Search PubMed⌕ Search

Biomedical subjects

A Irie

Publications and source records attributed to A Irie.

At least 55 records · Page 3Linked to original sources
PubMed1996

Abrogation of lung metastasis of human fibrosarcoma cells by ribozyme-mediated suppression of integrin alpha6 subunit expression.

The interaction of tumor cells with the basement membrane plays a crucial role in tumor metastasis. VLA-6 (alpha6beta1) integrin is one of the major surface receptors for the basement membrane, specifically recognizing laminin. To study the role of VLA-6 integrin in tumor invasion and metastasis, we synthesized a ribozyme that selectively degrades the integrin alpha6 subunit mRNA. The catalytic activity of the ribozyme was verified by in vitro cleavage of alpha6 subunit mRNA. Introduction of the anti-alpha6 ribozyme gene into the human fibrosarcoma cell line HT1080 yielded stable transfectants, which expressed a significantly reduced level of integrin alpha6 mRNA. Flow cytometric analysis showed that the surface expression of VLA-6, but not other integrins, was reduced by approximately 70% in transfected cells. Ribozyme-transfected cells were less adherent to laminin-coated substrata and less invasive into reconstituted basement membrane than mock-transfected cells. When injected i.v. into nude mice, ribozyme-transfected cells produced no lung metastasis in all except 1 of 35 mice, though mock-transfected cells produced multiple lung metastases in 22 of 29 mice. Our results indicate that VLA-6 integrin plays a critical role in tumor invasion and metastasis and may serve as a potential target for eradication of tumor metastasis in the lung.

Animals↗
PubMed1996

Induction of experimental bone metastasis in mice by transfection of integrin alpha 4 beta 1 into tumor cells.

Cell adhesion receptors (eg, integrins and CD44) play an important role in invasion and metastasis during tumor progression. The increase in integrin alpha 4 beta 1 expression on primary melanomas has been reported to significantly correlate with the development of metastases. alpha 4 beta 1 is a cell surface heterodimer that mediates cell-cell and cell-extracellular matrix interactions through adhesion to vascular cell adhesion molecule (VCAM)-1 and to the IIICS region of fibronectin. To test the effects of alpha 4 beta 1 expression on tumor cell metastasis, Chinese hamster ovary cells were transfected with human alpha 4 cDNA. Whereas alpha 4-negative Chinese hamster ovary cells developed only pulmonary metastasis, alpha 4-positive Chinese hamster ovary cells developed bone and pulmonary metastasis in 3 to 4 weeks when injected intravenously into nude mice. Bone metastasis was inhibited by antibody against alpha 4 or VCAM-1. Expression of alpha 3 beta 1, alpha 6 beta 1, or alpha V beta 1 did not induce bone metastasis. Expression of alpha 4 beta 1 also induced bone metastasis in K562 human erythroleukemia cells injected into SCID mice. These results demonstrate that alpha 4 beta 1 can induce tumor cell trafficking to bone, probably via interaction with VCAM-1 that is constitutively expressed on bone marrow stromal cells.

Animals↗
PubMed1995

Critical amino acid residues for ligand binding are clustered in a predicted beta-turn of the third N-terminal repeat in the integrin alpha 4 and alpha 5 subunits.

Integrin alpha 4 beta 1 is a receptor for vascular cell adhesion molecule (VCAM)-1 and fibronectin (CS-1). The alpha 4 beta 1-ligand interaction is involved in the pathogenesis of diseases and is, therefore, a therapeutic target. Here, we identified critical residues of alpha 4 for ligand binding using alanine-scanning mutagenesis of the previously localized putative ligand binding sites (residues 108-268). Among 43 mutations tested, mutations of Tyr187, Trp188 and Gly190 significantly inhibited cell adhesion to both VCAM-1 and CS-1. This inhibition was not due to any gross structural changes of alpha 4 beta 1. These critical residues are clustered in a predicted beta-turn structure (residues 181-190) of the third N-terminal repeat in alpha 4. The repeat does not contain divalent cation binding motifs. Notably, the mutations within the corresponding region of alpha 5 significantly reduced fibronectin-alpha 5 beta 1 interaction. These findings suggest that the predicted beta-turn structure could be ubiquitously involved in ligand binding of non-I domain integrins.

Amino Acid Sequence↗
PubMed1995

Selective coupling of prostaglandin E receptor EP3D to Gi and Gs through interaction of alpha-carboxylic acid of agonist and arginine residue of seventh transmembrane domain.

Prostaglandin (PG) E receptor EP3D is coupled to both Gi and Gs. To examine the roles of the interaction of alpha-carboxylic acid of PGE2 and its putative binding site, the arginine residue in the seventh transmembrane domain of EP3D, in receptor-G protein coupling, we have mutated the arginine residue to the noncharged glutamine. PGE2 with a negatively charged alpha-carboxylic acid and sulprostone, an EP3 agonist with a noncharged modified alpha-carboxylic acid, inhibited the forskolin-stimulated adenylate cyclase activity via Gi activation in the EP3D receptor in the same concentration-dependent manner. In contrast, the adenylate cyclase stimulation via Gs activation by sulprostone was much lower than that by PGE2. On the other hand, both PGE2 and sulprostone showed potent Gi activity but failed to show Gs activity in the mutant receptor. EP3D receptor showed a high affinity binding for PGE2 in the form coupled to either Gi or Gs. Although the mutant receptor showed high affinity binding when coupled to Gi, it lost high affinity binding in the condition of Gs coupling. Furthermore, sulprostone bound to the Gi-coupled EP3D receptor with higher affinity than the Gs-coupled receptor. Among various EP3 agonists, alpha-carboxylic acid-unmodified agonists showed both Gi and Gs activities, but the modified agonists showed only Gi activity. These findings suggest that the interaction between the alpha-carboxylic acid of PGE2 and the arginine residue of the receptor regulates the selectivity of the G protein coupling.

Abortifacient Agents, Nonsteroidal↗
PubMed1995

Characterization of neutral glycosphingolipids in rat lens.

Neutral glycosphingolipids were purified from non-cataractous lenses of Sprague-Dawley rats by a combination of solvent extraction, Folch's partition, and column chromatography using DEAE-Sephadex and Iatrobeads. Six major GSLs from monohexosylceramide to pentahexosylceramide were identified by sugar composition analysis, methylation analysis and glycosidase digestion. Structural relationships among the six neutral glycosphingolipids revealed metabolic pathways leading to the synthesis of Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide (IV3Gal alpha nLc4), instead of a Lewis(x) glycolipid (Gal beta 1- 4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1 ceramide, III3FucnLc4), from neolactotetraosylceramide (nLc4), together with isoglobotriaosylceramide (iGb3). The alpha-galactosyl epitope, Gal alpha 1-3Gal-R, is evolutionarily conserved in many types of cells of non-primate mammals, prosimians and New World monkeys, but not in those of Old World monkeys or humans. This evolution-related difference in carbohydrate epitopes suggests different cell-to-cell attachments, which may be mediated through cell surface glycosphingolipids, between rat and human lenses.

Animals↗
PubMed1995

[Radical prostatectomy in a patient with hemophilia B].

We describe our experience with radical prostatectomy in a patient with hemophilia B. whose factor IX activity was 7% and partial thromboplastin time was 55.9 sec. A total of 43,000 units of factor IX concentrate (PCC) was given perioperatively for 2 weeks. No abnormal hemorrhage was noted. An algorithm for perioperative management of hemophilia B patient is discussed.

Adenocarcinoma↗
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

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

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

[Long-term results and complications of intravesical instillation of bacillus Calmette-Guerin for prophylaxis of bladder cancer recurrence].

Since 1982, we treated 22 patients with superficial bladder cancer via intravesical bacillus Calmette-Guerin (Tokyo 172 strain) for prophylaxis against tumor recurrence. To determine the long-term efficacy and complications of BCG therapy, retrospective analysis was performed. The BCG therapy was initiated one to two weeks after complete transurethral resection of visible tumors. One hundred twenty mg or 80 mg of BCG suspended in 40 ml of 0.9% NaCl solution was instilled into the bladder once a week for 6 times, then monthly for 10 times. Of 22 patients, 10 tumor recurrences were recognized in 4 patients (mean followup 74 months). Recurrent free rate at 5 years was 78.5%. Common side effects were low grade fever and bladder irritability. Although these side effects were self-limiting, 15 patients (68%) refused instillation of BCG before completion of our protocol because of the bladder irritation. No relationship was observed between total dose of BCG instilled and tumor recurrence. Severe side effects such as permanent structural or functional alterations of the bladder or mycobacterial infection of other organs were not observed. Intravesical administration of Tokyo 172 strain of BCG seemed to be safe and useful for long-term prophylaxis of superficial bladder cancer recurrence.

Administration, Intravesical↗
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↗