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Biomedical subjects

S Ohtaki

Publications and source records attributed to S Ohtaki.

At least 109 records · Page 6Linked to original sources
PubMed1986

A murine monoclonal antibody derived from the immunization of human thyroglobulin reacts equally with L-thyroxine and reverse triiodo-L-thyronine and has a unique idiotype.

Murine monoclonal antibody (mAb 16.3.2) to human thyroglobulin which bound equally to various thyroglobulins derived from nine species was obtained from the fusion of C3H/He spleen cells sensitized with normal human thyroglobulin. Characterization of mAb 16.3.2 revealed that both L-thyroxine (T4) and reverse triiodo-L-thyroxine (rT3) were very efficient in the competitive binding inhibition test and that a molar ratio between T4 and rT3 needed for 50% inhibition of binding to immunized thyroglobulin was about 1:1. Further studies on the idiotype of mAb 16.3.2 using both binding and competitive binding inhibition tests showed that mAb 16.3.2 had a unique idiotype not cross-reacting with other monoclonal antibodies to thyroglobulins. Therefore, a possible explanation offered was that mAb 16.3.2 was endowed with a unique idiotype to be regulated by a distinct idiotype network from those of other mAbs.

Animals↗
PubMed1985

Thyroid peroxidase selects the mechanism of either 1- or 2-electron oxidation of phenols, depending on their substituents.

Unlike lactoperoxidase and horseradish peroxidase, thyroid peroxidase catalyzed the oxidation of hydroquinone mostly by way of 2-electron transfer. This conclusion could be derived from three independent experiments: ESR measurements of p-benzosemiquinone, trapping the unpaired electron by cytochrome c, and spectrophotometric analysis of catalytic intermediates of the enzymes. The 1-electron flux for hydroquinone oxidation was found to be 15-19% in the reaction of thyroid peroxidase, while it was nearly 100% in the reactions of lactoperoxidase and horseradish peroxidase. From the spectrophotometric analysis of the catalytic intermediates of enzyme, it was suggested that the mechanism of oxidation catalyzed by thyroid peroxidase changes from a 2-electron to a 1-electron type as the substituents at 2- and 6-positions of phenol become bulky or heavy. On the other hand, the mechanism was invariably a 1-electron type when the oxidation of phenols was catalyzed by lactoperoxidase or horseradish peroxidase. These three peroxidases all catalyzed 1-electron oxidation of ascorbate.

Animals↗
PubMed1985

Detection of a catalytic intermediate of peroxidase in hog thyroid microsomes.

A catalytic intermediate, Compound II of peroxidase was detected spectrophotometrically in thyroid microsomes. From comparison with the spectral data on purified thyroid peroxidase, the content of the peroxidase was estimated to be 0.019 nmol per mg of the microsomal protein, being about one-eighth of the amount of cytochrome b5. It was concluded that thyroid peroxidase exhibits the same peroxidase activity for guaiacol or ascorbate in the free and the microsome-bound forms.

Animals↗
PubMed1985

Purification of thyroid peroxidase by monoclonal antibody-assisted immunoaffinity chromatography.

A rapid method was developed for purification of hog thyroid peroxidase by immunoaffinity chromatography on a column of Sepharose 4B coupled to a monoclonal antibody to the peroxidase. The purified enzyme had a specific activity of 194 units/mg and showed the same absorption spectrum in the Soret and visible regions as that of the enzyme purified after trypsin treatment. The ratio of A413 nm to A280 nm was 0.24, being much less than that for the trypsinized enzymes. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it gave a broad protein band in the 100,000-dalton region. It is concluded that the preparation purified in this study represents a native form of thyroid peroxidase.

Animals↗
PubMed1985

Characterization of hog thyroid peroxidase.

Several fundamental properties of purified hog thyroid peroxidase (A413 nm/A280 nm = 0.55) were investigated in comparison with bovine lactoperoxidase. The Mr of thyroid peroxidase was 71,000. The prosthetic group of thyroid peroxidase was identified spectrophotometrically as protoheme IX after the enzyme was hydrolyzed with Pronase. Optical spectra of oxidized and reduced thyroid peroxidases and their complexes with azide and cyanide were very similar to lactoperoxidase, except that lactoperoxidase had two reduced forms with the Soret band either at 446 or 435 nm, and thyroid peroxidase lacked a reduced form having the 446-nm band. From comparison of their pyridine hemochrome spectra, epsilon mM at 413 nm of thyroid peroxidase was estimated to be 114, being the same as that of lactoperoxidase. The cyanide inhibition for the reaction of thyroid peroxidase was competitive with hydrogen peroxide and the inhibition constant was in rough accord with the dissociation constant of its cyanide complex measured from spectrophotometric titration. Azide inhibited the reaction with an inhibition constant which was about one one-thousandth of the dissociation constant for its spectrally discernible complex. The azide inhibition was not competitive with hydrogen peroxide and decreased as the reaction proceeded. Aminotriazole inhibited the reaction strongly, and the inhibition was augmented during the reaction. These inhibition patterns of azide and aminotriazole were more or less observed in the reaction of lactoperoxidase, but not in the case of horseradish peroxidase. Characteristics of animal peroxidases are discussed.

Animals↗
PubMed1985

Analyses of the specificity of mouse monoclonal antibodies to syngeneic and allogeneic thyroglobulins.

Fourteen mouse monoclonal antibodies (mAbs) to syngeneic and allogeneic thyroglobulins (Tgs) were prepared to study antigenic determinants, their variation among mouse Tgs, and cross-reactivity with various species Tgs. The binding pattern to the Tg panel classified mAbs into three groups but the clustering by competitive-binding inhibition with unlabeled mAbs suggested that at least five antigenic regions existed on the Tg molecule. Mouse Tgs showed no antigenic variation to our mAbs. The cross-reaction with other species Tgs was strictly limited to rat Tgs, excluding one mAb (8.1.1) which reacted with rat and human Tgs. The competitive-binding inhibition by various doses of Tgs substantiated the binding pattern to the Tg panel and furthermore demonstrated that both complete and partial cross-reactions existed.

Animals↗
PubMed1985

Clinical features of OKT4+/OKT8+ adult T-cell leukemia.

Adult T-cell leukemia (ATL) has a range of clinical characteristics. Phenotypically the leukemic cells usually express the helper/inducer associated antigen OKT4 with lack of OKT8. We have observed three patients with acute ATL cytologically indistinguishable from OKT4+/OKT8- ATL but whose neoplastic cells had the unusual phenotype, OKT3+, OKT4+, OKT6-, OKT8+ OKT9+/-, OKT11+, Tac+/-, TdT-. All patients had abnormal karyotypes and antibodies against anti-ATL associated antigens as well as proviral DNA of human T-cell leukemia virus in the leukemic cells. The clinical course was complicated by skin eruptions, hypercalcemia, pulmonary infection and disseminated intravascular coagulopathy. All died of complications shortly after diagnosis. The clinical features of these patients were similar to those of OKT4+/OKT8- ATL. However, their acute course suggests that co-expression surface antigens OKT4 and OKT8 may be a sign of aggressive nature of the disease with poor prognosis.

Aged↗
PubMed1985

Epitopic difference among rat thyroglobulins.

Epitopes on thyroglobulin (Tg) were examined using mouse monoclonal antibodies (mAbs). Two out of 6 mAbs indicated a remarkable difference in binding to three rat Tgs. Namely, they bound to one Tg as well as immunized Tg but could not bind to another at all. They could hardly bind to the third Tg. Another mAb was similar, to some extent, in binding to three rat Tgs as described above, although three other mAbs bound almost equally to three rat Tgs. Since competitive binding inhibition by Tgs supported the result of the binding study, the epitopic difference on rat Tgs was confirmed. Furthermore, competitive binding inhibition by unlabeled mAbs revealed that six mAbs recognized different epitopes individually. Therefore, there were at least two unique epitopes located on one rat Tg but not located on another. The relation between unique epitopes and thyroiditis in the rats used in the present study is also discussed.

Animals↗
PubMed1985

Identification of complex phenotype (OKT4+/OKT8+) on adult T-cell leukemia cells by sequential application of indirect rosette assay with protein A-coated ox red blood cells and immunoperoxidase technic.

The specific immunophenotypic characterization of the hematologic malignant diseases has been obtained most commonly by the immunofluorescent technic. It requires a microscope with illuminator or the expensive flow cytometer that usually precludes morphologic assessment. It was compared with the indirect rosette assay and the immunoperoxidase study, which allowed both immunologic and morphologic assessment. These three technics appeared to be comparable quantitatively, and the latter two technics utilizing cytospin preparations were applied sequentially to the leukemic cells of adult T-cell leukemia with a complex phenotype (OKT4+/OKT8+) and successfully displayed the double markers on each leukemic cell.

Animals↗
PubMed1985

Characterization of a monoclonal antibody to hog thyroid peroxidase and its use for immunohistochemical localization of the peroxidase in the thyroid gland.

A monoclonal antibody (30.1.2) to hog thyroid peroxidase was produced, purified, and characterized. The IgG of 30.1.2 formed an immune complex with the peroxidase in a 1:2 or 1:1 molar ratio depending on the IgG to antigen ratio in the incubation mixture. Immune complex formation did not inhibit the peroxidase activity, which was actually activated 2-fold in the 1:1 complex. Studies of the binding of the conjugate of the IgG or its Fab' with horseradish peroxidase to untreated and acetone-treated thyroid microsomes showed that the IgG conjugate could bind to only a very small portion of the total binding sites (thyroid peroxidase) present in untreated microsomes even after prolonged incubation. The binding of the Fab' conjugate to untreated microsomes, on the other hand, increased as the incubation time was increased, reaching 40% of the total sites after 20 h of incubation. These findings indicated that thyroid peroxidase is localized on the inner surface of the microsomal membranes and that the Fab' conjugate, but not the IgG conjugate, can slowly penetrate through the membrane barrier to reach the peroxidase. Immunohistochemical experiments using the Fab' conjugate as a probe revealed that most thyroid peroxidase in the thyroid gland is located in the endoplasmic reticulum and perinuclear cisternae of the follicular cell, although a small amount could occasionally be detected in the apical membrane including microvilli. In contrast to previous reports, no thyroid peroxidase could be found in other cellular structures such as Golgi apparatus and apical vesicles by the immunohistochemical technique employed.

Animals↗
PubMed1985

Thyroxine (T4) release from thyroglobulin and its T4-containing peptide by thyroid thiol proteases.

Two thiol proteases, TP-1 and TP-2, were purified 500- and 400-fold, respectively, from hog thyroid lysosomal extracts and examined for their activities as releasers of T4 1) from hog thyroglobulin, 2) a fragment (mol wt, approximately 15,000) prepared therefrom, and 3) a T4-containing peptide (19 amino acid residues) derived from the fragment, by measuring the liberation of free T4 with reversed phase HPLC. TP-1 released 8%, 55%, and 95% of the bound T4 present in thyroglobulin, the fragment, and the peptide, respectively, after incubation for 8 h. TP-2, on the other hand, released only 2% of the bound T4 in thyroglobulin and liberated no T4 from the fragment and the peptide. The release of T4 from thyroglobulin by the action of TP-1 was increased approximately 2-fold by the addition of TP-2. This synergistic effect suggested that TP-1 can release T4 from thyroglobulin much more efficiently after the protein has been partially degraded by the action of TP-2, which has only a poor T4-releasing activity. It also suggested that the T4-releasing activity of TP-1 markedly increases as the size of protein substrate is decreased. The amino acid composition of the T4-containing peptide was identical with that previously isolated from bovine thyroglobulin, and this peptide sequence has been shown to be derived from the NH2-terminal portion of the thyroglobulin. This suggests that TP-1 can release T4 at least from the NH2-terminal region of thyroglobulin.

Animals↗
PubMed1984

Iodination and oxidation of thyroglobulin catalyzed by thyroid peroxidase.

The kinetics of iodination and oxidation of hog thyroglobulin were studied with purified hog thyroid peroxidase and the results were compared with the reactions of free tyrosine. From Lineweaver-Burk plots and on the basis of a value of 0.83 for delta epsilon mM at 289 nm/iodine atom incorporated, the rate constant for transfer of an assumed enzyme-bound iodinium cation to thyroglobulin was estimated to be 6.7 X 10(7) and 2.3 X 10(7) M-1 s-1 in native (iodine content = 1.0%) and more iodinated (iodine content = 1.2%) thyroglobulins, respectively. This iodine-transferring reaction was stimulated by iodothyronines, similarly as observed in the reaction with free tyrosine. The iodination of thyroglobulin was inhibited by GSH, the inhibition being competitive with thyroglobulin. Thyroglobulin was oxidized in the presence of a thyroid peroxidase system without giving any appreciable change in absorbance around 300 nm. From stopped flow data, the oxidation was concluded to occur by way of two-electron transfer and the rate constant for the reaction of thyroid peroxidase Compound I with thyroglobulin was estimated to be 1.0 X 10(7) M-1 s-1. The stopped flow kinetic pattern was similar to that observed on the reaction with free tyrosine and monoiodotyrosine. About 6 mol of hydrogen peroxide were consumed per mol of thyroglobulin. Thyroid peroxidase catalyzed thyroglobulin-mediated oxidation of GSH, but lactoperoxidase did not.

Animals↗
PubMed1984

Clinical significance of autorosette-forming cells in T-cell lymphoma.

The capability to form rosettes with sheep erythrocytes (E), antibody-complement-sensitized ox erythrocytes (EAC) and autologous erythrocytes (ARFC), and surface immunoglobulin determinations were studied using 21 lymph nodes and one tonsil with pathologically-proven non-Hodgkin's lymphoma and 10 lymph nodes with benign pathology. Fourteen of 22 non-Hodgkin's lymphoma patients (64%) had a high incidence of E-rosette formation and they were further differentiated into ARFC-positive and ARFC-negative lymphomas. The clinicopathological findings of the latter were compatible with those of adult T-cell leukemia. ARFC-positive lymphoma was regarded as non-Hodgkin's lymphoma of T-cell type and one patient showed lymphoblastic lymphoma with high ARFC counts. ARFC counts were very low in B-cell and non-T, non-B lymphomas. The results from benign lymph nodes were too variable to draw any conclusion, although ARFC counts were relatively high in lymphadenitis and hyperplasia.

Humans↗
PubMed1984

Partial purification and characterization of two thiol proteases from hog thyroid lysosomes.

Two proteases were purified from lysosomal supernatants of hog thyroids. The first step of the purification by diethylaminoethyl cellulose chromatography separated the two proteases, which were shown to have quite similar properties with respect to 2-mercaptoethanol requirement and pH optima for benzoyl-L-arginine-2-naphthylamide (BANA) hydrolytic activity, and inhibition by sulfhydryl inhibitors. One of the proteases, designated as thiol protease-1 (TP-1), was further purified by gel filtration (Sephacryl S-200) and vigorously hydrolyzed BANA but not casein. BANA hydrolytic activity of TP-1 was 50% inhibited by 10(-5) M leupeptin and by 10(-7) M L-trans-epoxysuccinyl-leucylamido(4-amino)butane. A highly purified preparation of the other protease, designated as thiol protease-2 (TP-2), was obtained after gel filtration on Sephacryl S-200 and carboxymethyl cellulose chromatography. In contrast to TP-1, TP-2 exhibited high hydrolytic activity against both casein and BANA, and was more sensitive to leupeptin and L-trans-epoxysuccinyl-leucylamido(4-amino)butane, the concentrations for 50% inhibition being 10(-7) and 10(-8) M, respectively. Electrophoresis and chromatofocusing revealed that the two proteases had different isoelectric points. TP-1 could bind to concanavalin A Sepharose and hydrolyzed L-arginine-2-naphthylamide but not carbobenzoxy-L-arginyl-L-arginine methylcoumarylamide whereas TP-2 did not bind to concanavalin A Sepharose and hydrolyzed carbobenzoxy-L-arginyl-L-arginine methylcoumarylamide but not L-arginine-2-naphthylamide. These results suggested that TP-1 and TP-2 had properties similar to those of the liver lysosomal thiol proteases, cathepsins H and B, respectively.

Animals↗
PubMed1984

Increase in autorosette-forming lymphocytes in thyroid diseases.

A subpopulation of lymphocytes forming rosettes with autologous erythrocytes was studied on peripheral blood and thyroid tissues obtained from the patients with various thyroid diseases. The mean (+/-S.D.) percentage of autorosette-forming cells (ARFC) was 10.1(+/-5.5)% in the peripheral blood from patients with hyperthyroid Graves' disease, which was higher than that in normal subjects (5.6 +/- 2.8%), while the levels of ARFC in the peripheral blood from euthyroid patients with Graves' disease under treatment and Hashimoto's thyroiditis did not significantly differ from the normal level. The mean percentages of ARFC in the thyroid tissues from patients with Graves' disease and Hashimoto's thyroiditis were 14.7(+/-8.5) and 13.3(+/-7.8)%, respectively, which were higher than those in the peripheral blood from the same patients. Most of these cases with abnormally high levels of ARFC were accompanied with the abnormally low T cell to B cell ratios. The microscopic examination of the cytological materials from these patients showed an increased number of large stimulated lymphoid cells or lymphoblasts as compared with those who had few ARFC. These results suggest an increase in an activated T cell subset in the circulation and/or in the thyroid tissue, which is probably caused by active immune response to some stimuli.

Adolescent↗
PubMed1983

Use of inorganic salts to minimize serum interference in a sandwich enzyme immunoassay for human growth hormone using Fab'-horseradish peroxidase conjugate.

In a sandwich enzyme immunoassay (EIA) for human growth hormone (hGH) with anti-hGH Fab'-peroxidase conjugate, the effects of inorganic salts on serum interference were examined, and serum interference was eliminated by incubation of serum samples with anti-hGH IgG-coated polystyrene balls in the presence of 0.4 mol/l NaCl, avoiding the need for hGH-free serum. The sensitivity for hGH was 60 fg/tube or 3 ng/l of serum. No cross-reaction was observed with prolactin, chorionic gonadotropin or luteinizing, thyroid-stimulating and follicle-stimulating hormones. The coefficients of within-assay and between-assay variations were 2.8-6.5% and 4.8-8.7%, respectively. The regression equation and correlation coefficient to radioimmunoassay (RIA) were y(EIA) = 0.89x(RIA) + 0.11 and 0.98 (n = 100), respectively. hGH levels in normal male and female adult serum taken between 9:00 and 10:00 a.m. after overnight fasting and 1 h rest were 312 ng/l (range 53-940 ng/l; n = 10) and 662 ng/l (112-2195 ng/l; n = 13), respectively.

Adult↗