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Biomedical subjects

T Mizoguchi

Publications and source records attributed to T Mizoguchi.

At least 109 records · Page 6Linked to original sources
PubMed1993

Cloning and characterization of two cDNAs encoding casein kinase II catalytic subunits in Arabidopsis thaliana.

Two cDNA clones, ATCKA1 and ATCKA2, encoding casein kinase II (CKII) catalytic subunits, were cloned from Arabidopsis thaliana and their nucleotide sequences were determined. Both cDNAs contain 999 bp open reading frames and are 94% identical on the amino acid sequence level. The deduced amino acid sequences of ATCKA1 and ATCKA2 are very similar to that of the human CKII catalytic alpha subunit (72% homology). Northern blot analysis indicates that the ATCKA1 and ATCKA2 mRNAs are present in all plant organs, but that ATCKA1 transcript levels are quite low compared to those of ATCKA2. Genomic Southern blot analysis suggests that there are at least three CKII genes in the A. thaliana genome. We expressed the ATCKA1 and ATCKA2 cDNAs in Escherichia coli using a pET vector derivative and analyzed the expressed protein in vitro. The expressed ATCKA1 protein phosphorylated casein using either ATP or GTP. This activity was inhibited by heparin, indicating that the expressed protein has activity similar to those reported for animal and yeast CKII.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of carbonyl reductases from bovine liver cytosol and microsome. The cytosolic enzyme has a novel 3 alpha/17 beta-hydroxysteroid dehydrogenase activity.

1. Carbonyl reductase, which is distributed in both cytosolic and microsomal fractions in bovine liver, were purified to homogeneity on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis and shown to have molecular weights of 32 kDa and 68 kDa, respectively. 2. Both carbonyl reductases can catalyze the reduction of many carbonyl compounds including ketone, quinones and aldehyde with relatively low Km values. 3. From the absorption spectrum result, microsomal carbonyl reductase closely resembles cytochrome P-450 reductase. 4. Cytosolic carbonyl reductase is a novel enzyme which can act on both testosterone and androsterone at low concentration.

17-Hydroxysteroid Dehydrogenases↗
PubMed1993

Urinary thrombomodulin, its isolation and characterization.

Two major molecular forms of thrombomodulin fragments present in urine were isolated from human urine by four sequential steps of column chromatography. The apparent molecular weights of these thrombomodulins estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis were 98,000 (type I) and 90,000 (type II) with dithiothreitol, and 60,000 (type I) and 55,000 (type II) without dithiothreitol. The isoelectric points of the type I and type II molecules were 2.5 and 3.8, respectively. From sequence analyses, both forms were revealed to have identical 468 amino acid sequences, Ala1-Asp468, lacking 29 amino acids of the carboxyl-terminal sequence of intact cellular thrombomodulin. Major structural differences between type I and type II were observed in the carbohydrate composition: type II had less galactosamine content than type I. Both types were active in thrombin inhibition and protein C activation, although the activities were significantly less than those of intact cellular thrombomodulin. Type I had twice as much inhibitory activity on thrombin clotting activity as type II, whereas type II was more effective as a cofactor for thrombin-catalyzed protein C activation than type I.

Amino Acid Sequence↗
PubMed1993

Inhibition of bovine leukocyte thioltransferase by anti-inflammatory drugs and anti-histaminic drugs.

Thioltransferase was partially purified from bovine leukocyte using sonication, heat treatment at pH 5.1, Sephadex G-50 gel filtration and isoelectric focusing techniques. Isoelectric point (pI) of 8.3 for leukocyte thioltransferase was quite different from pI of 6.5 for erythrocyte enzyme. Bovine leukocyte thioltransferase was employed in a study on the influence of 12 anti-inflammatory drugs and 7 anti-histaminic drugs. Piroxicam which is a well-known anti-inflammatory drug demonstrated the most powerful inhibitory effect on enzyme activity. Inhibition of piroxicam was noncompetitive, and the Ki value measured 55.0 microM in the experiment involving bovine leukocyte enzyme. Tranilast which is a typical anti-histaminic drug most strongly inhibited the enzyme activity and the Ki value of the medicine was 20.3 microM (noncompetitive). Bovine liver and erythrocyte thioltransferases also were effectively inhibited by both medicines similar to leukocyte enzyme.

Animals↗
PubMed1993

Analysis of phosphorylation of wheat elongation factor 1 beta and beta' by casein kinase II.

The purified casein kinase II (CK II) from Arabidopsis thaliana phosphorylates wheat elongation factor 1 beta (EF-1 beta), but not elongation factor 1 beta' (EF-1 beta'), which lacks a serine residue in the conserved phosphorylation site. Both EF-1 beta and beta' subunits, with similar functions, seem to undergo different regulation despite the partial amino acid sequence of EF-1 beta being similar to that of EF-1 beta'.

Amino Acid Sequence↗
PubMed1993

Comparison of purified lens glutathione S-transferase isozymes from rabbit with other species.

Two glutathione S-transferase (GST) isozymes, GST-rl1 and GST-rl2, were purified from rabbit lenses and their properties were compared with those of other animals. GST-rl1 and GST-rl2 are dimeric enzymes whose subunit sizes are 24,000 and 21,500, respectively. The substrate specificities and inhibitor sensitivities of GST-rl1 and GST-rl2 are different from each other and from those of the isozymes from other animals. GST-rl1 immunologically crossreacted with the antibody against class mu GST (rat GST Yb1-Yb1), and GST-rl2 crossreacted with the antibody against class pi GST (rat GST Yp-Yp). N-Terminal amino acid sequences of GST-rl1 and GST-rl2 have great homology with other class mu and class pi enzymes, and thus indicate that they belong to class mu and class pi, respectively. Class pi GST-rl2 is inactivated by 1,2-naphthoquinone, an oxidized metabolite of naphthalene, but class mu GST-rl1 is insensitive to it. These results are similar to those of class pi pig lens GST and class mu bovine lens GST. Thus, the expression pattern of GST isozymes in lens varies with animal species, and may relate to their variation in sensitivity to oxidative stress.

Amino Acid Sequence↗
PubMed1993

Modulation of 3 alpha-hydroxysteroid dehydrogenase activity by the redox state of glutathione.

3 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.50), purified to homogeneity from rat liver, was strongly inactivated by incubation with a disulfide such as GSSG, L-cystine or L-cystamine, as well as an SH-reagent such as DTNB (5,5'-dithiobis(2-nitrobenzoic acid)), NEM (N-ethylmaleimide) or iodoacetic acid. The inactivation advanced with incubation time. Coenzyme (NADP+) completely protected the enzyme from this inactivation by disulfides, but neither of the substrates (androsterone and benzenedihydrodiol) did. The activity of inactivated enzyme was restored by treatment with thiols such as DTT (dithiothreitol) or GSH. In the GSH/GSSG redox buffer, the enzyme existed in an equilibrium between active (reduced) and inactive (oxidized) forms.

3-Hydroxysteroid Dehydrogenases↗
PubMed1993

[Successful weaning from mechanical ventilation in an aged subject with severe apneas due to brain damage].

A 73-year-old male was admitted to Tokyo University Hospital due to cardiac arrest secondary to ventricular arrhythmias. Although the patient survived after cardiopulmonary resuscitation, he needed mechanical ventilation. When we tried to wean the patient from mechanical ventilation, we found that he suffered from frequent sleep apneas of over 40 times/hour. To evaluate the severity and frequency of the apneas, we performed polysomnographic study on the patient under assist ventilation. The polysomnographic study revealed that frequency of apneas was very high but the magnitude of desaturation was not so severe during the night. Since we confirmed the lowest SaO2 in this patients was over 90% during day and night, we tried to wean from the patient from the ventilator again and succeeded. The clinical application and usefulness of nocturnal ventilatory monitoring for patients with mechanical ventilation was discussed.

Aged↗
PubMed1993

[Concentrations of multiple neurochemicals in the cerebrospinal fluid of patients with senile dementia and the relationship to alpha 1-antichymotrypsin].

The authors investigated the concentrations of multiple neurochemicals (6 kinds of catecholaminergic and 5 kinds of indolaminergic substances) in the lumbar cerebrospinal fluid (CSF) of patients with and without senile dementia (13 Alzheimer type (AD), 7 vascular type (VD), 11 Parkinson's disease (PD) and 9 non-demented controls (C)) by means of a neurochemical analyzing system (Neurochem, ESA). By means of the enzyme-linked immunosorbent assay (ELISA), we also determined the concentration of alpha 1-antichymotrypsin (ACT) in the CSF, which may be a possible diagnostic biochemical marker of the senile dementia of Alzheimer type. ACT in CSF was significantly higher in the AD group. It correlated negatively with Hasegawa's dementia scale (HDS) significantly. It also correlated negatively with the concentration of HVA significantly and showed tendency to correlate with the concentrations of dopamine and the ratio of kynurenine and tryptophan (KYN/TRP). Each dementia group showed characteristic concentration patterns of neurochemicals (DA, HVA, MHPG/NE, KYN/TRP, and 5-HIAA/5-HT). Our approach may provide a new quantitative method to diagnose geriatric neuropsychological diseases as well as senile dementia.

Aged↗
PubMed1993

[Reliability and validity of a Japanese version of the Dementia Behavior Disturbance Scale].

Since behavioral disturbance among patients with dementia is a great burden for their caregivers, quantification of behavioral disturbance is essential in determining disease severity and assessing the impact of the disease on caregivers. However, the method of its quantification for objective assessment is not established yet. We studied the reliability and validity of a Japanese version of the Dementia Behavior Disturbance Scale (DBD Scale) which was originally developed by Baumgarten et al. We also studied the relationship between DBD scores and the degree of burden felt by caregivers. Our subjects consisted of 27 cases with dementia (mean age 77.7 years), and 17 cases of patients with neurological disorders without dementia (76.8 years), and 10 institutionalized patients with dementia (82.3 years). The test-retest reliability, internal consistency, and inter-rater reliability were very good; the coefficient of correlation between DBD scores at the two interviews was 0.96, the coefficient of internal consistency was 0.95, and the intraclass correlation coefficient was 0.71 +/- 0.10. DBD scores correlated significantly with SPMSQ errors and caregivers' burden; r = 0.54 and 0.53, respectively. Our results indicate that the DBD Scale is highly reliable, and may be useful for objective assessment of behavioral disturbance and caregivers' burden.

Aged↗
PubMed1992

Characterization of a gene that encodes a homologue of protein kinase in Arabidopsis thaliana.

Cloning and analysis of a gene that encodes a homologue of protein kinase (PK) from Arabidopsis thaliana is reported. Oligodeoxyribonucleotides (oligos) corresponding to conserved regions in catalytic domains of various PKs were used for polymerase chain reaction (PCR) amplification with genomic DNA from A. thaliana as template, in an attempt to identify genes encoding PK in plants. We obtained several amplified DNA fragments that encoded part of a PK. We screened a genomic DNA library of A. thaliana with these oligos or PCR fragments as probes. Three genomic clones were obtained and one of them, named Atpk7, was sequenced and analyzed. Atpk7 was demonstrated by PCR to contain an intron. The mRNA transcribed from Atpk7 was detected predominantly in root tissue by Northern blot analysis. The transcription start point was determined by primer extension. The deduced amino acid (aa) sequence of the putative product of Atpk7 resembles those of S6 kinases, cyclic nucleotide-dependent PKs and calcium-dependent PKs. From this comparison of aa sequences, the ATPK7 protein is considered to be a member of a novel subfamily of Ser/Thr PKs in plants.

Amino Acid Sequence↗
PubMed1992

Characterization of bovine liver cytosolic 3 alpha-hydroxysteroid dehydrogenase and its aldo-keto reductase activity.

1. 3 alpha-Hydroxysteroid dehydrogenase was purified to homogeneity from bovine cytosolic fraction, which was monomeric and its molecular weight was estimated to be about 35 kDa. 2. The enzyme had ability to catalyze NADP(H)-dependent oxidoreduction of position 3 alpha-hydroxy and keto group of steroids and also could catalyze the reduction of some ketones and quinones. 3. In addition, benzenedihydrodiol was one of the substrates of dehydrogenase activity with NADP+. 4. Indomethacin, synthetic steroids and SH-reagents were potent inhibitors for this enzyme. 5. Inactivation of the enzyme by GSSG-treatment was restored to its original activity by the addition of DTT. 6. The presence of coenzyme, 0.33 mM NADP+, completely protected from the DTNB-inactivation. 7. Bovine liver cytosolic enzyme immunologically crossreacted with rat liver 3 alpha-hydroxysteroid dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

Purification and characterization of glutathione S-transferase isozymes in dog lens.

1. Two isozymes of glutathione S-transferase (GST-dl1 and GST-dl2) were purified to homogeneity from dog lens. 2. The subunit size and the isoelectric point were determined to be 24,000 and > pI 9.5 for GST-dl1 and 22,000 and pI 8.1 for GST-dl2. 3. It was judged that GST-dl1 is a class alpha enzyme and GST-dl2 belongs to class pi on the basis of their immunological properties and N-terminal amino acid sequences. 4. The expression pattern of glutathione S-transferase isoenzymes in dog lens is different from that in pig, rat and bovine lenses.

Amino Acid Sequence↗
PubMed1992

Study on human erythrocyte thioltransferase: comparative characterization with bovine enzyme and its physiological role under oxidative stress.

Thioltransferase, an enzyme which catalyzes the thiol/disulfide exchange reaction in the presence of GSH, was purified to homogeneity on 15% SDS-PAGE from human (36,000-fold purification) and bovine (23,000-fold) erythrocyte hemolysates. These enzymes had similar properties in their monomeric structures (M(r) = 11,000) and broad specificities for substrates ranging from low-molecular disulfides (S-sulfocysteine, cystamine, and cystine) to protein disulfides (trypsin and insulin). They were highly sensitive to SH-reagents (monoiodoacetic acid and mercuric chloride), but were protected from inactivation by the presence of disulfides (GSSG, cystamine, and cystine). Phosphofructokinase and pyruvate kinase that had been inactivated by disulfides were reactivated effectively by the addition of thioltransferase with GSH. In addition, disulfides in membrane proteins of human erythrocytes that have been oxidatively damaged by diamide treatment were reduced to the SH-free form more effectively by incubation with thioltransferase.

Amino Acids↗
PubMed1992

A novel dihydrodiol dehydrogenase in bovine liver cytosol: purification and characterization of multiple forms of dihydrodiol dehydrogenase.

Three enzymes (DD1, DD2, and DD3) having dihydrodiol dehydrogenase activity were purified to homogeneity from bovine cytosol. DD1 and DD2 were identified as 3 alpha-hydroxysteroid dehydrogenase and high-Km aldehyde reductase, respectively, as judged from their molecular weights, substrate specificities and inhibitor sensitivities. DD3 was a unique enzyme which could specifically catalyze the dehydrogenation of trans-benzenedihydrodiol and trans-naphthalenedihydrodiol without any activity toward the other tested alcohols, aldehydes, ketones, and quinones. The Km value of DD3 (0.18 mM) for benzenedihydrodiol was lower than those of other dihydrodiol dehydrogenases so far reported. DD3 immunologically crossreacted with DD1, but showed no crossreactivity with DD2. Additionally, DD3 was inhibited in a competitive manner, with a low Ki value of 1 microM, by androsterone, which was a good substrate for DD1. It was assumed that DD3 is a novel enzyme which is specific to dihydrodiols, exhibiting similarity to DD1 in immunological and structural properties.

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

Potentiation of the vincristine effect on P388 mouse leukemia cells by a newly synthesized dihydropyridine analogue, PAK-200.

A newly synthesized dihydropyridine analogue, 2-[benzyl(phenyl)amino]ethyl 1,4-dihydro-2,6-dimethyl-5-(5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorina n-2-yl)-1- (2-morpholinoethyl)-4-(3-nitrophenyl)-3-pyridinecarboxylate (PAK-200), at 1 microM completely reversed the resistance to vincristine in vincristine-resistant P388 mouse leukemia cells (P388/VCR), in vitro. PAK-200 at 2 microM inhibited the efflux of [3H]vincristine from P388/VCR and increased the accumulation of [3H]vincristine in P388/VCR to a level similar to that in P388 cells. P-Glycoprotein in membrane vesicles from P388/VCR cells was photolabeled with [3H]azidopine. The labeling was completely inhibited by 10 microM PAK-200. The calcium antagonistic activity of PAK-200 was about 1000 times lower than that of another dihydropyridine analogue, nicardipine. Experiments with P388 and P388/VCR-bearing mice showed that PAK-200 enhanced the effect of vincristine on both leukemia cells in vivo. These results suggest that PAK-200 interacts with P-glycoprotein and reverses drug resistance in P388 mouse leukemia cells in vitro, and that PAK-200 has an ability to potentiate the effect of vincristine on P388 mouse leukemia cells in vivo.

Animals↗