Five bovine polymorphic dinucleotide microsatellite loci (DIK021, DIK023, DIK024, DIK026 and DIK028).
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Animals↗
Biomedical subjects
Y Sugimoto
Publications and source records attributed to Y Sugimoto.
In situ hybridization studies of prostacyclin receptor mRNA expression in various mouse organs.
1. Expression of prostacyclin receptor (IP receptor) mRNA was examined in various mouse organs, and the cells expressing IP receptor mRNA were identified by in situ hybridization studies. Co-localization of mRNA for the IP receptor with that for preprotachykinin A (PPTA), a precursor protein for substance P, with mRNA for the prostaglandin E receptor subtypes (EP1, EP3 and EP4), and with renin mRNA, was examined by double in situ hybridization studies in the dorsal root ganglion and kidney, respectively. 2. IP receptor mRNA was expressed in the thymus and spleen. Expression in the thymus was found exclusively in the medulla, where mature thymocytes expressed transcripts for the IP receptor. Expression in the spleen was found as scattered signals over the white pulp and as punctate signals in the red pulp. The former was found in splenic lymphocytes and the latter in megakaryocytes. 3. IP receptor mRNA was also expressed in the vascular tissues of various organs such as the aorta, coronary arteries, pulmonary arteries and the cerebral arteries, where its expression was confined to smooth muscle cells. No expression was found in veins. In the kidney, IP receptor mRNA was detected in the interlobular arteries and glomerular arterioles but not in the juxtaglomerular (JG) cells which were labelled with the renin mRNA probe. 4. IP receptor mRNA was expressed in about 40% of the neurones in the dorsal root ganglion. Both small- and large-sized neurones were labelled but no labelling was found in the glia. Expression of PPTA mRNA was found in about 30% of total neurones. About 70% of these neurones expressed IP receptor mRNA, and about half of the IP receptor-positive neurones expressed PPTA mRNA. In addition to IP mRNA, mRNAs for EP1, EP3 and EP4 receptors were expressed in about 30%, 50% and 20%, respectively, of the dorsal root ganglion neurones. About 25%, 41% and 24% of the IP receptor-positive neurons co-expressed the EP1, EP3 and EP4 receptor, respectively. 5. These results not only verified IP receptor expression in various cells and tissues known to be sensitive to prostacyclin, but also revealed its expression in other systems, which urges the study of the actions of prostacyclin in these tissues. They also indicated that the actions of prostacyclin on blood vessels and platelets are mediated by the same type of receptor. Absence of IP receptor mRNA in the JG cells suggests that the action of prostacyclin on renin release may be indirect.
Animals↗
The effects of the serotonin1A receptor agonist buspirone on tolbutamide-induced hypoglycemia in rats.
The effects of the serotonin1A(5-HT1A) receptor agonist buspirone on hypoglycemia elicited by tolbutamide were investigated in rats. Buspirone, at doses not affecting plasma glucose levels, inhibited the hypoglycemic effects of tolbutamide. The inhibitory effects of buspirone on tolbutamide-induced hypoglycemia were antagonized by the 5-HT1A receptor antagonist pindolol. As tolbutamide is known to induce hypoglycemia by facilitating insulin release, the effects of buspirone on a tolbutamide-induced increase in serum insulin levels were also studied. However, buspirone did not affect tolbutamide-induced insulin release. Adrenodemedullation inhibited the effects of buspirone. These results suggest that buspirone inhibits tolbutamide-induced hypoglycemia mediated by the 5-HT1A receptor, and adrenaline release may be involved in the effects of buspirone.
Animals↗
Serotonin2 (5-HT2) receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) inhibits chlorpromazine- and haloperidol-induced hypothermia in mice.
Chlorpromazine- and haloperidol-induced hypothermia were examined in mice. The alpha 1 receptor agonist phenylephrine partially antagonized the hypothermia, while the dopamine D2 receptor agonist apomorphine did not inhibit it. The central serotonin2 (5-HT2) receptor agonist I-2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) strongly inhibited the chlorpromazine- and haloperidol-induced hypothermia. Both drugs at doses which can elicit hypothermia antagonized head twitch responses mediated by the central 5-HT2 receptor. These results suggest that the chlorpromazine- and haloperidol-induced hypothermia may be mediated by the blockade of the central 5-HT2 receptor.
Adrenergic alpha-1 Receptor Agonists↗
Effect of (Z)-11-[3-(dimethylamino) propylidene]-6,11-dihydrodibenz[b,e]oxepin-2-acetic acid hydrochloride on experimental allergic conjunctivitis and rhinitis in rats and guinea pigs.
The effect of KW-4679 (Z)-11-[3-(dimethylamino) propylidene]-6,11-dihydrodibenz [b,e] oxepin-2-acetic acid hydrochloride, CAS 140462-76-6; proposed INN: olopatadine) on experimental conjunctivitis and rhinitis was studied in comparison with that of ketotifen (CAS 34580-14-8) using guinea pigs and rats, respectively. KW-4679 was effective in inhibiting the antigen- and histamine-induced conjunctivitis by both oral and topical administrations. These effects of KW-4679 were somewhat more potent than those of ketotifen. KW-4679 as well as ketotifen was more effective in inhibiting the histamine-induced conjunctivitis than that seen in antigen-induced conjunctivitis when they were given topically. KW-4679 inhibited the increased dye leakage into the nasal cavity induced not only by antigen in actively sensitized rats but also by histamine perfusion in non-sensitized rats by oral and topical administrations. Similar to ketotifen, the effect of KW-4679 on histamine-induced increase in dye leakage was almost same as that induced by antigen. The potency of KW-4679 was higher than that of ketotifen in the increased dye leakage induced by both antigen and histamine perfusion.
Administration, Oral↗
Signal transductions of three isoforms of mouse prostaglandin E receptor EP3 subtype.
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Alternative Splicing↗
Conformational changes of the myosin heads during hydrolysis of ATP as analyzed by x-ray solution scattering.
We have shown for the first time that the myosin head (subfragment-1, S1), the energy-transducing component in the actomyosin motor system undergoes a distinct shape change during hydrolysis of ATP using x-ray solution scattering techniques. Among various analogs for intermediate states of the S1 ATPase cycle, the complexes with MgADP and vanadate (S1.ADP.Vi), MgADP and beryllium fluoride (S1.ADP.BeF3), or MgADP and aluminum fluoride (S1.ADP.AIF4) showed a shape change similar to that in the presence of MgATP, but the complexes with ATP gamma S (S1.ADP gamma S) and MgADP trapped by cross-linking with pPDM (S1.ADP-pPDM) seemed to have a shape similar to that of nucleotide-free S1. These results indicate that the shape of an S1**.ADP.Pi state is more rounded or bent than in other intermediate states of the S1 ATPase cycle. Such changes occur in light chain 2-deficient S1 and also in smooth muscle S1. However, MgADP-fluoride complexes with smooth muscle S1 (without phosphorylation of a regulatory light chain) seemed to have a structure similar to that of nucleotide-free S1. Analysis of x-ray scattering data indicated that a conformational change of S1 in the presence of MgATP might be caused by a hinge-like bending movement between the catalytic and regulatory domains. The global change of S1 is correlated with some specific changes of a nucleotide-binding moiety.
Adenosine Diphosphate↗
[In vivo drug-selectable markers in gene therapy].
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Animals↗
[Molecular structure and function of the prostanoid receptors].
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Amino Acid Sequence↗
Effects of intracerebroventricular injection of histamine and its related compounds on rectal temperature in mice.
Effects of intracerebroventricular injection of histamine and its related compounds on rectal temperature were studied in mice. Histamine (0.1-1.0 mu g) and histidine (500-1,000 mg/kg) caused a dose-related hypothermia. H1 agonist, 2-methylhistamine and 2-thiazolylethylamine also displayed a dose-dependent hypothermia. In addition, H2 agonists, 4-methylhistamine and dimaprit elicited a decrease in body temperature. Preinjection of not only H1-antagonists (diphenhydramine and chlorpheniramine) but also H2 antagonists (cimetidine and ranitidine) abolished histamine-induced hypothermia. Either intracerebroventricular or intraperitoneal injection of thioperamide, a histamine H3 antagonist, showed hypothermia. The hypothermic effect produced by intracerebroventricular injection of thioperamide was significantly blocked by (R)-alpha-methylhistamine, a selective H3 agonist. In addition, the effect induced by thioperamide was inhibited by H1 and H2 antagonists, indicating that the H3 receptor also participates in histamine-induced hypothermia.
Animals↗
Coexpression of a multidrug-resistance gene (MDR1) and herpes simplex virus thymidine kinase gene as part of a bicistronic messenger RNA in a retrovirus vector allows selective killing of MDR1-transduced cells
A new retroviral vector, pSXLC/pHa, was constructed to coexpress drug-selectable markers with a second gene of interest as a part of a bicistronic mRNA in a retroviral vector using an internal ribosome entry site (IRES) from encephalomyocarditis virus. This system was used to develop a new retroviral vector pHa-MDR-IRES-TK which expresses a single mRNA from which translation of the MDR1 gene is cap dependent and translation of the herpes simplex virus thymidine kinase gene is IRES dependent. The pHa-MDR-IRES-TK transfectants showed high levels of P-glycoprotein expression and multidrug resistance. More than 95% of the vincristine-resistant cells transfected or transduced with pHa-MDR-IRES-TK showed hypersensitivity to ganciclovir, which selects against cells expressing herpes simplex virus thymidine kinase. An amphotropic retrovirus titer of 7.8 x 10(4)/ml was obtained with this vector. This safety-modified vector should be useful for introducing the MDR1 gene into bone marrow cells to protect normal cells from the toxic effects of cancer chemotherapy because this vector allows the elimination of cancer cells that have been unintentionally transduced with the MDR1 vector.
Journal Article↗
Grb2/Ash binds directly to tyrosines 1068 and 1086 and indirectly to tyrosine 1148 of activated human epidermal growth factor receptors in intact cells.
The activation of receptor tyrosine kinases generates tyrosine-phosphorylated recognition motifs for the binding of signaling proteins containing Src homology 2 domains. We determined the binding sites of Grb2/Ash, an Src homology 2 domain-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing human EGF receptor mutants in which one of the autophosphorylation sites was retained. In intact cells, the amount of Grb2/Ash coimmunoprecipitated with mutant receptors retaining tyrosines 992, 1068, 1086, 1148, or 1173 was approximately 10, 85, 55, 50, or 20% of wild-type levels, respectively. The association of Grb2/Ash with in vitro autophosphorylated EGF receptor mutants was detectable in those retaining either tyrosines 1068 or 1086 but not in other mutants including those retaining tyrosine 1148. In peptide inhibition assay, phosphorylated peptides representing tyrosines 1068 and 1086 inhibited the binding of Grb2/Ash to in vitro autophosphorylated wild-type EGF receptors, whereas the other peptides representing tyrosines 992, 1148, and 1173 failed to inhibit the binding. Given that tyrosine 1148 of the activated EGF receptor is a major binding site of Shc (Okabayashi, Y., Kido, Y., Okutani, T., Sugimoto, Y., Sakaguchi, K., and Kasuga, M. (1994) J. Biol. Chem. 269, 18674-18678), these results indicate that tyrosines 1068 and 1086 of activated human EGF receptors are direct high affinity binding sites of Grb2/Ash and that tyrosine 1148 is an indirect binding site through Shc in intact cells.
Adaptor Proteins, Signal Transducing↗
Isolation and characterization of cDNA and genomic promoter region for a heat shock protein 30 from Aspergillus nidulans.
A cDNA encoding for a heat shock protein 30 (HSP30) of Aspergillus nidulans and the promoter region of its gene were analyzed for their primary structures. The promoter region had no heat shock element but possessed three inverted repeat sequences. Northern blot hybridization indicated that the expression of the HSP30 gene was high at a normal temperature and was slightly accelerated at elevated temperatures in A. nidulans cells. Although the deduced amino acid sequence of the A. nidulans HSP30 had a domain highly conserved among other small HSPs from different species, it showed a sequence homology of only 42% even in comparison with the most closely related molecular species, Neurospora crassa HSP30. These findings suggest that the present HSP30 belongs to a novel subfamily of low-molecular-weight HSPs.
Amino Acid Sequence↗
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↗
Somatic mutations of the MTS (multiple tumor suppressor) 1/CDK4l (cyclin-dependent kinase-4 inhibitor) gene in human primary non-small cell lung carcinomas.
Cytogenetic abnormalities of chromosome 9p21-22 have been documented in human non-small cell lung carcinomas (NSCLC). Recently, a candidate tumor suppressor gene, MTS1/CDK4l, was identified in this chromosomal region and frequent homozygous deletions of this gene were found in cancer cell lines derived from various types of tissues. We screened 64 primary NSCLCs, including 31 adenocarcinomas and 33 squamous cell carcinomas, for mutations in MTS1/CDK4l, and detected somatic mutations in 19 of the 64 tumors examined. Five were deletions and 14 were missense mutations. These results suggest that inactivation of MTS1/CDK4l plays an important role during carcinogenesis of NSCLC.
Adenocarcinoma↗
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↗
Tyrosines 1148 and 1173 of activated human epidermal growth factor receptors are binding sites of Shc in intact cells.
Autophosphorylation of receptor tyrosine kinases provides binding sites for signaling proteins containing Src homology 2 (SH2) domains. We determined the binding sites of Shc, SH2-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing EGF receptor mutants in which autophosphorylation sites, either alone or in combination, were replaced by phenylalanine. Binding of Shc to EGF receptor mutants lacking single tyrosine residues at 1148 or 1173 decreased by approximately 60 or approximately 15%, respectively, whereas other single point mutants bound the wild-type level of Shc. Binding of Shc markedly decreased in mutants lacking both tyrosine residues at 1148 and 1173. In peptide inhibition assay, phosphorylated nonameric peptide representing tyrosine 1148, DNPDpYQQDF, but not pentameric peptide, pYQQDF, inhibited the binding of glutathione S-transferase-Shc SH2 domain fusion protein to in vitro autophosphorylated EGF receptors, suggesting that N-terminal sequences adjacent to phosphotyrosine are necessary for the association of Shc. Based on results of peptide inhibition assays in which phosphorylated peptides representing tyrosines 992, 1148, and 1173 inhibited Shc binding to the receptor, we constructed another EGF receptor mutant in which one of these tyrosine residues was retained. The amount of Shc bound to mutant receptors retaining tyrosines 1148, 1173, or 992 was approximately 80, approximately 40, or approximately 10% of wild-type level, respectively. These results indicate that tyrosine 1148 of activated human EGF receptors is a major binding site of Shc and tyrosine 1173 is a secondary binding site in intact cells.
Amino Acid Sequence↗
Gene transfer of drug resistance genes. Implications for cancer therapy.
Two general approaches to the gene therapy of cancer have been proposed: (1) strategies that use exogenous genes to modify cancer cells so that they are less malignant or more susceptible to host defenses or to killing by exogenous agents; and (2) approaches that modify host cells so that they are more effective in eliminating cancer cells or more resistant to agents that are used to treat cancer. In both cases, the development of vectors that encode in vivo selectable phenotypes, such as drug resistance, would be extremely valuable because of the inherent inefficiency of gene transfer and the potential of such vectors to protect normal tissues against toxic agents. To allow the selection of cells in vivo that have been transduced with vectors for gene therapy, we have utilized the human multidrug resistance (MDR1) gene. The product of this gene is a 170,000-dalton glycoprotein known as P-glycoprotein, which acts as an energy-dependent efflux pump for a great many cytotoxic anticancer drugs, including doxorubicin, daunorubicin, etoposide, teniposide, actinomycin D, and taxol. Vectors encoding an MDR1 cDNA are able to transduce many cell types, including bone marrow cells, with high efficiency to allow selection of drug resistance in vitro and in vivo in mouse models. Thus, it should be possible to protect the bone marrow of patients undergoing intensive chemotherapy by transduction of their bone marrow with MDR1 vectors. Furthermore, the ability to select for the presence of the MDR1 cDNA in vivo means that it can be used to introduce otherwise nonselectable genes into the bone marrow for therapy of cancer and other diseases.
3T3 Cells↗