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Biomedical subjects

M Mizugaki

Publications and source records attributed to M Mizugaki.

At least 127 records · Page 7Linked to original sources
PubMed1991

Neocarzinostatin: selective tryptophan oxidation and neocarzinostatin-chromophore binding to apo-neocarzinostatin.

Neocarzinostatin (NCS), an antitumor protein antibiotic, is composed of apo-neocarzinostatin (apo-NCS) and neocarzinostatin-chromophore (NCS-chr), the principle of the biological activities of NCS. Apo-NCS having two tryptophan (Trp) residues at positions (39 and 83) was chemically modified by N-bromosuccinimide in a study on the correlation of the binding site(s) of NCS-chr. Selective oxidation of Trp residues was observed when NCS was titrated with N-bromosuccinimide. In contrast, non-selective oxidation of the two Trps on apo-NCS was observed and both Trp (39 and 83) of apo-NCS were titrated with N-bromosuccinimide. After selective oxidation, the remaining Trp residue of NCS was assigned as Trp (83). These results clearly indicate that the Trp (83) residue of apo-NCS changed from the "reactive type" to the "non-reactive type" after the binding of NCS-chr with apo-NCS. The fluorescence emission intensity of apo-NCS generated from the Trp (39) residue was quenched by NCS-chr. These data suggest that NCS-chr directly interacts with the Trp (39) residue and that a beta-sheeted loop containing the Trp (83) residue of apo-NCS changes the high-order structure upon binding with NCS-chr.

Amino Acid Sequence↗
PubMed1990

cDNA cloning of rat liver 2,4-dienoyl-CoA reductase.

cDNA clones of 2,4-dienoyl-CoA reductase were isolated from rat liver cDNA libraries constructed in phages lambda gt11 and lambda gt10. Hybrid selected translation analysis revealed that 2,4-dienoyl-CoA reductase was translated as a polypeptide with a molecular weight of about 36,000, which was about 3,000 molecular weight units larger than mature reductase. Sequencing analysis revealed that the open reading frame encoded a polypeptide consisting of 335 amino acid residues (predicted molecular weight = 36,132), which contained an N-terminal extension peptide of 34 amino acid residues (presequence) in addition to the mature enzyme. Thus, 2,4-dienoyl-CoA reductase is synthesized as a larger precursor polypeptide, and the N-terminal extension peptide may be acting as the mitochondrial import signal.

Amino Acid Sequence↗
PubMed1990

Purification of the multienzyme complex for fatty acid oxidation from Pseudomonas fragi and reconstitution of the fatty acid oxidation system.

The multienzyme complex for fatty acid oxidation was purified from Pseudomonas fragi, which was grown on oleic acid as the sole carbon source. This complex exhibited enoyl-CoA hydratase [EC 4.2.1.17], 3-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35], 3-oxoacyl-CoA thiolase [EC 2.3.1.16], cis-3,trans-2-enoyl-CoA isomerase [EC 5.3.3.3], and 3-hydroxyacyl-CoA epimerase [EC 5.1.2.3] activities. The molecular weight of the native complex was estimated to be 240,000. Two types of subunits, with molecular weights of 73,000 and 42,000, were identified. The complex was composed of two copies each of the 73,000- and 42,000-Da subunits. The beta-oxidation system was reconstituted in vitro using the multienzyme complex, acyl-CoA synthetase and acyl-CoA oxidase. This reconstituted system completely oxidized saturated fatty acids with acyl chains of from 4 to 18 carbon atoms as well as unsaturated fatty acids having cis double bonds extending from odd-numbered carbon atoms. However, unsaturated fatty acids having cis double bonds extending from even-numbered carbon atoms were not completely oxidized to acetyl-CoA: about 5 mol of acetyl-CoA was produced from 1 mol of linoleic or alpha-linolenic acid, and about 2 mol of acetyl-CoA from 1 mol of gamma-linolenic acid. These results suggested that the 3-hydroxyacyl-CoA epimerase in the complex was not operative. When the epimerase was by-passed by the addition of 2,4-dienoyl-CoA reductase to the reconstituted system, unsaturated fatty acids with cis double bonds extending from even-numbered carbon atoms were also completely degraded to acetyl-CoA.

Animals↗
PubMed1990

[Extra-weak chemiluminescence of drugs. X. Extra-weak chemiluminescence quenching component of rhubarb].

Active components contained in rhubarb (Rhei Rhizoma) that show a quenching effect of extra-week chemiluminescence (CL) arising from Maillard reaction, had been studied. As the result of fractionation and purification of rhubarb, CL quenching activity was found to exist mainly in the fraction of quinone derivatives as emodin, aloe-emodin or rhein. The authentic samples of these quinones showed high CL quenching activities.

Drugs, Chinese Herbal↗
PubMed1990

Replacement of L-T4 suppository in MMI treated rabbits.

L-T4 suppositories containing 50, 100 or 200 micrograms of L-T4 were given to rabbits treated with MMI. The serum T4 gradually increased within 3.5 and 6.5 hours following the use of 100 and 200 micrograms of L-T4 suppositories, respectively, without an acute increase in serum T3. The increase in serum T4 continued up to 48 hours. The area under the curve (AUC) for serum T4 was evidently dose-dependent. It is concluded from these results that the T4 suppository will be useful as a replacement therapy for patients with hypothyroidism.

Animals↗
PubMed1990

[Homicidal poisoning by aconite: report of a case from the viewpoint of clinical forensic medicine].

A case of homicidal poisoning by aconite is reported from the viewpoint of clinical forensic medicine and analytical chemistry. Jesaconitine was detected in the vomitus, stomach contents, plasma and urine at concentrations of 32.2, 5.48, 0.433 and 1.07 micrograms/ml, respectively. The total amount of jesaconitine in the stomach contents was 1.3 mg. Macroscopic autopsy revealed hemorrhagic pulmonary edema, and histologically, diffuse contraction-band necrosis was evident in the myocardium. Exogenous administration of adrenaline or endogenous release of catecholamines are known to induce hemorrhagic pulmonary edema and contraction-band necrosis, and aconitine is reported to cause release of endogenous catecholamines centrally and to induce neurogenic pulmonary edema and ventricular-type arrhythmia. Therefore, the chief cause of death in this case was considered to be aconite-induced centrogenic arrhythmia.

Aconitum↗
PubMed1990

[Molecular and immunological approach to hematological disease: detection and analysis of intracellular modified nucleosides by flow cytometry].

Modified nucleosides are components of ribosomal RNA (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA). 1-methyladenosine and pseudouridine are members of those modified nucleosides. The urinary concentration of 1-methyladenosine and pseudouridine of cancer patients are higher than that of healthy controls, and those compounds were reduced after effective chemotherapy. Thus those compounds might be expected to use as tumor markers. In this study cellular origin of 1-methyladenosine and pseudouridine were analysed about two tumor cell lines (HUT-102, THP-1), peripheral blood lymphocytes (PBL) from healthy adult and PBL under the phytohemagglutinin stimulation, by flow cytometric analysis and immunofluorescent staining of cellular RNA using monoclonal antibodies specific for 1-methyladenosine (AMA) and pseudouridine (APU). Both 1-methyladenosine and pseudouridine were detected in more than 90% of tumor cells above the thresholds of flow cytometric detection (Spectrum III, Ortho). The PBL under the PHA stimulation also tended to take the same way of the tumor cell lines, whereas few of the PBL contained 1-methyladenosine above the thresholds. According to the DNA analysis of those cell lines, high contents of the modified nucleosides in the cell might follow DNA synthesis, this leads to one reason for high levels of the urinary excretion of the modified nucleosides in cancer patient.

Adenosine↗
PubMed1989

Detection of elevated amounts of urinary pseudouridine in cancer patients by use of a monoclonal antibody.

Preparation of anti-pseudouridine monoclonal antibodies (MoAbs) and their applications for the quantitation of urinary pseudouridine in cancer patients are described. Seven MoAbs were selected. Five MoAbs were specific for pseudouridine and two MoAbs were cross-reactive with uridine. The most specific antibody, APU-6, was used in an enzyme-linked immunosorbent assay (ELISA) to determine urinary pseudouridine. Sensitivity was in the picomole range and the accuracy was nearly equal to that of the high performance liquid chromatography (HPLC) assay. The amount of pseudouridine in the urine of 28 healthy donors was 31.17 +/- 9.94 nmol/mumol creatinine. In 55% (35/63) of patients with cancer, urinary pseudouridine was elevated above the normal mean + 2 SD (51.04 nmol/mumol creatinine). Particularly, all of the patients (15/15) with leukemia and lymphoma had elevated levels of pseudouridine. These results suggest that urinary pseudouridine might be useful as a marker for leukemia and lymphoma.

Antibodies, Monoclonal↗
PubMed1989

Purification and characterization of N-ethylmaleimide reducing enzyme from Candida lipolytica.

N-Ethylmaleimide (NEM) reducing enzyme was purified to homogeneity from cell-free extracts of Candida lipolytica by chromatography techniques. The molecular weight of the native enzyme was estimated to be about 43,000 by gel filtration using Superose 12 and to be 47,000 by SDS-PAGE. This enzyme can use both NADPH and NADH as an electron donor, and catalyzes the reduction of the carbon-carbon double bond of five membered ring compounds which have two conjugated carbonyl groups on both sides of a double bond.

Amino Acids↗
PubMed1989

Characterization of secondary structure of neocarzinostatin apoprotein.

Characteristics of the secondary structure of neocarzinostatin apoprotein (apo-NCS) were examined by various means. Gaussian analysis of the Fourier-transform infrared (FT-IR) curve and curve-fitting of the circular dichroism (CD) spectrum for apo-NCS revealed that this peptide was abundant in beta-structures. In the presence of sodium dodecyl sulfate (SDS), the CD bands of NCS originating from phenylalanyl, tyrosyl, and cystinyl residues decreased, indicating a conformational change around the chromophore (CNS-chr). On the other hand, apo-NCS, in the SDS system, showed no change of these bands. We showed that the major parts of the protein moiety consist of beta-structures by measurements of the FT-IR and CD spectra of apo-NCS and a prediction of the secondary structure based on the amino acid sequence of the peptide. It seems that properties of the protein may be important to the hydrophobic interaction between NCS-chr and apo-NCS.

Amino Acid Sequence↗
PubMed1989

Feasibility of using [18O2]11-dehydrothromboxane B2 as an internal standard of immunoaffinity purification followed by gas chromatography/selected ion monitoring.

[1,1-18O2]11-Dehydrothromboxane B2 (11-dehydro-TXB2) was prepared by repetitive base-catalyzed hydrolysis of the lactone ring of its [1,1,11-18O3]-analogue, and evaluated for its suitability as an internal standard in gas chromatography/selected ion monitoring (GC/SIM) of 11-dehydro-TXB2. Use of the present [18O2]-analogue as an internal standard may make it a suitable candidate for specific immunoaffinity purification followed by GC/SIM.

Adult↗
PubMed1989

Synthesis of C-labeled imipramine and its biodistribution in mice: a potential tracer for positron emission tomography.

A tricyclic antidepressant, C-labeled imipramine was synthesized by N-methylation of desipramine with 11CH3I to assist in the imaging of the human imipramine receptor by positron emission tomography. The radiochemical yield after purification of 11C-imipramine by high performance liquid chromatography was 28-63% at a specific activity of 26-53 Ci/mmol. The time required for synthesis, including purification was 30 min from the end of 11CH3I trapping. The organ distribution of 11C-imipramine was investigated in mice at various times after i.v. injection. The main accumulation of radioactivity was in the kidney, followed by the lung and the heart. In the brain, the radioactivity levels in the hypothalamus and striatum were the highest and remained constant, differentiating them from other portions of the brain. Furthermore, the result of a binding assay with 3H-labeled imipramine suggested that the regional distribution of 11C-imipramine in the same mouse brain correlated to that of the high affinity imipramine binding site.

Animals↗
PubMed1989

Suppressive effect of 1-methyladenosine on the generation of chemiluminescence by mouse peritoneal macrophages stimulated with opsonized zymosan.

The preparation of an in vitro assay system for the immunosuppressive activities of modified nucleosides on macrophage (M phi) functions is described. Briefly, Listeria-elicited mouse peritoneal M phi s (Lm-M phi s) were treated with nucleosides in vitro for 18 hr at less than 1 mM concentration and chemiluminescence (CL) was measured after stimulation with opsonized zymosan. To confirm the usefulness of this assay (in vitro test), the immunosuppressive activities of 1-methyladenosine (m1 Ado) and adenosine (Ado) were determined by mouse Listeria infection (in vivo test), CL generation by M phi s obtained from the peritoneal cavity of nucleoside-treated mice (in vivo-in vitro test), and the in vitro test. The immunosuppressive activity of m1 Ado detected by the in vitro test was confirmed by the in vivo and the in vivo-in vitro tests. As for Ado, no immunosuppression was detected by the in vivo and the in vivo-in vitro tests through a potent suppressive effect was detected by the in vitro test. The ineffectiveness of Ado can be explained by the in vivo conversion of Ado to an inactive form. Thus, the proposed in vitro test seems to be useful, with the provision that the known in vivo metabolism is taken into consideration.

Adenosine↗
PubMed1989

Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase in rat liver. The effects of di-(2-ethylhexyl)phthalate, a peroxisome proliferator.

Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase (isomerase) was investigated in rat liver. Livers of di-(2-ethylhexyl)phthalate (DEHP)-treated or untreated rats were perfusion-fixed and embedded in Epon or Lowicryl K4M. By light microscopy, reaction deposits for the enzyme were present in the cytoplasmic granules of hepatocytes and interlobular bile duct epithelium. Weak staining was noted in sinus-lining cells. After administration of DEHP, the granular staining of the hepatocytes was markedly enhanced, whereas the staining reaction of the sinus-lining cells decreased. The isomerase staining pattern was quite similar to that of long-chain acyl-CoA dehydrogenase (a mitochondrial marker), but different from that of catalase (a peroxisomal marker). Under electron microscopy, gold particles for isomerase were seen to be confined mainly to mitochondria of the hepatocytes, the bile duct epithelial cells and sinus-lining cells. Peroxisomes were weakly labeled. After DEHP administration, the peroxisomes were markedly induced, but the mitochondria were not. Quantitative analysis showed that the induction of the peroxisomal isomerase was only 2-fold whereas the mitochondrial isomerase was enhanced about 5-fold, 40 times as high as the peroxisomal enzyme. The results show that the mitochondria are the main intracellular site for isomerase and the peroxisomes a minor site. The mitochondrial isomerase of the rat liver is markedly induced by peroxisome proliferators, DEHP and clofibrate.

Animals↗
PubMed1988

Purification and general properties of AMP deaminase from sheep brain.

AMP deaminase from sheep brain was purified to homogeneity on SDS-PAGE and its general properties were investigated. The native enzyme has a molecular weight of approximately 350,000 as estimated by gel filtration and it is composed of four identical subunits with a molecular weight of 85,000 each. The purified enzyme had a specific activity of 500 units/mg protein and shows a sigmoid-shaped AMP saturation curve in the presence of 100 mM KCl. This deaminase is strongly activated by ATP and inhibited by GTP. It slightly catalyzes the hydrolysis of adenosine monosulfate (AMS), dAMP, and adenosine phosphoramidate (APA). These catalytic properties resemble those of AMP deaminase from human liver.

AMP Deaminase↗
PubMed1988

Preparation of a monoclonal antibody specific for 1-methyladenosine and its application for the detection of elevated levels of 1-methyladenosine in urines from cancer patients.

A monoclonal antibody specific for a modified nucleoside, 1-methyladenosine, was prepared and characterized. This antibody, termed AMA-2, reacts with 1-methyladenosine and 1-methyladenine but not with other nucleosides, particularly methylated adenosines other than 1-methyladenosine and methylated guanosines, tested in this investigation. In our experiments, AMA-2 was used in an enzyme-linked immunosorbent assay (ELISA) system for the quantitation of the levels of 1-methyladenosine in urine. Sensitivity was in the picomole range and accuracy was nearly equal to that of the high-performance liquid chromatography (HPLC) assay system. Urinary levels of 1-methyladenosine in healthy donors and patients with various advanced cancers were determined by the inhibition ELISA. The amount of 1-methyladenosine in urine of 33 healthy donors was 1.91 +/- 0.66 nmol/mumol creatinine. In 54% (51/94) of patients, urinary 1-methyladenosine was elevated above the mean plus 2 standard deviations for the healthy donors (3.23 nmol/mumol creatinine). In patients with leukemia, esophageal cancer, stomach cancer, colon cancer, and bladder cancer, urinary levels of 1-methyladenosine were significantly elevated. In patients with leukemia, urinary 1-methyladenosine levels changed almost in parallel with the change in the clinical response during chemotherapy. These results suggest that urinary 1-methyladenosine might be useful in monitoring the effectiveness of therapy.

Adenosine↗