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Biomedical subjects

K Wada

Publications and source records attributed to K Wada.

At least 505 records · Page 28Linked to original sources
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↗
PubMed1992

Circulating thrombomodulin as a novel endothelial cell marker: comparison of its behavior with von Willebrand factor and tissue-type plasminogen activator.

Circulating thrombomodulin is a novel endothelial cell marker, which may reflect the endothelial injury. Plasma levels of thrombomodulin were quantitated by an enzyme-linked immunosorbent assay (ELISA) in patients with hematological malignancies, liver disease, diabetes mellitus, collagen disease, thrombotic disease, and disseminated intravascular coagulation (DIC), and the thrombomodulin values were compared with those of von Willebrand factor antigen (vWf:Ag) and tissue-type plasminogen activator (t-PA) which are released from stimulated or damaged endothelial cells. The mean plasma concentrations of thrombomodulin in these disease states were elevated as compared with healthy subjects. A relatively high mean thrombomodulin level was observed in DIC, liver disease, and collagen disease. Abnormally high thrombomodulin values (greater than normal mean value + 3 SD) were found in 32.3% of patients with hematological malignancies, 57.7% of patients with liver disease, 39.3% of patients with diabetes mellitus, 30.0% of patients with collagen disease, 23.1% of patients with thrombotic disease, and 69.0% of patients with DIC. Plasma concentrations of both vWf:Ag and t-PA were also elevated in these patients. On the whole, the plasma thrombomodulin concentration was positively correlated with vWf:Ag (r = 0.441, P less than 0.001) and t-PA (r = 0.398, P less than 0.001). These findings indicate that the elevation of plasma thrombomodulin is frequently seen in a variety of diseases and circulating thrombomodulin is possibly useful for evaluating the endothelial damage in selected disease states.

Biomarkers↗
PubMed1992

A synthetic analogue for the active site of plant-type ferredoxin: two different coordination isomers by a four-cys-containing [20]-peptide.

The (Fe2S2)2+ complex of an artificial 20-peptide ligand, Ac-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gly-Pro-Leu-Leu-T hr-Cys- Val-NH2, containing an invariant Cys-A-B-C-D-Cys-X-Y-Cys (A, B, C, D, X, Y = amino acid residues) fragment of plant-type ferredoxins was synthesized by a ligand exchange method with [Fe2S2(S-t-Bu)4]2-. 1H-nmr spectroscopic and electrochemical data of the complex indicate the presence of two coordination isomers. One of them having a Cys-X-Y-Cys bridging coordination to the two Fe(III) ions, has the (Fe2S2)2+ core environment similar to those of the denatured plant-type ferredoxins and exhibits a positive shifted redox potential at -0.64 V vs saturated colonel electrode (SCE) in N,N-dimethylformamide (DMF). Another isomer with the Cys-A-B-C-D-Cys bridging coordination shows a negative redox potential at -0.96 V vs SCE in DMF.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence of atpB, rbcL, trnR, dedB and psaI chloroplast genes from a fern Angiopteris lygodiifolia: a possible emergence of Spermatophyta lineage before the separation of Bryophyta and Pteridophyta.

To elucidate the evolutionary relationship between the Spermatophyta, Pteridophyta and Bryophyta, we cloned a fragment of chloroplast DNA from the fern Angiopteris lygodiifolia (Pteridophyta) and determined its nucleotide sequence. The fragment contained the atpB, rbcL, trnR-CCG, dedB and psaI genes. Comparisons of the deduced amino acid and nucleotide sequences of these genes from the three plant groups indicate that Angiopteris sequences are more closely related to those of Bryophyta species (85% identity on average) than to those of seed plants (76% identity on average), supporting a hypothesis that the Bryophyta and Pteridophyta diverged more recently from one another than their common progenitor diverged from that of the Spermatophyta.

Base Composition↗
PubMed1992

Effects of antiulcer drugs on phosphatidylcholine synthesis in isolated guinea pig gastric glands.

To better understand phosphatidylcholine synthesis in the stomach, we isolated guinea pig gastric glands and examined their [3H]choline incorporation into phosphatidylcholine in response to either antiulcer drugs such as geranylgeranylacetone (GGA) and H2-receptor antagonists or agents that cause phosphatidylcholine synthesis in other tissues. [3H]Choline incorporation was stimulated by GGA, palmitate, and 12-O-tetradecanoylphorbol-13-acetate (TPA). Dibutyryl cyclic-AMP had no effect. By contrast with GGA, famotidine, ranitidine, and cimetidine equipotently inhibited [3H]choline incorporation into phosphatidylcholine. GGA, palmitate, and TPA increased phosphatidyl-[3H]choline and decreased phosphoryl-[3H]choline as compared with control in tissues that had been pulsed with [3H]choline. On the other hand, no more decrease in [3H]choline incorporation at chase periods was observed in pulse-labeled glands in response to each H2-receptor antagonist. The particulate fraction of glands that had been incubated with GGA or palmitate had more CTP-phosphocholine cytidylyltransferase activity than that of glands incubated without agents. A decrease in choline kinase activity was not observed in the cytosolic fraction of glands that had been incubated with cimetidine. These results suggest that GGA and palmitate stimulate phosphatidylcholine synthesis by activating cytidylyltransferase, and H2-receptor antagonists may affect phosphatidylcholine synthesis by inhibiting choline uptake in the gastric glands.

Analysis of Variance↗
PubMed1992

M3 muscarinic receptors mediate pepsinogen secretion via polyphosphoinositide hydrolysis in guinea pig gastric chief cells.

The muscarinic receptor system involved in pepsinogen secretion from isolated guinea pig gastric chief cells was investigated by evaluating the effect of muscarinic receptor antagonists on carbamylcholine (CCh)-stimulated chief cell responses. CCh stimulated the hydrolysis of polyphosphoinositide in chief cells at the same concentrations as it stimulated pepsinogen secretion. Each of five different muscarinic receptor antagonists, atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), AF-DX116 and scopolamine, inhibited both pepsinogen secretion and inositol phosphate accumulation stimulated by graded concentrations of CCh. The pA2 values of the antagonists calculated from data on inositol phosphate accumulation and pepsinogen secretion (atropine = scopolamine = 4-DAMP greater than pirenzepine greater than AF-DX116) suggest that the muscarinic acetylcholine receptor in gastric chief cells is the M3 subtype. On the other hand, CCh did not affect the adenylate cyclase/cAMP signaling pathway in gastric chief cells. All pA2 values of the antagonists were also in agreement with the Ki values determined by [3H]NMS binding to chief cells. Furthermore, GTP gamma S reduced [3H]acetylcholine binding to chief cell membranes in a concentration-dependent manner. The present study, therefore, suggests that muscarinic M3 receptors, which may be coupled to a G protein, mediate pepsinogen secretion, probably by activation of the polyphosphoinositide second messenger system in guinea pig gastric chief cells.

Animals↗
PubMed1992

Estrogen binding sites in peripheral blood monocytes and effects of danazol on their sites in vitro.

1. This study was designed to investigate the presence of estrogen type I (high affinity, low capacity) and type II (low affinity, high capacity) binding sites in human peripheral blood monocytes and the effects of danazol on these sites. 2. These two types of estrogen binding sites existed in human peripheral blood monocytes. 3. Danazol bound to these sites in high concentration (10(-6) M, clinical serum concentration during danazol therapy) and decreased the number of both sites. 4. It is suggested that danazol has an anti-estrogenic action to the monocytes through the competition and suppression of estrogen binding sites as seen in the estrogen target organ.

Adult↗
PubMed1992

62Cu-labeled bifunctional radiopharmaceuticals with metabolizable ester groups.

Generator-produced positron emitting 62Cu-labeled bifunctional radiopharmaceuticals with metabolizable ester groups were synthesized and evaluated for their bifunctionality: the Cu chelating ability and the biochemical reactivity of the ester groups. In vitro studies showed high stability of the Cu chelate site (4,4-dimethyldithiosemicarbazone structure) and enzymatic hydrolysis of the ester site (ethylester or acetylester). The 62Cu-labeled compounds showed in vivo behavior which may be due to their ester hydrolysis.

Animals↗
PubMed1992

Application of the new zinc-62/copper-62 generator: an effective labeling method for 62Cu-PTSM.

A potential PET flow tracer, 62Cu-labeled pyruvaldehyde bis(N4-methylthiosemicarbazone) (62Cu-PTSM), was prepared using a new 62Zn/62Cu generator. With this 62Cu-labeling method based on a ligand exchange reaction, 62Cu-PTSM was quantitatively obtained by simple mixing of the generator eluate, 62Cu-glycine and PTSM solution for a few seconds. The glycine contained in the 62Cu-PTSM injectate had no significant effect in mouse biodistribution studies. The PET studies of 62Cu-PTSM showed brain images that were not visualized by 62Cu-labeled albumin (a plasma pool tracer), in the dog cranium.

Animals↗