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Biomedical subjects

Y Takeda

Publications and source records attributed to Y Takeda.

At least 19 recordsLinked to original sources
PubMed1992

Detection of topoisomerase I gene point mutation in CPT-11 resistant lung cancer cell line.

CPT-11, a recently developed topoisomerase I (Topo I) inhibitor, attracts the attention not only of basic researchers but also of clinicians because of its high antitumor activity. The CPT-11 resistant human lung cancer cell line, PC-7/CPT, showed 10-fold resistance compared to parental cell line, PC-7. The total activity of Topo I in the resistant cell line was one fourth that of the parental sensitive cell line. The Topo I from the resistant cells was also 5-fold more resistant to the inhibitory effect of CPT-11 than that of the parental cells. We speculated that the alteration of the Topo I gene may be responsible for the change in topoisomerase activity of the CPT-11 resistant cell line. Therefore, we analyzed the mutation of Topo I gene using the method of single strand conformation polymorphism of polymerase chain reaction and the reverse transcriptase. We divided Topo I cDNA into ten fragments which overlapped each other and covered whole coding sequences of the Topo I cDNA. We observed mobility shift of two fragments in the PC-7/CPT, suggesting the presence of some mutations in these fragments. We performed the direct-sequencing of these portions by the dideoxy chain termination method and observed an altered sequence having a G to A base change in PC-7/CPT. This base substitution results in replacement of the conserved threonine at 729 position with alanine. These results suggest that the point mutation of Topo I gene is related to the decreases of Topo I activity and the sensitivity to Topo I inhibitor in PC-7/CPT cells.

Antineoplastic Agents, Phytogenic↗
PubMed1992

A new endo-beta-galactosidase acting on the Gal beta 1-3Gal linkage of the proteoglycan linkage region.

A new type of endo-beta-galactosidase acting on the linkage region of peptidochondroitin sulfate was isolated from the mid-gut gland of the mollusk Patinopecten. The purification procedure included ammonium sulfate precipitation, Sephacryl S-200HR gel filtration, DEAE-Sephacel chromatography, and TSKgel Phenyl-5PW RP high performance liquid chromatography. The purified enzyme was free from exoglycosidases, sulfatases, and phosphatases. The specificity of the enzyme was as follows. 1) It acted on the internal galactoside linkage of sugar chains; 2) it specifically hydrolyzed the galactosylgalactose (Gal beta 1-3Gal) linkage, but not the galactosylxylose (Gal beta 1-4Xyl) linkage in the linkage region of peptidoglycans; 3) the enzyme activity was unaffected by the type of glycosaminoglycan, chondroitin sulfate, dermatan sulfate or heparan sulfate used as a substrate; 4) keratan sulfate and some oligosaccharides from glycolipid were not degraded by the enzyme. These properties of the endo-beta-galactosidase characterize it as a new endo-beta-galactosidase with unique specificity.

Animals↗
PubMed1992

Thermodynamics of Cro protein-DNA interactions.

Using a highly sensitive pulsed-flow microcalorimeter, we have measured the changes in enthalpy and determined the thermodynamic parameters delta H, delta S degree, delta G degree, and delta C(p) for Cro protein-DNA association reactions. The reactions studied include sequence-nonspecific DNA association and sequence-specific DNA associations involving single- and multiple-base alterations and/or single-amino acid alteration mutants. (i) The association of Cro protein with nonspecific DNA at 15 degrees C is characterized by delta H = +4.4 kcal.mol-1 (1 cal = 4.18J), delta S degrees = 49 cal.mol-1.K-1, delta G degrees = -9.7 kcal.mol-1, and delta Cp congruent to 0; the association with specific high-affinity operator OR3 DNA is characterized by delta H = +0.8 kcal.mol-1, delta S degree = 59 cal.mol-1.K-1, delta G degree = -16.1 kcal.mol-1, and delta Cp = -360 cal.mol-1.K-1, respectively. Both nonspecific and specific Cro-DNA associations are entropy-driven. (ii) Plots of delta H vs. delta Cp and delta S degree vs. delta Cp for the 20 association reactions studied fall into two correlation groups with linear slopes of +9.4 K and -20.5 K and of -0.03 and -0.14, respectively. These regression lines have common intercepts, at the delta H and delta S degree values of nonspecific association (where delta Cp congruent to 0). The results suggest that there are, at least, two distinct conformational subclasses in specific Cro-DNA complexes, stabilized by different combinations of enthalpic and entropic contributions. The delta G degree and delta Cp values form an approximately single linear correlation group as a consequence of compensatory contributions from delta H and delta S degree to delta G degree and to delta Cp. Cro protein-DNA associations share some similar thermodynamic properties with protein folding, but their overall energetics are quite different. Although the nonspecific complex is stabilized predominantly by electrostatic forces, it appears that H bonds, van der Waals contacts, hydrophobic effects, and charge interactions all contribute to the stability (delta G degree and delta Cp) of the specific complex. (iii) The variations in the values of the thermodynamic parameters are in general accord with our knowledge of the structure of the Cro-DNA complex.

Base Sequence↗
PubMed1992

A new type of mitogenic factor produced by Streptococcus pyogenes.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.

Amino Acid Sequence↗
PubMed1992

[11 beta-hydroxysteroid dehydrogenase and steroid receptors].

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD), as its name implies, is the enzyme responsible for the conversion of cortisol to cortisone, and of corticosterone to 11-dehydrocorticosterone. Ulick et al. reported the detailed investigation of a patient with the syndrome of apparent mineralocorticoid excess (AME), who had the stigmata of florid hyperaldosteronism but low normal or suppressed levels of renin and aldosterone. Such patients show marked abnormalities of cortisol metabolism. From a series of studies, the consensus grew that AME reflects the absence, or very low activity, of 11 beta-HSD in the kidney of affected patients. In addition to providing a framework for understanding the pathogenesis of AME, these studies prompted a re-evaluation of other areas of steroid in the kidney. Glycyrrhetinic acid, the active principle of liquorice and carbenoxolone, exerted its mineralocorticoid action not by a direct effect on mineralocorticoid receptors but by inhibiting renal 11 beta-HSD, thus producing a mild, drug-induced form of AME. Recently Monder et al. reported the cloning and expression of rat and human cDNA encoding corticosteroid 11 beta-dehydrogenase. The physiological role of 11 beta-HSD in conferring aldosterone-selectivity on otherwise non-selective type I receptors has been focused using the genetic method in addition to the biological ones.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1992

[Quantitative evaluation of liver function using 99mTc-GSA in rats with liver injury induced by ischemia-reperfusion].

We evaluated quantitatively the liver injury of rats induced by ischemia-reperfusion, using 99mTc-DTPA-Galactosyl-Human-Serum-Albumin (99mTc-GSA). The vessels of the left lobe were clamped for 5, 10, or 45 minutes followed by 15 minutes reperfusion. Then, 99mTc-GSA was intravenously administered (170 micrograms/kg body weight) to rats. Two compartment analysis was made on measurement curves in the heart and liver to obtain clearance parameters. Significant difference was observed between the ischemic group (clamped for 10 and for 45 minutes) and the control. These results suggest that 99mTc-GSA is useful in the estimation of liver injury produced by ischemia-reperfusion.

Animals↗
PubMed1992

Endothelin-1 release from the mesenteric arteries of cyclosporine-treated rats.

The release of endothelin-1 from mesenteric arteries from cyclosporine-treated rats was measured by a specific enzyme immunoassay after purification of the perfusate on an immunoaffinity column. Mesenteric arteries from cyclosporine-treated rats (25 mg/kg per day for 6 weeks) released a significantly larger amount of endothelin-1 than arteries from vehicle-treated control rats (P less than 0.05). Serum creatinine levels were not significantly different in the two groups. These findings indicate that cyclosporine is a potential inducer of endothelin release from the mesenteric artery.

Animals↗
PubMed1992

Molecular genetic analysis of HLA-DR and HLA-DQ genes among anti-U1-70-kd autoantibody positive connective tissue disease patients.

OBJECTIVE: We have recently found that the presence of autoantibodies against the 70-kd polypeptide of U1 RNP (U1-70-kd) is associated with HLA-DR4 and DR2. To further characterize this association, we performed a molecular genetic analysis of HLA-DR and DQ genes among patients with autoantibodies against U1-70-kd. METHODS: The polymerase chain reaction (PCR), sequence-specific oligonucleotide hybridization, and solid-phase direct DNA sequencing of PCR-amplified DNA were utilized to analyze HLA-DRB1, DRB5, DQA1, and DQB1 genes. RESULTS: A comprehensive analysis of HLA-DRB1, DRB5, DQA1, and DQB1 from 27 patients and controls identified shared amino acids FDYFYQA (Phe, Asp, Tyr, Phe, Tyr, Gln, Ala) at positions 26, 28, 30-32, 70, and 73 of HLA-DRB1 on disease-associated haplotypes. CONCLUSION: A common cluster of shared amino acids, or a shared epitope, identified within HLA-DRB1 among anti-U1-70-kd autoantibody positive connective tissue disease patients may be important in regulating an autoimmune response to the U1-70-kd antigen.

Amino Acid Sequence↗
PubMed1992

The replication origin of proplastid DNA in cultured cells of tobacco.

When tobacco suspension culture line BY2 cells in stationary phase are transferred into fresh medium, replication of proplastid DNA proceeds for 24 h in the absence of nuclear DNA replication. Replicative intermediates of the proplastid DNA concentrated by benzoylated, naphthoylated DEAE cellulose chromatography, were radioactively labelled and hybridized to several sets of restriction endonuclease fragments of tobacco chloroplast DNA. The intermediates hybridized preferentially to restriction fragments in the two large inverted repeats. Mapping of D-loops and of restriction fragment lengths by electron microscopy permitted the localization of the replication origin, which was close to the 23S rRNA gene in the inverted repeats. The replication origins in both segments of the inverted repeat in tobacco proplastid DNA were active in vivo.

Autoradiography↗
PubMed1992

Radioimmunolocalization of the monoclonal antibody J28 in early transformation stages in N-nitrosobis(2-hydroxypropyl)amine-induced pancreatic tumors in the Syrian golden hamster.

We describe the in vivo localization of radiolabeled mAb J28, a murine monoclonal antibody characterizing the oncodevelopmental human fetoacinar pancreatic (FAP) protein, at different stages of chemical induction of pancreatic tumors in the Syrian golden hamster. Before doing localization studies in this model, we looked at the cross-reactivity of mAb J28. Semiquantitative dot-blot analysis demonstrated that the antigen recognized in hamster pancreas has an oncodevelopmental expression pattern, while a molecular mass identical to that of human FAP was deduced from sodium dodecyl sulfate/polyacrylamide gel electrophoresis/nitrocellulose immunoblot. 125I-labeled mAb J28 was administered through micro-osmotic pumps to hamsters treated with N-nitrosobis(2-hydroxypropyl)amine (BHP). This was done at three intervals that roughly correspond to the latent period, pretumoral stages, and terminal cancerogenesis in two independent groups of hamsters. Both studies allowed similar results: (a) mAb J28 accumulated almost specifically in the pancreas; (b) maximal accumulation was associated with pleomorphic alterations of the acinar cell tissue at pretumoral stages; (c) no accumulation was found in the case of adenocarcinoma of the pancreas. It is concluded that FAP behaves as a marker of preneoplastic lesions, and therefore that radioimmunoimaging with mAb J28 might help with early diagnosis of pancreatic cancer.

Animals↗
PubMed1992

Clinicopathologic study of alcohol-like liver disease in non-alcoholics; non-alcoholic steatohepatitis and fibrosis.

Alcohol-like liver injury (ALLI) in non-alcoholics has not been elucidated in Japan. The present study attempted to characterize the clinicopathologic features of ALLI in routine liver biopsies. ALLI was found in 1% of 561 biopsy cases obtained from 1988 to May, 1991 at Kanazawa University Hospital. Laboratory data characteristically showed only a mild to moderate degree of dysfunction, and none of the cases exhibited jaundice. Hepatic histology showed a mild to moderate degree of perivenular, pericellular and/or portal stellate fibrosis with a varying degree of fatty change and inflammatory cell infiltration. Portal stellate fibrosis with a varying degree of cell infiltration was more severe than the centrilobular or pericellular fibrosis in all cases. Intralobular inflammatory cell infiltration was associated with spotty or single hepatocyte necrosis, but extensive hepatocyte necrosis was not observed. Neutrophil infiltration was absent or minimal, and lymphocytes predominated in all cases. Mallory bodies were rare and were found in a few hepatocytes of only one of the 7 cases. The above histologic findings in ALLI were very similar to those seen in liver disease in Japanese alcoholics, and were somewhat different from ALLI reported in Western countries. In cases in which hepatic fibrosis, characterized by pericellular, perivenular or portal stellate fibrosis dominated without apparent hepatic necrosis and inflammation, the term "non-alcoholic steatofibrosis" is more suitable to depict its liver histology, being very similar to the alcoholic fibrosis frequently seen in Japanese alcoholics.

Adult↗
PubMed1992

Determination of urinary 18-hydroxycortisol in the diagnosis of primary aldosteronism.

Urinary excretion of 18-hydroxycortisol (18-OHF), 18-hydroxycorticosterone (18-OHB) and aldosterone 18-glucuronide (Aldo-18-glu) was measured in 10 patients with primary aldosteronism; 5 with aldosterone-producing adenoma (APA) and 5 with idiopathic hyperaldosteronism (IHA), 10 patients with essential hypertension (EHT) and 11 normotensive subjects. In EHT patients, urinary 18-OHF (172 +/- 15 micrograms/24h) and 18-OHB (3.1 +/- 0.6 micrograms/24h) values were not significantly different from 18-OHF (142 +/- 35 micrograms/24h) and 18-OHB (3.6 +/- 0.5 micrograms/24h) in the controls. Urinary 18-OHF values were significantly higher in APA (640 +/- 213 micrograms/24h) when compared with controls and EHT, whereas 18-OHB (11.3 +/- 1.5 micrograms/24h) values were only slightly elevated. Both 18-OHF and 18-OHB were significantly increased in APA compared with 18-OHF (232 +/- 56 micrograms/24h) and 18-OHB (4.6 +/- 0.3 micrograms/24h) in IHA. The two urinary steroids, especially 18-OHF proved to be a useful marker for the diagnosis of APA, confirming the previous findings. Aldo-18-glu was not significantly different between APA and IHA. In normal subjects when sodium intake was restricted to 48meq/day for four days the urinary 18-OHF was increased two fold to 383 +/- 59 micrograms/24h (p less than 0.01 vs control period) associated with comparable rise in plasma renin activity. This suggests that the biosynthesis of 18-OHF is partly under control of renin-angiotensin axis in normal subjects.

18-Hydroxycorticosterone↗
PubMed1992

Mechanism of the membrane depolarization induced by oxidative stress in guinea-pig ventricular cells.

Mechanism of the membrane depolarization induced by oxidative stress was examined using ion-selective microelectrode and patch clamp techniques. In guinea-pig papillary muscles stimulated at 0.5 Hz, cumene hydroperoxide (CH) at a concentration of 300 microM decreased the resting membrane potential and shortened the action potential, concomitantly with muscle contracture. The membrane depolarization was not associated with a significant decrease in intracellular potassium ion activity, indicating that the depolarization is not due to a decrease in potassium equilibrium potential resulting from leak of intracellular K+. In isolated guinea-pig ventricular cells. CH (10-30 microM) consistently decreased the inward rectifier potassium current and slightly decreased the calcium current. In cell-attached patches CH inhibited the opening of the inward rectifier K+ channel without affecting the unit amplitude of the single channel current. Thus, the depolarization of the resting membrane induced by oxidative stress is, at least in part, due to the inhibition of the inward rectifier K+ channel activity, and may play an important role in the genesis of reperfusion-induced arrhythmias.

Animals↗
PubMed1992

GM-CSF and G-CSF stimulate the synthesis of histamine and putrescine in the hematopoietic organs in vivo.

Histamine and putrescine (a precursor of polyamines) are formed by histidine decarboxylase (HDC) and ornithine decarboxylase (ODC), respectively. Within a few hours after injection of a lipopolysaccharide (LPS) into mice, HDC is induced in the liver, spleen, lung and bone marrow, and ODC is induced in the liver, spleen and bone marrow. Since LPS is known to stimulate the production of various cytokines, the abilities of various cytokines to induce HDC and ODC in the tissues of mice were examined. IL-2, IL-6, IL-8, IFN gamma and M-CSF were ineffective. IL-1 alpha, IL-1 beta, TNF alpha and TNF beta induced HDC and ODC, as does LPS. On the other hand, GM-CSF and G-CSF induced HDC and ODC only in the spleen and bone marrow within a few hours after their injection. These results suggest that, in addition to their roles in inflammation or immune responses, HDC and ODC are also involved in an early stage of hematopoiesis.

Animals↗
PubMed1992

Protective effect of SPR-901 (RBS) on the decrease of peripheral leukocyte number in 5-fluorouracil-treated mice.

5-Fluorouracil (5-FU) induces a decrease in the number of peripheral leukocytes (leukopenia), which is one of the major obstacles in the chemotherapy of cancer. The number of peripheral leukocytes decreased by day 4 in mice injected i.p. with 130 mg/kg of 5-FU and recovered to the normal level by day 8. Such a decrease by 5-FU was prevented to some extent by the oral administration of 30 mg/kg/day of SPR-901. Proliferative responses of bone marrow cells to granulocyte/macrophage colony stimulating factor (GM-CSF) or granulocyte colony stimulating factor (G-CSF) were suppressed by 5-FU treatment and their recoveries were enhanced by SPR-901. The serum level of IL-6 in 5-FU-treated mice was increased by SPR-901. All of the mice treated with 300 mg/kg of 5-FU in combination with SPR-901 survived over 15 days, however, only 4 of 10 mice treated only with 300 mg/kg of 5-FU survived. These results suggest that SPR-901 acts on macrophages directly or indirectly, giving rise to the enhanced production of IL-1, IL-6, and other factors. Some of the factors derived from SPR-901 activated macrophages, perhaps mainly IL-6, act on the early stage of development of multipotent bone marrow progenitors synergistically with GM-CSF.

Animals↗
PubMed1992

Examination by site-directed mutagenesis of the amino acid residues of the thermostable direct hemolysin of Vibrio parahaemolyticus required for its hemolytic activity.

The amino acid sequences of eight variants of thermostable direct hemolysin (TDH) and a TDH-related hemolysin, deduced from nucleotide sequences, were compared. Fourteen amino acid residues, mainly within conserved regions, were chosen to prepare mutant TDHs with one or two amino-acid replacements by site-directed mutagenesis. Of the 25 mutant TDHs prepared, those with replacements of Trp65 (Trp65-His65, Trp65-Tyr65, Trp65-Leu65) or Leu66 (Leu66-Ser66) showed very significant reduction (more than 150-fold) of hemolytic activity. Several other mutant TDHs with replacements at Lys45, Arg46 and Gly90 showed weaker hemolytic activity than the wild-type TDH, but extents of reduction in hemolytic activity were much less than for mutant TDHs with replacements at Trp65 and Leu66. These results indicate that Trp65 and Leu66 are very important for the hemolytic activity of TDH.

Amino Acid Sequence↗