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K Wada

Publications and source records attributed to K Wada.

At least 19 recordsLinked to original sources
PubMed1992

N-acetylaspartylglutamate acts as an agonist upon homomeric NMDA receptor (NMDAR1) expressed in Xenopus oocytes.

The electrophysiological effects of N-acetylaspartylglutamate (NAAG), an endogenous peptide restrictively distributed in the central nervous system, were studied using Xenopus oocytes injected with RNAs transcribed from cloned glutamate receptor cDNAs. NAAG induced an inward current, dose dependently, in oocytes injected with RNA for an N-methyl-D-aspartate receptor subunit (NMDAR1). In contrast, the oocytes injected with RNAs for AMPA-selective glutamate receptors (GluR1, GluR3, GluR1+GluR2 and GluR2+GluR3) scarcely responded to NAAG, and the oocytes injected with RNA for kainate receptor (GluR6) did not respond to NAAG. The half-maximal response (ED50) value of NAAG on expressed NMDAR1 was 185 microM, which shows that NAAG is about 115-times less potent than L-glutamate (Glu), the ED50 of which value was 1.6 microM. The maximal current amplitude induced by NAAG was about 70% of that by Glu. NAAG-induced current in NMDAR1-injected oocytes was potentiated by glycine, dose-dependently antagonized by DL-2-amino-5-phosphonovaleric acid, and blocked by magnesium ions in a voltage-dependent fashion. These results suggest that NAAG is one of the endogenous agonists selective for NMDAR1.

2-Amino-5-phosphonovalerate↗
PubMed1992

Comparison of the inhibitory effects of the TXA2 receptor antagonist, vapiprost, and other antiplatelet drugs on arterial thrombosis in rats: possible role of TXA2.

The antithrombotic effect of the thromboxane A2 receptor antagonist, vapiprost, was compared with those of other antiplatelet drugs using an arterial thrombosis model which utilized photochemical reaction in the rat femoral artery. Vapiprost prolonged the time required to occlude the artery with thrombus and inhibited collagen-induced rat platelet aggregation in whole blood ex vivo, in a dose-dependent manner. The potency ranking of antithrombotic effect was vapiprost > ketanserin (serotonin 5-HT2 receptor antagonist) >> ticlopidine (inhibitor of ADP-induced platelet aggregation) = dipyridamole (adenosine uptake inhibitor) > aspirin (cyclooxygenase inhibitor). On the other hand, the ranking of antiplatelet effect was ticlopidine > or = vapiprost > or = aspirin. Ketanserin and dipyridamole were ineffective. Relative to their antiplatelet effect, vapiprost and ketanserin had powerful antithrombotic effects. It is possible that the potent antithrombotic effects of vapiprost and ketanserin in vivo reflect the ability of these drugs to inhibit mediator-induced vascular contractions in addition to platelet aggregation. The results of the present study also suggest that TXA2 may play an important role in thrombogenesis in rats.

Animals↗
PubMed1992

Expression of two types of neurofibromatosis type 1 gene transcripts in gastric cancers and comparison of GAP activities.

To understand the molecular mechanism of gastric tumorigenesis, the status of neurofibromatosis type 1 (NF1) gene was analyzed in human gastric cancer cell lines. Although the sequencing of the GTPase activating protein (GAP)-related region of NF1 (NF1-GRD) revealed no apparent mutation, the NF1-GRD transcript (type I) and that containing an additional 63 bp insert in the center of NF1-GRD (type II) were equally expressed in most gastric cancer cells. By contrast, type II was predominantly expressed in normal stomach mucosa. When these two types of NF1-GRD were bacterially expressed and their GAP activities were tested, both types of NF1-GRD similarly stimulated ras GTPase activity. However, arachidonic acid inhibited GAP activities of two types of NF1-GRD to different extents. These results suggest that the increased expression of type I NF1 protein may modulate ras-related signal transduction and it may be related to the control of the gastric cellular proliferation.

Arachidonic Acid↗
PubMed1992

Arterial thrombosis model with photochemical reaction in guinea-pig and its property.

We have already developed an arterial thrombosis model in the rat femoral artery which utilized photochemical reaction between systemically injected rose bengal and transillumination of a green light with 540 nm wave length from the outside of the vessel. In the present study, we applied this model to guinea-pigs in order to produce a more suitable thrombus model for evaluation of antithrombotic drugs which act on the prostaglandin cascade. In the guinea-pigs, the irradiated femoral artery was completely occluded in 7 min after the injection of rose bengal (10 mg/kg) in a similar manner to the rats. The processes of primary endothelial injury and the subsequent formation of thrombus during this manipulation were observed by the electron microscopy. Pretreatment with aspirin and Y-20811, a thromboxane synthetase inhibitor, significantly prolonged the time required for occlusion in the guinea-pigs, while these drugs were ineffective in the rats. The antithrombotic effect of vapiprost, a thromboxane A2 receptor antagonist, was more pronounced in the guinea-pigs than the rats. In conclusion, this model in guinea-pigs is more suitable for evaluating antithrombotic drugs, particularly, the action of which is exerted involving the prostaglandin cascade.

Animals↗
PubMed1992

p53 gene mutations in human gastric cancer: wild-type p53 but not mutant p53 suppresses growth of human gastric cancer cells.

To further investigate the role of p53 gene inactivation in gastric tumorigenesis, the mutational status of the p53 gene in primary human gastric cancer samples was examined. Reverse transcriptase polymerase chain reaction and subsequent direct sequencing of the p53 gene from gastric cancer samples revealed frequent point mutations of the p53 gene: some of these coincided with those previously identified in gastric cancer cell lines. In addition, both allelic deletion analysis using pYNZ 22 and polymerase chain reaction-restriction fragment length polymorphism analysis demonstrated an allelic deletion of the p53 gene in cancer tissue which contained a point mutation of the p53 gene in the remaining allele. Transfection of the wild-type or mutant p53 genes into gastric cancer cells showed that the wild-type but none of the mutated p53 genes suppressed the colony formation of gastric cancer cells. Furthermore, the incorporation of thymidine into DNA was reduced in cancer cells expressing the wild-type p53 gene. The glutathione S-transferase-wild type p53 fusion protein bound to simian virus 40 large T antigen in COS-1 cell lysate. None of the p53 fusion proteins containing mutations at codons 143, 175, 248, or 273 bound to simian virus 40 large T antigen. By contrast, two different mutant p53 fusion proteins containing mutations specifically observed in gastric cancer bound to simian virus 40 large T antigen. These results indicate that inactivation of the p53 gene through mutations and the allelic deletion may play an important role in gastric tumorigenesis. These mutations may cause a conformational change in the p53 protein resulting in the loss of the suppression by p53 of the growth of gastric cells, partly through disruption of the association of p53 protein with a cellular component.

Antigens, Viral, Tumor↗
PubMed1992

Protein degradation by the phosphoinositide-specific phospholipase C-alpha family from rat liver endoplasmic reticulum.

A 60-kDa protein homologous to phosphoinositide-specific phospholipase C-alpha was purified to apparent homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the rough endoplasmic reticulum of rat liver through three sequential chromatographies on DEAE Toyopearl 650, AF-heparin Toyopearl 650M, and TSK gel G3000SW. The purified protein was monomeric, with an M(r) of 60,000. Eight types of protein were further separated from the 60-kDa protein and named ER60A-ER60H according to the order of their elution from a TSK gel DEAE-5PW column. They were essentially identical in terms of immunochemical properties and the NH2-terminal amino acid sequence. The partial amino acid sequence of ER60F showed homology to that of phosphoinositide-specific phospholipase C-alpha. ER60A-ER60H showed no phosphoinositide-specific phospholipase C activity. However, ER60A-ER60H catalyzed cleavage of themselves and the endoplasmic reticulum proteins protein disulfide-isomerase and calreticulin. Proteolytic degradation was inhibited by p-chloromercuribenzoate. These results indicate that ER60A-ER60H comprise a group of endoplasmic reticulum resident proteins and show thiol group-related proteolytic activity.

Amino Acid Sequence↗
PubMed1992

A possible target protein for smg-25A/rab3A small GTP-binding protein.

The smg-25A/rab3A protein (smg p25A) is a small GTP-binding protein implicated in intracellular vesicle traffic, particularly in neurotransmitter release from the presynapse. In the present study, we attempted to identify a target protein in bovine brain crude membranes that might be interacted with the GTP-bound form of smg p25A. When the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of radioiodinated smg p25A and the crude membrane fraction of bovine brain were incubated with a cross-linker, disuccinimidyl suberate, and the sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography, one radioactive band with a M(r) of about 110,000 was detected. This radioactive band appeared to be composed of radioiodinated smg p25A and a molecule with a M(r) of about 86,000. This molecule, tentatively termed here smg p25A target, was extracted from the membranes by a detergent and highly purified by column chromatographies and sucrose density gradient ultracentrifugation. The purified smg p25A target was sensitive to heat boiling and tryptic digestion, indicating that smg p25A target is a protein molecule. The M(r) of the purified smg p25A target was estimated to be about 85,000-86,000 from SDS-PAGE and to be about 100,000 from the S value. The cross-linking of radioiodinated smg p25A with the purified smg p25A target was inhibited by the GTP gamma S-bound form of non-radioactive smg p25A with an IC50 of about 8 nM. The GDP-bound form of smg p25A was much less effective. Other small GTP-binding proteins, such as c-Ki-ras p21, rhoA p21, smg p21B, and rab11 p24 were ineffective. These results indicate that a protein with a M(r) of about 85,000-100,000 is a target for smg p25A.

Animals↗
PubMed1992

Biliary cystadenocarcinoma of the liver. A clinicopathologic and histochemical evaluation of nine cases.

Nine cases of biliary cystadenocarcinoma of the liver were studied, with emphasis on its clinicopathologic features, mucin profiles, and immunohistochemical characteristics. In general, the cystic tumors had protrusions that consisted of well-differentiated papillary adenocarcinoma cells with or without benign-appearing epithelial elements. In invading or metastatic foci, the carcinoma cells tended to show distinctive anaplastic changes. Tumor growth was confined to the cystic lesions in five cases (noninvasive type), whereas in four cases it extended to the hepatic parenchyma or neighboring organs (invasive type). There was a considerable difference between the two groups in terms of prognosis. In fact, the patients included in the group with the noninvasive type had no sign of tumor recurrence after an appropriate surgical procedure. With mucin histochemical and immunohistochemical approaches, positive reactions with carcinoembryonic antigen, tissue polypeptide antigen, carbohydrate 19-9, and Dupan-2 and the predominance of sialomucin were observed in most cases of biliary cystadenocarcinoma, indicating a similar cellular nature of cholangiocarcinoma.

Adenoma, Bile Duct↗
PubMed1992

Effect of aldose reductase inhibitor on the inhibition of platelet aggregation induced by diabetic rat plasma.

Plasma isolated from streptozotocin-induced diabetic rats inhibited platelet aggregation. Treatment of diabetic rats with an aldose reductase inhibitor, ONO-2235, which did not improve hyperglycemia, prevented the antiaggregating activity of plasma as did insulin treatment. Both treatments reduced the elevated sorbitol concentrations in erythrocytes. Although the factor(s) accounting for the antiaggregatory effect of diabetic plasma has not yet been characterized, it is possible that the accumulation of sorbitol in erythrocytes is related to the generation of the factor(s).

Aldehyde Reductase↗
PubMed1992

Isolation and characterization of a cDNA encoding Drosophila transcription factor TFIIB.

A Drosophila cDNA encoding a human transcription factor TFIIB homologue was isolated by PCR methods. The deduced amino acid sequence indicates 85% sequence similarity with human TFIIB, and the corresponding cDNA product expressed in Escherichia coli is interchangeable with human TFIIB for both basal and GAL4-VP16-induced transcription. Structural motifs including the direct repeats, basic repeats, and sigma sequence similarities are well conserved among Drosophila, human, and Xenopus TFIIB. However, the N-terminal region of each direct repeat is less conserved among the three species, suggesting the presence of two structural subdomains in the direct repeat. Moreover, the amino acid changes in the N-terminal subdomain produce altered positions of the conserved amino acids between the direct repeats. An overall similarity in general structural features between TFIIB and TFIID tau (the TATA-binding subunit of TFIID) was previously noted. However, in contrast to the sequence divergence reported for the N-terminal domains of TFIID tau from different species, the N-terminal sequence of TFIIB was highly conserved among the species. This suggests that TFIIB has a more rigid structure, consistent with its function as a "bridging" protein between TFIID and RNA polymerase II. Further implications of the TFIIB structure are discussed.

Amino Acid Sequence↗
PubMed1992

Structural characterization and expression of a brain specific gene encoding chick kainate binding protein.

The gene encoding chick kainate-binding protein (c-KBP), a member of the non-NMDA ionotropic glutamate receptor family has been isolated and characterized. The c-KBP gene is at least 13 kilobases long and contains 11 exons interrupted by 10 introns. Primer extension and RNase protection studies identified a major transcription initiation site located 117 bases upstream from the initiation methionine codon ATG. Consensus TATA and CCAAT sequences were detected in the putative promoter region. The structure of the c-KBP gene is strikingly different from that of other members of neurotransmitter-gated ion-channels (cloned at present) although the topology of c-KBP consists of four membrane-spanning domains, a structural characteristic of ionotropic receptor subunits. The c-KBP gene was found to be expressed at high levels in chick cerebellar Bergmann glia and at extremely low levels in the forebrain. The limited expression of the c-KBP gene raises important questions concerning the mechanisms governing the regulation of c-KBP gene transcription.

Animals↗
PubMed1992

Missense mutations and a deletion of the p53 gene in human gastric cancer.

To investigate the molecular pathogenesis of human gastric cancers the p53 gene, a suppressor oncogene, was analyzed in 12 human gastric cell lines. Southern blot and Northern blot analysis revealed a total deletion of p53 gene in KATO-III cells but no major abnormality of p53 gene in other cell lines. By the use of the reverse-transcriptase polymerase chain reaction and direct sequencing 7 cell lines showed point mutations of p53 gene resulting in amino-acid substitutions. Most of them were rare mutations which had not been observed in other types of cancers. One of these mutations was also detected through the use of PCR and oligomer-specific hybridization. Six out of 7 cell lines with mutations of p53 gene also lost one allele of chromosome 17p. Immunoblotting of cell lysates with an antibody specific to p53 demonstrated the absence of p53 protein in KATO-III cell. By contrast, the high levels of the p53 protein were observed in 5 cell lines all of which contained mutations of p53 gene. These results further suggest that the inactivation of p53 gene may play an important role in the transformation of gastric cells to the malignant phenotype. KATO-III cells might be a good model for studying the significance of the loss of p53 gene in cellular transformation.

Alleles↗
PubMed1992

Immunochemical characterization of the non-NMDA glutamate receptor using subunit-specific antibodies. Evidence for a hetero-oligomeric structure in rat brain.

Antibodies were made to synthetic peptides corresponding to sequences specific to the glutamate receptor (GluR) subunits, GluR1-4. The specificity of the antibodies was established by Western blotting using membranes of simian kidney cells (COS-7) transfected with GluR subunit DNA. Four antibodies were found to be selective for each of the four GluR subunits, and a fifth antibody recognized both GluR2 and 3. All five antibodies immunoadsorbed Triton X-100-solubilized rat brain [3H]AMPA binding activity and labeled an Mr = 108,000 band in samples of rat brain. The structure of the Triton X-100-solubilized GluR was studied using subunit-specific antibodies covalently attached to protein A-agarose and analyzing GluR subunits bound to the antibodies by Western blotting. Each of the four subunit-specific antibodies immunoadsorbed its respective GluR subunit as well as the other three forms of GluR, showing that the detergent solubilized GluR exists as hetero-oligomers composed of two or more of the four subunits. Evidence supporting a similar structure for membrane bound GluR was obtained using synaptic membranes chemically cross-linked with dithiobis(succinimidylpropionate). GluR was immunoaffinity-purified using the GluR2 and 3-selective antibody. This antibody, covalently attached to protein A-agarose, adsorbed 55% of [3H]AMPA binding activity, and after elution with 1 M KSCN, 22-37% of the binding activity was recovered. Analysis of the purified product showed a major immunoreactive band at Mr = 108,000, and silver staining identified the same major band and no additional polypeptides. The GluR receptor complex, therefore, appears to be made up exclusively of GluR1-4. In the purified GluR preparation, in addition to the Mr = 108,000 band, three higher molecular weight immunoreactive components were also detected. These bands migrated at Mr = 325,000, 470,000, and 590,000. Similar sized proteins were seen in the cross-linked synaptic membrane sample, with the Mr = 590,000 component being substantially enriched after cross-linking. The Mr = 590,000 band is the largest component detected, and it has a size consistent with its being a pentamer of the Mr = 108,000 protein.

Amino Acid Sequence↗
PubMed1992

Differential expression of two distinct forms of mRNA encoding members of a dipeptidyl aminopeptidase family.

We have identified two cDNAs encoding dipeptidyl aminopeptidase-like proteins (DPPXs) in both bovine and rat brains that have different N-terminal cytoplasmic domains but share an identical transmembrane domain and a long C-terminal extracellular domain. In both species, one of the cDNAs encodes a protein (designated DPPX-S) of 803 amino acid residues with a short cytoplasmic domain of 32 amino acids, and the other cDNA encodes a protein (designated DPPX-L) with a longer cytoplasmic domain--the bovine cDNA encodes 92 amino acids and the rat cDNA encodes 88 amino acids. The membrane topology of DPPX-S and -L is similar to that of other transmembrane peptidases, and DPPX-S share approximately 30% identity and 50% similarity with reported yeast and rat liver dipeptidyl aminopeptidase amino acid sequences, suggesting that DPPX is a member of the dipeptidyl aminopeptidase family. DPPX-S mRNA is expressed in brain and some peripheral tissues including kidney, ovary, and testis; in contrast, DPPX-L mRNA is expressed almost exclusively in brain. No transcripts for either form are found in heart, liver, or spleen. In situ hybridization studies show that the two transcripts have different distributions in the brain. DPPX-L mRNA is expressed in limited regions of brain with the highest level of expression in the medial habenula. More widespread expression is seen for DPPX-S mRNA. The differential distribution of mRNAs for the DPPX-S and -L suggests that these proteins are involved in the metabolism of certain localized peptides and that the cytoplasmic domain may play a key role in determining the physiological specificity of DPPX.

Amino Acid Sequence↗