RESUMO
The history of the study by our group of the generation, the role and the effects of H2O2 in the thyroid, is summarized. The relations with thyroid diseases are discussed: myxedematous cretinism, thyroiditis, thyroid cancer, congenital hypothyroiddism, are discussed. A new role of H2O2 in the chemorepulsion of bacteria is proposed.
Assuntos
Peróxido de Hidrogênio/metabolismo , Oxidases Duais , Humanos , NADPH Oxidases/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Nódulo da Glândula Tireoide/metabolismoRESUMO
Human rearranged RET/PTC3 (papillary thyroid carcinoma) proto-oncogene and high-risk human papillomavirus (HPV) type 16 E7 oncogene induces in the mouse a neoplastic transformation of thyroid follicular cells. We present a detailed immuno-histological study (170 mouse thyroids: RET/PTC3, E7, wild type, 2- to 10-month-old) with cell cycle proliferation and signalling pathway indicators. The characteristics of both models are different. There is an 'oncogene dependent' cellular signature, maintained at all studied ages in the E7 model, less in the RET/PTC3 model. During tumour development a large heterogeneity occurred in the Tg-RET/PTC3 model within a same tumour or within a same thyroid lobe. The Tg-E7 model was less heterogeneous, with a dominant goitrous pattern. The solid tumour already described in the RET/PTC3 models associated with cribriform patterns, suggested 'PTC spindle cell changes' as in humans PTC rather than the equivalent of the solid human PTC. Proliferation and apoptosis in the two thyroid models are related to the causal oncogene rather than reflect a general tumorigenic process. The thyroids of RET/PTC3 mice appeared as a partial and transient model of human PTCs, whereas the Tg-E7 mice do not belong to the usual PTC type.
Assuntos
Adenocarcinoma Papilar/patologia , Modelos Animais de Doenças , Proteínas E7 de Papillomavirus/genética , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias da Glândula Tireoide/patologia , Adenocarcinoma Papilar/genética , Adenocarcinoma Papilar/metabolismo , Animais , Ciclo Celular , Proliferação de Células , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Proto-Oncogene Mas , Transdução de Sinais , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismoRESUMO
CONTEXT: The long-lived thyroid cell generates, for the synthesis of thyroid hormones, important amounts of H2O2 that are toxic in other cell types. This review analyzes the protection mechanisms of the cell and the pathological consequences of disorders of this system. EVIDENCE ACQUISITION: The literature on H2O2 generation and disposal, thyroid hormone synthesis, and their control in the human thyroid is analyzed. EVIDENCE SYNTHESIS: In humans, H2O2 production by dual-oxidases and consequently thyroid hormone synthesis by thyroperoxidase are controlled by the phospholipase C-Ca2+-diacylglycerol arm of TSH receptor action. H2O2 in various cell types, and presumably in thyroid cells, is a signal, a mitogen, a mutagen, a carcinogen, and a killer. The various protection mechanisms of the thyroid cell against H2O2 are analyzed. They include the separation of the generating enzymes (dual-oxidases), their coupling to thyroperoxidase in a proposed complex, the thyroxisome, and H2O2 degradation systems. CONCLUSIONS: It is proposed that various pathologies can be explained, at least in part, by overproduction and lack of degradation of H2O2 (tumorigenesis, myxedematous cretinism, and thyroiditis) and by failure of the H2O2 generation or its positive control system (congenital hypothyroidism).
Assuntos
Peróxido de Hidrogênio/metabolismo , Doenças da Glândula Tireoide/metabolismo , Doenças da Glândula Tireoide/fisiopatologia , Glândula Tireoide/fisiologia , Animais , HumanosRESUMO
Cyclic nucleotide phosphodiesterase activities (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) were found in the 40,000 X g supernatant fraction of homogenates of Xenopus laevis oocytes. In the supernatant, the ratio of the specific activity of cyclic AMP phosphodiesterase to that of cyclic GMP phosphodiesterase was 1.1 at the 1 micro substrate level. Two phosphodiesterase forms were isolated by centrifugation on sucrose gradient: a 3-4 S form hydrolyzing specificity cyclic AMP and a 6-7 S form hydrolyzing both cyclic nucleotides (cyclic AMP and cyclic GMP). The activity of the 6-7 S phosphodiesterase was characterized by its activation by 0.1 micro M calmodulin purified from beef pancreas in the presence of 50 micro M CA2+. The calmodulin dependence of this form was completely abolished in the presence of 1 mM ethyleneglycobis(beta-aminoethyl ether)-N-N,N',N'-tetraacetic acid (EGTA). Trifluoperazine at 0.1 mM inhibited both the freshly prepared crude enzyme and the partially purified 6-7 S form. On the other hand, no effect of cyclic GMP at 3 micro M was observed on cyclic AMP hydrolysis in the case of the supernatant or that of the partially purified phosphodiesterases. These data show the presence of a calmodulin-dependent phosphodiesterase in the soluble fraction of X. laevis oocytes.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Proteínas de Ligação ao Cálcio/farmacologia , Calmodulina/farmacologia , Oócitos/enzimologia , Óvulo/enzimologia , Animais , Calmodulina/antagonistas & inibidores , Centrifugação com Gradiente de Concentração , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1 , Ácido Egtázico/farmacologia , Feminino , Fígado/enzimologia , Xenopus laevisRESUMO
OBJECTIVES: The H2O2 generating system of the thyrocyte and the O2- generating system of macrophages and leukocytes present numerous functional analogies. The main constituent enzymes belong to the NADPH oxidase (NOX) family (Duox/ThOX for the thyroid and NOX2 /gp91phox for the leukocytes and macrophages), and in both cell types, H2O2 generation is activated by the intracellular generation of Ca2+ and diacylglycerol signals. Nevertheless, although the controls involved in these two systems are similar, their mechanisms are different. The main factors controlling O2- production by NOX2 are the cytosolic proteins p67phox and p47phox, and Rac, a small GTP-binding protein. We have previously reported that there is no expression of p67phox and p47phox in thyrocytes. Here, we investigated whether Rac1 is an actor in the thyroid H2O2-generating system. DESIGN AND METHODS: Ionomycin- and carbamylcholine-stimulated H2O2 generation was measured in dog thyroid cells pretreated with the Clostridium difficile toxin B, which inhibits Rac proteins. Activation of Rac1 was measured in response to agents stimulating H2O2 production, using the CRIB domain of PAK1 as a probe in a glutathione S-transferase (GST) pull-down assay. RESULTS: Among the various agents inducing H2O2 generation in dog thyrocytes, carbamylcholine is the only one which activates Rac1, whereas phorbol ester and calcium increase alone have no effect, and cAMP inactivates it. Moreover, whereas toxin B inhibits the stimulation of O2- generation by phorbol ester in leukocytes, it does not inhibit H2O2 generation induced by carbamylcholine and ionomycin in dog thyrocytes. CONCLUSIONS: Unlike in leukocytes, Rac proteins do not play a role in H2O2 generation in thyroid cells. A different regulatory cascade for the control of H2O2 generation remains to be defined.
Assuntos
Peróxido de Hidrogênio/metabolismo , NADPH Oxidases/metabolismo , Glândula Tireoide/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Toxinas Bacterianas/farmacologia , Western Blotting , Carbacol/farmacologia , Cães , Ativação Enzimática , Ionomicina/farmacologia , Leucócitos/metabolismo , Microscopia de Fluorescência , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
In somatic cells, telomeres shorten with population doubling, thus limiting their capacity to divide. Telomerase, which synthesizes telomeric repeats, can compensate for such shortening. Telomerase activity is known to be absent from most somatic differentiated cells but is present in germline cells, immortal cell lines, or a large majority of malignant tumors. Autonomous thyroid adenomas are benign tumors composed of highly differentiated cells characterized by TSH-independent function and growth. Telomere length and telomerase activity were measured in autonomous and hypofunctioning adenomas and their surrounding tissues. A significant decrease of 3.8+/-1.0 kilobases (kb) was observed in the length of the terminal restriction fragments (TRF) in 12 autonomous adenomas (8.6+/-1.1 kb), compared with the TRF length of their surrounding tissues (12.4+/-1.6 kb). The same kind of decrease, 3.5+/-1.2 kb, was also observed in 16 hypofunctioning adenomas (12.3+/-1.7 kb in surrounding tissue and 8.8+/-1.6 kb in the adenomas). No telomerase activity was detected either in the 12 autonomous adenomas studied or in most of the quiescent tissues (10 of 12). Most of the hypofunctioning adenomas tested (15 of 16) did not display telomerase activity. These results suggest that the cells have undergone a higher number of cell divisions in the adenomas than in the surrounding tissue. Moreover, there is a larger spread of the TRF length distribution in autonomous adenomas than in the collateral tissue. This could reflect the heterogeneity in proliferation status of the cells in the nodule, some of which have reached the end of their life span, whereas others are still proliferating (but with no malignant potential for the autonomous adenomas). In conclusion, benign adenomas exhibit a shorter and more variable telomere length than the normal collateral quiescent tissue, with no telomerase activity to compensate this loss in telomere length.
Assuntos
Adenoma/enzimologia , Adenoma/genética , Telomerase/metabolismo , Telômero/genética , Neoplasias da Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/genética , Adenoma/fisiopatologia , Sequência de Bases/genética , Linhagem Celular Transformada , Humanos , Reação em Cadeia da Polimerase , Glândula Tireoide/enzimologia , Glândula Tireoide/fisiologia , Neoplasias da Glândula Tireoide/fisiopatologia , Nódulo da Glândula Tireoide/enzimologiaRESUMO
In animals, as well as in humans, the thyroid gland is made of active follicles, with cuboidal cells and hypofunctioning follicles, with flattened cells. In this study, the functional status of human follicles was dissected out, based on immunohistochemical detection of TSH receptor, Na(+)/I(-) symporter, pendrin, thyroperoxidase (TPO), thyroid oxidases (ThOXs), and T(4)-containing iodinated Tg (Tg-I). To ascertain that angiofollicular units exist in the human, we studied the microvascular bed of each follicle, in correlation with detection of vascular endothelial growth factor (VEGF), of nitric oxide synthase III, and of endothelin in normal and goitrous thyroids. In hypofunctioning follicles, pendrin, TPO, and ThOXs were not detected, and there was no Tg-I in the colloid. At the opposite, in active follicles, pendrin, TPO, and ThOXs were detected in thyrocytes, and Tg-I was present in the colloid. In normal and goitrous thyroids, the capillary networks surrounding active follicles were larger than those surrounding hypofunctioning follicles. Immunoreactivity for nitric oxide synthase III and endothelin was solely detected in active follicles. Only a few follicles in normal thyroids were immunostained for VEGF, regardless of their functional status. In multinodular goiters, VEGF was detected in contact with the extracellular matrix at the basal pole of the cells. In conclusion, the present study endorses the likelihood of angiofollicular units in the human thyroids. Vascular changes are related to the functional status of thyrocytes.
Assuntos
Bócio Nodular/patologia , Bócio Nodular/fisiopatologia , Iodo/metabolismo , Glândula Tireoide/fisiologia , Glândula Tireoide/fisiopatologia , Transporte Biológico/fisiologia , Capilares/anatomia & histologia , Fatores de Crescimento Endotelial/metabolismo , Endotelinas/metabolismo , Epitélio/metabolismo , Humanos , Imuno-Histoquímica , Linfocinas/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III , Proteínas/metabolismo , Valores de Referência , Glândula Tireoide/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Progress in biotechnology has provided useful tools for tracing proteins involved in thyroid hormone synthesis in vivo. Mono- or polyclonal antibodies are now available to detect on histological sections the Na(+)/I(-) symporter (NIS) at the basolateral pole of the cell, the putative iodide channel (pendrin) at the apical plasma membrane, thyroperoxidase (TPO), and members of the NADPH-oxidase family, thyroid oxidase 1 and 2 (ThOXs), part of the H(2)O(2)-generating system. The aim of this study was to correlate thyroglobulin (Tg) iodination with the presence of these proteins. Tg, T(4)-containing Tg, NIS, pendrin, TPO, ThOXs, and TSH receptor (TSHr) were detected by immunohistochemistry on tissue sections of normal thyroids and various benign and malignant thyroid disorders. Tg was present in all cases. T(4)-containing Tg was found in the adenomas, except in Hurthle cell adenomas. It was never detected in carcinomas. NIS was reduced in all types of carcinomas, whereas it was detected in noncancerous tissues. Pendrin was not expressed in carcinomas, except in follicular carcinomas, where weak staining persisted. TPO expression was present in insular, follicular carcinomas and in follicular variants of papillary carcinomas, but in a reduced percentage of cells. It was below the level of detection in papillary carcinomas. The H(2)O(2)-generating system, ThOXs, was found in all carcinomas and was even increased in papillary carcinomas. Its staining was apical in normal thyroids, whereas it was cytoplasmic in carcinomas. The TSHr was expressed in all cases, but the intensity of the staining was decreased in insular carcinomas. In conclusion, our work shows that all types of carcinomas lose the capacity to synthesize T(4)-rich, iodinated Tg. In follicular carcinomas, this might be due to a defect in iodide transport at the basolateral pole of the cell. In papillary carcinomas, this defect seems to be coupled to an altered apical transport of iodide and probably TPO activity. The TSHr persists in virtually all cases.
Assuntos
Carcinoma Papilar/metabolismo , Bócio/metabolismo , Iodo/metabolismo , Proteínas de Membrana Transportadoras , NADPH Oxidases , Tireoglobulina/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Biomarcadores , Carcinoma Papilar/patologia , Proteínas de Transporte/metabolismo , Oxidases Duais , Flavoproteínas/metabolismo , Bócio/patologia , Humanos , Imuno-Histoquímica , Iodeto Peroxidase/metabolismo , Receptores da Tireotropina/metabolismo , Transportadores de Sulfato , Simportadores/metabolismo , Neoplasias da Glândula Tireoide/patologia , Tiroxina/metabolismoRESUMO
Vasopressin has been shown previously to lower the glucagon-induced increase of cyclic AMP levels in isolated rat hepatocytes by way of an enhanced phosphodiesterase (EC 3.1.4.17) activity. Five phosphodiesterase inhibitors were tested for their ability to prevent vasopressin from lowering cyclic AMP levels in intact hepatocytes and for their inhibitory effect in vitro on soluble and particulate phosphodiesterase activities partially purified from hepatocytes. Three soluble activities have been separated by DEAE-cellulose chromatography: a phosphodiesterase hydrolyzing both cyclic AMP and cyclic GMP, a form stimulated by cyclic GMP and a cyclic AMP-specific activity. The absence of any statistically significant correlation between the in vivo (in intact cells) and the in vitro (on isolated phosphodiesterases) potencies of the inhibitors does not support a role for the cytosolic phosphodiesterases in mediating the vasopressin-induced decrease in cyclic AMP levels. No statistically significant correlation was observed between the inhibition of the vasopressin effect on cyclic AMP accumulation and the inhibition of phosphodiesterase activity either associated with the native plasma membranes or solubilized from these membranes with 0.4 M NaCl. In contrast, a statistically significant correlation was observed between the degree of inhibition of the vasopressin effect in the intact cells and the degree of inhibition of the intrinsic phosphodiesterase still associated with the plasma membranes after high-salt treatment. These data indicate that a phosphodiesterase activity integral to the plasma membrane is very likely involved in the negative control of cyclic AMP levels by vasopressin.
Assuntos
AMP Cíclico/metabolismo , Fígado/metabolismo , Diester Fosfórico Hidrolases/fisiologia , Vasopressinas/farmacologia , Animais , Membrana Celular/enzimologia , Glucagon/farmacologia , Fígado/efeitos dos fármacos , Masculino , Inibidores de Fosfodiesterase/farmacologia , Ratos , Ratos EndogâmicosRESUMO
The introduction of exogenous DNA into mammalian cells is commonly used to study the functions of gene products. However cells in primary culture are usually refractory to most transfection systems. Here we investigated the ability of a new lipid formulation, FuGENE 6 transfection reagent, to promote DNA uptake into dog and human thyroid cells in primary culture. Gene transfer was monitored by the expression of a Green Fluorescent Protein (GFP) reporter gene. We report that FuGENE 6 is particularly suited for the transfection of thyroid cells and does not interfere with their normal growth. Optimization of the experimental conditions, such as DNA amount, DNA/lipid ratio, cell density and incubation with the transfection mixture, was achieved by evaluating the percentage of GFP-expressing cells by FACS analysis. FuGENE 6 allowed us to obtain 8-15% thyrocytes expressing the reporter gene which represents an efficiency 100-fold superior to other transfection methods.
Assuntos
Expressão Gênica , Proteínas Luminescentes/genética , Glândula Tireoide/metabolismo , Transfecção/métodos , Animais , Células Cultivadas , Citomegalovirus/genética , DNA/metabolismo , Cães , Citometria de Fluxo , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes , Metabolismo dos Lipídeos , Regiões Promotoras GenéticasRESUMO
A crucial step in thyroid hormone synthesis is the oxidative coupling of iodide to thyroglobulin that is catalyzed by thyroperoxidase. The limiting factor of this reaction is the supply of hydrogen peroxide. The generation of hydrogen peroxide has been linked to an enzymatic system located at the apical pole of thyrocytes. This enzymatic activity is assumed to be exerted by NADPH oxidases encoded by two recently cloned genes hThOX1 and hThOX2. Both genes are expressed at high levels in thyrocytes. In this study we report the chromosomal organization of these two genes and the functional characterization of their respective promoter regions. The two human ThOX genes are arranged in a head to head configuration and are separated by a 16 kb-long region. Human ThOX1 and ThOX2 genes span 75 kb and are composed of 35 and 34 exons, respectively. The promoters of both genes do not resemble each other and differ from promoters of other known thyroid-specific genes. No TATA box is present in either ThOX gene promoter. Functional studies confirm that both promoters display significant transcriptional activities after transfection in differentiated thyroid cell lines. However, in contrast to that of thyroglobulin or Na(+)/I(-) symporter gene promoter, hThOX promoter activity is not restricted to thyroid cells. Additionally, functional studies show that both hThOX promoters are not positively controlled by cAMP.
Assuntos
Região 5'-Flanqueadora/genética , Flavoproteínas/genética , NADPH Oxidases/genética , Sequência de Bases , AMP Cíclico/farmacologia , Oxidases Duais , Flavoproteínas/química , Componentes do Gene , Humanos , Dados de Sequência Molecular , Estrutura Molecular , NADPH Oxidases/química , Regiões Promotoras Genéticas , Glândula Tireoide/citologia , Transcrição Gênica , TransfecçãoRESUMO
In FRTL-5 cells, cultured over a period of more than 3 years, different properties of the cells have been observed to undergo spontaneous changes in the course of aging, i.e. after an increase in the number of passages. This consists mainly in alterations in their morphological phenotype and in some of their functional properties. The morphology of the cells displayed a progressive disruption of the monolayer organization with a loss of cell-cell contacts and a marked rounding-up of the cells. The uptake of iodide was not modified nor was the expression of thyroglobulin (Tg) mRNA as determined at various time intervals in the course of the cells culturing. Estimation of the proliferation by counting the frequency of [3H]thymidine labeled nuclei revealed an age-related decline in the sensitivity to TSH mitogenic action associated with a reciprocal increase in the insulin synergistic effect. Aged cells (+/- 40 passages) lost their apoptosis sensitivity to the phosphatase inhibitor, okadaic acid (OA) but not to cycloheximide (CHX) and/or actinomycin D (act. D) exposure. Altogether these observations favor the existence of a shift towards transformed properties with only partial loss of differentiated functions.
Assuntos
Apoptose/fisiologia , Senescência Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Ácido Okadáico/farmacologia , Glândula Tireoide/fisiologia , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular , Ratos , Glândula Tireoide/patologia , Tireotropina/farmacologiaRESUMO
The function and the growth of adult thyroid gland is controlled by the opposite actions of thyrotropin (TSH) and iodide, the main substrate of the gland. Iodide deprivation leads to stimulation of the thyroid, improving the efficiency of iodide transport for hormone biosynthesis. We have investigated cell proliferation and thyroid specific gene expression 24 and 48 h after administering KI to dogs previously treated with goitrogens and perchlorate. In the hypothyroid dogs T3 and T4 serum levels decreased from 53 +/- 4 to < 30 ng/dl and from 1.6 +/- 0.6 to < 1 microg/dl respectively; TSH concentration increased from 0.16 +/- 0.02 to 2.7 +/- 0.4 ng/ml. After a 24 h moderate KI treatment (300 microg KI/dog of +/- 10 kg) serum T3 concentrations rose higher than the initial normal values, while T4 concentrations increased to reach values equivalent to the normal level. The high TSH concentration did not change significantly. The hyperplasia of the chronically stimulated thyroid resulting from goitrogens/NaClO4 treatment was not modified by this short term treatment with KI. In contrast, KI decreased the weight of the total gland and the level of cell proliferation, as determined by the fraction of cells incorporating BrdU. The effect of acute administration of KI on the expression of four major thyroid genes, the TSH receptor (TSHr), thyroglobulin (Tg), thyroperoxidase (TPO), and Na+/I- symporter (NIS) was analyzed by Northern blot. Tg, TPO and NIS mRNA expressions were up-regulated by chronic stimulation. The expression of the mRNAs of TSHr and Tg did not significantly differ between hyperstimulated and KI-treated dogs while TPO and NIS mRNA expression decreased after a 48 h KI treatment. TPO and NIS are therefore the only of these four genes whose expression is acutely modulated by iodide in vivo. Under TSH stimulation low doses of iodide resulted in: (1) decreased cell proliferation, (2) reestablished synthesis and secretion of thyroid hormones, (3) diminished TPO and NIS mRNA expression. Notably low doses of iodide under the same conditions had no effect on Tg and TSHr mRNA expression.
Assuntos
Proteínas de Transporte/genética , Divisão Celular/efeitos dos fármacos , Iodeto Peroxidase/genética , Proteínas de Membrana/genética , Iodeto de Potássio/farmacologia , RNA Mensageiro/metabolismo , Simportadores , Glândula Tireoide/efeitos dos fármacos , Animais , Northern Blotting , Cães , Expressão Gênica/efeitos dos fármacos , Hipotireoidismo/metabolismo , Percloratos/farmacologia , Propiltiouracila/farmacologia , Receptores da Tireotropina/genética , Compostos de Sódio/farmacologia , Tireoglobulina/genética , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Tireotropina/sangue , Tiroxina/sangue , Tri-Iodotironina/sangueRESUMO
Properties of sech/tanh and sin/cos half-passage RF pulses are discussed in view of their use in surface coil MR spectroscopy. We focus on the use of these pulses in a regime which is partially adiabatic, i.e. not strictly adiabatic off-resonance, while on-resonance the adiabaticity condition is fulfilled. It is shown that the frequencies of the singular points of the excitation profiles, as well as their number, depend on the B1 field. This leads to a signal intensity reduction from off-resonance spectral regions over much broader ranges than generally believed. We show in particular that with surface coil, sin/cos RF pulses may perform particularly well, providing optimal excitation on resonance and a desired attenuation over a broad spectral range off-resonance. This feature is applied for the in vivo detection of rat liver glycogen by means of 13C MR spectroscopy. Under suitable RF power conditions, a remarkable attenuation of the signals from the saturated carbons of the subcutaneous fat can be achieved.
Assuntos
Glicogênio Hepático/análise , Espectroscopia de Ressonância Magnética/métodos , Animais , RatosRESUMO
NADPH oxidases (NOXes) and dual oxidases (DUOXes) generate O2 (.-) and H2O2. Diphenyleneiodonium (DPI) inhibits the activity of these enzymes and is often used as a specific inhibitor. It is shown here that DPI, at concentrations similar to those which inhibit the generation of O2 derivatives, activated the efflux of radioiodide but not of its analog (99m)TcO4 (-) nor of the K(+) cation mimic (86)Rb(+) in thyroid cells, in the PCCl3 rat thyroid cell line and in COS cell lines expressing the iodide transporter NIS. Effects obtained with DPI, especially in thyroid cells, should therefore be interpreted with caution.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Proteínas de Ligação ao Cálcio/farmacologia , Calmodulina/farmacologia , Carbacol/farmacologia , AMP Cíclico/metabolismo , Glândula Tireoide/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , 4-(3-Butoxi-4-metoxibenzil)-2-imidazolidinona/farmacologia , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1 , Cães , Técnicas In Vitro , Cinética , Glândula Tireoide/efeitos dos fármacosAssuntos
1-Metil-3-Isobutilxantina/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Fígado/enzimologia , Teofilina/análogos & derivados , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Ligação Competitiva , GMP Cíclico/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Cinética , RatosAssuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Nucleotídeos Cíclicos/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/isolamento & purificação , 3',5'-GMP Cíclico Fosfodiesterases/isolamento & purificação , Glândulas Suprarrenais/enzimologia , Sítio Alostérico , Animais , Sítios de Ligação , Encéfalo/enzimologia , Calmodulina/fisiologia , Bovinos , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Ativação Enzimática , Cinética , Nucleotídeos Cíclicos/farmacologia , Ligação ProteicaAssuntos
Cálcio/farmacologia , Calmodulina/farmacologia , AMP Cíclico/fisiologia , Diester Fosfórico Hidrolases/metabolismo , Animais , Cálcio/fisiologia , Carbacol/farmacologia , GMP Cíclico/farmacologia , Cães , Ácido Egtázico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Norepinefrina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/farmacologia , Ratos , Glândula Tireoide/enzimologiaRESUMO
CREM (cAMP Response Element Modulator) transcription factors are involved in the cAMP-dependent transcriptional regulation of CRE-containing genes. Multiple CREM transactivators and repressors are generated from a single gene by alternative splicings and use of an alternative intronic promoter. Here we report the cloning and sequencing of the full-length dog CREM cDNA, corresponding to the CREMtau alpha splice variant. Amino acid sequence identity with mouse and human orthologs reached 94.5% and 91.0% respectively. Using the RNAse Protection Assay (RPA) method with three distinct probes, we analyzed the expression of the various CREM transcripts in several dog tissues. We showed that CREM transcription factors have a restricted tissue distribution and that the ratio between activators and repressors varies considerably from one tissue to another. Moreover, we amplified, by RT-PCR, a cDNA that corresponds to two new CREM isoforms and confirmed, by RPA experiments, the presence of these mRNAs in dog thyroid and in other tissues. These transcripts result from splicing of the gamma domain and encode potential CREM transactivators (CREMtau alphagamma and CREMtau2 alphagamma).