Search PubMed⌕ Search

Biomedical subjects

M Negishi

Publications and source records attributed to M Negishi.

At least 163 records · Page 9Linked to original sources
PubMed1996

[A basic study on gastric emptying test using barium grains and effect of drugs on gastric emptying in rats].

We reported an experimental study on a new non-invasive method for evaluation of gastric emptying by abdominal X-ray after administration of radiopaque barium grains. Adult male Wistar rats weighing around 200 g were used. After they were fasted for 24 hours, 1 ml of gruel mixed with 10 barium grains (1 mm diameter) was introduced into the rat stomach with a catheter. The rats were sacrificed at 30, 60, 90, 120 and 150 minutes after the introduction of the gruel. X-rays were taken at each time point and grains in the stomach were counted in the X-ray photographs. All barium grains were emptied from the stomach in 150 minutes. After incision of the abdomen, the residual gastric contents were weighed. A positive correlation was found between the grains in the stomach and the weight of the contents. We studied the effects of cisapride, scopolamine buthylbromide and enprostil on the gastric emptying time by this method. Cisapride accelerated gastric emptying, whereas scopolamine buthylbromide delayed it. A prostaglandin E2 analog, enprostil delayed the gastric emptying. This method was found to be a simple procedure which is outstanding for quantitative determination and useful in evaluating gastric emptying functions.

Animals↗
PubMed1995

Molecular mechanisms of diverse actions of prostanoid receptors.

This review summarizes recent advances in the molecular characterization of prostanoid receptors. Prostanoids exert versatile actions in diverse tissues and cells through specific cell surface receptors. Molecular biological studies revealed the primary structure of eight types and subtypes of prostanoid receptor from various species. These include the thromboxane A2 receptor, prostacyclin receptor, prostaglandin (PG) F receptor, PGD receptor and four subtypes of PGE receptors. They are coupled to different signal transduction systems. In addition, multiple isoforms of PGE receptor EP3 subtype have been identified in various species. They are produced through alternative RNA splicing from a single gene and differ only in their carboxy-terminal tails. These isoforms differ in the efficiency of G protein activation, in the specificity of coupling to G proteins or in sensitivity to desensitization. This molecular characterization is useful for understanding the diverse physiological roles of prostanoids.

Amino Acid Sequence↗
PubMed1995

The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and northern blot analysis.

A functional cDNA clone for the mouse prostaglandin (PG) E receptor EP2 subtype was isolated from a mouse cDNA library. The mouse EP2 receptor consists of 362 amino acid residues with seven putative transmembrane domains. [3H]PGE2 bound specifically to the membrane of Chinese hamster ovary cells stably expressing the cloned receptor. This binding was displaced by unlabeled prostanoids in the order of PGE2 = PGE1 >> iloprost, a stable PGI2 agonist > PGF2 alpha > PGD2. Binding was also inhibited by butaprost (an EP2 agonist) and to a lesser extent by M&B 28767 (an EP3 agonist), but not by sulprostone (an EP1 and EP3 agonist) or SC-19220 (an EP1 antagonist). PGE2 and butaprost increased the cAMP level in the Chinese hamster ovary cells in a concentration-dependent manner. Northern blot analysis revealed that EP2 mRNA is expressed most abundantly in the uterus, followed by the spleen, lung, thymus, ileum, liver, and stomach.

Amino Acid Sequence↗
PubMed1995

Involvement of protein kinase in delta 12-prostaglandin J2-induced expression of rat heme oxygenase-1 gene.

We recently identified the cis-regulatory element and its specific nuclear binding factors for delta 12-prostaglandin (PG) J2-induced expression of the rat heme oxygenase, HO-1 [Koizumi, T., Odani, N., Okuyama, T., Ichikawa, A. and Negishi, M. (1995) J. Biol. Chem. 270, in press]. Here we further examined the molecular mechanism underlying the delta 12-PGJ2-induced HO-1 gene expression. Protein kinase inhibitors, 2-aminopurine and staurosporine, suppressed the delta 12-PGJ2-induced HO-1 mRNA and the nuclear protein binding to the delta 12-PGJ2-responsive cis-regulatory element in rat basophilic leukemia cells. Furthermore, the nuclear protein binding to the element was suppressed by in vitro phosphatase treatment of the nuclear proteins from delta 12-PGJ2-treated cells. These findings suggest that delta 12-PGJ2 induces the expression of the HO-1 gene through phosphorylation of the nuclear proteins which bind to the delta 12-PGJ2-responsive element.

Animals↗
PubMed1995

Identification of a cis-regulatory element for delta 12-prostaglandin J2-induced expression of the rat heme oxygenase gene.

We recently reported that delta 12-prostaglandin (PG) J2 caused various cells to synthesize heme oxygenase, HO-1 (Koizumi, T., Negishi, M., and Ichikawa, A. (1992) Prostaglandins 43, 121-131). Here we examined the molecular mechanism underlying the delta 12-PGJ2-induced HO-1 synthesis. delta 12-PGJ2 markedly stimulated the promoter activity of the 5'-flanking region of the rat HO-1 gene from -810 to +101 in rat basophilic leukemia cells. From functional analysis of various deletion mutant genes we found that the delta 12-PGJ2-responsive element was localized in a region from -690 to -660, containing an E-box motif, which was essential for the delta 12-PGJ2-stimulated promoter activity. When the region containing the delta 12-PGJ2-responsive element was combined with a heterologous promoter, SV40 promoter, in the sense and antisense direction, the element showed an enhancer activity in response to delta 12-PGJ2. Gel mobility shift assays demonstrated that delta 12-PGJ2 specifically stimulated the binding of two nuclear proteins to the E-box motif of this region. These results indicate that delta 12-PGJ2 induces the expression of the rat HO-1 gene through nuclear protein binding to a specific element having an E-box motif.

Animals↗
PubMed1995

Molecular engineering of microsomal P450 2a-4 to a stable, water-soluble enzyme.

Peptitergented P450 2a-4 (Pepti-P450), a water-soluble form of the mouse microsomal P450 2a-4, was genetically engineered and expressed in Escherichia coli. The NH2-terminal hydrophobic sequence (positions 2 to 19) of Pepti-P450 was replaced by a peptitergent PD1, amphipathic peptide consisting of 24 residues (C. E. Schafmeister, L. J. Miercke, and R. M. Stroud (1993) Science 262, 734-738). The expression level of Pepti-P450 (90,000 molecules/cell) was at least four times greater than that of wild-type P450 2a-4. Since Pepti-P450 was quite stable and was expressed as a peripheral membrane protein, it can be easily purified from the membrane fraction treated with Na2CO3 without using any detergents during the chromatographic steps. The purified Pepti-P450 retained the spectral and catalytic properties of the unmodified enzyme with a similar Km value for steroid 15 alpha-hydroxylase activity (19.7 microM in comparison to 14.2 microM of the wild-type). Gel permeation chromatography showed that the purified Pepti-P450 in the detergent-free buffer was an oligomer with an approximate molecular mass of 450 kDa. The replacement of the hydrophobic anchor domain with an amphipathic helix such as peptitergent, therefore, may provide a general method for engineering membrane-bound P450s to soluble enzymes.

Amino Acid Sequence↗
PubMed1995

Selective coupling of prostaglandin E receptor EP3D to multiple G proteins depending on interaction of the carboxylic acid of agonist and arginine residue of seventh transmembrane domain.

Prostaglandin E receptor EP3D is coupled to stimulation and inhibition of adenylate cyclase and stimulation of phosphatidylinositol turnover. To examine the roles of the interaction of the carboxylic acid of an agonist 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 to the non-charged glutamine. TEI-3356, an EP3 agonist with a negatively charged the carboxylic acid, and TEI-4343, a non-charged methylester of TEI-3356, inhibited the forskolin-stimulated cAMP formation in the same concentration-dependent manner, but stimulation of basal cAMP formation and Ca2+ mobilization by TEI-4343 was much lower than that by TEI-3356. In the mutant receptor, both TEI-3356 and TEI-4343 showed the inhibition of forskolin-stimulated cAMP formation in the same profile, but did not stimulate basal cAMP formation or Ca2+ mobilization. These findings suggest that the interaction between the carboxylic acid of agonist and the arginine residue is important in signal transduction for adenylate cyclase stimulation and Ca2+ mobilization but not for adenylate cyclase inhibition.

Animals↗
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

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

Altering the regiospecificity of androstenedione hydroxylase activity in P450s 2a-4/5 by a mutation of the residue at position 481.

Mouse P450 2a-5 (coumarin 7-hydroxylase) acquires androstenedione (AD) hydroxylase activity by substituting Phe at position 209 with Asn. However, this mutant P450 2a-5 (F209N) and the corresponding mutant P450 2a-4 (L209N) exhibit different regiospecificites of androstenedione (AD) hydroxylase activity. While the former mutant catalyzes both AD 15 alpha- and 7 alpha-hydroxylase activities at similar rates, the latter mutant maintains the original high specificity of AD 15 alpha-hydroxylase activity. The AD hydroxylase activities in chimeric enzymes of the mutants L209N and F209N show that the regiospecificites are determined by the carboxy-terminal halves of the P450 molecules. Mutations at each of the four different residues within the carboxy-terminal halves indicate that the differences in regiospecificity are determined by the Val/Ala mutation at position 481. As the size of the hydrophobic amino acid at position 481 becomes larger (Ala < Val < Ile), the regiospecificities toward the C15 position of the AD molecule are dramatically increased. The regiospecificity is also increased by placing positively-charged Arg at position 481, although the remaining 15 alpha-hydroxylase activity in this mutant is considerably lower than the other P450s. The results indicate that the size of the residue at position 481 is a key factor in regulating the regiospecificity of AD hydroxylase activity in the P450s. Modeling AD in the substrate-heme pocket of bacterial P450 101A provided further support that residue 481 may reside near the steroid molecule so as to possibly affect the AD hydroxylase activity.

Amino Acid Sequence↗
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

Sexually dimorphic DNA demethylation in the promoter of the Slp (sex-limited protein) gene in mouse liver.

Mouse Slp, a duplicate of the fourth complement component (C4) gene, exhibits EDTA-independent complement activity with a hepatic expression that is male specific. To provide an underlying mechanism for the male-specific expression, we have analyzed the promoter activity of the various 5'-flanking sequences and CpG demethylation of the Slp gene. Transient transfections using HepG2 cells indicate that the element TTCCGGGC (nt -124 to -117) regulates the promoter activity. Moreover, CpG at position -121 of this regulatory element is demethylated to a much higher degree in males than in females. This sexually dimorphic DNA demethylation is consistent with the male-specific expression of the Slp gene in DBA/2 males. The regulatory element binds to the different TTCCGGGC-specific nuclear proteins depending on the methylation of the CpG site. In contrast, the corresponding CpG at position -119 of the C4 gene, which is expressed in both males and females, is demethylated at equal and high levels in both sexes. We therefore propose that the DNA demethylation and methylation-sensitive transcription factors may be a part of the regulatory mechanism for the male-specific expression of the Slp gene.

Animals↗
PubMed1995

Multiple steroid-binding orientations: alteration of regiospecificity of dehydroepiandrosterone 2- and 7-hydroxylase activities of cytochrome P-450 2a-5 by mutation of residue 209.

The mutation of Ala-117 to Val conferred dehydroepiandrosterone (DHEA) hydroxylase activity on cytochrome P-450 2a-4, with the production of both 2 alpha- and 7 alpha-hydroxyDHEA at similar rates. P-450 2a-5 which has Val at position 117, acquired high DHEA hydroxylase activity by mutation of Phe-209. Mutant F209L of P-450 2a-5 exhibited strong regiospecificity at the 2-position of the DHEA molecule with the production of 2 alpha-hydroxy DHEA as the major metabolite. On the other hand, mutant F209V of P-450 2a-5 showed the 7-position to be the major hydroxylation site, 7 beta-hydroxyDHEA and 7 alpha-OHDHEA being produced. Therefore the regiospecificity of DHEA hydroxylase activity of P-450 2a-5 is altered between the 2- and 7-position depending on the amino acid at position 209. Modelling of the DHEA molecule in the pocket of bacterial P-450cam showed that the steroid can be accommodated in at least two orientations for which the 2- or 7- position is near the sixth axial position of the haem. Moreover, these two orientations, which are of similar energy, can be interconverted by a 180 degrees rotation of the steroid molecule around its long axis. These results support the hypothesis that the steroid molecule in the pocket is in dynamic equilibrium with multiple binding orientations and that the equilibrium is apparently determined by a few critical residues including those at positions 117 and 209.

Aryl Hydrocarbon Hydroxylases↗
PubMed1995

A DNA methylation site in the male-specific P450 (Cyp 2d-9) promoter and binding of the heteromeric transcription factor GABP.

The Cyp 2d-9 gene encodes the male-specific steroid 16 alpha-hydroxylase in mouse liver and shares a conserved regulatory element (-100TTCCGGGC-93) with another male-specific Slp promoter. As shown with the Slp promoter (N. Yokomori, R. Moore, and M. Negishi, Proc. Natl. Acad. Sci. USA 92:1302-1306, 1995), the male-preferential demethylation also occurs at CpG/-97 in the Cyp 2d-9 promoter. The transcription factor which specifically binds to the demethylated element has been purified. The peptide sequences reveal that the factor consists of GABP alpha and GABP beta 1 with Ets and Notch motifs, respectively. Both DNase I footprinting and gel shift assays indicate that the bacterially expressed glutathione S-transferase-GABP fusion proteins bind to the regulatory element only when CpG/-97 is demethylated. Moreover, Cyp 2d-9 promoter is trans-activated by coexpression of GABP proteins in HepG2 cells. Given the additional results that CpG/-50 of the female-specific steroid 15 alpha-hydroxylase (Cyp 2a-4) promoter is preferentially demethylated in the females, the sex-specific expressions of the P450 genes correlate very well with DNA demethylation. We also conclude that GABP is a methylation-sensitive transcription factor and is a potential transcription activator of the male-specific Cyp 2d-9 promoter.

Amino Acid Sequence↗