Toichiro Hosoya

Allergenic epitopes of ovalbumin (OVA) in patients with hen's egg allergy : inhibition of basophil histamine release by haptenic ovalbumin peptide

We studied allergenic determinants that induce hypersensitivity to OVA, the major allergen in egg allergy, using immunoblot and histamine release assays. Immunoblot analysis demonstrated a part of the OVA epitope was in the C‐terminal region comprising residues 347‐385 (OVA347‐385). Histamine was released from basophils of a patient with egg allergy upon stimulation with the OVA fragment corresponding to OVA347–385. Furthermore, detailed epitope mapping using overlapping peptides (residues 347‐366, OVA‐A; residues 357‐376, OVA‐B; and residues 367‐385, OVA‐C) in the OVA 347‐385 region was carried out using the histamine release assay. In order for histamine release from basophils to occur, the allergen must possess two or more allergenic determinants located on the protein molecule at distances that would be equivalent to the distances between IgE molecules on the membrane surface. These results suggest that there are at least two epitopes that bind IgE antibodies on each OVA peptide. In addition, one epitope that binds IgE antibodies in two patients appears to reside in the haptenic peptide OVA357‐366 (OVA‐B1). The histamine release from basophils stimulated by OVA‐B was completely inhibited by OVA‐B1 in one of these patients. Similarly, OVA‐B1 inhibited the histamine release produced by OVA‐A in the other by more than 40%. These results suggest that haptenic synthetic peptides could regulate the allergic reaction in the effector phase if common epitope(s) recognized by IgE antibodies in the patients with egg allergy can be found. These are the first studies that provide an antigen‐specific approach to inhibiting histamine release from basophils by a haptenic peptide recognized by IgE antibodies in an allergic disorder.

Anti-thyroid peroxidase antibodies in sera from healthy subjects and from patients with chronic thyroiditis: differences in the ability to inhibit thyroid peroxidase activities

A significant percentage (6.4%) of healthy subjects was found to contain anti‐thyroid peroxidase (TPO) antibodies in their sera. However, in contrast with IgG from sera of patients with chronic thyroiditis, IgG from sera of healthy subjects did not inhibit TPO activities both in guaiacol and iodide assays. In addition, anti‐TPO antibodies from healthy subjects did not block the inhibition of enzyme activities by anti‐TPO antibodies from patients. These findings suggest that anti‐TPO antibodies from healthy subjects do not bind to the epitopes relating to substrate‐combining sites of TPO. Thus, the specificities of anti‐TPO antibodies in healthy subjects may differ from those in cases of chronic thyroiditis.

Anti-thyroglobulin autoantibodies in sera from patients with chronic thyroiditis and from healthy subjects: differences in cross-reactivity with thyroid peroxidase

A significant portion (about 12·7%) of healthy subjects was found to contain anti‐thyroglobulin (anti‐Tg) antibodies in their sera. We compared the binding activities of these antibodies and of anti‐Tg autoantibodies from sera of patients with chronic thyroiditis with human thyroid peroxidase (TPO). The results obtained by ELISA indicated that out of 10 healthy subjects with anti‐Tg antibodies, only four had anti‐Tg antibodies capable of binding to TPO, whereas anti‐Tg autoantibodies from almost all patients with chronic thyroiditis possessed high binding activities to TPO. By use of the immunoprecipitation method, it was also shown that although all anti‐Tg autoantibodies from patients precipitated TPO, a majority of anti‐Tg antibodies from healthy subjects could not precipitate TPO. Such findings cannot be ascribed to the differences in levels of anti‐Tg autoantibodies and anti‐TPO autoantibodies in sera and the differences in avidities of anti‐Tg antibodies in sera between healthy subjects and patients with chronic thyroiditis. Thus, it can be concluded that anti‐Tg antibodies from healthy subjects differ from those of patients with chronic thyroiditis with respect to TPO binding, probably due to difference in fine specificities of these anti‐Tg antibodies.

A common T-cell epitope between human thyroglobulin and human thyroid peroxidase is related to murine experimental autoimmune thyroiditis

We have investigated functional common T-cell epitopes between human thyroglobulin (hTg) and human thyroid peroxidase (hTPO) in mice. Four hTg peptides, Tg-P1, Tg-P2, Tg-P3 and Tg-P4, in which 5 amino acid residues are identical to those of hTPO, and 1 hTPO peptide, TPO-P4 relevant to Tg-P4, were prepared. Among these peptides, only Tg-P4 (residues 2730-2743) and TPO-P4 (residues 118-131) were highly antigenic and both peptides shared the common T-cell epitope. In addition, when the spleen cells from mice immunized with mouse Tg (mTg) were restimulated in vitro by Tg-P4 or TPO-P4 as well as by mTg, these cells transferred thyroiditis to naive recipient mice. These findings indicate that this common T-cell epitope between hTg and hTPO is immunogenic and related to the development of murine experimental autoimmune thyroiditis.

Binding of iodide to Arthromyces ramosus peroxidase investigated with X-ray crystallographic analysis, 1H and 127I NMR spectroscopy, and steady-state kinetics.

The site and characteristics of iodide binding to Arthromyces ramosus peroxidase were examined by x-ray crystallographic analysis, 1H and 127I NMR, and kinetic studies. X-ray analysis of an A. ramosus peroxidase crystal soaked in a KI solution at pH 5.5 showed that a single iodide ion is located at the entrance of the access channel to the distal side of the heme and lies between the two peptide segments, Phe90-Pro91-Ala92 and Ser151-Leu152-Ile153, 12.8 Å from the heme iron. The distances between the iodide ion and heme peripheral methyl groups were all more than 10 Å. The findings agree with the results obtained with 1H NMR in which the chemical shift and intensity of the methyl groups in the hyperfine shift region of A. ramosus peroxidase were hardly affected by the addition of iodide, unlike the case of horseradish peroxidase. Moreover, 127I NMR and steady-state kinetics showed that the binding of iodide depends on protonation of an amino acid residue with a pKa of about 5.3, which presumably is the distal histidine (His56), 7.8 Å away from the iodide ion. The mechanism of electron transfer from the iodide ion to the heme iron is discussed on the basis of these findings.

Synergistic stimulatory effect of glucocorticoid, EGF and insulin on the synthesis of ribosomal RNA and phosphorylation of nucleolin in primary cultured rat hepatocytes

Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1 microM) and/or for 20 h with dexamethasone (1 microM) before the end of incubation. The incorporation of [3H]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2-3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [32P]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin.

Congenital euthyroid goitre with impaired thyroglobulin transport

A case of congenital goitre with defective thyroglobulin (Tg) synthesis was studied from the clinical, biochemical and morphological perspectives. The patient, a 5.5‐year‐old boy, who was clinically euthyroid, showed a positive perchlorate discharge test (37.2%). However, the lodlna‐tion system seemed to be normal since radioiodine uptake into the thyroid was very high, and inspection of the H2O2‐generating system using thyroid slices and an assay for peroxidase activity In microsomes showed no abnormalities. On the other hand, virtually no Tg was detected In the serum, and the amount of Tg in thyroid tissue, estimated with gel electrophoresis, was below 10% of the normal value, the quality of Tg being unchanged. Morphological observations demonstrated the presence of Tg in the markedly distended rough endoplasmic reticulum of the cytoplasm of follicular cells and a lack of Tg in the colloid of the follicular lumen. These results suggest that the thyroid is defective in Tg synthesis, probably associated with Impaired transport of Tg from the cells to the lumen.

Effect of dexamethasone on nucleolar casein kinase II activity and phosphorylation of nucleolin in lymphosarcoma P1798 cells

Ribosomal RNA (rRNA) synthesis in murine P1798 lymphosarcoma cells is reversibly inhibited by glucocorticoids. The effects of dexamethasone upon nucleolin phosphorylation and upon the amount and activity of casein kinase II have been examined. P1798 cells were exposed to 0.1 microM dexamethasone for 36 h. Cells were labeled in vivo with [32P]orthophosphate followed by immunoprecipitation with anti-nucleolin antibody. Nucleolin phosphorylation was reduced by 60% in dexamethasone-treated cells. Nucleoli were isolated and labeled with [gamma-32P]ATP in vitro. Nucleolin protein was reduced to 40% of control in nuclei from dexamethasone-treated cells. Nucleolin phosphorylation was reduced to 20% of control. Nucleolar casein kinase II activity and protein were also reduced (30-55% and 35-50% of control, respectively) by treatment with dexamethasone. Cycloheximide (10 micrograms/ml for 3 h) reduced the amount and activity of casein kinase II, but did not cause a decrease in nucleolin protein. These observations are discussed relative to the hypothesis that glucocorticoids regulate the amount or activity of proteins of short biological half-life that are involved in the regulation of rRNA synthesis.

Regulation of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension

The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide (uptake and organification) when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03-0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor (EGF, 10(-9)M) and phorbol 12-myristate 13-acetate (PMA, 10(-8) M) completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.

Effects of mixed solvents on three elementary steps in the reactions of horseradish peroxidase and lactoperoxidase.

The effects of methanol, acetone, and ethylene glycol (up to 50% v/v) on elementary steps in the reactions of horseradish peroxidase (HRP) and lactoperoxidase (LPO) were studied by means of the stopped-flow method and the difference spectrum. The rate constant (k3,app) of the oxidation reaction of p-cresol with HRP compound II was remarkably reduced in the presence of organic solvents (to 2.3%, 1.8% and 9.4% of the original value in the presence of 50% (v/v) of methanol, acetone and ethylene glycol, respectively), then to a lesser degree were decreased the rate of the oxidation reaction with LPO compound II, and the rate of the oxidation reaction with HRP compound I. These reductions in the reaction rates were not due to competitive inhibition of the solvents, but considered to be related to the degree of exposure of the electron transfer route to the medium. While the rate constant of compound I formation (k1,app) was moderately affected by organic solvents in the case of HRP, the reaction rate with LPO was scarcely affected by organic solvents, being in harmony with the compact heme crevice which probably hampers penetration of solvent molecules. The rate constant (k2,i,app) of the oxidation reaction of an iodide ion by HRP compound I was also hardly affected by the solvents. On the basis of these facts, the mechanism of electron transfer from donors to compound I and compound II is discussed.