Search PubMed⌕ Search

Biomedical subjects

T Kotani

Publications and source records attributed to T Kotani.

At least 199 records · Page 11Linked to original sources
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↗
PubMed1987

Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNAs.

Two forms of human thyroid peroxidase cDNAs were isolated from a lambda gt11 cDNA library, prepared from Graves disease thyroid tissue mRNA, by use of oligonucleotides. The longest complete cDNA, designated phTPO-1, has 3048 nucleotides and an open reading frame consisting of 933 amino acids, which would encode a protein with a molecular weight of 103,026. Five potential asparagine-linked glycosylation sites are found in the deduced amino acid sequence. The second peroxidase cDNA, designated phTPO-2, is almost identical to phTPO-1 beginning 605 base pairs downstream except that it contains 1-base-pair difference and lacks 171 base pairs in the middle of the sequence. This results in a loss of 57 amino acids corresponding to a molecular weight of 6282. Interestingly, this 171-nucleotide sequence has GT and AG at its 5' and 3' boundaries, respectively, that are in good agreement with donor and acceptor splice site consensus sequences. Using specific oligonucleotide probes for the mRNAs derived from the cDNA sequences hTPO-1 and hTPO-2, we show that both are expressed in all thyroid tissues examined and the relative level of two mRNAs is different in each sample. These results suggest that two thyroid peroxidase proteins might be generated through alternate splicing of the same gene. By using somatic cell hybrid lines, the thyroid peroxidase gene was mapped to the short arm of human chromosome 2.

Amino Acid Sequence↗
PubMed1987

Characterization of thyroid follicular cell apical plasma membrane peroxidase using monoclonal antibody.

Thyroid peroxidase (TPO) located in the apical plasma membrane of follicular cells was investigated by means of a membrane-immunofluorescent technique. The epitope of TPO recognized by a murine monoclonal antibody (mAb 30.1.2) was identified on the apical membrane surface. Trypsinization removed TPO immunoreactivity and enzymatic activity after 60 min of incubation at 37 degrees C. The epitope reappeared on the apical membrane surface after short term culture for 120 min without the addition of TSH. With TSH the time required for reappearance was only 30 min. TPO activity was regenerated under both conditions. Since dibutyryl cyclic AMP could not accelerate the reappearance of the epitope, it was thought that TPO reappearance is mediated by other than the adenylate cyclase-cyclic AMP system.

Animals↗
PubMed1987

[NAD+-dependent 15-hydroxyprostaglandin dehydrogenase from the human term placenta].

A NAD+-dependent 15-hydroxyprostaglandin dehydrogenase was purified from human term placenta using monoclonal antibody-coupled Sepharose affinity chromatography. The monoclonal antibodies were prepared against the molecule which was purified according to the method of Jarabak et al. About 4,800-fold purification was achieved with a 3% yield. The highest specific activity was 1,890 mU/mg. The molecular weight of the enzyme determined by gel filtration was 49,000. Polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of two protein bands which were thought to be derived from the same enzyme molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed two main bands of which the molecular weights were 28,000 to 30,000 and 52,000 to 56,000. Electrophoretic analyses suggest a labile oligomeric structure that may relate to the instability of the enzyme activity, which contradicts the monomer theory reported by Jarabak et al.

Chromatography, Affinity↗
PubMed1986

Labile oligomeric structure of human placental 15-hydroxyprostaglandin dehydrogenase.

NAD-dependent 15-hydroxyprostaglandin dehydrogenase has been isolated from human term placenta. About 9,000-fold enrichment was achieved with a yield of 7.6%. Electrophoretic analyses suggested that glycerol stabilized an active structure of the enzyme, and sodium dodecyl sulfate might dissociate it. The instability of the enzyme activity may relate to its labile oligomeric structure which is easily dissociated into subunits.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Primary perianal actinomycosis over a thirty year period.

A 50-year-old Japanese man had had abscesses and draining fistulas in the perianal region. These lesions recurred, despite surgical treatment such as incision and drainage over a 30 year period. "Sulfur granules" were found in the pus from the abscess and Actinomyces israelii was cultured. Ampicillin-cloxacillin treatment lead to healing. The patient died 4 months later with a hepatoma and multiple metastases.

Abscess↗
PubMed1986

Detection of autoantibodies to thyroid peroxidase in autoimmune thyroid diseases by micro-ELISA and immunoblotting.

Serum autoantibodies to thyroid peroxidase (TPO) in patients with thyroid autoimmune diseases were studied by micro-ELISA and immunoblotting. Twenty-four patients, 15 with Graves' disease and 9 with Hashimoto's thyroiditis, whose serum titers were greater than 3200 on the microsomal hemagglutination test (except for 1 patient with a titer of 800) had autoantibodies to TPO. Both immunoglobulin G and M classes of autoantibodies were detected, with the former being more prominent. When TPO and thyroid microsomes were used as a target in a competitive binding inhibition test, the results suggested that TPO was a major thyroid microsomal antigen. On the other hand, immunoblotting analysis showed 3-4 bands in the 45-60K region stained by patients' sera in addition to human TPO with mol wt of 100K and 107K; only the latter 2 bands stained with antiporcine TPO antibody. In the majority of sera, TPO bands were clearer than others, although some sera showed the clearest band with a mol wt of 55K. These results indicate that patients with autoimmune thyroid disease often have autoantibodies to TPO that can be detected by micro-ELISA and immunoblotting, and that TPO is a major component of the thyroid microsomal antigen.

Animals↗
PubMed1986

Characterization of human thyroid peroxidase purified by monoclonal antibody-assisted chromatography.

A murine monoclonal antibody (mAb) to human thyroid peroxidase (TPO) was produced and used for purification of the enzyme. This report describes studies done to characterize the mAb and the purified human TPO. The mAb bound to human TPO at a molar ratio of 1:2 or 1:1 and to human thyroid microsomes with a dissociation constant of 1.2 nM. The mAb had no effect on TPO activity to catalyze guaiacol oxidation. As immunoglobulin G, the mAb consisted of a light chain (kappa) of 26 kilodaltons (kDa) and a heavy chain (gamma 1) of 53 kDa; its pI value was 5.9. Using an mAb immunoaffinity column, 85% of TPO activity was recovered from an ammonium sulfate precipitate of a human thyroid microsomal preparation solubilized with deoxycholate. The purified TPO had a specific activity of 164 guaiacol U/mg protein and an A413 nm/A280 nm ratio of 0.26. The enzyme showed bands only at 107 and 100 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TPO was very stable at alkaline pH, and its ability to catalyze iodide oxidation and tyrosine iodination was increased to the same extent as its ability to catalyze guaiacol oxidation. For comparison with a conventional method, the immunoaffinity-purified TPO was trypsinized and rechromatographed on the immunoaffinity column. The trypsinized enzyme had a specific activity of 336 U/mg and an A413 nm/A280nm ratio of 0.65. The absorption spectrum of the enzyme suggested that the prosthetic group of human TPO is protoheme IX. These results indicate the value of the immunoaffinity procedure for human TPO purification. The major advantages of this purification procedure are not only high yield and speed, but also recovery of intact enzyme.

Animals↗
PubMed1986

Immunoglobulin G subclasses of anti-thyroid peroxidase autoantibodies in human autoimmune thyroid diseases.

A distribution of immunoglobulin G (IgG) subclass of anti-thyroid peroxidase (TPO) autoantibodies was studied to know whether anti-TPO autoantibodies are closely implicated in the pathogenesis of human autoimmune thyroid diseases. As a result of analyzing 14 patients' sera, 7 with Graves' disease and 7 with Hashimoto's thyroiditis, anti-TPO autoantibodies were found to consist of mainly IgG1 subclass. Percentages of both IgG1 and IgG2 subclasses in IgG class of autoantibodies corresponded to those in the normal serum composition, whereas IgG3 subclass was scarcely contained in anti-TPO autoantibodies and IgG4 subclass markedly increased. It was thought that anti-TPO autoantibodies had a capability to lyse thyroid follicular cells by the mechanism of antibody-dependent complement-mediated cytolysis, because IgG1 and IgG2 subclasses of antibodies can fix complement and TPO locates in apical membrane surface of thyroid follicular cells. Comparing Graves' disease with Hashimoto's thyroiditis, mean percentages of both IgG1 and IgG2 subclasses of 2 groups were statistically different. Namely, sera of patients with Graves' disease had higher and lower mean percentages of IgG1 and IgG2 subclasses of autoantibodies, respectively, than those with Hashimoto's thyroiditis, though no plausible explanation for these differences can be offered at the present time.

Adult↗
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

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

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

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↗