ABSTRACT
Duox2 (and probably Duox1) is a glycoflavoprotein involved in thyroid hormone biosynthesis, as the thyroid H2O2 generator functionally associated with Tpo (thyroperoxidase). So far, because of the impairment of maturation and of the targeting process, transfecting DUOX into nonthyroid cell lines has not led to the expression of a functional H2O2-generating system at the plasma membrane. For the first time, we investigated the H2O2-generating activity in the particulate fractions from DUOX2- and DUOX1-transfected HEK293 and Chinese hamster ovary cells. The particulate fractions of these cells stably or transiently transfected with human or porcine DUOX cDNA demonstrate a functional NADPH/Ca2+-dependent H2O2-generating activity. The immature Duox proteins had less activity than pig thyrocyte particulate fractions, and their activity depended on their primary structures. Human Duox2 seemed to be more active than human Duox1 but only half as active as its porcine counterpart. TPO co-transfection produced a slight increase in the enzymatic activity, whereas p22(phox), the 22-kDa subunit of the leukocyte NADPH oxidase, had no effect. In previous studies on the mechanism of H2O2 formation, it was shown that mature thyroid NADPH oxidase does not release O2*- but H2O2. Using a spin-trapping technique combined with electron paramagnetic resonance spectroscopy, we confirmed this result but also demonstrated that the partially glycosylated form of Duox2, located in the endoplasmic reticulum, generates superoxide in a calcium-dependent manner. These results suggest that post-translational modifications during the maturation process of Duox2 could be implicated in the mechanism of H2O2 formation by favoring intramolecular superoxide dismutation.
Subject(s)
Calcium/metabolism , Flavoproteins/physiology , Hydrogen Peroxide/metabolism , Animals , Blotting, Western , CHO Cells , Catalysis , Cell Line , Cell Membrane/metabolism , Cells, Cultured , Cricetinae , DNA, Complementary/metabolism , Dual Oxidases , Electron Spin Resonance Spectroscopy , Endoplasmic Reticulum/metabolism , Flavoproteins/metabolism , Glycosylation , Green Fluorescent Proteins/metabolism , Humans , Hydrogen Peroxide/pharmacology , Leukocytes/enzymology , Magnetics , Models, Biological , Mutation , NADPH Oxidases/metabolism , Protein Processing, Post-Translational , Protein Structure, Tertiary , Spin Trapping , Superoxides/metabolism , Swine , Thyroid Gland/metabolism , Thyroid Hormones/metabolism , TransfectionABSTRACT
Thyroglobulin (Tg) is cleaved into several peptides during thyroid hormone synthesis, an oxidative process. P40, an iodinated C-terminal peptide from human Tg, has a molecular weight of about 40 kDa and contains two hormonogenic sites. P40 is the smallest peptide that is still recognized by monoclonal antibodies from mice immunized with human Tg directed against its immunodominant region. Since P40 also contains several T-cell epitopes, it is a good candidate for studying the primary events involved in the process of hormone synthesis leading to thyroid autoimmunity. The present results show that P40 is recognized by Tg antibodies from patients with thyroid disorders and induces Tg antibodies in CBA mice. P40 may therefore be involved in the autoimmune process, thus providing a useful tool for diagnostic and therapeutic purposes.
Subject(s)
Immunodominant Epitopes/immunology , Peptide Fragments/immunology , Thyroglobulin/immunology , Animals , Antibodies, Monoclonal/immunology , Epitopes, T-Lymphocyte/immunology , Female , Humans , Hydrogen Peroxide/chemistry , Immunodominant Epitopes/isolation & purification , Iron/chemistry , Mice , Peptide Fragments/isolation & purification , Thyroglobulin/chemistryABSTRACT
Uterine cervix cancer is the second most frequent female cancer after breast cancer in Morocco and represents a public health problem. Cervical cancer is highly linked to human papillomavirus (HPV) especially types 16 and 18 which are the highly oncogenic genotypes. To identify the contribution of HPV testing in the prevention of cervical cancer in Morocco, 147 biopsies collected at the Institut National d'Oncologie and 447 swabs from pathology laboratories and gynaecologist offices in Rabat were HPV analysed. HPV testing were made without any presumption of the histopathological diagnosis. A total of 147 paraffin-embedded biopsies and 447 exfoliated cervical samples were included. Based on histopathology results of the 147 biopsies, most cervical lesions were invasive carcinomas and non specific inflammations (NSI). With the molecular assay, HPV was detected in 91/147 (62%) patients. The high risk types 16 and 18 were found in 45% of the cases (41/91) and HPV 18 in 19% of the cases (17/91). Double infection with HPV 16 and 18 was found in 3 cases. Among the 447 swabs tested, 28 were HPV positives. Cytology results showed that 46% were inflammations (13/28). Among them, 10 patients had a NSI and only 3 patients had a cytology diagnosis of HPV infection. Based on these data, HPV testing should be associated to cervical cytology screening according to two algorithms established in function of the age of the patient and viral natural history. Combination of cytology and HPV testing allow identification of patient with high risk for development of high grade cervical lesions and improve cervical cancer prevention.
