Search PubMed⌕ Search

Biomedical subjects

M Kawata

Publications and source records attributed to M Kawata.

At least 199 records · Page 11Linked to original sources
PubMed1989

Effect of gastric acidity on bioavailability of N,N-dimethylcarbamoylmethylalpha, 2-dimethyl-5H-[1]benzopyrano[2,3-b]pyridine-7-acetate, a new prodrug-type anti-inflammatory agent.

The effect of gastric acidity on the bioavailability of N,N-dimethylcarbamoylmethyl alpha,2-dimethyl-5H-[1]benzopyrano[2,3-b] pyridine-7-acetate (1), a new anti-inflammatory agent, was investigated in gastric acidity-controlled beagle dogs. The dissolution rates of this compound in media of pH 1.2 and 3.0 were greater than those in media of pH 5.0 and 6.8. Reflecting these dissolution characteristics, the peak plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC0-12h) were reduced by shifting the gastric acidity to low levels (more than pH 6) with omeprazole treatment. In designing dosage forms of 1, it is necessary to develop pharmaceutical preparations whose bioavailability is not affected by the gastric acidity.

Animals↗
PubMed1989

[Dissolution properties and bioavailability of ground mixture of N,N-dimethylcarbamoylmethyl alpha, 2-dimethyl-5H-[1]benzopyrano[2,3-b]-pyridine-7-acetate with various pharmaceutical ingredients].

Ground mixture of N,N-dimethylcarbamoylmethyl alpha, 2-dimethyl-5H-[1]benzopyrano-[2,3-b]pyridine-7-acetate (1) with various pharmaceutical ingredients were prepared in order to investigate their dissolution behaviors and bioavailability. Taking into account the weakly basic property of 1, the dissolution rate was determined in the 2nd fluid (pH 6.8) of disintegration test, JP XI. Dissolution rates of the ground mixtures (1 : 1, w/w) of 1 with hydroxypropylcellulose-L (HPC-L), low substituted hydroxypropylcellulose (L-HPC) or lactose respectively, showed a significant increase compared with compound 1 alone. Three kinds of experimental fine granules were prepared; type A: produced from ground mixture of 1, HPC-L, L-HPC and lactose; type B: produced from physical mixture having the same composition as type A; type C: produced 1, L-HPC and lactose. In these fine granules, only type A exhibited pH-independent dissolution profiles. Bioavailability study was carried out in beagle dogs whose gastric acidity was controlled in advance to low levels by administration of omeprazole. The test was conducted in a cross over design. Reflecting their dissolution characteristics, type A granules showed better bioavailability than the others. These results suggest that grinding is useful for the improvement of the dissolution property and bioavailability of 1, a weakly basic compound.

Adjuvants, Pharmaceutic↗
PubMed1988

Phosphorylation by cyclic AMP-dependent protein kinase of a human platelet Mr 22,000 GTP-binding protein (smg p21) having the same putative effector domain as the ras gene products.

We have purified to near homogeneity a Mr 22,000 GTP-binding protein from human platelet membranes and identified it as the smg-21 gene product (smg p21), having the same putative effector domain as the ras gene products, which we have purified to near homogeneity from bovine brain membranes and characterized. This purified human platelet smg p21 was phosphorylated by cyclic AMP-dependent protein kinase. About one mol of phosphate was maximally incorporated into one mol of the protein. Only serine residue was phosphorylated. Both the guanosine 5'-(3-O-thio)-triphosphate (GTP gamma S)-bound and GDP-bound forms were phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affected neither its GTP gamma S-binding nor GTPase activity. Human platelet smg p21 was not phosphorylated by protein kinase C. A Mr 24,000 GTP-binding protein partially purified from human platelet membranes was not phosphorylated by cyclic AMP-dependent protein kinase or protein kinase C.

Animals↗
PubMed1988

Identification of a platelet Mr 22,000 GTP-binding protein as the novel smg-21 gene product having the same putative effector domain as the ras gene products.

We have recently purified a Mr 22,000 GTP-binding protein (G protein) to near homogeneity from human platelet membranes and characterized it (Ohmori, T., Kikuchi, A., Yamamoto, K., Kim, S. and Takai, Y. (1989) J. Biol. Chem. in press). This platelet G protein was present most abundantly among several G proteins in platelets and showed a Mr of about 22,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This platelet G protein showed kinetic and physical properties very similar to those of the novel smg-21 gene product, having the same putative effector domain as the ras gene products, which we have recently purified to near homogeneity from bovine brain membranes and characterized (Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y. and Takai, Y. (1988) J. Biol. Chem. in press). Moreover, the peptide map of the platelet G protein was identical with that of the smg-21 gene product and the partial amino acid sequence of the platelet G protein was identical with that of the smg-21 gene product. These results indicate that this human platelet G protein is the smg-21 gene product.

Amino Acid Sequence↗
PubMed1988

A novel small molecular weight GTP-binding protein with the same putative effector domain as the ras proteins in bovine brain membranes. Purification, determination of primary structure, and characterization.

In the present studies, we have purified a novel small Mr GTP-binding protein, designated as smg p21, to near homogeneity from bovine brain crude membranes, isolated the complementary DNA (cDNA) of this protein from a bovine brain cDNA library, determined the complete nucleotide and deduced amino acid sequences, and characterized the kinetic properties. The cDNA of smg p21 has an open reading frame encoding a protein of 184 amino acids with a calculated Mr of 20,987. The Mr of purified smg p21 is estimated to be about 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Homology search indicates that smg p21 is a novel protein with the consensus amino acid sequences for GTP/GDP-binding and GTPase domains but shares about 55% amino acid sequence homology with the human c-Ha-ras protein. Moreover, smg p21 has the same putative effector domain as the Ha-, Ki-, and N-ras proteins at the same position and the same consensus C-terminal sequence as in these ras proteins. Consistent with these structural properties, smg p21 binds specifically [35S] guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), GTP, and GDP with a Kd value for GTP gamma S of about 40 nM. smg p21 binds about 0.7 mol of GTP gamma S/mol of protein. [35S]GTP gamma S-binding to smg p21 is inhibited by pretreatment with N-ethylmaleimide.smg p21 hydrolyzes GTP to liberate Pi with a turnover number of about 0.007 min-1. These kinetic properties of smg p21 are similar to those of the c-ras proteins. These results suggest that smg p21 is a novel GTP-binding protein exerting action(s) similar or antagonistic to that (those) of the ras proteins.

Amino Acid Sequence↗
PubMed1988

Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes.

In the present studies, we attempted to purify the native molecular forms of the c-ras proteins (c-ras p21s) from bovine brain crude membranes and separated at least three GTP-binding proteins (G proteins) cross-reactive with the antibody recognizing all of Ha-, Ki-, and N-ras p21s. Among them, one G protein with a Mr of about 21,000 was highly purified and characterized. The Mr 21,000 G protein bound maximally about 0.6 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 30 nM. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by pretreatment with N-ethylmaleimide. Mr 21,000 G protein hydrolyzed GTP to liberate Pi with a turnover number of about 0.01 min-1. Mr 21,000 G protein was not copurified with the beta gamma subunits of the G proteins regulatory for adenylate cyclase. Mr 21,000 G protein was not recognized by the antibody against the ADP-ribosylation factor for Gs. The peptide map of Mr 21,000 G protein was different from those of the G proteins with Mr values of 25,000 and 20,000, designated as smg p25A and rho p20, respectively, which we have recently purified from bovine brain crude membranes. The partial amino acid sequence of Mr 21,000 G protein was identical with that of human c-Ki-ras 2B p21. These results indicate that Mr 21,000 G protein is bovine brain c-Ki-ras 2B p21 and that c-Ki-ras 2B p21 is present in bovine brain membranes.

Amino Acid Sequence↗
PubMed1988

Glycopeptides from murine teratocarcinoma cells: structure of the determinants recognised by Griffonia simplicifolia agglutinin I and by sera from patients with ovarian germ cell tumors.

High-molecular-weight glycopeptides synthesised by teratocarcinoma OTT6050 bear the binding site for Griffonia simplicifolia agglutinin I and are recognised by antibodies in the sera of patients with ovarian germ cell tumors. Digestion of the glycopeptides with endo-beta-D-galactosidase C abolished the lectin binding activity and the antigenic activity. Since the product of the enzymic digestion is alpha-D-Gal-(1----3)-D-Gal, it is concluded that the disaccharide structure is involved in the lectin binding site and the antigenic site.

Animals↗
PubMed1988

In situ hybridization analysis of osmotic stimulus-induced changes in ribosomal RNA in rat supraoptic nucleus.

A quantitative in situ hybridization analysis was used to investigate changes in levels of ribosomal RNA (rRNA) in neurons of the supraoptic nucleus (SON) of rats stimulated osmotically by giving 2% NaCl as drinking solution for 0 (control rats), 1, 4, and 14 days. The quantitation was autoradiographically accomplished by in situ hybridization with a nick-translated tritiated ribosomal DNA probe and with the use of computer-based image analysis system. The mean number of grains per neuron in the ventral SON was significantly increased: 1.8-fold for 1 day, 2.9-fold for 4 days, and 1.7-fold for 14 days of salt loading, whereas the mean number of grains per neuron in the dorsal SON was increased 1.3-fold for 1 day, 2.5-fold for 4 days, and 1.7-fold for 14 days. Kolmogorov-Smirnov analysis of frequency histograms of grains per neuron indicated that the amount of rRNA in neurons in the ventral and dorsal SON was significantly increased by osmotic stimulation. These increases were accompanied by increases in cell size. The subcellular location of hybridizable rRNA in magnocellular neurons was altered by osmotic stimulation. Following 1-14 days of salt-drinking, rRNAs appeared to be more unevenly distributed throughout the cytoplasm. These findings are consistent with the notion that hyperosmotic stimulation has a substantial effect on the expression of rRNA genes in neurons of both the ventral and dorsal SON.

Animals↗
PubMed1988

Nucleotide sequence of the thymidylate synthase B and dihydrofolate reductase genes contained in one Bacillus subtilis operon.

The nucleotide sequence of the thymidylate synthase B (thyB) and dihydrofolate reductase (dfrA) gene regions from wild-type and trimethoprim-resistant (TpR) mutant strains of Bacillus subtilis 168 was determined. The sequenced region contains two open reading frames, ORF1 and ORF2, which correspond to thyB and dfrA, respectively, and overlap by one nucleotide. The thyB-dfrA genes encode 267 and 168 amino acid polypeptides, respectively, and are present in the order of thyB - dfrA in 5'----3' orientation. This gene order differs from those which have been found in other organisms so far. S1 mapping analysis indicated that both genes were transcribed from a single promoter located upstream from the thyB gene. Thus, the genes belong to an operon. A nucleotide substitution from 'A' in the wild type to 'C' in the TpR mutant was located in the dfrA gene region, with predicted conversion of isoleucine-95 (wild type) to leucine-95 (mutant) in dihydrofolate reductase (DHFR). It is suggested that the affinity between DHFR and Tp is reduced by this alteration.

Amino Acid Sequence↗
PubMed1988

Atrial natriuretic polypeptide depresses angiotensin II induced excitation of neurons in the rat subfornical organ in vitro.

The effects of atrial natriuretic polypeptide (ANP) on the extracellularly recorded activity of neurons in the subfornical organ (SFO) were investigated in rat brain slice preparations by adding the peptide to the perfusion medium. Eight (14%) of 56 SFO neurons were inhibited and none of the cells were excited by ANP at 10(-7) M. Of 42 SFO neurons tested with both ANP and angiotensin II (AII) at 10(-7) M, 4 (10%) cells were inhibited by ANP and excited by AII, the remainder responded to either one or other of the peptides but not both or were unresponsive. In 13 (87%) of 15 SFO neurons, ANP at 10(-7) M depressed by more than 40% the excitation induced by AII at 10(-7) M, while ANP did not always depress the excitation induced by raising the extracellular potassium concentrations in 6 SFO cells tested. We conclude that ANP strongly depresses AII-induced excitation in all SFO neurons, although it has very weak inhibitory effects on spontaneous activity, thus ANP may act as a neuromodulator in the SFO.

Angiotensin II↗
PubMed1988

Purification and characterization of a novel GTP-binding protein with a molecular weight of 24,000 from bovine brain membranes.

At least six GTP-binding proteins (G proteins) with Mr values between 20,000 and 25,000 were extracted from the crude membrane fraction of bovine brain by sodium cholate and purified by successive column chromatographies of Ultrogel AcA-44, phenyl-Sepharose CL-4B, hydroxyapatite, and Mono Q HR5/5. One G protein with a Mr of about 24,000 (24K G) was purified to near homogeneity and characterized. 24K G bound maximally about 0.7 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 46 nM. [35S]GTP gamma S binding to this protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. 24K G hydrolyzed GTP to liberate Pi with a rate of about 40 mmol of Pi/mol of protein/min. 24K G did not associate with the beta gamma subunit of Go. 24K G was distinguishable on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the ras protein (ras p21) and ADP-ribosylation factor, the G protein with a Mr of about 21,000 serving as a cofactor for the cholera toxin-dependent ADP-ribosylation of Gs. 24K G was not recognized by the antibody against ras p21, ADP-ribosylation factor, the alpha subunit each of Gs, Gi, and Go, or tubulin. These results indicate that 24K G is a novel G protein and suggest that there are multiple forms of G proteins with small Mr values as is the case with the G proteins which have the alpha subunits with Mr values of about 40,000.

Animals↗
PubMed1988

Binding of ras p21 to bands 4.2 and 6 of human erythrocyte membranes.

The direct binding protein(s) of ras p21 was (were) investigated in inside-out vesicles of human erythrocyte ghosts using the pure v-Kirsten (Ki)-ras p21 synthesized in E. coli. The bound ras p21 was detected immunochemically using an anti-v-Ki-ras p21 monoclonal antibody, ras p21 bound to vesicles. Prior digestion of the vesicles with trypsin reduced this binding significantly. When ras p21 was laid over vesicle proteins immobilized on a nitrocellulose sheet by transfer from the gel of SDS-polyacrylamide gel electrophoresis, ras p21 bound to bands 4.2 and 6. ras p21 binding to these proteins was reduced by prior incubation of ras p21 with the purified band 4.2 or 6 protein. These results indicate that v-Ki-ras p21 can bind directly to bands 4.2 and 6 of human erythrocyte membranes as far as tested in an in vitro cell-free system.

Blood Proteins↗
PubMed1988

A method for parallel determination of choline acetyltransferase and muscarinic cholinergic receptors: application in aged-rat brain.

We have devised a method for the parallel determination of choline acetyltransferase (CAT) and muscarinic cholinergic receptor (mCh-R) in the same brain tissue. The method for CAT activity determination is more rapid, simplified, stable, and economical than the usual Fonnum's method. With our method, age-associated changes in CAT activity and mCh-R levels were examined. Although CAT activity hardly changed with age except in a few areas, mCh-R binding of aged-rats was markedly reduced in all areas. These results suggest that the change in mCh-R represents an age-associated biochemical change in the brain and that determination of CAT activity is not sufficient for the study of age-associated changes in the brain cholinergic system.

Aging↗
PubMed1988

In situ hybridization histochemistry with oxytocin synthetic oligonucleotide: strategy for making the probe and its application.

A 25 mer synthetic oligonucleotide, complementary to a specific region of the oxytocin-neurophysin preprohormone messenger RNA (mRNA), was designed for its application to in situ hybridization histochemistry. The probe was 3'-end labeled with [3H] deoxycytidine triphosphate (dCTP) by using terminal deoxynucleotidyl transferase, and hybridization of the labeled probe to the mRNA in the rat hypothalamus was visualized autoradiographically. Hybridization products were specifically localized in the dorsal part of the supraoptic nucleus and the peripheral part of the paraventricular nucleus. Not only is the oligomer designed useful for distinguishing oxytocin from vasopressin gene expressing neurons, but also it is proving useful for studies of estrogen-progesterone effects on neurons in the paraventricular nucleus. Thus, these results indicate that in situ hybridization histochemistry with synthetic oligonucleotide can be a valuable approach to measuring gene expression in hypothalamic neuroendocrine cells.

Animals↗
PubMed1988

Poly-N-acetyllactosamines synthesized by cultured Ehrlich carcinoma cells: application of endo-beta-galactosidase C for analysis of the terminal structure.

Poly-N-acetyllactosamines were prepared from Ehrlich carcinoma cells cultured in the presence of [14C]galactose. Methylation analysis indicated that 31% of the galactose was in the non-reducing end. Of it, 77% was cleaved by alpha-galactosidase, and 56% was released as a disaccharide by endo-beta-galactosidase C. Methylation analysis confirmed that the released disaccharide was mostly Gal alpha 1----3Gal. Therefore, Gal alpha 1----3Gal structure, not Gal alpha 1----3(Gal alpha 1----6)Gal structure, was the major alpha-galactosyl structure in the poly-N-acetyllactosamines synthesized. Furthermore, alpha-galactosidase digestion did not change the content of disubstituted galactosyl residues. Thus, Gal alpha 1----3(Gal alpha 1----6)Gal structure, which was suggested to be the sole non-reducing terminal structure of poly-N-acetyllactosamines of Ehrlich carcinoma cells, was not detected in significant amounts under the present experimental conditions.

Animals↗
PubMed1988

The capacity for differentiation and production of human chorionic gonadotropin and alpha-fetoprotein in human embryonal carcinoma cell lines.

The capacity for differentiation of five human embryonal carcinoma (EC) cell lines was studied by examination of the morphology of xenograft tumors in nude mice. In addition to morphology, differentiation into extraembyronic elements was studied by examining the evidence of human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP) production. The EC cell lines can be divided into two groups: pluripotent (NEC 14 and Tera 2) and nullipotent (NEC 8, NEC 15, and ITO-II) cell lines. In nude mice, the pluripotent NEC 14 and Tera 2 cells produced EC mixed with various somatic elements such as glandular, neuronal, and cartilaginous tissue. However, typical extraembryonic elements, such as choriocarcinoma and yolk sac elements, were not usually observed. Production of hCG and/or AFP was identified in the tumors by both radioimmunoassay and the immunoperoxidase reaction. These results indicate that some human EC cell lines retain the capacity for differentiation into somatic and extraembryonic elements and that hCG and/or AFP appears to be produced by transitional types of cells that still retain the morphology of EC cells.

Animals↗
PubMed1988

Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases.

We have isolated a DNA fragment from Bacillus subtilis 168 which, when present in a high-copy plasmid, inhibited production of extracellular alkaline and neutral proteases. The gene responsible for this activity was referred to as iep. The open reading frame of iep was found to be incomplete in the cloned DNA fragment. When the intact iep gene was reconstructed after the missing part of the iep gene had been cloned, it showed an enhancing effect on the production of the extracellular proteases. The open reading frame encodes a polypeptide of 229 amino acids with a molecular weight of ca. 25,866. Deletion of two amino acids from the N-terminal half of the putative iep protein resulted in dual effects, i.e., a decrease in the inhibitory activity shown by the incomplete iep gene and a slight increase in the enhancing activity shown by the complete iep gene. These results show that the iep gene product is a bifunctional protein, containing inhibitory and enhancing activities for the exoprotease production in the N-terminal and C-terminal regions, respectively. It was found by genetic and functional analyses that iep lies very close to sacU.

Amino Acid Sequence↗