate cyclase activity, while it induced concentration-dependent stimulation of phosphoinositide metabolism and caused an increase in the intracellular free Ca2+ concentration in a pertussis toxin-sensitive manner. PGE2 by itself did not evoke histamine release from the cells, but it markedly stimulated histamine release in concert with ionomycin, a Ca2+ ionophore. The PGE2-stimulated release was also completely blocked by pertussis toxin. Thus, the PGE receptor expressed on BNu-2cl3 mast cells is of the EP3 subtype and is linked to phosphoinositide metabolism via a pertussis toxin-sensitive G protein, and this activation leads to histamine release.

Adenylate Cyclase Toxin↗

PubMed1993

Activation of heat shock transcription factors by delta 12-prostaglandin J2 and its inhibition by intracellular glutathione.

We recently showed that delta 12-prostaglandin (PG) J2 bound to the thiol groups of nuclear proteins and stimulated the synthesis of a 67-kDa heat shock protein (HSP) in porcine aortic endothelial cells, and that intracellular glutathione (GSH) blocked this binding and HSP induction (Koizumi et al., Biochem Pharmacol 44: 1597-1602, 1992). In the present study, we examined the molecular mechanism underlying the induction of HSP by delta 12-PGJ2. Treatment of cells with delta 12-PGJ2 induced the activation of heat shock transcription factors (HSF) in a time- and concentration-dependent manner. Cycloheximide pretreatment inhibited this activation. Treatment of cells with buthionine sulfoximine, an inhibitor of GSH synthesis, depleted the intracellular GSH and enhanced the activation of HSF by delta 12-PGJ2, but treatment with GSH increased the intracellular GSH level and thus reduced the activation. Moreover, the thiol-reactive agents arsenite and diethylmaleate also induced the activation of HSF, and this activation was inhibited by GSH treatment and enhanced by buthionine sulfoximine treatment. These results taken together suggest that delta 12-PGJ2 binds to the thiol groups of nuclear proteins and activates HSF, leading to the synthesis of the 67-kDa HSP.

Animals↗

PubMed1993

Two isoforms of prostaglandin E receptor EP3 subtype. Different COOH-terminal domains determine sensitivity to agonist-induced desensitization.

We recently identified two isoforms of mouse prostaglandin (PG) E receptor EP3 subtype, EP3 alpha and EP 3 beta, which are produced by alternative splicing and different only in the carboxyl-terminal domain (Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, Y., T., Honda, A., Watabe, A., Hirata, M., Narumiya, S., and Ichikawa, A. (1993) J. Biol. Chem. 268, 2712-2718). We examined here agonist-induced desensitization of the two isoforms using Chinese hamster ovary cells stably expressing these isoforms. Exposure of the EP3 alpha isoform to PGE2 for 30 min did not change maximal response but increased PGE2 concentration needed to inhibit forskolin-induced cAMP accumulation in the cells. Further exposure of this isoform to PGE2 suppressed the maximal response as well as sensitivity to PGE2 in a time-dependent manner; after 24-h exposure, it elicited only 50% of the maximal response of the control cells. Consistent with these results, short term exposure sequestered the EP3 alpha isoform away from the cell surface and long term incubation decreased the total receptor number in the cells. In contrast, exposure of the EP3 beta isoform to PGE2 did not affect its dose-response curve for PGE2, and no sequestration or decrease in the receptor number was observed in this isoform. Thus, alternative splicing produced the two isoforms with different carboxyl-terminal domains, which are different in sensitivity to agonist-induced desensitization.

Alternative Splicing↗

PubMed1993

Fibroblast growth factor-dependent metabolism of hypoxanthine via the salvage pathway for purine synthesis in porcine aortic endothelial cells.

In this study we examined the metabolism of hypoxanthine in fibroblast growth factor (FGF)-stimulated porcine aortic endothelial cells (PAEC). Our previous report indicated that hypoxanthine in fetal bovine serum (FBS) was an essential component for both basal and FGF-dependent growth of PAEC (Hayashi et al., Exp Cell Res 185: 217-228, 1989). Besides hypoxanthine, the addition of various purine bases and purine nucleosides, but not xanthine, xanthosine or any pyrimidine metabolites, restored the limited growth of PAEC cultured in medium containing 10% dialyzed FBS in the presence or absence of FGF. The metabolism of [14C]hypoxanthine was compared in PAEC treated with and without FGF. Treatment of PAEC with FGF for 24 hr enhanced the radioactivity incorporation from [14C]hypoxanthine into both the acid-soluble and -insoluble fractions approximately 2-fold. Upon chromatographic analyses of hypoxanthine metabolites in the acid-soluble nucleotide fraction, it was found that in control PAEC hypoxanthine was largely metabolized to IMP, adenine nucleotides and uric acid, whereas in FGF-treated cells it was converted to ATP, ADP, GTP, xanthine and uric acid. The radioactivity of IMP was lowered in FGF-stimulated cells. The addition of FGF to PAEC increased phosphoribosyl pyrophosphate (PRPP) synthetase activity by approximately 8-fold and the PRPP content by approximately 2-fold, but it did not increase hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity or hypoxanthine transport. On the other hand, methotrexate, an inhibitor of de novo synthesis of purine, did not affect the growth of PAEC. Analyses of the rate of [14C]formate incorporation into total purine compounds showed that PAEC had a low capacity to synthesize purines de novo, which was not stimulated by FGF. These data indicate that FGF stimulates the synthesis of PRPP necessary for the salvage synthesis of purine nucleotides in conjunction with purine bases, e.g. hypoxanthine.

Animals↗

PubMed1993

Interleukin 3 promotes histamine synthesis in hematopoietic progenitors by increasing histidine decarboxylase mRNA expression.

Interleukin 3 (IL-3) is a potent stimulator of histamine production by cells from murine hematopoietic organs. We demonstrate herein that this phenomenon results from increased histidine decarboxylase (HDC: EC 4.1.1.22) activity in progenitor-enriched bone marrow cells (around 5% of the total bone marrow) isolated from the low density layers of a discontinuous Ficoll gradient. HDC levels are markedly enhanced after a 24 h incubation with IL-3 while a 4 h exposure results only in a slight activation. It results from increased expression of the mRNA coding for HDC, as assessed by Northern blot analysis after a 24 h incubation with IL-3. At the same time point and after a 4 h stimulation, we have evaluated the percentage of cells in this population which express HDC mRNA in response to IL-3, using in situ hybridization with the antisense riboprobe. We have thus established that enhanced HDC mRNA expression occurs in a small immature subset representing from 5 to 8% of the progenitor-enriched bone marrow cells.

Animals↗

PubMed1993

Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype.

A functional cDNA clone encoding mouse EP2 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse EP3 subtype PGE receptor cDNA. The mouse EP2 receptor consists of 513 amino acid residues with putative seven-transmembrane domains. In contrast to EP3 receptor, this receptor possesses long third intracellular loop and carboxyl-terminal tail. [3H] PGE2 specifically bound to the membrane of mammalian COS cells transfected with the cDNA. The binding to the membrane was displaced with unlabeled PG 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, and SC-19220, an EP1 antagonist. PGE2 markedly increased cAMP level in COS cells transfected with the cDNA. These results suggest that this receptor is EP2 subtype. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the abundant expression being observed in ileum, thymus, and mastocytoma P-815 cells.

Amino Acid Sequence↗