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Biomedical subjects

Y Sugimoto

Publications and source records attributed to Y Sugimoto.

At least 289 records · Page 16Linked to original sources
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

Deletion and site-directed mutagenesis of EF-hand domain of phospholipase C-delta 1: effects on its activity.

In order to elucidate a role of a putative EF-hand motif (144-172) in phospholipase C-delta 1 (PLC-delta 1), deletion and point mutation of the enzyme were performed and the mutated cDNAs were expressed in CHO cells and E. coli AD202 strain. Deletion of amino acid residues of 141-236 or 173-236 resulted in abolition of PLC activity. However, the decreased PLC activity to 15-20% by deletion of the EF-hand motif (144-172) was still Ca(2+)-dependent. Furthermore, mutants, in which conserved Asp153, Asp157, Glu164 or all these acidic amino acids in the EF-hand motif were replaced with alanine residues, showed nearly the same PLC activity and Ca(2+)-dependency as those of wild-type. These results suggest that the region containing the EF-hand motif may not play a role in regulation of Ca(2+)-sensitivity of PLC-delta 1, but is important for its activity.

Alanine↗
PubMed1995

Identification of prostaglandin E receptor 'EP2' cloned from mastocytoma cells EP4 subtype.

We previously cloned a cDNA for a mouse PGE receptor positively coupled to adenylate cyclase from mouse mastocytoma cells, and reported it as EP2 subtype of PGE receptor [Honda, A., Sugimoto, Y., Namba, T., Watabe, A., Irie, A., Negishi, M., Narumiya, S. and Ichikawa, A. (1993) J. Biol. Chem. 268, 7759-7762]. However, it is not sensitive to one of the EP2 agonists, butaprost. Recently another subtype of PGE receptor coupled to adenylate cyclase has been identified pharmacologically and named EP4. These findings have led us to examine whether the cloned receptor is the EP4 subtype. AH23848B, a selective EP4 antagonist, not only displaced the [3H]PGE2 binding to the cloned receptor but antagonized the PGE2-stimulated cAMP formation in the receptor. In contrast, EP2 specific agonist, butaprost and 19(R)OH-PGE2 neither bound to the receptor nor stimulated the cAMP formation. These results suggest that this receptor previously reported as 'EP2' subtype is identical to the pharmacologically defined EP4 subtype and not of EP2 subtype.

Adenylyl Cyclases↗
PubMed1995

Characterization of the signal transduction of prostaglandin E receptor EP1 subtype in cDNA-transfected Chinese hamster ovary cells.

We examined the signal transduction of mouse prostaglandin E receptor EP1 subtype using Chinese hamster ovary cells stably expressing the cloned EP1. Sulprostone, an EP1 agonist, induced a rapid increase in intracellular Ca2+ concentration in the EP1-expressing cells. Most of the increase was abolished by removal of extracellular Ca2+, and was insensitive to U-73122, a phospholipase C inhibitor. Sulprostone stimulated phosphatidylinositol hydrolysis, but this stimulation was abolished by removal of extracellular Ca2+, indicating that EP1-stimulated phosphatidylinositol hydrolysis is the result of extracellular Ca2+ influx. Thus, the signal transduction of EP1 is extracellular Ca2+ entry through a pathway independent of phospholipase C activation. We further examined the regulation of the signal transduction of EP1 having potential phosphorylation sites for either protein kinase C or protein kinase A. Short-term exposure of the cells to 12-O-tetradecanoylphorbol 13-acetate (TPA) completely suppressed the sulprostone-induced increase in intracellular Ca2+ concentration, while forskolin or dibutyryl cAMP did not affect it, suggesting that protein kinase C but not protein kinase A is involved in the regulation of the EP1 signal transduction. Furthermore, long-term exposure to TPA decreased PGE2 protein kinase A is involved in the regulation of the EP1 signal transduction. Furthermore, long-term exposure to TPA decreased PGE2 binding activity of EP1 due to the reduction of the EP1 mRNA level. Protein kinase C induces short- and long-term desensitization of EP1.

Animals↗
PubMed1995

EGF-induced activation of 70-kDa S6 kinase in CHO cells expressing human EGF receptors.

We investigated epidermal growth factor (EGF)-induced activation of 85-kDa/110-kDa phosphatidylinositol (PI)-3-kinase and 70-kDa S6 kinase in Chinese hamster ovary cells expressing the human EGF receptor. EGF-induced activation of p70 S6 kinase was comparable to that induced by insulin, whereas that of PI-3-kinase in anti-phosphotyrosine immunoprecipitates was very small compared with insulin. Wortmannin, a p85/p110 PI-3-kinase inhibitor, inhibited EGF-induced activation of p70 S6 kinase in a dose-dependent manner. Given that several proteins homologous to catalytic subunit of p85/p110 PI-3-kinase have been identified and that wortmannin inhibits distinct form of PI-3-kinase, the present results suggest that wortmannin-sensitive kinases that resemble catalytic subunit of p85/p110 PI-3-kinase may participate in the signaling pathway from EGF receptors to p70 S6 kinase.

Androstadienes↗
PubMed1995

cDNA cloning of a thromboxane A2 receptor from rat astrocytes.

A cDNA clone for rat thromboxane (TX) A2 receptor was obtained from cultured astrocytes. The cDNA encodes a protein of 341 amino acids with seven putative transmembrane domains. The receptor is homologous with mouse and human TXA2 receptors at 92.7% and 71.8%, respectively. Expression of messenger RNA for the receptor was examined by reverse transcriptase-polymerase chain reaction, showing ubiquitous expression in brain glial cells such as astrocytes, oligodendrocytes and microglia.

Amino Acid Sequence↗
PubMed1995

Correlation between complementation group for immortality and the cellular distribution of mortalin.

The dominance of cellular senescence over the immortal phenotype has been demonstrated by cell fusion experiments utilizing human and mouse cells. Mortalin, a novel 66-kDa member of the murine hsp70 family of proteins, has recently been identified as a marker of the mortal phenotype by virtue of its characteristic cytosolic distribution in mortal cells. Here we report the mortalin immunostaining observations on 21 human cell lines. These cell lines have previously been assigned by somatic cell hybridization analysis to one (18 lines) or more than one (3 lines) of the four complementation groups (A, B, C, and D) for immortalization. Four patterns of mortalin immunostaining were observed: granular-juxtanuclear cap, granular-gradient from nuclear to cell membrane, granular-juxtanuclear arch, and fibrous-perinuclear. In 17 of 18 cell lines assigned to a single complementation group, the mortalin staining corresponded with the complementation group. In two of the three cell lines previously assigned to multiple complementation groups, the mortalin staining corresponded to one of the assigned groups. Two cell lines, however, exhibited staining patterns which did not match to their assigned complementation groups. The basis of correlation between cellular distribution of mortalin and the complementation group remains unclear at present. However, the data (i) suggest that the intracellular distribution of mortalin can be used to distinguish mortal and immortal cells, confirming the association of mortalin with senescence; (ii) provide supportive evidence for the existence of at least four different pathways of immortalization in human cells; and (iii) indicate that mortalin is involved in processes that result in immortalization.

Animals↗
PubMed1995

Comparative pharmacodynamic analysis of TAT-59 and tamoxifen in rats bearing DMBA-induced mammary carcinoma.

TAT-59 suppressed the growth of DMBA-induced mammary tumors in rats earlier and more strongly than tamoxifen (TAM). After oral administration of the drugs, DP-TAT-59, one of the main metabolites of TAT-59, was found in 10- to 15-fold higher concentrations in both the tumor and blood compared to 4-OH-TAM, an active metabolite of TAM. In a 3-day antiuterotrophic test, every detected metabolite of TAT-59 showed stronger antiestrogenic activity than did TAM. In a competition assay, the affinity of the metabolites for estrogen receptors ranged from that of estradiol to that of TAM. These results suggest that the superior antiestrogenic activity of TAT-59 compared to TAM was either due to its higher penetration into tumor tissue or to the stronger antiestrogenic activity of its metabolites.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1995

Copresence of prostaglandin EP2 and EP3 receptors on gastric enterochromaffin-like cell carcinoid in African rodents.

BACKGROUND & AIMS: Prostaglandins (PGs) have important roles in the regulation of gastric acid secretion. The aim of this study was to examine the possible presence of PG receptors on the gastric enterochromaffin-like (ECL) carcinoid of Mastomys natalensis, which might be a useful model of normal ECL cells. METHODS: A [3H]PGE2 binding experiment was performed by using the ECL tumor membrane, and intracellular signal transduction was studied in the cells. In addition, Northern blot analysis using EP2 and EP3 receptor complementary DNAs was conducted. RESULTS: [3H]PGE2 specifically bound to the tumor cell membrane, and the binding was displaced by various PGs with a potency order of PGE1 = PGE2 > enprostil > PGF2 alpha. Although PGE1 and PGE2 stimulated 5'-cyclic adenosine monophosphate (cAMP) production, neither PGF2 alpha nor enprostil had any effect. On the other hand, all of PGE1, PGE2, PGF2 alpha, and enprostil attenuated the forskolin-induced cAMP production. Moreover, enprostil inhibited histamine release induced by forskolin. However, on pertussis toxin treatment, PGE2 paradoxically enhanced the forskolin-induced increase of cAMP production. Finally, the presence of EP2 and EP3 receptor messenger RNAs was confirmed by RNA blot analysis. CONCLUSIONS: The ECL carcinoid tumor cells of Mastomys seem to possess two subtypes of PGE receptor: EP2 linked to cAMP production and EP3 coupled with inhibitory guanosine 5'-triphosphate-binding proteins mediating the inhibition of cAMP production.

Animals↗
PubMed1995

The involvement of the peripheral 5-HT2A receptor in peripherally administered serotonin-induced hyperglycemia in rats.

The mechanism of the hyperglycemic response to intraperitoneally administered serotonin (5-HT) was studied in rats. 5-HT i.p.-induced hyperglycemia was strongly antagonized by the 5-HT2A receptor antagonist ketanserin. 5-HT did not affect the serum insulin levels and increased plasma glucagon levels only at the high dose of 10 mg/kg. 5-HT dose-dependently induced a remarkable increase in plasma adrenaline levels and these effects were antagonized by ketanserin. 5-HT-induced hyperglycemia was abolished by adrenodemedullation. These results suggest that the hyperglycemic effects of 5-HT are closely related to the release of adrenaline from the adrenal gland, mediated by 5-HT2A receptors.

Animals↗
PubMed1995

Inhibitory effects of bisbenzylisoquinoline alkaloids on induction of proinflammatory cytokines, interleukin-1 and tumor necrosis factor-alpha.

Bisbenzylisoquinoline alkaloids are known to affect immune responses as well as inflammatory responses, and have been used for the treatment of inflammatory symptoms in China. This study is aimed at elucidating the inhibitory effects of two alkaloids, fangchinoline and isotetrandrine, on the induction of the proinflammatory cytokines, interleukin-1 (IL-1), and tumor necrosis factor-alpha (TNF-alpha), by Staphylococcus aureus Cowan 1 (SAC)-stimulated human peripheral blood mononuclear cells. These two alkaloids inhibited cytokine production in a dose-dependent manner, and they inhibited it by more than 90% at 10 micrograms/ml at every time point examined. Of note was that these two alkaloids appeared to inhibit IL-1 beta production more effectively than IL-1 alpha production. When the levels of cytokine mRNA were measured by semiquantitative RT-PCR, these alkaloids reduced the levels of the mRNAs of IL-1 beta and TNF-alpha, but not that of beta 2-microglobulin, suggesting that these alkaloids may suppress cytokine transcription selectively.

Alkaloids↗
PubMed1995

Retroviral coexpression of a multidrug resistance gene (MDR1) and human alpha-galactosidase A for gene therapy of Fabry disease.

Human alpha-galactosidase A (alpha-Gal A; EC.3.2.1.22) is a lysosomal exoglycosidase encoded by a gene on Xq22. Deficiencies of this enzyme result in Fabry disease, an X-chromosome-linked recessive disorder that leads to premature death in affected males. For treatment of genetic diseases, we have developed a retroviral vector system, pSXLC/pHa, that enables coexpression of drug-selectable markers with a second nonselectable gene as part of a bicistronic message using the promoter from the Harvey murine sarcoma virus and an internal ribosomal entry site (IRES) from encephalomyocarditis virus. Retroviral vectors based on this system that carry the human alpha-Gal A cDNA either upstream (pHa-alpha Gal-IRES-MDR) or downstream (pHa-MDR-IRES-alpha Gal) from the IRES relative to the drug-selectable MDR1 (P-glycoprotein) cDNA were constructed. Each of eight independent vincristine-resistant, pHa-alpha Gal-IRES-MDR-transfected clones and all four vincristine-resistant, pHa-alpha Gal-IRES-MDR retrovirus-transduced clones showed significantly higher activity of alpha-Gal A than the parental cells. More than 50% of the vincristine-resistant, pHa-MDR-IRES-alpha Gal-transfected clones and all four vincristine-resistant, pHa-MDR-IRES-alpha Gal retrovirus-transduced clones showed significantly higher activity of alpha-Gal A than the parental cells. In these bicistronic vectors, the cDNA whose translation was cap-dependent (upstream) was expressed at higher levels than when the same cDNA was translated in an IRES-dependent manner (downstream). These vectors may prove useful in the gene therapy of Fabry disease.

3T3 Cells↗
PubMed1995

Drosophila melanogaster aldolase: characterization of the isozymes alpha, beta, and gamma generated from a single gene.

Three isozymic forms, alpha, beta, and gamma, of Drosophila melanogaster aldolase are produced from a single gene by alternative usage of the triple exons 4 (4 alpha, 4 beta, and 4 gamma) [Shaw-Lee et al. (1992) J. Biol. Chem. 267, 3959-3967; Kim et al. (1992) Mol. Cell. Biol. 12, 773-783; Kai et al. (1992) J. Biochem. 112,677-688]. The expression plasmids for the respective isozymes were transfected into Escherichia coli cells, and the isozymes alpha and beta were purified to homogeneity by a simple procedure, though isozyme gamma was only partially purified. These isozymes are active towards two substrates, fructose-1,6-bisphosphate (Fru-1,6-P2) and fructose-1-phosphate (Fru-1-P), with a preference for Fru-1,6-P2 over Fru-1-P, but they have different kcat/Km values towards these two substrates; isozyme alpha shows the highest value for Fru-1,6-P2. These isozymes show similarity in optimal pHs, thermal stability, and Km values for both Fru-1,6-P2 and Fru-1-P. They are composed of four identical subunits of 40 kDa, forming a tetramer with a molecular weight of approximately 160 kDa. The three isozymes are different in primary structure only at the carboxyl-terminal region encoded by the respective exon 4. Therefore, this region should be primarily responsible for the distinct characteristics of these isozymes.

Amino Acid Sequence↗
PubMed1995

Cations inhibit specifically type I 5 alpha-reductase found in human skin.

Steroid 5 alpha-reductase catalyzes the reduction of testosterone into the very potent androgen dihydrotestosterone. Previously, we showed that human type I 5 alpha-reductase is expressed mainly in the skin, whereas a type II 5 alpha-reductase is more specifically expressed in the prostate. To assess the possible differential effects of various cations on the two types of 5 alpha-reductase, we constructed expression vectors and transfected them into SW-13 cells, a human adrenal carcinoma cell line containing negligible endogenous 5 alpha-reductase activity. The expressed 5 alpha-reductases were analyzed for their sensitivity to Li, Ca, Cd, Cu, Mg, Mn, Ni, Zn, and Fe. The results showed that type I 5 alpha-reductase was strongly inhibited by Cd, Cu, and Zn and moderately inhibited by Ni and Fe, with 50% inhibitory concentration values of 0.9, 1.9, 2.0, 169.2, and 174.3 microM, respectively. In contrast, type II 5 alpha-reductase activity was inhibited only by Cu, with a 50% inhibitory concentration value of 19.2 microM. The data showed that cations could specifically control 5 alpha-reductase activity expression, which is more strongly inhibited in a target tissue, especially the skin.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1995

Infrequent somatic mutation of the MTS1 gene in primary bladder carcinomas.

We examined a candidate tumor suppressor gene on chromosome 9p21, MTS1/CDK4I (multiple tumor suppressor 1/cyclin-dependent kinase 4 inhibitor), which has been found to be mutated frequently in cell lines derived from bladder carcinomas, for somatic mutations in 39 primary bladder cancers by means of SSCP (single-stranded conformational polymorphism) and DNA sequencing. Mutations were detected in two of these carcinomas; one was a 61-base deletion and the other a 1-base deletion. In both cases the homologous allele was missing, indicating that "two-hit" mutation of the MTS1 gene had taken place in these tumors. The results indicated that inactivation of the MTS1 gene is likely to be a contributing factor in some, but not the majority of, bladder cancers.

Base Sequence↗