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Biomedical subjects

T Ito

Publications and source records attributed to T Ito.

At least 19 recordsLinked to original sources
PubMed1992

HPC-1 is associated with synaptotagmin and omega-conotoxin receptor.

Monoclonal antibodies were produced that recognize a membrane protein of 35,000 Da (p35) expressed in brain and adrenal medulla. They immunoprecipitated 50% of omega-conotoxin (omega-CgTX) receptor, a putative N-type calcium channel, solubilized from rat brain. Anti-synaptotagmin (p65) antibodies also immunoprecipitate omega-CgTX receptor (Leveque, C., Hoshino, T., David, P., Shoji-Kasai, Y., Leys, K., Omori, A., Lang, B., El Far, O., Sato, K., Martin-Moutot, N., Newsom-Davis, J., Takahashi, M., and Seagar, M.J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3625-3629); however, immunoprecipitation by anti-p35 antibodies and anti-synaptotagmin antibodies was not additive. Furthermore, both p35 and synaptotagmin were recovered in the immunoprecipitates with anti-synaptotagmin and anti-p35 antibodies, respectively, indicating that a population of omega-CgTX receptor exists as a ternary complex with synaptotagmin and p35. A cDNA coding p35 was isolated from a rat brain cDNA library by immuno-screening, and the primary structure of the protein was revealed to be identical to that of HPC-1 (Inoue, A., Obata, K., and Akagawa, K. (1992) J. Biol. Chem. 267, 10613-10619). HPC-1 has a putative transmembrane segment at the C terminus and four heptad motifs, which may be involved in protein-protein interaction. These results suggest that HPC-1 may play a role in neurotransmitter release from nerve terminals by associating with omega-CgTX-sensitive N-type calcium channel and synaptotagmin.

Amino Acid Sequence↗
PubMed1992

Semiquantitative analysis of immunoreactivities of tyrosine hydroxylase and aromatic L-amino acid decarboxylase in the locus coeruleus of desipramine-treated mice.

The influence of antidepressant drugs on catecholaminergic neuron groups of the mouse brain was studied immunohistochemically, using a microphotometric semiquantitative method. The immunoreactive level of tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) in the locus coeruleus (LC) was significantly decreased after 2 weeks of antidepressant administration. Our results suggest that long-term exposure to an antidepressant drug, desipramine, significantly affects intracellular catecholamine-synthesizing enzymes. These results may suggest some significant roles of the catecholaminergic neuronal groups to the action of the tricyclic antidepressant drugs.

Animals↗
PubMed1992

Structure of the bovine ETB endothelin receptor gene.

The structure of the gene encoding the bovine type B endothelin receptor (ETB) has been established and compared with those of other heptahelical receptors. The gene is present as a single copy in the bovine genome, as demonstrated by Southern blot analysis, and spans at least 36 kb. The coding region is divided into 7 exons separated by 6 introns, one of which is more than 23 kb in length. The exons correspond well to the structural domains of the receptor: the first exon encodes the first and second transmembrane domains, and each of the following transmembrane domains is encoded by a separate exon. The portion of the ETB protein sequence encoded by exon 3 is quite different from the corresponding ETA sequence, suggesting that this region is responsible for the distinct ligand specificities of the two receptor subtypes. The second intron interrupts the canonical Asp-Arg-Tyr sequence, which is located at the end of the third transmembrane domain of the heptahelical receptors, as with the substance P, substance K, dopamine D2 and dopamine D3 receptor genes. To map the 5' region of the gene and determine the start of transcription, primer-extended cDNAs were cloned and sequenced: multiple start sites were deduced with no apparent TATA box in the expected upstream region. Similar results were obtained by ribonuclease protection analysis.

Animals↗
PubMed1992

Ammonia: a possible promotor in Helicobacter pylori-related gastric carcinogenesis.

Helicobacter pylori (HP) has been shown to possibly be a pathogen of gastric carcinoma. HP has urease activity and produces ammonia in the stomach. In this study, the role of ammonia on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated in rats. After 24 weeks pretreatment with MNNG (83 mg/l), 0.01% ammonia or tap water as a drinking water was administered for 24 weeks. The ammonia-treated rats showed a significantly higher incidence of gastric cancer (percent of animals with tumors and number of tumors per rat). Ammonia would thus appear to have an important role in HP-related human gastric carcinogenesis.

Adenocarcinoma↗
PubMed1992

5-HT3 receptor antagonist effects of DAT-582, (R) enantiomer of AS-5370.

The serotonin 5-HT3 receptor antagonist effects of DAT-582, the (R) enantiomer of AS-5370 ((+/-)-N-[1-methyl-4-(3-methyl-benzyl)hexahydro-1H-1,4-diazepin-6- yl]-1H- indazole-3-carboxamide dihydrochloride), and its antipode were compared with those of AS-5370 and existing 5-HT3 receptor antagonists. In anesthetized rats, DAT-582 antagonized 2-methyl-5-HT-induced bradycardia with an ED50 value of 0.25 microgram/kg i.v., whereas the (S) enantiomer was without effect even at 1000 micrograms/kg i.v. In antagonizing the bradycardia, DAT-582 was as potent as granisetron, slightly more potent than AS-5370, and 2, 5 and 18 times more potent than ondansetron, ICS 205-903 and renzapride, respectively, although it was less potent than zacopride. DAT-582 inhibited cisplatin (10 mg/kg i.v.)-induced emesis in ferrets with an ED50 value of 3.2 micrograms/kg i.v. twice. The antiemetic activity of DAT-582 was more potent than that of the existing 5-HT3 receptor antagonists examined, except zacopride. In contrast, the (S) enantiomer had little effect at 1000 micrograms/kg i.v. twice. In isolated guinea-pig ileum, DAT-582 inhibited 5-HT-induced contractions with an IC50 value of 91 nM, whereas the (S) enantiomer hardly inhibited them even at 1000 nM. These results suggest that DAT-582, the (R) enantiomer of AS-5370, potently and selectively blocks 5-HT3 receptors.

Animals↗
PubMed1992

A novel blocker-PCR method for detection of rare mutant alleles in the presence of an excess amount of normal DNA.

A novel polymerase chain reaction method was developed to preferentially amplify a segment of DNA containing a base substitution mutation. This technique uses a pair of dideoxynucleotide-labeled oligonucleotides (18 mers) of normal sequences as blockers located between the two primers. By virtue of a subtle difference in the melting temperature between the blocker-normal DNA and blocker-mutant DNA hybrids, the method allows preferential amplification of the mutant DNA. We used the human N-ras gene as a model. Two different types of N-ras mutations could be effectively amplified when they were present with an excess amount of normal DNA at a ratio of 1:10(3). Furthermore, the sensitivity was increased 10-fold by using single strand conformation polymorphism analysis for the amplified products, and mutant DNA was detected in the presence of a 10(4) times excess amount of normal DNA.

Alleles↗
PubMed1992

Enalapril reduces the enhanced 1,2-diacylglycerol content and RNA synthesis in spontaneously hypertensive rat hearts before established hypertension.

There is evidence that cardiac hypertrophy in spontaneously hypertensive rats (SHR) occurs before the development of hypertension. 1,2-Diacylglycerol, which is thought to be a second messenger activating protein kinase C, is also produced in excess in SHR hearts at 4 weeks of age, before established hypertension. We determined myocardial 1,2-diacylglycerol content in SHR with and without prazosin and enalapril from 3 to 4 weeks of age. Hearts from untreated SHR had greater RNA and DNA synthesis and greater relative weights at 4 weeks of age than those from Wistar-Kyoto (WKY) rats. There was no difference in triglyceride content or phospholipid species between WKY rats and untreated SHR, except for a higher cholesterol content in SHR. Treatment of SHR with enalapril, but not prazosin, lowered not only 1,2-diacylglycerol content but also RNA synthesis to the levels of WKY rats. Moreover, fatty acids involved in 1,2-diacylglycerol were altered by enalapril despite the lack of a difference between WKY rats and untreated SHR. Prazosin did not have any effect on 1,2-diacylglycerol fatty acid composition. Enalapril may decrease cardiac hypertrophy in SHR by lowering myocardial 1,2-diacylglycerol production.

Animals↗
PubMed1992

Affinity chromatographic purification of bovine lung endothelin receptor using biotinylated endothelin and avidin-agarose.

Endothelin receptor was purified from bovine lung by affinity chromatography using biotinylated endothelin and avidin-agarose. Endothelin was biotinylated with sulphosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate, a reactive form of biotin with a cleavable spacer arm containing a disulphide bond designed for a simple elution by thiols. Starting from 3.5 kg of bovine lung, about 200 micrograms of pure receptor were obtained.

Amino Acid Sequence↗
PubMed1992

Modulation by NaCl of atrial natriuretic peptide receptor levels and cyclic GMP responsiveness to atrial natriuretic peptide of cultured vascular endothelial cells.

Type C atrial natriuretic peptide (ANP) receptor levels in cultured vascular endothelial cells were found to be very sensitive to NaCl and shown to be inversely related to the magnitude of ANP-induced cGMP response of the cells. Endothelial cells from bovine carotid artery were subcultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum (MEM-FBS) and in MEM-FBS plus 25 and 50 mM NaCl. Determination, after several passages, of ANP receptor levels in these cells by 125I-ANP binding assay and affinity labeling revealed a marked reduction in the number of type C receptor in the NaCl-treated cells, whereas type A receptor density was not affected. RNase protection assay to estimate the levels of type C receptor mRNA indicated that the reduction occurred at a pre-translational level. In spite of the decrease in type C receptor number and no significant change in type A receptor (i.e. particulate guanylate cyclase) levels, cGMP response of the NaCl-treated cells to ANP was greatly exaggerated; this sensitization was also observed in membrane preparations. Simple masking of type C ANP receptor with C-ANF (des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP), a ring-deleted ANP analog, did not produce any sensitization of the cGMP response to ANP; therefore, the above phenomenon cannot simply be explained by the clearance function of the type C receptor. Although whether the type C receptor depletion is directly related to the sensitization of the type A receptor/cyclase is not known, the phenomenon reported and characterized here will serve as a useful basis for elucidating ANP receptor regulation and activation.

Affinity Labels↗
PubMed1992

Intratumoral and whole-body distributions of C110 anti-carcinoembryonic antigen radioimmunotoxin after intraperitoneal and intravenous injection: a quantitative autoradiographic study.

The intratumoral and whole-body distributions of 90Y-labeled C110 anticarcinoembryonic antigen immunotoxin after i.p. and i.v. injection were compared by quantitative autoradiography. During in vitro incubation of spherical tumor nodules of LS174T human colon cancer (about 5 mm in diameter) in a medium containing C110 radioimmunotoxin (RIT), the direct penetration of the immunotoxin increased with time but was limited to the outer 300 microns of the tumor nodule after 12 h of incubation. In vivo experiments were performed in nude mice bearing LS174T xenografts as i.p. tumor nodules. Injection of C110 RIT i.p. resulted in a ring-like distribution, i.e., high uptake at the tumor periphery and considerably lower uptake at the tumor center (ratio of peripheral to central concentration, 7:1 at 1 day and 2:1 at 5 days). In contrast, i.v. injection provided a much smaller gradient in C110 RIT distribution from peripheral to central regions (ratio of peripheral to central concentration, 3:1 at 1 day and 1:1 at 5 days). Estimates of total tumor uptake of C110 RIT by quantitative autoradiography demonstrated almost equivalent tumor uptake after either i.p. or i.v. injection, while i.v. injection was associated with increased C110 RIT uptake in various normal organs, especially in the liver, as compared to i.p. injection. The results in this study suggest that (a) i.v. injection may produce more homogeneous distribution of C110 RIT in i.p. tumor nodules of LS174T but may also result in increased liver toxicity, and (b) i.p. injection may decrease C110 RIT exposure of normal tissues, which can reduce systemic toxicity, but may also produce more restricted intratumoral distribution of C110 RIT. In addition, current methods using a nude mouse model of i.p. tumor nodules and quantitative autoradiography allow us to assess intratumoral and whole-body distributions of radiolabeled immunoconjugates from various administration routes.

Animals↗
PubMed1992

Highly conserved hexamer, octamer and nonamer motifs are positive cis-regulatory elements of the wheat histone H3 gene.

Base substitution mutations were introduced into the promoter region of the wheat histone H3 gene, and promoter activity was assayed in stably transformed sunflower calli or in wheat protoplasts transfected transiently. At least four positive regulatory elements, a hexamer motif (ACGTCA), two octamer(-like) motifs of a direct (CcCGGATC) and a reverse (aATCCGCG) form, and a nonamer motif (CATCCAACG) were identified within the -185 region of the H3 promoter. Analyses of the type I element (CCACGTCACCaATCCGCG) consisting of the hexamer and reverse-oriented octamer motifs, and which is conserved in other plant histone genes as well, predicted the presence of an octamer-binding protein(s).

Base Sequence↗
PubMed1992

Unique association of p53 mutations with undifferentiated but not with differentiated carcinomas of the thyroid gland.

Thyroid neoplasms show a wide variety of lesions varying from slowly growing differentiated adenocarcinomas to rapidly proliferating undifferentiated carcinomas. There has been some histopathological evidence that the undifferentiated thyroid carcinomas are derived from differentiated carcinomas. Moreover, it is suspected that some genetic events might be associated with such changes. In the present study, mutations in the p53 gene were investigated by direct sequencing analysis after polymerase chain reaction amplification of exons 5 to 8, using paraffin-embedded primary tumors and cultured cells. No mutations in exons 5 to 8 were detected in 10 differentiated papillary adenocarcinomas, whereas 6 of 7 undifferentiated carcinomas were found to carry base substitution mutations. Sequencing analysis confirmed mutations at codons 135 (TGC----TGT), 141 (CCC----CCT), 178 (CAC----GAC), 213 (CGA----TGA), 248 (CGG----CAG, CGG----TGG), and 273 (CGT----TGT). The spectrum of mutations (G:C to A:T transitions in 7 of 8) might be a specific feature of the spontaneous cancers. The results strongly suggest that, in human thyroid glands, p53 mutations play a crucial role in the progression of differentiated carcinomas to undifferentiated ones.

Adenocarcinoma, Papillary↗
PubMed1992

[Signal contamination problem in three-dimensional localization technique of MR spectroscopy--experiment and an analysis of patient data].

Signal contamination from the surrounding volume was studied using the ISIS sequence, a three-dimensional localization technique. In phantom experiments, contamination decreased with the increase of Tr, regardless of where the contamination originated from. In 52 localized liver spectra with a Tr of 2 sec, PCr contamination was estimated at 3 to 4%, and contamination of ATP signals from the surrounding liver tissue at less than 1%, because of the relatively short T1 of ATP in the liver. In the spectra of brain neoplasms obtained using the simultaneous double-volume selection technique, the PCr ratio in the tumor relative to the control area was high in low grade astrocytoma (0.80, n =8) and low in others (0.45, n = 11). Considering contamination and spectral noise, a PCr ratio of less than 0.4 does not strongly suggest the existence of PCr in tumors. Among 25 cases whose therapeutic processes were monitored by MRS, signal disappearance was observed in two cases in the localized spectra after therapy. In both of them, tumor necrosis was proved pathologically and correlated well with spectroscopic findings. It was found that the ISIS technique suppressed signal contamination to the level of several percents, providing spectra of acceptable quality through localization.

Humans↗
PubMed1992

Sequence-specific DNA purification by triplex affinity capture.

A DNA isolation procedure was developed by using triple-helix formation and magnetic separation. In this procedure, target DNA is captured by a biotinylated oligonucleotide via intermolecular triplex formation, bound to streptavidin-coated magnetic beads, and recovered in double-stranded form by elution with a mild alkaline buffer that destabilizes the triple helix. The effectiveness of the procedure was demonstrated by a model experiment with an artificially reconstructed library and, also, by the isolation of (dT-dC)n.(dG-dA)n dinucleotide repeats from a human genomic library. This procedure provides a prototype for other triplex-mediated DNA isolation technologies.

Base Sequence↗
PubMed1992

A simple and rapid method for HLA-DQA1 genotyping by polymerase chain reaction-single strand conformation polymorphism and restriction enzyme cleavage analysis.

A simple and rapid method for identification of alleles at the human leucocyte antigen (HLA)-DQA1 locus is described. The polymorphic second exon of the HLA-DQA1 locus was amplified by the polymerase chain reaction (PCR) method. The amplified DNA was analyzed by single-strand conformation polymorphism (SSCP) and restriction enzyme cleavage assay. Using this method, the eight known DQA1 alleles could be distinguished from each other. This paper suggests that the method can be used for quick genotyping of DQA1 alleles, but detecting point mutations at various positions in a fragment as well as new HLA-DQA1 genotypes should also be possible.

Alleles↗