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Biomedical subjects

K Watanabe

Publications and source records attributed to K Watanabe.

At least 19 recordsLinked to original sources
PubMed1992

Effects of drugs acting on Cl(-)-HCO3- and Na(+)-H+ exchangers on acid secretion in the rat gastric mucosa sheet preparation.

The effects of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of the Cl(-)-HCO3- exchanger, and amiloride, an inhibitor of the Na(+)-H+ exchanger, on gastric acid secretion under basal conditions and after stimulation with bethanechol or dibutyryl cyclic AMP were studied in rat gastric mucosa sheet preparation. DIDS inhibited bethanechol-induced acid secretion in a dose-dependent manner, but amiloride had no effect. The stimulation of acid secretion by dibutyryl cyclic AMP plus 3-isobutyl-1-methylxanthine was also inhibited by DIDs, but not by amiloride. DIDS did not reduce basal acid secretion, and neither did amiloride. These results suggest that the Cl(-)-HCO3-exchanger in the basolateral membrane of the parietal cell plays an important role in stimulated gastric acid secretion and that the Na(+)-H+ exchanger is less important. In addition, these data show that DIDS inhibits stimulated gastric acid secretion irrespective of the secretagogue, but not basal gastric acid secretion.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Mouse hepatic microsomal oxidation of aliphatic aldehydes (C8 to C11) to carboxylic acids.

Addition of saturated and alpha, beta-unsaturated aliphatic aldehydes (C8 to C11) significantly increased NADPH oxidation with mouse hepatic microsomes, and the aldehydes themselves were oxidized to the corresponding carboxylic acids. When these aldehyde substrates were incubated similarly under oxygen-18 gas and the carboxylic acids formed were analyzed by GC-MS after methylation, it was indicated that oxygen-18 was significantly incorporated into the carboxylic acids formed from alpha, beta-unsaturated aldehydes, but not significantly into the carboxylic acids formed from saturated aldehydes. These results indicate that enzyme and/or mechanism responsible for the oxidation of these two types of aldehydes is different from each other.

Alcohols↗
PubMed1992

Protective effect of a selective endothelin receptor antagonist, BQ-123, in ischemic acute renal failure in rats.

To elucidate the pathophysiological role of endogenous endothelin (ET), we examined the effects of the newly synthesized ETA receptor-selective antagonist, BQ-123, on ischemic acute renal failure induced by bilateral clamping of renal artery and vein followed by reperfusion in rats. BQ-123, when given by i.v. infusion of 0.5 mg/kg per min for 2.5 h during the pre- and post-ischemic period, was found to prevent the decrease in creatinine clearance and increases in blood urea nitrogen, plasma creatinine and the fractional excretion of sodium. Morphological observation also showed an effect of BQ-123, i.e. prevention of proximal tubular (S3 segment) necrosis. At 2 h after the start of reperfusion, the ET-1 content in the kidney increased to its maximal level. At this time, the Ca2+ content in the mitochondrial fraction of the renal cortex increased, with a concomitant increase in blood urea nitrogen. However, these increases were limited by treatment with BQ-123. Thus, BQ-123 was effective to both prevent mitochondrial Ca2+ accumulation in the early phase of ischemic acute renal failure and protect proximal tubular cells from post-ischemic degeneration. We conclude that ET may be at least partially involved in the pathogenesis of tubular cell injury in this acute renal failure model.

Acute Kidney Injury↗
PubMed1992

Expression in human hepatocellular carcinoma of nucleoside diphosphate kinase, a homologue of the nm23 gene product.

BACKGROUND: Expression of nucleoside diphosphate (NDP) kinase, which is highly homologous to the nm23 gene product in a variety of species, has been found to be inversely associated with metastatic potential in human breast cancer. PURPOSE: The present study was conducted to clarify the association of NDP kinase expression with metastatic potential in human hepatocellular carcinoma. METHODS: The immunohistochemical expression of NDP kinase was analyzed in 30 patients with histopathologically proven hepatocellular carcinoma. These patients included nine with distant metastases and 21 without distant metastases. Tissue specimens were reacted with rabbit anti-rat NDP kinase antibody and stained by the biotin-streptavidin complex method. The relative staining intensities were evaluated by comparing primary tumor sites with adjacent nontumorous liver tissue or with metastatic sites, RESULTS: The expression of NDP kinase in primary sites in patients with distant metastases was significantly less intense than that in patients without distant metastases (P = .018). NDP kinase was expressed significantly less intensely in metastatic sites than in primary sites (P = .005). The intensity of NDP kinase expression did not statistically correlate with tumor size or number of lesions in the liver, histopathological classification of tumor, associated liver diseases, hepatitis virus markers, or tumor markers. CONCLUSION: These results suggest that the reduced expression of NDP kinase is closely associated with distant metastatic potential in hepatocellular carcinoma. IMPLICATIONS: It is possible that both NDP kinase and the nm23 gene product may be active in the progression and differentiation of tumor cells and that their reduced expression induces a high metastatic potential in tumor cells. Studies using Northern blotting or in situ hybridization should be planned to confirm our findings.

Adult↗
PubMed1992

A monoclonal antibody-based enzyme immunoassay for human GMP-140/P-selectin.

Two hybridoma cell lines producing monoclonal antibodies WGA-1 and PL7-6, reactive only with thrombin-stimulated human platelet have been established. Both these antibodies were investigated for their specific reactivity against GMP-140, based on the amino acid composition analysis of immunopurified antigen and N terminal amino acid sequencing of its protease fragments. A two-site enzyme immunoassay for quantification of human GMP-140 was developed using WGA-1 monoclonal antibody immobilized on 96-well microplates and horseradish peroxidase-labeled PL7-6 monoclonal antibody as detector. The assay was able to measure GMP-140 in serum and plasma with a sensitivity of about 5 ng/ml and a precision better than 10%. This assay will be useful for the detection of GMP-140 derived from platelets or endothelium in biological fluids and tissue extracts.

Amino Acid Sequence↗
PubMed1992

Extraordinarily stable mini-hairpins: electrophoretical and thermal properties of the various sequence variants of d(GCGAAAGC) and their effect on DNA sequencing.

A small DNA fragment having a characteristic sequence d(GCGAAAGC) has been shown to form an extraordinarily stable mini-hairpin structure and to have an unusually rapid mobility in polyacrylamide gel electrophoresis, even when containing 7M urea. Here, we have studied the stability of the various sequence variants of d(GCGAAAGC) and the corresponding RNA fragments. Many such sequence variants form stable mini-hairpins in a similar manner to the d(GCGAAAGC) sequence. The RNA fragment, r(GCGAAAGC) also forms a mini-hairpin structure with less stability. The DNA mini-hairpins with GAAA or GAA loop are much more stable than DNA and RNA mini-hairpins with other loop sequence so far as has been examined. The stability difference between DNA and RNA mini-hairpins may be deduced to the stem structures formed by DNA (B form) and RNA (A form). The stable hairpins consisting of the GCGAAAGC sequence cause strong band compression on the sequencing gel. This phenomenon should be carefully considered in DNA sequencing.

Base Sequence↗
PubMed1992

Isolation and characterization of a gene encoding rat nucleoside diphosphate kinase.

Two overlapping genomic clones for a rat nucleoside diphosphate kinase (NDP kinase) have been isolated and characterized. Complete sequencing of the genomic segment including the whole coding region for the enzyme revealed that the gene consists of four exons spanning 5.5 kilobase pairs. Primer extension analyses and ribonuclease protection assays indicated that the transcription may start from multiple sites with the major initiation site at 3 base pairs upstream from the translation initiation site, Met-1. Neither CAAT-box nor TATA-box could be assigned for each transcription initiation site, whereas five putative Sp1-binding sites (GC-boxes) were present in the 5'-flanking region. These features of the NDP kinase gene represent those of housekeeping genes. In genomic Southern blotting using a full-length rat NDP kinase cDNA as a probe, many positively hybridized fragments were detected. In support of this, five possible processed pseudogenes were identified in different DNA segments although many other NDP kinase-related genomic fragments remained to be characterized. These results demonstrate that the NDP kinase gene may consist of a multiple gene family.

Amino Acid Sequence↗
PubMed1992

Fluorescence studies on the interaction of adenine with ricin A-chain.

Ricin A-chain, an N-glycosidase that attacks 28S rRNA at a highly conserved adenine residue, has a unique tryptophan (Trp-211) in the putative active site cleft. Fluorescence spectroscopy revealed that specific binding of adenine to the A-chain caused a large enhancement of Trp-211 fluorescence (70%) and a concomitant red shift of the emission spectrum (8 nm). A Scatchard plot of the fluorescence enhancement data was not linear, indicating that the environment of Trp-211 was altered by heterogeneous binding of adenines. These results, taken together with the protective effect of adenine on the ribosome-inactivation by ricin A-chain, suggest that at least two adenines bind to the active site cleft.

Adenine↗
PubMed1992

The rat peptidylarginine deiminase-encoding gene: structural analysis and the 5'-flanking sequence.

Genomic clones of the rat peptidylarginine deiminase (PAD)-encoding gene (PAD) were isolated, and the gene organization was analyzed by restriction mapping and nucleotide sequencing. The PAD spans more than 50 kb and contains 16 exons and 15 introns. The lengths of the introns from 0.5 kb to more than 16.5 kb. A 1.7-kb sequence in the 5'-flanking region was determined. S1 nuclease mapping revealed two putative cap sites 79 and 81 bp upstream from the N-terminal ATG codon of PAD, which had been determined by amino acid sequence analysis. This ATG was confirmed to be the translation start site, since no other ATG codon was found in the open reading frame downstream from the cap sites. The 5'-flanking sequence contains four potential SP1-binding sites, a putative Pit-1/GHF-1-binding site, four short sequences either identical or homologous to the sequences in the promoter regions of rat or human growth hormone encoding genes, as well as a sequence similar to an estrogen-responsive element. However, neither a typical TATAA box, nor CCAAT box is present. These results provide important clues for elucidating the mechanism of female-specific and/or sex cycle-dependent gene expression.

Amino Acid Sequence↗
PubMed1992

The T-loop region of animal mitochondrial tRNA(Ser)(AGY) is a main recognition site for homologous seryl-tRNA synthetase.

Recognition sites of bovine mitochondrial serine tRNA specific for condons AGY [tRNA(Ser) (AGY)] by the cognate mitochondrial seryl-tRNA synthetase were studied using a range of tRNA(Ser)(AGY) variants which were obtained by the in vitro transcription of synthetic tRNA genes with T7 RNA polymerase. Base replacements in the anticodon and discriminator sites did not affect serine acceptance. However, deletion and/or replacement in the T-loop region completely deprived the variants of their charging activities. Point mutation experiments in this region also showed that the adenosine residue in the middle of the T-loop (position 58), which is involved in tertiary interaction between the T-loop and the truncated D-arm [de Bruijn and Klug, 1983] played a significant role in the recognition process by the synthetase.

Animals↗
PubMed1992

Effect of the higher-order structure of tRNAs on the stability of hybrids with oligodeoxyribonucleotides: separation of tRNA by an efficient solution hybridization.

In the course of developing a method to purify a single tRNA species efficiently, we have examined hybridization efficiencies between some tRNAs and short oligodeoxyribonucleotide probes both by the filter and solution hybridization methods without denaturants. The hybridization efficiencies varied considerably among probes which are complementary to different regions of the tRNAs, although there was little efficiency variation in the probes toward DNA substrates including the same nucleotide sequence. This efficiency variation was shown to be due to tRNA-specific higher-order structures as well as a hypermodified nucleotide in the anticodon loop. Characterization of the tRNA-probe hybrids by both nondenaturing gel electrophoresis and chemical modification showed the existence of two stable hybridizing states as a function of ionic strength. Our results indicate that RNA molecules with a number of intramolecular base pairings are able to form stable hybrids with complementary sequences under nondenaturing conditions. On the basis of these data, an appropriate probe was designed to successfully purify yeast tRNA(Phe) by making a tRNA(Phe)-probe hybrid, which has a longer retention time in hydroxyapatite high performance liquid chromatography than the tRNA(Phe) itself.

Animals↗
PubMed1992

The embryonic pineal body as a multipotent organ.

The repertoire of differentiating potency of mammalian and avian pineal cells has been examined utilizing cell culture technique. Skeletal muscle fibers are differentiated from pineal cells of the rat under the usual culture condition and from those of quail under hypertonic conditions. Myogenesis of pineal cells may be explained from the ontogeny of the pineal body. Anlagen of a pineal body are situated in bilateral cephalic neural folds, which also supply multipotent neural crest cells. In some conditions, almost all quail pineal cells are able to differentiate into pigmented epithelial cells and/or lens cells. Opsin containing cells found in culture of rat pineal cells may be in a similar category reflecting the "third eye": the phylogenetic ancestor of the pineal body of avian and mammalian species. Neuron-like cells have also been reported and neuronal morphology has been intensified under the effect of testicular hyaluronidase. The cytodifferentiation described above is suggested to be different expressions of a single type of progenitor cells in the pineal body. In relation to multipotentiality of pineal cells, the original differentiating state of pineal cells is interesting; it has been found that tyrosinase is expressed from the beginning of pineal formation and that its expression is stage-specific (during embryonic period) and site-specific (predominance in the dorsal half of the pineal body and in the apical cytoplasm of the pineal cell). In the 8 day quail embryo used for culture studies, three differentiating states as to tyrosinase are noticed. However, the distinction may be apparent, as even the cells negative in tyrosinase in this stage are still ready to express tyrosinase in the suitable culture condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Cholinergic/dopaminergic interaction in the rat striatum assessed from drug-induced repetitive oral movements.

The role of striatal dopaminergic/cholinergic interactions in the regulation of oral behaviour in rats was studied using methods which resolve distinct patterns of jaw movements, allowing a more accurate quantitative and qualitative analysis. Both dopamine and acetylcholine receptor agonists given either systemically or into the ventral striatum induced repetitive oral movements. However, the cholinergic movements differed from dopaminergic movements as to pattern of activity. Oral movements induced by apomorphine (0.2 mg/kg i.v.) were potentiated by carbachol (0.1 microgram/0.2 microliters) injected into the dorsal striatum, while inhibition was observed when carbachol was injected into the ventral striatum. Pilocarpine (4 mg/kg)-induced oral movements were reduced by injecting flupentixol (10 micrograms/0.2 microliters), but not a combination of SKF 38393 (3 micrograms)+quinpirole (10 micrograms/0.2 microliter), into either the dorsal or the ventral striatum. Oral movements induced by the injection of carbachol (1 microgram/0.2 microliter) into the ventral striatum were enhanced by previous injection of this combination of dopamine receptor agonists into the same site and were inhibited by flupentixol. These results suggest that cholinergic and dopaminergic oral movements are separate behaviors and that the striatal dopamine/acetylcholine interaction in their regulation is neither simply antagonistic or synergistic, nor reciprocal.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1992

Identification and isolation of endogenous insect phenoloxidase inhibitors.

Inhibitors of phenoloxidase were identified in pupae of the housefly, Musca domestica L. The phenoloxidase inhibitors were purified from final instar pupae of the housefly by a combination of ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and reverse-phase high performance liquid chromatography. The potent phenoloxidase inhibitors were heat-stable low molecular weight peptides with an inhibition constant of nM range. To the best of our knowledge, this is the first time that endogenous phenoloxidase inhibitors have been identified among the insects, and probably also among the invertebrates. It is likely that the inhibitors play a central role in regulating the action of active phenoloxidases and will also serve as important tools for understanding the structures and functions of phenoloxidases, as well as their role in insect metamorphosis.

Amino Acid Sequence↗
PubMed1992

Immunochemical characterization of a cytochrome P450 isozyme and a protein purified from liver microsomes of male guinea pigs and their roles in the oxidative metabolism of delta 9-tetrahydrocannabinol by guinea pig liver microsomes.

A protein (designated as protein-B) was purified from liver microsomes of adult male guinea pigs by an affinity chromatography with omega-aminooctyl Sepharose 4B, followed by HPLC using DEAE-5PW and hydroxyapatite columns which had been used to purify a cytochrome P450 (P450) isozyme (P450-A) from the same subcellular fraction (Narimatsu et al., Biochem Biophys Res Commun 172: 607-613, 1990). Protein-B had a molecular mass of 49 kDa in SDS-PAGE, but did not show absorbance at 417 nm for heme. Further, it did not show any oxidative activities towards aniline (AN), d-benzphetamine (d-BP), p-nitroanisole (p-NA) or delta 9-tetrahydrocannabinol (delta 9-THC) in a reconstituted system including dilauroylphosphatidylcholine, NADPH-P450 reductase, and cytochrome b5. However, antiserum against protein-B raised in rabbits suppressed liver microsomal oxidative activities towards d-BP and p-NA dose-dependently. The antibody decreased delta 9-THC oxidative activity most effectively, but did not decrease AN hydroxylation activity. Antiserum against P450-A suppressed all the activities towards these four substrates, especially towards delta 9-THC, in liver microsomes of male guinea pigs. Moreover, reconstitution with hemin made it possible for protein-B to produce some oxidative activity toward delta 9-THC. These results suggest that protein-B is also a cytochrome P450 isozyme which has lost a heme moiety during purification steps. Both P450-A and protein-B could have a role as cytochrome P450 isozymes in the oxidative metabolism of drugs, especially that of delta 9-THC by the liver microsomes of adult male guinea pigs.

Amino Acid Sequence↗
PubMed1992

High-performance liquid chromatography and post-column derivatization with diphenyl-1-pyrenylphosphine for fluorimetric determination of triacylglycerol hydroperoxides.

Triacylglycerol monohydroperoxides (TG-mHPO) were selectively detected at the picomole levels after post-column reaction with diphenyl-1-pyrenylphosphine (DPPP). TG-mHPO were separated on two types of reserved-phase columns, an ODS column and a phenylated silica gel column, which were useful for determining TG-mHPO at their molecular species levels and their class levels, respectively. After the separation, DPPP solution was mixed with the eluent followed by reaction in a stainless-steel coil 20 m x 0.5 mm I.D. at 80 degrees C, then the fluorescence intensity of DPPP oxide was measured (lambda ex. 352 nm, lambda em. 380 nm). Using these systems, TG-mHPO were determined in the range 2-1000 pmol. The relative standard deviations were 2.3-2.8%.

Chromatography, High Pressure Liquid↗
PubMed1992

Molecular cloning and expression of a major surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis in Escherichia coli.

A major immunodominant surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis 381 has been purified and its amino-terminal amino acid sequence has been determined. Using oligonucleotide probes corresponding to the sequence, we identified a recombinant plasmid clone carrying a single 4.2-kb BamHI fragment from pUC19 libraries of P. gingivalis. The BamHI fragment transferred to the bacteriophage T7 RNA polymerase/promoter expression vector system produced a slightly larger (77-kDa) protein, a precursor form, immunoreactive to the antibody against the 75-kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 75-kDa protein. Genomic Southern analysis revealed a single copy of the 75-kDa protein gene per genome among all P. gingivalis strains tested, and that no homologous genes are present in other black-pigmented Bacteroides species. These observations suggest that the 75-kDa protein gene may be useful as a specific DNA probe to classify or to detect this organism.

Amino Acid Sequence↗