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S Ohtaki

Publications and source records attributed to S Ohtaki.

At least 73 records · Page 4Linked to original sources
PubMed1990

Extracellular ATP-induced production of hydrogen peroxide in porcine thyroid cells.

Hydrogen peroxide (H2O2) is an essential substrate for the peroxidase reaction in thyroid hormone biosynthesis. We demonstrated the production of H2O2 from porcine thyroid cells stimulated with extracellular ATP, using a scopoletin-horseradish peroxidase (HRP) system. Incubation of isolated cells for 1 day in the presence of 10% (v/v) newborn calf serum was necessary for the detection of induction by ATP of H2O2 production. The rate of H2O2 production induced by the addition of ATP increased in a dose-dependent manner, and the concentration of ATP required for half-maximum stimulation was about 10 mumol/l. ADP and GTP were also effective, but only at higher concentrations than ATP. In the absence of extracellular Ca2+, the production rate was very low. Production of H2O2 from thyroid cells was also measured by a method which discriminated between H2O2 and superoxide anion (O2-); in this, diacetyldeuteroheme-substituted HRP was employed as the trapping agent for both O2 metabolites. The thyroid cells produced H2O2, but not O2-, when the cells were stimulated by extracellular ATP.

Adenosine Triphosphate↗
PubMed1990

[Thyroid peroxidase].

Thyroid Peroxidase (TPO) is a key enzyme in the synthesis of thyroid hormone and is a major thyroid microsomal antigen corresponding to anti-microsomal autoantibodies in thyroid autoimmune diseases. We studied the autoantigenicity, thyroiditogenicity and gene structure of TPO. In micro-ELISA using human TPO as a target, all sera from patients with anti-microsomal antibodies contained IgG class of antibodies to TPO and some sera had IgM class of antibodies. The competitive inhibition test revealed that TPO is the major thyroid microsomal antigen. Experimental murine thyroiditis was successfully induced by the immunization of porcine TPO. Susceptibility of thyroiditis in each strain was very different from that of thyroiditis induced by thyroglobulin. T-cell line specific for porcine TPO could mediate thyroid lesions. Two kinds of full length cDNAs to human TPO were isolated from cDNA library which was constructed from mRNA purified from thyroid with Graves' disease. The longer one consisted of 3,048 nucleotides and its open-reading-frame was likely to encode 933 amino acids. The shorter one lacked 171 nucleotides at the middle portion of the longer one. The structure-gene for human TPO was located on 2q and consisted of 17 exons. One hundred and seventy-one nucleotides deleted in the shorter cDNA exactly corresponded to the 10th exon.

Animals↗
PubMed1989

Characterization of rat and human steroid sulfatases.

Rat and human steroid sulfatases were purified from liver and placenta, respectively, by the same procedure. The rat and human enzymes were solubilized with Triton X-100, and purified by immunoaffinity chromatography with a monoclonal antibody showing high binding activities to both the enzymes. They were further purified by high-pressure anion-exchange chromatography to compare their structural and catalytic properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that both enzymes had a molecular weight of 62,000. The enzymes had similar amino acid compositions and amino-terminal amino acid sequences. Significant differences of the optimum pH, Michaelis constant and maximum velocity were observed between these enzymes. The optimum pH of each enzyme varied from 6.0 to 8.0, depending on substrates and with or without Triton X-100. In detergent-free media, steroid sulfates competitively inhibited the ability of these enzymes to hydrolyze 4-nitrophenyl sulfate. In media containing Triton X-100, on the other hand, the inhibition types of the steroid sulfates on the hydrolyzing activities of the rat and human enzymes were noncompetitive- and mixed-types, respectively.

Amino Acid Sequence↗
PubMed1989

Thyroglobulin-mediated one- and two-electron oxidations of glutathione and ascorbate in thyroid peroxidase systems.

The one- or two-electron oxidation of thyroglobulin by the thyroid peroxidase system was found to be regulated by the iodine content of thyroglobulin. The catalytic intermediate of thyroid peroxidase observed at steady state of the reaction was Compound I and II when the iodine content in thyroglobulin was 0.2 and 0.7%, respectively, apparent rate constants for the rate-limiting steps being estimated at 4.7 x 10(7) and 4.8 x 10(4) M-1 S-1. The thyroglobulin-mediated oxidation of GSH occurred by way of two-electron transfer at 0.2% iodine content and by way of one-electron transfer at 0.7% iodine content. The spin-trapping experiment with 5,5-dimethyl-1-pyrroline-N-oxide showed that glutathione radicals were formed in the latter reaction but not in the former. In the reactions of thyroid peroxidase, the one- and two-electron oxidations of ascorbate were also mediated by 0.2 and 0.7% iodine thyroglobulins, respectively. The reactions were analyzed and mimicked with the use of p-cresol and p-acetaminophenol as a mediator in the reactions of lactoperoxidase and thyroid peroxidase.

Acetaminophen↗
PubMed1989

cDNA-directed expression of human thyroid peroxidase.

A human thyroid peroxidase cDNA, hTPO-1 [(1987) Proc. Natl. Acad. Sci. USA 84, 5555-5559], was expressed in human Hep G2 cells using a vaccinia virus cDNA-expression system. When examined by immunoblot analysis, the level of hTPO-1 protein expression reached a maximum approx. 24 h after infection and remained at a similar level up to 72 h post-infection. The expressed protein was enzymatically active as measured by guaiacol oxidation. Monoclonal antibody-assisted immunoaffinity column chromatography was used for partial purification of vaccinia-expressed hTPO-1, resulting in more than 300-fold higher specific activity and a measurable difference spectrum of the hTPO-1 (Fe3+)-CN complex.

Blotting, Western↗
PubMed1989

Structure of the human thyroid peroxidase gene: comparison and relationship to the human myeloperoxidase gene.

All exons of the human thyroid peroxidase gene were cloned from phage and cosmid libraries and sequenced, including 2599 base pairs of upstream DNA. The gene contains 17 exons and covers at least 150 kilobase pairs of chromosome 2. The transcription start site was identified by both S1 mapping and primer extension; a typical TATA box was found 25 bases upstream of the putative start site. A comparison of the gene structures of thyroid peroxidase and a granulocyte protein, myeloperoxidase, revealed that the positions of the 3rd through 11th exon-intron junctions in thyroid peroxidase coincide exactly with those of the 2nd through 11th exon-intron junctions in myeloperoxidase except the 7th myeloperoxidase junction, that does not have any counterpart in thyroid peroxidase. The amino acid codon separation pattern in each junction is well conserved between both enzymes. Four exons, unique to thyroid peroxidase, are located at the 3' end of the gene (exons 13-16), each of which encompasses a different protein module. Three of these modules, representing exons 13, 14, and 15, bear significant similarities to C4b-beta 2 glycoprotein, the EGF-LDL receptor, and a typical transmembrane domain, respectively. The genes coding for these modules were probably fused to an ancestral peroxidase gene to generate the present thyroid peroxidase gene. The data suggest that intron loss, and/or insertion, and exon shuffling have played important roles in the evolution of the thyroid peroxidase gene.

Amino Acid Sequence↗
PubMed1989

Resonance Raman characterization of hog thyroid peroxidase. An SERRS study.

Resonance Raman (RR) spectra of hog thyroid peroxidase (TPO) were observed for the first time and compared with those of lactoperoxidase (LPO) and horseradish peroxidase (HRP). Since TPO purified by monoclonal antibody-assisted immunoaffinity chromatography was strongly fluorescent, the surface enhancement technique using Ag colloid adsorption was used for the oxidized form, but ordinary RR spectra could be obtained for the reduced form. The RR spectra of TPO were distinct from those of HRP in both the oxidized and reduced states and indicated the presence of six-coordinated iron-protoporphyrin.

Animals↗
PubMed1989

Superoxide anion is the initial product in the hydrogen peroxide formation catalyzed by NADPH oxidase in porcine thyroid plasma membrane.

The plasma membrane fraction from porcine thyroid is known to exhibit an NADPH-dependent production of hydrogen peroxide (H2O2), which is utilized for the oxidative biosynthesis of thyroid hormones catalyzed by thyroid peroxidase. The H2O2 formation is cyanide-insensitive, ATP-activatable, and Ca2+-dependent (Nakamura, Y., Ogihara, S., and Ohtaki, S. (1987) J. Biochem. (Tokyo) 102, 1121-1132). It remains unknown, however, whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of superoxide anion (O2-). We therefore attempted to analyze the mechanism of H2O2 formation by utilizing a new method for the simultaneous measurement of O2- and H2O2, in which diacetyldeuteroheme-substituted horseradish peroxidase was employed as the trapping agent for both oxygen metabolites. When NADPH was incubated with the membrane fraction in the presence of the heme-substituted peroxidase, a massive O2 consumption was observed together with the formation of compound III, and O2- adduct of the peroxidase. The amounts of compound III formed and O2 consumed were stoichiometric with each other, while formation of compound II, an indicative of H2O2, was not observed during the reaction. On the other hand, when an excess amount of superoxide dismutase was included in the reaction mixture, compound II was produced with complete suppression of the compound III formation. NADH minimally supported both O2 consumption and formation of compound III or II. These results indicate that the NADPH oxidase in the plasma membrane of thyroid produces O2- as the primary metabolite of O2 and hence that H2O2 required for the thyroid hormone synthesis provided through the dismutation of O2-.

Animals↗
PubMed1989

A monoclonal antibody to rat liver arylsulfatase C and its application in immunohistochemistry.

We purified arylsulfatase C from rat liver microsomes and prepared a monoclonal antibody (P42C2) to the purified enzyme. By SDS-PAGE and immunoblotting analysis using P42C2, the molecular weight of the purified enzyme and of the enzyme in liver and kidney microsomes were estimated at 62,000 daltons. P42C2 caused little inhibition of arylsulfatase C activity, and was bound only slightly to liver microsomes. Localization of arylsulfatase C was studied at the light and electron microscopic level by the indirect immunoperoxidase method using P42C2. In rat liver, arylsulfatase C was detected mainly in the hepatocytes, and less frequently in endothelial cells, Kupffer's cells, and Ito's cells. In rat kidney, strong staining was observed in the straight portions of the proximal tubules. The podocytes, interstitial cells, endothelial cells, and epithelial cells of Henle's thin limbs were stained faintly. By electron microscopy, arylsulfatase C was found localized on the membranes of the endoplasmic reticulum and nuclear envelopes in these cells. These immunohistochemical findings agree with the localization demonstrated by an enzyme-histochemical method which we had previously developed.

Animals↗
PubMed1989

Simian adenovirus type 7 (SA-7) induces tumours of nerve-supporting or paraneural cell origin in newborn hamsters.

Simian adenovirus type 7 (SA-7) was found to induce tumours originating from nerve-supporting or paraneural cells in newborn hamsters, regardless of injection site or tissues. SA-7 induces glioblastomas characterized by definite localization (subependymal regions) and its main cell type, bipolar spongioblast-like cells, in the brain of hamsters inoculated as newborns. When the eyes of newborn hamsters were directly inoculated, SA-7 failed to induce retinoblastoma (0/27), but retro or peri-bulbar SA-7 tumours frequently occurred in tissues closely related to the peripheral nerve apparatus, including the oculomotor nerve or ciliary ganglion. These tumour cells were situated like stromal cells in these nerve tissues. The histological features of the orbital tumours were similar to those of SA-7-induced subcutaneous tumours but not to brain tumours. In contrast with other hamster brain tumours induced by human adenovirus type 12 or human papova JC virus, medulloepithelioma or medulloblastoma, SA-7 induced tumours exhibit distinctive histological and localization characteristics.

Adenoviridae↗
PubMed1988

Immunoelectron microscopy of the hormonogenic sites of the thyroglobulin molecule.

Hog thyroglobulin was treated with the monoclonal anti-thyroglobulin antibody, 16.3.2, which recognizes the thyroxine residues of thyroglobulin. Size-exclusion high-performance liquid chromatography of the immune complexes indicated that there were 3.5 immunoreactable thyroxine residues/mol of 19 S thyroglobulin. The immune complexes were negatively stained and observed under an electron microscope. Under antigen excess conditions, the complexes were demonstrated as branched chains in which thyroglobulin molecules were linked by antibody molecules. When the antibody was in excess, we found many single thyroglobulin molecules each connecting to 2-3 antibody molecules. Based on the morphology of the immune complexes, we conclude that thyroxine residues are formed preferentially on the molecular surface and localized near both ends of the elongated molecule of 19 S thyroglobulin and possibly at one side of the middle of the molecule.

Animals↗
PubMed1988

Molecular mechanism of iodide transport by thyroid plasmalemmal vesicles: cooperative sodium activation and asymmetrical affinities for the ions on the outside and inside of the vesicles.

The 125I- uptake by plasmalemmal vesicles from porcine thyroid was measured by a Millipore filtration method using 2 mM ClO4- as a reaction stopper. Effective uptake occurred in the presence of high concentrations of extravesicular Na+ (Na+o). In the presence of Na-ionophores such as monensin and nigericin, no uptake was observed and the accumulated I- was released. The initial rate of I- uptake increased with the concentration of extravesicular I- (I-o) according to simple saturation kinetics and [I-o] giving a half-maximum rate of about 5 microM. The dependence of the rate on [Na+o] showed cooperativity with a Hill coefficient of 1.8, and a KNa value of 0.0064 M2, suggesting that the binding of at least 2 Na+ ions to a carrier molecule was required to transport an I- ion. Further kinetic data were consistent with a mechanism in which bindings of the ions were rapid and the Na+ binding occurred prior to the I- binding. Intravesicular Na+ inhibited the I- uptake and the inhibition constant (KiNa) was about 4 mM, independently of [I-o] and [Na+o]. Intravesicular I- inhibited the I- uptake with an apparent KiI value of about 100 microM. The results suggest that the differences in the Na+- and I- -binding modes between outside and inside of the vesicles are important factors causing the I- uptake against its concentration gradient.

Animals↗
PubMed1988

Experimental activation of latent cytomegalovirus infection of the captive bred (F1) cynomolgus monkeys by live or killed varicella-zoster virus inoculated under immunosuppression.

F1 cynomolgus monkeys bred in captivity and thought to be "SPF" had latent cytomegalovirus (CMV) infection although less frequently than in wild-born monkeys. Latent CMV could be activated under severe immunosuppression by inoculation with a certain strain of varicella-zoster virus (VZV) as shown in a previous study. We carried out further experiments using live and formalin-inactivated VZV in F1 monkeys which were sero-positive for CMV, but not for any other viruses. The results showed that both live and inactivated VZV were equivalent in permitting activation of latent CMV, suggesting that the VZV inoculum merely played an immunogenic, but not any virological, role in this case. Captive F1 monkeys, however, carried fewer CMV, and/or resisted CMV reactivation more than the wild monkeys in the previous studies, judging from the time required for generalized CMV infection to become expressed.

Animals↗
PubMed1988

A circulating thyroid peroxidase-like substance in healthy human peripheral blood.

Circulating thyroid peroxidase (TPO)-like substance in healthy human peripheral blood was studied to clarify the immunological role of TPO. By using a highly sensitive radioimmunoassay system combined with murine monoclonal antibodies, TPO-like substance was measurable in 6 out of 84 sera. Characterization of this circulating substance by gel filtration revealed that the molecular weight of a major peak corresponded to that of trypsinized TPO. From these findings it is possible that peripheral blood lymphocytes are exposed to a low level of TPO as well as to thyroglobulin.

Adult↗