THE ASSESSMENT OF ZINC STATUS BY THE ZINC TOLERANCE-TEST IN THYROID-DISEASE

Zinc status was evaluated in 12 hyperthyroid and in 7 hypothyroid patients in comparison with 8 euthyroid individuals by the oral zinc tolerance test and by the determination of urinary zinc excretion. Hyperthyroid patients presented a basal serum concentration similar to that of euthyroid individuals but greater urinary zinc excretion, indicating the occurrence of zinc depletion from tissues to the blood stream caused by the catabolism inherent in the hyperthyroid state. Hyperthyroidism also caused lower zinc assimilation by tissues after zinc ingestion. Hypothyroid individuals present lower basal zinc levels in serum than euthyroid and hyperthyroid individuals and urinary zinc excretion similar to that of euthyroid individuals. No changes in the parameters measured were observed after zinc load, suggesting reduced or delayed intestinal absorption and zinc assimilation by tissues. The present data are indicative of zinc deficiency, the lower intestinal zinc absorption probably being one of the contributing factors.

Immunocytochemical localization of heparin in secretory granules of rat peritoneal mast cells using a monoclonal anti-heparin antibody (ST-1)

We performed immunogold labeling with an ST-1 monoclonal antibody (IgM), specific for intact heparin, to define the subcellular localization of heparin in mast cells. Rat peritoneal mast cells were fixed by a modified Karnovsky method and embedded in Araldite. Ultrathin sections were first treated with sodium periodate and then sequentially incubated with MAb ST-1, rabbit anti-mouse IgM, and protein A-gold. By transmission electron microscopy, gold particles were localized inside cytoplasmic granules of peritoneal mast cells. In contrast, with the same procedure, no labeling was observed in mast cells from rat intestinal mucosa. Control sections of rat peritoneal or intestinal mucosa mast Mast cells cells treated with an irrelevant MAb (IgM) did not show any labeling. Treatment with nitrous Heparin acid abolished the reactivity of MAb ST-1 with peritoneal mast cells. These results Granules show that different mast cells can be identified regarding their heparin content by immunochemical procedures using MAb ST-1.

Trypanosoma cruzi: identification and characterization of a novel ribosomal protein L27 (TcrL27) that cross-reacts with an affinity-purified anti-Sm antibody

Small nuclear ribonucleoproteins (snRNPs)are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-Sm serum used in the library screening as well as by chagasic and systemic lupus erythemathosus (SLE) sera. Our results suggest that the T. cruzi L27 ribosomal protein may be involved in autoimmunity of Chagas disease.

MODULATION OF PARASITEMIA AND ANTIBODY-RESPONSE TO TRYPANOSOMA-CRUZI BY CYCLOPHOSPHAMIDE IN CALOMYS-CALLOSUS (RODENTIA, CRICETIDAE)

Calomys callosus a wild rodent, previously described as harboring Trypanosoma cruzi, has a low susceptibility to infection by this protozoan.Experiments were designed to evaluate the contribution of the immune response to the resistance to T. cruzi infection exhibited by C. callosus. Animals were submitted to injections of high (200 mg/kg body weight) and low (20 mg/kg body weight) doses of cyclophosphamide on days -1 or -1 and +5, and inoculated with 4 x 10(3) T. cruzi on day O. Parasitemia, mortality and antibody response as measured by direct agglutination of trypomastigotes were observed. Two hundred mg doses of cyclophosphamide resulted in higher parasitemia and mortality as well as in suppression of the antibody response. A single dose of 20 mg enhanced antibody levels on the 20th day after infection, while an additional dose did not further increase antibody production. Parasitemia levels were not depressed, but rather increased in both these groups as compared to untreated controls. Passive transfer of hyperimmune C. callosus anti-T. cruzi serum to cyclophosphamide immunosuppressed animals resulted in lower parasitemia and mortality rates. These results indicate that the immune response plays an important role in the resistance of C. callosus to T. cruzi.

Triiodothyronine mimics the effects of estrogen in breast cancer cell lines

MCF-7 (estrogen receptor positive - ER(+)) and MDA-MB-231 (estrogen receptor negative - ER(-)) are human breast cancer cell lines which express functional thyroid hormone receptors (c-erb A alpha 1 and c-erb beta 1) as indicated by stimulation of mitochondrial alpha-glycerophosphate dehydrogenase. In MCF-7, mimicking E(2), T-3 stimulated growth in a dose-dependent (10(10) M-10(-8) M) manner, induced the expression of progesterone receptor and growth factor TGF alpha mRNAs and inhibited that of TGF beta mRNA; T-3 also increased progesterone binding and LDH5 isozyme activities. None of these effects were observed in (ER(-)) MDA-MB-231 cells. 10(-6) M tamoxifen (TAM) reverted growth stimulation, suppressed progesterone receptor and TGF alpha mRNA induction and restored TGF beta mRNA to control levels in T-3-treated MCF-7 cells. That T-3 is acting in MCF-7 cells via its binding to ER is suggested by the immunoprecipitation of pre-bound I-125-T-3 from MCF-7 nuclear extracts by an ER-specific monoclonal antibody and by the displacement of H-3-estradiol binding to ER by radioinert T-3. Copyright (C) 1996 Elsevier B.V. Ltd.

Clinical features and genetic analysis of four Brazilian kindreds with resistance to thyroid hormone

Objective Resistance to thyroid hormone (RTH) is a dominantly inherited syndrome of reduced tissue responsiveness to thyroid hormone usually due to mutations located in the ligand-binding domain and adjacent hinge region of the thyroid hormone receptor beta (TR beta). In the present report we describe the clinical and laboratory characteristics and the genetic analysis of patients with this rare disorder from a Brazilian population.Patients Four unrelated Brazilian families with diagnosis of RTH were studied. Age at diagnosis varied from 14 months to 29 years.Results All affected individuals were clinically euthyroid, except for one patient who presented immediately after birth with hyperthyroidism. All individuals had tachycardia and goitre, elevated concentrations of free thyroid hormones and reduced sensitivity to thyroid hormone. Direct sequencing analysis of the TR beta gene revealed four previously reported mutations: c.949G -> A, c.1313G -> A, c.1357C -> A and c.1358dupC in families A, B, C and D, respectively.Conclusion the present report shows that the frequent mutations described in the thyroid hormone receptor worldwide are also present in the Brazilian population, which is characterized by a variable ethnic background.

Peroxidase from peach fruit: Thermal stability

Peroxidase from peach fruit was purified 28.9-fold by DEAE-cellulose, Sephadex G-100 and hydroxylapatite chromatography. The purified enzyme showed only one peak of activity with an optimum pH of 5.0 and temperature of 40 degreesC. The calculated activation energy (Ea) for the reaction was 7.97 kcal/mol. The enzyme was heat-labile in the temperature range of 60 to 80 degreesC with a fast inactivation at 80 degreesC. PAGE of the inactivation course at 70 degreesC showed only one band of activity. Different sugars increased the heat stability of the activity in the following order: sucrose>lactose>glucose>fructose. Measurement of residual activity showed a stabilizing effect of sucrose at various temperature/sugar concentrations (10 to 40%, w/w) with the Ea for inactivation increasing with sucrose concentration from 0 to 20% (w/w). After inactivation at 70 degreesC and 75 degreesC the enzyme was able to be reactivated by up to 40% of the initial activity when stared at 30 degreesC.